研究者詳細 - 豊國　伸哉

Iron-catalyzed oxidative stress compromises cancer promotional effect of BRCA2 haploinsufficiency through mitochondria-targeted ferroptosis. 査読有り国際誌 Open Access

Maeda Y, Motooka Y, Akatsuka S, Tanaka H, Mashimo T, Toyokuni S

Redox biology 85 巻頁： 103739 2025年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Biology

Pathogenic variants in BRCA2 are hereditary risks for various cancers, including breast, ovary, pancreas and prostate. Genomic instability due to insufficient homologous recombination is thought as responsible for carcinogenesis. Reportedly, endogenous or exogenous aldehydes, including formaldehyde and acetaldehyde, suppress BRCA2 function. However, molecular sequences how BRCA2 insufficiency leads to carcinogenesis remains unelucidated. To assess whether Fenton reaction-based oxidative stress is a promotional risk factor of carcinogenesis in BRCA2 haploinsufficiency, we here applied iron-induced renal carcinogenesis to a newly established rat heterozygous mutation model of Brca2 (mutant, T1942Kfs/+; MUT). Rat MUT model, despite significant increase in spontaneous malignant tumors, showed no promotional effect on renal carcinogenesis induced by ferric nitrilotriacetate (Fe-NTA) in contrast to our previous study using Brca1 mutant rats. Array-based comparative genome hybridization of renal cell carcinoma in MUT revealed significant increase in the frequency of homozygous Cdkn2A deletion. Whereas acute-phase analysis of the kidney after single or 1-week Fe-NTA administration to MUT showed suppressed lipid peroxidation, consistent with ferroptosis-resistance, ferroptosis and regeneration of tubular cells were coexistent with higher cytoplasmic catalytic Fe(II) levels in the subacute phase of MUT after 3-week Fe-NTA administration. Mechanistically, mitochondrial dysfunction with excess iron, promoted by insufficient BRCA2 presumably for maintaining DNA integrity, eventually initiated ferroptotic process. In conclusion, iron-dependent oxidative stress plays double-edged roles either for cell death or proliferation in carcinogenesis and its biological consequences are distinct between BRCA2 and BRCA1 haploinsufficiency. Our results suggest that iron-catalyzed oxidative stress is not a major driving force of carcinogenesis in BRCA2 pathogenic variants.

DOI： 10.1016/j.redox.2025.103739

LRP2 is a potential molecular target for nonsyndromic pathological myopia 査読有り国際誌 Open Access

Delaunay, K; Picard, E; Lassiaz, P; Jonet, L; Cannaya, V; Ruiz-Moreno, JM; Kojima, K; Vorum, H; Honore, B; Medrano, JR; Cehofski, LJ; Pussard, E; Kozyraki, R; Torriglia, A; Cases, O; Behar-Cohen, F

JCI INSIGHT 10 巻 ( 15 ) 2025年8月

Terrestrial iron sulfide minerals induce distinct regulation of intracellular redox homeostasis and iron assimilation. 査読有り国際誌 Open Access

Lyu Q, Kouketsu Y, Tazaki A, Kato M, Motooka Y, Toyokuni S

Ecotoxicology and environmental safety 298 巻頁： 118327 2025年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Ecotoxicology and Environmental Safety

Repeated exposure to airborne terrestrial natural minerals may cause pneumoconiosis and lung cancer, among which iron sulfide is identified as an aggravating factor. In the biological system, iron-sulfur cluster is an inorganic cofactor that is evolutionarily conserved in all the living organisms. Whereas ferrous iron catalyzes the generation of hydroxyl radicals, sulfur is indispensable as a component of antioxidants, such as glutathione. Imbalanced redox homeostasis contributes to oxidative stress, causing ferroptosis, an iron-dependent regulated necrosis characterized by lipid peroxidation, resulting in various disorders. We undertook this study to understand the cellular regulatory mechanisms against major terrestrial minerals containing iron and sulfur from the viewpoint of cellular redox. We used fundamental iron sulfide minerals collected from natural sources to treat human macrophage and fibroblast cells and investigated the biological responses. Alterations in sulfane sulfur, glutathione and iron have been analyzed using either specific fluorescent probes or inductively coupled plasma mass spectrometry. Iron sulfide microparticles with high Fe/S ratio (pyrrhotite; Fe<inf>1-X</inf>S) induced more reactive sulfane species and glutathione, with less catalytic iron inside cells, whereas the mineral with low Fe/S ratio (pyrite; FeS<inf>2</inf>) exhibited the opposite effects. Notably both showed cytotoxicity, where pyrite caused ferroptosis but pyrrhotite led to non-ferroptotic disruption. Furthermore, assimilated cellular excess iron was secreted via CD63(+) exosome containing iron-loaded ferritin to the extracellular space with higher iron content in pyrrhotite. Our findings suggest that iron and sulfur work complementarily in maintaining intracellular redox homeostasis, which would be crucial to understand the associated pathology.

DOI： 10.1016/j.ecoenv.2025.118327

Selective Killing of Human Breast Cancer Cells by Plasma-Treated L-Arginine Solution 査読有り国際誌 Open Access

Du, LY; Miron, C; Kondo, T; Kumagai, J; Mizuno, M; Toyokuni, S; Kajiyama, H; Hori, M; Tanaka, H

PLASMA PROCESSES AND POLYMERS 22 巻 ( 4 ) 2025年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plasma Processes and Polymers

l-arginine is a nonessential amino acid that plays an important role in normal immune function and can be used as an adjuvant treatment for cancers. The antitumor effect of nonequilibrium atmospheric pressure plasma treated l-arginine solutions (PTA) was investigated in this research. The cancer-specific killing effect of PTA on human breast epithelial cells (MCF-10A) and human breast cancer cells (MCF-7) was proved. Liquid chromatography-tandem mass spectrometry was used to analyze the organic compounds formed in PTA, and their effects were individually evaluated by MTS assay, revealing that some compounds inhibited cell proliferation but lacked selectivity. PTA's selective cytotoxicity may result from a mixture of various compounds formed in plasma combined in a certain concentration.

DOI： 10.1002/ppap.202400192

Selenoprotein-mediated redox regulation shapes the cell fate of HSCs and mature lineages. 査読有り国際誌 Open Access

Aoyama Y, Yamazaki H, Nishimura K, Nomura M, Shigehiro T, Suzuki T, Zang W, Tatara Y, Ito H, Hayashi Y, Koike Y, Fukumoto M, Tanaka A, Zhang Y, Saika W, Hasegawa C, Kasai S, Kong Y, Minakuchi Y, Itoh K, Yamamoto M, Toyokuni S, Toyoda A, Ikawa T, Takaori-Kondo A, Inoue D

Blood 145 巻 ( 11 ) 頁： 1149 - 1163 2025年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Blood

The maintenance of cellular redox balance is crucial for cell survival and homeostasis and is disrupted with aging. Selenoproteins, comprising essential antioxidant enzymes, raise intriguing questions about their involvement in hematopoietic aging and potential reversibility. Motivated by our observation of messenger RNA downregulation of key antioxidant selenoproteins in aged human hematopoietic stem cells (HSCs) and previous findings of increased lipid peroxidation in aged hematopoiesis, we used selenocysteine transfer RNA (tRNASec) gene (Trsp) knockout (KO) mouse model to simulate disrupted selenoprotein synthesis. This revealed insights into the protective roles of selenoproteins in preserving HSC stemness and B-lineage maturation, despite negligible effects on myeloid cells. Notably, Trsp KO exhibited B lymphocytopenia and reduced HSCs’ self-renewal capacity, recapitulating certain aspects of aged phenotypes, along with the upregulation of aging-related genes in both HSCs and pre-B cells. Although Trsp KO activated an antioxidant response transcription factor NRF2, we delineated a lineage-dependent phenotype driven by lipid peroxidation, which was exacerbated with aging yet ameliorated by ferroptosis inhibitors such as vitamin E. Interestingly, the myeloid genes were ectopically expressed in pre-B cells of Trsp KO mice, and KO pro-B/pre-B cells displayed differentiation potential toward functional CD11b+ fraction in the transplant model, suggesting that disrupted selenoprotein synthesis induces the potential of B-to-myeloid switch. Given the similarities between the KO model and aged wild-type mice, including ferroptosis vulnerability, impaired HSC self-renewal and B-lineage maturation, and characteristic lineage switch, our findings underscore the critical role of selenoprotein-mediated redox regulation in maintaining balanced hematopoiesis and suggest the preventive potential of selenoproteins against aging-related alterations.

DOI： 10.1182/blood.2024025402

Wavelength-dependency of the impact of light on proliferation and DNA damage of corneal cells in vitro 査読有り国際誌 Open Access

Francon, A; Behar-Cohen, F; Torriglia, A

JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 264 巻 2025年3月

Silicone oil, an intraocular surgical adjuvant, induces retinal ferroptosis. 査読有り国際誌 Open Access

Shimizu H, Tanaka H, Tazaki A, Yamada K, Suzumura A, Ota J, Ushio-Watanabe N, Zheng H, Kataoka K, Hara H, Nishikawa Y, Yasukawa T, Suzuma K, Terasaki H, Nishiguchi KM, Kato M, Toyokuni S, Kaneko H

Free radical biology & medicine 228 巻頁： 33 - 43 2025年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Vitrectomy with silicone oil (SO) endotamponade is an effective treatment for vision-threatening retinal diseases. However, unexplained vision impairment has been reportedly critical side effects. Previously, we reported that the eyes with ocular toxoplasmosis showed retinal ferroptosis with the clinical sign of reduced intravitreal iron (Fe). We also found that total iron levels in sub-silicone oil fluid (SOF) in eyes with SO endotamponade were significantly reduced. We hypothesized that the cause of complications related to SO endotamponade is retinal ferroptosis and that low total iron in SOF is a secondary change that occurs similarly to the changes in ocular toxoplasmosis. In this study, we measured total iron levels in ocular fluid from patients, rabbits with SO endotamponade. Retinal iron taken up from the SOF was evaluated using laser ablation inductively coupled plasma mass spectrometry in human and rabbit eyes. Retinal ferroptosis was confirmed by immunohistochemistry of 4-hydrox-2-nonenal-modified proteins, FeRhoNox-1 staining, western blotting and RT-PCR. We found low total iron levels in the SOF, increased oxidative stress and Fe uptake from the SOF into the retinae of human and rabbit eyes, as well as decreased GPx4 expression, increased FeRhoNox-1 signals and altered Fe-related gene expression in SO-filled rabbit eyes. Of note, the target of ferroptosis was Müller cells. We generated an in vitro silicone oil-filled eye model using MIO-M1 cells (a human Müller cell line). The in vitro SO-filled eye model showed decreased GPx4 expression and increased intracellular catalytic Fe(II), an increase in ferroptosis, prevention of cell death by ferrostatin-1, a ferroptosis inhibitor, and altered Fe-related gene expression. These results indicate that the cause of complications related to SO endotamponade was the induction of retinal (Müller cell) ferroptosis, which can be prevented by ferrostatin-1.

DOI： 10.1016/j.freeradbiomed.2024.12.039

Repeated exposure to low doses of light induces retinal damage in vivo in a wavelength-dependent manner 査読有り国際誌 Open Access

Françon, A; Jonet, L; Behar-Cohen, F; Torriglia, A

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 290 巻 2025年1月

Decoding the molecular enigma behind asbestos and fibrous nanomaterial-induced carcinogenesis. 国際誌 Open Access

Toyokuni S, Kong Y

Journal of occupational health 67 巻 ( 1 ) 頁： n/a 2025年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Occupational Health

Objectives: The natural fibrous mineral, asbestos, has been useful in industry for many centuries. In the 1960s, epidemiology recognized the association between asbestos exposure and mesothelioma, and in 1987 the International Agency for Research on Cancer designated all kinds of asbestos as Group 1 carcinogens. However, various scientific enigmas remained regarding the molecular mechanisms of asbestos-induced mesothelial carcinogenesis. This review article was undertaken to reveal and summarize recent discoveries to resolve those enigmas. Methods: We collected recent important findings from our own laboratory and others to explain why mesothelial cells are the target for asbestos-induced carcinogenesis and what are the key molecular mechanisms. Results: The long incubation period of 30-40 years for mesothelial carcinogenesis after asbestos exposure allows the asbestos fibers to go through the pulmonary parenchyma from the central to peripheral portions and ultimately reach the parietal mesothelium by piercing visceral pleura. Asbestos fibers have affinity for hemoglobin and histones, thus accumulating iron on the surface while traveling through the lung. Mesothelial cells are phagocytic cells, engulfing iron-coated asbestos fibers. Accordingly, homozygous deletion of the p16INK4a tumor suppressor gene, a signature of excess iron-induced carcinogenesis, is acquired through oxidative DNA damage. Recently, exosome-dependent iron transfer from asbestos-fed macrophages to mesothelial cells was reported. Similar molecular mechanisms are observed with multiwalled carbon nanotubes of ∼50-nm diameter. Conclusions: Physical dimensions, biopersistence, and affinity to iron/histones are essential for fibrous material to be carcinogenic to mesothelial cells. Therefore, local iron reduction may be a strategy to prevent mesothelial carcinogenesis.

DOI： 10.1093/joccuh/uiae064

BRCA1 haploinsufficiency impairs iron metabolism to promote chrysotile-induced mesothelioma via ferroptosis resistance 査読有り国際誌

Luo, YG; Toyokuni, S

CANCER SCIENCE 116 巻頁： 193 - 193 2025年1月

Complementary Interplay of Iron and Sulfur: Implications for Cellular Redox Regulation 査読有り国際誌

Lyu, QY; Toyokuni, S; Kouketsu, Y

CANCER SCIENCE 116 巻頁： 1388 - 1388 2025年1月

BRCA1 haploinsufficiency interferes mitochondria under Fenton reaction-based carcinogenesis 査読有り国際誌

Kong, Y; Mashimo, T; Imaoka, T; Toyokuni, S

CANCER SCIENCE 116 巻頁： 648 - 648 2025年1月

Homozygous slc25a20 zebrafish mutant reveals insights into carnitine-acylcarnitine translocase deficiency pathogenesis. 査読有り国際誌 Open Access

Hishida R, Ishiguro K, Yamanaka T, Toyokuni S, Matsui H

Molecular genetics and metabolism reports 41 巻頁： 101165 2024年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Molecular Genetics and Metabolism Reports

The SLC25A20 gene encodes carnitine-acylcarnitine translocase (CACT), facilitating the transport of long-chain acylcarnitine required for energy production via β-oxidation into the mitochondria. Loss-of-function mutations in this gene lead to CACT deficiency, a rare autosomal recessive disorder of fatty acid metabolism characterized by severe symptoms including cardiomyopathy, hepatic dysfunction, rhabdomyolysis, hypoketotic hypoglycemia, and hyperammonemia, often resulting in neonatal mortality. Here, we utilized CRISPR/Cas9 gene editing to isolate slc25a20 mutant zebrafish. Homozygous mutants displayed significant lethality, with the majority succumbing before reaching maturity. However, we identified a notably rare homozygous individual that survived into adulthood, prompting a histological examination. Firstly, we observed adipose tissue accumulation at various sites in the homozygous mutant. The mutant heart exhibited hypertrophy, along with degenerated myocardial and muscle cells containing numerous eosinophilic nuclei. Additionally, we found no large oil droplet vacuoles in the mutant liver; however, the hepatocytes displayed numerous small vacuoles resembling lipid droplets. Iron deposition was evident in the spleen and parts of the liver. Overall, our slc25a20 zebrafish mutant displayed tissue pathologies analogous to human CACT deficiency, suggesting its potential as a pathological model contributing to the elucidation of pathogenesis and the improvement/development of therapies for CACT deficiency.

DOI： 10.1016/j.ymgmr.2024.101165

New era of plasma dentistry. 査読有り国際誌

Sato K, Tanaka H, Okazaki Y, Hori M, Hibi H, Toyokuni S

Free radical research 58 巻 ( 12 ) 頁： 868 - 874 2024年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Plasma, which was coined by Irving Langmuir in 1928, is the fourth physical state after the solid/liquid/gas phases. Low-temperature plasma (LTP) is a contradictory condition that involves high energy with free radicals at near-body temperatures and was developed through engineering in the 1990’s. Research on LTP in engineering and medical fields has rapidly developed since the 2000’s. LTP can be applied through direct or indirect exposure, and there are advantages to both methods. In the medical field, LTP has been found to exert several effects, such as wound healing, hemostasis and anticancer effects, mainly based on different levels of oxidative stress. In the dental field, studies have been performed on LTP applications for general dental procedures, such as restorative, periodontal and prosthodontic procedures, and for oral cancer treatment. Many studies have demonstrated the effectiveness of LTP. Compared with other organs, the anatomical characteristic of the oral cavity is easy direct observation, which is highly advantageous for clinical applications. Due to its good accessibility and efficiency, plasma dentistry is expected to be applied to various dental applications in clinics in the near future.

DOI： 10.1080/10715762.2024.2446323

Deficiency in glutathione peroxidase 4 (GPX4) results in abnormal lens development and newborn cataract. 査読有り国際誌 Open Access

Wei Z, Hao C, Radeen KR, Hao Z, Kettimuthu K, Maner-Smith K, Toyokuni S, Fan X

Proceedings of the National Academy of Sciences of the United States of America 121 巻 ( 48 ) 頁： e2407842121 2024年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

The human lens is composed of a monolayer of lens epithelial cells (LECs) and elongated fibers that align tightly but are separated by the plasma membrane. The integrity of the lens plasma membrane is crucial for maintaining lens cellular structure, homeostasis, and transparency. Glutathione peroxidase 4 (GPX4), a selenoenzyme, plays a critical role in protecting against lipid peroxidation. This study aims to elucidate the role of GPX4 in lens plasma membrane stability during lens development using in vitro, ex vivo, and in vivo systems. Our findings reveal that GPX4 deficiency triggers lens epithelial apoptosis-independent but ferroptosis-mediated cell death. Blocking lens GPX4 activity during ex vivo culture induces lens opacification, LEC death, and disruption of lens fiber cell arrangement. Deletion of lens-specific Gpx4 results in significant unsaturated phospholipid loss and an increase in oxidized phospholipids. Consequently, lenses with Gpx4 deficiency exhibit massive disruption of lens fiber cell structure, significant loss of LECs via ferroptosis, and formation of newborn cataracts. Remarkably, administering the lipid peroxidation inhibitor, liproxstatin-1, to pregnant mothers at embryonic days 9.5 significantly prevents lipid peroxidation, LEC death, and lens developmental defects. Our study unveils the crucial role of GPX4 in lens development and transparency, and also provides a successful intervention approach to prevent lens developmental defects through lipid peroxidation inhibition.

DOI： 10.1073/pnas.2407842121

Low-temperature plasma as a strategy to achieve SDGs. 査読有り国際誌 Open Access

Tanaka H, Ishikawa K, Toyokuni S

Free radical research 58 巻 ( 10 ) 頁： 594 - 595 2024年10月

Glyburide confers neuroprotection against age-related macular degeneration (AMD) 査読有り国際誌 Open Access

Picard, E; Youale, J; Hyman, MJ; Xie, ED; Achiedo, S; Kaufmann, GT; Moir, J; Daruich, A; Crisanti, P; Torriglia, A; Polak, M; Behar-Cohen, F; Skondra, D; Berdugo, M

TRANSLATIONAL RESEARCH 272 巻頁： 81 - 94 2024年10月

Ferroptosis in health and disease. 査読有り国際共著国際誌 Open Access

Berndt C, Alborzinia H, Amen VS, Ayton S, Barayeu U, Bartelt A, Bayir H, Bebber CM, Birsoy K, Böttcher JP, Brabletz S, Brabletz T, Brown AR, Brüne B, Bulli G, Bruneau A, Chen Q, DeNicola GM, Dick TP, Distéfano A, Dixon SJ, Engler JB, Esser-von Bieren J, Fedorova M, Friedmann Angeli JP, Friese MA, Fuhrmann DC, García-Sáez AJ, Garbowicz K, Götz M, Gu W, Hammerich L, Hassannia B, Jiang X, Jeridi A, Kang YP, Kagan VE, Konrad DB, Kotschi S, Lei P, Le Tertre M, Lev S, Liang D, Linkermann A, Lohr C, Lorenz S, Luedde T, Methner A, Michalke B, Milton AV, Min J, Mishima E, Müller S, Motohashi H, Muckenthaler MU, Murakami S, Olzmann JA, Pagnussat G, Pan Z, Papagiannakopoulos T, Pedrera Puentes L, Pratt DA, Proneth B, Ramsauer L, Rodriguez R, Saito Y, Schmidt F, Schmitt C, Schulze A, Schwab A, Schwantes A, Soula M, Spitzlberger B, Stockwell BR, Thewes L, Thorn-Seshold O, Toyokuni S, Tonnus W, Trumpp A, Vandenabeele P, Vanden Berghe T, Venkataramani V, Vogel FCE, von Karstedt S, Wang F, Westermann F, Wientjens C, Wilhelm C, Wölk M, Wu K, Yang X, Yu F, Zou Y, Conrad M

Redox biology 75 巻頁： 103211 2024年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Biology

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells’ susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with – or caused by – ferroptosis.

DOI： 10.1016/j.redox.2024.103211

Growth promotion and disease tolerance in strawberry plants with plasma-activated Ringer’s lactate solution 査読有り国際誌

Sakai K., Matsubara Y., Hashizume H., Tanaka H., Toyokuni S., Hori M.

Acta Horticulturae 3 巻 ( 1404 ) 頁： 917 - 922 2024年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Acta Horticulturae

Recently, plasma-activated solutions have been attempted on biotic regulations mainly in medical aspects. In this study, effect of plasma-activated Ringer’s lactate solution (PAL) on disease (anthracnose, Fusarium wilt) tolerance and antifungal function in strawberry plants was investigated. Runner-derived strawberry (Fragaria × ananassa Duch. ‘Benihoppe’) plants were treated with Fuji-γ-PAL and Strong PAL (×100, ×300 dilution) to leaves or roots. Then, plants were inoculated with Colletotrichum fructicola (MAFF306788, 105 cfu mL-1); causable pathogen of anthracnose, or Fusarium oxysporum f. sp. fragariae (NRF0903, 106 cfu mL-1); pathogen of Fusarium wilt. Increase in dry weight of shoots occurred in Strong PAL 100-treated plants. Incidence and severity in symptoms of anthracnose and Fusarium wilt were alleviated in most of the PAL-treated plants in both diseases. In this case, Strong-PAL showed relatively higher suppression effect than Fuji-γ-PAL in both diseases. As for antifungal aspects in vitro using Czapek-Doc medium, both PALs suppressed Fusarium propagation; Strong PAL showed higher suppression effect compared to Fuji-γ-PAL. On the other hand, Strong PAL 100-treated plants showed higher DPPH radical scavenging activity in leaves among the treatments. These findings suggest that PAL induce growth promotion and disease tolerance in strawberry plants, and these effects differ with the method of PAL treatment.

DOI： 10.17660/ActaHortic.2024.1404.125

特集フェロトーシス(鉄依存性細胞死)--そのメカニズムの解明と,治療への応用フェロトーシス抵抗性と発がん国際誌

前田勇貴, 豊國伸哉

医学のあゆみ 290 巻 ( 2 ) 頁： 174 - 177 2024年7月

Phototoxicity of low doses of light and influence of the spectral composition on human RPE cells 査読有り国際誌 Open Access

Françon, A; Delaunay, K; Jaworski, T; Lebon, C; Picard, E; Youale, J; Behar-Cohen, F; Torriglia, A

SCIENTIFIC REPORTS 14 巻 ( 1 ) 2024年3月

Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria. 査読有り国際誌 Open Access

Mi D, Yanatori I, Zheng H, Kong Y, Hirayama T, Toyokuni S

Free radical research 58 巻 ( 4 ) 頁： 261 - 275 2024年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here, we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S-transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC–MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane. Although direct association was not observed between PCBP2 and SFXN3 with co-immunoprecipitation, proximity ligation assay demonstrated proximal localization of PCBP2 with TOM20 and there was a direct binding between TOM20 and SFXN3. Single knockdown either of PCBP2 and SFXN3 in K562 leukemia cells significantly decreased mitochondrial catalytic Fe(II) and mitochondrial maximal respiration. SFXN3 but not MFRN1 knockout (KO) in mouse embryonic fibroblasts decreased FBXL5 and heme oxygenase-1 (HO-1) but increased transferrin uptake and induced ferritin, indicating that mitochondrial iron entry through SFXN3 is distinct. MFRN1 KO revealed more intense mitochondrial Fe(II) deficiency than SFXN3 KO. Insufficient mitochondrial heme synthesis was evident under iron overload both with SFXN3 and MFRN KO, which was partially reversed by HO-1 inhibitor. Conversely, SFXN3 overexpression caused cytosolic iron deficiency with mitochondrial excess Fe(II), which further sensitized HeLa cells to RSL3-induced ferroptosis. In conclusion, we discovered a novel pathway of iron entry into mitochondria from cytosol through PCBP2-TOM20-SFXN3 axis.

DOI： 10.1080/10715762.2024.2340711

Analysis of spontaneous pancreatic endocrine tumors in Gcggfp/gfp mice 査読有り国際誌

Hori, M; Maeda, K; Imai, T; Hippo, Y; Toyokuni, S; Hayashi, Y

CANCER SCIENCE 115 巻頁： 181 - 181 2024年3月

Tracking BRCA1 haploinsufficiency focusing mitochondrial metabolism under Fenton reactionbased carcinogenesis 査読有り国際誌

Kong, YY; Mashimo, T; Imaoka, T; Toyokuni, S

CANCER SCIENCE 115 巻頁： 195 - 195 2024年3月

Role of iron, oxygen and ferroptosis in tumor evolutionary biology 査読有り国際誌

Toyokuni, S; Kong, YY; Luo, YG; Zheng, H; Motooka, Y; Imaoka, T; Akatsuka, S

CANCER SCIENCE 115 巻頁： 907 - 907 2024年3月

Involvement of iron-sulphur cofactors in regulation of mammalian THP-1 macrophage iron homeostasis and redox status 査読有り国際誌

Lyu, QY; Toyokuni, S; Kouketsu, Y

CANCER SCIENCE 115 巻頁： 908 - 908 2024年3月

Genomic distribution of 8-oxoguanines in the kidney genome of ferric nitrilotriacetate -treated mice 査読有り国際誌

Akatsuka, S; Toyokuni, S

CANCER SCIENCE 115 巻頁： 1279 - 1279 2024年3月

Effect of aging and PARP inhibitor, olaparib on ovarian function with BRCA1 mutant model rats 査読有り国際誌

Sonehara, R; Motooka, Y; Mashimo, T; Imaoka, T; Kajiyama, H; Toyokuni, S

CANCER SCIENCE 115 巻頁： 170 - 170 2024年3月

BRCA2 germline mutation promotes follicle development and induces diminished ovarian reserve 査読有り国際誌

Tanaka, H; Motooka, Y; Maeda, Y; Mashimo, T; Kajiyama, H; Toyokuni, S

CANCER SCIENCE 115 巻頁： 172 - 172 2024年3月

Epstein-Barr virus lytic gene BNRF1 promotes B-cell lymphomagenesis via IFI27 upregulation. 査読有り国際誌 Open Access

Sagou K, Sato Y, Okuno Y, Watanabe T, Inagaki T, Motooka Y, Toyokuni S, Murata T, Kiyoi H, Kimura H

PLoS pathogens 20 巻 ( 2 ) 頁： e1011954 2024年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plos Pathogens

Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.

DOI： 10.1371/journal.ppat.1011954

Cold Atmospheric Pressure Plasma-Activated Liquids for Cancer Treatment 査読有り国際誌

Miron, C; Hiromasa, T; Britun, N; Hashizume, H; Ishikawa, K; Du, LY; Yamakawa, T; Kurebayashi, Y; Kondo, T; Kondo, H; Kajiyama, H; Toyokuni, S; Mizuno, M; Hori, M

ADVANCES IN DIGITAL HEALTH AND MEDICAL BIOENGINEERING, VOL 3, EHB-2023 111 巻頁： 150 - 162 2024年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Ifmbe Proceedings

Low-temperature plasma (LTP) is a promising tool that can assist in cancer treatment. Plasma-treated liquids have shown a selective cytotoxic effect on cancer cells attributed to reactive species formed in discharge, but this mechanism is not completely understood. In this study, the chemical compounds formed by plasma irradiation of Ringer’s lactate solutions and their effects on non-tumorigenic breast epithelial cells (MCF-10A) and breast cancer cells (MCF-7) were investigated. Among these compounds, glyceric acid increased the cell viability by more than two-fold compared to the control samples for MCF-10A and MCF-7 cells, whereas the tricarballylic acid had a pronounced cytotoxic effect on the cells when incubated with solutions of 0.6 mM to 50 mM concentrations. A good selectivity in killing the cancer cells was observed when the chemical compounds identified in plasma-treated liquids (such as ethyl acetate, glyceric acid, and tricarballylic acid) were used in a mixture solution of a certain concentration. The results show that the LTP-generated chemical compounds have both, stimulatory and inhibitory effects on cell viability, and the best selectivity is achieved only when these compounds are combined in a certain amount and have a synergistic effect, completely killing only the cancer cells.

DOI： 10.1007/978-3-031-62523-7_17

Role of Iron, Oxygen and Ferroptosis in Tumor Evolutionary Biology 査読有り国際誌

Toyokuni, S; Kong, Y; Luo, Y; Zheng, H; Motooka, Y; Akatsuka, S

FREE RADICAL BIOLOGY AND MEDICINE 208 巻頁： S112 - S112 2023年11月

Retinal ferroptosis as a critical mechanism for the induction of retinochoroiditis during ocular toxoplasmosis. 査読有り国際誌 Open Access

Yamada K, Tazaki A, Ushio-Watanabe N, Usui Y, Takeda A, Matsunaga M, Suzumura A, Shimizu H, Zheng H, Ariefta NR, Yamamoto M, Hara H, Goto H, Sonoda KH, Nishiguchi KM, Kato M, Nishikawa Y, Toyokuni S, Kaneko H

Redox biology 67 巻頁： 102890 2023年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Biology

Toxoplasmosis is a major infectious disease, affecting approximately one-third of the world's population; its main clinical manifestation, ocular toxoplasmosis (OT), is a severe sight-threatening disease. Nevertheless, the diagnosis of OT is based on clinical findings, which needs improvement, even with biochemical tests, such as polymerase chain reaction and antibody detections. Furthermore, the efficacy of OT-targeted treatment is limited; thus, additional measures for diagnosis and treatments are needed. Here, we for the first time report a significantly reduced iron concentration in the vitreous humor (VH) of human patients infected with OT. To obtain further insights into molecular mechanisms, we established a mouse model of T. gondii infection, in which intravitreally injected tracer 57Fe, was accumulated in the neurosensory retina. T. gondii-infected eyes showed increased lipid peroxidation, reduction of glutathione peroxidase-4 expression and mitochondrial deformity in the photoreceptor as cristae loss. These findings strongly suggest the involvement of ferroptotic process in the photoreceptor of OT. In addition, deferiprone, an FDA-approved iron chelator, reduced the iron uptake but also ameliorated toxoplasma-induced retinochoroiditis by reducing retinal inflammation. In conclusion, the iron levels in the VH could serve as diagnostic markers and iron chelators as potential treatments for OT.

DOI： 10.1016/j.redox.2023.102890

Ferroptosis contributes to multiple sclerosis and its pharmacological targeting suppresses experimental disease progression. 査読有り国際誌 Open Access

Van San E, Debruyne AC, Veeckmans G, Tyurina YY, Tyurin VA, Zheng H, Choi SM, Augustyns K, van Loo G, Michalke B, Venkataramani V, Toyokuni S, Bayir H, Vandenabeele P, Hassannia B, Vanden Berghe T

Cell death and differentiation 30 巻 ( 9 ) 頁： 2092 - 2103 2023年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Death and Differentiation

Multiple sclerosis (MS) is a chronic autoimmune disorder characterized by central nervous (CNS) demyelination resulting in axonal injury and neurological deficits. Essentially, MS is driven by an auto-amplifying mechanism of inflammation and cell death. Current therapies mainly focus on disease modification by immunosuppression, while no treatment specifically focuses on controlling cell death injury. Here, we report that ferroptosis, an iron-catalyzed mode of regulated cell death (RCD), contributes to MS disease progression. Active and chronic MS lesions and cerebrospinal fluid (CSF) of MS patients revealed several signs of ferroptosis, reflected by the presence of elevated levels of (labile) iron, peroxidized phospholipids and lipid degradation products. Treatment with our candidate lead ferroptosis inhibitor, UAMC-3203, strongly delays relapse and ameliorates disease progression in a preclinical model of relapsing-remitting MS. In conclusion, the results identify ferroptosis as a detrimental and targetable factor in MS. These findings create novel treatment options for MS patients, along with current immunosuppressive strategies. [Figure not available: see fulltext.]

DOI： 10.1038/s41418-023-01195-0

Organic decomposition and synthesis reactions in lactated solution exposed to nonequilibrium atmospheric pressure plasma 査読有り国際誌 Open Access

Liu, Y; Ishikawa, K; Tanaka, H; Miron, C; Kondo, T; Nakamura, K; Mizuno, M; Kajiyama, H; Toyokuni, S; Hori, M

PLASMA PROCESSES AND POLYMERS 20 巻 ( 5 ) 2023年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plasma Processes and Polymers

Lactate is used in the food and pharmaceutical industries and is a crucial intermediate for synthesis. Plasma-activated lactate (PAL) in Ringer's solution was recently shown to have effective antitumor action. Small molecule aldehydes, ketones, and organic acids were produced from lactate during plasma exposure, and five-membered conjugated lactone isomers of furanone (C<inf>5</inf>H<inf>6</inf>O<inf>2</inf>) were detected formed by interactions of lactate or its fragments with •OH, organic radicals, and H<inf>2</inf>O<inf>2</inf>. 2,3-Dimethyl-tartaric acid may be the effective component in PAL for the selective killing of cancer but not normal cells and possible pathways for its synthesis are provided. Aqueous reaction mechanisms are explained, including dehydration, esterification, hydrolysis, and dimerization. This study will help develop novel cancer therapies and further plasma organic chemistry.

DOI： 10.1002/ppap.202200193

Commentary on "Mechanisms of asbestos-induced carcinogenesis" published in 2009. 査読有り国際誌 Open Access

Toyokuni S

Nagoya journal of medical science 85 巻 ( 1 ) 頁： 13 - 15 2023年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nagoya Journal of Medical Science

Respiratory exposure to asbestos fibers has been associated with diffuse malignant mesothelioma (DMM) in humans. Despite advancements in the molecular analyses of human DMM and the development of animal models, the carcinogenic mechanisms of the disease remain unclear. There are basically three hypotheses regarding the pathogenesis of asbestos-induced DMM, which may be summarized as follows: (1) the “oxidative stress theory” is based on the fact that phagocytic cells that engulf asbestos fibers produce large amounts of free radicals due to their inability to digest the fibers, and epidemiological studies indicating that iron-containing asbestos fibers appear more carcinogenic; (2) the “chromosome tangling theory” postulates that asbestos fibers damage chromosomes when cells divide; and (3) the “theory of adsorption of many specific proteins as well as carcinogenic molecules” states that asbestos fibers in vivo concentrate proteins or chemicals including the components of cigarette smoke. Elucidation of the major mechanisms underlying DMM would be helpful for the development of novel strategies to prevent DMM induction in people who have already been exposed to asbestos

DOI： 10.18999/nagjms.85.1.13

Toward the next century of the Nagoya Journal of Medical Science: message from the Editor-in-chief. 査読有り国際誌 Open Access

Toyokuni S

Nagoya journal of medical science 85 巻 ( 1 ) 頁： 1 - 4 2023年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nagoya Journal of Medical Science

It is my great pleasure and honor as the Editor-in-chief to congratulate the centennial anniversary of the Nagoya Journal of Medical Science in 2023 with all the editorial board members and the technical staffs as well as the authors and the readers. One hundred years are quite a long period, and the persistent publication from a medical school of national university in Japan has not been so easy. Especially, the publication was suspended from 1940 to 1950 due to the World War II.

DOI： 10.18999/nagjms.85.1.1

BRCA1 haploinsufficiency promotes chromosomal amplification under renal carcinogenesis through ferroptosis-resistance 査読有り国際誌

Kong, YY; Akatsuka, S; Motooka, Y; Zheng, H; Shiraki, Y; Mashimo, T; Imaoka, T; Toyokuni, S

CANCER SCIENCE 114 巻頁： 1534 - 1534 2023年2月

A/J mice exhibit a high susceptibility to ferric nitrilotriacetate-induced RCC through the resistance to ferroptosis 査読有り国際誌

Akatsuka, S; Cheng, Z; Toyokuni, S

CANCER SCIENCE 114 巻頁： 1315 - 1315 2023年2月

Carcinogenic genome toxicity of environmental factors, asbestos and talc in ovary 査読有り国際誌

Motooka, Y; Onimaru, K; Suzuki, HI; Katabuchi, M; Kondoh, E; Tashiro, H; Katabuchi, H; Toyokuni, S

CANCER SCIENCE 114 巻頁： 1085 - 1085 2023年2月

Validation of reduced ovarian reserve in BRCA1 haploinsufficient rats 査読有り国際誌

Sonehara, R; Motooka, Y; Kajiyama, H; Mashimo, T; Imaoka, T; Toyokuni, S

CANCER SCIENCE 114 巻頁： 2041 - 2041 2023年2月

Precision cancer prevention 査読有り国際誌

Toyokuni, S; Takayama, T

CANCER SCIENCE 114 巻頁： 1528 - 1528 2023年2月

Ferroptosis-resistance in rodent cancer models 査読有り国際誌

Toyokuni, S; Kong, Y; Akatsuka, S; Motooka, Y; Zheng, H; Mashimo, T; Imaoka, T

CANCER SCIENCE 114 巻頁： 912 - 912 2023年2月

Ferroptosis resistance is induced by BRCA1 deficiency in asbestos-induced mesothelioma 査読有り国際誌

Luo, YG; Motooka, Y; Akatsuka, S; Mashimo, T; Imaoka, T; Toyokuni, S

CANCER SCIENCE 114 巻頁： 1308 - 1308 2023年2月

Cooperation of Poly(rC)-binding Proteins 1/2 and Glutathione in Ferroptosis Induced by Plasmaactivated Ringer's Lactate 査読有り国際誌

Zheng, H; Jiang, L; Lyu, QY; Akatsuka, S; Motooka, Y; Sekido, Y; Nakamura, K; Tanaka, H; Ishikawa, K; Kajiyama, H; Mizuno, M; Hori, M; Toyokuni, S

CANCER SCIENCE 114 巻頁： 1340 - 1340 2023年2月

Cooperation of Poly(rC)-binding Proteins 1/2 and Glutathione in Ferroptosis Induced by Plasma-activated Ringer's Lactate 査読有り国際誌

Zheng, H; Jiang, L; Lyu, QY; Akatsuka, S; Motooka, Y; Sekido, Y; Nakamura, K; Tanaka, H; Ishikawa, K; Kajiyama, H; Mizuno, M; Hori, M; Toyokuni, S

CANCER SCIENCE 114 巻頁： 749 - 749 2023年2月

Plasma activated Ringer's lactate solution. 査読有り国際誌 Open Access

Tanaka H, Mizuno M, Ishikawa K, Miron C, Okazaki Y, Toyokuni S, Nakamura K, Kajiyama H, Hori M

Free radical research 57 巻 ( 1 ) 頁： 14 - 20 2023年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Low-temperature plasma (LTP) has been widely used in life science. Plasma-activated solutions were defined as solutions irradiated with LTP, and water, medium, and Ringer’s solutions have been irradiated with LTP to produce plasma-activated solutions. They contain chemical compounds produced by reactions among LTP, air, and solutions. Reactive oxygen and nitrogen species (RONS) are major components in plasma-activated solutions and recent studies revealed that plasma-activated organic compounds are produced in plasma-activated Ringer’s lactate solution (PAL). Many in vitro and in vivo studies demonstrated that PAL exhibits anti-tumor effects on cancers, and biochemical analyses revealed intracellular molecular mechanisms of cancer cell death by PAL.

DOI： 10.1080/10715762.2023.2182663

Non-thermal plasma elicits ferrous chloride-catalyzed DMPO-OH. 査読有り国際誌

Okazaki Y, Ito N, Tanaka H, Hori M, Toyokuni S

Free radical research 56 巻 ( 9-10 ) 頁： 595 - 606 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Non-thermal plasma (NTP) induces the generation of reactive oxygen species (ROS) and reactive nitrogen species, such as hydroxyl radicals (•OH), hydrogen peroxide (H<inf>2</inf>O<inf>2</inf>), singlet oxygen, superoxide, ozone, and nitric oxide, at near-physiological temperatures. These molecules promote blood coagulation, wound healing, disinfection, and selective cancer cell death. Based on these evidences, clinical trials of NTP have been conducted for treating chronic wounds and head and neck cancers. Although clinical applications have progressed, the stoichiometric quantification of NTP-induced ROS remains unclear in the liquid phase in the presence of FeCl<inf>2</inf> or FeCl<inf>3</inf> in combination with biocompatible reducing agents, which may modulate the final biological effects of NTP. In this study, we employed electron paramagnetic resonance spectroscopy to quantify ROS using spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and H<inf>2</inf>O<inf>2</inf>, using luminescent probe in the presence of FeCl<inf>2</inf> or FeCl<inf>3</inf>. NTP-induced DMPO-OH levels were elevated 10–100 µM FeCl<inf>2</inf> or 500 and 1000 µM FeCl<inf>3</inf>. NTP-induced DMPO-OH with 10 µM FeCl<inf>2</inf> or FeCl<inf>3</inf> was significantly scavenged by ascorbate, α-tocopherol, dithiothreitol, reduced glutathione, or oxidized glutathione, whereas dehydroascorbate was ineffective in 2 mM DMPO. NTP-induced H<inf>2</inf>O<inf>2</inf> was significantly degraded by 100 µM FeCl<inf>2</inf> and FeCl<inf>3</inf> in an iron-dependent manner. Meanwhile, decomposition of H<inf>2</inf>O<inf>2</inf> by catalase decayed DMPO-OH efficiently in the presence of iron, indicating iron causes DMPO-OH production and degradation simultaneously. These results suggest that NTP-induced DMPO-OH is generated by the H<inf>2</inf>O<inf>2</inf>-consuming, iron-dependent Fenton reaction and ferryl intermediates. The potential iron-mediated ROS production by NTP is also discussed to clarify the interaction between NTP-induced ROS and biomolecules.

DOI： 10.1080/10715762.2022.2157272

Editorial: Centennial anniversary of vitamin E discovery. 査読有り国際誌

Toyokuni S, Noguchi N, Niki E

Free radical biology & medicine 183 巻頁： 125 - 126 2022年4月

BRCA1 deficiency regulates the metabolism of iron and oxidative stress-induced carcinogenesis in the kidney 査読有り国際誌

Kong, YY; Motooka, Y; Akatsuka, S; Mashimo, T; Imaoka, T; Toyokuni, S

CANCER SCIENCE 113 巻頁： 336 - 336 2022年2月

Visualization of physiological ferroptosis 査読有り国際誌

Zheng, H; Toyokuni, S

CANCER SCIENCE 113 巻頁： 1547 - 1547 2022年2月

The differences between cigarette smoke and electronic cigarette smoke in inducing oxidative stress 査読有り国際誌

Lyu, QY; Toyokuni, S

CANCER SCIENCE 113 巻頁： 1652 - 1652 2022年2月

Pathologic findings toward the concept of cancer as ferroptosis-resistance 査読有り国際誌

Toyokuni, S; Zheng, H; Kong, YY; Luo, YG; Motooka, Y

CANCER SCIENCE 113 巻頁： 661 - 661 2022年2月

Iron overload led by genital talc exposure contributes to ovarian carcinogenesis 査読有り国際誌

Motooka, Y; Ito, F; Tashiro, H; Katabuchi, H; Toyokuni, S

CANCER SCIENCE 113 巻頁： 725 - 725 2022年2月

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation. 査読有り国際誌 Open Access

Omori S, Tsugita M, Hoshikawa Y, Morita M, Ito F, Yamaguchi SI, Xie Q, Noyori O, Yamaguchi T, Takada A, Saitoh T, Toyokuni S, Akiba H, Nagata S, Kinoshita K, Nakayama M

Cell reports 34 巻 ( 6 ) 頁： 108734 - 108734 2021年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Reports

Needle-like multi-walled carbon nanotubes (MWCNTs) are engulfed by macrophages, which cause inflammation and asbestosis-like pathogenesis in rodents; however, how macrophages recognize MWCNTs remains unclear. Omori et al. demonstrate that Tim4 recognizes MWCNTs through aromatic-aromatic interactions and mediates macrophage phagocytosis leading to granulomas.

DOI： 10.1016/j.celrep.2021.108734

Ferroptosis and Oxidative DNA Damage 査読有り国際誌

Toyokuni, S; Yanatori, I

DNA DAMAGE, DNA REPAIR AND DISEASE, VOL. 1 14 巻頁： 140 - 153 2021年

Induction of cancer cell-specific ferroptosis by non-thermal plasma exposure 査読有り国際誌

Okazaki, Y; Toyokuni, S

JAPANESE JOURNAL OF APPLIED PHYSICS 59 巻 ( 11 ) 2020年11月

Chemical reaction mechanism in non-thermal plasma from the viewpoint of oxidative stress toward clinical cancer applications 査読有り国際共著国際誌

Toyokuni, S; Shi, L; Okazaki, Y; Richardson, DR

FREE RADICAL BIOLOGY AND MEDICINE 100 巻頁： S131 - S131 2016年11月

CANCER AS A FERROTOXIC DISEASE: WHAT WE HAVE LEARNED FROM ANIMAL STUDIES TOWARD ITS PREVENTION 査読有り国際誌

Toyokuni, S, Jiang, L, Okazaki, Y, Akatsuka, S

AMERICAN JOURNAL OF HEMATOLOGY 88 巻 ( 5 ) 頁： E168 - E169 2013年5月

Mysterious link between iron overload and CDKN2A/2B. 国際誌 Open Access

Toyokuni S

Journal of clinical biochemistry and nutrition 48 巻 ( 1 ) 頁： 46 - 9 2011年1月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

Persistent oxidative stress has been associated with carcinogenesis. Iron overload is considered one such condition that causes oxidative stress. Epidemiological studies support a close link between iron overload and carcinogenesis. Reportedly, regular semiannual phlebotomies reduced cancer risk in an otherwise normal population. More specifically, genetic hemochromatosis, chronic viral hepatitis, ovarian endometriosis and asbestosis induce iron overload, which can lead to hepatocellular carcinoma, ovarian carcinoma or mesothelioma in humans. Through a combination of animal experiments and microarray analyses, homozygous deletion of CDKN2A/2B has been recognized as one of the major target genes involved in iron overload-induced carcinogenesis. CDKN2A/2B are the second most frequently inactivated tumor suppressing genes in human cancers. Currently, when infection is becoming sufficiently controlled worldwide, iron regulation may be the next target for human longevity. ©2011 JCBN.

DOI： 10.3164/jcbn.11-001FR

Asbestos-induced Carcinogenesis is Oxidative Stress-dependent 査読有り国際誌

Toyokuni, S; Jiang, L; Nagai, H; Ohara, H; Okazaki, Y; Akatsuka, S; Yamashita, Y

FREE RADICAL BIOLOGY AND MEDICINE 49 巻頁： S72 - S72 2010年

Spaciotemporal alteration of 8-hydroxy-2'-deoxyguanosine levels in cardiomyocytes after myocardial infarction in rats. 査読有り国際誌 Open Access

Miwa S, Toyokuni S, Nishina T, Nomoto T, Hiroyasu M, Nishimura K, Komeda M

Free radical research 36 巻 ( 8 ) 頁： 853 - 8 2002年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Temporary or persistent heart failure is one of the major complications after myocardial infarction (MI). In order to elucidate the pathogenesis of MI, we studied the spaciotemporal alteration of 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in cardiomyocytes in a rat model of ligation of the left anterior descending branch of the coronary artery. The lethality in this model was 18%. Hearts were dissected at 0, 3, 6, 12, 24, 48h, and 1, 2, 4, 6 weeks after the operation. The cardiac level of 8-OHdG was evaluated biochemically as well as by immunohistochemistry with monoclonal antibody N45.1. Three to 6h after ligation, the 8-OHdG levels were increased in the cardiomyocytes of MI (six-fold) and peri-MI (four-fold) areas. After 24h, the myocardium in the MI area was necrotized, and thereafter the 8-OHdG level decreased. 8-OHdG levels in the myocardium of peri-MI areas returned once to a normal level, but were significantly increased at 2-4 weeks along with the appearance of apoptotic cardiomyocytes in this area. The heart after MI has been generally considered as clinically stable after four weeks. However, cardiomyocytes near the infarcted area were oxidatively stressed even after four weeks when the affected lesion was extensive. The present data support the use of supplementary antioxidant therapies to save functional myocardium after MI. (213 words).

DOI： 10.1081/1071576021000005285

Endogenous formation of protein adducts with carcinogenic aldehydes - Implications for oxidative stress 査読有り国際誌

Ichihashi K, Osawa T, Toyokuni S, Uchida K

JOURNAL OF BIOLOGICAL CHEMISTRY 276 巻 ( 26 ) 頁： 23903 - 23913 2001年6月

Antioxidant alpha-tocopherol improves glycemic control of GK rats, a model of type 2 diabetes 査読有り国際誌

Ihara Y, Yamada Y, Toyokuni S, Ban N, Kuroe A, Seino Y

DIABETES 49 巻頁： A429 - A429 2000年5月

4-hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress - Identification of proteasomes as target molecules 査読有り国際共著国際誌

Okada K, Wangpoengtrakul C, Osawa T, Toyokuni S, Tanaka K, Uchida K

JOURNAL OF BIOLOGICAL CHEMISTRY 274 巻 ( 34 ) 頁： 23787 - 23793 1999年8月

8-hydroxy-2'-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure (vol 107, pg 733, 1996) 査読有り国際誌

Hattori, Y, Nishigori, C, Tanaka, T, Uchida, K, Nikaido, O, Osawa, T, Hiai, H, Imamura, S, Toyokuni, S

JOURNAL OF INVESTIGATIVE DERMATOLOGY 108 巻 ( 2 ) 頁： 237 - 237 1997年2月

ACUTE NEPHROTOXICITY OF A CARCINOGENIC IRON CHELATE - SELECTIVE-INHIBITION OF A PROTEOLYTIC CONVERSION OF ALPHA(2U)-GLOBULIN TO THE KIDNEY FATTY-ACID-BINDING PROTEIN 査読有り国際誌

UCHIDA K, FUKUDA A, KAWAKISHI S, TOYOKUNI S, HIAI H, IKEDA S, HORIO F

FEBS LETTERS 357 巻 ( 2 ) 頁： 165 - 167 1995年1月

Susceptibility of Brca1((L63X/+)) rat to ovarian reserve dissipation by chemotherapeutic agents to breast cancer. 査読有り国際誌 Open Access

Kaseki S, Sonehara R, Motooka Y, Tanaka H, Nakamura T, Osuka S, Akatsuka S, Kajiyama H, Mashimo T, Imaoka T, Toyokuni S

Cancer science 116 巻 ( 4 ) 頁： 1139 - 1152 2025年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

BRCA1 is one of the causative genes for hereditary breast and ovarian cancer syndrome with a high risk of early-onset breast cancer. Whereas olaparib (OLA), an inhibitor of poly-ADP-ribose polymerase, has been applied as adjuvant therapy to those cancer patients, its effect on ovarian reproductive function remains unelucidated. Recently, a rat model (MUT; Brca1(L63X/+) mutation) mimicking a human BRCA1 pathogenic variant has been established. Using this model, we evaluated the effects of OLA on ovarian reproductive function in comparison with the wild-type (WT) rats. MUT showed a significantly reduced number of primordial follicles and subfertility in accordance with aging. Oxidative stress was significantly elevated in the young MUT granulosa cells (GCs) accompanied by increased mTOR but decreased PTEN signals. OLA administration in MUT further decreased primordial follicles, with gene set enrichment analysis, indicating upregulated DNA repair pathways. Furthermore, a combination of OLA and cyclophosphamide (CPA) induced empty primordial follicles, recognized as CPA-induced severe ovarian toxicity. Whereas OLA + CPA caused greater reduction in primordial follicles both in MUT and WT in comparison with CPA alone, MUT ovaries were more susceptible to oxidative stress, potentially depleting primordial follicles via activation of GCs and inducing oocyte death due to accumulated DNA damage by OLA treatment. Our findings in this preclinical model underscore the importance of evaluating ovarian reserve prior to chemotherapy by performing reproductive consultation with female patients with BRCA1 pathogenic variants.

DOI： 10.1111/cas.16412

Heterozygous mutation in BRCA2 induces accelerated age-dependent decline in sperm quality with male subfertility in rats. 査読有り国際誌 Open Access

Motooka Y, Tanaka H, Maeda Y, Katabuchi M, Mashimo T, Toyokuni S

Scientific reports 15 巻 ( 1 ) 頁： 447 - 447 2025年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Tumor suppressor BRCA2 executes homologous recombination to repair DNA double-strand breaks in collaboration with RAD51, involving exon 11 and 27. Exon 11 constitutes a region where pathogenic variants (PVs) accumulate, and mutations in this region are known to contribute to carcinogenesis. However, the impact of the heterozygous PVs of BRCA2 exon 11 on the life quality beyond cancer risk, including male fertility, remains unclear. Here, we established a rat model with a frameshift on the seventh BRC repeat in Brca2 exon 11 (Brca2+/p.T1942fs), which is homologous to human BRCA2+/p.T1974fs, using CRISPR/Cas9 system. Our analyses revealed that the heterozygous rats with the PV in the BRCA2 exon 11 showed increased DNA double-strand breaks and apoptosis in spermatogonia and spermatocytes, accelerated testicular germ cell loss, and deterioration in sperm quality according with aging, ultimately resulting in early male reproductive dysfunction. Of note, these alterations in testes and sperm, including DNA fragmentation in spermatozoa, were observed from completion of sexual maturation. The present findings suggest that it is crucial to consider not only cancer risk but also potential declines in reproductive capacity in men carrying BRCA2 exon 11 PVs. Further investigation is warranted to determine whether similar traits appear in humans.

DOI： 10.1038/s41598-024-84184-8

細胞外微粒子が鉄代謝と酸化ストレスに及ぼす影響と発がんへの関与査読有り国際誌

本岡大社, 豊國伸哉

大気環境学会誌 60 巻 ( 1 ) 頁： A32 - A38 2025年1月

BRCA2 mutant induce ferroptosis resistance via AKT and promote cell cycle under Fenton reaction-based carcinogenesis 査読有り国際誌

Maeda, Y; Mashimo, T; Toyokuni, S

CANCER SCIENCE 116 巻頁： 875 - 875 2025年1月

Cold plasma irradiation of chitosan: A straight pathway to selective antitumor therapy. 査読有り国際誌 Open Access

Miron C, Andreica B, Iftime MM, Fifere A, Yamakawa T, Toyokuni S, Mizuno M, Tartau LM, Bejan A, Motooka Y, Kondo T, Sava I, Harabagiu V, Kumagai J, Tanaka A, Tanaka H, Marin L, Hori M

International journal of biological macromolecules 281 巻 ( Pt 4 ) 頁： 136513 - 136513 2024年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Biological Macromolecules

Plasma-activated chitosan (PAC) colloids for cancer treatment were obtained by using the cold atmospheric plasma technique. Chitosan solutions were irradiated by plasma ignited in argon gas and in a mixture of argon with nitrogen and oxygen gases in certain ratios. The structural modifications of chitosan and the chemical species generated in plasma were investigated by EPR, LC-MS/MS, XRD, DLS, and TGA methods. The cell viability test showed a selective cytotoxic effect on human breast carcinoma cells (MCF-7), while the human mammary epithelial cells (MCF-10A) were left unharmed. The cytotoxic effect was attributed mainly to chitooligosaccharides, but also to a synergistic effect with other compounds generated in very low concentrations in plasma, such as glyceric acid, ethyl acetate, or tricarballylic acid. The plasma irradiation improved the antioxidant activity and mucoadhesivity, while not affecting the hemocompatibility investigated by a standard hemolysis ex vivo test on mice blood. Moreover, the in vivo biocompatibility investigation at intraperitoneal administration of PAC in mice showed no statistically significant changes in the hematologic, biochemical, and immune system parameters, and no morphologic alterations of the liver and kidney. All these data indicate the cold plasma activation of chitosan as a straight method to produce biocompatible, antitumor systems.

DOI： 10.1016/j.ijbiomac.2024.136513

Effects of autophagy on the selective death of human breast cancer cells exposed to plasma-activated Ringer's lactate solution. 査読有り国際誌

Yamakawa T, Tanaka A, Miron C, Nakamura K, Kajiyama H, Toyokuni S, Mizuno M, Hori M, Tanaka H

Free radical research 58 巻 ( 11 ) 頁： 758 - 769 2024年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Plasma-activated Ringer’s lactate (PAL) solution prepared by irradiating an intravenous solution with a non-equilibrium atmospheric pressure plasma is a potential new cancer therapy having no side effects. However, the induction of autophagy to avoid cell death has been confirmed to occur following exposure to PAL solution. It is thought that the antitumor effect of PAL solution could be weakened by this process, which is meant to maintain homeostasis in cells and assists tumorigenesis. Thus, it would be helpful to devise PAL-based cancer therapies that inhibit autophagy. Unfortunately, it is not yet clear which substances in PAL solution promote autophagy. The present work examined the mechanism by which PAL solution induces autophagy when treating MCF-7 human breast cancer cells. Autophagy was found to be temporarily induced upon exposure to PAL solution, suggesting that this effect contributes to cell proliferation. Although autophagy is associated with reactive oxygen and nitrogen species and/or acidic environments, in this study, significant autophagy was observed using a PAL solution diluted 1/256x without these stressors. Acetate, glyoxylate and 2,3-dimethyltartrate in the PAL solution were determined to promote autophagy. Interestingly, 2,3-dimethyltartrate was found to either induce cell death or autophagy depending on the concentration.

DOI： 10.1080/10715762.2024.2433965

増大特集学術研究支援の最先端 Ⅱ.先端モデル動物支援プラットフォーム(AdAMS) ⅱ.病理形態解析支援てんかん,神経変性症および慢性腎臓病モデル動物としてのTxn1遺伝子変異ラットの樹立査読有り国際誌

大守(川﨑) 伊織, 大内田守, 真下知士, 豊國伸哉

生体の科学 75 巻 ( 5 ) 頁： 454 - 455 2024年10月

増大特集学術研究支援の最先端 Ⅱ.先端モデル動物支援プラットフォーム(AdAMS) ⅱ.病理形態解析支援経験豊富な病理専門医が研究の方向性を指南する(総論) 査読有り国際誌

豊國伸哉

生体の科学 75 巻 ( 5 ) 頁： 438 - 439 2024年10月

Ferroptosis is a targetable detrimental factor in metabolic dysfunction-associated steatotic liver disease. 国際誌 Open Access

Peleman C, Hellemans S, Veeckmans G, Arras W, Zheng H, Koeken I, Van San E, Hassannia B, Walravens M, Kayirangwa E, Beyene NT, Van Herck MA, De Vos WH, Pintelon I, van Nassauw L, Oosterlinck B, Smet A, Vits L, Dirinck E, Verrijken A, De Man J, Van Eyck A, Kwanten WJ, Vonghia L, Driessen A, Augustyns K, Toyokuni S, De Winter B, Van Steenkiste C, Francque S, Vanden Berghe T

Cell death and differentiation 31 巻 ( 9 ) 頁： 1113 - 1126 2024年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Death and Differentiation

There is an unmet clinical need for pharmacologic treatment for metabolic dysfunction-associated steatotic liver disease (MASLD). Hepatocyte cell death is a hallmark of this highly prevalent chronic liver disease, but the dominant type of cell death remains uncertain. Here we report that ferroptosis, an iron-catalyzed mode of regulated cell death, contributes to MASLD. Unsupervised clustering in a cohort of biopsy-proven MASLD patients revealed a subgroup with hepatic ferroptosis signature and lower glutathione peroxidase 4 (GPX4) levels. Likewise, a subgroup with reduced ferroptosis defenses was discerned in public transcriptomics datasets. Four weeks of choline-deficient L-amino acid-defined high-fat diet (CDAHFD) induced MASLD with ferroptosis in mice. Gpx4 overexpression did not affect steatohepatitis, instead CDAHFD protected from morbidity due to hepatocyte-specific Gpx4 knockout. The ferroptosis inhibitor UAMC-3203 attenuated steatosis and alanine aminotransferase in CDAHFD and a second model, i.e., the high-fat high-fructose diet (HFHFD). The effect of monounsaturated and saturated fatty acids supplementation on ferroptosis susceptibility was assessed in human HepG2 cells. Fat-laden HepG2 showed a drop in ferroptosis defenses, increased phosphatidylglycerol with two polyunsaturated fatty acid (PUFA) lipid tails, and sustained ferroptosis sensitivity. In conclusion, this study identified hepatic ferroptosis as a detrimental factor in MASLD patients. Unexpectedly, non-PUFA supplementation to hepatocytes altered lipid bilayer composition to maintain ferroptosis sensitivity. Based on findings in in vivo models, ferroptosis inhibition represents a promising therapeutic target in MASLD. (Figure presented.)

DOI： 10.1038/s41418-024-01348-9

Ferroptosis induced by plasma-activated Ringer's lactate solution prevents oral cancer progression. 国際誌 Open Access

Sato K, Yang M, Nakamura K, Tanaka H, Hori M, Nishio M, Suzuki A, Hibi H, Toyokuni S

Oral diseases 30 巻 ( 6 ) 頁： 3912 - 3924 2024年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Oral Diseases

Objective: This study aimed to investigate the effect of plasma-activated Ringer's lactate solution (PAL) on oral squamous cell carcinoma (OSCC) cells and carcinogenic processes with a particular focus on iron and collagenous matrix formation. Materials and Methods: We used three OSCC cell lines, one keratinocyte cell line, and two fibroblast lines, and cell viability assays, immunoblotting, flow cytometry, and transmission electron microscopy were performed to evaluate the effect and type of cell death. The effect of PAL treatment on lysyl oxidase (LOX) expression was investigated in vitro and in vivo. Tamoxifen-inducible Mob1a/b double-knockout mice were used for the in vivo experiment. Results: PAL killed OSCC cells more effectively than the control nontumorous cells and suppressed cell migration and invasion. Ferroptosis occurred and the protein level of LOX was downregulated in cancer cells in vitro and in vivo. Additionally, PAL improved the survival rate of mice and suppressed collagenous matrix formation. Conclusions: We demonstrated that PAL specifically kills OSCC cells and that ferroptosis occurs in vitro and in vivo. Furthermore, PAL can prevent carcinogenesis and improve the survival rate of oral cancer, especially tongue cancer, by changing collagenous matrix formation via LOX suppression.

DOI： 10.1111/odi.14827

A guideline on the molecular ecosystem regulating ferroptosis. 国際誌 Open Access

Dai E, Chen X, Linkermann A, Jiang X, Kang R, Kagan VE, Bayir H, Yang WS, Garcia-Saez AJ, Ioannou MS, Janowitz T, Ran Q, Gu W, Gan B, Krysko DV, Zhu X, Wang J, Krautwald S, Toyokuni S, Xie Y, Greten FR, Yi Q, Schick J, Liu J, Gabrilovich DI, Liu J, Zeh HJ, Zhang DD, Yang M, Iovanna J, Kopf M, Adolph TE, Chi JT, Li C, Ichijo H, Karin M, Sankaran VG, Zou W, Galluzzi L, Bush AI, Li B, Melino G, Baehrecke EH, Lotze MT, Klionsky DJ, Stockwell BR, Kroemer G, Tang D

Nature cell biology 26 巻 ( 9 ) 頁： 1447 - 1457 2024年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Cell Biology

Ferroptosis, an intricately regulated form of cell death characterized by uncontrolled lipid peroxidation, has garnered substantial interest since this term was first coined in 2012. Recent years have witnessed remarkable progress in elucidating the detailed molecular mechanisms that govern ferroptosis induction and defence, with particular emphasis on the roles of heterogeneity and plasticity. In this Review, we discuss the molecular ecosystem of ferroptosis, with implications that may inform and enable safe and effective therapeutic strategies across a broad spectrum of diseases.

DOI： 10.1038/s41556-024-01360-8

BRCA2変異は酸化ストレス発がん環境下でAKTを介したフェロトーシス抵抗性を誘導し細胞周期を促進する(BRCA2 mutant induce ferroptosis resistance via AKT and promote cell cycle under Fenton reaction-based carcinogenesis) 査読有り国際誌

前田勇貴, 真下知士, 豊國伸哉, 豊國伸哉

日本癌学会総会記事 83回巻頁： P - 2013 2024年9月

Special issue: Extracellular fine particles in life, death and disease. 国際誌

Toyokuni S

Archives of biochemistry and biophysics 757 巻頁： 110027 2024年7月

Heat-treated and/or lysozyme-treated Enterococcus faecalis (FK-23) improves the progression of renal disease in a unilateral ischemia-reperfusion injury rat model. 国際誌 Open Access

Takemura S, Minamiyama Y, Ito N, Yamamoto A, Ichikawa H, Nakagawa K, Toyokuni S, Osada-Oka M, Yoshikawa T

Journal of clinical biochemistry and nutrition 75 巻 ( 1 ) 頁： 78 - 89 2024年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

The prevalence of chronic kidney disease (CKD) is increasing owing to the elderly population. Here, we investigated the effects of heat-treated Enterococcus faecalis (FK-23) and lysozyme-treated FK-23 (LFK) on the progression of CKD in rats. A CKD model was established using male Wistar rats by subjecting them to right nephrectomy (1K), followed by ischemia and reperfusion (IR). FK-23 or LFK was fed ad libitum as a mixed diet after right nephrectomy. Animals subjected to renal ischemia-reperfusion injury (IRI) showed increased plasma creatinine and blood urea nitrogen levels. Furthermore, in the kidneys, collagen accumulation and α-smooth muscle actin, indicative of fibroblast activation and fibrosis-related gene and protein expression, increased 3 weeks after IRI. FK-23 and LFK suppressed the increase in the mRNA levels of some of these genes. The increase in oxidative stress markers, 4-hydroxy-2-nonenal, endothelial nitric oxide synthase, and nitrotyrosine in the kidney, as well as increased plasma uremic toxins after IRI, were also ameliorated by FK-23 and LFK. Metagenomic analysis of fecal samples revealed that gut microbial alteration caused by IRI was also ameliorated by LFK treatment. These results suggest that Enterococcus faecalis ingredients may improve CKD progression by suppressing oxidative stress and correcting the balance of the intestinal microflora.

DOI： 10.3164/jcbn.24-29

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis. 国際誌 Open Access

Chen X, Tsvetkov AS, Shen HM, Isidoro C, Ktistakis NT, Linkermann A, Koopman WJH, Simon HU, Galluzzi L, Luo S, Xu D, Gu W, Peulen O, Cai Q, Rubinsztein DC, Chi JT, Zhang DD, Li C, Toyokuni S, Liu J, Roh JL, Dai E, Juhasz G, Liu W, Zhang J, Yang M, Liu J, Zhu LQ, Zou W, Piacentini M, Ding WX, Yue Z, Xie Y, Petersen M, Gewirtz DA, Mandell MA, Chu CT, Sinha D, Eftekharpour E, Zhivotovsky B, Besteiro S, Gabrilovich DI, Kim DH, Kagan VE, Bayir H, Chen GC, Ayton S, Lünemann JD, Komatsu M, Krautwald S, Loos B, Baehrecke EH, Wang J, Lane JD, Sadoshima J, Yang WS, Gao M, Münz C, Thumm M, Kampmann M, Yu D, Lipinski MM, Jones JW, Jiang X, Zeh HJ, Kang R, Klionsky DJ, Kroemer G, Tang D

Autophagy 20 巻 ( 6 ) 頁： 1213 - 1246 2024年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Autophagy

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results. Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

DOI： 10.1080/15548627.2024.2319901

Fatal COVID-19 pulmonary disease involves ferroptosis. 国際誌 Open Access

Qiu B, Zandkarimi F, Saqi A, Castagna C, Tan H, Sekulic M, Miorin L, Hibshoosh H, Toyokuni S, Uchida K, Stockwell BR

Nature communications 15 巻 ( 1 ) 頁： 3816 2024年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Communications

Ferroptosis is a form of cell death that has been associated with different diseases. Here the authors describe an association of ferroptosis with COVID-19 pulmonary pathologies in both patient samples and hamster model and suggest that the dysregulation in iron and lipid metabolism could provide targets to reduce pathology.

DOI： 10.1038/s41467-024-48055-0

特集活性酸素と酸化ストレスがんとフェロトーシス国際誌

豊國伸哉

医学のあゆみ 289 巻 ( 4 ) 頁： 263 - 266 2024年4月

Protocol for the isolation of GFP-expressing ferroptosis-dependent extracellular vesicles in in vitro cell culture models. 査読有り国際誌 Open Access

Ito F, Yanatori I, Kato K, Toyokuni S

STAR protocols 5 巻 ( 1 ) 頁： 102892 - 102892 2024年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：STAR Protocols

Extracellular vesicles (EVs) are complex structures that transport various DNA, RNA, and protein. Recently, new EV secretion mechanisms have been identified through the iron regulatory system in mammalian cells. We revealed that ferroptosis increases EV secretion, which is named ferroptosis-dependent EVs (FedEVs). Here, we describe a step-by-step procedure to isolate GFP-expressing FedEVs for in vitro analysis. The FedEVs are further analyzed by imaging and flow cytometry analysis. For complete details on the use and execution of this protocol, please refer to Ito et al.1

DOI： 10.1016/j.xpro.2024.102892

Elaborate cooperation of poly(rC)-binding proteins 1/2 and glutathione in ferroptosis induced by plasma-activated Ringer's lactate. 国際誌 Open Access

Jiang L, Zheng H, Ishida M, Lyu Q, Akatsuka S, Motooka Y, Sato K, Sekido Y, Nakamura K, Tanaka H, Ishikawa K, Kajiyama H, Mizuno M, Hori M, Toyokuni S

Free radical biology & medicine 214 巻頁： 28 - 41 2024年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Reactive species are involved in various aspects of neoplastic diseases, including carcinogenesis, cancer-specific metabolism and therapeutics. Non-thermal plasma (NTP) can directly provide reactive species, by integrating atmospheric and interjacent molecules as substrates, to represent a handy strategy to load oxidative stress in situ. NTP causes apoptosis and/or ferroptosis specifically in cancer cells of various types. Plasma-activated Ringer's lactate (PAL) is another modality at the preclinical stage as cancer therapeutics, based on more stable reactive species. PAL specifically kills malignant mesothelioma (MM) cells, employing lysosomal ·NO as a switch from autophagy to ferroptosis. However, the entire molecular mechanisms have not been elucidated yet. Here we studied cytosolic iron regulations in MM and other cancer cells in response to PAL exposure. We discovered that cells with higher catalytic Fe(II) are more susceptible to PAL-induced ferroptosis. PAL caused a cytosolic catalytic Fe(II)-associated pathology through iron chaperones, poly (rC)-binding proteins (PCBP)1/2, inducing a disturbance in glutathione-regulated iron homeostasis. PCBP1/NCOA4-mediated ferritinophagy started at a later phase, further increasing cytosolic catalytic Fe(II), ending in ferroptosis. In contrast, PCBP2 after PAL exposure contributed to iron loading to mitochondria, leading to mitochondrial dysfunction. Therapeutic effect of PAL was successfully applied to an orthotopic MM xenograft model in mice. In conclusion, PAL can selectively sensitize MM cells to ferroptosis by remodeling cytoplasmic iron homeostasis, where glutathione and PCBPs play distinct roles, resulting in lethal ferritinophagy and mitochondrial dysfunction. Our findings indicate the clinical application of PAL as a ferroptosis-inducer and the potential of PCBPs as novel targets in cancer therapeutics.

DOI： 10.1016/j.freeradbiomed.2024.02.001

BRCA2 mutation promotes ferroptosis resistance via suppression of p53 査読有り国際誌

Maeda, Y; Mashimo, T; Toyokuni, S

CANCER SCIENCE 115 巻頁： 504 - 504 2024年3月

Brca2((p.T1942fs/+)) dissipates ovarian reserve in rats through oxidative stress in follicular granulosa cells. 国際誌 Open Access

Tanaka H, Motooka Y, Maeda Y, Sonehara R, Nakamura T, Kajiyama H, Mashimo T, Toyokuni S

Free radical research 58 巻 ( 2 ) 頁： 130 - 143 2024年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Pathogenic variants of BRCA1/2 constitute hereditary breast and ovarian cancer (HBOC) syndrome, and BRCA1/2 mutant is a risk for various cancers. Whereas the clinical guideline for HBOC patients has been organized for the therapy and prevention of cancer, there is no recommendation on the female reproductive discipline. Indeed, the role of BRCA1/2 pathogenic variants in ovarian reserve has not been established due to the deficiency of appropriate animal models. Here, we used a rat model of Brca2(p.T1942fs/+) mutant of Sprague-Dawley strain with CRISPR-Cas9 editing to evaluate ovarian reserve in females. Fertility and ovarian follicles were evaluated and anti-Müllerian hormone (AMH) was measured at 8–32 weeks of age with a comparison between the wild-type and the mutant rats (MUT). MUT revealed a significantly smaller number of deliveries with fewer total pups. Furthermore, MUT showed a significant decrease in primordial follicles at 20 weeks and a low AMH level at 28 weeks. RNA-sequencing of the ovary at 10 weeks detected acceleration of the DNA damage repair pathway, which was accompanied by oxidative stress-induced DNA double-strand breaks, a decrease in PTEN, and an increase in mTOR in follicular granulosa cells. In conclusion, Brca2(p.T1942fs/+) dissipates primordial follicles via early activation of granulosa cells through oxidative stress, leading to earlier termination of fertility.

DOI： 10.1080/10715762.2024.2320405

Formalin-Fixed Paraffin-Embedded Proteomics of Malignant Mesothelioma and New Candidate Biomarkers Thioredoxin and Superoxide Dismutase 2 for Immunohistochemistry. 国際誌 Open Access

Hiratsuka T, Yoshizawa A, Endo T, Yamamoto T, Toyokuni S, Tsuruyama T

Laboratory investigation; a journal of technical methods and pathology 104 巻 ( 2 ) 頁： 100299 2024年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Laboratory Investigation

The pathogenesis of malignant mesothelioma (MM) has been extensively investigated, focusing on stress derived from reactive oxygen species. We aimed to identify diagnostic biomarkers of MM by analyzing proteins in formalin-fixed paraffin-embedded specimens using liquid chromatography-mass spectrometry. We extracted proteins from formalin-fixed paraffin-embedded sections of MM tissues (n = 7) and compared their profiles with those of benign mesothelial tissues (n = 4) and alveolar tissue (n = 1). Proteomic data were statistically assessed and profiled using principal component analysis. We were successful in the classification of MM and healthy tissue. The levels of superoxide dismutase 2 (SOD2), an enzyme that converts superoxide anion into oxygen and hydrogen peroxide, and thioredoxin (TXN), which plays a crucial role in reducing disulfide bonds in proteins, primarily contributed to the classification. Other redox-related proteins, such as pyruvate dehydrogenase subunit X, and ceruloplasmin also contributed to the classification. Protein-protein interaction analysis demonstrated that these proteins play essential roles in MM pathogenesis. Immunohistochemistry revealed that TXN levels were significantly lower, whereas SOD2 levels were significantly higher in MM and lung cancer tissues than in controls. Proteomic profiling suggested that MM tissues experienced increased exposure to hydrogen peroxide and other reactive oxygen species. Combining immunohistochemistry for TXN and SOD2 allows for differentiation among MM, lung cancer, and control tissues; hence, TXN and SOD2 may be promising MM biomarkers and therapeutic targets.

DOI： 10.1016/j.labinv.2023.100299

BRCA2の生殖細胞変異は卵胞発育を促進し卵巣予備能の低下を惹起する(BRCA2 germline mutation accelerates follicle maturation, leading to reduced ovarian reserve) 査読有り国際誌

田中秀明, 本岡大社, 前田勇貴, 真下知士, 梶山広明, 豊國伸哉

日本病理学会会誌 113 巻 ( 1 ) 頁： 428 - 429 2024年2月

病理形態学から発がんメカニズムの本質に迫る環境中の微粒子であるアスベストやタルクによる卵巣発がん(Mechanisms of carcinogenesis revealed by pathomorphology Ovarian carcinogenesis induced by environmental particulates: asbestos and talc) 査読有り国際誌

本岡大社, 豊國伸哉

日本病理学会会誌 113 巻 ( 1 ) 頁： 194 - 194 2024年2月

マイクロプラスチック曝露によるラット子宮内膜症の増悪(Growth of rat endometriosis is accelerated by microplastics exposure) 査読有り国際誌

片渕充沙子, 本岡大社, 田代浩徳, 近藤英治, 豊國伸哉

日本病理学会会誌 113 巻 ( 1 ) 頁： 429 - 429 2024年2月

シクロホスファミドとオラパリブによる卵巣毒性はBRCA1変異で増強される(Ovarian follicle toxicity of Cyclophosphamide and Olaparib in BRCA1 mutant rats) 査読有り国際誌

可世木聡, 本岡大社, 中村智子, 梶山広明, 豊國伸哉

日本病理学会会誌 113 巻 ( 1 ) 頁： 428 - 428 2024年2月

BRCA2変異はラットモデルにおいて精子の質の低下と精巣の退行性変化をきたし男性不妊を惹起する査読有り国際誌

界外誠, 本岡大社, 豊國伸哉

日本病理学会会誌 113 巻 ( 1 ) 頁： 479 - 479 2024年2月

BRCA2変異のフェロトーシス抵抗性獲得因子の探索査読有り国際誌

前田勇貴, 真下知士, 豊國伸哉

日本病理学会会誌 113 巻 ( 1 ) 頁： 292 - 292 2024年2月

Science and Applications of Plasma-Activated Solutions: Current Trends and Future Directions 査読有り国際誌

Tanaka H., Mizuno M., Ishikawa K., Miron C., Okazaki Y., Toyokuni S., Nakamura K., Kajiyama H., Ito M., Hori M.

Plasma Medicine 14 巻 ( 1 ) 頁： 67 - 76 2024年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plasma Medicine

Recent research into plasma-activated solutions has unveiled their considerable potential across a range of applications, including cancer treatment, industrial processes, and environmental management. This review summarizes key findings from recent studies on the effects of plasma-activated solutions, focusing on their ability to induce cell death in glioblastoma cells, enhance ethanol production in yeast, and convert organic compounds into environmentally friendly substances. The review highlights the mechanisms by which plasma-activated solutions exert their effects, such as oxidative stress induction and cellular metabolic alterations. Addition-ally, it discusses the implications of these findings for overcoming challenges in cancer therapy, improving agricultural practices, and developing novel environmental technologies. The ongoing advancements in plasma technology offer promising avenues for future research and applications, underscoring its transformative potential in both scientific and practical domains.

DOI： 10.1615/PlasmaMed.v14.i1.50

芳香族アミン類MOCAのDNA酸化損傷に関する研究－ラット肝臓における8 -ヒドロキシ-2’-デオキシグアノシンの検討－国際誌

Tetrachloroaurate (III)-induced oxidation increases nonthermal plasma-induced aldehydes 査読有り国際誌 Open Access

Okazaki, Y; Yoshitake, J; Ito, N; Sasaki, K; Tanaka, H; Hori, M; Shibata, T; Toyokuni, S

ADVANCES IN REDOX RESEARCH 9 巻頁： 100074 - 100074 2023年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Advances in Redox Research

Research on nonthermal plasma (NTP) devices first began almost five decades ago. NTP devices discharge electrons, positive ions, ultraviolet light, reactive oxygen species (ROS) and reactive nitrogen species (RNS) at near-physiological temperatures. Advances in plasma science have enabled the manipulation of ROS/RNS irradiation for medical applications. During preclinical stages and in human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral, and biofilm-related infections, wound healing, and cancer therapy. Previously, tetrachloroaurate (III) increased NTP-induced oxidative stress that was attenuated by reduced and oxidized glutathione, indicating that the presence of interactions between metal ions and biomolecules may modulate biological effects. In this study, using 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) as a spin probe in electron paramagnetic resonance (EPR), we observed that the tetrachloroaurate-induced M4PO-X spin adduct was significantly suppressed by ascorbate and α-tocopherol, while dehydroascorbate (DHA) and Trolox were ineffective. Tetrachloroaurate-induced lipid peroxidation, which was measured by 2-thiobarbituric acid-reactive substances (TBARS) in combination with NTP exposure, was suppressed by ascorbate, α-tocopherol and Trolox, while DHA was ineffective. Furthermore, N-acetyl-L-cysteine and dithiothreitol efficiently suppressed tetrachloroaurate-induced M4PO-X spin adduct and lipid peroxidation. LC‒MS/MS analyzes identified hexanal that was significantly elevated by NTP exposure and/or tetrachloroaurate. However, 25 and 250 μM ascorbate did not significantly suppress the formation of aldehydes, such as acetaldehyde, hexanal, nonanal, nonenal, and 4‑hydroxy-2-nonenal. Further studies are warranted to elucidate the redox reactions between metal ions, including gold (III), and biomolecules to expand the possibility of NTP application in medicine and agriculture.

DOI： 10.1016/j.arres.2023.100074

Generation and measurement of low-temperature plasma for cancer therapy: a historical review. 査読有り国際誌 Open Access

Ishikawa K, Takeda K, Yoshimura S, Kondo T, Tanaka H, Toyokuni S, Nakamura K, Kajiyama H, Mizuno M, Hori M

Free radical research 57 巻 ( 3 ) 頁： 239 - 270 2023年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

This review provides a description of the historical background of the development of biological applications of low-temperature plasmas. The generation of plasma, methods and devices, plasma sources, and measurements of plasma properties, such as electron dynamics and chemical species generation in both gaseous and aqueous phases, were assessed. Currently, direct irradiation methods for plasma discharges contacting biological surfaces, such as the skin and teeth, are related to plasma biological interactions. Indirect methods using plasma-treated liquids are based on plasma–liquid interactions. The use of these two methods is rapidly increasing in preclinical studies and cancer therapy. The authors address the prospects for further developments in cancer therapeutic applications by understanding the interactions between the plasma and living organisms.

DOI： 10.1080/10715762.2023.2230351

Three-Dimensional Regulation of Ferroptosis at the Intersection of Iron, Sulfur, and Oxygen Executing Scrap and Build Toward Evolution. 査読有り国際誌

Toyokuni S, Kong Y, Zheng H, Maeda Y, Katabuchi M, Motooka Y

Antioxidants & redox signaling 39 巻 ( 10-12 ) 頁： 807 - 815 2023年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Antioxidants and Redox Signaling

Significance: Iron is an essential element for every life on earth as a primary media for electron flow. Sulfur compounds as sulfhydryls counteract catalytic activity of iron whereas sulfur overdose is also toxic. In aerobic organisms, oxygen is the major media for electron transfer with higher intracellular mobility, which cooperates with the iron system. Based on the importance of iron, there is no active pathway to excrete iron outside the body in higher species. Whereas bacterial infection causes a scramble for iron in situ, cancer can be the outcome of the side effects of long use of iron and oxygen. Recent Advances: Ferroptosis is a recently coined cell death, defined as catalytic Fe(II)-dependent regulated necrosis accompanied by lipid peroxidation. Researchers recently recognized that ferroptosis is involved in a variety of physiological and pathological contexts, including embryonic erythropoiesis, aging, neurodegeneration and cancer cell death. Alternatively, carcinogenesis is a process to obtain iron addiction with ferroptosisresistance, based on rodent animal studies. Critical Issues: Here we propose that ferroptosis is three-dimensionally regulated by iron, sulfur and oxygen, which correspond to oxidants, antioxidants and membrane fluidity with susceptibility to lipid peroxidation, respectively. Future Directions: Whereas life attempts to prevent ferroptosis, ferroptotic cells eventually emit iron-loaded ferritin as extracellular vesicles to maintain monopoly of iron. Antioxid. Redox Signal. 39, 807–815.

DOI： 10.1089/ars.2022.0142

New iron export pathways acting via holo-ferritin secretion. 査読有り国際誌

Yanatori I, Kishi F, Toyokuni S

Archives of biochemistry and biophysics 746 巻頁： 109737 - 109737 2023年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Ferritin is a spherical nanocage protein for iron storage, composed of 24 light- or heavy-polypeptide chain subunits. A single ferritin molecule can carry up to 4500 iron atoms in its core, which plays an important role in suppressing intracellular iron toxicity. Serum ferritin levels are used as a marker for the total amount of iron stored in the body. Most serum ferritin is iron-free (apo-ferritin) and it is unclear how ferritin is released from cells. Ferritin is secreted into serum via extracellular vesicles (EVs) or the secretory autophagy pathway but not via the classical endoplasmic reticulum (ER)-to-Golgi secretion pathway. We recently discovered that the level of tetraspanin CD63, a common EV marker, is post-transcriptionally regulated by the intracellular iron level and both CD63 and ferritin expression is induced by iron loading. Ferritin is incorporated into CD63(+)-EVs through the ferritin-specific autophagy adapter molecule, NCOA4, and then secreted from cells. EV production differs drastically depending on cell type and physiological conditions. Extracellular matrix detached cells express pentaspanin prominin 2 and prominin 2(+)-EVs secrete ferritin independently of NCOA4 trafficking. Ferritin is tightly bound to iron in EVs and functions as an iron-carrier protein in the extracellular environment. Cells can suppress ferroptosis by secreting holo-ferritin, which reduces intracellular iron concentration. However, this exposes the neighboring cells receiving the secreted holo-ferritin to a large excess of iron. This results in cellular toxicity through increased generation of reactive oxygen species (ROS). Here we review the machinery by which ferritin is incorporated into EVs and its role as an intercellular communication molecule.

DOI： 10.1016/j.abb.2023.109737

Iron links endogenous and exogenous nanoparticles. 査読有り国際誌 Open Access

Toyokuni S, Kong Y, Katabuchi M, Maeda Y, Motooka Y, Ito F, Yanatori I

Archives of biochemistry and biophysics 745 巻頁： 109718 - 109718 2023年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Current progress in biology and medical science is based on the observation at the level of nanometers via electron microscopy and computation. Of note, the size of most cells in higher species exists in a limited range from 5 to 50 μm. Recently, it was demonstrated that endogenous extracellular nanoparticles play a role in communication among various cellular types in a variety of contexts. Among them, exosomes in serum have been established as biomarkers for human diseases by analyzing the cargo molecules. No life on the earth can survive without iron. However, excess iron can be a risk for carcinogenesis in rodents and humans. Nano-sized molecules may cause unexpected bioeffects, including carcinogenesis, which is a process to establish cellular iron addiction with ferroptosis-resistance. Asbestos and carbon nanotubes are the typical examples, leading to carcinogenesis by the alteration of iron metabolism. Recently, we found that CD63, one of the representative markers of exosomes, is under the regulation of iron-responsive element/iron-regulatory protein system. This is a safe strategy to share excess iron in the form of holo-ferritin between iron-sufficient and -deficient cells. On the other hand, damaged cells may secrete holo-ferritin-loaded exosomes as in the case of macrophages in ferroptosis after asbestos exposure. These holo-ferritin-loaded exosomes can cause mutagenic DNA damage in the recipient mesothelial cells. Thus, there is an iron link between exogenous and endogenous nanoparticles, which requires further investigation for better understanding and the future applications.

DOI： 10.1016/j.abb.2023.109718

BRCA1変異モデルラットの卵巣機能に対する加齢とPARP阻害剤オラパリブの影響(Effect of aging and PARP inhibitor, olaparib on ovarian function with BRCA1 mutant model rats) 査読有り国際誌

曽根原玲菜, 本岡大社, 真下知士, 今岡達彦, 梶山広明, 豊國伸哉

日本癌学会総会記事 82回巻頁： 170 - 170 2023年9月

腫瘍進化生物学における鉄、酸素、フェロトーシスの役割(Role of iron, oxygen and ferroptosis in tumor evolutionary biology) 査読有り国際誌

豊國伸哉, 孔穎怡, 羅亜光, 鄭好, 本岡大社, 今岡達彦, 赤塚慎也

日本癌学会総会記事 82回巻頁： 907 - 907 2023年9月

BRCA2の生殖細胞変異は卵胞発育を促進し卵巣予備能の低下を惹起する(BRCA2 germline mutation promotes follicle development and induces diminished ovarian reserve) 査読有り国際誌

田中秀明, 本岡大社, 前田勇貴, 真下知士, 梶山広明, 豊國伸哉

日本癌学会総会記事 82回巻頁： 172 - 172 2023年9月

BRCA2変異はp53活性を抑制しフェロトーシス抵抗性を獲得する(BRCA2 mutation promotes ferroptosis resistance via suppression of p53) 査読有り国際誌

前田勇貴, 真下知士, 豊國伸哉

日本癌学会総会記事 82回巻頁： 504 - 504 2023年9月

Txn1 mutation is a monogenic cause of chronic kidney disease associated with mitochondrial dysfunction in rats

Iori Ohmori, Mamoru Ouchida, Yoshiko Hada, Haruhito A. Uchida, Shinya Toyokuni, Tomoji Mashimo

BioRxiv 2023年8月

Ferroptosis As Ultimate Target of Cancer Therapy. 査読有り国際誌

Motooka Y, Toyokuni S

Antioxidants & redox signaling 39 巻 ( 1-3 ) 頁： 206 - 223 2023年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Antioxidants and Redox Signaling

Significance: The significance of ferroptosis in cancer therapeutics has now been unveiled. Specific ferroptosis inducers are expected as a promising strategy for cancer treatment, especially in cancers with epithelial mesenchymal transition and possibly in cancers with activated Hippo signaling pathways, both of which cause resistance to traditional chemotherapy but tend to show ferroptosis susceptibility. Recent Advances: Ferroptosis is a new form of regulated non-apoptotic cell death, which is characterized by iron-dependent lipid peroxidation, leading eventually to plasma membrane rupture. Its core mechanisms have been elucidated, consisting of a driving force as catalytic Fe(II)-dependent Fenton reaction and an incorporation of polyunsaturated fatty acids to membrane phospholipids via peroxisome-dependent and -independent pathways, and suppressing factors as prevention of lipid peroxidation with glutathione peroxidase 4 and direct membrane repair via coenzyme Q10 and ESCRT-III pathways. Critical Issues: Developments of ferroptosis inducers are in progress by nanotechnology-based drugs or by innovative engineering devices. Especially, low-temperature (non-thermal) plasma is a novel technology at the preclinical stage. The exposure can induce ferroptosis selectively in cancer cells rich in catalytic Fe(II). Future Directions: We also summarize and discuss the recently uncovered responsible molecular mechanisms in association with iron metabolism, ferroptosis and cancer therapeutics. Targeting ferroptosis in addition to the current therapeutic modalities would be important to cure advanced-stage cancer. Antioxid. Redox Signal. 39, 206-223.

DOI： 10.1089/ars.2022.0048

Carbon nanotube recognition by human Siglec-14 provokes inflammation. 査読有り国際誌

Yamaguchi SI, Xie Q, Ito F, Terao K, Kato Y, Kuroiwa M, Omori S, Taniura H, Kinoshita K, Takahashi T, Toyokuni S, Kasahara K, Nakayama M

Nature nanotechnology 18 巻 ( 6 ) 頁： 628 - 636 2023年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Nanotechnology

For the design and development of innovative carbon nanotube (CNT)-based tools and applications, an understanding of the molecular interactions between CNTs and biological systems is essential. In this study, a three-dimensional protein-structure-based in silico screen identified the paired immune receptors, sialic acid immunoglobulin-like binding lectin-5 (Siglec-5) and Siglec-14, as CNT-recognizing receptors. Molecular dynamics simulations showed the spatiotemporally stable association of aromatic residues on the extracellular loop of Siglec-5 with CNTs. Siglec-14 mediated spleen tyrosine kinase (Syk)-dependent phagocytosis of multiwalled CNTs and the subsequent secretion of interleukin-1β from human monocytes. Ectopic in vivo expression of human Siglec-14 on mouse alveolar macrophages resulted in enhanced recognition of multiwalled CNTs and exacerbated pulmonary inflammation. Furthermore, fostamatinib, a Syk inhibitor, blocked Siglec-14-mediated proinflammatory responses. These results indicate that Siglec-14 is a human activating receptor recognizing CNTs and that blockade of Siglec-14 and the Syk pathway may overcome CNT-induced inflammation.

DOI： 10.1038/s41565-023-01363-w

Sleep-wake patterns are altered with age, Prdm13 signaling in the DMH, and diet restriction in mice. 査読有り国際誌 Open Access

Tsuji S, Brace CS, Yao R, Tanie Y, Tada H, Rensing N, Mizuno S, Almunia J, Kong Y, Nakamura K, Furukawa T, Ogiso N, Toyokuni S, Takahashi S, Wong M, Imai SI, Satoh A

Life science alliance 6 巻 ( 6 ) 2023年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Life Science Alliance

Old animals display significant alterations in sleep–wake patterns such as increases in sleep fragmentation and sleep propensity. Here, we demonstrated that PR-domain containing protein 13 (Prdm13)+ neurons in the dorsomedial hypothalamus (DMH) are activated during sleep deprivation (SD) in young mice but not in old mice. Chemogenetic inhibition of Prdm13+ neurons in the DMH in young mice promotes increase in sleep attempts during SD, suggesting its involvement in sleep control. Furthermore, DMH-specific Prdm13-knockout (DMH-Prdm13-KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during SD. These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Dietary restriction (DR), a well-known anti-aging intervention in diverse organisms, ameliorated age-associated sleep fragmentation and increased sleep attempts during SD, whereas these effects of DR were abrogated in DMHPrdm13-KO mice. Moreover, overexpression of Prdm13 in the DMH ameliorated increased sleep attempts during SD in old mice. Therefore, maintaining Prdm13 signaling in the DMH might play an important role to control sleep–wake patterns during aging.

DOI： 10.26508/lsa.202301992

Matrigel-based organoid culture of malignant mesothelioma reproduces cisplatin sensitivity through CTR1. 査読有り国際誌 Open Access

Ito F, Kato K, Yanatori I, Maeda Y, Murohara T, Toyokuni S

BMC cancer 23 巻 ( 1 ) 頁： 487 - 487 2023年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BMC Cancer

Organoids are a three-dimensional (3D) culture system that simulate actual organs. Therefore, tumor organoids are expected to predict precise response to chemotherapy in patients. However, to date, few studies have studied the drug responses in organoids of malignant mesothelioma (MM). The poor prognosis of MM emphasizes the importance of establishing a protocol for generating MM-organoid for research and clinical use. Here, we established murine MM organoids from p53 +/- or wild-type C57BL/6 strain by intraperitoneal injection either with crocidolite or carbon nanotube. Established MM-organoids proliferated in Matrigel as spheroids. Subcutaneous injection assays revealed that the MM-organoids mimicked actual tissue architecture and maintained the original histological features of the primary MM. RNA sequencing and pathway analyses revealed that the significant expressional differences between the 2D- and 3D-culture systems were observed in receptor tyrosine kinases, including IGF1R and EGFR, glycosylation and cholesterol/steroid metabolism. MM-organoids exhibited a more sensitive response to cisplatin through stable plasma membrane localization of a major cisplatin transporter, copper transporter 1/Slc31A1 (Ctr1) in comparison to 2D-cultures, presumably through glycosylation and lipidation. The Matrigel culture system facilitated the localization of CTR1 on the plasma membrane, which simulated the original MMs and the subcutaneous xenografts. These results suggest that the newly developed protocol for MM-organoids is useful to study strategies to overcome chemotherapy resistance to cisplatin.

DOI： 10.1186/s12885-023-10966-4

BRCA1 haploinsufficiency impairs iron metabolism to promote chrysotile-induced mesothelioma via ferroptosis resistance. 査読有り国際誌 Open Access

Luo Y, Akatsuka S, Motooka Y, Kong Y, Zheng H, Mashimo T, Imaoka T, Toyokuni S

Cancer science 114 巻 ( 4 ) 頁： 1423 - 1436 2023年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Malignant mesothelioma (MM) is still a social burden associated with asbestos exposure. Local iron accumulation thereby represents the major pathogenesis, followed by oxidative DNA strand breaks and genomic alterations in the mesothelium. BRCA1 is a critical component of homologous recombination repair directed to DNA double-stranded breaks, whereas BRCA1 germline mutation is an established risk for breast/ovarian cancer, its role in MM development remains to be elucidated. Murine Brca1 mutant models so far have not reproduced human phenotypes. However, a rat Brca1 mutant model (Mut; L63X/+) recently reproduced them at least partially. Here we describe the differential induction of MM in Brca1 mutant rats by intraperitoneal injection of chrysotile or crocidolite. Only Mut males injected with chrysotile revealed a promotional effect on mesothelial carcinogenesis in comparison with wild-type and/or females, with all the MMs Brca1 haploinsufficient. Array-based comparative genomic hybridization of MMs disclosed a greater extent of chromosomal deletions in Brca1 mutants, including Cdkn2a/2b accompanied by Tfr2 amplification, in comparison with wild-type tumors. Mutant MMs indicated iron metabolism dysregulation, such as an increase in catalytic Fe(II) and Ki67-index as well as a decrease in Fe(III) and ferritin expression. Simultaneously, mutant MMs revealed ferroptosis resistance by upregulation of Slc7A11 and Gpx4. At an early carcinogenic stage of 4 weeks, induced Brca1 expression in mesothelial cells was significantly suppressed in chrysotile/Mut in comparison with crocidolite/Mut, whereas significant preference to iron with a decrease in Fe(III) has been already established. In conclusion, chrysotile exposure can be a higher risk for MM in BRCA1 mutant males, considering the rat results.

DOI： 10.1111/cas.15705

Field-effect transistor antigen/antibody-TMDs sensors for the detection of COVID-19 samples. 査読有り国際誌 Open Access

Canton-Vitoria R, Sato K, Motooka Y, Toyokuni S, Liu Z, Kitaura R

Nanoscale 15 巻 ( 9 ) 頁： 4570 - 4580 2023年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nanoscale

We fabricated sensors by modifying the surface of MoS<inf>2</inf> and WS<inf>2</inf> with COVID-19 antibodies and investigated their characteristics, including stability, reusability, sensitivity, and selectivity. Thiols and disulfanes in antibodies strongly interact with vacant Mo or W sites of MoS<inf>2</inf> or WS<inf>2</inf>, yielding durable devices that are stable for several days in the air or water. More importantly, detachment of the antibodies is suppressed even during the aggressive cleaning process of the devices at pH 3, which allows reusing the same device in several experiments without appreciable loss of sensitivity. Therefore, the nanodevice may be employed in samples of different patients. Further, we found a limit of detection (LOD) of 1 fg ml−1 at room temperature, time responses of 1 second, and selectivity against interferences such as KLH protein or Albumin.

DOI： 10.1039/d2nr06630k

BRCA2生殖細胞系列変異が卵巣予備能低下へ及ぼす影響について査読有り国際誌

田中秀明, 本岡大社, 前田勇貴, 真下知士, 梶山広明, 豊國伸哉

日本病理学会会誌 112 巻 ( 1 ) 頁： 370 - 370 2023年3月

卵巣における細胞外小胞を介した鉄代謝と酸化ストレス応答の解析査読有り国際誌

加藤大暉, 本岡大社, 豊國伸哉

日本病理学会会誌 112 巻 ( 1 ) 頁： 382 - 382 2023年3月

タルクによる細胞外小胞を介した卵巣の鉄代謝の撹乱と発がん性査読有り国際誌

本岡大社, 加藤大暉, 豊國伸哉

日本病理学会会誌 112 巻 ( 1 ) 頁： 256 - 256 2023年3月

BRCA2変異は腎臓において鉄代謝を変化させ、フェロトーシス抵抗性を獲得する査読有り国際誌

前田勇貴, 真下知士, 豊國伸哉

日本病理学会会誌 112 巻 ( 1 ) 頁： 338 - 338 2023年3月

小林沙穂, 本岡大社, 柏木裕呂樹, 豊國伸哉

労働安全衛生研究 16 巻 ( 1 ) 頁： 45 - 49 2023年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：独立行政法人労働者健康安全機構労働安全衛生総合研究所

芳香族アミン類の一種である3,3’-ジクロロ-4,4’-ジアミノジフェニルメタン（MOCA）は，ウレタン樹脂の硬化剤等に産業利用される一方，国際がん研究機関によりグループ1（ヒトに発がん性あり）に分類され，労働者の健康影響が懸念されている．MOCAの発がん過程には，肝臓をはじめとした各臓器で代謝される際に生じる活性酸素種（ROS）やDNA付加体によるDNA損傷が関係すると考えられているが，その中でもROSによって引き起こされるDNA酸化的損傷である8-ヒドロキシ-2’ -デオキシグアノシン（8-オキソグアニン：8-OHdG）は，高頻度に発生し，DNA複製時のG→Tの点突然変異を惹起する．しかし，実験動物においてMOCAばく露が実際に変異原性の高い8-OHdGを引き起こすか調べた研究は我々の知る限り存在しない． そこで本研究では，ラットを用い，肝臓におけるMOCAばく露が十分に期待できる経口投与法により，0（対照）, 0.4, 2, 10, 50mg/kg/日の用量で週3回2週間の反復ばく露を実施し，その標的臓器である肝臓について，病理解析及び8-OHdGの生成レベルを検討した．その結果，病理所見では，50mg/kg/日投与群において空胞変性が認められた一方， 8-OHdGは，0.4mg/kg/日投与群を除いて用量依存的な若干の増加傾向がみられたが，いずれも有意な差は認められなかった．以上から，少なくとも8-OHdGはMOCAにより引き起こされる発がんメカニズムの主原因ではないと推察される．

DOI： 10.2486/josh.josh-2022-0022-ta

Cancer-specific cytotoxicity of Ringer's acetate solution irradiated by cold atmospheric pressure plasma. 査読有り国際誌 Open Access

Miron C, Ishikawa K, Kashiwagura S, Suda Y, Tanaka H, Nakamura K, Kajiyama H, Toyokuni S, Mizuno M, Hori M

Free radical research 57 巻 ( 2 ) 頁： 91 - 104 2023年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Cold atmospheric pressure plasmas are promising medical tools that can assist in cancer treatment. While the medical pathology mechanism is substantially understood, knowledge of the contribution of reactive species formed in plasma and the mode of activation of biochemical pathways is insufficient. Herein, we present a concept involving antitumoral plasma-activated organics, which is envisaged to increase cytotoxicity levels against cancer cells. Ringer′s acetate solution was irradiated by low-temperature plasma at atmospheric pressure and possible reaction pathways of the compound generation are presented. The chemical compounds formed by plasma treatment and their effects on non-tumorigenic breast epithelial cells (MCF-10A) and breast cancer cells (MCF-7) were investigated. The cell viability results have shown that plasma-derived compounds have both, stimulatory and inhibitory effects on cell viability, depending on the concentration of the generated compounds in the irradiated liquids. Previous studies have shown that oxidative stresses involving reactive oxygen and nitrogen species (RONS) can be used to kill cancer cells. Hence, while RONS offers promising first-step killing effects, cell viability results have shown that plasma-derived compounds, such as acetic anhydride and ethyl acetate, have the potential to play important roles in plasma-based cancer therapy.

DOI： 10.1080/10715762.2023.2201390

Environmental impact on carcinogenesis under BRCA1 haploinsufficiency. 査読有り国際誌 Open Access

Toyokuni S, Kong Y, Motooka Y, Akatsuka S

Genes and environment : the official journal of the Japanese Environmental Mutagen Society 45 巻 ( 1 ) 頁： 2 - 2 2023年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Genes and Environment

Cancer is the primary cause of human mortality in Japan since 1981. Although numerous novel therapies have been developed and applied in clinics, the number of deaths from cancer is still increasing worldwide. It is time to consider the strategy of cancer prevention more seriously. Here we propose a hypothesis that cancer can be side effects of long time-use of iron and oxygen and that carcinogenesis is an evolution-like cellular events to obtain “iron addiction with ferroptosis-resistance” where genes and environment interact each other. Among the recognized genetic risk factors for carcinogenesis, we here focus on BRCA1 tumor suppressor gene and how environmental factors, including daily life exposure and diets, may impact toward carcinogenesis under BRCA1 haploinsufficiency. Although mice models of BRCA1 mutants have not been successful for decades in generating phenotype mimicking the human counterparts, a rat model of BRCA1 mutant was recently established that reasonably mimics the human phenotype. Two distinct categories of oxidative stress, one by radiation and one by iron-catalyzed Fenton reaction, promoted carcinogenesis in Brca1 rat mutants. Furthermore, mitochondrial damage followed by alteration of iron metabolism finally resulted in ferroptosis-resistance of target cells in carcinogenesis. These suggest a possibility that cancer prevention by active pharmacological intervention may be possible for BRCA1 mutants to increase the quality of their life rather than preventive mastectomy and/or oophorectomy.

DOI： 10.1186/s41021-023-00258-5

Exposure of low-temperature plasma after vaccination in tongue promotes systemic IgM induction against spike protein of SARS-CoV-2. 査読有り国際誌 Open Access

Sato K, Fujii K, Tanaka H, Hori M, Hibi H, Toyokuni S

Free radical research 57 巻 ( 1 ) 頁： 30 - 37 2023年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

COVID-19 has been pandemic since 2020 with persistent generation of new variants. Cellular receptor for SARS-CoV-2 is angiotensin-converting enzyme 2 (ACE2), where transmembrane serine protease-2 (TMPRSS2) is essential for viral internalization. We recently reported abundant expression of ACE2 and TMPRSS2 in the oral cavity of humans and mice. Therefore, oral cavity may work for COVID-19 infection gates. Here we undertook to evaluate whether vaccination in the tongue harbors any merit in comparison to subcutaneous injection. Low-temperature plasma (LTP) is the fourth physical state of matters with ionization above gas but at body temperature. LTP provides complex chemistry, eventually supplying oxidative and/or nitrosative stress on the interface. LTP-associated cellular death has been reported to cause apoptosis and/or ferroptosis. However, there is few data available on immunogenicity retention after LTP exposure. We therefore studied the effect of LTP exposure after the injection of keyhole limpet hemocyanin (KLH) or spike 2 protein of SARS-CoV-2 to the tongue of six-week-old male BALB/c mice, compared to subcutaneous vaccination. Whereas LTP did not change the expression of ACE2 and TMPRSS2 in the tongue, repeated LTP exposure after tongue vaccination significantly promoted systemic and specific IgM production at day 11. In contrast, repeated LTP exposure after subcutaneous vaccination of KLH decreased systemic IgM production. Of note, tongue injection produced significantly higher titer of IgM and IgG in the case of KLH. In conclusion, LTP significantly reinforced humoral immunity by IgM after tongue injection. Vaccination to the tongue can be a novel strategy to acquire immediate immunity.

DOI： 10.1080/10715762.2023.2190486

Low-temperature plasma as magic wand to differentiate between the good and the evil. 査読有り国際誌 Open Access

Toyokuni S, Zheng H, Kong Y, Sato K, Nakamura K, Tanaka H, Okazaki Y

Free radical research 57 巻 ( 1 ) 頁： 38 - 46 2023年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Plasma is the fourth physical state of matter, characterized by an ionized gaseous mixture, after solid, liquid, and gas phases, and contains a wide array of components such as ions, electrons, radicals, and ultraviolet ray. Whereas the sun and thunder are typical natural plasma, recent progress in the electronics enabled the generation of body-temperature plasma, designated as low-temperature plasma (LTP) or non-thermal plasma since the 1990s. LTP has attracted the attention of researchers for possible biological and medical applications. All the living species on earth utilize water as essential media for solvents and molecular transport. Thus, biological application of LTP naturally intervenes water whether LTP is exposed directly or indirectly, where plasma-activated lactate (PAL) is a standard, containing H<inf>2</inf>O<inf>2</inf>, NO<inf>2</inf>− and other identified molecules. Electron spin resonance and immunohistochemical studies demonstrated that LTP exposure is a handy method to load local oxidative stress. Cancer cells are characterized by persistent self-replication and high cytosolic catalytic Fe(II). Therefore, both direct exposure of LTP and PAL can provide higher damage to cancer cells in comparison to non-tumorous cells, which has been demonstrated in a variety of cancer types. The cell death mode is either apoptosis or ferroptosis, depending on the cancer-type. Thus, LTP and PAL are expected to work as an additional cancer therapy to the established guideline protocols, especially for use in somatic cavities or surgical margins.

DOI： 10.1080/10715762.2023.2190860

Extracellular fine particles and carcinogenesis 査読有り国際誌

Toyokuni S.

Seikagaku 95 巻 ( 2 ) 頁： 177 - 183 2023年

担当区分：最終著者,　責任著者記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Seikagaku

外因性微粒子や感染に伴う炎症反応は体内の鉄分布を大きく変え，細胞外の鉄を減らすように作用するため，細胞内鉄過剰が発生する．過剰鉄は発がんリスクである．鉄は細胞増殖に必須な酵素の補因子であるが，過剰鉄はフェントン反応により変異性DNA傷害を起こし，発がんの基盤となるフェロトーシス抵抗性を持った細胞の進化・選択を促進する．血清フェリチン値は体内鉄貯蔵の指標だがその分泌機構は不明であった．我々は最近，ヒト細胞において細胞外小胞（EV）のマーカーCD63が鉄代謝に固有なIRP/IREを介して転写後制御を受け，鉄が十分あるときには鉄充填フェリチンをEVとして分泌することを明らかにした．一方，アスベストに曝露されたマクロファージはフェロトーシスに陥るが，そのとき同様の鉄充填フェリチンをEVとして放出し，それを受け取った中皮細胞に変異性傷害を与えることを見いだした．

DOI： 10.14952/SEIKAGAKU.2023.950177

Thioredoxin deficiency increases oxidative stress and causes bilateral symmetrical degeneration in rat midbrain. 査読有り国際誌 Open Access

Ohmori I, Ouchida M, Imai H, Ishida S, Toyokuni S, Mashimo T

Neurobiology of disease 175 巻頁： 105921 - 105921 2022年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Neurobiology of Disease

Thioredoxin, encoded by Txn1, acts as a critical antioxidant in the defense against oxidative stress by regulating the dithiol/disulfide balance of interacting proteins. The role of thioredoxin in the central nervous system (CNS) is largely unknown. A phenotype-driven study of N-ethyl-N-nitrosourea-mutated rats with wild-running seizures revealed the importance of Txn1 mutations in CNS degeneration. Genetic mapping identified Txn1-F54L in the epileptic rats. The insulin-reducing activity of Txn1-F54L was approximately one-third of that of the wild-type (WT). Bilateral symmetrical vacuolar degeneration in the midbrain, mainly in the thalamus and the inferior colliculus, was observed in the Txn1-F54L rats. The lesions displayed neuronal and oligodendrocytic cell death. Neurons in Txn1-F54L rats showed morphological changes in the mitochondria. Vacuolar degeneration peaked at five weeks of age, and spontaneous repair began at seven weeks. The TUNEL assay showed that fibroblasts derived from homozygotes were susceptible to cell death under oxidative stress. In five-week-old WT rats, energy metabolism in the thalamus was significantly higher than that in the cerebral cortex. In conclusion, in juvenile rats, Txn1 seems to play an essential role in reducing oxidative stress in the midbrains with high energy metabolism.

DOI： 10.1016/j.nbd.2022.105921

RhoA and vigilin are candidates for immunohistochemical markers for epithelioid malignant mesothelioma. 国際誌 Open Access

Hiratsuka T, Yamamoto T, Yoshizawa A, Toyokuni S, Tsuruyama T

Scientific reports 12 巻 ( 1 ) 頁： 18519 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Diagnostic markers of malignant mesothelioma (MM) have been extensively investigated. Immunohistochemistry (IHC) markers, such as calretinin, have been used for pathologic diagnosis. However, more diagnostic markers are required to improve the specificity and sensitivity of pathologic diagnosis. This study proposed two proteins as diagnostic markers for epithelioid MM. One is RhoA, an MM mutation-susceptible locus-derived protein, and another is vigilin, a lung small cell carcinoma marker. IHC was performed using 93 MM (epithelioid, 71 cases; sarcomatoid, 13 cases; and biphasic, 9 cases), 64 lung adenocarcinoma (LAC), 60 lung squamous cell carcinoma (LSC), and 14 normal mesothelial (NM) tissues. The majority of epithelioid MM cases were positive for both RhoA and vigilin, whereas both IHCs showed lower stainability in biphasic and sarcomatoid MM. Besides, both IHCs showed significantly higher stainability for RhoA and vigilin in epithelioid MM than in LAC and LSC (p < 0.05). Chi-square tests showed that both RhoA and vigilin IHC positive rate in epithelioid MM was not significantly different from that of calretinin (p > 0.05). In the differential diagnosis of MM from lung cancer, the accuracy and specificity of RhoA, vigilin, and calretinin staining were almost equivalent. Further, H-score test showed that there was no significant difference between RhoA versus calretinin and vigilin versus calretinin in IHC positivity in epithelioid MM (p > 0.05). In conclusion, RhoA and vigilin may be candidates for immunohistochemical markers for epithelioid MM.

DOI： 10.1038/s41598-022-20334-0

Molecular hydrogen has a positive impact on pregnancy maintenance through enhancement of mitochondrial function and immunomodulatory effects on T cells. 査読有り国際誌

Aoki C, Imai K, Mizutani T, Sugiyama D, Miki R, Koya Y, Kobayashi T, Ushida T, Iitani Y, Nakamura N, Owaki T, Nishikawa H, Toyokuni S, Kajiyama H, Kotani T

Life sciences 308 巻頁： 120955 - 120955 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Life Sciences

Aims: Molecular hydrogen (H<inf>2</inf>) has attracted growing interest because of its implications in various diseases. However, the molecular mechanisms underlying the remarkable effect of a small amount of H<inf>2</inf> remain elusive. No knowledge has been available on the role of H<inf>2</inf> in the etiology of pregnancy disorders or its direct influence on human immune cells. Since maternal immunity, T cells in particular, plays a critical role in pregnancy maintenance. We investigated the effects of H<inf>2</inf> on T cells and its relation to preterm birth (PTB). Main methods: Exhaled H<inf>2</inf> concentrations in pregnant women were measured and correlated with cytokine concentrations in maternal and umbilical cord blood. H<inf>2</inf> was added to T cells collected from healthy donors, and differentiation and proliferation were examined. Energy metabolism was also examined. H<inf>2</inf> was administered to mice and cytokine expression was compared. Key findings: Our prospective observational study revealed that maternal production of H<inf>2</inf> is significantly lower in pregnant women with PTB, suggesting its potential as a biomarker for predicting PTB. We found that H<inf>2</inf> has clear associations with several maternal cytokines, and acts as an immunomodulator by exerting mitochondrial function in human T cells. Moreover, in vivo administration of H<inf>2</inf> to pregnant mice regulated inflammatory responses and reduced PTB caused by T cell activation, which further supports the notion that H<inf>2</inf> may contribute to prolonged gestation through its immunomodulatory effect. Significance: Measuring maternal H<inf>2</inf>-production could be a potential clinical tool in the management of PTB, and H<inf>2</inf> may have positive impact on pregnancy maintenance.

DOI： 10.1016/j.lfs.2022.120955

BRCA1のハプロ不全は発がん過程において、フェロトーシス抵抗性を獲得することにより染色体増幅を促進する(BRCA1 haploinsufficiency promotes carcinogenic chromosomal amplification by ferroptosis-resistance)

On 'Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation' by Robert L. Heath and Lester Packer. 査読有り国際誌

Toyokuni S, Kong Y, Mi D

Archives of biochemistry and biophysics 726 巻頁： 109133 - 109133 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

This commentary concerns a highly cited paper by Robert L Heath and Lester Packer in Archives of Biochemistry and Biophysics published in 1968. Chloroplasts are organelles in algae and plants that use light energy for carbon fixation and oxygen production. These authors discovered that isolated chloroplasts exposed to visible light undergo a cyclic peroxidation of tri-unsaturated fatty acids, contributing to the double-edged sword concept of electron transfer reactions.

DOI： 10.1016/j.abb.2022.109133

Iron as spirit of life to share under monopoly. Open Access

Toyokuni S, Kong Y, Zheng H, Maeda Y, Motooka Y, Akatsuka S

Journal of clinical biochemistry and nutrition 71 巻 ( 2 ) 頁： 78 - 88 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

Any independent life requires iron to survive. Whereas iron deficiency causes oxygen insufficiency, excess iron is a risk for cancer, generating a double-edged sword. Iron metabolism is strictly regulated via specific systems, including iron-responsive element (IRE)/iron regulatory proteins (IRPs) and the corresponding ubiquitin ligase FBXL5. Here we briefly reflect the history of bioiron research and describe major recent advancements. Ferroptosis, a newly coined Fe(II)-dependent regulated necrosis, is providing huge impact on science. Carcinogenesis is a process to acquire ferroptosis-resistance and ferroptosis is preferred in cancer therapy due to immunogenicity. Poly(rC)-binding proteins 1/2 (PCBP1/2) were identified as major cytosolic Fe(II) chaperone proteins. The mechanism how cells retrieve stored iron in ferritin cores was unraveled as ferritinophagy, a form of autophagy. Of note, ferroptosis may exploit ferritinophagy during the progression. Recently, we discovered that cellular ferritin secretion is through extracellular vesicles (EVs) escorted by CD63 under the regulation of IRE/IRP system. Furthermore, this process was abused in asbestos-induced mesothelial carcinogenesis. In summary, cellular iron metabolism is tightly regulated by multisystem organizations as surplus iron is shared through ferritin in EVs among neighbor and distant cells in need. However, various noxious stimuli dramatically promote cellular iron uptake/storage, which may result in ferroptosis.

DOI： 10.3164/jcbn.22-43

Association of alcohol intake and female gender with high expression of TMPRSS2 in tongue as potential risk for SARS-CoV-2 infection. 国際誌 Open Access

Sato K, Fujii K, Yamamoto N, Ichimura N, Yamaguchi S, Yamada H, Hibi H, Toyokuni S

Journal of clinical biochemistry and nutrition 71 巻 ( 2 ) 頁： 129 - 135 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

COVID-19 is pandemic since 2020 and further information is necessary on the risk factors associated with the infection of SARS-CoV-2. As an entry mechanism, SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as receptor and transmembrane serine protease 2 (TMPRSS2) to activate fusion with host plasma membrane. Because dysgeusia is an early symptom of COVID-19, we here studied the expression of ACE2 and TMPRSS2 in the tongue and the associated tissues of mice and humans with immunohistochemistry and immunoblot analysis. ACE2 expression was low in the human tongue but was observed in the squamous epithelium, perineurium, arterial wall, salivary glands as well as taste buds. In contrast, mice showed high expression. In sharp contrast, TMPRSS2 expression was high in all the cells mentioned above in humans but relatively low in mice except for salivary glands. We then performed semiquantitation of immunohistochemistry data of human ACE2 and TMPRSS2 and analyzed for age, sex, alcohol intake, and smoking habit with logistic regression analysis. We found that alcohol intake and female gender were the significant risk factors for increasing TMPRSS2 expression. In conclusion, TMPRSS2 is an important factor to be considered regarding SARS-CoV-2 entry and amplification in the oral cavity, which is promoted through drinking habit.

DOI： 10.3164/jcbn.21-172

環境因子であるタルクおよびアスベストの卵巣発がん性とゲノム毒性の検討(Carcinogenic genome toxicity of environmental factors, asbestos and talc in ovary)

本岡大社, 鬼丸洸, 鈴木洋, 片渕充沙子, 近藤英治, 田代浩徳, 片渕秀隆, 豊國伸哉

日本癌学会総会記事 81回巻頁： P - 2007 2022年9月

プラズマ活性化された乳酸リンゲルに誘導されたフェロトーシスにおけるpcbp1/2とグルタチオンとの協同作用(Cooperation of Poly(rC)-binding Proteins 1/2 and Glutathione in Ferroptosis Induced by Plasma-activated Ringer's Lactate)

鄭好, 蒋麗, 呂沁穎, 赤塚慎也, 本岡大社, 関戸好孝, 中村香江, 田中宏昌, 石川健治, 梶山広明, 水野正明, 堀勝, 豊國伸哉

日本癌学会総会記事 81回巻頁： E - 2004 2022年9月

個別化予防医療・先制医療 BRCA1のハプロ不全は、腎臓発がん過程において、フェロトーシス抵抗性を獲得することにより染色体増幅を促進する(BRCA1 haploinsufficiency promotes chromosomal amplification under renal carcinogenesis through ferroptosis-resistance)

孔穎怡, 赤塚慎也, 本岡大社, 鄭好, 白木之浩, 真下知士, 今岡達彦, 豊國伸哉

日本癌学会総会記事 81回巻頁： S20 - 7 2022年9月

孔穎怡, 赤塚慎也, 本岡大社, 鄭好, 白木之浩, 真下知士, 今岡達彦, 豊國伸哉

Biomedical Research on Trace Elements 33 巻 ( 1 ) 頁： 130 - 130 2022年9月

BRCA1遺伝子ハプロ不全モデルラットを用いた卵巣予備能低下の検証(Validation of reduced ovarian reserve in BRCA1 haploinsufficient rats.)

曽根原玲菜, 本岡大社, 梶山広明, 真下知士, 今岡達彦, 豊國伸哉

日本癌学会総会記事 81回巻頁： P - 3012 2022年9月

BRCA1欠損はフェロトーシス抵抗性を誘導することによりアスベスト誘発性腹膜中皮腫を促進する(Ferroptosis resistance is induced by BRCA1 deficiency in asbestos-induced mesothelioma)

羅亜光, 本岡大社, 赤塚慎也, 真下知士, 今岡達彦, 豊國伸哉

日本癌学会総会記事 81回巻頁： P - 2003 2022年9月

環境因子であるタルクおよびアスベストの卵巣発がん性とゲノム毒性の検討(Carcinogenic genome toxicity of environmental factors, asbestos and talc in ovary)

本岡大社, 鬼丸洸, 鈴木洋, 片渕充沙子, 近藤英治, 田代浩徳, 片渕秀隆, 豊國伸哉

日本癌学会総会記事 81回巻頁： P - 2007 2022年9月

BRCA1のハプロ不全は発がん過程において、フェロトーシス抵抗性を獲得することにより染色体増幅を促進する(BRCA1 haploinsufficiency promotes carcinogenic chromosomal amplification by ferroptosis-resistance) 査読有り国際誌

孔穎怡, 赤塚慎也, 本岡大社, 鄭好, 白木之浩, 真下知士, 今岡達彦, 豊國伸哉

Biomedical Research on Trace Elements 33 巻 ( 1 ) 頁： 130 - 130 2022年9月

孔穎怡, 赤塚慎也, 本岡大社, 鄭好, 白木之浩, 真下知士, 今岡達彦, 豊國伸哉

日本癌学会総会記事 81回巻頁： S20 - 7 2022年9月

BRCA1 haploinsufficiency promotes chromosomal amplification under Fenton reaction-based carcinogenesis through ferroptosis-resistance. 査読有り国際誌 Open Access

Kong Y, Akatsuka S, Motooka Y, Zheng H, Cheng Z, Shiraki Y, Mashimo T, Imaoka T, Toyokuni S

Redox biology 54 巻頁： 102356 - 102356 2022年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Biology

Germline-mutation in BRCA1 tumor suppressor gene is an established risk for carcinogenesis not only in females but also in males. Deficiency in the repair of DNA double-strand breaks is hypothesized as a responsible mechanism for carcinogenesis. However, supporting data is insufficient both in the mutation spectra of cancers in the patients with BRCA1 germline-mutation and in murine knockout/knock-in models of Brca1 haploinsufficiency. Furthermore, information on the driving force toward carcinogenesis in BRCA1 mutation carriers is lacking. Here we applied Fenton reaction-based renal carcinogenesis to a rat heterozygously knockout model of BRCA1 haploinsufficiency (mutant [MUT] model; L63X/+). Rat MUT model revealed significant promotion of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Array-based comparative genome hybridization of the RCCs identified significant increase in chromosomal amplification, syntenic to those in breast cancers of BRCA1 mutation carriers, including c-Myc, in comparison to those in the wild-type. Subacute-phase analysis of the kidney after repeated Fe-NTA treatment in the MUT model revealed dysregulated iron metabolism with mitochondrial malfunction assessed by expression microarray and electron microscopy, leading to renal tubular proliferation with iron overload. In conclusion, we for the first time demonstrate that biallelic wild-type BRCA1 provides more robust protection for mitochondrial metabolism under iron-catalyzed oxidative stress, preventing the emergence of neoplastic cells with chromosomal amplification. Our results suggest that oxidative stress via excess iron is a major driving force for carcinogenesis in BRCA1 haploinsufficiency, which can be a target for cancer prevention and therapeutics.

DOI： 10.1016/j.redox.2022.102356

Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers. 査読有り国際誌 Open Access

Soyama H, Nishio M, Otani J, Sakuma T, Takao S, Hara S, Masuda T, Mimori K, Toyokuni S, Lydon JP, Nakao K, Nishina H, Fukumoto T, Maehama T, Suzuki A

Proceedings of the National Academy of Sciences of the United States of America 119 巻 ( 29 ) 頁： e2123134119 2022年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Breast cancer is the most frequent malignancy in women worldwide. Basal-like breast cancer (BLBC) is the most aggressive form of this disease, and patients have a poor prognosis. Here, we present data suggesting that the Hippo-transcriptional coactivator with PDZ-binding motif (TAZ) pathway is a key driver of BLBC onset and progression. Deletion of Mob1a/b in mouse mammary luminal epithelium induced rapid and highly reproducible mammary tumorigenesis that was dependent on TAZ but not yes-associated protein 1 (YAP1). In situ early-stage BLBC-like malignancies developed in mutant animals by 2 wk of age, and invasive BLBC appeared by 4 wk. In a human estrogen receptor+ luminal breast cancer cell line, TAZ hyperactivation skewed the features of these luminal cells to the basal phenotype, consistent with the aberrant TAZ activation frequently observed in human precancerous BLBC lesions. TP53 mutation is rare in human precancerous BLBC but frequent in invasive BLBC. Addition of Trp53 deficiency to our Mob1a/b-deficient mouse model enhanced tumor grade and accelerated cancer progression. Our work justifies targeting the Hippo-TAZ pathway as a therapy for human BLBC, and our mouse model represents a powerful tool for evaluating candidate agents.

DOI： 10.1073/pnas.2123134119

Hepatocyte growth factor derived from senescent cells attenuates cell competition-induced apical elimination of oncogenic cells. 査読有り国際誌 Open Access

Igarashi N, Miyata K, Loo TM, Chiba M, Hanyu A, Nishio M, Kawasaki H, Zheng H, Toyokuni S, Kon S, Moriyama K, Fujita Y, Takahashi A

Nature communications 13 巻 ( 1 ) 頁： 4157 - 4157 2022年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Communications

Cellular senescence and cell competition are important tumor suppression mechanisms that restrain cells with oncogenic mutations at the initial stage of cancer development. However, the link between cellular senescence and cell competition remains unclear. Senescent cells accumulated during the in vivo aging process contribute toward age-related cancers via the development of senescence-associated secretory phenotype (SASP). Here, we report that hepatocyte growth factor (HGF), a SASP factor, inhibits apical extrusion and promotes basal protrusion of Ras-mutated cells in the cell competition assay. Additionally, cellular senescence induced by a high-fat diet promotes the survival of cells with oncogenic mutations, whereas crizotinib, an inhibitor of HGF signaling, provokes the removal of mutated cells from mouse livers and intestines. Our study provides evidence that cellular senescence inhibits cell competition-mediated elimination of oncogenic cells through HGF signaling, suggesting that it may lead to cancer incidence during aging.

DOI： 10.1038/s41467-022-31642-4

Diluted aqueous extract of heat-not-burn tobacco product smoke causes less oxidative damage in fibroblasts than conventional cigarette. 国際誌 Open Access

Lyu Q, Jiang L, Zheng H, Hayashi S, Sato K, Toyokuni S

Journal of clinical biochemistry and nutrition 71 巻 ( 1 ) 頁： 55 - 63 2022年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

Smoke from conventional cigarettes (C-cigarettes) contains various reactive oxygen species and toxic chemicals, which potentially cause oxidative damage not only to airways but also to the whole body, leading eventually to diseases, including emphysema, advanced atherosclerosis, and cancer. Many heat-not-burn tobacco products (HTPs) have been commercialized recently in Japan to maintain the smoking population by advertising that HTPs are less toxic. However, there were few studies reported from neutral organizations whether HTPs are indeed less damaging. To evaluate the potential capacity of HTPs to induce oxidative stress, we here compared two different HTPs with two types of C-cigarettes, using human fibroblast IMR90SV cells and 5% aqueous extracts in 10-ml phosphate-buffered saline (50-ml smoke/10 s). HTPs exhibited significantly lower oxidative toxicity in comparison to C-cigarettes. Whereas C-cigarettes induced ferroptosis in fibroblasts, the effects of HTPs were significantly reduced by measuring the levels of peroxides, pro-inflammatory cytokine expression, autophagy, catalytic Fe(II) and 8-hydroxy-2'-deoxyguanosine. Notably, major portions of C-cigarettes-induced pathogenic responses were inhibited by catalase. However, HTPs still induced p62 autophagy-adaptor at 5%-dilution and caused lethal effects to fibroblasts with undiluted solution. In conclusion, HTPs smoke per se can be toxic despite less toxicity in comparison to C-cigarettes, which warrants further investigation.

DOI： 10.3164/jcbn.21-134

Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. 国際誌 Open Access

Murphy MP, Bayir H, Belousov V, Chang CJ, Davies KJA, Davies MJ, Dick TP, Finkel T, Forman HJ, Janssen-Heininger Y, Gems D, Kagan VE, Kalyanaraman B, Larsson NG, Milne GL, Nyström T, Poulsen HE, Radi R, Van Remmen H, Schumacker PT, Thornalley PJ, Toyokuni S, Winterbourn CC, Yin H, Halliwell B

Nature metabolism 4 巻 ( 6 ) 頁： 651 - 662 2022年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Metabolism

Multiple roles of reactive oxygen species (ROS) and their consequences for health and disease are emerging throughout biological sciences. This development has led researchers unfamiliar with the complexities of ROS and their reactions to employ commercial kits and probes to measure ROS and oxidative damage inappropriately, treating ROS (a generic abbreviation) as if it were a discrete molecular entity. Unfortunately, the application and interpretation of these measurements are fraught with challenges and limitations. This can lead to misleading claims entering the literature and impeding progress, despite a well-established body of knowledge on how best to assess individual ROS, their reactions, role as signalling molecules and the oxidative damage that they can cause. In this consensus statement we illuminate problems that can arise with many commonly used approaches for measurement of ROS and oxidative damage, and propose guidelines for best practice. We hope that these strategies will be useful to those who find their research requiring assessment of ROS, oxidative damage and redox signalling in cells and in vivo.

DOI： 10.1038/s42255-022-00591-z

Cytotoxicity of plasma-irradiated lactate solution produced under atmospheric airtight conditions and generation of the methyl amino group 査読有り国際誌

Ito, D; Iwata, N; Ishikawa, K; Nakamura, K; Hashizume, H; Miron, C; Tanaka, H; Kajiyama, H; Toyokuni, S; Mizuno, M; Hori, M

APPLIED PHYSICS EXPRESS 15 巻 ( 5 ) 2022年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Applied Physics Express

Ringer's lactate solution was irradiated with non-equilibrium plasma under airtight conditions. The plasma-activated lactate (PAL) was produced with argon, oxygen, and nitrogen gases following purging of Ar. Cytotoxicity could be controlled by diluting PAL, and a killing effect was selectively obtained on cancer cells compared to normal cells for Ar+O2+N2 PALs. Nonetheless, cytotoxicity was partly reproduced by similar concentrations of H2O2 and NO2- in the PALs. The organics produced by plasma irradiation to lactate were investigated using nuclear magnetic resonance, and the generation of methyl amino species was confirmed.

DOI： 10.35848/1882-0786/ac6360

CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury

PCBP2 knockdown promotes ferroptosis in malignant mesothelioma. 査読有り国際誌 Open Access

Yue L, Luo Y, Jiang L, Sekido Y, Toyokuni S

Pathology international 72 巻 ( 4 ) 頁： 242 - 251 2022年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

Malignant mesothelioma (MM) is still increasing worldwide. The pathogenesis depends on asbestos-induced iron accumulation, which eventually leads to ferroptosis-resistance of mesothelial cells via somatic mutations. Poly (rC)-binding proteins 1 and 2 (PCBP1/2) are recently recognized cytosolic Fe(II) chaperones. Here we studied the role of PCBP1/2 in rat/human mesothelial and MM cells as well as rat/human MM specimens. Normal peritoneal mesothelial cells in rats exhibited PCBP1 but not PCBP2 immunopositivity whereas primary/immortalized mesothelial cells showed PCBP1/2 immunopositivity. Rat MM specimens induced by intraperitoneal injection of chrysotile, including in situ lesion, revealed PCBP1/2 immunopositivity (90% for both) in the nucleus and cytoplasm with a tendency of higher expression in epithelioid subtype. Knockdown of PCBP2 but not PCBP1 significantly decreased both TfR1 and FTH expression in MM cells with inhibition of proliferation, indicating stagnation of intracellular iron transport. Erastin, a cysteine-deprivation type ferroptosis inducer, decreased the expression of both PCBP1/2 in MM cells. Furthermore, PCBP2 knockdown significantly increased the sensitivity of MM cells to erastin-induced ferroptosis with increased catalytic Fe(II). In conclusion, PCBP2 works for ferroptosis-resistance not only during mesothelial carcinogenesis but also in MM, which warrants further investigation as a novel therapeutic target.

DOI： 10.1111/pin.13209

タルクは曝露卵巣に鉄過剰状態とフェロトーシス抵抗性を誘導し発がんに関わる

井上陽太, 本岡大社, 豊國伸哉

日本病理学会会誌 111 巻 ( 1 ) 頁： 353 - 353 2022年3月

アスベストはヘモグロビンを吸着し卵巣表層上皮に鉄過剰状態を形成することで卵巣がん形成に関わる

本岡大社, 豊國伸哉

日本病理学会会誌 111 巻 ( 1 ) 頁： 249 - 249 2022年3月

Sato Yuki, Oguchi Akiko, Fukushima Yuji, Masuda Kyoko, Toriu Naoya, Taniguchi Keisuke, Yoshikawa Takahisa, Cui Xiaotong, Kondo Makiko, Hosoi Takeshi, Komidori Shota, Shimizu Yoko, Fujita Harumi, Jiang Li, Kong Yingyi, Yamanashi Takashi, Seita Jun, Yamamoto Takuya, Toyokuni Shinya, Hamazaki Yoko, Hattori Masakazu, Yoshikai Yasunobu, Boor Peter, Floege Jürgen, Kawamoto Hiroshi, Murakawa Yasuhiro, Minato Nagahiro, Yanagita Motoko

Journal of Clinical Investigation 132 巻 ( 2 ) 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Tertiary lymphoid tissues (TLTs) facilitate local T- and B-cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here we identify TNF superfamily CD153-CD30 signaling between two unique age-dependent lymphocyte subpopulations, CD153⁺PD-1⁺CD4⁺ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL21 and IFNγ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153-CD30 signaling in TLT formation and propose targeting CD153-CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.

CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury. 国際誌

Sato Y, Oguchi A, Fukushima Y, Masuda K, Toriu N, Taniguchi K, Yoshikawa T, Cui X, Kondo M, Hosoi T, Komidori S, Shimizu Y, Fujita H, Jiang L, Kong Y, Yamanashi T, Seita J, Yamamoto T, Toyokuni S, Hamazaki Y, Hattori M, Yoshikai Y, Boor P, Floege J, Kawamoto H, Murakawa Y, Minato N, Yanagita M

The Journal of clinical investigation 132 巻 ( 2 ) 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Investigation

Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling between 2 unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL-21 and IFN-γ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis, and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153/CD30 signaling in TLT formation and propose targeting the CD153/CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.

DOI： 10.1172/JCI146071

CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury

Journal of Clinical Investigation 132 巻 ( 2 ) 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Enhancement of ethanol production and cell growth in budding yeast by direct irradiation of low-temperature plasma 査読有り国際誌 Open Access

Tanaka, H; Matsumura, S; Ishikawa, K; Hashizume, H; Ito, M; Nakamura, K; Kajiyama, H; Kikkawa, F; Ito, M; Ohno, K; Okazaki, Y; Toyokuni, S; Mizuno, M; Hori, M

JAPANESE JOURNAL OF APPLIED PHYSICS 61 巻 ( SA ) 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Applied Physics

Ethanol production by budding yeast was compared between direct and indirect plasma irradiation. We observed enhancement of ethanol production and cell growth not by indirect plasma irradiation but by direct plasma irradiation. Glucose consumption was increased in budding yeast by direct plasma irradiation. Extracellular flux analysis revealed that glycolytic activity in the budding yeast was elevated by direct plasma irradiation. These results suggest that direct plasma irradiation enhances ethanol production in budding yeast by elevating the glycolytic activity.

DOI： 10.35848/1347-4065/ac2037

Ferroptosis resistance determines high susceptibility of murine A/J strain to iron-induced renal carcinogenesis. 査読有り国際誌 Open Access

Cheng Z, Akatsuka S, Li GH, Mori K, Takahashi T, Toyokuni S

Cancer science 113 巻 ( 1 ) 頁： 65 - 78 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Cancer susceptibility is a critical factor in the understanding of carcinogenesis. Intraperitoneal (i.p.) injection of an iron chelate, ferric nitrilotriacetate (Fe-NTA), produces hydroxyl radicals via Fenton reaction to induce ferroptosis in renal proximal tubules. Rats or mice subjected to repeated i.p. injections of Fe-NTA develop renal cell carcinoma (RCC). To elucidate the molecular mechanisms that cause susceptibility to renal carcinogenesis, we first established an inter-strain difference in the susceptibility to Fe-NTA-induced renal carcinogenesis in mice. Based on a previous observation of a low incidence of RCC with this model in C57BL/6J strain mice, we investigated A/J strain mice here, which demonstrated significantly higher susceptibility to Fe-NTA-induced renal carcinogenesis. Homozygous deletion of the Cdkn2a/2b tumor suppressor locus was detected for the first time in A/J strain mice. Focusing on ferroptosis and iron metabolism, we explored the mechanisms involved that lead to the difference in RCC development. We compared the protective responses in the kidney of A/J and C57BL/6J strains after Fe-NTA treatment. After 3-week Fe-NTA treatment, A/J mice maintained higher levels of expression of glutathione peroxidase 4 and xCT (SLC7A11), leading to a lower level of lipid peroxidation. Simultaneously, A/J mice had decreased expression of transferrin receptor and increased expression of ferritin to greater degrees than C57BL/6 mice. After a single Fe-NTA injection, higher levels of oxidative cell damage and cytosolic catalytic Fe(II) were observed in C57BL/6J mice, accompanied by a greater increase in lipocalin-2. Lipocalin-2 deficiency significantly decreased oxidative renal damage. Our results suggest that a genetic trait favoring ferroptosis resistance contributes to high susceptibility to Fe-NTA-induced RCC in A/J strain.

DOI： 10.1111/cas.15175

Tetrachloroaurate (III)-induced oxidation increases non-thermal plasma-induced oxidative stress. 査読有り国際誌

Okazaki Y, Sasaki K, Ito N, Tanaka H, Matsumoto KI, Hori M, Toyokuni S

Free radical research 56 巻 ( 1 ) 頁： 17 - 27 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Non-thermal plasma (NTP) devices have been explored for medical applications. NTP devices discharge electrons, positive ions, ultraviolet (UV), reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as the hydroxyl radical (•OH), singlet oxygen (1O<inf>2</inf>), superoxide (O<inf>2</inf>•-), hydrogen peroxide (H<inf>2</inf>O<inf>2</inf>), ozone, and nitric oxide, at near-physiological temperature. At preclinical stages or in human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral, and biofilm-related infections, wound healing, and cancer cell death. Here, we observed that ferric, vanadium, and gold(III) ions significantly elevated lipid peroxidation, which was measured by 2-thiobarbituric acid-reactive substances (TBARS) in combination with NTP exposure. Using 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) as a spin probe in electron paramagnetic resonance (EPR), we observed that tetrachloroaurate (III) yielded an M4PO-X spin adduct. Tetrachloroaurate-induced oxidation was attenuated efficiently by reduced (GSH) and oxidized glutathione (GSSG), while glycine (Gly), and L-glutamate (Glu), components of GSH, were ineffective. Furthermore, GSH and GSSG efficiently suppressed tetrachloroaurate-induced lipid peroxidation, while Gly and Glu were ineffective in suppressing TBARS elevation. These results indicate that tetrachloroaurate-induced oxidation is attenuated by GSH as well as GSSG. Further studies are warranted to elucidate the redox reactions between metal ions and biomolecules to advance the clinical application of NTP.

DOI： 10.1080/10715762.2022.2026348

Double-edged Sword Role of Iron-loaded Ferritin in Extracellular Vesicles. 査読有り国際誌 Open Access

Toyokuni S, Kong Y, Zheng H, Mi D, Katabuchi M, Motooka Y, Ito F

Journal of cancer prevention 26 巻 ( 4 ) 頁： 244 - 249 2021年12月

Embryonal erythropoiesis and aging exploit ferroptosis. 査読有り国際誌 Open Access

Zheng H, Jiang L, Tsuduki T, Conrad M, Toyokuni S

Redox biology 48 巻頁： 102175 - 102175 2021年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Biology

Ferroptosis is a form of regulated cell necrosis, as a consequence of Fe(II)-dependent lipid peroxidation. Although ferroptosis has been linked to cancer cell death, neurodegeneration and reperfusion injury, physiological roles of ferroptosis have not been elucidated to date mostly due to the lack of appropriate methodologies. Here, we show that 4-hydroxy-2-nonenal (HNE)-modified proteins detected by a HNEJ-1 mouse monoclonal antibody is a robust immunohistochemical technology to locate ferroptosis in tissues in combination with morphological nuclear information, based on various models of ferroptosis, including erastin-induced cysteine-deprivation, conditional Gpx4 knockout and Fe(II)-dependent renal tubular injury, as well as other types of regulated cell death. Specificity of HNEJ-1 with ferroptosis was endorsed by non-selective identification of HNE-modified proteins in an Fe(II)-dependent renal tubular injury model. We further comprehensively searched for signs of ferroptosis in different developmental stages of Fischer-344 rats from E9.5–2.5 years of age. We observed that there was a significant age-dependent increase in ferroptosis in the kidney, spleen, liver, ovary, uterus, cerebellum and bone marrow, which was accompanied by iron accumulation. Not only phagocytic cells but also parenchymal cells were affected. Epidermal ferroptosis in ageing SAMP8 mice was significantly promoted by high-fat or carbohydrate-restricted diets. During embryogenesis of Fischer-344 rats, we found ferroptosis in nucleated erythrocytes at E13.5, which disappeared in enucleated erythrocytes at E18.5. Administration of a ferroptosis inhibitor, liproxstatin-1, significantly delayed erythrocyte enucleation. Therefore, our results demonstrate for the first time the involvement of ferroptosis in physiological processes, such as embryonic erythropoiesis and aging, suggesting the evolutionally acquired mechanism and the inevitable side effects, respectively.

DOI： 10.1016/j.redox.2021.102175

Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin. 査読有り国際誌 Open Access

Ito F, Kato K, Yanatori I, Murohara T, Toyokuni S

Redox biology 47 巻頁： 102174 - 102174 2021年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Biology

Asbestos-associated diseases remain a social burden worldwide. Our previous studies identified asbestos-induced iron-rich milieu for mesothelial cells with ceaseless macrophage ferroptosis. However, molecular mechanisms how this mutagenic milieu influences mesothelial cells have not been elucidated yet. Here, we propose a novel mechanism that extracellular vesicles (EVs) mediate asbestos-associated mutagenic factors to mesothelial cells. In a mice model of intraperitoneal crocidolite injection, mutagenic milieu highly expressed CD63, an exosomal marker. We then used a GFP-CD63 labeled THP-1 macrophage model exposed to crocidolite/iron, which generated EVs under ferroptotic process. We observed that MeT-5A mesothelial cells can receive and internalize these EVs. Furthermore, we comprehensively analyzed the ferroptosis-dependent EVs (FedEVs) for transported proteins and identified ferritin heavy/light chains as major components. Therefore, we inferred that FedEVs transport iron from ferroptotic macrophages to mesothelial cells. RNA sequencing revealed that the mesothelial cells receiving higher amounts of the FedEVs were mitotic, especially at the S and G2/M phases, by the use of Fucci mesothelial cells. Nuclear 8-hydroxy-2′-deoxyguanosine and γ-H2AX were significantly increased in the recipient mesothelial cells after exposure to FedEVs. Collectively, we here demonstrate a novel mechanism that FedEVs act as a key mutagenic mediator by transporting iron, which contribute to asbestos-induced mesothelial carcinogenesis.

DOI： 10.1016/j.redox.2021.102174

Mitochondrial involvement in the development and progression of diseases. 査読有り国際誌 Open Access

Valacchi G, Pecorelli A, Toyokuni S

Archives of biochemistry and biophysics 711 巻頁： 109006 - 109006 2021年10月

CD63 is regulated by iron via the IRE-IRP system and is important for ferritin secretion by extracellular vesicles. 査読有り国際誌 Open Access

Yanatori I, Richardson DR, Dhekne HS, Toyokuni S, Kishi F

Blood 138 巻 ( 16 ) 頁： 1490 - 1503 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Blood

Extracellular vesicles (EVs) transfer functional molecules between cells. CD63 is a widely recognized EV marker that contributes to EV secretion from cells. However, the regulation of its expression remains largely unknown. Ferritin is a cellular iron storage protein that can also be secreted by the exosome pathway, and serum ferritin levels classically reflect body iron stores. Iron metabolism–associated proteins such as ferritin are intricately regulated by cellular iron levels via the iron responsive element-iron regulatory protein (IRE-IRP) system. Herein, we present a novel mechanism demonstrating that the expression of the EV-associated protein CD63 is under the regulation of the IRE-IRP system. We discovered a canonical IRE in the 5′ untranslated region of CD63 messenger RNA that is responsible for regulating its expression in response to increased iron. Cellular iron loading caused a marked increase in CD63 expression and the secretion of CD63+ EVs from cells, which were shown to contain ferritin-H and ferritin-L. Our results demonstrate that under iron loading, intracellular ferritin is transferred via nuclear receptor coactivator 4 (NCOA4) to CD63+ EVs that are then secreted. Such iron-regulated secretion of the major iron storage protein ferritin via CD63+ EVs, is significant for understanding the local cell-to-cell exchange of ferritin and iron.

DOI： 10.1182/blood.2021010995

Mice lacking DYRK2 exhibit congenital malformations with lung hypoplasia and altered Foxf1 expression gradient. 査読有り国際誌 Open Access

Yogosawa S, Ohkido M, Horii T, Okazaki Y, Nakayama J, Yoshida S, Toyokuni S, Hatada I, Morimoto M, Yoshida K

Communications biology 4 巻 ( 1 ) 頁： 1204 - 1204 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Communications Biology

Congenital malformations cause life-threatening diseases in pediatrics, yet the molecular mechanism of organogenesis is poorly understood. Here we show that Dyrk2-deficient mice display congenital malformations in multiple organs. Transcriptome analysis reveals molecular pathology of Dyrk2-deficient mice, particularly with respect to Foxf1 reduction. Mutant pups exhibit sudden death soon after birth due to respiratory failure. Detailed analyses of primordial lungs at the early developmental stage demonstrate that Dyrk2 deficiency leads to altered airway branching and insufficient alveolar development. Furthermore, the Foxf1 expression gradient in mutant lung mesenchyme is disrupted, reducing Foxf1 target genes, which are necessary for proper airway and alveolar development. In ex vivo lung culture system, we rescue the expression of Foxf1 and its target genes in Dyrk2-deficient lung by restoring Shh signaling activity. Taken together, we demonstrate that Dyrk2 is essential for embryogenesis and its disruption results in congenital malformation.

DOI： 10.1038/s42003-021-02734-6

Role of ferroptosis in nanofiber-induced carcinogenesis 国際誌

Toyokuni Shinya, Ito Fumiya, Motooka Yashiro

Metallomics Research 1 巻 ( 1 ) 頁： rev-14 - rev-21 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japan Society for Biomedical Research on Trace Elements

Biopersistent nanofibers with specified physical dimension are unexpected human carcinogens whether they are natural or synthetic. Asbestos, a natural fibrous mineral, is classified as a definite human carcinogen (IARC Group 1) to cause malignant mesothelioma (MM) and lung cancer. Multi-walled carbon nanotube of 50 nm-diameter was defined in 2014 as a possible carcinogen (IARC Group 2B) toward MM, fortunately with no authorized patients thus far. Carcinogenic mechanism of asbestos has been a mystery for a long time. It is now recognized that asbestos goes through lung parenchyma by collecting hemoglobin-derived iron to reach pleural cavity, which takes several decades. Iron-loaded asbestos can induce oxidative damage directly to mesothelial cells, carcinogenesis-target cells lining somatic cavities. Recently, it was clarified that surrounding stromal environment are as important for mesothelial carcinogenesis. The novel concept here is ceaseless ferroptosis of macrophages, which forms a Fe(II)-dependent stromal mutagenic milieu indirectly for mesothelial cells and indeed is a revised understanding of frustrated phagocytosis. Deposition of foreign materials eventually causes iron accumulation in situ due to the innate characteristic of preserving iron inside cells. Nanofiber-induced carcinogenesis may be involved in other human carcinogenesis, including ovarian cancer. Alternatively, iron excess can be an optimal target of cancer prevention and cancer treatment.

DOI： 10.11299/metallomicsresearch.mr202102

Txn1 mutation causes epilepsy associated with vacuolar degeneration in the midbrain

Iori Ohmori, Mamoru Ouchida, Hirohiko Imai, Saeko Ishida, Shinya Toyokuni, Tomoji Mashimo

bioRxiv 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cold Spring Harbor Laboratory

<title>Abstract</title>Thioredoxin (TXN), encoded by <italic>Txn1</italic>, acts as a critical antioxidant in the defense against oxidative stress by regulating the dithiol/disulfide balance of interacting proteins. The role of TXN in the central nervous system (CNS) is largely unknown. A phenotype-driven study of <italic>N</italic>-ethyl-<italic>N</italic>-nitrosourea-mutated rats with running seizures at around five-week of age revealed the relevance of <italic>Txn1</italic> mutations to CNS disorders. Genetic mapping identified <italic>Txn1</italic>-F54L in epileptic rats. The insulin-reducing activity of <italic>Txn1</italic>-F54L rats was approximately one-third that of the wild-type. Vacuolar degeneration in the midbrain, mainly in the thalamus and the inferior colliculus, was observed in the <italic>Txn1</italic>-F54L rats. The lesions displayed neuronal and oligodendrocyte cell death. Neurons in <italic>Txn1</italic>-F54L rats showed morphological changes in the mitochondria. Vacuolar degeneration began at three weeks of age, and spontaneous repair began at seven weeks; a dramatic change from cell death to repair occurred in the midbrain during a restricted period. In conclusion, <italic>Txn1</italic> is essential for the development of the midbrain in juvenile rats.

DOI： 10.1101/2021.10.07.463470

Plasma-activated Ringer's lactate solution inhibits the cellular respiratory system in HeLa cells 査読有り国際誌

Tanaka H., Maeda S., Nakamura K., Hashizume H., Ishikawa K., Ito M., Ohno K., Mizuno M., Motooka Y., Okazaki Y., Toyokuni S., Kajiyama H., Kikkawa F., Hori M.

Plasma Processes and Polymers 18 巻 ( 10 ) 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plasma Processes and Polymers

Nonequilibrium atmospheric pressure plasma has enabled a variety of new applications in medicine, agriculture, and other industries. It is particularly noteworthy that plasma itself and/or plasma-activated culture medium have been shown to preferentially kill various cancer cells. We have previously developed a plasma-activated Ringer's lactate solution (PAL) for use as a new cancer treatment. In this study, behaviors of extracellular and intracellular reactive oxygen and nitrogen species in the cellular respiratory system of PAL-treated HeLa cells were investigated using an extracellular flux analyzer and a probe to measure mitochondrial membrane potential. In PAL-treated HeLa cells, extracellular hydrogen peroxide in PAL was found to be responsible for the induction of intracellular hydrogen peroxide and apoptosis, while other components in PAL are responsible for the induction of non-H<inf>2</inf>O<inf>2</inf> intracellular ROS and non-apoptotic cell death, which should be clarified by further experiments. We believe that these are long-lived species derived from plasma-activated lactates. Furthermore, we found that the plasma-activated lactates inhibited glycolysis and the tricarboxylic acid (TCA) cycle, but not the electron transport chain in HeLa cells. These results suggest that PAL induces multiple modes of cell death, including apoptosis through hydrogen peroxide, and non-apoptotic cell death associated with the impairment of mitochondrial functions (glycolysis and TCA cycle). These findings shed light on the novel mechanism underlying plasma-activated lactate-induced cell death.

DOI： 10.1002/ppap.202100056

Defective biosynthesis of ascorbic acid in Sod1-deficient mice results in lethal damage to lung tissue 国際誌

Low temperature plasma irradiation products of sodium lactate solution that induce cell death on U251SP glioblastoma cells were identified. 査読有り国際誌 Open Access

Tanaka H, Hosoi Y, Ishikawa K, Yoshitake J, Shibata T, Uchida K, Hashizume H, Mizuno M, Okazaki Y, Toyokuni S, Nakamura K, Kajiyama H, Kikkawa F, Hori M

Scientific reports 11 巻 ( 1 ) 頁： 18488 - 18488 2021年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Low-temperature plasma is being widely used in the various fields of life science, such as medicine and agriculture. Plasma-activated solutions have been proposed as potential cancer therapeutic reagents. We previously reported that plasma-activated Ringer’s lactate solution exhibited selective cancer-killing effects, and that the plasma-treated L-sodium lactate in the solution was an anti-tumor factor; however, the components that are generated through the interactions between plasma and L-sodium lactate and the components responsible for the selective killing of cancer cells remain unidentified. In this study, we quantified several major chemical products, such as pyruvate, formate, and acetate, in plasma-activated L-sodium lactate solution by nuclear magnetic resonance analysis. We further identified novel chemical products, such as glyoxylate and 2,3-dimethyltartrate, in the solution by direct infusion-electrospray ionization with tandem mass spectrometry analysis. We found that 2,3-dimethyltartrate exhibited cytotoxic effects in glioblastoma cells, but not in normal astrocytes. These findings shed light on the identities of the components that are responsible for the selective cytotoxic effect of plasma-activated solutions on cancer cells, and provide useful data for the potential development of cancer treatments using plasma-activated L-sodium lactate solution.

DOI： 10.1038/s41598-021-98020-w

女性器へのタルクの曝露は鉄過剰環境を形成し卵巣がんの発がんに関わる

本岡大社, 伊藤文哉, 田代浩徳, 片渕秀隆, 豊國伸哉

日本癌学会総会記事 80回巻頁： [E1 - 6] 2021年9月

変革する病理学:形態診断から普遍的研究プラットフォームへ実験病理学に起源を持つがんのフェロトーシス抵抗性

豊國伸哉, Zheng Hao, Kong Yingyi, Yaguang Luo, 本岡大社

日本癌学会総会記事 80回巻頁： [S10 - 6] 2021年9月

BRCA1欠損は腎臓における鉄代謝の変化と酸化ストレスによる腎発がんに関わる

孔穎怡, 本岡大社, 赤塚慎也, 真下知士, 今岡達彦, 豊國伸哉

日本癌学会総会記事 80回巻頁： [P2 - 1] 2021年9月

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer. 査読有り国際誌 Open Access

Miyata K, Imai Y, Hori S, Nishio M, Loo TM, Okada R, Yang L, Nakadai T, Maruyama R, Fujii R, Ueda K, Jiang L, Zheng H, Toyokuni S, Sakata T, Shirahige K, Kojima R, Nakayama M, Oshima M, Nagayama S, Seimiya H, Hirota T, Saya H, Hara E, Takahashi A

Proceedings of the National Academy of Sciences of the United States of America 118 巻 ( 35 ) 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

DOI： 10.1073/pnas.2025647118

Prenatal Molecular Hydrogen Administration Ameliorates Several Findings in Nitrofen-Induced Congenital Diaphragmatic Hernia. 査読有り国際誌 Open Access

Miura M, Imai K, Tsuda H, Miki R, Tano S, Ito Y, Hirako-Takamura S, Moriyama Y, Ushida T, Iitani Y, Nakano-Kobayashi T, Toyokuni S, Kajiyama H, Kotani T

International journal of molecular sciences 22 巻 ( 17 ) 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Molecular Sciences

Oxidative stress plays a pathological role in pulmonary hypoplasia and pulmonary hypertension in congenital diaphragmatic hernia (CDH). This study investigated the effect of molecular hydrogen (H2), an antioxidant, on CDH pathology induced by nitrofen. Sprague-Dawley rats were divided into three groups: control, CDH, and CDH + hydrogen-rich water (HW). Pregnant dams of CDH + HW pups were orally administered HW from embryonic day 10 until parturition. Gasometric evaluation and histological, immunohistochemical, and real-time polymerase chain re-action analyses were performed. Gasometric results (pH, pO2, and pCO2 levels) were better in the CDH + HW group than in the CDH group. The CDH + HW group showed amelioration of alveo-larization and pulmonary artery remodeling compared with the CDH group. Oxidative stress (8-hydroxy-2′-deoxyguanosine-positive-cell score) in the pulmonary arteries and mRNA levels of pro-tein-containing pulmonary surfactant that protects against pulmonary collapse (surfactant protein A) were significantly attenuated in the CDH + HW group compared with the CDH group. Overall, prenatal H2 administration improved respiratory function by attenuating lung morphology and pulmonary artery thickening in CDH rat models. Thus, H2 administration in pregnant women with diagnosed fetal CDH might be a novel antenatal intervention strategy to reduce newborn mortality due to CDH.

DOI： 10.3390/ijms22179500

Role and management of oxidative stress in human disease. 査読有り国際誌 Open Access

Checker R, Sharma D, Sandur SK, Toyokuni S

Free radical research 55 巻 ( 8 ) 頁： 755 - 757 2021年8月

Non-thermal plasma-induced DMPO-OH yields hydrogen peroxide. 国際誌

Okazaki Y, Tanaka H, Matsumoto KI, Hori M, Toyokuni S

Archives of biochemistry and biophysics 705 巻頁： 108901 - 108901 2021年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Recent developments in electronics have enabled the medical applications of non-thermal plasma (NTP), which elicits reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as hydroxyl radical (●OH), hydrogen peroxide (H<inf>2</inf>O<inf>2</inf>), singlet oxygen (1O<inf>2</inf>), superoxide (O<inf>2</inf>●-), ozone, and nitric oxide at near-physiological temperatures. In preclinical studies or human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral and biofilm-related infections, wound healing, and cancer cell death. To elucidate the solution-phase biological effects of NTP in the presence of biocompatible reducing agents, we employed electron paramagnetic resonance (EPR) spectroscopy to quantify ●OH using a spin-trapping probe, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); 1O<inf>2</inf> using a fluorescent probe; and O<inf>2</inf>●- and H<inf>2</inf>O<inf>2</inf> using luminescent probes in the presence of thiols or tempol. NTP-induced ●OH was significantly scavenged by dithiothreitol (DTT), reduced glutathione (GSH), and oxidized glutathione (GSSG) in 2 or 5 mM DMPO. NTP-induced O<inf>2</inf>●- was significantly scavenged by 10 μM DTT and GSH, while 1O<inf>2</inf> was not efficiently scavenged by these compounds. GSSG degraded H<inf>2</inf>O<inf>2</inf> more effectively than GSH and DTT, suggesting that the disulfide bonds reacted with H<inf>2</inf>O<inf>2</inf>. In the presence of 1–50 mM DMPO, NTP-induced H<inf>2</inf>O<inf>2</inf> quantities were unchanged. The inhibitory effect of tempol concentration (50 and 100 μM) on H<inf>2</inf>O<inf>2</inf> production was observed in 1 and 10 mM DMPO, whereas it became ineffective in 50 mM DMPO. Furthermore, DMPO-OH did not interact with tempol. These results suggest that DMPO and tempol react competitively with O<inf>2</inf>●-. Further studies are warranted to elucidate the interaction between NTP-induced ROS and biomolecules.

DOI： 10.1016/j.abb.2021.108901

Lysosomal nitric oxide determines transition from autophagy to ferroptosis after exposure to plasma-activated Ringer's lactate. 国際誌 Open Access

Jiang L, Zheng H, Lyu Q, Hayashi S, Sato K, Sekido Y, Nakamura K, Tanaka H, Ishikawa K, Kajiyama H, Mizuno M, Hori M, Toyokuni S

Redox biology 43 巻頁： 101989 - 101989 2021年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Biology

Non-thermal plasma (NTP), an engineered technology to generate reactive species, induces ferroptosis and/or apoptosis specifically in various-type cancer cells. NTP-activated Ringer's lactate (PAL) is another modality for cancer therapy at preclinical stage. Here we found that PAL induces selective ferroptosis of malignant mesothelioma (MM) cells, where non-targeted metabolome screening identified upregulated citrulline-nitric oxide (.NO) cycle as a PAL target .NO probe detected biphasic peaks transiently at PAL exposure with time-dependent increase, which was responsible for inducible .NO synthase (iNOS) overexpression through NF-κB activation. .NO and lipid peroxidation occupied lysosomes as a major compartment with increased TFEB expression. Not only ferrostatin-1 but inhibitors for .NO and/or iNOS could suppress this ferroptosis. PAL-induced ferroptosis accompanied autophagic process in the early phase, as demonstrated by an increase in essential amino acids, LC3B-II, p62 and LAMP1, transforming into the later phase with boosted lipid peroxidation. Therefore, .NO-mediated lysosomal impairment is central in PAL-induced ferroptosis.

DOI： 10.1016/j.redox.2021.101989

L-Dehydroascorbate efficiently degrades non-thermal plasma-induced hydrogen peroxide 国際誌

Okazaki Y.

Archives of Biochemistry and Biophysics 700 巻 2021年3月

Preclinical Verification of the Efficacy and Safety of Aqueous Plasma for Ovarian Cancer Therapy. 国際誌 Open Access

Nakamura K, Yoshikawa N, Mizuno Y, Ito M, Tanaka H, Mizuno M, Toyokuni S, Hori M, Kikkawa F, Kajiyama H

Cancers 13 巻 ( 5 ) 頁： 1 - 15 2021年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancers

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. The major cause of EOC’s lethality is that intraperitoneal recurrence occurs with high frequency due to occult metastasis. We had demonstrated that plasma-activated medium (PAM) exerts a metastasis-inhibitory effect on ovarian cancer in vitro and in vivo. Here we investigated how PAM inhibits intraperitoneal metastasis. We studied PAM’s inhibition of micro-dissemination onto the omentum by performing in vivo imaging in combination with a sequential histological analysis. The results revealed that PAM induced macrophage infiltration into the disseminated lesion. The iNOS-positive signal was co-localized at the macrophages in the existing lesion, indicating that PAM might induce M1-type macrophages. This may be another mechanism of the antitumor effect through a PAM-evoked immune response. Intraperitoneal lavage with plasma-activated lactate Ringer’s solution (PAL) significantly improved the overall survival rate in an ovarian cancer mouse model. Our results demonstrated the efficiency and practicality of aqueous plasma for clinical applications.

DOI： 10.3390/cancers13051141

Role of ferroptosis in carcinogenesis and tumor biology

Toyokuni Shinya

FREE RADICAL BIOLOGY AND MEDICINE 165 巻 2021年3月

Mechanism of asbestos-induced carcinogenesis via dysregulation of redox-active iron 国際誌

Ito Fumiya, Toyokuni Shinya

CANCER SCIENCE 112 巻頁： 998 - 998 2021年2月

Mechanism of asbestos-induced carcinogenesis via dysregulation of redox-active iron 国際誌

Ito Fumiya, Toyokuni Shinya

CANCER SCIENCE 112 巻頁： 998 - 998 2021年2月

Signature analysis of genomic mutations in asbestos-induced rat mesothelioma

Akatsuka Shinya, Jiang Li, Elzawahry Asmaa, Kato Mamoru, Totsuka Yukari, Shibata Tatsuhiro, Toyokuni Shinya

CANCER SCIENCE 112 巻頁： 610 - 610 2021年2月

Asbestos and talc contribute to ovarian carcinogenesis via iron overload

Motooka Yashiro, Ito Fumiya, Tashiro Hironori, Katabuchi Hidetaka, Toyokuni Shinya

CANCER SCIENCE 112 巻頁： 245 - 245 2021年2月

Role of redox-active metals for the prevention and treatment of cancer in the era of precision medicine

Toyokuni Shinya, Richardson Des R.

CANCER SCIENCE 112 巻頁： 996 - 996 2021年2月

Homma T.

Free Radical Biology and Medicine 162 巻頁： 255 - 265 2021年1月

Prognostic significance of the MDM2/HMGA2 ratio and histological tumor grade in dedifferentiated liposarcoma 国際誌

Yamashita K.

Genes Chromosomes and Cancer 60 巻 ( 1 ) 頁： 26 - 37 2021年1月

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 国際誌 Open Access

Klionsky, DJ; Abdel-Aziz, AK; Abdelfatah, S; Abdellatif, M; Abdoli, A; Abel, S; Abeliovich, H; Abildgaard, MH; Abudu, YP; Acevedo-Arozena, A; Adamopoulos, IE; Adeli, K; Adolph, TE; Adornetto, A; Aflaki, E; Agam, G; Agarwal, A; Aggarwal, BB; Agnello, M; Agostinis, P; Agrewala, JN; Agrotis, A; Aguilar, P; Ahmad, ST; Ahmed, ZM; Ahumada-Castro, U; Aits, S; Aizawa, S; Akkoc, Y; Akoumianaki, T; Akpinar, HA; Al-Abd, AM; Al-Akra, L; Al-Gharaibeh, A; Alaoui-Jamali, MA; Alberti, S; Alcocer-Gómez, E; Alessandri, C; Ali, M; Al-Bari, MAA; Aliwaini, S; Alizadeh, J; Almacellas, E; Almasan, A; Alonso, A; Alonso, GD; Altan-Bonnet, N; Altieri, DC; Alves, S; da Costa, CA; Alzaharna, MM; Amadio, M; Amantini, C; Amaral, C; Ambrosio, S; Amer, AO; Ammanathan, V; An, ZY; Andersen, SU; Andrabi, SA; Andrade-Silva, M; Andres, AM; Angelini, S; Ann, D; Anozie, UC; Ansari, MY; Antas, P; Antebi, A; Antón, Z; Anwar, T; Apetoh, L; Apostolova, N; Araki, T; Araki, Y; Arasaki, K; Araújo, WL; Araya, J; Arden, C; Arévalo, MA; Arguelles, S; Arias, E; Arikkath, J; Arimoto, H; Ariosa, AR; Armstrong-James, D; Arnauné-Pelloquin, L; Aroca, A; Arroyo, DS; Arsov, I; Artero, R; Asaro, DML; Aschner, M; Ashrafizadeh, M; Ashur-Fabian, O; Atanasov, AG; Au, AK; Auberger, P; Auner, HW; Aurelian, L; Autelli, R; Avagliano, L; Avalos, Y; Aveic, S; Aveleira, CA; AvinWittenberg, T; Aydin, Y; Ayton, S; Ayyadevara, S; Azzopardi, M; Baba, M; Backer, JM; Backues, SK; Bae, DH; Bae, ON; Bae, SH; Baehrecke, EH; Baek, A; Baek, SH; Baek, SH; Bagetta, G; Bagniewska-Zadworna, A; Bai, H; Bai, J; Bai, XY; Bai, YD; Bairagi, N; Baksi, S; Balbi, T; Baldari, CT; Balduini, W; Ballabio, A; Ballester, M; Balazadeh, S; Balzan, R; Bandopadhyay, R; Banerjee, S; Banerjee, S; Bao, Y; Baptista, MS; Baracca, A; Barbati, C; Bargiela, A; Barilà, D; Barlow, PG; Barmada, SJ; Barreiro, E; Barreto, GE; Bartek, J; Bartel, B; Bartolome, A; Barve, GR; Basagoudanavar, SH; Bassham, DC; Bast, RC ; Basu, A; Batoko, H; Batten, I; Baulieu, EE; Baumgarner, BL; Bayry, J; Beale, R; Beau, I; Beaumatin, F; Bechara, LRG; Beck, GR ; Beers, MF; Begun, J; Behrends, C; Behrens, GMN; Bei, R; Bejarano, E; Bel, S; Behl, C; Belaid, A; Belgareh-Touzé, N; Bellarosa, C; Belleudi, F; Perez, MB; Bello-Morales, R; Beltran, JSD; Beltran, S; Benbrook, DM; Bendorius, M; Benitez, BA; Benito-Cuesta, I; Bensalem, J; Berchtold, MW; Berezowska, S; Bergamaschi, D; Bergami, M; Bergmann, A; Berliocchi, L; Berlioz-Torrent, C; Bernard, A; Berthoux, L; Besirli, CG; Besteiro, S; Betin, VM; Beyaert, R; Bezbradica, JS; Bhaskar, K; Bhatia-Kissova, I; Bhattacharya, R; Bhattacharya, S; Bhattacharyya, S; Bhuiyan, MS; Bhutia, SK; Bi, LR; Bi, XL; Biden, TJ; Bijian, K; Billes, VA; Binart, N; Bincoletto, C; Birgisdottir, AB; Bjorkoy, G; Blanco, G; Blas-Garcia, A; Blasiak, J; Blomgran, R; Blomgren, K; Blum, JS; Boada-Romero, E; Boban, M; BoeszeBattaglia, K; Boeuf, P; Boland, B; Bomont, P; Bonaldo, P; Bonam, SR; Bonfili, L; Bonifacino, JS; Boone, BA; Bootman, MD; Bordi, M; Borner, C; Bornhauser, BC; Borthakur, G; Bosch, J; Bose, S; Botana, LM; Botas, J; Boulanger, CM; Boulton, ME; Bourdenx, M; Bourgeois, B; Bourke, NM; Bousquet, G; Boya, P; Bozhkov, P; Bozi, LHM; Bozkurt, TO; Brackney, DE; Brandts, CH; Braun, RJ; Braus, GH; Bravo-Sagua, R; Bravo-San Pedro, JM; Brest, P; Bringer, MA; Briones-Herrera, A; Broaddus, VC; Brodersen, P; Alvarez, ÉMC; Brodsky, JL; Brody, SL; Bronson, PG; Bronstein, JM; Brown, CN; Brown, RE; Brum, PC; Brumell, JH; Brunetti-Pierri, N; Bruno, D; Bryson-Richardson, RJ; Bucci, C; Buchrieser, C; Bueno, M; Buitrago-Molina, LE; Buraschi, S; Buch, S; Buchan, JR; Buckingham, EM; Budak, H; Budini, M; Bultynck, G; Burada, F; Burgoyne, JR; Burón, MI; Bustos, V; Büttner, S; Butturini, E; Byrd, A; Cabas, I; Cabrera-Benitez, S; Cadwell, K; Cai, JJ; Cai, L; Cai, Q; Cairó, M; Calbet, JA; Caldwell, GA; Caldwell, KA; Call, JA; Calvani, R; Calvo, AC; Barrera, MCR; Camara, NO; Camonis, JH; Camougrand, N; Campanella, M; Campbell, EM; Campbell-Valois, FX; Campello, S; Campesi, I; Campos, JC; Camuzard, O; Cancino, J; de Almeida, DC; Canesi, L; Caniggia, I; Canonico, B; Cantí, C; Cao, B; Caraglia, M; Caramés, B; Carchman, EH; Cardenal-Muñoz, E; Cardenas, C; Cardenas, L; Cardoso, SM; Carew, JS; Carle, GF; Carleton, G; Carloni, S; Carmona-Gutierrez, D; Carneiro, LA; Carnevali, O; Carosi, JM; Carra, S; Carrier, A; Carrier, L; Carroll, B; Carter, AB; Carvalho, AN; Casanova, M; Casas, C; Casas, J; Cassioli, C; Castillo, EF; Castillo, K; Castillo-Lluva, S; Castoldi, F; Castori, M; Castro, AF; Castro-Caldas, M; Castro-Hernandez, J; Castro-Obregon, S; Catz, SD; Cavadas, C; Cavaliere, F; Cavallini, G; Cavinato, M; Cayuela, ML; Rica, PC; Cecarini, V; Cecconi, F; Cechowska-Pasko, M; Cenci, S; Ceperuelo-Mallafré, V; Cerqueira, JJ; Cerutti, JM; Cervia, D; Cetintas, VB; Cetrullo, S; Chae, HJ; Chagin, AS; Chai, CY; Chakrabarti, G; Chakrabarti, O; Chakraborty, T; Chakraborty, T; Chami, M; Chamilos, G; Chan, DW; Chan, EYW; Chan, ED; Chan, HYE; Chan, HH; Chan, H; Chan, MT; Chan, YS; Chandra, PK; Chang, CP; Chang, CM; Chang, HC; Chang, K; Chao, J; Chapman, T; Charlet-Berguerand, N; Chatterjee, S; Chaube, SK; Chaudhary, A; Chauhan, S; Chaum, E; Checler, F; Cheetham, ME; Chen, CS; Chen, GC; Chen, JF; Chen, LL; Chen, LL; Chen, L; Chen, ML; Chen, MK; Chen, N; Chen, Q; Chen, RH; Chen, S; Chen, W; Chen, WQ; Chen, XM; Chen, XW; Chen, X; Chen, Y; Chen, YG; Chen, YY; Chen, YQ; Chen, YJ; Chen, YQ; Chen, ZS; Chen, Z; Chen, ZH; Chen, ZJ; Chen, ZX; Cheng, HH; Cheng, J; Cheng, SY; Cheng, W; Cheng, XD; Cheng, XT; Cheng, YY; Cheng, ZY; Chen, Z; Cheong, H; Cheong, JK; Chernyak, B; Cherry, S; Cheung, CFR; Cheung, CHA; Cheung, KH; Chevet, E; Chi, RJ; Chiang, AKS; Chiaradonna, F; Chiarelli, R; Chiariello, M; Chica, N; Chiocca, S; Chiong, M; Chiou, SH; Chiramel, A; Chiurchiù, V; Cho, DH; Choe, SK; Choi, AMK; Choi, ME; Choudhury, KR; Chow, NS; Chu, CT; Chua, JP; Chua, JJE; Chung, H; Chung, KP; Chung, S; Chung, SH; Chung, YL; Cianfanelli, V; Ciechomska, IA; Cifuentes, M; Cinque, L; Cirak, S; Cirone, M; Clague, MJ; Clarke, R; Clementi, E; Coccia, EM; Codogno, P; Cohen, E; Cohen, MM; Colasanti, T; Colasuonno, F; Colbert, RA; Colell, A; Coll, NS; Collins, MO; Colombo, M; Colón-Ramos, DA; Combaret, L; Comincini, S; Cominetti, MR; Consiglio, A; Conte, A; Conti, F; Contu, VR; Cookson, MR; Coombs, KM; Coppens, I; Corasaniti, MT; Corkery, DP; Cordes, N; Cortese, K; Costa, MD; Costantino, S; Costelli, P; Coto-Montes, A; Crack, PJ; Crespo, JL; Criollo, A; Crippa, V; Cristofani, R; Csizmadia, T; Cuadrado, A; Cui, B; Cui, J; Cui, YX; Cui, Y; Culetto, E; Cumino, AC; Cybulsky, A; Czaja, MJ; Czuczwar, SJ; D'Adamo, S; D'Amelio, M; D'Arcangelo, D; D'Lugos, AC; D'Orazi, G; da Silva, JA; Dafsari, HS; Dagda, RK; Dagdas, Y; Daglia, M; Dai, X; Dai, Y; Dai, YY; Dal Col, J; Dalhaimer, P; Dalla Valle, L; Dallenga, T; Dalmasso, G; Damme, M; Dando, I; Dantuma, NP; Darling, AL; Das, H; Dasarathy, S; Dasari, SK; Dash, S; Daumke, O; Dauphinee, AN; Davies, JS; Dávila, VA; Davis, RJ; Davis, T; Naidu, SD; De Amicis, F; De Bosscher, K; De Felice, F; De Franceschi, L; De Leonibus, C; Barbosa, MGD; De Meyer, GRY; De Milito, A; De Nunzio, C; De Palma, C; De Santi, M; De Virgilio, C; De Zio, D; Debnath, J; DeBosch, BJ; Decuypere, J; Deehan, MA; Deflorian, G; DeGregori, J; Dehay, B; Del Rio, G; Delaney, JR; Delbridge, LMD; Delorme-Axford, E; Delpino, MV; Demarchi, F; Dembitz, V; Demers, ND; Deng, HB; Deng, ZQ; Dengjel, J; Dent, P; Denton, D; DePamphilis, ML; Der, CJ; Deretic, V; Descoteaux, A; Devis, L; Devkota, S; Devuyst, O; Dewson, G; Dharmasivam, M; Dhiman, R; di Bernardo, D; Di Cristina, M; Di Domenico, F; Di Fazio, P; Di Fonzo, A; Di Guardo, G; Di Guglielmo, GM; Di Leo, L; Di Malta, C; Di Nardo, A; Di Rienzo, M; Di Sano, F; Diallinas, G; Diao, JJ; Diaz-Araya, G; Díaz-Laviada, I; Dickinson, JM; Diederich, M; Dieudé, M; Dikic, I; Ding, SP; Ding, WX; Dini, L; Dinic, J; Dinic, M; Dinkova-Kostova, AT; Dionne, MS; Distler, JHW; Diwan, A; Dixon, IMC; Djavaheri-Mergny, M; Dobrinski, I; Dobrovinskaya, O; Dobrowolski, R; Dobson, RCJ; Emre, SD; Donadelli, M; Dong, B; Dong, XN; Dong, ZW; Ii, GWD; Dotsch, V; Dou, H; Dou, J; Dowaidar, M; Dridi, S; Drucker, L; Du, AL; Du, CG; Du, GW; Du, HN; Du, LL; du Toit, A; Duan, SB; Duan, XQ; Duarte, SP; Dubrovska, A; Dunlop, EA; Dupont, N; Durán, R; Dwarakanath, BS; Dyshlovoy, SA; Ebrahimi-Fakhari, D; Eckhart, L; Edelstein, CL; Efferth, T; Eftekharpour, E; Eichinger, L; Eid, N; Eisenberg, T; Eissa, NT; Eissa, S; Ejarque, M; El Andaloussi, A; El-Hage, N; El-Naggar, S; Eleuteri, AM; El-Shafey, ES; Elgendy, M; Eliopoulos, AG; Elizalde, MM; Elks, PM; Elsasser, HP; Elsherbiny, ES; Emerling, BM; Emre, NCT; Eng, CH; Engedal, N; Engelbrecht, AM; Engelsen, AST; Enserink, JM; Escalante, R; Esclatine, A; Escobar-Henriques, M; Eskelinen, EL; Espert, L; Eusebio, MO; Fabrias, G; Fabrizi, C; Facchiano, A; Facchiano, F; Fadeel, B; Fader, C; Faesen, AC; Fairlie, WD; Falcó, A; Falkenburger, BH; Fan, DP; Fan, J; Fan, YB; Fang, EF; Fang, YS; Fang, YQ; Fanto, M; Farfel-Becker, T; Faure, M; Fazeli, G; Fedele, AO; Feldman, AM; Feng, D; Feng, JC; Feng, LF; Feng, YB; Feng, YC; Feng, W; Araujo, TF; Ferguson, TA; Fernandez-Checa, JC; FernándezVeledo, S; Fernie, AR; Ferrante, AW Jr; Ferraresi, A; Ferrari, MF; Ferreira, JCB; Ferro-Novick, S; Figueras, A; Filadi, R; Filigheddu, N; FilippiChiela, E; Filomeni, G; Fimia, GM; Fineschi, V; Finetti, F; Finkbeiner, S; Fisher, EA; Fisher, PB; Flamigni, F; Fliesler, SJ; Flo, TH; Florance, I; Florey, O; Florio, T; Fodor, E; Follo, C; Fon, EA; Forlino, A; Fornai, F; Fortini, P; Fracassi, A; Fraldi, A; Franco, B; Franco, R; Franconi, F; Frankel, LB; Friedman, SL; Fröhlich, LF; Frühbeck, G; Fuentes, JM; Fujiki, Y; Fujita, N; Fujiwara, Y; Fukuda, M; Fulda, S; Furic, L; Furuya, N; Fusco, C; Gack, MU; Gaffke, L; Galadari, S; Galasso, A; Galindo, MF; Kankanamalage, SG; Galluzzi, L; Galy, V; Gammoh, N; Gan, BY; Ganley, IG; Gao, F; Gao, H; Gao, MH; Gao, P; Gao, SJ; Gao, WT; Gao, XB; Garcera, A; Garcia, MN; Garcia, VE; García-Del Portillo, F; Garcia-Escudero, V; GarciaGarcia, A; Garcia-Macia, M; García-Moreno, D; Garcia-Ruiz, C; García-Sanz, P; Garg, AD; Gargini, R; Garofalo, T; Garry, RF; Gassen, NC; Gatica, D; Ge, L; Ge, WZ; Geiss-Friedlander, R; Gelfi, C; Genschik, P; Gentle, IE; Gerbino, V; Gerhardt, C; Germain, K; Germain, M; Gewirtz, DA; Afshar, EG; Ghavami, S; Ghigo, A; Ghosh, M; Giamas, G; Giampietri, C; Giatromanolaki, A; Gibson, GE; Gibson, SB; Ginet, V; Giniger, E; Giorgi, C; Girao, H; Girardin, SE; Giridharan, M; Giuliano, S; Giulivi, C; Giuriato, S; Giustiniani, J; Gluschko, A; Goder, V; Goginashvili, A; Golab, J; Goldstone, DC; Golebiewska, A; Gomes, LR; Gomez, R; Gómez-Sánchez, R; Gomez-Puerto, MC; Gomez-Sintes, R; Gong, Q; Goni, FM; González-Gallego, J; Gonzalez-Hernandez, T; Gonzalez-Polo, RA; Gonzalez-Reyes, JA; González-Rodríguez, P; Goping, IS; Gorbatyuk, MS; Gorbunov, N; Gorojod, RM; Gorski, SM; Goruppi, S; Gotor, C; Gottlieb, RA; Gozes, I; Gozuacik, D; Graef, M; Gräler, MH; Granatiero, V; Grasso, D; Gray, JP; Green, DR; Greenhough, A; Gregory, SL; Griffin, EF; Grinstaff, MW; Gros, F; Grose, C; Gross, AS; Gruber, F; Grumati, P; Grune, T; Gu, XY; Guan, JL; Guardia, CM; Guda, K; Guerra, F; Guerri, C; Guha, P; Guillén, C; Gujar, S; Gukovskaya, A; Gukovsky, I; Gunst, J; Günther, A; Guntur, AR; Guo, CY; Guo, C; Guo, HQ; Guo, LW; Guo, M; Gupta, P; Fernandez, AF; Gupta, SK; Gupta, S; Gupta, VB; Gupta, V; Gustafsson, AB; Gutterman, DD; Ranjitha, HB; Haapasalo, A; Haber, JE; Hadano, S; Hafrén, AJ; Haidar, M; Hall, BS; Halldén, G; Hamacher-Brady, A; Hamann, A; Hamasaki, M; Han, WD; Hansen, M; Hanson, P; Hao, ZJ; Harada, M; Harhaji-Trajkovic, L; Hariharan, N; Haroon, N; Harris, J; Hasegawa, T; Nagoor, NH; Haspel, JA; Haucke, V; Hawkins, WD; Hay, BA; Haynes, CM; Hayrabedyan, SB; Hays, TS; He, CC; He, Q; He, RR; He, YW; He, YY; Heakal, Y; Heberle, AM; Hejtmancik, JF; Helgason, GV; Henkel, V; Herb, M; Hergovich, A; Herman-Antosiewicz, A; Hernández, A; Hernandez, C; Hernandez-Diaz, S; Hernandez-Gea, V; Herpin, A; Herreros, J; Hervás, JH; Hesselson, D; Hetz, C; Heussler, VT; Higuchi, Y; Hilfiker, S; Hill, JA; Hlavacek, WS; Ho, EA; Ho, IHT; Ho, PWL; Ho, S; Ho, WY; Hobbs, GA; Hochstrasser, M; Hoet, PHM; Hofius, D; Hofman, P; Höhn, A; Holmberg, C; Hombrebueno, JR; Hong, CW; Hong, YR; Hooper, L; Hoppe, T; Horos, R; Hoshida, Y; Hsin, IL; Hsu, HY; Hu, B; Hu, D; Hu, LF; Hu, MC; Hu, RG; Hu, W; Hu, YC; Hu, ZW; Hua, F; Hua, JL; Hua, YQ; Huan, CM; Huang, CH; Huang, CS; Huang, CX; Huang, CL; Huang, HS; Huang, K; Huang, MLH; Huang, R; Huang, S; Huang, TZ; Huang, X; Huang, YJ; Huber, TB; Hubert, V; Hubner, CA; Hughes, SM; Hughes, WE; Humbert, M; Hummer, G; Hurley, JH; Hussain, S; Hussain, S; Hussey, PJ; Hutabarat, M; Hwang, HY; Hwang, S; Ieni, A; Ikeda, F; Imagawa, Y; Imai, Y; Imbriano, C; Imoto, M; Inman, DM; Inoki, K; Iovanna, J; Iozzo, R; Ippolito, G; Irazoqui, JE; Iribarren, P; Ishaq, M; Ishikawa, M; Ishimwe, N; Isidoro, C; Ismail, N; Issazadeh-Navikas, S; Itakura, E; Ito, D; Ivankovic, D; Ivanova, S; Iyer, AK; Izquierdo, JM; Izumi, M; Jäättelä, M; Jabir, MS; Jackson, WT; Jacobo-Herrera, N; Jacomin, AC; Jacquin, E; Jadiya, P; Jaeschke, H; Jagannath, C; Jakobi, AJ; Jakobsson, J; Janji, B; JansenDürr, P; Jansson, PJ; Jantsch, J; Januszewski, S; Jassey, A; Jean, S; JeltschDavid, H; Jendelova, P; Jenny, A; Jensen, TE; Jessen, N; Jewell, JL; Ji, J; Jia, LJ; Jia, R; Jiang, LW; Jiang, Q; Jiang, RC; Jiang, T; Jiang, XJ; Jiang, Y; Jimenez-Sanchez, M; Jin, EJ; Jin, FY; Jin, HC; Jin, L; Jin, LQ; Jin, MY; Jin, S; Jo, EK; Joffre, C; Johansen, T; Johnson, GVW; Johnston, SA; Jokitalo, E; Jolly, MK; Joosten, LAB; Jordan, J; Joseph, B; Ju, DW; Ju, JS; Ju, JF; Juárez, E; Judith, D; Juhász, G; Jun, Y; Jung, CH; Jung, S; Jung, YK; Jungbluth, H; Jungverdorben, J; Just, S; Kaarniranta, K; Kaasik, A; Kabuta, T; Kaganovich, D; Kahana, A; Kain, R; Kajimura, S; Kalamvoki, M; Kalia, M; Kalinowski, DS; Kaludercic, N; Kalvari, I; Kaminska, J; Kaminskyy, VO; Kanamori, H; Kanasaki, K; Kang, C; Kang, R; Kang, SS; Kaniyappan, S; Kanki, T; Kanneganti, TD; Kanthasamy, AG; Kanthasamy, A; Kantorow, M; Kapuy, O; Karamouzis, M; Karim, MR; Karmakar, P; Katare, RG; Kato, M; Kaufmann, SHE; Kauppinen, A; Kaushal, GP; Kaushik, S; Kawasaki, K; Kazan, K; Ke, PY; Keating, DJ; Keber, U; Kehrl, JH; Keller, KE; Keller, CW; Kemper, JK; Kenific, CM; Kepp, O; Kermorgant, S; Kern, A; Ketteler, R; Keulers, TG; Khalfin, B; Khalil, H; Khambu, B; Khan, SY; Khandelwal, VKM; Khandia, R; Kho, W; Khobrekar, N; Khuansuwan, S; Khundadze, M; Killackey, SA; Kim, D; Kim, DR; Kim, DH; Kim, DE; Kim, EY; Kim, EK; Kim, H; Kim, HS; Kim, HR; Kim, JH; Kim, JK; Kim, JH; Kim, J; Kim, JH; Kim, KI; Kim, PK; Kim, SJ; Kimball, SR; Kimchi, A; Kimmelman, AC; Kimura, T; King, MA; Kinghorn, KJ; Kinsey, CG; Kirkin, V; Kirshenbaum, LA; Kiselev, SL; Kishi, S; Kitamoto, K; Kitaoka, Y; Kitazato, K; Kitsis, RN; Kittler, JT; Kjaerulff, O; Klein, PS; Klopstock, T; Klucken, J; Knovelsrud, H; Knorr, RL; Ko, BB; Ko, F; Ko, JL; Kobayashi, H; Kobayashi, S; Koch, I; Koch, JC; Koenig, U; Kögel, D; Koh, YH; Koike, M; Kohlwein, SD; Kocaturk, NM; Komatsu, M; König, J; Kono, T; Kopp, BT; Korcsmaros, T; Korkmaz, G; Korolchuk, V; Korsnes, MS; Koskela, A; Kota, J; Kotake, Y; Kotler, ML; Kou, YJ; Koukourakis, M; Koustas, E; Kovacs, AL; Kovács, T; Koya, D; Kozako, T; Kraft, C; Krainc, D; Krämer, H; Krasnodembskaya, AD; Kretz-Remy, C; Kroemer, G; Ktistakis, NT; Kuchitsu, K; Kuenen, S; Kuerschner, L; Kukar, T; Kumar, A; Kumar, A; Kumar, D; Kumar, D; Kumar, S; Kume, S; Kumsta, C; Kundu, CN; Kundu, M; Kunnumakkara, AB; Kurgan, L; Kutateladze, TG; Kutlu, O; Kwak, S; Kwon, HJ; Kwon, TK; Kwon, YT; Kyrmizi, I; La Spada, A; Labonté, P; Ladoire, S; Laface, I; Lafont, F; Lagace, DC; Lahiri, V; Lai, ZB; Laird, AS; Lakkaraju, A; Lamark, T; Lan, SH; Landajuela, A; Lane, DJR; Lane, JD; Lang, CH; Lange, C; Langer, R; Lapaquette, P; Laporte, J; LaRusso, NF; Lastres-Becker, I; Lau, WCY; Laurie, GW; Lavandero, S; Law, BYK; Law, HKW; Layfield, R; Le, WD; Le Stunff, H; Leary, AY; Lebrun, JJ; Leck, LYW; Leduc-Gaudet, JP; Lee, C; Lee, CP; Lee, DH; Lee, EB; Lee, EF; Lee, GM; Lee, HJ; Lee, HK; Lee, JM; Lee, JS; Lee, JA; Lee, JY; Lee, JH; Lee, M; Lee, MG; Lee, MJ; Lee, MS; Lee, SY; Lee, SJ; Lee, SY; Lee, SB; Lee, WH; Lee, YR; Lee, YH; Lee, Y; Lefebvre, C; Legouis, R; Lei, YL; Lei, YC; Leikin, S; Leitinger, G; Lemus, L; Leng, SL; Lenoir, O; Lenz, G; Lenz, HJ; Lenzi, P; León, Y; Leopoldino, AM; Leschczyk, C; Leskelä, S; Letellier, E; Leung, CT; Leung, PS; Leventhal, JS; Levine, B; Lewis, PA; Ley, K; Li, B; Li, DQ; Li, JM; Li, J; Li, J; Li, K; Li, LW; Li, M; Li, M; Li, M; Li, M; Li, MC; Li, PL; Li, MQ; Li, Q; Li, S; Li, TG; Li, W; Li, WM; Li, X; Li, YP; Li, Y; Li, ZQ; Li, ZY; Li, ZY; Lian, JQ; Liang, CY; Liang, QR; Liang, WC; Liang, YH; Liang, YT; Liao, GH; Liao, LJ; Liao, MZ; Liao, YF; Librizzi, M; Lie, PPY; Lilly, MA; Lim, HJ; Lima, TRR; Limana, F; Lin, C; Lin, CW; Lin, DS; Lin, FC; Lin, JDD; Lin, KM; Lin, KH; Lin, LT; Lin, PH; Lin, Q; Lin, SF; Lin, SJ; Lin, WY; Lin, XY; Lin, YX; Lin, YS; Linden, R; Lindner, P; Ling, SC; Lingor, P; Linnemann, AK; Liou, Y; Lipinski, MM; Lipovsek, S; Lira, VA; Lisiak, N; Liton, PB; Liu, C; Liu, CH; Liu, CF; Liu, CH; Liu, F; Liu, H; Liu, HS; Liu, HF; Liu, HF; Liu, J; Liu, J; Liu, JL; Liu, LY; Liu, LH; Liu, ML; Liu, Q; Liu, W; Liu, WD; Liu, XH; Liu, XD; Liu, XG; Liu, X; Liu, XD; Liu, YF; Liu, Y; Liu, Y; Liu, YY; Liu, YL; Livingston, JA; Lizard, G; Lizcano, JM; Ljubojevic-Holzer, S; LLeonart, ME; Llobet-Navàs, D; Llorente, A; Lo, CH; Lobato-Márquez, D; Long, Q; Long, YC; Loos, B; Loos, JA; López, MG; López-Doménech, G; López-Guerrero, JA; López-Jiménez, AT; López-Valero, I; Lorenowicz, MJ; Lorente, M; Lorincz, P; Lossi, L; Lotersztajn, S; Lovat, PE; Lovell, JF; Lovy, A; Lu, G; Lu, HC; Lu, JH; Lu, JJ; Lu, MJ; Lu, SY; Luciani, A; Lucocq, JM; Ludovico, P; Luftig, MA; Luhr, M; Luis-Ravelo, D; Lum, JJ; Luna-Dulcey, L; Lund, AH; Lund, VK; Lünemann, JD; Lüningschrör, P; Luo, HL; Luo, RC; Luo, SQ; Luo, Z; Luparello, C; Lüscher, B; Luu, L; Lyakhovich, A; Lyamzaev, KG; Lystad, AH; Lytvynchuk, L; Ma, AC; Ma, CL; Ma, MX; Ma, NF; Ma, QH; Ma, XL; Ma, YY; Ma, ZY; MacDougald, OA; Macian, F; MacIntosh, GC; MacKeigan, JP; Macleod, KF; Maday, S; Madeo, F; Madesh, M; Madl, T; Madrigal-Matute, J; Maeda, A; Maejima, Y; Magarinos, M; Mahavadi, P; Maiani, E; Maiese, K; Maiti, P; Maiuri, MC; Majello, B; Major, MB; Makareeva, E; Malik, F; Mallilankaraman, K; Malorni, W; Maloyan, A; Mammadova, N; Man, GCW; Manai, F; Mancias, JD; Mandelkow, EM; Mandell, MA; Manfredi, AA; Manjili, MH; Manjithaya, R; Manque, P; Manshian, BB; Manzano, R; Manzoni, C; Mao, K; Marchese, C; Marchetti, S; Marconi, AM; Marcucci, F; Mardente, S; Mareninova, OA; Margeta, M; Mari, M; Marinelli, S; Marinelli, O; Mariño, G; Mariotto, S; Marshall, RS; Marten, MR; Martens, S; Martin, APJ; Martin, KR; Martin, S; Martin, S; Martín-Segura, A; Martín-Acebes, MA; Martin-Burriel, I; Martin-Rincon, M; Martin-Sanz, P; Martina, JA; Martinet, W; Martinez, A; Martinez, A; Martinez, J; Velazquez, MM; Martinez-Lopez, N; Martinez-Vicente, M; Martins, DO; Lange, Ü; Lopez-Perez, O; Martins, JO; Martins, WK; Martins-Marques, T; Marzetti, E; Masaldan, S; Masclaux-Daubresse, C; Mashek, DG; Massa, V; Massieu, L; Masson, GR; Masuelli, L; Masyuk, A; Masyuk, T; Matarrese, P; Matheu, A; Matoba, S; Matsuzaki, S; Mattar, P; Matte, A; Mattoscio, D; Mauriz, JL; Mauthe, M; Mauvezin, C; Maverakis, E; Maycotte, P; Mayer, J; Mazzoccoli, G; Mazzoni, C; Mazzulli, JR; McCarty, N; McDonald, C; McGill, MR; McKenna, SL; McLaughlin, B; McLoughlin, F; McNiven, MA; McWilliams, TG; Mechta-Grigoriou, F; Medeiros, TC; Medina, DL; Megeney, LA; Megyeri, K; Mehrpour, M; Mehta, JL; Meijer, AJ; Meijer, AH; Mejlvang, J; Meléndez, A; Melk, A; Memisoglu, G; Mendes, AF; Meng, D; Meng, F; Meng, T; Menna-Barreto, R; Menon, MB; Mercer, C; Mercier, AE; Mergny, JL; Merighi, A; Merkley, SD; Merla, G; Meske, V; Mestre, AC; Metur, SP; Meyer, C; Meyer, H; Mi, WY; Mialet-Perez, J; Miao, JY; Micale, L; Miki, Y; Milan, E; Milczarek, M; Miller, DL; Miller, S; Miller, S; Millward, SW; Milosevic, I; Minina, EA; Mirzaei, H; Mirzaei, HR; Mirzaei, M; Mishra, A; Mishra, N; Mishra, PK; Marjanovic, MM; Misasi, R; Misra, A; Misso, G; Mitchell, C; Mitou, G; Miura, T; Miyamoto, S; Miyazaki, M; Miyazaki, M; Miyazaki, T; Miyazawa, K; Mizushima, N; Mogensen, TH; Mograbi, B; Mohammadinejad, R; Mohamud, Y; Mohanty, A; Mohapatra, S; Möhlmann, T; Mohmmed, A; Moles, A; Moley, KH; Molinari, M; Mollace, V; Müller, AB; Mollereau, B; Mollinedo, F; Montagna, C; Monteiro, MJ; Montella, A; Montes, LR; Montico, B; Mony, VK; Compagnoni, GM; Moore, MN; Moosavi, MA; Mora, AL; Mora, M; Morales-Alamo, D; Moratalla, R; Moreira, P; Morelli, E; Moreno, S; Moreno-Blas, D; Moresi, V; Morga, B; Morgan, AH; Morin, F; Morishita, H; Moritz, OL; Moriyama, M; Moriyasu, Y; Morleo, M; Morselli, E; Moruno-Manchon, JF; Moscat, J; Mostowy, S; Motori, E; Moura, AF; Moustaid-Moussa, N; Mrakovcic, M; MuciñoHernández, G; Mukherjee, A; Mukhopadhyay, S; Levy, JMM; Mulero, V; Muller, S; Münch, C; Munjal, A; Munoz-Canoves, P; Muñoz-Galdeano, T; Münz, C; Murakawa, T; Muratori, C; Murphy, BM; Murphy, JP; Murthy, A; Myöhänen, TT; Mysorekar, IU; Mytych, J; Nabavi, SM; Nabissi, M; Nagy, P; Nah, J; Nahimana, A; Nakagawa, I; Nakamura, K; Nakatogawa, H; Nandi, SS; Nanjundan, M; Nanni, M; Napolitano, G; Nardacci, R; Narita, M; Nassif, M; Nathan, I; Natsumeda, M; Naude, RJ; Naumann, C; Naveiras, O; Navid, F; Nawrocki, ST; Nazarko, TY; Nazio, F; Negoita, F; Neill, T; Neisch, AL; Neri, LM; Netea, MG; Neubert, P; Neufeld, TP; Neumann, D; Neutzner, A; Newton, PT; Ney, PA; Nezis, IP; Ng, CCW; Ng, TB; Nguyen, HTT; Nguyen, LT; Ni, HM; Cheallaigh, CN; Ni, Z; Nicolao, MC; Nicoli, F; Nieto-Diaz, M; Nilsson, P; Ning, S; Niranjan, R; Nishimune, H; Niso-Santano, M; Nixon, RA; Nobili, A; Nobrega, C; Noda, T; Nogueira-Recalde, U; Nolan, TM; Nombela, I; Novak, I; Novoa, B; Nozawa, T; Nukina, N; Nussbaum-Krammer, C; Nylandsted, J; O'Donovan, TR; O'Leary, SM; O'Rourke, EJ; O'Sullivan, MP; O'Sullivan, TE; Oddo, S; Oehme, I; Ogawa, M; Ogier-Denis, E; Ogmundsdottir, MH; Ogretmen, B; Oh, GT; Oh, SH; Oh, YJ; Ohama, T; Ohashi, Y; Ohmuraya, M; Oikonomou, V; Ojha, R; Okamoto, K; Okazawa, H; Oku, M; Oliván, S; Oliveira, JMA; Ollmann, M; Olzmann, JA; Omari, S; Omary, MB; Önal, G; Ondrej, M; Ong, SB; Ong, SG; Onnis, A; Orellana, JA; Orellana-Muñoz, S; Ortega-Villaizan, MD; Ortiz-Gonzalez, XR; Ortona, E; Osiewacz, HD; Osman, AHK; Osta, R; Otegui, MS; Otsu, K; Ott, C; Ottobrini, L; Ou, JHJ; Outeiro, TF; Oynebraten, I; Ozturk, M; Pagès, G; Pahari, S; Pajares, M; Pajvani, UB; Pal, R; Paladino, S; Pallet, N; Palmieri, M; Palmisano, G; Palumbo, C; Pampaloni, F; Pan, LF; Pan, QJ; Pan, WL; Pan, X; Panasyuk, G; Pandey, R; Pandey, UB; Pandya, V; Paneni, F; Pang, SY; Panzarini, E; Papademetrio, DL; Papaleo, E; Papinski, D; Papp, D; Park, EC; Park, HT; Park, JM; Park, J; Park, JT; Park, J; Park, SC; Park, SY; Parola, AH; Parys, JB; Pasquier, A; Pasquier, B; Passos, JF; Pastore, N; Patel, HH; Patschan, D; Pattingre, S; Pedraza-Alva, G; Pedraza-Chaverri, J; Pedrozo, Z; Pei, G; Pei, JM; Peled-Zehavi, H; Pellegrini, JM; Pelletier, J; Peñalva, MA; Peng, D; Peng, Y; Penna, F; Pennuto, M; Pentimalli, F; Pereira, CM; Pereira, GJS; Pereira, LC; de Almeida, LP; Perera, ND; PerezOliva, AB; Pérez-Pérez, ME; Periyasamy, P; Perl, A; Perrotta, C; Perrotta, I; Pestell, RG; Petersen, M; Petrache, I; Petrovski, G; Pfirrmann, T; Pfister, AS; Philips, JA; Pi, HF; Picca, A; Pickrell, AM; Picot, S; Pierantoni, GM; Pierdominici, M; Pierre, P; Pierrefite-Carle, V; Pierzynowska, K; Pietrocola, F; Pietruczuk, M; Pignata, C; PimentelMuiños, FX; Pinar, M; Pinheiro, RO; Pinkas-Kramarski, R; Pinton, P; Pircs, K; Piya, S; Pizzo, P; Plantinga, TS; Platta, HW; Plaza-Zabala, A; Plomann, M; Plotnikov, EY; Plun-Favreau, H; Pluta, R; Pocock, R; Pöggeler, S; Pohl, C; Poirot, M; Poletti, A; Ponpuak, M; Popelka, H; Popova, B; Porta, H; Alcon, SP; Portilla-Fernandez, E; Post, M; Potts, MB; Poulton, J; Powers, T; Prahlad, V; Prajsnar, TK; Praticò, D; Prencipe, R; Priault, M; ProikasCezanne, T; Promponas, VJ; Proud, CG; Puertollano, R; Puglielli, L; Pulinilkunnil, T; Puri, D; Puri, R; Puyal, J; Qi, XP; Qi, YM; Qian, WB; Qiang, L; Qiu, Y; Quadrilatero, J; Quarleri, J; Raben, N; Rabinowich, H; Ragona, D; Ragusa, MJ; Rahimi, N; Rahmati, M; Raia, V; Raimundo, N; Rajasekaran, NS; Rao, SR; Rami, A; Ramírez-Pardo, I; Ramsden, DB; Randow, F; Rangarajan, PN; Ranieri, D; Rao, H; Rao, L; Rao, R; Rathore, S; Ratnayaka, JA; Ratovitski, EA; Ravanan, P; Ravegnini, G; Ray, SK; Razani, B; Rebecca, V; Reggiori, F; Régnier-Vigouroux, A; Reichert, AS; Reigada, D; Reiling, JH; Rein, T; Reipert, S; Rekha, RS; Ren, HM; Ren, J; Ren, WC; Renault, T; Renga, G; Reue, K; Rewitz, K; Ramos, BRD; Riazuddin, SA; Ribeiro-Rodrigues, TM; Ricci, JE; Ricci, R; Riccio, V; Richardson, D; Rikihisa, Y; Risbud, M; Risueño, RM; Ritis, K; Rizza, S; Rizzuto, R; Roberts, HC; Roberts, LD; Robinson, KJ; Roccheri, MC; Rocchi, S; Rodney, GG; Rodrigues, T; Silva, VRR; Rodriguez, A; Rodriguez-Barrueco, R; Rodriguez-Henche, N; Rodriguez-Rocha, H; Roelofs, J; Rogers, RS; Rogov, VV; Rojo, A; Rolka, K; Romanello, V; Romani, L; Romano, A; Romano, PS; Romeo-Guitart, D; Romero, LC; Romero, M; Roney, JC; Rongo, C; Roperto, S; Rosenfeldt, MT; Rosenstiel, P; Rosenwald, AG; Roth, KA; Roth, L; Roth, S; Rouschop, KMA; Roussel, BD; Roux, S; Rovere-Querini, P; Roy, A; Rozieres, A; Ruano, D; Rubinsztein, DC; Rubtsova, MP; Ruckdeschel, K; Ruckenstuhl, C; Rudolf, E; Rudolf, R; Ruggieri, A; Ruparelia, AA; Rusmini, P; Russell, RR; Russo, GL; Russo, M; Russo, R; Ryabaya, OO; Ryan, KM; Ryu, KY; Sabater-Arcis, M; Sachdev, U; Sacher, M; Sachse, C; Sadhu, A; Sadoshima, J; Safren, N; Saftig, P; Sagona, AP; Sahay, G; Sahebkar, A; Sahin, M; Sahin, O; Sahni, S; Saito, N; Saito, S; Saito, T; Sakai, R; Sakai, Y; Sakamaki, JI; Saksela, K; Salazar, G; Salazar-Degracia, A; Salekdeh, GH; Saluja, AK; Sampaio-Marques, B; Sanchez, MC; Sanchez-Alcazar, JA; Sanchez-Vera, V; Sancho-Shimizu, V; Sanderson, JT; Sandri, M; Santaguida, S; Santambrogio, L; Santana, MM; Santoni, G; Sanz, A; Sanz, P; Saran, S; Sardiello, M; Sargeant, TJ; Sarin, A; Sarkar, C; Sarkar, S; Sarrias, MR; Sarkar, S; Sarmah, DT; Sarparanta, J; Sathyanarayan, A; Sathyanarayanan, R; Scaglione, KM; Scatozza, F; Schaefer, L; Schafer, ZT; Schaible, UE; Schapira, AH; Scharl, M; Schatzl, HM; Schein, CH; Scheper, W; Scheuring, D; Schiaffino, MV; Schiappacassi, M; Schindl, R; Schlattner, U; Schmidt, O; Schmitt, R; Schmidt, SD; Schmitz, I; Schmukler, E; Schneider, A; Schneider, BE; Schober, R; Schoijet, AC; Schott, MB; Schramm, M; Schröder, B; Schuh, K; Schüller, C; Schulze, RJ; Schürmanns, L; Schwamborn, JC; Schwarten, M; Scialo, F; Sciarretta, S; Scott, MJ; Scotto, KW; Scovassi, AI; Scrima, A; Scrivo, A; Sebastian, D; Sebti, S; Sedej, S; Segatori, L; Segev, N; Seglen, PO; Seiliez, I; Seki, E; Selleck, SB; Sellke, FW; Perez-Lara, A; Selsby, JT; Sendtner, M; Senturk, S; Seranova, E; Sergi, C; Serra-Moreno, R; Sesaki, H; Settembre, C; Setty, SRG; Sgarbi, G; Sha, O; Shacka, JJ; Shah, JA; Shang, DT; Shao, CS; Shao, F; Sharbati, S; Sharkey, LM; Sharma, D; Sharma, G; Sharma, K; Sharma, P; Sharma, S; Shen, HM; Shen, HT; Shen, JG; Shen, M; Shen, WL; Shen, ZN; Sheng, R; Sheng, Z; Sheng, ZH; Shi, JJ; Shi, XB; Shi, YH; Shiba-Fukushima, K; Shieh, J; Shimada, Y; Shimizu, S; Shimozawa, M; Shintani, T; Shoemaker, CJ; Shojaei, S; Shoji, I; Shravage, B; Shridhar, V; Shu, CW; Shu, HB; Shui, K; Shukla, AK; Shutt, TE; Sica, V; Siddiqui, A; Sierra, A; Sierra-Torre, V; Signorelli, S; Sil, P; Silva, BJD; Silva, JD; Silva-Pavez, E; Silvente-Poirot, S; Simmonds, RE; Simon, AK; Simon, HU; Simons, M; Singh, A; Singh, LP; Singh, R; Singh, S; Singh, SK; Singh, SB; Singh, S; Singh, SP; Sinha, D; Sinha, RA; Sinha, S; Sirko, A; Sirohi, K; Sivridis, EL; Skendros, P; Skirycz, A; Slaninová, I; Smaili, SS; Smertenko, A; Smith, MD; Soenen, SJ; Sohn, EJ; Sok, SPM; Solaini, G; Soldati, T; Soleimanpour, SA; Soler, RM; Solovchenko, A; Somarelli, JA; Sonawane, A; Song, FY; Song, HK; Song, JX; Song, KH; Song, ZY; Soria, LR; Sorice, M; Soukas, AA; Soukup, SF; Sousa, D; Sousa, N; Spagnuolo, PA; Spector, SA; Bharath, MMS; St Clair, D; Stagni, V; Staiano, L; Stalnecker, CA; Stankov, M; Stathopulos, PB; Stefan, K; Stefan, SM; Stefanis, L; Steffan, JS; Steinkasserer, A; Stenmark, H; Sterneckert, J; Stevens, C; Stoka, V; Storch, S; Stork, B; Strappazzon, F; Strohecker, AM; Stupack, DG; Su, HX; Su, LY; Su, LX; SuarezFontes, AM; Subauste, CS; Subbian, S; Subirada, P; Sudhandiran, G; Sue, CM; Sui, XB; Summers, C; Sun, GC; Sun, J; Sun, K; Sun, MX; Sun, QM; Sun, Y; Sun, ZJ; Sunahara, KKS; Sundberg, E; Susztak, K; Sutovsky, P; Suzuki, H; Sweeney, G; Symons, JD; Sze, SCW; Szewczyk, NJ; Tabolacci, C; Tacke, F; Taegtmeyer, H; Tafani, M; Tagaya, M; Tai, HR; Tait, SWG; Takahashi, Y; Takats, S; Talwar, P; Tam, C; Tam, SY; Tampellini, D; Tamura, A; Tan, CT; Tan, EK; Tan, YQ; Tanaka, M; Tanaka, M; Tang, D; Tang, JF; Tang, TS; Tanida, I; Tao, ZP; Taouis, M; Tatenhorst, L; Tavernarakis, N; Taylor, A; Taylor, GA; Taylor, JM; Tchetina, E; Tee, AR; Tegeder, I; Teis, D; Teixeira, N; Teixeira-Clerc, F; Tekirdag, KA; Tencomnao, T; Tenreiro, S; Tepikin, A; Testillano, PS; Tettamanti, G; Tharaux, P; Thedieck, K; Thekkinghat, AA; Thellung, S; Thinwa, JW; Thirumalaikumar, VP; Thomas, SM; Thomes, PG; Thorburn, A; Thukral, L; Thum, T; Thumm, M; Tian, L; Tichy, A; Till, A; Timmerman, V; Titorenko, V; Todi, S; Todorova, K; Toivonen, JM; Tomaipitinca, L; Tomar, D; Tomas-Zapico, C; Tong, BCK; Tong, C; Tong, X; Tooze, SA; Torgersen, ML; Torii, S; Torres-López, L; Torriglia, A; Towers, CG; Towns, R; Toyokuni, S; Trajkovic, V; Tramontano, D; Tran, Q; Travassos, LH; Trelford, CB; Tremel, S; Trougakos, IP; Tsao, BP; Tschan, MP; Tse, HF; Tse, TF; Tsugawa, H; Tsvetkov, AS; Tumbarello, DA; Tumtas, Y; Tuñón, MJ; Turcotte, S; Turk, B; Turk, V; Turner, BJ; Tuxworth, R; Tyler, JK; Tyutereva, E; Uchiyama, Y; UgunKlusek, A; Uhlig, HH; Ulasov, I; Umekawa, M; Ungermann, C; Unno, R; Urbe, S; Uribe-Carretero, E; Üstün, S; Uversky, VN; Vaccari, T; Vaccaro, M; Vahsen, BF; Vakifahmetoglu-Norberg, H; Valdor, R; Valente, MJ; Valko, A; Vallee, RB; Valverde, AM; Van den Berghe, G; van Der Veen, S; Van Kaer, L; van Loosdregt, J; van Wijk, SJL; Vandenberghe, W; Vanhorebeek, I; Vannier-Santos, MA; Vannini, N; Vanrell, MC; Vantaggiato, C; Varano, G; Varela-Nieto, I; Varga, M; Vasconcelos, MH; Vats, S; Vavvas, DG; VegaNaredo, I; Vega-Rubin-de-Celis, S; Velasco, G; Velázquez, AP; Vellai, T; Vellenga, E; Velotti, F; Verdier, M; Verginis, P; Vergne, I; Verkade, P; Verma, M; Verstreken, P; Vervliet, T; Vervoorts, J; Vessoni, AT; Victor, VM; Vidal, M; Vidoni, C; Vieira, OV; Vierstra, RD; Viganó, S; Vihinen, H; Vijayan, V; Vila, M; Vilar, M; Villalba, JM; Villalobo, A; Villarejo-Zori, B; Villarroya, F; Villarroya, J; Vincent, O; Vindis, C; Viret, C; Viscomi, MT; Visnjic, D; Vitale, I; Vocadlo, DJ; Voitsekhovskaja, OV; Volonté, C; Volta, M; Vomero, M; Von Haefen, C; Vooijs, MA; Voos, W; Vucicevic, L; Wade-Martins, R; Waguri, S; Waite, KA; Wakatsuki, S; Walker, DW; Walker, MJ; Walker, SA; Walter, J; Wandosell, FG; Wang, B; Wang, CY; Wang, C; Wang, CR; Wang, CW; Wang, CY; Wang, D; Wang, FY; Wang, F; Wang, FM; Wang, GS; Wang, H; Wang, H; Wang, HX; Wang, HG; Wang, JR; Wang, JG; Wang, J; Wang, JD; Wang, K; Wang, LR; Wang, LM; Wang, MH; Wang, MQ; Wang, NB; Wang, PW; Wang, PP; Wang, P; Wang, P; Wang, QJ; Wang, Q; Wang, QK; Wang, QA; Wang, WT; Wang, WY; Wang, XN; Wang, XJ; Wang, Y; Wang, YC; Wang, YZ; Wang, YY; Wang, YH; Wang, YP; Wang, Y; Wang, YQ; Wang, Z; Wang, ZY; Wang, ZG; Warnes, G; Warnsmann, V; Watada, H; Watanabe, E; Watchon, M; Weaver, TE; Wegrzyn, G; Wehman, AM; Wei, HF; Wei, L; Wei, TT; Wei, YJ; Weiergräber, OH; Weihl, CC; Weindl, G; Weiskirchen, R; Wells, A; Wen, RXH; Wen, X; Werner, A; Weykopf, B; Wheatley, SP; Whitton, JL; Whitworth, AJ; Wiktorska, K; Wildenberg, ME; Wileman, T; Wilkinson, S; Willbold, D; Williams, B; Williams, RSB; Williams, RL; Williamson, PR; Wilson, RA; Winner, B; Winsor, NJ; Witkin, SS; Wodrich, H; Woehlbier, U; Wollert, T; Wong, E; Wong, JH; Wong, RW; Wong, VKW; Wong, WWL; Wu, AG; Wu, CB; Wu, J; Wu, JF; Wu, KK; Wu, M; Wu, SY; Wu, SZ; Wu, SY; Wu, SF; Wu, WKK; Wu, XH; Wu, XQ; Wu, YW; Wu, YH; Xavier, RJ; Xia, HG; Xia, LX; Xia, ZY; Xiang, G; Xiang, J; Xiang, ML; Xiang, W; Xiao, B; Xiao, GZ; Xiao, HY; Xiao, HT; Xiao, J; Xiao, L; Xiao, S; Xiao, Y; Xie, BM; Xie, CM; Xie, M; Xie, YX; Xie, ZP; Xie, ZL; Xilouri, M; Xu, CF; Xu, E; Xu, HX; Xu, J; Xu, JR; Xu, L; Xu, WW; Xu, XL; Xue, Y; Yakhine-Diop, SMS; Yamaguchi, M; Yamaguchi, O; Yamamoto, A; Yamashina, S; Yan, SM; Yan, SJ; Yan, Z; Yanagi, Y; Yang, CB; Yang, DS; Yang, H; Yang, HT; Yang, H; Yang, JM; Yang, J; Yang, JY; Yang, L; Yang, L; Yang, M; Yang, PM; Yang, Q; Yang, S; Yang, S; Yang, SF; Yang, WN; Yang, WY; Yang, XY; Yang, XS; Yang, Y; Yang, Y; Yao, HH; Yao, SG; Yao, XQ; Yao, YG; Yao, YM; Yasui, T; Yazdankhah, M; Yen, PM; Yi, C; Yin, XM; Yin, YH; Yin, ZY; Yin, ZY; Ying, MD; Ying, Z; Yip, CK; Yiu, SPT; Yoo, YH; Yoshida, K; Yoshii, SR; Yoshimori, T; Yousefi, B; Yu, BX; Yu, HY; Yu, J; Yu, J; Yu, L; Yu, ML; Yu, SW; Yu, VC; Yu, WH; Yu, ZP; Yu, Z; Yuan, JY; Yuan, LQ; Yuan, SL; Yuan, SSF; Yuan, YG; Yuan, ZQ; Yue, JB; Yue, ZY; Yun, J; Yung, RL; Zacks, DN; Zaffagnini, G; Zambelli, VO; Zanella, I; Zang, QS; Zanivan, S; Zappavigna, S; Zaragoza, P; Zarbalis, KS; Zarebkohan, A; Zarrouk, A; Zeitlin, SO; Zeng, JL; Zeng, JD; Zerovnik, E; Zhan, LX; Zhang, B; Zhang, DD; Zhang, HL; Zhang, H; Zhang, H; Zhang, HH; Zhang, HF; Zhang, HY; Zhang, H; Zhang, HL; Zhang, JB; Zhang, JH; Zhang, JP; Zhang, KLYB; Zhang, LSW; Zhang, L; Zhang, LS; Zhang, L; Zhang, LY; Zhang, MH; Zhang, P; Zhang, S; Zhang, W; Zhang, XN; Zhang, XW; Zhang, XL; Zhang, XY; Zhang, X; Zhang, XX; Zhang, XD; Zhang, Y; Zhang, YJ; Zhang, Y; Zhang, YD; Zhang, YM; Zhang, YY; Zhang, YC; Zhang, Z; Zhang, ZG; Zhang, ZB; Zhang, ZH; Zhang, ZY; Zhang, ZL; Zhao, HB; Zhao, L; Zhao, S; Zhao, TB; Zhao, XF; Zhao, Y; Zhao, YC; Zhao, YL; Zhao, YT; Zheng, GP; Zheng, K; Zheng, L; Zheng, SZ; Zheng, XL; Zheng, Y; Zheng, ZG; Zhivotovsky, B; Zhong, Q; Zhou, A; Zhou, B; Zhou, CF; Zhou, G; Zhou, H; Zhou, H; Zhou, HB; Zhou, J; Zhou, J; Zhou, J; Zhou, JY; Zhou, KL; Zhou, RJ; Zhou, XJ; Zhou, YS; Zhou, YH; Zhou, YB; Zhou, ZY; Zhou, Z; Zhu, BL; Zhu, CL; Zhu, GQ; Zhu, HN; Zhu, HX; Zhu, H; Zhu, WG; Zhu, YP; Zhu, YS; Zhuang, HX; Zhuang, XH; Zientara-Rytter, K; Zimmermann, CM; Ziviani, E; Zoladek, T; Zong, WX; Zorov, DB; Zorzano, A; Zou, WP; Zou, Z; Zou, ZZ; Zuryn, S; Zwerschke, W; Brand-Saberi, B; Dong, XC; Kenchappa, CS; Li, ZG; Lin, Y; Oshima, S; Rong, YG; Sluimer, JC; Stallings, CL; Tong, CK

AUTOPHAGY 17 巻 ( 1 ) 頁： 1 - 382 2021年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Autophagy

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

DOI： 10.1080/15548627.2020.1797280

Cancer Treatments Using Low-Temperature Plasma. 査読有り国際誌

Tanaka H, Mizuno M, Ishikawa K, Toyokuni S, Kajiyama H, Kikkawa F, Hori M

Current medicinal chemistry 28 巻 ( 41 ) 頁： 8549 - 8558 2021年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Current Medicinal Chemistry

Low-temperature plasma (LTP) is a partially ionized gas that contains elec-trons, ions, radicals, light, etc. Recently, the bio-medical application of LTP has become a hot topic in plasma science and biological science. Cancer treatment with plasma is the most challenging topic in plasma bio-medical applications. Many in vitro and in vivo ex-periments have been conducted to investigate the anti-tumor effects of LTP. Extracellular reactive oxygen and nitrogen species (RONS) in plasma-activated solutions are key factors for the anti-tumor effects, and amino acid modifications by LTP may affect cellular responses. Intracellular RONS are also key factors for the anti-tumor effects. Various signaling pathways, such as p53 signaling pathways, survival and proliferation signaling pathways, and oxidative stress-dependent signaling pathways are activated by LTP.

DOI： 10.2174/0929867328666210629121731

山口慎一朗, 守田匡伸, 伊藤文哉, 謝祺琳, 豊國伸哉, 中山勝文

日本毒性学会学術年会 48.1 巻 ( 0 ) 頁： O-4 - 4 2021年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本毒性学会

カーボンナノチューブ（CNTs）は次世代ナノ材料として期待されているものの、その毒性の懸念から国際化学物質事務局ChemSecがCNTsをSIN (Substitute It Now)リストに加えたため、今後のCNTsの使用継続について国際的に大きく議論されている。多層CNTs（MWCNTs）は生体内に入ると主にマクロファージに取り込まれ、そのマクロファージ細胞死およびNLRP3インフラマソーム活性化によるIL-1β分泌が毒性発現に関与すると考えられているが、マクロファージがどのように細胞表面上でMWCNTsを認識するのかはよく分かっていない。本研究で我々は貪食受容体スクリーニングによりT cell immunoglobulin mucin 4（Tim4）およびTim1がMWCNTsを認識することを見出した。CRISPR/Cas9システムによって作製したTim4-/- Tim1-/- マウスやTim4-/- マウス由来腹腔マクロファージを用いた解析から、Tim4はマクロファージによるMWCNTsの貪食、およびその結果起きるIL-1β分泌に関与することが判明した。さらにTim4-/- マウス、あるいは抗Tim4モノクローナル抗体を全投与した野生型マウスにおいて、MWCNTs腹腔投与による横隔膜中皮細胞層の肉芽種形成が有意に抑制されていたことから、Tim4はMWCNTs曝露によるin vivo炎症応答に関与することが示唆された。一方、Tim1はヒト肺細胞株によるMWCNTsの認識に関与することが判明した。以上の結果は、Tim4およびTim1はMWCNTs受容体として機能することを示す。

DOI： 10.14869/toxpt.48.1.0_o-4

The new role of poly (rC)-binding proteins as iron transport chaperones: Proteins that could couple with inter-organelle interactions to safely traffic iron 国際誌

The new era for research on polyphenols and food factors 国際誌

Oteiza P.I.

Archives of Biochemistry and Biophysics 696 巻 2020年12月

The new era for redox research. 国際誌 Open Access

Naito Y, Uchida K, Toyokuni S

Free radical research 54 巻 ( 11-12 ) 頁： 787 - 789 2020年12月

Neural stem cell-specific ITPA deficiency causes neural depolarization and epilepsy. 国際誌 Open Access

Koga Y, Tsuchimoto D, Hayashi Y, Abolhassani N, Yoneshima Y, Sakumi K, Nakanishi H, Toyokuni S, Nakabeppu Y

JCI insight 5 巻 ( 22 ) 2020年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Jci Insight

Inosine triphosphate pyrophosphatase (ITPA) hydrolyzes inosine triphosphate (ITP) and other deaminated purine nucleotides to the corresponding nucleoside monophosphates. In humans, ITPA deficiency causes severe encephalopathy with epileptic seizure, microcephaly, and developmental retardation. In this study, we established neural stem cell–specific Itpa–conditional KO mice (ItpacKO mice) to clarify the effects of ITPA deficiency on the neural system. The Itpa-cKO mice showed growth retardation and died within 3 weeks of birth. We did not observe any microcephaly in the Itpa-cKO mice, although the female Itpa-cKO mice did show adrenal hypoplasia. The Itpa-cKO mice showed limb-clasping upon tail suspension and spontaneous and/or audiogenic seizure. Whole-cell patch-clamp recordings from entorhinal cortex neurons in brain slices revealed a depolarized resting membrane potential, increased firing, and frequent spontaneous miniature excitatory postsynaptic current and miniature inhibitory postsynaptic current in the Itpa-cKO mice compared with ITPA-proficient controls. Accumulated ITP or its metabolites, such as cyclic inosine monophosphates, or RNA containing inosines may cause membrane depolarization and hyperexcitability in neurons and induce the phenotype of ITPA-deficient mice, including seizure.

DOI： 10.1172/jci.insight.140229

Carcinogenesis as Side Effects of Iron and Oxygen Utilization: From the Unveiled Truth toward Ultimate Bioengineering. 国際誌 Open Access

Toyokuni S, Kong Y, Cheng Z, Sato K, Hayashi S, Ito F, Jiang L, Yanatori I, Okazaki Y, Akatsuka S

Cancers 12 巻 ( 11 ) 頁： 1 - 20 2020年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancers

Evolution from the first life on earth to humans took ~3.8 billion years. During the time there have been countless struggles among the species. Mycobacterium tuberculosis was the last major uncontrollable species against the human public health worldwide. After the victory with antibiotics, cancer has become the leading cause of death since 1981 in Japan. Considering that life inevitably depends on ceaseless electron transfers through iron and oxygen, we believe that carcinogenesis is intrinsically unavoidable side effects of using iron and oxygen. Many animal models unequivocally revealed that excess iron is a risk for carcinogenesis. This is supported by a variety of human epidemiological data on cancer risk and prognosis. Cancer is basically a disease of the genome with persistently activated oncogenes and inactivated tumor suppressor genes through which iron addiction with ferroptosis-resistance is maintained. Engineering has made a great advance in the past 50 years. In particular, nanotechnology is distinct in that the size of the engineered molecules is similar to that of our biomolecules. While some nano-molecules are found carcinogenic, there are principles to avoid such carcinogenicity with a smart possibility to use nano-molecules to specifically kill cancer cells. Non-thermal plasma is another modality to fight against cancer.

DOI： 10.3390/cancers12113320

Induction of cancer cell-specific ferroptosis by non-thermal plasma exposure 国際誌

Okazaki Y.

Japanese Journal of Applied Physics 59 巻 ( 11 ) 2020年11月

Yanatori I.

Biochimica et Biophysica Acta - General Subjects 1864 巻 ( 11 ) 2020年11月

Adjusted multiple gases in the plasma flow induce differential antitumor potentials of plasma-activated solutions 国際誌

Nakamura K.

Plasma Processes and Polymers 17 巻 ( 10 ) 2020年10月

Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice 国際誌

Nishio M.

Cancer Science 111 巻 ( 10 ) 頁： 3576 - 3587 2020年10月

Novel ovarian endometriosis model causes infertility via iron-mediated oxidative stress in mice 国際誌

Hayashi S.

Redox Biology 37 巻 2020年10月

アスベストとタルクは鉄過剰環境を形成し卵巣がんの発がんに関わる

本岡大社, 伊藤文哉, 田代浩徳, 片渕秀隆, 豊國伸哉

日本癌学会総会記事 79回巻頁： OJ1 - 1 2020年10月

Asbestos conceives Fe(II)-dependent mutagenic stromal milieu through ceaseless macrophage ferroptosis and β-catenin induction in mesothelium 国際誌

Ito F.

Redox Biology 36 巻 2020年9月

Connective tissue growth factor produced by cancer-associated fibroblasts correlates with poor prognosis in epithelioid malignant pleural mesothelioma 国際誌

Ohara Y.

Oncology Reports 44 巻 ( 3 ) 頁： 838 - 848 2020年9月

Role of carbonic anhydrases in ferroptosis-resistance 国際誌

Li Z.

Archives of Biochemistry and Biophysics 689 巻 2020年8月

Ferroptosis at the crossroads of infection, aging and cancer 国際誌

Toyokuni S.

Cancer Science 111 巻 ( 8 ) 頁： 2665 - 2671 2020年8月

Non-thermal plasma-activated lactate solution kills U251SP glioblastoma cells in an innate reductive manner with altered metabolism 国際誌

Ishikawa Kenji, Hosoi Yugo, Tanaka Hiromasa, Jiang Li, Toyokuni Shinya, Nakamura Kae, Kajiyama Hiroaki, Kikkawa Fumitaka, Mizuno Masaaki, Hori Masaru

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 688 巻 2020年7月

Overexpression of miR-199/214 is a distinctive feature of iron-induced and asbestos-induced sarcomatoid mesothelioma in rats 国際誌

Okazaki Y.

Cancer Science 111 巻 ( 6 ) 頁： 2016 - 2027 2020年6月

Frequent homozygous deletion of Cdkn2a/2b in tremolite-induced malignant mesothelioma in rats 査読有り国際誌

Okazaki Y.

Cancer Science 111 巻 ( 4 ) 頁： 1180 - 1192 2020年4月

Mth1 deficiency provides longer survival upon intraperitoneal crocidolite injection in female mice 査読有り国際誌

Funahashi S.

Free Radical Research 54 巻 ( 2-3 ) 頁： 195 - 205 2020年3月

アスベストは卵巣表層上皮に鉄過剰状態を形成し卵巣がんの発がんに関わる査読有り国際誌

本岡大社, 伊藤文哉, 豊國伸哉

日本病理学会会誌 109 巻 ( 1 ) 頁： 314 - 314 2020年3月

Plasma-activated medium promotes autophagic cell death along with alteration of the mTOR pathway 国際誌

Yoshikawa Nobuhisa, Liu Wenting, Nakamura Kae, Yoshida Kosuke, Ikeda Yoshiki, Tanaka Hiromasa, Mizuno Masaaki, Toyokuni Shinya, Hori Masaru, Kikkawa Fumitaka, Kajiyama Hiroaki

SCIENTIFIC REPORTS 10 巻 ( 1 ) 2020年1月

Augmented oxidative stress increases 8-oxoguanine preferentially in the transcriptionally active genomic regions 査読有り国際誌

Akatsuka S.

Free Radical Research 2020年

Oxidative stress-dependent and -independent death of glioblastoma cells induced by non-thermal plasma-exposed solutions 査読有り国際誌

Tanaka Hiromasa, Mizuno Masaaki, Katsumata Yuko, Ishikawa Kenji, Kondo Hiroki, Hashizume Hiroshi, Okazaki Yasumasa, Toyokuni Shinya, Nakamura Kae, Yoshikawa Nobuhisa, Kajiyama Hiroaki, Kikkawa Fumitaka, Hori Masaru

SCIENTIFIC REPORTS 9 巻 ( 1 ) 頁： 13657 2019年9月

How iron is handled in the course of heme catabolism: Integration of heme oxygenase with intracellular iron transport mechanisms mediated by poly (rC)-binding protein-2 国際誌

Yanatori I.

Archives of Biochemistry and Biophysics 672 巻頁： 108071 2019年9月

Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia 査読有り国際誌

Li Z.

Redox Biology 26 巻頁： 101297 - 101297 2019年9月

アスベストは鉄過剰環境を形成し卵巣癌の発癌に関わる(Asbestos contributes to ovarian carcinogenesis via iron overload) 査読有り国際誌

本岡大社, 伊藤文哉, 田代浩徳, 片渕秀隆, 豊國伸哉

日本癌学会総会記事 78回巻頁： E - 1077 2019年9月

L-Dehydroascorbic acid recycled by thiols efficiently scavenges non-thermal plasma-induced hydroxyl radicals 査読有り国際誌

Okazaki Yasumasa, Tanaka Hiromasa, Hori Masaru, Toyokuni Shinya

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 669 巻頁： 87 - 95 2019年7月

アスベストの曝露は上皮性悪性卵巣腫瘍の発癌に関与する査読有り国際誌

水野勇太, 本岡大社, 豊國伸哉

日本病理学会会誌 108 巻 ( 1 ) 頁： 460 - 461 2019年4月

A scrutiny of circulating microRNA biomarkers for drug-induced tubular and glomerular injury in rats.

Toxicology 415 巻頁： 26-36 2019年3月

Non-thermal plasma-activated medium modified metabolomic profiles in the glycolysis of U251SP glioblastoma 国際誌

Kurake Naoyuki, Ishikawa Kenji, Tanaka Hiromasa, Hashizume Hiroshi, Nakamura Kae, Kajiyama Hiroaki, Toyokuni Shinya, Kikkawa Fumitaka, Mizuno Masaaki, Hori Masaru

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 662 巻頁： 83 - 92 2019年2月

Twist1 was detected in mesenchymal cells of mammary fibroadenoma and invasive components of breast carcinoma in rats.

Funahashi S, Okazaki Y, Nagai H, Chew SH, Ogawa K, Toyoda T, Cho YM, Toyokuni S

Journal of toxicologic pathology 32 巻 ( 1 ) 頁： 19-26 2019年1月

Nonthermal plasma specifically kills oral squamous cell carcinoma cells in a catalytic Fe(II)dependent manner 査読有り国際誌

Sato K.

Journal of Clinical Biochemistry and Nutrition 65 巻 ( 4 ) 頁： 8 - 15 2019年

Iron Metabolism and Ferroptosis 査読有り国際誌

Toyokuni Shinya, Yanatori Izumi

FERROPTOSIS IN HEALTH AND DISEASE 頁： 27 - 41 2019年

尿細管障害と糸球体障害に対する新規バイオマーカーとしての血中microRNAの検討

香川匠, Tomáš ZÁRYBNICKÝ, 大見貴尚, 白井勇司, 豊國伸哉, 織田進吾, 横井毅

日本毒性学会学術年会 46 巻 ( 0 ) 頁： P - 233 2019年

掲載種別：研究論文（学術雑誌）出版者・発行元：日本毒性学会

[Introduction] Drug-induced acute kidney injury (AKI) is a frequent cause of adverse drug reaction. Serum creatinine (CRE) and blood urea nitrogen (BUN) are widely used as standard biomarkers for kidney injury; however, the sensitivity and specificity are considered to be low. In recent years, circulating microRNA (miRNAs) have been attracting considerable attention as novel biomarkers for organ injury, but there are currently no established miRNA biomarkers for drug-induced AKI. The present study aimed to identify plasma miRNAs that may enable early and specific detection of drug-induced tubular and glomerular injury through next-generation sequencing analysis. [Methods] Six-week old male Sprague-Dawley rats were administered cisplatin and gentamicin to induce tubular injury. To create glomerular injury models, puromycin and doxorubicin were administered, and these models were always accompanied by tubular damage. Small RNA-sequencing was performed to analyze time-dependent changes in the plasma miRNA profiles. [Results and Discussion] In the differential analysis, miR-3473 was specifically up-regulated in the glomerular injury models. miR-143-3p and miR-122-5p were commonly down-regulated in all models, and the changes were earlier than the traditional biomarkers, such as plasma CRE and BUN. These data indicated that changes in the specific miRNAs in plasma may enable the early and sensitive detection of tubular and glomerular injuries. The present study suggests the potential utility of plasma miRNAs in the early and type-specific detection of drug-induced AKI.

DOI： 10.14869/toxpt.46.1.0_P-233

Polymer coating on carbon nanotubes into Durobeads is a novel strategy for human environmental safety.

Chemiluminescence imaging of UVA induced reactive oxygen species in mouse skin using L-012 as a probe 国際誌

Liu Jiao-Li, Xue Qiao, Liu Chen-Guang, Bai Feng-Wu, Wada Satoshi, Wang Jin-Ye

FREE RADICAL RESEARCH 52 巻 ( 11-12 ) 頁： 1424 - 1431 2018年12月

Multifaceted roles of Ptger2 (Prostaglandin E receptor 2) in asbestos-induced inflammation and malignant mesothelioma

Jiang Li, Akatsuka Shinya, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 830-830 2018年12月

Inflammatory microenvironment derived from asbestos increases mutagenesis to repairing mesothelial cell

Ito Fumiya, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 830 - 830 2018年12月

Ferroptosis in Cancer Research

Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 195-195 2018年12月

Critical role of Hippo signaling pathway at the onset of squamous cell carcinomas

Suzuki Akira, Nishio Miki, Omori Hirofumi, To Yoko, Maehama Tomohiko, Aono Yukari, Kiyono Tohru, Taguchi Kenichi, Masuda Muneyuki, Toyokuni Shinya, Tashiro Hironori, Katabuchi Hidetaka

CANCER SCIENCE 109 巻頁： 781-781 2018年12月

Asbestos exposure as a possible cause of ovarian carcinogenesis

Yashiro Motooka, Ito Fumiya, Tashiro Hironori, Katabuchi Hidetaka, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 255-255 2018年12月

Analysis of the involvement of cancer-associated fibroblasts in the progression of malignant mesothelioma

Ohara Yuuki, Enomoto Atsushi, Takahashi Masahide, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 454-454 2018年12月

Analysis of the involvement of cancer-associated fibroblasts in the progression of malignant mesothelioma 国際誌

Ohara Yuuki, Enomoto Atsushi, Takahashi Masahide, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 454-454 2018年12月

Ferroptosis in Cancer Research 国際誌

Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 195-195 2018年12月

Critical role of Hippo signaling pathway at the onset of squamous cell carcinomas 国際誌

Suzuki Akira, Nishio Miki, Omori Hirofumi, To Yoko, Maehama Tomohiko, Aono Yukari, Kiyono Tohru, Taguchi Kenichi, Masuda Muneyuki, Toyokuni Shinya, Tashiro Hironori, Katabuchi Hidetaka

CANCER SCIENCE 109 巻頁： 781-781 2018年12月

Asbestos exposure as a possible cause of ovarian carcinogenesis 国際誌

Yashiro Motooka, Ito Fumiya, Tashiro Hironori, Katabuchi Hidetaka, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 255-255 2018年12月

Inflammatory microenvironment derived from asbestos increases mutagenesis to repairing mesothelial cell 国際誌

Ito Fumiya, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 830-830 - 830 2018年12月

Multifaceted roles of Ptger2 (Prostaglandin E receptor 2) in asbestos-induced inflammation and malignant mesothelioma 国際誌

Jiang Li, Akatsuka Shinya, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 830-830 2018年12月

Ito F, Hisashi H, Toyokuni S

Nagoya journal of medical science 80 巻 ( 4 ) 頁： 597 - 604 2018年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nagoya Journal of Medical Science

Carbon nanotubes (CNTs) have attracted much business interest in industrial applications due to their high electrical and heat conductivities while being both durable and versatile. However, multiwall CNTs (MWCNTs) of ~ 50 nm diameter (NT50) have been shown to cause mesothelioma in rodents after direct exposure to mesothelial cells, and thus were classified as a Group 2B carcinogen to humans, which requires considerable regulations for use. In contrast, tangled MWCNTs of ~ 15 nm diameter (NTtngl) are not carcinogenic to rats, indicating that the physical dimension linked with mesothelial cellular uptake is an important factor for human environmental risk. In the present study, hypothesizing that dustability is another distinct risk factor, for the first time, we evaluated the toxicity of CNT granules (Durobeads) that were generated with a polymer coating to mesothelial cells. Polymer coating induced prominent agglomeration and significantly suppressed the dustability of CNTs in a dose-dependent manner, with a 10% polymer coating resulting in 730 times less dustability. These CNT granules revealed significantly lower mesothelial uptake and cytotoxicity in comparison to NT50 in in vitro assays. Similarly, in in vivo analyses, CNT granules induced limited peritoneal inflammation 4 weeks after intraperitoneal injection, whereas NT50 caused severe fibrosing inflammation. Previously, we demonstrated that the severity of inflammation by intraperitoneal injection in the subacute studies are in agreement with the mesothelial carcinogenicity by CNTs. Therefore, we suggest that adding a polymer coating to CNTs provides another smart strategy for the safe use of CNTs.

DOI： 10.18999/nagjms.80.4.597

Global overexpression of divalent metal transporter 1 delays crocidolite-induced mesothelial carcinogenesis in male mice.

Funahashi S, Okazaki Y, Nishiyama T, Ohyoshi H, Yasui H, Nishida K, Matsui S, Toyokuni S

Free radical research 頁： 1-10 2018年10月

Iron addiction with ferroptosis-resistance in asbestos-induced mesothelial carcinogenesis: Toward the era of mesothelioma prevention.

Toyokuni S

Free radical biology & medicine 2018年10月

Molecular mechanisms of non-thermal plasma-induced effects in cancer cells.

Tanaka H, Mizuno M, Ishikawa K, Toyokuni S, Kajiyama H, Kikkawa F, Hori M

Biological chemistry 2018年10月

A special issue of SFRR Asia: Cross talk between free radicals and mitochondria in health and disease.

Liu J, Toyokuni S, Surh YJ

Free radical research 頁： 1-51 2018年9月

新たながん治療法に向けたプラズマ生物学の進展国際誌

豊國伸哉

応用物理学会学術講演会講演予稿集 2018.2 巻 ( 0 ) 頁： 37 - 37 2018年9月

動物モデルを用いたがん研究扁平上皮癌発症におけるHippo経路の役割(Animal models in cancer research Critical role of Hippo signaling pathway at the onset of squamous cell carcinomas)

鈴木聡, 西尾美希, 大森裕文, 藤庸子, 前濱朝彦, 青野ゆかり, 清野透, 田口健一, 益田宗幸, 豊國伸哉, 田代浩徳, 片渕秀隆

日本癌学会総会記事 77回巻頁： 1110 - 1110 2018年9月

アスベストが卵巣癌を起こす可能性についてクロシドライトは卵巣表層上皮におけるDNAの二本鎖切断を惹起する(Asbestos exposure as a possible cause of ovarian carcinogenesis)

本岡大社, 伊藤文哉, 田代浩徳, 片渕秀隆, 豊國伸哉

日本癌学会総会記事 77回巻頁： 118 - 118 2018年9月

Development of a novel monoclonal antibody against 4-hydroxy-2E,6Z-dodecadienal (4-HDDE)-protein adducts: Immunochemical application in quantitative and qualitative analyses of lipid peroxidation in vitro and ex vivo. 国際誌

Uchida K, Shibata T, Toyokuni S, Daniel B, Zarkovic K, Zarkovic N, Sasson S

Free radical biology & medicine 124 巻頁： 12 - 20 2018年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Non-enzymatic peroxidation of polyunsaturated fatty acids (PUFA) results in the formation of various α,β-unsaturated aldehydes, of which 4-hydroxyalkenals are abundant. The propensity of n-6 PUFA, such as linoleic acid, γ-linolenic acid and arachidonic acid, to undergo radical-induced peroxidation and generate 4-hydroxy-2E-nonenal (4-HNE) has been widely demonstrated. The ability of the latter to form covalent adducts with macromolecules and modify cellular functions has been linked to numerous pathological processes. Concomitantly, evidence has accumulated on specific signaling properties of low concentrations of 4-HNE that may induce hormetic and protective responses to peroxidation stress in cells. It has long been known that peroxidation of PUFA, and particularly arachidonic acid, also give rise to 4-hydroxy-2E,6Z-dodecadienal (4-HDDE), which is more chemically reactive than 4-HNE. Few studies on 4-HDDE revealed its ability to avidly interact covalently with electronegative moieties in macromolecules and to its ability to selectively activate the transcriptional regulator Peroxisome Proliferator-Activated Receptor (PPAR)-β/δ. The research on 4-HDDE has been impeded due to the lack of available pure 4-HDDE and antibodies that recognize 4-HDDE-modified epitopes in proteins. The purpose of this study was to employ an established procedure to synthesize 4-HDDE and use it to create and characterize a monoclonal antibody against 4-HDDE-modified proteins and establish its application for ELISA and immunohistochemical analysis of cells and tissues and further expand lipid peroxidation research.

DOI： 10.1016/j.freeradbiomed.2018.05.079

Non-thermal plasma as a simple ferroptosis inducer in cancer cells: A possible role of ferritin 国際誌

Furuta Takahiro, Shi Lei, Toyokuni Shinya

PATHOLOGY INTERNATIONAL 68 巻 ( 7 ) 頁： 442 - 443 2018年7月

Administration of molecular hydrogen during pregnancy improves behavioral abnormalities of offspring in a maternal immune activation model. 査読有り国際誌

Imai K, Kotani T, Tsuda H, Nakano T, Ushida T, Iwase A, Nagai T, Toyokuni S, Suzumura A, Kikkawa F

Scientific reports 8 巻 ( 1 ) 頁： 9221 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

The aim of the present study was to investigate long-term outcomes of the offspring in a lipopolysaccharide (LPS)-induced maternal immune activation (MIA) model and the effect of maternal molecular hydrogen (H2) administration. We have previously demonstrated in the MIA mouse model that maternal administration of H2 attenuates oxidative damage and neuroinflammation, including induced pro-inflammatory cytokines and microglial activation, in the fetal brain. Short-term memory, sociability and social novelty, and sensorimotor gating were evaluated using the Y-maze, three-chamber, and prepulse inhibition (PPI) tests, respectively, at postnatal 3 or 4 weeks. The number of neurons and oligodendrocytes was also analyzed at postnatal 5 weeks by immunohistochemical analysis. Offspring of the LPS-exposed dams showed deficits in short-term memory and social interaction, following neuronal and oligodendrocytic loss in the amygdala and cortex. Maternal H2 administration markedly attenuated these LPS-induced abnormalities. Moreover, we evaluated the effect of H2 on LPS-induced astrocytic activation, both in vivo and in vitro. The number of activated astrocytes with hypertrophic morphology was increased in LPS-exposed offspring, but decreased in the offspring of H2-administered dams. In primary cultured astrocytes, LPS-induced pro-inflammatory cytokines were attenuated by H2 administration. Overall, these findings indicate that maternal H2 administration exerts neuroprotective effects and ameliorates MIA-induced neurodevelopmental deficits of offspring later in life.

DOI： 10.1038/s41598-018-27626-4

Acute fulminant invasive pulmonary aspergillosis in an immunocompetent host: An autopsy case report. 査読有り国際誌

Ohara Y, Ito T, Ito M, Yamashita K, Toyokuni S

Medical mycology case reports 20 巻頁： 39 - 42 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Medical Mycology Case Reports

A 62-year-old previously healthy male who was a welder/smoker/drinker was admitted to Kani Tono Hospital for severe hypoxemia (Day 0). Initial physical and radiological examinations suggested an acute exacerbation of chronic obstructive pulmonary disease. However, respiratory failure developed rapidly, and he died on Day + 4. Aspergillus fumigatus was identified after his death, and he was diagnosed with invasive pulmonary aspergillosis. The clinical and pathological features are precisely described with pathogenetic considerations.

DOI： 10.1016/j.mmcr.2018.02.002

An autopsy case report: Differences in radiological images correlate with histology in Erdheim–Chester disease 査読有り国際誌

Ohara Y., Kato S., Yamashita D., Satou A., Shimoyama Y., Hamaie C., Sato M., Ban N., Yamamoto K., Yamada T., Kawai H., Ohshima K., Nakamura S., Toyokuni S.

Pathology International 68 巻 ( 6 ) 頁： 374 - 381 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

p16 activation caused by oncogenic mutations may represent oncogene-induced senescence (OIS), a protective mechanism against oncogenic events. However, OIS can contribute to tumor development via tissue remodeling in some tumors. Erdheim–Chester disease (ECD), a rare non-Langerhans cell histiocytosis, is one such tumor. Its clinical and histological features vary, making it difficult to diagnose. Herein, we describe an autopsy of an ECD patient. The patient underwent radiological examinations, including 18F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography (PET/CT), bone scintigraphy and CT. A biopsy from the lesion with the highest FDG accumulation confirmed the presence of foamy macrophages, a diagnostic clue for ECD. Based on this finding and the clinical features, ECD was diagnosed. However, the patient died from heart dysfunction. After the autopsy, each radiologically different site showed various histological findings regarding the morphology of macrophages, fibrosis, inflammation, and p16 expression. OIS-induced histological progression can cause certain changes observed in radiological images. In addition, in order to evaluate the increase in glucose metabolism, which can affect FDG accumulation, the expression of glucose transporter 1 and hexokinase II was also analyzed. Summarizing the radio-histological correlation can help further both the understanding and diagnosis of ECD.

DOI： 10.1111/pin.12663

タルクへの曝露は卵巣表層上皮細胞内の2価鉄イオンの増加に寄与し卵巣癌の発癌に関わる

本岡大社, 伊藤文哉, 田代浩徳, 片渕秀隆, 豊國伸哉

Biomedical Research on Trace Elements 29 巻 ( 1 ) 頁： 71 - 71 2018年6月

Ferroptosis in carcinogenesis and tumor biology 国際誌

Toyokuni Shinya

FREE RADICAL BIOLOGY AND MEDICINE 120 巻頁： S19-S19 - S19 2018年5月

Expression of P-REX2a is associated with poor prognosis in endometrial malignancies. 国際誌

Takeshita S, Yamashita Y, Shiomi K, Suzuki N, Yoshida J, Naiki-Ito A, Suzuki S, Akatsuka S, Toyokuni S, Takahashi T, Mase S, Arakawa A, Sugiura-Ogasawara M, Takahashi S

Oncotarget 9 巻 ( 37 ) 頁： 24778 - 24786 2018年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Oncotarget

P-REX2a is a PTEN inhibitor that also activates Rac 1. No associations with P-REX2a and human endometrial cancers have been reported to date. In this study, we immunohistochemically analyzed 155 uterine endometrial malignancies for P-REX2a expression. The P-REX2a-positive tumors displayed worse prognosis independent of PTEN expression. Then, we transduced either P-REX2a expression vector or short hairpin RNAs targeting P-REX2a into 2 uterine endometrioid carcinoma cell lines, OMC-2 and JHUEM-14. Ectopic expression of P-REX2a led to increased cell proliferation only in the PTEN-expressing OMC-2 cells but did not show any change in the PTEN-negative JHUEM-14 cells or the P-REX2a-knockdown cells. Induction of P-REX2a increased and knockdown of P-REX2a decreased cell migration in both cell lines. Then, we performed expression microarray analysis using these cells, and pathway analysis revealed that the expression of members of the GPCR downstream pathway displayed the most significant changes induced by the knockdown of P-REX2a. Immunohistochemical analysis revealed that Vav1, a member of the GPCR downstream pathway, was expressed in 139 of the 155 endometrial tumors, and the expression levels of Vav1 and P-REX2a showed a positive correlation (r = 0.44, p < 0.001). In conclusion, P-REX2a enhanced cell motility via the GPCR downstream pathway independently of PTEN leading to progression of uterine endometrioid malignancies and poor prognosis of the patients.

DOI： 10.18632/oncotarget.25349

Superiority of rat over murine model for studies on the evolution of cancer genome.

Akatsuka S, Li GH, Toyokuni S

Free radical research 頁： 1-5 2018年5月

Effect of molecular hydrogen on uterine inflammation during preterm labour. 査読有り国際誌

Nakano T, Kotani T, Imai K, Iitani Y, Ushida T, Tsuda H, Li H, Iwase A, Toyokuni S, Kikkawa F

Biomedical reports 8 巻 ( 5 ) 頁： 454 - 460 2018年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biomedical Reports

Intrauterine inflammation causes preterm birth and is associated with complications in preterm neonates. Thus, strategies aimed at suppressing inflammation are expected to be effective for reducing the risk of preterm birth and associated complications. Our previous studies demonstrated that molecular hydrogen (H2), an anti-inflammatory agent, prevented inflammation-induced impairment in foetal brain and lung tissues in lipopolysaccharide (LPS)-induced rodent models. However, it remains unclear whether H2 is capable of inhibiting preterm labour. The aim of the current study was therefore to investigate the effect of H2 on inflammation-induced preterm labour. Pregnant ICR (CD-1) mice were divided into three groups: Control, LPS and H2 water (HW) + LPS. In the control and LPS groups, vehicle and LPS, respectively, were intraperitoneally injected on embryonic day 15.5. In the HW + LPS group, HW was administered 24 h prior to LPS injection. The time from LPS administration to parturition was compared between the LPS and HW + LPS groups. Maternal uterus was collected 6 h after LPS injection and the transcript levels of pro-inflammatory cytokines, contractile-associated proteins (CAPs), matrix metalloproteinase-3 (Mmp3) and endothelin-1 (Et1) were assessed by reverse transcription-quantitative polymerase chain reaction. The protein levels of cyclooxygenase-2 (Cox2) were also evaluated by immunohistochemistry. The time from LPS administration to parturition in the HW + LPS group was significantly increased compared with that in the LPS group (33.5±3.4 vs. 18.3±8.8 h, respectively, P=0.020). H2 administration also resulted in significantly higher progesterone levels compared with LPS treatment alone (P=0.002). The transcript levels of pro-inflammatory cytokines, CAPs, Mmp3 and Et1 in the uteri of the LPS group were significantly higher than those in the control group (all P<0.05). In turn, all these levels with the exception of interleukin-8 and Mmp3 were significantly lower in the HW + LPS group compared with those in the LPS group (all P<0.05). The protein levels of Cox2 in the LPS group were also significantly increased compared with those in the control (P<0.001) and HW + LPS (P=0.003) groups. These results suggest that inflammation-induced changes in the uterus may be ameliorated through maternal H2 administration. Preventive H2 administration may therefore represent an effective strategy for the suppression of inflammation during preterm labour.

DOI： 10.3892/br.2018.1082

Connective tissue growth factor-specific monoclonal antibody inhibits growth of malignant mesothelioma in an orthotopic mouse model. 査読有り国際誌

Ohara Y, Chew SH, Misawa N, Wang S, Somiya D, Nakamura K, Kajiyama H, Kikkawa F, Tsuyuki Y, Jiang L, Yamashita K, Sekido Y, Lipson KE, Toyokuni S

Oncotarget 9 巻 ( 26 ) 頁： 18494 - 18509 2018年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Oncotarget

Malignant mesothelioma is an aggressive neoplasm with no particularly effective treatments. We previously reported that overexpression of connective tissue growth factor (CTGF/CCN2) promotes mesothelioma growth, thus suggesting it as a novel molecular target. A human monoclonal antibody that antagonizes CTGF (FG-3019, pamrevlumab) attenuates malignant properties of different kinds of human cancers and is currently under clinical trial for the treatment of pancreatic cancer. This study reports the effects of FG-3019 on human mesothelioma in vitro and in vivo. We analyzed the effects of FG-3019 on the proliferation, apoptosis, migration/invasion, adhesion and anchorage-independent growth in three human mesothelioma cell lines, among which ACC-MESO-4 was most efficiently blocked with FG-3019 and was chosen for in vivo experiments. We also evaluated the coexistent effects of fibroblasts on mesothelioma in vitro, which are also known to produce CTGF in various pathologic situations. Coexistent fibroblasts in transwell systems remarkably promoted the proliferation and migration/invasion of mesothelioma cells. In orthotopic nude mice model, FG-3019 significantly inhibited mesothelioma growth. Histological analyses revealed that FG-3019 not only inhibited the proliferation but also induced apoptosis in both mesothelioma cells and fibroblasts. Our data suggest that FG-3019 antibody therapy could be a novel additional choice for the treatment of mesothelioma.

DOI： 10.18632/oncotarget.24892

Osteogenic differentiation in dedifferentiated liposarcoma: a study of 36 cases in comparison to the cases without ossification. 査読有り国際誌

Yamashita K, Kohashi K, Yamada Y, Ishii T, Nishida Y, Urakawa H, Ito I, Takahashi M, Inoue T, Ito M, Ohara Y, Oda Y, Toyokuni S

Histopathology 72 巻 ( 5 ) 頁： 729 - 738 2018年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Histopathology

Aims: Ossification is found occasionally in dedifferentiated liposarcoma (DDLPS). The aims of this study were to elucidate whether the formed bone tissue is usually produced by tumour cells or by reactive non-neoplastic cells, and to reveal the clinicopathological characteristics of DDLPS with ossification. Methods and results: We examined 36 cases of ossified DDLPS by comparing them to 31 cases of non-ossified DDLPS. MDM2 amplification was confirmed in osteocytes and/or osteoblastic cells in all but one ossified DDLPS cases (27 of 28) using fluorescence in-situ hybridisation, although the morphological impression of ossification appeared to be mainly metaplastic (27 of 36) or high-grade osteosarcoma-like (six of 36). The bone tissue was often formed predominantly at the periphery of the DDLPS area near the well-differentiated liposarcoma component (18 of 36), and an organised structure such as bone marrow-like differentiation was not uncommon (12 of 36). According to a modified French Fédération Nationale des Centers de Lutte Contre le Cancer (FNCLCC) grading system, ossified DDLPS tended to be lower grade than non-ossified DDLPS (mean grade: 1.88 and 2.15, respectively). Ossification in DDLPS was associated significantly with shorter local recurrence-free survival by multivariate analysis (P = 0.02347), but metaplastic-appearing ossification tended to be associated with longer overall survival (P = 0.1400). Conclusions: The bone tissue formed in DDLPS was mainly neoplastic regardless of its morphology and maturity, which highlighted the osteogenic differentiation of the tumour cells. DDLPS patients with osteogenic differentiation tended to suffer from earlier local recurrences, which did not necessarily lead to poor life outcomes.

DOI： 10.1111/his.13421

脱分化型脂肪肉腫における骨形成の特徴と予後との関連

山下享子, 孝橋賢一, 山田裕一, 伊藤以知郎, 小田義直, 豊國伸哉

日本病理学会会誌 107 巻 ( 1 ) 頁： 291 - 291 2018年4月

Phlebotomy as a preventive measure for crocidolite-induced mesothelioma in male rats. 査読有り国際誌

Ohara Y, Chew SH, Shibata T, Okazaki Y, Yamashita K, Toyokuni S

Cancer science 109 巻 ( 2 ) 頁： 330 - 339 2018年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Malignant mesothelioma (MM) is a rare but socially important neoplasm due to its association with asbestos exposure. Malignant mesothelioma is difficult to diagnose at an early stage, yet there are no particularly effective treatments available at the advanced stage, thus necessitating efficient strategies to prevent MM in individuals already exposed to asbestos. We previously showed that persistent oxidative damage caused by foreign body reaction and affinity of asbestos both to hemoglobin and histones is one of the major pathogeneses. Accordingly, as an effective strategy to prevent asbestos-induced MM, we undertook the use of an iron chelator, deferasirox, which decreased the epithelial–mesenchymal transition in a crocidolite-induced rat MM model. However, this agent may show adverse effects. Here, we studied the effects of iron removal by phlebotomy as a realistic measure on the same rat model. We injected a total of 5 mg crocidolite i.p. to F1 hybrid rats between the Fischer-344 and Brown-Norway strains at the age of 6 weeks. We repeated weekly or biweekly phlebotomy of 6-8 mL/kg/time from 10 to 60 weeks of age. The animals were observed until 120 weeks. In male rats, phlebotomy significantly decreased the weight and nuclear grade of MM, and modestly reduced the associated ascites and the fraction of more malignant sarcomatoid subtype. Weekly phlebotomy prolonged long-term survival. Our results indicate that appropriate phlebotomy may be a practical preventive measure to attenuate the initiation and promotion capacity of asbestos towards MM by reducing iron in individuals exposed to asbestos.

DOI： 10.1111/cas.13460

Phlebotomy attenuates the growth of malignant mesothelioma on rat model

Ohara, Y; Toyokuni, S

CANCER SCIENCE 109 巻頁： 781 - 781 2018年1月

Molecular mechanisms in oxidative stress-induced carcinogenesis

Toyokuni, S

CANCER SCIENCE 109 巻頁： 163 - 163 2018年1月

Ferric nitrilotriacetate induced renal tumorigenesis in MUTYH deficient mice

Akatsuka, S; Li, GH; Sakumi, K; Nakabeppu, Y; Futakuchi, M; Suzuki, H; Toyokuni, S

CANCER SCIENCE 109 巻頁： 167 - 167 2018年1月

Ferric nitrilotriacetate induced renal tumorigenesis in MUTYH deficient mice 国際誌

Akatsuka Shinya, Li Guang-Hua, Sakumi Kunihiko, Nakabeppu Yusaku, Futakuchi Mitsuru, Suzuki Hiromu, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 167-167 2018年1月

Molecular mechanisms in oxidative stress-induced carcinogenesis 国際誌

Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 163-163 2018年1月

Phlebotomy attenuates the growth of malignant mesothelioma on rat model 国際誌

Ohara Yuuki, Toyokuni Shinya

CANCER SCIENCE 109 巻頁： 781-781 2018年1月

Glioblastoma Cell Lines Display Different Sensitivities to Plasma-Activated Medium 国際誌

Tanaka H., Mizuno M., Ishikawa K., Takeda K., Hashizume H., Nakamura K., Utsumi F., Kajiyama H., Okazaki Y., Toyokuni S., Akiyama S., Maruyama S., Kikkawa F., Hori M.

IEEE Transactions on Radiation and Plasma Medical Sciences 2 巻 ( 2 ) 頁： 99 - 102 2018年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：IEEE Transactions on Radiation and Plasma Medical Sciences

Plasma-activated medium (PAM) is a novel chemotherapy that induces reactive oxygen species (ROS) and cell death in a wide range of cancer cell types, suggesting that PAM may be a promising therapeutic option for cancer treatment. However, dose response experiments suggest that PAM sensitivity is cell line specific. We examined the sensitivities of three glioblastoma cell lines to PAM, and found a wide variation in cell killing that was linked to differences in PAM induced ROS and apoptosis. These results indicate that the PAM sensitivity of glioblastoma cells, and potentially cancer cells more generally, is heterogeneous and likely to be dependent on the regulation of apoptosis and antioxidant pathways in target cells.

DOI： 10.1109/trpms.2017.2721973

State of the art in medical applications using non-thermal atmospheric pressure plasma 査読有り国際誌

Tanaka H., Ishikawa K., Mizuno M., Toyokuni S., Kajiyama H., Kikkawa F., Metelmann H.R., Hori M.

Reviews of Modern Plasma Physics 1 巻 ( 1 ) 2017年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Reviews of Modern Plasma Physics

Plasma medical science is a novel interdisciplinary field that combines studies on plasma science and medical science, with the anticipation that understanding the scientific principles governing plasma medical science will lead to innovations in the field. Non-thermal atmospheric pressure plasma has been used for medical treatments, such as for cancer, blood coagulation, and wound healing. The interactions that occur between plasma and cells/tissues have been analyzed extensively. Direct and indirect treatment of cells with plasma has broadened the applications of non-thermal atmospheric pressure plasma in medicine. Examples of indirect treatment include plasma-assisted immune-therapy and plasma-activated medium. Controlling intracellular redox balance may be key in plasma cancer treatment. Animal studies are required to test the effectiveness and safety of these treatments for future clinical applications.

DOI： 10.1007/s41614-017-0004-3

Fenton reaction-induced renal carcinogenesis in Mutyh-deficient mice exhibits less chromosomal aberrations than the rat model. 国際誌

Phlebotomy as a preventive measure for crocidolite-induced mesothelioma in male rats. 査読有り国際誌

Ohara Y, Chew SH, Shibata T, Okazaki Y, Yamashita K, Toyokuni S

Cancer Science 2017年11月

Fenton reaction-induced renal carcinogenesis in Mutyh-deficient mice exhibits less chromosomal aberrations than the rat model. 査読有り

Li GH, Akatsuka S, Chew SH, Jiang L, Nishiyama T, Sakamoto A, Takahashi T, Futakuchi M, Suzuki H, Sakumi K, Nakabeppu Y, Toyokuni S.

Pathology International 67 巻 ( 11 ) 頁： 564-574 2017年11月

Role of catalytic iron and oxidative stress in nitrofen-induced congenital diaphragmatic hernia and its amelioration by Saireito (TJ-114). 査読有り国際誌

Hirako S, Tsuda H, Ito F, Okazaki Y, Hirayama T, Nagasawa H, Nakano T, Imai K, Kotani T, Kikkawa F, Toyokuni S

Journal of clinical biochemistry and nutrition 61 巻 ( 3 ) 頁： 176 - 182 2017年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

Congenital diaphragmatic hernia (CDH) is a life-threatening neonatal disease that leads to lung hypoplasia and pulmonary hypertension. We recently found that maternal prenatal administration of Saireito (TJ-114) ameliorates fetal CDH in a nitrofen-induced rat model. Here, we studied the role of iron and oxidative stress in neonates of this model and in lung fibroblasts IMR90-SV in asso-ciation with nitrofen and Saireito. We observed increased immuno- staining of 8-hydroxy-2'-deoxyguanosine in the lungs of neonates with CDH, which was ameliorated by maternal Saireito intake. Pulmonary transferrin receptor expression was significantly decreased in both CDH and CDH after Saireito in comparison to normal controls, indicating functional lung immaturity, whereas catalytic Fe(ll) and pulmonaiy DMT1/ferroportin expression remained constant among the three groups. Saireito revealed a dose-dependent scavenging capacity with electron spin resonance spin trapping in vitro against hydroxyl radicals but not against superoxide. Finally, nitrofen revealed dose-dependent cytotoxicity to IMR90-SV cells, accompanied by an increase in oxidative stress, as seen by 5(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and catalytic Fe(ll). Saireito ameliorated all of these in IMR90-SV cells. In conclusion, catalytic Fe(ll)-dependent oxidative stress by nitrofen may be the pathogenic cause of CDH, and the antioxidative activity of Saireito is at least partially responsible for improving nitrof en-induced CDH.

DOI： 10.3164/jcbn.17-17

Li GH, Akatsuka S, Chew SH, Jiang L, Nishiyama T, Sakamoto A, Takahashi T, Futakuchi M, Suzuki H, Sakumi K, Nakabeppu Y, Toyokuni S

Pathology international 67 巻 ( 11 ) 頁： 564 - 574 2017年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

Oxidative stress including iron excess has been associated with carcinogenesis. The level of 8-oxoguanine, a major oxidatively modified base in DNA, is maintained very low by three distinct enzymes, encoded by OGG1, MUTYH and MTH1. Germline biallelic inactivation of MUTYH represents a familial cancer syndrome called MUTYH-associated polyposis. Here, we used Mutyh-deficient mice to evaluate renal carcinogenesis induced by ferric nitrilotriacetate (Fe-NTA). Although the C57BL/6 background is cancer-resistant, a repeated intraperitoneal administration of Fe-NTA induced a high incidence of renal cell carcinoma (RCC; 26.7%) in Mutyh-deficient mice in comparison to wild-type mice (7.1%). Fe-NTA treatment also induced renal malignant lymphoma, which did not occur without the Fe-NTA treatment in both the genotypes. Renal tumor-free survival after Fe-NTA treatment was marginally different (P = 0.157) between the two genotypes. Array-based comparative genome hybridization analyses revealed, in RCC, the loss of heterozygosity in chromosomes 4 and 12 without p16INKA inactivation; these results were confirmed by a methylation analysis and showed no significant difference between the genotypes. Lymphomas showed a preference for genomic amplifications. Dlk1 inactivation by promoter methylation may be involved in carcinogenesis in both tumors. Fe-NTA-induced murine RCCs revealed significantly less genomic aberrations than those in rats, demonstrating a marked species difference.

DOI： 10.1111/pin.12598

Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. 国際誌

Cell 171 巻 ( 2 ) 頁： 273 - 285 2017年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell

Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death. Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.

DOI： 10.1016/j.cell.2017.09.021

Osteogenic differentiation in dedifferentiated liposarcoma: a study of 36 cases in comparison to the cases without ossification. 査読有り

Yamashita K, Kohashi K, Yamada Y, Ishii T, Nishida Y, Urakawa H, Ito I, Takahashi M, Inoue T, Ito M, Ohara Y, Oda Y, Toyokuni S.

Histopathology 2017年10月

Osteogenic differentiation in dedifferentiated liposarcoma: a study of 36 cases in comparison to the cases without ossification. 査読有り国際誌

Yamashita K, Kohashi K, Yamada Y, Ishii T, Nishida Y, Urakawa H, Ito I, Takahashi M, Inoue T, Ito M, Ohara Y, Oda Y, Toyokuni S

Histopathology 2017年10月

The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer. 査読有り

Yanatori I, Richardson DR, Toyokuni S, Kishi F.

Journal of Biological Chemistry 292 巻 ( 32 ) 頁： 13205-13229 2017年8月

The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer. 国際誌

Yanatori I, Richardson DR, Toyokuni S, Kishi F

The Journal of biological chemistry 292 巻 ( 32 ) 頁： 13205 - 13229 2017年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)–NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron. Moreover, intestinal iron is incorporated as ferrous iron, which is transported via the iron importer, divalent metal transporter 1 (DMT1). Recently, we demonstrated that the iron chaperone poly(rC)-binding protein 2 (PCBP2) can directly receive ferrous iron from DMT1 or transfer iron to the iron exporter, ferroportin 1. To promote intracellular iron flux, an iron chaperone may be essential for receiving iron generated by heme catabolism, but this hypothesis is untested so far. Herein, we demonstrate that HO1 binds to PCBP2, but not to other PCBP family members, namely PCBP1, PCBP3, or PCBP4. Interestingly, HO1 formed a complex with either CPR or PCBP2, and it was demonstrated that PCBP2 competes with CPR for HO1 binding. Using PCBP2-deletion mutants, we demonstrated that the PCBP2 K homology 3 domain is important for the HO1/PCBP2 interaction. In heme-loaded cells, heme prompted HO1–CPR complex formation and decreased the HO1/PCBP2 interaction. Furthermore, in vitro reconstitution experiments with purified recombinant proteins indicated that HO1 could bind to PCBP2 in the presence of heme, whereas loading of PCBP2 with ferrous iron caused PCBP2 to lose its affinity for HO1. These results indicate that ferrous iron released from heme can be bound by PCBP2 and suggest a model for an integrated heme catabolism and iron transport metabolon.

DOI： 10.1074/jbc.M117.776021

The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer. 査読有り国際誌

Yanatori I, Richardson DR, Toyokuni S, Kishi F

Journal of Biological Chemistry 292 巻 ( 32 ) 頁： 13205-13229 2017年8月

IRON OVERLOAD ENHANCED CELL DEATH INDUCED BY CARCINOGENIC FIBROUS MATERIALS

Ito Fumiya, Shi Lei, Toyokuni Shinya

AMERICAN JOURNAL OF HEMATOLOGY 92 巻 ( 8 ) 頁： E365 - E365 2017年8月

IRON OVERLOAD ENHANCED CELL DEATH INDUCED BY CARCINOGENIC FIBROUS MATERIALS 国際誌

Fumiya Ito, Lei Shi, Shinya Toyokuni

AMERICAN JOURNAL OF HEMATOLOGY 92 巻 ( 8 ) 頁： E365 - E365 2017年8月

Novel Intraperitoneal Treatment With Non-Thermal Plasma-Activated Medium Inhibits Metastatic Potential of Ovarian Cancer Cells. 査読有り

Nakamura K, Peng Y, Utsumi F, Tanaka H, Mizuno M, Toyokuni S, Hori M, Kikkawa F, Kajiyama H.Sci

Scientific Reports 7 巻 ( 1 ) 頁： 6085 2017年7月

Novel Intraperitoneal Treatment With Non-Thermal Plasma-Activated Medium Inhibits Metastatic Potential of Ovarian Cancer Cells. 国際誌

Nakamura K, Peng Y, Utsumi F, Tanaka H, Mizuno M, Toyokuni S, Hori M, Kikkawa F, Kajiyama H

Scientific reports 7 巻 ( 1 ) 頁： 6085 - 6085 2017年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Non-thermal atmospheric pressure plasma has been proposed as a new therapeutic tool for cancer treatment. Recently, plasma-activated medium (PAM) has been widely studied in various cancer types. However, there are only few reports demonstrating the anti-tumour effects of PAM in an animal model reflecting pathological conditions and the accompanying mechanism. Here we investigated the inhibitory effect of PAM on the metastasis of ovarian cancer ES2 cells in vitro and in vivo. We demonstrated that ES2 cell migration, invasion and adhesion were suppressed by PAM at a certain PAM dilution ratio, whereas cell viability remained unaffected. In an in vivo mouse model of intraperitoneal metastasis, PAM inhibited peritoneal dissemination of ES2 cells, resulting in prolonged survival. Moreover, we assessed the molecular mechanism and found that MMP-9 was decreased by PAM. On further investigation, we also found that PAM prevented the activation of the MAPK pathway by inhibiting the phosphorylation of JNK1/2 and p38 MAPK. These findings indicate that PAM inhibits the metastasis of ovarian cancer cells through reduction of MMP-9 secretion, which is critical for cancer cell motility. Our findings suggest that PAM intraperitoneal therapy may be a promising treatment option for ovarian cancer.

DOI： 10.1038/s41598-017-05620-6

Astaxanthin ameliorates ferric nitrilotriacetate-induced renal oxidative injury in rats. 査読有り

Okazaki Y, Okada S, Toyokuni S.

Journal of Clinical Biochemistry and Nutrition 61 巻 ( 1 ) 頁： 18-24 2017年7月

Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy. 査読有り

Shi L, Ito F, Wang Y, Okazaki Y, Tanaka H, Mizuno M, Hori M, Hirayama T, Nagasawa H, Richardson DR, Toyokuni S.

Free Radical Biology & Medicine 108 巻頁： 904-917 2017年7月

Astaxanthin ameliorates ferric nitrilotriacetate-induced renal oxidative injury in rats.

Okazaki Y, Okada S, Toyokuni S

Journal of clinical biochemistry and nutrition 61 巻 ( 1 ) 頁： 18 - 24 2017年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

Daily intake of vegetables can reduce the risk of cancer and lifestyle-related diseases. However, supplementary intake of p-carotene alone has been reported to increase the risk of lung cancer in male cigarette smokers and people who were exposed to asbestos. The mechanism of the antioxidative properties of carotenoids in vivo, especially under oxidative stress conditions, still remains unclear. To investigate the antioxidant properties of dietary compounds, we examined the effects of chemically modified astaxanthin (Ax-C-8) using a rat model of ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative injury. Ax-C-8 demonstrated lethally toxic effects on the rats in a dose-dependent manner. Following supplementation with Ax-C-8 (0.02%, w/w) for 30 days, the rats were euthanized 1, 4 and 24 h after injection of Fe-NTA. After 4 h, Ax-C-8 pretreatment suppressed the elevation of creatinine and blood urea nitrogen and protected the rats from renal tubular necrosis and the formation of 4-hydroxy-2-nonenal-modified proteins. After 24 h, pretreatment with Ax-C-8 maintained the renal antioxidant enzyme levels and renal tubules. Here, we demonstrate the antioxidant effects of Ax-C-8 against Fe-NTA-induced oxidative injury in rats receiving a regular diet. These data suggest that dietary intake of astaxanthin may be useful for the prevention of renal tubular oxidative damage.

DOI： 10.3164/jcbn.16-114

Iron and thiol redox signaling in cancer: An exquisite balance to escape ferroptosis. 査読有り国際誌

Toyokuni S, Ito F, Yamashita K, Okazaki Y, Akatsuka S

Free radical biology & medicine 108 巻頁： 610 - 626 2017年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Epidemiological data indicate a constant worldwide increase in cancer mortality, although the age of onset is increasing. Recent accumulation of genomic data on human cancer via next-generation sequencing confirmed that cancer is a disease of genome alteration. In many cancers, the Nrf2 transcription system is activated via mutations either in Nrf2 or Keap1 ubiquitin ligase, leading to persistent activation of the genes with antioxidative functions. Furthermore, deep sequencing of passenger mutations is clarifying responsible cancer causative agent(s) in each case, including aging, APOBEC activation, smoking and UV. Therefore, it is most likely that oxidative stress is the principal initiating factor in carcinogenesis, with the involvement of two essential molecules for life, iron and oxygen. There is evidence based on epidemiological and animal studies that excess iron is a major risk for carcinogenesis, suggesting the importance of ferroptosis-resistance. Microscopic visualization of catalytic Fe(II) has recently become available. Although catalytic Fe(II) is largely present in lysosomes, proliferating cells harbor catalytic Fe(II) also in the cytosol and mitochondria. Oxidative stress catalyzed by Fe(II) is counteracted by thiol systems at different functional levels. Nitric oxide, carbon monoxide and hydrogen (per)sulfide modulate these reactions. Mitochondria generate not only energy but also heme/iron sulfur cluster cofactors and remain mostly dysfunctional in cancer cells, leading to Warburg effects. Cancer cells are under persistent oxidative stress with a delicate balance between catalytic iron and thiols, thereby escaping ferroptosis. Thus, high-dose L-ascorbate and non-thermal plasma as well as glucose/glutamine deprivation may provide additional benefits as cancer therapies over preexisting therapeutics.

DOI： 10.1016/j.freeradbiomed.2017.04.024

Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy. 国際誌

Shi L, Ito F, Wang Y, Okazaki Y, Tanaka H, Mizuno M, Hori M, Hirayama T, Nagasawa H, Richardson DR, Toyokuni S

Free radical biology & medicine 108 巻頁： 904 - 917 2017年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Non-thermal plasma (NTP) is a potential new therapeutic modality for cancer. However, its mechanism of action remains unclear. Herein, we studied the effect of NTP on mesothelioma cells and fibroblasts to understand its anti-proliferative efficacy. Interestingly, NTP demonstrated greater selective anti-proliferative activity against mesothelioma cells relative to fibroblasts than cisplatin, which is used for mesothelioma treatment. The anti-proliferative effect of NTP was enhanced by pre-incubation with the cellular iron donor, ferric ammonium citrate (FAC), and inhibited by iron chelation using desferrioxamine (DFO). Three oxidative stress probes (CM-H2DCFDA, MitoSOX and C11-BODIPY) demonstrated reactive oxygen species (ROS) generation by NTP, which was inhibited by DFO. Moreover, NTP decreased transferrin receptor-1 and increased ferritin-H and -L chain expression that was correlated with decreased iron-regulatory protein expression and RNA-binding activity. This regulation was potentially due to increased intracellular iron in lysosomes, which was demonstrated via the Fe(II)-selective probe, HMRhoNox-M, and was consistent with autophagic-induction. Immunofluorescence using LysoTracker and Pepstatin A probes demonstrated increased cellular lysosome content, which was confirmed by elevated LAMP1 expression. The enhanced lysosomal biogenesis after NTP could be due to the observed increase in fluid-phase endocytosis and early endosome formation. These results suggest NTP acts as a stressor, which results in increased endocytosis, lysosome content and autophagy. In fact, NTP rapidly increased autophagosome formation, as judged by increased LC3B-II expression, which co-localized with LAMP1, indicating autophagolysosome formation. Autophagic-induction by NTP was confirmed using electron microscopy. In summary, NTP acts as a cellular stressor to rapidly induce fluid-phase endocytosis, lysosome biogenesis and autophagy.

DOI： 10.1016/j.freeradbiomed.2017.04.368

Astaxanthin ameliorates ferric nitrilotriacetate-induced renal oxidative injury in rats. 査読有り国際誌

Okazaki Y, Okada S, Toyokuni S

Journal of Clinical Biochemistry and Nutrition 61 巻 ( 1 ) 頁： 18-24 2017年7月

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics. 査読有り

Yoshimaru T, Aihara K, Komatsu M, Matsushita Y, Okazaki Y, Toyokuni S, Honda J, Sasa M, Miyoshi Y, Otaka A, Katagiri T.

Scientific Reports 7 巻 ( 1 ) 頁： 1821 2017年5月

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics. 国際誌

Yoshimaru T, Aihara K, Komatsu M, Matsushita Y, Okazaki Y, Toyokuni S, Honda J, Sasa M, Miyoshi Y, Otaka A, Katagiri T

Scientific reports 7 巻 ( 1 ) 頁： 1821 2017年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Estradiol (E2) and the oestrogen receptor-alpha (ERα) signalling pathway play pivotal roles in the proliferative activity of breast cancer cells. Recent findings show that the brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERα activity-regulator synthetic peptide (ERAP: 165-177 amino acids), derived from α-helical BIG3 sequence, resulted in a significant anti-tumour effect. However, the duration of this effect was very short for viable clinical application. We developed the chemically modified ERAP using stapling methods (stapledERAP) to improve the duration of its antitumour effects. The stapledERAP specifically inhibited the BIG3-PHB2 interaction and exhibited long-lasting suppressive activity. Its intracellular localization without the membrane-permeable polyarginine sequence was possible via the formation of a stable α-helix structure by stapling. Tumour bearing-mice treated daily or weekly with stapledERAP effectively prevented the BIG3-PHB2 interaction, leading to complete regression of E2-dependent tumours in vivo. Most importantly, combination of stapledERAP with tamoxifen, fulvestrant, and everolimus caused synergistic inhibitory effects on growth of breast cancer cells. Our findings suggested that the stapled ERAP may be a promising anti-tumour drug to suppress luminal-type breast cancer growth.

DOI： 10.1038/s41598-017-01951-6

Significance of low mTORC1 activity in defining the characteristics of brain tumor stem cells. 査読有り

Han YP, Enomoto A, Shiraki Y, Wang SQ, Wang X, Toyokuni S, Asai N, Ushida K, Ara H, Ohka F, Wakabayashi T, Ma J, Natsume A, Takahashi M.

Nero Oncology 19 巻 ( 5 ) 頁： 636-647 2017年5月

Significance of low mTORC1 activity in defining the characteristics of brain tumor stem cells. 国際誌

Han YP, Enomoto A, Shiraki Y, Wang SQ, Wang X, Toyokuni S, Asai N, Ushida K, Ara H, Ohka F, Wakabayashi T, Ma J, Natsume A, Takahashi M

Neuro-oncology 19 巻 ( 5 ) 頁： 636 - 647 2017年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Neuro-Oncology

Background. The signifcance of mammalian target of rapamycin complex 1 (mTORC1) activity in the maintenance of cancer stem cells (CSCs) remains controversial. Previous fndings showed that mTORC1 activation depleted the population of leukemia stem cells in leukemia, while maintaining the stemness in pancreatic CSCs. The purpose of this study was to examine the currently unknown role and signifcance of mTORC1 activity in brain tumor stem cells (BTSCs). Methods. Basal mTORC1 activity and its kinetics were investigated in BTSC clones isolated from patients with glioblastoma and their differentiated progenies (DIFFs). The effects of nutrient deprivation and the mTORC1 inhibitors on cell proliferation were compared between the BTSCs and DIFFs. Tissue sections from patients with brain gliomas were examined for expression of BTSC markers and mTORC1 activity by immunohistochemistry. Results. BTSCs presented lower basal mTORC1 activity under each culture condition tested and a more rapid decline of mTORC1 activity after nutrient deprivation than observed in DIFFs. The self-renewal capacity of BTSCs was unaffected by mTORC1 inhibition, whereas it effectively suppressed DIFF proliferation. In agreement, immunohistochemical staining of glioma tissues revealed low mTORC1 activity in tumor cells positive for BTSC markers. In in vitro culture, BTSCs exhibited resistance to the antitumor agent temozolomide. Conclusions. Our fndings indicated the importance of low mTORC1 activity in maintaining the undifferentiated state of BTSCs, implicating the relevance of manipulating mTORC1 activity when developing future strategies that target BTSCs.

DOI： 10.1093/neuonc/now237

Primary extraskeletal osteosarcoma: a clinicopathological study of 18 cases focusing on MDM2 amplification status. 査読有り国際誌

Yamashita K, Kohashi K, Yamada Y, Nishida Y, Urakawa H, Oda Y, Toyokuni S

Human pathology 63 巻頁： 63 - 69 2017年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Pathology

Extraskeletal osteosarcoma (ESOS) is an uncommon malignant neoplasm. Most ESOSs are high grade, although some low-grade cases have been reported. A few cases of ESOS with MDM2 amplification have also been reported, suggesting some similarity to skeletal low-grade osteosarcoma such as parosteal osteosarcoma, where MDM2 is often amplified. However, the frequency of low-grade cases and cases with MDM2 amplification among ESOSs remains unknown, and their relationship is unclear. To clarify this, we examined 18 primary ESOS cases clinically, pathologically, and genetically, focusing on their MDM2 amplification status. Our cases comprised 10 men and 8 women whose mean age was 58.6 years; the most common site of the lesion was the thigh and buttock. There were one histologically low-grade case evaluated by biopsy specimen with an aggressive course and 2 relatively low-grade cases whose lesions were of low grade for the most part. MDM2 amplification status was revealed by fluorescence in situ hybridization in all 18 cases; 2 patients—histologically intermediate- and high-grade cases—were found to have MDM2 amplification. In conclusion, this study indicates that histologically low-grade and relatively low-grade cases of ESOS are not always associated with MDM2 amplification. The ESOS case with MDM2 amplification could be high grade, although MDM2-amplified dedifferentiated liposarcoma with osteogenic differentiation should be ruled out in making the diagnosis.

DOI： 10.1016/j.humpath.2017.02.007

In response to Sharing different perspectives to understand asbestos-induced carcinogenesis: Acomment to Jiang et al. (2016) by Alessandro Francesco Gualtieri (2017) 国際誌

Toyokuni Shinya, Jiang Li

CANCER SCIENCE 108 巻 ( 5 ) 頁： 1089 - 1090 2017年5月

Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma. 国際誌

Chew SH, Okazaki Y, Akatsuka S, Wang S, Jiang L, Ohara Y, Ito F, Saya H, Sekido Y, Toyokuni S

Free radical biology & medicine 106 巻頁： 91 - 99 2017年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

CD44 exists as a standard (CD44s) isoform and different variant isoforms (CD44v) due to alternative splicing. While the complex nature of these different isoforms has not been fully elucidated, CD44v expression has been shown to exert oncogenic effects by promoting tumor progression, metastasis and resistance of tumor cells to chemotherapy. One of the CD44v isoforms, CD44v8-10, was recently shown to protect cancer cells from oxidative stress by increasing the synthesis of glutathione (GSH). However, data regarding CD44 isoform expression in malignant mesothelioma (MM) are still lacking. Here, we show that most of the MM cell lines express both the CD44s and CD44v isoforms, in contrast to non-tumorigenic mesothelial cells, which express only CD44s. Moreover, we show here that these MM cell lines are positive for CD44 variable exon 9, with CD44v8-10 among the variant isoforms expressed. The expression of CD44 variable exon 9 was found to be statistically associated with NF2 inactivation, a common occurrence in MM. Knockdown of CD44 reduced the protein level of xCT, a cystine transporter, and increased oxidative stress. However, an increase in GSH was also observed and was associated with enhanced chemoresistance in CD44-knockdown cells. Increased GSH was mediated by the Nrf2/AP-1-induced upregulation of GCLC, a subunit of the enzyme catalyzing GSH synthesis. Our results thus suggest that the response to CD44 depletion is cell type-dependent and, in cases such as MM cells, compensatory pathway(s) might be activated rheostatically to account for the loss of CD44 and counteract enhanced oxidative stress.

DOI： 10.1016/j.freeradbiomed.2017.02.011

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor. 査読有り

Shikata Y, Yoshimaru T, Komatsu M, Katoh H, Sato R, Kanagaki S, Okazaki Y, Toyokuni S, Tashiro E, Ishikawa S, Katagiri T, Imoto M.

Cancer Science 108 巻 ( 4 ) 頁： 785-794 2017年4月

Iron and thiol redox signaling in cancer: An exquisite balance to escape ferroptosis. 査読有り

Toyokuni S, Ito F, Yamashita K, Okazaki Y, Akatsuka S.

Free Radical Biology & Medicine 108 巻頁： 610-626 2017年4月

Iron and thiol redox signaling in cancer: An exquisite balance to escape ferroptosis. 査読有り国際誌

Toyokuni S, Ito F, Yamashita K, Okazaki Y, Akatsuka S

Free Radical Biology & Medicine 108 巻頁： 610-626 2017年4月

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor. 国際誌

Shikata Y, Yoshimaru T, Komatsu M, Katoh H, Sato R, Kanagaki S, Okazaki Y, Toyokuni S, Tashiro E, Ishikawa S, Katagiri T, Imoto M

Cancer science 108 巻 ( 4 ) 頁： 785 - 794 2017年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.

DOI： 10.1111/cas.13175

Primary extraskeletal osteosarcoma: a clinicopathological study of 18 cases focusing on MDM2 amplification status. 査読有り

Yamashita K, Kohashi K, Yamada Y, Nishida Y, Urakawa H, Oda Y, Toyokuni S.

Human Pathology 63 巻頁： 63-69 2017年2月

Primary extraskeletal osteosarcoma: a clinicopathological study of 18 cases focusing on MDM2 amplification status. 査読有り国際誌

Yamashita K, Kohashi K, Yamada Y, Nishida Y, Urakawa H, Oda Y, Toyokuni S

Human Pathology 63 巻頁： 63-69 2017年2月

今月の特集2 微量金属元素と生体機能-メタロミクス研究から臨床検査へがんと微量金属元素

伊藤文哉, 豊國伸哉

臨床検査 61 巻 ( 2 ) 頁： 168 - 173 2017年2月

Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma. 査読有り

Chew SH, Okazaki Y, Akatsuka S, Wang S, Jiang L, Ohara Y, Ito F, Saya H, Sekido Y, Toyokuni S.

Free Radical Biology & Medicine 106 巻頁： 91-99 2017年2月

Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma. 査読有り国際誌

Chew SH, Okazaki Y, Akatsuka S, Wang S, Jiang L, Ohara Y, Ito F, Saya H, Sekido Y, Toyokuni S

Free Radical Biology & Medicine 106 巻頁： 91-99 2017年2月

Clinicopathological and Molecular Features of Dedifferentiated Liposarcoma with Ossification: A Comparative Study with Dedifferentiated Liposarcoma without Ossification and Extraskeletal Osteosarcoma

Yamashita Kyoko, Kohashi Kenichi, Yamada Yuichi, Oda Yoshinao, Toyokuni Shinya

MODERN PATHOLOGY 30 巻頁： 27A-27A 2017年2月

Clinicopathological and Molecular Features of Dedifferentiated Liposarcoma with Ossification: A Comparative Study with Dedifferentiated Liposarcoma without Ossification and Extraskeletal Osteosarcoma

Yamashita Kyoko, Kohashi Kenichi, Yamada Yuichi, Oda Yoshinao, Toyokuni Shinya

LABORATORY INVESTIGATION 97 巻頁： 27A - 27A 2017年2月

Clinicopathological and Molecular Features of Dedifferentiated Liposarcoma with Ossification: A Comparative Study with Dedifferentiated Liposarcoma without Ossification and Extraskeletal Osteosarcoma 国際誌

Kyoko Yamashita, Kenichi Kohashi, Yuichi Yamada, Yoshinao Oda, Shinya Toyokuni

LABORATORY INVESTIGATION 97 巻頁： 27A - 27A 2017年2月

Kyoko Yamashita, Kenichi Kohashi, Yuichi Yamada, Yoshinao Oda, Shinya Toyokuni

MODERN PATHOLOGY 30 巻頁： 27A - 27A 2017年2月

Future perspective of strategic non-thermal plasma therapy for cancer treatment.

Kajiyama H, Utsumi F, Nakamura K, Tanaka H, Toyokuni S, Hori M, Kikkawa F

Journal of clinical biochemistry and nutrition 60 巻 ( 1 ) 頁： 33 - 38 2017年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

The therapeutic effects of non-thermal plasma are expected in the medical fields, including hemostasis, vascularization, prevention of organ adhesion, and cell proliferation. Cancer is an internal enemy arising from normal tissue in the body. The prognosis of metastatic and recurrent cancers is still poor despite advances in medicine. To apply non-thermal plasma in cancer treatment is now on going. The mechanism of the proliferation-inhibitory effect of plasma is reactive nitrogen oxide species/reactive oxygen species production in cells. There are a number of problems to be overcome, such as existence of intrinsic reactive oxygen species/ reactive nitrogen species scavengers and the shallow infiltration of plasma on tumor surface. The current reviews makes referral to the study results of plasma therapy clarified so far, the possibility of its application in the future.

DOI： 10.3164/jcbn.16-65

豊國伸哉

日本毒性学会学術年会 44 巻 ( 0 ) 頁： S11 - S11 2017年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本毒性学会

地球の生命体において鉄は必須であり、高等生物の生命は鉄・酸素・食物によって紡がれる。細胞内で電子の流れを作り制御された化学反応を行うのが酸素の主な役割であるが、体内に取り込まれた酸素の数％は活性酸素・フリーラジカルとなり生体分子に切断・修飾・重合などの傷害を与える。この反応は２価鉄触媒によるFenton反応として1894年より認識されていた。当初放射線の生物作用として理解されたが、1968年SOD発見により活性酸素・フリーラジカルの化学反応が細胞内で常時発生していることが認識されるようになった。発がんは1980年代に始まるがん遺伝子・がん抑制遺伝子の概念の確立により、論理的に理解されるようになった。発がん要因分類として、環境因子・習慣・職業曝露・食習慣・感染症・慢性炎症・遺伝的要因などが列挙され、それを防ぐような手立てが取られてきた。しかし日本では1981年以降、がんが死因の第１位であり右上りである。2014年には英国男性で初めてがんが死因の第１位になった。死因としてのがんの独走は今のがん予防法に大きな疑問を投げかける。私はこれまでの研究より、がんの独走を私たちが酸素と鉄を使用する宿命と理解したい。Wild typeのラットにFenton反応を起こすことでヒトのがんのゲノム変化と酷似したがんが発生することはこの仮説を強く支持する。アスベストや多層カーボンナノチューブによる発がんも異物発がんで基本的には過剰鉄を介するものであり、ラットとヒトで極めて類似したゲノム変化が見られることも注目に値する。これまでの観察により、これらの動物発がんは1ヶ月程度までの初期変化と正比例関係にあることは、毒性学にとって極めて重要であると考えたい。鉄の制御こそが今、がん制御に重要であると考えられる。鉄は一旦、血液内に入ると、体外への積極的な排泄経路はない。50才を過ぎると基礎代謝も低下し、女性は閉経を迎え、鉄が余分になる。私が年２回の全血献血を推奨する所以である。

DOI： 10.14869/toxpt.44.1.0_SL5

The emerging role of progesterone receptor membrane component 1 (PGRMC1) in cancer biology. 国際誌

がんと微量金属元素 (Cancer and trace metal element)

伊藤文哉, 豊國伸哉

臨床検査Journal of clinical laboratory medicine 61 巻 2017年

がんと微量金属元素 (Cancer and trace metal element) 国際誌

伊藤文哉, 豊國伸哉

臨床検査Journal of clinical laboratory medicine 61 巻 2017年

Future perspective of strategic non-thermal plasma therapy for cancer treatment. 査読有り

Kajiyama H, Utsumi F, Nakamura K, Tanaka H, Toyokuni S, Hori M, Kikkawa F.

Journal of Clinical Biochemistry and Nutrition 60 巻 ( 1 ) 頁： 33-38 2016年12月

Future perspective of strategic non-thermal plasma therapy for cancer treatment. 査読有り国際誌

Kajiyama H, Utsumi F, Nakamura K, Tanaka H, Toyokuni S, Hori M, Kikkawa F

Journal of Clinical Biochemistry and Nutrition 60 巻 ( 1 ) 頁： 33-38 2016年12月

Editorial: The cutting edge of zinc biology. 国際誌

Kambe T, Fukada T, Toyokuni S

Archives of biochemistry and biophysics 611 巻頁： 1 - 2 2016年12月

Cahill MA, Jazayeri JA, Catalano SM, Toyokuni S, Kovacevic Z, Richardson DR

Biochimica et biophysica acta 1866 巻 ( 2 ) 頁： 339 - 349 2016年12月

Molecular hydrogen ameliorates several characteristics of preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model 査読有り国際誌

Ushida, T; Kotani, T; Tsuda, H; Imai, K; Nakano, T; Hirako, S; Ito, Y; Li, H; Mano, Y; Wang, J; Miki, R; Yamamoto, E; Iwase, A; Bando, YK; Hirayama, M; Ohno, K; Toyokuni, S; Kikkawa, F

FREE RADICAL BIOLOGY AND MEDICINE 101 巻 ( 16 ) 頁： 524 - 533 2016年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) has been shown to have therapeutic potential in various oxidative stress-related diseases. The aim of this study is to investigate the effect of H2 on preeclampsia. We used the reduced utero-placental perfusion pressure (RUPP) rat model, which has been widely used as a model of preeclampsia. H2 water (HW) was administered orally ad libitum in RUPP rats from gestational day (GD) 12–19, starting 2 days before RUPP procedure. On GD19, mean arterial pressure (MAP) was measured, and samples were collected. Maternal administration of HW significantly decreased MAP, and increased fetal and placental weight in RUPP rats. The increased levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and diacron reactive oxygen metabolites as a biomarker of reactive oxygen species in maternal blood were decreased by HW administration. However, vascular endothelial growth factor level in maternal blood was increased by HW administration. Proteinuria, and histological findings in kidney were improved by HW administration. In addition, the effects of H2 on placental villi were examined by using a trophoblast cell line (BeWo) and villous explants from the placental tissue of women with or without preeclampsia. H2 significantly attenuated hydrogen peroxide-induced sFlt-1 expression, but could not reduce the expression induced by hypoxia in BeWo cells. H2 significantly attenuated sFlt-1 expression in villous explants from women with preeclampsia, but not affected them from normotensive pregnancy. The prophylactic administration of H2 attenuated placental ischemia-induced hypertension, angiogenic imbalance, and oxidative stress. These results support the theory that H2 has a potential benefit in the prevention of preeclampsia.

DOI： 10.1016/j.freeradbiomed.2016.10.491

Preclinical Use of CTGF-Specific Monoclonal Antibody for the Treatment of Malignant Mesothelioma 国際誌

Ohara, Y; Wang, SQ; Jiang, L; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE 100 巻頁： S127 - S127 2016年11月

Urokinase-type plasminogen activator receptor promotes proliferation and invasion with reduced cisplatin sensitivity in malignant mesothelioma. 査読有り国際誌

Wang S, Jiang L, Han Y, Chew SH, Ohara Y, Akatsuka S, Weng L, Kawaguchi K, Fukui T, Sekido Y, Yokoi K, Toyokuni S

Oncotarget 7 巻 ( 43 ) 頁： 69565 - 69578 2016年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Oncotarget

Malignant mesothelioma (MM) is a rare neoplasm associated with asbestos exposure. The prognosis of MM is poor because it is aggressive and highly resistant to chemotherapy. Using a rat model of asbestos-induced MM, we found elevated urokinase-type plasminogen activator receptor (uPAR; Plaur) expression in rat tissues, which was associated with poor prognosis. The proliferation, migration and invasion of MM cells were suppressed by uPAR knockdown and increased by overexpression experiments, irrespective of urokinase-type plasminogen activator (uPA; Plau) levels. More importantly, we found that uPAR expression is associated with sensitivity to cisplatin in MM through the PI3K/AKT pathway, which was demonstrated with specific inhibitors, LY294002 and Akti-1/2. uPAR knockdown significantly increased sensitivity to cisplatin whereas its overexpression significantly decreased cisplatin sensitivity. Furthermore, sera and tissues from MM patients showed significantly high uPAR levels, which suggested the pathogenic role of uPAR in the tumor biology of human MM. In conclusion, our findings indicate that uPAR levels are associated with malignant characteristics and cisplatin sensitivity of MM. In addition to the potential use of uPAR as a prognostic marker, the combination of uPAR abrogation and cisplatin may reveal a promising therapeutic approach for MM.

DOI： 10.18632/oncotarget.11829

Molecular hydrogen ameliorates several characteristics of preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model. 査読有り

Ushida T, Kotani T, Tsuda H, Imai K, Nakano T, Hirako S, Ito Y, Li H, Mano Y, Wang J, Miki R, Yamamoto E, Iwase A, Bando YK, Hirayama M, Ohno K, Toyokuni S, Kikkawa F.

Free Radic Biol Med. pii: S0891-5849 巻 ( 16 ) 頁： 30978-9. 2016年10月

Molecular hydrogen ameliorates several characteristics of preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model. 査読有り国際誌

Ushida T, Kotani T, Tsuda H, Imai K, Nakano T, Hirako S, Ito Y, Li H, Mano Y, Wang J, Miki R, Yamamoto E, Iwase A, Bando YK, Hirayama M, Ohno K, Toyokuni S, Kikkawa F

Free Radic Biol Med. pii: S0891-5849 巻 ( 16 ) 頁： 30978-9. 2016年10月

Non-thermal plasma prevents progression of endometriosis in mice. 査読有り

Ishida C, Mori M, Nakamura K, Tanaka H, Mizuno M, Hori M, Iwase A, Kikkawa F, Toyokuni S.

Free Radical Research 50 巻 ( 10 ) 頁： 1131-1139 2016年10月

Non-thermal plasma prevents progression of endometriosis in mice. 査読有り国際誌

Ishida C, Mori M, Nakamura K, Tanaka H, Mizuno M, Hori M, Iwase A, Kikkawa F, Toyokuni S

Free radical research 50 巻 ( 10 ) 頁： 1131 - 1139 2016年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Endometriosis is observed in ∼10% of reproductive age women. Ovarian endometriosis not only causes dysmenorrhea but also causes infertility and a high risk of adenocarcinoma. Due to its scattered nature, complete surgical resection is difficult. Endometriosis consists of glandular and stromal cells. Previously, we showed that endometrial stromal cells (ESCs) play a role in the protection against pathologic events caused by monthly repeated hemorrhage. Here, we undertook a preclinical study of non-thermal plasma (NTP) as a surgical treatment of endometriosis. Epithelial cells were most sensitive to NTP-activated medium in vitro, whereas ectopic ESCs were most resistant. We then transplanted excised uteruses into BALB/c mice from donors of the same strain with estradiol supplementation. Four weeks after the transplantation, we exposed NTP to each endometriotic lesion after laparotomy. Immunohistochemical analysis revealed that immediately after NTP exposure, epithelial cells exhibited significantly higher levels of nuclear immunostaining for 8-hydroxy-2′-deoxyguanosine than did stromal cells. Four weeks after NTP exposure, the total surface area consisting of endometriotic cysts was significantly smaller with less epithelial proliferative activity than the helium-exposed control, whereas the number of endometriotic lesions had not changed. Therefore, NTP exposure may be useful to prevent the progression and recurrence of endometriosis.

DOI： 10.1080/10715762.2016.1211273

Special issue for the 7th Biennial Meeting of Society for Free Radical Research-Asia (SFRR-Asia 2015 Thailand). 査読有り国際共著国際誌

Ungsurungsie M, Surh YJ, Toyokuni S, Davies MJ

Free radical research 50 巻 ( 10 ) 頁： 1045 - 1046 2016年10月

Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450. 査読有り国際誌

Zong C, Garner CE, Huang C, Zhang X, Zhang L, Chang J, Toyokuni S, Ito H, Kato M, Sakurai T, Ichihara S, Ichihara G

Toxicology letters 258 巻頁： 249 - 258 2016年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Toxicology Letters

Neurotoxicity of 1-bromopropane (1-BP) has been reported in both human cases and animal studies. To date, neurotoxicity of 1-BP has been induced in rats but not in mice due to the lethal hepatotoxicity of 1-BP. Oxidization by cytochromes P450 and conjugation with glutathione (GSH) are two critical metabolism pathways of 1-BP and play important roles in toxicity of 1-BP. The aim of the present study was to establish a murine model of 1-BP neurotoxicity, by reducing the hepatotoxicity of 1-BP with 1-aminobenzotriazole (1-ABT); a commonly used nonspecific P450s inhibitor. The results showed that subcutaneous or intraperitoneal injection of 1-ABT at 50 mg/kg body weight BID (100 mg/kg BW/day) for 3 days, inhibited about 92–96% of hepatic microsomal CYP2E1 activity, but only inhibited about 62–64% of CYP2E1 activity in brain microsomes. Mice treated with 1-ABT survived even after exposure to 1200 ppm 1-BP for 4 weeks and histopathological studies showed that treatment with 1-ABT protected mice from 1-BP-induced hepatic necrosis, hepatocyte degeneration, and hemorrhage. After 4-week exposure to 1-BP, the brain weight of 1-ABT(+)/1200 ppm 1-BP group was decreased significantly. In 1-ABT-treated groups, expression of hippocampal Ran protein and cerebral cortical GRP78 was dose-dependently increased by exposure to 1-BP. We conclude that the control of hepatic P450 activity allows the observation of effects of 1-BP on the murine brain at a higher concentration by reduction of hepatotoxicity. The study suggests that further experiments with liver-specific control of P450 activity using gene technology might provide better murine models for 1-bromopropane-induced neurotoxicity.

DOI： 10.1016/j.toxlet.2016.07.006

Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells. 査読有り国際共著国際誌

Shi L, Wang Y, Ito F, Okazaki Y, Tanaka H, Mizuno M, Hori M, Richardson DR, Toyokuni S

Archives of biochemistry and biophysics 605 巻頁： 109 - 16 2016年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Non-thermal plasma (NTP) is a recently developed technology that elicits a variety of biological effects. This includes cancer cell-specific cytotoxicity, which is mainly attributed to the regional generation of reactive oxygen species (ROS). We studied the effects of NTP on malignant mesothelioma (MM) and its modulation by L-ascorbate. L-ascorbate is a major water-soluble anti-oxidant in vivo, but its pro-oxidant activity in vitro has been well recognized. Thus, the effects of ascorbate on the efficacy of NTP is important to examine. NTP exposure dose-dependently killed MM cells, whereas MM cells tolerated 1 mM L-ascorbate. However, brief pre-treatment with a pharmacological dose (250–750 μM) of L-ascorbate immediately prior to NTP exposure significantly increased its cytotoxicity in a dose-dependent manner, which was inhibited by the iron chelator, deferoxamine. However, paradoxically, this potentiating effect of L-ascorbate was completely abolished by a prolonged 4 h pre-incubation with L-ascorbate (500 μM). MM cytotoxicity induced by NTP was associated with immediate oxidative stress evaluated by 2′,7′-dichlorodihydrofluorecein diacetate, which was followed by an increase in the expression of the autophagosome marker, LC3B-II. In conclusion, MM can be a target for NTP treatment and L-ascorbate can increase or decrease its efficacy depending on the length of the pre-incubation period.

DOI： 10.1016/j.abb.2016.05.016

Low-temperature plasma in biology and medicine 査読有り国際誌

Hori M.

Archives of Biochemistry and Biophysics 605 巻頁： 1 - 2 2016年9月

Antenatal Saireito (TJ-114) Can Improve Pulmonary Hypoplasia and Pulmonary Vascular Remodeling in Nitrofen-Induced Congenital Diaphragmatic Hernia. 査読有り

Hirako S, Tsuda H, Kotani T, Sumigama S, Mano Y, Nakano T, Imai K, Li H, Toyokuni S, Kikkawa F.

Phytother Res. 30 巻 ( 9 ) 頁： 1474-80 2016年9月

Urokinase-type plasminogen activator receptor promotes proliferation and invasion with reduced cisplatin sensitivity in malignant mesothelioma. 査読有り

Wang S, Jiang L, Han Y, Hwu Chew S, Ohara Y, Akatsuka S, Weng L, Kawaguchi K, Fukui T, Sekido Y, Yokoi K, Toyokuni S.

Oncotarget. 2016 Sep 2. 頁： 11829 2016年9月

Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450. 査読有り

Zong C, Garner CE, Huang C, Zhang X, Zhang L, Chang J, Toyokuni S, Ito H, Kato M, Sakurai T, Ichihara S, Ichihara G.

Toxicol Lett. 258 巻頁： 249-58 2016年9月

Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells. 査読有り

Shi L, Wang Y, Ito F, Okazaki Y, Tanaka H, Mizuno M, Hori M, Richardson DR, Toyokuni S.

Arch Biochem Biophys. 605 巻頁： 106-16 2016年9月

Antenatal Saireito (TJ-114) Can Improve Pulmonary Hypoplasia and Pulmonary Vascular Remodeling in Nitrofen-Induced Congenital Diaphragmatic Hernia. 査読有り国際誌

Hirako S, Tsuda H, Kotani T, Sumigama S, Mano Y, Nakano T, Imai K, Li H, Toyokuni S, Kikkawa F

Phytotherapy research : PTR 30 巻 ( 9 ) 頁： 1474 - 80 2016年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Phytotherapy Research

Congenital diaphragmatic hernia (CDH) can induce lung hypoplasia and pulmonary hypertension and is associated with high mortality. The purpose of this study is to examine the efficacy and safety of antenatal Saireito (TJ-114), a traditional Japanese herbal medicine, in a rat CDH model. Sprague-Dawley rats were exposed to an herbicide (nitrofen, 100 mg) on embryonic day 9 (E9) to induce CDH, and antenatal Saireito (2000 mg/kg/day) was orally administered from E10 to E20. On E21, fetuses were delivered. Antenatal Saireito significantly decreased the incidence of CDH (p < 0.01), increased lung volume (p < 0.01), improved alveolarization and pulmonary artery remodeling using histological analysis, and improved respiratory function using gasometric analysis (pH; p < 0.05, and PCO2; p < 0.01). In addition, antenatal Saireito significantly decreased endothelin-1 and endothelin receptor A expression in the pulmonary arteries. Taken together, our results demonstrated that antenatal Saireito can improve fetal pulmonary hypoplasia and pulmonary vascular remodeling and, as a result, can improve respiratory function in a rat CDH model. Copyright © 2016 John Wiley & Sons, Ltd.

DOI： 10.1002/ptr.5645

Molecular hydrogen suppresses activated Wnt/beta-catenin signaling 査読有り国際誌

Lin Yingni, Ohkawara Bisei, Ito Mikako, Misawa Nobuaki, Miyamoto Kentaro, Takegami Yasuhiko, Masuda Akio, Toyokuni Shinya, Ohno Kinji

SCIENTIFIC REPORTS 6 巻 ( 6 ) 頁： 31986 2016年8月

Contrasting intra- and extracellular distribution of catalytic ferrous iron in ovalbumin-induced peritonitis. 査読有り国際誌

Ito F, Nishiyama T, Shi L, Mori M, Hirayama T, Nagasawa H, Yasui H, Toyokuni S

Biochemical and biophysical research communications 476 巻 ( 4 ) 頁： 600 - 606 2016年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Iron is an essential nutrient for every type of life on earth. However, excess iron is cytotoxic and can lead to an increased cancer risk in humans. Catalytic ferrous iron [Fe(II)] is an initiator of the Fenton reaction, which causes oxidative stress by generating hydroxyl radicals. Recently, it became possible to localize catalytic Fe(II) in situ with a turn-on fluorescent probe, RhoNox-1. Here, we screened each organ/cell of rats to globally evaluate the distribution of catalytic Fe(II) and found that eosinophils showed the highest abundance. In various cells, lysosomes were the major organelle, sharing ∼40–80% of RhoNox-1 fluorescence. We then used an ovalbumin-induced allergic peritonitis model to study the dynamics of catalytic Fe(II). Peritoneal lavage revealed that the total iron contents per cell were significantly decreased, whereas an increase in the number of inflammatory cells (macrophages, neutrophils, eosinophils and lymphocytes) resulted in an increased total iron content of the peritoneal inflammatory cells. Notably, macrophages, eosinophils and neutrophils exhibited significantly increased catalytic Fe(II) with increased DMT1 expression and decreased ferritin expression, though catalytic Fe(II) was significantly decreased in the peritoneal lavage fluid. In conclusion, catalytic Fe(II) in situ more directly reflects cellular activity and the accompanying pathology than total iron does.

DOI： 10.1016/j.bbrc.2016.06.003

Molecular hydrogen suppresses activated Wnt/β-catenin signaling. 査読有り

Lin Y, Ohkawara B, Ito M, Misawa N, Miyamoto K, Takegami Y, Masuda A, Toyokuni S, Ohno K.

Sci Rep. 25 巻 ( 6 ) 頁： 31986 2016年8月

Contrasting intra- and extracellular distribution of catalytic ferrous iron in ovalbumin-induced peritonitis. 査読有り

Ito F, Nishiyama T, Shi L, Mori M, Hirayama T, Nagasawa H, Yasui H, Toyokuni S.

Biochem Biophys Res Commun. 476 巻 ( 4 ) 頁： 600-6 2016年8月

Iron function and carcinogenesis 査読有り国際誌

Akatsuka S., Toyokuni S.

Nihon rinsho. Japanese journal of clinical medicine 74 巻 ( 7 ) 頁： 1168 - 1175 2016年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nihon rinsho. Japanese journal of clinical medicine

Though iron is an essential micronutrient for humans, the excess state is acknowledged to be associated with oncogenesis. For example, iron overload in the liver of the patients with hereditary hemocromatosis highly increases the risk of hepatocellular carcinoma. Also, as to asbestos-related mesothelioma, such kinds of asbestos with a higher iron content are considered to be more carcinogenic. Iron is a useful element, which enables fundamental functions for life such as oxygen carrying and electron transport. However, in the situation where organisms are unable to have good control of it, iron turns into a dangerous element which catalyzes generation of reactive oxygen. In this review, I first outline the relationships between iron and cancer in general, then give an explanation about iron-related animal carcinogenesis models.

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics. 国際誌

The emerging role of progesterone receptor membrane component 1 (PGRMC1) in cancer biology. 査読有り

Cahill MA, Jazayeri JA, Catalano SM, Toyokuni S, Kovacevic Z, Richardson DR.

Biochim Biophys Acta. 2016 Jul 22. pii: S0304-419X 巻 ( 16 ) 頁： 30049-X. 2016年7月

Dual preventive benefits of iron elimination by desferal in asbestos-induced mesothelial carcinogenesis 査読有り国際誌

Jiang Li, Chew Shan-Hwu, Nakamura Kosuke, Ohara Yuuki, Akatsuka Shinya, Toyokuni Shinya

CANCER SCIENCE 107 巻 ( 7 ) 頁： 908 - 915 2016年7月

Possible therapeutic option of aqueous plasma for refractory ovarian cancer 査読有り国際誌

Kajiyama Hiroaki, Utsumi Fumi, Nakamura Kae, Tanaka Hiromasa, Mizuno Masaaki, Toyokuni Shinya, Hori Masaru, Kikkawa Fumitaka

CLINICAL PLASMA MEDICINE 4 巻 ( 1 ) 頁： 14 - 18 2016年7月

The emerging role of progesterone receptor membrane component 1 (PGRMC1) in cancer biology. 査読有り国際誌

Cahill MA, Jazayeri JA, Catalano SM, Toyokuni S, Kovacevic Z, Richardson DR

Biochim Biophys Acta. 2016 Jul 22. pii: S0304-419X 巻 ( 16 ) 頁： 30049-X. 2016年7月

[Iron function and carcinogenesis].

Akatsuka S, Toyokuni S

Nihon rinsho. Japanese journal of clinical medicine 74 巻 ( 7 ) 頁： 1168 - 75 2016年7月

[Iron function and carcinogenesis].

Akatsuka S, Toyokuni S

Nihon rinsho. Japanese journal of clinical medicine 74 巻 ( 7 ) 頁： 1168 - 75 2016年7月

Variable susceptibility of ovarian cancer cells to non-thermal plasma-activated medium. 査読有り

35 巻 ( 6 ) 頁： 3169-77 2016年6月

Dual preventive benefits of iron elimination by desferal in asbestos-induced mesothelial carcinogenesis. 査読有り

Jiang L, Chew SH, Nakamura K, Ohara Y, Akatsuka S, Toyokuni S.

Cancer Sci. 2016 Jul;107(7):908-15. 107 巻 ( 7 ) 頁： 908-15 2016年6月

Dual preventive benefits of iron elimination by desferal in asbestos-induced mesothelial carcinogenesis. 査読有り国際誌

Jiang L, Chew SH, Nakamura K, Ohara Y, Akatsuka S, Toyokuni S

Cancer Sci. 2016 Jul;107(7):908-15. 107 巻 ( 7 ) 頁： 908-15 2016年6月

Variable susceptibility of ovarian cancer cells to non-thermal plasma-activated medium. 査読有り国際誌

Utsumi F, Kajiyama H, Nakamura K, Tanaka H, Mizuno M, Toyokuni S, Hori M, Kikkawa F

Oncology reports 35 巻 ( 6 ) 頁： 3169 - 77 2016年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Oncology Reports

Non- Thermal atmospheric pressure plasma has been widely studied in recent years in many fields,including cancer treatment. However,its efficiency for inducing apoptosis sometimes varies depending on the cell species and experimental conditions. The aim of this study was to elucidate what causes these differences in responses to plasma treatment. Using four ovarian cancer cell lines,the cell density had a markedly negative impact on the proliferation inhibition rate (PIR) and it was more obvious in OVCAR-3 and NOS2 cells. Furthermore,TOV21G and ES-2 cells were drastically sensitive to plasma- Activated medium (PAM) compared with the other two cell lines. We demonstrated that the proportion of reactive oxygen species and cell number had a marked impact on the effect of PAM against ovarian cancer cells. Additionally it was suggested that the morphological features of cells were also closely related to the sensitivity of cancer cells to the plasma treatment.

DOI： 10.3892/or.2016.4726

The origin and future of oxidative stress pathology: From the recognition of carcinogenesis as an iron addiction with ferroptosis-resistance to non-thermal plasma therapy 査読有り国際誌

Toyokuni S.

Pathology International 66 巻 ( 5 ) 頁： 245 - 259 2016年5月

The origin and future of oxidative stress pathology: From the recognition of carcinogenesis as an iron addiction with ferroptosis-resistance to non-thermal plasma therapy. 招待有り査読有り国際誌

Toyokuni S

Pathol Int. 66 巻 ( 5 ) 頁： 245-259 2016年5月

Oxidative stress as an iceberg in carcinogenesis and cancer biology 査読有り国際誌

Toyokuni Shinya

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 595 巻頁： 46 - 49 2016年4月

Oxidative stress as an iceberg in carcinogenesis and cancer biology. 査読有り

Toyokuni S.

Arch Biochem Biophys. 2016 Apr 1;595:46-9. 595 巻頁： 46-9 2016年4月

Oxidative stress as an iceberg in carcinogenesis and cancer biology. 査読有り国際誌

Toyokuni S

Arch Biochem Biophys. 2016 Apr 1;595:46-9. 595 巻頁： 46-9 2016年4月

Tribute issue: Helmut Sies and oxidative stress: Venit, vidit, vicit 査読有り国際誌

Forman H.

Archives of Biochemistry and Biophysics 595 巻 2016年4月

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics.

Kalinowski DS, Stefani C, Toyokuni S, Ganz T, Anderson GJ, Subramaniam NV, Trinder D, Olynyk JK, Chua A, Jansson PJ, Sahni S, Lane DJ, Merlot AM, Kovacevic Z, Huang ML, Lee CS, Richardson DR.

Biochim Biophys Acta. 1863 巻 ( 4 ) 頁： 727-748 2016年4月

Kalinowski DS, Stefani C, Toyokuni S, Ganz T, Anderson GJ, Subramaniam NV, Trinder D, Olynyk JK, Chua A, Jansson PJ, Sahni S, Lane DJ, Merlot AM, Kovacevic Z, Huang ML, Lee CS, Richardson DR

Biochimica et biophysica acta 1863 巻 ( 4 ) 頁： 727 - 48 2016年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochimica et Biophysica Acta - Molecular Cell Research

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics.

DOI： 10.1016/j.bbamcr.2016.01.026

Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis. 査読有り

Wang Y, Okazaki Y, Shi L, Kohda H, Tanaka M, Taki K, Nishioka T, Hirayama T, Nagasawa H, Yamashita Y, Toyokuni S.

Cancer Sci. 107 巻 ( 3 ) 頁： 250-257 2016年3月

Pain-reducing anesthesia prevents oxidative stress in human term placenta. 査読有り

Tsuzuki Y, Yamashita Y, Hattori Y, Hua Li G, Akatsuka S, Kotani T, Kikkawa F, Naiki-Ito A, Takahashi S, Nishiwaki K, Toyokuni S.

J Clin Biochem Nutr. 58 巻 ( 2 ) 頁： 156-60 2016年3月

Pain-reducing anesthesia prevents oxidative stress in human term placenta. 査読有り国際誌

Tsuzuki Y, Yamashita Y, Hattori Y, Hua Li G, Akatsuka S, Kotani T, Kikkawa F, Naiki-Ito A, Takahashi S, Nishiwaki K, Toyokuni S

Journal of clinical biochemistry and nutrition 58 巻 ( 2 ) 頁： 156 - 60 2016年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

Anesthesia is sometimes used for the reduction of maternal pain in normal human term labor, but whether the drugs affect oxidative stress remains unclear. The placenta serves as an interface between the maternal and fetal vasculature. In this study, we immunohistochemically analyzed two markers for oxidative stress, namely 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal-modified proteins (HNE), using placentas from 21 cases of normal tansvaginal delivery (V group), 20 Caesarean sections (C group), and 17 normal transvaginal deliveries with epidural anesthesia (E group). 8-OHdG staining in the nuclei of trophoblasts lining the chorionic villi was significantly stronger in the V group either compared with the C or E group (p<0.001), without significant differences in the C and E groups (p = 0.792). Moderate to intense staining by HNE of the intravascular serum of chorionic villi vasculature was frequently observed in the placentas from the V group, but less frequently of those in either C or E groups (p<0.001), nor the p value comparing the C and E groups was significant (p = 0.128) for HNE staining. Our results suggest that although the role of oxidative stress and its influences on fetal state in the placenta in labor remains unclear, it seems to be lessened by epidural anesthesia.

DOI： 10.3164/jcbn.15-138

Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis. 査読有り国際誌

Wang Y, Okazaki Y, Shi L, Kohda H, Tanaka M, Taki K, Nishioka T, Hirayama T, Nagasawa H, Yamashita Y, Toyokuni S

Cancer science 107 巻 ( 3 ) 頁： 250 - 7 2016年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Multi-wall carbon nanotubes (MWCNT) are a form of flexible fibrous nanomaterial with high electrical and thermal conductivity. However, 50-nm MWCNT in diameter causes malignant mesothelioma (MM) in rodents and, thus, the International Agency of Research on Cancer has designated them as a possible human carcinogen. Little is known about the molecular mechanism through which MWCNT causes MM. To elucidate the carcinogenic mechanisms of MWCNT in mesothelial cells, we used a variety of lysates to comprehensively identify proteins specifically adsorbed on pristine MWCNT of different diameters (50 nm, NT50; 100 nm, NT100; 150 nm, NT150; and 15 nm/tangled, NTtngl) using mass spectrometry. We identified >400 proteins, which included hemoglobin, histone, transferrin and various proteins associated with oxidative stress, among which we selected hemoglobin and transferrin for coating MWCNT to further evaluate cytotoxicity, wound healing, intracellular catalytic ferrous iron and oxidative stress in rat peritoneal mesothelial cells (RPMC). Cytotoxicity to RPMC was observed with pristine NT50 but not with NTtngl. Coating NT50 with hemoglobin or transferrin significantly aggravated cytotoxicity to RPMC, with an increase in cellular catalytic ferrous iron and DNA damage also observed. Knockdown of transferrin receptor with ferristatin II decreased not only NT50 uptake but also cellular catalytic ferrous iron. Our results suggest that adsorption of hemoglobin and transferrin on the surface of NT50 play a role in causing mesothelial iron overload, contributing to oxidative damage and possibly subsequent carcinogenesis in mesothelial cells. Uptake of NT50 at least partially depends on transferrin receptor 1. Modifications of NT50 surface may decrease this human risk.

DOI： 10.1111/cas.12865

Neuroprotective potential of molecular hydrogen against perinatal brain injury via suppression of activated microglia. 査読有り

Imai K, Kotani T, Tsuda H, Mano Y, Nakano T, Ushida T, Li H, Miki R, Sumigama S, Iwase A, Hirakawa A, Ohno K, Toyokuni S, Takeuchi H, Mizuno T, Suzumura A, Kikkawa F.

Free Radic Biol Med. 91 巻頁： 154-163 2016年2月

Neuroprotective potential of molecular hydrogen against perinatal brain injury via suppression of activated microglia. 査読有り国際誌

Imai K, Kotani T, Tsuda H, Mano Y, Nakano T, Ushida T, Li H, Miki R, Sumigama S, Iwase A, Hirakawa A, Ohno K, Toyokuni S, Takeuchi H, Mizuno T, Suzumura A, Kikkawa F

Free radical biology & medicine 91 巻頁： 154 - 63 2016年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Exposure to inflammation in utero is related to perinatal brain injury, which is itself associated with high rates of long-term morbidity and mortality in children. Novel therapeutic interventions during the perinatal period are required to prevent inflammation, but its pathogenesis is incompletely understood. Activated microglia are known to play a central role in brain injury by producing a variety of pro-inflammatory cytokines and releasing oxidative products. The study is aimed to investigate the preventative potential of molecular hydrogen (H2), which is an antioxidant and anti-inflammatory agent without mutagenicity. Pregnant ICR mice were injected with lipopolysaccharide (LPS) intraperitoneally on embryonic day 17 to create a model of perinatal brain injury caused by prenatal inflammation. In this model, the effect of maternal administration of hydrogen water (HW) on pups was also evaluated. The levels of pro-inflammatory cytokines, oxidative damage and activation of microglia were determined in the fetal brains. H2 reduced the LPS-induced expression of pro-inflammatory cytokines, oxidative damage and microglial activation in the fetal brains. Next, we investigated how H2 contributes to neuroprotection, focusing on microglia, using primary cultured microglia and neurons. H2 prevented LPS- or cytokine-induced generation of reactive oxidative species by microglia and reduced LPS-induced microglial neurotoxicity. Finally, we identified several molecules influenced by H2, involved in the process of activating microglia. These results suggested that H2 holds promise for the prevention of inflammation related to perinatal brain injury.

DOI： 10.1016/j.freeradbiomed.2015.12.015

Plasma with high electron density and plasma-activated medium for cancer treatment 査読有り国際誌

Tanaka H., Mizuno M., Ishikawa K., Kondo H., Takeda K., Hashizume H., Nakamura K., Utsumi F., Kajiyama H., Kano H., Okazaki Y., Toyokuni S., Akiyama S., Maruyama S., Yamada S., Kodera Y., Kaneko H., Terasaki H., Hara H., Adachi T., Iida M., Yajima I., Kato M., Kikkawa F., Hori M.

Clinical Plasma Medicine 3 巻 ( 2 ) 頁： 72 - 76 2015年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Clinical Plasma Medicine

Cancer treatment using non-thermal atmospheric pressure plasma is a brand new and challenging approach for cancer therapy. Conventional cancer therapies are surgery, radio-therapy, and chemo-therapy. We propose plasma-therapy as the fourth cancer therapy. Plasma cancer therapy involves direct plasma treatment of cancers including melanomas, head and neck cancer, pancreatic cancer and liver metastasis, and indirect plasma treatment of cancers by using plasma irradiated solutions such as plasma-activated medium (PAM).We have been recently studying plasma cancer therapy using target cancers such as ovarian cancers, brain tumors, gastric cancers and skin cancers. We have developed a plasma source with ultrahigh electron density, which we have applied to these cancer cells. In addition, we found that plasma-irradiated medium itself can kill these cancer cells. This medium was termed plasma-activated medium (PAM). In vitro and in vivo studies have suggested that PAM is an important tool for cancer therapy especially for disseminated cancers that are currently untreatable.Although many dramatic therapeutic effects of plasma therapy on cancer cells have been reported, the molecular mechanisms of the anti-tumor effects of plasma remain to be elucidated. The greatest challenge for plasma medical science is to understand the complex system that mediates plasma inputs resulting in physiological outputs such as cell death of cancer cells and proliferation of normal cells. Intracellular molecular mechanisms of PAM are also being intensively studied in order to understand the mode of action of PAM. In this review, we summarize the latest understanding of plasma cancer treatments.

DOI： 10.1016/j.cpme.2015.09.001

プラズマ活性溶液の細胞影響 (小特集プラズマが誘導する生体応答とそのバイオ・医療応用)

Ovarian endometriosis-associated stromal cells reveal persistently high affinity for iron. 査読有り

Mori M, Ito F, Shi L, Wang Y, Ishida C, Hattori Y, Niwa M, Hirayama T, Nagasawa H, Iwase A, Kikkawa F, Toyokuni S.

Redox Biol. 6 巻頁： 578-586 2015年12月

Cancer therapy using non-thermal atmospheric pressure plasma with ultra-high electron density 査読有り国際誌

Tanaka, H; Mizuno, M; Toyokuni, S; Maruyama, S; Kodera, Y; Terasaki, H; Adachi, T; Kato, M; Kikkawa, F; Hori, M

PHYSICS OF PLASMAS 22 巻 ( 12 ) 頁： 122004 2015年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Physics of Plasmas

Cancer therapy using non-thermal atmospheric pressure plasma is a big challenge in plasma medicine. Reactive species generated from plasma are key factors for treating cancer cells, and thus, non-thermal atmospheric pressure plasma with high electron density has been developed and applied for cancer treatment. Various cancer cell lines have been treated with plasma, and non-thermal atmospheric plasma clearly has anti-tumor effects. Recent innovative studies suggest that plasma can both directly and indirectly affect cells and tissues, and this observation has widened the range of applications. Thus, cancer therapy using non-thermal atmospheric pressure plasma is promising. Animal experiments and understanding the mode of action are essential for clinical application in the future. A new academic field that combines plasma science, the biology of free radicals, and systems biology will be established.

DOI： 10.1063/1.4933402

Ovarian endometriosis-associated stromal cells reveal persistently high affinity for iron. 査読有り国際誌

Mori M, Ito F, Shi L, Wang Y, Ishida C, Hattori Y, Niwa M, Hirayama T, Nagasawa H, Iwase A, Kikkawa F, Toyokuni S

Redox biology 6 巻頁： 578 - 586 2015年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Biology

Ovarian endometriosis is a recognized risk for infertility and epithelial ovarian cancer, presumably due to iron overload resulting from repeated hemorrhage. To find a clue for early detection and prevention of ovarian endometriosis-associated cancer, it is mandatory to evaluate catalytic (labile) ferrous iron (catalytic Fe(II)) and to study iron manipulation in ovarian endometriotic lesions. By the use of tissues from women of ovarian endometriosis as well as endometrial tissue from women with and without endometriosis, we for the first time performed histological analysis and cellular detection of catalytic Fe(II) with a specific fluorescent probe (HMRhoNox-M), and further evaluated iron transport proteins in the human specimens and in co-culture experiments using immortalized human eutopic/ectopic endometrial stromal cells (ESCs) in the presence or absence of epithelial cells (EpCs). The amounts of catalytic Fe(II) were higher in ectopic endometrial stromal cells (ecESCs) than in normal eutopic endometrial stromal cells (n-euESCs) both in the tissues and in the corresponding immortalized ESCs. ecESCs exhibited higher transferrin receptor 1 expression both in vivo and in vitro and lower ferroportin expression in vivo than n-euESCs, leading to sustained iron uptake. In co-culture experiments of ESCs with iron-loaded EpCs, ecESCs received catalytic ferrous iron from EpCs, but n-euESCs did not. These data suggest that ecESC play a protective role for cancer-target epithelial cells by collecting excess iron, and that these characteristics are retained in the immortalized ecESCs.

DOI： 10.1016/j.redox.2015.10.001

田中宏昌, 水野正明, 豊國伸哉, 丸山彰一, 小寺泰弘, 足立哲夫, 寺崎浩子, 加藤昌志, 吉川史隆, 堀勝

プラズマ・核融合学会誌 91 巻 ( 12 ) 頁： 776 - 779 2015年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：プラズマ・核融合学会

近年,非平衡大気圧プラズマの医療・バイオ応用が盛んに研究されている.プラズマに含まれる電子,イオン,ラジカル,光など様々な要因が複雑に細胞及び細胞を取り巻く環境に影響を及ぼし,細胞応答を引き起こしていると考えられている.最近,プラズマ照射された溶液(これはプラズマ活性溶液と名付けられた)ががん細胞にプログラム細胞死として知られるアポトーシスを誘導することがわかり,プラズマ活性溶液が細胞に及ぼす影響が活発に研究されている.脳腫瘍培養細胞においてはプラズマ活性溶液が生存・増殖シグナル伝達経路を抑制することによりアポトーシスへと導くことが明らかになった.これまでに,プラズマ活性溶液は脳腫瘍,卵巣がん,胃がん,非小肺がん細胞など様々ながん細胞に有効であり,抗癌剤耐性卵巣がん細胞に関しても有効であることがわかり,従来のがん治療法では困難な腹膜播種等の播種性のがん治療への応用が期待される.ここではこれまで研究で明らかになりつつあるプラズマ活性溶液の細胞影響とその分子機構について紹介する.

プラズマ活性溶液の細胞影響 (小特集プラズマが誘導する生体応答とそのバイオ・医療応用)

田中宏昌, 水野正明, 豊國伸哉, 丸山彰一, 小寺泰弘, 足立哲夫, 寺崎浩子, 加藤昌志, 吉川史隆, 堀勝

プラズマ・核融合学会誌 91 巻 ( 12 ) 頁： 776 - 779 2015年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：プラズマ・核融合学会

Hepatic distribution of GST cannot explain the gap between humans and rodents for induction of cholangiocarcioma following exposure to dichloropropane

Maternal molecular hydrogen administration on lipopolysaccharide-induced mouse fetal brain injury 査読有り国際誌

Nakano Tomoko, Kotani Tomomi, Mano Yukio, Tsuda Hiroyuki, Imai Kenji, Ushida Takafumi, Li Hua, Miki Rika, Sumigama Seiji, Sato Yoshiaki, Iwase Akira, Hirakawa Akihiro, Asai Masato, Toyokuni Shinya, Kikkawa Fumitaka

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 57 巻 ( 3 ) 頁： 178 - 182 2015年11月

Maternal molecular hydrogen administration on lipopolysaccharide-induced mouse fetal brain injury. 査読有り

Nakano T, Kotani T, Mano Y, Tsuda H, Imai K, Ushida T, Li H, Miki R, Sumigama S, Sato Y, Iwase A, Hirakawa A, Asai M, Toyokuni S, Kikkawa F.

J Clin Biochem Nutr. 57 巻 ( 3 ) 頁： 178-182 2015年11月

A trial to find appropriate animal models of dichloropropaneinduced cholangiocarcinoma based on the hepatic distribution of glutathione S-transferases

Zhang Lingyi, Zong Cai, Ichihara Sahoko, Naito Hisao, Toyokuni Shinya, Kumagai Shinji, Ichihara Gaku

JOURNAL OF OCCUPATIONAL HEALTH 57 巻 ( 6 ) 頁： 548 - 554 2015年11月

Zhang, L; Zong, C; Ichihara, S; Naito, H; Toyokuni, S; Kumagai, S; Ichihara, G

TOXICOLOGY LETTERS 238 巻 ( 2 ) 頁： S245 - S245 2015年10月

印刷会社で集団発生した胆管癌の最近の知見

熊谷信二, 久保正二, 竹村茂一, 田中肖吾, 木下正彦, 濱野玄弥, 伊藤得路, 青田哲尚, 江田将樹, 中沼安二, 圓藤吟史, 市原学, 張霊逸, 豊國伸哉

産業医学レビュー 28 巻 ( 2 ) 頁： 131 - 156 2015年9月

Malignant mesothelioma as an oxidative stress-induced cancer: An update. 査読有り国際誌

Chew SH, Toyokuni S

Free radical biology & medicine 86 巻頁： 166 - 78 2015年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Abstract Malignant mesothelioma (MM) is a relatively rare cancer that occurs almost exclusively following respiratory exposure to asbestos in humans. Its pathogenesis is closely associated with iron overload and oxidative stress in mesothelial cells. On fiber exposure, mesothelial cells accumulate fibers simultaneously with iron, which either performs physical scissor function or catalyzes free radical generation, leading to oxidative DNA damage such as strand breaks and base modifications, followed by activation of intracellular signaling pathways. Chrysotile, per se without iron, causes massive hemolysis and further adsorbs hemoglobin. Exposure to indigestible foreign materials also induces chronic inflammation, involving consistent generation of free radicals and subsequent activation of NALP3 inflammasomes in macrophages. All of these contribute to mesothelial carcinogenesis. Genomic alterations most frequently involve homozygous deletion of INK4A/4B, and other pathways such as Hippo and TGF-β pathways are also affected in MM. Recently, analyses of familial MM sorted out BAP1 as a novel responsible tumor suppressor gene, whose function is not fully elucidated. Five-year survival of mesothelioma is still ~8%, and this cancer is increasing worldwide. Connective tissue growth factor, a secretory protein creating a vicious cycle mediated by β-catenin, has been recognized as a hopeful target for therapy, especially in sarcomatoid subtype. Recent research outcomes related to microRNAs and cancer stem cells also offer additional novel targets for the treatment of MM. Iron reduction as chemoprevention of mesothelioma is helpful at least in an animal preclinical study. Integrated approaches to fiber-induced oxidative stress would be necessary to overcome this currently fatal disease.

DOI： 10.1016/j.freeradbiomed.2015.05.002

Aging rather than sun exposure is a major determining factor for the density of miR-125b-positive epidermal stem cells in human skin. 査読有り国際誌

Toyokuni S, Jiang L, Wang S, Hirao A, Wada T, Soh C, Toyama K, Kawada A

Pathology international 65 巻 ( 8 ) 頁： 415 - 9 2015年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

Sunlight exposure and aging are two major factors in the deterioration of skin function. In the present study, we used eighty formalin-fixed human skin samples from sun-exposed and unexposed areas from old and young individuals to evaluate the presence of miR-125b-positive epidermal stem cells (ESCs) by in situ hybridization. miR-125b-positive ESCs were detected in the basal layer of the epidermis. The density of miR-125b-positive ESCs was significantly associated with age rather than sun exposure, whereas the density of miR-125b-positive ESCs tended to decrease in the sun-exposed area. These data suggest the potential use of miR-125b as a surrogate marker for the quality of epidermal cells.

DOI： 10.1111/pin.12320

Receptor role of the annexin A2 in the mesothelial endocytosis of crocidolite fibers 査読有り国際誌

Yamashita Kyoko, Nagai Hirotaka, Toyokuni Shinya

LABORATORY INVESTIGATION 95 巻 ( 7 ) 頁： 749 - 764 2015年7月

Chemical conversion of human fibroblasts into neuronal cells: dawn of future clinical trials. Open Access

Toyokuni S

Journal of clinical biochemistry and nutrition 56 巻 ( 3 ) 頁： 165 - 165 2015年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

Major causes of human death in developed countries are atherosclerosis(1) and carcinogenesis.(2) Atherosclerosis finally leads to a variety of diseases such as myocardial infarction and cerebral infarction. The essence of these pathologic processes is largely due to free radical reactions or oxidative stress due to our persistent use of oxygen in the presence of iron,(3–5) and the products can be literally rust in our body.(6) Heart may be replaced with transplantation from another person with brain death under his/her own permission, but it is currently impossible to transplant brain. Additionally, highly ethical issues, severe donor deficiency and immunological rejections are always present with transplantation.(7) If brand-new cells and organs of any type to be replaced and of their own immunophenotype were freely available, all the people on earth would benefit from this novel technology. Induced pluripotent stem cells can be established from differentiated somatic cells by the forced induction of four transcription factors: Oct3/4, Klf4, Sox2 and c-Myc.(8) However, premature termination of reprogramming might lead to cancer development through altered epigenetic regulation.(9) Recently, the concept of directly converting one type of somatic cell into another has attracted much attention.(10) However, one of the major drawbacks linked to the current strategies is that they are also based on ectopic expression of important developmental genes (transcription factors, etc.),(11) which might cause unexpected undesired effects. In the present issue of Journal of Clinical Biochemistry and Nutrition, Dai et al.(12) for the first time describe a highly efficient chemical conversion of human fibroblasts to neuronal cells with defined six chemical compounds (Fig. 1). Their protocol used five inhibitors to block distinct signaling pathways including transforming growth factor (TGF)-β (SMAD pathway), bone morphologic protein (BMP), glycogen synthase kinase (GSK)-3β, MEK-ERK and p53, together with a cAMP stimulator. Moreover, the authors emphasize that this protocol shortens the time required for differentiation into neuronal cells to 3 weeks in comparison to the reported protocols(10,13) with ~90% efficiency. Fig. 1 Chemical conversion of human fibroblasts into neuronal cells. Refer to text for details. I believe that this is the dawn of regenerative neurology. In a few years or so, human clinical trials would be performed by the use of this technique. Target diseases would be neurodegenerative diseases(14) such as Alzheimer’s and Parkinson diseases, motor neuron diseases such as amyotropic lateral sclerosis, and cerebral infarction/hemorrhage. Simultaneously, we always have to consider how we can load these new neuronal cells with long-time experience and memory of the recipient if necessary.

DOI： 10.3164/jcbn.56-3-editorial

Plasma-Activated Solution: Mechanism of Action, Clinical Application, and Industrialization 査読有り国際誌

Tanaka H., Mizuno M., Toyokuni S., Maruyama S., Kodera Y., Kikkawa F., Hori M.

Fukuoka igaku zasshi = Hukuoka acta medica 106 巻 ( 4 ) 頁： 71 - 76 2015年4月

プラズマ活性溶液 : 作用機序解明と臨床応用・産業化を目指して

田中宏昌, 水野正明, 豊國伸哉, 丸山彰一, 小寺泰弘, 吉川史隆, 堀勝

福岡醫學雜誌 106 巻 ( 4 ) 頁： 71 - 6 2015年4月

Asbestos and multi-walled carbon nanotubes generate distinct oxidative responses in inflammatory cells. 査読有り国際誌

Funahashi S, Okazaki Y, Ito D, Asakawa A, Nagai H, Tajima M, Toyokuni S

Journal of clinical biochemistry and nutrition 56 巻 ( 2 ) 頁： 111 - 7 2015年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

Asbestos exposure is considered a social burden bycausing meso-thelioma. Despite the use of synthetic materials, multi-walled carbon nanotubes (MWCNTs) are similar in dimension to asbestos and produce mesothelioma in animals. The role of inflammatory cells in mesothelial carcinogenesis remains unclear. Here, we evaluated the differences in inflammatory cell responses following exposure to these fibrous materials using a luminometer and L-012 (8-amino-5-chloro-7-phenylpyrido[3,4-dlpyridazine-1,4-(2H,3H) dione) to detect reactive oxygen species (ROS). Rat peripheral blood or RAW264.7 cells were used to assess the effects on neutro-phils and macrophages, respectively. Crocidolite and amosite induced significant ROS generation by neutrophils with a peak at 10 min, whereas that of chrysotile was ∼25% of the crocidolite/ amosite response. MWCNTs with different diameters (∼15, 50, 115 and 145 nm) and different carcinogenicity did not induce significant ROS in peripheral blood. However, the MWCNTs induced a comparable amount of ROS in RAW264.7 cells to that following asbestos treatment. The peaks for MWCNTs (0.5-1.5 h) were observed earlier than those for asbestos (1-5 h). Apocynin and superoxide dismutase significantly inhibited ROS generation for each fiber, suggesting an involvement of NADPH oxidase and superoxide. Thus, asbestos and MWCNTs induce different oxida-tive responses in inflammatory cells, indicating the importance of mesothelial cell evaluation for carcinogenesis.

DOI： 10.3164/jcbn.14-92

血栓内膜摘除術後に体外循環から離脱できずに死亡した慢性血栓塞栓性肺高血圧症の一剖検例

山下享子, 榎本篤, 大島英揮, 平敷安希博, 近藤隆久, 豊国伸哉, 中村栄男

日本病理学会会誌 104 巻 ( 1 ) 頁： 336 - 336 2015年3月

Possible involvement of iron-induced oxidative insults in neurodegeneration. 査読有り国際誌

Asano T, Koike M, Sakata S, Takeda Y, Nakagawa T, Hatano T, Ohashi S, Funayama M, Yoshimi K, Asanuma M, Toyokuni S, Mochizuki H, Uchiyama Y, Hattori N, Iwai K

Neuroscience letters 588 巻 ( 1 ) 頁： 29 - 35 2015年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Neuroscience Letters

Involvement of iron in the development of neurodegenerative disorders has long been suggested, and iron that cannot be stored properly is suggested to induce iron toxicity. To enhance iron uptake and suppress iron storage in neurons, we generated transgenic (Tg) mice expressing iron regulatory protein 2 (IRP2), a major regulator of iron metabolism, in a neuron-specific manner. Although very subtle, IRP2 was expressed in all regions of brain examined. In the Tg mice, mitochondrial oxidative insults were observed including generation of 4-hydroxynonenal modified proteins, which appeared to be removed by a mitochondrial quality control protein Parkin. Inter-crossing of the Tg mice to Parkin knockout mice perturbed the integrity of neurons in the substantia nigra and provoked motor symptoms. These results suggest that a subtle, but chronic increase in IRP2 induces mitochondrial oxidative insults and accelerates neurodegeneration in a mouse model of Parkinson's disease. Thus, the IRP2 Tg may be a useful tool to probe the roles of iron-induced mitochondrial damages in neurodegeraration research.

DOI： 10.1016/j.neulet.2014.12.052

MINIMAL INFLAMMOGENICITY OF PRISTINE SINGLE-WALL CARBON NANOTUBES 査読有り国際誌

Shinya Toyokuni, Li Jiang, Ryo Kitaura, Hisanori Shinohara

NAGOYA JOURNAL OF MEDICAL SCIENCE 77 巻 ( 1-2 ) 頁： 195 - 202 2015年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NAGOYA UNIV, SCH MED

Carbon nanotubes (CNTs) are a novel synthetic material comprising only carbon atoms. Based on its rigidity, its electrical and heat conductivity and its applicability to surface manufacturing, this material is expected to have numerous applications in industry. However, due to the material's dimensional similarity to asbestos fibers, its carcinogenicity was hypothesized during the last decade, and indeed, we have shown that multi- wall CNTs (MWCNTs) of 50 nm in diameter are potently carcinogenic to mesothelial cells after intraperitoneal injection. Additionally, we suggested that inflammogenicity after intraperitoneal injection can predict mesothelial carcinogenesis. However, few data have been published on the intraperitoneal inflammogenicity of single-wall CNTs (SWCNTs). Here, we conducted a series of studies on SWCNTs using both intraperitoneal injection into rats and MeT5A mesothelial cells. Intraperitoneal injection of 10 mg SWCNTs caused no remarkable inflammation in the abdominal cavity, and the exposure of MeT5A cells to up to 25 mu g/cm(2) SWCNTs did not alter proliferation. MWCNTs of 50 nm in diameter were used as a positive control, and tangled MWCNTs of 15 nm in diameter were used as a negative control. The results suggest that SWCNTs are a low- risk material with respect to mesothelial carcinogenesis.

A trial to find appropriate animal models of dichloropropane‐induced cholangiocarcinoma based on the hepatic distribution of glutathione S‐transferases 国際誌

Minimal inflammogenicity of pristine single-wall carbon nanotubes.

Toyokuni S, Jiang LI, Kitaura R, Shinohara H

Nagoya journal of medical science 77 巻 ( 1-2 ) 頁： 195 - 202 2015年2月

Minimal inflammogenicity of pristine single-wall carbon nanotubes.

Toyokuni S, Jiang LI, Kitaura R, Shinohara H

Nagoya journal of medical science 77 巻 ( 1-2 ) 頁： 195 - 202 2015年2月

MINIMAL INFLAMMOGENICITY OF PRISTINE SINGLE-WALL CARBON NANOTUBES

Toyokuni Shinya, Jiang Li, Kitaura Ryo, Shinohara Hisanori

NAGOYA JOURNAL OF MEDICAL SCIENCE 77 巻 ( 1-2 ) 頁： 195 - 202 2015年2月

Catalytic ferrous iron in amniotic fluid as a predictive marker of human maternal-fetal disorders. 査読有り

Hattori Y, Mukaide T, Jiang L, Kotani T, Tsuda H, Mano Y, Sumigama S, Hirayama T, Nagasawa H, Kikkawa F, Toyokuni S

Journal of clinical biochemistry and nutrition 56 巻 ( 1 ) 頁： 57 - 63 2015年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

Amniotic fluid contains numerous biomolecules derived from fetus and mother, thus providing precious information on pregnancy. Here, we evaluated oxidative stress of human amniotic fluid and measured the concentration of catalytic Fe(II). Amniotic fluid samples were collected with consent from a total of 89 subjects in Nagoya University Hospital, under necessary medical interventions: normal pregnancy at term, normal pregnancy at the 2nd trimester, preterm delivery with maternal disorders but without fetal disorders, congenital diaphragmatic hernia, fetal growth restriction, pregnancy-induced hypertension, gestational diabetes mellitus, Down syndrome and trisomy 18. Catalytic Fe(II) and oxi-dative stress markers (8-hydroxy-2'-deoxyguanosine, 8-OHdG; dityrosine) were determined with RhoNox-1 and specific antibodies, respectively, using plate assays. Levels of 8-OHdG and dityrosine were higher in the 3rd trimester compared with the 2nd trimester in normal subjects, and the abnormal groups generally showed lower levels than the controls, thus suggesting that they represent fetal metabolic activities. In contrast, catalytic Fe(II) was higher in the 2nd trimester than the 3rd trimester in the normal subjects, and overall the abnormal groups showed higher levels than the controls, suggesting that high catalytic Fe(II) at late gestation reflects fetal pathologic alterations. Notably, products of H2O2 and catalytic Fe(II) remained almost constant in amniotic fluid.

DOI： 10.3164/jcbn.14-82

Napsin A is a specific marker for ovarian clear cell adenocarcinoma. 査読有り国際誌

Yamashita Y, Nagasaka T, Naiki-Ito A, Sato S, Suzuki S, Toyokuni S, Ito M, Takahashi S

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 28 巻 ( 1 ) 頁： 111 - 7 2015年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Modern Pathology

Ovarian clear cell adenocarcinoma has a relatively poor prognosis among the ovarian cancer subtypes because of its high chemoresistance. Differential diagnosis of clear cell adenocarcinoma from other ovarian surface epithelial tumors is important for its treatment. Napsin A is a known diagnostic marker for lung adenocarcinoma, and expression of napsin A is reported in a certain portion of thyroid and renal carcinomas. However, napsin A expression in ovarian surface epithelial tumors has not previously been examined. In this study, immunohistochemical analysis revealed that in 71 of 86 ovarian clear cell adenocarcinoma patients (83%) and all of the 13 patients with ovarian clear cell adenofibroma, positive napsin A staining was evident. No expression was observed in 30 serous adenocarcinomas, 11 serous adenomas or borderline tumors, 19 endometrioid adenocarcinomas, 22 mucinous adenomas or borderline tumors, 10 mucinous adenocarcinomas, or 3 yolk sac tumors of the ovary. Furthermore, expression of napsin A was not observed in the normal surface epithelium of the ovary, epithelia of the fallopian tubes, squamous epithelium, endocervical epithelium, or the endometrium of the uterus. Therefore, we propose that napsin A is another sensitive and specific marker for distinguishing ovarian clear cell tumors (especially adenocarcinomas) from other ovarian tumors.

DOI： 10.1038/modpathol.2014.61

Chemical conversion of human fibroblasts into neuronal cells: dawn of future clinical trials. 招待有り

Toyokuni S.

J Clin Biochem Nutr 56 巻頁： 165 2015年

Application of intermittent microwave irradiation to western blot analysis. 査読有り

Liu YT, Toyokuni S.

Methods Mol Biol 1314 巻頁： 185-190 2015年

Aging rather than sun exposure is a major determining factor for the density of miR-125b-positive epidermal stem cells in human skin. 査読有り

Toyokuni S, Jiang L, Wang S, Hirao A, Wada T, Soh C, Tohyama K and Kawada A.

Pathol Int 65 巻頁： 415-419 2015年

Malignant mesothelioma as an oxidative stress-induced cancer: An update. 査読有り

Chew SH and Toyokuni S.

Free Radic Biol Med 86 巻頁： 166-178 2015年

Maternal molecular hydrogen treatment attenuates lipopolysaccharide-induced rat fetal lung injury. 査読有り

Hattori Y, Kotani T, Tsuda H, Mano Y, Rei T, Li H, Hirako S, Ushida T, Imai K, Nakano T, Sato Y, Miki R, Sumigama S, Iwase A, Toyokuni S and Kikkawa F.

Free Radic Res 49 巻頁： 1026-1037 2015年

Possible involvement of iron-induced oxidative insults in neurodegeneration. 査読有り

Asano T, Koike M, Sakata S, Takeda Y, Nakagawa T, Hatano T, Ohshi S, Funayama M, Yoshimi K, Sanuma M, Toyokuni S, Mochizuki H, Uchiyama Y, Hattori N and Iwai K.

Neurosci Lett 588 巻頁： 29-35 2015年

Receptor role of annexin A2 in the mesothelial endocytosis of crocidolite fibers. 査読有り

Yamashita K, Nagai H and Toyokuni S.

Lab Invest 95 巻頁： 749-764 2015年

Minimal inflammogenicity of pristine single-wall carbon nanotubes. 査読有り

Toyokuni S, Jiang L, Kitaura R and Shinohara S.

Nagoya J Med Sci 77 巻頁： 195-202 2015年

Asbestos and multi-walled carbon nanotubes generate distinct oxidative responses in inflammatory cells. 査読有り

Funahashi F, Okazaki Y, Ito D, Asakawa A, Nagai H, Tajima M and Toyokuni S.

J Clin Biochem Nutr 56: 111-117, 2015. 56 巻頁： 111-117 2015年

Catalytic ferrous iron in amniotic fluid as a predictive marker of human maternal-fetal disorders. 査読有り

Hattori Y, Mukaide T, Jiang L, Kotani T, Tsuda H, Mano Y, Sumigama S, Hirayama T, Nagasawa H, Kikkawa F and Toyokuni S.

J Clin Biochem Nutr 56: 57-63, 2015. 56 巻頁： 57-63 2015年

Napsin A is a specific marker for ovarian clear cell adenocarcinoma. 査読有り

Yamashita Y, Nagasaka T, Naiki-Ito A, Sato S, Suzuki S, Toyokuni S, Ito M and Takahashi S.

Mod Pathol 28 巻頁： 111-117 2015年

A trial to find appropriate animal models of dichloropropane-induced cholangiocarcinoma based on the hepatic distribution of glutathione S-transferases. 査読有り

Zhang L, Zong C, Ichihara S, Naito H, Toyokuni S, Kumagai S, Ichihara G.

J Occup Health. 57 巻 ( 6 ) 頁： 548-554 2015年

Zhang L., Zong C., Ichihara S., Naito H., Toyokuni S., Kumagai S., Ichihara G.

ｊｏｕｒｎａｌ　ｏｆ　Ｏｃｃｕｐａｔｉｏｎａｌ　Ｈｅａｌｔｈ 57 巻 ( 6 ) 頁： 548 - 554 2015年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本産業衛生学会

Determinants of workplace violence against clinical physicians in hospitals: Jeng-Cheng WU, et al. Department of Urology, Taipei Medical University Hospital, Taiwan-Objectives: Workplace violence in the health sector is a worldwide concern. Physicians play an essential role in health-care teamwork; thus, understanding how organizational factors influence workplace violence against physicians is critical. Methods: A total of 189 physicians from three public hospitals and one private hospital in Northern Taiwan completed a survey, and the response rate was 47.1%. This study was approved by the institutional review board of each participating hospital. The 189 physicians were selected from the Taipei area, Taiwan. Results: The results showed that 41.5% of the respondents had received at least one workplace-related physical or verbal violent threat, and that 9.8% of the respondents had experienced at least one episode of sexual harassment in the 3 months before the survey. Logistic regression analysis revealed that physicians in psychiatry or emergency medicine departments received more violent threats and sexual harassment than physicians in other departments. Furthermore, physicians with a lower workplace safety climate (OR=0.89; 95% CI=0.81-0.98) and more job demands (OR=1.15; 95% CI=1.02-1.30) were more likely to receive violent threats. Conclusions: This study found that workplace violence was associated with job demands and the workplace safety climate. Therefore, determining how to develop a workplace safety climate and ensure a safe job environment for physicians is a crucial management policy issue for health-care systems.

DOI： 10.1539/joh.15-0085-oa

Application of intermittent microwave irradiation to western blot analysis. 査読有り国際共著国際誌

Liu YT, Toyokuni S

Methods Mol Biol 1314 巻頁： 185-190 2015年

Chemical conversion of human fibroblasts into neuronal cells: dawn of future clinical trials. 招待有り国際誌

Toyokuni S

J Clin Biochem Nutr 56 巻頁： 165 2015年

Maternal molecular hydrogen treatment attenuates lipopolysaccharide-induced rat fetal lung injury. 査読有り国際誌

Hattori Y, Kotani T, Tsuda H, Mano Y, Tu L, Li H, Hirako S, Ushida T, Imai K, Nakano T, Sato Y, Miki R, Sumigama S, Iwase A, Toyokuni S, Kikkawa F

Free radical research 49 巻 ( 8 ) 頁： 1026 - 37 2015年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Maternal inflammation is associated with spontaneous preterm birth and respiratory impairment among premature infants. Recently, molecular hydrogen (H2) has been reported to have a suppressive effect on oxidative stress and inflammation. The aim of this study was to evaluate the effects of H2 on fetal lung injury caused by maternal inflammation. Cell viability and the production of interleukin-6 (IL-6) and reactive oxygen species (ROS) were examined by treatment with lipopolysaccharide (LPS) contained in ordinal or H2-rich medium (HM) using a human lung epithelial cell line, A549. Pregnant Sprague Dawley rats were divided into three groups: Control, LPS, and HW + LPS groups. Rats were injected with phosphate-buffered saline (Control) or LPS intraperitoneally (LPS) on gestational day 19 and provided H2 water (HW) ad libitum for 24 h before LPS injection (HW + LPS). Fetal lung samples were collected on day 20, and the levels of apoptosis, oxidative damage, IL-6, and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry. The number of apoptotic cells, and levels of ROS and IL-6 were significantly increased by LPS treatment, and repressed following cultured with HM in A549 cells. In the rat models, the population positive for cleaved caspase-3, 8-hydroxy-2′-deoxyguanosine, IL-6, and VEGF was significantly increased in the LPS group compared with that observed in the Control group and significantly decreased in the HW + LPS group. In this study, LPS administration induced apoptosis and oxidative damage in fetal lung cells that was ameliorated by maternal H2 intake. Antenatal H2 administration may decrease the pulmonary mobility associated with inflammation in premature infants.

DOI： 10.3109/10715762.2015.1038257

Minimal inflammogenicity of pristine single-wall carbon nanotubes 査読有り国際誌

Toyokuni S., Jiang L., Kitaura R., Shinohara H.

Nagoya Journal of Medical Science 77 巻 ( 1-2 ) 頁： 195 - 202 2015年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nagoya Journal of Medical Science

Carbon nanotubes (CNTs) are a novel synthetic material comprising only carbon atoms. Based on its rigidity, its electrical and heat conductivity and its applicability to surface manufacturing, this material is expected to have numerous applications in industry. However, due to the material's dimensional similarity to asbestos fibers, its carcinogenicity was hypothesized during the last decade, and indeed, we have shown that multi-wall CNTs (MWCNTs) of 50 nm in diameter are potently carcinogenic to mesothelial cells after intraperitoneal injection. Additionally, we suggested that inflammogenicity after intraperitoneal injection can predict mesothelial carcinogenesis. However, few data have been published on the intraperitoneal inflammogenicity of single-wall CNTs (SWCNTs). Here, we conducted a series of studies on SWCNTs using both intraperitoneal injection into rats and MeT5A mesothelial cells. Intraperitoneal injection of 10 mg SWCNTs caused no remarkable inflammation in the abdominal cavity, and the exposure of MeT5A cells to up to 25 μg/cm2 SWCNTs did not alter proliferation. MWCNTs of 50 nm in diameter were used as a positive control, and tangled MWCNTs of 15 nm in diameter were used as a negative control. The results suggest that SWCNTs are a low-risk material with respect to mesothelial carcinogenesis.

アスベストによる中皮腫発がん機構の解明とナノマテリアルのリスク評価

Application of Intermittent Microwave Irradiation to Western Blot Analysis. 査読有り国際誌

Liu YT, Toyokuni S

Methods in molecular biology (Clifton, N.J.) 1314 巻頁： 185 - 90 2015年

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Methods in Molecular Biology

We established a shortened protocol for Western blot analysis using intermittent microwave irradiation. With this method, the procedure is completed within 1 h after applying the primary antibody, and thus greatly saves time. This procedure appears to be applicable to any antibody based on our experience of several years.

DOI： 10.1007/978-1-4939-2718-0_20

Receptor role of annexin A2 in the mesothelial endocytosis of crocidolite fibers. 査読有り

Yamashita K, Nagai H, Toyokuni S

Lab Invest 95 巻頁： 749-764 2015年

豊國伸哉

日本毒性学会学術年会 42 巻 ( 0 ) 頁： S4 - 2 2015年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本毒性学会

現在、日本人死因の第１位はがんである。産業・経済の発展を重視するあまり、リスク評価が十分になされないまま、ナノマテリアルが社会に多量に持ち込まれ、がんの原因となったことを忘れてはならない。それがアスベストであり、白石綿・青石綿・茶石綿が世界中で多量に使用された。日本では2006年に全面禁止となったが、アジア諸国を中心に現在も産生・使用されている。日本の中皮腫発生ピークは2025年と予想されており、今後10万人以上が中皮腫で死亡すると試算され、現在発生数が増加している。ラットで上記３種のアスベストの腹腔内10mg投与により中皮腫発がん実験を行った。２年の経過でほぼ全動物に中皮腫が発生した。アスベスト投与部の中皮細胞や貪食細胞に著明な鉄沈着を認め、Fenton反応促進性のニトリロ三酢酸の追加投与により、中皮腫発生が有意に早くなった。93％の腫瘍でCdkn2a/2bのホモ欠損を認めた。アスベスト発がんでは局所の過剰鉄病態が重要なことが示唆された。このような背景のもと、すでに中皮腫の危険性の報告のあった多層カーボンナノチューブ（MWCNT）の評価を行った。MWCNTは軽量・高強度で熱伝導性が高く、導体・半導体になることからすでに使用されているが、物理的形状は石綿に酷似している。直径が15/50/115/150 nmのMWCNTを使用し中皮細胞毒性実験ならびに、上記と同様のラットを使用した発がん実験を行った。中皮細胞への毒性と発がん性はほぼ一致し、直径50 nmのMWCNTの発がん性が最も高かった。Cdkn2a/2bのホモ欠損をほぼ全例で認めた。このことは、剛性が高い50 nm直径のMWCNTは特に注意して扱うべきことを示唆している(IARC Group 2B)。ヒトにおいて体腔に繊維が到達することはそう容易ではないと考えられるが、感染症が克服され、ますます長寿化が進む現在、新素材の十分なリスク評価とリスク管理は重要と考える。参考文献：1. Nagai H et al. Proc Natl Acad Sci USA 108: E1330, 2011; 2. Jiang L, et al. J Pathol 228: 366, 2012; 3. Toyokuni S. Adv Drug Deliv Rev 65: 298, 2013

DOI： 10.14869/toxpt.42.1.0_S4-2

グルタチオンＳ－トランスフェラーゼの肝臓内における分布は、 1，2－ジクロロプロパン曝露による胆管癌誘導作用におけるヒトとげっ歯類の違いを説明できない国際誌

Lingyi ZHANG, Cai ZONG, 市原佐保子, 内藤久雄, 豊國伸哉, 熊谷信二, 市原学

日本毒性学会学術年会 42 巻 ( 0 ) 頁： P - 165 2015年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本毒性学会

Purpose: 1,2-Dichloropropane(DCP) is thought to induce cholangiocarcinoma(CCA) among Japanese printing workers in 2013. However no studies have shown DCP-induced CCA in rodents. Five kinds of rodents were exposed to DCP to find an appropriate animal model for DCP-induced CCA. Methods: First, 12 C57BL/6J mice, Balb/cA mice, F344 rats, Syrian Hamsters and Guinea Pigs were divided into 4 groups equally and exposed to DCP at 0, 300, 1000 and 3000 ppm respectively. Then 32 Balb/cA mice and Syrian Hamsters were divvied into 4 groups equally and exposed to DCP at 0, 200, 400 and 800ppm respectively. After the last exposure livers were dissected out for immunohistochemistry with anti- GSTT1, GSTM1, GSTPi antibodies. Result: Either in control or exposed group all of the animals expressed GSTT1 both at liver cells and bile duct cells. Conclusion: GSTT1 expression cannot explain the gap between human and rodents in the case of DCP inducing CCA.

DOI： 10.14869/toxpt.42.1.0_P-165

１-ブロモプロパン神経毒性のマウスモデルの確立

宗才, Edwin GARNER, 黄晋彦, 張霊逸, 張堯, 櫻井敏博, 豊國伸哉, 市原佐保子, 市原学

日本毒性学会学術年会 42 巻 ( 0 ) 頁： P - 13 2015年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本毒性学会

【目的】オゾン層破壊物質代替物質1-ブロモプロパン（1BP）はヒトとラットで神経毒性と生殖毒性を引き起こすことがわかっている。雄ラットは1000 ppmの1BPに1日8時間、５から７週間の曝露でも生存するのに対し、最近の我々の研究で雄C57BL6/JJclマウスは300 ppmの1BPに7日間曝露後、肝臓の壊死を引き起こし、結果として生存できず、神経毒性影響が検出できなかった。本研究はサイトクロームP450の阻害剤、1ーアミノベンゾトライアゾール（ABT）を用い、1BPが引き起こす肝障害作用を抑制することによって、マウスを用いた1BP神経毒性モデルの確立を試みるとともに、P450による1BPの酸化が生殖毒性のパラメータにあたる影響を明らかにする。 【方法】42匹のC57BL/6JJclマウスを6匹ずつ7群に分け、4群にABT 50mg/kgを皮下注射で1日2回投与し、0、50、250、1200 ppmで1BP８時間の曝露を28日間行った。残る3群には生理食塩水を注射し、0、50、250 ppmの1BPに1日8時間、28日間曝露した。曝露終、麻酔下で臓器を剖出した。 【結果】ABT-・1BP 250 ppm群で、肝臓で壊死、炎症、肝臓細胞の変性像が見られた。一方、ABT+マウスでは、1200 ppmを含むすべての群で病理的な変化が見られなかった。脳重量は、ABT＋・1200ppm群でABT＋・0ppm群に比して有意に減少した。ABT-・1BP250 ppmのグループで、精子数がABT-・1BP 0ppm群に比して減少したが、ABT＋・1BP250ppm群およびABT＋・1BP50ppm群においてABT＋・1BP0ppm群に比して精子数の変化は無かった。精嚢と前立腺はABTの投与の有無に関わらず、250 ppmの濃度で、それぞれの0ppm群に対して重量が減少した。 【考察】ABT投与は1-BPによる肝毒性を抑制した。本研究はP450が1-BPの代謝と毒性発現において重要な役割を担っていることを明らかにした。この方法によりマウスの1-BP神経毒性モデルの作成が期待される。

DOI： 10.14869/toxpt.42.1.0_P-13

鉄過剰症による発がんの分子機構　特集：鉄代謝制御機構と鉄過剰症

豊國伸哉

病理と臨床 70 巻頁： 337 - 242 2015年

Minimal inflammogenicity of pristine single-wall carbon nanotubes 査読有り国際誌

Toyokuni S

Nagoya Journal of Medical Science 77 巻 ( 1-2 ) 頁： 195 - 202 2015年

Genome-wide profiling of 8-oxoguanine reveals its association with spatial positioning in nucleus. 査読有り国際誌

Yoshihara M, Jiang L, Akatsuka S, Suyama M, Toyokuni S

DNA research : an international journal for rapid publication of reports on genes and genomes 21 巻 ( 6 ) 頁： 603 - 12 2014年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：DNA Research

8-Oxoguanine (8-oxoG) is one of the most common DNA lesions generated by reactive oxygen species. In this study, we analysed the genome-wide distribution profile of 8-oxoG by combining immunoprecipitation by antibodies specific for the DNA fragments containing 8-oxoG with a microarray that covers rat genome. Genome-wide mapping of 8-oxoG in normal rat kidney revealed that 8-oxoG is preferentially located at gene deserts. We did not observe differences in 8-oxoG levels between groups of genes with high and low expression, possibly because of the generally low 8-oxoG levels in genic regions compared with gene deserts. The distribution of 8-oxoG and lamina-associated domains (LADs) were strongly correlated, suggesting that the spatial location of genomic DNA in the nucleus determines the susceptibility to oxidative modifications. One possible explanation for high 8-oxoG levels in LADs is that the nuclear periphery is more susceptible to the oxidative damage caused by the extra-nuclear factors. Moreover, LADs have a rather compact conformation, which may limit the recruitment of repair components to the modified bases.

DOI： 10.1093/dnares/dsu023

Plasma Medical Science for Cancer Therapy: Toward Cancer Therapy Using Nonthermal Atmospheric Pressure Plasma 査読有り国際誌

Tanaka, H; Mizuno, M; Ishikawa, K; Takeda, K; Nakamura, K; Utsumi, F; Kajiyama, H; Kano, H; Okazaki, Y; Toyokuni, S; Maruyama, S; Kikkawa, F; Hori, M

IEEE TRANSACTIONS ON PLASMA SCIENCE 42 巻 ( 12 ) 頁： 3760 - 3764 2014年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：IEEE Transactions on Plasma Science

We have been developing novel ultrahigh density atmospheric pressure plasma sources and succeeded in the selective killing ovarian cancer cells against normal ones. Furthermore, we have found out the plasma-activated medium (PAM) also killed glioblastoma brain tumor cells selectively against normal ones and the chemical products in the PAM have long lifetime healing effects. To clarify the mechanism, interactions of plasma with the organism and the medium where the organism belongs were investigated on the viewpoint of intracellular molecular mechanism.

DOI： 10.1109/TPS.2014.2353659

Direct exposure of non-equilibrium atmospheric pressure plasma confers simultaneous oxidative and ultraviolet modifications in biomolecules. 査読有り国際誌

Okazaki Y, Wang Y, Tanaka H, Mizuno M, Nakamura K, Kajiyama H, Kano H, Uchida K, Kikkawa F, Hori M, Toyokuni S

Journal of clinical biochemistry and nutrition 55 巻 ( 3 ) 頁： 207 - 15 2014年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

Thermal plasmas and lasers are used in medicine to cut and ablate tissues and for coagulation. Non-equilibrium atmospheric pressure plasma (NEAPP) is a recently developed, non-thermal technique with possible biomedical applications. Although NEAPP reportedly generates reactive oxygen/nitrogen species, electrons, positive ions, and ultraviolet radiation, little research has been done into the use of this technique for conventional free radical biology. Recently, we developed a NEAPP device with high electron density. Electron spin resonance spin-trapping revealed .OH as a major product. To obtain evidence of NEAPP-induced oxidative modifications in biomolecules and standardize them, we evaluated lipid peroxidation and DNA modifications in various in vitro and ex vivo experiments. Conjugated dienes increased after exposure to linoleic and α-linolenic acids. An increase in 2-thiobarbituric acid-reactive substances was also observed after exposure to phosphatidylcholine, liposomes or liver homogenate. Direct exposure to rat liver in saline produced immunohistochemical evidence of 4-hydroxy-2-nonenal- and acrolein-modified proteins. Exposure to plasmid DNA induced dose-dependent single/double strand breaks and increased the amounts of 8-hydroxy-2′-deoxyguanosine and cyclobutane pyrimidine dimers. These results indicate that oxidative biomolecular damage by NEAPP is dose-dependent and thus can be controlled in a site-specific manner. Simultaneous oxidative and UV-specific DNA damage may be useful in cancer treatment.

DOI： 10.3164/jcbn.14-40

Histological detection of catalytic ferrous iron with the selective turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model. 査読有り国際誌

Mukaide T, Hattori Y, Misawa N, Funahashi S, Jiang L, Hirayama T, Nagasawa H, Toyokuni S

Free radical research 48 巻 ( 9 ) 頁： 990 - 5 2014年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Iron overload of a chronic nature has been associated with a wide variety of human diseases, including infection, carcinogenesis, and atherosclerosis. Recently, a highly specific turn-on fluorescent probe (RhoNox-1) specific to labile ferrous iron [Fe(II)], but not to labile ferric iron [Fe(III)], was developed. The evaluation of Fe(II) is more important than Fe(III) in vivo in that Fe(II) is an initiating component of the Fenton reaction. In this study, we applied this probe to frozen sections of an established Fenton reaction-based rat renal carcinogenesis model with an iron chelate, ferric nitrilotriacetate (Fe-NTA), in which catalytic iron induces the Fenton reaction specifically in the renal proximal tubules, presumably after iron reduction. Notably, this probe reacted with Fe(II) but with neither Fe(II)-NTA, Fe(III) nor Fe(III)-NTA in vitro. Prominent red fluorescent color was explicitly observed in and around the lumina of renal proximal tubules 1 h after an intraperitoneal injection of 10-35 mg iron/kg Fe-NTA, which was dose-dependent, according to semiquantitative analysis. The RhoNox-1 signal colocalized with the generation of hydroxyl radicals, as detected by hydroxyphenyl fluorescein (HPF). The results demonstrate the transformation of Fe(III)-NTA to Fe(II) in vivo in the Fe-NTA-induced renal carcinogenesis model. Therefore, this probe would be useful for localizing catalytic Fe(II) in studies using tissues. © 2014 Informa UK, Ltd.

DOI： 10.3109/10715762.2014.898844

FREE FERROUS IRON IN AMNIOTIC FLUID AS A PREDICTIVE MARKER OF FETAL DISORDERS IN PREGNANCY 査読有り国際誌

Hattori, Y; Kotani, T; Nakano, T; Mano, Y; Sumigama, S; Tsuda, H; Usida, T; Nagasawa, H; Toyokuni, S; Kikkawa, F

PLACENTA 35 巻 ( 9 ) 頁： A71 - A71 2014年9月

Connective tissue growth factor and β-catenin constitute an autocrine loop for activation in rat sarcomatoid mesothelioma. 査読有り国際誌

Jiang L, Yamashita Y, Chew SH, AKatsuka S, Ukai S, Wang S, Nagai H, Okazaki Y, Takahashi T, Toyokuni S

J Pathol 233 巻 ( 4 ) 頁： 402 - 414 2014年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Pathology

Due to the formerly widespread use of asbestos, malignant mesothelioma (MM) is increasingly frequent worldwide. MM is classified into epithelioid (EM), sarcomatoid (SM), and biphasic subtypes. SM is less common than EM but is recognized as the most aggressive type of MM, and these patients have a poor prognosis. To identify genes responsible for the aggressiveness of SM, we induced EM and SM in rats, using asbestos, and compared their transcriptomes. Based on the results, we focused on connective tissue growth factor (Ctgf), whose expression was significantly increased in SM compared with EM; EM itself exhibited an increased expression of Ctgf compared with normal mesothelium. Particularly in SM, Ctgf was a major regulator of MM proliferation and invasion through activation of the β-catenin-TCF-LEF signalling pathway, which is autocrine and formed a positive feedback loop via LRP6 as a receptor for secreted Ctgf. High Ctgf expression also played a role in the epithelial-mesenchymal transition in MM. Furthermore, Ctgf is a novel serum biomarker for both early diagnosis and determining the MM prognosis in rats. These data link Ctgf to SM through the LRP6-GSK3β-β-catenin-TCF-Ctgf autocrine axis and suggest Ctgf as a therapeutic target. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DOI： 10.1002/path.4377

Expression of chromobox homolog 7 (CBX7) is associated with poor prognosis in ovarian clear cell adenocarcinoma via TRAIL-induced apoptotic pathway regulation. 査読有り国際誌

Shinjo K, Yamashita Y, Yamamoto E, Akatsuka S, Uno N, Kamiya A, Niimi K, Sakaguchi Y, Nagasaka T, Takahashi T, Shibata K, Kajiyama H, Kikkawa F, Toyokuni S

International journal of cancer 135 巻 ( 2 ) 頁： 308 - 18 2014年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Cancer

Ovarian cancer is the most lethal gynecologic malignancy, and clear cell adenocarcinoma of the ovary (OCCA), in particular, has a relatively poor prognosis among the ovarian cancer subtypes because of its high chemoresistance. Chromobox (CBX) 7 is a polycomb repressive complex 1 component that prolongs the lifespan of normal human cells by downregulating the INK4a/ARF expression which promotes cell-cycle progression. However, recent reports studying the relationship between CBX7 expression and patient survival have differed regarding the tumor cell origins, and the precise role of CBX7 in human carcinomas remains obscure. In this study, we analyzed CBX7 expression by immunohistochemistry in 81 OCCA patients and evaluated its association with their clinical outcomes. Both the overall and progression-free survival rates of the CBX7-positive patients were significantly shorter than those of the CBX7-negative patients (p < 0.05). CBX7 knockdown experiments using two OCCA cell lines, TOV21G and KOC-7C, revealed that cell viability was significantly reduced compared to the control cells (p < 0.001). Expression microarray analysis revealed that apoptosis-related genes, particularly tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were significantly upregulated in CBX7 knockdown cells (p < 0.01). We further confirmed that CBX7 knockdown resulted in TRAIL-induced apoptosis in the OCCA cells. Thus, in this study, we showed for the first time that CBX7 was associated with a decreased OCCA prognosis. We also successfully demonstrated that the TRAIL pathway is a novel target for CBX7 expression modulation in these cells, and therapeutic agents utilizing the TRAIL pathway may be particularly effective for targeted OCCA therapy. What's new? Ovarian cancer is the most lethal gynecologic malignancy, with clear celladenocarcinoma of the ovary (OCCA) having a particularly poor prognosis due to high chemoresistance. Chromobox homolog 7 (CBX7) is a polycomb group transcriptional repressor whose role in human cancer remains controversial. Here, the authors showed for the first time that CBX7 expression is related to worse prognosis in OCCA. Furthermore, knockdown of CBX7 in vitro induced apoptosis in OCCA cell lines, possibly via regulation of the TRAIL-pathway. The findings thus indicate CBX7 as a good prognostic marker, andthe TRAIL-pathway as a potential target for OCCA diagnosis and therapy. © 2013 UICC.

DOI： 10.1002/ijc.28692

Lack of presence of the human cytomegalovirus in human glioblastoma. 査読有り国際誌

Yamashita Y, Ito Y, Isomura H, Takemura N, Okamoto A, Motomura K, Tsujiuchi T, Natsume A, Wakabayashi T, Toyokuni S, Tsurumi T

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 27 巻 ( 7 ) 頁： 922 - 9 2014年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Modern Pathology

Recent reports have indicated human cytomegalovirus (HCMV) to be associated with human glioblastoma carcinogenesis. In established examples of viral carcinogenesis, viral DNA and one or more of its products have been detected in most tumor cells of biopsies in the majority of cases. To test whether HCMV is associated with human glioblastoma based on this criterion, we measured the number of viral DNA molecules per cell in both frozen and paraffin-embedded tumor biopsies from 58 patients using real-time quantitative PCR (QPCR). Immunohistochemical and fluorescence in situ hybridization (FISH) to detect HCMV proteins and genome was performed in 10 cases using formalin-fixed paraffin-embedded glioblastoma tissues. Southern blotting using DNA extracted from four glioblastoma cell lines together with immunoblotting using the four cell lines and five glioblastoma tissue samples were also performed. We further confirmed the immunoblot bands using liquid chromatography-tandem mass spectrometry assay. As a result, HCMV DNA was not detected in the tumor cells from any of the glioblastoma cases by QPCR detecting two different HCMV genes, in clear contrast to samples from patients with HCMV infection. Southern blotting and immunoblotting of cell lines and FISH using paraffin sections were all negative. However, immunoblotting and immunohistochemistry using tissue samples were partly positive, but HCMV proteins were not detected by proteomic analysis, suggesting false positivity of the analyses. As our QPCR analysis could detect 10 copies of HCMV DNA mixed with DNA extracted from 10 4 HCMV-negative cells, we conclude that HCMV is not persistent, at least in the tumor cells, of developed human glioblastoma. © 2014 USCAP, Inc All rights reserved.

DOI： 10.1038/modpathol.2013.219

Dexamethasone palmitate ameliorates macrophages-rich graft-versus-host disease by inhibiting macrophage functions. 査読有り国際誌

Nishiwaki S, Nakayama T, Murata M, Nishida T, Terakura S, Saito S, Kato T, Mizuno H, Imahashi N, Seto A, Ozawa Y, Miyamura K, Ito M, Takeshita K, Kato H, Toyokuni S, Nagao K, Ueda R, Naoe T

PloS one 9 巻 ( 5 ) 頁： e96252 2014年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type. © 2014 Nishiwaki et al.

DOI： 10.1371/journal.pone.0096252

Maternal molecular hydrogen administration ameliorates rat fetal hippocampal damage caused by in utero ischemia-reperfusion. 査読有り国際誌

Mano Y, Kotani T, Ito M, Nagai T, Ichinohashi Y, Yamada K, Ohno K, Kikkawa F, Toyokuni S

Free radical biology & medicine 69 巻頁： 324 - 30 2014年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Molecular hydrogen (H2) scavenges hydroxyl radicals. Recently, H2 has been reported to prevent a variety of diseases associated with oxidative stress in model systems and in humans. Here, we studied the effects of H2 on rat fetal hippocampal damage caused by ischemia and reperfusion (IR) on day 16 of pregnancy with the transient occlusion of the bilateral utero-ovarian arteries. Starting 2 days before the operation, we provided the mothers with hydrogen-saturated water ad libitum until vaginal delivery. We observed a significant increase in the concentration of H 2 in the placenta after the oral administration of hydrogen-saturated water to the mothers, with less placental oxidative damage after IR in the presence of H2. Neonatal growth retardation was observed in the IR group, which was alleviated by the H2 administration. We analyzed the neuronal cell damage in the CA1 and CA3 areas of the hippocampus at day 7 after birth by immunohistochemical analysis of the 8-oxo-7,8-dihydro-2.©2014 ElsevierInc.Allrightsreserved.

DOI： 10.1016/j.freeradbiomed.2014.01.037

Ovarian mucinous tumors arising from mature cystic teratomas-a molecular genetic approach for understanding the cellular origin 査読有り国際誌

Fujii, K; Yamashita, Y; Yamamoto, T; Takahashi, K; Hashimoto, K; Miyata, T; Kawai, K; Kikkawa, F; Toyokuni, S; Nagasaka, T

HUMAN PATHOLOGY 45 巻 ( 4 ) 頁： 717 - 724 2014年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Pathology

Mucinous tumors of the ovary are frequently associated with mature cystic teratomas, and it has been speculated that the mucinous tumors arise from teratoma components. The cellular origins of mature cystic teratomas are believed to be post-meiotic ovarian germ cells, and the analysis of microsatellite markers such as short tandem repeats is suitable for determining the cellular origin of tumors. In this study, we analyzed 3 ovarian mature cystic teratomas, all of which were associated with simultaneous ovarian mucinous tumors within the same ovary. Two of the 3 mucinous tumors were intestinal-type and the other was endocervical type. A laser capture microdissection technique was used to separate the epithelial component of the mucinous tumor, the components of the mature cystic teratoma, and control ovarian somatic tissue. Using short tandem repeat analysis based on 6 markers (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), we could distinguish the germ cell (homozygous) or somatic (heterozygous) origin of a given component in each sample. The epithelial components of the intestinal-type mucinous tumors in cases 1 and 2 were homozygous, and the epithelial component in case 3 (endocervical type) was heterozygous. All teratomatous components were homozygous, and the control components were heterozygous. In addition, we analyzed 3 mature cystic teratomas without mucinous tumors, and all 3 were homozygous in the tumor component. Our data suggest that the origin of mucinous tumors in the ovary may differ among histological subtypes, and intestinal-type mucinous tumors may arise from mature cystic teratomas, although endocervical-type mucinous tumors may not. © 2014 Elsevier Inc.

DOI： 10.1016/j.humpath.2013.10.031

From Nagoya to the world 査読有り国際誌

Toyokuni S.

Nagoya Journal of Medical Science 76 巻 ( 1-2 ) 頁： 1 - 2 2014年3月

From Nagoya to the world 国際誌

Toyokuni S

Nagoya Journal of Medical Science 76 巻 ( 1-2 ) 頁： 1 - 2 2014年3月

RAT MODEL DEMONSTRATES A HIGH RISK OF TREMOLITE BUT A LOW RISK OF ANTHOPHYLLITE FOR MESOTHELIAL CARCINOGENESIS 査読有り

Dilinuer Aierken, Yasumasa Okazaki, Shan Hwu Chew, Akihiro Sakai, Yue Wang, Hirotaka Nagai, Nobuaki Misawa, Norihiko Kohyama, Shinya Toyokuni

NAGOYA JOURNAL OF MEDICAL SCIENCE 76 巻 ( 1-2 ) 頁： 149 - 160 2014年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NAGOYA UNIV, SCH MED

Asbestos was abundantly used in industry during the last century. Currently, asbestos confers a heavy social burden due to an increasing number of patients with malignant mesothelioma ( MM), which develops after a long incubation period. Many studies have been conducted on the effects of the asbestos types that were most commonly used for commercial applications. However, there are few studies describing the effects of the less common types, or minor asbestos. We performed a rat carcinogenesis study using Japanese tremolite and Afghan anthophyllite. Whereas more than 50% of tremolite fibers had a diameter of < 500 nm, only a small fraction of anthophyllite fibers had a diameter of < 500 nm. We intraperitoneally injected 1 or 10 mg of asbestos into F1 Fischer-344/Brown-Norway rats. In half of the animals, repeated intraperitoneal injections of nitrilotriacetate (NTA), an iron chelator to promote Fenton reaction, were performed to evaluate the potential involvement of iron overload. Tremolite induced MM with a high incidence (96% with 10 mg; 52% with 1 mg), and males were more susceptible than females. Histology was confirmed using immunohistochemistry, and most MMs were characterized as the sarcomatoid or biphasic subtype. Unexpectedly NTA showed an inhibitory effect in females. In contrast, anthophyllite induced no MM after an observation period of 550 days. The results suggest that the carcinogenicity of anthophyllite is weaker than formerly reported, whereas that of tremolite is as potent as major asbestos as compared with our previous data.

Rat model demonstrates a high risk of tremolite but a low risk of anthophyllite for mesothelial carcinogenesis.

Aierken D, Okazaki Y, Chew SH, Sakai A, Wang Y, Nagai H, Misawa N, Kohyama N, Toyokuni S

Nagoya journal of medical science 76 巻 ( 1-2 ) 頁： 149 - 60 2014年2月

Rat model demonstrates a high risk of tremolite but a low risk of anthophyllite for mesothelial carcinogenesis.

Aierken D, Okazaki Y, Chew SH, Sakai A, Wang Y, Nagai H, Misawa N, Kohyama N, Toyokuni S

Nagoya journal of medical science 76 巻 ( 1-2 ) 頁： 149 - 60 2014年2月

RAT MODEL DEMONSTRATES A HIGH RISK OF TREMOLITE BUT A LOW RISK OF ANTHOPHYLLITE FOR MESOTHELIAL CARCINOGENESIS

Aierken Dilinuer, Okazaki Yasumasa, Chew Shan Hwu, Sakai Akihiro, Wang Yue, Nagai Hirotaka, Misawa Nobuaki, Kohyama Norihiko, Toyokuni Shinya

NAGOYA JOURNAL OF MEDICAL SCIENCE 76 巻 ( 1-2 ) 頁： 149 - 160 2014年2月

Rat model demonstrates a high risk of tremolite but a low risk of anthophyllite for mesothelial carcinogenesis. 査読有り国際誌

Aierken D., Okazaki Y., Chew S.H., Sakai A., Wang Y., Nagai H., Misawa N., Kohyama N., Toyokuni S.

Nagoya journal of medical science 76 巻 ( 1-2 ) 頁： 149 - 160 2014年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nagoya journal of medical science

Asbestos was abundantly used in industry during the last century. Currently, asbestos confers a heavy social burden due to an increasing number of patients with malignant mesothelioma (MM), which develops after a long incubation period. Many studies have been conducted on the effects of the asbestos types that were most commonly used for commercial applications. However, there are few studies describing the effects of the less common types, or minor asbestos. We performed a rat carcinogenesis study using Japanese tremolite and Afghan anthophyllite. Whereas more than 50% of tremolite fibers had a diameter of < 500 nm, only a small fraction of anthophyllite fibers had a diameter of < 500 nm. We intraperitoneally injected 1 or 10 mg of asbestos into F1 Fischer-344/Brown-Norway rats. In half of the animals, repeated intraperitoneal injections of nitrilotriacetate (NTA), an iron chelator to promote Fenton reaction, were performed to evaluate the potential involvement of iron overload. Tremolite induced MM with a high incidence (96% with 10 mg; 52% with 1 mg), and males were more susceptible than females. Histology was confirmed using immunohistochemistry, and most MMs were characterized as the sarcomatoid or biphasic subtype. Unexpectedly NTA showed an inhibitory effect in females. In contrast, anthophyllite induced no MM after an observation period of 550 days. The results suggest that the carcinogenicity of anthophyllite is weaker than formerly reported, whereas that of tremolite is as potent as major asbestos as compared with our previous data.

Cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis through dysregulated adipocytokine production. 査読有り国際誌

Chew SH, Okazaki Y, Nagai H, Misawa N, Akatsuka S, Yamashita K, Jiang L, Yamashita Y, Noguchi M, Hosoda K, Sekido Y, Takahashi T, Toyokuni S

Carcinogenesis 35 巻 ( 1 ) 頁： 164 - 72 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Carcinogenesis

Like many other human cancers, the development of malignant mesothelioma is closely associated with a chronic inflammatory condition. Both macrophages and mesothelial cells play crucial roles in the inflammatory response caused by asbestos exposure. Here, we show that adipocytes can also contribute to asbestos-induced inflammation through dysregulated adipocytokine production. 3T3-L1 preadipocytes were differentiated into mature adipocytes prior to use. These cells took up asbestos fibers (chrysotile, crocidolite and amosite) but were more resistant to asbestos-induced injury than macrophages and mesothelial cells. Expression microarray analysis followed by reverse transcription-PCR revealed that adipocytes respond directly to asbestos exposure with an increased production of proinflammatory adipocytokines [e.g. monocyte chemoattractant protein-1 (MCP-1)], whereas the production of anti-inflammatory adipocytokines (e.g. adiponectin) is suppressed. This was confirmed in epididymal fat pad of mice after intraperitoneal injection of asbestos fibers. Such dysregulated adipocytokine production favors the establishment of a proinflammatory environment. Furthermore, MCP-1 marginally promoted the growth of MeT-5A mesothelial cells and significantly enhanced the wound healing of Y-MESO-8A and Y-MESO-8D human mesothelioma cells. Our results suggest that increased levels of adipocytokines, such as MCP-1, can potentially contribute to the promotion of mesothelial carcinogenesis through the enhanced recruitment of inflammatory cells as well as a direct growth and migration stimulatory effect on mesothelial and mesothelioma cells. Taken together, our findings support a potential cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis. © The Author 2013. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/carcin/bgt267

Iron overload as a major targetable pathogenesis of asbestos-induced mesothelial carcinogenesis 査読有り国際誌 Open Access

Toyokuni, S

REDOX REPORT 19 巻 ( 1 ) 頁： 1 - 7 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Report

Few people expected that asbestos, a fibrous mineral, would be carcinogenic to humans. In fact, asbestos is a definite carcinogen in humans, causing a rare but aggressive cancer called malignant mesothelioma (MM). Mesothelial cells line the three somatic cavities and thus do not face the outer surface, but reduce the friction among numerous moving organs. MM has several characteristics: extremely long incubation period of 30-40 years after asbestos exposure, difficulty in clinical diagnosis at an early stage, and poor prognosis even under the current multimodal therapies. In Japan, 'Kubota shock' attracted considerable social attention in 2005 for asbestos-induced mesothelioma and, thereafter, the government enacted a law to provide the people suffering from MM a financial allowance. Several lines of recent evidence suggest that the major pathology associated with asbestos-induced MM is local iron overload, associated with asbestos exposure. Preclinical studies to prevent MM after asbestos exposure with iron reduction are in progress. In addition, novel target genes in mesothelial carcinogenesis have been discovered with recently recognized mesothelioma-prone families. Development of an effective preventive strategy is eagerly anticipated because of the long incubation period for MM. © W. S. Maney & Son Ltd 2014.

DOI： 10.1179/1351000213Y.0000000075

As a host society to SFRRI 2014 in Kyoto. Open Access

Handa O, Naito Y, Toyokuni S

Journal of clinical biochemistry and nutrition 54 巻 ( 1 ) 頁： 1 - 1 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

It is our great honor to host the 17th Biennial Meeting of Society for Free Radical Research International (SFRRI 2014) that is being held at the Kyoto International Conference Center, in Kyoto, Japan on March 23rd–26th, 2014. Previously we had this biennial meeting twice in Kyoto, in the year 1988 and 2000. This meeting has a great meaning not only for every member in SFRRI but also for those in SFRR Japan, since the SFRRI 1998 meeting was a key event behind the establishment of SFRR Japan. Since 1998, SFRR Japan has expanded the number of the members and has developed a variety of original research activities. The International Scientific Program Committee (ISPC) representing each region of SFRR has worked hard all together to develop what we believe will be an exciting and informative meeting to both young and senior participants. As a result, the congress will include award lectures and symposia on novel aspects of free radical research, which was democratically selected after the application during December 2012 and January 2013 by the voting of ISPC. Concurrent symposia will address in-depth and cutting-edge scientific topics in free radical chemistry, biology and medicine. In addition, the meeting will have four plenary lectures presented by Prof. Rafael Radi (Uruguay), Prof. Harold. M Swartz (USA), Prof. Arturo Zychlinsky (Germany) and Prof. Hideyuki Saya (Japan). Each lecture would reveal novel aspects of free radical research. It has been our sincere wish to make the meeting affordable and attractive especially to young scientists. There will be several awards from each region of SFRR as well as from this meeting. Sunrise lecture for the beginner of free radical research will be presented. Several satellite meetings are now planned in order to allow participants with special interests to meet. The congress venue, Kyoto city, is a UNESCO World Heritage site with a long and rich history going back over 1,200 years and was the original imperial capital of Japan. With more than 3,000 temples and shrines, full of traditional Japanese culture, with cherry blossoms presumably in full bloom, Kyoto city will deeply inspire your sixth sense. Besides traditional atmosphere, Kyoto city is thriving and exciting modern city with excellent facilities and easy links to the rest of the world. We believe that SFRRI 2014 meeting will be rich in science, and a great opportunity to hear cutting edge advances in the free radical research, as well as an opportunity to renew old friendships and establish new collaborations and associations. We look forward to meeting all of you in Kyoto in March 2014.

DOI： 10.3164/jcbn.54-1E1

Cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis through dysregulated adipocytokine production. 査読有り

Chew SH, Okazaki Y, Nagai H, Misawa N, Akatsuka S, Yamashita K, Jiang L, Noguchi M, Hosoda K, Sekido Y, Takahashi T and Toyokuni S.

Carcinogenesis 35 巻頁： 164-172 2014年

Histological detection of catalytic ferrous iron with the selective turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model. 査読有り

Mukaide T, Hatori Y, Misawa N, Funahashi S, Jiang L, Hirayama T, Nagasawa H and Toyokuni S.

Free Radic Res 48 巻頁： 990-995 2014年

Rat model demonstrates a high risk of tremolite but a low risk of anthophyllite for mesothelial carcinogenesis. 査読有り

Aierken D, Okazaki Y, Chew SH, Sakai A, Wang Y, Nagai H, Misawa N, Kohyama N and Toyokuni S.

Nagoya J Med Sci 76 巻頁： 149-160 2014年

Maternal molecular hydrogen administration ameliorates rat fetal hippocampal damage by in ut ero ischemia-reperfusion. 査読有り

Mano Y, Kotani T, Ito M, Nagai T, Ichinohashi Y, Yamada K, Ohno K, Kikkawa F and Toyokuni S.

Free Radic Biol Med 69 巻頁： 324-330 2014年

Lack of presence of the human cytomegalovirus in human glioblastoma. Modern Pathol 27: 922-929, 2014. 査読有り

Yamashita Y, Ito Y, Isomura H, Takemura N, Okamoto A, Motomura K, Tsujiuchi T, Natsume A, Wakabayashi T, Toyokuni S and Tsurumi T.

Modern Pathology 27 巻頁： 922-929 2014年

Ovarian mucinous tumors arising from mature cystic teratomas-a molecular genetic approach for understanding the cellular origin. 査読有り

Fujii K, Yamashita Y, Yamamoto T, Takahashi K, Hashimoto K, Miyata T, Kawai K, Kikkawa F, Toyokuni S, Nagasaka T.

Human Pathol 45 巻頁： 717-724 2014年

Iron as a major targetable pathogenesis of asbestos-induced mesothelial carcinogenesis. 査読有り

Toyokuni, S.

Redox Rep. 19 巻頁： 1-7 2014年

Dexamethasone palmitate ameliorates macrophages-1 rich graft-versus-host disease by inhibiting macrophage functions. 査読有り

Nishiwaki S, Nakayama T, Murata M, Nishida T, Terakura S, Saito S, Kato T. Mizuno H, Imahashi N, Seto S, Ozawa Y, Miyamura K, Ito M, Takeshita K, Kato H, Toyokuni S, Nagao K, Ueda R and Naoe T.

PLoS ONE 9 巻頁： e96252 2014年

Preclinical evaluation of an O(6)-methylguanine-DNA methyltransferase-siRNA/liposome complex administered by convection-enhanced delivery to rat and porcine brains. 査読有り

Tsujiuchi T, Natsume A, Motomura K, Kondo G, Ranjit M, Hachisu R, Sugimura I, Tomita S, Takehara I, Woolley M, Barua NU, Gill SS, Bienemann AS, Yamashita Y, Toyokuni S, Wakabayashi T.

Am J Transl Res 15 巻頁： 169-178 2014年

Direct exposure of non-equilibrium atmospheric pressure plasma confers simultaneous oxidative and ultraviolet modifications in biomolecules. 査読有り

Okazaki Y, Wang Y, Tanaka H, Mizuno M, Nakamura K, Kajiyama H, Kano H, Uchida K, Kikkawa F, Hori M and Toyokuni S.

J Clin Biochem Nutr 55 巻頁： 207-215 2014年

Plasma medical science for cancer therapy using non-thermal atomospheric pressure plasma. 査読有り

Tanaka H, Mizuno M, Ishikawa K, Takeda K, Nakamura K, Utsumi F, Kajiyama H, Nano H, Okazaki Y, Toyokuni S, Maruyama S, Kikkawa F and Hori M.

IEEE Trans Plasma Sci 42 巻頁： 3760-3764 2014年

Iron and thiols as two major players in carcinogenesis: friends or foes? 招待有り査読有り

Toyokuni S.

Front Pharmacol 5 巻頁： 200 2014年

Genome-wide profiling of 8-oxoguanine reveals its association with spatial positioning in nucleus. 査読有り

Yoshihara M, Jiang L, Akatsuka S, Suyama M and Toyokuni S.

DNA Res 21 巻頁： 603-612 2014年

Iron and thiols as two major players in carcinogenesis: friends or foes? 査読有り国際誌 Open Access

Toyokuni S

Frontiers in pharmacology 5 巻頁： 200 - 200 2014年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Pharmacology

Iron is the most abundant metal in the human body and mainly works as a cofactor for proteins such as hemoglobin and various enzymes. No independent life forms on earth can survive without iron. However, excess iron is intimately associated with carcinogenesis by increasing oxidative stress via its catalytic activity to generate hydroxyl radicals. Biomolecules with redox-active sulfhydryl function(s) (thiol compounds) are necessary for the maintenance of mildly reductive cellular environments to counteract oxidative stress, and for the execution of redox reactions for metabolism and detoxification. Involvement of glutathione S-transferase and thioredoxin has long attracted the attention of cancer researchers. Here, I update recent findings on the involvement of iron and thiol compounds during carcinogenesis and in cancer cells. It is now recognized that the cystine/glutamate transporter (antiporter) is intimately associated with ferroptosis, an iron-dependent, non-apoptotic form of cell death, observed in cancer cells, and also with cancer stem cells; the former with transporter blockage but the latter with its stabilization. Excess iron in the presence of oxygen appears the most common known mutagen. Ironically, the persistent activation of antioxidant systems via genetic alterations in Nrf2 and Keap1 also contributes to carcinogenesis. Therefore, it is difficult to conclude the role of iron and thiol compounds as friends or foes, which depends on the quantity/distribution and induction/flexibility, respectively. Avoiding further mutation would be the most helpful strategy for cancer prevention, and myriad of efforts are being made to sort out the weaknesses of cancer cells. © 2014 Toyokuni.

DOI： 10.3389/fphar.2014.00200

Iron and thiols as two major players in carcinogenesis: friends or foes? 招待有り査読有り

Toyokuni S

Front Pharmacol 5 巻頁： 200 2014年

Preclinical evaluation of an O(6)-methylguanine-DNA methyltransferase-siRNA/liposome complex administered by convection-enhanced delivery to rat and porcine brains.

Tsujiuchi T, Natsume A, Motomura K, Kondo G, Ranjit M, Hachisu R, Sugimura I, Tomita S, Takehara I, Woolley M, Barua NU, Gill SS, Bienemann AS, Yamashita Y, Toyokuni S, Wakabayashi T

American journal of translational research 6 巻 ( 2 ) 頁： 169 - 78 2014年

Preclinical evaluation of an O6-methylguanine-DNA methyltransferase-siRNA/liposome complex administered by convection-enhanced delivery to rat and porcine brains 査読有り国際誌

Tsujiuchi T., Natsume A., Motomura K., Kondo G., Ranjit M., Hachisu R., Sugimura I., Tomita S., Takehara I., Woolley M., Barua N.U., Gill S.S., Bienemann A.S., Yamashita Y., Toyokuni S., Wakabayashi T.

American Journal of Translational Research 6 巻 ( 2 ) 頁： 169 - 178 2014年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Translational Research

The main determinant of glioblastoma (GBM) resistance to temozolomide (TMZ) is thought to be O6-methylguanine-DNA methyltransferase (MGMT), which is a DNA-repair enzyme that removes alkyl groups from the O6-position of guanine. Previously, we reported that a MGMT-siRNA/cationic liposome complex exerted a clear synergistic antitumor effect in combination with TMZ. Translation to a clinical setting might be desirable for reinforcing the efficacy of TMZ therapy for GBM. In this study, we aim to evaluate the safety of MGMT-siRNA/cationic liposome complexes and determine whether the convection-enhanced delivery of these complexes is suitable for clinical use by undertaking preclinical testing in laboratory animals. No significant adverse events were observed in rats receiving infusions of MGMT-siRNA/cationic liposome complex directly into the brain with or without TMZ administration. A pig which received the complex administered by CED also showed no evidence of neurological dysfunction or histological abnormalities. However, the complex did not appear to achieve effective distribution by CED in either the rat or the porcine brain tissue. Considering these results together, we concluded that insufficient distribution of cationic liposomes was achieved for tumor treatment by CED.

Preclinical evaluation of an O-6-methylguanine-DNA methyltransferase-siRNA/liposome complex administered by convection-enhanced delivery to rat and porcine brains 査読有り

Takashi Tsujiuchi, Atsushi Natsume, Kazuya Motomura, Goro Kondo, Melissa Ranjit, Rei Hachisu, Itsuro Sugimura, Shinpei Tomita, Isao Takehara, Max Woolley, Neil U. Barua, Steven S. Gill, Alison S. Bienemann, Yoriko Yamashita, Shinya Toyokuni, Toshihiko Wakabayashi

AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH 6 巻 ( 2 ) 頁： 169 - 178 2014年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：E-CENTURY PUBLISHING CORP

The main determinant of glioblastoma (GBM) resistance to temozolomide (TMZ) is thought to be O-6-methylguanine-DNA methyltransferase (MGMT), which is a DNA-repair enzyme that removes alkyl groups from the O-6-position of guanine. Previously, we reported that a MGMT-siRNA/cationic liposome complex exerted a clear synergistic antitumor effect in combination with TMZ. Translation to a clinical setting might be desirable for reinforcing the efficacy of TMZ therapy for GBM. In this study, we aim to evaluate the safety of MGMT-siRNA/cationic liposome complexes and determine whether the convection-enhanced delivery of these complexes is suitable for clinical use by undertaking preclinical testing in laboratory animals. No significant adverse events were observed in rats receiving infusions of MGMT-siRNA/cationic liposome complex directly into the brain with or without TMZ administration. A pig which received the complex administered by CED also showed no evidence of neurological dysfunction or histological abnormalities. However, the complex did not appear to achieve effective distribution by CED in either the rat or the porcine brain tissue. Considering these results together, we concluded that insufficient distribution of cationic liposomes was achieved for tumor treatment by CED.

脳梗塞発症前の運動はsuperoxide dismutase活性を増加させ脳梗塞障害を軽減する

野口泰司, 石田和人, 濱川みちる, 玉越敬悟, 高松泰行, 戸田拓弥, 加藤寛聡, 早稲田雄也, 赤塚慎也, 豊國伸哉

理学療法学Supplement 2013 巻 ( 0 ) 頁： 0007 - 0007 2014年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本理学療法士協会

【はじめに，目的】脳梗塞の後遺症に苦しむ患者数の増大から,脳梗塞の予防的介入が重要視されている。動物実験では,脳梗塞発症前に一定期間運動を行うことで,脳梗塞後の運動機能障害が軽減し,脳梗塞体積も減少するという報告がなされている（Wang RY. et al., 2001）。この作用機序について,我々の研究室では脳梗塞の二次的傷害の主要な要因である酸化ストレスに着目し,脳梗塞発症前に運動を行うことで酸化ストレス産物（8-OHdG，4-HNE修飾タンパク）の生成が軽減されることを報告し,酸化ストレス抑制の関連を示唆した（Hamakawa M. et al., 2013）。しかし,この酸化ストレス抑制の機序は明らかになっていない。そこで,本研究では脳内の主要な抗酸化ストレス物質の1つであるsuperoxide dismutase（SOD）に着目し,事前の運動による脳梗塞障害軽減効果の作用機序を検討することを目的とする。【方法】実験動物にはWistar系雄性ラット（5週齢）を用いた。無作為にSham群（n=6）,運動＋sham群（n=6）,脳梗塞群（n=8）,運動+脳梗塞群（n=12）の4群に分け,運動＋sham群と運動＋脳梗塞群は3週間のトレッドミル運動（15m/min，30分/日）を毎日行った。Sham群と脳梗塞群は走行させずにトレッドミル装置内に曝露させた。3週間後,脳梗塞群と運動＋脳梗塞群に対し,小泉法により90分間左中大脳動脈を閉塞することで脳梗塞モデル作成手術を施した。手術24時間後に,感覚-運動機能に関し,麻痺の重症度の評価としてneurological deficits（ND）を,前肢の感覚運動機能の評価としてlimb placing test（LP）を,前肢の協調運動機能の評価としてladder testを,歩行時のバランス能力の評価としてbeam walking test（BW）を行った。その直後に脳梗塞周囲の大脳皮質感覚運動野を採取し,SOD-Assay kit-WST（同仁化学研究所）を用いてSOD活性を測定した。また,SODの遺伝子発現について,real-time PCR法によりSOD1（Cu，Zn-SOD）,SOD2（Mn-SOD）,SOD3（EC-SOD）のmRNA発現量を定量化した。統計学的解析はSPSS ver. 16.0を用い,感覚－運動機能評価に関しND，LP，BWについてはMann-Whitney U testを,ladder testについてはStudent's t-testを行った。また,SOD活性,SOD1，2，3の遺伝子発現については一元配置分散分析にて比較し,事後検定としてTukey's testを行った。統計学的検定における有意水準は5%未満とした。【倫理的配慮，説明と同意】本研究における前処置は名古屋大学動物実験指針に従って実施した。【結果】運動＋脳梗塞群は脳梗塞群に比べて,ND，LP，ladder testにおいて有意に障害が軽度であった（p＜0.05）。一方でBWでは群間に有意差は認められなかった。またSOD活性は,運動＋脳梗塞群が脳梗塞群に比べ有意に高値を示した（p＜0.05）。さらにSOD1は,運動＋脳梗塞群が脳梗塞群に比べ有意に発現が高かった（p＜0.05）。SOD2，3は群間に有意差は認められなかった。【考察】脳梗塞モデル作成前に3週間のトレッドミル運動を行うことで,脳梗塞後の感覚－運動機能障害が軽減することが示された。またSOD活性およびSOD1発現量の増加が示された。これらの結果より,事前に運動を行うことは,脳梗塞時のSOD発現,活性が促進され,虚血/再灌流により生じる大量の活性酸素を迅速に消去し,酸化ストレスを抑制する作用があると考えられる。よって,脳梗塞発症前の運動が及ぼす障害軽減効果には,SODの抗酸化ストレス能に伴う神経保護作用が関与していることが示唆された。【理学療法学研究としての意義】脳梗塞発症前の運動による脳梗塞障害軽減効果の作用機序の一端を分子生物学的に示した。これらの結果は,脳梗塞の予防として推奨されている運動の効果を科学的に検討し,予防医療分野における理学療法のさらなる発展に寄与するものと考える。

DOI： 10.14900/cjpt.2013.0007

【職業・環境発がん:メカニズムと病理】繊維性ナノマテリアルの発がん機構

豊國伸哉

病理と臨床 32 巻頁： 632 - 637 2014年

Preclinical evaluation of an O-6-methylguanine-DNA methyltransferase-siRNA/liposome complex administered by convection-enhanced delivery to rat and porcine brains

Tsujiuchi Takashi, Natsume Atsushi, Motomura Kazuya, Kondo Goro, Ranjit Melissa, Hachisu Rei, Sugimura Itsuro, Tomita Shinpei, Takehara Isao, Woolley Max, Barua Neil U., Gill Steven S., Bienemann Alison S., Yamashita Yoriko, Toyokuni Shinya, Wakabayashi Toshihiko

AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH 6 巻 ( 2 ) 頁： 169 - 178 2014年

Genotoxicity and carcinogenicity risk of carbon nanotubes. 査読有り国際誌

Toyokuni S

Advanced drug delivery reviews 65 巻 ( 15 ) 頁： 2098 - 110 2013年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Advanced Drug Delivery Reviews

Novel materials are often commercialized without a complete assessment of the risks they pose to human health because such assessments are costly and time-consuming; additionally, sometimes the methodology needed for such an assessment does not exist. Carbon nanotubes have the potential for widespread application in engineering, materials science and medicine. However, due to the needle-like shape and high durability of multiwalled carbon nanotubes (MWCNTs), concerns have been raised that they may induce asbestos-like pathogenicity when inhaled. Indeed, experiments in rodents supported this hypothesis. Notably, the genetic alterations in MWCNT-induced rat malignant mesothelioma were similar to those induced by asbestos. Single-walled CNTs (SWCNTs) cause mitotic disturbances in cultured cells, but thus far, there has been no report that SWCNTs are carcinogenic. This review summarizes the recent noteworthy publications on the genotoxicity and carcinogenicity of CNTs and explains the possible molecular mechanisms responsible for this carcinogenicity. The nanoscale size and needle-like rigid structure of CNTs appear to be associated with their pathogenicity in mammalian cells, where carbon atoms are major components in the backbone of many biomolecules. Publishing adverse events associated with novel materials is critically important for alerting people exposed to such materials. CNTs still have a bright future with superb economic and medical merits. However, appropriate regulation of the production, distribution and secondary manufacturing processes is required, at least to protect the workers. © 2013 Elsevier B.V.

DOI： 10.1016/j.addr.2013.05.011

Deferasirox Induces Mesenchymal-Epithelial Transition in Crocidolite-Induced Mesothelial Carcinogenesis in Rats 国際誌

Toyokuni, S; Nagai, H; Okazaki, Y; Chew, SH; Yasui, H

FREE RADICAL BIOLOGY AND MEDICINE 65 巻頁： S25 - S25 2013年11月

Deferasirox induces mesenchymal-epithelial transition in crocidolite-induced mesothelial carcinogenesis in rats. 査読有り国際誌

Nagai H, Okazaki Y, Chew SH, Misawa N, Yasui H, Toyokuni S

Cancer prevention research (Philadelphia, Pa.) 6 巻 ( 11 ) 頁： 1222 - 30 2013年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Prevention Research

Asbestos was used worldwide in huge quantities in the past century. However, because of the unexpected carcinogenicity to mesothelial cells with an extremely long incubation period, many countries face this long-lasting social problem. Mesothelioma is often diagnosed in an advanced stage, for which no effective therapeutic protocols are yet established. We previously reported on the basis of animal experiments that the major pathology in asbestos-induced mesothelial carcinogenesis is local iron overload. Here, we undertook to find an effective strategy to prevent, delay, or lower the malignant potential of mesothelioma during asbestos-induced carcinogenesis. We used intraperitoneal injections of crocidolite to rats. We carried out a 16-week study to seek the maximal-tolerated intervention for iron reduction via oral deferasirox administration or intensive phlebotomy. Splenic iron deposition was significantly decreased with either method, and we found that Perls' iron staining in spleen is a good indicator for iron reduction. We injected a total of 10 mg crocidolite at the age of six weeks, and the preventive measures were via repeated oral administration of 25 to 50 mg/kg/d deferasirox or weekly to bimonthly phlebotomy of 4 to 10 mL/kg/d. The animals were observed until 110 weeks. Deferasirox administration significantly increased the fraction of less malignant epithelioid subtype. Although we found a slightly prolonged survival in deferasirox-treated female rats, larger sample size and refinement of the current protocol are necessary to deduce the cancer-preventive effects of deferasirox. Still, our results suggest deferasirox serves as a potential preventive strategy in people already exposed to asbestos via iron reduction. ©2013 AACR.

DOI： 10.1158/1940-6207.CAPR-13-0244

From nagoya to the world 査読有り国際誌

Toyokuni S.

Nagoya Journal of Medical Science 75 巻 ( 1-2 ) 頁： 1 - 2 2013年9月

From nagoya to the world 国際誌

Toyokuni S

Nagoya Journal of Medical Science 75 巻 ( 1-2 ) 頁： 1 - 2 2013年9月

Intraperitoneal administration of tangled multiwalled carbon nanotubes of 15 nm in diameter does not induce mesothelial carcinogenesis in rats. 査読有り国際誌

Nagai H, Okazaki Y, Chew SH, Misawa N, Miyata Y, Shinohara H, Toyokuni S

Pathology international 63 巻 ( 9 ) 頁： 457 - 62 2013年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

Multiwalled carbon nanotubes (MWCNTs) have attracted public attention not only for their potential applications in engineering and materials science but also for possible environmental risks. MWCNTs share similar properties with asbestos, a definite human carcinogen causing malignant mesothelioma (MM), in that they are both biopersistent thin fibers with a high aspect ratio. Certain types of MWCNTs do induce MM in animal experiments. Though there are many different types of MWCNTs awaiting use in industry, there is little evidence about what types of MWCNTs present a high risk for MMin vivo. We have previously shown that the diameter of MWCNTs is one of the critical factors for mesothelial injury, which eventually leads to MM. Because of the extensive commercial use of MWCNTs, the properties of MWCNTs that determine carcinogenic activity should be clarified. Here we report that a high dose (10mg) of a tangled form of pristine MWCNT (with a diameter of 15nm) did not induce MM after intraperitoneal administration in rats, which were followed for up to 3 years after injection. This observation strengthens our previous finding that the rigidity, diameter, length and surface properties of MWCNTs are important factors in MM induction in vivo. © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

DOI： 10.1111/pin.12093

Reduction of diabetes-induced renal oxidative stress by a cantaloupe melon extract/gliadin biopolymers, oxykine, in mice (vol 23, pg 85, 2005) 査読有り国際誌

Naitoa, Y; Akagiri, S; Uchiyama, K; Kokura, S; Yoshida, N; Hasegawa, G; Nakamura, N; Ichikawa, H; Toyokuni, S; Ijichi, T; Yoshikawa, T

BIOFACTORS 39 巻 ( 5 ) 頁： 589 - 589 2013年9月

CD146 and insulin-like growth factor 2 mRNA-binding protein 3 predict prognosis of asbestos-induced rat mesothelioma. 査読有り国際誌

Okazaki Y, Nagai H, Chew SH, Li J, Funahashi S, Tsujimura T, Toyokuni S

Cancer science 104 巻 ( 8 ) 頁： 989 - 95 2013年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Malignant mesothelioma (MM), which is associated with asbestos exposure, is one of the most deadly tumors in humans. Early MM is concealed in the serosal cavities and lacks specific clinical symptoms. For better treatment, early detection and prognostic markers are necessary. Recently, CD146 and insulin-like growth factor 2 mRNA-binding protein 3 (IMP3) were reported as possible positive markers of MM to distinguish from reactive mesothelia in humans. However, their application on MM of different species and its impact on survival remain to be elucidated. To disclose the utility of these molecules as early detection and prognostic markers of MM, we injected chrysotile or crocidolite intraperitoneally to rats, thus obtaining 26 peritoneal MM and establishing 11 cell lines. We immunostained CD146 and IMP3 using paraffin-embedded tissues and cell blocks and found CD146 and IMP3 expression in 58% (15/26) and 65% (17/26) of MM, respectively, but not in reactive mesothelia. There was no significant difference in both immunostainings for overexpression among the three histological subtypes of MM and the expression of CD146 and IMP3 was proportionally associated. Furthermore, the overexpression of CD146 and/or IMP3 was proportionally correlated with shortened survival. These results suggest that CD146 and IMP3 are useful diagnostic and prognostic markers of MM. © 2013 Japanese Cancer Association.

DOI： 10.1111/cas.12185

Lewis y antigen is expressed in oral squamous cell carcinoma cell lines and tissues, but disappears in the invasive regions leading to the enhanced malignant properties irrespective of sialyl-Lewis x. 査読有り国際誌

Hotta H, Hamamura K, Yamashita K, Shibuya H, Tokuda N, Hashimoto N, Furukawa K, Yamamoto N, Hattori H, Toyokuni S, Ueda M, Furukawa K

Glycoconjugate journal 30 巻 ( 6 ) 頁： 585 - 97 2013年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Glycoconjugate Journal

Expression and implication of carbohydrate antigens in squamous cell carcinomas (SCCs) in oral cavity was examined. In the cell lines, type 2H and Lewis y antigens were markedly expressed. In the tissues from SCC patients and benign disorders, type 2H was highly expressed in hyperplasia (96.4 %), displasia (92.9 %) and SCC (100 %). Lewis y was, in turn, expressed mainly in cancer tissues (91.3 %), suggesting that Lewis y is a cancer-associated antigen. Normal oral mucosa showed no expression of these blood group antigens. Surprisingly, Lewis y antigen disappeared in the invasion sites where Ki-67 was definitely stained. Over-expression of Lewis y with manipulation of a fucosyltransferase cDNA resulted in suppression of cell growth and invasion, and knockdown of Lewis y also brought about increased cell growth and invasion. In either situations, no changes in the expression of sialyl-Lewis x could be found. Lowered tumor growth and invasion into surrounding tissues were also shown in Lewis y-positive SCC grafts in nu/nu mice. All these results together with alternative staining between Lewis y and Ki-67 in cancer tissues and FUT1 transfectants suggested that loss of Lewis y is a crucial event for the late stage of SCCs. © 2012 Springer Science+Business Media New York.

DOI： 10.1007/s10719-012-9458-2

Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells. 査読有り国際誌

Yamashita K, Nagai H, Kondo Y, Misawa N, Toyokuni S

Journal of clinical biochemistry and nutrition 53 巻 ( 1 ) 頁： 27 - 35 2013年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

Exposure to asbestos fibers increases the risk of mesothelioma in humans. One hypothetical carcinogenic mechanism is that asbestos fibers may directly induce mutations in mesothelial cells. Although the uptake of asbestos fibers by mesothelial cells is recognized, methods for the quantification of the uptake have not been well established. In the present study, we evaluated two distinct methods, using crocidolite fibers and MeT5A mesothelial cells. One method is histological evaluation using the cell-block technique, which allows for the direct cross-sectional observation of cells and fibers. We found the bright field observation with ×1000 magnification (oil-immersion) of the sample with Kemechtrot staining was most suitable for this purpose. The other method is flow cytometric analysis, which permits the evaluation of a much larger number of cells. We observed that the side scatter (SSC) increased with the intracellular fibers, and that the "mean SSC ratio (treated/control)" was useful for quantification. We could collect the cells with abundant internalized crocidolite fibers by sorting. Results of the two methodologies were correlated well in the experiments. The quantities of internalized fibers increased with incubation time and loaded dosage, but they were inversely associated with cellular density in culture. © 2013 JCBN.

DOI： 10.3164/jcbn.12-104

Repeated short-term daily exercise ameliorates oxidative cerebral damage and the resultant motor dysfunction after transient ischemia in rats. 査読有り

Hamakawa M, Ishida A, Tamakoshi K, Shimada H, Nakashima H, Noguchi T, Toyokuni S, Ishida K

Journal of clinical biochemistry and nutrition 53 巻 ( 1 ) 頁： 8 - 14 2013年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

Long-term exercise prior to brain ischemia enhances the activities of antioxidant enzymes and leads to a significant reduction in brain damage and neurological deficits in rats subjected to transient middle cerebral artery occlusion. However, it has not been established whether relatively short-term exercise generates similar results following middle cerebral artery occlusion. We aimed to determine whether short-term exercise could reduce oxidative damage and prevent sensori-motor dysfunction. Male Wistar rats were subjected to perform daily exercise on a treadmill for 30 min at a speed of 15 m/min for 3 weeks, followed by a 90-min middle cerebral artery occlusion. Animals were assessed after middle cerebral artery occlusion for neurological deficits and sensori-motor function. Brain tissues were processed to evaluate infarct volume and oxidative damage. Oxidative stress was assessed using immunohistochemistry for 4-hydroxy-2-nonenalmodified proteins and 8-hydroxy-2'-deoxyguanosine. Antioxidant enzymes were evaluated using immunohistochemistry for thioredoxin and activity assay for superoxide dismutase. Exercise for 3 weeks decreased the severity of paralysis and impairment in forelimb motor coordination. Furthermore, exercise had effect on superoxide dismutase and reduced the infarct volume and the number of cells immunopositive for 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2'-deoxyguanosine. Our results suggest that pre-conditioning treadmill exercise for 3 weeks is useful for ameliorating ischemia-induced brain injury. © 2013 JCBN.

DOI： 10.3164/jcbn.12-72

Multispecificity of immunoglobulin M antibodies raised against advanced glycation end products: involvement of electronegative potential of antigens. 査読有り国際誌

Chikazawa M, Otaki N, Shibata T, Miyashita H, Kawai Y, Maruyama S, Toyokuni S, Kitaura Y, Matsuda T, Uchida K

The Journal of biological chemistry 288 巻 ( 19 ) 頁： 13204 - 14 2013年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

Background: Advanced glycation end products (AGEs) can act as neoantigens to trigger immune responses. Results: Natural IgM antibodies against AGEs recognize multiple molecules, including DNA and chemically modified proteins. Conclusion: There is a close relationship between the formation of AGEs and innate immune responses. Significance: Our findings highlight AGEs and related modified proteins as a source of multispecific natural antibodies. Copyright © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M113.452177

DOI： 10.1074/jbc.m113.452177

CANCER AS A FERROTOXIC DISEASE: WHAT WE HAVE LEARNED FROM ANIMAL STUDIES TOWARD ITS PREVENTION 国際誌

Toyokuni, S; Jiang, L; Okazaki, Y; Akatsuka, S

AMERICAN JOURNAL OF HEMATOLOGY 88 巻 ( 5 ) 頁： E168 - E169 2013年5月

Met is the most frequently amplified gene in endometriosis-associated ovarian clear cell adenocarcinoma and correlates with worsened prognosis. 査読有り国際誌

Yamashita Y, Akatsuka S, Shinjo K, Yatabe Y, Kobayashi H, Seko H, Kajiyama H, Kikkawa F, Takahashi T, Toyokuni S

PloS one 8 巻 ( 3 ) 頁： e57724 2013年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

Clear cell adenocarcinoma of the ovary (OCC) is a chemo-resistant tumor with a relatively poor prognosis and is frequently associated with endometriosis. Although it is assumed that oxidative stress plays some role in the malignant transformation of this tumor, the characteristic molecular events leading to carcinogenesis remain unknown. In this study, an array-based comparative genomic hybridization (CGH) analysis revealed Met gene amplification in 4/13 OCC primary tumors and 2/8 OCC cell lines. Amplification of the AKT2 gene, which is a downstream component of the Met/PI3K signaling pathway, was also observed in 5/21 samples by array-based CGH analysis. In one patient, both the Met and AKT2 genes were amplified. These findings were confirmed using fluorescence in situ hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemistry. In total, 73 OCC cases were evaluated using real-time quantitative PCR; 37.0% demonstrated Met gene amplification (>4 copies), and 8.2% had AKT2 amplification. Furthermore, stage 1 and 2 patients with Met gene amplification had significantly worse survival than patients without Met gene amplification (p<0.05). Met knockdown by shRNA resulted in reduced viability of OCC cells with Met amplification due to increased apoptosis and cellular senescence, suggesting that the Met signaling pathway plays an important role in OCC carcinogenesis. Thus, we believe that targeted inhibition of the Met pathway may be a promising treatment for OCC. © 2013 Yamashita et al.

DOI： 10.1371/journal.pone.0057724

FROM NAGOYA TO THE WORLD 国際誌

Toyokuni, S

NAGOYA JOURNAL OF MEDICAL SCIENCE 75 巻 ( 1-2 ) 頁： 1 - 2 2013年2月

Protective effect of cardioplegia with poly (ADP-ribose) polymerase-1 inhibitor against myocardial ischemia-reperfusion injury: in vitro study of isolated rat heart model. 査読有り国際誌

Yamazaki K, Tanaka S, Sakata R, Miwa S, Oriyanhan W, Takaba K, Minakata K, Marui A, Ikeda T, Toyokuni S, Komeda M, Ueda K

Journal of enzyme inhibition and medicinal chemistry 28 巻 ( 1 ) 頁： 143 - 7 2013年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Enzyme Inhibition and Medicinal Chemistry

Poly(ADP-ribose) polymerase (PARP)-1 inhibitor has been suggested to attenuate the ischemia-reperfusion injury. We investigated the protective effect of the cardioplegia with a PARP-1 inhibitor, 4-hydoxyquinazoline (4-HQ), against myocardial ischemia-reperfusion injury. Isolated rat hearts were perfused on a Langendorff apparatus and cardioplegically arrested for 90 min by perfusion with St. Thomas' Hospital solution (ST-solution). In the Group ST (n = 8), the hearts were arrested with the ST-solution alone. The Group HQ (n = 8) were treated with the ST-solution containing 4-HQ (10 μM) for cardioplegia. During reperfusion, the Group HQ showed significantly greater functional recovery of +dp/dtmax (p = 0.005) and lower enzymatic leakage (p < 0.01). NAD+ levels were also preserved higher in the Group HQ (p < 0.01). Immunohistochemical study revealed lesser extents of oxidative stress and apoptosis, in the Group HQ. Thus, addition of 4-HQ in the cardioplegia may provide a new intervention for myocardial protection against ischemia-reperfusion injury by decreasing NAD+ consumption and suppressing oxidative stress. © 2013 Informa UK, Ltd.

DOI： 10.3109/14756366.2011.642373

FROM NAGOYA TO THE WORLD 国際誌

Toyokuni, S

NAGOYA JOURNAL OF MEDICAL SCIENCE 75 巻 ( 1-2 ) 頁： 1 - 2 2013年2月

Deferasirox induces mesenchymal-epithelial transition in crocidolite-induced mesothelila carcinogenesis in rats. 査読有り

Nagai H, Okazaki Y, Chew SH, Misawa N, Yasui H and Toyokuni S.

Cancer Prev Res 6 巻 ( 11 ) 頁： 1222-1230 2013年

Genotoxicity and carcinogenicity risk of carbon nanotubes. 査読有り

Toyokuni S.

Adv Drug Deliver Rev 2013年

Repeated short-term daily exercise ameliorates oxidative cerebral damage and the resultant motor dysfunction after transient ischemia in rats. 査読有り

Hamakawa M, Ishida A, Tamakoshi K, Shimada H, Nakashima H, Noguchi T, Toyokuni S and Ishida K.

J Clin Biochem Nutr 53 巻頁： 8-14 2013年

CD146 and IMP3 predict prognosis of asbestos-induced rat mesothelioma. 査読有り

Okazaki Y, Nagai H, Chew SH, Jiang L, Funahashi S, Tsujimura T and Toyokuni S.

Cnacer Sci 104 巻頁： 989-995 2013年

Multi-specificity of IgM antibodies raised against advanced glycation end products: Involvement of electronegative potential of antigens. 査読有り

Chikazawa M, Otaki N, Shibata T, Miyashita H, Kawai Y, Maruyama S, Toyokuni S, Kitaura Y, Matsuda T, Uchida K.

J Biol Chem 288 巻頁： 13204-13214 2013年

Met is the most frequently amplified gene in endometriosis-associated ovarian clear cell adenocarcinoma and correlates with worsened prognosis. 査読有り

Yamashita Y, Akatsuka S, Shinjo K, Yatabe Y, Kobayashi H, Seko H, Kajiyama H, Kikkawa F, Takahashi T and Toyokuni S.

PLoS One 8 巻 ( 3 ) 頁： e57724 2013年

Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells. 査読有り

Yamashita K, Nagai H, Kondo Y, Misawa N and Toyokuni S.

J Clin Biochem Nutr 53 巻頁： 27-35 2013年

Metamorphosis of mesothelial cells with active horizontal motility in tissue culture. 査読有り

Nagai H, Chew SH, Okazaki Y, Funahashi S, Namba T, Kato T, Enomoto A, Jiang L, Akatsuka S and Toyokuni S.

Sci Rep 3 巻頁： 1144 2013年

Protective effect of cardioplegia with poly(ADP-ribose)polymerase-1 inhibitor aganst myocardial ischemia-reperfusion injury: in vitro study if isolated rat heart model. 査読有り

Yamazaki K, Tanaka S, Sakata R, Miwa S, Oriyanhan W, Takaba K, Minakata K, Marui A, Ikeda T, Toyokuni S, Komeda M and Ueda K.

J Enzyme Inhibition Medicinal Chem 28 巻頁： 143-147 2013年

Hotta H, Hamamura K, Yamashita K, Shibuya H, Tokuda N, Hashimoto N, Furukawa K, Yamamoto N, Hattori H, Toyokuni S, Ueda M, Furukawa K.

Glycoconj J 30 巻頁： 585-597 2013年

Intraperitoneal administration of tangled multiwalled carbon nanotubes of 15 nm in diameter does not induce mesothelial carcinogenesis in rats. 査読有り

Nagai H, Okazaki Y, Chew SH, Misawa N, Miyata Y, Shinohara N and Toyokuni S.

Pathol. Int. 63 巻頁： 457-462 2013年

Metamorphosis of mesothelial cells with active horizontal motility in tissue culture. 査読有り国際誌

Nagai H, Chew SH, Okazaki Y, Funahashi S, Namba T, Kato T, Enomoto A, Jiang L, Akatsuka S, Toyokuni S

Scientific reports 3 巻頁： 1144 2013年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Mesothelial cells, which have diverse roles in physiology and pathology, constitute the mesothelium along with connective tissue and the basement membrane; the mesothelium serves to shield the somatic cavities. After mesothelial injury, mesothelial cells undergo tissue recovery. However, the mechanism of mesothelial regeneration remains poorly understood. In this study, we used confocal time-lapse microscopy to demonstrate that transformed mesothelial cells (MeT5A) and mouse peritoneal mesothelial cells can randomly migrate between cells in cell culture and in ex vivo tissue culture, respectively. Moreover, peritoneal mesothelial cells changed their morphology from a flattened shape to a cuboidal one prior to the migration. Conversely, MDCKII epithelial cells forming tight cell-cell contacts with one another do not alter the arrangement of adjacent cells during movement. Our evidence complements the current hypotheses of mesothelial regeneration and suggests that certain types of differentiated mesothelial cells undergo morphological changes before initiating migration to repair injured sites.

DOI： 10.1038/srep01144

Genotoxicity and carcinogenicity risk of carbon nanotubes. 査読有り

Toyokuni S

Adv Drug Deliver Rev 2013年

Multi-specificity of IgM antibodies raised against advanced glycation end products: Involvement of electronegative potential of antigens. 査読有り

Chikazawa M, Otaki N, Shibata T, Miyashita H, Kawai Y, Maruyama S, Toyokuni S, Kitaura Y, Matsuda T, Uchida K

J Biol Chem 288 巻頁： 13204-13214 2013年

ナノ材料（CNT）の物性と発がん性国際誌

豊國伸哉

日本毒性学会学術年会 40 巻 ( 0 ) 頁： 1044 2013年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本毒性学会

1981年以降，日本人死因の第1位はがんである。喫煙や特定の感染症が発がんリスクとして同定された。しかし，産業・経済を重視するあまり，リスク評価が十分になされず，ナノマテリアルが社会に多量に持ち込まれ，がんの原因となったことも忘れてはならない。それが繊維状鉱物のアスベストであり白石綿・青石綿・茶石綿が使用された。日本では2006年に禁止となったが，アジアの諸国やロシアなどでは今も使用されている。日本の中皮腫発生ピークは2025年で今後40年間に10万人以上の方が中皮腫で死亡すると試算されている。ラットを使用して上記３種の石綿で，腹腔内10mg投与により中皮腫発がん実験を行った。2年の経過でほぼ全動物に中皮腫が発生した。石綿投与に伴い，同部の中皮細胞や貪食細胞に著明な鉄沈着を認め，Fenton反応促進性のニトリロ三酢酸の追加投与でどの石綿でも中皮腫発生が早くなった。93％の腫瘍でCdkn2A/2Bのホモ欠損を認めた。アスベスト発がんでは局所の過剰鉄病態が重要なことが示唆された。このような背景のもと，すでに中皮腫の危険性の報告のあった多層カーボンナノチューブ（CNT）の評価を行った。CNTは軽量・高強度で熱伝導性が高く導体・半導体になることからすでに電池・液晶パネルのマテリアルとして使用されているが，形状は石綿に酷似している。直径が15/50/115/150nmのCNTを使用し中皮細胞毒性実験と上記と同様のラットを使用した発がん実験を行った。中皮細胞への毒性と発がん性はほぼ一致し，50nmの発がん性が最も高かった。Cdkn2A/2Bのホモ欠損をほぼ全例で認めた。このことは，剛性が高い50nm直径のCNTは特に注意して扱うべきことを示唆している。一方，石綿はendocytosisで中皮細胞に取り込まれるが，CNTは突き刺さり入ることも明らかになった。ヒトで体腔に繊維が到達することはそう簡単ではないと考えられるが，ますます長寿化が進む現在，十分なリスク評価が必要と考えられる。

DOI： 10.14869/toxpt.40.1.0.1044.0

脳梗塞発症前の運動による脳梗塞障害軽減効果及びその作用機序の検討〜酸化ストレスに着目して〜国際誌

野口泰司, 濱川みちる, 玉越敬悟, 戸田拓弥, 豊國伸哉, 石田和人

理学療法学Supplement 2012 巻 ( 0 ) 頁： 48101465 - 48101465 2013年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本理学療法士協会

【はじめに、目的】脳梗塞発症時には活性酸素やフリーラジカルが大量に産生され、その酸化ストレスは組織障害を拡大させ脳梗塞を増悪させる因子である。一方で、健常なラットに長期間運動させると、脳内で抗酸化物質が増加し酸化ストレスの減少がもたらされることが報告されており、運動は脳内の抗酸化作用を高めることが示されている。また、先行研究では脳梗塞モデルラット作成前に一定期間の運動を行うと、運動していないラットに比べて、脳梗塞後の梗塞体積が減少し運動機能障害も軽減するといった脳梗塞障害軽減効果が報告されている。しかし、この運動により高まる脳内の抗酸化作用が、脳梗塞時の酸化ストレスに影響を及ぼしているかどうかは不明である。そこで本研究では、脳梗塞発症前の運動による脳梗塞障害軽減効果およびその作用機序を、脳内の抗酸化作用に着目して検討することを目的とする。【方法】実験動物にはWistar系雄性ラット（5 週齢）を用いた。無作為に（1）Sham群、（2）運動＋sham群、（3）脳梗塞群、（4）運動＋脳梗塞群に分け、脳梗塞＋運動群と運動＋sham群は3 週間のトレッドミル運動（15 m/min，30 分/日）を毎日行った。脳梗塞群とSham群は走行させずにトレッドミル装置内に暴露させた（1 日30 分間）。3 週間後、運動＋脳梗塞群と脳梗塞群に対し、小泉法による脳梗塞モデル作成手術を施行した。手術24 時間後に、運動-感覚機能評価として、麻痺の重症度を評価するneurological deficits（ND）、歩行時のバランス能力の評価としてbeam walking（BW）、はしごの上の歩行における前肢の協調運動機能の評価としてladder test、前肢の感覚運動機能の評価としてlimb placing（LP）を行った。その直後に脳を採取し、TTC染色により非梗塞半球体積に対する梗塞体積の割合を算出した。また、酸化ストレス関連指標として、脂質過酸化の指標である4-hydroxy-2-nonenal（4-HNE）とDNAの酸化的損傷の指標となる8-Hydroxydeoxyguanosine（8-OHdG）、抗酸化酵素であるthioredoxin（TRX）の免疫組織化学染色を行い、陽性細胞数及び光学濃度を計測した。またSOD Assay kit-WST（同仁化学研究所）を用いて抗酸化酵素であるsuperoxide dismutase（SOD）活性を計測した。【倫理的配慮、説明と同意】本研究における全処置は名古屋大学動物実験指針に従って実施した。【結果】運動+脳梗塞群は脳梗塞群に比べて、NDとladder testのスコアが低値となり有意に障害が軽度であった。LPとBWは群間に有意差は認められなかった。また、梗塞体積割合は運動＋脳梗塞群の方が脳梗塞群よりも有意に小さかった。4-HNE 及び8-OHdG陽性細胞数は、運動＋脳梗塞群の方が脳梗塞群に比べ有意に少なかった。一方、TRXの光学濃度は群間に有意な差は認められなかったが、SOD活性は運動＋脳梗塞群が脳梗塞群に比べ高値を示す傾向にあった。【考察】脳梗塞モデル作成前に3 週間のトレッドミル運動を継続することで、脳梗塞後の麻痺の重症度および前肢の協調運動機能障害が軽減すること、梗塞体積が縮小すること、また脳梗塞時の脂質やDNAに対する酸化ストレスを抑制することが示された。さらに抗酸化酵素であるSOD活性が高められることが示された。これらの結果より、脳梗塞前の運動による脳梗塞の障害軽減効果には酸化ストレスの抑制とSODの活性化が関与していることが示唆された。【理学療法学研究としての意義】脳梗塞発症前の運動による脳梗塞障害軽減効果を行動学的、組織化学的に示した。加えて、運動による酸化ストレスの抑制と抗酸化作用の増加の関連も示され、その作用機序の一端を明らかにした。これらの結果は、脳梗塞の予防として推奨されている運動の効果を科学的に検討し、予防医療分野における理学療法のさらなる発展に寄与するものと考える。

DOI： 10.14900/cjpt.2012.0.48101465.0

がんモデルマウス・ラットライブラリ　第7回　中皮腫モデル

CD146 and IMP3 predict prognosis of asbestos-induced rat mesothelioma. 査読有り

Okazaki Y, Nagai H, Chew SH, Jiang L, Funahashi S, Tsujimura T, Toyokuni S

Cnacer Sci 104 巻頁： 989-995 2013年

豊國伸哉

細胞工学 32 巻頁： 220 - 224 2013年

The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma. 査読有り国際誌

Elshazley M, Sato M, Hase T, Yamashita R, Yoshida K, Toyokuni S, Ishiguro F, Osada H, Sekido Y, Yokoi K, Usami N, Shames DS, Kondo M, Gazdar AF, Minna JD, Hasegawa Y

International journal of cancer 131 巻 ( 12 ) 頁： 2820 - 31 2012年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Cancer

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT-5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT-5A but not in ACC-MESO-1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in two MPM cell lines (ACC-MESO-1 and H290) but not in MeT-5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21WAF1/CIP1 and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC-MESO-1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM. Copyright © 2012 UICC.

DOI： 10.1002/ijc.27598

YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes 査読有り国際誌

Mizuno, T; Murakami, H; Fujii, M; Ishiguro, F; Tanaka, I; Kondo, Y; Akatsuka, S; Toyokuni, S; Yokoi, K; Osada, H; Sekido, Y

ONCOGENE 31 巻 ( 49 ) 頁： 5117 - 5122 2012年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Oncogene

Malignant mesothelioma (MM) shows frequent inactivation of the neurofibromatosis type 2 (NF2)-tumor-suppressor gene. Recent studies have documented that the Hippo signaling pathway, a downstream cascade of Merlin (a product of NF2), has a key role in organ size control and carcinogenesis by regulating cell proliferation and apoptosis. We previously reported that MMs show overexpression of Yes-associated protein (YAP) transcriptional coactivator, the main downstream effector of the Hippo signaling pathway, which results from the inactivation of NF2, LATS2 and/or SAV1 genes (the latter two encoding core components of the mammalian Hippo pathway) or amplification of YAP itself. However, the detailed roles of YAP remain unclear, especially the target genes of YAP that enhance MM cell growth and survival. Here, we demonstrated that YAP-knockdown inhibited cell motility, invasion and anchorage-independent growth as well as cell proliferation of MM cells in vitro. We analyzed genes commonly regulated by YAP in three MM cell lines with constitutive YAP-activation, and found that the major subsets of YAP-upregulating genes encode cell cycle regulators. Among them, YAP directly induced the transcription of CCND1 and FOXM1, in cooperation with TEAD transcription factor. We also found that knockdown of CCND1 and FOXM1 suppressed MM cell proliferation, although the inhibitory effects were less evident than those of YAP knockdown. These results indicate that constitutive YAP activation in MM cells promotes cell cycle progression giving more aggressive phenotypes to MM cells. © 2012 Macmillan Publishers Limited All rights reserved.

DOI： 10.1038/onc.2012.5

Distinct affinity of nuclear proteins to the surface of chrysotile and crocidolite. 査読有り国際誌

Kubo Y, Takenaka H, Nagai H, Toyokuni S

Journal of clinical biochemistry and nutrition 51 巻 ( 3 ) 頁： 221 - 6 2012年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

The inhalation of asbestos is a risk factor for the development of malignant mesothelioma and lung cancer. Based on the broad surface area of asbestos fibers and their ability to enter the cytoplasm and nuclei of cells, it was hypothesized that proteins that adsorb onto the fiber surface play a role in the cytotoxicity and carcinogenesis of asbestos fibers. However, little is known about which proteins adsorb onto asbestos. Previously, we systematically identified asbestos-interacting proteins and classified them into eight sub-categories: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. Here, we report an adsorption profile of proteins for the three commercially used asbestos compounds: chrysotile, crocidolite and amosite. We quantified the amounts of adsorbed proteins by analyzing the silver-stained gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis with ImageJ software, using the bands for amosite as a standard. We found that histones were most adsorptive to crocidolite and that chromatin-binding proteins were most adsorptive to chrysotile. The results suggest that chrysotile and crocidolite directly interact with chromatin structure through different mechanisms. Furthermore, RNA-binding proteins preferably interacted with chrysotile, suggesting that chrysotile may interfere with transcription and translation. Our results provide novel evidence demonstrating that the specific molecular interactions leading to carcinogenesis are different between chrysotile and crocidolite. ©2012 JCBN.

DOI： 10.3164/jcbn.12-39

Iron overload signature in chrysotile-induced malignant mesothelioma

Jiang L., Akatsuka S., Nagai H., Chew S.H., Ohara H., Okazaki Y., Yamashita Y., Yoshikawa Y., Yasui H., Ikuta K., Sasaki K., Kohgo Y., Hirano S., Shinohara Y., Kohyama N., Takahashi T., Toyokuni S.

Journal of Pathology 228 巻 ( 3 ) 頁： 366 - 377 2012年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Pathology

Exposure to asbestos is a risk for malignant mesothelioma (MM) in humans. Among the commercially used types of asbestos (chrysotile, crocidolite, and amosite), the carcinogenicity of chrysotile is not fully appreciated. Here, we show that all three asbestos types similarly induced MM in the rat peritoneal cavity and that chrysotile caused the earliest mesothelioma development with a high fraction of sarcomatoid histology. The pathogenesis of chrysotile-induced mesothelial carcinogenesis was closely associated with iron overload: repeated administration of an iron chelator, nitrilotriacetic acid, which promotes the Fenton reaction, significantly reduced the period required for carcinogenesis; massive iron deposition was found in the peritoneal organs with high serum ferritin; and homozygous deletion of the CDKN2A/2B/ARF tumour suppressor genes, the most frequent genomic alteration in human MM and in iron-induced rodent carcinogenesis, was observed in 92.6% of the cases studied with array-based comparative genomic hybridization. The induced rat MM cells revealed high expression of mesoderm-specific transcription factors, Dlx5 and Hand1, and showed an iron regulatory profile of active iron uptake and utilization. These data indicate that chrysotile is a strong carcinogen when exposed to mesothelia, acting through the induction of local iron overload. Therefore, an intervention to remove local excess iron might be a strategy to prevent MM after asbestos exposure. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DOI： 10.1002/path.4075

Asbestos surface provides a niche for oxidative modification: A novel role of free radicals in carcinogenesis

Nagai, H; Okazaki, Y; Jiang, L; Akatsuka, S; Yamashita, Y; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE 53 巻頁： S51 - S52 2012年9月

Bovine lactoferrin ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats. 査読有り

Okazaki Y, Kono I, Kuriki T, Funahashi S, Fushimi S, Iqbal M, Okada S, Toyokuni S

Journal of clinical biochemistry and nutrition 51 巻 ( 2 ) 頁： 84 - 90 2012年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：一般社団法人　日本酸化ストレス学会

Milk provides a well-balanced source of amino acids and other ingredients. One of the functional ingredients in milk is lactoferrin (LF). LF presents a wide variety of bioactivities and functions as a radical scavenger in models using iron-ascorbate complexes and asbestos. Human clinical trials of oral LF administration for the prevention of colon polyps have been successful and demonstrated that dietary compounds exhibit direct interactions. However, antioxidative properties of LF in distant organs require further investigation. To study the antioxidant property of LF, we employed bovine lactoferrin (bLF) using the rat model of ferric nitrilotriacetate (Fe-NTA)-induced renal tubular oxidative injury. We fed rats with bLF (0.05%, w/w) in basal chow for 4 weeks and sacrificed them after Fe-NTA treatment. After intraperitoneal administration of 9.0 mg iron/kg Fe-NTA for 4 and 24 h, bLF pretreatment suppressed elevation of serum creatinine and blood urea nitrogen levels. In addition, we observed protective effects against renal oxidative tubular damage and maintenance of antioxidant enzyme activities in the bLF-pretreated group. We thus demonstrated the antioxidative effect of bLF against Fe-NTA-induced renal oxidative injury. These results suggest that LF intake is useful for the prevention of renal tubular oxidative damage mediated by iron. ©2012 JCBN.

DOI： 10.3164/jcbn.11-100

Iron overload signature in chrysotile-induced malignant mesothelioma. 査読有り

Jiang L, Akatsuka S, Nagai H, Chew SH, Ohara H, Okazaki Y, Yamashita Y, Yoshikawa Y, Yasui H, Ikuta K, Sasaki K, Kohgo Y, Hirano S, Shinohara Y, Kohyama N, Takahashi T, Toyokuni S

J Pathol 228 巻頁： 366-377 2012年8月

Differences and similarities between carbon nanotubes and asbestos fibers during mesothelial carcinogenesis: shedding light on fiber entry mechanism. 査読有り国際誌 Open Access

Nagai H, Toyokuni S

Cancer science 103 巻 ( 8 ) 頁： 1378 - 90 2012年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

The emergence of nanotechnology represents an important milestone, as it opens the way to a broad spectrum of applications for nanomaterials in the fields of engineering, industry and medicine. One example of nanomaterials that have the potential for widespread use is carbon nanotubes, which have a tubular structure made of graphene sheets. However, there have been concerns that they may pose a potential health risk due to their similarities to asbestos, namely their high biopersistence and needle-like structure. We recently found that despite these similarities, carbon nanotubes and asbestos differ in certain aspects, such as their mechanism of entry into mesothelial cells. In the study, we showed that non-functionalized, multi-walled carbon nanotubes enter mesothelial cells by directly piercing through the cell membrane in a diameter- and rigidity-dependent manner, whereas asbestos mainly enters these cells through the process of endocytosis, which is independent of fiber diameter. In this review, we discuss the key differences, as well as similarities, between asbestos fibers and carbon nanotubes. We also summarize previous reports regarding the mechanism of carbon nanotube entry into non-phagocytic cells. As the entry of fibers into mesothelial cells is a crucial step in mesothelial carcinogenesis, we believe that a comprehensive study on the differences by which carbon nanotubes and asbestos fibers enter into non-phagocytic cells will provide important clues for the safer manufacture of carbon nanotubes through strict regulation on fiber characteristics, such as diameter, surface properties, length and rigidity. © 2012 Japanese Cancer Association.

DOI： 10.1111/j.1349-7006.2012.02326.x

Mitochondria and free radical studies on health, disease and pollution 招待有り国際誌

Majima, HJ; Toyokuni, S

FREE RADICAL RESEARCH 46 巻 ( 8 ) 頁： 925 - 6 2012年8月

The ferroimmunomodulatory role of ectopic endometriotic stromal cells in ovarian endometriosis. 査読有り国際誌

Kobayashi H, Yamashita Y, Iwase A, Yoshikawa Y, Yasui H, Kawai Y, Uchida K, Uno N, Akatsuka S, Takahashi T, Kikkawa F, Toyokuni S

Fertility and sterility 98 巻 ( 2 ) 頁： 415 - 22 2012年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Fertility and Sterility

Objective: To understand the role of ectopic endometriotic stromal cells in ovarian endometriosis (OEM) and the associated risks for infertility and carcinogenesis. Design: Analyses of secreted proteins and gene expression using immortalized eutopic/ectopic endometrial(-otic) stromal cells from OEM. Setting: University. Patient(s): Women with and without OEM. Intervention(s): Samples of endometrial(-otic) tissue from women with or without OEM. Main Outcome Measure(s): Immunohistochemical analysis of oxidative stress in OEM, gene expression profiles, and the identification of secreted proteins by mass spectrometry in immortalized endometrial(-otic) stromal cells. Result(s): 4-Hydroxy-2-nonenal-modified proteins and carboxymethyllysine were abundant in the stroma, rather than epithelia, of OEM patients, indicating the presence of oxidative stress. Immortalized ectopic endometriotic stromal cells exhibited high IRP1/IRP2/HIF-1β expression and contained lower amounts of iron and copper than their eutopic counterparts. Expression profiles, in combination with protein identification, revealed that complement component 3 (C3) and pentraxin-3 (PTX3) are the major proteins secreted from immortalized ectopic endometriotic stromal cells. Complement-3/PTX3 promoted the secretion of various cytokines by THP1 macrophage cells and thus supported M1 differentiation. Conclusion(s): Immortalized ectopic endometriotic stromal cells in OEM predominantly secrete C3 and PTX3 and exhibit a differential regulation of iron metabolism. Copyright © 2012 American Society for Reproductive Medicine, Published by Elsevier Inc.

DOI： 10.1016/j.fertnstert.2012.04.047

Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells. 査読有り国際誌

Horio M, Sato M, Takeyama Y, Elshazley M, Yamashita R, Hase T, Yoshida K, Usami N, Yokoi K, Sekido Y, Kondo M, Toyokuni S, Gazdar AF, Minna JD, Hasegawa Y

Annals of surgical oncology 19 Suppl 3 巻 ( Suppl 3 ) 頁： S634-45 - S645 2012年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Annals of Surgical Oncology

Background: The role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear. Methods: The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference-mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity. Results: Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent. Conclusions: RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown. © 2011 Society of Surgical Oncology.

DOI： 10.1245/s10434-011-2142-0

In vitro transformation of mouse testis cells by oncogene transfection. 査読有り

Morimoto H, Lee J, Tanaka T, Ishii K, Toyokuin S, Kanatsu-Shinohara M and Shinohara T.

Biol Reprod 86 巻 ( 5 ) 頁： 1-11 2012年5月

FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation. 査読有り国際誌

Ishii K, Kanatsu-Shinohara M, Toyokuni S, Shinohara T

Development (Cambridge, England) 139 巻 ( 10 ) 頁： 1734 - 43 2012年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Development

Fibroblast growth factor 2 (FGF2) and glial cell line-derived neurotrophic factor (GDNF) are required to recapitulate spermatogonial stem cell (SSC) self-renewal in vitro. Although studies have revealed the role of the GDNF signaling pathway in SSCs, little is known about how FGF2 is involved. In the present study, we assessed the role of the FGF2 signaling pathway using a mouse germline stem (GS) cell culture system that allows in vitro expansion of SSCs. Adding GDNF or FGF2 induced phosphorylation of MAPK1/3, and adding the MAP2K1 inhibitor PD0325091 reduced GS cell proliferation and MAPK1/3 phosphorylation. Moreover, GS cells transfected with an activated form of Map2k1 not only upregulated Etv5 and Bcl6b gene expression, but also proliferated in an FGF2-independent manner, suggesting that they act downstream of MAP2K1 signaling to drive SSC self-renewal. Although GS cells transfected with Map2k1, Etv5 or Bcl6b showed normal spermatogonial markers, transplanting GS cells expressing Bcl6b into infertile mouse testes resulted in the formation of a germ cell tumor, suggesting that excessive self-renewal signals causes tumorigenic conversion. These results show that FGF2 depends on MAP2K1 signaling to drive SSC self-renewal via upregulation of the Etv5 and Bcl6b genes. © 2012. Published by The Company of Biologists Ltd.

DOI： 10.1242/dev.076539

In vitro transformation of mouse testis cells by oncogene transfection. 査読有り国際誌

Morimoto H, Lee J, Tanaka T, Ishii K, Toyokuni S, Kanatsu-Shinohara M, Shinohara T

Biology of reproduction 86 巻 ( 5 ) 頁： 148, 1 - 11 2012年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming. © 2012 by the Society for the Study of Reproduction, Inc.

DOI： 10.1095/biolreprod.111.095307

過剰鉄と活性酸素を介した発がん機構と予防・治療への展開

豊國伸哉

臨床血液 53 巻 ( 4 ) 頁： 401 - 408 2012年4月

過剰鉄と活性酸素を介した発がん機構と予防・治療への展開国際誌

豊國伸哉

臨床血液 53 巻 ( 4 ) 頁： 401 - 408 2012年4月

Genome-wide assessment of oxidatively generated DNA damage. 査読有り国際誌

Akatsuka S, Toyokuni S

Free radical research 46 巻 ( 4 ) 頁： 523 - 30 2012年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

In the tide of science nouveau after the completion of genome projects of various species, there appeared a movement to understand an organism as a system rather than the sum of cells directed for certain functions. With the advent and spread of microarray techniques, systematic and comprehensive genome-wide approaches have become reasonably possible and more required on the investigation of DNA damage and the subsequent repair. The immunoprecipitation- based technique combined with high-density microarrays or next-generation sequencing is one of the promising methods to provide access to such novel research strategies. Oxygen is necessary for most of the life on earth for electron transport. However, reactive oxygen species are inevitably generated, giving rise to steady-state levels of DNA damage in the genome, that may cause mutations leading to cancer, ageing and degenerative diseases. Previously, we showed that there are many factors involved in the genomic distribution of oxidatively generated DNA damage including chromosome territory, and proposed this sort of research area as oxygenomics. Recently, RNA is also recognized as a target of this kind of modification. © 2012 Informa UK, Ltd.

DOI： 10.3109/10715762.2011.633212

[Carcinogenic mechanisms through excess iron and reactive oxygen species and its application to cancer prevention and therapy].

Toyokuni S

[Rinsho ketsueki] The Japanese journal of clinical hematology 53 巻 ( 4 ) 頁： 401 - 8 2012年4月

[Carcinogenic mechanisms through excess iron and reactive oxygen species and its application to cancer prevention and therapy].

Toyokuni S

[Rinsho ketsueki] The Japanese journal of clinical hematology 53 巻 ( 4 ) 頁： 401 - 8 2012年4月

Age- and sun exposure-dependent differences in 8-hydroxy-2′-deoxyguanosine and Nε-(carboxymethyl)lysine in human epidermis 査読有り国際誌

Kawada, A; Date, A; Toyokuni, S

JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY 66 巻 ( 4 ) 頁： AB174 - AB174 2012年4月

[Carcinogenic mechanisms through excess iron and reactive oxygen species and its application to cancer prevention and therapy]. 査読有り国際誌

Toyokuni S.

[Rinshō ketsueki] The Japanese journal of clinical hematology 53 巻 ( 4 ) 頁： 401 - 408 2012年4月

多層カーボンナノチューブの直径と剛性は、中皮細胞傷害と中皮腫形成に重要な因子である

永井裕崇, 岡崎泰昌, 周珊瑚, 三澤伸明, 山下依子, 赤塚慎也, 石原敏和, 山下享子, 吉川豊, 安井裕之, 蒋麗, 高橋隆, 市原学, 宮田耕充, 篠原久典, 豊國伸哉

日本病理学会会誌 101 巻 ( 1 ) 頁： 280 - 280 2012年3月

Array CGH Analysis Reveals Amplification of Met and AKT2 in Clear Cell Carcinoma of the Ovary 査読有り国際誌

Yamashita, Y; Akatsuka, S; Yatabe, Y; Toyokuni, S

LABORATORY INVESTIGATION 92 巻頁： 302A - 302A 2012年2月

Array CGH Analysis Reveals Amplification of Met and AKT2 in Clear Cell Carcinoma of the Ovary 査読有り国際誌

Yamashita, Y; Akatsuka, S; Yatabe, Y; Toyokuni, S

MODERN PATHOLOGY 25 巻頁： 302A - 302A 2012年2月

FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b genes through MAP2K1 activation. 査読有り

Ishii K, Kanatsu-Shinohara M, Toyokuni S and Shinohara T.

Development 139 巻頁： 1734-1743 2012年

Differences and similarities between carbon nanotubes and asbestos fibers during mesothelial carcinogenesis. 査読有り

Nagai H and Toyokuni S.

Cancer Sci 103 巻頁： 1378-1390 2012年

The ferroimmunomodulatory role of ectopic endometriotic stromal cells in ovarian endometriosis. 査読有り

Kobayashi H, Yamashita Y, Iwase A, Yoshikawa Y, Yasui H, Kawai Y, Uchida K, Uno N, Akatsuka S, Takahashi T, Kikkawa F, Toyokuni S.

Fertil Steril 98 巻頁： 415-422 2012年

Mitochondria and free radical studies on health, disease and pollusion (editorial). 招待有り

Majima HJ and Toyokuni S.

Free Radic Res 46 巻頁： 925-926 2012年

Distinct affinity of nuclear proteins to the surface of chrysotile and crocidolite. 招待有り査読有り

Kubo Y, Takenaka H, Nagai H and Toyokuni S.

J Clin Biochem Nutr 51 巻頁： 221-226 2012年

The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma. 査読有り

Elshazley M, Sato M, Hase T, Yamashita R, Yoshida K, Toyokuni S, Ishiguro F, Osada H, Sekido Y, Yokoi K, Usami N, Shames DS, Kondo M, Gazdar AF, Minna JD and Hasegawa Y.

INt J Cancer 131 巻頁： 2820-2831 2012年

YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle promoting genes. 査読有り

Mizuno T, Murakami H, Fujii M, Ishiguro F, Tanaka I, Kondo Y, Akatsuka S, Toyokuni S and Sekido Y.

Oncogene 31 巻頁： 5117-5122 2012年

Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothalioma cells. 査読有り

Horio M, Sato M, Takayama Y, Elshazley M, Yamashita R, Hase T, Yoshida K, Usami N, Yokoi K, Sekido Y, Kondo M, Toyokuni S, Gazdar AF, Minna JD and Hasegawa Y.

Ann Surg Oncol 19 巻 ( Suppl 3 ) 頁： 634-645 2012年

Genome-wide assessment of oxidatively generated DNA damage. 査読有り

Akatsuka S and Toyokuni S.

Free Radic Res 46 巻頁： 523-530 2012年

Bovine lactoferrin ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats. 査読有り

Okazaki Y, Kono H, Kuriki R, Funahashi S, Fushimi S, Iqbal M, Okada S and Toyokuni S.

J Clin Biochem Nutr 51 巻頁： 84-90 2012年

Differences and similarities between carbon nanotubes and asbestos fibers during mesothelial carcinogenesis. 査読有り

Nagai H, Toyokuni S

Cancer Sci 103 巻頁： 1378-1390 2012年

FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b genes through MAP2K1 activation. 査読有り

Ishii K, Kanatsu-Shinohara M, Toyokuni S, Shinohara T

Development 139 巻頁： 1734-1743 2012年

Fenton reaction induced cancer in wild type rats recapitulates genomic alterations observed in human cancer. 査読有り国際誌

Akatsuka S, Yamashita Y, Ohara H, Liu YT, Izumiya M, Abe K, Ochiai M, Jiang L, Nagai H, Okazaki Y, Murakami H, Sekido Y, Arai E, Kanai Y, Hino O, Takahashi T, Nakagama H, Toyokuni S

PloS one 7 巻 ( 8 ) 頁： e43403 2012年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis. © 2012 Akatsuka et al.

DOI： 10.1371/journal.pone.0043403

過剰鉄と活性酸素を介した発がん機構と予防・治療への展開

豊國伸哉

臨床血液 53 巻 ( 4 ) 頁： 401 - 408 2012年

酸化ストレスによるゲノム変化と発がん

赤塚慎也, 豊國伸哉

実験医学 vol.30 巻頁： 2779 - 2785 2012年

Asbestos surface provides a niche for oxidative modification

NAGAI Hirotaka, ISHIHARA Toshikazu, LEE Wen-Hua, OHARA Hiroki, OKAZAKI Yasumasa, OKAWA Katsuya, TOYOKUNI Shinya

Cancer science 102 巻 ( 12 ) 頁： 2118 - 2125 2011年12月

Diameter and rigidity of multiwalled carbon nanotubes are critical factors in mesothelial injury and carcinogenesis. 査読有り国際誌

Proceedings of the National Academy of Sciences of the United States of America 108 巻 ( 49 ) 頁： E1330-8 - 8 2011年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Multiwalled carbon nanotubes (MWCNTs) have the potential for widespread applications in engineering and materials science. However, because of their needle-like shape and high durability, concerns have been raised that MWCNTs may induce asbestos-like pathogenicity. Although recent studies have demonstrated that MWCNTs induce various types of reactivities, the physicochemical features of MWCNTs that determine their cytotoxicity and carcinogenicity in mesothelial cells remain unclear. Here, we showed that the deleterious effects of nonfunctionalized MWCNTs on human mesothelial cells were associated with their diameter-dependent piercing of the cell membrane. Thin MWCNTs (diameter ∼ 50 nm) with high crystallinity showed mesothelial cell membrane piercing and cytotoxicity in vitro and subsequent inflammogenicity and mesotheliomagenicity in vivo. In contrast, thick (diameter ∼ 150 nm) or tangled (diameter ∼ 2-20 nm) MWCNTs were less toxic, inflammogenic, and carcinogenic. Thin and thick MWCNTs similarly affected macrophages. Mesotheliomas induced by MWCNTs shared homozygous deletion of Cdkn2a/2b tumor suppressor genes, similar to mesotheliomas induced by asbestos. Thus, we propose that different degrees of direct mesothelial injury by thin and thick MWCNTs are responsible for the extent of inflammogenicity and carcinogenicity. This work suggests that control of the diameter of MWCNTs could reduce the potential hazard to human health.

DOI： 10.1073/pnas.1110013108

Asbestos surface provides a niche for oxidative modification. 査読有り国際誌

Nagai H, Ishihara T, Lee WH, Ohara H, Okazaki Y, Okawa K, Toyokuni S

Cancer science 102 巻 ( 12 ) 頁： 2118 - 25 2011年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Asbestos is a potent carcinogen associated with increased risks of malignant mesothelioma and lung cancer in humans. Although the mechanism of carcinogenesis remains elusive, the physicochemical characteristics of asbestos play a role in the progression of asbestos-induced diseases. Among these characteristics, a high capacity to adsorb and accommodate biomolecules on its abundant surface area has been linked to cellular and genetic toxicity. Several previous studies identified asbestos-interacting proteins. Here, with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry, we systematically identified proteins from various lysates that adsorbed to the surface of commercially used asbestos and classified them into the following groups: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. The surfaces of crocidolite and amosite, two iron-rich types of asbestos, caused more protein scissions and oxidative modifications than that of chrysotile by in situ-generated 4-hydroxy-2-nonenal. In contrast, we confirmed the intense hemolytic activity of chrysotile and found that hemoglobin attached to chrysotile, but not silica, can work as a catalyst to induce oxidative DNA damage. This process generates 8-hydroxy-2′-deoxyguanosine and thus corroborates the involvement of iron in the carcinogenicity of chrysotile. This evidence demonstrates that all three types of asbestos adsorb DNA and specific proteins, providing a niche for oxidative modification via catalytic iron. Therefore, considering the affinity of asbestos for histones/DNA and the internalization of asbestos into mesothelial cells, our results suggest a novel hypothetical mechanism causing genetic alterations during asbestos-induced carcinogenesis. © 2011 Japanese Cancer Association.

DOI： 10.1111/j.1349-7006.2011.02087.x

miR-375 is activated by ASH1 and inhibits YAP1 in a lineage-dependent manner in lung cancer. 査読有り国際誌

Nishikawa E, Osada H, Okazaki Y, Arima C, Tomida S, Tatematsu Y, Taguchi A, Shimada Y, Yanagisawa K, Yatabe Y, Toyokuni S, Sekido Y, Takahashi T

Cancer research 71 巻 ( 19 ) 頁： 6165 - 73 2011年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Research

Lung cancers with neuroendocrine (NE) features are often very aggressive but the underlying molecular mechanisms remain elusive. The transcription factor ASH1/ASCL1 is a master regulator of pulmonary NE cell development that is involved in the pathogenesis of lung cancers with NE features (NE-lung cancers). Here we report the definition of the microRNA miR-375 as a key downstream effector of ASH1 function in NE-lung cancer cells. miR-375 was markedly induced by ASH1 in lung cancer cells where it was sufficient to induce NE differentiation. miR-375 upregulation was a prerequisite for ASH1-mediated induction of NE features. The transcriptional coactivator YAP1 was determined to be a direct target of miR-375. YAP1 showed a negative correlation with miR-375 in a panel of lung cancer cell lines and growth inhibitory activities in NE-lung cancer cells. Our results elucidate an ASH1 effector axis in NE-lung cancers that is functionally pivotal in controlling NE features and the alleviation from YAP1-mediated growth inhibition. ©2011 AACR.

DOI： 10.1158/0008-5472.CAN-11-1020

Age-and sun exposure-dependent differences in 8-hydroxy-2′-deoxyguanosine and Nε-(carboxymethyl)lysine in human epidermis 査読有り国際誌

Toyokuni, S; Hirao, A; Wada, T; Nagai, R; Date, A; Yoshii, T; Akatsuka, S; Yamashita, Y; Kawada, A

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 49 巻 ( 2 ) 頁： 121 - 124 2011年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

Aging and exposure to sunlight are two major factors in the deterioration of skin function. In this study, thirty-six fixed human skin samples from sun-exposed and unexposed areas from young and old individuals were used to evaluate the localization of oxidative stress according to levels and distribution of 8-hydroxy-2′-deoxyguanosine and N ε- (carboxymethyl)lysine in samples using immunohistochemistry. In the epidermis of the young, negligible amounts of 8-hydroxy-2′-deoxyguanosine and N ε-(carboxymethyl)lysine were detected in unexposed areas, whereas nuclear 8-hydroxy-2′-deoxyguanosine and cytoplasmic N ε-(carboxymethyl)lysine were increased in the lower epidermis in sun-exposed areas. In contrast, the aged presented prominent nuclear 8-hydroxy-2′-deoxyguanosine and nuclear N ε-(carboxymethyl) lysine in the epidermis of unexposed areas, concomitant with dermal increase in N ε-(carboxymethyl)lysine. However, the immunostaining of 8-hydroxy-2′-deoxyguanosine and N ε-(carboxymethyl)lysine revealed a decrease in the epidermis of sun-exposed areas in the aged. These results suggest an age-dependent difference in the adaptation and protective mechanisms of the epidermis against sunlight-associated oxidative stress, thus necessitating distinct standards for evaluation in each age group. Further investigation is warranted to elucidate underlying molecular mechanisms. ©2011 JCBN.

DOI： 10.3164/jcbn.11-05

BMAL1 depletion represses growth of mesothelioma through induction of mitotic catastrophe 査読有り国際共著国際誌

Elshazley, MHKA; Hasegawa, Y; Sato, M; Hase, T; Yoshida, K; Toyokuni, S; Seikido, Y; Yokoi, K; Usami, N; Kondo, M

CANCER RESEARCH 71 巻 2011年9月

MET Amplification is a Molecular Hallmark in Endometriosis-associated Ovarian Clear Cell Carcinoma and Correlates With Worse Prognosis 査読有り国際誌

Yamashita, Y; Yatabe, Y; Akatsuka, S; Kajiyama, H; Kikkawa, F; Takahashi, T; Toyokuni, S

EUROPEAN JOURNAL OF CANCER 47 巻頁： S529 - S529 2011年9月

Elucidation of asbestos-induced mesothelial carcinogenesis toward its prevention 国際誌 Open Access

Jiang L., Toyokuni S.

Genes and Environment 33 巻 ( 1 ) 頁： 4 - 9 2011年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Genes and Environment

Human exposure to asbestos fibers has been associated with diffuse malignant mesothelioma (DMM) in the pleural and abdominal cavity. Despite advancements in the molecular analyses of human cases of DMM and animal models, the understanding of the carcinogenic mechanisms remains still limited. There are basically three hypotheses regarding the pathogenesis of asbestos-induced DMM, which may be integrated as follows; (1) the "oxidative stress theory" is based on the fact that phagocytic cells that engulf asbestos fibers produce large amounts of reactive oxygen species (ROS) due to their inability to digest the fibers, and that iron contained in crocidolite and amosite fibers works as a catalyst for the generation of ROS, (2) the "chromosome tangling theory" postulates that asbestos fibers impair the equivalent distribution of chromosomes during mitosis, and (3) the "theory of adsorbing many specific proteins as well as carcinogenic molecules" states that asbestos fibers in vivo concentrate specific proteins or chemicals including the components of cigarette smoke and radioactive chemical element. Recent studies suggest that local iron overload is a key event. Elucidation of the major mechanisms underlying DMM would be helpful for the development of strategies to prevent DMM generation in people who have been exposed to asbestos. © The Japanese Environmental Mutagen Society.

DOI： 10.3123/jemsge.33.4

[Archives of "comprehensive approach on asbestos-related diseases" supported by the "special coordination funds for promoting science and technology (H18-1-3-3-1)"-- overview of group research project, care and specimen registration, cellular characteristics of mesothelioma and immunological effects of asbestos]. 査読有り Open Access

Iron as a target of chemoprevention for longevity in humans. 査読有り国際誌

Toyokuni S

Free radical research 45 巻 ( 8 ) 頁： 906 - 17 2011年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Iron is universally abundant and no life can exist without it. However, iron levels should be maintained within a narrow range. Iron deficiency causes anaemia, whereas excessive iron increases cancer risk, presumably by free radical generation. Several pathological conditions such as genetic haemochromatosis, chronic viral hepatitis B and C, conditions related to asbestos fibre exposure and ovarian endometriosis have been recognized as iron overload-associated conditions that also increase human cancer risks. Iron's carcinogenicity has been documented in animal experiments. Surprisingly, these studies have revealed that the homozygous deletion of CDKN2A/2B is a major hallmark of iron-induced carcinogenesis. Recently, the hormonal regulation of iron metabolism has been elucidated. A commonly hypothesized mechanism may be the lack of any iron disposal pathway other than for bleeding and a mechanism of iron re-uptake as catechol chelate has been discovered. Iron overload in neurons via the ferroportin block may play a role in Alzheimer's disease. Furthermore, a recent epidemiological study reported that iron reduction by phlebotomy was associated with decreased cancer risks in a general population. Given that the required amounts of iron decrease during ageing, the fine control of body iron stores would be a wise strategy for chemoprevention of several diseases. © 2011 Informa UK, Ltd.

DOI： 10.3109/10715762.2011.564170

Dietary Fish Oil Promotes Colonic Apoptosis and Mitochondrial Proton Leak in Oxidatively Stressed Mice 査読有り国際誌

Fan, YY; Ran, QT; Toyokuni, S; Okazaki, Y; Callaway, ES; Lupton, JR; Chapkin, RS

CANCER PREVENTION RESEARCH 4 巻 ( 8 ) 頁： 1267 - 74 2011年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Prevention Research

An alteration of mitochondrial function can result in disruption of redox homeostasis and is associated with abnormal cancer cell growth. Manganese superoxide dismutase (SOD2) and glutathione peroxidase 4 (Gpx4) are two of the most important antioxidant defense enzymes that protect cells against oxidative stress. We had previously shown that n-3 polyunsaturated fatty acids (PUFA) promote colonocyte apoptosis, a marker of colon cancer risk, in part by enhancing phospholipid oxidation. To elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, we fed heterozygous SOD2Het, Gpx4Het, and transgenic Gpx4Tg mice diets containing either 15% corn oil by weight (CO, enriched in n-6 PUFA) or 3.5% CO + 11.5% fish oil (FO, enriched in n-3 PUFA) for 4 weeks. Our data showed that (i) genetic predeposition to oxidative stress facilitates apoptosis in the mouse colon (Gpx4Het > SOD2Het > Wt > Gpx4Tg), (ii) dietary n-3 PUFA have an additive effect on the induction of apoptosis in Gpx4Het and SOD2Het mice; and (iii) dietary n-3 PUFA reverse the phenotype in oxidatively protected Gpx4Tg mice by elevating apoptosis to a level observed in wild-type (Wt; control) animals. Complimentary experiments examining colonic mitochondrial bioenergetic profiles indicate that FO-fed mice exhibit a significantly (P < 0.05) increased respiration-induced proton leak relative to control CO treatment. This finding was consistent with a loss of membrane potential in response to chronic oxidative stress and supports the contention that n-3 PUFA alter mitochondrial metabolic activity, thereby enhancing apoptosis and reducing colon cancer risk. ©2011 AACR.

DOI： 10.1158/1940-6207.CAPR-10-0368

The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication 査読有り国際誌

Isomura, H; Stinski, MF; Murata, T; Yamashita, Y; Kanda, T; Toyokuni, S; Tsurumi, T

JOURNAL OF VIROLOGY 85 巻 ( 13 ) 頁： 6629 - 6644 2011年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Virology

The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed. © 2011, American Society for Microbiology.

DOI： 10.1128/JVI.00384-11

YAP TRANSCRIPTION COACTIVATOR INDUCES MALIGNANT MESOTHELIOMA CELL PROLIFERATION BY UP-REGULATING CELL CYCLE PROGRESSION 査読有り国際誌

Mizuno, T; Murakami, H; Fujii, M; Ishiguro, F; Kondo, Y; Akatsuka, S; Toyokuni, S; Yokoi, K; Osada, H; Sekido, Y

JOURNAL OF THORACIC ONCOLOGY 6 巻 ( 6 ) 頁： S707 - S708 2011年6月

Otsuki T., Nakano T., Hasegawa S., Okada M., Tsujimura T., Sekido Y., Toyokuni S., Nishimoto H., Fukuoka K., Tanaka F., Kumagai N., Maeda M., Nishimura Y.

Nihon eiseigaku zasshi. Japanese journal of hygiene 66 巻 ( 3 ) 頁： 543 - 552 2011年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Nihon eiseigaku zasshi. Japanese journal of hygiene

The research project entitled "Comprehensive approach on asbestos-related diseases" supported by the "Special Coordination Funds for Promoting Science and Technology (H18-1-3-3-1)" began in 2006 and was completed at the end of the Japanese fiscal year of 2010. This project included four parts; (1) malignant mesothelioma (MM) cases and specimen registration, (2) development of procedures for the early diagnosis of MM, (3) commencement of clinical investigations including multimodal approaches, and (4) basic research comprising three components; (i) cellular and molecular characterization of mesothelioma cells, (ii) immunological effects of asbestos, and (iii) elucidation of asbestos-induced carcinogenesis using animal models. In this special issue of the Japanese Journal of Hygiene, we briefly introduce the achievements of our project. The second and third parts and the third component of the fourth part are described in other manuscripts written by Professors Fukuoka, Hasegawa, and Toyokuni. In this manuscript, we introduce a brief summary of the first part "MM cases and specimen registration", the first component of the fourth part "Cellular and molecular characterization of mesothelioma cells" and the second component of the fourth part "Immunological effects of asbestos". In addition, a previous special issue presented by the Study Group of Fibrous and Particulate Substances (SGFPS) (chaired by Professor Otsuki, Kawasaki Medical School, Japan) for the Japanese Society of Hygiene and published in Environmental Health and Preventive Medicine Volume 13, 2008, included reviews of the aforementioned first component of the fourth part of the project. Taken together, our project led medical investigations regarding asbestos and MM progress and contributed towards the care and examination of patients with asbestos-related diseases during these five years. Further investigations are required to facilitate the development of preventive measures and the cure of asbestos-related diseases, particularly in Japan, where asbestos-related diseases are predicted to increase in the next 10 to 20 years.

DOI： 10.1265/jjh.66.543

[Mechanisms of asbestos-induced carcinogenesis]. 国際誌 Open Access

Toyokuni S, Jiang L, Hu Q, Nagai H, Okazaki Y, Akatsuka S, Yamashita Y

Nihon eiseigaku zasshi. Japanese journal of hygiene 66 巻 ( 3 ) 頁： 562 - 7 2011年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Nihon eiseigaku zasshi. Japanese journal of hygiene

Several types of fibrous stone called asbestos have been an unexpected cause of human cancer in the history. This form of mineral is considered precious in that they are heat-, friction-, and acid-resistant, are obtained easily from mines, and can be modified to any form with many industrial merits. However, it became evident that the inspiration of asbestos causes a rare cancer called malignant mesothelioma. Because of the long incubation period, the peak year for malignant mesothelioma is expected to be 2025 in Japan. Thus, it is necessary to elucidate the mechanisms of asbestos-induced mesothelial carcinogenesis. In this review, we summarize the cutting edge results of our 5-year project funded by a MEXT grant, in which local iron deposition and the characteristics of mesothelial cells are the key issues.

DOI： 10.1265/jjh.66.562

Roles of ASH1-miR-375 pathway in development of lung cancers with neuroendocrine features

Hirotaka, O; Nishikawa, E; Arima, C; Okazaki, Y; Tomida, S; Tatematsu, Y; Taguchi, A; Shimada, Y; Yanagisawa, K; Toyokuni, S; Sekido, Y; Takahashi, T

CANCER RESEARCH 71 巻 2011年4月

XCR1 expression and biased VH gene usage are distinct features of diffuse large B-cell lymphoma initially manifesting in the bone marrow. 査読有り国際誌

Yamashita Y, Kajiura D, Tang L, Hasegawa Y, Kinoshita T, Nakamura S, Akatsuka S, Toyokuni S, Mori N

American journal of clinical pathology 135 巻 ( 4 ) 頁： 556 - 64 2011年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Clinical Pathology

A total of 29 cases of diffuse large B-cell lymphoma initially manifesting in the bone marrow (BM-DLBCL) were analyzed for VH gene sequence, and expression microarray of chemokines and chemokine receptors and immunohistochemical analysis were done. Seminested polymerase chain reaction (PCR) and sequencing analyses of 18 cases revealed that the VH gene usage in 6 cases was restricted to VH3-7, in 3 cases to V H4-34, and in 2 cases to VH4-39, which were all previously reported to be autoreactive. In total, 14 of 18 VH genes were those associated with autoimmune diseases, including VH3-21, V H3-23, and VH3-48. Furthermore, cDNA microarray analysis specific for chemokine and chemokine receptors revealed that chemokine receptor XCR1 expression was significantly elevated in the BM-DLBCL cases (P < 05), which was confirmed by quantitative reverse transcriptase-PCR and immunohistochemical analysis. Expression of the chemokine receptor XCR1 and frequent usage of autoreactive VH genes seem to be distinct characteristics of BM-DLBCL. © American Society for Clinical Pathology.

DOI： 10.1309/AJCPCTDC5PY3LXBP

LATS2 is a tumor suppressor gene of malignant mesothelioma

Murakami H.

Cancer Research 71 巻 ( 3 ) 頁： 873 - 883 2011年2月

LATS2 is a tumor suppressor gene of malignant mesothelioma.

Murakami H, Mizuno T, Taniguchi T, Fujii M, Ishiguro F, Fukui T, Akatsuka S, Horio Y, Hida T, Kondo Y, Toyokuni S, Osada H, Sekido Y

Cancer research 71 巻 ( 3 ) 頁： 873 - 83 2011年2月

Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats. 査読有り国際誌

Ohara H, Akatsuka S, Nagai H, Liu YT, Jiang L, Okazaki Y, Yamashita Y, Nakamura T, Toyokuni S

Free radical research 45 巻 ( 2 ) 頁： 211 - 20 2011年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

By using a rat model of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA), this study performed genome-wide analysis to identify target genes during carcinogenesis. It screened for genes with decreased expression in RCCs, with simultaneous loss of heterozygosity, eventually to focus on the fibulin-5 (fbln5) gene. Oxidative damage via Fe-NTA markedly increased Fbln5 in the proximal tubules. RCCs presented lower levels of Fbln5. However, a fraction of RCCs presenting pulmonary metastasis revealed significantly higher levels of Fbln5 than those without metastasis, accompanied by immunopositivity of RCC cells and myofibroblast proliferation. Experiments revealed that RCC cell lines showed lower expression of fbln5 than its non-transformed counterpart NRK52E, but that fbln5 transfection to RCC cell lines changed neither proliferation nor migration/invasion. The data suggest that Fbln5 plays a role not only in the tissue repair and remodelling after renal tubular oxidative damage but also in RCC metastasis, presumably as a cytokine. © 2011 Informa UK, Ltd.

DOI： 10.3109/10715762.2010.523702

LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma 査読有り

Hideki Murakami, Tetsuya Mizuno, Tetsuo Taniguchi, Makiko Fujii, Futoshi Ishiguro, Takayuki Fukui, Shinya Akatsuka, Yoshitsugu Horio, Toyoaki Hida, Yutaka Kondo, Shinya Toyokuni, Hirotaka Osada, Yoshitaka Sekido

CANCER RESEARCH 71 巻 ( 3 ) 頁： 873 - 883 2011年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

Malignant mesothelioma (MM) is an aggressive neoplasm associated with asbestos exposure. We carried out genome-wide array-based comparative genomic hybridization analysis with 14 MM cell lines. Three cell lines showed overlapping homozygous deletion at chromosome 13q12, which harbored the LATS2 (large tumor suppressor homolog 2) gene. With 6 other MM cell lines and 25 MM tumors, we found 10 inactivating homozygous deletions or mutations of LATS2 among 45 MMs. LATS2 encodes a serine/threonine kinase, a component of the Hippo tumor-suppressive signaling pathway, and we transduced LATS2 in MM cells with its mutation. Transduction of LATS2 inactivated oncoprotein YAP, a transcriptional coactivator, via phosphorylation, and inhibited MM cell growth. We also analyzed LATS2 immunohistochemically and found that 13 of 45 MM tumors had low expression of LATS2. Because NF2 is genetically mutated in 40% to 50% of MM, our data indicate that Hippo pathway dysregulation is frequent in MM cells with inactivation of LATS2 or an upstream regulator of this pathway, Merlin, which is encoded by NF2. Thus, our results suggest that the inactivation of LATS2 is one of the key mechanisms for constitutive activation of YAP, which induces deregulation of MM cell proliferation. Cancer Res; 71(3); 873-83. (C)2011 AACR.

DOI： 10.1158/0008-5472.CAN-10-2164

3-Morpholinosydnonimine participates in the attenuation of neointima formation via inhibition of annexin A2-mediated vascular smooth muscle cell migration 国際誌

CD8+CD122+ regulatory T cells (Tregs) and CD4+ Tregs cooperatively prevent and cure CD4+ cell-induced colitis. 査読有り国際誌 Open Access

Endharti AT, Okuno Y, Shi Z, Misawa N, Toyokuni S, Ito M, Isobe K, Suzuki H

Journal of immunology (Baltimore, Md. : 1950) 186 巻 ( 1 ) 頁： 41 - 52 2011年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Immunology

We identified CD8+CD122+ regulatory T cells (Tregs) and demonstrated their importance in the maintenance of immune homeostasis and in the recovery from experimental autoimmune encephalomyelitis. In this paper, we show that CD8+CD122+ Tregs effectively prevent and cure colitis in a mouse model. In our experiments, colitis was induced in lymphocyte-deficient RAG-2-/- mice by transferring CD4 +CD45RBhigh cells that were excluded with CD4+ Tregs. Cotransfer of CD8+CD122+ cells clearly suppressed the development of colitis, and this suppressive effect was similar to that of CD4+CD45RBlow cells that were mostly CD4+ Tregs. CD8+CD122+ cells obtained from IL-10-/- mice were unable to suppress colitis, indicating that IL-10 is an important effect-transmitting factor in the suppression of colitis. CD8 +CD122+ cells showed a suppressive effect when they were transferred 4 wk after CD4+CD45RBhigh cells, indicating the therapeutic potential of CD8+CD122+ cells. A mixture of CD8+CD122+ cells and CD4+CD45RB low cells was far more effective than single Tregs, indicating the synergistic effect of these Tregs. These overall findings demonstrate the potential role of CD8+ Tregs, and possibly together with CD4 + Tregs, in the medical care of inflammatory bowel disease patients. Copyright © 2010 by The American Association of Immunologists, Inc.

DOI： 10.4049/jimmunol.1000800

Won, KJ; Lee, P; Jung, SH; Jiang, X; Lee, CK; Lin, HY; Kang, H; Lee, HM; Kim, J; Toyokuni, S; Kim, B

PROTEOMICS 11 巻 ( 2 ) 頁： 193 - 201 2011年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proteomics

3-Morpholinosydnonimine (SIN-1) affects vascular smooth muscle cell migration and proliferation, processes essential for atherosclerosis. However, the mechanism by which SIN-1 exerts these effects has not been elucidated. We used 2-DE followed by MALDI-TOF/TOF MS to identify responses in protein expression to SIN-1 in rat aortic smooth muscle. Platelet-derived growth factor-BB increased cell migration and proliferation in rat aortic smooth muscle cells, and subsequent SIN-1 treatment inhibited it. Administration of SIN-1 in vivo attenuated neointima formation in balloon-injured rat carotid arteries. Proteomic analysis showed that glutathione peroxidase and 40S ribosomal protein S12 were differentially expressed in aortic strips exposed to SIN-1. Expression of annexin A2 was decreased by SIN-1. Platelet-derived growth factor-BB-induced cell migration was increased and inhibited in rat aortic smooth muscle cells with overexpression and knockdown of annexin A2 gene, respectively. The expression of annexin A2 was increased in vascular neointima compared with the intact control, which was inhibited by SIN-1 treatment. These results demonstrate that SIN-1 may attenuate vascular neointima formation by inhibiting annexin A2-mediated migration. Therefore, annexin A2 may be a potential target for therapeutic strategies for atherosclerosis. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/pmic.200900834

XCR1 expression and biased VH gene usage are distinct features of diffuse large B-cell lymphoma initially manifesting in the bone marrow. 査読有り

Yamashita Y, Kajiura D, Tang L, Hasegawa Y, Kinoshita T, Nakamura S, Akatsuka S, Toyokuni S and Mori N.

Am J Clin Pathol 135 巻頁： 556-564 2011年

Iron as a target of chemoprevention for longevity in humans. 査読有り

Toyokuni S.

Free Radic Res 45 巻頁： 906-917 2011年

The human cytomegalovirus gene products essential for late viral gene expression assemble into prereplication complexes before viral DNA replication. 査読有り

Isomura H, Stinski MF, Murata T, Yamashita Y, Kanda T, Toyokuni S and Tsurumi T.

J Virol 85 巻頁： 6629-6644 2011年

Dietary fish oil promotes colonic apoptosis and mitochondrial proton leak in oxidatively stressed mice. 査読有り

Fan YY, Ran Q, Toyokuni S, Okazaki Y, Callaway ES, Lupton JR, and Chapkin RS.

Cancer Prev Res 4 巻頁： 1267-1274 2011年

Age- and sun exposure-dependent differences in 8-hydroxy-2'-deoxyguanosine and Nε-(carboxymethyl)lysine in human epidermis. 査読有り

Toyokuni S, Hirao A, Wada T, Nagai R, Date A, Yoshii T, Akatsuka S, Yamashita Y, and Kawada A.

J Clin Biochem Nutr 49 巻頁： 1-4 2011年

Mysterious link between iron overload and CDKN2A/2B. 査読有り

Toyokuni S.

J 48 巻頁： 46-49 2011年

LATS2 is a tumor suppressor gene of malignancy mesothelioma. 査読有り

Murakami H, Mizuno T, Taniguchi T, Fujii M, Ishiguro F, Fukui T, Akatsuka S, Horio Y, HIda T, Kondo Y, Toyokuni S, Osada H and Sekido Y.

Cancer Res 71 巻頁： 873-883 2011年

Asbetos provides a niche for oxidative modification. 査読有り

Nagai H, Ishihara T, Lee WH, Ohara H, Okazaki Y, Okawa K and Toyokuni S.

Cancer Sci 102 巻頁： 2118-2125 2011年

miR-375 is activated by ASH1 and inhibits YAP1 in a lineage dependent manner in lung cancer. 査読有り

Nishikawa E, Osada H, Okazaki Y, Arima C, Tomida S, Tatematsu Y, Taguchi A, Shimada Y, Yanagisawa K, Yatabe Y, Toyokuni S, Sekido Y and Takahashi T

Cancer Res 71 巻頁： 6165-6173 2011年

Oxidative DNA Damage and Carcinogenesis

Jiang, L; Toyokuni, S

FREE RADICAL BIOLOGY IN DIGESTIVE DISEASES 29 巻頁： 55 - 63 2011年

Iron as a target of chemoprevention for longevity in humans. 査読有り

Toyokuni S

Free Radic Res 45 巻頁： 906-917 2011年

Mysterious link between iron overload and CDKN2A/2B. 査読有り

Toyokuni S

J 48 巻頁： 46-49 2011年

LATS2 is a tumor suppressor gene of malignant mesothelioma.

Murakami Hideki, Mizuno Tetsuya, Taniguchi Tetsuo, Fujii Makiko, Ishiguro Futoshi, Fukui Takayuki, Akatsuka Shinya, Horio Yoshitsugu, Hida Toyoaki, Kondo Yutaka, Toyokuni Shinya, Osada Hirotaka, Sekido Yoshitaka

Cancer research 71 巻 ( 3 ) 2011年

記述言語：英語掲載種別：研究論文（学術雑誌）

:Malignant mesothelioma (MM) is an aggressive neoplasm associated with asbestos exposure. We carried out genome-wide array-based comparative genomic hybridization analysis with 14 MM cell lines. Three cell lines showed overlapping homozygous deletion at chromosome 13q12, which harbored the LATS2 (large tumor suppressor homolog 2) gene. With 6 other MM cell lines and 25 MM tumors, we found 10 inactivating homozygous deletions or mutations of LATS2 among 45 MMs. LATS2 encodes a serine/threonine kinase, a component of the Hippo tumor-suppressive signaling pathway, and we transduced LATS2 in MM cells with its mutation. Transduction of LATS2 inactivated oncoprotein YAP, a transcriptional coactivator, via phosphorylation, and inhibited MM cell growth. We also analyzed LATS2 immunohistochemically and found that 13 of 45 MM tumors had low expression of LATS2. Because NF2 is genetically mutated in 40% to 50% of MM, our data indicate that Hippo pathway dysregulation is frequent in MM cells with inactivation of LATS2 or an upstream regulator of this pathway, Merlin, which is encoded by NF2. Thus, our results suggest that the inactivation of LATS2 is one of the key mechanisms for constitutive

DOI： 10.1158/0008-5472.CAN-10-2164

脳梗塞発症前の運動の効果及び酸化ストレスに着目した作用機序の検討

濱川みちる, 嶋田悠, 中島宏樹, 石田章真, 玉越敬悟, 豊國伸哉, 石田和人

理学療法学Supplement 2010 巻 ( 0 ) 頁： AcOF1030 - AcOF1030 2011年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本理学療法士協会

【目的】脳梗塞時には活性酸素やフリーラジカルが大量に産生され、酸化ストレスとして組織損傷を拡大させるため、酸化ストレスは脳梗塞を増悪させる主要な因子である。一方、健常なラットを長期間運動させると、脳内で抗酸化酵素が増加し酸化ストレスの減少をもたらすことが報告されており、運動は脳内の抗酸化作用を高めることが示されている。また、先行研究では、脳梗塞モデルラット作成前に一定期間の運動をさせると、運動していないラットに比べて、脳梗塞後の梗塞体積が減少し運動機能障害も軽減するといった脳梗塞障害軽減効果が報告されている。しかし、運動により高まる脳内の抗酸化作用が、脳梗塞時の酸化ストレスに影響を及ぼしているかどうかは不明である。そこで本研究では、脳梗塞発症前の運動の効果および作用機序を酸化ストレスに着目して検討することを目的とした。 【方法】実験動物には Wistar系雄性ラット（5週齢）を用いた。無作為に運動群と非運動群に分け、運動群は3週間トレッドミル運動（15 m / min、30分/日）を毎日行った。非運動群は走行させずにトレッドミル装置内に暴露させた（1日30分間）。3週間後、各群に小泉法による脳梗塞モデル作成手術を施行した。手術24時間後に、運動機能評価として、麻痺の重症度を評価する Neurological Deficits（ND）、歩行時のバランス能力の評価として Beam Walking（BW）、はしご上の歩行における前肢の協調運動機能を評価するLadder test、前肢の感覚運動機能の評価として Limb Placing（LP）を行った。その直後に脳を採取し、TTC染色により非梗塞半球体積に対する梗塞体積の割合を算出した。また、酸化ストレスの指標として、酸化ストレスによる細胞膜障害の指標となる 4-hydroxy-2-nonenal [4-HNE]及びDNA障害の指標となる 8-Hydroxydeoxyguanosine[8-OHdG]の免疫染色を行った。統計学的検定には、対応のない t検定と Mann-WhitneyのU検定を用い、有意水準は5％未満とした。 【説明と同意】本研究は、名古屋大学医学部保健学科動物実験委員会の承諾を得て実施した（承認番号：022-028号）。 【結果】脳梗塞手術24時間後の運動機能のうち、 ND（麻痺の重症度）と Ladder test（前肢の協調運動機能障害）は運動群の方が非運動群に比べて軽度で、有意差が認められた（p < 0.05）。一方、 LP（前肢の感覚機能）や BW（歩行時の後肢機能）は群間に有意差は認められなかった。また、梗塞体積割合は、運動群の方が非運動群よりも有意に小さく（p < 0.05）、脳梗塞発症前の運動はその後の脳梗塞による組織損傷を軽減することが示された。酸化ストレス指標のうち、過酸化脂質の指標である4-HNEの免疫染色では、非運動群に比べて運動群は陽性細胞が少ない傾向にあった。また、DNAの酸化的損傷を示す8-OHdGは、梗塞側では運動群の方が陽性細胞が少ない傾向にあったが、非梗塞側は両群とも陽性細胞は見られなかった。 【考察】本研究は、脳梗塞発症前に3週間運動を継続することが、その後の脳梗塞の障害に及ぼす効果を酸化ストレスに着目して検討した。その結果、脳梗塞前に運動を行った運動群は非運動群と比較して、脳梗塞後の梗塞体積が減少し運動機能障害が軽減され、先行研究の結果と一致した。また、脳梗塞による細胞膜及びDNAに対する酸化ストレスが運動群で減少したことから、運動によって高められる脳内の抗酸化作用は、正常時だけでなく、脳梗塞時の酸化ストレスに対しても抑制効果をもたらす可能性が示唆された。これらのことから、3週間の運動が脳梗塞後の障害軽減効果をもたらした作用機序として、脳内の抗酸化作用を高めて脳梗塞の酸化ストレスを抑制したことにより、組織損傷が軽減し、運動機能障害が改善されたと考えられる。しかし、今回の運動条件がどの程度の抗酸化作用を有しているかは確認していないため、今後検討していく必要がある。 【理学療法学研究としての意義】本研究は、健常時の運動習慣の有無が脳梗塞を発症した場合の障害程度に及ぼす影響について検討した基礎研究であり、発症前の運動習慣が脳梗塞の予後を改善する可能性を示唆している。また、今後理学療法士が活躍の場を広げ予防医療の分野に携わる際、運動を推奨することの科学的根拠となる有用な知見である。

DOI： 10.14900/cjpt.2010.0.AcOF1030.0

Diameter and rigidity of multi-walled carbon nanotubes are critical factors in mesothelial injury and carcinogenesis.

Oxidative DNA Damage and Carcinogenesis

Jiang Li, Toyokuni Shinya

FREE RADICAL BIOLOGY IN DIGESTIVE DISEASES 29 巻頁： 55 - 63 2011年

Nagai, H; Okazaki, Y; Chew, S; Misawa, N; Yamashita, Y; Akatsuka, S; Ishihara, T; Yamashita, K; Yoshikawa, Y; Yasui, H; Jiang, L; Ohara, H; Takahashi, T; Ichihara, G; Miyata, Y; Shinohara, H; Toyokuni, S

MOLECULAR BIOLOGY OF THE CELL 22 巻 2011年

Oxidative DNA damage and carcinogenesis

Jiang L., Toyokuni S.

Free Radical Biology in Digestive Diseases 29 巻頁： 55 - 63 2010年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology in Digestive Diseases

Carcinogenesis follows multi-step processes involving both genetic alteration and increased cell proliferation. Oxidative stress can occur via overproduction of reactive oxygen and nitrogen species through either endogenous or exogenous insults. Oxidative stress is always associated with inflammation, radiation, reperfusion, and iron overload. Epidemiological observations have shown that oxidative stress is one of the major pathologic mechanisms for cancer, the top-ranked cause of human mortality worldwide. During carcinogenesis, the unregulated or prolonged production of cellular oxidants has been linked to mutation through generation of oxidative DNA damage. Furthermore, signal transduction pathways are activated by reactive species, and they lead to the transcription of genes involved in cellular growth regulatory and stress protection pathways. This review examines the involvement of oxidative stress in the carcinogenic process starting from its history, and presents future perspectives.

DOI： 10.1159/000319941

Oxidative DNA damage and carcinogenesis

Jiang L., Toyokuni S.

Frontiers of Gastrointestinal Research 29 巻頁： 55 - 63 2010年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers of Gastrointestinal Research

DOI： 10.1159/0003199419

A Beverage Containing Fermented Black Soybean Ameliorates Ferric Nitrilotriacetate-Induced Renal Oxidative Damage in Rats 査読有り

Yasumasa Okazaki, Mohammad Iqbal, Norito Kawakami, Yorihiro Yamamoto, Shinya Toyokuni, Shigeru Okada

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 47 巻 ( 3 ) 頁： 198 - 207 2010年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

It is beneficial to seek scientific basis for the effects of functional foods. Natural pigments derived from plants are widely known as possible antioxidants. Black soybean contains a larger amount of anthocyanins than regular soybean. Here we studied the anti-oxidative effect of a beverage obtained via citric acid fermentation of black soybean (BBS), using a rat model of renal oxidative injury induced by a renal carcinogen, ferric nitrilotriacetate. BBS (10 ml/kg) was orally administered 30 min before ferric nitrilotriacetate treatment. Renal lipid peroxidation was significantly suppressed in the BBS-pretreated animals concomitant with decrease in 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2`-deoxyguanosine. Maintenance of renal activities of antioxidative enzymes including catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glucose-6-phosphate dehydrogenase and quinone reductase was significantly better in the BBS-pretreated rats. Elevation of serum creatinine and urea nitrogen was significantly suppressed in the BBS-pretreated rats. These data suggest that dietary intake of BBS is useful for the prevention of renal tubular oxidative damage mediate by iron, and warrant further investigation.

A beverage containing fermented black soybean ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats

Okazaki Y., Iqbal M., Kawakami N., Yamamoto Y., Toyokuni S., Okada S.

Journal of Clinical Biochemistry and Nutrition 47 巻 ( 3 ) 頁： 198 - 207 2010年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

It is beneficial to seek scientific basis for the effects of functional foods. Natural pigments derived from plants are widely known as possible antioxidants. Black soybean contains a larger amount of anthocyanins than regular soybean. Here we studied the antioxidative effect of a beverage obtained via citric acid fermentation of black soybean (BBS), using a rat model of renal oxidative injury induced by a renal carcinogen, ferric nitrilotriacetate. BBS (10 ml/kg) was orally administered 30 min before ferric nitrilotriacetate treatment. Renal lipid peroxidation was significantly suppressed in the BBS-pretreated animals concomitant with decrease in 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2′-deoxyguanosine. Maintenance of renal activities of antioxidative enzymes including catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glucose-6-phosphate dehydrogenase and quinone reductase was significantly better in the BBS-pretreated rats. Elevation of serum creatinine and urea nitrogen was significantly suppressed in the BBS-pretreated rats. These data suggest that dietary intake of BBS is useful for the prevention of renal tubular oxidative damage mediate by iron, and warrant further investigation.

DOI： 10.3164/jcbn.10-52

A Beverage Containing Fermented Black Soybean Ameliorates Ferric Nitrilotriacetate-Induced Renal Oxidative Damage in Rats

Okazaki Yasumasa, Iqbal Mohammad, Kawakami Norito, Yamamoto Yorihiro, Toyokuni Shinya, Okada Shigeru

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 47 巻 ( 3 ) 頁： 198 - 207 2010年11月

Biopersistent fiber-induced inflammation and carcinogenesis: lessons learned from asbestos toward safety of fibrous nanomaterials. 査読有り国際誌

Nagai H, Toyokuni S

Archives of biochemistry and biophysics 502 巻 ( 1 ) 頁： 1 - 7 2010年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Nano-sized durable fibrous materials such as carbon nanotubes have raised safety concerns similar to those raised by asbestos. However, the mechanism by which particulates with ultrafine structure cause inflammation and ultimately cancer (e.g. malignant mesothelioma and lung cancer) is largely unknown. This is partially because the particulates are not uniform and they vary in a plethora of factors. Such variances include length, diameter, surface area, density, shape, contaminant metals (including iron) and crystallinity. Each of these factors is involved in particulate toxicity both in vitro and in vivo. Thus, the elicited biological responses are incredibly complicated. Various kinds of fibers were evaluated with different cells, animals and methods. The aim of this review is to concisely summarize previous reports from the standpoint that activation of macrophages and mesothelial injury are the two major mechanisms of inflammation and possibly cancer. Importantly, these two mechanisms appear to be interacting with each other. However, there is a lack of data on the interplay of macrophage and mesothelium especially in vivo. Since fibrous nanomaterials present potential applications in various fields, it is necessary to develop standard evaluation methods to minimize risks for human health. © 2010 Elsevier Inc.

DOI： 10.1016/j.abb.2010.06.015

Genome-Scale Approaches to Investigate Oxidative DNA Damage 査読有り国際誌

Akatsuka Shinya, Toyokuni Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 47 巻 ( 2 ) 頁： 91 - 97 2010年9月

Genome-Scale Approaches to Investigate Oxidative DNA Damage 査読有り

Shinya Akatsuka, Shinya Toyokuni

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 47 巻 ( 2 ) 頁： 91 - 97 2010年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

In the trend of biological science after the completion of the human genome project, appreciation of an organism as a system rather than the sum of many molecular functions is necessary. On the investigation of DNA damage and repair, therefore, the orientation toward systematic and comprehensive genome-scale approaches is rapidly growing. The immuno-precipitation-based technique combined with high-density microarrays is one of the promising methods to provide access to such novel research strategies. We propose this sort of research area as oxygenomics.

Genome-scale approaches to investigate oxidative DNA damage

Akatsuka S., Toyokuni S.

Journal of Clinical Biochemistry and Nutrition 47 巻 ( 2 ) 頁： 91 - 97 2010年9月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

In the trend of biological science after the completion of the human genome project, appreciation of an organism as a system rather than the sum of many molecular functions is necessary. On the investigation of DNA damage and repair, therefore, the orientation toward systematic and comprehensive genome-scale approaches is rapidly growing. The immunoprecipitation-based technique combined with high-density microarrays is one of the promising methods to provide access to such novel research strategies. We propose this sort of research area as oxygenomics.

DOI： 10.3164/jcbn.10-38R

Suppression of SLC11A2 expression is essential to maintain duodenal integrity during dietary iron overload. 査読有り国際誌 Open Access

Shirase T, Mori K, Okazaki Y, Itoh K, Yamamoto M, Tabuchi M, Kishi F, Jiang L, Akatsuka S, Nakao K, Toyokuni S

The American journal of pathology 177 巻 ( 2 ) 頁： 677 - 85 2010年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Pathology

Iron is essential for the survival of mammals, but iron overload causes fibrosis and carcinogenesis. Reduced iron absorption and regulated release into circulation in duodenal mucosa constitute two major mechanisms of protection against dietary iron overload; however, their relative contribution remains elusive. To study the significance of the former process, we generated SLC11A2 transgenic mice (TGs) under the control of the chicken β-actin promoter. TGs were viable and fertile, and displayed no overt abnormalities up to 20 months. No significant difference in iron concentration was observed in major solid organs between TGs and their wild-type littermates, suggesting that increased number of iron transporters does not lead to increased iron absorption. To test the sensitivity to iron overload, TGs and wild-type mice were fed with an iron-rich diet containing 2% ferric citrate. Iron supplementation caused suppression of endogenous duodenal SLC11A2 expression, down-regulation of duodenal ferroportin, and overexpression of hepatic hepcidin, precluding excessive iron uptake both in the TGs and wild-type mice. However, iron-treated TGs revealed increased mortality, resulting from oxidative mucosal damage leading to hemorrhagic erosion throughout the whole intestinal area. These findings suggest that reduced iron release from duodenal cells into circulation plays a role in mitigating excessive iron uptake from the diet and that finely regulated duodenal absorption is essential to protect intestinal mucosa from iron-induced oxidative damage. Copyright © American Society for Investigative Pathology.

DOI： 10.2353/ajpath.2010.090823

TOWARD A NEW ERA OF THE NAGOYA JOURNAL OF MEDICAL SCIENCE: MESSAGE FROM THE NEW EDITOR-IN-CHIEF 国際誌

Toyokuni Shinya

NAGOYA JOURNAL OF MEDICAL SCIENCE 72 巻 ( 3-4 ) 頁： 107 - 109 2010年8月

Toward a new era of the Nagoya journal of medical science: message from the new editor-in-chief.

Toyokuni S

Nagoya journal of medical science 72 巻 ( 3-4 ) 頁： 107 - 9 2010年8月

Toward a new era of the Nagoya journal of medical science: message from the new editor-in-chief. 査読有り国際誌

Toyokuni S.

Nagoya journal of medical science 72 巻 ( 3-4 ) 頁： 107 - 109 2010年8月

Lipid peroxidation generates body odor component trans-2-nonenal covalently bound to protein in vivo. 査読有り国際誌 Open Access

Ishino K, Wakita C, Shibata T, Toyokuni S, Machida S, Matsuda S, Matsuda T, Uchida K

The Journal of biological chemistry 285 巻 ( 20 ) 頁： 15302 - 15313 2010年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-N∈-3-[(hept-1-enyl)-4-hexylpyridinium] lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenallysine adducts were indeed formed during the lipid peroxidation- mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M109.068023

A Novel Method for Efficient Collection of Normal Mesothelial Cells In Vivo 査読有り国際誌

Jiang Li, Yamashita Yoriko, Toyokuni Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 46 巻 ( 3 ) 頁： 265 - 268 2010年5月

A Novel Method for Efficient Collection of Normal Mesothelial Cells In Vivo 査読有り

Li Jiang, Yoriko Yamashita, Shinya Toyokuni

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 46 巻 ( 3 ) 頁： 265 - 268 2010年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

Asbestos-induced mesothelioma is a challenging social problem in many countries, and oxidative stress via iron is closely associated with its carcinogenesis. Mesothelioma is thought to originate from the mesothelial cells that cover the somatic cavity such as pleural, pericardial and peritoneal cavities. They are single layered and so flat that it is extremely difficult to obtain pure mesothelial cells as control samples from experimental animals. Here we describe a novel method to collect mesothelial cells from animals by the use of simple equipments. Surface of the most organs including lung, spleen and liver are covered with a single layer of mesothelial cells. Scraping the surface of those organs with razor blades after snap-freeze in liquid nitrogen satisfactorily confers almost pure population of mesothelial cells. This simple method would be helpful for obtaining mesothelial control samples from animals to elucidate the molecular mechanisms of a variety of mesothelial pathology.

A novel method for efficient collection of normal mesothelial cells in vivo

Jiang L., Yamashita Y., Toyokuni S.

Journal of Clinical Biochemistry and Nutrition 46 巻 ( 3 ) 頁： 265 - 268 2010年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

DOI： 10.3164/jcbn.10-01

n-3 polyunsaturated fatty acids promote apoptosis in oxidatively stressed transgenic mouse models 国際誌

リンパ腫ゲノムにおけるマイクロサテライト多型不安定性集積の空間統計学解析

鶴山竜昭, リチャード・カシンスキー, 赤塚慎也, 今井裕紀子, 竹内晴哉, 尾関宗孝, 奥野知子, 豊國伸哉, 玉木敬二

日本法医学雑誌 64 巻 ( 1 ) 頁： 54 - 54 2010年5月

Fan, YY; Toyokuni, S; Callaway, ES; Ran, Q; Lupton, JR; Chapkin, RS

CANCER RESEARCH 70 巻 2010年4月

A Quantitative trait locus responsible for inducing B-cell lymphoblastic lymphoma is a hotspot for microsatellite instability

KASZYNSKI Richard H., AKATSUKA Shinya, HIRATSUKA Takuya, JIN Guang, OZEKI Munetaka, OKUNO Tomoko, NAKAMURA Takuro, MANABE Toshiaki, TAKAKUWA Tetsuya, HIAI Hiroshi, TOYOKUNI Shinya, TAMAKI Keiji, TSURUYAMA Tatsuaki

Cancer science 101 巻 ( 3 ) 頁： 800 - 805 2010年3月

Homozygous deletion of CDKN2A/2B is a hallmark of iron-induced high-grade rat mesothelioma. 査読有り国際誌 Open Access

Hu Q, Akatsuka S, Yamashita Y, Ohara H, Nagai H, Okazaki Y, Takahashi T, Toyokuni S

Laboratory investigation; a journal of technical methods and pathology 90 巻 ( 3 ) 頁： 360 - 73 2010年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Laboratory Investigation

In humans, mesothelioma has been linked to asbestos exposure, especially crocidolite and amosite asbestos, which contain high amounts of iron. Previously, we established a rat model of iron-induced peritoneal mesothelioma with repeated intraperitoneal injections of iron saccharate and an iron chelator, nitrilotriacetate. Here, we analyze these mesotheliomas using array-based comparative genomic hybridization (aCGH) and gene expression profiling by microarray. Mesotheliomas were classified into two distinct types after pathologic evaluation by immunohistochemistry. The major type, epithelioid mesothelioma (EM), originated in the vicinity of tunica vaginalis testis, expanded into the upper peritoneal cavity and exhibited papillary growth and intense podoplanin immunopositivity. The minor type, sarcomatoid mesothelioma (SM), originated from intraperitoneal organs and exhibited prominent invasiveness and lethality. Both mesothelioma types showed male preponderance. SMs revealed massive genomic alterations after aCGH analysis, including homozygous deletion of CDKN2A/2B and amplification of ERBB2 containing region, whereas EMs showed less genomic alterations. Uromodulin was highly expressed in most of the cases. After 4-week treatment, iron deposition in the mesothelia was observed with 8-hydroxy-2′-deoxyguanosine formation. These results not only show two distinct molecular pathways for iron-induced peritoneal mesothelioma, but also support the hypothesis that oxidative stress by iron overload is a major cause of CDKN2A/2B homozygous deletion. © 2010 USCAP, Inc All rights reserved.

DOI： 10.1038/labinvest.2009.140

A quantitative trait locus responsible for inducing B-cell lymphoblastic lymphoma is a hotspot for microsatellite instability. 査読有り国際共著国際誌 Open Access

Kaszynski RH, Akatsuka S, Hiratsuka T, Jin G, Ozeki M, Okuno T, Nakamura T, Manabe T, Takakuwa T, Hiai H, Toyokuni S, Tamaki K, Tsuruyama T

Cancer science 101 巻 ( 3 ) 頁： 800 - 5 2010年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

While the molecular mechanisms underlying microsatellite instability (MSI) have been exhaustively investigated, identifying the patterns of MSI distribution within diverse cancer genomes has remained an elusive issue. In the present study, we conducted genome-wide MSI screening in B-cell lymphoblastic lymphomas (B-LBL) which spontaneously develop in the SL/Kh strain of mice. Tumor samples harvested from 16 mice were investigated using a framework map consisting of 150 microsatellite markers spaced at increments of roughly 0.5-3.0 centimorgans, spanning the entirety of mouse chromosomes (mus musculus chromosomes [MMU]) 3-6. MMU3 contains a quantitative trait locus (QTL), Bomb1 (bone marrow pre-B1), known to induce an aberrant expansion of pre-B cells in bone marrow prior to the onset of B-LBL in SL/Kh mice. The remaining chromosomes were selected on the basis of those most closely resembling MMU3 in terms of total estimated length (maximum variance 10 Mb). MSI was confirmed at 2≤ markers in DNA derived from tumor tissues in 15 SL/Kh mice (93.7%), while healthy splenic DNA samples screened in parallel were consistently negative for MSI. The overall MSI incidence was significantly higher on MMU3 compared with MMU4-6 (P = 0.031). Additionally, by applying spatial point pattern analysis combined with a 1-D version of Ripley's K-function, we successfully demonstrated the predilection of MSI-susceptible loci to structure a massive cluster within the Bomb1 locus. Our study is the first to suggest that a QTL concomitantly serves as a hotspot for MSI-susceptible loci and sheds new light on somatic cancer genetics. © 2010 Japanese Cancer Association.

DOI： 10.1111/j.1349-7006.2009.01437.x

前駆B芽球性リンパ腫におけるマイクロサテライト不安定性解析とポイントパターン解析の応用

鶴山竜昭, リチャード・カシンスキー, 赤塚慎也, 中村卓郎, 真鍋敏明, 日合弘, 玉木敬二, 豊國伸哉

日本病理学会会誌 99 巻 ( 1 ) 頁： 217 - 217 2010年3月

平成20年度学術大会科学技術振興研究発表「悪性中皮腫」より : 概要と基礎研究からのアプローチ

大槻剛巳, 中野孝司, 長谷川誠紀, 岡田守人, 辻村亨, 関戸好孝, 豊國伸哉, 西本寛, 福岡和也, 田中文啓

日本職業・災害医学会会誌 = Japanese journal of occupational medicine and traumatology 58 巻 ( 1 ) 頁： 1 - 8 2010年1月

*Homozygous deletion of CDKN2A/2B is a hallmark of iron-induced high-grade rat mesothelioma. 査読有り

Hu Q, Akatsuka S, Yamashita Y, Ohara H, Nagai H, Okazaki Y, Takahashi T, Toyokuni S.

Lab. Invest. 90 巻頁： 360-372 2010年1月

Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats. 査読有り

Ohara H, Akatsuka S, Nagai H, Liu YT, Jiang L, Okazaki Y, Yamashita Y, Nakamura T and Toyokuni S.

Free Radic Res 頁： (in press) 2010年

A quantitative trait locus for inducing B-cell lymphoblastic lymphoma is a hot spot for microsatellite instability. 査読有り

Kaszynski RH, Akatsuka S, Hiratsuka T, Jin G, Ozeki M, Okuno T, Nakamura T, Manabe T, Takakuwa T, Hiai H, Toyokuni S, Tamaki K and Tsuruyama T.

Cancer Sci 101 巻頁： 800-805 2010年

CD8+CD122+ Regulatory T Cells and CD4+ Treg Cooperatively Prevent and Cure CD4+ cell-induced Colitis. 査読有り

Endharti AT, Okuno Y, Shi Z, Misawa N, Toyokuni S, Ito M, Isobe KI and Suzuki H.

186 巻頁： 41-52 2010年

Toward a new era of the Nagoya Journal of Medical Science: message from the new editor-in-chief. 招待有り

Toyokuni S.

Nagoy J Med Sci 72 巻頁： 107-109 2010年

A beverage containing fermented black soybean ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats. 査読有り

Okazaki Y, Iqbal M, Kawakami N, Yamamoto Y, Toyokuni S and Okada S.

J Clin Biochem Nutr 47 巻頁： 198-207 2010年

Biopersistent fiber-induced inflammation and carcinogenesis: lessons learned from asbestos toward safety of fibrous nanomaterials. 査読有り

Nagai H and Toyokuni S.

Arch Biochem Biophys 502 巻頁： 1-7 2010年

Genome-scale approaches to investigate oxidative DNA damage. 査読有り

Akatsuka S and Toyokuni S.

J Clin Biochem Nutr 47 巻頁： 91-97 2010年

Lipid peroxidation generates a body odor component trans-2-nonenal covalently bound to protein in vivo. 査読有り

Ishino K, Wakita C, Shibata T, Toyokuni S, Machida S, Matsuda S, Matsuda T and Uchida K.

J Biol Chem 285 巻頁： 15302-15313 2010年

Suppression of SLC11A2 expression is essential to maintain duodenal integrity during dietary iron overload. 査読有り

Shirase T, Mori K, Okazaki Y, Itoh K, Yamamoto M, Tabuchi M, Kishi F, Jiang L, Akatsuka S, Nakao K and Toyokuni S.

Am J Pathol 177 巻頁： 677-685 2010年

Novel method for efficient collection of normal mesothelial cells in vivo. 査読有り

Jiang L, Yamashita Y and Toyokuni S.

J Clin Biochem Nutr 46 巻頁： 265-268 2010年

Homozygous Deletion of CDKN2A/2B is a Hallmark of Iron-induced High-grade Rat Mesothelioma 査読有り国際誌

Okazaki, Y; Hu, QA; Akatsuka, S; Ohara, H; Nagai, H; Takahashi, T; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE 49 巻頁： S67 - S67 2010年

Biopersistent fiber-induced inflammation and carcinogenesis: lessons learned from asbestos toward safety of fibrous nanomaterials. 査読有り

Nagai H, Toyokuni S

Arch Biochem Biophys 502 巻頁： 1-7 2010年

CD8+CD122+ Regulatory T Cells and CD4+ Treg Cooperatively Prevent and Cure CD4+ cell-induced Colitis. 査読有り

Endharti AT, Okuno Y, Shi Z, Misawa N, Toyokuni S, Ito M, Isobe KI, Suzuki H

J Immunol 186 巻頁： 41-52 2010年

Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats. 査読有り

Ohara H, Akatsuka S, Nagai H, Liu YT, Jiang L, Okazaki Y, Yamashita Y, Nakamura T, Toyokuni S

Free Radic Res 頁： (in press) 2010年

Toward a new era of the Nagoya Journal of Medical Science: message from the new editor-in-chief. 招待有り

Toyokuni S

Nagoy J Med Sci 72 巻頁： 107-109 2010年

脳梗塞モデルラット作成前に実施する運動の効果および作用機序の検討:～抗酸化作用に着目して～国際誌

濱川みちる, 石田章真, 玉越敬悟, 嶋田悠, 中島宏樹, 豊國伸哉, 石田和人

東海北陸理学療法学術大会誌 26 巻 ( 0 ) 頁： 35 - 35 2010年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：東海北陸理学療法学術大会

【目的】脳梗塞モデル作成前の運動には、梗塞体積減少、運動機能改善などの脳梗塞の障害軽減効果があると報告されている。また、運動は抗酸化作用を高めることが報告されているが、抗酸化作用が脳梗塞の障害軽減効果にいかに関与するかについては不明である。そこで本研究では、脳梗塞発症前に運動を行うことの効果および作用機序を抗酸化作用に着目して検討する。【方法】実験動物にはWistar系雄性ラット(5週齢)を用いた。無作為に運動群と非運動群に分け、運動群はトレッドミル運動(15 m / min、30分/日)を3週間毎日行った。非運動群は1日30分間走行させずにトレッドミル装置内に暴露させた。介入終了の翌日、各群に脳梗塞モデル作成手術を施行した。手術24時間後に、麻痺の重症度を評価するNeurological Deficits(ND)、歩行時のバランス能力の評価としてBeam Walking(BW)、はしご上の歩行における前肢の協調運動機能を評価するLadder test、前肢の感覚運動機能の評価としてLimb Placing(LP)を行った。その直後に脳を採取し、TTC染色により非梗塞半球体積に対する梗塞体積の割合を算出した。また、酸化ストレス産物の指標として4-hydroxy-2-nonenal (4-HNE)及び8-Hydroxydeoxyguanosine(8-OHdG)の免疫染色を行った。統計学的検定には、対応のないt検定とMann-WhitneyのU検定を用い、有意水準は5％未満とした。【結果】NDとladder testにおいて、運動群が非運動群と比較して有意に障害が軽減した（p < 0.05）。また、他の機能評価や梗塞体積については群間に有意差は認められなかった。免疫染色では、非梗塞側半球で運動群に比べ非運動群は4-HNE陽性細胞が多くみられた。しかし、8-OHdG染色では差を認めなかった。【まとめ】脳梗塞前の運動は、梗塞後の運動機能改善効果をもたらしたものの、梗塞体積は非運動群と同程度であった。しかし、脂質過酸化の指標である4-HNE陽性細胞は非運動群の非梗塞側でより多く見られ、運動による酸化ストレス抑制効果が示された。 本研究は科研費（22500456）の助成を受けたものである。

DOI： 10.11529/thpt.26.0.35.0

S1-3 アスベスト誘発中皮腫の発がん機構の解明とその予防への応用(シンポジウム1:「生活習慣・環境因子とバイオマーカー」)

豊國伸哉

日本環境変異原学会大会プログラム・要旨集 ( 38 ) 頁： 75 2009年11月

ITPase-deficient mice show growth retardation and die before weaning. 査読有り

Behrmanech M, Sakumi K, Abolhassani N, Toyokuni S, Oka S, Ohnishi YN, Tsuchimoto D, Nakabeppu Y.

Cell Death Differ. 16 巻頁： 1315-1322 2009年10月

ITPase-deficient mice show growth retardation and die before weaning 査読有り国際誌 Open Access

Behmanesh, M; Sakumi, K; Abolhassani, N; Toyokuni, S; Oka, S; Ohnishi, YN; Tsuchimoto, D; Nakabeppu, Y

CELL DEATH AND DIFFERENTIATION 16 巻 ( 10 ) 頁： 1315 - 1322 2009年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Death and Differentiation

Inosine triphosphate pyrophosphatase (ITPase), the enzyme that hydrolyzes ITP and other deaminated purine nucleoside triphosphates to the corresponding purine nucleoside monophosphate and pyrophosphate, is encoded by the Itpa gene. In this study, we established Itpa knockout (KO) mice and used them to show that ITPase is required for the normal organization of sarcomeres in the heart. Itpa-/- mice died about 2 weeks after birth with features of growth retardation and cardiac myofiber disarray, similar to the phenotype of the cardiac α-actin KO mouse. Inosine nucleotides were found to accumulate in both the nucleotide pool and RNA of Itpa-/- mice. These data suggest that the role of ITPase in mice is to exclude ITP from the ATP pool, and the main target substrate of this enzyme is rITP. Our data also suggest that cardiomyopathy, which is mainly caused by mutations in sarcomeric protein-encoding genes, is also caused by a defect in maintaining the quality of the ATP pool, which is an essential requirement for sarcomere function.

DOI： 10.1038/cdd.2009.53

Potent antioxidative acticity of Vineatrol® 30 grapevine-shoot extract. 査読有り

Müller C, Ullmann K, Wilkins A, Winterhalter P, Toyokuni S and Steinberg P.

Biosci Biotechnol Biochem 73 巻頁： 1831-1836 2009年8月

Elucidation of asbestos-induced carcinogenesis and its application to prevention, diagnosis and treatment in relation to iron 国際誌 Open Access

Toyokuni S.

Japanese Journal of Lung Cancer 49 巻 ( 4 ) 頁： 362 - 367 2009年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Lung Cancer

Objective. Respiratory exposure to asbestos has been associated with mesothelioma in humans. However, its carcinogenic mechanism is still unclear. Methods. Here we studied the ability of chrysotile, crocidolite and amosite fibers to induce oxidative DNA damage and the modifying factors using 4 distinct approaches. Results. Electron spin resonance analyses showed that crocidolite and amosite containing high amounts of iron, but not chrysotile, catalyzed hydroxyl radical formation in the presence of hydrogen peroxide, which was enhanced by an iron chelator, nitrilotriacetic acid, and suppressed by Desferal®. Iron chelators, such as citrate, ATP and GTP, did not inhibit this reaction. Second, we used time-lapse videomicroscopy to evaluate how cells deal with asbestos fibers. RAW264.7 cells, MeT-5A and HeLa cells engulfed asbestos fibers, which reached not only cytoplasm but also the nucleus. Third, we utilized supercoiled plasmid DNA to evaluate the ability of each type of asbestos to induce DNA double strand breaks (DSBs). Crocidolite and amosite, but not chrysotile, induced DNA DSBs in the presence of iron chelators. We cloned the fragments to identify break ends. DSBs tended to occur within repeat sequences and between two G:C sequences. Finally, intraperitoneal administration of each type of asbestos to rats induced not only formation of nuclear 8-hydroxy-2′-deoxyguanosine in the mesothelium but also significant iron deposits in the spleen. Conclusion. Together with the established carcinogenicity of intraperitoneal chrysotile, our data suggest that asbestos-associated catalytic iron, whether constitutional or induced by other mechanisms, plays a role in asbestos-induced carcinogenesis and that chemoprevention may be possible through targeting the catalytic iron. © 2009 The Japan Lung Cancer Society.

DOI： 10.2482/haigan.49.362

Potent Antioxidative Activity of Vineatrol®30 Grapevine-shoot Extract 査読有り国際誌 Open Access

Müller, C; Ullmann, K; Wilkens, A; Winterhalter, P; Toyokuni, S; Steinberg, P

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 73 巻 ( 8 ) 頁： 1831 - 1836 2009年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioscience, Biotechnology and Biochemistry

The health promoting effects of a grapevine-shoot extract named Vineatrol®30, which contains resveratrol (Resv) as well as considerable amounts of Resv oligomers, have recently been investigated. In the present study, we analyzed the free radical scavenging capacity, the ability to inhibit lipid peroxidation, and the capacity to enhance the human glutathione peroxidase 1 (GPx) and the human superoxide dismutase 1 (SOD) gene promoter activities of Vineatrol®30. Vineatrol®30 was able to scavenge the 2,2'-azinobis-3- ethylbenzothiazoline-6-sulfonic acid radical cation and led to concentration-dependent inhibition of lipid peroxidation, Vineatrol®30 not being superior to Resv alone in both cases. Vineatrol®30 also enhanced the gene promoter activities of human GPx and SOD expressed in V79 cells, whereas this effect could not be demonstrated for Resv. In summary, the results presented in this study show that the Vineatrol®30 grapevine-shoot extract is a free radical scavenger and potent antioxidant at non-cytotoxic concentrations.

DOI： 10.1271/bbb.90213

B細胞性芽球型リンパ腫ゲノムにおけるマイクロサテライト不安定性(Microsatellite instability in the genome of B-lymphoblastic lymphoma)

リチャード・カシンスキー, 鶴山竜昭, 河野文彦, 平塚拓也, 尾関宗孝, 豊國伸哉, 日合弘, 玉木敬二

日本癌学会総会記事 68回巻頁： 163 - 163 2009年8月

Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation. 査読有り国際誌 Open Access

Lee J, Kanatsu-Shinohara M, Morimoto H, Kazuki Y, Takashima S, Oshimura M, Toyokuni S, Shinohara T

Cell stem cell 5 巻 ( 1 ) 頁： 76 - 86 2009年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Stem Cell

Spermatogonial stem cells (SSCs) undergo self-renewal division and support spermatogenesis. Although several cytokines coordinate to drive SSC self-renewal, little is known about the mechanisms underlying this process. We investigated the molecular mechanism by reconstructing SSC self-renewal in vitro without exogenous cytokines. Activation of Ras or overexpression of cyclins D2 and E1, both of which were induced by Ras, enabled long-term self-renewal of cultured spermatogonia. SSCs with activated Ras responded properly to differentiation signals and underwent spermatogenesis, whereas differentiation was abrogated in cyclin transfectants after spermatogonial transplantation. Both Ras- and cyclin-transfected cells produced seminomatous tumors, suggesting that excessive self-renewing stimulus induces oncogenic transformation. In contrast, cells that overexpressed cyclin D1 or D3 failed to make germ cell colonies after transplantation, which indicated that cyclin expression pattern is an important determinant to long-term SSC recolonization. Thus, the Ras-cyclin D2 pathway regulates the balance between tissue maintenance and tumorigenesis in the SSC population. © 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.stem.2009.04.020

Abnormal DNA methyltransferase expression in mouse germline stem cells results in spermatogenic defects. 査読有り

Takashima S, Takehashi M, Lee J, Chuma S, Okano M, Hata K, Suetake I, Nakatsuji N, Miyoshi H, Tajima S, Tanaka Y, Toyokuni S, Sasaki H, Kanatsu-Shinohara M, Shinohara S.

81 巻頁： 155-164 2009年7月

Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation. 査読有り

Lee J, Kanatsu-Shinohara M, Morimoto H, Kazuki Y, Takashima S, Oshimura M, Toyokuni S and Shinohara T.

Cell Stem Cell 5 巻頁： 76-86 2009年7月

Abnormal DNA methyltransferase expression in mouse germline stem cells results in spermatogenic defects. 査読有り国際誌

Takashima S, Takehashi M, Lee J, Chuma S, Okano M, Hata K, Suetake I, Nakatsuji N, Miyoshi H, Tajima S, Tanaka Y, Toyokuni S, Sasaki H, Kanatsu-Shinohara M, Shinohara T

Biology of reproduction 81 巻 ( 1 ) 頁： 155 - 64 2009年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Although spermatogonial stem cells (SSCs) are committed to spermatogenesis, they may also convert to an embryonic stem cell-like pluripotent state at a low frequency. Because changes in DNA methylation patterns are associated with this conversion, we examined the effect of manipulating DNA methyltransferase (Dnmt) expression on the fate of cultured SSCs, germline stem (GS) cells. Dnmt1 knockdown induced apoptosis in GS cells, which was attenuated by the loss of Trp53. In contrast, GS cells proliferated normally in vitro after Dnmt3a/Dnmt3b ablation or during Dnmt3l overexpression. However, Dnmt3a/Dnmt3b double-mutant cells showed hypomethylation in the SineB1 repetitive sequence, and Dnmt3l-overexpressing cells showed hypermethylation in major and minor satellite sequences; neither cell type formed teratomas and completed spermatogenesis following transplantation into the seminiferous tubules. Although genetic manipulation did not increase the conversion of GS cells to a pluripotent state, these results underscore the important role of DNMTs in survival and spermatogenic differentiation in SSCs. © 2009 by the Society for the Study of Reproduction, Inc.

DOI： 10.1095/biolreprod.108.074708

Proapoptotic Effects of Dietary (n-3) Fatty Acids Are Enhanced in Colonocytes of Manganese-Dependent Superoxide Dismutase Knockout Mice 国際誌 Open Access

Fan, YY; Zhan, Y; Aukema, HM; Davidson, LA; Zhou, L; Callaway, E; Tian, Y; Weeks, BR; Lupton, JR; Toyokuni, S; Chapkin, RS

JOURNAL OF NUTRITION 139 巻 ( 7 ) 頁： 1328 - 1332 2009年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Nutrition

We recently demonstrated that (n-3) PUFA trigger the induction of apoptosis in the colon by enhancing phospholipid oxidation and mitochondrial Ca 2+ accumulation. To further elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, a 2 x 2 experiment was designed using both wild type (control) and manganese-dependent superoxide dismutase (SOD2) heterozygous knockout mice (SOD2+/-), which exhibit increased mitochondrial oxidative stress. Mice were fed diets differing only in the type of fat [corn oil or fish oil containing (n-3) PUFA] at 15% by weight for 4 wk. Dietary (n-3) PUFA treatment enhanced (22%) apoptosis in colonic crypts. In addition, SOD2 haploinsufficiency enhanced (20%) apoptosis, which was further increased (36%) by (n-3) PUFA feeding. Dietary lipid source and genotype interactively modulated nitrotyrosine levels (P = 0.027) and inflammation (P = 0.032). These findings demonstrate that the proapoptotic effects of (n-3) PUFA are enhanced in oxidatively stressed SOD2+/- mice. Thus, (n-3) PUFA appear to promote an oxidation-reduction imbalance in the intestine, which may directly or indirectly trigger apoptosis and thereby reduce colon cancer risk. © 2009 American Society for Nutrition.

DOI： 10.3945/jn.109.106203

The potential role of reactive oxygen species in mammary neoplasm 査読有り国際誌

Arafah M., Khabaz M., Toyokuni S.

Australian Journal of Basic and Applied Sciences 3 巻 ( 3 ) 頁： 2949 - 2954 2009年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Australian Journal of Basic and Applied Sciences

The current study investigated the presence of oxidative stress markers (8-OHdG and HNE) in breast carcinoma in females from Saudi Arabia, and to examine the growth modulatory effects of HNE (induce cell growth inhibition) by measuring the expression of Ki-67 protein (cell proliferation antigen). Furthermore, to record if there is any significant prognostic indicator of these markers. In order to estimate the level of oxidative damage in breast cancer, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (HNE) expressions were investigated in breast carcinoma and benign lesions of breast, and were estimated by semi-quantitative immunohistochemical method. The observations of the present study showed that the level of expressions of 8-OHdG and HNE were significantly higher than those in controls. Reduction of oxidative stress is thought to be a very important measure for primary prevention of breast carcinoma. © 2009, INSInet Publication.

The potential role of reactive oxygen species in mammary neoplasm 国際誌

Arafah M, Khabaz M, Toyokuni S

Australian Journal of Basic and Applied Sciences 3 巻 ( 3 ) 頁： 2949 - 2954 2009年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Australian Journal of Basic and Applied Sciences

Effect of edaravone, a novel free radical scavenger, supplemented to cardioplegia on myocardial function after cardioplegic arrest: in vitro study of isolated rat heart 査読有り

Pro-apoptotic effects of dietary (n-3) fatty acids are enhanced in colonocytes of SOD2 knockout mice. 査読有り

Fan YY, Zhan Y, Aukema HM, Davidson LA, Zhou L, Callaway E, Tian Y, Weeks BR, Lupton JR, Toyokuni S, Chapkin RS.

J. Nutr. 139 巻頁： 1328-1332 2009年5月

Effect of edaravone, a novel free radical scavenger, supplemented to cardioplegia on myocardial function after cardioplegic arrest: in vitro study of isolated rat heart. 査読有り

Yamazaki K, Miwa S, Toyokuni S, Nemoto S, Oriyanhan W, Takaba K, Saji Y, Marui A, Nishina T, Ikeda T and Komeda M.

Heart Vessels 24 巻頁： 228-235 2009年5月

Yamazaki, K; Miwa, S; Toyokuni, S; Nemoto, S; Oriyanhan, W; Takaba, K; Saji, Y; Marui, A; Nishina, T; Ikeda, T; Komeda, M

HEART AND VESSELS 24 巻 ( 3 ) 頁： 228 - 235 2009年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Heart and Vessels

Cardioplegic arrest has been the main mechanism of myocardial protection during open-heart surgery; however, it causes myocardial injury during ischemia-reperfusion. Free radical scavengers are widely known to attenuate ischemia-reperfusion injury in various settings. We investigated the effects of edaravone, a novel free radical scavenger that was originally used for cerebral protection, on myocardial function during ischemia-reperfusion after cardioplegic arrest. Rat hearts were excised and perfused using Langendorff apparatus. The hearts were cardioplegically arrested for 90 min using St. Thomas' Hospital cardioplegic solution (ST solution) at 4°C every 45 min and then reperfused for 20 min. The hearts were divided into 4 groups (n = 13 in each group). In Group ST, the hearts were arrested using the ST solution alone. In Groups L, M, and H, the hearts were arrested using the ST solution supplemented with a low-dose (1 μM), moderate dose (10 μM), and high dose (100 μM) of edaravone, respectively. Left ventricular function (+dp/dt max) and the levels of the cardiac enzymes released were measured before and after cardioplegic arrest. At the end of the study, the water content and the tissue oxidative stress (8-hydroxy-2′-deoxyguanosine) of the heart were measured. During reperfusion, the edaravone-treated groups showed a greater functional recovery with regard to the +dp/dtmax (P < 0.05). The lactate level was the lowest (P < 0.01) in Group M. The water content of the hearts in the edaravone-treated groups was significantly lower (P< 0.05) than that in Group ST. Oxidative stress was significantly lower (P< 0.01) in the edaravone-treated hearts than in Group ST, and it was the lowest in Group M. The addition of edaravone to the cardioplegic solution ameliorates the impairment in myocardial function by reducing the oxidative stress after cardioplegic arrest. In this study, the maximum improvement in the myocardial function was achieved by addition of a moderate dose (10 μ) of edaravone. © Springer Japan 2009.

DOI： 10.1007/s00380-008-1106-1

Pro-apoptotic effects of dietary (n-3) fatty acids are enhanced in colonocytes of SOD2 knockout mice. 査読有り国際誌

Fan YY, Zhan Y, Aukema HM, Davidson LA, Zhou L, Callaway E, Tian Y, Weeks BR, Lupton JR, Toyokuni S, Chapkin RS

J. Nutr. 139 巻頁： 1328-1332 2009年5月

Granulocyte colony-stimulating factor protects cardiac mitochondria in the early phase of cardiac injury. 査読有り

Hiraumi Y, Iwai-Kanai E, Baba S, Yui Y, Kamitsuji Y, Mizushima Y, Matsubara H, Watanabe M, Watanabe KI, Toyokuni S, Matsubara H, Nakahata T, Adachi S.

Am. J. Physiol. Heart Circ. Physiol. 296 巻頁： H823-H832 2009年3月

Granulocyte colony-stimulating factor protects cardiac mitochondria in the early phase of cardiac injury 査読有り国際誌 Open Access

Hiraumi, Y; Iwai-Kanai, E; Baba, S; Yui, Y; Kamitsuji, Y; Mizushima, Y; Matsubara, H; Watanabe, M; Watanabe, K; Toyokuni, S; Matsubara, H; Nakahata, T; Adachi, S

AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY 296 巻 ( 3 ) 頁： H823 - H832 2009年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Physiology - Heart and Circulatory Physiology

Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardiopro- tective role in several models of cardiac injury, clinical use of this drug in cardiac patients has been controversial. Here, we tested, in vivo and in vitro, the effect of G-CSF on cardiac mitochondria, which play a key role in determining cardiac cellular fate and function. Mild stimulation of C57/BL6 mice with doxorubicin (Dox) did not induce cardiac apoptosis or fibrosis but did induce damage to mitochondrial organization of the myocardium as observed through an electron microscope. Cardiac catheterization and echocardiography revealed that Dox did not alter cardiac systolic function or left ventricular size but did reduce diastolic function, an early sign of cardiac damage. Treatment with G-CSF attenuated significantly the damage to mitochondrial organization and rescued diastolic function. In an in vitro model for rat neonatal cardiomyocytes, a subapoptotic dose of Dox induced severe mitochondrial damage, including marked swelling of the cardiac mitochondria and/or decreased mitochondrial membrane potential. These mitochondrial changes were completely blocked by pretreatment with G-CSF. In addition, G-CSF dramatically improved ATP generation, which rescued Dox-impaired mitochondrial electron transport and oxygen consumption mainly through complex IV. These findings clearly indicate that G-CSF protects cardiac mitochondria, which are key organelles in the determination of cardiac cellular fate, in the early phase of cardiac injury. © 2009 the American Physiological Society.

DOI： 10.1152/ajpheart.00774.2008

Mechanisms of asbestos-induced carcinogenesis.

Toyokuni S.

Nagoya J. Med. Sci. 71 巻頁： 1-10 2009年2月

MECHANISMS OF ASBESTOS-INDUCED CARCINOGENESIS 国際誌

Toyokuni Shinya

NAGOYA JOURNAL OF MEDICAL SCIENCE 71 巻 ( 1-2 ) 頁： 1 - 10 2009年2月

MECHANISMS OF ASBESTOS-INDUCED CARCINOGENESIS

TOYOKUNI S.

Nagoya Journal of Medical Science 71 巻 ( 1 ) 頁： 1 - 10 2009年2月

掲載種別：研究論文（学術雑誌）出版者・発行元：名古屋大学

Respiratory exposure to asbestos fibers has been associated with diffuse malignant mesothelioma (DMM) in humans. Despite advancements in the molecular analyses of human DMM and the development of animal models, the carcinogenic mechanisms of the disease remain unclear. There are basically three hypotheses regarding the pathogenesis of asbestos-induced DMM, which may be summarized as follows: (1) the "oxidative stress theory" is based on the fact that phagocytic cells that engulf asbestos fibers produce large amounts of free radicals due to their inability to digest the fibers, and epidemiological studies indicating that iron-containing asbestos fibers appear more carcinogenic; (2) the "chromosome tangling theory" postulates that asbestos fibers damage chromosomes when cells divide; and (3) the "theory of adsorption of many specific proteins as well as carcinogenic molecules" states that asbestos fibers in vivo concentrate proteins or chemicals including the components of cigarette smoke. Elucidation of the major mechanisms underlying DMM would be helpful for the development of novel strategies to prevent DMM induction in people who have already been exposed to asbestos.

DOI： 10.18999/nagjms.71.1-2.1

Mechanisms of asbestos-induced carcinogenesis. 査読有り国際誌

Toyokuni S.

Nagoya journal of medical science 71 巻 ( 1-2 ) 頁： 1 - 10 2009年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nagoya journal of medical science

Respiratory exposure to asbestos fibers has been associated with diffuse malignant mesothelioma (DMM) in humans. Despite advancements in the molecular analyses of human DMM and the development of animal models, the carcinogenic mechanisms of the disease remain unclear. There are basically three hypotheses regarding the pathogenesis of asbestos-induced DMM, which may be summarized as follows: (1) the "oxidative stress theory" is based on the fact that phagocytic cells that engulf asbestos fibers produce large amounts of free radicals due to their inability to digest the fibers, and epidemiological studies indicating that iron-containing asbestos fibers appear more carcinogenic; (2) the "chromosome tangling theory" postulates that asbestos fibers damage chromosomes when cells divide; and (3) the "theory of adsorption of many specific proteins as well as carcinogenic molecules" states that asbestos fibers in vivo concentrate proteins or chemicals including the components of cigarette smoke. Elucidation of the major mechanisms underlying DMM would be helpful for the development of novel strategies to prevent DMM induction in people who have already been exposed to asbestos.

Role of iron in carcinogenesis : Cancer as a ferrotoxic disease

Mechanisms of asbestos-induced carcinogenesis.

Toyokuni S

Nagoya journal of medical science 71 巻 ( 1-2 ) 頁： 1 - 10 2009年2月

Mechanisms of asbestos-induced carcinogenesis.

Toyokuni S

Nagoya J. Med. Sci. 71 巻頁： 1-10 2009年2月

TOYOKUNI Shinya

Cancer science 100 巻 ( 1 ) 頁： 9 - 16 2009年1月

Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inhibitor FK228 through AIF translocation. 査読有り国際誌 Open Access

Watanabe M, Adachi S, Matsubara H, Imai T, Yui Y, Mizushima Y, Hiraumi Y, Watanabe K, Kamitsuji Y, Toyokuni SY, Hosoi H, Sugimoto T, Toguchida J, Nakahata T

International journal of cancer 124 巻 ( 1 ) 頁： 55 - 67 2009年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Cancer

Malignant rhabdoid tumors (MRT) exhibit a very poor prognosis because of their resistance to chemotherapeutic agents and new therapies are needed for the treatment of this cancer. Here, we show that the histone deacetylase (HDAC) inhibitor FK228 (depsipeptide) has an antitumor effect on MRT cells both in vitro and in vivo. FK228 is a unique cyclic peptide and is among the most potent inhibitors of both Class I and Class II HDACs. FK228 inhibited proliferation and induced apoptosis in all MRT cell lines tested. Preincubation with the pancaspase inhibitor zVAD-fmk did not completely rescue FK228-induced cell death, although it did inhibit apoptosis. Transmission electron microscopy (TEM) showed that FK228 could stimulate MRT cells to undergo apoptosis, necrosis or autophagy. FK228 converted unconjugated microtubule-associated protein light chain 3 (LC3-I) to conjugated light chain 3 (LC3-II) and induced localization of LC3 to autophagosomes. Apoptosis inducing factor (AIF), which plays a role in caspase-independent cell death, translocated to the nucleus in response to FK228 treatment. Moreover, small interfering RNA (siRNA) targeting of AIF prevented the morphological changes associated with autophagy and redistribution of LC3 to autophagosomes. Disrupting autophagy with chloroquine treatment enhanced FK228-induced cell death. In vivo, FK228 caused a reduction in tumor size and induced autophagy in tumor tissues. Using immunoelectron microscopy, we confirmed AIF translocation into the nucleus of FK228-induced autophagic cells in vivo. Thus, FK228 is a novel candidate for an antitumor agent for MRT cells. © 2008 Wiley-Liss, Inc.

DOI： 10.1002/ijc.23897

*Role of iron in carcinogenesis: Cancer as a ferrotoxic disease. 招待有り査読有り

Toyokuni S.

Cancer Sci. 100 巻頁： 6-16 2009年1月

Genome-wide analysis identifies a tumor suppressor role for aminoacylase 1 in iron-induced rat renal cell carcinoma. 査読有り

Zhong Y, Onuki J, Yamasaki T. Ogawa O, Akatsuka S and Toyokuni S.

Carcinogenesis 30 巻頁： 158-164 2009年1月

Biased Immunoglobulin VH Recombination and Increased Expression of Chemokine Receptor XCR1 Are Distinct Features of Bone Marrow-Derived Diffuse Large B-Cell Lymphoma 国際誌

Yamashita, Y; Kajiura, D; Nakamura, S; Toyokuni, S; Mori, N

LABORATORY INVESTIGATION 89 巻頁： 292A - 292A 2009年1月

Genome-wide analysis identifies a tumor suppressor role for aminoacylase 1 in iron-induced rat renal cell carcinoma. 査読有り国際誌

Zhong Y, Onuki J, Yamasaki T, Ogawa O, Akatsuka S, Toyokuni S

Carcinogenesis 30 巻 ( 1 ) 頁： 158 - 64 2009年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Carcinogenesis

A growing number of studies indicate a link between oxidative stress and cancer. We previously developed a rat model of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA). Here, we performed a genome-wide analysis to study characteristics of genomic alteration and identify putative genes involved in the development of Fe-NTA-induced RCCs. Array-based comparative genomic hybridization analyses revealed a chromosomal loss spanning chromosome 8 in most of the RCCs studied, with a common deletion at 8q31-32, which was confirmed by loss of heterozygosity (LOH) analysis. Studies of gene expression in RCCs or following Fe-NTA treatment revealed globally decreased transcription levels of 34 genes derived from chromosome 8 that are expressed in the kidney. Among them, the aminoacylase 1 (Acy1) gene, which maps to 8q32 and is highly expressed in the kidney, displayed a significantly decreased level of expression in RCCs. Significant amounts of the Acy1 protein were detected in the cytoplasm as well as in the nuclei of renal proximal tubular cells of untreated rats. Transfection of Acy1 into RCC cell lines inhibited proliferation and colony formation on soft agar. An increased number of apoptotic cells were observed following Acy1 transfection. The rat 8q31-32 chromosomal region corresponds to human 3p21.31-24.1, a hot spot where LOH is frequently found in various human cancers. Thus, Fe-NTA-induced renal tumor model is ideal for studying the link between deletions within this region and tumor formation. Our data demonstrate that Acy1 functions as a tumor suppressor in this rat RCC model. © The Author 2008. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/carcin/bgn255

Biased Immunoglobulin VH Recombination and Increased Expression of Chemokine Receptor XCR1 Are Distinct Features of Bone Marrow-Derived Diffuse Large B-Cell Lymphoma 国際誌

Yamashita, Y; Kajiura, D; Nakamura, S; Toyokuni, S; Mori, N

MODERN PATHOLOGY 22 巻頁： 292A - 292A 2009年1月

Role of iron in carcinogenesis: cancer as a ferrotoxic disease. 査読有り国際誌 Open Access

Toyokuni S

Cancer science 100 巻 ( 1 ) 頁： 9 - 16 2009年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Iron is abundant universally. During the evolutionary processes, humans have selected iron as a carrier of oxygen inside the body. However, iron works as a double-edged sword, and its excess is a risk for cancer, presumably via generation of reactive oxygen species. Thus far, pathological conditions such as hemochromatosis, chronic viral hepatitis B and C, exposure to asbestos fibers, as well as endometriosis have been recognized as iron overload-associated risks for human cancer. Indeed, iron is carcinogenic in animal experiments. These reports unexpectedly revealed that there are target genes in iron-induced carcinogenesis and that iron-catalyzed oxidative DNA damage is not random in.vivo. Several iron transporters and hepcidin, a peptide hormone regulating iron metabolism, were discovered in the past decade. Furthermore, a recent epidemiological study reported that iron reduction by phlebotomy decreased cancer risk in the apparently normal population. These results warrant reconsideration of the role of iron in carcinogenesis and suggest that fine control of body iron stores would be a wise strategy for cancer prevention. © 2009 Japanese Cancer Association.

DOI： 10.1111/j.1349-7006.2008.01001.x

*Role of iron in carcinogenesis: Cancer as a ferrotoxic disease. 招待有り査読有り

Toyokuni S

Cancer Sci. 100 巻頁： 6-16 2009年1月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. 招待有り

Liu YT, Toyokuni S.

Methods Mol. Biol. 536 巻頁： 307-312 2009年

Lipid Peroxidation Generates a Body Odor Component Trans-2-Nonenal Covalently Bound to Protein in vivo 国際誌

Ishino Kousuke, Wakita Chika, Shibata Takahiro, Toyokuni Shinya, Machida Sachiko, Matsuda Shun, Matsuda Tomonari, Uchida Koji

FREE RADICAL BIOLOGY AND MEDICINE 47 巻頁： S76 - S76 2009年

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. 招待有り

Liu YT, Toyokuni S

Methods Mol. Biol. 536 巻頁： 307-312 2009年

Lipid Peroxidation Generates a Body Odor Component Trans-2-Nonenal Covalently Bound to Protein in vivo 査読有り

Kousuke Ishino, Chika Wakita, Takahiro Shibata, Shinya Toyokuni, Sachiko Machida, Shun Matsuda, Tomonari Matsuda, Koji Uchida

FREE RADICAL BIOLOGY AND MEDICINE 47 巻頁： S76 - S76 2009年

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. 査読有り国際誌

Liu YT, Toyokuni S

Methods in molecular biology (Clifton, N.J.) 536 巻頁： 307 - 12 2009年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Methods in molecular biology (Clifton, N.J.)

We established a shortened protocol for western blot analysis using intermittent microwave irradiation. With this method, the procedure is completed within 1 h after applying the primary antibody, and thus greatly saves time. This procedure appears to be applicable to any antibody based on our experience of several years.

DOI： 10.1007/978-1-59745-542-8_33

S1-3 アスベスト誘発中皮腫の発がん機構の解明とその予防への応用(シンポジウム1:「生活習慣・環境因子とバイオマーカー」)

豊國伸哉

日本環境変異原学会大会プログラム・要旨集 ( 38 ) 2009年

Characteristics and modifying factors of asbestos-induced oxidative DNA damage

JIANG Li, NAGAI Hirotaka, OHARA Hiroki, HARA Shigeo, TACHIBANA Mitsuhiro, HIRANO Seishiro, SHINOHARA Yasushi, KOHYAMA Norihiko, AKATSUKA Shinya, TOYOKUNI Shinya

Cancer science 99 巻 ( 11 ) 頁： 2142 - 2151 2008年11月

*Characteristics and modifying factors of asbestos-induced oxidative DNA damage. 査読有り

Jiang L, Nagai H; Ohara H, Hara S, Tachibana M, Hirano S, Shinohara Y, Kohyama N, Akatsuka S and Toyokuni S.

Cancer Sci. 99 巻 ( 11 ) 頁： 2142-2151 2008年11月

Characteristics and modifying factors of asbestos-induced oxidative DNA damage. 査読有り国際誌 Open Access

Jiang L, Nagai H, Ohara H, Hara S, Tachibana M, Hirano S, Shinohara Y, Kohyama N, Akatsuka S, Toyokuni S

Cancer science 99 巻 ( 11 ) 頁： 2142 - 51 2008年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Respiratory exposure to asbestos has been linked with mesothelioma in humans. However, its carcinogenic mechanism is still unclear. Here we studied the ability of chrysotile, crocidolite and amosite fibers to induce oxidative DNA damage and the modifying factors using four distinct approaches. Electron spin resonance analyses revealed that crocidolite and amosite containing high amounts of iron, but not chrysotile, catalyzed hydroxyl radical generation in the presence of H2O2, which was enhanced by an iron chelator, nitrilotriacetic acid, and suppressed by desferal. Natural iron chelators, such as citrate, adenosine 5′-triphosphate and guanosine 5′-triphosphate, did not inhibit this reaction. Second, we used time-lapse video microscopy to evaluate how cells cope with asbestos fibers. RAW264.7 cells, MeT-5 A and HeLa cells engulfed asbestos fibers, which reached not only cytoplasm but also the nucleus. Third, we utilized supercoiled plasmid DNA to evaluate the ability of each asbestos to induce DNA double strand breaks (DSB). Crocidolite and amosite, but not chrysotile, induced DNA DSB in the presence of iron chelators. We cloned the fragments to identify break sites. DSB occurred preferentially within repeat sequences and between two G:C sequences. Finally, i.p. administration of each asbestos to rats induced not only formation of nuclear 8-hydroxy-2′-deoxyguanosine in the mesothelia, spleen, liver and kidney but also significant iron deposits in the spleen. Together with the established carcinogenicity of i.p. chrysotile, our data suggest that asbestos-associated catalytic iron, whether constitutional or induced by other mechanisms, plays an important role in asbestos-induced carcinogenesis and that chemoprevention may be possible through targeting the catalytic iron. © 2008 Japanese Cancer Association.

DOI： 10.1111/j.1349-7006.2008.00934.x

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias. 査読有り国際誌 Open Access

Kamitsuji Y, Kuroda J, Kimura S, Toyokuni S, Watanabe K, Ashihara E, Tanaka H, Yui Y, Watanabe M, Matsubara H, Mizushima Y, Hiraumi Y, Kawata E, Yoshikawa T, Maekawa T, Nakahata T, Adachi S

Cell death and differentiation 15 巻 ( 11 ) 頁： 1712 - 22 2008年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Death and Differentiation

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl+) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

DOI： 10.1038/cdd.2008.107

Increased susceptibility of chronic ulcerative colitis-induced carcinoma development in DNA repair enzyme Ogg1 deficient mice 査読有り国際誌 Open Access

Liao, J; Seril, DN; Lu, GG; Zhang, M; Toyokuni, S; Yang, AL; Yang, GY

MOLECULAR CARCINOGENESIS 47 巻 ( 8 ) 頁： 638 - 646 2008年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Molecular Carcinogenesis

Ogg1 DNA repair enzyme recognizes and excises oxidative stress-caused 8-hydroxyl-deoxyguanosine (8-OHdG) from GC base-pairs. Ogg1 knockout mice are phenotypically normal, but exhibit elevated levels of 8-OHdG in nuclear and mitochondrial DNA, as well as moderately elevated mutagenesis and spontaneous lung tumors and UV-induced skin tumors. To elucidate the mechanistic role of inflammation-caused oxidative stress in carcinogenesis, the development of chronic ulcerative colitis (UC)-induced carcinoma in Ogg1 knockout mice was studied using a dextran sulfate sodium (DSS)-induced UC model without the use of a carcinogen. Ogg1 (-/-), Ogg1 (+/-), and wild type C57BL/6 mice were subjected to long-term, cyclic DSS treatment to induce chronic UC and carcinogenesis. In wild type C57BL/6 control mice after 15 cycles of DSS treatment, colorectal adenocarcinoma incidence was 24.1% (7/29 mice), with a tumor volume of 27.9 ± 5.2 mm3. Ogg1 (-/-) mice showed significantly increased adenocarcinoma development in the colon with a tumor incidence of 57.1% (12 of 21 mice, P < 0.05) and a tumor volume of 35.1 ± 6.1 mm3. Ogg1 mice (+/-) also exhibited significantly increased tumor development in the colon with a tumor incidence of 50.0% (13/26 mice) and a tumor volume of 29.1 ± 7.2 mm3. Histopathologic analyses revealed that colorectal tumors were well-differentiated tubular adenocarcinomas or mucinous carcinoma and adjacent colonic mucosa showed mild to moderate chronic UC. Using immunohistochemical approaches, Ogg1 (-/-) and (+/-) mice exhibited similar numbers and staining intensities of macrophages in UC areas as seen in Ogg1 (+/+) mice, but markedly increased numbers and staining intensities of 8-OHdG positive inflammatory and epithelial cells. These results provide important evidence on the relationship between inflammation-caused oxidative stress, DNA repair enzyme Ogg1, and carcinogenesis. © 2008 Wiley-Liss, Inc.

DOI： 10.1002/mc.20427

Molecular mechanisms of oxidative stress-induced carcinogenesis: from epidemiology to oxygenomics. 査読有り国際誌 Open Access

Toyokuni S

IUBMB life 60 巻 ( 7 ) 頁： 441 - 7 2008年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：IUBMB Life

Oxidative stress is associated with inflammation, radiation, reperfusion, and iron overload. Epidemiological observations have shown that oxidative stress is one of the major sources of carcinogenesis, the top-ranked cause of human mortality worldwide. In situations of oxidative stress, reactive oxygen and nitrogen species contribute to the alteration of genome information, presumably followed by selection of the adapted proliferating cells in a given environment. Recent data suggest that common molecular mechanisms exist in oxidative stress-induced carcinogenesis, including p16INK4A inactivation. Thus far, oxidative DNA damage in the genome as a cause of mutation has been recognized to be randomly distributed based on in vitro experiments, while localization of oxidative DNA damage in vivo has not been pursued. However, using a novel technique based on DNA immunoprecipitation in combination with genome information, we now know that the localization of oxidative DNA damage is not random in vivo. We propose to call this rather novel research area "oxygenomics." Many signaling pathways start from the recognition of DNA damage. Thus, possible underlying principles should be elucidated in association with each cell type, the genomic location of the damage with its transcriptional activity as well as the chromatin status determining the epigenetic effect. © 2008 IUBMB.

DOI： 10.1002/iub.61

Contents of endometriotic cysts, especially the high concentration of free iron, are a possible cause of carcinogenesis in the cysts through the iron-induced persistent oxidative stress.

Protein N-acylation: H2O2-mediated covalent modification of protein by lipid peroxidation-derived saturated aldehydes. 査読有り国際誌

Ishino K, Shibata T, Ishii T, Liu YT, Toyokuni S, Zhu X, Sayre LM, Uchida K

Chemical research in toxicology 21 巻 ( 6 ) 頁： 1261 - 70 2008年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Research in Toxicology

Various lines of evidence indicate that the oxidative modification of protein and the subsequent accumulation of the degenerated proteins have been found in cells and tissues during aging, oxidative stress, and in a variety of pathological states. The critical agents that give rise to this protein degeneration may be represented by aldehydes. Although the covalent modification of proteins by aldehydes alone has been well-studied, the effect of reactive oxygen species, such as H2O2, upon aldehyde modification of the protein has received little attention. We have now established a unique protein modification in which H2O2 and, to a lesser extent, alkyl hydroperoxides mediate the binding of alkanals to the lysine residues of protein to generate structurally unusual N-acylation products. Upon the reaction of a lysine-containing peptide, Nα-benzoylglycyl- lysine, with hexanal in the presence of H2O2, a product containing one molecule of hexanal per peptide was detected. On the basis of the chemical and spectroscopic evidence, the product was identified to be the acylation product, Nε-hexanoyllysine. H2O2 mediated the N-acylation of the lysine derivative by the saturated aldehydes of 1-6 carbons in length. The H2O2-mediated acylation of the protein was immunochemically confirmed by reaction of the proteins with hexanal in the presence of H2O2. Furthermore, the enhanced N-acylations (N-acetylation and N-hexanoylation) were also observed in the kidney of rats exposed to ferric nitrilotriacetate, a well-characterized inducer of oxidative stress. Mechanistic studies using a phosphonium lysine derivative suggest a Baeyer-Villiger-like reaction proceeding through peroxide addition to the aldehyde Schiff base. These data suggest that the hydroperoxides, including H2O2, might be involved not only in the oxidative modification of protein but also in the covalent binding of the saturated aldehydes to proteins under oxidative stress. © 2008 American Chemical Society.

DOI： 10.1021/tx800080x

Pluripotency of a single spermatogonial stem cell in mice. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Lee J, Inoue K, Ogonuki N, Miki H, Toyokuni S, Ikawa M, Nakamura T, Ogura A, Shinohara T

Biology of reproduction 78 巻 ( 4 ) 頁： 681 - 7 2008年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Although pluripotent stem cells were recently discovered in postnatal testis, attempts to analyze their developmental potential have led to conflicting claims that spermatogonial stem cells are pluripotent or that they lose spermatogenic potential after conversion into pluripotent stem cells. To examine this issue, we analyzed the developmental fate of a single spermatogonial stem cell that appeared during transfection experiments. After transfection of a neomycin-resistance gene into germline stem cells, we obtained an embryonic stem-like, multipotent germline stem cell line. Southern blot analysis revealed that the germline stem and multipotent germline stem clones have the same transgene integration pattern, demonstrating their identical origin. The two lines, however, have different DNA methylation patterns. The multipotent germline stem cells formed chimeras after blastocyst injection but did not produce sperm after germ cell transplantation, whereas the germline stem cells could produce only spermatozoa and did not differentiate into somatic cells. Interestingly, the germline stem cells expressed several transcription factors (Pou5f1, Sox2, Myc, and Klf4) required for reprogramming fibroblasts into a pluripotent state, suggesting that they are potentially pluripotent. Thus, our study provides evidence that a single spermatogonial stem cell can acquire pluripotentiality but that conversion into a pluripotent cell type is accompanied by loss of spermatogenic potential. © 2008 by the Society for the Study of Reproduction, Inc.

DOI： 10.1095/biolreprod.107.066068

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model 査読有り国際誌

Hakozaki, T; Date, A; Yoshii, T; Toyokuni, S; Yasui, H; Sakurai, H

ARCHIVES OF DERMATOLOGICAL RESEARCH 300 巻 ( SUPPL. 1 ) 頁： S51 - S56 2008年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Dermatological Research

Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, β-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2′-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with β-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O 2.- in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses. © 2007 Springer-Verlag.

DOI： 10.1007/s00403-007-0804-3

豊國伸哉

日本衞生學雜誌 63 巻 ( 2 ) 頁： 282 2008年3月

酸化ストレス発がんの標的遺伝子について

豊國伸哉

日本衞生學雜誌 63 巻 ( 2 ) 頁： 282 - 282 2008年3月

Yamaguchi K, Mandai M, Toyokuni S, Hamanishi J, Higuchi T, Takakura K, Fujii S

Clinical cancer research : an official journal of the American Association for Cancer Research 14 巻 ( 1 ) 頁： 32 - 40 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Clinical Cancer Research

Purpose: Endometriotic cysts are known to transforminto ovarian cancers, such as clear cell and endometrioid carcinomas. We hypothesized that an iron-rich environment produced by the repetition of hemorrhage in the endometriotic cysts during the reproductive period may play a crucial role in carcinogenesis in the cysts through the iron-induced persistent oxidative stress. Experimental Design: Contents of human ovarian cysts, including 21 endometriotic cysts, 4 clear cell carcinomas, and 11 nonendometriotic cysts,were analyzed for the concentrations of free "catalytic" iron, lactose dehydrogenase, potential antioxidant, lipid peroxide, and 8-hydroxy-2′- deoxyguanosine (8-OHdG). Iron deposition and 8-OHdG levels were also analyzed histologically. Reactive oxygen species and the mutagenicity of the contents in endometriotic cyst were determined in vitro. Results: The concentration of free iron in endometriotic cysts (100.9 mmol/L) was significantly higher than that in nonendometriotic cysts (0.075 mmol/L; P < 0.01). The average concentrations of lactose dehydrogenase, potential antioxidant, lipidperoxide, and 8-OHdG were also significantly higher in endometriotic cysts (P < 0.01). There was a correlation between the concentration of free iron and that of 8-OHdG (P < 0.01). Histologically, we could observe iron deposits more abundantly in endometriotic cysts than in nonendometriotic cysts (P < 0.01). The level of 8-OHdG in carcinoma associated with endometriosis was higher than that of carcinoma without endometriosis (P < 0.05). In vitro analyses showed that the contents of endometriotic cyst couldproducemore reactiveoxygen species and could induce genemutations more frequently than the contents in the other cysts. Conclusions: Abundant free iron in the contents of endometriotic cysts was strongly associated with greater oxidative stress and frequent DNA mutations. A long-standing history of the RBCs accumulated in the ovarian endometriotic cysts during the reproductive period produces oxidative stress that is a possible cause for the malignant change of the endometriotic cyst. © 2008 American Association for Cancer Research.

DOI： 10.1158/1078-0432.CCR-07-1614

Overexpression of CYP3A aggravates endotoxin-induced liver injury in hypophysectomized female rats

TAKEMURA Shigekazu, MINAMIYAMA Yukiko, TOYOKUNI Shinya, IMAOKA Susumu, HAI Seikan, KUBO Shoji, HIROHASHI Kazuhiro, FUNAE Yoshihiko, OKADA Shigeru

Hepatology research : the official journal of the Japan Society of Hepatology 38 巻 ( 1 ) 頁： 70 - 78 2008年1月

Contents of endometriotic cysts, especially the high concentration of free iron, are a possible cause of carcinogenesis in the cysts through the iron-induced persistent oxidative stress

Yamaguchi Ken, Mandai Masaki, Toyokuni Shinya, Hamanishi Junzo, Higuchi Toshihiro, Takakura Kenji, Fujii Shingo

CLINICAL CANCER RESEARCH 14 巻 ( 1 ) 頁： 32 - 40 2008年1月

Overexpression of CYP3A aggravates endotoxin-induced liver injury in hypophysectomized female rats 査読有り国際誌

Takemura, S; Minamiyama, Y; Toyokuni, S; Imaoka, S; Hai, S; Kubo, S; Hirohashi, K; Funae, Y; Okada, S

HEPATOLOGY RESEARCH 38 巻 ( 1 ) 頁： 70 - 78 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Hepatology Research

Aim: CYP3A2 is a male-specific isoform of cytochrome P450 enzyme which is expressed abundantly in male rats but not in intact female rats. Having previously reported that hepatic CYP3A2 promotes lipopolysaccharide (LPS)-induced liver injury in male rats, we further examined the impact of CYP3A on LPS-induced liver injury by comparing intact and hypophysectomized female rats. In hypophysectomized female rats, phenobarbital (PB), a cytochrome P450 inducer, markedly increased the hepatic content and activity of CYP3A1/2, but did not do so in intact rats. CYP2B1 increased to similar levels in PB-treated hypophysectomized and intact rats. Methods: Rats were administered 10 mg/kg LPS intravenously and some were given PB for threedays before LPS injection. Liver injury was analyzed 8h after LPS injection. Results: PB-LPS increased plasma alanine aminotransferase significantly more in hypophysectomized female rats than in intact female rats. Ketoconazole, a CYP3A inhibitor, inhibited the increase of liver injury. Hepatic 8-hydroxydeoxyguanosine in nuclei and 4-hydroxy-2-nonenal-modified proteins, measured to evaluate oxidative stress by LPS treatment, increased markedly more in PB-treated, hypophysectomized female rats, than in intact female rats. Conclusion: Overexpression of CYP3A aggravated LPS-induced liver injury in rats, apparently through the formation of reactive oxygen species. © 2007 The Japan Society of Hepatology.

DOI： 10.1111/j.1872-034X.2007.00212.x

Contents of endometriotic cysts, especially the high concentration of free iron, are a possible cause of carcinogenesis in the cysts through the iron-induced persistent oxidative stress 査読有り

Ken Yamaguchi, Masaki Mandai, Shinya Toyokuni, Junzo Hamanishi, Toshihiro Higuchi, Kenji Takakura, Shingo Fujii

CLINICAL CANCER RESEARCH 14 巻 ( 1 ) 頁： 32 - 40 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

Purpose: Endometriotic cysts are known to transform into ovarian cancers, such as clear cell and enclometrioid carcinomas. We hypothesized that an iron-rich environment produced by the repetition of hemorrhage in the endometriotic cysts during the reproductive period may play a crucial role in carcinogenesis in the cysts through the iron-induced persistent oxidative stress.
Experimental Design: Contents of human ovarian cysts, including 21 enclometriotic cysts, 4 clear cell carcinomas, and 11 nonendometriotic cysts, were analyzed for the concentrations of free "catalytic" iron, lactose clehydrogenase, potential antioxiclant, lipid peroxide, and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Iron deposition and 8-OHdG levels were also analyzed histologically. Reactive oxygen species and the mutagenicity of the contents in enclometriotic cyst were determined in vitro.
Results: The concentration of free iron in endometriotic cysts (100.9 mmol/L) was significantly higher than that in nonendometriotic cysts (0.075 mmol/L; P < 0.01). The average concentrations of lactose dehydrogenase, potential antioxiclant, lipid peroxide, and 8-OHdG were also significantly higher in enclometriotic cysts (P < 0.01).There was a correlation between the concentration of free iron and that of 8-OHdG (P < 0.01). Histologically, we could observe iron deposits more abundantly in endometriotic cysts than in nonendometriotic cysts (P < 0.01).The level of 8-OHdG in carcinoma associated with endometriosis was higher than that of carcinoma without endometriosis (P < 0.05). In vitro analyses showed that the contents of enclometriotic cyst could produce more reactive oxygen species and could induce gene mutations more frequently than the contents in the other cysts.
Conclusions: Abundant free iron in the contents of enclometriotic cysts was strongly associated with greater oxidative stress and frequent DNA mutations. A long-standing history of the RBCs accumulated in the ovarian endometriotic cysts during the reproductive period produces oxidative stress that is a possible cause for the malignant change of the endometriotic cyst.

Association of microRNA-34a overexpression with proliferation is cell type-dependent

A reporter gene assay to prove the ability of natural compounds to modulate gluthathione peroxidase, superoxide dismutase and catalase gene promoters in V79 cells. 査読有り

Ullmann K, Wiencierz A M, Müller C, Thierbach R, Steege A, Toyokuni S and Steinberg P.

Free Radic. Res. 42 巻頁： 746-753 2008年

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias. 査読有り

Kamitsuji Y, Kuroda J, Kimura S, Toyokuni S, Watanabe K, Ashihara E, Tanaka H, Yui Y, Watanabe M, Matsubara H, Mizushima Y, Hiraumi Y, Kawata E, Yoshikawa T, Maekawa T, Nakahata T, Adachi S.

Cell Death Differ. 15 巻頁： 1712-1722 2008年

Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inihibitor FK228 through AIF translocation. 査読有り

Watanabe M, Adachi S, Matsubara H, Imai T, Yui Y, Mizushima Y, Hiraumi Y, Watanabe KI, Kamitsuji Y, Toyokuni S, Hosoi H, Sugimoto T, Tokuchida J and Nakahata T.

Int. J. Cancer 124 巻頁： 55-67 2008年

Genome-wide analysis identifies a tumor suppressor role for aminoacylase 1 in iron-induced rat renal cell carcinoma. 査読有り

Zhong Y, Onuki J, Yamasaki T. Ogawa O, Akatsuka S and Toyokuni S.

Carcinogenesis 頁： in press 2008年

Protein N-acylation: H2O2-mediated covalent modification of protein by lipid peroxidation-derived saturated aldehydes. 査読有り

Ishino K, Shibata T, Ishii T, Liu YT, Toyokuni S, Zhu X, Sayre LM and Uchida K.

Chem. Res. Toxicol. 21 巻頁： 1261-1270 2008年

Overexpression of CYP3A aggravates endotoxin-induced liver injury in hypophysectomized female rats. 査読有り

Takemura S, Minamiyama Y, Toyokuni S, Imaoka S, Hai S, Kubo S, Hirohashi K, Funae Y and Okada S.

Hepatol. Res. 38 巻頁： 70-78 2008年

Pluripotency of a single spermatogonial stem cell. 査読有り

Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Yoshida S, Toyokuni S, Lee J, Ogura A and Shinohara T.

Biol. Reprod. 78 巻頁： 681-687 2008年

Contents of endometriotic cysts, especially the high concentration of free iron, are a possible cause of carcinogenesis in the cysts through the iron-induced persistent oxidative stress. 査読有り

Yamaguchi K, Mandai M, Toyokuni S, Hamanishi J, Higuchi T, Takakura K and Fujii S.

Clin. Cancer Res. 14 巻頁： 32-40 2008年

Molecular mechanisms of oxidative stress-induced carcinogenesis: from epidemiology to oxygenomics. 招待有り査読有り

Toyokuni S.

IUBMB Life 60 巻頁： 441-447 2008年

Increased susceptibility of chronic ulcerative colitis-induced carcinoma development in DNA repair enzyme Ogg1 deficient mice. 査読有り

Liao J, Seril DN, Lu GG, Zhang M, Toyokuni S, Yang AL and Yang GY.

Mol. Carcinog. 47 巻頁： 638-646 2008年

A high-throughput reporter gene assay to prove the ability of natural compounds to modulate glutathione peroxidase, superoxide dismutase and catalase gene promoters in V79 cells 査読有り国際誌

Ullmann, K; Wiencierz, AM; Müller, C; Thierbach, R; Steege, A; Toyokuni, S; Steinberg, P

FREE RADICAL RESEARCH 42 巻 ( 8 ) 頁： 746 - 753 2008年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

The aim of the study was to establish a 96-well microtiter plate-based reporter gene assay to test the influence of natural compounds on the promoter activities of rat catalase, human glutathione peroxidase and human superoxide dismutase expressed in V79 cells. Luciferase expression vectors with the promoter regions of the genes coding for the three above-mentioned enzymes were constructed and transfected into V79 cells. Thereafter the ability of sodium ascorbate, L-carnitine, catechin, epigallocatechin gallate, genistein, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate and Trolox to enhance the promoter activities was evaluated. Genistein, paraquat and quercetin led to a statistically significant increase in the glutathione peroxidase and superoxide dismutase gene promoter activities. None of the compounds tested enhanced the catalase gene promoter activity. The reporter gene assay described in this report is easy to perform, fast and allows one to test a high number of compounds and different concentrations of a single compound at the same time.

DOI： 10.1080/10715760802337273

Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inihibitor FK228 through AIF translocation. 査読有り

Watanabe M, Adachi S, Matsubara H, Imai T, Yui Y, Mizushima Y, Hiraumi Y, Watanabe KI, Kamitsuji Y, Toyokuni S, Hosoi H, Sugimoto T, Tokuchida J, Nakahata T

Int. J. Cancer 124 巻頁： 55-67 2008年

Molecular mechanisms of oxidative stress-induced carcinogenesis: from epidemiology to oxygenomics. 招待有り査読有り

Toyokuni S

IUBMB Life 60 巻頁： 441-447 - 447 2008年

DUTTA Khokon Kumar, ZHONG Yi, LIU Yu-Ting, YAMADA Takuji, AKATSUKA Shinya, HU Qian, YOSHIHARA Minako, OHARA Hiroki, TAKEHASHI Masanori, SHINOHARA Takashi, MASUTANI Hiroshi, ONUKI Janice, TOYOKUNI Shinya

Cancer science 98 巻 ( 12 ) 頁： 1845 - 1852 2007年12月

Production of knockout mice by gene targeting in multipotent germline stem cells. 査読有り国際誌 Open Access

Takehashi M, Kanatsu-Shinohara M, Miki H, Lee J, Kazuki Y, Inoue K, Ogonuki N, Toyokuni S, Oshimura M, Ogura A, Shinohara T

Developmental biology 312 巻 ( 1 ) 頁： 344 - 52 2007年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Developmental Biology

Spermatogonial stem cells can convert into embryonic stem (ES) cell-like multipotent germline stem (mGS) cells in vitro and produce germline chimeras by blastocyst microinjection. Although homologous recombination was previously demonstrated in mGS cells, spermatogenesis was not found in chimeras, suggesting that they are not competent for germline modification. Here we conducted detailed analysis of chimeric animals to determine whether mGS cells retain germline potential after genetic manipulation. Spermatozoa that were deficient in the occludin gene could be recovered from animals that were chimeric with mGS cells that underwent homologous recombination. The phenotypes of the occludin knockout (KO) mice were similar to those reported for KO mice produced using ES cells, and the animals showed growth retardation, gastritis and male infertility. Furthermore, we found that heterozygous mGS cells acquire two copies of the G418-resistant genes and become homozygous for the targeted allele by culturing at high concentrations of G418. Cytogenetic analysis showed that the aneuploid mGS cells observed during genetic manipulation were trisomic for chromosome 8 or 11, which is a common chromosomal abnormality in ES cells. Thus, mGS cells can be used to produce KO animals, and this novel method of germline manipulation may prove useful in diverse mammalian species. © 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2007.09.029

Association of microRNA-34a overexpression with proliferation is cell type-dependent. 査読有り国際誌 Open Access

Dutta KK, Zhong Y, Liu YT, Yamada T, Akatsuka S, Hu Q, Yoshihara M, Ohara H, Takehashi M, Shinohara T, Masutani H, Onuki J, Toyokuni S

Cancer science 98 巻 ( 12 ) 頁： 1845 - 52 2007年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

Recently Welch et al. reported that microRNA (miRNA)-34a functions as a potential tumor suppressor in neuroblastoma cells (Oncogene 26: 5017-22, 2007). Here, we conversely show that miRNA-34a supports cell proliferation in rat oxidative stress-induced renal carcinogenesis and is overexpressed in various types of human cancers. While searching for genetically unstable chromosomal areas in rat renal carcinogenesis, we found the miRNA-34 family reciprocally overexpressed in chromosomal areas with frequent allelic loss. By in situ hybridization and reverse transcription-polymerase chain reaction, cerebral neurons and Purkinje cells showed the highest expression of a major type, miRNA-34a, followed by a variety of endocrine cells and proliferating cells including germinal center lymphocytes and mouse embryonic fibroblasts and stem cells. In contrast, normal renal tubules, hepatocytes and myocardial cells showed faint expression. After 3 weeks of ferric nitrilotriacetate (Fe-NTA)-induced oxidative stress, regenerating renal proximal tubular cells showed high miRNA-34a expression. All of the Fe-NTA-induced rat renal carcinomas and an array of human cancers (151 positive cases of 177) showed high expression of miRNA-34a. Furthermore, knockdown of miRNA-34a with small interfering RNA significantly suppressed proliferation not only of renal carcinoma cells but also of HeLa and MCF7 cells. These results indicate that miRNA-34a overexpression, an acquired trait during carcinogenesis, supports cell proliferation in the majority of cancers suggesting an unexpected link in the cellular metabolism between cancer and neuronal and/or endocrine cells, which warrants further investigation. © 2007 Japanese Cancer Association.

DOI： 10.1111/j.1349-7006.2007.00619.x

Chronic oxidative stress causes amplification and overexpression of ptprz1 protein tyrosine phosphatase to activate beta-catenin pathway

Liu Yu-Ting, Shang Donghao, Akatsuka Shinya, Ohara Hiroki, Dutta Khokon Kumar, Mizushima Katsura, Naito Yuji, Yoshikawa Toshikazu, Izumiya Masashi, Abe Kouichiro, Nakagama Hitoshi, Noguchi Noriko, Toyokuni Shinya

AMERICAN JOURNAL OF PATHOLOGY 171 巻 ( 6 ) 頁： 1978 - 1988 2007年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Pathology

Ferric nitrilotriacetate induces oxidative renal tubular damage via Fenton-reaction, which subsequently leads to renal cell carcinoma (RCC) in rodents. Here, we used gene expression microarray and array-based comparative genomic hybridization analyses to find target oncogenes in this model. At the common chromosomal region of amplification (4q22) in rat RCCs, we found ptprz1, a tyrosine phosphatase (also known as protein tyrosine phosphatase ζ or receptor tyrosine phosphatase β) highly expressed in the RCCs. Analyses revealed genomic amplification up to eightfold. Despite scarcity in the control kidney, the amounts of PTPRZ1 were increased in the kidney after 3 weeks of oxidative stress, and mRNA levels were increased 16∼552-fold in the RCCs. Network analysis of the expression revealed the involvement of the β-catenin pathway in the RCCs. In the RCCs, dephosphorylated β-catenin was translocated to nuclei, resulting in the expression of its target genes cyclin D1, c-myc, c-jun, fra-1, and CD44. Furthermore, knockdown of ptprz1 with small interfering RNA (siRNA), in FRCC-001 and FRCC-562 cell lines established from the induced RCCs, decreased the amounts of nuclear β-catenin and suppressed cellular proliferation concomitant with a decrease in the expression of target genes. These results demonstrate that chronic oxidative stress can induce genomic amplification of ptprz1, activating β-catenin pathways without the involvement of Wnt signaling for carcinogenesis. Thus, iron-mediated persistent oxidative stress confers an environment for gene amplification. Copyright © American Society for Investigative Pathology.

DOI： 10.2353/ajpath.2007.070741

Chronic oxidative stress causes amplification and overexpression of ptprz1 protein tyrosine phosphatase to activate beta-catenin pathway 査読有り

Yu-Ting Liu, Donghao Shang, Shinya Akatsuka, Hiroki Ohara, Khokon Kumar Dutta, Katsura Mizushima, Yuji Naito, Toshikazu Yoshikawa, Masashi Izumiya, Kouichiro Abe, Hitoshi Nakagama, Noriko Noguchi, Shinya Toyokuni

AMERICAN JOURNAL OF PATHOLOGY 171 巻 ( 6 ) 頁： 1978 - 1988 2007年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC INVESTIGATIVE PATHOLOGY, INC

Ferric nitrilotriacetate induces oxidative renal tubular damage via Fenton-reaction, which subsequently leads to renal cell carcinoma (RCC) in rodents. Here, we used gene expression microarray and array-based comparative genomic hybridization analyses to find target oncogenes in this model. At the common chromosomal region of amplification (4q22) in rat RCCs, we found ptprz1, a tyrosine phosphatase (also known as protein tyrosine phosphatase zeta or receptor tyrosine phosphatase beta) highly expressed in the RCCs. Analyses revealed genomic amplification up to eightfold. Despite scarcity in the control kidney, the amounts of PTPRZ1 were increased in the kidney after 3 weeks of oxidative stress, and mRNA levels were increased 16 similar to 552-fold in the RCCs. Network analysis of the expression revealed the involvement of the beta-catenin pathway in the RCCs. in the RCCs, dephosphorylated beta-catenin was translocated to nuclei, resulting in the expression of its target genes cyclin D1, c-myc, c-jun, fra-1, and CD44. Furthermore, knockdown of ptprz1 with small interfering RNA (siRNA), in FRCC-001 and FRCC-562 cell lines established from the induced RCCs, decreased the amounts of nuclear beta-catenin and suppressed cellular proliferation concomitant with a decrease in the expression of target genes. These results demonstrate that chronic oxidative stress can induce genomic amplification of ptprz1, activating beta-catenin pathways without the involvement of Wnt signaling for carcinogenesis. Thus, iron-mediated persistent oxidative stress confers an environment for gene amplification.

DOI： 10.2353/ajpath.2007-070741

Oxidative stress is related to the formation of Antoni B patterns and eosinophilic hyaline droplets in schwannomas

FasL expression in hepatic antigen-presenting cells and phagocytosis of apoptotic T cells by FasL+ Kupffer cells are indicators of rejection activity in human liver allografts. 査読有り国際誌 Open Access

Miyagawa-Hayashino A, Tsuruyama T, Egawa H, Haga H, Sakashita H, Okuno T, Toyokuni S, Tamaki K, Yamabe H, Manabe T, Uemoto S

The American journal of pathology 171 巻 ( 5 ) 頁： 1499 - 508 2007年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Pathology

Fas-Fas ligand (FasL) interaction and apoptosis are important in the mechanism of allograft rejection. However, the interaction between donor and recipient cells, specifically focusing on antigen-presenting cells (APCs), under various conditions is poorly understood in human liver allografts. FasL expression on APCs, its association with apoptosis, and the origin of apoptotic lymphocytes in human liver allografts were assessed by immunohistochemistry and in situ hybridization. We found increased expression of FasL on Kupffer cells (KCs) and endothelium in acute cellular rejection (w = 20) and to lesser extent in chronic rejection (n = 6) and septic cholangitis (n = 5) compared with stable grafts and normal controls. In addition, the graft specificity of infiltrating T cells was confirmed by polymerase chain reaction examination of T-cell receptor-γ loci. T-cell apoptosis occurred at a higher rate in acute cellular rejection than in chronic rejection or septic cholangitis. The number of apoptotic bodies derived from recipient lymphocytes correlated with the severity of rejection and was reversed by treatment. FasL+ KCs phagocytosed CD4+ interferon-γ+ T cells, rather dian CD4+ interleukin-4+ T cells, suggesting a role of KCs in regulating CD4+ T-cell subset differentiation. In conclusion, our data suggest that FasL expression on APCs and phagocytosis of apoptotic T cells by FasL+ KCs are indicators of rejec tion activity in human liver allografts. Copyright © American Society for Investigative Pathology.

DOI： 10.2353/ajpath.2007.070027

Overexpression of a transcription factor LYL1 induces T- and B-cell lymphoma in mice. 査読有り国際誌

Zhong Y, Jiang L, Hiai H, Toyokuni S, Yamada Y

Oncogene 26 巻 ( 48 ) 頁： 6937 - 47 2007年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Oncogene

LYL1, a member of the class II basic helix-loop-helix transcription factors, is aberrantly expressed in a fraction of human T-cell acute lymphoblastic leukemia. Here, we generated transgenic mice ubiquitously overexpressing LYL1 using a construct expressing full-length cDNA driven by a human elongation factor 1α promoter. Four independent lines exhibiting high LYL1 expression were established. Of these transgenic mice, 96% displayed loss of hair with a short kinked tail. Furthermore, 30% of them developed malignant lymphoma, with an average latent period of 352 days. In these mice, histological examination revealed tumor cell infiltration in multiple organs and immunohistochemical analysis showed that the infiltrated tumor cells were either CD3 or CD45R/B220-positive; fluorescence-activated cell sorter analysis indicated that each tumor consisted either of mainly CD4, CD8 double-positive T cells or mature B cells; the clonality of LYL1-induced lymphoma was confirmed by T-cell receptor rearrangement and immunoglobulin heavy-chain gene rearrangement analyses. Mammalian two-hybrid analysis and luciferase assay suggested that excess LYL1 blocked the dimerization of E2A and thus inhibited the regulatory activity of E2A on the CD4 promoter. Reverse transcription-polymerase chain reaction results showed that the expression of certain E2A/HEB target genes was downregulated. Taken together, our results provide direct evidence that aberrant expression of LYL1 plays a role in lymphomagenesis. © 2007 Nature Publishing Group All rights reserved.

DOI： 10.1038/sj.onc.1210494

Pathological investigation of oxidative stress in the post-genomic era. 招待有り査読有り国際誌

Toyokuni S, Akatsuka S

Pathology international 57 巻 ( 8 ) 頁： 461 - 73 2007年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

Aerobes, including humans, are consistently exposed to oxidative stress by consuming oxygen. The biological significance of oxidative stress via reactive oxygen and nitrogen species consists of two stages: reversible redox regulation and irreversible oxidative molecular damage, which are sometimes intermingled. During the past decade, many signaling cascades associated with oxidative stress have been discovered. An interaction between Keap1 and the Nrf2 transcription factor is among the most fundamental mechanisms of the defense system against oxidative or similar stress. Furthermore, it became apparent that reactive oxygen species are actively produced through enzymes such as xanthine oxidoreductase and nicotinamide adenine dinucleotide phosphate, reduced (NADPH) oxidases in non-phagocytic cells as well. The role of α-tocopherol solely as an anti-oxidant was also questioned. Now there is a long list of pathological states implicating oxidative stress. At the same time, genome projects on various species have been completed. These efforts convincingly led to a new era of oxidative stress investigation, contributing powerful strategies to select candidate genes or biomolecules. Herein are reviewed recent advances and novel concepts in this field, including oxygenomics. These fruitful results may lead to more accurate and useful pathological diagnosis and more efficient prophylaxis and therapeutic interventions on human diseases. © 2007 The Authors.

DOI： 10.1111/j.1440-1827.2007.02127.x

アレイCGH法を用いた酸化ストレス誘発ラット腎癌における染色体変化の解析(Analysis of chromosomal alterations in oxidative stress-induced rat renal carcinomas using array-based CGH)

赤塚慎也, 泉谷昌志, 阿部浩一郎, 劉玉亭, 胡茜, 大原浩貴, 落合雅子, 中釜斉, 豊國伸哉

日本癌学会総会記事 66回巻頁： 343 - 344 2007年8月

YOKOO Hideaki, OISHI Takuma, ISODA Koji, NAKAZATO Yoichi, TOYOKUNI Shinya

Neuropathology : official journal the Japanese Society of Neuropathology 27 巻 ( 3 ) 頁： 237 - 244 2007年6月

Analysis of genes associated with karyomegalic cells in rat kidney after ferric nitilotriacetate treatment

Lee, WH; Toyokuni, S

AMERICAN JOURNAL OF HEMATOLOGY 82 巻 ( 6 ) 頁： 602 - 602 2007年6月

Novel approach for elucidation of iron-induced carcinogenesis: Oxygenomics and micro-RNA

Toyokuni, S; Dutta, KK; Akatsuka, S

AMERICAN JOURNAL OF HEMATOLOGY 82 巻 ( 6 ) 頁： 512 - 512 2007年6月

Oxidative stress is related to the formation of Antoni B patterns and eosinophilic hyaline droplets in schwannomas 査読有り国際誌

Yokoo, H; Oishi, T; Isoda, K; Nakazato, Y; Toyokuni, S

NEUROPATHOLOGY 27 巻 ( 3 ) 頁： 237 - 244 2007年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Neuropathology

Schwannomas, particularly of vestibular origin, often accompany degenerative hypocellular areas known as Antoni B patterns; however, the detailed mechanism is uncertain. Eosinophilic hyaline droplets (EHD), the substantial nature of which are autophagic vacuoles, preferentially appear in acoustic schwannomas and distribute around areas of Antoni B. We investigated their common background using schwannomas with (15 cases) or without (10 cases) EHD, and demonstrated that EHD showed selective immunoreactivity with an anti-nitrotyrosine antibody, suggesting the overproduction of nitric oxide in this condition. The expression of inducible nitric oxide synthase was emphasized in infiltrating macrophages around hyalinized vessels. Protein-bound 4-hydroxy 2-nonenal, another oxidative stress marker, was detected in Antoni B tissue, but not in EHD. Antibodies to cleaved caspase-3 and single strand DNA, indicators of apoptosis, did not label tumors cells in Antoni B areas as well as EHD-bearing cells. The morphology and the mitotically static state of EHD-laden cells are phenotypically similar to autophagic cell death; however, autophagy in normal cells is a cell survival strategy against starvation, so the possibility remains that EHD are formed in that context. In either case, schwannomas may show a characteristic autophagic change by an endogenous mechanism. Tumor growth in a narrow intracranial space and resultant ischemia by self-oppression were postulated to be an initial event, because ischemia-reperfusion injury is a major source of reactive oxygen species and ischemia is also a potent trigger of autophagy as well as of tissue degeneration. Moreover, potential roles of chemokines and hemosiderosis are discussed. © 2007 Japanese Society of Neuropathology.

DOI： 10.1111/j.1440-1789.2007.00772.x

Analysis of genes associated with karyo-megalic cells in rat kidney after ferric nitilotriacetate treatment 国際誌

Novel approach for elucidation of iron-induced carcinogenesis: Oxygenomics and micro-RNA 国際誌

Toyokuni, S, Dutta, KK, Akatsuka, S

AMERICAN JOURNAL OF HEMATOLOGY 82 巻 ( 6 ) 頁： 512 - 512 2007年6月

Analysis of genes associated with karyomegalic cells in rat kidney after ferric nitilotriacetate treatment 査読有り国際共著国際誌

Lee, WH, Toyokuni, S

AMERICAN JOURNAL OF HEMATOLOGY 82 巻 ( 6 ) 頁： 602 - 602 2007年6月

Akt mediates self-renewal division of mouse spermatogonial stem cells. 査読有り国際誌 Open Access

Lee J, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Kimura T, Nakano T, Ogura A, Shinohara T

Development (Cambridge, England) 134 巻 ( 10 ) 頁： 1853 - 9 2007年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Development

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (P13K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of P13K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the P13K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.

DOI： 10.1242/dev.003004

Adenovirus-mediated gene delivery into mouse spermatogonial stem cells. 査読有り国際誌 Open Access

Takehashi M, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Ogura A, Shinohara T

Proceedings of the National Academy of Sciences of the United States of America 104 巻 ( 8 ) 頁： 2596 - 601 2007年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49-76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells. © 2007 by The National Academy of Sciences of the USA.

DOI： 10.1073/pnas.0609282104

Adult pancreatic islets require differential pax6 gene dosage. 査読有り国際誌 Open Access

Hamasaki A, Yamada Y, Kurose T, Ban N, Nagashima K, Takahashi A, Fujimoto S, Shimono D, Fujiwara M, Toyokuni S, Seino Y, Inagaki N

Biochemical and biophysical research communications 353 巻 ( 1 ) 頁： 40 - 6 2007年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Pax6, a paired homeodomain transcription factor, plays crucial roles in morphogenesis of eye, central nervous system, and pancreatic islets. Recently, heterozygosity for pax6 mutation has been reported in some individuals with glucose intolerance and aniridia. To investigate the role of pax6 for pancreatic islet function, we examined the pancreatic phenotype of small eye rat strain (rSey2) with a point mutation in the pax6 locus resulting in truncated PAX6 proteins. Analyses of the insulin secretory profile of heterozygous rSey2/+ revealed that insulin secretion is significantly increased in response to membrane-depolarizing stimuli such as arginine, tolbutamide, and KCl. The processes of insulin granule exocytosis were suggested to be enhanced in rSey2/+. On the other hand, pancreatic insulin and glucagon content and islet architecture in rSey2/+ showed no significant differences compared to wild-type. These findings indicate differential requirements for pax6 gene dosage in displaying function and maintaining architecture of adult pancreatic islets. © 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.11.105

Generation of insulin-secreting cells from pancreatic acinar cells of animal models of type 1 diabetes 査読有り国際誌

Okuno, M; Minami, K; Okumachi, A; Miyawaki, K; Yokoi, N; Toyokuni, S; Seino, S

AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 292 巻 ( 1 ) 頁： E158 - E165 2007年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Physiology - Endocrinology and Metabolism

We recently found that pancreatic acinar cells isolated from normal adult mouse can transdifferentiate into insulin-secreting cells in vitro. Using two different animal models of type 1 diabetes, we show here that insulin-secreting cells can also be generated from pancreatic acinar cells of rodents in the diabetic state with absolute insulin deficiency. When pancreatic acinar cells of streptozotocin-treated mice were cultured in suspension in the presence of epidermal growth factor and nicotinamide under low-serum condition, expressions of insulin genes gradually increased. In addition, expressions of other pancreatic hormones, including glucagon, somatostatin, and pancreatic polypeptide, were also induced. Analysis by the Cre/loxP-based direct cell lineage tracing system revealed that these newly made cells originated from amylase-expressing pancreatic acinar cells. Insulin secretion from the newly made cells was significantly stimulated by high glucose and other secretagogues. In addition, insulin-secreting cells were generated from pancreatic acinar cells of Komeda diabetesprone rats, another animal model of type 1 diabetes. The present study demonstrates that insulin-secreting cells can be generated by transdifferentiation from pancreatic acinar cells of rodents in the diabetic state and further suggests that pancreatic acinar cells represent a potential source of autologous transplantable insulin-secreting cells for treatment of type 1 diabetes. Copyright © 2007 the American Physiological Society.

DOI： 10.1152/ajpendo.00180.2006

Leukemia inhibitory factor enhances formation of germ cell colonies in neonatal mouse testis culture. 国際誌 Open Access

Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Yoshida S, Toyokuni S, Lee J, Ogura A, Shinohara T

Biology of reproduction 76 巻 ( 1 ) 頁： 55 - 62 2007年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogonial stem cells continuously divide in the testis to support spermatogenesis throughout the life of adult male animals. Although very few spermatogonial stem cells are present in vivo, we recently succeeded in expanding these cells in vitro. Germ cells from postnatal testes were able to proliferate in the presence of several types of cytokines, and they formed uniquely shaped colonies of spermatogonia (germline stem or GS cells). These cells reinitiated normal spermatogenesis when transplanted into seminiferous tubules. However, much remains unknown about the contributions of cytokines to successful stem cell culture. In the present study, we examined the role of leukemia inhibitory factor (LIF) in GS cell culture. We found that the addition of LIF to newborn testis cell culture enhances the formation of germ cell colonies. Ciliary neurotrophic factor, but not oncostatin M, had the same effect, although they both bind to the IL-6ST (gp130) receptor. On the other hand, GS cells could be established from pup or adult testes in the absence of LIF. No phenotypic or functional difference was found between CS cells established from different stages, and normal offspring were born from pup-derived GS cells that had been maintained in the absence of LIF, indicating that LIF per se is not involved in the self-renewal of GS cells. These results demonstrate that LIF is useful in the initiation of GS cell culture and suggest that LIF or a related cytokine is involved in the maturation of gonocytes into spermatogonia. © 2007 by the Society for the Study of Reproduction, Inc.

DOI： 10.1095/biolreprod.106.055863

*Chronic oxidative stress causes amplification and overexpresson of ptprz1 protein tyrosine phosphatase to activate beta-catenin pathway. 査読有り

Liu YT, Shang DH, Akatsuka S, Ohara H, Dutta KK, Mizushima K, Naito Y, Yoshikawa T, Izumiya M, Abe K, Nakagama H, Noguchi N and Toyokuni S.

Am. J. Pathol. 171 巻頁： 1978-1988 2007年

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model. 査読有り

Hakozaki T, Date A, Yoshii T, Toyokuni S, Yasui H and Sakurai H.

J. Dermatol. Res. 300 (Supplement 1) 巻頁： S51-56 2007年

Generation of insulin-secreting cells from pancreatic acinar cells of animal models of type 1 diabetes. 査読有り

Okuno M, Minami K, Okumachi A, Miyawaki K, Yokoi N, Toyokuni S, Seino S.

Am. J. Physiol. Endocrinol. Metab. 292 巻頁： E158-165 2007年

Oxidative stress is related to formation of Antoni B pattern and eosinophilic hyaline droplets in schwannoma. 査読有り

Yokoo H, Oishi T, Isoda, K, Nakazato Y and Toyokuni S.

Neuropathology 27 巻頁： 237-244 2007年

Angiotensin II, oxidative stress, and extracellular matrix degradation during transition to LV failure in rats with hypertension. 査読有り

Takenaka H, Kihara Y, Iwanaga Y, Onozawa Y, Toyokuni S, Kita T.

J. Mol. Cell. Cardiol. 41 巻頁： 989-997 2007年

Leukemia inhibitory factor enhances formation of germ cell colonies in neonatal mouse testis culture.

Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Yoshida S, Toyokuni S, Lee J, Ogura A and Shinohara T.

Biol. Reprod. 76 巻頁： 55-62 2007年

Adult pancreatic islets require differential pax6 gene dosage. 査読有り

Hamasaki A, Yamada Y, Kurose T, Ban N, Nagashima K, Takahashi A, Fujimoto S, Shimono D, Fujiwara M, Toyokuni S, Seino Y and Inagaki N.

Biochem. Biophys. Res. Commun. 353 巻頁： 40-46 2007年

Adenovirus-mediated gene delivery into mouse spermatogonial stem cells. 査読有り

Takehashi M, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Ogura A, and Shinohara T.

Proc. Natl. Acad. Sci. USA 104 巻頁： 2596-2601 2007年

Pathological investigation of oxidative stress in the post-genomic era. 招待有り査読有り

Toyokuni S and Akatsuka S.

Pathol. Int. 57 巻頁： 461-473 2007年

Overexpressin of a transcription factor LYL-1 induces T- and B-cell lymphoma in mice. 査読有り

Zhong Y, Li J, Hiai H, Toyokuni S* and Yamada Y* (*corresponding author).

Oncogene 26 巻頁： 6937-6947 2007年

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element. 査読有り

Mizobuchi N, Hoseki J, Kubota H, Toyokuni S, Nozaki J, Naitoh M, Koizumi A and Nagata K.

Cell Struct. Funct. 32 巻頁： 41-50 2007年

Akt mediates self-renewal division of mouse spermatogonial stem cells. 査読有り

Lee J, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Kimura T, Nakano T, Ogura A and Shinohara T.

Development 134 巻頁： 1853-1859 2007年

Association of microRNA-34a overexpression with proliferation is cell type-dependent. 査読有り

Dutta KK, Zhong Y, Liu YT, Yamada T, Akatsuka S, Hu Q, Yoshihara M, Ohara H, Takehashi M, Shinohara T, Masutani H, Onuki J and Toyokuni S.

Cancer Sci. 98 巻頁： 1845-1852 2007年

FasL expression in hepatic antigen-presenting cells and phagocytosis of apoptotic T cells by FasL+ Kupffer cells are indicators of rejection activity in human liver allografts. 査読有り

Miyagawa-Hayashino A, Tsuruyama T, Egawa H, Haga H, Sakashita H, Okuno T, Toyokuni S, Tamaki K, Yamabe H, Manabe T and Uemoto S.

Am J. Pathol. 171 巻頁： 1499-1508 2007年

Production of knockout mice by gene targeting in multipotent germline stem cells. 査読有り

Takehashi M, Kanatsu-Shinohara M, Miki H, Lee J, Kazuki Y, Inoue K, Ogonuki N, Toyokuni S, Oshimura M, Ogura A and Shinohara T.

Dev. Biol. 312 巻頁： 344-352 2007年

Lee, WH; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE 43 巻頁： S24 - S24 2007年

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element.

Mizobuchi N, Hoseki J, Kubota H, Toyokuni S, Nozaki J, Naitoh M, Koizumi A, Nagata K

Cell structure and function 32 巻 ( 1 ) 頁： 41 - 50 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Structure and Function

Arginine rich, mutated in early stage of tumors (ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum (ER) stress. Here, we show that the mouse homologue of ARMET is an 18-kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70-family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE-II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE-II-dependent gene and likely contributes to quality control of proteins in the ER. © 2007 by Japan Society for Cell Biology.

DOI： 10.1247/csf.07001

Chronic oxidative stress causes amplification and overexpresson of ptprzl protein tyrosine phosphatase to activate beta-catenin pathway

Liu, Y; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE 43 巻頁： S154 - S155 2007年

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model. 査読有り

Hakozaki T, Date A, Yoshii T, Toyokuni S, Yasui H, Sakurai H

J. Dermatol. Res. 300 (Supplement 1) 巻頁： S51-56 2007年

Oxidative stress is related to formation of Antoni B pattern and eosinophilic hyaline droplets in schwannoma. 査読有り

Yokoo H, Oishi T, Isoda, K, Nakazato Y, Toyokuni S

Neuropathology 27 巻頁： 237-244 2007年

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element 査読有り

Naomi Mizobuchi, Jun Hoseki, Hiroshi Kubota, Shinya Toyokuni, Jun-ichi Nozaki, Motoko Naitoh, Akio Koizumi, Kazuhiro Nagata

CELL STRUCTURE AND FUNCTION 32 巻 ( 1 ) 頁： 41 - 50 2007年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPAN SOC CELL BIOLOGY

Arginine rich, mutated in early stage of tumors ( ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum ( ER) stress. Here, we show that the mouse homologue of ARMET is an 18- kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70- family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE- II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE- II- dependent gene and likely contributes to quality control of proteins in the ER.

Novel screening method for ultraviolet protection : Combination of a human skin-equivalent model and 8-hydroxy-2'-deoxyguanosine

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element

Mizobuchi Naomi, Hoseki Jun, Kubota Hiroshi, Toyokuni Shinya, Nozaki Jun-ichi, Naitoh Motoko, Koizumi Akio, Nagata Kazuhiro

CELL STRUCTURE AND FUNCTION 32 巻 ( 1 ) 頁： 41 - 50 2007年

Angiotensin II, oxidative stress, and extracellular matrix degradation during transition to LV failure in rats with hypertension. 査読有り

Takenaka H, Kihara Y, Iwanaga Y, Onozawa Y, Toyokuni S, Kita T

J. Mol. Cell. Cardiol. 41 巻頁： 989-997 - 997 2007年

TOYOKUNI Shinya, YASUI Hiroyuki, DATE Akira, HAKOZAKI Tomohiro, AKATSUKA Shinya, KOHDA Haruyasu, YOSHII Takashi, SAKURAI Hiromu

Pathology international 56 巻 ( 12 ) 頁： 760 - 762 2006年12月

Angiotensin II, oxidative stress, and extracellular matrix degradation during transition to LV failure in rats with hypertension. 査読有り国際誌

Takenaka H, Kihara Y, Iwanaga Y, Onozawa Y, Toyokuni S, Kita T

Journal of molecular and cellular cardiology 41 巻 ( 6 ) 頁： 989 - 97 2006年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Molecular and Cellular Cardiology

Angiotensin II (Ang-II) plays pivotal roles in the progression of left ventricular (LV) remodeling in diseased hearts; it remains to be elucidated how Ang-II links to degradation of the extracellular matrix (ECM). Using hypertensive Dahl salt-sensitive rats that show the distinctive transition from concentric LV hypertrophy to LV remodeling, we chronically treated them with an angiotensin type-1 receptor blocker (telmisartan 5 mg/kg/day, ARB group) or vehicle (0.5% CMC, CHF group). During the process of LV remodeling, we assessed, (1) in-vivo LV shape and function; (2) animal survival; (3) amounts of ECM in LV using a scanning electron microscope (SEM); (4) mRNA (by real time RT-PCR) and protein (by immunoblotting) levels in LV of NADPH oxidase, glutathione peroxidase-1 (GPX-1), and matrix metalloproteinase (MMP)-2, -9, and -13; (5) immunohistochemical staining of myocardial 4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine; (6) nuclear factor kappa-B (NFκB) protein levels in the nuclear extract; and (7) endogenous activities of MMP-2 and -9 by an antibody capture method. Compared with CHF, ARB group showed an improvement of survival and preserved LV shape and function, and ECM density in SEM that was accompanied by decreases in oxidative stress-mediated protein degenerations, activities of GPX-1, NADPH oxidase, NFκB, and MMP-2, -9, and -13. Local activation of Ang-II in hypertrophic LV triggers MMP-mediated ECM degradation, namely LV remodeling, at least in part, through NADPH oxidase-induced oxidative stress and the subsequent NFκB activation. © 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yjmcc.2006.07.019

Deletion and single nucleotide substitution at G:C in the kidney of gpt delta transgenic mice after ferric nitrilotriacetate treatment

Novel screening method for ultraviolet protection:: Combination of a human skin-equivalent model and 8-hydroxy-2′-deoxyguanosine 査読有り国際誌

Toyokuni, S; Yasui, H; Date, A; Hakozaki, T; Akatsuka, S; Kohda, H; Yoshii, T; Sakurai, H

PATHOLOGY INTERNATIONAL 56 巻 ( 12 ) 頁： 760 - 762 2006年12月

JIANG Li, ZHONG Yi, AKATSUKA Shinya, LIU Yu-Ting, DUTTA Khokon Kumar, LEE Wen-Hua, ONUKI Janice, MASUMURA Ken-ichi, NOHMI Takehiko, TOYOKUNI Shinya

Cancer science 97 巻 ( 11 ) 頁： 1159 - 1167 2006年11月

Deletion and single nucleotide substitution at G:C in the kidney of gpt delta transgenic mice after ferric nitrilotriacetate treatment. 査読有り国際誌 Open Access

Jiang L, Zhong Y, Akatsuka S, Liu YT, Dutta KK, Lee WH, Onuki J, Masumura K, Nohmi T, Toyokuni S

Cancer science 97 巻 ( 11 ) 頁： 1159 - 67 2006年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Science

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces oxidative renal proximal tubular damage that subsequently leads to a high incidence of renal cell carcinoma in rodents, presenting an intriguing model of free radical-induced carcinogenesis. In the present study, we used gpt delta transgenic mice, which allow efficient detection of point mutations and deletions in vivo, to evaluate the mutation spectra, in association with the formation of 8-oxoguanine and acrolein-modified adenine during the first 3 weeks of carcinogenesis. Immunohistochemical analysis revealed the highest levels of 8-oxoguanine and acrolein-modifed adenine in the renal proximal tubules after 1 week of repeated administration. DNA immunoprecipitation and quantitative polymerase chain reaction analysis showed that the relative abundance of 8-oxoguanine and acrolein-modified adenine at the gpt reporter gene were increased at the first week in the kidney. Similarly, in both 6-thioguanine and Spi- selections performed on the renal specimens after Fe-NTA administration, the mutant frequencies were increased in the Fe-NTA-treated mice at the first week. Further analyzes of 79 mutant clones and 93 positive plaques showed a high frequency of G:C pairs as preferred targets for point mutation, notably G:C to C:G transversion-type mutation followed by deletion, and of large-size (>1 kilobase) deletions with short homologous sequences in proximity to repeated sequences at the junctions. The results demonstrate that the iron-based Fenton reaction is mutagenic in vivo in the renal tubular cells and induces characteristic mutations. © 2006 Japanese Cancer Association.

DOI： 10.1111/j.1349-7006.2006.00301.x

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis. 査読有り国際誌 Open Access

Akatsuka S, Aung TT, Dutta KK, Jiang L, Lee WH, Liu YT, Onuki J, Shirase T, Yamasaki K, Ochi H, Naito Y, Yoshikawa T, Kasai H, Tominaga Y, Sakumi K, Nakabeppu Y, Kawai Y, Uchida K, Yamasaki A, Tsuruyama T, Yamada Y, Toyokuni S

The American journal of pathology 169 巻 ( 4 ) 頁： 1328 - 42 2006年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Pathology

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N 6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate- induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress. Copyright © American Society for Investigative Pathology.

DOI： 10.2353/ajpath.2006.051280

Overexpression of LYL1 gene induces T- and B-cell leukemia in mice

Alpha-tocopherol induces calnexin in renal tubular cells: another protective mechanism against free radical-induced cellular damage. 査読有り国際誌 Open Access

Lee WH, Akatsuka S, Shirase T, Dutta KK, Jiang L, Liu YT, Onuki J, Yamada Y, Okawa K, Wada Y, Watanabe A, Kohro T, Noguchi N, Toyokuni S

Archives of biochemistry and biophysics 453 巻 ( 2 ) 頁： 168 - 78 2006年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Pre-administration of α-tocopherol is protective against oxidative renal tubular damage and subsequent carcinogenesis by ferric nitrilotriacetate (Fe-NTA) in rats. We searched for mechanisms other than the scavenging effect of α-tocopherol with microarray analyses, which implicated calnexin, a chaperone for glycoproteins. Renal mRNA levels of calnexin significantly increased 3 h after an injection of Fe-NTA in rats fed a standard diet whereas those fed an α-tocopherol-supplemented diet showed an increase prior to injection, but after injection showed a decrease in renal calnexin mRNA levels, with unaltered protein levels. In experiments using LLC-PK1 cells, addition of α-tocopherol was protective against oxidative stress by H2O2, concomitant with calnexin induction. Knockdown of calnexin by siRNA significantly reduced this protection. Furthermore, COS-7 cells transfected with the calnexin gene were more resistant to H2O2. Together with the fact that α-tocopherol induced N-acetylglucosaminyltransferase 3, our data suggest that α-tocopherol modifies glycoprotein metabolism partially by conferring mild ER stress. This adds another molecular mechanism of α-tocopherol toward cancer prevention. © 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2006.07.002

Zhong, Y; Jiang, L; Toyokuni, S; Hiai, H; Yamada, Y

EXPERIMENTAL HEMATOLOGY 34 巻 ( 9 ) 頁： 47 - 47 2006年9月

Overexpression of LYL1 gene induces T- and B-cell leukemia in mice 国際誌

Zhong, Y, Jiang, L, Toyokuni, S, Hiai, H, Yamada, Y

EXPERIMENTAL HEMATOLOGY 34 巻 ( 9 ) 頁： 47 - 47 2006年9月

Historical aspects of free radical chemistry and biology: What's new in oxidative stress-induced carcinogenesis and its prevention? 査読有り国際誌

Toyokuni Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 39 巻 ( 1 ) 頁： 1 - 2 2006年7月

Historical aspects of free radical chemistry and biology: What's new in oxidative stress-induced carcinogenesis and its prevention?

Toyokuni S.

Journal of Clinical Biochemistry and Nutrition 39 巻 ( 1 ) 頁： 1 - 2 2006年7月

Novel aspects of oxidative stress-associated carcinogenesis. 査読有り

Toyokuni S

Antioxidants & redox signaling 8 巻 ( 7-8 ) 頁： 1373 - 7 2006年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Antioxidants and Redox Signaling

Oxidative stress is associated with carcinogenesis. Reactive oxygen and nitrogen species contribute to the accumulation of mutations in the genome, presumably followed by selective processes. Recent data suggest that preferred signaling pathways exist for oxidative stress-associated carcinogenesis. Whether this completely depends on random mutations induced by reactive species or whether instead some fragile genomic loci are sensitive to oxidative damage in association with changes of transcriptional activity or other topologic or non-topologic effects remains to be explored. Reliable markers for oxidative stress as well as for oxidative stress-induced preneoplastic lesions must be established. © Mary Ann Liebert, Inc.

DOI： 10.1089/ars.2006.8.1373

What has been learned from the studies of oxidative stress-induced carcinogenesis: Proposal of the concept of oxygenomics

Toyokuni S., Akatsuka S.

Journal of Clinical Biochemistry and Nutrition 39 巻 ( 1 ) 頁： 3 - 10 2006年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

Epidemiological studies have demonstrated that oxidative stress associated with a variety of pathological conditions is one of the major causes of carcinogenesis. Reactive oxygen and nitrogen species contribute to genomic alterations, presumably followed by selection of the best-adapted proliferating cells in a given environment. Recent data suggest that there exist common signaling pathways for oxidative stress-associated carcinogenesis. So far, oxidative DNA damage has been assumed to be randomly distributed based on in vitro experiments, and localization of oxidative DNA damage in the genome in vivo has rarely been studied. However, by the use of novel techniques in combination with constructed genome databases, it was found that the localization of oxidative DNA appears to be not random in vivo. We propose to call this rather novel research area "oxygenomics". Not a few signaling pathways start from the recognition of DNA damage. Possible underlying principles should be elucidated in association with cell type, the function of each genomic location, and its transcriptional activity as well as chromatin status determining epigenetic information. Furthermore, this concept may contribute to the development of novel oxidative stress biomarkers. Thus, oxygenomics is a promising research area.

DOI： 10.3164/jcbn.39.3

What has been learned from the studies of oxidative stress-induced carcinogenesis: proposal of the concept of oxygenomics 査読有り

Shinya Toyokuni, Shinya Akatsuka

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 39 巻 ( 1 ) 頁： 3 - 10 2006年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

Differences in oxidative stress dependence between gastric adenocarcinoma subtypes.

What has been learned from the studies of oxidative stress-induced carcinogenesis: proposal of the concept of oxygenomics

Toyokuni Shinya, Akatsuka Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 39 巻 ( 1 ) 頁： 3 - 10 2006年7月

Historical aspects of free radical chemistry and biology: What's new in oxidative stress-induced carcinogenesis and its prevention? 査読有り国際誌

Toyokuni Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 39 巻 ( 1 ) 頁： 1 - 2 2006年7月

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Ikawa M, Takehashi M, Ogonuki N, Miki H, Inoue K, Kazuki Y, Lee J, Toyokuni S, Oshimura M, Ogura A, Shinohara T

Proceedings of the National Academy of Sciences of the United States of America 103 巻 ( 21 ) 頁： 8018 - 23 2006年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis. © 2006 by The National Academy of Sciences of the USA.

DOI： 10.1073/pnas.0601139103

ヒト大腸癌予後因子としてのNKT細胞腫瘍内浸潤

鶴山竜昭, 橘強, 小野寺久, 幸田晴康, 豊國伸哉, 日合弘, 山邉博彦, 玉木敬二, 真鍋俊明

日本病理学会会誌 95 巻 ( 1 ) 頁： 213 - 213 2006年4月

Anchorage-independent growth of mouse male germline stem cells in vitro. 査読有り国際誌

Kanatsu-Shinohara M, Inoue K, Lee J, Miki H, Ogonuki N, Toyokuni S, Ogura A, Shinohara T

Biology of reproduction 74 巻 ( 3 ) 頁： 522 - 9 2006年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogenesis originates from a small number of spermatogonial stem cells that reside on the basement membrane and undergo self-renewal division to support spermatogenesis throughout the life of adult animals. Although the recent development of a technique to culture spermatogonial stem cells allowed reproduction of self-renewal division in vitro, much remains unknown about how spermatogonial stem cells are regulated. In this study, we found that spermatogonial stem cells could be cultured in an anchorage-independent manner, which is characteristic of stem cells from other types of self-renewing tissues. Although the cultured cells grew slowly (doubling time, ∼4.7 days), they expressed markers of spermatogonia, and crew exponentially for at least 5 months to achieve 1.5 × 1010-fold expansion. The cultured cells underwent spermatogenesis following transplantation into the seminiferous tubules of infertile animals and fertile offspring were obtained by microinsemination of germ cells that had developed within the testes of recipients of the cultured cells. These results indicate that spermatogonial stem cells can undergo anchorage-independent, self-renewal division, and suggest that stem cells have the common property to survive and proliferate in the absence of exogenous substrata. © 2006 by the Society for the Study of Reproduction, Inc.

DOI： 10.1095/biolreprod.105.046441

Differences in oxidative stress dependence between gastric adenocarcinoma subtypes

Bancel Brigitte, Esteve Jacques, Souquet Jean-Christophe, Toyokuni Shinya, Ohshima Hiroshi, Pignatelli Brigitte

WORLD JOURNAL OF GASTROENTEROLOGY 12 巻 ( 7 ) 頁： 1005 - 1012 2006年2月

Bancel B, Esteve J, Souquet JC, Toyokuni S, Ohshima H, Pignatelli B

World journal of gastroenterology 12 巻 ( 7 ) 頁： 1005 - 12 2006年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：World Journal of Gastroenterology

Aim: To investigate the extent of oxidative stress in preneoplastic and neoplastic gastric mucosa in relation to their pathological criteria and histological subtypes. Methods: A total of 104 gastric adenocarcinomas from 98 patients (88 infiltrative and 16 intraepithelial tumors) were assessed immunohistochemically for expression of iNOS and occurrence of nitrotyrosine (NTYR)-containing proteins and 8-hydroxy-2′-deoxyguanosine (8-OH-dG)-containing DNA, as markers of NO production and damages to protein and DNA. Results: Tumor cells staining for iNOS, NTYR and 8-OH-dG were detected in 41%, 62% and 50% of infiltrative carcinoma, respectively. The three markers were shown for the first time in intraepithelial carcinoma. The expression of iNOS was significantly more frequent in tubular carcinoma (TC) compared to diffuse carcinoma (DC) (54% vs 18%; P=0.008) or in polymorphous carcinoma (PolyC) (54% vs 21%; P=0.04). NTYR staining was obviously more often found in TC than that in PolyC (72% vs 30%; P=0.03). There was a tendency towards a higher rate of iNOS staining when distant metastasis (pM) was present. In infiltrative TC, the presence of oxidative stress markers was not significantly correlated with histological grade, density of inflammation, the depth of infiltration (pT), lymph nodes dissemination (pN) and pathological stages (pTNM). Conclusion: The iNOS-oxidative pathway may play an important role in TC, but moderately in PolyC and DC. DNA oxidation and protein nitration occur in the three subtypes. Based on the significant differences of NTYR levels, TC and PolyC appear as two distinct subtypes. © 2006 The WJG Press. All rights reserved.

DOI： 10.3748/wjg.v12.i7.1005

An N-glycan structure correlates with pulmonary metastatic ability of cancer cells 査読有り国際誌 Open Access

Sakuma, K; Fujimoto, I; Hitoshi, S; Tanaka, F; Ikeda, T; Tanabe, K; Toyokuni, S; Wada, H; Mio, T; Mishima, M; Ikenaka, K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 340 巻 ( 3 ) 頁： 829 - 835 2006年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

N-Glycan structures on the surface of cancer cells have diverse structures and play significant roles in metastatic process. However, little is known about their roles in organ-selective metastasis. Our study revealed that an α1,6-fucosylated biantennary N-glycan structure designated A2G2F is characteristic of lungs, with far more abundant expression in normal human and murine lungs than in other organs. In this study, we further examined the role of A2G2F in pulmonary metastasis. We stained metastatic cancers by α1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed α1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific α1,6-fucosylated N-glycan structure in pulmonary metastatic cancer was identified to be A2G2F. Using a B16 melanoma cell metastasis model, we showed that A2G2F-rich B16 cells formed more pulmonary metastatic nodules than A2G2F-poor cells. Our results suggest that A2G2F plays a critical role in pulmonary metastasis. © 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.12.072

Differences in oxidative stress dependence between gastric adenocarcinoma subtypes 査読有り

Brigitte Bancel, Jacques Esteve, Jean-Christophe Souquet, Shinya Toyokuni, Hiroshi Ohshima, Brigitte Pignatelli

WORLD JOURNAL OF GASTROENTEROLOGY 12 巻 ( 7 ) 頁： 1005 - 1012 2006年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：W J G PRESS

AIM: To investigate the extent of oxidative stress in pre-neoplastic and neoplastic gastric mucosa in relation to their pathological criteria and histological subtypes.
METHODS: A total of 104 gastric adenocarcinomas from 98 patients (88 infiltrative and 16 intraepithelial tumors) were assessed immunohistochemically for expression of iNOS and occurrence of nitrotyrosine (NTYR)-containing proteins and 8-hydroxy-2'-deoxyguanosine (8-OH-dG)-containing DNA, as markers of NO production and damages to protein and DNA.
RESULTS: Tumor cells staining for NOS, NTYR and 8-OH-dG were detected in 41%, 62% and 50% of infiltrative carcinoma, respectively. The three markers were shown for the first time in intraepithelial carcinoma. The expression of iNOS was significantly more frequent in tubular carcinoma (TC) compared to diffuse carcinoma (DC) (54% vs 18%; P = 0.008) or in polymorphous carcinoma (PolyC) (54% vs 21%; P = 0.04). NTYR staining was obviously more often found in TC than that in PolyC (72% vs 30%; P = 0.03). There was a tendency towards a higher rate of iNOS staining when distant metastasis (pM) was present. In infiltrative TC, the presence of oxidative stress markers was not significantly correlated with histological grade, density of inflammation, the depth of infiltration (pT), lymph nodes dissemination (pN) and pathological stages (pTNM).
CONCLUSION: The iNOS-oxidative pathway may play an important role in TC, but moderately in PolyC and DC. DNA oxidation and protein nitration occur in the three subtypes. Based on the significant differences of NTYR levels, TC and PolyC appear as two distinct subtypes. (C) 2006 The WJG Press. All rights reserved.

Involvement of aminoacylase 1 in ferric nitrilotriacetate (Fe-NTA)-induced rat renal cell carcinoma

A novel screening method for ultraviolet protection: combination of a human skin-equivalent model and 8-hydroxy-2'-deoxyguanosine. 査読有り

Toyokuni S, Yasui H, Date A, Hakozaki T, Akatsuka S, Kohda H, Yoshii T and Sakurai H.

Pathol. Int. 56 巻頁： 760-762 2006年

Anchorage-independent growth of mouse male germline stem cells in vitro. 査読有り

Shinohara M, Inoue K, Lee J, Miki H, Ogonuki N, Toyokuni S, Ogura A and Shinohara T.

Biol. Reprod. 74 巻頁： 522-529 2006年

An N-glycan structure correlates with pulmonary metastatic ability of cancer cells. 査読有り

Sakuma K, Fujimoto I, Hitoshi S, Tanaka F, Ikeda T, Tanabe K, Toyokuni S, Wada H, Mio T, Mishima M, Ikenaka K.

Biochem. Biophys. Res. Commun. 349 巻頁： 829-835 2006年

Novel aspects of oxidative stress-associated carcinogenesis. 査読有り

Toyokuni S.

Antiox. Redox Signal. 8 巻頁： 1373-1377 2006年

Differences in oxidative stress dependence between gastric adenocarcinoma subtypes. 査読有り

Bancel B, Esteve J, Souquet JC, Toyokuni S, Ohshima H, Pignatelli B.

World J. Gastroenterol. 12 巻頁： 1005-1012 2006年

What has been learned from the studies of oxidative stress-induced carcinogenesis: proposal of the concept of oxygenomics. 査読有り

Toyokuni S, Akatsuka S.

J. Clin. Biochem. Nutr. 39 巻頁： 3-10 2006年

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. 査読有り

Kanatsu-Shinohara M, Ikawa M, Takehashi M, Ogonuki N, Miki H, Inoue K, Kazuki Y, Lee J, Toyokuni S, Oshimura M, Ogura A, Shinohara T.

Proc. Natl. Acad. Sci. USA 103 巻頁： 8018-8023 2006年

alfa-Tocopherol induces calnexin in renal tubular cells: Another protective mechanism against free radical-induced cellular damage. 査読有り

Lee W-H, Akatsuka S, Shirase T, Dutta KK, Jiang L, Liu Y-T, Onuki J, Yamada Y, Okawa K, Wada Y, Watanabe A, Kohro T, Noguchi N and Toyokuni S.

Arch. Biochem. Biophys. 453 巻頁： 168-178 2006年

*Contrasting Genome-wide Distribution of 8-Hydroxyguanine and Acrolein-modified Adenine during Oxidative Stress-induced Renal Carcinogenesis. 査読有り

Akatsuka S, Aung TT, Dutta KK, Jiang L, Lee W-H, Liu Y-T, Onuki J, Shirase T, Yamasaki K, Ochi H, Naito Y, Yoshikawa T, Kasai H, Tominaga Y, Sakumi K, Nakabeppu Y, Kawai Y, Uchida K, Yamasaki A, Tsuruyama T, Yamada Y and Toyokuni S.

Am. J. Pathol. 169 巻頁： 1328-1342 2006年

Deletion and single nucleotide substitution at G:C are major mutations in the kidney of gpt delta transgenic mice after the Fenton reaction. 査読有り

Jiang L, Zhong Y, Akatsuka S, Liu Y-T, Dutta KK, Lee W-H, Onuki J, Masumura K, Nohmi T and Toyokuni S.

Cancer Sci. 97 巻頁： 1159-1167 2006年

Onuki, J; Liu, Y; Lee, W; Jiang, L; Dutta, KK; Akatsuka, S; Toyokuni, S

FREE RADICAL RESEARCH 40 巻頁： S128 - S128 2006年

Iron-Induced Carcinogenesis 査読有り国際誌

Toyokuni, S

OXIDATIVE STRESS, DISEASE AND CANCER 頁： 789 - 805 2006年

Novel aspects of oxidative stress-associated carcinogenesis

Toyokuni Shinya

ANTIOXIDANTS & REDOX SIGNALING 8 巻 ( 7-8 ) 頁： 1373 - 1377 2006年

酸化ストレスと癌国際誌

豊國伸哉

日本トキシコロジー学会学術年会 33 巻 ( 0 ) 頁： 15 - 15 2006年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：日本毒性学会

「フリーラジカル・活性酸素による負荷から、抗酸化分子・抗酸化酵素・修復酵素などを介した消去・防御・修復能を差し引いたもの」が酸化ストレスの概念である。ここでは、発がんおよびがん領域における酸化ストレス研究の現状ならびにどのようなアプローチが可能かに関して新旧併せて紹介する。私たちは、鉄キレート剤投与によりFenton反応を尿細管にひき起こす腎傷害・発がんモデルを詳細に検討してきた。通常のパラフィン包埋標本上で酸化ストレスの可視化が可能となったのは90年代になってからであった。フリーラジカル反応で特異的に発生しかつ標本内に固定される epitopeに対する抗体を作製がなされた。脂質過酸化最終産物の一つである4-hydroxy-2-nonenalがMichael付加反応で蛋白と反応したhemiacetal構造、DNA修飾塩基である8-hydroxy-2'-deoxyguanosineやacrolein-deoxyadenosineが代表的 epitope である。これらの方法は既に定法として酸化ストレス病態の解析に広く使用されている。フリーラジカルはその反応性の高さより近傍にある分子と反応するため、反応選択性は低いと考えられてきた。私たちは、上記腎がんモデルにおいてF1動物を使用して遺伝学的解析を行い、主な標的遺伝子のひとつがp16INK4A がん抑制遺伝子であることを見いだした。しかもその変化は初期に発生する。発がんにおいては選択という要素があるが、フリーラジカルの反応は生体内において、特異的な表現型となりうることが判明してきた。最後に、がん発生の起点であるゲノム情報の変化に関して私たちがoxygenomicsと呼ぶ新しいアプローチを紹介する。 参考文献：1. Toyokuni S. Pathol Int 49: 91, 1999. 2. Tanaka T et al. Oncogene 18, 3793, 1999. 3. Hiroyasu et al. 160: 419, 2002. 4. Dutta KK et al. Lab Invest 85: 768, 2005.

DOI： 10.14869/toxp.33.0.15.0

Molecular characteristics of ferric nitrilotriacetate-induced deletions and base substitutions in the kidney of gpt delta transgenic mice

Jiang, L; Zhong, Y; Akatsuka, S; Onuki, J; Masumura, K; Nohmi, T; Toyokuni, S

FREE RADICAL RESEARCH 40 巻頁： S128 - S128 2006年

Iron-Induced Carcinogenesis

Toyokuni Shinya

OXIDATIVE STRESS, DISEASE AND CANCER 頁： 789 - 805 2006年

Deletion and single nucleotide substitution at G:C are major mutations in the kidney of gpt delta transgenic mice after the Fenton reaction. 査読有り

Jiang L, Zhong Y, Akatsuka S, Liu Y-T, Dutta KK, Lee W-H, Onuki J, Masumura K, Nohmi T, Toyokuni S

Cancer Sci. 97 巻頁： 1159-1167 2006年

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis

Akatsuka S., Aung T. T., Dutta K. K., Jiang L., Liu Y., Lee W., Onuki J., Shirase T., Naito Y., Yoshikawa T., Uchida K., Toyokuni S.

FREE RADICAL RESEARCH 40 巻頁： S129 - S129 2006年

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis 査読有り

S. Akatsuka, T. T. Aung, K. K. Dutta, L. Jiang, Y. Liu, W. Lee, J. Onuki, T. Shirase, Y. Naito, T. Yoshikawa, K. Uchida, S. Toyokuni

FREE RADICAL RESEARCH 40 巻頁： S129 - S129 2006年

Imidapril, an angiotensin-converting enzyme inhibitor, reduces diabetes-induced renal oxidative damage in mice

Naito Y., Uchiyama K., Akagiri S., Aoi W., Hasegawa G., Nakamura N., Kokura S., Yoshida N., Ichikawa H., Toyokuni S., Yoshikawa T.

Journal of Clinical Biochemistry and Nutrition 37 巻 ( 1 ) 頁： 29 - 37 2005年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Clinical Biochemistry and Nutrition

The aim of the present study was to determine whether imidapril, an angiotensin-converting enzyme inhibitor, prevents diabetic nephropathy and to determine the effect of imidapril on oxidative damage in the diabetic glomeruli in mice. We used female BKS.cg-m+Leprdb/+Leprdb (db/db) mice, a rodent model of type 2 diabetes, and their non-diabetic db/m littermates. The mice were divided into the following four groups: non-diabetic db/m, diabetic db/db, and diabetic db/db treated with imidapril at doses of 1 mg/kg and 5 mg/kg. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments. Histological and 8-OHdG immunohistochemical studies were performed for 12 weeks from the beginning of treatment. After 12 weeks of treatment, the levels of blood glucose and the body weight were not significantly different between the imidapril-treated group and the non-treated db/db group. The systolic blood pressure was significantly increased in db/db mice compared with db/m mice. The increased blood pressure was significantly reduced by the treatment with imidapril at a dose of 5 mg/kg. The relative mesangial area calculated by the mesangial area/total glomerular area ratio was significantly ameliorated in the imidapril-treated group compared with the non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with imidapril in a dose-dependent manner. The 8-OHdG immunoreactive cells in the glomeruli of non-treated db/db mice were more numerous than in the imidapril-treated db/db mice. In conclusion, imidapril prevented the progression of diabetic nephropathy in mice, not only by decreasing the glomerular pressure but also by decreasing the oxidative stress in glomerular cells.

DOI： 10.3164/jcbn.37.29

Susceptibility of actin to modification by 4-hydroxy-2-nonenal

Susceptibility of actin to modification by 4-hydroxy-2-nonenal. 査読有り国際誌

Ozeki M, Miyagawa-Hayashino A, Akatsuka S, Shirase T, Lee WH, Uchida K, Toyokuni S

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 827 巻 ( 1 ) 頁： 119 - 26 2005年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, reacts with histidine, lysine or cysteine residues of proteins to form hemiacetal Michael adducts and thus interferes with the functions of the proteins. Here we undertook to identify HNE-modified proteins in the target organ of a ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis model with histidine-specific HNEJ-2 antibody. Immunoaffinity column separation and sequencing identified one of the major modified proteins as actin. To further explore the characteristics of actin as an HNE acceptor, we produced four novel monoclonal antibodies against HNE-modified keyhole limpet hemocyanin. All these antibodies (HNEJ-1, 3-5) recognized histidine adducts, but were different from HNEJ-2 in recognizing lysine and cysteine adducts to some extent. Actin, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), metallothionein and superoxide dismutase were treated in vitro with HNE and evaluated with these antibodies. The results revealed that actin was most sensitive to HNE modification and metallothionein most resistant. Furthermore, the residue-specificity of GAPDH was in accord with that shown by our recent mass spectrometry data. Immunohistochemistry with the antibodies revealed cytoplasmic staining with or without nuclear staining in the renal proximal tubules after Fe-NTA administration. The results suggest that actin is a major target protein for HNE modification in vivo, and that our monoclonal antibodies are useful for evaluating the HNE adducts produced. © 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jchromb.2005.02.025

OZEKI Munetaka, MIYAGAWA HAYASHINO Aya, AKATSUKA Shinya, SHIRASE Tomoyuki, LEE Wen-hua, UCHIDA Koji, TOYOKUNI Shinya

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 827 巻 ( 1 ) 頁： 119 - 126 2005年11月

Partial correction of abnormal cardiac development in caspase-8-deficient mice by cardiomyocyte expression of p35

Yajima N, Yamada S, Morisaki T, Toyokuni S, Yonehara S, Sakamaki K

TRANSGENIC RESEARCH 14 巻 ( 5 ) 頁： 593 - 604 2005年10月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Transgenic Research

Baculovirus p35 protein protects cells from apoptotic cell death by inhibiting caspase activation. We have established transgenic mouse lines specifically expressing p35 in cardiomyocytes, and primary cardiomyocytes isolated from these mice exhibit resistance to staurosporine-induced apoptosis. In a previous study, we observed defects in heart formation associated with abdominal hemorrhage and cardiomyocyte cell death in caspase-8-deficent animals. In order to better understand the etiology of the cardiac defects and embryonic lethality in caspase-8-deficient mice, we crossed these mice with the p35 transgenic animals. Although the newly generated mice still died in utero and exhibited some cardiac defects, cardiomyocyte apoptosis was suppressed and ventricular trabeculation was restored. Thus, cardiomyocyte expression of p35 prevented cell death induced by staurosporine or caspase-8 deficiency. Additionally, our data suggest that caspase-8 plays multiple roles in cardiac development. © Springer 2005.

DOI： 10.1007/s11248-005-4350-5

Partial correction of abnormal cardiac development in caspase-8-deficient mice by cardiomyocyte expression of p 35. 国際誌

Nobuyuki Yajima, Shu-Ichi Yamada, Takayuki Morisaki, Shinya Toyokuni, Shin Yonehara, Kazuhiro Sakamaki

Transgenic research 14 巻 ( 5 ) 頁： 593 - 604 2005年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Baculovirus p 35 protein protects cells from apoptotic cell death by inhibiting caspase activation. We have established transgenic mouse lines specifically expressing p 35 in cardiomyocytes, and primary cardiomyocytes isolated from these mice exhibit resistance to staurosporine-induced apoptosis. In a previous study, we observed defects in heart formation associated with abdominal hemorrhage and cardiomyocyte cell death in caspase-8-deficient animals. In order to better understand the etiology of the cardiac defects and embryonic lethality in caspase-8-deficient mice, we crossed these mice with the p 35 transgenic animals. Although the newly generated mice still died in utero and exhibited some cardiac defects, cardiomyocyte apoptosis was suppressed and ventricular trabeculation was restored. Thus, cardiomyocyte expression of p 35 prevented cell death induced by staurosporine or caspase-8 deficiency. Additionally, our data suggest that caspase-8 plays multiple roles in cardiac development.

Nestin-positive cells in adult pancreas express amylase and endocrine precursor Cells.

Helicobacter felis-induced gastritis was suppressed in mice overexpressing thioredoxin-1. 国際誌 Open Access

Kawasaki K, Nishio A, Nakamura H, Uchida K, Fukui T, Ohana M, Yoshizawa H, Ohashi S, Tamaki H, Matsuura M, Asada M, Nishi T, Nakase H, Toyokuni S, Liu W, Yodoi J, Okazaki K, Chiba T

Laboratory investigation; a journal of technical methods and pathology 85 巻 ( 9 ) 頁： 1104 - 17 2005年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Laboratory Investigation

Thioredoxin-1 (TRX-1) is a redox-active protein involved in scavenging reactive oxygen species and regulating redox-sensitive transcription factors. TRX-1 is induced in various inflammatory conditions and shows cytoprotective action. We investigated the roles of TRX-1 in the host defense mechanism against Helicobacter felis (H. felis) infection. Transgenic (TG) mice overexpressing human TRX-1 and wild-type (WT) mice were orally inoculated with H. felis. After 2 months, histology, oxidative damage, and gene expression of several cytokines, including macrophage inflammatory protein-2 (MIP-2), a murine equivalent to interleukin (IL)-8, in the gastric mucosa were investigated. Furthermore, the effects of TRX-1 on oxidative stress and neutrophil migration were studied both in vivo and in vitro. The gastric mucosa was thickened in H. felis-infected WT mice, but not in infected TRX-1-TG mice. Histologically, all H. felis-infected WT mice developed moderate-to-severe gastritis, whereas the development of gastritis was significantly suppressed in infected TRX-1-TG mice. Oxidative damage markers, 8-hydroxy-2′-deoxyguanosine and malondialdehyde, increased in the stomach of infected WT mice, but not TRX-1-TG mice. Upregulation of IL-1β and tumor necrosis factor-α gene expression in H. felis-infected TRX-1-TG mice was significantly lower than in WT mice. However, upregulation of MIP-2 and IL-7 was not different between the two groups. TRX-1 suppressed oxidative cytotoxicity and DNA damage, and inhibited neutrophil migration both in vivo and in vitro. The present study suggests that overexpression of TRX-1 suppresses H. felis-induced gastritis by inhibiting chemotaxis of neutrophils and reducing oxidative stress. © 2005 USCAP, Inc All Rights Reserved.

DOI： 10.1038/labinvest.3700305

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture. 査読有り国際誌

Kanatsu-Shinohara M, Ogonuki N, Iwano T, Lee J, Kazuki Y, Inoue K, Miki H, Takehashi M, Toyokuni S, Shinkai Y, Oshimura M, Ishino F, Ogura A, Shinohara T

Development (Cambridge, England) 132 巻 ( 18 ) 頁： 4155 - 63 2005年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Development

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved ∼1085-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.

DOI： 10.1242/dev.02004

Germline niche transplantation restores fertility in infertile mice. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Ogura A, Shinohara T

Human reproduction (Oxford, England) 20 巻 ( 9 ) 頁： 2376 - 82 2005年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Reproduction

Background: Stem cells interact closely with their microenvironment or niche, and abnormalities in niche compromise the self-renewing tissue. In testis, for example, Sertoli cells interact with germ cells, and defects in Sertoli cells compromises spermatogenesis, leading to male infertility. However, it has not been possible to restore spermatogenesis from endogenous stem cells in infertile testis with environmental defects. Methods and results: When healthy Sertoli cells from infertile white spotting (W) mouse were transplanted into the seminiferous tubules of infertile Steel (Sl) mouse testis that had defective Sertoli cells, spermatogenesis occurred from Sl stem cells in the recipient testis. On average, 1.1% of the recipient tubules showed spermatogenesis. Furthermore, in a microinsemination experiment with germ cells that developed in the testis, we obtained four normal offspring from 114 successfully injected oocytes. Conclusions: This study demonstrates that defects in male germline microenvironment can be corrected by Sertoli cell transplantation. Although further improvements are required to enhance the low efficiency of spermatogenesis, the ability to correct environmental defect by niche transplantation has important implications in developing new strategies for treating incurable disorders in self-renewing tissues. © The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

DOI： 10.1093/humrep/dei096

Nestin-positive cells in adult pancreas express amylase and endocrine precursor cells

Ueno H, Yamada Y, Watanabe R, Mukai E, Hosokawa M, Takahashi A, Hamasaki A, Fujiwara H, Toyokuni S, Yamaguchi M, Takeda J, Seino Y

PANCREAS 31 巻 ( 2 ) 頁： 126 - 131 2005年8月

Ueno H, Yamada Y, Watanabe R, Mukai E, Hosokawa M, Takahashi A, Hamasaki A, Fujiwara H, Toyokuni S, Yamaguchi M, Takeda J, Seino Y

Pancreas 31 巻 ( 2 ) 頁： 126 - 31 2005年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pancreas

The neural precursor cell-specific marker nestin is expressed in fetal and adult pancreas, but its role is not fully understood. Using nestin-enhanced green fluorescent protein (EGFP) transgenic mice and fluorescence activated cell sorter, we characterized nestin-positive cells in adult mice pancreas. EGFP mRNA- and protein-positive cells expressed amylase, a pancreatic exocrine marker. Interestingly, EGFP mRNA-negative and protein-positive cells expressed insulin, glucagon, somatostatin and pancreatic polypeptide, pancreatic endocrine markers. These findings demonstrate that nestin-positive cells comprise a portion of pancreatic exocrine cells and suggest that they can be differentiated into pancreatic endocrine cells. Copyright © 2005 by Lippincott Williams & Wilkins.

DOI： 10.1097/01.mpa.0000172564.80921.f7

Nestin-positive cells in adult pancreas express amylase and endocrine precursor Cells. 国際誌

Hiroyuki Ueno, Yuichiro Yamada, Rie Watanabe, Eri Mukai, Masaya Hosokawa, Akira Takahashi, Akihiro Hamasaki, Hideya Fujiwara, Shinya Toyokuni, Masahiro Yamaguchi, Jun Takeda, Yutaka Seino

Pancreas 31 巻 ( 2 ) 頁： 126 - 31 2005年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

Establishment of a diabetic mouse model with progressive diabetic nephropathy.

Inada A, Nagai K, Arai H, Miyazaki J, Nomura K, Kanamori H, Toyokuni S, Yamada Y, Bonner-Weir S, Weir GC, Fukatsu A, Seino Y

The American journal of pathology 167 巻 ( 2 ) 頁： 327 - 36 2005年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Pathology

Although diabetic animal models exist, no single animal model develops renal changes identical to those seen in humans. Here we show that transgenic mice that overexpress inducible cAMP early repressor (ICER Iγ) in pancreatic β cells are a good model to study the pathogenesis of diabetic nephropathy. Although ICER Iγ transgenic mice exhibit extremely high blood glucose levels throughout their lives, they survive long enough to develop diabetic nephropathy. Using this model we followed the progress of diabetic renal changes compared to those seen in humans. By 8 weeks of age, the glomerular filtration rate (GFR) was already increased, and glomerular hypertrophy was prominent. At 20 weeks, GFR reached its peak, and urine albumin excretion rate was elevated. Finally, at 40 weeks, diffuse glomerular sclerotic lesions were prominently accompanied by increased expression of collagen type IV and laminin and reduced expression of matrix metalloproteinase-2. Nodular lesions were absent, but glomerular basement membrane thickening was prominent. At this point, GFR declined and urinary albumin excretion rate increased, causing a nephrotic state with lower serum albumin and higher serum total cholesterol. Thus, similar to human diabetic nephropathy, ICER Iγ transgenic mice exhibit a stable and progressive phenotype of diabetic kidney disease due solely to chronic hyperglycemia without other modulating factors. Copyright © American Society for Investigative Pathology.

DOI： 10.1016/s0002-9440(10)62978-1

Establishment of a diabetic mouse model with progressive diabetic nephropathy

Inada A, Nagai K, Arai H, Miyazaki J, Nomura K, Kanamori H, Toyokuni S, Yamada Y, Bonner-Weir S, Weir GC, Fukatsu A, Seino Y

AMERICAN JOURNAL OF PATHOLOGY 167 巻 ( 2 ) 頁： 327 - 336 2005年8月

Establishment of a diabetic mouse model with progressive diabetic nephropathy 査読有り

A Inada, K Nagai, H Arai, J Miyazaki, K Nomura, H Kanamori, S Toyokuni, Y Yamada, S Bonner-Weir, GC Weir, A Fukatsu, Y Seino

AMERICAN JOURNAL OF PATHOLOGY 167 巻 ( 2 ) 頁： 327 - 336 2005年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC INVESTIGATIVE PATHOLOGY, INC

Although diabetic animal models exist, no single animal model develops renal changes identical to those seen in humans. Here we show that transgenic mice that overexpress inducible cAMP early repressor (ICER I gamma) in pancreatic beta cells are a good model to study the pathogenesis of diabetic nephropathy. Although ICER I gamma transgenic mice exhibit extremely high blood glucose levels throughout their lives, they survive long enough to develop diabetic nephropathy. Using this model we followed the progress of diabetic renal changes compared to those seen in humans. By 8 weeks of age, the glomerular filtration rate (GFR) was already increased, and glomerular hypertrophy was prominent. At 20 weeks, GFR reached its peak, and urine albumin excretion rate was elevated. Finally, at 40 weeks, diffuse glomerular sclerotic lesions were prominently accompanied by increased expression of collagen type IV and laminin and reduced expression of matrix metalloproteinase-2. Nodular lesions were absent, but glomerular basement membrane thickening was prominent. At this point, GFR declined and urinary albumin excretion rate increased, causing a nephrotic state with lower serum albumin and higher serum total cholesterol. Thus, similar to human diabetic nephropathy, ICER I gamma transgenic mice exhibit a stable and progressive phenotype of diabetic kidney disease due solely to chronic hyperglycemia without other modulating factors.

Annexin II overexpression correlates with a poor prognosis in surgically resected non-small-cell lung cancer.

Mitochondrial dysfunction is related to necrosis-like programmed cell death induced by A23187 in CEM cells. 査読有り国際誌 Open Access

Hamahata K, Adachi S, Matsubara H, Okada M, Imai T, Watanabe K, Toyokuni SY, Ueno M, Wakabayashi S, Katanosaka Y, Akiba S, Kubota M, Nakahata T

European journal of pharmacology 516 巻 ( 3 ) 頁： 187 - 96 2005年6月

記述言語：英語掲載種別：学位論文（博士）出版者・発行元：European Journal of Pharmacology

We have previously reported that calcium ionophore A23187 differentially induces necrosis in CEM cells, a T-lymphoblastic leukemia cell line, and apoptosis in HL60 cells, a promyelocytic leukemia cell line. Stimulation with VP16, however, induces typical apoptosis in both cell lines. Necrosis in CEM cells, characterized by cell shrinkage and clustering, began within 5 min of treatment. Swelling of the mitochondria, lumpy chromatin condensation and intact plasma membranes were evident by electron microscopy. These A23187-mediated changes in CEM cells were suppressed by clonazepam or CGP37157, inhibitors of the mitochondrial Na+/Ca2+ exchanger. The changes, however, were not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. In both CEM and HL60 cells, intra-cellular calcium increased with similar amplitude within 1 min of treatment with 2 μM A23187. Intra-mitochondrial calcium increased with clonazepam pre-treatment alone in both CEM and HL60 cells. However, intra-mitochondrial calcium did not change drastically in response to A23187 in CEM or HL60 cells, either untreated or pre-treated with clonazepam. A23187 induces necrosis in CEM cells concurrent with mitochondrial dysfunction, which is independent of the mitochondrial permeability transition, but affected by intra-mitochondrial calcium, while HL60 cells lack these early changes. Differences in the responses to A23187 between these two cell lines might derive from differences in the susceptibility of the mitochondrial membrane to rapid increases in intra-cellular calcium. © 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2005.04.018

Ichikawa, M; Mio, T; Toyokuni, S; Tanaka, F; Wada, H; Mishima, M; Yanagihara, K

JOURNAL OF CLINICAL ONCOLOGY 23 巻 ( 16 ) 頁： 682S - 682S 2005年6月

Annexin II overexpression correlates with a poor prognosis in surgically resected non-small-cell lung cancer. 査読有り国際誌

Ichikawa, M, Mio, T, Toyokuni, S, Tanaka, F, Wada, H, Mishima, M, Yanagihara, K

JOURNAL OF CLINICAL ONCOLOGY 23 巻 ( 16 ) 頁： 682S - 682S 2005年6月

Two distinct mechanisms for loss of thioredoxin-binding protein-2 in oxidative stress-induced renal carcinogenesis. 査読有り国際誌 Open Access

Dutta KK, Nishinaka Y, Masutani H, Akatsuka S, Aung TT, Shirase T, Lee WH, Yamada Y, Hiai H, Yodoi J, Toyokuni S

Laboratory investigation; a journal of technical methods and pathology 85 巻 ( 6 ) 頁： 798 - 807 2005年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Laboratory Investigation

Thioredoxin is a major component of thiol-reducing system. Recently, we identified thioredoxin-binding protein-2 (TBP-2) as a negative regulator of thioredoxin. Here, we report the role of TBP-2 in oxidative renal tubular injury and the subsequent carcinogenesis by ferric nitrilotriacetate. TBP-2 was abundantly expressed in the rat kidney. Immunohistochemical analysis revealed that TBP-2 was present in association with nuclei and mitochondrial intermembrane space in the proximal tubular cells and coimmunoprecipitated with cytochrome c. After acute oxidative tubular damage, TBP-2 protein, but not messenger RNA, markedly decreased, demonstrating shortened half-life of this protein. Most cases of the induced renal cell carcinoma showed undetectable levels of TBP-2 protein, which was associated with the methylation of CpG island in the promoter region. Genome sequence analyses identified the poly-A tract in the 3′ untranslated region as a mutation hot spot in this rather nonselective environment. Collectively, the amounts of TBP-2 protein were inversely associated with proliferation of tubular cells, as evaluated by proliferating cell nuclear antigen. These results suggest that loss of TBP-2 is essential for proliferation of not only neoplastic but also non-neoplastic renal tubular cells, and that TBP-2 is a target gene in oxidative stress-induced renal carcinogenesis by ferric nitrilotriacetate. © 2005 USCAP, Inc All rights reserved.

DOI： 10.1038/labinvest.3700280

Transcriptional control of fetal liver hematopoiesis: dominant negative effect of the overexpression of the LIM domain mutants of LMO2. 査読有り国際誌 Open Access

Terano T, Zhong Y, Toyokuni S, Hiai H, Yamada Y

Experimental hematology 33 巻 ( 6 ) 頁： 641 - 51 2005年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Experimental Hematology

Objective. The LIM-finger protein LMO2 forms a transcription factor complex with other hematopoietic regulator proteins, such as TAL1 (SCL), LDB1, GATA1, 2, and 3, in the promoters of several erythroid genes. To elucidate the functional role of two LIM domains in LMO2, we introduced deletion or mutation in each of the LIM domains and analyzed their phenotypic effects on the hematopoietic system when overexpressed in vivo or in vitro. Materials and Methods. Protein interactions of LIM-modified LMO2 constructs with TAL1, LDB1, and GATAs were examined in an immunoprecipitation assay. In vivo hematopoiesis in transgenic mice with wild-type and LIM-modified Lmo2 was studied morphologically and by measuring the progenitor cells in fetal liver. Their effects on the erythroid differentiation of the dimethylsulfoxide (DMSO)-induced murine erythroleukemia (MEL) cells were evaluated. Results. Deletion of the LIM2 domain, but not of the LIM1 domain, abolished its binding of GATA proteins. Overexpression of wild-type LMO2 is known to have dominant negative inhibitory effects on erythropoietic development. Enforced expression of LMO2 constructs with mutant or absent LIM2 but with an intact LIM1 domain resulted in fetal death, small livers and hearts, and decreased hematopoiesis, as well as a hypoplastic thymus. DMSO-induced erythroid differentiation of the MEL cells was inhibited by the overexpressed LMO2 with mutant LIM2 but not by the LMO2 with modified LIM1. Conclusion. Overexpression of the LMO2 with modified LIM2 inhibited hematopoiesis probably by interfering with the formation of the physiological complex or by replacing the functional LMO2 with mutants with reduced affinity to GATA proteins. In this experiment, no evident effect of the LMO2 with modified LIM1 could be observed. © 2005 International. Society for Experimental Hematology. Published by Elsevier Inc.

DOI： 10.1016/j.exphem.2005.01.018

Analysis of rat insulin II promoter-ghrelin transgenic mice and rat glucagon promoter-ghrelin transgenic mice. 査読有り国際誌 Open Access

Iwakura H, Hosoda K, Son C, Fujikura J, Tomita T, Noguchi M, Ariyasu H, Takaya K, Masuzaki H, Ogawa Y, Hayashi T, Inoue G, Akamizu T, Hosoda H, Kojima M, Itoh H, Toyokuni S, Kangawa K, Nakao K

The Journal of biological chemistry 280 巻 ( 15 ) 頁： 15247 - 56 2005年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

We developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The pancreatic tissue ghrelin concentration measured by C-terminal radioimmunoassay (RIA) and plasma desacyl ghrelin concentration of RIP-G Tg were about 1000 and 3.4 times higher than those of nontransgenic littermates, respectively. The pancreatic tissue n-octanoylated ghrelin concentration measured by N-terminal RIA and plasma n-octanoylated ghrelin concentration of RIP-G Tg were not distinguishable from those of nontransgenic littermates. RIP-G Tg showed suppression of glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion, pancreatic insulin mRNA and peptide levels, β cell mass, islet architecture, and GLUT2 and PDX-1 immunoreactivity in RIP-G Tg pancreas were not significantly different from those of nontransgenic littermates. Islet batch incubation study did not show suppression of insulin secretion of RIP-G Tg in vitro. The insulin tolerance test showed lower tendency of blood glucose levels in RIP-G Tg. Taking lower tendency of triglyceride level of RIP-G Tg into consideration, these results may indicate that the suppression of insulin secretion is likely due to the effect of desacyl ghrelin on insulin sensitivity. RGP-G Tg, in which the pancreatic tissue ghrelin concentration measured by C-RIA was about 50 times higher than that of nontransgenic littermates, showed no significant changes in insulin secretion, glucose metabolism, islet mass, and islet architecture. The present study raises the possibility that desacyl ghrelin may have influence on glucose metabolism. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M411358200

その他リンク： http://orcid.org/0000-0001-8033-0736

CYP3A induction more impairs endotoxemic liver injury via reactive oxygen species in rats

Minamiyama, Y; Takemura, S; Kodai, S; Toyokuni, S; Yoshikawa, T; Okada, S

GASTROENTEROLOGY 128 巻 ( 4 ) 頁： A709 - A709 2005年4月

Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions. 査読有り国際誌

Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Ogura A, Shinohara T

Biology of reproduction 72 巻 ( 4 ) 頁： 985 - 91 2005年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogonial stem cells are the only stem cells in the body that transmit genetic information to the next generation. These cells can be cultured for extended periods in the presence of serum and feeder cells. However, little is known about factors that regulate self-renewal division of spermatogonial stem cells. In this investigation we examined the possibility of establishing culture systems for spermatogonial stem cells that lack serum or a feeder cell layer. Spermatogonial stem cells could expand in serum-free conditions on mouse embryonic fibroblasts (MEFs), or were successfully cultivated without feeder cells on a laminin-coated plate. However, they could not expand when both serum and feeder cells were absent. Although the cells cultured on laminin differed phenotypically from those on feeder cells, they grew exponentially for at least 6 mo, and produced normal, fertile progeny following transplantation into infertile mouse testis. This culture system will provide a new opportunity for understanding the regulatory mechanism that governs spermatogonial stem cells. © 2005 by the Society for the Study of Reproduction, Inc.

DOI： 10.1095/biolreprod.104.036400

CYP3A induction more impairs endotoxemic liver injury via reactive oxygen species in rats 国際誌

Minamiyama, Y, Takemura, S, Kodai, S, Toyokuni, S, Yoshikawa, T, Okada, S

GASTROENTEROLOGY 128 巻 ( 4 ) 頁： A709 - A709 2005年4月

多発隆起型早期胆嚢癌の1切除例

浦井俊二, 早雲孝信, 羽生泰樹, 河村卓二, 塩せいじ, 池浦司, 吉野琢哉, 平井潔, 豊國伸哉

日本消化器病學會雜誌 = The Japanese journal of gastro-enterology 102 巻 ( 3 ) 頁： 340 - 345 2005年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：一般財団法人日本消化器病学会

症例は75歳，女性．スクリーニングの腹部超音波検査で胆嚢内に複数の隆起性病変を認め，入院となった．CT，ERCPなどの所見より隆起型胆嚢癌と診断し，開腹胆摘術を行った．病理組織学的に3個の乳頭腺癌を認め，深達度は最大の腫瘍がmp，他はmであった．全割切片による検索で上皮内癌を認め，癌周囲には異形成をともなっていた．Ki-67標識率は固有上皮部2.6％，異形成部9.7％，上皮内癌部26.6％，癌部36.9％であった．p53蛋白過剰発現を認めなかった．

DOI： 10.11405/nisshoshi.102.340

Selenium Supplementation Rescues Cardiac Output in Patients with Severe Heart Failure who are Intractable to ACE Inhibitors and β-Blockers(Heart Failure, Clinical 14 (M), The 69th Annual Scientific Meeting of the Japanese Circulation Society)

Hiraoka Yuuji, Kihara Yasuki, Sarazawa Katsuhiko, Inai Yoshihito, Kitagawa Motoaki, Takahashi Nobuki, Hamanaka Ichirou, Yamazaki Taketoshi, Akagi Tadasu, Hama Norio, Tanabe Masato, Sakurai Hiromu, Toyokuni Shinya, Ueda Kinzou

Circulation journal : official journal of the Japanese Circulation Society 69 巻 ( 0 ) 頁： 310 2005年3月

Circulation journal : official journal of the Japanese Circulation Society 69 巻 ( 0 ) 2005年3月

Genetic selection of mouse male germline stem cells in vitro: offspring from single stem cells. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Toyokuni S, Shinohara T

Biology of reproduction 72 巻 ( 1 ) 頁： 236 - 40 2005年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogenesis originates from a small population of spermatogonial stem cells. These cells are believed to divide infinitely and support spermatogenesis throughout life in the male. In this investigation, we examined the possibility of deriving transgenic offspring from single spermatogonial stem cells. Spermatogonial stem cells were transfected in vitro with a plasmid vector containing a drug resistant gene. Stably transfected stem cell clones were isolated by in vitro drug selection; these clones were expanded and used to produce transgenic progeny following spermatogonial transplantation into infertile recipients. An average of 49% of the offspring carried the transgene, and the recipient mice continued to produce monoclonal transgenic progeny a year after transplantation. Thus, a somatic cell-based genetic approach can be used to modify and select clones of spermatogonial stem cells in a manner similar to embryonic stem cells. The feasibility of genetic selection using postnatal spermatogonial stem cells demonstrates their extensive proliferative potential and provides the opportunity to develop new methods for generating stable animal transgenics or for germline gene therapy.

DOI： 10.1095/biolreprod.104.035659

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis. 査読有り国際誌 Open Access

Chuma S, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Hosokawa M, Nakatsuji N, Ogura A, Shinohara T

Development (Cambridge, England) 132 巻 ( 1 ) 頁： 117 - 22 2005年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Development

Primordial germ cells (PGCs) are derived from a population of pluripotent epiblast cells in mice. However, little is known about when and how PGCs acquire the capacity to differentiate into functional germ cells, while keeping the potential to derive pluripotent embryonic germ cells and teratocarcinomas. In this investigation, we show that epiblast cells and PGCs can establish colonies of spermatogenesis after transfer into postnatal seminiferous tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination using spermatozoa or spermatids derived from PGCs harvested from fetuses as early as 8.5 days post coitum. Thus, fetal male germ cell development is remarkably flexible, and the maturation process, from epiblast cells through PGCs to postnatal spermatogonia, can occur in the postnatal testicular environment. Primordial germ cell transplantation techniques will also provide a novel tool to assess the developmental potential of PGCs, such as those manipulated in vitro or recovered from embryos harboring lethal mutations.

DOI： 10.1242/dev.01555

Reduction of dibetes-induced renal oxidative stress by a cantaloupe melon extract/gliadin biopolymers, oxykine, in mice. 査読有り

Naito Y, Akagiri S, Uchiyama K, Kokura S, Yoshida N, Hasegawa G, Nakamura N, Ichikawa H, Toyokuni S, Ijichi T, Yoshikawa T.

Biofactors 23 巻頁： 85-98 2005年

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture. 査読有り

Kanatsu-Shinohara M, Ogonuki N, Iwano T, Lee J, Kazuki Y, Inoue K, Miki H, Takehashi M, Toyokuni S, Shinkai Y, Oshimura M, Ishino F, Ogura A, Shinohara T.

Developmnet 132 巻頁： 4155-4163 2005年

Establishment of a diabetic mouse model with progressive diabetic nephropathy. 査読有り

Inada A, Nagai K, Arai H, Miyazaki J, Nomura K, Kanamori H, Toyokuni S, Yamada Y, Bonner-Weir S, Weir GC, Fukatsu A, Seino Y.

Am. J. Pathol. 167 巻頁： 327-336 2005年

Free radical-induced carcinogenesis: Target genes and fragile genome sites

Toyokuni, S; Akatsuka, S; Aung, TT; Dutta, KK

FREE RADICAL RESEARCH 39 巻頁： S30 - S30 2005年

Imidapril, an angiotensin-converting enzyme inhibitor, reduces diabetes-induced renal oxidative damage in mice

Naito Y, Uchiyama K, Akagiri S, Aoi W, Hasegawa G, Nakamura N, Kokura S, Yoshida N, Ichikawa H, Toyokuni S, Yoshikawa T

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 37 巻 ( 1 ) 頁： 29 - 37 2005年

Reduction of diabetes-induced renal oxidative stress by a cantaloupe melon extract/gliadin biopolymers, oxykine, in mice

Naito Y, Akagiri S, Uchiyama K, Kokura S, Yoshida N, Hasegawa G, Nakamura N, Ichikawa H, Toyokuni S, Ijichi T, Yoshikawa T

BIOFACTORS 23 巻 ( 2 ) 頁： 85 - 95 2005年

Reduction of diabetes-induced renal oxidative stress by a cantaloupe melon extract/gliadin biopolymers, oxykine, in mice.

Naito Y, Akagiri S, Uchiyama K, Kokura S, Yoshida N, Hasegawa G, Nakamura N, Ichikawa H, Toyokuni S, Ijichi T, Yoshikawa T

BioFactors (Oxford, England) 23 巻 ( 2 ) 頁： 85 - 95 2005年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BioFactors

Oxidative stress is implicated as an important mechanism by which diabetes causes nephropathy. Oxykine is the cantaloupe melon extract rich in vegetal superoxide dismutase covered by polymeric films of wheat matrix gliadin. In this study, we examined whether chronic oral administration of oxykine could prevent the progression of diabetic nephropathy induced by oxidative stress using preclinical rodent model of type 2 diabetes. We used female db/db mice and their non-diabetic db/m littermates. The mice were divided into the following three groups: non-diabetic db/m; diabetic db/db, and diabetic db/db treated with oxykine. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments. Histological and 8-OHdG immunohistochemical studies were preformed on 12 weeks from the beginning of treatment. After 12 weeks of treatment, the levels of blood glucose and the body weight were not significantly different between the oxykine-treated group and the non-treated db/db group, however both groups kept significantly high levels rather than db/m mice. The relative mesangial area calculated by mesangial area/total glomerular area ratio was significantly ameliorated in the oxykine treated group compared with non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with oxykine. The 8-OHdG immunoreactive cells in the glomeruli of non-treated db/db mice were more numerous than that of oxykine-treated db/db mice. In this study, treatment of oxykine ameliorated the progression and acceleration of diabetic nephropathy for rodent model of type 2 diabetes. These results indicated that the oxykine reduced the diabetes-induced oxidative stress and renal mesangial cell injury. In conclusion, oxykine might be a novel approach for the prevention of diabetes nephropathy. © 2005 - IOS Press and the authors. All rights reserved.

DOI： 10.1002/biof.5520230204

Reduction of diabetes-induced renal oxidative stress by a cantaloupe melon extract/gliadin biopolymers, oxykine, in mice 査読有り

Y Naito, S Akagiri, K Uchiyama, S Kokura, N Yoshida, G Hasegawa, N Nakamura, H Ichikawa, S Toyokuni, T Ijichi, T Yoshikawa

BIOFACTORS 23 巻 ( 2 ) 頁： 85 - 95 2005年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：IOS PRESS

Oxidative stress is implicated as an important mechanism by which diabetes causes nephropathy. Oxykine is the cantaloupe melon extract rich in vegetal superoxide dismutase covered by polymeric films of wheat matrix gliadin. In this study, we examined whether chronic oral administration of oxykine could prevent the progression of diabetic nephropathy induced by oxidative stress using preclinical rodent model of type 2 diabetes. We used female db/db mice and their non-diabetic db/m littermates. The mice were divided into the following three groups: non-diabetic db/m; diabetic db/db, and diabetic db/db treated with oxykine. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments. Histological and 8-OHdG immunohistochemical studies were preformed on 12 weeks from the beginning of treatment. After 12 weeks of treatment, the levels of blood glucose and the body weight were not significantly different between the oxykine-treated group and the non-treated db/db group, however both groups kept significantly high levels rather than db/m mice. The relative mesangial area calculated by mesangial area/total glomerular area ratio was significantly ameliorated in the oxykine treated group compared with non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with oxykine. The 8-OHdG immunoreactive cells in the glomeruli of non-treated db/db mice were more numerous than that of oxykine-treated db /db mice. In this study, treatment of oxykine ameliorated the progression and acceleration of diabetic nephropathy for rodent model of type 2 diabetes. These results indicated that the oxykine reduced the diabetes-induced oxidative stress and renal mesangial cell injury. In conclusion, oxykine might be a novel approach for the prevention of diabetes nephropathy.

Imidapril, an angiotensin-converting enzyme inhibitor, reduces diabetes-induced renal oxidative damage in mice 査読有り

Y Naito, K Uchiyama, S Akagiri, W Aoi, G Hasegawa, N Nakamura, S Kokura, N Yoshida, H Ichikawa, S Toyokuni, T Yoshikawa

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 37 巻 ( 1 ) 頁： 29 - 37 2005年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

The aim of the present study was to determine whether imidapril, an angiotensin-converting enzyme inhibitor, prevents diabetic nephropathy and to determine the effect of imidapril on oxidative damage in the diabetic glomeruli in mice. We used female BKS.cg-m+Lepr(db)/+Lepr(db) (db/db) mice, a rodent model of type 2 diabetes, and their non-diabetic db/m littermates. The mice were divided into the following four groups: non-diabetic db/m, diabetic db/db, and diabetic db/db treated with imidapril at doses of 1 mg/kg and 5 mg/kg. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8OHdG) were measured during the experiments. Histological and 8-OHdG immunohistochemical studies were performed for 12 weeks from the beginning of treatment. After 12 weeks of treatment, the levels of blood glucose and the body weight were not significantly different between the imidapril-treated group and the non-treated db/db group. The systolic blood pressure was significantly increased in db/db mice compared with db/m mice. The increased blood pressure was significantly reduced by the treatment with imidapril at a dose of 5 mg/kg. The relative mesangial area calculated by the mesangial area/total glomerular area ratio was significantly ameliorated in the imidapril-treated group compared with the non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with imidapril in a dose-dependent manner. The 8-OHdG immunoreactive cells in the glomeruli of non-treated db/db mice were more numerous than in the imidapril-treated db/db mice. In conclusion, imidapril prevented the progression of diabetic nephropathy in mice, not only by decreasing the glomerular pressure but also by decreasing the oxidative stress in glomerular cells.

Novel mechanism of matrix metalloproteinase inhibitor in the treatment of heart failure:: Direct inhibition of myocardial oxidant stress and NF-κB activation in rats

Control of the peri-operative oxidative stress enables fast recovery from myocardial ischemic damage in cardic surgery. 査読有り

Takaba K, Matsumura K, Miwa S, Yamazaki K, Nishina K, Nomoto T, Toyokuni S, Hyon SH, Ikeda T, Komeda M

International Redox Network 2005（2005.11.9－11. kyoto） 2005年

Generation of pluripotent stem cells from neonatal mouse testis. 査読有り国際誌

Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, Oshimura M, Heike T, Nakahata T, Ishino F, Ogura A, Shinohara T

Cell 119 巻 ( 7 ) 頁： 1001 - 12 2004年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.

DOI： 10.1016/j.cell.2004.11.011

Generation of pluripotent stem cells from neonatal mouse testis

Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, Oshimura M, Heike T, Nakahata T, Ishino F, Ogura A, Shinohara T

CELL 119 巻 ( 7 ) 頁： 1001 - 1012 2004年12月

オキシカインによる糖尿病性腎症の進展抑制効果査読有り

市川寛, 赤桐里美, 内藤裕二, 内山和彦, 吉田憲正, 豊國伸哉, 吉川敏一

日本病態栄養学会誌 7 巻 ( 4 ) 頁： 375 - 375 2004年11月

Takenaka, H; Kihara, Y; Iwanaga, Y; Onozawa, Y; Toyokuni, S; Kita, T

CIRCULATION 110 巻 ( 17 ) 頁： 514 - 515 2004年10月

Novel mechanism of matrix metalloproteinase inhibitor in the treatment of heart failure: Direct inhibition of myocardial oxidant stress and NF-kappa B activation in rats 国際誌

Takenaka H, Kihara Y, Iwanaga Y, Onozawa Y, Toyokuni S, Kita T

CIRCULATION 110 巻 ( 17 ) 頁： 514 - 515 2004年10月

Novel mechanism of matrix metalloproteinase inhibitor in the treatment of heart failure:: Direct inhibition of myocardial oxidant stress and NF-κB activation in rats 国際誌

Takenaka, H, Kihara, Y, Iwanaga, Y, Onozawa, Y, Toyokuni, S, Kita, T

CIRCULATION 110 巻 ( 17 ) 頁： 514 - 515 2004年10月

Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo. 国際誌 Open Access

Kanatsu-Shinohara M, Toyokuni S, Shinohara T

Biology of reproduction 71 巻 ( 4 ) 頁： 1202 - 7 2004年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogonial stem cells are the only stem cells in the postnatal body that can transmit parental genetic information to the offspring, making them an attractive target cell population for animal transgenesis. Although transgenic mice and rats were recently produced by retrovirus transduction of these cells in vitro, with transplantation of the transduced cells into infertile recipients, the difficulty of restoring fertility and preparing recipients using spermatogonial transplantation limits practical application of the technique. In this article, we describe a novel approach for producing transgenic animals by transducing spermatogonial stem cells in vivo using a retrovirus vector. Microinjection of retrovirus into immature seminiferous tubules resulted in the direct transduction of spermatogonial stem cells in situ, and the animals produced transgenic offspring after mating with females. Transgenic mice were produced in C57BL/6, BALB/C, A, and C3H backgrounds, with an average efficiency of 2.8%. The transgene was transmitted stably and expressed in the next generation. The technique overcomes the drawback of the in vitro-transduction approach, and will be useful as a novel method for producing transgenic animals as well as providing a means for analyzing the self-renewal and differentiation processes of spermatogonial stem cells in vivo.

DOI： 10.1095/biolreprod.104.031294

Cyp3A induction aggravates endotoxemic liver injury via reactive oxygen species in male rats 査読有り国際誌

Minamiyama, Y; Takemura, S; Toyokuni, S; Imaoka, S; Funae, Y; Hirohashi, K; Yoshikawa, T; Okada, S

FREE RADICAL BIOLOGY AND MEDICINE 37 巻 ( 5 ) 頁： 703 - 712 2004年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

We carried out this experiment to evaluate the relationship between isoforms of cytochrome P450 (P450) and liver injury in lipopolysaccharide (LPS)-induced endotoxemic rats. Male rats were intraperitoneally administered phenobarbital (PB), a P450 inducer, for 3 days, and 1 day later, they were intravenously given LPS. PB significantly increased P450 levels (200% of control levels) and the activities (300-400% of control) of the specific isoforms (CYP), CYP3A2 and CYP2B1, in male rats. Plasma AST and ALT increased slightly more in PB-treated rats than in PB-nontreated (control) rats with LPS treatment. Furthermore, either troleandomycin or ketoconazole, specific CYP3A inhibitors, significantly inhibited LPS-induced liver injury in control and PB-treated male rats. To evaluate the oxidative stress in LPS-treated rats, in situ superoxide radical detection using dihydroethidium (DHE), hydroxy-2-nonenal (HNE)-modified proteins in liver microsomes and 8-hydroxydeoxyguanosine (8-OHdG) in liver nuclei were measured in control and PB-treated rats. DHE signal intensity, levels of HNE-modified proteins, and 8-OHdG increased significantly in PB-treated rats. LPS further increased DHE intensity, HNE-modified proteins, and 8-OHdG levels in normal and PB-treated groups. CYP3A inhibitors also inhibited the increases in these items. Our results indicate that the induction or preservation of CYP isoforms further promotes LPS-induced liver injury through mechanisms related to oxidative stress. In particular, CYP3A2 of P450 isoforms made an important contribution to this LPS-induced liver injury. © 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2004.05.022

Gastric inhibitory polypeptide is the major insulinotropic factor in K-ATP null mice 査読有り国際誌

Tsukiyama K, Yamada Y, Miyawaki K, Hamasaki A, Nagashima K, Hosokawa M, Fujimoto S, Takahashi A, Toyoda K, Toyokuni S, Oiso Y, Seino Y

EUROPEAN JOURNAL OF ENDOCRINOLOGY 151 巻 ( 3 ) 頁： 407 - 412 2004年9月

Gastric inhibitory polypeptide is the major insulinotropic factor in K(ATP) null mice.

Tsukiyama K, Yamada Y, Miyawaki K, Hamasaki A, Nagashima K, Hosokawa M, Fujimoto S, Takahashi A, Toyoda K, Toyokuni S, Oiso Y, Seino Y

European journal of endocrinology 151 巻 ( 3 ) 頁： 407 - 12 2004年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：European Journal of Endocrinology

Objective: ATP-sensitive K+ (KATP) channels in pancreatic β-cells are crucial in the regulation of glucose-induced insulin secretion. Recently, KATP channel-deficient mice were generated by genetic disruption of Kir6.2, the pore-forming component of KATP channels, but the mice still showed a significant insulin response after oral glucose loading in vivo. Gastric inhibitory polypeptide (GIP) is a physiological incretin that stimulates insulin release upon ingestion of nutrients. To determine if GIP is the insulinotropic factor in insulin secretion in KATP channel-deficient mice, we generated double-knockout Kir6.2 and GIP receptor null mice and compared them with Kir6.2 knockout mice. Methods: Double-knockout mice were generated by intercrossing Kir6.2-knockout mice with GIP receptor-knockout mice. An oral glucose tolerance test, insulin tolerance test and batch incubation study of pancreatic islets were performed on double-knockout mice and Kir6.2-knockout mice. Results: Fasting glucose and insulin levels were similar in both groups. After oral glucose loading, blood glucose levels of double-knockout mice became elevated compared with Kir6.2-knockout mice, especially at 15min (345±10mg/dl vs 294±20mg/dl, P<0.05 and 30min (453±20 mg/dl vs 381 ± 26 mg/dl, P < 0.05). The insulin response was almost completely lost in double-knockout mice, although insulin secretion from isolated islets was stimulated by another incretin, glucagon-like peptide-1 in the double-knockout mice. Double-knockout mice and Kir6.2-knockout mice were similarly insulin sensitive as assessed by the insulin tolerance test. Conclusion: GIP is the major insulinotropic factor in the secretion of insulin in response to glucose load in KATP channel-deficient mice. © 2004 Society of the European Journal of Endocrinology.

DOI： 10.1530/eje.0.1510407

Gastric inhibitory polypeptide is the major insulinotropic factor in K(ATP) null mice. 査読有り国際誌

Katsushi Tsukiyama, Yuichiro Yamada, Kazumasa Miyawaki, Akihiro Hamasaki, Kazuaki Nagashima, Masaya Hosokawa, Shimpei Fujimoto, Akira Takahashi, Kentaro Toyoda, Shinya Toyokuni, Yutaka Oiso, Yutaka Seino

European journal of endocrinology 151 巻 ( 3 ) 頁： 407 - 12 2004年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

OBJECTIVE: ATP-sensitive K(+) (K(ATP)) channels in pancreatic beta-cells are crucial in the regulation of glucose-induced insulin secretion. Recently, K(ATP) channel-deficient mice were generated by genetic disruption of Kir6.2, the pore-forming component of K(ATP) channels, but the mice still showed a significant insulin response after oral glucose loading in vivo. Gastric inhibitory polypeptide (GIP) is a physiological incretin that stimulates insulin release upon ingestion of nutrients. To determine if GIP is the insulinotropic factor in insulin secretion in K(ATP) channel-deficient mice, we generated double-knockout Kir6.2 and GIP receptor null mice and compared them with Kir6.2 knockout mice. METHODS: Double-knockout mice were generated by intercrossing Kir6.2-knockout mice with GIP receptor-knockout mice. An oral glucose tolerance test, insulin tolerance test and batch incubation study of pancreatic islets were performed on double-knockout mice and Kir6.2-knockout mice. RESULTS: Fasting glucose and insulin levels were similar in both groups. After oral glucose loading, blood glucose levels of double-knockout mice became elevated compared with Kir6.2-knockout mice, especially at 15 min (345+/-10 mg/dl vs 294+/-20 mg/dl, P<0.05) and 30 min (453+/-20 mg/dl vs 381+/-26 mg/dl, P<0.05). The insulin response was almost completely lost in double-knockout mice, although insulin secretion from isolated islets was stimulated by another incretin, glucagon-like peptide-1 in the double-knockout mice. Double-knockout mice and Kir6.2-knockout mice were similarly insulin sensitive as assessed by the insulin tolerance test. CONCLUSION: GIP is the major insulinotropic factor in the secretion of insulin in response to glucose load in K(ATP) channel-deficient mice.

PAX6 regulates islet β-cell function

Prevention of myocardial reperfusion injury by poly(ADP-ribose) synthetase inhibitor, 3-aminobenzamide, in cardioplegic solution: in vitro study of isolated rat heart model. 国際誌 Open Access

Yamazaki K, Miwa S, Ueda K, Tanaka S, Toyokuni S, Unimonh O, Nishimura K, Komeda M

European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery 26 巻 ( 2 ) 頁： 270 - 5 2004年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：European Journal of Cardio-thoracic Surgery

Objective: Cardioplegic arrest remains the method of choice for myocardial protection in cardiac surgery. Poly(adenosine 5′-diphosphate-ribose) synthetase (PARS) inhibitor has been suggested to attenuate the ischemia-reperfusion injury in myocardial infarction by preventing energy depletion associated with oxidative stress. We investigated the efficacy of a cardioplegic solution containing a PARS inhibitor, 3-aminobenzamide (3-AB), for myocardial protection against ischemia-reperfusion injury caused by cardioplegic arrest. Methods: Isolated hearts were set on a Langendorff apparatus and perfused. The hearts were arrested for 90 min with a cardioplegic solution given at 30-min intervals and then reperfused for 20 min. The hearts of rat in the 3-AB(-) group (n=8) were perfused with a standard cardioplegic solution and terminal warm cardoplegia, whereas the 3-AB(+) group (n=8) received these solutions supplemented with 3-AB (100 μM). Left ventricular function and release of cardiac enzymes were monitored before and after cardioplegic arrest. After reperfusion, NAD+ (nicotinamide-adenine dinucleotide) levels were assessed, and the tissues were examined immunohistochemically for oxidative stress and apoptosis. Results: During reperfusion, the 3-AB(+) group showed significantly higher (P=0.005) dp/dt and lower creatine phosphokinase (CPK) level and glucotamic-oxaloacetic transaminase (GOT) in the effluent (CPK; P=0.003, GOT; P<0.001). The cardiomyocytes of the 3-AB(+) group also preserved a higher NAD+ level (P<0.001). Immunohistochemical study of oxidative stress revealed a lesser extent (P=0.007) of nuclear staining and a lower fraction of apoptosis in the 3-AB(+) group. Conclusion: Cardioplegic solution supplemented with 3-AB provides efficient myocardial protection in cardioplegic ischemic reperfusion by suppressing oxidative stress and overactivation of PARS. © 2004 Published by Elsevier B.V.

DOI： 10.1016/j.ejcts.2004.04.044

Proteasome inhibitor, bortezomib, potently inhibits the growth of adult T-cell leukemia cells both in vivo and in vitro. 査読有り国際誌 Open Access

Satou Y, Nosaka K, Koya Y, Yasunaga JI, Toyokuni S, Matsuoka M

Leukemia 18 巻 ( 8 ) 頁： 1357 - 63 2004年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Leukemia

Adult T-cell leukemia (ATL) is a fatal neoplasm derived from CD4-positive T-lymphocytes, and regardless of intensive chemotherapy, its mean survival time is less than 1 year. Nuclear factor-κB (NF-κB) activation was reported in HTLV-I associated cells, and has been implicated in oncogenesis and resistance to anticancer agents and apoptosis. We studied the effect of a proteasome inhibitor, bortezomib (formerly known as PS-341), on ATL cells in vitro and in vivo. Bortezomib could inhibit the degradation Of IκBα in ATL cells, resulting in suppression of NF-κB and induction of cell death in ATL cells in vitro. Susceptibilities to bortezomib were well correlated with NF-κB activation, suggesting that suppression of the NF-κB pathway was implicated in the cell death induced by bortezomib. Although the majority of the cell death was apoptosis, necrotic cell death was observed in the presence of a caspase inhibitor, z-VAD-fmk. When bortezomib was administered into SCID mice bearing tumors, it suppressed tumor growth in vivo, showing that bortezomib was effective against ATL cells in vivo. These studies revealed that bortezomib is highly effective against ATL cells in vitro and in vivo by induction of apoptosis, and its clinical application might improve the prognosis of patients with this fatal disease. © 2004 Nature Publishing Group All rights reserved.

DOI： 10.1038/sj.leu.2403400

その他リンク： http://orcid.org/0000-0002-7939-2080

Regulation of mouse spermatogonial stem cell self-renewing division by the pituitary gland. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Morimoto T, Toyokuni S, Shinohara T

Biology of reproduction 70 巻 ( 6 ) 頁： 1731 - 7 2004年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogenesis originates in spermatogonial stem cells, which have the unique mode of replication. It is considered that a single stem cell can produce two stem cells (self-renewing division), one stem and one differentiating (asymmetric division), or two differentiating cells (differentiating division). However, little is known regarding how each type of division is regulated. In this investigation, we focused on the analysis of self-renewing division and examined the effect of the pituitary gland using two models of stem cell self-renewing division. In the first experiment using newborn mice, the administration of GnRH-analogue, which represses the release of gonadotropin, reduced the number of stem cells during postnatal testicular development, suggesting that the pituitary gland enhances stem cell self-renewing division. In the second experiment, however, the number of stem cells increased dramatically in hypophysectomized adult recipients after spermatogonial transplantation. Thus, the pituitary gland affects the self-renewing division of stem cells, but these contradictory results suggest that its role may be different depending on the stage of the testicular development.

DOI： 10.1095/biolreprod.103.025668

Hamasaki, A; Yamada, Y; Nagashima, K; Fujimoto, S; Ban, N; Toyokuni, S; Kurose, T; Seino, Y

DIABETES 53 巻頁： A394 - A394 2004年6月

Role of reactive oxygen species in skin carcinogenesis

Nishigori C, Hattori Y, Toyokuni S

ANTIOXIDANTS & REDOX SIGNALING 6 巻 ( 3 ) 頁： 561 - 570 2004年6月

Redox control of carcinogenesis and tumor biology

Toyokuni S

ANTIOXIDANTS & REDOX SIGNALING 6 巻 ( 3 ) 頁： 481 - 482 2004年6月

Role of reactive oxygen species in skin carcinogenesis.

Nishigori C, Hattori Y, Toyokuni S

Antioxidants & redox signaling 6 巻 ( 3 ) 頁： 561 - 70 2004年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Antioxidants and Redox Signaling

Reactive oxygen species (ROS) are associated not only with initiation, but also with promotion and progression in the multistage carcinogenesis model. In the present review, we will focus on the involvement of ROS in skin carcinogenesis, especially that induced by ultraviolet (UV) radiation. UV-specific DNA damage has been well studied thus far. However, recent reports have revealed the previously unknown participation of oxidative stress in UV-induced skin carcinogenesis. Indeed, in addition to transition-type mutations at dipyrimidine sites, G:C to T:A transversions, which may be induced by the presence of 8-oxoguanine during DNA replication, are frequently observed in the ras oncogene and p53 tumor suppressor gene in human skin cancers of sun-exposed areas and in UV-induced mouse skin cancers. Recent studies have shown that not only UV-B, but also UV-A is involved in UV-induced carcinogenesis. A wide variety of biological phenomena other than direct influence by UV, such as inflammatory and immunological responses and oxidative modifications of DNA and proteins, appear to play roles in UV-induced skin carcinogenesis. Furthermore, it has become clear that genetic diseases such as xeroderma pigmentosum show deficient repair of oxidatively modified DNA lesions. The involvement of ROS in skin carcinogeneisis caused by arsenic and chemical carcinogens will also be discussed.

DOI： 10.1089/152308604773934314

Redox control of carcinogenesis and tumor biology.

Toyokuni S

Antioxidants & redox signaling 6 巻 ( 3 ) 頁： 481 - 2 2004年6月

Role of reactive oxygen species in skin carcinogenesis. 国際誌

Chikako Nishigori, Yukari Hattori, Shinya Toyokuni

Antioxidants & redox signaling 6 巻 ( 3 ) 頁： 561 - 70 2004年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Analysis of ghrelin transgenic mice driven by rat insulin II promoter and transgenic mice driven by rat glucagon promoter

PAX6 regulates islet beta-cell function 国際誌

Hamasaki A, Yamada Y, Nagashima K, Fujimoto S, Ban N, Toyokuni S, Kurose T, Seino Y

DIABETES 53 巻頁： A394 - A394 2004年6月

Iwakura, H; Hosoda, K; Son, C; Fujikura, J; Tomita, T; Noguchi, M; Takaya, K; Masuzaki, H; Ogawa, Y; Hayashi, T; Inoue, G; Akamizu, T; Hosoda, H; Kojima, M; Itoh, H; Toyokuni, S; Kangawa, K; Nakao, K

DIABETES 53 巻頁： A357 - A357 2004年6月

PAX6 regulates islet β-cell function 国際誌

Hamasaki, A, Yamada, Y, Nagashima, K, Fujimoto, S, Ban, N, Toyokuni, S, Kurose, T, Seino, Y

DIABETES 53 巻頁： A394 - A394 2004年6月

Redox regulation of annexin 2 and its implications for oxidative stress-induced renal carcinogenesis and metastasis. 国際誌

Tomoyuki Tanaka, Shinya Akatsuka, Munetaka Ozeki, Tomoyuki Shirase, Hiroshi Hiai, Shinya Toyokuni

Oncogene 23 巻 ( 22 ) 頁： 3980 - 9 2004年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal damage leading to a high incidence of renal cell carcinoma (RCC) in rats. Differential display analysis of such RCCs revealed elevated expression of annexin 2 (Anx2), a substrate for kinases and a receptor for tissue-type plasminogen activator and plasminogen. We conducted this study to clarify the significance of Anx2 in Fenton reaction-based carcinogenesis. Messenger RNA and protein levels of Anx2 were increased time-dependently in the rat kidney after Fe-NTA administration as well as in LLC-PK1 cells after exposure to H2O2. The latter was inhibited by pretreatment with N-acetylcysteine, pyrrolidine dithiocarbamate or catalase. Immunohistochemistry revealed negligible staining in the normal renal proximal tubules, but strong staining in regenerating proximal tubules, karyomegalic cells and RCCs. Metastasizing RCCs showed higher Anx2 protein levels. Anx2 was phosphorylated at serine and tyrosine residues in these cells and coimmunoprecipitated with phosphorylated actin. Overexpression of Anx2 induced a higher cell proliferation rate in LLC-PK1 cells. In contrast, a decrease in proliferation leading to apoptosis was observed after Anx2 antisense treatment to cell lines established from Fe-NTA-induced RCCs. These results suggest that Anx2 is regulated by redox status, and that persistent operation of this adaptive mechanism plays a role in the proliferation and metastasis of oxidative stress-induced cancer.

Redox regulation of annexin 2 and its implications for oxidative stress-induced renal carcinogenesis and metastasis

Tanaka T, Akatsuka S, Ozeki M, Shirase T, Hiai H, Toyokuni S

ONCOGENE 23 巻 ( 22 ) 頁： 3980 - 3989 2004年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Oncogene

DOI： 10.1038/sj.onc.1207555

Neuroprotection by hyperbaric oxygenation after experimental focal cerebral ischemia monitored by MRI. 国際誌

Wolf-Ruediger Schäbitz, Holger Schade, Sabine Heiland, Rainer Kollmar, Jürgen Bardutzky, Nils Henninger, Harald Müller, Ulrich Carl, Shinya Toyokuni, Clemens Sommer, Stefan Schwab

Stroke 35 巻 ( 5 ) 頁： 1175 - 9 2004年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Hyperbaric oxygenation (HBO) after focal cerebral ischemia reduces infarct size and improves outcome when applied early after stroke. Here, we evaluated effects of HBO on permanent focal cerebral ischemia and applied magnetic resonance imaging (MRI) monitoring to study lesion evolution. METHODS: Rats underwent permanent middle cerebral artery occlusion (MCAO). Two hours later, animals were treated with HBO (100% O(2)/2 atm; n=17) for 1 hour or treated with room air (n=17). Animals underwent serial MRI studies (DWI, PI, T2) beginning 90 minutes after MCAO. Neuroscore was assessed (5-point rating scale). Animals were euthanized and brains were 2,3,5-triphenyltetrazolium chloride (TTC)-stained for infarct volume calculation 120 hours after MCAO. Immunohistochemistry was performed with antibodies against c-FOS and 4-hydroxy-2-nonenal-modified proteins (HNE) to check for effects of oxidative stress caused by HBO treatment. RESULTS: HBO reduced infarct volume by 38% (P<0.001). As shown by MRI, neuroprotection began 5 hours after ischemia and remained effective for 5 days. The relative regional cerebral blood flow was not different between groups at 3.5 and 5 hours after occlusion. There was less neurological deficit in HBO-treated animals compared with controls (P<0.05). Lipid peroxidation of cerebral vessels after HBO treatment as measured by HNE staining and pattern of c-FOS induction were not significantly different between groups at 3.5 and 8 hours after ischemia. CONCLUSIONS: As monitored by MRI HBO treatment reversed ischemic lesion size between 3 and 5 hours after ischemia and achieved a long-lasting neuroprotective effect without significant oxidative damage.

Neuroprotection by hyperbaric oxygenation after experimental focal cerebral ischemia monitored by MRI

Schabitz WR, Schade H, Heiland S, Kollmar R, Bardutzky J, Henninger N, Muller H, Carl U, Toyokuni S, Sommer C, Schwab S

STROKE 35 巻 ( 5 ) 頁： 1175 - 1179 2004年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Stroke

Background - Hyperbaric oxygenation (HBO) after focal cerebral ischemia reduces infarct size and improves outcome when applied early after stroke. Here, we evaluated effects of HBO on permanent focal cerebral ischemia and applied magnetic resonance imaging (MRI) monitoring to study lesion evolution. Methods - Rats underwent permanent middle cerebral artery occlusion (MCAO). Two hours later, animals were treated with HBO (100% O2/2 atm; n=17) for 1 hour or treated with room air (n=17). Animals underwent serial MRI studies (DWI, PI, T2) beginning 90 minutes after MCAO. Neuroscore was assessed (5-point rating scale). Animals were euthanized and brains were 2,3,5-triphenyltetrazolium chloride (TTC)-stained for infarct volume calculation 120 hours after MCAO. Immunohistochemistry was performed with antibodies against c-FOS and 4-hydroxy-2-nonenal-modified proteins (HNE) to check for effects of oxidative stress caused by HBO treatment. Results - HBO reduced infarct volume by 38% (P<0.001). As shown by MRI, neuroprotection began 5 hours after ischemia and remained effective for 5 days. The relative regional cerebral blood flow was not different between groups at 3.5 and 5 hours after occlusion. There was less neurological deficit in HBO-treated animals compared with controls (P<0.05). Lipid peroxidation of cerebral vessels after HBO treatment as measured by HNE staining and pattern of c-FOS induction were not significantly different between groups at 3.5 and 8 hours after ischemia. Conclusion - As monitored by MRIHBO treatment reversed ischemic lesion size between 3 and 5 hours after ischemia and achieved a long-lasting neuroprotective effect without significant oxidative damage.

DOI： 10.1161/01.STR.0000125868.86298.8e

Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens

HIROYASU Makoto, AKATSUKA Shinya, SHIRASE Tomoyuki, TODA Yoshinobu, HIAI Hiroshi, TOYOKUNI Shinya

Pathology international 54 巻 ( 4 ) 頁： 251 - 255 2004年4月

Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens. 国際誌

Makoto Hiroyasu, Shinya Akatsuka, Tomoyuki Shirase, Yoshinobu Toda, Hiroshi Hiai, Shinya Toyokuni

Pathology international 54 巻 ( 4 ) 頁： 251 - 5 2004年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

Although the human genome project has been completed, the functions of many genes remain undetermined. In situ hybridization (ISH) is a key method for identifying cells in which a given messenger RNA is transcribed. Paraffin-embedded specimens remain precious materials for research, but preservation of high-quality RNA in these specimens is not expected unless ample caution was taken during fixation. Peptide nucleic acid (PNA) is a recently developed hybrid molecule with genetic information that has high stability and high affinity to the complementary DNA or RNA. We applied a PNA probe to mRNA ISH of liver specimens obtained by autopsy and embedded in paraffin 28-48 years ago. An 18-mer PNA probe for glyceraldehyde 3-phosphate dehydrogenase was used. Staining was then analyzed in association with morphology by hematoxylin and eosin staining, and with the time between death of the patient and tissue fixation. Notably, specimens fixed with formalin and embedded in paraffin 48 years ago yielded excellent results if the time before fixation was short enough (<8 h). There was a significant inverse correlation between the intensity of ISH staining and the time before fixation. Oligonucleotide PNA probe, albeit at high cost, would increase the value of paraffin-embedded specimens in storage for use in human medical research.

Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens.

Overexpression of inducible cyclic AMP early repressor inhibits transactivation of genes and cell proliferation in pancreatic beta cells. 国際誌 Open Access

Inada A, Hamamoto Y, Tsuura Y, Miyazaki J, Toyokuni S, Ihara Y, Nagai K, Yamada Y, Bonner-Weir S, Seino Y

Molecular and cellular biology 24 巻 ( 7 ) 頁： 2831 - 41 2004年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Molecular and Cellular Biology

Transcriptional control mediated by the cyclic AMP-responsive element (CRE) represents an important mechanism of gene regulation. To test our hypothesis that increased inducible cyclic AMP early repressor (ICER) Iγ inhibits function of CRE-binding proteins and thus disrupts CRE-mediated transcription in pancreatic β cells, we generated transgenic mice with β-cell-directed expression of ICER Iγ, a powerful repressor that is greatly increased in diabetes. Three transgenic lines clearly show that increased ICER Iγ expression in β cells results in early severe diabetes. From birth islets were severely disorganized with a significantly increased proportion of α cells throughout the islet. Diabetes results from the combined effects of impaired insulin expression and a decreased number of β cells. The decrease in β cells appears to result from impaired proliferation rather than from increased apoptosis after birth. Cyclin A gene expression is impaired by the strong inhibition of ICER; the suppression of cyclin A results in a substantially decreased proliferation of β cells in the postnatal period. These results suggest that CRE and CRE-binding factors have an important role in pancreatic β-cell physiology not only directly by regulation of gene trans-activation but also indirectly by regulation of β-cell mass.

DOI： 10.1128/MCB.24.7.2831-2841.2004

Hiroyasu M, Akatsuka S, Shirase T, Toda Y, Hiai H, Toyokuni S

Pathology international 54 巻 ( 4 ) 頁： 251 - 5 2004年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

DOI： 10.1111/j.1440-1827.2004.01620.x

A novel mechanism for imatinib mesylate-induced cell death of BCR-ABL-positive human leukemic cells: Caspase-independent, necrosis-like programmed cell death mediated by serine protease activity 査読有り

Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens

Hiroyasu M, Akatsuka S, Shirase T, Toda Y, Hiai H, Toyokuni S

PATHOLOGY INTERNATIONAL 54 巻 ( 4 ) 頁： 251 - 255 2004年4月

Okada M., Adachi S., Imai T., Watanabe K.I., Toyokuni S.Y., Ueno M., Zervos A.S., Kroemer G., Nakahata T.

Blood 103 巻 ( 6 ) 頁： 2299 - 2307 2004年3月

記述言語：日本語掲載種別：学位論文（博士）出版者・発行元：Blood

Caspase-independent programmed cell death can exhibit either an apoptosis-like or a necrosis-like morphology. The ABL kinase inhibitor, imatinib mesylate, has been reported to induce apoptosis of BCR-ABL-positive cells in a caspase-dependent fashion. We investigated whether caspases alone were the mediators of imatinib mesylate-induced cell death. In contrast to previous reports, we found that a broad caspase inhibitor, zVAD-fmk, failed to prevent the death of imatinib mesylate-treated BCR-ABL-positive human leukemic cells. Moreover, zVAD-fmk-preincubated, imatinib mesylate-treated cells exhibited a necrosis-like morphology characterized by cellular pyknosis, cytoplasmic vacuolization, and the absence of nuclear signs of apoptosis. These cells manifested a loss of the mitochondrial transmembrane potential, indicating the mitochondrial involvement in this caspase-independent necrosis. We excluded the participation of several mitochondrial factors possibly involved in caspase-independent cell death such as apoptosis-inducing factor, endonuclease G, and reactive oxygen species. However, we observed the mitochondrial release of the serine protease Omi/HtrA2 into the cytosol of the cells treated with imatinib mesylate or zVAD-fmk plus imatinib mesylate. Furthermore, serine protease inhibitors prevented the caspase-independent necrosis. Taken together, our results suggest that imatinib mesylate induces a caspase-independent, necrosis-like programmed cell death mediated by the serine protease activity of Omi/HtrA2. © 2004 by The American Society of Hematology.

DOI： 10.1182/blood-2003-05-1605

A novel mechanism for imatinib mesylate-induced cell death of BCR-ABL-positive human leukemic cells: caspase-independent, necrosis-like programmed cell death mediated by serine protease activity

Okada M, Adachi S, Imai T, Watanabe K, Toyokuni SY, Ueno M, Zervos AS, Kroemer G, Nakahata T

BLOOD 103 巻 ( 6 ) 頁： 2299 - 2307 2004年3月

PJ-628 Oxidant Stress-Mediated Myocardial Cell Injury in Selenium-Deficient Mice : Age-Dependent Roles of Glutathion Peroxidase Inhibition(Cardiomyopathy, Basic and Clinical 2 (M) : PJ105)(Poster Session (Japanese))

Hiraoka Yuuji, Okada Ikutaro, Tamura Takashi, Akagi Tadasu, Hama Norio, Tanabe Masato, Suzuki Joji, Fujimori Mariko, Takahashi Nobuki, Shimizu Takayuki, Sarazawa Katsuhiko, Shinnmura Tetuo, Toyokuni Shinya, Kihara Yasuki, Nakajima Hisayoshi, Inoue Hiroshi

Circulation journal : official journal of the Japanese Circulation Society 68 巻 ( 0 ) 頁： 634 2004年3月

フリーラジカルによる発がんの標的遺伝子

豊國伸哉

日本衞生學雜誌 59 巻 ( 2 ) 頁： 105 2004年3月

Circulation journal : official journal of the Japanese Circulation Society 68 巻 ( 0 ) 2004年3月

Role of phenobarbital-inducible cytochrome P450s as a source of active oxygen species in DNA-oxidation 査読有り国際誌

Imaoka, S; Osada, M; Minamiyama, Y; Yukimura, T; Toyokuni, S; Takemura, S; Hiroi, T; Funae, Y

CANCER LETTERS 203 巻 ( 2 ) 頁： 117 - 125 2004年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Letters

We investigated the biological effects of the active oxygen produced by P450s. First, we identified which isoforms of P450 efficiently produced active oxygen using electron spin resonance. Eight forms of P450 purified from rat liver were used. Of these, CYP1A2, 2B1, 2C11 and 3A2 produced hydroxyl radicals efficiently. Phenobarbital (PB) which is a typical inducer of CYP2B1 and 3A2 induced production of hydroxyl radicals by rat liver and ketoconazole, an inhibitor of P450, inhibited production of hydroxyl radicals in vitro. PB is a tumor promoter as well as the P450-inducer. We investigated oxidation of the genomic DNA by the hydroxyl radicals produced by PB-inducible P450 in vitro and in vivo. 8-Hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of DNA oxidation in vivo was assayed by HPLC. PB strongly induced the production of 8-OHdG in the rat liver. While ketoconazole inhibited the production of 8-OHdG in vivo. These results suggest that active oxygen produced by P450 oxidized genomic DNA and induction of P450 increased oxidative stress that may contribute to tumor initiation and promotion. © 2003 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.canlet.2003.09.009

Adrenomedullin infusion attenuates myocardial ischemia/reperfusion injury through the phosphatidylinositol 3-kinase/Akt-dependent pathway. 国際誌

Hiroyuki Okumura, Noritoshi Nagaya, Takefumi Itoh, Ichiro Okano, Jun Hino, Kenji Mori, Yoshitane Tsukamoto, Hatsue Ishibashi-Ueda, Senri Miwa, Keiichi Tambara, Shinya Toyokuni, Chikao Yutani, Kenji Kangawa

Circulation 109 巻 ( 2 ) 頁： 242 - 8 2004年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Infusion of adrenomedullin (AM) has beneficial hemodynamic effects in patients with heart failure. However, the effect of AM on myocardial ischemia/reperfusion remains unknown. METHODS AND RESULTS: Male Sprague-Dawley rats were exposed to a 30-minute period of ischemia induced by ligation of the left coronary artery. They were randomized to receive AM, AM plus wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), or saline for 60 minutes after coronary ligation. Hemodynamics and infarct size were examined 24 hours after reperfusion. Myocardial apoptosis was also examined 6 hours after reperfusion. The effect of AM on Akt phosphorylation in cardiac tissues was examined by Western blotting. Intravenous administration of AM significantly reduced myocardial infarct size (28+/-4% to 16+/-1%, P<0.01), left ventricular end-diastolic pressure (19+/-2 to 8+/-2 mm Hg, P<0.05), and myocardial apoptotic death (19+/-2% to 9+/-4%, P<0.05). Western blot analysis showed that AM infusion accelerated Akt phosphorylation in cardiac tissues and that pretreatment with wortmannin significantly attenuated AM-induced Akt phosphorylation. Moreover, pretreatment with wortmannin abolished the beneficial effects of AM: a reduction of infarct size, a decrease in left ventricular end-diastolic pressure, and inhibition of myocardial apoptosis after ischemia/reperfusion. CONCLUSIONS: Short-term infusion of AM significantly attenuated myocardial ischemia/reperfusion injury. These cardioprotective effects are attributed mainly to antiapoptotic effects of AM via a PI3K/Akt-dependent pathway.

Adrenomedullin infusion attenuates myocardial ischemia/reperfusion injury through the phosphatidylinositol 3-kinase/Akt-dependent pathway

Okumura H, Nagaya N, Itoh T, Okano I, Hino J, Mori K, Tsukamoto Y, Ishibashi-Ueda H, Miwa S, Tambara K, Toyokuni S, Yutani C, Kangawa K

CIRCULATION 109 巻 ( 2 ) 頁： 242 - 248 2004年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Circulation

Background-Infusion of adrenomedullin (AM) has beneficial hemodynamic effects in patients with heart failure. However, the effect of AM on myocardial ischemia/reperfusion remains unknown. Methods and Results-Male Sprague-Dawley rats were exposed to a 30-minute period of ischemia induced by ligation of the left coronary artery. They were randomized to receive AM, AM plus wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), or saline for 60 minutes after coronary ligation. Hemodynamics and infarct size were examined 24 hours after reperfusion. Myocardial apoptosis was also examined 6 hours after reperfusion. The effect of AM on Akt phosphorylation in cardiac tissues was examined by Western blotting. Intravenous administration of AM significantly reduced myocardial infarct size (28±4% to 16±1%, P<0.01), left ventricular end-diastolic pressure (19±2 to 8±2 mm Hg, P<0.05), and myocardial apoptotic death (19±2% to 9±4%, P<0.05). Western blot analysis showed that AM infusion accelerated Akt phosphorylation in cardiac tissues and that pretreatment with wortmannin significantly attenuated AM-induced Akt phosphorylation. Moreover, pretreatment with wortmannin abolished the beneficial effects of AM: a reduction of infarct size, a decrease in left ventricular end-diastolic pressure, and inhibition of myocardial apoptosis after ischemia/reperfusion. Conclusions-Short-term infusion of AM significantly attenuated myocardial ischemia/reperfusion injury. These cardioprotective effects are attributed mainly to antiapoptotic effects of AM via a PI3K/Akt-dependent pathway.

DOI： 10.1161/01.CIR.0000109214.30211.7C

Target genes and genomic areas in free radical-induced carcinogenesis

CD9 is a surface marker on mouse and rat male germline stem cells. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Toyokuni S, Shinohara T

Biology of reproduction 70 巻 ( 1 ) 頁： 70 - 5 2004年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogenesis is dependent on a small population of stem cells. Despite the biological significance of spermatogonial stem cells, their analysis has been hampered by their scarcity. However, spermatogonial stem cells can be enriched by selection with an antibody against cell-surface molecules. In this investigation, we searched for new antigens expressed on spermatogonial stem cells. Using the spermatogonial transplantation technique, we examined expression of the CD9 molecule, which is commonly expressed on stem cells of other tissues. Selection of both mouse and rat testis cells with anti-CD9 antibody resulted in 5- to 7-fold enrichment of spermatogonial stem cells from intact testis cells, indicating that CD9 is commonly expressed on spermatogonial stem cells of both species. Therefore, CD9 may be involved in the common machinery in stem cells of many self-renewing tissues, and the identification of a common surface antigen on spermatogonial stem cells of different species has important implications for the development of a technique to enrich stem cells from other mammalian species.

DOI： 10.1095/biolreprod.103.020867

Target genes in free radical-induced carcinogenesis

Toyokuni S

SFRR: PROCEEDINGS OF THE XII BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 頁： 181 - 185 2004年

Toyokuni, S; Akatsuka, S; Shirase, T; Than, TA

FREE RADICAL BIOLOGY AND MEDICINE 36 巻頁： S29 - S29 2004年

Target genes in free radical-induced carcinogenesis 査読有り

S Toyokuni

SFRR: PROCEEDINGS OF THE XII BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 頁： 181 - 185 2004年

記述言語：英語掲載種別：研究論文（国際会議プロシーディングス）出版者・発行元：MEDIMOND PUBLISHING CO

Ferric nitrilotriacetate-induced renal carcinogenesis presents an intriguing model demonstrating what happens if repeated Fenton reaction occurs in vivo. We have asked a question whether there is any target gene in this carcinogenesis model. With a genetic approach, we found that p15 and p16 tumor suppressor genes are one of the major targets. Furthermore, allelic loss of p16 was present as early as three weeks after the start of the experiments. In this model, alterations in expressional levels of several genes were also observed by differential display analysis. Among them, annexin 2 was overexpressed in renal cell carcinoma, and was regulated by oxidative stress. Persistent operation of this adaptive mechanism plays a role in the proliferation and metastasis of oxidative stress-induced cancer.

Edaravone, a novel free radical scavenger, protects myocardium against oxidative stress after cardioplegic arrest

Formation of acrolein-derived 2'-deoxyadenosine adduct in an iron-induced carcinogenesis model. 査読有り国際誌 Open Access

Kawai Y, Furuhata A, Toyokuni S, Aratani Y, Uchida K

The Journal of biological chemistry 278 巻 ( 50 ) 頁： 50346 - 54 2003年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation. It shows facile reactivity toward DNA to form an exocyclic DNA adduct. To verify the formation of acrolein-derived DNA adduct under oxidative stress in vivo, we raised a novel monoclonal antibody (mAb21) against the acrolein-modified DNA and found that the antibody most significantly recognized an acrolein-modified 2′-deoxyadenosine. On the basis of chemical and spectroscopic evidence, the major antigenic product of mAb21 was the 1,N6-propano-2′ -deoxyadenosine adduct. The exposure of rat liver epithelial RL34 cells to acrolein resulted in a significant accumulation of the acrolein-2′ -deoxyadenosine adduct in the nuclei. Formation of this adduct under oxidative stress in vivo was immunohistochemically examined in rats exposed to ferric nitrilotriacetate, a carcinogenic iron chelate that specifically induces oxidative stress in the kidneys of rodents. It was observed that the acrolein-2′-deoxyadenosine adduct was formed in the nuclei of the proximal tubular cells, the target cells of this carcinogenesis model. The same cells were stained with a monoclonal antibody 5F6 that recognizes an acrolein-lysine adduct, by which cytosolic accumulation of acrolein-modified proteins appeared. Similar results were also obtained from myeloperoxidase knockout mice exposed to the iron complex, suggesting that the myeloperoxidase-catalyzed oxidation system might not be essential for the generation of acrolein in this experimental animal carcinogenesis model. The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.

DOI： 10.1074/jbc.M309057200

Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Shinohara T

Human reproduction (Oxford, England) 18 巻 ( 12 ) 頁： 2660 - 7 2003年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Reproduction

Background: The development of a spermatogonial transplantation technique has provided new possibilities for the treatment of male infertility. Previous studies have shown that spermatogonial stem cells could reinitiate spermatogenesis after cryopreservation and reintroduction into the seminiferous tubules of infertile recipient males, and this raised the possibility of banking frozen stem cells for male infertility treatment. It remains unknown, however, whether germ cells from freeze-thawed stem cells are fertile, leaving the possibility that the procedure compromises the integrity of the stem cells. Methods and results: Dissociated mouse testis cells were cryopreserved and transplanted into infertile recipient testes. The freeze-thawed testis cell populations contained higher concentrations of stem cells than fresh testis cell populations. Offspring were obtained from freeze-thawed stem cells transplanted into infertile males, and fertility restoration was more efficient in immature (5-10 days old) than in mature (6-12 weeks old) recipients. However, offspring were also obtained from infertile adult recipients using in-vitro microinsemination. Conclusions: This first successful application of frozen stem cell technology in the production of offspring by spermatogonial transplantation suggests the superiority of immature recipients for clinical applications. Thus, the combination of cryopreservation and transplantation of stem cells is a promising approach to overcome male infertility.

DOI： 10.1093/humrep/deg483

Effects of the phenolic contents of Mauritian endemic plant extracts on promoter activities of antioxidant enzymes. 査読有り国際誌

Toyokuni S, Tanaka T, Kawaguchi W, Fang NR, Ozeki M, Akatsuka S, Hiai H, Aruoma OI, Bahorun T

Free radical research 37 巻 ( 11 ) 頁： 1215 - 24 2003年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Oxidative stress has been associated with a variety of pathologic conditions in humans. Increasing the transcriptional activities of antioxidant enzymes might be a strategy to prevent oxidative stress-associated diseases such as atherosclerosis and cancer. In the present paper, we studied the effects of extracts from 12 Mauritian endemic plants on the promoter activities of antioxidant enzymes; Cu, Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and glutathione dismutase (GPx). The levels of total phenolic compounds, total flavonoids, and proanthocyanidins were measured. Four luciferase expression vectors (pGL3-Basic) with promoter region of each enzyme were constructed, transfected to COS7 cells followed by an exposure to each extract (25 μg/ml, 24 h, non-toxic dose). Thereafter, luciferase activities were evaluated in comparison with a control luciferase vector with a herpes simplex virus thymidine kinase promoter. Mauritian endemic plants contained high amounts of total phenols, flavonoids and proanthocyanidins. Total phenols and flavonoids were proportionally associated with Cu,Zn-SOD promoter activity, whereas they were inversely correlated with catalase promoter activity. These results suggest that the chemopreventive potentials of the extracts might reside in their abilities to modulate the expression of the antioxidant enzyme genes.

DOI： 10.1080/10715760310001598150

Yamazaki, K; Miwa, S; Toyokuni, S; Horii, T; Unimonh, O; Sasahashi, N; Takaba, K; Komeda, M

CIRCULATION 108 巻 ( 17 ) 頁： 175 - 175 2003年10月

Edaravone, a novel free radical scavenger, protects myocardium against oxidative stress after cardioplegic arrest 国際誌

Yamazaki, K, Miwa, S, Toyokuni, S, Horii, T, Unimonh, O, Sasahashi, N, Takaba, K, Komeda, M

CIRCULATION 108 巻 ( 17 ) 頁： 175 - 175 2003年10月

Novel aspects in free radical chemistry and biology 査読有り国際誌

Toyokuni, S

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 417 巻 ( 1 ) 頁： 2 - 2 2003年9月

Long-term proliferation in culture and germline transmission of mouse male germline stem cells. 査読有り国際誌

Mito Kanatsu-Shinohara, Narumi Ogonuki, Kimiko Inoue, Hiromi Miki, Atsuo Ogura, Shinya Toyokuni, Takashi Shinohara

Biology of reproduction 69 巻 ( 2 ) 頁： 612 - 6 2003年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC STUDY REPRODUCTION

Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>10(14)-fold)and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.

DOI： 10.1095/biloreprod.103.017012

Long-term proliferation in culture and germline transmission of mouse male germline stem cells

Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T

BIOLOGY OF REPRODUCTION 69 巻 ( 2 ) 頁： 612 - 616 2003年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogenesis is a complex process that originates in a small population of spermatogonial stem cells. Here we report the in vitro culture of spermatogonial stem cells that proliferate for long periods of time. In the presence of glial cell line-derived neurotrophic factor, epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor, gonocytes isolated from neonatal mouse testis proliferated over a 5-month period (>1014-fold) and restored fertility to congenitally infertile recipient mice following transplantation into seminiferous tubules. Long-term spermatogonial stem cell culture will be useful for studying spermatogenesis mechanism and has important implications for developing new technology in transgenesis or medicine.

DOI： 10.1095/biolreprod.103.017012

Involvement of death receptor Fas in germ cell degeneration in gonads of Kit-deficient Wv/Wv mutant mice. 査読有り国際誌 Open Access

Sakata S, Sakamaki K, Watanabe K, Nakamura N, Toyokuni S, Nishimune Y, Mori C, Yonehara S

Cell death and differentiation 10 巻 ( 6 ) 頁： 676 - 86 2003年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Death and Differentiation

Kit and its ligand stem cell factor (SCF) play a fundamental role in hematopoiesis, melanogenesis and gametogenesis. Homozygous Wv mutant mice with a mutation in kit show abnormalities in these cell lineages. Fas is a member of the death receptor family inducing apoptosis. In this study, we generated double-mutant mice (Wv/Wv:Fas-/-) and analyzed histologically their reproductive organs. In testes and ovaries of the double-mutant mice, testicular germ cells and oocytes were detected, respectively, whereas the same-aged Wv/Wv mice contained neither cells. In addition, inhibition of Kit signals by administration of anti-Kit mAb, which induces degeneration of testicular germ cells in vivo in wild-type mice, did not cause degeneration in Fas-deficient mice. In testicular germ cells of Wv/Wv mutant mice, an increase of Fas expression was observed in spermatogonia. Further, in vitro treatment with SCF was shown to downregulate Fas on fibroblasts expressing exogenous Kit through activation of PI3-kinase/Akt. All the results clearly indicate that Fas-mediated apoptosis is involved in germ cell degeneration accompanied by defects in Kit-mediated signals, and Kit signaling negatively regulates Fas-mediated apoptosis in vivo.

DOI： 10.1038/sj.cdd.4401215

Evidence for oxidative stress in NSAID-induced colitis in IL10-/- mice. 国際誌

Seiko Narushima, Douglas R Spitz, Larry W Oberley, Shinya Toyokuni, Toshio Miyata, Carol A Gunnett, Garry R Buettner, Juan Zhang, Hanan Ismail, Richard G Lynch, Daniel J Berg

Free radical biology & medicine 34 巻 ( 9 ) 頁： 1153 - 66 2003年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

The goal of this study was to evaluate for evidence of oxidative stress in colonic inflammation in a novel model of inflammatory bowel disease, nonsteroidal anti-inflammatory drug- (NSAID-) treated interleukin-10-deficient (IL10(-/-)) mice. IL10(-/-) and wild-type (wt) mice were treated with a nonselective NSAID (piroxicam, 200 ppm in the diet) for 2 weeks to induce colitis, and parameters for oxidative stress in the colonic tissues were evaluated. Mean chemiluminescence enhanced with lucigenin in the colons from IL10(-/-) mice treated with piroxicam was more than 5-fold higher than that of the control wt group. Chemiluminescence was inhibited with diphenylethylene iodinium, but not allopurinol, indomethacin, or N-omega-nitro-L-arginine, indicating that flavin-containing enzymes were the source of the reactive oxygen species. Colonic aconitase activity in NSAID-treated IL10(-/-) mice decreased to 50% of the activity of control mice. There was no difference in the total glutathione levels in the colonic mucosa among the groups; however, glutathione disulfide levels were approximately 2-fold greater in the colon of NSAID-treated IL10(-/-) mice as compared with control groups. Immunohistochemistry studies of colons from NSAID-treated IL10(-/-) mice demonstrated intense staining with two antibodies that recognize advanced glycation endproducts formed through glycation and oxidation: anticarboxymethylysine and antipentosidine. The epithelial cells and lamina propria cells in the colons of NSAID-treated IL10(-/-) mice showed immunostaining with antinitrotyrosine, indicating the presence of reactive nitrogen species. Colonic epithelium of IL10(-/-) mice with colitis showed moderate immunostaining for 8-hydroxy-2'-deoxyguanosine in the nuclei. NSAID-treated IL10(-/-) mice treated with diphenylene idodonium chloride (DPI), an irreversible inhibitor of flavoprotein enzymes, experienced significantly reduced inflammation. Taken together, these results strongly indicate the presence of oxidative stress in the inflammatory bowel disease in NSAID-treated IL10(-/-) mice and suggests a role for oxidative stress in the pathophysiology of this model of inflammatory bowel disease.

Evidence for oxidative stress in NSAID-induced colitis in IL10(-/-) mice

Narushima S, Spitz DR, Oberley LW, Toyckuni S, Miyata T, Gunnett CA, Buettner GR, Zhang J, Ismail H, Lynch RG, Berg DJ

FREE RADICAL BIOLOGY AND MEDICINE 34 巻 ( 9 ) 頁： 1153 - 1166 2003年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

The goal of this study was to evaluate for evidence of oxidative stress in colonic inflammation in a novel model of inflammatory bowel disease, nonsteroidal anti-inflammatory drug- (NSAID-) treated interleukin-10-deficient (IL10-/-) mice. IL10-/- and wild-type (wt) mice were treated with a nonselective NSAID (piroxicam, 200 ppm in the diet) for 2 weeks to induce colitis, and parameters for oxidative stress in the colonic tissues were evaluated. Mean chemiluminescence enhanced with lucigenin in the colons from IL10-/- mice treated with piroxicam was more than 5-fold higher than that of the control wt group. Chemiluminescence was inhibited with diphenylethylene iodinium, but not allopurinol, indomethacin, or N-ω-nitro-L-arginine, indicating that flavin-containing enzymes were the source of the reactive oxygen species. Colonic aconitase activity in NSAID-treated IL10-/- mice decreased to 50% of the activity of control mice. There was no difference in the total glutathione levels in the colonic mucosa among the groups; however, glutathione disulfide levels were approximately 2-fold greater in the colon of NSAID-treated IL10-/- mice as compared with control groups. Immunohistochemistry studies of colons from NSAID-treated IL10-/- mice demonstrated intense staining with two antibodies that recognize advanced glycation endproducts formed through glycation and oxidation: anticarboxymethylysine and antipentosidine. The epithelial cells and lamina propria cells in the colons of NSAID-treated IL10-/- mice showed immunostaining with antinitrotyrosine, indicating the presence of reactive nitrogen species. Colonic epithelium of IL10-/- mice with colitis showed moderate immunostaining for 8-hydroxy-2′-deoxyguanosine in the nuclei. NSAID-treated IL10-/- mice treated with diphenylene idodonium chloride (DPI), an irreversible inhibitor of flavoprotein enzymes, experienced significantly reduced inflammation. Taken together, these results strongly indicate the presence of oxidative stress in the inflammatory bowel disease in NSAID-treated IL10-/- mice and suggests a role for oxidative stress in the pathophysiology of this model of inflammatory bowel disease. © 2003 Elsevier Inc.

DOI： 10.1016/S0891-5849(03)00065-0

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis

Functional assessment of self-renewal activity of male germline stem cells following cytotoxic damage and serial transplantation. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Toyokuni S, Morimoto T, Matsui S, Honjo T, Shinohara T

Biology of reproduction 68 巻 ( 5 ) 頁： 1801 - 7 2003年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

Spermatogenesis is dependent on a small population of stem cells. Although stem cells are believed to expand infinitely, there is little functional evidence regarding whether spermatogonial stem cells can increase in their number. Using the spermatogonial transplantation technique, we evaluated the proliferative potential of spermatogonial stem cells in two models of regeneration. After busulfan injection to deplete stem cells, the surviving stem cells were able to expand by at least 15.8-fold within 2 mo. On the other hand, a serial transplantation study indicated that one transplanted stem cell was able to expand by 3.8-and 12-fold within 2 and 4 mo, respectively. These results provide direct functional evidence for the expansion of stem cells and establish the basis for further characterization of the stem cell self-renewal process.

DOI： 10.1095/biolreprod.102.012575

TOYOKUNI Shinya, KAWAGUCHI Waka, AKATSUKA Shinya, HIROYASU Makoto, HIAI Hiroshi

Pathology international 53 巻 ( 4 ) 頁： 259 - 261 2003年4月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis

TOYOKUNI Shinya, KAWAGUCHI Waka, AKATSUKA Shinya, HIROYASU Makoto, HIAI Hiroshi

Pathology international 53 巻 ( 4 ) 頁： 259 - 261 2003年4月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis

Toyokuni S, Kawaguchi W, Akatsuka S, Hiroyasu M, Hiai H

PATHOLOGY INTERNATIONAL 53 巻 ( 4 ) 頁： 259 - 261 2003年4月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis.

Toyokuni S, Kawaguchi W, Akatsuka S, Hiroyasu M, Hiai H

Pathology international 53 巻 ( 4 ) 頁： 259 - 61 2003年4月

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. 国際誌

Shinya Toyokuni, Waka Kawaguchi, Shinya Akatsuka, Makoto Hiroyasu, Hiroshi Hiai

Pathology international 53 巻 ( 4 ) 頁： 259 - 61 2003年4月

Glycine ameriorates lung reprefusion injury after cold preservation in an ex vivo rat lung model.

Omasa M, Fukuse T, Toyokuni S, Mizutani Y, Yoshida H, Ikeyama K, Hasegawa S, Wada H

TRANSPLANTATION 75 巻 ( 5 ) 頁： 591 - 598 2003年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Transplantation

Background. The role of glycine has not been investigated in lung ischemia-reperfusion injury after cold preservation. Furthermore, the role of apoptosis after reperfusion following cold preservation has not been fully understood. Methods. Lewis rats were divided into three groups (n=6 each). In the GLY(-) and GLY(+) groups, isolated lungs were preserved for 15 hr at 4°C after a pulmonary artery (PA) flush using our previously developed preservation solution (ET-K; extracellular-type trehalose containing Kyoto), with or without the addition of glycine (5 mM). In the Fresh group, isolated lungs were reperfused immediately after a PA flush with ET-K. They were reperfused for 60 min with an ex vivo perfusion model. Pulmonary function, oxidative stress, apoptosis, and tumor necrosis factor (TNF)-α expression were assessed after reperfusion. Results. Shunt fraction and peak inspiratory pressure after reperfusion in the GLY(-) group were significantly higher than those in the GLY(+) and Fresh groups. Oxidative damage and apoptosis in the alveolar epithelial cells of the GLY(-) group, assessed by immunohistochemical staining and quantification of 8-hydroxy-2′-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method, were significantly higher than those of the GLY(+) and Fresh groups. There were correlations among shunt fraction, oxidative damage, and apoptosis. There was no expression of TNF-α messenger RNA in all groups evaluated by the reverse transcription-polymerase chain reaction. Conclusions. Glycine attenuates ischemia/reperfusion injury after cold preservation by reducing oxidative damage and suppressing apoptosis independent of TNF-α in this model. The suppression of apoptosis might ameliorate lung function after reperfusion.

DOI： 10.1097/01.TP.0000053200.98125.14

Glycine ameliorates lung reperfusion injury after cold preservation in an ex vivo rat lung model. 国際誌

Mitsugu Omasa, Tatsuo Fukuse, Shinya Toyokuni, Yoichi Mizutani, Hiroshi Yoshida, Kazuyuki Ikeyama, Seiki Hasegawa, Hiromi Wada

Transplantation 75 巻 ( 5 ) 頁： 591 - 8 2003年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: The role of glycine has not been investigated in lung ischemia-reperfusion injury after cold preservation. Furthermore, the role of apoptosis after reperfusion following cold preservation has not been fully understood. METHODS: Lewis rats were divided into three groups (n=6 each). In the GLY(-) and GLY(+) groups, isolated lungs were preserved for 15 hr at 4 degrees C after a pulmonary artery (PA) flush using our previously developed preservation solution (ET-K; extracellular-type trehalose containing Kyoto), with or without the addition of glycine (5 mM). In the Fresh group, isolated lungs were reperfused immediately after a PA flush with ET-K. They were reperfused for 60 min with an ex vivo perfusion model. Pulmonary function, oxidative stress, apoptosis, and tumor necrosis factor (TNF)-alpha expression were assessed after reperfusion. RESULTS: Shunt fraction and peak inspiratory pressure after reperfusion in the GLY(-) group were significantly higher than those in the GLY(+) and Fresh groups. Oxidative damage and apoptosis in the alveolar epithelial cells of the GLY(-) group, assessed by immunohistochemical staining and quantification of 8-hydroxy-2'-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method, were significantly higher than those of the GLY(+) and Fresh groups. There were correlations among shunt fraction, oxidative damage, and apoptosis. There was no expression of TNF-alpha messenger RNA in all groups evaluated by the reverse transcription-polymerase chain reaction. CONCLUSIONS: Glycine attenuates ischemia/reperfusion injury after cold preservation by reducing oxidative damage and suppressing apoptosis independent of TNF-alpha in this model. The suppression of apoptosis might ameliorate lung function after reperfusion.

Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway.

Tumor initiating activity of Helicobacter pylori water extract on mouse skin carcinogenesis 査読有り国際誌

Ishikawa, T; Yoshida, N; Tokuda, H; Ichiishi, E; Kuchide, M; Kokura, S; Naito, Y; Toyokuni, S; Nishino, H; Yoshikawa, T

CANCER LETTERS 191 巻 ( 1 ) 頁： 41 - 47 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Letters

Helicobacter pylori (H. pylori) infection has been associated with gastric carcinogenesis, but responsible and detail mechanisms are insufficient by the absence of adequate data. To obtain direct evidence regarding the carcinogenicity of H. pylori, we investigated the initiating and promoting activity of H. pylori water extract (HPE) in two-stage mouse skin carcinogenesis model. HPE treatment, as an initiation, significantly enhanced tumor formation compared with control group. Moreover, HPE treatment increased production of 8-hydroxydeoxyguanosine in epidermal cells and HPE-initiated/TPA-promoted papillomas demonstrated a point mutation of the Ha-ras gene. These results suggest an initiating activity of HPE on two-stage mouse skin carcinogenesis. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3835(02)00628-6

Structural basis of protein-bound endogenous aldehydes. Chemical and immunochemical characterizations of configurational isomers of a 4-hydroxy-2-nonenal-histidine adduct. 査読有り国際誌 Open Access

Hashimoto M, Sibata T, Wasada H, Toyokuni S, Uchida K

The Journal of biological chemistry 278 巻 ( 7 ) 頁： 5044 - 51 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. In the present study, we characterized configurational isomers of a HNE-Nα-acetylhistidine adduct by NMR spectroscopy and by molecular orbital calculations. In addition, we raised monoclonal antibodies against (R)-HNE-histidine and (S)-HNE-histidine adducts, characterized their specificities, and examined in vivo localizations of each adduct under oxidative stress. To facilitate structural characterization of the configurational isomers of an HNE-histidine adduct, we prepared the (R)-HNE-histidine and (S)-HNE-histidine adducts by incubating Nα-acetylhistidine with each HNE enantiomer, both of which provided two peaks (Ra and Rb from (R)-HNE-histidine and Sa and Sb from (S)-HNE-histidine adducts) in reversed-phase high-performance liquid chromatography. The NMR analysis showed that each peak was a mixture of two diastereomers. In addition, the analysis of the nuclear Overhauser effect enabled the determination of configurations of the eight isomers. The relative amounts of these isomers in the NMR analysis correlated with the relative energies calculated by molecular orbital methods. On the other hand, using (R)-HNE-modified and (S)-HNE-modified keyhole limpet hemocyanins as the antigens, we raised the monoclonal antibodies, mAbR310 and mAbS412, which enantioselectively recognized the (R)-HNE-histidine and (S)-HNE-histidine adducts, respectively. Among the mixtures (Ra, Rb, Sa, and Sb) of diastereomers, mAbR310 showed the highest immunoreactivity to Rb (the mixture of 2R,4S,5R and 2S,4S,5R isomers), whereas mAbS412 preferentially recognized Sa (the mixture of 2R,4S,5S and 2S,4S,5S isomers). The presence of (R)-HNE and (S)-HNE epitopes in vivo was immunohistochemically examined in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate, by which nuclear and cytosolic stainings with mAbR310 and mAbS412, respectively, were detected.

DOI： 10.1074/jbc.M210129200

Kuroda K, Han H, Tani S, Tanigaki K, Tun T, Furukawa T, Taniguchi Y, Kurooka H, Hamada Y, Toyokuni S, Honjo T

Immunity 18 巻 ( 2 ) 頁： 301 - 12 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Immunity

We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless.

DOI： 10.1016/s1074-7613(03)00029-3

Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway

Kuroda K, Han H, Tani S, Tanigaki K, Tun T, Furukawa T, Taniguchi Y, Kurooka H, Hamada Y, Toyokuni S, Honjo T

IMMUNITY 18 巻 ( 2 ) 頁： 301 - 312 2003年2月

Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. 国際誌

Kazuki Kuroda, Hua Han, Shoichi Tani, Kenji Tanigaki, Tin Tun, Takahisa Furukawa, Yoshihito Taniguchi, Hisanori Kurooka, Yoshio Hamada, Shinya Toyokuni, Tasuku Honjo

Immunity 18 巻 ( 2 ) 頁： 301 - 12 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice. 査読有り国際誌

Aoi W, Naito Y, Sakuma K, Kuchide M, Tokuda H, Maoka T, Toyokuni S, Oka S, Yasuhara M, Yoshikawa T

Antioxidants & redox signaling 5 巻 ( 1 ) 頁： 139 - 44 2003年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Antioxidants and Redox Signaling

Dietary antioxidants may attenuate oxidative damage from strenuous exercise in various tissues. Beneficial effects of the antioxidant astaxanthin have been demonstrated in vitro, but not yet in vivo. We investigated the effect of dietary supplementation with astaxanthin on oxidative damage induced by strenuous exercise in mouse gastrocnemius and heart. C57BL/6 mice (7 weeks old) were divided into groups: rested control, intense exercise, and exercise with astaxanthin supplementation. After 3 weeks of exercise acclimation, both exercise groups ran on a treadmill at 28 m/min until exhaustion. Exercise-increased 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-2′-deoxyguanosine in gastrocnemius and heart were blunted in the astaxanthin group. Increases in plasma creatine kinase activity, and in myeloperoxidase activity in gastrocnemius and heart, also were lessened by astaxanthin. Astaxanthin showed accumulation in gastrocnemius and heart from the 3 week supplementation. Astaxanthin can attenuate exercise-induced damage in mouse skeletal muscle and heart, including an associated neutrophil infiltration that induces further damage.

DOI： 10.1089/152308603321223630

Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice

Aoi W, Naito Y, Sakuma K, Kuchide M, Tokuda H, Maoka T, Toyokuni S, Oka S, Yasuhara M, Yoshikawa T

ANTIOXIDANTS & REDOX SIGNALING 5 巻 ( 1 ) 頁： 139 - 144 2003年2月

Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis. 査読有り国際誌 Open Access

Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Honjo T, Shinohara T

Biology of reproduction 68 巻 ( 1 ) 頁： 167 - 73 2003年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biology of Reproduction

The testis is one of several immune-privileged organs and is known for its unique ability to support allogeneic or xenogeneic tissue transplants. We investigated the possibility of deriving offspring from mice that underwent transplantation with allogeneic male germ line stem cells in the testis. Although mature adult mice rejected allogeneic germ cells and were infertile, offspring were obtained by intracytoplasmic germ cell injection using partially differentiated donor cells. In contrast, complete spermatogenesis occurred when allogeneic germ cells were transplanted into immature pup testes. Tolerance induction by monoclonal antibody administration allowed the pup transplant recipients to produce allogeneic offspring by natural mating, whereas no spermatozoa were found in the epididymis of untreated recipients. Thus, these results indicate that a histoincompatible recipient can serve as a "surrogate father" to propagate the genetic information of heterologous male donors.

DOI： 10.1095/biolreprod.102.008516

Birth of offspring following transplantation of cyropreserved immature testicular pieces and in-vitro microinsemination 査読有り国際誌

Shinohara T., Inoue K., Ogonuki N., Kanatsu-Shinohara M., Miki H., Nakata K., Kurome M., Nagashima H., Toyokuni S., Kogishi K., Honjo T., Ogura A.

Human Reproduction 17 巻 ( 12 ) 頁： 3039 - 3045 2002年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Reproduction

Background: Fertility protection is an urgent clinical problem for prepubertal male oncology patients who undergo either chemotherapy or radiotherapy. As these patients do not have mature sperm to be frozen, there is as yet no effective method to preserve their fertility. Methods and results: Single pieces of immature mouse (1.5 × 1.5 × 1.5 mm) or rabbit (2.0 × 2.0 × ≃3.0 mm) testis were cryopreserved, thawed and transplanted into mouse testes. Histological techniques were used to determine the presence of spermatogenesis, which was restored in both mouse and rabbit testicular pieces, and led to the production of mature sperm after both cryopreservation and syngeneic or xenogeneic transplantation into mouse testes. Using sperm developed in the frozen-thawed transplants, mouse offspring were born after in-vitro microinsemination. Furthermore, rabbit offspring were obtained using rabbit sperm that developed in fresh transplants in a xenogeneic surrogate mouse. Conclusions: This approach of 'testicular tissue banking' is a promising technique for the preservation of fertility in prepubertal male oncology patients. Xenogeneic transplantation into immunodeficient mice may provide a system for study spermatogenic failure in infertile men.

Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination

Shinohara, T; Inoue, K; Ogonuki, N; Kanatsu-Shinohara, M; Miki, H; Nakata, K; Kurome, M; Nagashima, H; Toyokuni, S; Kogishi, K; Honjo, T; Ogura, A

HUMAN REPRODUCTION 17 巻 ( 12 ) 頁： 3039 - 3045 2002年12月

A novel method of "preparative" myocardial protection using green tea polyphenol in oral uptake

Miwa, S; Saito, M; Yamazaki, K; Toyokuni, S; Hyon, SH; Fujita, M; Komeda, M

CIRCULATION 106 巻 ( 19 ) 頁： 345 - 345 2002年11月

Oxydative stress persists in nearby LV area after myocardial infarction (MI) and shows two peaks

Miwa, S; Toyokuni, S; Tanaka, S; Nishina, T; Nomoto, T; Yamazaki, K; Ueda, K; Nishimura, K

CIRCULATION 106 巻 ( 19 ) 頁： 312 - 312 2002年11月

Oxydative stress persists in nearby LV area after myocardial infarction (MI) and shows two peaks 国際誌

Miwa, S, Toyokuni, S, Tanaka, S, Nishina, T, Nomoto, T, Yamazaki, K, Ueda, K, Nishimura, K

CIRCULATION 106 巻 ( 19 ) 頁： 312 - 312 2002年11月

A novel method of "preparative" myocardial protection using green tea polyphenol in oral uptake 査読有り国際誌

Miwa, S, Saito, M, Yamazaki, K, Toyokuni, S, Hyon, SH, Fujita, M, Komeda, M

CIRCULATION 106 巻 ( 19 ) 頁： 345 - 345 2002年11月

Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart. 査読有り国際誌

Sakamaki K, Inoue T, Asano M, Sudo K, Kazama H, Sakagami J, Sakata S, Ozaki M, Nakamura S, Toyokuni S, Osumi N, Iwakura Y, Yonehara S

Cell death and differentiation 9 巻 ( 11 ) 頁： 1196 - 206 2002年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cell Death and Differentiation

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5-E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.

DOI： 10.1038/sj.cdd.4401090

その他リンク： http://orcid.org/0000-0003-0548-6452

Comparison between high-performance liquid chromatography and enzyme-linked Immunosorbent assay for the determination of 8-hydroxy-2 '-deoxyguanosine in human urine

Shimoi K, Kasai H, Yokota N, Toyokuni S, Kinae N

CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION 11 巻 ( 8 ) 頁： 767 - 770 2002年8月

Vitamin D receptor activity and prevention of colonic hyperproliferation and oxidative stress

Kallay E, Bareis P, Bajna E, Kriwanek S, Bonner E, Toyokuni S, Cross HS

FOOD AND CHEMICAL TOXICOLOGY 40 巻 ( 8 ) 頁： 1191 - 1196 2002年8月

Protective effect of colored rice over white rice on fenton reaction-based renal lipid peroxidation in rats (vol 36, pg 583, 2002)

Toyokuni, S; Itani, T; Morimitsu, Y; Okada, K; Ozeki, M; Kondo, S; Uchida, K; Osawa, T; Hiai, H; Tashiro, T

FREE RADICAL RESEARCH 36 巻 ( 8 ) 2002年8月

Comparison between high-performance liquid chromatography and enzyme-linked immunosorbent assay for the determination of 8-hydroxy-2'-deoxyguanosine in human urine. 国際誌

Kayoko Shimoi, Hiroshi Kasai, Naoko Yokota, Shinya Toyokuni, Naohide Kinae

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 11 巻 ( 8 ) 頁： 767 - 70 2002年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

8-Hydroxy-2'-deoxyguanosine (8-OH-dG), which has been regarded as a potential marker of oxidative DNA damage induced by reactive oxygen species, was measured in human urine by a commercial ELISA using a monoclonal antibody N45.1 and by high-performance liquid chromatography (HPLC) coupled to an electrochemical detector (HPLC-ECD) to evaluate whether the ELISA system is applicable to human monitoring studies. The urine samples were collected from 120 healthy men ages 18-58 in a steel-manufacturing company. A good correlation (r = 0.833; P < 0.0001) was observed between the two methods on HPLC-purified 8-OH-dG fractions from 23 urine samples. The mean value (+/-SD) of 8-OH-dG (microg/g creatinine) was 5.47 (+/-2.97) by HPLC-ECD assay and 5.50 (+/-2.36) by ELISA. However, the correlation (r) between the two methods on 120 original urine samples was 0.460 [P < 0.001; mean value (+/-SD) of 8-OH-dG (microg/g creatinine) was 4.46 (+/-2.03) by the HPLC assay and 9.33 (+/-3.23) by ELISA]. ELISA estimates were about 2-fold higher than the HPLC estimates on original urine. For an unknown reason, 10% of the urine samples showed more than a 4-fold increase in value by ELISA. It is suggested that the ELISA system is applicable for comparative human monitoring studies. Prepurification of 'samples is required to determine the absolute value of 8-OH-dG in individual urine samples by ELISA.

Vitamin D receptor activity and prevention of colonic hyperproliferation and oxidative stress

Comparison between high-performance liquid chromatography and enzyme-linked immunosorbent assay for the determination of 8-hydroxy-2'-deoxyguanosine in human urine.

Shimoi K, Kasai H, Yokota N, Toyokuni S, Kinae N

Vitamin D receptor activity and prevention of colonic hyperproliferation and oxidative stress 査読有り国際誌

Kallay E, Bareis P, Bajna E, Kriwanek S, Bonner E, Toyokuni S, Cross HS

FOOD AND CHEMICAL TOXICOLOGY 40 巻 ( 8 ) 頁： 1191 - 1196 2002年8月

Erratum: Protective effect of colored rice over white rice on fenton reaction-based renal lipid peroxidation in rats (Free Radical Research vol. 36 (5) (583-592)) 査読有り国際誌

Toyokuni S., Itani T., Morimitsu Y., Okada K., Ozeki M., Kondo S., Uchida K., Osawa T., Hiai H., Tashiro T.

Free Radical Research 36 巻 ( 8 ) 2002年8月

Inhibition of gastric inhibitory polypeptide signaling prevents obesity. 査読有り国際誌

Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H, Fujimoto S, Oku A, Tsuda K, Toyokuni S, Hiai H, Mizunoya W, Fushiki T, Holst JJ, Makino M, Tashita A, Kobara Y, Tsubamoto Y, Jinnouchi T, Jomori T, Seino Y

Nature medicine 8 巻 ( 7 ) 頁： 738 - 42 2002年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Medicine

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr−/−) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr−/−, Lepob/Lepob) generated by crossbreeding Gipr−/− and obese ob/ob (Lepob/Lepob) mice gained less weight and had lower adiposity than Lepob/Lepob mice. The Gipr−/− mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.

DOI： 10.1038/nm727

Kállay E., Bareis P., Bajna E., Kriwanek S., Bonner E., Toyokuni S., Cross H.

Food and Chemical Toxicology 40 巻 ( 8 ) 頁： 1191 - 1196 2002年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Food and Chemical Toxicology

Unimpaired vitamin D action has been implicated in human cancer prevention. We have previously demonstrated the effectiveness of 1α-dihydroxyvitamin D3 (1,25-D3) to reduce proliferation and increase differentiation in human colon cancer cells. The aim of this study was to investigate, on the one hand, expression of the vitamin D receptor (VDR) and of 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase) in human normal and malignant colonic tissue and, on the other hand, to determine consequences of reduced or lacking VDR action in a VDR knockout mouse model. In low-grade malignancies of the human colon we found increased VDR and 1α-hydroxylase mRNA expression. However, in late-stage high-grade tumors the vitamin D system is severely compromised. In the mouse colon we found an inverse relationship between VDR levels and proliferation in colon descendens, a tissue known to be specifically affected by nutrients during carcinogenesis. Expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was significantly augmented with complete loss of VDR. These data suggest that genomic 1,25-D3 action is necessary to protect against nutrition-linked hyperproliferation and oxidative DNA damage. © 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0278-6915(02)00030-3

Carvedilol decreases elevated oxidative stress in human failing myocardium 査読有り国際誌 Open Access

Nakamura, K; Kusano, K; Nakamura, Y; Kakishita, M; Ohta, K; Nagase, S; Yamamoto, M; Miyaji, K; Saito, H; Morita, H; Emori, T; Matsubara, H; Toyokuni, S; Ohe, T

CIRCULATION 105 巻 ( 24 ) 頁： 2867 - 2871 2002年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Circulation

Background - Oxidative stress has been implicated in the pathogenesis of heart failure. However, direct evidence of oxidative stress generation in the human failing myocardium has not been obtained. Furthermore, the effect of carvedilol, a vasodilating β-blocker with antioxidant activity, on oxidative stress in human failing hearts has not been assessed. This study was therefore designed to determine whether levels of lipid peroxides are elevated in myocardia of patients with dilated cardiomyopathy (DCM) and whether carvedilol reduces the lipid peroxidation level. Methods and Results - Endomyocardial biopsy samples obtained from 23 patients with DCM and 13 control subjects with normal cardiac function were studied immunohistochemically for the expression of 4-hydroxy-2-nonenal (HNE)-modified protein, which is a major lipid peroxidation product. Expression of HNE-modified protein was found in all myocardial biopsy samples from patients with DCM. Expression was distinct in the cytosol of cardiac myocytes. Myocardial HNE-modified protein levels in patients with DCM were significantly increased compared with the levels in control subjects (P<0.0001). Endomyocardial biopsy samples from 11 patients with DCM were examined before and after treatment (mean, 9±4 months) with carvedilol (5 to 30 mg/d; mean dosage, 22±8 mg/d). After treatment with carvedilol, myocardial HNE-modified protein levels decreased by 40% (P<0.005) along with amelioration of heart failure. Conclusions - Oxidative stress is elevated in myocardia of patients with heart failure. Administration of carvedilol resulted in a decrease in the oxidative stress level together with amelioration of cardiac function.

DOI： 10.1161/01.CIR.0000018605.14470.DD

Peroxynitrite-mediated stress is associated with proliferation of human metastatic colorectal carcinoma in the liver.

Peroxynitrite-mediated stress is associated with proliferation of human metastatic colorectal carcinoma in the liver

Kondo S, Toyokuni S, Tsuruyama T, Ozeki M, Tachibana T, Echizenya M, Hiai H, Onodera H, Imamura M

CANCER LETTERS 179 巻 ( 1 ) 頁： 87 - 93 2002年5月

Kondo S, Toyokuni S, Tsuruyama T, Ozeki M, Tachibana T, Echizenya M, Hiai H, Onodera H, Imamura M

Cancer letters 179 巻 ( 1 ) 頁： 87 - 93 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Letters

3-Nitrotyrosine (3-NT), a product of peroxynitrite reaction, is abundantly observed in hepatocytes adjacent to human metastatic colorectal carcinoma. To elucidate its biological significance, we undertook to identify nitric oxide (NO)-producing cells and apoptosis under oxidative stress. We observed strong inducible NO-synthase (iNOS) immunoreactivity in the hepatocytes adjacent to metastatic tumor, revealing an identical pattern to 3-NT immunostaining. Furthermore, intense 3-NT immunostaining of hepatocytes was associated with apoptosis whereas carcinoma cells near those hepatocytes presented high proliferating-cell nuclear antigen. Our results suggest that contact of metastatic tumor induces apoptosis in adjacent hepatocytes through peroxynitrite, thus permitting the proliferation of cancer cells. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/s0304-3835(01)00859-x

Peroxynitrite-mediated stress is associated with proliferation of human metastatic colorectal carcinoma in the liver. 国際誌

Shohei Kondo, Shinya Toyokuni, Tatsuaki Tsuruyama, Munetaka Ozeki, Tsuyoshi Tachibana, Michiko Echizenya, Hiroshi Hiai, Hisashi Onodera, Masayuki Imamura

Cancer letters 179 巻 ( 1 ) 頁： 87 - 93 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Isolation and characterization of annexin 2 pseudogene in Rattus norvegicus.

Isolation and characterization of annexin 2 pseudogene in Rattus norvegicus

Ozeki M, Hoshino S, Hiai H, Toyokuni S

GENE 289 巻 ( 1-2 ) 頁： 185 - 190 2002年5月

Ozeki M, Hoshino S, Hiai H, Toyokuni S

Gene 289 巻 ( 1-2 ) 頁： 185 - 90 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Gene

Annexin 2 is a calcium-regulated, phospholipid-binding protein present in endothelial cells, macrophages and some tumor cells. Annexin 2 is a substrate for a variety of protein kinases, and plays roles in the regulation of endocytosis, exocytosis and thrombolysis. We have determined the nucleotide sequence of a rat genomic DNA fragment that hybridized to a rat annexin 2 DNA complementary to RNA (cDNA) probe. Sequence analysis revealed that it was an intronless rat annexin 2, consisting of a start-to-stop-codon-length copy of the processed transcript. This pseudogene contained 33 point mutations and two deletion sites in the coding region as compared with the cDNA, and thus displayed typical features of a retroposon. Transitions were more frequent than transversions, and the most frequent type of mutation was G to A transition. We isolated a phage clone that contained a functional rat annexin 2 genomic fragment including coding exons 3 and 4. Polymerase chain reaction and subsequent sequence analysis revealed an intron of approximately 4 kbp at the same site as in humans and mice. Whereas the annexin 2 gene or its cDNA homologues have been detected in various species from Xenopus to humans, its pseudogene has been reported only in humans. In the present study, we demonstrated the presence of an annexin 2 pseudogene in rats. © 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/s0378-1119(02)00549-8

Isolation and characterization of annexin 2 pseudogene in Rattus norvegicus. 国際誌

Munetaka Ozeki, Shinsuke Hoshino, Hiroshi Hiai, Shinya Toyokuni

Gene 289 巻 ( 1-2 ) 頁： 185 - 90 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Hepatocyte nuclear factor-1 alpha recruits the transcriptional co-activator p300 on the GLUT2 gene promoter

Ban N, Yamada Y, Someya Y, Miyawaki K, Ihara Y, Hosokawa M, Toyokuni S, Tsuda K, Seino Y

DIABETES 51 巻 ( 5 ) 頁： 1409 - 1418 2002年5月

Inhibitory effect of a novel water-soluble vitamin E derivative on atherosclerosis in rabbits

Yoshida N, Murase H, Kunieda T, Toyokuni S, Tanaka T, Terao J, Naito Y, Tanigawa T, Yoshikawa T

ATHEROSCLEROSIS 162 巻 ( 1 ) 頁： 111 - 117 2002年5月

Protective effect of colored rice over white rice on Fenton reaction-based renal lipid peroxidation in rats. 国際誌

Shinya Toyokuni, Tomio Itani, Yasujiro Morimitsu, Kunihiko Okada, Munetaka Ozeki, Shohei Kondo, Koji Uchida, Toshihiko Osawa, Hiroshi Hiai, Toru Tashiro

Free radical research 36 巻 ( 5 ) 頁： 583 - 92 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Rice has been one of the most important grains. While polished white rice is favored, colored strains of rice, red, or black, have been maintained for religious purposes in Japan. We studied whether feeding of unpolished colored rice instead of white rice ameliorates oxidative renal tubular damage in rats induced by ferric nitrilotriacetate. Whereas renal lipid peroxidation was exacerbated in white rice-fed group in comparison with standard chow group, this exacerbation was not observed in red or black rice-fed groups. These changes were dependent on the proportion of colored rice to standard chow in the diet. Cyanidin 3-O-beta-D-glucoside was detectable neither in the serum nor kidney after one week of colored rice diet, but serum protocatechuic acid was significantly increased after black rice diet. There was a generalized decrease in the renal glutathione peroxidase activity in rice diet groups. Renal enzymatic activities of superoxide dismutase, glutathione S-transferase and NAD(P)H quinone reductase were not associated with the levels of lipid peroxidation. However, renal catalase activity was significantly increased in black rice-fed groups. These may partly explain the antioxidative effect. Furthermore, colored strains of rice are rich in proteins. Thus, our data warrants further investigation of the antioxidative effect of colored rice.

Protective effect of colored rice over white rice on Fenton reaction-based renal lipid peroxidation in rats

Toyokuni S, Itani T, Morimitsu Y, Okada K, Ozeki M, Kondo S, Uchida K, Osawa T, Hiai H, Tashiro T

FREE RADICAL RESEARCH 36 巻 ( 5 ) 頁： 583 - 592 2002年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Rice has been one of the most important grains. While polished white rice is favored, colored strains of rice, red, or black, have been maintained for religious purposes in Japan. We studied whether feeding of unpolished colored rice instead of white rice ameliorates oxidative renal tubular damage in rats induced by ferric nitrilotriacetate. Whereas renal lipid peroxidation was exacerbated in white rice-fed group in comparison with standard chow group, this exacerbation was not observed in red or black rice-fed groups. These changes were dependent on the proportion of colored rice to standard chow in the diet. Cyanidin 3-O-β-D-glucoside was detectable neither in the serum nor kidney after one week of colored rice diet, but serum protocatechuic acid was significantly increased after black rice diet. There was a generalized decrease in the renal glutathione peroxidase activity in rice diet groups. Renal enzymatic activities of superoxide dismutase, glutathione S-transferase and NAD(P)H quinone reductase were not associated with the levels of lipid peroxidation. However, renal catalase activity was significantly increased in black rice-fed groups. These may partly explain the antioxidative effect. Furthermore, colored strains of rice are rich in proteins. Thus, our data warrants further investigation of the antioxidative effect of colored rice.

DOI： 10.1080/10715760290025960

Inhibitory effect of a novel water-soluble vitamin E derivative on atherosclerosis in rabbits.

Yoshida N, Murase H, Kunieda T, Toyokuni S, Tanaka T, Terao J, Naito Y, Tanigawa T, Yoshikawa T

Atherosclerosis 162 巻 ( 1 ) 頁： 111 - 7 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Atherosclerosis

A novel vitamin E derivative that is freely soluble in water, 2-(α-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), was evaluated for ability to inhibit development of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits or cholesterol-loaded New Zealand White rabbits. Although TMG rapidly entered the circulation blood after oral administration, the blood TMG concentration remained low, while neither TMG nor its metabolites appeared in the low-density lipoprotein (LDL) fraction. TMG did not decrease serum total cholesterol and the various lipoprotein-associated cholesterol fractions (very LDL-, or high-density lipoprotein- (HDL) cholesterol). TMG reduced the serum concentration of thiobarbituric acid-reactive substances (TBARS; an index of lipid peroxidation) in cholesterol-loaded rabbits but not WHHL rabbits. Nonetheless, TMG inhibited aortic atherosclerosis as effectively as probucol in both models. Our results indicate that TMG opposes progression of atherosclerosis not only by preventing oxidation of LDL, but also by presently unknown mechanisms. Even an antioxidant with no uptake by LDL apparently can inhibit development of atherosclerosis despite a very low serum concentration. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/s0021-9150(01)00702-x

Hepatocyte nuclear factor-1alpha recruits the transcriptional co-activator p300 on the GLUT2 gene promoter. 国際誌

Nobuhiro Ban, Yuichiro Yamada, Yoshimichi Someya, Kazumasa Miyawaki, Yu Ihara, Masaya Hosokawa, Shinya Toyokuni, Kinsuke Tsuda, Yutaka Seino

Diabetes 51 巻 ( 5 ) 頁： 1409 - 18 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Mutations in the hepatocyte nuclear factor (HNF)-1alpha gene have been linked to subtype 3 of maturity-onset diabetes of the young (MODY), a disease characterized by a primary defect in insulin secretion. Here we show that the human GLUT2 gene is closely regulated by HNF-1alpha via sequences downstream of the transcriptional start site by interaction with transcriptional co-activator p300. The promoter region of the human GLUT2 gene was subcloned into luciferase expression plasmids that were transfected together with HNF-1alpha expression plasmid into a pancreatic beta-cell line, HIT-T15, to evaluate transcriptional activities. HNF-1alpha enhanced human GLUT2 promoter activity sixfold. Site-direct mutagenesis and footprint analyses showed that the HNF-1alpha binding site (+200 to +218) is critical in human GLUT2 gene expression. Furthermore, mammalian two-hybrid and immunoprecipitation studies revealed the transactivation domain of HNF-1alpha (amino acids 391-540) to interact with both the NH(2)-terminal region (amino acids 180-662) and the COOH-terminal region (amino acids 1,818-2,079) of p300. These findings demonstrated that HNF-1alpha binds to the 5'-untranslated region of GLUT2 and that p300 acts as a transcriptional co-activator for HNF-1alpha. In addition, these results provided new insight into the regulatory function of HNF-1alpha by suggesting a molecular basis for human GLUT2 gene expression.

Hepatocyte nuclear factor-1alpha recruits the transcriptional co-activator p300 on the GLUT2 gene promoter.

Ban N, Yamada Y, Someya Y, Miyawaki K, Ihara Y, Hosokawa M, Toyokuni S, Tsuda K, Seino Y

Diabetes 51 巻 ( 5 ) 頁： 1409 - 18 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Diabetes

Mutations in the hepatocyte nuclear factor (HNF)-1α gene have been linked to subtype 3 of maturity-onset diabetes of the young (MODY), a disease characterized by a primary defect in insulin secretion. Here we show that the human GLUT2 gene is closely regulated by HNF-1α via sequences downstream of the transcriptional start site by interaction with transcriptional co-activator p300. The promoter region of the human GLUT2 gene was subcloned into luciferase expression plasmids that were transfected together with HNF-1α expression plasmid into a pancreatic β-cell line, HIT-T15, to evaluate transcriptional activities. HNF-1α enhanced human GLUT2 promoter activity sixfold. Site-direct mutagenesis and footprint analyses showed that the HNF-1α binding site (+200 to +218) is critical in human GLUT2 gene expression. Furthermore, mammalian two-hybrid and immunoprecipitation studies revealed the transactivation domain of HNF-1α (amino acids 391-540) to interact with both the NH2-terminal region (amino acids 180-662) and the COOH-terminal region (amino acids 1,818-2,079) of p300. These findings demonstrated that HNF-1α binds to the 5′-untranslated region of GLUT2 and that p300 acts as a transcriptional co-activator for HNF-1α. In addition, these results provided new insight into the regulatory function of HNF-1α by suggesting a molecular basis for human GLUT2 gene expression.

DOI： 10.2337/diabetes.51.5.1409

Inhibitory effect of a novel water-soluble vitamin E derivative on atherosclerosis in rabbits. 国際誌

Norimasa Yoshida, Hironobu Murase, Tsutomu Kunieda, Shinya Toyokuni, Tomoyuki Tanaka, Junji Terao, Yuji Naito, Toru Tanigawa, Toshikazu Yoshikawa

Atherosclerosis 162 巻 ( 1 ) 頁： 111 - 7 2002年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

A novel vitamin E derivative that is freely soluble in water, 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), was evaluated for ability to inhibit development of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits or cholesterol-loaded New Zealand White rabbits. Although TMG rapidly entered the circulation blood after oral administration, the blood TMG concentration remained low, while neither TMG nor its metabolites appeared in the low-density lipoprotein (LDL) fraction. TMG did not decrease serum total cholesterol and the various lipoprotein-associated cholesterol fractions (very LDL-, or high-density lipoprotein- (HDL) cholesterol). TMG reduced the serum concentration of thiobarbituric acid-reactive substances (TBARS; an index of lipid peroxidation) in cholesterol-loaded rabbits but not WHHL rabbits. Nonetheless, TMG inhibited aortic atherosclerosis as effectively as probucol in both models. Our results indicate that TMG opposes progression of atherosclerosis not only by preventing oxidation of LDL, but also by presently unknown mechanisms. Even an antioxidant with no uptake by LDL apparently can inhibit development of atherosclerosis despite a very low serum concentration.

子宮内膜細胞診疑陽性例の検討 : 構造異型を加味した判定基準を主体に

Increase in oxidative stress in kidneys of diabetic Akita mice 査読有り国際誌

Ueno Y, Horio F, Uchida K, Naito M, Nomura H, Kato Y, Tsuda T, Toyokuni S, Osawa T

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 巻 ( 4 ) 頁： 869 - 872 2002年4月

Increase in oxidative stress in kidneys of diabetic Akita mice. 国際誌

Ueno Y, Horio F, Uchida K, Naito M, Nomura H, Kato Y, Tsuda T, Toyokuni S, Osawa T

Bioscience, biotechnology, and biochemistry 66 巻 ( 4 ) 頁： 869 - 72 2002年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本農芸化学会

New biomarkers for oxidative damage, were used to identify whether hyperglycemia caused oxidative stress in diabetic Akita mice. At 13 weeks of age, the tissues of these mice were obtained, and the levels of Nε-(hexanonyl)lysine (HEL) and dityrosine (DT) were measured, these being related to lipid peroxide-derived protein covalent modification and protein cross-linking. The levels of HEL and DT in the kidneys of Akita mice were significantly increased compared with the control mice without any accumulation of thiobarbituric acid reactive substances and 4-hydroxy-2-nonenal-modified protein. Immunopositive staining was clearly observed in the kidneys of the Akita mice when using the anti-HEL antibody or anti-DT antibody. These results suggest that hyperglycemia in Akita mice induced oxidative stress and increased these markers in the kidneys. © 2002 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

DOI： 10.1271/bbb.66.869

Increase in oxidative stress in kidneys of diabetic Akita mice 査読有り国際誌

Ueno Y, Horio F, Uchida K, Naito M, Nomura H, Kato Y, Tsuda T, Toyokuni S, Osawa T

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 巻 ( 4 ) 頁： 869 - 872 2002年4月

清水恵子, 小椋聖子, 小林八郎, 豊國伸哉, 則松良明, 森谷卓也, 桜井幹己

日本臨床細胞学会雑誌 41 巻 ( 2 ) 頁： 89 - 97 2002年3月

子宮内膜細胞診疑陽性例の検討 : 構造異型を加味した判定基準を主体に

清水恵子, 小椋聖子, 小林八郎, 豊國伸哉, 則松良明, 森谷卓也, 桜井幹己

日本臨床細胞学会雑誌 41 巻 ( 2 ) 頁： 89 - 97 2002年3月

Adenovirus-mediated gene delivery and in vitro microinsemination produce offspring from infertile male mice. 査読有り国際誌

Kanatsu-Shinohara M, Ogura A, Ikegawa M, Inoue K, Ogonuki N, Tashiro K, Toyokuni S, Honjo T, Shinohara T

Proceedings of the National Academy of Sciences of the United States of America 99 巻 ( 3 ) 頁： 1383 - 8 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Sertoli cells play a pivotal role in spermatogenesis through their interactions with germ cells. To set up a strategy for treating male infertility caused by Sertoli cell dysfunction, we developed a Sertoli cell gene transfer system by using an adenovirus vector, which maintained long-term transgene expression in the testes of infertile mice. Introduction of an adenovirus carrying the mouse Steel (Sl) gene into Sertoli cells restored partial spermatogenesis in infertile Steel/Steeldickie (Sl/Sld) mutant mouse testes. Although these males remained infertile, round spermatids and spermatozoa from the testes produced normal fertile offspring after intracytoplasmic injection into oocytes. None of the offspring showed evidence of germ line transmission of adenoviral DNA. Thus, we demonstrate a successful treatment for infertility by using a gene therapy vector. Therefore, adenovirus-mediated gene delivery into Sertoli cells not only provides an efficient and convenient means for studying germ cell-Sertoli cell interactions through manipulation of the germ cell microenvironment in vivo, but also is a useful method to treat male infertility resulting from a Sertoli cell defect.

DOI： 10.1073/pnas.022646399

Thioredoxin as a biomarker for oxidative stress in patients with rheumatoid arthritis.

Adenovirus-mediated gene delivery and in vitro microinsemination produce offspring from infertile male mice

Kanatsu-Shinohara M, Ogura A, Ikegawa M, Inoue K, Ogonuki N, Tashiro K, Toyokuni S, Honjo T, Shinohara T

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 99 巻 ( 3 ) 頁： 1383 - 1388 2002年2月

Specific allelic loss of p16(INK4A) tumor suppressor gene after weeks of iron-mediated oxidative damage during rat renal carcinogenesis

Hiroyasu M, Ozeki M, Kohda H, Echizenya M, Tanaka T, Hiai H, Toyokuni S

AMERICAN JOURNAL OF PATHOLOGY 160 巻 ( 2 ) 頁： 419 - 424 2002年2月

Thioredoxin as a biomarker for oxidative stress in patients with rheumatoid arthritis

Jikimoto T, Nishikubo Y, Koshiba M, Kanagawa S, Morinobu S, Morinobu A, Saura R, Mizuno K, Kondo S, Toyokuni S, Nakamura H, Yodoi J, Kumagai S

MOLECULAR IMMUNOLOGY 38 巻 ( 10 ) 頁： 765 - 772 2002年2月

Jikimoto T, Nishikubo Y, Koshiba M, Kanagawa S, Morinobu S, Morinobu A, Saura R, Mizuno K, Kondo S, Toyokuni S, Nakamura H, Yodoi J, Kumagai S

Molecular immunology 38 巻 ( 10 ) 頁： 765 - 72 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Molecular Immunology

There is no doubt that oxidative stress occurs in patients with rheumatoid arthritis (RA) and play an important role in both inflammation and destruction of RA joints. Thioredoxin (TRX) is a ubiquitous redox-active protein and is known to be induced in several cells against oxidative stress and to be secreted extracellularly. To clarify whether plasma thioredoxin levels could be a marker for oxidative stress in patients with RA, we measured plasma TRX levels in patients with RA using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and investigated its relationship to TRX concentrations in the inflammatory joints. We have found that the plasma TRX levels of RA patients were significantly higher than those of normal subjects (86.8 ± 54.1 ng/ml versus 38.6 ± 18.5 ng/ml, P <0.0001). The plasma levels were correlated with the disease activity of RA and also with serum C-reactive protein (CRP) values (P <0.01). The concentration of TRX in synovial fluid (SF) from RA was 353.3 ± 220.1 ng/ml (mean ± S.D.) which was significantly higher than that in SF from osteoarthritis patients (70.6 ± 31.0 ng/ml, P <0.0001). The SF TRX concentration was significantly correlated with the number of leukocytes infiltrating in SF and with the serum CRP levels. The serum TRX levels were significantly positively correlated with the SF TRX concentrations in RA patients (P <0.05). By the histological examination for synovial tissue of RA patients, TRX was shown to be present on the surface of synovial lining layer as well as in the leukocytes. Moreover, urinary excretion of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage by endogenously generated oxygen radicals, was significantly higher in RA patients than in healthy subjects (11.55 ± 4.71 versus 7.76 ± 2.26 ng/mg creatinine, P <0.0001). Plasma TRX levels were significantly correlated with urinary excretion of 8-OHdG (P <0.005). We concluded that plasma TRX level is a new biomarker for the disease activity of RA and may reflect higher levels of oxidative stress in RA patients. © 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/s0161-5890(01)00113-4

Specific allelic loss of p16 (INK4A) tumor suppressor gene after weeks of iron-mediated oxidative damage during rat renal carcinogenesis. 国際誌

Makoto Hiroyasu, Munetaka Ozeki, Haruyasu Kohda, Michiko Echizenya, Tomoyuki Tanaka, Hiroshi Hiai, Shinya Toyokuni

The American journal of pathology 160 巻 ( 2 ) 頁： 419 - 24 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Oxidative tissue damage has been shown to be associated with carcinogenesis. In human cancers p16(INK4A) is one of the most frequently mutated tumor suppressor genes. The present study used the ferric nitrilotriacetate (Fe-NTA)-induced rat renal carcinogenesis model to determine whether oxidative damage can cause specific allelic loss of p16 (INK4A). By the use of fluorescent in situ hybridization in combination with imprint cytology at single-cell resolution, we found that the number of renal tubular cells with aneuploidy (1 or 3 signals) at the p16(INK4A) locus was significantly and specifically increased (1 week, 37.2 +/- 2.3%; 3 weeks, 37.8 +/- 1.3% vs control, 22.5 +/- 1.9%; mean +/- SE, N = 8; P < 0.001 and P < 0.0001, respectively) after repeated intraperitoneal administration of 5 to10 mg of iron/kg in the form of Fe-NTA for 3 weeks. No increase in aneuploidy was observed at the loci of either the p53 or vhl tumor suppressor gene. Furthermore, the increase in the cells with 3 signals was followed by a continuous increase in those with 1 signal. Therefore, the p16 (INK4A) locus is specifically vulnerable to oxidative damage, leading to its allelic loss within weeks, presumably due to a deficiency in the replication of both the alleles.

Specific allelic loss of p16 (INK4A) tumor suppressor gene after weeks of iron-mediated oxidative damage during rat renal carcinogenesis.

Hiroyasu M, Ozeki M, Kohda H, Echizenya M, Tanaka T, Hiai H, Toyokuni S

The American journal of pathology 160 巻 ( 2 ) 頁： 419 - 24 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Pathology

Oxidative tissue damage has been shown to be associated with carcinogenesis. In human cancers p16INK4A is one of the most frequently mutated tumor suppressor genes. The present study used the ferric nitrilotriacetate (Fe-NTA)-induced rat renal carcinogenesis model to determine whether oxidative damage can cause specific allelic loss of p16INK4A. By the use of fluorescent in situ hybridization in combination with imprint cytology at single-cell resolution, we found that the number of renal tubular cells with aneuploidy (1 or 3 signals) at the p16INK4A locus was significantly and specifically increased (1 week, 37.2 ± 2.3%; 3 weeks, 37.8 ± 1.3% vs control, 22.5 ± 1.9%; mean ± SE, N = 8; P < 0.001 and P < 0.0001, respectively) after repeated intraperitoneal administration of 5 to10 mg of iron/kg in the form of Fe-NTA for 3 weeks. No increase in aneuploidy was observed at the loci of either the p53 or vhl tumor suppressor gene. Furthermore, the increase in the cells with 3 signals was followed by a continuous increase in those with 1 signal. Therefore, the p16INK4A locus is specifically vulnerable to oxidative damage, leading to its allelic loss within weeks, presumably due to a deficiency in the replication of both the alleles.

DOI： 10.1016/S0002-9440(10)64860-2

Thioredoxin as a biomarker for oxidative stress in patients with rheumatoid arthritis. 国際誌

Takumi Jikimoto, Yuko Nishikubo, Masahiro Koshiba, Sugayo Kanagawa, Sahoko Morinobu, Akio Morinobu, Ryuichi Saura, Kosaku Mizuno, Shohei Kondo, Shinya Toyokuni, Hajime Nakamura, Junji Yodoi, Shunichi Kumagai

Molecular immunology 38 巻 ( 10 ) 頁： 765 - 72 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

There is no doubt that oxidative stress occurs in patients with rheumatoid arthritis (RA) and play an important role in both inflammation and destruction of RA joints. Thioredoxin (TRX) is a ubiquitous redox-active protein and is known to be induced in several cells against oxidative stress and to be secreted extracellularly. To clarify whether plasma thioredoxin levels could be a marker for oxidative stress in patients with RA, we measured plasma TRX levels in patients with RA using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and investigated its relationship to TRX concentrations in the inflammatory joints. We have found that the plasma TRX levels of RA patients were significantly higher than those of normal subjects (86.8 +/-54.1 ng/ml versus 38.6 +/-18.5 ng/ml, P<0.0001). The plasma levels were correlated with the disease activity of RA and also with serum C-reactive protein (CRP) values (P<0.01). The concentration of TRX in synovial fluid (SF) from RA was 353.3 +/- 220.1 ng/ml (mean +/- S.D.) which was significantly higher than that in SF from osteoarthritis patients (70.6 +/- 31.0 ng/ml, P<0.0001). The SF TRX concentration was significantly correlated with the number of leukocytes infiltrating in SF and with the serum CRP levels. The serum TRX levels were significantly positively correlated with the SF TRX concentrations in RA patients (P<0.05). By the histological examination for synovial tissue of RA patients, TRX was shown to be present on the surface of synovial lining layer as well as in the leukocytes.Moreover, urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage by endogenously generated oxygen radicals, was significantly higher in RA patients than in healthy subjects (11.55 +/- 4.71 versus 7.76 +/- 2.26 ng/mg creatinine, P<0.0001). Plasma TRX levels were significantly correlated with urinary excretion of 8-OHdG (P<0.005). We concluded that plasma TRX level is a new biomarker for the disease activity of RA and may reflect higher levels of oxidative stress in RA patients.

Target genes in fenton reaction-induced carcinogenisis: A novel concept

Heat shock preconditioning reduces the formation of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal modified proteins in ischemia-reperfused liver of rats. 査読有り国際誌

Yamagami K, Yamamoto Y, Toyokuni S, Hata K, Yamaoka Y

Free radical research 36 巻 ( 2 ) 頁： 169 - 76 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Heat shock preconditioning (HSPC) is a promising strategy for providing ischemic tolerance. The objective of this study is to investigate the effectiveness of HSPC in preventing oxidative damage of cellular proteins and DNA during ischemia-reperfusion of the liver. Male Wistar rats were divided into a heat shock group (group HS) and control (group C). Forty-eight hours prior to ischemia, rats in group HS received HSPC at 42°C for 15 min. All rats received hepatic warm ischemia for 30 min and subsequent reperfusion. The formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) modified proteins in liver tissue, survival rate of the animals, and changes in biochemical and histological parameters were compared between groups. Heat shock protein 72 was produced only in group HS. The 7-day survival of rats was significantly better in group HS (10/10) than in group C (5/10) (p <0.01). The serum release of alanine aminotransferase (n =10, p < 0.01) and the concentration of adenosine triphosphate in liver tissue (n = 10, p < 0.01) 40 min after reperfusion was significantly better in group HS than in group C. The formation of 8-OHdG in liver tissue measured by high-performance liquid chromatography was suppressed in group HS (p < 0.01). The production of HNE-modified proteins as determined by Westem-blot analysis was also decreased in group HS. These results were also confirmed by immunohistochemical analysis. As determined by levels of 8-OHdG and HNE-modified proteins produced during ischemia-reperfusion of the liver, HSPC reduced the oxidative injury of cellular proteins and DNA in the liver tissue.

DOI： 10.1080/10715760290006385

Amelioration of cisplatin toxicity by a fermented grain food product

Minamiyama Y, Takemura S, Toyokuni S, Nishino Y, Yamasaki K, Hai S, Yamamoto S, Okada S

BIOFACTORS 16 巻 ( 3-4 ) 頁： 105 - 115 2002年

Iron and carcinogenesis: from Fenton reaction to target genes. 国際誌

Toyokuni S

Redox report : communications in free radical research 7 巻 ( 4 ) 頁： 189 - 97 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Report

Reactive oxygen species (ROS) have been shown to be associated with a wide variety of pathological phenomena such as carcinogenesis, inflammation, radiation and reperfusion injury. Iron, the most abundant transition metal ion in our body, may work as a catalyst for the generation of ROS in pathological conditions. In the past few years, there have been great advances in the understanding of iron metabolism. These include the discoveries of iron transporters and the gene responsible for hereditary hemochromatosis. Iron overload has been shown to be associated with carcinogenesis. We recently identified the major target genes (p16INK4A and p15INK4B tumor suppressor genes, which encode cyclin-dependent kinase inhibitors) in a ferric nitrilotriacetate-induced rat renal carcinogenesis model, in which the Fenton reaction is induced in the renal proximal tubules. Allelic loss of the p16 gene occurs early in carcinogenesis and specifically at the p16 loci as compared with other tumor suppressor genes. This led to the novel concept of 'genomic sites vulnerable to the Fenton reaction'. Here, recent new findings on iron metabolism are reviewed and the concept of the vulnerable sites explored. More effort to link iron metabolism with human carcinogenesis is anticipated.

DOI： 10.1179/135100002125000596

Toyokuni, S; Hiroyasu, M; Ozeki, M; Hiai, H

FREE RADICAL BIOLOGY AND MEDICINE 33 巻頁： S177 - S177 2002年

Target genes in Fenton reaction-induced carcinogenesis: A novel concept

Toyokuni, S; Hiroyasu, M; Ozeki, M; Hiai, H

XI BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 頁： 459 - 462 2002年

Radiation-induced kidney injury: a role for chronic oxidative stress?

Robbins MEC, Zhao WL, Davis CS, Toyokuni S, Bonsib SM

MICRON 33 巻 ( 2 ) 頁： 133 - 141 2002年

Novel surrogate end-point biomarker to evaluate agents for use in the chemoprevention of reactive oxygen species-associated cancer. 査読有り国際誌

Hiroyasu M, Ozeki M, Miyagawa-Hayashino A, Fujiwara Y, Hiai H, Toyokuni S

Redox report : communications in free radical research 7 巻 ( 5 ) 頁： 335 - 8 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Redox Report

Cancer chemoprevention is the use of chemical agents to inhibit, delay or reverse carcinogenesis. We established a novel method to evaluate agents for use in the chemoprevention of reactive oxygen species (ROS)-associated cancer. Induction of renal cell carcinoma in rats by ferric nitrilotriacetate (Fe-NTA) is an established model of ROS-associated cancer. We recently identified the p16INK4A tumor suppressor gene as one of the major target genes in this model, and showed by the use of in situ hybridization that allelic loss of p16INK4A occurs in the increased fraction of renal tubular cells within a few weeks. In the present study, we tested whether diets including green tea powder or a processed grain food are effective chemopreventive agents in this animal model. Consumption of these modified diets led to a significant decrease in the fraction of aneuploid cells after 1 week of repeated Fe-NTA administration. A decrease in renal lipid peroxidation after a single administration of Fe-NTA was also observed. Therefore, intake of green tea or processed grain foods stabilizes p16INK4A in the genome, at least in this model, and might be helpful for the prevention of ROS-associated cancer. This novel method is versatile, and may work as a surrogate end-point biomarker for screening the usefulness of agents for cancer chemoprevention.

DOI： 10.1179/135100002125000938

子宮内膜細胞診疑陽性例の検討構造異型を加味した判定基準を主体に:構造異型を加味した判定基準を主体に

清水恵子, 小椋聖子, 小林八郎, 豊國伸哉, 則松良明, 森谷卓也, 桜井幹己

日本臨床細胞学会雑誌 41 巻 ( 2 ) 頁： 89 - 94 2002年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：特定非営利活動法人日本臨床細胞学会

目的:子宮内膜細胞診は判定が困難な分野として認識されており, さらに各施設により採取法, 検体処理法, 疑陽性とする細胞診断基準が異なっている.今回われわれは当院に於ける細胞診疑陽性例の判定基準を主体にした検討 (特に構造異型を加味した判定基準に対する検討) を行い若干の知見を得たので報告する. 方法:1995年5月から2000年9月までの疑陽性例のうち組織診が施行された84件を対象とし, 細胞異型を中心に判定した期間 (期間A) と細胞異型に構造異型を加味して判定した期問 (期間B) の成績の差異について検討した.標本作製法はエンドサイト直接塗抹法で期間BではRPMI液による採取器具洗浄を加えた. 成績:検討症例84件の組織診断の内訳は期間Aでは良性23件, 増殖症21件, 腺癌7件, 期間Bでは良性11件, 増殖症21件, 腺癌1件であった.検体不良率は, 期間Aでは32.9%, 期間Bでは20%であった. 結論:細胞異型に構造異型を加味して判定した期間では, 細胞異型を中心に判定した期間に比べて増殖症の正診率が向上し腺癌の疑陽性率は減少した.また, 直接塗抹標本に器具洗浄標本を加えることにより検体不良率の減少がみられた.

DOI： 10.5795/jjscc.41.89

Radiation-induced kidney injury: a role for chronic oxidative stress?

Target genes in fenton reaction-induced carcinogenisis: A novel concept 国際誌

Toyokuni, S, Hiroyasu, M, Ozeki, M, Hiai, H

FREE RADICAL BIOLOGY AND MEDICINE 33 巻頁： S177 - S177 2002年

Target genes in Fenton reaction-induced carcinogenesis: A novel concept 国際誌

Toyokuni, S, Hiroyasu, M, Ozeki, M, Hiai, H

XI BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 頁： 459 - 462 2002年

Robbins ME, Zhao W, Davis CS, Toyokuni S, Bonsib SM

Micron (Oxford, England : 1993) 33 巻 ( 2 ) 頁： 133 - 41 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Micron

Kidney irradiation clearly leads to a progressive reduction in function associated with concomitant glomerulosclerosis and/or tubulointerstitial fibrosis. However, the particular cell types, mediators and/or mechanisms involved in the development and progression of radiation nephropathy remain ill defined. Angiotensin II (Ang II) plays a major pathogenic role; administration of Ang II blockers markedly abrogates the severity of radiation nephropathy in experimental models. Both ionizing radiation and Ang II signal via generation of reactive oxygen species (ROS). Thus, we hypothesized that localized kidney irradiation might lead to a chronic oxidative stress. In view of the difficulty in measuring ROS in vivo we adopted an indirect immunohistochemical approach in which we used a monoclonal antibody specific for 8hydroxy-2′-deoxyguanosine (8-OHdG), one of the most commonly used markers of DNA oxidation. The right kidney of 7-8 week-old male Sprague-Dawley rats was removed. Five to 6 weeks later the remaining hypertrophied kidney was irradiated with single doses of 0-20.0 Gy X-rays. Groups of rats, three per dose, were killed at 4, 8, 16 and 24 weeks post-irradiation, their kidneys fixed, and sections stained with the 8-OHdG-specific antibody N45.1. For quantitation of glomerular DNA oxidation with the N45.1 antibody stained sections, 50 glomeruli/animal were counted. The presence of any intensely stained nuclei within the glomerular tuft was scored as positive. Quantitation of tubular DNA oxidation employed a 10 × 10 point ocular grid. Sections were examined at 400 magnification; 250 tubular profiles were counted. All tubules with any nuclear staining were scored as positive. Sham-irradiated kidneys showed little evidence of DNA oxidation over the experimental period. In contrast, localized kidney irradiation led to a marked, dose-independent increase in glomerular and tubular cell nuclear DNA oxidation. This increase was evident at the first time point studied, i.e. 4 weeks after irradiation, and persisted for up to 24 weeks postirradiation. DNA oxidation in the irradiated kidney was only seen in apparently viable glomerular and tubular cells. Thus, while from 16 to 24 weeks post-irradiation structural alterations had progressed to glomerular sclerosis and tubular atrophy, positive staining for 8-OHdG was not observed in severely atrophic tubules. Similarly, fewer positive staining cells were noted in glomeruli undergoing sclerosis, while none were seen in totally sclerotic glomeruli. These data support the hypothesis that renal irradiation is associated with a chronic and persistent oxidative stress. © 2001 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/s0968-4328(01)00006-3

Radiation-induced kidney injury: a role for chronic oxidative stress? 国際誌

Mike E Robbins, Weiling Zhao, Charles S Davis, Shinya Toyokuni, Stephen M Bonsib

Micron (Oxford, England : 1993) 33 巻 ( 2 ) 頁： 133 - 41 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）

Kidney irradiation clearly leads to a progressive reduction in function associated with concomitant glomerulosclerosis and/or tubulointerstitial fibrosis. However, the particular cell types, mediators and/or mechanisms involved in the development and progression of radiation nephropathy remain ill defined. Angiotensin II (Ang II) plays a major pathogenic role; administration of Ang II blockers markedly abrogates the severity of radiation nephropathy in experimental models. Both ionizing radiation and Ang II signal via generation of reactive oxygen species (ROS). Thus, we hypothesized that localized kidney irradiation might lead to a chronic oxidative stress. In view of the difficulty in measuring ROS in vivo we adopted an indirect immunohistochemical approach in which we used a monoclonal antibody specific for 8-hydroxy-2'-deoxyguanosine (8-OHdG), one of the most commonly used markers of DNA oxidation. The right kidney of 7-8 week-old male Sprague-Dawley rats was removed. Five to 6 weeks later the remaining hypertrophied kidney was irradiated with single doses of 0-20.0 Gy X-rays. Groups of rats, three per dose, were killed at 4, 8, 16 and 24 weeks post-irradiation, their kidneys fixed, and sections stained with the 8-OHdG-specific antibody N45.1. For quantitation of glomerular DNA oxidation with the N45.1 antibody stained sections, 50 glomeruli/animal were counted. The presence of any intensely stained nuclei within the glomerular tuft was scored as positive. Quantitation of tubular DNA oxidation employed a 10 x 10 point ocular grid. Sections were examined at 400 magnification; 250 tubular profiles were counted. All tubules with any nuclear staining were scored as positive.Sham-irradiated kidneys showed little evidence of DNA oxidation over the experimental period. In contrast, localized kidney irradiation led to a marked, dose-independent increase in glomerular and tubular cell nuclear DNA oxidation. This increase was evident at the first time point studied, i.e. 4 weeks after irradiation, and persisted for up to 24 weeks postirradiation. DNA oxidation in the irradiated kidney was only seen in apparently viable glomerular and tubular cells. Thus, while from 16 to 24 weeks post-irradiation structural alterations had progressed to glomerular sclerosis and tubular atrophy, positive staining for 8-OHdG was not observed in severely atrophic tubules. Similarly, fewer positive staining cells were noted in glomeruli undergoing sclerosis, while none were seen in totally sclerotic glomeruli. These data support the hypothesis that renal irradiation is associated with a chronic and persistent oxidative stress.

Comparison between high-performance liquid chromatography and enzyme-linked immunosorbent assay for the determination of 8-hydroxy-2′-deoxyguanosine in human urine 査読有り国際誌

Shimoi K., Kasai H., Yokota N., Toyokuni S., Kinae N.

Cancer Epidemiology Biomarkers and Prevention 11 巻 ( 8 ) 頁： 767 - 770 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Epidemiology Biomarkers and Prevention

8-Hydroxy-2′-deoxyguanosine (8-OH-dG), which has been regarded as a potential marker of oxidative DNA damage induced by reactive oxygen species, was measured in human urine by a commercial ELISA using a monoclonal antibody N45.1 and by high-performance liquid chromatography (HPLC) coupled to an electrochemical detector (HPLCECD) to evaluate whether the ELISA system is applicable to human monitoring studies. The urine samples were collected from 120 healthy men ages 18-58 in a steel-manufacturing company. A good correlation (r = 0.833; P < 0.0001) was observed between the two methods on HPLC-purified 8- OH-dG fractions from 23 urine samples. The mean value (±SD) of 8-OH-dG (μg/g creatinine) was 5.47 (±2.97) by HPLC-ECD assay and 5.50 (±2.36) by ELISA. However, the correlation (r) between the two methods on 120 original urine samples was 0.460 [P < 0.001; mean value (±SD) of 8-OH-dG (μg/g creatinine) was 4.46 (±2.03) by the HPLC assay and 9.33 (±3.23) by ELISA]. ELISA estimates were about 2-fold higher than the HPLC estimates on original urine. For an unknown reason, 10% of the urine samples showed more than a 4-fold increase in value by ELISA. It is suggested that the ELISA system is applicable for comparative human monitoring studies. Prepurification of 'samples is required to determine the absolute value of 8-OH-dG in individual urine samples by ELISA.

Amelioration of cisplatin toxicity by a fermented grain food product.

Birth of offspring transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination

Shinohara, T, Inoue, K, Ogonuki, N, Kanatsu-Shinohara, M, Miki, H, Nakata, K, Kurome, M, Nagashima, H, Toyokuni, S, Kogishi, K, Honjo, T, Ogura, A

Human Reproduction 17 巻頁： 3039-3045 - 3045 2002年

Minamiyama Y, Takemura S, Toyokuni S, Nishino Y, Yamasaki K, Hai S, Yamamoto S, Okada S

BioFactors (Oxford, England) 16 巻 ( 3-4 ) 頁： 105 - 15 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BioFactors

The most noticeable hypothesis regarding the pathogenesis of cisplatin toxicity, seen mainly in kidney and intestine, is oxidative stress, an imbalance between free-radical generating cisplatin and radical scavenging systems. This paper describes the role of the antioxidant system in cisplatin-induced toxicity and the protective effect by a processed grain food (Antioxidant Biofactor: AOB®), which has been shown to exhibit strong antioxidant activity. Male Fischer 344 rats were used. They were pre-fed either a basal diet (control, 15 g/day) or the diet supplemented with AOB® to provide 6.5% or 20% of total diet throughout the experiment. Cisplatin (5 mg/kg, i.v.) was administered at the start of the experiment, and the animals were sacrificed 5 days later. Blood urea nitrogen (BUN) and plasma creatinine, NO2- and NO3- (NOx) were determined from the plasma. The levels of 4-hydroxy-2-nonenal (a lipid peroxidation product), 8-hydroxy-deoxyguanosine (8-OHdG, an oxidatively modified DNA adduct) and nitrotyrosine were histologically analyzed. The cisplatin administration resulted in a loss of body weight and elevations of BUN, serum creatinine and NOx levels, whereas AOB supplement reversed these effects. The severe morphological damages induced in the kidney and intestine by the cisplatin administration were markedly improved in the AOB group. The levels of lipid peroxidation, 8-OHdG, and nitrotyrosine all paralleled the morphological damage. The AOB effect was dose dependent. In conclusion, the present study suggests that certain food additives like AOB may be of benefit against the side effects of cisplatin.

DOI： 10.1002/biof.5520160306

Amelioration of cisplatin toxicity by a fermented grain food product. 査読有り国際誌

Yukiko Minamiyama, Shigekazu Takemura, Shinya Toyokuni, Yoshihiro Nishino, Keiichi Yamasaki, Seikan Hai, Satoshi Yamamoto, Shigeru Okada

BioFactors (Oxford, England) 16 巻 ( 3-4 ) 頁： 105 - 15 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）

The most noticeable hypothesis regarding the pathogenesis of cisplatin toxicity, seen mainly in kidney and intestine, is oxidative stress, an imbalance between free-radical generating cisplatin and radical scavenging systems. This paper describes the role of the antioxidant system in cisplatin-induced toxicity and the protective effect by a processed grain food (Antioxidant Biofactor: AOB), which has been shown to exhibit strong antioxidant activity. Male Fischer 344 rats were used. They were pre-fed either a basal diet (control, 15 g/day) or the diet supplemented with AOB to provide 6.5% or 20% of total diet throughout the experiment. Cisplatin (5 mg/kg, i.v.) was administered at the start of the experiment, and the animals were sacrificed 5 days later. Blood urea nitrogen (BUN) and plasma creatinine, NO2(-) and NO3(-) (NOx) were determined from the plasma. The levels of 4-hydroxy-2-nonenal (a lipid peroxidation product), 8-hydroxy-deoxyguanosine (8-OHdG, an oxidatively modified DNA adduct) and nitrotyrosine were histologically analyzed. The cisplatin administration resulted in a loss of body weight and elevations of BUN, serum creatinine and NOx levels, whereas AOB supplement reversed these effects. The severe morphological damages induced in the kidney and intestine by the cisplatin administration were markedly improved in the AOB group. The levels of lipid peroxidation, 8-OHdG, and nitrotyrosine all paralleled the morphological damage. The AOB effect was dose dependent. In conclusion, the present study suggests that certain food additives like AOB may be of benefit against the side effects of cisplatin.

酸化ストレス発がんの分子機構とその標的遺伝子

豊國伸哉

環境変異原研究 23 巻 ( 3 ) 頁： 177 - 182 2001年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本環境変異原学会

Oxidative stress has been associated with carcinogenesis. In 1982, our laboratory established an oxys-tress-induced carcinogenesis model of rodent kidney by the use of an iron chelate, ferric nitrilotriacetate. This model is unique in that 1) only carcinoma, not sarcoma, is induced, 2) the induced tumor is of high malignant potential to kill the animal by pulmonary metastasis, peritoneal invasion or rupture of the primary tumor, and, 3) increase in a variety of covalently modified products including 8-oxoguanine, thymine-tyrosine cross-link, 4-hydroxy-2-nonenal and its modified proteins are demonstrated in its early stage. Since in general free radicals are not considered to react with specific target molecules in contrast with immune reaction, we undertook to answer the question whether there is any specific target gene (s) in this carcinogenesis model. By genetically analyzing F_1 hybrid rats, we found two chromosomal areas that showed a high incidence of loss of heterozygosity, which lead to a finding that p15^<INK4B>/p16^<INK4A> tumor suppressor genes are one of the major pathways responsible for this oxystress-induced cancer. Furthermore, by screening 84,000 transcripts by differential display techniques, we found that at least 15 genes associated with stress-response or cellular proliferation are differentially expressed in this model. We believe that not only genetic changes but also epigenetic changes play a role in oxidative stress-induced carcinogenesis.

酸化ストレス発がんの分子機構とその標的遺伝子

豊國伸哉

環境変異原研究 23 巻 ( 3 ) 頁： 177 - 182 2001年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本環境変異原学会

Activation of lectin-like oxidized low-density lipoprotein receptor-1 induces apoptosis in cultured neonatal rat cardiac myocytes

Iwai-Kanai E., Hasegawa K., Sawamura T., Fujita M., Yanazume T., Toyokuni S., Adachi S., Kihara Y., Sasayama S.

Circulation 104 巻 ( 24 ) 頁： 2948 - 2954 2001年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Circulation

Background - Lectin-like oxidized LDL receptor-1 (LOX-1) was originally identified as a receptor expressed predominantly in endothelial cells. LOX-1 can also be expressed in other cell types, and the activation of the LOX-1 pathway has been implicated in apoptosis. There have been no reports, however, about LOX-1 expression in cardiac myocytes or regulation of myocardial cell apoptosis by LOX-1. Methods and Results - In primary cardiac myocytes from neonatal rats, immunohistochemical analyses using a specific monoclonal antibody against LOX-1 demonstrated that LOX-1 expression was markedly induced by stimulation with norepinephrine and endothelin-1. LOX-1 expression was upregulated in cardiac myocytes as well as in vessel walls of failing rat hearts in vivo. In the presence of a low concentration of oxidized LDL that did not induce apoptosis by itself, artificial overexpression of LOX-1 in cardiac myocytes in culture resulted in apoptosis. LOX-1 overexpression induced activation of p38 mitogen-activated protein kinase (MAPK) and oxidative stress in cardiac myocytes, as demonstrated by an increase in positive immunostaining for 8-hydroxy-2′-deoxyguanosine. Inhibition of p38 MAPK by cotransfection of a dominant-negative form of MKK6 as well as by administration of a specific inhibitor, SB203580 or FR167653, almost completely blocked the induction of apoptosis by LOX-1 activation. Antioxidant catalase also blocked LOX-1-induced apoptosis as well as activation of p38 MAPK. Conclusions - These findings demonstrate that LOX-1 expression in cardiac myocytes is induced by neurohormonal factors activated in heart failure and that LOX-1-dependent apoptosis in these cells requires p38 MAPK, a component of oxidant stress-sensitive signaling pathways.

DOI： 10.1161/hc4901.100381

境界悪性卵巣粘液性腫瘍の再発との細胞学的鑑別が困難であった子宮頸部上皮内腺癌の1例

Activation of lectin-like oxidized low-density lipoprotein receptor-1 induces apoptosis in cultured neonatal rat cardiac myocytes

Iwai-Kanai E, Hasegawa K, Sawamura T, Fujita M, Yanazume T, Toyokuni S, Adachi S, Kihara Y, Sasayama S

CIRCULATION 104 巻 ( 24 ) 頁： 2948 - 2954 2001年12月

Activation of lectin-like oxidized low-density lipoprotein receptor-1 induces apoptosis in cultured neonatal rat cardiac myocytes 査読有り

Iwai-Kanai E, Hasegawa K, Sawamura T, Fujita M, Araki M, Yanazume T, Toyokuni S, Adachi S, Kihara Y, Sasayama S

Circulation 104 巻 ( 24 ) 頁： 2948 - 2954 2001年12月

小椋聖子, 清水恵子, 小林八郎, 豊國伸哉, 桜井幹己

日本臨床細胞学会雑誌 40 巻 ( 6 ) 頁： 636 - 640 2001年11月

境界悪性卵巣粘液性腫瘍の再発との細胞学的鑑別が困難であった子宮頸部上皮内腺癌の1例

小椋聖子, 清水恵子, 小林八郎, 豊國伸哉, 桜井幹己

日本臨床細胞学会雑誌 40 巻 ( 6 ) 頁： 636 - 640 2001年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：特定非営利活動法人日本臨床細胞学会

55歳,1経妊1経産.卵巣粘液性腫瘍の再発との鑑別が困難であった子宮頸部上皮内腺癌の1例を経験した.3年前に右卵巣腫瘍破裂で右付属器摘出術が施行され,右卵巣境界悪性粘液性腫瘍,腸上皮型,Ic期と診断され,外来経過観察中であった.子宮頸部擦過細胞診で異型腺細胞集団が認められ,子宮頸部腺系病変あるいは卵巣粘液性腫瘍の再発が疑われ,単純子宮全摘術と左付属器摘出術を施行した.摘出標本の組織診で子宮頸部上皮内腺癌と診断され,子宮体部,左付属器に悪性所見は認められなかった.婦人科領域の重複癌の診断では,細胞所見のみならず臨床所見や画像診断を加味した総合的判断が必要と思われた

Role of Helicobacter pylori infection in gastroduodenal injury and gastric prostaglandin synthesis during long term/low dose aspirin therapy: A prospective placebo-controlled, double-blind randomized trial 査読有り国際誌

A novel selectin blocker alleviates oxidative stress of lung reperfusion injury. 国際誌 Open Access

Yoshimura T, Kawashima M, Nakamura T, Isowa N, Bando T, Hasegawa S, Kondo H, Toyokuni S, Wada H

The Journal of surgical research 101 巻 ( 1 ) 頁： 91 - 8 2001年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Surgical Research

Background. The importance of reactive oxygen species released through interaction of leukocyte/endothelial cell in ischemia-reperfusion injury of the lung is not yet fully understood. A novel selectin blocker, OJ-R9188, which inhibits the interaction, may alleviate oxidative stress and pulmonary dysfunction after warm ischemia-reperfusion. Materials and methods. Rat lungs were reperfused at 37°C for 60 min with an ex vivo model and were divided into three groups (n = 10). In the fresh group, lungs were reperfused immediately after harvest. In the OJ-R (-) and OJ-R (+) groups, lungs were reperfused after warm ischemia at 37°C for 90 min. In the OJ-R (+) group, rats received 100 μg per body of OJ-R9188 intravenously 10 min before the harvest. The electron spin resonance method was used to assess the direct scavenging activity of OJ-R9188. Results. Both shunt fractions and wet/dry weight ratios of the lung tissue after reperfusion in the OJ-R (+) group were significantly lower than those in the OJ-R (-) group. Oxidative DNA damage in the alveolar wall of the OJ-R (+) group, assessed by immunohistochemical quantitation of 8-hydroxy-2′-deoxyguanosine, was significantly lower than that of the OJ-R (-) group. Immunostaining of 3-nitro-L-tyrosine, which represents nitric oxide-mediated oxidative damage, was also more intense in the OJ-R (-) group than in the OJ-R (+) group. Direct scavenging activity of OJ-R9188 was not observed, and the number of leukocytes infiltrated to the lung tissue as seen by myeloperoxidase activity was not different between the OJ-R (-) and OJ-R (+) groups. Conclusions. A novel selectin blocker, OJ-R9188, improves pulmonary function after warm ischemia-reperfusion and alleviates reperfusion-induced oxidative alveolar damage. © 2001 Academic Press.

DOI： 10.1006/jsre.2001.6258

Characterization of a vitamin D receptor knockout mouse as a model of colorectal hyperproliferation and DNA damage

Kallay E, Pietschmann P, Toyokuni S, Bajna E, Hahn P, Mazzucco K, Bieglmayer C, Kato S, Cross HS

CARCINOGENESIS 22 巻 ( 9 ) 頁： 1429 - 1435 2001年9月

Characterization of a vitamin D receptor knockout mouse as a model of colorectal hyperproliferation and DNA damage

Kallay E, Pietschmann P, Toyokuni S, Bajna E, Hahn P, Mazzucco K, Bieglmayer C, Kato S, Cross HS

CARCINOGENESIS 22 巻 ( 9 ) 頁： 1429 - 1435 2001年9月

Curcumin and especially tetrahydrocurcumin ameliorate oxidative stress-induced renal injury in mice 査読有り国際誌

Okada K, Wangpoengtrakul C, Tanaka T, Toyokuni S, Uchida K, Osawa T

JOURNAL OF NUTRITION 131 巻 ( 8 ) 頁： 2090 - 2095 2001年8月

Curcumin and especially tetrahydrocurcumin ameliorate oxidative stress-induced renal injury in mice 査読有り国際誌

Okada K, Wangpoengtrakul C, Tanaka T, Toyokuni S, Uchida K, Osawa T

JOURNAL OF NUTRITION 131 巻 ( 8 ) 頁： 2090 - 2095 2001年8月

Pignatelli B., Bancel B., Plummer M., Toyokuni S., Patricot L., Ohshima H.

American Journal of Gastroenterology 96 巻 ( 6 ) 頁： 1751 - 1757 2001年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Gastroenterology

OBJECTIVES: Whether gastric infection with Helicobacter pylori increases the risk of gastric mucosal injury during long term/low dose aspirin therapy is unknown. We examined whether H. pylori infection enhances upper GI mucosal damage, assessed endoscopically, in volunteers given low dose aspirin. We studied 61 healthy men and women, 29 with and 32 without active H. pylori infection. METHODS: We treated volunteers for 45 days with a placebo or aspirin (either 81 mg every day or 325 mg every 3 days). Gastroduodenal mucosal damage was then assessed by endoscopy, as was gastric histology and ex vivo gastric mucosal prostaglandin E2 and F2α synthesis rates. RESULTS: Erosive disease from low dose aspirin (erosions and/or ulcers) occurred in 50% of H. pylori-infected volunteers and in 16% of their noninfected counterparts (p = 0.02). Aspirin caused a significantly higher average mucosal injury score in the gastric antrum in H. pylori-infected participants than in noninfected subjects (p = 0.03), and two H. pylori-infected subjects developed antral gastric ulcers. Subjects with H. pylori gastritis treated with the placebo had nearly 50% higher gastric mucosal prostaglandin (E2 plus F2α) synthesis rates than their noninfected counterparts (108 ± 6 ng/g/min versus 75 ± 6 ng/g/min, p < 0.001). Aspirin reduced mucosal prostaglandin synthesis to similar levels in infected and noninfected participants. CONCLUSIONS: Long term/low dose aspirin therapy led to more gastric mucosal damage when H. pylori gastritis was present than when it was absent, despite similar degrees of gastric mucosal prostaglandin depletion. © 2001 by Am. Coll. of Gastroenterology.

DOI： 10.1016/S0002-9270(01)02491-1

Oxidative DNA damage in cultured cells and rat lungs by carcinogenic nickel compounds

Oxidative DNA damage in cultured cells and rat lungs by carcinogenic nickel compounds

Kawanishi S, Inoue S, Oikawa S, Yamashita N, Toyokuni S, Kawanishi M, Nishino K

FREE RADICAL BIOLOGY AND MEDICINE 31 巻 ( 1 ) 頁： 108 - 116 2001年7月

Kawanishi S., Inoue S., Oikawa S., Yamashita N., Toyokuni S., Kawanishi M., Nishino K.

Free Radical Biology and Medicine 31 巻 ( 1 ) 頁： 108 - 116 2001年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

DNA damage in cultured cells and in lungs of rats induced by nickel compounds was investigated to clarify the mechanism of nickel carcinogenesis. DNA strand breaks in cultured cells exposed to nickel compounds were measured by using a pulsed field gel electrophoresis technique. Among nickel compounds (Ni3S2, NiO (black), NiO (green), and NiSO4), only Ni3S2, which is highly carcinogenic, induced lesions of both double- and single-stranded DNA in cultured human cells (Raji and HeLa cells). Treatment of cultured HeLa cells with Ni3S2 (10 μg/ml) induced a 1.5-fold increase in 8-hydroxy-2′-deoxyguanosine (8-OH-dG) compared with control, whereas NiO (black), NiO (green), and NiSO4 did not enhance the generation of 8-OH-dG. Intratracheal instillation of Ni3S2, NiO(black), and NiO(green) to Wistar rats increased 8-OH-dG in the lungs significantly. NiSO4 induced a smaller but significant increase in 8-OH-dG. Histological studies showed that all the nickel compounds used induced inflammation in lungs of the rats. Nitric oxide (NO) generation in phagocytic cells induced by Ni3S2, NiO(black), and NiO(green) was examined using macrophage cell line RAW 264.7 cells. NO generation in RAW 264.7 cells stimulated with lipopolysaccharide was enhanced by all nickel particles. Two mechanisms for nickel-induced oxidative DNA damage have been proposed as follows: all the nickel compounds used induced indirect damage through inflammation, and Ni3S2 also showed direct oxidative DNA damage through H2O2 formation. This double action may explain relatively high carcinogenic risk of Ni3S2. © 2001 Elsevier Science Inc.

DOI： 10.1016/S0891-5849(01)00558-5

Oxidative DNA damage in cultured cells and rat lungs by carcinogenic nickel compounds 査読有り

S Kawanishi, S Inoue, S Oikawa, N Yamashita, S Toyokuni, M Kawanishi, K Nishino

FREE RADICAL BIOLOGY AND MEDICINE 31 巻 ( 1 ) 頁： 108 - 116 2001年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

DNA damage in cultured cells and in lungs of rats induced by nickel compounds was investigated to clarify the mechanism of nickel carcinogenesis. DNA strand breaks in cultured cells exposed to nickel compounds were measured by using a pulsed held gel electrophoresis technique. Among nickel compounds (Ni3S2, NiO (black), NiO (green), and NiSO4), only Ni3S2, which is highly carcinogenic, induced lesions of both double- and single-stranded DNA in cultured human cells (Raji and HeLa cells). Treatment of cultured HeLa cells with Ni3S2 (10 mug/ml) induced a 1.5-fold increase in 8-hydroxy-2'-deoxyguanosine (8-OH-dG) compared with control, whereas NiO (black), NiO (green), and NiSO4 did not enhance the generation of 8-OH-dG. Intratracheal instillation of Ni3S2, NiO(black), and NiO(green) to Wistar rats increased 8-OH-dG in the lungs significantly. NiSO4 induced a smaller but significant increase in 8-OH-dG. Histological studies showed that all the nickel compounds used induced inflammation in lungs of the rats. Nitric oxide (NO) generation in phagocytic cells induced by Ni3S2, NiO(black). and NiO(green) was examined using macrophage cell line RAW 264.7 cells. NO generation in RAW 264.7 cells stimulated with lipopolysaccharide was enhanced by all nickel particles. Two mechanisms for nickel-induced oxidative DNA damage have been proposed as follows: all the nickel compounds used induced indirect damage through inflammation, and Ni3S2 also showed direct oxidative DNA damage through H2O2 formation. This double action may explain relatively high carcinogenic risk of Ni3S2. (C) 2001 Elsevier Science Inc.

Endogenous formation of protein adducts with carcinogenic aldehydes: Implications for oxidative stress

Endogenous formation of protein adducts with carcinogenic aldehydes - Implications for oxidative stress 査読有り国際誌

Ichihashi K, Osawa T, Toyokuni S, Uchida K

JOURNAL OF BIOLOGICAL CHEMISTRY 276 巻 ( 26 ) 頁： 23903 - 23913 2001年6月

Ichihashi K., Osawa T., Toyokuni S., Uchida K.

Journal of Biological Chemistry 276 巻 ( 26 ) 頁： 23903 - 23913 2001年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

In the present study, we characterize the covalent modification of a protein by crotonaldehyde, a representative carcinogenic aldehyde, and describe the endogenous production of this aldehyde in vivo. The crotonaldehyde preferentially reacted with the lysine and histidine residues of bovine serum albumin and generated a protein-linked carbonyl derivative. Upon incubation with the histidine and lysine derivatives, crotonaldehyde predominantly generated β-substituted butanal adducts of histidine and lysine and N ε-(2,5-dimethyl-3-formyl-3,4-dehydropiperidino)lysine (dimethyl-FDP-lysine) as the putative carbonyl derivatives generated in the crotonaldehyde-modified protein. To verify the endogenous formation of crotonaldehyde in vivo, we raised the monoclonal antibody (mAb82D3) against the crotonaldehyde-modified protein and found that it cross-reacted with the protein-bound 2-alkenals, such as crotonaldehyde, 2-pentenal, and 2-hexenal. The anti-2-alkenal antibody recognized multiple crotonaldehyde-lysine adducts, including dimethyl-FDP-lysine and an unknown product, which showed the greatest immunoreactivity with the antibody. On the basis of the chemical and spectroscopic evidence, the major antigenic product was determined to be a novel Schiff base-derived crotonaldehyde-lysine adduct, Nε -(5-ethyl-2-methylpyridinium)lysine (EMP-lysine). It was found that the lysine residues that had disappeared in the protein treated with crotonaldehyde were partially recovered by EMP-lysine. The presence of immunoreactive materials with mAb82D3 in vivo was demonstrated in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate. In addition, the observations that the metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of proteins resulted in an increase in the antigenicity of the protein indicated that lipid peroxidation represents a potential pathway for the formation of crotonaldehyde/2-alkenals in vivo. These data suggest that the formation of carcinogenic aldehydes during lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.

DOI： 10.1074/jbc.M101947200

Difference in Thioredoxin Expression in Viral Myocarditis in Inbred Strains of Mice

Difference in thioredoxin expression in viral myocarditis in inbred strains of mice

Miyamoto M, Kishimoto C, Shioji K, Nakamura H, Toyokuni S, Nakayama Y, Kita M, Yodoi J, Sasayama S

JAPANESE CIRCULATION JOURNAL-ENGLISH EDITION 65 巻 ( 6 ) 頁： 561 - 564 2001年6月

Helicobacter pylori eradication attenuates oxidative stress in human gastric mucosa

Pignatelli B, Bancel B, Plummer M, Toyokuni S, Patricot LM, Ohshima H

AMERICAN JOURNAL OF GASTROENTEROLOGY 96 巻 ( 6 ) 頁： 1758 - 1766 2001年6月

Difference in thioredoxin expression in viral myocarditis in inbred strains of mice 国際誌

Miyamoto M, Kishimoto C, Shioji K, Nakamura H, Toyokuni S, Nakayama Y, Kita M, Yodoi J, Sasayama S

JAPANESE CIRCULATION JOURNAL-ENGLISH EDITION 65 巻 ( 6 ) 頁： 561 - 564 2001年6月

Helicobacter pylori eradication attenuates oxidative stress in human gastric mucosa 国際誌

Pignatelli B, Bancel B, Plummer M, Toyokuni S, Patricot LM, Ohshima H

AMERICAN JOURNAL OF GASTROENTEROLOGY 96 巻 ( 6 ) 頁： 1758 - 1766 2001年6月

MIYAMOTO Miki, KISHIMOTO Chiharu, SHIOJI Keisuke, NAKAMURA Hajime, TOYOKUNI Shinya, NAKAYAMA Yukie, KITA Masakazu, YODOI Junji, SASAYAMA Shigetake

Japanese circulation journal 65 巻 ( 6 ) 頁： 561 - 564 2001年5月

掲載種別：研究論文（学術雑誌）出版者・発行元：社団法人日本循環器学会

Redox regulating mechanisms may be involved in the pathogenesis of viral myocarditis and thioredoxin (TRX) is a small multifunctional protein that contains a redox active sequence. The present study investigated the histopathology and characteristics of TRX expression in acute coxsackievirus B3 myocarditis in inbred strains of mice (severe myocarditis in DBA/2 mice, moderate myocarditis in BALB/c mice and mild myocarditis in C57BL/6 mice). Thioredoxin was upregulated and its expression correlated with the severity of the disease. In addition, 8-hydroxy-2'-deoxyguanosine, which is an established marker for oxidative stress, was concominantly positive in damaged myocytes. Thus, TRX may be specifically induced by the acute inflammatory stimuli in murine viral myocarditis, and the severity and development of acute viral myocarditis may be regulated by the cellular redox state. (Jpn Circ J 2001; 65: 561-564)

Difference in Thioredoxin Expression in Viral Myocarditis in Inbred Strains of Mice

MIYAMOTO Miki, KISHIMOTO Chiharu, SHIOJI Keisuke, NAKAMURA Hajime, TOYOKUNI Shinya, NAKAYAMA Yukie, KITA Masakazu, YODOI Junji, SASAYAMA Shigetake

Japanese circulation journal 65 巻 ( 6 ) 頁： 561 - 564 2001年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：社団法人日本循環器学会

Spontaneous loss-of-function mutations of the 8-oxoguanine DNA glycosylase gene in mice and exploration of the possible implication of the gene in senescence

Mori M., Toyokuni S., Kondo S., Kasai H., Naiki H., Toichi E., Hosokawa M., Higuchi K.

Free Radical Biology and Medicine 30 巻 ( 10 ) 頁： 1130 - 1136 2001年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

8-Oxoguanine is one of the major premutagenic oxidative base legions in vivo and is suspected to play a crucial role in various pathophysiological processes, such as cancer and aging. Mammalian 8-oxoguanine DNA glycosylase (OGG1) is thought to play a major role in the removal of 8-oxoguanine adducts in vivo. We have identified several inbred mouse strains with a spontaneous mutation, OGG1-R336H or double mutations, OGG1-R304W/R336H. R304W mutation caused a complete loss of OGG1 activity, while the R336H mutation led to disruption of nuclear localization of the enzyme although the activity remained normal. Among the double mutants was SAMP1, which exhibits accelerated senescence and short lifespan. We assessed the possible implication of the mutant OGG1 and 8-oxoguanine in aging utilizing SAMP1 mice. SAMP1 retained 1.5- to 1.9-fold increase in 8-oxoguanine level of hepatic nuclear DNA as compared with normal mice, until at least 12 months of age. A genetic association study, however, indicated that the mutant Ogg1 gene per se is not responsible for the accelerated senescence and short lifespan of SAMP1. Mutant OGG1 may be associated with pathologic conditions in other mouse strains. © 2001 Elsevier Science Inc.

DOI： 10.1016/S0891-5849(01)00511-1

Angiosarcoma of the scalp: Absence of vascular endothelial cadherin in primary and metastatic lesions

Spontaneous loss-of-function mutations of the 8-oxoguanine DNA glycosylase gene in mice and exploration of the possible implication of the gene in senescence

Mori M, Toyokuni S, Kondo S, Kasai H, Naiki H, Toichi E, Hosokawa M, Higuchi K

FREE RADICAL BIOLOGY AND MEDICINE 30 巻 ( 10 ) 頁： 1130 - 1136 2001年5月

Tanioka M., Ikoma A., Morita K., Fujii H., Toda K.I., Takahashi K., Tanaka T., Nishigori C., Jin G., Higashi S., Toyokuni S., Miyachi Y.

British Journal of Dermatology 144 巻 ( 2 ) 頁： 380 - 383 2001年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：British Journal of Dermatology

A 73-year-old man with angiosarcoma of the scalp died about 1 year after disease onset, despite systemic and topical administration of recombinant interleukin-2. Histopathology showed typical changes of endothelial cells with very sparse lymphocytic infiltration into the tumour. An autopsy revealed that the primary site penetrated cranial bone and invaded vertically into the subarachnoid space. Multiple metastases to lung, chest wall, vertebrae and ribs were also found. On immunofluorescence staining, the expression of vascular endothelial cadherin, which is present in normal endothelium, was absent from both primary and metastatic sites. This may have promoted local invasion and metastasis.

DOI： 10.1046/j.1365-2133.2001.04032.x

Pericardial fluid from patients with ischemic heart disease induces myocardial cell apoptotis via an oxidant stress-sensitive p38 mitogen-activated protein kinase pathway. 査読有り国際誌

Iwakura A, Fujita M, Hasegawa K, Toyokuni S, Sawamura T, Nohara R, Sasayama S, Komeda M

Journal of molecular and cellular cardiology 33 巻 ( 3 ) 頁： 419 - 30 2001年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Molecular and Cellular Cardiology

Factors produced by the heart are accumulated at high concentrations in pericardial fluid. We recently reported that pericardial fluid from patients with ischemic heart disease induces apoptosis in an F2 cell line. To characterize factors in pericardial fluid from patients with ischemic heart disease, we investigated signaling pathways by which this pericardial fluid induces apoptosis in cardiac myocytes. Pericardial fluid from patients with ischemic heart disease markedly increased the percentage of TUNEL-positive myocytes compared with fetal bovine serum. Apoptosis was also confirmed by ladder formation and morphologic features. Apoptosis mediated by this pericardial fluid occurs as readily in cardiac myocytes prepared from neonatal mice nullizygous for p53 as in wild-type littermates. This indicates that p53 is not required for this process. We have found that pericardial fluid from ischemic heart disease elicits a robust increase in phosphorylation of p38 mitogen-activated protein kinase. Specific inhibition of the p38 mitogen-activated protein kinase pathway with SB 203580 almost completely blocked apoptosis mediated by pericardial fluid from ischemic heart disease. Activation of p38 mitogen-activated protein kinase is caused by cellular stress, including oxidants. We have also found that anti-oxidant catalase inhibited pericardial fluid-induced activation of p38 mitogen-activated protein kinase and apoptosis. These findings demonstrate that myocardial cell apoptosis induced by pericardial fluid from patients with ischemic heart disease is mediated by an oxidant stress-sensitive p38 mitogen-activated protein kinase pathway. A possible application of SB 203580 to preserve cardiac function in patients with ischemic heart disease should be discussed.

DOI： 10.1006/jmcc.2000.1314

Difference in Thioredoxin Expression in Viral Myocarditis in Inbred Strains of Mice

Angiosarcoma of the scalp: absence of vascular endothelial cadherin in primary and metastatic lesions

Tanioka M, Ikoma A, Morita K, Fujii H, Toda KI, Takahashi K, Tanaka T, Nishigori C, Jin G, Higashi S, Toyokuni S, Miyachi Y

BRITISH JOURNAL OF DERMATOLOGY 144 巻 ( 2 ) 頁： 380 - 383 2001年2月

Angiosarcoma of the scalp: absence of vascular endothelial cadherin in primary and metastatic lesions

Tanioka M, Ikoma A, Morita K, Fujii H, Toda KI, Takahashi K, Tanaka T, Nishigori C, Jin G, Higashi S, Toyokuni S, Miyachi Y

BRITISH JOURNAL OF DERMATOLOGY 144 巻 ( 2 ) 頁： 380 - 383 2001年2月

An electron spin resonance study on alkylperoxyl radical in thin-sliced renal tissues from ferric nitrilotriacetate-treated rats: the effect of alpha-tocopherol feeding

Toyokuni S, Masumizu T, Ozeki M, Kondo S, Hiroyasu M, Kohno M, Hiai H

FREE RADICAL RESEARCH 35 巻 ( 3 ) 頁： 245 - 255 2001年

Miyamoto Miki, Kishimoto Chiharu, Shioji Keisuke, Nakamura Hajime, Toyokuni Shinya, Nakayama Yukie, Kita Masakazu, Yodoi Junji, Sasayama Shigetake

JAPANESE CIRCULATION JOURNAL 65 巻 ( 6 ) 頁： 561 - 564 2001年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：社団法人日本循環器学会

DOI： 10.1253/jcj.65.561

境界悪性卵巣粘液性腫ようの再発との細胞学的鑑別が困難であった子宮頚部上皮内腺癌の１例国際誌

小椋聖子, 清水恵子, 小林八郎, 豊國伸哉, 桜井幹己

日本臨床細胞学会雑誌 40 巻 ( 6 ) 頁： 636 - 640 2001年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：特定非営利活動法人日本臨床細胞学会

背景:近年, 診断法や治療法の進歩に伴う治癒率の向上とともに, 婦人科領域の重複癌発生率が上昇している. 婦人科領域の細胞診断の際に重複癌を疑う像に遭遇することがあるが, その判定に苦慮することは少なくない. 今回われわれは, 境界悪性卵巣粘液性腫瘍の再発との鑑別が困難であった子宮頸部上皮内腺癌の1例を報告する. 症例:55歳, 女性. 3年前に右卵巣腫瘍破裂のため, 右付属器摘出術が施行され, 右卵巣境界悪性粘液性腫瘍, 腸上皮型, Ic期と診断された. 外来での経過観察中, 子宮頸部擦過細胞診にて異型腺細胞集団が認められ, 子宮頸部腺系病変あるいは卵巣粘液性腫瘍の再発が疑われた. 単純子宮全摘術および左付属器摘出術が施行され, 摘出標本の組織診で子宮頸部上皮内腺癌と診断された. 子宮体部, 左付属器に悪性所見は認められなかった. 結論:婦人科領域における重複癌の診断においては, 細胞所見のみならず臨床所見や画像診断を加味した総合的な判断が必要である.

DOI： 10.5795/jjscc.40.636

Iron-induced oxidative damage in colon carcinoma (Caco-2) cells

Núñez M., Tapia V., Toyokuni S., Okada S.

Free Radical Research 34 巻 ( 1 ) 頁： 57 - 68 2001年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Intestinal epithelial cells have an active apical iron uptake system that is involved in the regulated absorption of iron. By the action of this system, intestinal cells acquire increasing amounts of iron with time. Since intracellular reactive iron is a source of free radicals and a possible cause of colon carcinoma, this study analyzed the oxidative damages generated by iron accumulation in Caco-2 cells. Cells cultured with increasing concentrations of iron increased both total intracellular iron and the reactive iron pool, despite an active IRE/IRP system, which regulates intracellular iron levels. Increasing concentrations of iron resulted in increased protein oxidative damage, as shown by the immunoreactivity for 4-hydroxy-2-nonenal-modified proteins, and markedly induced DNA oxidation determined by 8-hydroxy-2′-deoxyguanidine production. Iron also impaired cell viability, resulting in increased cell death after 6 days of culture. In summary, iron accumulation by intestinal Caco-2 cells correlated with oxidative damage to proteins and DNA. Oxidative damage finally resulted in loss of cell viability. The Fe-induced oxidative damage observed may be relevant in understanding the cascade of events associated with iron-mediated colon carcinogenesis.

DOI： 10.1080/10715760100300061

Iron-induced oxidative damage in colon carcinoma (Caco-2) cells 査読有り

MT Nunez, Tapia, V, S Toyokuni, S Okada

FREE RADICAL RESEARCH 34 巻 ( 1 ) 頁： 57 - + 2001年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：HARWOOD ACAD PUBL GMBH

Intestinal epithelial cells have an active apical iron uptake system that is involved in the regulated absorption of iron. By the action of this system, intestinal cells acquire increasing amounts of iron with time. Since intracellular reactive iron is a source of free radicals and a possible cause of colon carcinoma, this study analyzed the oxidative damages generated by iron accumulation in Caco-2 cells. Cells cultured with increasing concentrations of iron increased both total intracellular iron and the reactive iron pool, despite an active IRE/IRP system, which regulates intracellular iron levels. Increasing concentrations of iron resulted in increased protein oxidative damage, as shown by the immunoreactivity for 4-hydroxy-2-nonenal-modified proteins, and markedly induced DNA oxidation determined by 8-hydroxy-2'-deoxyguanidine production. Iron also impaired cell viability, resulting in increased cell death after 6 days of culture. In summary, iron accumulation by intestinal Caco-2 cells correlated with oxidative damage to proteins and DNA. Oxidative damage finally resulted in loss of cell viability. The Fe-induced oxidative damage observed may be relevant in understanding the cascade of events associated with iron-mediated colon carcinogenesis.

Iron-induced oxidative damage in colon carcinoma (Caco-2) cells

Nunez MT, Tapia V, Toyokuni S, Okada S

FREE RADICAL RESEARCH 34 巻 ( 1 ) 頁： 57 - + 2001年

Helicobacter pylori eradication attenuates oxidative stress in human gastric mucosa 国際誌

Pignatelli B., Bancel B., Plummer M., Toyokuni S., Patricot L., Ohshima H.

American Journal of Gastroenterology 96 巻 ( 6 ) 頁： 1758 - 1766 2001年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Gastroenterology

OBJECTIVE: Helicobacter pylori infection causes gastric diseases, but the responsible mechanisms are not completely understood. They can involve DNA and tissue damage induced by reactive oxygen and nitrogen species. Our aim is to investigate the effects of bacterial eradication on oxidative stress by measuring changes of relevant markers. METHODS: Antral biopsies were obtained from 34 patients with chronic atrophic gastritis and peptic ulcer disease before and after bacterial eradication. The expression of inducible nitric oxide synthase (iNOS) and levels of nitrotyrosine (NTYR) and 8-hydroxy-2′-deoxyguanosine were assessed immunohistochemically as markers of nitric oxide (NO) production and of damage to proteins and DNA, respectively. RESULTS: Before treatment, the percentages of patients with staining were: 56 for iNOS in inflammatory cells, 79 and 61 for NTYR and 8-hydroxy-2′-deoxyguanosine in foveolar cells, respectively, and 82 for 8-hydroxy-2′-deoxyguanosine in lymphoid follicles. NTYR staining was associated with the intensity of inflammation (p = 0.04) and gastritis activity (p = 0.07). The prevalence of 8-hydroxy-2′-deoxyguanosine tended to be associated with that of NTYR. After successful H. pylori eradication, the prevalence of iNOS and NTYR (in mild gastritis) staining decreased (p < 0.001 and p < 0.06, respectively). 8-Hydroxy-2′-deoxyguanosine staining disappeared in 24% of cases but appeared in 18% of previously negative cases despite eradication. CONCLUSION: Targets of oxidative stress associated with H. pylori infection are inflammatory and deep foveolar cells and lymphoid follicles. This is the first report of 8-hydroxy-2′-deoxyguanosine localization in gastric mucosa. Oxidative stress is reduced by bacterial eradication in the first stages of mild gastritis. Moderate-severe gastritis may be a step that is reversible for iNOS, but partly irreversible for NTYR and 8-hydroxy-2′-deoxyguanosine. © 2001 Am. Coll. of Gastroenterology.

DOI： 10.1111/j.1572-0241.2001.03869.x

Helicobacter pylori eradication attenuates oxidative stress in human gastric mucosa

Pignatelli B., Bancel B., Plummer M., Toyokuni S., Patricot L.M., Ohshima H.

American Journal of Gastroenterology 96 巻 ( 6 ) 頁： 1758 - 1766 2001年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Gastroenterology

DOI： 10.1016/S0002-9270(01)02432-7

Curcumin and especially tetrahydrocurcumin ameliorate oxidative stress-induced renal injury in mice

Okada K., Wangpoengtrakul C., Tanaka T., Toyokuni S., Uchida K., Osawa T.

Journal of Nutrition 131 巻 ( 8 ) 頁： 2090 - 2095 2001年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Nutrition

Protective effects of curcumin (U1), one of the major yellow pigments in turmeric and its derivative, tetrahydrocurcumin (THU1), against ferric nitrilotriacetate (Fe-NTA)-induced oxidative renal damage were studied in male ddY mice. Single Fe-NTA treatment (5 mg Fe/kg body intraperitoneally) transiently causes oxidative stress, as shown by the accumulation of lipid peroxidation products and 8-hydroxy-2′-deoxyguanosine in the kidney. Mice were fed with a diet containing 0.5 g/100 g U1 or THU1 for 4 wk. THU1 significantly inhibited 2-thiobarbituric acid reactive substances and 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2′-deoxyguanosine formation in the kidney; U1 inhibited only 4-hydroxy-2-nonenal-modified protein formation. To elucidate the mechanisms of protection by U1 and THU1, the pharmacokinetics and radical-scavenging capacities of U1 and THU1 were investigated by HPLC and electron spin resonance spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide, respectively. Induction of antioxidant enzymes was also investigated. The amounts of THU1 and its conjugates (as sulfates and glucuronides) in the liver and serum were larger in the THU1 group than in the U1 group. The amounts of U1 and its conjugates were small even in the U1 group. These results suggest that THU1 is more easily absorbed from the gastrointestinal tract than U1. Furthermore, THU1 induced antioxidant enzymes, such as glutathione peroxidase, glutathione S-transferase and NADPH: quinone reductase, as well as or better than U1 and scavenged Fe-NTA-induced free radicals in vitro better than U1. These results suggest that U1 is converted to THU1 in vivo and that THU1 is a more promising chemopreventive agent.

DOI： 10.1093/jn/131.8.2090

Characterization of a vitamin D receptor knockout mouse as a model of colorectal hyperproliferation and DNA damage

Kallay E., Pietschmann P., Toyokuni S., Bajna E., Hahn P., Mazzucco K., Bieglmayer C., Kato S., Cross H.S.

Carcinogenesis 22 巻 ( 9 ) 頁： 1429 - 1435 2001年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Carcinogenesis

The vitamin D receptor knockout (VDR-KO) mouse presents with a skeletal phenotype typical for complete lack of genomic 1,25-dihydroxycholecalciferol effects. Our previous data from human colorectal tissue suggest that the steroid hormone and its receptor may have protective function against tumour progression. In order to investigate the relevance of the vitamin D system for pre-malignant site-directed changes in the colon, we characterized the amount and site-specific distribution of the VDR along the large intestine in wild-type (WT), heterozygote (HT) and KO mice. We also evaluated expression of proliferating cell nuclear antigen (PCNA), of cyclin D1 and the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative stress. In colon ascendens, proliferative cells were dispersed all along the crypt and expression levels of all three markers were high in WT mice. A decrease of VDR expression did not affect expression significantly. In colon descendens, however, fewer proliferative cells were solely located in the lower third of the crypt, and an inverse relationship between VDR reduction, PCNA positivity and cyclin D1 expression was found in HT and KO mice. In parallel to enhanced proliferation a highly significant increase of 8-OHdG positivity occurred. Therefore, the sigmoid colon of VDR-KO mice, fed on an appropriate lactose/calcium-enriched diet to alleviate impaired calcium homeostasis-related phenotypic changes, is an excellent model for investigating induction and prevention of pre-malignant changes in one of the hotspots for human colorectal cancer incidence.

DOI： 10.1093/carcin/22.9.1429

An electron spin resonance study on alkylperoxyl radical in thin-sliced renal tissues from ferric nitrilotriacetate-treated rats: The effect of α-tocopherol feeding

Toyokuni S., Masumizu T., Ozeki M., Kondo S., Hiroyasu M., Kohno M., Hiai H.

Free Radical Research 35 巻 ( 3 ) 頁： 245 - 255 2001年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

Formation of excess free radical causes cellular oxidative stress, which has been shown to be associated with a variety of pathologic conditions. While electron spin resonance (ESR) spectroscopy has been the only method to demonstrate the presence of free radicals, its application to tissue samples has been challenging. We report here the successful ESR detection in thin-sliced fresh tissues or frozen sections in a rat model. Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal tubular damage that ultimately leads to high incidence of renal carcinoma in rodents. Twenty minutes after administration of 5 mg iron/kg Fe-NTA to rats, a thin-slice of the kidney was mounted on a tissue-type cell and analyzed by ESR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An ESR signal from alkylperoxyl radical adduct was obtained, and the signal was inversely proportional to renal α-tocopherol content which was modulated through diet. Furthermore, we undertook ex vivo study using frozen sections. Fe-NTA (1mM) was added to a rat kidney frozen section for 10 min. After washing the specimen was mounted on a tissue-type cell and analyzed with ESR spin trapping using DMPO. Alkylperoxyl radical signal was dependent on thickness, incubation time and renal tissue levels of α-tocopherol, and was reduced by preincubation with catalase or dimethyl sulfoxide but not with α-tocopherol outside tissue. This versatile method facilitates identification of free radicals in pathologic conditions, and may be useful for selection of antioxidants.

DOI： 10.1080/10715760100300781

An electron spin resonance study on alkylperoxyl radical in thin-sliced renal tissues from ferric nitrilotriacetate-treated rats: the effect of alpha-tocopherol feeding

Toyokuni S, Masumizu T, Ozeki M, Kondo S, Hiroyasu M, Kohno M, Hiai H

FREE RADICAL RESEARCH 35 巻 ( 3 ) 頁： 245 - 255 2001年

Enhanced hepatic lipid peroxidation in patients with primary biliary cirrhosis 査読有り国際誌 Open Access

Kawamura K., Kobayashi Y., Kageyama F., Kawasaki T., Nagasawa M., Toyokuni S., Uchida K., Nakamura H.

American Journal of Gastroenterology 95 巻 ( 12 ) 頁： 3596 - 3601 2000年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Gastroenterology

OBJECTIVE: The mechanisms responsible for hepatic injury have not been fully clarified in primary biliary cirrhosis (PBC). It has recently been suggested that hepatic lipid peroxidation may be involved in the pathogenesis of PBC. The aims of the current study were to determine whether patients with PBC have evidence of enhanced hepatic lipid peroxidation and to evaluate its relationship to clinicopathological features. METHODS: Immunohistochemical detection of 4-hydroxynonenal (HNE) protein adducts was performed in the liver biopsies of 20 patients with PBC. Histological stages of PBC were evaluated. Orcein or Victoria blue staining was performed for detection of copper-associated proteins. The size of bile ducts was defined as the smallest diameter between the subepithelial basement membranes. RESULTS: All 20 patients had immunodetectable HNE protein adducts in the cytoplasm of damaged, but also intact, biliary cells. The mean diameter of bile ducts with HNE protein adducts was smaller than those without the adducts (61.0 ± 1.9 vs 122.5 ± 24.4 μm, respectively, p < 0.01). Out of 20 patients, 6 (30%) also had immunodetectable HNE protein adducts in hepatocytes preferentially located around the portal tracts. Most of the patients with hepatocytic HNE protein adducts had copper-associated protein granules in hepatocytes around the portal tracts and were classified as histological stage 3, whereas all of the patients without the adducts lacked copper-associated protein granules and were classified as histological stage 1 or 2. The patients with hepatocytic HNE protein adducts had higher levels of serum total bilirubin than did those without the adducts (2.9 ± 0.9 vs 0.7 ± 0.1 mg/dl, respectively, p < 0.01). CONCLUSIONS: Hepatic lipid peroxidation can occur in PBC and may be an early event in bile duct destruction. At advanced stages of PBC, hepatocellular lipid peroxidation may play a role in hepatocyte injury during cholestasis. (C) 2000 by Am. Coll. of Gastroenterology.

DOI： 10.1016/S0002-9270(00)02093-1

Activating transcripton factor-2 is a positive regulator in CaM kinase IV-induced human insulin gene expression

Enhanced hepatic lipid peroxidation in patients with primary biliary cirrhosis 国際誌

Kawamura, K; Kobayashi, Y; Kageyama, F; Kawasaki, T; Nagasawa, M; Toyokuni, S; Uchida, K; Nakamura, H

AMERICAN JOURNAL OF GASTROENTEROLOGY 95 巻 ( 12 ) 頁： 3596 - 3601 2000年12月

Enhanced hepatic lipid peroxidation in patients with primary biliary cirrhosis 査読有り国際誌

Kawamura K, Kobayashi Y, Kageyama F, Kawasaki T, Nagasawa M, Toyokuni S, Uchida K, Nakamura H

AMERICAN JOURNAL OF GASTROENTEROLOGY 95 巻 ( 12 ) 頁： 3596 - 3601 2000年12月

Ban N., Yamada Y., Someya Y., Ihara Y., Adachi T., Kubota A., Watanabe R., Kuroe A., Inada A., Miyawaki K., Sunaga Y., Shen Z.P., Iwakura T., Tsukiyama K., Toyokuni S., Tsuda K., Seino Y.

Diabetes 49 巻 ( 7 ) 頁： 1142 - 1148 2000年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Diabetes

Insulin plays a crucial role in the regulation of glucose-homeostasis, and its synthesis is regulated by several stimuli. The transcription of the human insulin gene, enhanced by an elevated intracellular concentration of calcium ions, was completely blocked by Ca2+/calmodulin-dependent protein kinase inhibitor. The activity o the transcription factor activating transcription factor-2 (ATF-2), which binds to the cAMP responsive elements of the human insulin gene, was enhanced by Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). Mutagenesis studies showed that Thr69, Thr71, and Thr73 of ATF-2 are all required for activation by CaMKIV. CaMKIV- induced ATF-2 transcriptional activity was not altered by activation of c-Jun NH2-terminal protein kinase (JNK) or p38 mitogen-activated protein (MAP) kinase. Furthermore, when transfected into rat primary cultured islets, ATF-2 enhanced glucose-induced insulin promoter activity, whereas cAMP response element-binding protein (CREB) repressed it. These results suggest a mechanism in which ATF-2 regulates insulin gene expression in pancreatic β- cells, with the transcriptional activity of ATF2 being increased by an elevated concentration of calcium ions.

DOI： 10.2337/diabetes.49.7.1142

Activating transcription factor-2 is a positive regulator in CaM kinase IV - Induced human insulin gene expression

Ban N, Yamada Y, Someya Y, Ihara Y, Adachi T, Kubota A, Watanabe R, Kuroe A, Inada A, Miyawaki K, Sunaga Y, Shen ZP, Iwakura T, Tsukiyama K, Toyokuni S, Tsuda K, Seino Y

DIABETES 49 巻 ( 7 ) 頁： 1142 - 1148 2000年7月

Activating transcription factor-2 is a positive regulator in CaM kinase IV-induced human insulin gene expression. 国際誌

N Ban, Y Yamada, Y Someya, Y Ihara, T Adachi, A Kubota, R Watanabe, A Kuroe, A Inada, K Miyawaki, Y Sunaga, Z P Shen, T Iwakura, K Tsukiyama, S Toyokuni, K Tsuda, Y Seino

Diabetes 49 巻 ( 7 ) 頁： 1142 - 8 2000年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Insulin plays a crucial role in the regulation of glucose-homeostasis, and its synthesis is regulated by several stimuli. The transcription of the human insulin gene, enhanced by an elevated intracellular concentration of calcium ions, was completely blocked by Ca2+/calmodulin-dependent protein kinase inhibitor. The activity of the transcription factor activating transcription factor-2 (ATF-2), which binds to the cAMP responsive elements of the human insulin gene, was enhanced by Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). Mutagenesis studies showed that Thr69, Thr71, and Thr73 of ATF-2 are all required for activation by CaMKIV. CaMKIV-induced ATF-2 transcriptional activity was not altered by activation of cJun NH2-terminal protein kinase (JNK) or p38 mitogen-activated protein (MAP) kinase. Furthermore, when transfected into rat primary cultured islets, ATF-2 enhanced glucose-induced insulin promoter activity, whereas cAMP response element-binding protein (CREB) repressed it. These results suggest a mechanism in which ATF-2 regulates insulin gene expression in pancreatic beta-cells, with the transcriptional activity of ATF-2 being increased by an elevated concentration of calcium ions.

Detection of lipofuscin-like fluorophore in oxidized human low-density lipoprotein. 4-Hydroxy-2-nonenal as a potential source of fluorescent chromophore

Heat-shock preconditioning reduces oxidative protein denaturation and ameliorates liver injury by carbon tetrachloride in rats 査読有り国際誌

Yamamoto H, Yamamoto Y, Yamagami K, Kume M, Kimoto S, Toyokuni S, Uchida K, Fukumoto M, Yamaoka Y

RESEARCH IN EXPERIMENTAL MEDICINE 199 巻 ( 6 ) 頁： 309 - 318 2000年6月

Expression of stress-response and cell proliferation genes in renal cell carcinoma induced by oxidative stress

Tanaka T, Kondo S, Iwasa Y, Hiai H, Toyokuni S

AMERICAN JOURNAL OF PATHOLOGY 156 巻 ( 6 ) 頁： 2149 - 2157 2000年6月

Heat-shock preconditioning reduces oxidative protein denaturation and ameliorates liver injury by carbon tetrachloride in rats 査読有り国際誌

Yamamoto H, Yamamoto Y, Yamagami K, Kume M, Kimoto S, Toyokuni S, Uchida K, Fukumoto M, Yamaoka Y

RESEARCH IN EXPERIMENTAL MEDICINE 199 巻 ( 6 ) 頁： 309 - 318 2000年6月

Expression of stress-response and cell proliferation genes in renal cell carcinoma induced by oxidative stress

Tanaka T, Kondo S, Iwasa Y, Hiai H, Toyokuni S

AMERICAN JOURNAL OF PATHOLOGY 156 巻 ( 6 ) 頁： 2149 - 2157 2000年6月

Effects of geranyl-geranyl-acetone administration before heat shock preconditioning for conferring tolerance against ischemia-reperfusion injury in rat livers

Yamagami K, Yamamoto Y, Ishikawa Y, Yonezawa K, Toyokuni S, Yamaoka Y

JOURNAL OF LABORATORY AND CLINICAL MEDICINE 135 巻 ( 6 ) 頁： 465 - 475 2000年6月

Itakura K., Oya-Ito T., Osawa T., Yamada S., Toyokuni S., Shibata N., Kobayashi M., Uchida K.

FEBS Letters 473 巻 ( 2 ) 頁： 249 - 253 2000年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：FEBS Letters

It has recently been shown that the lipid peroxidation product 4- hydroxy-2-nonenal (HNE) forms a fluorescent hydroxyiminodihydropyrrole derivative with the ε-amino group of lysine residue. In this study, we raised a monoclonal antibody (mAb2C12) directed to the fluorophore-protein conjugate and found that the antibody was specific to the chromophore structure of the compound. Immunohistochemical analysis of atherosclerotic lesions from the human aorta showed that the fluorophore was indeed present in the lesions, in which intense positivity was primarily associated with macrophage-derived foam cells and thickening of the neointima of the arterial walls. Antigenic materials were also detected in the oxidatively modified low-density lipoprotein (LDL) with Cu2+ and in the oxidatively modified bovine serum albumin with an iron/linoleic acid autoxidation system, indicating that the HNE, which originated from the peroxidation of polyunsaturated fatty acids, could be a potential source of the fluorescent chromophore in oxidized LDL. (C) 2000 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(00)01539-8

Detection of lipofuscin-like fluorophore in oxidized human low-density lipoprotein - 4-Hydroxy-2-nonenal as a potential source of fluorescent chromophore

Itakura K, Oya-Ito T, Osawa T, Yamada S, Toyokuni S, Shibata N, Kobayashi M, Uchida K

FEBS LETTERS 473 巻 ( 2 ) 頁： 249 - 253 2000年5月

Antioxidant alpha-tocopherol ameliorates glycemic control of GK rats, a model of type 2 diabetes. 査読有り国際誌

Ihara Y, Yamada Y, Toyokuni S, Miyawaki K, Ban N, Adachi T, Kuroe A, Iwakura T, Kubota A, Hiai H, Seino Y

FEBS letters 473 巻 ( 1 ) 頁： 24 - 6 2000年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FEBS Letters

We have shown recently that oxidative stress by chronic hyperglycemia damages the pancreatic β-cells of GK rats, a model of non-obese type 2 diabetes, which may worsen diabetic condition and suggested the administration of antioxidants as a supportive therapy. To determine if natural antioxidant α-tocopherol (vitamin E) has beneficial effects on the glycemic control of type 2 diabetes, GK rats were fed a diet containing 0, 20 or 500 mg/kg diet α-tocopherol. Intraperitoneal glucose tolerance test revealed a significant increment of insulin secretion at 30 min and a significant decrement of blood glucose levels at 30 and 120 min after glucose loading in the GK rats fed with high α-tocopherol diet. The levels of glycated hemoglobin A1c, an indicator of glycemic control, were also reduced. Vitamin E supplementation clearly ameliorated diabetic control of GK rats, suggesting the importance of not only dietary supplementation of natural antioxidants but also other antioxidative intervention as a supportive therapy of type 2 diabetic patients. Copyright (C) 2000 Federation of European Biochemical Societies.

DOI： 10.1016/s0014-5793(00)01489-7

Antioxidant α-tocopherol improves glycemic control of GK rats, a model of type 2 diabetes

Ihara, Y; Yamada, Y; Toyokuni, S; Ban, N; Kuroe, A; Seino, Y

DIABETES 49 巻頁： A429 - A429 2000年5月

Upregulation of thioredoxin (TRX) expression in giant cell myocarditis in rats. 査読有り国際誌 Open Access

Shioji K, Kishimoto C, Nakamura H, Toyokuni S, Nakayama Y, Yodoi J, Sasayama S

FEBS letters 472 巻 ( 1 ) 頁： 109 - 13 2000年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FEBS Letters

To examine the possible involvement of a redox regulating mechanism in the pathogenesis of immune-mediated myocarditis, myocarditis was induced by immunization of porcine cardiac myosin in rats and immunohistochemistry and Western blot for thioredoxin (TRX) were performed. Immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor kappa-B (NF-κB) was also performed. TRX was upregulated in the acute stage, but not in the chronic stage, and the expression was correlated with the severity of the disease. Damaged myocytes were strongly immunostained for 8-OHdG and NF-κB. Thus, TRX may be specifically induced by acute inflammatory stimuli, and the development of acute immune-mediated myocarditis may be regulated by the cellular redox state via TRX. Copyright (C) 2000 Federation of European Biochemical Societies.

DOI： 10.1016/s0014-5793(00)01446-0

Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C 国際誌

Upregulation of thioredoxin (TRX) expression in giant cell myocarditis in rats

Shioji K, Kishimoto C, Nakamura H, Toyokuni S, Nakayama Y, Yodoi J, Sasayama S

FEBS LETTERS 472 巻 ( 1 ) 頁： 109 - 113 2000年4月

Kageyama, F; Kobayashi, Y; Kawasaki, T; Toyokuni, S; Uchida, K; Nakamura, H

AMERICAN JOURNAL OF GASTROENTEROLOGY 95 巻 ( 4 ) 頁： 1041 - 1050 2000年4月

Overexpression of the hOGG1 gene and high 8-hydroxy-2 '-deoxyguanosine (8-OHdG) lyase activity in human colorectal carcinoma: Regulation mechanism of the 8-OHdG level in DNA

Kondo S, Toyokuni S, Tanaka T, Hiai H, Onodera H, Kasai H, Imamura M

CLINICAL CANCER RESEARCH 6 巻 ( 4 ) 頁： 1394 - 1400 2000年4月

Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C 査読有り国際誌 Open Access

Kageyama F., Kobayashi Y., Kawasaki T., Toyokuni S., Uchida K., Nakamura H.

American Journal of Gastroenterology 95 巻 ( 4 ) 頁： 1041 - 1050 2000年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Gastroenterology

OBJECTIVES: Hepatic iron deposition has been reported in chronic hepatitis C (CH-C), and iron-induced lipid peroxidation may be involved in the pathogenesis of CH-C. The aims of the present study were: 1) to determine whether patients with CH-C have evidence of enhanced hepatic lipid peroxidation and to evaluate its relation to iron status, compared with that in patients with chronic hepatitis B (CH-B); and 2) to assess the effect of interferon (IFN) therapy on hepatic iron and lipid peroxidation. METHODS: In the liver biopsies of 40 patients with CH-C and 26 patients with CH-B, immunohistochemical detection of 4-hydroxy-2-nonenal (HNE)-protein adducts for evaluation of lipid peroxidation was performed, and hepatic iron status was biochemically and histologically assessed. In 16 CH-C patients with normal serum transaminases and undetectable serum HCV-RNA >6 months after the end of IFN treatment (responders) and in 11 nonresponders, hepatic HNE- protein adducts and siderosis were evaluated in pre- and posttreatment liver biopsies. RESULTS: Hepatocytic HNE-protein adducts and iron deposits were more abundant in the patients with CH-C than in those with CH-B. No correlation was found between the levels of hepatocytic HNE-protein adducts and hepatic iron status in either of the two groups. In the responders to IFN treatment for CH-C, hepatocytic HNE-protein adducts disappeared or attenuated with improvement of hepatic siderosis after the treatment, whereas IFN treatment did not improve hepatocytic expression of HNE-protein adducts and hepatic siderosis in the nonresponders. CONCLUSIONS: Patients with CH-C have evidence of enhanced hepatic iron accumulation and lipid peroxidation compared to those with CH-B. In CH-C, hepatic siderosis and lipid peroxidation are improved with successful IFN treatment. These results suggest that hepatic lipid peroxidation and iron may potentially play contributory roles in the pathogenesis of CH-C. (C) 2000 by Am. Coll. of Gastroenterology.

DOI： 10.1016/S0002-9270(00)00771-1

Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C 国際誌

Kageyama, F, Kobayashi, Y, Kawasaki, T, Toyokuni, S, Uchida, K, Nakamura, H

AMERICAN JOURNAL OF GASTROENTEROLOGY 95 巻 ( 4 ) 頁： 1041 - 1050 2000年4月

Overexpression of the hOGG1 gene and high 8-hydroxy-2'-deoxyguanosine (8-OHdG) lyase activity in human colorectal carcinoma: Regulation mechanism of the 8-OHdG level in DNA 査読有り国際誌

Kondo S., Toyokuni S., Tanaka T., Hiai H., Onodera H., Kasai H., Imamura M.

Clinical Cancer Research 6 巻 ( 4 ) 頁： 1394 - 1400 2000年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Clinical Cancer Research

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidatively modified lesions in DNA. Our previous study (Kondo et al., Free Radic. Biol. Med., 27: 401-410, 1999) revealed that human colorectal carcinoma cells are oxidatively stressed based on 8-OHdG determination. To elucidate 8-OHdG metabolism and its clinical significance in colorectal carcinoma, we studied the 8-OHdG repair system in DNA by measuring specific lyase activity and hOGG1 expression using quantitative-competitive reverse transcription-PCR. In addition, we searched for the presence of mutations and single nucleotide polymorphisms of the hOGG1 gene by single-strand conformational polymorphism and sequencing analyses. It was found that 8- OHdG-specific lyase activity and hOGG1 expression were significantly up- regulated in carcinoma, and a proportional association between 8-OHdG levels and either 8-OHdG lyase activity (r = 0.641, P < 0.05) or hOGG1 expression (r = 0.702, P < 0.05) was present. Whereas no difference was detected in the 8- OHdG level between early- and advanced-stage cancer, lyase activity (1.2- fold) and hOGG1 expression (1.6-fold) were significantly increased in advanced-stage cancer. No mutation was found in the 25 tumors examined. Three kinds of single nucleotide polymorphism were observed, including that of codon 326 (Ser/Cys) in exon 7. However, there was no correlation between any of the three polymorphic patterns and either 8-OHdG level or lyase activity. These results suggest that increased 8-OHdG levels in colorectal carcinoma are attributed to increased formation and are maintained by induced 8-OHdG repair activity at appropriate high levels. Our results may offer a unique approach in the development of preventive and therapeutic interventions as well as new insights into the pathogenesis of colorectal carcinoma.

Formation of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal- modified proteins in rat liver after ischemia-reperfusion: Distinct localization of the two oxidatively modified products

Overexpression of the hOGG1 gene and high 8-hydroxy-2 '-deoxyguanosine (8-OHdG) lyase activity in human colorectal carcinoma: Regulation mechanism of the 8-OHdG level in DNA

Kondo S, Toyokuni S, Tanaka T, Hiai H, Onodera H, Kasai H, Imamura M

CLINICAL CANCER RESEARCH 6 巻 ( 4 ) 頁： 1394 - 1400 2000年4月

Overexpression of the hOGG1 gene and high 8-hydroxy-2'-deoxyguanosine (8-OHdG) lyase activity in human colorectal carcinoma: regulation mechanism of the 8-OHdG level in DNA.

Kondo S, Toyokuni S, Tanaka T, Hiai H, Onodera H, Kasai H, Imamura M

Clinical cancer research : an official journal of the American Association for Cancer Research 6 巻 ( 4 ) 頁： 1394 - 400 2000年4月

Cytoprotective effects of nitroglycerin in ischemia-reperfusion-induced lung injury

Kawashima M, Bando T, Nakamura T, Isowa N, Liu MY, Toyokuni S, Hitomi S, Wada H

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 161 巻 ( 3 ) 頁： 935 - 943 2000年3月

Yamagami K., Yamamoto Y., Kume M., Ishikawa Y., Yamaoka Y., Hiai H., Toyokuni S.

Antioxidants and Redox Signaling 2 巻 ( 1 ) 頁： 127 - 136 2000年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Antioxidants and Redox Signaling

Ischemia-reperfusion (IR) injury is an intractable process associated not only with therapeutic recanalization of vessels, but also with partial resection or transplantation of solid organs including liver. To develop methods for predicting the degree of hepatic IR injury and further to identify injured cells, we studied the formation of 8-hydroxy-2'-deoxy- guanosine (8-OHdG) and 4-hydroxy-2-nonenal (HNE)-modified proteins in the normothermic hepatic IR model of rats using immunohistochemistry, high- performance liquid chromatography (HPLC) determination and Western blot. The Pringle maneuver for either 15 or 30 min duration produced reversible or lethal damage, respectively. The levels of both products were significantly increased in proportion to ischemia duration 40 min after reperfusion, suggesting the involvement of hydroxyl radicals. Increased immunoreactivity of 8-OHdG was observed not only in the nuclei of hepatocytes but also in those of bile canalicular and endothelial cells. However, immunoreactivity of HNE-modified proteins was detected in the cytoplasm of hepatocytes, which was confirmed by Western blot, and in addition, in the nuclei of hepatocytes after severe injury. Thus, localization of the two oxidatively modified products was not identical. Our data suggest that these two products could be used for the assessment of hepatic IR injury in tissue, but that the biological significance of the two products might be different.

DOI： 10.1089/ars.2000.2.1-127

Serum 4-Hydroxy-2-Nonenal-Modified Albumin Is Elevated in Patients with Type 2 Diabetes Mellitus 査読有り国際誌

Toyokuni Shinya, Yamada Satoshi, Kashima Minoru, Ihara Yu, Yamada Yuichiro, Tanaka Tomoyuki, Hiai Hiroshi, Seino Yutaka, Uchida Koji

ANTIOXIDANTS & REDOX SIGNALING 2 巻 ( 4 ) 頁： 681 - U65 2000年

Formation of 8-Hydroxy-2 '-Deoxyguanosine and 4-Hydroxy-2-Nonenal-Modified Proteins in Rat Liver after Ischemia-Reperfusion: Distinct Localization of the Two Oxidatively Modified Products

Yamagami Kazuhiko, Yamamoto Yuzo, Kume Makoto, Ishikawa Yasuhide, Yamaoka Yoshio, Hiai Hiroshi, Toyokuni Shinya

ANTIOXIDANTS & REDOX SIGNALING 2 巻 ( 1 ) 頁： 127 - 136 2000年

Serum 4-hydroxy-2-nonenal-modified albumin is elevated in patients with type 2 diabetes mellitus 査読有り国際誌

Toyokuni S., Yamada S., Kashima M., Ihara Y., Yamada Y., Tanaka T., Hiai H., Seino Y., Uchida K.

Antioxidants and Redox Signaling 2 巻 ( 4 ) 頁： 681 - 685 2000年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Antioxidants and Redox Signaling

4-Hydroxy-2-nonenal (HNE) is one of the major lipid peroxidation products with cytotoxic and mutagenic activity. It further reacts with protein residues such as histidine to generate stable Michael adducts. To evaluate the status of oxidative stress in the serum of type 2 diabetes mellitus, we constructed a sandwich enzyme-linked immunosorbent assay to measure serum HNE-modified albumin by the use of a specific monoclonal antibody (HNEJ-2) against HNE-histidine adducts as well as an antibody against human serum albumin. Serum of type 2 diabetes outpatients revealed significantly higher levels of HNE-modified albumin (736.1 ± 34.2 pmol/ml, n = 54) than the matched nondiabetics (611.4 ± 39.1 pmol/ml, n = 30; means ± SEM; p = 0.018). However, no significant correlation was observed in diabetic outpatients between the levels of HNE-modified albumin and clinical parameters such as fasted blood glucose, HbA1c, diabetes duration, or complications. Our data demonstrated the increased formation of serum HNE-modified albumin in type 2 diabetic outpatients in the milieu between liver and vascular lumina, indicating the presence of oxidative stress.

DOI： 10.1089/ars.2000.2.4-681

Expression of stress-response and cell proliferation genes in renal cell carcinoma induced by oxidative stress

Tanaka T., Kondo S., Iwasa Y., Hiai H., Toyokuni S.

American Journal of Pathology 156 巻 ( 6 ) 頁： 2149 - 2157 2000年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Pathology

Ferric nitrilotriacetate induces oxidative damage in renal proximal tubules that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. In search of genes specifically involved in oxystress-induced carcinogenesis, we have applied a modified fluorescent differential display technique to the tumors and an established cell line as well as their non- neoplastic counterparts. We screened approximately 84,000 products. Reverse Northern blotting confirmed differential expression of 20 transcripts, which showed either significant increase, decrease or lack of expression in the RCCs. Five cDNA clones encoded novel products of unknown function. Fifteen cDNA clones were identified by homology search, which included annexin II, Y- box binding protein, ribosomal proteins, heat shock proteins, DNA polymerase, nonmuscle caldesmon (increased); protein tyrosine phosphatase (decreased); selenoprotein P, stromal cell-derived factor 1, intestinal trefoil protein, nicotinamide adenine dinucleotide, reduced form (NADH) dehydrogenase, and insulin-like growth factor binding protein 7 (deleted). Most of the identified genes were associated with stress-response or cellular proliferation. These results suggest that multiple, interactive genetic pathways are involved in carcinogenesis induced by oxidative stress.

DOI： 10.1016/S0002-9440(10)65085-7

Effects of geranyl-geranyl-acetone administration before heat shock preconditioning for conferring tolerance against ischemia-reperfusion injury in rat livers

Yamagami K., Yamamoto Y., Ishikawa Y., Yonezawa K., Toyokuni S., Yamaoka Y.

Journal of Laboratory and Clinical Medicine 135 巻 ( 6 ) 頁： 465 - 475 2000年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Laboratory and Clinical Medicine

The effect of geranyl-geranyl-acetone (GGA) administration before heat shock preconditioning on heat shock protein (HSP) 72 induction and on the acquisition of tolerance against ischemia-reperfusion injury was studied in rat livers. Male Wistar rats were divided into four groups: a control group (group C); a GGA group (group G); a simple heat shock group (group VH); and a heat shock with GGA premedication group (group GH). Five-, 10-, and 15-minute periods of heat shock preconditioning at 42°C were performed in groups VH and GH. Subgroups were determined according to the period of heat shock exposure. After a 48-hour recovery, rats in groups C, VH5, VH15, and GH5 received a 30-minute period of hepatic ischemia. Induction of HSP72, survival rates, and changes in biochemical and histologic parameters were compared among the groups. Five-minute heat shock preconditioning was not enough to induce HSP72. However, livers in group GH5 expressed approximately the same amount of HSP72 as those in group VH15. The expression of HSP72 in group GH15 was stronger than that found in group VH15. The degree and location of HSP72 expression were not different between groups GH5 and VH15. Seven-day survival was significantly better in groups GH5 (16/16) and VH15 (15/16) than in group C (8/16) or VH5 (9/16). The recovery of adenosine triphosphate in liver tissue was faster, and the release of liver-related enzymes during reperfusion was lower in groups GH5 and VH15 than in group C or VH5. Administration of GGA before heat shock preconditioning augmented the induction of HSP72 by decreasing the threshold for triggering the stress response.

DOI： 10.1067/mlc.2000.106806

Cytoprotective effects of nitroglycerin in ischemia-reperfusion-induced lung injury

Kawashima M., Bando T., Nakamura T., Isowa N., Liu M., Toyokuni S., Hitomi S., Wada H.

American Journal of Respiratory and Critical Care Medicine 161 巻 ( 3 I ) 頁： 935 - 943 2000年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Respiratory and Critical Care Medicine

Prevention of ischemia-reperfusion (IR) injury is crucial for successful lung transplantation. We investigated whether a nitric oxide donor, nitroglycerin (NTG), could suppress the oxidative stress of IR injury and improve pulmonary function after reperfusion in an ex vivo rat lung perfusion model. In Fresh group of animals, the lungs were flushed with perfusate, followed immediately by reperfusion, and no lung injury was observed. In NTG- and NTG+ groups of animals, the lungs were flushed with perfusate alone or perfusate containing NTG, respectively. Harvested lung and heart blocks from these latter two groups were immersed in the corresponding perfusate at 4°C for 15 h, and were then reperfused for 60 min. Reperfusion induced pulmonary edema in the NTG- group, but not in the NTG + group. Shunt fractions in NTG+ group were significantly lower than in the NTG- group throughout reperfusion. NTG had no effect on pulmonary arterial pressure or myeloperoxidase activity. In contrast, oxidative DNA damage assessed immunohistochemically with a monoclonal antibody against 8-hydroxy-2'-deoxyguanosine (8-OHdG) was significantly increased in the NTG- group, in the order alveolar epithelium > pulmonary endothelium > bronchial epithelium. NTG treatment significantly decreased staining with the anti-8-OHdG antibody in all three areas Of tissue. Therefore, administration of NTG attenuates the oxidative stress of IR injury, and may improve pulmonary function after reperfusion.

DOI： 10.1164/ajrccm.161.3.9905003

Fine-needle aspiration cytology of malignant hemangiopericytoma of the salivary gland: A case report

Shimizu K., Ogura S., Kobayashi T.K., Kushima R., Toyokuni S., Iwasa Y., Sakurai M.

Diagnostic Cytopathology 21 巻 ( 6 ) 頁： 398 - 401 1999年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Diagnostic Cytopathology

A 79-yr-old woman presented with a 5-yr history of swelling of the left cheek. The fine-needle aspiration (FNA) smear showed a spindle-cell neoplasm with capillaries and benign endothelial cells. The spindle cells possessed pleomorphic, hyperchromatic elongated nuclei and a moderate amount of ill- defined cytoplasm. They also showed papillary arcades surrounded and encased by relatively small ovoid to short spindle cells. Subsequent surgical excision confirmed the presence of malignant hemangiopericytoma (HP). Immunohistochemical studies on the histologic section using vimentin were strongly positive, consistent with HP. To the best of our knowledge, this is the second published report of FNA cellular features of malignant HP of the salivary gland. Besides delineating the FNA cellular features of HP of the salivary gland, the present case illustrates the value of using immunohistochemical approaches.

DOI： 10.1002/(SICI)1097-0339(199912)21:6<398::AID-DC6>3.0.CO;2-V

Fine-needle aspiration cytology of malignant hemangiopericytoma of the salivary gland: A case report

Shimizu K, Ogura S, Kobayashi TK, Kushima R, Toyokuni S, Iwasa Y, Sakurai M

DIAGNOSTIC CYTOPATHOLOGY 21 巻 ( 6 ) 頁： 398 - 401 1999年12月

Fine-needle aspiration cytology of malignant hemangiopericytoma of the salivary gland: A case report 査読有り

K Shimizu, S Ogura, TK Kobayashi, R Kushima, S Toyokuni, Y Iwasa, M Sakurai

DIAGNOSTIC CYTOPATHOLOGY 21 巻 ( 6 ) 頁： 398 - 401 1999年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

A 79-yr-old woman presented with a 5-yr history of swelling of the left cheek. The fine-needle aspiration (FNA) smear show ed a spindle-cell neoplasm with capillaries and benign endothelial cells. The spindle cells possessed pleomorphic, hyperchromatic elongated nuclei and a moderate amount of ill-defined cytoplasm. They also showed papillary arcades surrounded and encased by relatively: small ovoid to short spindle cells. Subsequent surgical excision confirmed the presence of malignant hemangiopericytoma (HP). Immunohistochemical studies on the histologic section casing vimentin were strongly positive, consistent with HP To the best of our knowledge, this is the second published report of FNA cellular features of malignant HP of the salivary gland. Besides delineating the FNA cellular of HP of the salivary gland the present case illustrates the value of using immunohistochemical approaches.

Heat-shock preconditioning reduces oxidative protein denaturation and ameliorates liver injury by carbon tetrachloride in rats

Yamamoto H., Yamamoto Y., Yamagami K., Kume M., Kimoto S., Toyokuni S., Uchida K., Fukumoto M., Yamaoka Y.

Research in Experimental Medicine 199 巻 ( 6 ) 頁： 309 - 318 1999年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Research in Experimental Medicine

Membrane lipids and cytosolic proteins are major targets of oxidative injury. This study examined the effect of heat-shock preconditioning associated with the induction of heat-shock protein 72 on liver injury, from the aspect of lipid peroxidation and protein denaturation after carbon tetrachloride (CCl4) administration in rats - one of the representative oxidative injuries. Male Wistar rats were divided into two groups, group HS (preconditioned by heat exposure) and group C (not preconditioned). Expression of HSP72 in the liver tissue was confirmed by Western blot analysis. After a 48-h recovery period, all rats were given CCl4 intragastrically. Liver damage was assessed by measuring serum liver-related enzyme levels and adenine nucleotide concentration in the liver tissue. Lipid peroxidation and protein denaturation were evaluated by measuring tiobarbituric acid reactive substances (TBARS) and by immunohistochemical staining of 4-hydroxy-2-nonenal(HNE)-modified proteins in the liver. Survival rates of the rats after CCl4 administration were also compared. Expression of HSP72 was clearly detected in group HS, but not in group C. Heat-shock preconditioning significantly improved the survival rate, suppressed the increase in liver-related enzyme levels and maintained adenosine triphosphate levels (P < 0.01 each). HNE-modified proteins - denatured proteins by free radical attack - were significantly less stained in group HS than in group C (P < 0.05). However, TBARS levels did not differ between groups. Because heat-shock preconditioning did not alter TBARS levels but reduced HNE-modified proteins in association with the expression of HSP72, it is suggested that HSP72 did not prevent lipid peroxidation but decreased the lipid peroxidation-induced denaturation of proteins. This seemed to be a mechanism of heat-shock preconditioning to ameliorate oxidative liver injury.

DOI： 10.1007/s004339900040

P-58 Ontogenesis of Anti-Oxidative Enzymes and Effects of Oxygen Stress on Developing Mouse Embryos.

Enhanced hepatic lipid peroxidation in patients with primary biliary cirrhosis. 国際誌

Kawamura, K; Kageyama, F; Kobayashi, Y; Kawasaki, T; Toyokuni, S; Uchida, K

HEPATOLOGY 30 巻 ( 4 ) 頁： 543A - 543A 1999年10月

Enhanced hepatic lipid peroxidation in patients with primary biliary cirrhosis. 国際誌

Kawamura, K, Kageyama, F, Kobayashi, Y, Kawasaki, T, Toyokuni, S, Uchida, K

HEPATOLOGY 30 巻 ( 4 ) 頁： 543A - 543A 1999年10月

KOBAYASHI M., NAKAMURA H., YODOI J., TOYOKUNI S., SHIOTA K.

Congenital anomalies 39 巻 ( 3 ) 頁： 175 1999年9月

P-58 Ontogenesis of Anti-Oxidative Enzymes and Effects of Oxygen Stress on Developing Mouse Embryos.

KOBAYASHI M, NAKAMURA H, YODOI J, TOYOKUNI S, SHIOTA K

Congenital anomalies 39 巻 ( 3 ) 1999年9月

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2'-deoxyguanosine

Toyokuni S., Sagripanti J.

Free Radical Research 31 巻 ( 2 ) 頁： 123 - 128 1999年9月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Research

The interaction of chelators and reducing agents is of particular importance in understanding iron-associated pathology since catalytic iron undergoes cyclic reduction and oxidation in vivo. Therefore, we treated plasmid DNA with free or chelated Fe(III) in the presence of biological reductants, and simultaneously measured the number of single strand breaks (SSBs) and oxidative base modification (8-hydroxy-2'-deoxyguanosine; 8-OHdG) by quantitative gel electrophoresis and HPLC with electrochemical detection, respectively. Production of SSBs and 8-OHdG was linearly correlated suggesting that these true different lesions share a common chemical mechanism. The levels of both lesions were enhanced when Fe(III) was chelated to citrate or nitrilotriacetic acid. Reducing agents showed different potency in inducing DNA damage catalyzed by chelated iron (L-ascorbate > L-cysteine > H2O2). Chelation increased SSB formation by ~ 8-fold and 8-OHdG production by ~ 4-fold. The ratio of SSB/8-OHdG catalyzed by chelated iron, which is twice as high as by unchelated iron, indicates that chelation affects iron-catalyzed oxidative DNA damage in a specific way favoring strand breakage over base modification. Since iron is mostly chelated in biological systems, the production of genomic and mitochondrial DNA damage, particularly strand breaks, in diseases involving iron overload is likely to be higher than previously predicted from studies using unchelated iron.

DOI： 10.1080/10715769900301631

Localization of hydroxynonenal protein adducts in normal human kidney and selected human kidney cancers

Oberley TD, Toyokuni S, Szweda LI

FREE RADICAL BIOLOGY AND MEDICINE 27 巻 ( 5-6 ) 頁： 695 - 703 1999年9月

Localization of hydroxynonenal protein adducts in normal human kidney and selected human kidney cancers 査読有り

TD Oberley, S Toyokuni, LI Szweda

FREE RADICAL BIOLOGY AND MEDICINE 27 巻 ( 5-6 ) 頁： 695 - 703 1999年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

Both polyclonal and monoclonal antibodies to 4-hydroxy-2-nonenal (HNE) protein adducts were used to identify lipid peroxidation products in normal human kidney and in selected human kidney cancers using immunoperoxidase techniques at the light microscopic level and immunogold techniques at the ultrastructural level. HNE protein adducts were detected in most cell types in normal kidney, although in highly variable amounts. All six morphologic types of renal tumors examined showed some staining with antibodies to HNE protein adducts, although the intensity of staining Varied considerably depending on tumor type. Renal oncocytoma and the granular cell variant of renal adenocarcinoma both showed greater cytoplasmic staining for HNE protein adducts than the other tumors examined; these tumors both contain high numbers of mitochondria and suggest that mitochondria are a major source of lipid peroxidation products. To test this possibility, immunogold ultrastructural analysis was performed. HNE protein adducts were identified in nuclei and mitochondria in both normal proximal tubule and three types of renal carcinoma examined; these results localize oxidative damage at the subcellular level in both benign and malignant epithelium to nuclei and mitochondria. In conclusion, HNE protein adducts occur in kidneys in both normal and tumor cells, although immunomorphologic analyses suggest less HNE protein adducts in tumor cells. (C) 1999 Elsevier Science Inc.

Association between M/L55-polymorphism of paraoxonase enzyme and oxidative DNA damage in patients with type 2 diabetes mellitus and in control subjects

Malin R., Rantalaiho V., Huang X.H., Wirta O., Pasternack A., Leinonen J.S., Alho H., Jokela H., Koivula T., Tanaka T., Okada K., Ochi H., Toyokuni S., Lehtimäki T.

Human Genetics 105 巻 ( 1-2 ) 頁： 179 - 180 1999年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Human Genetics

The paraoxonase enzyme (PON) gene polymorphism causes a change of methionine (M-allele) to leucine (L-allele). PON may reduce low density lipoprotein oxidation and prevent atherosclerosis. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a sensitive index of oxidative DNA damage. We have studied the association between the PON genotypes and the urinary excretion of 8-OHdG. The study population consisted of 93 Finnish type 2 diabetes patients and 106 non-diabetic control subjects. The 24-h excretion of 8-OHdG was significantly higher in diabetic patients than in control subjects (P < 0.001). In control subjects, the ratio of the 8-OHdG/glomerular filtration rate increased in order of genotype from MM to ML to LL (P < 0.0412). These results suggest that lipid peroxidation may have an effect on DNA oxidation.

DOI： 10.1007/s004399900074

4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules

4-hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress - Identification of proteasomes as target molecules 査読有り国際誌

Okada K, Wangpoengtrakul C, Osawa T, Toyokuni S, Tanaka K, Uchida K

JOURNAL OF BIOLOGICAL CHEMISTRY 274 巻 ( 34 ) 頁： 23787 - 23793 1999年8月

Okada K., Wangpoengtrakul C., Osawa T., Toyokuni S., Tanaka K., Uchida K.

Journal of Biological Chemistry 274 巻 ( 34 ) 頁： 23787 - 23793 1999年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for these effects because of their ability to damage cellular components such as membranes, proteins, and DNA. In the present study, we provide evidence that oxidative stress causes a transient impairment of intracellular proteolysis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment with the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress, as monitored by accumulation of lipid peroxidation products and 8-hydroxy-2'- deoxyguanosine, in the kidney of mice. In addition, transient accumulation of HNE-modified proteins in the kidney was also found by competitive enzyme- linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitinated suggested a crucial role of proteasomes in the metabolism of HNE-modified proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent elimination of HNE-modified proteins. We found that, among three proteolytic activities (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase activities in the kidney were transiently diminished in accordance with the accumulation of HNE-modified proteins during oxidative stress. The loss of proteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an immunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impairment of ubiquitin/proteasome-dependent intracellular proteolysis.

DOI： 10.1074/jbc.274.34.23787

Persistent oxidative stress in human colorectal carcinoma, but not in adenoma

Persistent oxidative stress in human colorectal carcinoma, but not in adenoma

Kondo S, Toyokuni S, Iwasa Y, Tanaka T, Onodera H, Hiai H, Imamura M

FREE RADICAL BIOLOGY AND MEDICINE 27 巻 ( 3-4 ) 頁： 401 - 410 1999年8月

Kondo S., Toyokuni S., Iwasa Y., Tanaka T., Onodera H., Hiai H., Imamura M.

Free Radical Biology and Medicine 27 巻 ( 3-4 ) 頁： 401 - 410 1999年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Few studies have been conducted focusing on a potential role of reactive oxygen species in tumor cell metabolism. Here we studied human colorectal adenocarcinomas and adenomas to determine whether oxidative stress is imposed on cancer cells in vivo and used specific antibodies against 8-hydroxy-2'- deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE)-modified proteins, and 3- nitro-L-tyrosine (3-NT) to determine whether there is an association between oxidative stress and cellular proliferation. Higher levels of oxidative modifications in DNA and proteins were observed in carcinoma cells, but not in adenoma cells, than in the corresponding nontumorous epithelial cells by immunohistochemistry as well as high-performance liquid chromatography (HPLC)-based 8-OHdG determination. The fraction of proliferating cell nuclear antigen-positive cells was proportionally associated in adenocarcinomas with the staining intensities of 8-OHdG and 3-NT. Furthermore, Western blot analysis of the proteins extracted from carcinoma cells revealed several specific proteins modified by HNE or peroxynitrite. Thus we concluded that colorectal carcinoma, but not adenoma cells, are exposed to more oxidative stress than their corresponding nontumorous epithelial cells, regardless of clinical stage and histology, and further that the oxidative stress in carcinoma cells might stimulate cellular proliferation.

DOI： 10.1016/S0891-5849(99)00087-8

High incidence of allelic loss on chromosome 5 and inactivation of p15(INK4B) and p16(INK4A) tumor suppressor genes in oxystress-induced renal cell carcinoma of rats

The role of oxidative DNA damage in human arsenic carcinogenesis: Detection of 8-hydroxy-2 '-deoxyguanosine in arsenic-related Bowen's disease

Matsui M, Nishigori C, Toyokuni S, Takada J, Akaboshi M, Ishikawa M, Imamura S, Miyachi Y

JOURNAL OF INVESTIGATIVE DERMATOLOGY 113 巻 ( 1 ) 頁： 26 - 31 1999年7月

The role of oxidative DNA damage in human arsenic carcinogenesis: Detection of 8-hydroxy-2 '-deoxyguanosine in arsenic-related Bowen's disease

Matsui M, Nishigori C, Toyokuni S, Takada J, Akaboshi M, Ishikawa M, Imamura S, Miyachi Y

JOURNAL OF INVESTIGATIVE DERMATOLOGY 113 巻 ( 1 ) 頁： 26 - 31 1999年7月

High incidence of allelic loss on chromosome 5 and inactivation of p15(INK4B) and p16(INK4A) tumor suppressor genes in oxystress-induced renal cell carcinoma of rats

Tanaka T, Iwasa Y, Kondo S, Hiai H, Toyokuni S

ONCOGENE 18 巻 ( 25 ) 頁： 3793 - 3797 1999年6月

Tanaka T., Iwasa Y., Kondo S., Hiai H., Toyokuni S.

Oncogene 18 巻 ( 25 ) 頁： 3793 - 3797 1999年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Oncogene

Ferric nitrilotriacetate induces oxidative damage in renal proximal tubules, a consequence of Fenton-like reaction, that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. In order to find common genetic alterations in this oxystress-induced carcinogengesis model, RCCs were produced in F1 hybrid rats between Wistar and Long-Evans strains and genomes were screened for loss of heterozygosity (LOH) with microsatellite polymorphic markers by PCR. Five consecutive markers on chromosome 5 (D5Mgh5, D5Mit9, D5Mgh6, D5Mit11 and D5Mit6) showed LOH in ≥ 40% of the RCCs. As possible candidate tumor suppressor genes on chromosome 5, p15(INK4B) and p16(INK4A) were investigated for genetic alteration and aberrant methylation by Southern blot, PCR/SSCP/sequencing and methylation-specific PCR. Genetic alteration (homozygous or hemizygous deletion with or without point mutation) or aberrant methylation were found in 30.7 and 53.8% of the RCC cases, respectively, which was proportionally associated with the histological nuclear grade and metastatic activity. Our data suggest that inactivation of p15 and p16 genes could be one of the major pathways responsible for oxystress-induced carcinogenesis.

DOI： 10.1038/sj.onc.1202707

A 1-hour enzyme-linked immunosorbent assay for quantitation of acrolein- and hydroxynonenal-modified proteins by epitope-bound casein matrix method

High incidence of allelic loss on chromosome 5 and inactivation of p15(INK4B) and p16(INK4A) tumor suppressor genes in oxystress-induced renal cell carcinoma of rats

Tanaka T, Iwasa Y, Kondo S, Hiai H, Toyokuni S

ONCOGENE 18 巻 ( 25 ) 頁： 3793 - 3797 1999年6月

A 1-hour enzyme-linked immunosorbent assay for quantitation of acrolein- and hydroxynonenal-modified proteins by epitope-bound casein matrix method 査読有り国際誌

Satoh K, Yamada S, Koike Y, Igarashi Y, Toyokuni S, Kumano T, Takahata T, Hayakari M, Tsuchida S, Uchida K

ANALYTICAL BIOCHEMISTRY 270 巻 ( 2 ) 頁： 323 - 328 1999年6月

Satoh K., Yamada S., Koike Y., Igarashi Y., Toyokuni S., Kumano T., Takahata T., Hayakari M., Tsuchida S., Uchida K.

Analytical Biochemistry 270 巻 ( 2 ) 頁： 323 - 328 1999年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Analytical Biochemistry

A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for quantitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was developed. Microtiter plate wells were precoated and blocked simultaneously with epitope-bound bovine caseins as matrix proteins, and aldehyde-modified proteins were quantitated by a competition assay with a monoclonal antibody specific for acrolein-modified lysine or HNE-modified histidine epitopes. Minimal reaction times required for the coating/blocking; first monoclonal antibody and the peroxidase-conjugated second antibody binding steps were 3, 3, and 7 min, respectively, the former two steps being found to be or akin to diffusion-rate-limiting reactions. The convenient ELISA should find an application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may also be advantageous for the quantitation of various other biologically important haptenic molecules.

DOI： 10.1006/abio.1999.4073

Intranuclear distribution of 8-hydroxy-2'-deoxyguanosine: An immunocytochemical study

A 1-hour enzyme-linked immunosorbent assay for quantitation of acrolein- and hydroxynonenal-modified proteins by epitope-bound casein matrix method 査読有り国際誌

Satoh K, Yamada S, Koike Y, Igarashi Y, Toyokuni S, Kumano T, Takahata T, Hayakari M, Tsuchida S, Uchida K

ANALYTICAL BIOCHEMISTRY 270 巻 ( 2 ) 頁： 323 - 328 1999年6月

Intranuclear distribution of 8-hydroxy-2 '-deoxyguanosine: An immunocytochemical study

Toyokuni S, Iwasa Y, Kondo S, Tanaka T, Ochi H, Hiai H

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 47 巻 ( 6 ) 頁： 833 - 835 1999年6月

Toyokuni S., Iwasa Y., Kondo S., Tanaka T., Ochi H., Hiai H.

Journal of Histochemistry and Cytochemistry 47 巻 ( 6 ) 頁： 833 - 835 1999年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Histochemistry and Cytochemistry

We used immunofluorescence staining (monoclonal antibody N45.1) with cytological imprinting to study changes in the intranuclear distribution of 8-hydroxy-2'-deoxyguanosine in renal cells of male Wistar rats after oxidative stress by ferric nitrilotriacetate. In the control proximal tubule cells, small spherical signals were uniformly distributed throughout the nuclei. Under oxidative stress, immunofluorescence intensity was increased, especially near nuclear membrane. In cells with nuclear shrinkage or deformity, intense, diffuse signals throughout the nuclei were observed. Our results suggest that specific nuclear sites are vulnerable to oxidative DNA damage and that diffuse intense signals precede cell death after oxidative stress.

DOI： 10.1177/002215549904700613

Reactive oxygen species-induced molecular damage and its application in pathology

Intranuclear distribution of 8-hydroxy-2 '-deoxyguanosine: An immunocytochemical study

Toyokuni S, Iwasa Y, Kondo S, Tanaka T, Ochi H, Hiai H

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 47 巻 ( 6 ) 頁： 833 - 835 1999年6月

Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes 査読有り国際誌

Ihara Y, Toyokuni S, Uchida K, Odaka H, Tanaka T, Ikeda H, Hiai H, Seino Y, Yamada Y

DIABETES 48 巻 ( 4 ) 頁： 927 - 932 1999年4月

Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes 査読有り国際誌

Ihara Y, Toyokuni S, Uchida K, Odaka H, Tanaka T, Ikeda H, Hiai H, Seino Y, Yamada Y

DIABETES 48 巻 ( 4 ) 頁： 927 - 932 1999年4月

Toyokuni S.

Pathology International 49 巻 ( 2 ) 頁： 91 - 102 1999年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

Recent studies have clarified that reactive oxygen species (ROS) are involved in a diversity of biological phenomena including radiation damage, carcinogenesis, ischemia-reperfusion injury, diabetes mellitus and neurodegenerative diseases. The breakthrough of these fruitful accomplishments was the discovery of an enzyme, superoxide dismutase, by McCord and Fridovich in 1968. In the 1970s and 80s, biochemists and radiation biologists were attracted by the role of ROS in its irreversible damage to biological molecules. In the 1990s, ROS were further found to be a reversible modulator of protein structure as well, and this led to a recent rapid data accumulation on the association of ROS and transcription factors. At the same time, methods to localize ROS-induced damage in paraffin-embedded tissues have been established. This owes to a successful production of antibodies against covalently modified structures specific for ROS-induced damage. The epitopes include 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins. The present article reviews histochemical and immunohistochemical methods to localize ROS-induced damage in tissues and cells, further comments on the association of ROS with transcription factors, and shows a prospective view of ROS-induced carcinogenesis.

DOI： 10.1046/j.1440-1827.1999.00829.x

Protection of ischemia-reperfusion lung against oxidative stress by nitroglycerin.

Formation of 4-hydroxy-2-nonenal-modified proteins and 3-nitro-L-tyrosine in rat island skin flaps during and after ischemia 査読有り国際誌

Um SC, Suzuki S, Toyokuni S, Uchida K, Hiai H, Nishimura Y

ANNALS OF PLASTIC SURGERY 42 巻 ( 3 ) 頁： 293 - 298 1999年3月

Kawashima, M; Bando, T; Nakamura, T; Yoshimura, T; Hanaoka, N; Hirata, T; Fukuse, T; Hasegawa, S; Toyokuni, S; Hitomi, S; Wada, H

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 159 巻 ( 3 ) 頁： A543 - A543 1999年3月

Protection of ischemia-reperfusion lung against oxidative stress by nitroglycerin. 国際誌

Kawashima, M, Bando, T, Nakamura, T, Yoshimura, T, Hanaoka, N, Hirata, T, Fukuse, T, Hasegawa, S, Toyokuni, S, Hitomi, S, Wada, H

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 159 巻 ( 3 ) 頁： A543 - A543 1999年3月

Formation of 4-hydroxy-2-nonenal-modified proteins and 3-nitro-L- tyrosine in rat island skin flaps during and after ischemia 査読有り国際誌

Um S.C., Suzuki S., Toyokuni S., Uchida K., Hiai H., Nishimura Y.

Annals of Plastic Surgery 42 巻 ( 3 ) 頁： 293 - 298 1999年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Annals of Plastic Surgery

4-Hydroxy-2-nonenal (HNE)-modified proteins and 3-nitro-L-tyrosine were evaluated as a specific marker of reactive oxygen species (ROS)- and nitric oxide (NO)-mediated peroxynitrite-induced tissue injuries in ischemic and reperfused skin flap by Western blot analysis. Specimens were taken from island skin flaps of rats during the following three conditions: ischemia only, 5 hours of ischemia and reperfusion, and 10 hours of ischemia and reperfusion. HNE-modified proteins and 3-nitro-L-tyrosine increased with ischemic time (3, 6, and 10 hours postischemia). In the reperfused skin flap after both 5 and 10 hours of ischemia, HNE-modified proteins and 3-nitro-L- tyrosine were increased 3 hours postreperfusion, and they reached a maximum 6 hours after reperfusion. HNE-modified proteins and 3-nitro-L-tyrosine 1 hour postreperfusion were higher with 10 hours ischemia-reperfusion than with 5 hours ischemia-reperfusion. These results indicate (1) that ROS- and NO- induced peroxynitrite-mediated cytotoxicity in ischemic flaps is dependent on the ischemic period and (2) that ROS- and NO-induced peroxynitrite-mediated cytotoxicity occurs during an early stage of reperfusion if the ischemic period is long.

DOI： 10.1097/00000637-199903000-00011

Reactive oxygen species-induced molecular damage and its application in pathology

Toyokuni Shinya

Pathology international 49 巻 ( 2 ) 頁： 91 - 102 1999年2月

Reactive oxygen species-induced molecular damage and its application in pathology

Toyokuni S

PATHOLOGY INTERNATIONAL 49 巻 ( 2 ) 頁： 91 - 102 1999年2月

The 4-hydroxy-2-nonenal-derived fluorescent pigment 国際誌

Uchida, K; Itakura, K; Oya-Ito, T; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE 27 巻頁： S100 - S100 1999年

The malondialdehyde-derived fluorescent pigment 国際誌

Uchida, K; Sakamoto, S; Itakura, K; Kumazawa, S; Nakayama, T; Toyokuni, S; Osawa, T

FREE RADICAL BIOLOGY AND MEDICINE 27 巻頁： S100 - S100 1999年

Detection of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1 and its application

Toyokuni, S

ADVANCES IN DNA DAMAGE AND REPAIR 302 巻頁： 395 - 405 1999年

hOGG1 overexpression and high 8-Oxo-2'-deoxyguanosine lyase activity in human colorectal carcinoma

Toyokuni, S; Kondo, S; Tanaka, T; Hiai, H; Onodera, H; Imamura, M

FREE RADICAL BIOLOGY AND MEDICINE 27 巻頁： S149 - S149 1999年

Oxidative stress in the human stomach infected with Helicobacter pylori:: effect of bacterial eradication

Pignatelli, B; Li, CQ; Bancel, B; Toyokuni, S; Ohshima, H

2ND INTERNATIONAL CONGRESS ON GASTROENTEROLOGICAL CARCINOGENESIS 頁： 39 - 42 1999年

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2 '-deoxyguanosine

Toyokuni S, Sagripanti JL

FREE RADICAL RESEARCH 31 巻 ( 2 ) 頁： 123 - 128 1999年

Association between M/L55-polymorphism of paraoxonase enzyme and oxidative DNA damage in patients with type 2 diabetes mellitus and in control subjects

Malin R, Rantalaiho V, Huang XH, Wirta O, Pasternack A, Leinonen JS, Alho H, Jokela H, Koivula T, Tanaka T, Okada K, Ochi H, Toyokuni S, Lehtimaki T

HUMAN GENETICS 105 巻 ( 1-2 ) 頁： 179 - 180 1999年

Squamous cell carcinoma in thyroid papillary carcinoma 国際誌 Open Access

Minamino M., Nakamura A., Hujisawa T., Tsuji H., Inoue T., Ohnishi S., Yamashita T., Toyokuni S.

Practica Otologica 92 巻 ( 3 ) 頁： 283 - 286 1999年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Practica Otologica

We report the case of a 62-year-old man with a squamous cell carcinoma of the thyroid gland which had invaded the hypopharynx. Primary squamous cell carcinoma of the thyroid gland is extremely rare. Its clinical course is very aggressive and the prognosis is poor. The patient underwent a radical resection of the thyroid, hypopharynx, larynx and resection of the trachea and esophagus was conducted concomitantly with bilateral neck dissection. Reconstruction of the terminal tracheostomy in the anterior mediastinum was created using a pectoralis major muscle flap.

DOI： 10.5631/jibirin.92.283

The role of oxidative DNA damage in human arsenic carcinogenesis: Detection of 8-hydroxy-2'-deoxyguanosine in arsenic-related Bowen's disease

Matsui M., Nishigori C., Toyokuni S., Takada J., Akaboshi M., Ishikawa M., Imamura S., Miyachi Y.

Journal of Investigative Dermatology 113 巻 ( 1 ) 頁： 26 - 31 1999年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Investigative Dermatology

Arsenic is widely distributed in nature in the form of either metalloids or chemical compounds, which cause a variety of pathologic conditions including cutaneous and visceral malignancies. Recently, reactive oxygen species have been hypothesized to be one of the causes of arsenic-induced carcinogenesis. 8-Hydroxy-2'-deoxyguanosine is one of the major reactive oxygen species-induced DNA base-modified products that is widely accepted as a sensitive marker of oxidative DNA damage. We studied the presence of 8- hydroxy-2'-deoxyguanosine by immunohistochemistry using N45.1 monoclonal antibody in 28 cases of arsenic-related skin neoplasms and arsenic keratosis as well as in 11 cases of arsenic-unrelated Bowen's diseases. The frequency of 8-hydroxy-2'-deoxyguanosine positive cases was significantly higher in arsenic-related skin neoplasms (22 of 28; 78%) than in arsenic-unrelated Bowen's disease (one of 11; 9%) (p < 0.001 by χ2 test). 8-Hydroxy-2'- deoxyguanosine was also detected in normal tissue adjacent to the arsenic- related Bowen's disease lesions. Furthermore, arsenic was detected by neutron activation analysis in the deparaffined skin tumor samples of arsenic- related disease (four of five; 80%), whereas arsenic was not detected in control samples. Our results strongly suggest the involvement of reactive oxygen species in arsenic-induced human skin cancer.

DOI： 10.1046/j.1523-1747.1999.00630.x

Hyperglycemia causes oxidative stress in pancreatic β-cells of GK rats, a model of type 2 diabetes

The 4-hydroxy-2-nonenal-derived fluorescent pigment 国際誌

Uchida, K, Itakura, K, Oya-Ito, T, Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE 27 巻頁： S100 - S100 1999年

The malondialdehyde-derived fluorescent pigment 国際誌

Uchida, K, Sakamoto, S, Itakura, K, Kumazawa, S, Nakayama, T, Toyokuni, S, Osawa, T

FREE RADICAL BIOLOGY AND MEDICINE 27 巻頁： S100 - S100 1999年

Oxidative stress in the human stomach infected with Helicobacter pylori: effect of bacterial eradication 国際誌

Pignatelli B, Li CQ, Bancel B, Toyokuni S, Ohshima H

2ND INTERNATIONAL CONGRESS ON GASTROENTEROLOGICAL CARCINOGENESIS 頁： 39 - 42 1999年

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2 '-deoxyguanosine

Toyokuni S, Sagripanti JL

FREE RADICAL RESEARCH 31 巻 ( 2 ) 頁： 123 - 128 1999年

Ihara Y., Toyokuni S., Uchida K., Odaka H., Tanaka T., Ikeda H., Hiai H., Seino Y., Yamada Y.

Diabetes 48 巻 ( 4 ) 頁： 927 - 932 1999年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Diabetes

Reactive oxygen species are involved in a diversity of biological phenomena such as inflammation, carcinogenesis, aging, and atherosclerosis. We and other investigators have shown that the level of 8-hydroxy-2'- deoxyguanosine (8-OHdG), a marker for oxidative stress, is increased in either the urine or the mononuclear cells of the blood of type 2 diabetic patients. However, the association between type 2 diabetes and oxidative stress in the pancreatic β-cells has not been previously described. We measured the levels of 8-OHdG and 4-hydroxy-2-nonenal (HNE)-modified proteins in the pancreatic β-cells of GK rats, a model of nonobese type 2 diabetes. Quantitative immunohistochemical analyses with specific antibodies revealed higher levels of 8-OHdG and HNE-modified proteins in the pancreatic β-cells of GK rats than in the control Wistar rats, with the levels increasing proportionally with age and fibrosis of the pancreatic islets. We further investigated whether the levels of 8-OHdG and HNE-modified proteins would be modified in the pancreatic β-cells of GK rats fed with 30% sucrose solution or 50 ppm of voglibose (α-glucosidase inhibitor). In the GK rats, the levels of 8-OHdG and HNE-modified proteins, as well as islet fibrosis, were increased by sucrose treatment but reduced by voglibose treatment. These results indicate that the pancreatic β-cells of GK rats are oxidatively stressed, and that chronic hyperglycemia might be responsible for the oxidative stress observed in the pancreatic β-cells.

DOI： 10.2337/diabetes.48.4.927

hOGG1 overexpression and high 8-Oxo-2'-deoxyguanosine lyase activity in human colorectal carcinoma 国際誌

Toyokuni, S, Kondo, S, Tanaka, T, Hiai, H, Onodera, H, Imamura, M

FREE RADICAL BIOLOGY AND MEDICINE 27 巻頁： S149 - S149 1999年

Detection of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1 and its application 国際誌

Toyokuni, S

ADVANCES IN DNA DAMAGE AND REPAIR 302 巻頁： 395 - 405 1999年

Association between M/L55-polymorphism of paraoxonase enzyme and oxidative DNA damage in patients with type 2 diabetes mellitus and in control subjects

Malin R, Rantalaiho V, Huang XH, Wirta O, Pasternack A, Leinonen JS, Alho H, Jokela H, Koivula T, Tanaka T, Okada K, Ochi H, Toyokuni S, Lehtimaki T

HUMAN GENETICS 105 巻 ( 1-2 ) 頁： 179 - 180 1999年

Oxidative stress in the human stomach infected with Helicobacter pylori:: effect of bacterial eradication 国際誌

Pignatelli, B, Li, CQ, Bancel, B, Toyokuni, S, Ohshima, H

2ND INTERNATIONAL CONGRESS ON GASTROENTEROLOGICAL CARCINOGENESIS 頁： 39 - 42 1999年

卵巣粘液性腫瘍の術中腫瘍捺印細胞像の検討 : 境界悪性例を中心に

清水恵子, 小椋聖子, 村田匡好, 高尾由美, 豊國伸哉, 桜井幹己

日本臨床細胞学会雑誌 37 巻 ( 6 ) 頁： 583 - 590 1998年11月

卵巣粘液性腫瘍の術中腫瘍捺印細胞像の検討 : 境界悪性例を中心に

清水恵子, 小椋聖子, 村田匡好, 高尾由美, 豊國伸哉, 桜井幹己

日本臨床細胞学会雑誌 37 巻 ( 6 ) 頁： 583 - 590 1998年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：特定非営利活動法人日本臨床細胞学会

1990年〜1997年迄に術中腫瘍捺印細胞診が施行された卵巣粘液性腫瘍は34例で,良性が16例,境界悪性が12例,悪性が6例であった.術中腫瘍捺印細胞診の正診率は,良性と悪性に関しては100%であったが,境界悪性では75%で誤判定例は全て良性と判定していた.著者等は境界悪性例を中心とした細胞像と,誤判定を防ぐための新しい圧挫標本の併用について検討した.境界悪性腫瘍の細胞像の特徴は,背景が比較的綺麗で,良性例に比べて重積性が増し,乳頭状の集団やボール状の集団が出現していることであった.個々の細胞異型に関しては,良性例に比べてN/C比の増大や,核クロマチン増量,核小体の肥大が部分的に観察されるものの著明ではなく,悪性例とは区別が可能と思われた.良性と誤判定した例は,上皮性成分の出現数の少なさがその最大の原因と思われ,圧挫標本の作製を加えることにより正診率の向上が期待できるものと思われた

Improvement in hepatic lipid peroxidation in patients with complete response to interferon-α treatment for chronic hepatitis C. 国際誌

Kageyama, F; Kobayashi, Y; Kawasaki, T; Toyokuni, S; Uchida, K; Nakamura, H

HEPATOLOGY 28 巻 ( 4 ) 頁： 250A - 250A 1998年10月

Role of cytochrome P450 isoforms and free radicals in endotoxemic liver injury.

Minamiyama, Y; Takemura, S; Imaoka, S; Toyokuni, S; Funae, Y; Kinoshita, H; Inoue, M

HEPATOLOGY 28 巻 ( 4 ) 頁： 251A - 251A 1998年10月

Role of cytochrome P450 isoforms and free radicals in endotoxemic liver injury. 査読有り

Minamiyama Y, Takemura S, Imaoka S, Toyokuni S, Funae Y, Kinoshita H, Inoue M

HEPATOLOGY 28 巻 ( 4 ) 頁： 251A - 251A 1998年10月

Improvement in hepatic lipid peroxidation in patients with complete response to interferon-alpha treatment for chronic hepatitis C. 査読有り国際誌

Kageyama F, Kobayashi Y, Kawasaki T, Toyokuni S, Uchida K, Nakamura H

HEPATOLOGY 28 巻 ( 4 ) 頁： 250A - 250A 1998年10月

Improvement in hepatic lipid peroxidation in patients with complete response to interferon-α treatment for chronic hepatitis C. 国際誌

Kageyama, F, Kobayashi, Y, Kawasaki, T, Toyokuni, S, Uchida, K, Nakamura, H

HEPATOLOGY 28 巻 ( 4 ) 頁： 250A - 250A 1998年10月

Heat shock preconditioning ameliorates liver injury following normothermic ischemia-reperfusion in steatotic rat livers

Yamagami K, Yamamoto Y, Kume M, Kimoto S, Yamamoto H, Ozaki N, Yamamoto M, Shimahara Y, Toyokuni S, Yamaoka Y

JOURNAL OF SURGICAL RESEARCH 79 巻 ( 1 ) 頁： 47 - 53 1998年9月

Heat shock preconditioning ameliorates liver injury following normothermic ischemia-Reperfusion in steatotic rat livers

Yamagami K., Yamamoto Y., Kume M., Kimoto S., Yamamoto H., Ozaki N., Yamamoto M., Shimahara Y., Toyokuni S., Yamaoka Y.

Journal of Surgical Research 79 巻 ( 1 ) 頁： 47 - 53 1998年9月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Surgical Research

The decreased tolerance of steatotic livers to warm ischemia complicates liver surgery. The efficacy of heat shock preconditioning in steatotic livers to lessen ischemia-reperfusion injury was studied in rats. Steatotic liver was produced in Lewis rats with a choline-deficient diet. Rats with steatotic livers were divided into a heat shock preconditioned group (group HS) and a control group (group C). All rats received 45 min of hepatic warm ischemia. Survival rates and changes in biochemical and histological parameters were compared in both groups. Heat shock protein 72 (HSP72) was produced only in group HS. The 7-day survival of the rats after warm ischemic intervention was significantly better in group HS (13/15) than in group C (5/15) (P < 0.01). The concentration of ATP in liver tissue (n = 10, P < 0.01) and serum levels of aspartate aminotransferase (n = 10, P < 0.05), alanine aminotransferase (n = 10, P < 0.01), and lactic dehydrogenase (n = 10, P < 0.01) at 40 min reperfusion were also significantly better in group HS than in group C. Histological examination at 40 min reperfusion showed severe sinusoidal congestion, hepatocyte necrosis, and increased positivity to 4-hydroxy-2- nonenal-modified proteins in group C livers; these signs were markedly suppressed in group HS livers. The data indicate that heat shock preconditioning provides the steatotic rat liver with significant tolerance to warm ischemia-reperfusion injury.

DOI： 10.1006/jsre.1998.5403

Development of High-grade Renal Cell Carcinomas in Rats Independently of Somatic Mutations in the Tsc2 and VHL Tumor Suppressor Genes

TOYOKUNI Shinya, OKADA Kunihiko, KONDO Shohei, NISHIOKA Hiroaki, TANAKA Tomoyuki, NISHIYAMA Yasuyuki, HINO Okio, HIAI Hiroshi

Japanese journal of cancer research : gann 89 巻 ( 8 ) 頁： 814 - 820 1998年8月

Development of High-grade Renal Cell Carcinomas in Rats Independently of Somatic Mutations in the Tsc2 and VHL Tumor Suppressor Genes 国際誌

TOYOKUNI Shinya, OKADA Kunihiko, KONDO Shohei, NISHIOKA Hiroaki, TANAKA Tomoyuki, NISHIYAMA Yasuyuki, HINO Okio, HIAI Hiroshi

Japanese journal of cancer research : gann 89 巻 ( 8 ) 頁： 814 - 820 1998年8月

Development of high-grade renal cell carcinomas in rats independently of somatic mutations in the Tsc2 and VHL tumor suppressor genes

Toyokuni S, Okada K, Kondo S, Nishioka H, Tanaka T, Nishiyama Y, Hino O, Hiai H

JAPANESE JOURNAL OF CANCER RESEARCH 89 巻 ( 8 ) 頁： 814 - 820 1998年8月

A processed grain food inhibits hepatic injury in endotoxemic rats

Minamiyama Y, Takemura S, Toyokuni S, Tanimoto Y, Sato EF, Inoue M

JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY 44 巻 ( 4 ) 頁： 547 - 559 1998年8月

Development of high-grade renal cell carcinomas in rats independently of somatic mutations in the Tsc2 and VHL tumor suppressor genes

Toyokuni S., Okada K., Kondo S., Nishioka H., Tanaka T., Nishiyama Y., Hino O., Hiai H.

Japanese Journal of Cancer Research 89 巻 ( 8 ) 頁： 814 - 820 1998年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Cancer Research

Ferric nitrilotriacetate (Fe-NTA) induces renal proximal tubular damage that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. The RCCs are characterized by 1) high incidence of pulmonary metastasis and peritoneal invasion, 2) high incidence of tumor-associated mortality and 3) possible involvement of reactive oxygen species in carcinogenesis. The present study investigated the possible role of Tsc2 and VHL tumor suppressor genes in this model. Thirty-four Fe-NTA-induced primary RCCs and 20 other primary or metastatic tumors of rats were searched for genetic alteration in all the coding exons of both genes by polymerase chain reaction-single-strand-conformation polymorphism analysis and sequencing in conjunction with morphological evaluation. In the Fe-NTA-induced RCCs, frequency of metastasis or invasion was proportionally associated with the nuclear grade of the tumor (grades 1-3). Only one Fe-NTA-induced RCC of grade 1 revealed missense mutations with loss of heterozygosity in exon 10 of the Tsc2 gene (codons 334, GTG (Val) to GCG (Ala), and 336, TAT (Tyr) to CAT (His)). No mutation was found in the VHL gene. The results suggest that 1) high-grade RCCs can develop in the absence of mutations in the Tsc2 and VHL genes in rats, and that 2) Tsc2 gene somatic mutation can nonetheless be one of the causes of non-Eker rat RCCs.

DOI： 10.1111/j.1349-7006.1998.tb00633.x

Oxidative stress and cancer: The role of redox regulation

Development of high-grade renal cell carcinomas in rats independently of somatic mutations in the Tsc2 and VHL tumor suppressor genes

Toyokuni S, Okada K, Kondo S, Nishioka H, Tanaka T, Nishiyama Y, Hino O, Hiai H

JAPANESE JOURNAL OF CANCER RESEARCH 89 巻 ( 8 ) 頁： 814 - 820 1998年8月

Toyokuni S.

Biotherapy 11 巻 ( 2-3 ) 頁： 147 - 154 1998年7月

Over-expression of glutathione S-transferase Yp isozyme and concomitant down-regulation of Ya isozyme in renal cell carcinoma of rats induced by ferric nitrilotriacetate 査読有り国際誌

Tanaka T, Nishiyama Y, Okada K, Satoh K, Fukuda A, Uchida K, Osawa T, Hiai H, Toyokuni S

CARCINOGENESIS 19 巻 ( 5 ) 頁： 897 - 903 1998年5月

Oxidative stress in pancreatic β-cells of GK rat, a model of non-obese NIDDM

Ihara, Y; Yamada, Y; Uchida, K; Toyokuni, S; Seino, Y

DIABETES 47 巻頁： A263 - A263 1998年5月

Oxidative stress in pancreatic beta-cells of GK rat, a model of non-obese NIDDM 国際誌

Ihara Y, Yamada Y, Uchida K, Toyokuni S, Seino Y

DIABETES 47 巻頁： A263 - A263 1998年5月

Over-expression of glutathione S-transferase Yp isozyme and concomitant down-regulation of Ya isozyme in renal cell carcinoma of rats induced by ferric nitrilotriacetate

Tanaka T., Nishiyama Y., Okada K., Satoh K., Fukuda A., Uchida K., Osawa T., Hiai H., Toyokuni S.

Carcinogenesis 19 巻 ( 5 ) 頁： 897 - 903 1998年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Carcinogenesis

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage, a consequence of iron-catalysed Fenton-like reactions, that finally leads to a high incidence of renal cell carcinoma (RCC) in rodents. Glutathione S-transferase (GST) is a family of enzymes that play an important role in detoxification of hydrophobic and electrophilic molecules, and has been associated with putative preneoplastic foci of rat hepatocarcinogenesis and chemotherapy-resistance of human cancers. Our previous study revealed an induction of π-class glutathione S-transferase (Yp) mRNA in the kidney 3 h after administration of Fe-NTA. In the present study, expression of GST isozymes were further investigated in the Fe-NTA-induced RCCs of rats which are characterized by (1) high incidence of metastasis and invasion, (2) high incidence of tumour-associated mortality, and (3) possible involvement of reactive oxygen species in carcinogenesis. In the Fe-NTA-induced RCCs, the levels of α-class GST (Ya) mRNA and proteins were markedly decreased with no apparent change in the copy number of the gene. In contrast, GST-Yp mRNA and proteins were significantly increased in the RCCs while the total GST enzymatic activity was decreased. Immunohistochemical analysis revealed intense staining of GST-Yp not only in the primary RCCs and its metastatic sites, but also in their non-tumorous part of proximal tubules. The contrastive expression of GST isozymes in this renal carcinogenesis model suggests an alteration of its transcription mechanisms and warrants further investigation of this particular detoxifying enzyme from the viewpoint of reactive oxygen species-induced carcinogenesis.

DOI： 10.1093/carcin/19.5.897

Immunohistochemical detection of 4-hydroxy-2-nonenal-modified-protein adducts in human alcoholic liver diseases

Ohhira M, Ohtake T, Matsumoto A, Saito H, Ikuta K, Fujimoto Y, Ono M, Toyokuni S, Kohgo Y

ALCOHOLISM-CLINICAL AND EXPERIMENTAL RESEARCH 22 巻 ( 3 ) 頁： 145S - 149S 1998年5月

Immunohistochemical detection of 4-hydroxy-2-nonenal-modified-protein adducts in human alcoholic liver diseases 査読有り

M Ohhira, T Ohtake, A Matsumoto, H Saito, K Ikuta, Y Fujimoto, M Ono, S Toyokuni, Y Kohgo

ALCOHOLISM-CLINICAL AND EXPERIMENTAL RESEARCH 22 巻 ( 3 ) 頁： 145S - 149S 1998年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：LIPPINCOTT WILLIAMS & WILKINS

4-Hydroxy-2-nonenal (HNE) is one of the major components of lipid peroxidation product and has been shown to react with proteins to form HNE-protein adducts. HNE-protein adducts are relatively stable and can be used as a marker of radical-mediated cellular damage. We report herein the immunohistochemical analysis of HNE-protein adducts in human alcoholic liver diseases using a specific monoclonal antibody HNEJ-2. Cytoplasm of hepatocytes and bile duct epithelia was positively stained for HNE-protein adducts, and the nucleus was negligibly stained. The immunohistochemical intensity of hepatocytes was classified into three groups: strong, moderate, and faint staining. Strong staining was found in 43% of alcoholic liver diseases and in 4% of viral liver diseases. Hepatocytes of alcoholic liver diseases contained a higher amount of HNE-protein adducts than those of viral liver diseases, and the difference was statistically significant (p = 0.005; chi(2) test). Semiquantitative analysis of the histological intensities of HNE-protein adducts and iron indicated a significant positive correlation (p = 0.084; Spearman's rank correlation). The localization of HNE-protein adducts and iron in hepatocytes appeared to be identical. These data suggested the correlation between HNE-protein adducts and iron. Our results indicate that HNE-protein adducts, a marker of oxidative stress-induced damage, are increased in human alcoholic liver damage, and that hepatic siderosis may act on the production of free radicals.

Significant deposition of 4-hydroxy-2-nonenal-modified-protein adducts in human alcoholic liver disease; Positive correlation with parenchymal iron. 国際誌

Oxidative stress in pancreatic β-cells of GK rat, a model of non-obese NIDDM 国際誌

Ihara, Y, Yamada, Y, Uchida, K, Toyokuni, S, Seino, Y

DIABETES 47 巻頁： A263 - A263 1998年5月

Ohhira, M; Ohtake, T; Matsumoto, A; Saito, H; Kawashima, T; Ikuta, K; Tanaka, K; Fujimoto, Y; Ono, M; Toyokuni, S; Kohgo, Y

GASTROENTEROLOGY 114 巻 ( 4 ) 頁： A1315 - A1315 1998年4月

The role of oxidative DNA damage in human arsenic carcinogenesis: Detection of 8-OHdG in arsenic related Bowen's disease.

Matsui, M; Nishigori, C; Toyokuni, S; Takada, J; Akaboshi, M; Ochi, H; Ishikawa, M; Imamura, S

JOURNAL OF INVESTIGATIVE DERMATOLOGY 110 巻 ( 4 ) 頁： 526 - 526 1998年4月

The role of oxidative DNA damage in human arsenic carcinogenesis: Detection of 8-OHdG in arsenic related Bowen's disease. 国際誌

Matsui, M, Nishigori, C, Toyokuni, S, Takada, J, Akaboshi, M, Ochi, H, Ishikawa, M, Imamura, S

JOURNAL OF INVESTIGATIVE DERMATOLOGY 110 巻 ( 4 ) 頁： 526 - 526 1998年4月

Enhanced hepatic lipid peroxidation in chronic hepatitis C 査読有り国際誌

Kageyama F., Kobayashi Y., Koide S., Murohisa G., Otake M., Shimizu E., Sasada Y., Suzuki F., Kawasaki T., Nakamura H., Toyokuni S., Uchida K.

Japanese Pharmacology and Therapeutics 26 巻 ( SUPPL. 2 ) 頁： 149 - 152 1998年4月

Enhanced hepatic lipid peroxidation in chronic hepatitis C 査読有り国際誌

Kageyama F, Kobayashi Y, Koide S, Murohisa G, Otake M, Shimizu E, Sasada Y, Suzuki F, Kawasaki T, Nakamura H, Toyokuni S, Uchida K

Japanese Pharmacology and Therapeutics 26 巻 ( SUPPL. 2 ) 頁： 149 - 152 1998年4月

Involvement of nitric oxide in survival of random pattern skin flap

Um SC, Suzuki S, Toyokuni S, Kim BM, Tanaka T, Hiai H, Nishimura Y

PLASTIC AND RECONSTRUCTIVE SURGERY 101 巻 ( 3 ) 頁： 785 - 792 1998年3月

Involvement of nitric oxide in survival of random pattern skin flap

Um S.C., Suzuki S., Toyokuni S., Kim B.M., Tanaka T., Hiai H., Nishimura Y.

Plastic and Reconstructive Surgery 101 巻 ( 3 ) 頁： 785 - 792 1998年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Plastic and Reconstructive Surgery

A free radical gas, nitric oxide, has many useful functions when produced under physiological conditions by neurons and endothelial cells. However, excess nitric oxide has been reported to exert cytotoxic effects by direct toxicity or by reaction with superoxide. The effect of nitric oxide on the microcirculation in the periphery of a flap remains unclear, and its effect on flap survival is also unknown because nitric oxide has a dual action. Thus, we attempted to clarify the effect of nitric oxide on survival of rat random pattern skin flaps by the use of an endothelial constitutive nitric oxide synthase inhibitor (IP administration of 50 mg/kg N(G)-nitro-L- arginine) and the substrate of nitric oxide synthase (IP administration of 1 g/kg L-arginine). Three kinds of experiments were done using a total number of 120 animals: (1) time course measurement of blood flow in the flap periphery was performed using a laser Doppler flowmeter (30 rats), (2) tile length of the surviving area of flaps was measured 1 week after raising the flap (60 rats), and (3) Western blot analysis was used to determine the time course of changes ill tile amount of endothelial constitutive nitric oxide synthase and the formation of 3-nitro-L-tyrosine, which is it marker of peroxynitrite-mediated (i.e., nitric oxide-dependent) tissue damage (30 rats). Inhibition of endothelial constitutive nitric oxide synthase by N(G)- nitro-L-arginine significantly decreased the length of the surviving area of skin flap (p < 0.01 compared with the control), which was associated with a decrease in tim blood flow of the proximal portion of the flap. On the other hand, exogenous L-arginine increased the survival length of skin flap significantly (p < 0.01 compared with the control), which was associated with all increase in blood flow of the distal portion of the flap even though there was nitric oxide-mediated oxidative tissue damage. These results suggest that nitric oxide produced by endothelial constitutive nitric oxide synthase plays a role in maintaining circulation in the skin flap periphery and that L-arginine administration contributes to reduction of ischemic necrosis in the skin flap.

DOI： 10.1097/00006534-199803000-00030

Major targets of ROS-induced cancer in rats are p15/p16 oncosuppressor genes

Toyokuni, S; Tanaka, T; Iwasa, Y; Kondo, S; Hiai, H

FREE RADICAL BIOLOGY AND MEDICINE 25 巻頁： S119 - S119 1998年

A processed grain food AOB inhibits hepatic injury in endotoxemic rats

Minamiyama, Y; Takemura, S; Toyokuni, S; Tanimoto, Y; Sato, EF; Inoue, M

BIOLOGICAL OXIDANTS AND ANTIOXIDANTS: MOLECULAR MECHANISMS AND HEALTH EFFECTS 頁： 349 - 357 1998年

A processed grain food inhibits hepatic injury in endotoxemic rats 査読有り

Minamiyama Y., Takemura S., Toyokuni S., Tanimoto Y., Sato E.F., Inoue M.

Journal of Nutritional Science and Vitaminology 44 巻 ( 4 ) 頁： 547 - 559 1998年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Nutritional Science and Vitaminology

Although various antioxidants have been tested as therapeutics for endotoxemic subjects, the results of their efficacy are conflicting. Antioxidant biofactor (AOB) is a unique processed grain food that exhibits strong antioxidant activity (Minamiyama et al, J Nutr Sci Vitaminol, 40: 467- 477, 1994). The present study was carried out to test the effect of AOB on hepatic injury in rats induced by lipopolysaccharide (LPS). Intravenous administration of LPS induced liver injury with a concomitant increase in hepatic generation of nitric oxide (NO) and 4-hydroxy-2-nonenal (HNE) modified proteins in the control group. The administration of AOB significantly inhibited the LPS-induced hepatic injury and generation of HNE- modified proteins and increased the survival rate of endotoxemic rats without affecting NO generation and plasma levels of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ). AOB scavenged superoxide radicals without affecting NO production by LPS-stimulated macrophage cell line J 774.2 cells. AOB also inhibited lipid peroxidation induced by LPS in the cells. These results suggested that AOB might scavenge superoxide radicals and decrease toxic metabolites including HNE, thereby inhibiting liver injury in endotoxemic rats.

DOI： 10.3177/jnsv.44.547

その他リンク： https://jlc.jst.go.jp/DN/JALC/00085275768?from=CiNii

Oxidative stress and cancer: The role of redox regulation

Toyokuni S

BIOTHERAPY 11 巻 ( 2-3 ) 頁： 147 - 154 1998年

卵巣粘液性腫ようの術中腫よう捺印細胞像の検討境界悪性例を中心に:境界悪性例を中心に国際誌

清水恵子, 小椋聖子, 村田匡好, 高尾由美, 豊國伸哉, 桜井幹己

日本臨床細胞学会雑誌 37 巻 ( 6 ) 頁： 583 - 590 1998年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：特定非営利活動法人日本臨床細胞学会

卵巣腫瘍は良悪性の術前診断が困難なことが多く, 当院では術中腫瘍捺印細胞診を積極的に施行している. 1990年から1997年の8年間に術中腫瘍捺印細胞診が施行された卵巣粘液性腫瘍は34例で, 良性が16例, 境界悪性が12例, 悪性が6例であった. 術中腫瘍捺印細胞診の正診率は, 良性と悪性に関しては100%であったが, 境界悪性では75%で誤判定例はすべて良性と判定していた. 今回, われわれは境界悪性例を中心とした細胞像と, 誤判定を防ぐための新しい圧挫標本の併用について検討したので報告する. 境界悪性腫瘍の細胞像の特徴は, 背景が比較的きれいで, 良性例に比べて重積性が増し, 乳頭状の集団やボール状の集団が出現していることであった. 個々の細胞異型に関しては, 良性例に比べてN/C比の増大や, 核クロマチン増量, 核小体の肥大が部分的に観察されるものの著明ではなく, 悪性例とは区別が可能と思われた. 良性と誤判定した例については, 上皮性成分の出現i数の少なさがその最大の原因と思われ, 圧挫標本の作製を加えることにより正診率の向上が期待できるものと思われた.

DOI： 10.5795/jjscc.37.583

Immunohistochemical detection of 4-hydroxy-2-nonenal-modified-protein adducts in human alcoholic liver diseases

Oxidative stress and cancer: The role of redox regulation 査読有り

S Toyokuni

BIOTHERAPY 11 巻 ( 2-3 ) 頁： 147 - 154 1998年

Ohhira M., Ohtake T., Matsumoto A., Saito H., Ikuta K., Fujimoto Y., Ono M., Toyokuni S., Kohgo Y.

Alcoholism: Clinical and Experimental Research 22 巻 ( 3 SUPPL. 1 ) 1998年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Alcoholism: Clinical and Experimental Research

4-Hydroxy-2-nonenal (HNE) is one of the major components of lipid peroxidation product and has been shown to react with proteins to form HNE-protein adducts. HNE-protein adducts are relatively stable and can be used as a marker of radical-mediated cellular damage. We report herein the immunohistochemical analysis of HNE-protein adducts in human alcoholic liver diseases using a specific monoclonal antibody HNEJ-2. Cytoplasm of hepatocytes and bile duct epithelia was positively stained for HNE-protein adducts, and the nucleus was negligibly stained. The immunohistochemical intensity of hepatocytes was classified into three groups: strong, moderate, and faint staining. Strong staining was found in 43% of alcoholic liver diseases and in 4% of viral liver diseases. Hepatocytes of alcoholic liver diseases contained a higher amount of HNE-protein adducts than those of viral liver diseases, and the difference was statistically significant (p = 0.005; χ2 test). Semiquantitative analysis of the histological intensities of HNE-protein adducts and iron indicated a significant positive correlation (p = 0.084; Spearman's rank correlation). The localization of HNE-protein adducts and iron in hepatocytes appeared to be identical. These data suggested the correlation between HNE-protein adducts and iron. Our results indicate that HNE-protein adducts, a marker of oxidative stress-induced damage, are increased in human alcoholic liver damage, and that hepatic siderosis may act on the production of free radicals. Copyright © 1998 by The Research Society on Alcoholism.

DOI： 10.1111/acer.1998.22.s3_part1.145s

New biomarker evidence of oxidative DNA damage in patients with non-insulin-dependent diabetes mellitus

A processed grain food AOB inhibits hepatic injury in endotoxemic rats 査読有り国際誌

Minamiyama, Y, Takemura, S, Toyokuni, S, Tanimoto, Y, Sato, EF, Inoue, M

BIOLOGICAL OXIDANTS AND ANTIOXIDANTS: MOLECULAR MECHANISMS AND HEALTH EFFECTS 頁： 349 - 357 1998年

Leinonen J., Lehtimäki T., Toyokuni S., Okada K., Tanaka T., Hiai H., Ochi H., Laippala P., Rantalaiho V., Wirta O., Pasternack A., Alho H.

FEBS Letters 417 巻 ( 1 ) 頁： 150 - 152 1997年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：FEBS Letters

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. We have investigated oxidative DNA damage in patients with non-insulin-dependent diabetes mellitus (NIDDM) by urinary 8-OHdG assessments. We determined the total urinary excretion of and OHdG from 24 h urine samples of 81 NIDDM patients 9 years after the initial diagnosis and of 100 non-diabetic control subjects matched for age and gender. The total 24 h urinary excretion of 8-OHdG was markedly higher in NIDDM patients than in control subjects (68.2 ± 39.4 μg vs, 49.6 ± 37.7 μg, P= 0.001). High glycosylated hemoglobin was associated with a high level of urinary 8-OHdG. The increased excretion of urinary and OHdG is seen as indicating an increased systemic level of oxidative DNA damage in NIDDM patients.

DOI： 10.1016/S0014-5793(97)01273-8

Protein modification by lipid peroxidation products: Formation of malondialdehyde-derived N(ε)(2-propenal)lysine in proteins

New biomarker evidence of oxidative DNA damage in patients with non-insulin-dependent diabetes mellitus

Leinonen J, Lehtimaki T, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H, Laippala P, Rantalaiho V, Wirta O, Pasternack A, Alho H

FEBS LETTERS 417 巻 ( 1 ) 頁： 150 - 152 1997年11月

Protein modification by lipid peroxidation products: Formation of malondialdehyde-derived N epsilon-(2-propenal)lysine in proteins 査読有り国際誌

Uchida K, Sakai K, Itakura K, Osawa T, Toyokuni S

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 346 巻 ( 1 ) 頁： 45 - 52 1997年10月

Uchida K., Sakai K., Itakura K., Osawa T., Toyokuni S.

Archives of Biochemistry and Biophysics 346 巻 ( 1 ) 頁： 45 - 52 1997年10月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Malondialdehyde (MDA), a naturally occurring dialdehyde produced in the membrane lipid peroxidation, is known to react with lysine residues of proteins, but the MDA-lysine adducts generated in the proteins have not been characterized adequately. In the present study, we provide evidence that the enaminal-type MDA-lysine adduct, N(ε)-(2-propenal)lysine, is formed in human low-density lipoprotein (LDL) upon reaction with MDA or Cu2+. We found that the incubation of N(α)acetyllysine with MDA generated N(α)-acetyl-N(ε)- (2-propenal)lysine as the predominant product. In addition, a polyclonal antiserum raised against the MDA-modified protein was found to contain antibody populations that could be purified by affinity gel prepared by covalent attachment of N(α)-acetyl-N(ε)-(2-propenal)lysine. It was concluded that the affinity-purified anti-N(ε)-(2-propenal) lysine antibody was highly specific to the enaminal derivative of both lysine residues and phosphatidylethanolamine, based on the observations that (i) MDA was the only aldehyde which generated immunoreactive materials in proteins; (ii) among structurally defined MDA-lysine adducts tested, the antibody recognized the enaminal adduct only; and (iii) immunoreactivity to N(2-propenal)serine was still significant but much weaker than its reactivity to N-(2- propenal)ethanol- amine. Furthermore, analysis of antibody recognition sites with a variety of N-(2-propenal)alkylamines revealed that the mono-specific antibody recognized the N-2-propenal-N-ethyl moiety [-(CH2)2-NH-CH=CH - CHO] of enaminal adducts. Determination by a competitive enzyme-linked immunosorbent assay demonstrated that N(ε)-(2-propenal)lysine accounted for 33.7 and 3.1% of the lysine residues that disappeared during in vitro incubation of LDL with MDA and Cu2+, respectively. These results suggest that N(ε)-(2-propenal)lysine represents a major form of MDA covalently attached to proteins.

DOI： 10.1006/abbi.1997.0266

Impact of the protection from free radical injury in heat shock preconditioning against ischemia-reperfusion injury in steatotic rat livers.

Protein modification by lipid peroxidation products: Formation of malondialdehyde-derived N epsilon-(2-propenal)lysine in proteins 査読有り国際誌

Uchida K, Sakai K, Itakura K, Osawa T, Toyokuni S

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 346 巻 ( 1 ) 頁： 45 - 52 1997年10月

Enhanced hepatic lipid peroxidation in chronic hepatitis C. 国際誌

Kageyama, F; Kobayashi, Y; Kawasaki, T; Nakamura, H; Toyokuni, S; Uchida, K

HEPATOLOGY 26 巻 ( 4 ) 頁： 79 - 79 1997年10月

Yamagami, K; Yamamoto, Y; Kume, M; Kimoto, S; Ozaki, N; Toyokuni, S; Yamaoka, Y

HEPATOLOGY 26 巻 ( 4 ) 頁： 844 - 844 1997年10月

Impact of the protection from free radical injury in heat shock preconditioning against ischemia-reperfusion injury in steatotic rat livers. 国際誌

Yamagami, K, Yamamoto, Y, Kume, M, Kimoto, S, Ozaki, N, Toyokuni, S, Yamaoka, Y

HEPATOLOGY 26 巻 ( 4 ) 頁： 844 - 844 1997年10月

Enhanced hepatic lipid peroxidation in chronic hepatitis C. 査読有り国際誌

Kageyama F, Kobayashi Y, Kawasaki T, Nakamura H, Toyokuni S, Uchida K

HEPATOLOGY 26 巻 ( 4 ) 頁： 79 - 79 1997年10月

Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: Independence of tubular necrosis and sulfhydryl depletion

Tanaka T., Nishiyama Y., Okada K., Hirota K., Matsui M., Yodoi J., Hiai H., Toyokuni S.

Laboratory Investigation 77 巻 ( 2 ) 頁： 145 - 155 1997年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Laboratory Investigation

Adult T-cell leukemia-derived factor (ADF), originally defined as an interleukin-2 receptor inducer, is a human thioredoxin (TRX). ADF/TRX is a conserved multifunctional reductant presumably associated with redox regulation of the cellular environment; it works in vitro as a cytokine, free radical scavenger, activator of transcription factors, and substrate for several enzymes. In the present series of experiments, we studied the expression and intracellular localization of ADF/TRX in mouse kidney from two different renal tubular injury models: a free radical-associated model with ferric nitrilotriacetate (Fe-NTA) and a free radical-independent model with HgCI2. Markers of oxidative damage, such as 8-hydroxy-2'-deoxyguanosine, thiobarbituric acid-reactive substances, and 4-hydroxy-2-nonenal-modified proteins, were significantly increased in kidney from male C57BL/6 mice 1 hour after a single intraperitoneal injection of Fe-NTA (5 mg iron/kg). However, in kidney from mice given a subcutaneous injection of HgCI2 (5 mg Hg/kg), none of these markers were increased, despite tubular necrosis with sulfhydryl depletion. In the Fe-NTA model only, Northern and Western blot analyses of the kidney revealed induction of ADF/TRX (> 2.5-fold) after 16 hours and translocation of ADF/TRX from the cytoplasmic to nuclear fraction (> 3.5-fold) after 24 hours. Immunohistochemistry showed a catchy distribution of ADF/TRX in the normal renal proximal tubules. In ex vivo experiments using serial normal kidney frozen sections, it was found mat renal proximal tubules with low expression of ADF/FRX were more vulnerable to oxidative stress mediated by Fe-NTA. Collectively, these data suggest that: (a) ADF/TRX is induced and translocated to nuclei by oxidative damage mediated by Fe-NTA in the renal proximal tubules: (b) induction of ADF/TRX is independent of tubular necrosis or sulfhydryl depletion; and (c) ADF/TRX is involved in the cellular defense mechanisms in vive against oxidative damage.

Biomarker evidence of DNA oxidation in lung cancer patients: Association of urinary 8-hydroxy-2'-deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment

Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: Independence of tubular necrosis and sulfhydryl depletion

Tanaka T, Nishiyama Y, Okada K, Hirota K, Matsui M, Yodoi J, Hiai H, Toyokuni S

LABORATORY INVESTIGATION 77 巻 ( 2 ) 頁： 145 - 155 1997年8月

Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: independence of tubular necrosis and sulfhydryl depletion.

Tanaka T, Nishiyama Y, Okada K, Hirota K, Matsui M, Yodoi J, Hiai H, Toyokuni S

Laboratory investigation; a journal of technical methods and pathology 77 巻 ( 2 ) 頁： 145 - 55 1997年8月

Erhola M., Toyokuni S., Okada K., Tanaka T., Hiai H., Ochi H., Uchida K., Osawa T., Nieminen M.M., Alho H., Kellokumpu-Lehtinen P.

FEBS Letters 409 巻 ( 2 ) 頁： 287 - 291 1997年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：FEBS Letters

Ratios of urinary 8-hydroxy-2'-deoxyguanosine to urinary creatinine (8-OHdG/creatinine) have been considered as a good biological indicator of DNA oxidation. Urinary 8-OHdG/creatinine levels of lung cancer patients were evaluated by enzyme-linked immunosorbent assay using a monoclonal antibody N45.1 during radiotherapy and chemotherapy. An increase in urinary 8-OHdG/creatinine was found in non-small-cell carcinoma (non-SCC) patients during the course of radiotherapy. SCC patients showed higher levels of urinary 8-OHdG/creatinine than the controls. Furthermore, SCC patients with complete or partial response to the chemotherapy showed a significant decrease in urinary 8-OHdG/creatinine while patients with no change or progressive disease showed an increase.

DOI： 10.1016/S0014-5793(97)00523-1

Protection by αG-Rutin, a Water-soluble Antioxidant Flavonoid, against Renal Damage in Mice Treated with Ferric Nitrilotriacetate

Biomarker evidence of DNA oxidation in lung cancer patients: Association of urinary 8-hydroxy-2'-deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment

Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H, Uchida K, Osawa T, Nieminen MM, Alho H, KellokumpuLehtinen P

FEBS LETTERS 409 巻 ( 2 ) 頁： 287 - 291 1997年6月

Biomarker evidence of DNA oxidation in lung cancer patients: Association of urinary 8-hydroxy-2'-deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment 査読有り国際共著国際誌

Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H, Uchida K, Osawa T, Nieminen MM, Alho H, KellokumpuLehtinen P

FEBS LETTERS 409 巻 ( 2 ) 頁： 287 - 291 1997年6月

SHIMOI Kayoko, SHEN Bingrong, TOYOKUNI Shinya, MOCHIZUKI Rika, FURUGORI Michiyo, KINAE Naohide

Japanese journal of cancer research : gann 88 巻 ( 5 ) 頁： 453 - 460 1997年5月

Protection by αG-Rutin, a Water-soluble Antioxidant Flavonoid, against Renal Damage in Mice Treated with Ferric Nitrilotriacetate

SHIMOI Kayoko, SHEN Bingrong, TOYOKUNI Shinya, MOCHIZUKI Rika, FURUGORI Michiyo, KINAE Naohide

Japanese journal of cancer research : gann 88 巻 ( 5 ) 頁： 453 - 460 1997年5月

Protection by alpha G-rutin, a water-soluble antioxidant flavonoid, against renal damage in mice treated with ferric nitrilotriacetate

Shimoi K, Shen BR, Toyokuni S, Mochizuki R, Furugori M, Kinae N

JAPANESE JOURNAL OF CANCER RESEARCH 88 巻 ( 5 ) 頁： 453 - 460 1997年5月

Protection by αG-Rutin, a water-soluble antioxidant flavonoid, against renal damage in mice treated with ferric nitrilotriacetate

Shimoi K., Shen B., Toyokuni S., Mochizuki R., Furugori M., Kinae N.

Japanese Journal of Cancer Research 88 巻 ( 5 ) 頁： 453 - 460 1997年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Cancer Research

The protective effect of αG-Rutin against ferric nitrilotriacetate (Fe-NTA)-induced renal damage was studied in male ICR mice. Fe-NTA induces renal lipid peroxidation, leading to a high incidence of renal cell carcinoma in rodents. Administration of αG-Rutin (50 μmol as rutin/kg) by gastric intubation 30 min after i.p. injection of Fe-NTA (7 mg Fe/kg) most effectively suppressed renal lipid peroxidation. Repeated i.p. injection of Fe-NTA (2 mg Fe/kg/day for the first 3 days and 3 mg Fe/kg/day for 12 days, 5 days a week) causes subacute nephrotoxicity as revealed by induction of karyomegalic cells in renal proximal tubules. A protective effect was observed in mice given αG-Rutin 30 min after each Fe-NTA treatment. To elucidate the mechanism of protection by αG-Rutin, the pharmacokinetics and hydroxyl radical-scavenging effect of αG-Rutin were investigated by HPLC analysis and by electron spin resonance (ESR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), respectively. When mice were given αG-Rutin (50 μmol as rutin/kg) by gastric intubation, rapid absorption into the circulation was observed. The plasma concentration of αG-Rutin reached the highest level 30 min after oral administration and then decreased to the control level within 60 min. αG-Rutin inhibited the formation of DMPO-OH in a concentration-dependent manner. Further, chelating activity of αG-Rutin to ferric ions was shown by spectrophotometric analysis. These results suggest that absorbed αG-Rutin works as an antioxidant in vivo either by scavenging reactive oxygen species or by chelating ferric ions and this serves to prevent oxidative renal damage in mice treated with Fe-NTA.

DOI： 10.1111/j.1349-7006.1997.tb00403.x

Copper and iron-induced oxidative damage in non-tumor bearing LEC rats

Protection by alpha G-rutin, a water-soluble antioxidant flavonoid, against renal damage in mice treated with ferric nitrilotriacetate

Shimoi K, Shen BR, Toyokuni S, Mochizuki R, Furugori M, Kinae N

JAPANESE JOURNAL OF CANCER RESEARCH 88 巻 ( 5 ) 頁： 453 - 460 1997年5月

Copper and iron-induced oxidative damage in non-tumor bearing LEC rats 査読有り国際誌

Ma YX, Zhang DX, Kawabata T, Kiriu T, Toyokuni S, Uchida K, Okada S

PATHOLOGY INTERNATIONAL 47 巻 ( 4 ) 頁： 203 - 208 1997年4月

Ma Yuxiang, Zhang Daxian, Kawabata Teruyuki

Pathology international 47 巻 ( 4 ) 頁： 203 - 208 1997年4月

Copper and iron-induced oxidative damage in non-tumor bearing LEC rats 査読有り

YX Ma, DX Zhang, T Kawabata, T Kiriu, S Toyokuni, K Uchida, S Okada

PATHOLOGY INTERNATIONAL 47 巻 ( 4 ) 頁： 203 - 208 1997年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BLACKWELL SCIENCE

The copper and iron status in the liver of non-tumor bearing Long-Evans Cinnamon (LEC) rats (average age 17 months) was investigated. A direct quantitation of loosely-bound copper anti iron was also investigated by using a chelating agent, nitrilotriacetic acid (NTA-chelatable free copper and iron). Besides the total copper and iron contents, the level of NTA-chelatable free copper was also higher in LEC rats than in LEA rats (P<0.05). But for the free iron level there was no significant difference between the two rat groups (P>0.05). The formation of thiobarbituric acid-reactive substances was higher in LEC rats than in LEA mts (P<0.01). The 4-hydroxy-2-nonenal (HNE)-modified proteins were also clearly demonstrated in LEC rat liver. The copper and iron which produced the most important effect in the process of oxidative damage in LEC rats could not be distinguished. Even though free copper, which could induce free radical injuries, was increased in LEC rats, neither tumor-induction nor preneoplastic lesions in the experimental LEC rats were observed. therefore it is speculated that the elevation of a free iron is another important factor. Copper and iron, both important transition metals In the body, may participate in the induction of DNA damage and oncogenesis.

Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model 査読有り国際誌

Toyokuni S, Tanaka T, Hatton Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T

LABORATORY INVESTIGATION 76 巻 ( 3 ) 頁： 365 - 374 1997年3月

Toyokuni S, Tanaka T, Hatton Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T

LABORATORY INVESTIGATION 76 巻 ( 3 ) 頁： 365 - 374 1997年3月

Quantitative immunohistochemical determination of 8-hydroxy-2'- deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model 査読有り国際誌

Toyokuni S., Tanaka T., Hattori Y., Nishiyama Y., Yoshida A., Uchida K., Hiai H., Ochi H., Osawa T.

Laboratory Investigation 76 巻 ( 3 ) 頁： 365 - 374 1997年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Laboratory Investigation

The DNA base-modified product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most commonly used markers for the evaluation of oxidative DNA damage. A monoclonal antibody specific for 8-OHdG (N45.1) was characterized end applied in quantitative immunohistochemistry. N45.1 recognized both the modified base and deoxyribose structure of 8-OHdG and required a concentration two orders higher of 8-hydroxyguanosine as a competitor in the ELISA. In addition, N45.1 did not cross-react with the original four deoxyribonucleosides, other DNA base-modified products such as 8-hydroxy-2'- deoxyadenosine and O6-methyl-2'-deoxyguanosine, or urine components such as uric acid, creatine, and creatinine. A ferric nitrilotriacetate-induced rat renal carcinogenesis model was used for the evaluation of quantitative immunohistochemistry. The 8-OHdG index of quantitative immunohistochemistry, as analyzed by NIH Image freeware, correlated reasonably well with the 8- OHdG amount determined by high-performance liquid chromatography with an electrochemical detector-except for a difference in peak time, which could be attributed to the selection of target location. The present method has advantages over the high-performance liquid chromatography/electrochemical detector, gas chromatography/mass spectrometry, and 32P-postlabeling methods in that it allows localization of 8-OHdG to be specified without the risk of artifactual production of 8-OHdG during the DNA extraction and hydrolytic processes.

Overexpression of Integrin-associated Protein (CD47) in Rat Kidney Treated with a Renal Carcinogen, Ferric Nitrilotriacetate

Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model.

Toyokuni S, Tanaka T, Hattori Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T

Laboratory investigation; a journal of technical methods and pathology 76 巻 ( 3 ) 頁： 365 - 74 1997年3月

NISHIYAMA Yasuyuki, TANAKA Tomoyuki, NAITOH Hiroshi, MORI Chisato, FUKUMOTO Manabu, HIAI Hiroshi, TOYOKUNI Shinya

Japanese journal of cancer research : gann 88 巻 ( 2 ) 頁： 120 - 128 1997年2月

Overexpression of Integrin-associated Protein (CD47) in Rat Kidney Treated with a Renal Carcinogen, Ferric Nitrilotriacetate

NISHIYAMA Yasuyuki, TANAKA Tomoyuki, NAITOH Hiroshi, MORI Chisato, FUKUMOTO Manabu, HIAI Hiroshi, TOYOKUNI Shinya

Japanese journal of cancer research : gann 88 巻 ( 2 ) 頁： 120 - 128 1997年2月

Induction of a wide range of C<inf>2-12</inf> aldehydes and C<inf>7-12</inf> acyloins in the kidney of wistar rats after treatment with a renal carcinogen, ferric nitrilotriacetate

Toyokuni S., Luo X.P., Tanaka T., Uchida K., Hiai H., Lehotay D.C.

Free Radical Biology and Medicine 22 巻 ( 6 ) 頁： 1019 - 1027 1997年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis associated with lipid peroxidation and oxidative DNA damage that finally leads to a high incidence of renal cell carcinoma in rodents. In the present study, we investigated what kinds of C2-12 saturated and unsaturated aldehydes and C7-12 acyloins, metabolites of saturated aldehydes, are produced in the kidney and liver within 24 h after single ip administration of 15 mg Fe/kg of Fe-NTA, or after repeated (1 or 3 wk) ip administration of 5-10 mg Fe/kg of Fe-NTA. Amounts of twenty one aldehydes and five acyloins were determined by capillary column gas chromatography-negative-ion chemical ionization mass spectrometry with ammonia as reagent gas. Most of the aldehydes and all the acyloins measured revealed a significant dose-dependent increase 1 to 3 h after single administration in the kidney, among which 4-hydroxy-2-nonenal (HNE) showed the highest increase (27.3-fold) and malondialdehyde (MDA) was the most abundant aldehyde (2.40 nmol/100 mg wet tissue). In the liver, however, the increase in aldehydes and acyloins was less prominent. After repeated administration of Fe-NTA, only 9 aldehydes (ethanal; furfural; trans, trans- 2,4-heptadienal; nonanal; trans-2, cis-6 nonadienal; HNE; decanal; trans-4, cis-4-decenal; MDA) and 4 acyloins (3-hydroxyheptan-2-one; 3-hydroxyoctan-2- one; 3-hydroxynonan-2-one; 3-hydroxydodecan-2-one) showed a significant increase. Immunohistochemistry further demonstrated an increased amount of HNE-modified and MDA-modified proteins in the renal proximal tubules after repeated Fe-NTA administration. Some of the aldehydes measured such as HNE and MDA are reportedly cytotoxic, genotoxic and mutagenic. Accumulation of these aldehydes may play a role in this renal carcinogenesis model.

DOI： 10.1016/S0891-5849(96)00489-3

8-hydroxy-2'-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure (vol 107, pg 733, 1996)

Hattori, Y; Nishigori, C; Tanaka, T; Uchida, K; Nikaido, O; Osawa, T; Hiai, H; Imamura, S; Toyokuni, S

JOURNAL OF INVESTIGATIVE DERMATOLOGY 108 巻 ( 2 ) 頁： 237 - 237 1997年2月

Overexpression of integrin-associated protein (CD47) in rat kidney treated with a renal carcinogen, ferric nitrilotriacetate

Nishiyama Y, Tanaka T, Naitoh H, Mori C, Fukumoto M, Hiai H, Toyokuni S

JAPANESE JOURNAL OF CANCER RESEARCH 88 巻 ( 2 ) 頁： 120 - 128 1997年2月

Induction of a wide range of C-2-12 aldehydes and C-7-12 acyloins in the kidney of Wistar rats after treatment with a renal carcinogen, ferric nitrilotriacetate 査読有り国際誌

Toyokuni S, Luo XP, Tanaka T, Uchida K, Hiai H, Lehotay DC

FREE RADICAL BIOLOGY AND MEDICINE 22 巻 ( 6 ) 頁： 1019 - 1027 1997年

口蓋原発低悪性型筋上皮腫の1例

喜多淳, 岩井大, 豊國伸哉, 柳田昌宏, 川崎英子, 南野雅之, 中嶋安彬, 山下敏夫

耳鼻と臨床 43 巻 ( 1 ) 頁： 30 - 33 1997年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：JIBI TO RINSHO KAI

硬口蓋に原発した極めて稀な小唾液腺由来の悪性筋上皮腫の症例を経験した. 病理組織学的に筋上皮細胞由来であり, 悪性である細胞分裂像に乏しい点で低悪性型筋上皮腫と診断した. 手術的摘出と術後の放射線照射を行った. 性筋上皮腫はこれまでの報告例がなく, 治療方針は定まつていないのが現状であるが, 一般の扁平上皮癌に比し放射線療法や化学療法の効果が低いことは, 他の腺系悪性腫瘍と同様の性状であると思われる. したがつて, 放射線療法や化学療法はあくまで補助的な治療であり, 手術的摘出が優先されると思われる.

DOI： 10.11334/jibi1954.43.1_30

Overexpression of integrin-associated protein (CD47) in rat kidney treated with renal carcinogen, ferric nitrilotriacetate

Nishiyama Y., Tanaka T., Naitoh H., Mori C., Fukumoto M., Hiai H., Toyokuni S.

Japanese Journal of Cancer Research 88 巻 ( 2 ) 頁： 120 - 128 1997年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Cancer Research

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis, a consequence of free radical-associated damage, that ultimately leads to a polycystic change of the renal cortex and a high incidence of renal cell carcinoma (RCC) in rodents. The differential display technique was used to search for inducible genes in the kidney of male Wistar rats treated with Fe-NTA and in the induced RCCs. Six fragments were selected that showed specific quantitative changes in mRNA. Two of them exhibited similar patterns in northern blots as well. One fragment showed a high homology (89%) to murine integrin-associated protein (IAP; CD47). We thus cloned rat IAP cDNA including the entire coding region for use in further analysis. Rat IAP cDNA showed a 21-amino-acid deletion that was also observed in human, but not in mouse. Northern blots revealed that IAP was consistently overexpressed in non-tumorous parts of the kidney (2.4-fold increase, n = 9, P < 0.0001) as compared with matched controls 1 to 2 years after Fe-NTA treatment. IAP overexpression of more than 2.9-fold was found in 25% (2/8) of RCCs studied, and was limited to cases of a high histological grade and lung metastasis. Unexpectedly, IAP expression was higher in the non-tumorous part of the kidney after Fe-NTA treatment (2.8-fold) than in RCC (1.5-fold) in each case (n = 4, P < 0.05). Abundant expression of IAP mRNA in the renal tubular epithelium after Fe-NTA treatment and RCC cells was observed by in situ hybridization. The results suggest that IAP overexpression may be associated with Fe-NTA-induced renal cortical tubular damage and regeneration that lead to a polycystic state, and with tumor progression and metastasis of the induced RCCs.

DOI： 10.1111/j.1349-7006.1997.tb00356.x

Induction of a wide range of C-2-12 aldehydes and C-7-12 acyloins in the kidney of Wistar rats after treatment with a renal carcinogen, ferric nitrilotriacetate 査読有り

S Toyokuni, XP Luo, T Tanaka, K Uchida, H Hiai, DC Lehotay

FREE RADICAL BIOLOGY AND MEDICINE 22 巻 ( 6 ) 頁： 1019 - 1027 1997年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis associated with lipid peroxidation and oxidative DNA damage that finally leads to a high incidence of renal cell carcinoma in rodents. In the present study, we investigated what kinds of C-2-12 saturated and unsaturated aldehydes and C-7-12 acyloins, metabolites of saturated aldehydes, are produced in the kidney and liver within 24 h after single ip administration of 15 mg Fe/kg of Fe-NTA, or after repeated (1 or 3 wk) ip administration of 5-10 mg Fe/kg of Fe-NTA. Amounts of twenty one aldehydes and five acyloins were determined by capillary column gas chromatography-negative-ion chemical ionization mass spectrometry with ammonia as reagent gas. Most of the aldehydes and all the acyloins measured revealed a significant dose-dependent increase 1 to 3 h after single administration in the kidney, among which 4-hydroxy-2-nonenal (HNE) showed the highest increase (27.3-fold) and malondialdehyde (MDA) was the most abundant aldehyde (2.40 nmol/100 mg wet tissue). In the liver, however, the increase in aldehydes and acyloins was less prominent. After repeated administration of Fe-NTA, only 9 aldehydes (ethanal; furfural; trans,trans-2,4-heptadienal; nonanal; trans-2,cis-6-nonadienal; HNE; decanal; trans-4,cis-4-decenal; MDA) and 4 acyloins (3-hydroxyheptan-2-one; 3-hydroxyoctan-2-one; 3-hydroxynonan-2-one; 3-hydroxydodecan-2-one) showed a significant increase. Immunohistochemistry further demonstrated an increased amount of HNE-modified and MDA-modified proteins in the renal proximal tubules after repeated Fe-NTA administration. Some of the aldehydes measured such as HNE and MDA are reportedly cytotoxic, genotoxic and mutagenic. Accumulation of these aldehydes may play a role in this renal carcinogenesis model. Copyright (C) 1997 Elsevier Science Inc.

Induction and nuclear translocation of thioredoxin by oxidative damage in the mouse kidney: Independence of tubular necrosis and sulfhydryl depletion 査読有り

Tanaka T, Nishiyama Y, Okada K, Hirota K, Matsui M, Yodoi J, Hiai H, Toyokuni S

Laboratory Investigation 77 巻 ( 2 ) 頁： 145 - 155 1997年

Copper and iron-induced oxidative damage in non-tumor bearing LEC rats

Ma Y., Zhang D., Kawabata T., Kiriu T., Toyokuni S., Uchida K., Okada S.

Pathology International 47 巻 ( 4 ) 頁： 203 - 208 1997年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

The copper and iron status in the liver of non-tumor bearing Long-Evans Cinnamon (LEC) rats (average age 17 months) was investigated. A direct quantitation of loosely-bound copper and iron was also investigated by using a chelating agent, nitrilotriacetic acid (NTA-chelatable free copper and iron). Besides the total copper and iron contents, the level of NTA- chelatable free copper was also higher in LEC rats than in LEA rats (P<0.05). But for the free iron level there was no significant difference between the two rat groups (P>0.05). The formation of thiobarbituric acid-reactive substances was higher in LEC rate than in LEA rats (P<0.01). The 4-hydroxy- 2-nonenal (HNE)-modified proteins were also clearly demonstrated In LEC rat liver. The copper and iron which produced the most important effect in the process of oxidative damage in LEC rats could not be distinguished. Even though free copper, which could induce free radical injuries, was increased in LEC rats, neither tumor-induction nor preneoplastic lesions in the experimental LEC rats were observed. Therefore it is speculated that the elevation of s free iron is another important factor. Copper and iron, both important transition metals in the body, may participate in the induction of DNA damage and oncogenesis.

DOI： 10.1111/j.1440-1827.1997.tb04481.x

Prevention by 2-Mercaptoethane Sulfonate and N-Acetylcysteine of Renal Oxidative Damage in Rats Treated with Ferric Nitrilotriacetate

8-hydroxy-2'-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure 査読有り国際誌

Hattori Y, Nishigori C, Tanaka T, Uchida K, Nikaido O, Osawa T, Hiai H, Imamura S, Toyokuni S

JOURNAL OF INVESTIGATIVE DERMATOLOGY 107 巻 ( 5 ) 頁： 733 - 737 1996年11月

8-hydroxy-2'-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure 査読有り国際誌

Hattori Y, Nishigori C, Tanaka T, Uchida K, Nikaido O, Osawa T, Hiai H, Imamura S, Toyokuni S

JOURNAL OF INVESTIGATIVE DERMATOLOGY 107 巻 ( 5 ) 頁： 733 - 737 1996年11月

UMEMURA Takashi, HASEGAWA Ryuichi, SAI-KATO Kimie, NISHIKAWA Akiyoshi, FURUKAWA Fumio, TOYOKUNI Shinya, UCHIDA Koji, INOUE Tohru, KUROKAWA Yuji

Japanese journal of cancer research : gann 87 巻 ( 9 ) 頁： 882 - 886 1996年9月

Prevention by 2-Mercaptoethane Sulfonate and N-Acetylcysteine of Renal Oxidative Damage in Rats Treated with Ferric Nitrilotriacetate

UMEMURA Takashi, HASEGAWA Ryuichi, SAI-KATO Kimie, NISHIKAWA Akiyoshi, FURUKAWA Fumio, TOYOKUNI Shinya, UCHIDA Koji, INOUE Tohru, KUROKAWA Yuji

Japanese journal of cancer research : gann 87 巻 ( 9 ) 頁： 882 - 886 1996年9月

Prevention by 2-mercaptoethane sulfonate and N-acetylcysteine of renal oxidative damage in rats treated with ferric nitrilotriacetate 査読有り国際誌

Umemura T, Hasegawa R, SaiKato K, Nishikawa A, Furukawa F, Toyokuni S, Uchida K, Inoue T, Kurokawa Y

JAPANESE JOURNAL OF CANCER RESEARCH 87 巻 ( 9 ) 頁： 882 - 886 1996年9月

Prevention by 2-mercaptoethane sulfonate and N-acetylcysteine of renal oxidative damage in rats treated with ferric nitrilotriacetate

Umemura T., Hasegawa R., Sai-Kato K., Nishikawa A., Furukawa F., Toyokuni S., Uchida K., Inoue T., Kurokawa Y.

Japanese Journal of Cancer Research 87 巻 ( 9 ) 頁： 882 - 886 1996年9月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Cancer Research

Ferric nitrilotriacetate (Fe-NTA) is a renal toxicant and carcinogen in rats and mice. We found that its administration results in formation of 4-hydroxy-2-nonenal (HNE) in the renal proximal tubule cells of rats, and 8-hydroxydeoxyguanosine (8-OHdG) adducts in their DNA, suggesting a role for oxidative stress. Since 2-mercaptoethane sulfonate (MESNA) and N-acetylcysteine (NAC), administered orally, have been shown to increase the kidney levels of free thiol groups, their influence on the renal toxicity and carcinogenicity induced by Fe-NTA was examined in the present study. Male Wistar rats were intraperitoneally injected with Fe-NTA (12 mg Fe/kg), and MESNA (100 mg/kg) or NAC (200 mg/kg) was given orally 1 h before and 1 h after this treatment. The animals were killed for tissue analyses 3 h after the Fe-NTA exposure. In accord with our previous reports, HNE-modified protein was detected in the proximal tubules of Fe-NTA-treated rats by means of immunohistochemistry. Likewise, levels of 8-OHdG in the renal nuclear DNA, lipid peroxides as thiobarbituric acid-reactive substances in the kidneys, and blood urea nitrogen and creatinine in the serum were significantly increased by the Fe-NTA treatment. All of these changes were completely inhibited by oral administration of MESNA or NAG. These results suggest that both of these compounds can prevent the oxidative stress induced by Fe-NTA.

DOI： 10.1111/j.1349-7006.1996.tb02115.x

Prevention by 2-mercaptoethane sulfonate and N-acetylcysteine of renal oxidative damage in rats treated with ferric nitrilotriacetate 査読有り国際誌

Umemura T, Hasegawa R, SaiKato K, Nishikawa A, Furukawa F, Toyokuni S, Uchida K, Inoue T, Kurokawa Y

JAPANESE JOURNAL OF CANCER RESEARCH 87 巻 ( 9 ) 頁： 882 - 886 1996年9月

Induction of renal cell carcinoma in male Wistar rats treated with cupric nitrilotriacetate

Toyokuni S, Tanaka T, Nishiyama Y, Okamoto K, Nakashima Y, Hamazaki S, Okada S, Hiai H

LABORATORY INVESTIGATION 75 巻 ( 2 ) 頁： 239 - 248 1996年8月

Induction of renal cell carcinoma in male Wistar rats treated with cupric nitrilotriacetate 査読有り国際誌

Toyokuni S., Tanaka T., Nishiyama Y., Okamoto K., Nakashima Y., Hamazaki S., Okada S., Hiai H.

Laboratory Investigation 75 巻 ( 2 ) 頁： 239 - 248 1996年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Laboratory Investigation

Systemic administration of copper, an essential trace metal in the human body, has never been reported to be carcinogenic in animals. We investigated the induction of tumors by the cupric complex of nitrilotriacetic acid (Cu- NTA) in male Wistar rats. Thirty-two animals received ip injections of Cu- NTA, 3 to 5 mg of copper/kg body weight 5 days a week for 12 weeks, and were kept under close observation. For comparison, 31 animals received ip injections of ferric nitrilotriacetate (Fe-NTA), 5 to 10 mg of iron/kg body weight, and 16 animals received nitrilotriacetic acid (NTA) alone at the molar dose equivalent to Cu-NTA for the same period of time. Sixteen animals were left untreated as controls. Fourteen animals in the Cu-NTA group died of hepatic failure during the treatment period, and renal cell carcinoma (RCC) was induced in eight animals (25%). Of these, four animals died of either pulmonary metastasis or intraperitoneal hemorrhage. A total of 12 RCC were obtained, of which six tumors were ≤5 mm. The Cu-NTA group yielded fewer RCC and required a longer latent period for their incubation than the Fe-NTA group. Furthermore, the Cu-NTA group showed one hepatocellular carcinoma and one high-grade sarcoma of hepatic origin. No renal or hepatic tumor was observed in the NTA or control groups. The nontumorous part of the kidney treated with Cu-NTA presented hemosiderosis caused by copper-induced hemolytic anemia. This is the first report that systemic administration of copper compounds can induce malignant tumors in animals. Not only copper but also iron may play a role in the Cu-NTA-induced renal carcinogenesis model.

Oxidative stress response in iron-induced renal carcinogenesis: Acute nephrotoxicity mediates the enhanced expression of glutathione S-transferase Yp isozyme

Induction of renal cell carcinoma in male Wistar rats treated with cupric nitrilotriacetate

Toyokuni S, Tanaka T, Nishiyama Y, Okamoto K, Nakashima Y, Hamazaki S, Okada S, Hiai H

LABORATORY INVESTIGATION 75 巻 ( 2 ) 頁： 239 - 248 1996年8月

Induction of renal cell carcinoma in male Wistar rats treated with cupric nitrilotriacetate.

Toyokuni S, Tanaka T, Nishiyama Y, Okamoto K, Nakashima Y, Hamazaki S, Okada S, Hiai H

Laboratory investigation; a journal of technical methods and pathology 75 巻 ( 2 ) 頁： 239 - 48 1996年8月

Oxidative stress response in iron-induced renal carcinogenesis: Acute nephrotoxicity mediates the enhanced expression of glutathione S-transferase Yp isozyme 査読有り国際誌

Fukuda A, Osawa T, Oda H, Toyokuni S, Satoh K, Uchida K

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 329 巻 ( 1 ) 頁： 39 - 46 1996年5月

Fukuda A., Osawa T., Oda H., Toyokuni S., Satoh K., Uchida K.

Archives of Biochemistry and Biophysics 329 巻 ( 1 ) 頁： 39 - 46 1996年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces acute renal proximal tubular necrosis, a consequence of free radical-mediated oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we investigated the free radical-induced oxidative stress response in this carcinogenesis model, focusing on the expression of glutathione S-transferases (GSTs) which catalyze the conjugation of reactive chemicals with glutathione and play an important role in protecting cells. A single intraperitoneal Fe-NTA treatment (15 mg Fe/kg body weight) induced a rapid oxidative stress, which was monitored by the accumulation of lipid peroxidation products and the loss of sulfhydryl contents in the kidneys, resulting in a 30% reduction of GST activity 1 h after an Fe-NTA treatment. The enzyme activity returned to the control level after 16 h. The immunoblot analysis of GST isozymes demonstrated that the level of α-class GSTs (GST-Ya and GST-Yc) and π- class GST (GST-Yp), major GST isozymes constitutively produced in the kidney, decreased immediately within 1 h of the Fe-NTA treatment. The onset of the recovery of GST-Yp protein levels was detected 3 h after the Fe-NTA treatment. The enhanced production of GST-Yp in gene expression was evident in the drastic elevation of mRNA levels and these increases coincided with a substantial rise in the GST activity and protein levels. The α-class GSTs were not inducible by treatment with Fe-NTA. The immunohistochemical analysis demonstrated that the expression of GST-Yp was strongly induced in the regenerating proximal tubular cells. A steady accumulation of GST-Yp protein was observed in the subacute toxicity experiments with multiple injections of Fe-NTA. These results suggest that the enhanced expression of GST-Yp is important in mediating cell repairs or increasing the resistance to subsequent injury.

DOI： 10.1006/abbi.1996.0189

Overexpression of human mutT homologue gene messenger RNA in renal-cell carcinoma: Evidence of persistent oxidative stress in cancer

Overexpression of human mutT homologue gene messenger RNA in renal-cell carcinoma: Evidence of persistent oxidative stress in cancer

Okamoto K, Toyokuni S, Kim WJ, Ogawa O, Kakehi Y, Arao S, Hiai H, Yoshida O

INTERNATIONAL JOURNAL OF CANCER 65 巻 ( 4 ) 頁： 437 - 441 1996年2月

Okamoto K., Toyokuni S., Kim W., Ogawa O., Kakehi Y., Arao S., Hiai H., Yoshida O.

International Journal of Cancer 65 巻 ( 4 ) 頁： 437 - 441 1996年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Cancer

Data regarding oxidatively modified DNA bases suggest that cancer cells are more exposed to oxidative stress than adjacent non-tumorous tissue. This novel concept may contribute to the understanding of certain aspects of tumor biology such as activated transcription factors, genetic instability, chemotherapy resistance and metastasis. We therefore tested this concept in human renal-cell carcinomas (RCCs) by evaluating the expression of hMTHI, an enzyme preventing the misincorporation into DNA of 8-oxo-dGTP (8-oxo-7,8 -dihydrodeoxyguanosine triphosphate), an oxidized form of dGTP in the nucleotide pool. The expression of hMTHI messenger RNA (mRNA) in RCC was significantly higher than that in adjacent nontumorous kidney. Moreover, advanced-stage tumors showed significantly higher hMTHI mRNA expression than early-stage tumors, and there was a modest linear correlation between hMTHI expression and c-myc expression. The results provide logical support for the concept of 'persistent oxidative stress in cancer' and suggest a role of hMTHI mRNA level as prognostic marker.

DOI： 10.1002/(SICI)1097-0215(19960208)65:4<437::AID-IJC7>3.0.CO;2-Y

Oxidative stress response in iron-induced acute nephrotoxicity: Enhanced expression of heat shock protein 90

Overexpression of human mutT homologue gene messenger RNA in renal-cell carcinoma: Evidence of persistent oxidative stress in cancer

Okamoto K, Toyokuni S, Kim WJ, Ogawa O, Kakehi Y, Arao S, Hiai H, Yoshida O

INTERNATIONAL JOURNAL OF CANCER 65 巻 ( 4 ) 頁： 437 - 441 1996年2月

Oxidative stress response in iron-induced acute nephrotoxicity: Enhanced expression of heat shock protein 90 査読有り国際誌

Fukuda A, Osawa T, Oda H, Tanaka T, Toyokuni S, Uchida K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 219 巻 ( 1 ) 頁： 76 - 81 1996年2月

Fukuda A., Osawa T., Oda H., Tanaka T., Toyokuni S., Uchida K.

Biochemical and Biophysical Research Communications 219 巻 ( 1 ) 頁： 76 - 81 1996年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Iron overload with ferric nitrilotriacetate (Fe-NTA) induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage, that leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we determined the proteins preferentially produced in response to the Fe-NTA-induced oxidative injury. A single intraperitoneal Fe-NTA treatment led to the enhanced production of a number of proteins with molecular masses of 85-95 kDa. These included heat shock protein 90 (HSP90) as determined by immunoprecipitation. The enhanced production of HSP90 was prominent in the renal tubular cells. Steady accumulation of HSP90 was observed in the subacute toxicity experiments with multiple injections of Fe-NTA, suggesting that the enhanced production of HSP90 is important in increasing resistance to subsequent injury caused by the Fe-NTA-induced oxidative stress.

DOI： 10.1006/bbrc.1996.0184

Iron-induced carcinogenesis: The role of redox regulation

Oxidative stress response in iron-induced acute nephrotoxicity: Enhanced expression of heat shock protein 90 査読有り国際誌

Fukuda A, Osawa T, Oda H, Tanaka T, Toyokuni S, Uchida K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 219 巻 ( 1 ) 頁： 76 - 81 1996年2月

Association between 8-hydroxy-2'-deoxyguanosine formation and DNA strand breaks mediated by copper and iron

Toyokuni S, Sagripanti JL

FREE RADICAL BIOLOGY AND MEDICINE 20 巻 ( 6 ) 頁： 859 - 864 1996年

Iron-induced carcinogenesis: The role of redox regulation

Toyokuni S

FREE RADICAL BIOLOGY AND MEDICINE 20 巻 ( 4 ) 頁： 553 - 566 1996年

Toyokuni S.

Free Radical Biology and Medicine 20 巻 ( 4 ) 頁： 553 - 566 1996年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Redox cycling is a characteristic of transition metals such as iron. Iron is hypothesized to have been actively involved in the birth of primitive life on earth through the generation of reducing equivalents in the presence of UV light. Iron is an essential metal in mammals for oxygen transport by hemoglobin and for the function of many enzymes including catalase and cytochromes. However, the 'free' or 'catalytic' form of iron mediates the production of reactive oxygen species via the Fenton reaction and induces oxidative stress. Serum 'free' iron is observed in rare situations such as in severe hemochromatosis in which serum transferrin is saturated. However, it is known that superoxide can release 'free' iron from ferritin and hemosiderin in the cell. 'Free' iron is quite cytotoxic as well as mutagenic and carcinogenic. Iron compounds were first reported to induce sarcomas in rats by Richmond in 1959. Thereafter, several iron-induced carcinogenesis models were established, including the ferric nitrilotriacetate model by Okada and colleagues. Iron may have a role in the carcinogenic process of other transition metals such as copper and nickel, or other kinds of carcinogens such as nitrosamine and even virus-induced carcinogenesis. In humans, genetic hemochromatosis and asbestosis are two major diseases associated with iron-induced carcinogenesis. There is an increasing number of reports of an association between increased body iron stores and increased risk of cancer. Iron-induced oxidative stress results in two possible consequences: (1) redox regulation failure that leads to lipid peroxidation and oxidative DNA and protein damage; (2) redox regulation that activates a variety of reducing and oxystress-protective mechanisms via signal transduction. Both consequences appear to play a role in iron-induced carcinogenesis.

DOI： 10.1016/0891-5849(95)02111-6

Association between 8-hydroxy-2′-deoxyguanosine formation and DNA strand breaks mediated by copper and iron

Toyokuni S., Sagripanti J.

Free Radical Biology and Medicine 20 巻 ( 6 ) 頁： 859 - 864 1996年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

Oxidative DNA damage is involved in diverse biological phenomena and consists of several kinds of lesions, mainly, strand breaks, base modifications, and DNA-protein crosslinking. However, little is known about the existence of a chemical relationship among them or the ratio by which these different types of lesions are produced. In the present study we investigated whether a relationship exists between DNA strand breakage and base modification. We selected cupric [Cu(II)] and ferric [Fe(III)] ions for this study because these transition metals are active catalysts of DNA damage in vivo. Supercoiled plasmid DNA pZ189 was treated with Cu(II) or Fe(III) in the presence of different reducing agents. We measured in each sample both the number of DNA single-strand breaks (SSB) by quantitative electrophoresis and the amount of a modified DNA base, 8-hydroxy-2′-deoxyguanosine (8-OHdG) by HPLC with simultaneous electrochemical (EC) and spectrophotometric detection. The number of DNA SSBs produced was linearly related to the number of 8-OHdG present. The regression of the number of SSBs as a function of the number of 8-OHdG is expressed by the equation [SSBs] = b × [8-OHdG], where b = 1.7, 2.0, 2,7, 1.7, and 9.4, for Cu(II) in the presence of H2O2, L-cysteine and L-ascorbate, and for Fe(III) in the presence of H2O2 and L-ascorbate, respectively. The linear correlation we observed between the production of SSB and 8-OHdG mediated by Fe(III) and by Cu(II) suggests that these products may arise via a common chemical mechanism and could allow an easier and more precise estimation of DNA breakage.

DOI： 10.1016/0891-5849(95)02184-1

8-Hydroxy-2'-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure

Hattori Y., Nishigori C., Tanaka T., Uchida K., Nikaido O., Osawa T., Hiai H., Imamura S., Toyokuni S.

Journal of Investigative Dermatology 107 巻 ( 5 ) 頁： 733 - 737 1996年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Investigative Dermatology

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a mutation-prone (G:C to T:A transverion) DNA base-modified product generated by reactive oxygen species or photodynamic action. G:C to T:A transversions are observed in the p53 and ras genes of UVB-induced skin cancers of mice and in squamous and basal cell carcinomas of human skin exposed to sun-light. In the current study, 8-OHdG formation was evaluated in the epidermis of hairless mice after repeated exposure to UVB, and possible mechanisms involved were studied. Exposure of hairless mice to either 3.4 [2 minimal erythema dose (MED)] or 16.8 (10 MED) kJ/m2 of UVB three times a week for 2 wk induced a 2.5- or 6.1-fold increase, respectively, in the levels of 8-OHdG in DNA, compared to the unexposed controls. An immunohistochemical method using a monoclonal antibody specific for 8-OHdG showed stronger and more extensive staining in the nuclei of UV-irradiated epidermal cells than in those of nonirradiated cells. Western blots probed with antibodies against 4-hydroxy-2-nonenal-modified proteins confirmed the involvement of reactive oxygen species in the epidermal damage induced by chronic UVB exposure. 3-Nitro-L-tyrosine was detected in western blots in a concentration-dependent manner, suggesting that peroxynitrite derived from the reaction of nitric oxide and superoxide, both of which were probably released from inflammatory cells, was involved in modifying the DNA bases. Therefore, the formation of 8-OHdG after UVB exposure appears to be regulated by at least three pathways: photodynamic action, lipid peroxidation, and inflammation and may play a role in sunlight-induced skin carcinogenesis.

DOI： 10.1111/1523-1747.ep12365625

Characterization of epitopes recognized by 4-Hydroxy-2-nonenal specific antibodies

Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies 査読有り国際誌

Uchida K, Itakura K, Kawakishi S, Hiai H, Toyokuni S, Stadtman ER

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 324 巻 ( 2 ) 頁： 241 - 248 1995年12月

Uchida K., Itakura K., Kawakishi S., Hiai H., Toyokuni S., Stadtman E.

Archives of Biochemistry and Biophysics 324 巻 ( 2 ) 頁： 241 - 248 1995年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

In the present study, we have raised anti-peptide antibodies directed to the major membrane lipid peroxidation product 4-hydroxy-2-nonenal (HNE) attached covalently to histidine, and their specificities were compared with those of the polyclonal antibodies (anti-HNE-protein antibodies) raised against HNE-treated keyhole limpet hemocyanin (K. Uchidaet al.(1993)Proc. Natl. Acad. Sci. USA90, 8742-8746). The anti-HNE-histidyl peptide antibodies (anti-HNE-histidine antibodies) were prepared by immunizing rabbits with a HNE-conjugated heptapeptide (Gly3-His-Gly3amide) coupled to the carrier protein. The antisera were purified on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide (Ala3-His-Ala3amide). Among the structurally defined 4-hydroxy-2-alkenal-amino acid adducts tested, binding of anti-HNE-histidine antibodies to the HNE-treated protein was not only inhibited by HNE-histidine, HNE-cysteine, and HNE-lysine, but also by 4-hydroxy-2-octenal-histidine and 4-hydroxy-2-decenal-histidine adducts. Cross-reactivity studies revealed that both anti-HNE-protein antibodies had the highest affinity for the HNE-treated protein and that neither of the antibodies cross-reacted with the protein treated with aldehydes including malondialdehyde, 1-hexanal, 2-hexenal, or 2-nonenal. These results suggest that the dominant epitope recognized by antibodies is the 2-CH3(CH2)n-5-hydroxytetrahydrofuran (n≥ 3) moiety of the Michael adducts. The immunohistochemical analysis of atherosclerotic lesions of human aorta demonstrated that these antibodies reacted strongly with granular cytoplasmic elements of foam cells and weakly with elements in the surrounding sclerotic stroma. © 1995 Academic Press. All rights reserved.

DOI： 10.1006/abbi.1995.0036

Low Incidence of Point Mutations in H‐, K‐ and N‐ras Oncogenes and p53 Tumor Suppressor Gene in Renal Cell Carcinoma and Peritoneal Mesothelioma of Wistar Rats Induced by Ferric Nitrilotriacetate

Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies 査読有り国際誌

Uchida K, Itakura K, Kawakishi S, Hiai H, Toyokuni S, Stadtman ER

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 324 巻 ( 2 ) 頁： 241 - 248 1995年12月

Low incidence of point mutations in H-, K- and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate

Nishiyama Y, Suwa H, Okamoto K, Fukumoto M, Hiai H, Toyokuni S

JAPANESE JOURNAL OF CANCER RESEARCH 86 巻 ( 12 ) 頁： 1150 - 1158 1995年12月

Nishiyama Y., Suwa H., Okamoto K., Fukumoto M., Hiai H., Toyokuni S.

Japanese Journal of Cancer Research 86 巻 ( 12 ) 頁： 1150 - 1158 1995年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Cancer Research

An iron chelate, ferric nitrilotriacetate (Fe‐NTA), induces renal proximal tubular damage, a consequence of iron‐catalyzed free radical reactions, that finally leads to a high incidence of renal cell carcinoma (RCC) in rodents. Previous studies have identified, within 24 h after administration of Fe‐NTA, lipid peroxidation products, aldehyde‐modified proteins and a variety of modified DNA bases such as 8‐hydroxyguanine that may be mutagenic in vivo. In the present study, pathological features of the RCCs were studied, and, in an effort to correlate them with carcinogen‐specific molecular events in Fe‐NTA‐induced carcinogenesis, the H‐, K‐ and N‐ras oncogenes and the p53 tumor suppressor gene were investigated for the presence of mutations. Fe‐NTA‐induced RCCs showed similarity to human RCCs in that they are often invasive, metastatic and fatal. None (0 of 12) of the tumors had mutation in codons 12, 13 and 61 of the H‐, K‐ and N‐ras genes by direct sequencing. Only one (1 of 12) tumor with high grade histology revealed a CGC‐to‐CTC (Arg to Leu) transversion in codon 246 of the p53 gene by the use of single strand conformation polymorphism (SSCP) analysis and direct sequencing. High expression of mutant p53 protein was confirmed by Western blotting and immunohistochemistry. Study of three peritoneal mesotheliomas induced by Fe‐NTA revealed no mutation in ras and p53 genes. These results suggest that the ras and p53 genes are not the major targets of mutation in Fe‐NTA‐induced carcinogenesis of kidney and mesothelium. Instead, p53 mutation may work for potentiation of malignant character in Fe‐NTA‐induced renal carcinogenesis. Copyright © 1995, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1349-7006.1995.tb03308.x

Low incidence of point mutations in H-, K- and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of wistar rats induced by ferric nitrilotriacetate 査読有り国際誌

Nishiyama Y., Suwa H., Okamoto K., Fukumoto M., Hiai H., Toyokuni S.

Japanese Journal of Cancer Research 86 巻 ( 12 ) 頁： 1150 - 1158 1995年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Journal of Cancer Research

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage, a consequence of iron-catalyzed free radical reactions, that finally leads to a high incidence of renal cell carcinoma (RCC) in rodents. Previous studies have identified, within 24 h after administration of Fe-NTA, lipid peroxidation products, aldehyde-modified proteins and a variety of modified DNA bases such as 8-hydroxyguanine that may be mutagenic in vivo. In the present study, pathological features of the RCCs were studied, and, in an effort to correlate them with carcinogen-specific molecular events in Fe-NTA-induced carcinogenesis, the H-, K- and N-ras oncogenes and the p53 tumor suppressor gene were investigated for the presence of mutations. Fe-NTA-induced RCCs showed similarity to human RCCs in that they are often invasive, metastatic and fatal. None (0 of 12) of the tumors had mutation in codons 12,13 and 61 of the H-, K- and N-ras genes by direct sequencing. Only one (1 of 12) tumor with high grade histology revealed a CGC-to-CTC (Arg to Leu) transversion in codon 246 of the p53 gene by the use of single strand conformation polymorphism (SSCP) analysis and direct sequencing. High expression of mutant p53 protein was confirmed by Western blotting and immunohistochemistry. Study of three peritoneal mesotheliomas induced by Fe-NTA revealed no mutation in ras and p53 genes. These results suggest that the ras and p53 genes are not the major targets of mutation in Fe-NTA-induced carcinogenesis of kidney and mesothelium. Instead, p53 mutation may work for potentiation of malignant character in Fe-NTA-induced renal carcinogenesis. © 1995.

DOI： 10.1016/0910-5050(96)85255-0

Treatment of wistar rats with a renal carcinogen, ferric nitrilotriacetate, causes dna‐protein cross‐linking between thymine and tyrosine in their renal chromatin

Low incidence of point mutations in H-, K- and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate

Nishiyama Y, Suwa H, Okamoto K, Fukumoto M, Hiai H, Toyokuni S

JAPANESE JOURNAL OF CANCER RESEARCH 86 巻 ( 12 ) 頁： 1150 - 1158 1995年12月

TREATMENT OF WISTAR RATS WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE, CAUSES DNA-PROTEIN CROSS-LINKING BETWEEN THYMINE AND TYROSINE IN THEIR RENAL CHROMATIN

TOYOKUNI S, MORI T, HIAI H, DIZDAROGLU M

INTERNATIONAL JOURNAL OF CANCER 62 巻 ( 3 ) 頁： 309 - 313 1995年7月

Toyokuni S., Mori T., Hiai H., Dizdaroglu M.

International Journal of Cancer 62 巻 ( 3 ) 頁： 309 - 313 1995年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Cancer

Ferric nitrilotriacetate (Fe‐NTA) induces renal proximal tubular damage associated with lipid peroxidation and oxidative DNA base modifications that finally leads to a high incidence of renal adenocarcinoma in rodents. In the present study, we report on the in vivo formation of DNA‐protein cross‐links (DPCs) involving thymine and tyrosine in the renal chromatin of Wistar rats treated with single or repeated i.p. administration of Fe‐NTA. Analyses of chromatin samples by gas chromatography/mass spectrometry revealed a significant increase in the amount of 3‐[(1, 3‐dihydro‐2, 4‐dioxopyrimidin‐5‐yl)‐methyl]‐L‐tyrosine (Thy‐Tyr cross‐link) 24 and 48 hr after the administration of Fe‐NTA. At 19th day of Fe‐NTA treatment, the amount of Thy‐Tyr cross‐link decreased to the control level, indicating the presence of cellular repair activity. Thy‐Tyr cross‐link may play a role in the genetic alteration of this renal carcinogenesis model, since mitoses for regeneration of renal proximal tubules were closely associated with the increase in DPCs. © 1995 Wiley‐Liss Inc. Copyright © 1995 Wiley‐Liss, Inc., A Wiley Company

DOI： 10.1002/ijc.2910620313

Degradation of DNA in dried tissues by atmospheric oxygen

TREATMENT OF WISTAR RATS WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE, CAUSES DNA-PROTEIN CROSS-LINKING BETWEEN THYMINE AND TYROSINE IN THEIR RENAL CHROMATIN

TOYOKUNI S, MORI T, HIAI H, DIZDAROGLU M

INTERNATIONAL JOURNAL OF CANCER 62 巻 ( 3 ) 頁： 309 - 313 1995年7月

4-HYDROXY-2-NONENAL-TRAPPING ELISA - DIRECT EVIDENCE FOR THE RELEASE OF A CYTOTOXIC ALDEHYDE FROM OXIDIZED LOW-DENSITY LIPOPROTEINS 査読有り国際誌

UCHIDA K, OSAWA T, HIAI H, TOYOKUNI S

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 212 巻 ( 3 ) 頁： 1068 - 1073 1995年7月

DEGRADATION OF DNA IN DRIED TISSUES BY ATMOSPHERIC OXYGEN

MATSUO S, TOYOKUNI S, OSAKA M, HAMAZAKI S, SUGIYAMA T

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 208 巻 ( 3 ) 頁： 1021 - 1027 1995年3月

Matsuo S., Toyokuni S., Osaka M., Hamazaki S., Sugiyama T.

Biochemical and Biophysical Research Communications 208 巻 ( 3 ) 頁： 1021 - 1027 1995年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Recently, DNA of extinct creatures have been brought into analyses. The difficulty in this field of research is DNA degradation. Oxidative damage is considered to be one of the causes of the DNA degradation. We studied how DNA in dried tissue was affected by atmospheric oxygen using freeze-dried rat liver as a model. In tissues exposed to oxygen, DNA degradation occurred within several months and the amount of 8-hydroxy-2′-deoxyguanosine in DNA rapidly increased. The DNA degradation was inhibited by lipid extraction prior to the exposure to atmospheric oxygen. Purified lambda phage DNA was not affected by oxygen. Cellular DNA and RNA were degraded slowly in nitrogen air. These results suggest that both atmospheric and endogenous oxygens play a role in DNA degradation in dried tissues. © 1995 Academic Press, Inc.

DOI： 10.1006/bbrc.1995.1436

A Renal carcinogen ferric nitrilotriacetate mediates a temporary accumulation of aldehyde-modified proteins within cytosolic compartment of rat-Kidney

DEGRADATION OF DNA IN DRIED TISSUES BY ATMOSPHERIC OXYGEN

MATSUO S, TOYOKUNI S, OSAKA M, HAMAZAKI S, SUGIYAMA T

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 208 巻 ( 3 ) 頁： 1021 - 1027 1995年3月

A RENAL CARCINOGEN FERRIC NITRILOTRIACETATE MEDIATES A TEMPORARY ACCUMULATION OF ALDEHYDE-MODIFIED PROTEINS WITHIN CYTOSOLIC COMPARTMENT OF RAT-KIDNEY 査読有り国際誌

UCHIDA K, FUKUDA A, KAWAKISHI S, HIAI H, TOYOKUNI S

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 317 巻 ( 2 ) 頁： 405 - 411 1995年3月

Uchida K., Fukuda A., Kawakishi S., Hiai H., Toyokuni S.

Archives of Biochemistry and Biophysics 317 巻 ( 2 ) 頁： 405 - 411 1995年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Iron overload with ferric nitrilotriacetate (Fe-NTA) induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. A recent immunohistochemical study has revealed that the materials immunoreactive to the antibody against the aldehyde (4-hydroxy-2-nonenal)-modified proteins are generated in the renal proximal tubules of rats treated with Fe-NTA (S. Toyokuni et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620). Here we present further evidences that cytosolic proteins modified with lipid peroxidation-derived aldehydes (4-hydroxy-2-nonenal and malondialdehyde) are indeed accumulated in the kidney of rats treated with Fe-NTA. A single intraperitoneaI Fe-NTA treatment (15 or 30 mg Fe/kg body wt) induced a rapid accumulation of thiobarbituric acid-reactive substances, a direct consequence of membrane lipid peroxidation, mostly within the cytosolic compartment of rat kidney. The accumulation of lipid peroxidation-derived aldehydes was associated with an apparent accumulation (from 67.9 nmol in untreated controls to 83.3 nmol/mg protein in Fe-NTA-treated rats at 3 h after treatment) and subsequent elimination of aberrant proteins as assessed by a 2, 4-dinitrophenylhydrazine postlabeling assay. Immunoblot analysis with two different polyclonal antibodies against 4-hydroxy-2-nonenal-modified and malondialdehyde-modified proteins detected a temporary accumulation of aldehyde-modified proteins in the cytosol. An immunohistochemical technique with serial sectioning revealed that immunoreactivities of these antibodies were observed in the identical portion of the renal proximal tubules. The implications of these data in relation to the nephrotoxicity of Fe-NTA-are discussed. © 1995 Academic Press. All rights reserved.

DOI： 10.1006/abbi.1995.1181

A RENAL CARCINOGEN FERRIC NITRILOTRIACETATE MEDIATES A TEMPORARY ACCUMULATION OF ALDEHYDE-MODIFIED PROTEINS WITHIN CYTOSOLIC COMPARTMENT OF RAT-KIDNEY 査読有り

K UCHIDA, A FUKUDA, S KAWAKISHI, H HIAI, S TOYOKUNI

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 317 巻 ( 2 ) 頁： 405 - 411 1995年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS

Iron overload with ferric nitrilotriacetate (Fe-NTA) induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. A recent immunohistochemical study has revealed that the materials immunoreactive to the antibody against the aldehyde (4-hydroxy-2-nonenal)-modified proteins are generated in the renal proximal tubules of rats treated with Fe-NTA (S. Toyokuni et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620). Here we present further evidences that cytosolic proteins modified with lipid peroxidation-derived aldehydes (4-hydroxy-2-nonenal and malondialdehyde) are indeed accumulated in the kidney of rats treated with Fe-NTA. A single intraperitoneaI Fe-NTA treatment (15 or 30 mg Fe/kg body wt) induced a rapid accumulation of thiobarbituric acid-reactive substances, a direct consequence of membrane lipid peroxidation, mostly within the cytosolic compartment of rat kidney. The accumulation of lipid peroxidation-derived aldehydes was associated with an apparent accumulation (from 67.9 nmol in untreated controls to 83.3 nmol/mg protein in Fe-NTA-treated rats at 3 h after treatment) and subsequent elimination of aberrant proteins as assessed by a 2,4-dinitrophenylhydrazine postlabeling assay. Immunoblot analysis with two different polyclonal antibodies against 4-hydroxy-2-nonenal-modified and malondialdehyde-modified proteins detected a temporary accumulation of aldehyde-modified proteins in the cytosol. An immunohistochemical technique with serial sectioning revealed that immunoreactivities of these antibodies were observed in the identical portion of the renal proximal tubules. The implications of these data in relation to the nephrotoxicity of Fe-NTA-are discussed. (C) 1995 Academic Press, Inc.

The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct

THE MONOCLONAL-ANTIBODY SPECIFIC FOR THE 4-HYDROXY-2-NONENAL HISTIDINE ADDUCT 査読有り国際誌

TOYOKUNI S, MIYAKE N, HIAI H, HAGIWARA M, KAWAKISHI S, OSAWA T, UCHIDA K

FEBS LETTERS 359 巻 ( 2-3 ) 頁： 189 - 191 1995年2月

Toyokuni S., Miyake N., Hiai H., Hagiwara M., Kawakishi S., Osawa T., Uchida K.

FEBS Letters 359 巻 ( 2-3 ) 頁： 189 - 191 1995年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：FEBS Letters

Monoclonal antibodies directed against proteins modified with the major membrane lipid peroxidation product, 4-hydroxy-2-nonenal, have been established and characterized. The monoclonal antibodies specific for HNE-modified proteins were raised by immunizing mice with a HNE-keyhole limpet hemocyanin conjugate. The resulting five monoclonal antibodies (mAbs HNEJ-1-5) recognized HNE-modified bovine serum albumin (BSA), but not native BSA in Western blot studies. Of the five mAbs, HNEJ-2 exhibited the highest affinity for HNE-modified proteins and a much higher affinity for the HNE-histidine adduct than the HNE-lysine or HNE-cysteine adducts. mAb HNEJ-2 did not cross-react with proteins that had been treated with other aldehydes, such as 1-hexenal, 2-hexenal, 4-hydroxy-2-hexenal, 2-nonenal, formaldehyde, or glutaraldehyde. These results suggest that the major epitope recognized by mAb HNEJ-2 is the Michael addition-type HNE-histidine adduct. © 1995.

DOI： 10.1016/0014-5793(95)00033-6

Cytotoxic and mutagenic effects of ferric nitrilotriacetate on L5178Y mouse lymphoma cells

CYTOTOXIC AND MUTAGENIC EFFECTS OF FERRIC NITRILOTRIACETATE ON L5178Y MOUSE LYMPHOMA-CELLS

TOYOKUNI S, SAGRIPANTI JL, HITCHINS VM

CANCER LETTERS 88 巻 ( 2 ) 頁： 157 - 162 1995年1月

CYTOTOXIC AND MUTAGENIC EFFECTS OF FERRIC NITRILOTRIACETATE ON L5178Y MOUSE LYMPHOMA-CELLS

TOYOKUNI S, SAGRIPANTI JL, HITCHINS VM

CANCER LETTERS 88 巻 ( 2 ) 頁： 157 - 162 1995年1月

Toyokuni S., Sagripanti J., Hitchins V.

Cancer Letters 88 巻 ( 2 ) 頁： 157 - 162 1995年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Letters

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis that leads to a high incidence of renal adenocarcinoma in rodents. Others have shown that Fe-NTA induces modified DNA base products both in vitro and in vivo. However, Fe-NTA is negative in the Ames Salmonella test with or without S9 activation. The goal of this project was to determine if Fe-NTA is cytotoxic and mutagenic using the L5178Y (TK+/-) mouse lymphoma assay. Our experiments showed a relationship between the concentration of Fe-NTA (0 to 1 mM) and the decrease in relative survival. An exposure-dependent increase in the number of mutations was observed with increasing concentrations of Fe-NTA. At 14% relative survival, there was about a 4-fold increase in mutations (trifluorothymidine resistance) over unexposed, control cells. Ferric nitrate or nitrilotriacetic acid alone induced a relatively tow 1.5- or 1.1-fold increase in mutation, respectively. Our results establish that Fe-NTA is mutagenic in the L5178Y mouse lymphoma assay system. © 1995.

DOI： 10.1016/0304-3835(94)03641-U

Persistent oxidative stress in cancer

PERSISTENT OXIDATIVE STRESS IN CANCER

TOYOKUNI S, OKAMOTO K, YODOI J, HIAI H

FEBS LETTERS 358 巻 ( 1 ) 頁： 1 - 3 1995年1月

Toyokuni S., Okamoto K., Yodoi J., Hiai H.

FEBS Letters 358 巻 ( 1 ) 頁： 1 - 3 1995年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：FEBS Letters

DNA of cancers such as renal cell carcinoma and mammary invasive ductal carcinoma, is persistently exposed to more oxidative stress than that of adjacent nornal tissue. We suggest that the concept of 'persistent oxidative stress in cancer' may open up a new research area, explaining part of the characteristic tumor biology of cancer such as activated transcription factors and proto-oncogenes, genomic instability, chemotherapy-resistance, invasion and metastasis. © 1995.

DOI： 10.1016/0014-5793(94)01368-B

Acute nephrotoxicity of a carcinogenic iron chelate Selective inhibition of a proteolytic conversion of α<inf>2U</inf>-globulin to the kidney fatty acid-binding protein

PERSISTENT OXIDATIVE STRESS IN CANCER

TOYOKUNI S, OKAMOTO K, YODOI J, HIAI H

FEBS LETTERS 358 巻 ( 1 ) 頁： 1 - 3 1995年1月

ACUTE NEPHROTOXICITY OF A CARCINOGENIC IRON CHELATE - SELECTIVE-INHIBITION OF A PROTEOLYTIC CONVERSION OF ALPHA(2U)-GLOBULIN TO THE KIDNEY FATTY-ACID-BINDING PROTEIN 査読有り国際誌

UCHIDA K, FUKUDA A, KAWAKISHI S, TOYOKUNI S, HIAI H, IKEDA S, HORIO F

FEBS LETTERS 357 巻 ( 2 ) 頁： 165 - 167 1995年1月

Uchida K., Fukuda A., Kawakishi S., Toyokuni S., Hiai H., Ikeda S., Horio F.

FEBS Letters 357 巻 ( 2 ) 頁： 165 - 167 1995年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：FEBS Letters

The mechanism of acute nephrotoxicity of an iron chelate in vivo has been investigated. Administration of a renal carcinogen ferric nitrolotriacetate (Fe-NTA) (15 mg Fe/kg body weight, intraperitoneally) led to selective loss of a renal protein with an apparent molecular mass of 17 kDa. Analysis of the 17 kDa protein by NH2-terminal sequence demonstrated its identity over 16 NH2-terminal residues as a kidney fatty acid-binding protein (k-FABP) that is a proteolytically modified form of α2U-globulin, a major urinary protein of adult male rats. An immunochemical study using anti-α2U-globulin polyclonal antibodies confirmed that a single injection of Fe-NTA led to a decrease in k-FABP levels. However, a 19-kDa protein identical to the α2U-globulin progressively appeared in the kidney, suggesting that the proteolytic processing of α2U-globulin in the renal proximal tubules was suppressed by the treatment with Fe-NTA. By monitoring k-FABP and its precursor α2U-globulin, it was determined that repeated exposure to Fe-NTA caused suppression of both proteolytic and endocytotic activity of the kidney. The implications of these data in relation to the nephrotoxicity of Fe-NTA are discussed. © 1995.

DOI： 10.1016/0014-5793(94)01330-4

4-Hydroxy-2-nonenal-trapping elisa: Direct evidence for the release of a cytotoxic aldehyde from oxidized low density lipoproteins

Uchida K., Osawa T., Hiai H., Toyokuni S.

Biochemical and Biophysical Research Communications 212 巻 ( 3 ) 頁： 1068 - 1073 1995年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Among the aldehydes that originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-nonenal (HNE) is thought to be largely responsible for cytopathological effects observed during oxidative stress. Taking advantage of the fact that HNE is very reactive with proteins and forms stable Michael addition-type adducts, a novel immunochemical procedure for quantifying “free” HNE has been developed. The method designated as “HNE-trapping ELISA” is based on the detection of HNE trapped by a protein that has been coated in the immunoplate. The HNE-derived epitopes generated in the coating protein are then detected by the ELISA using a monoclonal antibody (mAbHNEJ-2) specific to the haptenic groups of the HNE-protein conjugates. Using this method, we determined that a considerable amount of HNE was released from human plasma low density lipoproteins (LDL) treated with copper ions or endothelial cells. © 1995 by Academic Press, Inc.

DOI： 10.1006/bbrc.1995.2078

Michael Addition-Type 4-Hydroxy-2-nonenal Adducts in Modified Low-Density Lipoproteins: Markers for Atherosclerosis

MICHAEL ADDITION-TYPE 4-HYDROXY-2-NONENAL ADDUCTS IN MODIFIED LOW-DENSITY LIPOPROTEINS - MARKERS FOR ATHEROSCLEROSIS 査読有り国際誌

UCHIDA K, TOYOKUNI S, NISHIKAWA K, KAWAKISHI S, ODA H, HIAI H, STADTMAN ER

BIOCHEMISTRY 33 巻 ( 41 ) 頁： 12487 - 12494 1994年10月

MICHAEL ADDITION-TYPE 4-HYDROXY-2-NONENAL ADDUCTS IN MODIFIED LOW-DENSITY LIPOPROTEINS - MARKERS FOR ATHEROSCLEROSIS 査読有り国際誌

UCHIDA K, TOYOKUNI S, NISHIKAWA K, KAWAKISHI S, ODA H, HIAI H, STADTMAN ER

BIOCHEMISTRY 33 巻 ( 41 ) 頁： 12487 - 12494 1994年10月

Uchida K., Toyokuni S., Nishikawa K., Kawakishi S., Oda H., Hiai H., Stadtman E.

Biochemistry 33 巻 ( 41 ) 頁： 12487 - 12494 1994年10月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemistry

It has been proposed that plasma low-density lipoprotein (Lall-trans-bRDL) undergoes oxidative modification before it can give rise to foam cells in atherosclerosis. Oxidation of LDL generates a variety of reactive aldehyde products including 4-hydroxy-2-nonenal (HNE), which may covalently attach to the LDL apolipoproteins. We here present direct evidence that HNE derivatization of LDL forms Michael additiontype adducts of HNE with histidine and lysine residues of apolipoprotein B-100 (apo B) and also demonstrate the utility of an antibody specific to the HNE adducts generated in the LDL treated with HNE or oxidatively modified by Cu2+ or cultured endothelial cells. HNE adducts present in the LDL that had been treated with HNE were attested to be Michael addition-type adducts on the basis of the fact that incubation of LDL with 1 mM HNE (2 h, 37 °C) resulted primarily in the formation of Michael addition-type HNE-histidine (39.9 mol/mol of LDL) and HNE-lysine (19.3 mol/mol of LDL) adducts. An enzyme-linked immunosorbent assay (ELISA) and an SDS-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot analysis of HNE-modified LDL demonstrated that these HNE adducts were detectable with the HNE-specific antibody affinity-purified with the Michael adduct (HNE-histidine) as a ligand. The following lines of evidence indicated the presence of Michael addition-type HNE adducts in the oxidatively modified LDL in vitro: (i) Amino acid analysis of LDL that had been treated with Cu2+ (24 h, 37 °C) demonstrated the presence of a Michael addition-type HNE-histidine adduct (7-9 mol/mol of LDL). (ii) Competitive ELISA demonstrated the inhibition of antibody binding to HNE-modified LDL by the addition of Cu2+- oxidized LDL. (iii) SDS-PAGE/immunoblot analysis of the Cu2+-oxidized LDL demonstrated the appearance of HNE adducts, which were abolished by the addition of a HNE-modified histidyl peptide. Furthermore, HNE adducts were detected in the LDL that had been treated with cultured bovine aortic endothelial cells (24 h, 37 °C). Taken together, this evidence suggests that the Michael addition-type HNE adducts may be reliable markers for atherosclerosis. © 1994, American Chemical Society. All rights reserved.

DOI： 10.1021/bi00207a016

Formation of 8‐hydroxy‐2′‐deoxyguanosine and 4‐hydroxy‐2‐nonenal‐modified proteins in human renal‐cell carcinoma

FORMATION OF 8-HYDROXY-2'-DEOXYGUANOSINE AND 4-HYDROXY-2-NONENAL-MODIFIED PROTEINS IN HUMAN RENAL-CELL CARCINOMA 査読有り国際誌

OKAMOTO K, TOYOKUNI S, UCHIDA K, OGAWA O, TAKENEWA J, KAKEHI Y, KINOSHITA H, HATTORINAKAKUKI Y, HIAI H, YOSHIDA O

INTERNATIONAL JOURNAL OF CANCER 58 巻 ( 6 ) 頁： 825 - 829 1994年9月

Okamoto K., Toyokuni S., Uchida K., Ogawa O., Takenewa J., Kakehi Y., Kinoshita H., Hattori‐Nakakuki Y., Hiai H., Yoshida O.

International Journal of Cancer 58 巻 ( 6 ) 頁： 825 - 829 1994年9月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Cancer

To study the possible involvement of reactive oxygen species (ROS) in the tumor biology of human renal‐cell carcinoma (RCC), we analvzed 35 cases of RCC for 2 parameters of oxidative damage: 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a mutation‐prone DNA‐ base ‐modified product, was measured by means of high‐performance liquid chromatography (HPLC) with an electrochemical (EC) detector, and 4‐hydroxy‐2‐nonenal (HNE)‐modified proteins were measured with a poly‐clonal antibody against HNE‐modified proteins. A 54% higher content of 8‐OHdG was found in RCC than in the corresponding non‐tumorous kidney, suggesting that the DNA of RCC is more exposed to ROS than is the DNA of non‐tumorous kidneys. Immunohistochemistry for HNE‐modified proteins showed a distinct staining pattern of fine to coarse granularity in the cytoplasm of RCC (n = 15), implying that lipid peroxidation products are located in cytoplasmic organelles. These results suggest that RCC constitutionally elaborates more ROS than is produced by the non‐tumorous parts of kidneys. No correlation was found between clinical stage, histology, age or sex and the 2 parameters examined. Copyright © 1994 Wiley‐Liss, Inc., A Wiley Company

DOI： 10.1002/ijc.2910580613

Formation of 8-Hydroxy-2′-Deoxyguanosine in epidermis of hairless mice exposed to near-UV

FORMATION OF 8-HYDROXY-2'-DEOXYGUANOSINE AND 4-HYDROXY-2-NONENAL-MODIFIED PROTEINS IN HUMAN RENAL-CELL CARCINOMA 査読有り国際誌

OKAMOTO K, TOYOKUNI S, UCHIDA K, OGAWA O, TAKENEWA J, KAKEHI Y, KINOSHITA H, HATTORINAKAKUKI Y, HIAI H, YOSHIDA O

INTERNATIONAL JOURNAL OF CANCER 58 巻 ( 6 ) 頁： 825 - 829 1994年9月

FORMATION OF 8-HYDROXY-2'-DEOXYGUANOSINE IN EPIDERMIS OF HAIRLESS MICE EXPOSED TO NEAR-UV

HATTORINAKAKUKI Y, NISHIGORI C, OKAMOTO K, IMAMURA S, HIAI H, TOYOKUNI S

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 201 巻 ( 3 ) 頁： 1132 - 1139 1994年6月

Hattori-Nakakuki Y., Nishigori C., Okamoto K., Imamura S., Hiai H., Toyokuni S.

Biochemical and Biophysical Research Communications 201 巻 ( 3 ) 頁： 1132 - 1139 1994年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Six-week-old male albino hairless mice (Hos: Hr-1) were exposed to a near-ultraviolet (UV) fluorescent sun lamp (33.5 kJ/m2/hr; wave length > 270 nm with a peak at 312.5 nm) to investigate the induction of oxidative DNA damage in epidermal cells. Significantly higher levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) were detected in a dose-dependent manner in epidermis of mice exposed to near-UV than in those of control animals. The ratio of 8-OHdG in near-UV-exposed/unexposed control was 2.08 ± 0.19 after 168 kJ/m2 exposure, P < 0.01; 3.49 ± 0.36 after 335 kJ/m2 exposure, P < 0.01 (means ± SE). The levels of 8-OHdG decreased with time after near-UV exposure, suggesting the presence of removal and/or repair mechanisms. This is the first report that oxidative DNA base modification is induced in vivo in epidermal cells by near-UV exposure. Oxidative DNA base modification may be one of the causes of sunlight-induced skin carcinogenesis. © 1994 Academic Press, Inc.

DOI： 10.1006/bbrc.1994.1823

Increased 8-hydroxydeoxyguanosine in kidney and liver of rats continuously exposed to copper

FORMATION OF 8-HYDROXY-2'-DEOXYGUANOSINE IN EPIDERMIS OF HAIRLESS MICE EXPOSED TO NEAR-UV

HATTORINAKAKUKI Y, NISHIGORI C, OKAMOTO K, IMAMURA S, HIAI H, TOYOKUNI S

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 201 巻 ( 3 ) 頁： 1132 - 1139 1994年6月

INCREASED 8-HYDROXYDEOXYGUANOSINE IN KIDNEY AND LIVER OF RATS CONTINUOUSLY EXPOSED TO COPPER

TOYOKUNI S, SAGRIPANTI JL

TOXICOLOGY AND APPLIED PHARMACOLOGY 126 巻 ( 1 ) 頁： 91 - 97 1994年5月

Toyokuni S., Sagripanti J.

Toxicology and Applied Pharmacology 126 巻 ( 1 ) 頁： 91 - 97 1994年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Toxicology and Applied Pharmacology

Copper is a ubiquitous metal in the environment, it is a component of dental casting gold alloys and dental amalgams, and it is a main component in some intrauterine contraceptive devices (IUDs). Since copper materials implanted in the human body corrode and release ions into the surrounding tissue, the potential toxicity caused by contact of this metal with bodily fluids needs to be evaluated. We implanted male Wistar rats with osmotic mini pumps that continuously administered saline, CuCl2, or a copper chelate, cupric nitrilotriacetate (Cu-NTA), at a rate of 4 mg copper/kg body wt/day. This experimental design maintained serum copper concentrations at a level 30-70% (CuCl2) or 100-120% (Cu-NTA) higher than in untreated controls. At different times postimplantation, we measured the levels of 8-hydroxydeoxyguanosine (8-OHdG) in DNA of kidney, liver, and tissue surrounding the pump implant, since production of 8-OHdG has been associated with mutagenesis and carcinogenesis. Hepatic and renal levels of 8-OHdG in CuCl2- or Cu-NTA-treated animals were significantly higher than in control animals. In contrast, histopathologic changes in kidneys and livers of rats exposed to CuCl2 and Cu-NTA were limited to mild changes involving hepatic focal necrosis and slightly increased mitotic activity in the renal proximal tubules. These observations suggest that levels of 8-OHdG could be an early marker of copper toxicity. It is unlikely that the high levels of copper at which we observed DNA modification will be encountered after occupational or environmental exposure. A different situation could be found around medical devices that include copper, particularly IUDs, where the amount of copper administered in our experiments could be released in the uterus of women after a few months of continued IUD use. © 1994 Academic Press, Inc.

DOI： 10.1006/taap.1994.1094

DNA base modifications in renal chromatin of wistar rats treated with a renal carcinogen, ferric nitrilotriacetate

INCREASED 8-HYDROXYDEOXYGUANOSINE IN KIDNEY AND LIVER OF RATS CONTINUOUSLY EXPOSED TO COPPER

TOYOKUNI S, SAGRIPANTI JL

TOXICOLOGY AND APPLIED PHARMACOLOGY 126 巻 ( 1 ) 頁： 91 - 97 1994年5月

DNA-BASE MODIFICATIONS IN RENAL CHROMATIN OF WISTAR RATS TREATED WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE

TOYOKUNI S, MORI T, DIZDAROGLU M

INTERNATIONAL JOURNAL OF CANCER 57 巻 ( 1 ) 頁： 123 - 128 1994年4月

DNA-BASE MODIFICATIONS IN RENAL CHROMATIN OF WISTAR RATS TREATED WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE

TOYOKUNI S, MORI T, DIZDAROGLU M

INTERNATIONAL JOURNAL OF CANCER 57 巻 ( 1 ) 頁： 123 - 128 1994年4月

Toyokuni S., Mori T., Dizdaroglu M.

International Journal of Cancer 57 巻 ( 1 ) 頁： 123 - 128 1994年4月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Cancer

Ferric nitrilotriacetate (Fe‐NTA) causes renal proximal tubular necrosis, a consequence of iron ion‐mediated free‐radical‐associated damage, that finally leads to a high incidence of renal ad en oca re in dm a in male rats and mice. We have investigated the levels of typical hydroxyl radical‐induced DNA base modifications in renal chromatin of male Wistar rats treated with a single or repeated administrations of Fe‐NTA. Five pyrimidine‐derived and 5 purine‐derived modified DNA bases were identified and quantified by gas chromatography/mass spectrometry with selected‐ion monitoring. The modified bases were 5‐hydroxy‐5‐methylhydantoin, 5‐(hydroxymethyl)uracil, 5‐hydroxy‐cytosine, thymine glycol, 5,6‐dihydroxyuraril, 4,6‐diamino‐5‐form‐amidopyrimidine, 8‐hydroxyadenine, xanthine, 2‐hydroxyadenine and 8‐hydroxyguanine. The amounts of most of these compounds were significantly increased over control levels in renal chromatin of Fe‐NTA‐treated rats as measured 3 and 24 hr after treatment. Elevated levels of modified bases were accompanied by proximal tubular necrosis. On the 19th day, however, accumulation of modified DNA bases was not observed. Morphologically, scattered karyomegaiic cells were seen in the proximal tubules, but necrosis was rarely found. Some of the identified DNA base lesions are known to be promutagenic., although others have not been investigated. Presence of modified DNA bases concomitant with necrosis and regeneration of the renal proximal tubules may be a critical step in Fe‐NTA‐induced carcinogenesis. © Wiley‐Liss, Inc. Copyright © 1994 Wiley‐Liss, Inc., A Wiley Company

DOI： 10.1002/ijc.2910570122

Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate

FORMATION OF 4-HYDROXY-2-NONENAL-MODIFIED PROTEINS IN THE RENAL PROXIMAL TUBULES OF RATS TREATED WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE 査読有り国際誌

TOYOKUNI S, UCHIDA K, OKAMOTO K, HATTORINAKAKUKI Y, HIAI H, STADTMAN ER

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 巻 ( 7 ) 頁： 2616 - 2620 1994年3月

Toyokuni S., Uchida K., Okamoto K., Hattori-Nakakuki Y., Hiai H., Stadtman E.

Proceedings of the National Academy of Sciences of the United States of America 91 巻 ( 7 ) 頁： 2616 - 2620 1994年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces proximal tubular necrosis, a consequence of lipid peroxidation, that finally leads to a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation as monitored by formation of thiobarbituric acid-reactive substances and free 4- hydroxy-2-nonenal (HNE) was observed in the kidney homogenates of rats treated with Fe-NTA. Based on the fact that HNE is capable of reacting with cellular proteins, we attempted to detect the localization of HNE-modified proteins in rat kidney tissues with an immunohistochemical procedure. By means of an immunohistochemical technique using polyclonal antibody against the HNE-modified proteins, it was shown that HNE-modified proteins are formed in the target cells of this carcinogenesis model. HNE-modified proteins were detected in the renal proximal tubules 1 hr after i.p. administration of Fe- NTA (15 mg of iron per kg). Intense positivity was found in the cells with degeneration. After 6 hr, the level of HNE-protein conjugates decreased due to the subsequent necrosis. The intensity of the immunochemical reaction with HNE-modified proteins increased in parallel with an increase in the amounts of thiobartituric acid-reactive substances and free HNE that were found. Furthermore, histochemical detection of aldehydes by cold Schiff's reagent demonstrated that location of aldehydes was identical to that of the HNE- modified proteins determined by immunohistochemical procedures. It would thus appear that the production of HNE, a genotoxic and mutagenic aldehyde, and its reaction with proteins may play a role in Fe-NTA-induced renal carcinogenesis.

DOI： 10.1073/pnas.91.7.2616

FORMATION OF 4-HYDROXY-2-NONENAL-MODIFIED PROTEINS IN THE RENAL PROXIMAL TUBULES OF RATS TREATED WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE 査読有り国際誌

TOYOKUNI S, UCHIDA K, OKAMOTO K, HATTORINAKAKUKI Y, HIAI H, STADTMAN ER

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 巻 ( 7 ) 頁： 2616 - 2620 1994年3月

ABNORMAL BONE-MARROW B-CELL DIFFERENTIATION IN PRE-B LYMPHOMA-PRONE SL KH MICE

OKAMOTO K, YAMADA Y, OGAWA MS, TOYOKUNI S, NAKAKUKI Y, IKEDA H, YOSHIDA O, HIAI H

CANCER RESEARCH 54 巻 ( 2 ) 頁： 399 - 402 1994年1月

GENETIC PREDISPOSITION TO PRE-B LYMPHOMAS IN SL KH STRAIN MICE

YAMADA Y, MATSUSHIRO H, OGAWA MS, OKAMOTO K, NAKAKUKI Y, TOYOKUNI S, FUKUMOTO M, HIAI H

CANCER RESEARCH 54 巻 ( 2 ) 頁： 403 - 407 1994年1月

ABNORMAL BONE-MARROW B-CELL DIFFERENTIATION IN PRE-B LYMPHOMA-PRONE SL KH MICE

OKAMOTO K, YAMADA Y, OGAWA MS, TOYOKUNI S, NAKAKUKI Y, IKEDA H, YOSHIDA O, HIAI H

CANCER RESEARCH 54 巻 ( 2 ) 頁： 399 - 402 1994年1月

Genetic predisposition to pre-B lymphomas in SL/Kh strain mice.

Yamada Y, Matsushiro H, Ogawa MS, Okamoto K, Nakakuki Y, Toyokuni S, Fukumoto M, Hiai H

Cancer research 54 巻 ( 2 ) 頁： 403 - 7 1994年1月

Abnormal bone marrow B-cell differentiation in pre-B lymphoma-prone SL/Kh mice.

Okamoto K, Yamada Y, Ogawa MS, Toyokuni S, Nakakuki Y, Ikeda H, Yoshida O, Hiai H

Cancer research 54 巻 ( 2 ) 頁： 399 - 402 1994年1月

Genetic Predisposition to Pre-B Lymphomas in SL/Kh Strain Mice 査読有り国際誌

Yamada Y., Matsushiro H., Ogawa M.S., Okamoto K., Nakakuki Y., Toyokuni S., Fukumoto M., Hiai H.

Cancer Research 54 巻 ( 2 ) 頁： 403 - 407 1994年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Research

Genetic predisposition of SL/Kh mice to spontaneous pre-B lymphomas was investigated in crosses between SL/Kh and NFS/N, another inbred strain of mice lacking endogenous ecotropic provirus and spontaneous lymphoma. (SL/Kh x NFS/N) F! hybrids developed lymphomas similar to those in SL/Kh but at a lower frequency and with a longer latent period. Of 83 backcross mice to NFS/N, 22 developed hemopoietic tumors: 8 were diffuse lymphoblastic lymphomas; 2 were myeloid leukemias arising by 12 months of age; and 12 were follicular center cell lymphomas found later in life. Of 6 endogenous ecotropic proviruses in SL/Kh, 2 were expressed in (SL/Kh x NFS) Fx backcrossed to NFS. One, encoded by a 27-kilobase EcoSl fragment, was closely linked to Gpi-1” on chromosome 7 and its expression seemed to be a prerequisite for the occurrence of all types of hemopoietic tumors. Microsatellite analysis of the backcross generation revealed multiple host genetic factors determining susceptibility to tumors. An allele derived from SL/Kh, mapped in the major histocompatibility locus on chromosome 17, was essential for development of early onset tumors. This locus was designated as Esl-1 (early lymphoma of SL-1). On the other hand, follicular center cell lymphomas developed mostly in the backcross mice homozygous for the NFS/N derived allele at the D4MIT17-linked locus, designated as foc-1 (follicular center cell lymphoma-1), on chromosome 4. © 1994, American Association for Cancer Research. All rights reserved.

Genetic Predisposition to Pre-B Lymphomas in SL/Kh Strain Mice 国際誌

Yamada Y, Matsushiro H, Ogawa M.S, Okamoto K, Nakakuki Y, Toyokuni S, Fukumoto M, Hiai H

Cancer Research 54 巻 ( 2 ) 頁： 403 - 407 1994年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Research

Abnormal Bone Marrow B-Cell Differentiation in Pre-B Lymphoma-prone SL/Kh Mice 査読有り国際誌

Okamoto K., Yamada Y., Ogawa M.S., Toyokuni S., Nakakuki Y., Ikeda H.

Cancer Research 54 巻 ( 2 ) 頁： 399 - 402 1994年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Research

SL/Kh mice spontaneously develop pre-B lymphomas with surface phenotypes of B220+, BP-1+, Thy-1-, and surface immunoglobulin negative. The immunoglobulin heavy chain of lymphoma is clonally rearranged but the light chain gene remains in germline configuration. Studying prelymphoma stage SL/Kh bone marrow (BM), we found unusual multiclonal expansion of BP-1+ pre-B cells [34.8 + 5.8% (mean - SD)] by 4 weeks of age, whereas there were far fewer of such cells in most other laboratory strains (8 - 5%). The BP-1+ cells did not express surface immunoglobulin, Thy-1.1, or c-kit. Therefore, they seemed to belong to the pre-B II category. Increased numbers of BP-1+ cells were seen in Fx hybrids between SL/Kh and NFS/N; thus it was apparently a dominant heritable property of SL/Kh mice. Emergence of this population was independent of expression of endogenous ecotropic virus, since they were present in BMs of the Yt hybrid to C4W (Fv-4r) and were not inhibited by neonatal injection of maternal resistance factor. In the radiation chimeras SL/Kh - BALB/c, BP-1+ cells appeared abundantly (29.0 - 3.8%), whereas in the reciprocal chimeras BALB/c - SL/Kh, for fewer (5.5 - 2.3%) appeared. Therefore, expansion of BP-1+ cells in prelymphomatous BM is a property of SL/Kh stem cells rather than BM microenvironments. © 1994, American Association for Cancer Research. All rights reserved.

ALTERATION OF THE DEOXYGUANINE NUCLEOTIDES TO 8-HYDROXYDEOXYGUANOSINE IN HUMAN RENAL-CELL CARCINOMA

OKAMOTO, K; YOSHIDA, O; HATTORINAKAKUKI, Y; HIAI, H; TOYOKUNI, S

FRONTIERS OF REACTIVE OXYGEN SPECIES IN BIOLOGY AND MEDICINE 1058 巻頁： 447 - 448 1994年

FORMATION OF 4-HYDROXY-2-NONENAL-MODIFIED PROTEINS IN THE RENAL PROXIMAL TUBULES OF RATS TREATED WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE

TOYOKUNI, S; UCHIDA, K; OKAMOTO, K; HATTORINAKAKUKI, Y; HIAI, H; STADTMAN, ER

FRONTIERS OF REACTIVE OXYGEN SPECIES IN BIOLOGY AND MEDICINE 1058 巻頁： 171 - 172 1994年

FORMATION OF 4-HYDROXY-2-NONENAL-MODIFIED PROTEINS IN THE RENAL PROXIMAL TUBULES OF RATS TREATED WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE 国際誌

TOYOKUNI, S, UCHIDA, K, OKAMOTO, K, HATTORINAKAKUKI, Y, HIAI, H, STADTMAN, ER

FRONTIERS OF REACTIVE OXYGEN SPECIES IN BIOLOGY AND MEDICINE 1058 巻頁： 171 - 172 1994年

ALTERATION OF THE DEOXYGUANINE NUCLEOTIDES TO 8-HYDROXYDEOXYGUANOSINE IN HUMAN RENAL-CELL CARCINOMA 査読有り国際誌

OKAMOTO, K, YOSHIDA, O, HATTORINAKAKUKI, Y, HIAI, H, TOYOKUNI, S

FRONTIERS OF REACTIVE OXYGEN SPECIES IN BIOLOGY AND MEDICINE 1058 巻頁： 447 - 448 1994年

ABNORMAL BONE-MARROW B-CELL DIFFERENTIATION IN PRE-B LYMPHOMA-PRONE SL/KH MICE

OKAMOTO, K; YAMADA, Y; SHIMADA, M; TOYOKUNI, S; NAKAKUKI, Y; HIAI, H

EXPERIMENTAL HEMATOLOGY 21 巻 ( 8 ) 頁： 1126 - 1126 1993年8月

GENETIC PREDISPOSITION TO PRE-B LYMPHOMAS IN SL/KH STRAIN MICE

YAMADA, Y; SHIMADA, M; OKAMOTO, K; TOYOKUNI, S; NAKAKUKI, Y; FUKUMOTO, M; HIAI, H

EXPERIMENTAL HEMATOLOGY 21 巻 ( 8 ) 頁： 1118 - 1118 1993年8月

INDUCTION OF OXIDATIVE SINGLE-STRAND AND DOUBLE-STRAND BREAKS IN DNA BY FERRIC CITRATE

TOYOKUNI S, SAGRIPANTI JL

FREE RADICAL BIOLOGY AND MEDICINE 15 巻 ( 2 ) 頁： 117 - 123 1993年8月

INDUCTION OF OXIDATIVE SINGLE-STRAND AND DOUBLE-STRAND BREAKS IN DNA BY FERRIC CITRATE 査読有り

S TOYOKUNI, JL SAGRIPANTI

FREE RADICAL BIOLOGY AND MEDICINE 15 巻 ( 2 ) 頁： 117 - 123 1993年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

The relative risk of primary hepatocellular carcinoma in genetic hemochromatosis (GH) is estimated at over 200 times as that of control populations. Recently, ferric ion chelated to citrate (Fe-citrate) was identified as the major non-transferrin-bound iron in the serum of GH patients. We investigated whether low concentration of Fe-citrate plus reductant could damage supercoiled plasmid DNA under physiological pH and ionic strength. Incubation of Fe-citrate with either H2O2, L-ascorbate, or L-cysteine induced single- and double-strand breaks in supercoiled plasmid pZ189 in a concentration- and time-dependent fashion. DNA strand breaks produced by Fe-citrate plus H2O2 increased at reduced pH (less-than-or-equal-to 6.9). Catalase and free radical scavengers inhibited the DNA breakage produced by Fe-citrate in combination with each reductant, suggesting that H2O2 and finally .OH are responsible DNA damaging species. The catalytic ability of Fe-citrate to induce DNA strand breaks, particularly double-strand breaks (DSBs), may contribute to the carcinogenic processes observed in GH.

Induction of oxidative single- and double-strand breaks in DNA by ferric citrate

GENETIC PREDISPOSITION TO PRE-B LYMPHOMAS IN SL/KH STRAIN MICE 国際誌

YAMADA, Y, SHIMADA, M, OKAMOTO, K, TOYOKUNI, S, NAKAKUKI, Y, FUKUMOTO, M, HIAI, H

EXPERIMENTAL HEMATOLOGY 21 巻 ( 8 ) 頁： 1118 - 1118 1993年8月

Toyokuni S., Sagripanti J.

Free Radical Biology and Medicine 15 巻 ( 2 ) 頁： 117 - 123 1993年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Free Radical Biology and Medicine

The relative risk of primary hepatocellular carcinoma in genetic hemochromatosis (GH) is estimated at over 200 times as that of control populations. Recently, ferric ion chelated to citrate (Fe-citrate) was identified as the major non-transferrin-bound iron in the serum of GH patients. We investigated whether low concentration of Fe-citrate plus reductant could damage supercoiled plasmid DNA under physiological pH and ionic strength. Incubation of Fe-citrate with either H2O2, l-ascorbate, or l-cysteine induced single- and double-strand breaksin supercoiled plasmid pZ189 in a concentration- and time-dependent fashion. DNA strand breaks produced by Fe-citrate plus H2O2 increased at reduced pH (≤ 6.9). Catalase and free radical scavengers inhibited the DNA breakage produced by Fe-citrate in combination with each reductant, suggesting that H2O2 and finally .OH are responsible DNA damaging species. The catalytic ability of Fe-citrate to induce DNA strand breaks, particularly double-strand breaks (DSBs), may contribute to the carcinogenic processes observed in GH. © 1993.

DOI： 10.1016/0891-5849(93)90050-5

Glutathione cycle dependency of ferric nitrilotriacetate-induced lipid peroxidation in mouse proximal renal tubules

ABNORMAL BONE-MARROW B-CELL DIFFERENTIATION IN PRE-B LYMPHOMA-PRONE SL/KH MICE 査読有り国際誌

OKAMOTO, K, YAMADA, Y, SHIMADA, M, TOYOKUNI, S, NAKAKUKI, Y, HIAI, H

EXPERIMENTAL HEMATOLOGY 21 巻 ( 8 ) 頁： 1126 - 1126 1993年8月

GLUTATHIONE CYCLE DEPENDENCY OF FERRIC NITRILOTRIACETATE-INDUCED LIPID-PEROXIDATION IN MOUSE PROXIMAL RENAL TUBULES

OKADA S, MINAMIYAMA Y, HAMAZAKI S, TOYOKUNI S, SOTOMATSU A

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 301 巻 ( 1 ) 頁： 138 - 142 1993年2月

GLUTATHIONE CYCLE DEPENDENCY OF FERRIC NITRILOTRIACETATE-INDUCED LIPID-PEROXIDATION IN MOUSE PROXIMAL RENAL TUBULES

OKADA S, MINAMIYAMA Y, HAMAZAKI S, TOYOKUNI S, SOTOMATSU A

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 301 巻 ( 1 ) 頁： 138 - 142 1993年2月

Okada S., Minamiyama Y., Hamazaki S., Toyokuni S., Sotomatsu A.

Archives of Biochemistry and Biophysics 301 巻 ( 1 ) 頁： 138 - 142 1993年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

Parenteral administration of ferric nitrilotriacetate (Fe-NTA), a known carcinogen in mouse and rat kidneys, enhances iron-dependent lipid peroxidation (LP) and causes acute renal tubular necrosis. We assume that filtered Fe-NTA in vivo is rapidly reduced by cysteine, a component of glutathione which is hydrolyzed by γ-GTP and dipeptidase, and that this reduced iron initiates lipid peroxidation in the lumen. In addition, the fatty acid composition of phospholipids between the cortex and the medulla may differ, because only the proximal tubules (which are located mainly in the cortex) are known to be vulnerable to LP. We tested these assumptions in the present study. Gas chromatographic determination of fatty acid composition in five male and five female 6-week-old normal ddY mice showed the ratio of polyunsaturated fatty acids to saturated fatty acids plus C18:1, a single double-bond fatty acid, to be 0.98 ± 0.08 (av ± SD) in the male cortex and 1.00 ± 0.08 in the female cortex. In the male and female medulla, however, it was 0.78 ± 0.09 (P < 0.05, vs cortex) and 0.68 ± 0.04 (P < 0.01, vs cortex), respectively. Pretreatment of the animals with buthionine sulfoximine, a glutathione synthetase inhibitor, and a procedure that reduces total glutathione content in the kidneys, suppressed LP. Reduced thiobarbituric acid reactive substances were also observed in animals treated with AT-125, a γ-GTP inhibitor, and in animals with immature γ-GTP activity. These results are consistent with our assumptions. © 1993 Academic Press, Inc.

DOI： 10.1006/abbi.1993.1125

DNA single- and double-strand breaks produced by ferric nitrilotriacetate in relation to renal tubular carcinogenesis

DNA SINGLE-STRAND AND DOUBLE-STRAND BREAKS PRODUCED BY FERRIC NITRILOTRIACETATE IN RELATION TO RENAL TUBULAR CARCINOGENESIS

TOYOKUNI S, SAGRIPANTI JL

CARCINOGENESIS 14 巻 ( 2 ) 頁： 223 - 227 1993年2月

DNA SINGLE-STRAND AND DOUBLE-STRAND BREAKS PRODUCED BY FERRIC NITRILOTRIACETATE IN RELATION TO RENAL TUBULAR CARCINOGENESIS

TOYOKUNI S, SAGRIPANTI JL

CARCINOGENESIS 14 巻 ( 2 ) 頁： 223 - 227 1993年2月

Toyokuni S., Sagripanti J.

Carcinogenesis 14 巻 ( 2 ) 頁： 223 - 227 1993年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Carcinogenesis

Fe(III) bound to a chelator, nitrilotriacetate (NTA), has been reported to induce a high frequency of adenocarcinoma localized to the proximal tubules of the kidney in rodents. In order to examine possible mechanisms for the carcinogenic activity, we investigated the in vitro production of single- and double-strand breaks in DNA mediated by iron alone or Fe-NTA chelate using supercoiled plasmid pZ189. Neither Fe(III) nor NTA alone broke DNA. Fe(III) plus NTA together mediated the efficient oxidative production of DNA single-and double-strand breaks in the presence of reducing agents (ascorbate » H2O2 > cysteine). The Fe(III): NTA ratio (1:4) that was found to be optimal for DNA strand breakage was similar to the ratio that produced adenocarcinomas in rodents. Maximal Fe-NTA-mediated DNA damage in vitro was induced under conditions of neutral pH, low ionic strength, presence of reducing agent and absence of albumin. These conditions are present exclusively in the cortical proximal tubules of the kidney, the only location where toxicity and carcinogenicity of Fe-NTA has been observed. Thus, localized DNA damage may explain the anatomic site preferred by Fe-NTA-induced carcinogenesis. © 1993 Oxford University Press.

DOI： 10.1093/carcin/14.2.223

CHARACTERIZATION OF IMMORTALIZED CELL-LINES ISOLATED FROM A SOFT-TISSUE TUMOR-DERIVED FROM A RABBIT SUBCHRONICALLY EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1-G

NING, JT; BUSHAR, G; LI, L; TOYOKUNI, S; THOMAS, D; CRESS, L; CZERSKA, EM; SWICORD, ML; ELSON, E; DAVIS, CC

ELECTRICITY AND MAGNETISM IN BIOLOGY AND MEDICINE 頁： 405 - 408 1993年

DOSE-DEPENDENT HEALTH-EFFECTS EXHIBITED IN RABBITS EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1 G AND 5 G

NING, JT; CZERSKA, EM; CASAMENTO, J; TOYOKUNI, S; SWICORD, ML; STCHARLES, C; EINSMANN, FE; PERRY, B; ELSON, E; DAVIS, CC

ELECTRICITY AND MAGNETISM IN BIOLOGY AND MEDICINE 頁： 845 - 848 1993年

PATHOLOGICAL EVALUATION OF RABBITS SUBCHRONICALLY EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1 G AND 5 G

NING, JT; TOYOKUNI, S; THOMAS, D; CZERSKA, EM; CASAMENTO, J; SWICORD, ML; DAVIS, CC; ELSON, E

ELECTRICITY AND MAGNETISM IN BIOLOGY AND MEDICINE 頁： 835 - 838 1993年

PATHOLOGICAL EVALUATION OF RABBITS SUBCHRONICALLY EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1 G AND 5 G 国際誌

NING, JT, TOYOKUNI, S, THOMAS, D, CZERSKA, EM, CASAMENTO, J, SWICORD, ML, DAVIS, CC, ELSON, E

ELECTRICITY AND MAGNETISM IN BIOLOGY AND MEDICINE 頁： 835 - 838 1993年

DOSE-DEPENDENT HEALTH-EFFECTS EXHIBITED IN RABBITS EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1 G AND 5 G 国際誌

NING, JT, CZERSKA, EM, CASAMENTO, J, TOYOKUNI, S, SWICORD, ML, STCHARLES, C, EINSMANN, FE, PERRY, B, ELSON, E, DAVIS, CC

ELECTRICITY AND MAGNETISM IN BIOLOGY AND MEDICINE 頁： 845 - 848 1993年

CHARACTERIZATION OF IMMORTALIZED CELL-LINES ISOLATED FROM A SOFT-TISSUE TUMOR-DERIVED FROM A RABBIT SUBCHRONICALLY EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1-G 査読有り国際共著国際誌

NING, JT, BUSHAR, G, LI, L, TOYOKUNI, S, THOMAS, D, CRESS, L, CZERSKA, EM, SWICORD, ML, ELSON, E, DAVIS, CC

ELECTRICITY AND MAGNETISM IN BIOLOGY AND MEDICINE 頁： 405 - 408 1993年

IRON-MEDIATED DNA DAMAGE - SENSITIVE DETECTION OF DNA STRAND BREAKAGE CATALYZED BY IRON

TOYOKUNI S, SAGRIPANTI JL

JOURNAL OF INORGANIC BIOCHEMISTRY 47 巻 ( 3-4 ) 頁： 241 - 248 1992年8月

REPORT OF A PATIENT WITH POEMS-TAKATSUKI-CROW-FUKASE SYNDROME ASSOCIATED WITH FOCAL SPINAL PACHYMENINGEAL AMYLOIDOSIS

TOYOKUNI S, EBINA Y, OKADA S, YAMABE H, ISHIKAWA T, UCHIYAMA T, YODOI J, UCHINO H, TAKATSUKI K

CANCER 70 巻 ( 4 ) 頁： 882 - 886 1992年8月

Report of a patient with POEMS/takatsuki/crow‐fukase syndrome associated with focal spinal pachymeningeal amyloidosis

Toyokuni S., Ebina Y., Okada S., Yamabe H., Ishikawa T., Uchiyama T., Yodoi J., Uchino H., Takatsuki K.

Cancer 70 巻 ( 4 ) 頁： 882 - 886 1992年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer

The authors present the results of an autopsy of a 67‐year‐old Japanese man with POEMS/Takatsuki/Crow‐Fukase syndrome (P/T/CFS) diagnosed in 1972. Each component of the syndrome was gradually recognized after the resection of lumbar vertebral solitary plasma‐cytoma in 1967. The patient died in 1989 of generalized infection and renal failure. Autopsy revealed in the vertebral canal between the fifth and seventh thoracic vertebrae dorsal pachymeningeal fibrosis, with prominent amyloid deposition that oppressed the spinal cord. This condition was consistent with the final neurologic manifestation of the patient, bilateral motor and sensory disturbance below the sixth thoracic level. Myelopathy remained clinically unnoticed because neurologic disturbance had begun as peripheral polyneuropathy. Normo‐cellular marrow with heterogeneously scattered lambda light chain‐positive plasma cells and degeneration of the myelinated fibers of sciatic nerve also were observed. This is the first report of focal spinal amyloidosis associated with P/T/CFS. Cancer 1992; 70:882–886. Copyright © 1992 American Cancer Society

DOI： 10.1002/1097-0142(19920815)70:4<882::AID-CNCR2820700426>3.0.CO;2-R

Iron-mediated DNA damage: Sensitive detection of DNA strand breakage catalyzed by iron

REPORT OF A PATIENT WITH POEMS-TAKATSUKI-CROW-FUKASE SYNDROME ASSOCIATED WITH FOCAL SPINAL PACHYMENINGEAL AMYLOIDOSIS

TOYOKUNI S, EBINA Y, OKADA S, YAMABE H, ISHIKAWA T, UCHIYAMA T, YODOI J, UCHINO H, TAKATSUKI K

CANCER 70 巻 ( 4 ) 頁： 882 - 886 1992年8月

IRON-MEDIATED DNA DAMAGE - SENSITIVE DETECTION OF DNA STRAND BREAKAGE CATALYZED BY IRON

TOYOKUNI S, SAGRIPANTI JL

JOURNAL OF INORGANIC BIOCHEMISTRY 47 巻 ( 3-4 ) 頁： 241 - 248 1992年8月

肝未分化肉腫の1例

奥山隆三, 豊國伸哉, 岡田茂, 中嶋安彬, 山辺博彦, 韓秀〓, 森敬一郎, 小沢和惠

日臨細胞会誌 31 巻頁： 89 - 90 1992年

肝未分化肉腫の１例

奥山隆三, 豊國伸哉, 岡田茂, 中嶋安彬, 山辺博彦, 韓秀絃, 森敬一郎, 小沢和恵

日本臨床細胞学会雑誌 31 巻 ( 1 ) 頁： 89 - 90 1992年

肝未分化肉腫の1例

奥山隆三, 豊國伸哉, 岡田茂, 中嶋安彬, 山辺博彦, 韓秀〓, 森敬一郎, 小沢和惠

日臨細胞会誌 31 巻頁： 89 - 90 1992年

Toyokuni S., Sagripanti J.

Journal of Inorganic Biochemistry 47 巻 ( 1 ) 頁： 241 - 248 1992年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Inorganic Biochemistry

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 μM Fe(III)-NTA and 100 μM L-ascorbate induced detectable DNA strand breaks after 30 min at 24°C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload. © 1992.

DOI： 10.1016/0162-0134(92)84069-Y

Erratum: Combined histochemical and biochemical analysis of sex hormone dependence of ferric nitrilotriacetate-induced renal lipid peroxidation in ddY mice (Cancer Research (September 1, 1990) (5574-5580)) 査読有り国際誌

Toyokuni

Cancer Research 52 巻 ( 21 ) 1992年

Histochemical detection of burn-induced lipid peroxidation in sebaceous glands of rat skin. 国際誌

Kitajima T, Hamanaka H, Miyachi Y, Ono Y, Moriguchi T, Toyokuni S, Imamura S

The Journal of dermatology 18 巻 ( 7 ) 頁： 393 - 6 1991年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Dermatology

Increased lipid peroxide levels both in skin and serum have been reported after cutaneous thermal injury. However, it still remains unclear where lipid peroxides are produced at the site of burned skin. In the present study, a histochemical method using cold Schiff's reagent was applied in order to detect the localization of lipid peroxide. Schiff positivity was detected in sebaceous glands, and the extent of positivity seemed to correlate with the serum lipid peroxide levels. These results may suggest that lipid peroxides produced in sebaceous glands after thermal injury enter the blood stream and are partially responsible for the elevated serum lipid peroxide levels.

DOI： 10.1111/j.1346-8138.1991.tb03104.x

SEX-DIFFERENCES IN THE LOCALIZATION AND SEVERITY OF FERRIC NITRILOTRIACETATE-INDUCED LIPID-PEROXIDATION IN THE MOUSE KIDNEY

OKADA, S; FUKUNAGA, Y; HAMAZAKI, S; YAMADA, Y; TOYOKUNI, S

ACTA PATHOLOGICA JAPONICA 41 巻 ( 3 ) 頁： 221 - 226 1991年3月

SEX-DIFFERENCES IN THE LOCALIZATION AND SEVERITY OF FERRIC NITRILOTRIACETATE-INDUCED LIPID-PEROXIDATION IN THE MOUSE KIDNEY

OKADA S, FUKUNAGA Y, HAMAZAKI S, YAMADA Y, TOYOKUNI S

ACTA PATHOLOGICA JAPONICA 41 巻 ( 3 ) 頁： 221 - 226 1991年3月

Sex Differences in the Localization and Severity of Ferric Nitrilotriacetate‐induced Lipid Peroxidation in the Mouse Kidney 国際誌

Okada S., Fukunaga Y., Hamazaki S., Yamada Y., Toyokuni S.

Pathology International 41 巻 ( 3 ) 頁： 221 - 226 1991年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Pathology International

Male mice are much more susceptible than female mice to acute renal proximal tubular necrosis as well as the carcinogenic effect induced by an iron chelate, ferric nitrilotriacetate (Fe‐NTA). In the present study, iron‐promoted lipid peroxidation was analyzed histochemically in frozen kidney sections using cold Schiffs reagent and biochemically by measuring thiobarbituric acid‐reactive substance in the kidney. When untreated mouse frozen kidney sections were exposed to ascorbic acid‐Fe‐NTA (0.8 mM, 0.2 mM and 0.4 mM) in vitro for 40 min and washed, diffuse Schiff positivity was obtained along the proximal tubules, and no sex differences were evident. When non‐reduced Fe‐NTA (0.2 mM and 0.4 mM) was layered on untreated kidney sections from males, about half of the cortical proximal tubules showed a positive reaction with Schiffs reagent, whereas in females the proximal tubules of the outer stripe of the outer medulla were positive. In an ex vivo study, 30 to 40 min after i.p. injection of Fe‐NTA (5 mg Fe/kg), Schiff‐positive areas corresponded to those observed in the in vitro non‐reduced Fe‐NTA study in each sex. However, in males, the positive tubular cells gradually became necrotic, whereas in females the positivity disappeared with time and there was no tubular necrosis. Results of the thiobarbituric acid test were correlated with the above findings. In conclusion, the localization and severity of FeNTA induced lipid peroxidation in the tubules differed between the sexes. We suspect that these results reflect functional heterogeneity in the ability of the tubules to reduce iron. Copyright © 1991, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1440-1827.1991.tb01651.x

Congenital myotonic dystrophy with progressive edema and hypoproteinemia

CONGENITAL MYOTONIC-DYSTROPHY WITH PROGRESSIVE EDEMA AND HYPOPROTEINEMIA

FUJII T, YORIFUJI T, OKUNO T, TOYOKUNI S, OKADA S, MIKAWA H

BRAIN & DEVELOPMENT 13 巻 ( 1 ) 頁： 58 - 60 1991年

CONGENITAL MYOTONIC-DYSTROPHY WITH PROGRESSIVE EDEMA AND HYPOPROTEINEMIA

FUJII T, YORIFUJI T, OKUNO T, TOYOKUNI S, OKADA S, MIKAWA H

BRAIN & DEVELOPMENT 13 巻 ( 1 ) 頁： 58 - 60 1991年

Fujii T., Yorifuji T., Okuno T., Toyokuni S., Okada S., Mikawa H.

Brain and Development 13 巻 ( 1 ) 頁： 58 - 60 1991年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Brain and Development

We report a patient with congenital myotonic dystrophy who had progressive edema and hypoproteinemia. An atrioseptal defect and patent ductus arteriosus were noted and were considered to be the cause of the right heart failure and edema. Although urinary protein levels were minimal, infusion of albumin did not improve the hypoproteinemia. Administration of dexamethasone increased the serum protein level, but the edema was not ameliorated. Autopsy revealed a slight lymphatic dilatation in the small intestine, suggesting protein-losing enteropathy. © 1991, All rights reserved.

DOI： 10.1016/S0387-7604(12)80300-7

A case of biopsy‐proven myocarditis progressing to autopsy‐proven dilated cardiomyopathy

A CASE OF BIOPSY-PROVEN MYOCARDITIS PROGRESSING TO AUTOPSY-PROVEN DILATED CARDIOMYOPATHY

MATOBA Y, MATSUMORI A, OHTANI H, TOMINAGA M, FUJIWARA H, TOYOKUNI S, TAKAHASHI K, MIDORIKAWA O, KAWAI C

CLINICAL CARDIOLOGY 13 巻 ( 10 ) 頁： 732 - 737 1990年10月

Matoba Y., Matsumori A., Ohtani H., Tominaga M., Fujiwara H., Kawai C., Toyokuni S., Midorikawa O., Takahashi K.

Clinical Cardiology 13 巻 ( 10 ) 頁： 732 - 737 1990年10月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Clinical Cardiology

A 22‐year‐old man presented with congestive heart failure following flulike symptoms. The diagnosis of acute myocarditis was confirmed by endomyocardial biopsy, which revealed mild infiltration of inflammatory cells. A favorable response to beta‐adrenergic receptor blockade was seen, and the patient was discharged without symptoms. Five months later, however, congestive heart failure recurred, and intracardiac thrombi were demonstrated. The patient died after two months. Postmortem examination revealed left ventricular dilatation with slight interstitial fibrosis; the diagnosis was dilated cardiomyopathy. Thus, progression of biopsy‐proven myocarditis to dilated cardiomyopathy 10 months after the onset of disease was documented. Copyright © 1990 Wiley Periodicals, Inc.

DOI： 10.1002/clc.4960131013

A CASE OF BIOPSY-PROVEN MYOCARDITIS PROGRESSING TO AUTOPSY-PROVEN DILATED CARDIOMYOPATHY

MATOBA Y, MATSUMORI A, OHTANI H, TOMINAGA M, FUJIWARA H, TOYOKUNI S, TAKAHASHI K, MIDORIKAWA O, KAWAI C

CLINICAL CARDIOLOGY 13 巻 ( 10 ) 頁： 732 - 737 1990年10月

COMBINED HISTOCHEMICAL AND BIOCHEMICAL-ANALYSIS OF SEX-HORMONE DEPENDENCE OF FERRIC NITRILOTRIACETATE-INDUCED RENAL LIPID-PEROXIDATION IN DDY-MICE

TOYOKUNI S, OKADA S, HAMAZAKI S, MINAMIYAMA Y, YAMADA Y, LIANG P, FUKUNAGA Y, MIDORIKAWA O

CANCER RESEARCH 50 巻 ( 17 ) 頁： 5574 - 5580 1990年9月

COMBINED HISTOCHEMICAL AND BIOCHEMICAL-ANALYSIS OF SEX-HORMONE DEPENDENCE OF FERRIC NITRILOTRIACETATE-INDUCED RENAL LIPID-PEROXIDATION IN DDY-MICE

TOYOKUNI S, OKADA S, HAMAZAKI S, MINAMIYAMA Y, YAMADA Y, LIANG P, FUKUNAGA Y, MIDORIKAWA O

CANCER RESEARCH 50 巻 ( 17 ) 頁： 5574 - 5580 1990年9月

Combined Histochemical and Biochemical Analysis of Sex Hormone Dependence of Ferric Nitrilotriacetate-induced Renal Lipid Peroxidation in ddY Mice 査読有り国際誌

Toyokuni S., Okada S., Hamazaki S., Minamiyama Y., Yamada Y., Liang P., Fukunaga Y., Midorikawa O.

Cancer Research 50 巻 ( 17 ) 頁： 5574 - 5580 1990年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cancer Research

Ferric nitrilotriacetate (Fe-NTA) induces renal proximal tubular necrosis, a consequence of lipid peroxidation, that finally leads to a high incidence of renal adenocarcinoma in rats and mice. Male animals are much more susceptible than female animals to both effects. Moreover, the distribution of the susceptible proximal tubules is different between male and female animals. The present study investigated the effects of castration and sex hormones on Fe-NTA-induced renal lipid peroxidation. Male and female ddY mice were either left untreated, castrated, and/or treated with testosterone or estriol. Histochemical (reactivity to cold SchifTs reagent) and biochemical (thiobarbSturic acid-reactive substance) evaluations were performed 1 h after the i.p. injection of Fe-NTA (5 mg iron/kg). Testosterone treatment and/or oophorectomy increased the Schiff pos-itivity of the renal cortical proximal tubules and the amount of thiobar-bituric acid-reactive substance (testosterone-treated female > intact female, P < 0.005; castrated female > intact female, P < 0.1; castrated and testosterone-treated female >intact female, P < 0.005). In contrast, estriol treatment and/or orchiectomy decreased the Schiff positivity of the renal cortical proximal tubules and the amount of thiobarbituric acid-reactive substance (estriol-treated male < intact male, P < 0.01; castrated male < intact male, P < 0.01; castrated and estriol-treated male < intact male, P < 0.005). Estradiol treatment produced similar results to estriol treatment (estradiol-treated male <intact male, P < 0.005). Castration and/or administration of the opposite sex hormone reversed the sex difference in the distribution of proximal tubules susceptible to lipid peroxidation. However, the i.v. injection to male mice, 5 min prior to the Fe-NTA treatment, of conjugated estrogen that is promptly excreted via the urine produced no significant effect. Thus, altered metabolic pathways rather than the direct scavenging activity of estrogens seem to be involved in the sex hormone-dependent difference of lipid peroxidation. Genetically determined sex hormone status appears to have influenced the incidence of Fe-NTA-induced renal adenocarcinoma in intact animals. © 1990, American Association for Cancer Research. All rights reserved.

Thiobarbituric acid-reactive substance formation of rat kidney brush border membrane vesicles induced by ferric nitrilotriacetate

Combined histochemical and biochemical analysis of sex hormone dependence of ferric nitrilotriacetate-induced renal lipid peroxidation in ddY mice.

Toyokuni S, Okada S, Hamazaki S, Minamiyama Y, Yamada Y, Liang P, Fukunaga Y, Midorikawa O

Cancer research 50 巻 ( 17 ) 頁： 5574 - 80 1990年9月

THIOBARBITURIC ACID-REACTIVE SUBSTANCE FORMATION OF RAT-KIDNEY BRUSH-BORDER MEMBRANE-VESICLES INDUCED BY FERRIC NITRILOTRIACETATE

HAMAZAKI S, OKADA S, TOYOKUNI S, MIDORIKAWA O

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 274 巻 ( 2 ) 頁： 348 - 354 1989年11月

Hamazaki S., Okada S., Toyokuni S., Midorikawa O.

Archives of Biochemistry and Biophysics 274 巻 ( 2 ) 頁： 348 - 354 1989年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

An iron chelate, ferric nitrilotriacetate (Fe3+-NTA), is nephrotoxic and also carcinogenic to the kidney in experimental animals. Iron-promoted lipid peroxidation in the proximal tubules is thought to be responsible for the pathologic process. In the present study, iron-promoted lipid peroxidation, with thiobarbituric acid (TBA) formation as an indication, in the tubular surface was simulated in vitro using rat kidney brush border membrane vesicles and the results were compared with those using linoleate micelles and rat liver microsomal lipid liposomes. Addition of ascorbate, cysteine, or dithiothreitol to the Fe3+-NTA solution resulted in consumption of dissolved oxygen and promoted the lipid peroxidation in the micelles and in the liposomes. In contrast, addition of glutathione to the Fe3+-NTA solution caused only sluggish oxygen consumption and far less peroxidation in these lipid systems. When the brush border membrane vesicles were used for the peroxidation substrate, Fe3+-NTA and glutathione could promote TBA formation at a rate comparable to that elicited by Fe3+-NTA with cysteine or dithiothreitol. Acivicin, a γ-glutamyl transpeptidase inhibitor, suppressed the peroxidation of the brush border membrane vesicles promoted by Fe3+-NTA and glutathione. These results suggest the following mechanism of proximal tubular cell lipid peroxidation promoted by Fe-NTA: Fe3+-NTA filtered through glomeruli is rapidly reduced by cysteine and Fe2+-NTA starts lipid peroxidation at the site, leading to proximal tubular necrosis. Cysteine is amply supplied by the decomposition of glutathione within the lumen by the action of γ-glutamyl transpeptidase and dipeptidase situated at the proximal tubular brush border membrane. © 1989.

DOI： 10.1016/0003-9861(89)90448-7

Induction of mesothelioma by intraperitoneal injections of ferric saccharate in male wistar rats

INDUCTION OF MESOTHELIOMA BY INTRAPERITONEAL INJECTIONS OF FERRIC SACCHARATE IN MALE WISTAR RATS

OKADA S, HAMAZAKI S, TOYOKUNI S, MIDORIKAWA O

BRITISH JOURNAL OF CANCER 60 巻 ( 5 ) 頁： 708 - 711 1989年11月

THIOBARBITURIC ACID-REACTIVE SUBSTANCE FORMATION OF RAT-KIDNEY BRUSH-BORDER MEMBRANE-VESICLES INDUCED BY FERRIC NITRILOTRIACETATE 査読有り

S HAMAZAKI, S OKADA, S TOYOKUNI, O MIDORIKAWA

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 274 巻 ( 2 ) 頁： 348 - 354 1989年11月

Okada S., Hamazaki S., Toyokuni S., Midorikawa O.

British Journal of Cancer 60 巻 ( 5 ) 頁： 708 - 711 1989年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：British Journal of Cancer

Iron appears to play a major role in catalysing free radical production, leading to lipid peroxidation and DNA damage. We, therefore, investigated the effect of colloidal iron deposited in the peritoneum. Wistar male rats were given either ferric saccharate, ferric saccharate and nitrilotriacetic acid (NTA), NTA or saline. NTA was shown previously to ‘free’ iron to promote lipid peroxidation and an iron chelate of NTA is known to be carcinogenic to the kidney. Iron at a dose of 5 mg kg-1 day-1, and saline at a dose of 0.5 ml day-1 were injected i.p. for 3 months. NTA at a dose of 83.5 mg kg-1 day1 was give i.p for 5 months. All the rats were killed about a year later for histological examination. In nine of the 19 rats treated with ferric saccharate, mesothelial tumors were induced in the serosa of the tunica vaginalis or the length of the spermatic cord. Among rats treated with ferric saccharate and NTA, seven had localised mesotheliomas in the above locations and six had wide-spread peritoneal mesotheliomas. No mesothelial tumors developed in either NTA treated or saline treated rats. No pleural mesotheliomas were found in any group. These findings add to the evidence that iron is involved in some carcinogenic processes. © The Macmillan Press Ltd., 1989.

DOI： 10.1038/bjc.1989.344

Oxygen reduction and lipid peroxidation by iron chelates with special reference to ferric nitrilotriacetate

INDUCTION OF MESOTHELIOMA BY INTRAPERITONEAL INJECTIONS OF FERRIC SACCHARATE IN MALE WISTAR RATS

OKADA S, HAMAZAKI S, TOYOKUNI S, MIDORIKAWA O

BRITISH JOURNAL OF CANCER 60 巻 ( 5 ) 頁： 708 - 711 1989年11月

OXYGEN REDUCTION AND LIPID-PEROXIDATION BY IRON CHELATES WITH SPECIAL REFERENCE TO FERRIC NITRILOTRIACETATE

HAMAZAKI S, OKADA S, LI JL, TOYOKUNI S, MIDORIKAWA O

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 272 巻 ( 1 ) 頁： 10 - 17 1989年7月

OXYGEN REDUCTION AND LIPID-PEROXIDATION BY IRON CHELATES WITH SPECIAL REFERENCE TO FERRIC NITRILOTRIACETATE 査読有り

S HAMAZAKI, S OKADA, JL LI, S TOYOKUNI, O MIDORIKAWA

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 272 巻 ( 1 ) 頁： 10 - 17 1989年7月

Hamazaki S., Okada S., Li J., Toyokuni S., Midorikawa O.

Archives of Biochemistry and Biophysics 272 巻 ( 1 ) 頁： 10 - 17 1989年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Archives of Biochemistry and Biophysics

A certain iron chelate, ferric nitrilotriacetate (Fe3+-NTA) is nephrotoxic and also carcinogenic to the kidney in mice and rats, a distinguishing feature not shared by other iron chelates tested so far. Iron-promoted lipid peroxidation is thought to be responsible for the initial events. We examined its ability to initiate lipid peroxidation in vitro in comparison with that of other ferric chelates. Chelation of Fe2+ by nitrilotriacetate (NTA) enhanced the autoxidation of Fe2+. In the presence of Fe2+-NTA, lipid peroxidation occurred as measured by the formation of conjugated diene in detergent-dispersed linoleate micelles, and by the formation of thiobarbituric acid-reactive substances in the liposomes of rat liver microsomal lipids. Addition of ascorbic acid to Fe3+-NTA solution promoted dose-dependent consumption of dissolved oxygen, which indicates temporary reduction of iron. On reduction, Fe3+-NTA initiated lipid peroxidation both in the linoleate micelles and in the liposomes. Fe3+-NTA also initiated NADPH-dependent lipid peroxidation in rat liver microsomes. Although other chelators used (deferoxamine, EDTA, diethylenetriaminepentaacetic acid, ADP) enhanced autoxidation, reduction by ascorbic acid, or in vitro lipid peroxidation of linoleate micelles or liposomal lipids, NTA was the sole chelator that enhanced all the reactions. © 1989.

DOI： 10.1016/0003-9861(89)90188-4

CIRRHOSIS OF THE LIVER INDUCED BY CUPRIC NITRILOTRIACETATE IN WISTAR RATS - AN EXPERIMENTAL-MODEL OF COPPER TOXICOSIS

TOYOKUNI S, OKADA S, HAMAZAKI S, FUJIOKA M, LI JL, MIDORIKAWA O

AMERICAN JOURNAL OF PATHOLOGY 134 巻 ( 6 ) 頁： 1263 - 1274 1989年6月

CIRRHOSIS OF THE LIVER INDUCED BY CUPRIC NITRILOTRIACETATE IN WISTAR RATS - AN EXPERIMENTAL-MODEL OF COPPER TOXICOSIS 査読有り

S TOYOKUNI, S OKADA, S HAMAZAKI, M FUJIOKA, JL LI, O MIDORIKAWA

AMERICAN JOURNAL OF PATHOLOGY 134 巻 ( 6 ) 頁： 1263 - 1274 1989年6月

Cirrhosis of the liver induced by cupric nitrilotriacetate in Wistar rats. An experimental model of copper toxicosis.

Toyokuni S, Okada S, Hamazaki S, Fujioka M, Li JL, Midorikawa O

The American journal of pathology 134 巻 ( 6 ) 頁： 1263 - 74 1989年6月

Cirrhosis of the liver induced by cupric nitrilotriacetate in Wistar rats: an experimental model of copper toxicosis. 査読有り

Toyokuni S, Okada S, Hamazaki S, Fujioka M, Li J-L and Midorikawa O.

Am. J. Pathol. 134 巻頁： 1263-1274 1989年

Oxygen reduction and lipid peroxidation by iron chelates with special reference to ferric nitrilotriacetate. 査読有り

Hamazaki S, Okada S, Li J-L, Toyokuni S and Midorikawa O.

Arch. Biochem. Biophys. 272 巻頁： 10-17 1989年

Nasal T-Cell Lymphoma Associated with Double Gammopathy; a Case Report 国際誌 Open Access

Omori K., Kojima H., Nonomura M., Toyokuni S., Kanaji M.

Practica Oto-Rhino-Laryngologica 82 巻 ( 12 ) 頁： 1739 - 1744 1989年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Practica Oto-Rhino-Laryngologica

Nasal T-cell lymphoma associated with double gammopathy (IgG and IgA) was found in a 78-year-old man with a necrotic tumor in his right nasal cavity. He was treated with radiotherapy and CVP chemotherapy but the lymphoma relapsed in his left nasal cavity 1 year later. He was treated again with the same therapy and is now alive and tumor-free. Two serum M-components (IgG and IgA) were detected through his clinical course. To our knowledge, this is the first report of T-cell lymphoma associated with double gammopathy. The significance of M-components in T-cell lymphocytic proliferation remains unclear, but it may be that the M-components appear because of the inappropriate regulation of the T-B immune-response system in the presence of T-cell lymphoma. Histology in this case is also noteworthy. The first nasal tumor showed granulation and necrotic tissue with plasma cell infiltration; it was diagnosed as non-Hodgkin diffuse lymphoma, mixed type, by surface marker analysis. This indicates that immuno-histochemical study is indispensable for the diagnosis of nasal non-healing granuloma with necrosis. © 1989, The Society of Practical Otolaryngology. All rights reserved.

DOI： 10.5631/jibirin.82.1739

Renal changes induced by chronic oral administration of potassium bromate of ferric nitrilotriacetate in Wistar rats 査読有り国際誌

Nakano K., Okada S., Toyokuni S., Midorikawa O.

Japanese Archives of Internal Medicine 36 巻 ( 2 ) 頁： 41 - 47 1989年

Cirrhosis of the liver induced by cupric nitrilotriacetate in Wistar rats. An experimental model of copper toxicosis 査読有り国際誌

Toyokuni S., Okada S., Hamazaki S., Fujioka M., Li J.L., Midorikawa O.

American Journal of Pathology 134 巻 ( 6 ) 頁： 1263 - 1274 1989年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Pathology

Rats intraperitoneally injected with a daily dose of cupric nitrilotriacetate (Cu-NTA), which contained 4 to 7 mg of copper/kg body weight, showed submassive liver necrosis, hemolytic anemia, and acute renal tubular necrosis at the beginning of the experiment and intermittently after 4 weeks of injections. All rats that survived over 8 weeks exhibited liver fibrosis with portal-portal, portal-central, and central-central bridging. In all rats that survived over 16 weeks, micronodular cirrhosis of the liver or extensive liver fibrosis was observed. The copper content of the cirrhotic/fibrotic liver was above 250 μg/g dry weight. Electron-microscopic x-ray analysis at day 93 revealed that copper stored in secondary lysosomes was always accompanied by a proportional amount of sulfur (correlation coefficient, 0.98; P < 0.005). An experimental model of copper toxicosis in terms of copper-induced cirrhosis of the liver was established with exogenous copper chelated by nitrilotriacetate.