Faculty Profiles - TOYOKUNI Shinya

Silicone oil, an intraocular surgical adjuvant, induces retinal ferroptosis. Reviewed International journal

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 Vol. 228 page： 33 - 43 2025.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Decoding the molecular enigma behind asbestos and fibrous nanomaterial-induced carcinogenesis. Reviewed International journal

Toyokuni S, Kong Y

Journal of occupational health Vol. 67 ( 1 ) 2025.1

Heterozygous mutation in BRCA2 induces accelerated age-dependent decline in sperm quality with male subfertility in rats. Reviewed International journal Open Access

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

Scientific reports Vol. 15 ( 1 ) page： 447 2025.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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

New era of plasma dentistry. Reviewed International journal

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

Free radical research Vol. 58 ( 12 ) page： 868 - 874 2024.12

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International coauthorship International journal

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 Vol. 121 ( 48 ) page： e2407842121 2024.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Effects of autophagy on the selective death of human breast cancer cells exposed to plasma-activated Ringer's lactate solution. Reviewed International coauthorship International journal

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

Free radical research Vol. 58 ( 11 ) page： 758 - 769 2024.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Low-temperature plasma as a strategy to achieve SDGs. Reviewed International journal

Tanaka H, Ishikawa K, Toyokuni S

Free radical research Vol. 58 ( 10 ) page： 594 - 595 2024.10

Ferroptosis is a targetable detrimental factor in metabolic dysfunction-associated steatotic liver disease. Reviewed International coauthorship International journal 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 Vol. 31 ( 9 ) page： 1113 - 1126 2024.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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 in health and disease. Reviewed International coauthorship International journal 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 Vol. 75 page： 103211 2024.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Ferroptosis induced by plasma-activated Ringer's lactate solution prevents oral cancer progression. Reviewed International journal Open Access

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

Oral diseases Vol. 30 ( 6 ) page： 3912 - 3924 2024.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International coauthorship International journal

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 Vol. 26 ( 9 ) page： 1447 - 1457 2024.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Growth promotion and disease tolerance in strawberry plants with plasma-activated Ringer’s lactate solution Reviewed International journal

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

Acta Horticulturae Vol. 3 ( 1404 ) page： 917 - 922 2024.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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 International journal

前田勇貴, 豊國伸哉

医学のあゆみ Vol. 290 ( 2 ) page： 174 - 177 2024.7

Special issue: Extracellular fine particles in life, death and disease. Reviewed International journal

Toyokuni S

Archives of biochemistry and biophysics Vol. 757 page： 110027 2024.7

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis. Reviewed International journal 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 Vol. 20 ( 6 ) page： 1213 - 1246 2024.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International coauthorship International journal 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 Vol. 15 ( 1 ) page： 3816 2024.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria. Reviewed International journal Open Access

Danyang Mi, Izumi Yanatori, Hao Zheng, Yingyi Kong, Tasuku Hirayama, Shinya Toyokuni

Free radical research Vol. 58 ( 4 ) page： 261 - 275 2024.3

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

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. Though 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

Elaborate cooperation of poly(rC)-binding proteins 1/2 and glutathione in ferroptosis induced by plasma-activated Ringer's lactate. Reviewed International journal 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 Vol. 214 page： 28 - 41 2024.3

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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((p.T1942fs/+)) dissipates ovarian reserve in rats through oxidative stress in follicular granulosa cells. Reviewed International journal Open Access

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

Free radical research Vol. 58 ( 2 ) page： 130 - 143 2024.2

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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

Science and Applications of Plasma-Activated Solutions: Current Trends and Future Directions Reviewed International coauthorship International journal

Hiromasa Tanaka, Masaaki Mizuno, Kenji Ishikawa, Camelia Miron, Yasumasa Okazaki, Shinya Toyokuni, Kae Nakamura, Hiroaki Kajiyama, Masafumi Ito, Masaru Hori

Plasma Medicine Vol. 14 ( 1 ) page： 67 - 76 2024

Language：English Publishing type：Research paper (scientific journal)

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

Cold Atmospheric Pressure Plasma-Activated Liquids for Cancer Treatment Reviewed International coauthorship International journal

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

IFMBE Proceedings Vol. 111 IFMBE page： 150 - 162 2024

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Selective Killing of Human Breast Cancer Cells by Plasma-Treated L-Arginine Solution Reviewed International coauthorship International journal Open Access

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

Plasma Processes and Polymers 2024

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Retinal ferroptosis as a critical mechanism for the induction of retinochoroiditis during ocular toxoplasmosis. Reviewed International journal 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 Vol. 67 page： 102890 2023.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Three-dimensional regulation of ferroptosis at the intersection of iron, sulfur and oxygen executing scrap and build toward evolution. Reviewed International journal

Shinya Toyokuni, Yingyi Kong, Hao Zheng, Yuki Maeda, Misako Katabuchi, Yashiro Motooka

Antioxidants & redox signaling Vol. 39 ( 10-12 ) page： 807 - 815 2023.10

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

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. 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 ferroptosis-resistance, based on rodent animal studies. 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. Whereas life attempts to prevent ferroptosis, ferroptotic cells eventually emit iron-loaded ferritin as extracellular vesicles to maintain monopoly of iron.

DOI： 10.1089/ars.2022.0142

Carbon nanotube recognition by human Siglec-14 provokes inflammation. Reviewed International journal

Shin-Ichiro Yamaguchi, Qilin Xie, Fumiya Ito, Kazuki Terao, Yoshinobu Kato, Miki Kuroiwa, Satoshi Omori, Hideo Taniura, Kengo Kinoshita, Takuya Takahashi, Shinya Toyokuni, Kota Kasahara, Masafumi Nakayama

Nature nanotechnology Vol. 18 ( 6 ) page： 628 - 636 2023.6

Language：English Publishing type：Research paper (scientific journal)

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

BRCA1 haploinsufficiency impairs iron metabolism to promote chrysotile-induced mesothelioma via ferroptosis-resistance. Reviewed International journal Open Access

Yaguang Luo, Shinya Akatsuka, Yashiro Motooka, Yingyi Kong, Hao Zheng, Tomoji Mashimo, Tatsuhiko Imaoka, Shinya Toyokuni

Cancer science Vol. 114 ( 4 ) page： 1423 - 1436 2023.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

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-strand 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 thus 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 to 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 to wild-type tumors. Mutant MMs indicated iron metabolism dysregulation, such as increase in catalytic Fe(II) and Ki67-index as well as 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 to crocidolite/Mut whereas significant preference to iron with 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

Commentary on "Mechanisms of asbestos-induced carcinogenesis" published in 2009. Reviewed International journal

Toyokuni S

Nagoya journal of medical science Vol. 85 ( 1 ) page： 13 - 15 2023.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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

Cancer-specific cytotoxicity of Ringer’s acetate solution irradiated by cold atmospheric pressure plasma Reviewed International journal

Camelia Miron, Kenji Ishikawa, Satoshi Kashiwagura, Yuki Suda, Hiromasa Tanaka, Kae Nakamura, Hiroaki Kajiyama, Shinya Toyokuni, Masaaki Mizuno, Masaru Hori

Free Radical Research Vol. 57 ( 2 ) page： 91 - 104 2023.2

Environmental impact on carcinogenesis under BRCA1 haploinsufficiency. Reviewed International journal Open Access

Toyokuni S, Kong Y, Motooka Y, Akatsuka S

Genes and environment : the official journal of the Japanese Environmental Mutagen Society Vol. 45 ( 1 ) page： 2 2023.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal

Kotaro Sato, Kouki Fujii, Hiromasa Tanaka, Masaru Hori, Hideharu Hibi, Shinya Toyokuni

Free radical research Vol. 57 ( 1 ) page： 30 - 37 2023.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

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

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

Shinya Toyokuni, Yingyi Kong, Hao Zheng, Yuki Maeda, Yashiro Motooka, Shinya Akatsuka

Journal of clinical biochemistry and nutrition Vol. 71 ( 2 ) page： 78 - 88 2022.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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 multi-system 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 Reviewed International journal Open Access

Kotaro Sato, Koki Fujii, Noriyuki Yamamoto, Norihisa Ichimura, Satoshi Yamaguchi, Hirohisa Yamada, Hideharu Hibi, Shinya Toyokuni

Journal of Clinical Biochemistry and Nutrition Vol. 71 ( 2 ) page： 129 - 135 2022.9

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：The Society for Free Radical Research Japan

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 semi-quantitation 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

BRCA1 haploinsufficiency promotes chromosomal amplification under Fenton reaction-based carcinogenesis through ferroptosis-resistance. Reviewed International journal Open Access

Yingyi Kong, Shinya Akatsuka, Yashiro Motooka, Hao Zheng, Zhen Cheng, Yukihiro Shiraki, Tomoji Mashimo, Tatsuhiko Imaoka, Shinya Toyokuni

Redox biology Vol. 54 page： 102356 - 102356 2022.8

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

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

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

Qinying Lyu, Li Jiang, Hao Zheng, Shotaro Hayashi, Kotaro Sato, Shinya Toyokuni

Journal of Clinical Biochemistry and Nutrition Vol. 71 ( 1 ) page： 55 - 63 2022.7

Language：English Publishing type：Research paper (scientific journal) Publisher：The Society for Free Radical Research Japan

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

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation. Reviewed International journal Open Access

Satoshi Omori, Misato Tsugita, Yasuto Hoshikawa, Masanobu Morita, Fumiya Ito, Shin-Ichiro Yamaguchi, Qilin Xie, Osamu Noyori, Tomoya Yamaguchi, Ayato Takada, Tatsuya Saitoh, Shinya Toyokuni, Hisaya Akiba, Shigekazu Nagata, Kengo Kinoshita, Masafumi Nakayama

Cell reports Vol. 34 ( 6 ) page： 108734 - 108734 2021.2

Language：English Publishing type：Research paper (scientific journal)

Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.

DOI： 10.1016/j.celrep.2021.108734

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1. Reviewed International coauthorship International journal Open Access

Daniel J Klionsky, Amal Kamal Abdel-Aziz, Sara Abdelfatah, Mahmoud Abdellatif, Asghar Abdoli, Steffen Abel, Hagai Abeliovich, Marie H Abildgaard, Yakubu Princely Abudu, Abraham Acevedo-Arozena, Iannis E Adamopoulos, Khosrow Adeli, Timon E Adolph, Annagrazia Adornetto, Elma Aflaki, Galila Agam, Anupam Agarwal, Bharat B Aggarwal, Maria Agnello, Patrizia Agostinis, Javed N Agrewala, Alexander Agrotis, Patricia V Aguilar, S Tariq Ahmad, Zubair M Ahmed, Ulises Ahumada-Castro, Sonja Aits, Shu Aizawa, Yunus Akkoc, Tonia Akoumianaki, Hafize Aysin Akpinar, Ahmed M Al-Abd, Lina Al-Akra, Abeer Al-Gharaibeh, Moulay A Alaoui-Jamali, Simon Alberti, Elísabet Alcocer-Gómez, Cristiano Alessandri, Muhammad Ali, M Abdul Alim Al-Bari, Saeb Aliwaini, Javad Alizadeh, Eugènia Almacellas, Alexandru Almasan, Alicia Alonso, Guillermo D Alonso, Nihal Altan-Bonnet, Dario C Altieri, Élida M C Álvarez, Sara Alves, Cristine Alves da Costa, Mazen M Alzaharna, Marialaura Amadio, Consuelo Amantini, Cristina Amaral, Susanna Ambrosio, Amal O Amer, Veena Ammanathan, Zhenyi An, Stig U Andersen, Shaida A Andrabi, Magaiver Andrade-Silva, Allen M Andres, Sabrina Angelini, David Ann, Uche C Anozie, Mohammad Y Ansari, Pedro Antas, Adam Antebi, Zuriñe Antón, Tahira Anwar, Lionel Apetoh, Nadezda Apostolova, Toshiyuki Araki, Yasuhiro Araki, Kohei Arasaki, Wagner L Araújo, Jun Araya, Catherine Arden, Maria-Angeles Arévalo, Sandro Arguelles, Esperanza Arias, Jyothi Arikkath, Hirokazu Arimoto, Aileen R Ariosa, Darius Armstrong-James, Laetitia Arnauné-Pelloquin, Angeles Aroca, Daniela S Arroyo, Ivica Arsov, Rubén Artero, Dalia Maria Lucia Asaro, Michael Aschner, Milad Ashrafizadeh, Osnat Ashur-Fabian, Atanas G Atanasov, Alicia K Au, Patrick Auberger, Holger W Auner, Laure Aurelian, Riccardo Autelli, Laura Avagliano, Yenniffer Ávalos, Sanja Aveic, Célia Alexandra Aveleira, Tamar Avin-Wittenberg, Yucel Aydin, Scott Ayton, Srinivas Ayyadevara, Maria Azzopardi, Misuzu Baba, Jonathan M Backer, Steven K Backues, Dong-Hun Bae, Ok-Nam Bae, Soo Han Bae, Eric H Baehrecke, Ahruem Baek, Seung-Hoon Baek, Sung Hee Baek, Giacinto Bagetta, Agnieszka Bagniewska-Zadworna, Hua Bai, Jie Bai, Xiyuan Bai, Yidong Bai, Nandadulal Bairagi, Shounak Baksi, Teresa Balbi, Cosima T Baldari, Walter Balduini, Andrea Ballabio, Maria Ballester, Salma Balazadeh, Rena Balzan, Rina Bandopadhyay, Sreeparna Banerjee, Sulagna Banerjee, Ágnes Bánréti, Yan Bao, Mauricio S Baptista, Alessandra Baracca, Cristiana Barbati, Ariadna Bargiela, Daniela Barilà, Peter G Barlow, Sami J Barmada, Esther Barreiro, George E Barreto, Jiri Bartek, Bonnie Bartel, Alberto Bartolome, Gaurav R Barve, Suresh H Basagoudanavar, Diane C Bassham, Robert C Bast Jr, Alakananda Basu, Henri Batoko, Isabella Batten, Etienne E Baulieu, Bradley L Baumgarner, Jagadeesh Bayry, Rupert Beale, Isabelle Beau, Florian Beaumatin, Luiz R G Bechara, George R Beck Jr, Michael F Beers, Jakob Begun, Christian Behrends, Georg M N Behrens, Roberto Bei, Eloy Bejarano, Shai Bel, Christian Behl, Amine Belaid, Naïma Belgareh-Touzé, Cristina Bellarosa, Francesca Belleudi, Melissa Belló Pérez, Raquel Bello-Morales, Jackeline Soares de Oliveira Beltran, Sebastián Beltran, Doris Mangiaracina Benbrook, Mykolas Bendorius, Bruno A Benitez, Irene Benito-Cuesta, Julien Bensalem, Martin W Berchtold, Sabina Berezowska, Daniele Bergamaschi, Matteo Bergami, Andreas Bergmann, Laura Berliocchi, Clarisse Berlioz-Torrent, Amélie Bernard, Lionel Berthoux, Cagri G Besirli, Sebastien Besteiro, Virginie M Betin, Rudi Beyaert, Jelena S Bezbradica, Kiran Bhaskar, Ingrid Bhatia-Kissova, Resham Bhattacharya, Sujoy Bhattacharya, Shalmoli Bhattacharyya, Md Shenuarin Bhuiyan, Sujit Kumar Bhutia, Lanrong Bi, Xiaolin Bi, Trevor J Biden, Krikor Bijian, Viktor A Billes, Nadine Binart, Claudia Bincoletto, Asa B Birgisdottir, Geir Bjorkoy, Gonzalo Blanco, Ana Blas-Garcia, Janusz Blasiak, Robert Blomgran, Klas Blomgren, Janice S Blum, Emilio Boada-Romero, Mirta Boban, Kathleen Boesze-Battaglia, Philippe Boeuf, Barry Boland, Pascale Bomont, Paolo Bonaldo, Srinivasa Reddy Bonam, Laura Bonfili, Juan S Bonifacino, Brian A Boone, Martin D Bootman, Matteo Bordi, Christoph Borner, Beat C Bornhauser, Gautam Borthakur, Jürgen Bosch, Santanu Bose, Luis M Botana, Juan Botas, Chantal M Boulanger, Michael E Boulton, Mathieu Bourdenx, Benjamin Bourgeois, Nollaig M Bourke, Guilhem Bousquet, Patricia Boya, Peter V Bozhkov, Luiz H M Bozi, Tolga O Bozkurt, Doug E Brackney, Christian H Brandts, Ralf J Braun, Gerhard H Braus, Roberto Bravo-Sagua, José M Bravo-San Pedro, Patrick Brest, Marie-Agnès Bringer, Alfredo Briones-Herrera, V Courtney Broaddus, Peter Brodersen, Jeffrey L Brodsky, Steven L Brody, Paola G Bronson, Jeff M Bronstein, Carolyn N Brown, Rhoderick E Brown, Patricia C Brum, John H Brumell, Nicola Brunetti-Pierri, Daniele Bruno, Robert J Bryson-Richardson, Cecilia Bucci, Carmen Buchrieser, Marta Bueno, Laura Elisa Buitrago-Molina, Simone Buraschi, Shilpa Buch, J 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Villalba, Antonio Villalobo, Beatriz Villarejo-Zori, Francesc Villarroya, Joan Villarroya, Olivier Vincent, Cecile Vindis, Christophe Viret, Maria Teresa Viscomi, Dora Visnjic, Ilio Vitale, David J Vocadlo, Olga V Voitsekhovskaja, Cinzia Volonté, Mattia Volta, Marta Vomero, Clarissa Von Haefen, Marc A Vooijs, Wolfgang Voos, Ljubica Vucicevic, Richard Wade-Martins, Satoshi Waguri, Kenrick A Waite, Shuji Wakatsuki, David W Walker, Mark J Walker, Simon A Walker, Jochen Walter, Francisco G Wandosell, Bo Wang, Chao-Yung Wang, Chen Wang, Chenran Wang, Chenwei Wang, Cun-Yu Wang, Dong Wang, Fangyang Wang, Feng Wang, Fengming Wang, Guansong Wang, Han Wang, Hao Wang, Hexiang Wang, Hong-Gang Wang, Jianrong Wang, Jigang Wang, Jiou Wang, Jundong Wang, Kui Wang, Lianrong Wang, Liming Wang, Maggie Haitian Wang, Meiqing Wang, Nanbu Wang, Pengwei Wang, Peipei Wang, Ping Wang, Ping Wang, Qing Jun Wang, Qing Wang, Qing Kenneth Wang, Qiong A Wang, Wen-Tao Wang, Wuyang Wang, Xinnan Wang, Xuejun Wang, Yan Wang, Yanchang Wang, Yanzhuang Wang, Yen-Yun Wang, Yihua Wang, Yipeng Wang, Yu Wang, Yuqi Wang, Zhe Wang, Zhenyu Wang, Zhouguang Wang, Gary Warnes, Verena Warnsmann, Hirotaka Watada, Eizo Watanabe, Maxinne Watchon, Anna Wawrzyńska, Timothy E Weaver, Grzegorz Wegrzyn, Ann M Wehman, Huafeng Wei, Lei Wei, Taotao Wei, Yongjie Wei, Oliver H Weiergräber, Conrad C Weihl, Günther Weindl, Ralf Weiskirchen, Alan Wells, Runxia H Wen, Xin Wen, Antonia Werner, Beatrice Weykopf, Sally P Wheatley, J Lindsay Whitton, Alexander J Whitworth, Katarzyna Wiktorska, Manon E Wildenberg, Tom Wileman, Simon Wilkinson, Dieter Willbold, Brett Williams, Robin S B Williams, Roger L Williams, Peter R Williamson, Richard A Wilson, Beate Winner, Nathaniel J Winsor, Steven S Witkin, Harald Wodrich, Ute Woehlbier, Thomas Wollert, Esther Wong, Jack Ho Wong, Richard W Wong, Vincent Kam Wai Wong, W Wei-Lynn Wong, An-Guo Wu, Chengbiao Wu, Jian Wu, Junfang Wu, Kenneth K Wu, Min Wu, Shan-Ying Wu, Shengzhou Wu, Shu-Yan Wu, Shufang Wu, William K K Wu, Xiaohong Wu, Xiaoqing Wu, Yao-Wen Wu, Yihua Wu, Ramnik J Xavier, Hongguang Xia, Lixin Xia, Zhengyuan Xia, Ge Xiang, Jin Xiang, Mingliang Xiang, Wei Xiang, Bin Xiao, Guozhi Xiao, Hengyi Xiao, Hong-Tao Xiao, Jian Xiao, Lan Xiao, Shi Xiao, Yin Xiao, Baoming Xie, Chuan-Ming Xie, Min Xie, Yuxiang Xie, Zhiping Xie, Zhonglin Xie, Maria Xilouri, Congfeng Xu, En Xu, Haoxing Xu, Jing Xu, JinRong Xu, Liang Xu, Wen Wen Xu, Xiulong Xu, Yu Xue, Sokhna M S Yakhine-Diop, Masamitsu Yamaguchi, Osamu Yamaguchi, Ai Yamamoto, Shunhei Yamashina, Shengmin Yan, Shian-Jang Yan, Zhen Yan, Yasuo Yanagi, Chuanbin Yang, Dun-Sheng Yang, Huan Yang, Huang-Tian Yang, Hui Yang, Jin-Ming Yang, Jing Yang, Jingyu Yang, Ling Yang, Liu Yang, Ming Yang, Pei-Ming Yang, Qian Yang, Seungwon Yang, Shu Yang, Shun-Fa Yang, Wannian Yang, Wei Yuan Yang, Xiaoyong Yang, Xuesong Yang, Yi Yang, Ying Yang, Honghong Yao, Shenggen Yao, Xiaoqiang Yao, Yong-Gang Yao, Yong-Ming Yao, Takahiro Yasui, Meysam Yazdankhah, Paul M Yen, Cong Yi, Xiao-Ming Yin, Yanhai Yin, Zhangyuan Yin, Ziyi Yin, Meidan Ying, Zheng Ying, Calvin K Yip, Stephanie Pei Tung Yiu, Young H Yoo, Kiyotsugu Yoshida, Saori R Yoshii, Tamotsu Yoshimori, Bahman Yousefi, Boxuan Yu, Haiyang Yu, Jun Yu, Jun Yu, Li Yu, Ming-Lung Yu, Seong-Woon Yu, Victor C Yu, W Haung Yu, Zhengping Yu, Zhou Yu, Junying Yuan, Ling-Qing Yuan, Shilin Yuan, Shyng-Shiou F Yuan, Yanggang Yuan, Zengqiang Yuan, Jianbo Yue, Zhenyu Yue, Jeanho Yun, Raymond L Yung, David N Zacks, Gabriele Zaffagnini, Vanessa O Zambelli, Isabella Zanella, Qun S Zang, Sara Zanivan, Silvia Zappavigna, Pilar Zaragoza, Konstantinos S Zarbalis, Amir Zarebkohan, Amira Zarrouk, Scott O Zeitlin, Jialiu Zeng, Ju-Deng Zeng, Eva Žerovnik, Lixuan Zhan, Bin Zhang, Donna D Zhang, Hanlin Zhang, Hong Zhang, Hong Zhang, Honghe Zhang, Huafeng Zhang, Huaye Zhang, Hui Zhang, Hui-Ling Zhang, Jianbin Zhang, Jianhua Zhang, Jing-Pu Zhang, Kalin Y B Zhang, Leshuai W Zhang, Lin Zhang, Lisheng Zhang, Lu Zhang, Luoying Zhang, Menghuan Zhang, Peng Zhang, Sheng Zhang, Wei Zhang, Xiangnan Zhang, Xiao-Wei Zhang, Xiaolei Zhang, Xiaoyan Zhang, Xin Zhang, Xinxin Zhang, Xu Dong Zhang, Yang Zhang, Yanjin Zhang, Yi Zhang, Ying-Dong Zhang, Yingmei Zhang, Yuan-Yuan Zhang, Yuchen Zhang, Zhe Zhang, Zhengguang Zhang, Zhibing Zhang, Zhihai Zhang, Zhiyong Zhang, Zili Zhang, Haobin Zhao, Lei Zhao, Shuang Zhao, Tongbiao Zhao, Xiao-Fan Zhao, Ying Zhao, Yongchao Zhao, Yongliang Zhao, Yuting Zhao, Guoping Zheng, Kai Zheng, Ling Zheng, Shizhong Zheng, Xi-Long Zheng, Yi Zheng, Zu-Guo Zheng, Boris Zhivotovsky, Qing Zhong, Ao Zhou, Ben Zhou, Cefan Zhou, Gang Zhou, Hao Zhou, Hong Zhou, Hongbo Zhou, Jie Zhou, Jing Zhou, Jing Zhou, Jiyong Zhou, Kailiang Zhou, Rongjia Zhou, Xu-Jie Zhou, Yanshuang Zhou, Yinghong Zhou, Yubin Zhou, Zheng-Yu Zhou, Zhou Zhou, Binglin Zhu, Changlian Zhu, Guo-Qing Zhu, Haining Zhu, Hongxin Zhu, Hua Zhu, Wei-Guo Zhu, Yanping Zhu, Yushan Zhu, Haixia Zhuang, Xiaohong Zhuang, Katarzyna Zientara-Rytter, Christine M Zimmermann, Elena Ziviani, Teresa Zoladek, Wei-Xing Zong, Dmitry B Zorov, Antonio Zorzano, Weiping Zou, Zhen Zou, Zhengzhi Zou, Steven Zuryn, Werner Zwerschke, Beate Brand-Saberi, X Charlie Dong, Chandra Shekar Kenchappa, Zuguo Li, Yong Lin, Shigeru Oshima, Yueguang Rong, Judith C Sluimer, Christina L Stallings, Chun-Kit Tong

Autophagy Vol. 17 ( 1 ) page： 1 - 382 2021.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Chemical reaction mechanism in non-thermal plasma from the viewpoint of oxidative stress toward clinical cancer applications Reviewed International coauthorship International journal

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

FREE RADICAL BIOLOGY AND MEDICINE Vol. 100 page： S131 - S131 2016.11

CANCER AS A FERROTOXIC DISEASE: WHAT WE HAVE LEARNED FROM ANIMAL STUDIES TOWARD ITS PREVENTION Reviewed International journal

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

AMERICAN JOURNAL OF HEMATOLOGY Vol. 88 ( 5 ) page： E168 - E169 2013.5

Mysterious link between iron overload and CDKN2A/2B International journal Open Access

Toyokuni Shinya

Journal of Clinical Biochemistry and Nutrition Vol. 48 ( 1 ) page： 46 - 9 2011.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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.

DOI： 10.3164/jcbn.11-001FR

Asbestos-induced Carcinogenesis is Oxidative Stress-dependent Reviewed International journal

Shinya Toyokuni, Li Jiang, Hirotaka Nagai, Hiroki Ohara, Yasumasa Okazaki, Shinya Akatsuka, Yoriko Yamashita

FREE RADICAL BIOLOGY AND MEDICINE Vol. 49 page： S72 - S72 2010

Spaciotemporal alteration of 8-hydroxy-2'-deoxyguanosine levels in cardiomyocytes after myocardial infarction in rats. Reviewed International journal Open Access

Senri Miwa, Shinya Toyokuni, Takeshi Nishina, Takuya Nomoto, Makoto Hiroyasu, Kazunobu Nishimura, Masashi Komeda

Free radical research Vol. 36 ( 8 ) page： 853 - 8 2002.8

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

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, 48 h, 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 Reviewed International journal

Ichihashi K, Osawa T, Toyokuni S, Uchida K

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276 ( 26 ) page： 23903 - 23913 2001.6

Antioxidant alpha-tocopherol improves glycemic control of GK rats, a model of type 2 diabetes Reviewed International journal

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

DIABETES Vol. 49 page： A429 - A429 2000.5

4-hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress - Identification of proteasomes as target molecules Reviewed International coauthorship International journal

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

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274 ( 34 ) page： 23787 - 23793 1999.8

8-hydroxy-2'-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure (vol 107, pg 733, 1996) Reviewed International journal

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

JOURNAL OF INVESTIGATIVE DERMATOLOGY Vol. 108 ( 2 ) page： 237 - 237 1997.2

ACUTE NEPHROTOXICITY OF A CARCINOGENIC IRON CHELATE - SELECTIVE-INHIBITION OF A PROTEOLYTIC CONVERSION OF ALPHA(2U)-GLOBULIN TO THE KIDNEY FATTY-ACID-BINDING PROTEIN Reviewed International journal

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

FEBS LETTERS Vol. 357 ( 2 ) page： 165 - 167 1995.1

Susceptibility of Brca1((L63X/+)) rat to ovarian reserve dissipation by chemotherapeutic agents to breast cancer. Reviewed International journal 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 2025.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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 Reviewed International journal

本岡大社, 豊國伸哉

大気環境学会誌 Vol. 60 ( 1 ) page： A32 - A38 2025.1

Homozygous slc25a20 zebrafish mutant reveals insights into carnitine-acylcarnitine translocase deficiency pathogenesis. Reviewed International journal

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

Molecular genetics and metabolism reports Vol. 41 page： 101165 2024.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Cold plasma irradiation of chitosan: A straight pathway to selective antitumor therapy. Reviewed International coauthorship International journal 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 Vol. 281 ( Pt 4 ) page： 136513 2024.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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 Reviewed International journal

大守(川﨑) 伊織, 大内田守, 真下知士, 豊國伸哉

生体の科学 Vol. 75 ( 5 ) page： 454 - 455 2024.10

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) Reviewed International journal

豊國伸哉

生体の科学 Vol. 75 ( 5 ) page： 438 - 439 2024.10

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

Takemura Shigekazu, Minamiyama Yukiko, Ito Norihiko, Yamamoto Atsushi, Ichikawa Hiroshi, Nakagawa Kanako, Toyokuni Shinya, Osada-Oka Mayuko, Yoshikawa Toshikazu

Journal of Clinical Biochemistry and Nutrition Vol. 75 ( 1 ) page： 78 - 89 2024.7

Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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

特集活性酸素と酸化ストレスがんとフェロトーシス International journal

豊國伸哉

医学のあゆみ Vol. 289 ( 4 ) page： 263 - 266 2024.4

Protocol for the isolation of GFP-expressing ferroptosis-dependent extracellular vesicles in in vitro cell culture models. Reviewed International journal Open Access

Fumiya Ito, Izumi Yanatori, Katsuhiro Kato, Shinya Toyokuni

STAR protocols Vol. 5 ( 1 ) page： 102892 - 102892 2024.3

Language：English Publishing type：Research paper (scientific journal)

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

Epstein-Barr virus lytic gene BNRF1 promotes B-cell lymphomagenesis via IFI27 upregulation. Reviewed International journal Open Access

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

PLoS pathogens Vol. 20 ( 2 ) page： e1011954 2024.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Formalin-Fixed Paraffin-Embedded Proteomics of Malignant Mesothelioma and New Candidate Biomarkers Thioredoxin and Superoxide Dismutase 2 for Immunohistochemistry. Reviewed International journal

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

Laboratory investigation; a journal of technical methods and pathology Vol. 104 ( 2 ) page： 100299 2024.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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) Reviewed International journal

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 Vol. 113 ( 1 ) page： 428 - 429 2024.2

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 Reviewed International journal

界外誠, 本岡大社, 豊國伸哉

日本病理学会会誌 Vol. 113 ( 1 ) page： 479 - 479 2024.2

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) Reviewed International journal

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 Vol. 113 ( 1 ) page： 428 - 428 2024.2

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) Reviewed International journal

本岡大社, 豊國伸哉

日本病理学会会誌 Vol. 113 ( 1 ) page： 194 - 194 2024.2

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) Reviewed International journal

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 Vol. 113 ( 1 ) page： 429 - 429 2024.2

Tetrachloroaurate (III)–induced oxidation increases nonthermal plasma-induced aldehydes Reviewed International journal Open Access

Yasumasa Okazaki, Jun Yoshitake, Nanami Ito, Kanako Sasaki, Hiromasa Tanaka, Masaru Hori, Takahiro Shibata, Shinya Toyokuni

Advances in Redox Research Vol. 9 page： 100074 - 100074 2023.12

Generation and measurement of low-temperature plasma for cancer therapy: a historical review Reviewed International journal Open Access

Kenji Ishikawa, Keigo Takeda, Shinji Yoshimura, Takashi Kondo, Hiromasa Tanaka, Shinya Toyokuni, Kae Nakamura, Hiroaki Kajiyama, Masaaki Mizuno, Masaru Hori

Free Radical Research Vol. 57 ( 3 ) page： 239 - 270 2023.12

New iron export pathways acting via holo-ferritin secretion. Reviewed International journal Open Access

Izumi Yanatori, Fumio Kishi, Shinya Toyokuni

Archives of biochemistry and biophysics Vol. 746 page： 109737 - 109737 2023.9

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed International journal Open Access

Shinya Toyokuni, Yingyi Kong, Misako Katabuchi, Yuki Maeda, Yashiro Motooka, Fumiya Ito, Izumi Yanatori

Archives of biochemistry and biophysics Vol. 745 page： 109718 - 109718 2023.9

Language：English Publishing type：Research paper (scientific journal)

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

Ferroptosis contributes to multiple sclerosis and its pharmacological targeting suppresses experimental disease progression. Reviewed International journal 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 Vol. 30 ( 9 ) page： 2092 - 2103 2023.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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) Reviewed International journal

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 Vol. 82回 page： 170 - 170 2023.9

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) Reviewed International journal

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 Vol. 82回 page： 172 - 172 2023.9

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) Reviewed International journal

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 Vol. 82回 page： 907 - 907 2023.9

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 2023.8

Ferroptosis as ultimate target of cancer therapy. Reviewed International journal

Yashiro Motooka, Shinya Toyokuni

Antioxidants & redox signaling Vol. 39 ( 1-3 ) page： 206 - 223 2023.7

Language：English Publishing type：Research paper (scientific journal)

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, whereas suppressing factors are the prevention of lipid peroxidation by glutathione peroxidase 4 to protect lipid peroxidation and direct membrane repair via coenzyme Q10 and ESCRT-III pathways. 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. 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 which are generally rich in catalytic Fe(II). Here we summarize and discuss the recently uncovered responsible molecular mechanisms in association with iron metabolism, ferroptosis and cancer therapeutics. Finally, we also highlight the current classification of ferroptosis inducers.

DOI： 10.1089/ars.2022.0048

Sleep-wake patterns are altered with age, Prdm13 signaling in the DMH, and diet restriction in mice. Reviewed International journal Open Access

Shogo Tsuji, Cynthia S Brace, Ruiqing Yao, Yoshitaka Tanie, Hirobumi Tada, Nicholas Rensing, Seiya Mizuno, Julio Almunia, Yingyi Kong, Kazuhiro Nakamura, Takahisa Furukawa, Noboru Ogiso, Shinya Toyokuni, Satoru Takahashi, Michael Wong, Shin-Ichiro Imai, Akiko Satoh

Life science alliance Vol. 6 ( 6 ) 2023.6

Language：English Publishing type：Research paper (scientific journal)

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 DMH-Prdm13-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. Reviewed International journal Open Access

Fumiya Ito, Katsuhiro Kato, Izumi Yanatori, Yuki Maeda, Toyoaki Murohara, Shinya Toyokuni

BMC cancer Vol. 23 ( 1 ) page： 487 - 487 2023.5

Language：English Publishing type：Research paper (scientific journal)

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

Organic decomposition and synthesis reactions in lactated solution exposed to nonequilibrium atmospheric pressure plasma Reviewed International journal

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

Plasma Processes and Polymers Vol. 20 ( 5 ) 2023.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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 (C5H6O2) were detected formed by interactions of lactate or its fragments with •OH, organic radicals, and H2O2. 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

Field-effect transistor antigen/antibody-TMDs sensors for the detection of COVID-19 samples. Reviewed International journal Open Access

Ruben Canton-Vitoria, Kotaro Sato, Yashiro Motooka, Shinya Toyokuni, Zheng Liu, Ryo Kitaura

Nanoscale Vol. 15 ( 9 ) page： 4570 - 4580 2023.3

Language：English Publishing type：Research paper (scientific journal)

We fabricated sensors by modifying the surface of MoS2 and WS2 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 MoS2 or WS2, 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 Reviewed International journal

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 Vol. 112 ( 1 ) page： 370 - 370 2023.3

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 Reviewed International journal

加藤大暉, 本岡大社, 豊國伸哉

日本病理学会会誌 Vol. 112 ( 1 ) page： 382 - 382 2023.3

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 Reviewed International journal

本岡大社, 加藤大暉, 豊國伸哉

日本病理学会会誌 Vol. 112 ( 1 ) page： 256 - 256 2023.3

DNA oxidative damage caused by the aromatic amine MOCA —Examination of 8-hydroxy-2’-deoxyguanosine levels in rat liver— International journal

KOBAYASHI Saho, MOTOOKA Yashiro, KASHIWAGI Hiroki, TOYOKUNI Shinya

Journal of Occupational Safety and Health Vol. 16 ( 1 ) page： 45 - 49 2023.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：National Institute of Occupational Safety and Health, Japan

MOCA (4,4’-methylenebis (2-chloroaniline)) , an aromatic amine, is industrially used as a curing agent for urethane resins; however, it is classified as a Group 1 compound (carcinogenic to humans) by the International Agency for Research on Cancer, and there is concern regarding its health effects on workers. The mechanism underlying MOCA-mediated carcinogenesis is thought to be related to DNA damage caused by reactive oxygen species (ROS) and DNA adducts, which are mainly generated during metabolism in the liver. 8-Oxoguanine (8-OHdG), a product of ROS-induced oxidation, occurs at high frequency and can induce G→T transversion mutations during DNA replication. However, to the best of our knowledge, no study has examined whether MOCA induces the formation of highly mutagenic 8-OHdG in experimental animals. Here, F344 rats were orally exposed to 0, 0.4, 2, 10, or 50 mg/kg/day MOCA three times a week for 2 weeks; this is expected to exert toxicity through the hepatic metabolism of MOCA. Livers obtained from these animals were examined for pathology and 8-OHdG levels. In pathological sections, vacuolar degeneration was observed with 50 mg/kg/day MOCA. Further, MOCA-induced 8-OHdG levels showed a slight increasing trend, except at 0.4 mg/kg/day. However, none of these differences were significant. Thus, 8-OHdG is unlikely the main cause of carcinogenesis.

DOI： 10.2486/josh.josh-2022-0022-ta

Toward the next century of the Nagoya Journal of Medical Science: message from the Editor-in-chief. Reviewed International journal

Toyokuni S

Nagoya journal of medical science Vol. 85 ( 1 ) page： 1 - 4 2023.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Plasma activated Ringer's lactate solution. Reviewed International journal

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

Free radical research Vol. 57 ( 1 ) page： 14 - 20 2023.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Low-temperature plasma as magic wand to differentiate between the good and the evil. Reviewed International journal

Shinya Toyokuni, Hao Zheng, Yingyi Kong, Kotaro Sato, Kae Nakamura, Hiromasa Tanaka, Yasumasa Okazaki

Free radical research Vol. 57 ( 1 ) page： 38 - 46 2023.1

Language：English Publishing type：Research paper (scientific journal)

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 H2O2, NO2- 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 Reviewed International journal

Toyokuni Shinya

Vol. 95 ( 2 ) page： 177 - 183 2023

Thioredoxin deficiency increases oxidative stress and causes bilateral symmetrical degeneration in rat midbrain. Reviewed International journal Open Access

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

Neurobiology of disease Vol. 175 page： 105921 - 105921 2022.12

Language：English Publishing type：Research paper (scientific journal)

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. International journal Open Access

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

Scientific reports Vol. 12 ( 1 ) page： 18519 2022.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal

Chieko Aoki, Kenji Imai, Teruyuki Mizutani, Daisuke Sugiyama, Rika Miki, Yoshihiro Koya, Tomoko Kobayashi, Takafumi Ushida, Yukako Iitani, Noriyuki Nakamura, Taro Owaki, Hiroyoshi Nishikawa, Shinya Toyokuni, Hiroaki Kajiyama, Tomomi Kotani

Life sciences Vol. 308 page： 120955 - 120955 2022.11

Language：English Publishing type：Research paper (scientific journal)

AIMS: Molecular hydrogen (H2) has attracted growing interest because of its implications in various diseases. However, the molecular mechanisms underlying the remarkable effect of a small amount of H2 remain elusive. No knowledge has been available on the role of H2 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 H2 on T cells and its relation to preterm birth (PTB). MAIN METHODS: Exhaled H2 concentrations in pregnant women were measured and correlated with cytokine concentrations in maternal and umbilical cord blood. H2 was added to T cells collected from healthy donors, and differentiation and proliferation were examined. Energy metabolism was also examined. H2 was administered to mice and cytokine expression was compared. KEY FINDINGS: Our prospective observational study revealed that maternal production of H2 is significantly lower in pregnant women with PTB, suggesting its potential as a biomarker for predicting PTB. We found that H2 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 H2 to pregnant mice regulated inflammatory responses and reduced PTB caused by T cell activation, which further supports the notion that H2 may contribute to prolonged gestation through its immunomodulatory effect. SIGNIFICANCE: Measuring maternal H2-production could be a potential clinical tool in the management of PTB, and H2 may have positive impact on pregnancy maintenance.

DOI： 10.1016/j.lfs.2022.120955

Commentary for an article on photooxidation in isolated chloroplasts. Reviewed International journal Open Access

Shinya Toyokuni, Yingyi Kong, Danyang Mi

Archives of biochemistry and biophysics Vol. 726 page： 109133 - 109133 2022.9

Language：English Publishing type：Research paper (scientific journal)

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

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 Vol. 81回 page： P - 2007 2022.9

Non-thermal plasma elicits ferrous chloride-catalyzed DMPO-OH. Reviewed International journal

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

Free radical research Vol. 56 ( 9-10 ) page： 595 - 606 2022.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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 (H2O2), 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 FeCl2 or FeCl3 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 H2O2, using luminescent probe in the presence of FeCl2 or FeCl3. NTP-induced DMPO-OH levels were elevated 10–100 µM FeCl2 or 500 and 1000 µM FeCl3. NTP-induced DMPO-OH with 10 µM FeCl2 or FeCl3 was significantly scavenged by ascorbate, α-tocopherol, dithiothreitol, reduced glutathione, or oxidized glutathione, whereas dehydroascorbate was ineffective in 2 mM DMPO. NTP-induced H2O2 was significantly degraded by 100 µM FeCl2 and FeCl3 in an iron-dependent manner. Meanwhile, decomposition of H2O2 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 H2O2-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

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)

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 Vol. 81回 page： E - 2004 2022.9

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 Vol. 81回 page： S20 - 7 2022.9

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 Vol. 33 ( 1 ) page： 130 - 130 2022.9

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 Vol. 81回 page： P - 3012 2022.9

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 Vol. 81回 page： P - 2003 2022.9

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 Vol. 81回 page： P - 2007 2022.9

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) Reviewed International journal

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 Vol. 33 ( 1 ) page： 130 - 130 2022.9

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 Vol. 81回 page： S20 - 7 2022.9

Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers Reviewed International journal Open Access

Hirotoshi Soyama, Miki Nishio, Junji Otani, Toshiko Sakuma, Shintaro Takao, Shigeo Hara, Takaaki Masuda, Koshi Mimori, Shinya Toyokuni, John P. Lydon, Kazuwa Nakao, Hiroshi Nishina, Takumi Fukumoto, Tomohiko Maehama, Akira Suzuki

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vol. 119 ( 29 ) page： e2123134119 2022.7

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

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. Reviewed International journal Open Access

Nanase Igarashi, Kenichi Miyata, Tze Mun Loo, Masatomo Chiba, Aki Hanyu, Mika Nishio, Hiroko Kawasaki, Hao Zheng, Shinya Toyokuni, Shunsuke Kon, Keiji Moriyama, Yasuyuki Fujita, Akiko Takahashi

Nature communications Vol. 13 ( 1 ) page： 4157 - 4157 2022.7

Language：English Publishing type：Research paper (scientific journal)

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

Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. International journal 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 Vol. 4 ( 6 ) page： 651 - 662 2022.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Daiki Ito, Naoyuki Iwata, Kenji Ishikawa, Kae Nakamura, Hiroshi Hashizume, Camelia Miron, Hiromasa Tanaka, Hiroaki Kajiyama, Shinya Toyokuni, Masaaki Mizuno, Masaru Hori

APPLIED PHYSICS EXPRESS Vol. 15 ( 5 ) 2022.5

Language：English Publishing type：Research paper (scientific journal) Publisher：IOP Publishing Ltd

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+O-2+N-2 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

PCBP2 knockdown promotes ferroptosis in malignant mesothelioma. Reviewed International journal Open Access

Lin Yue, Yaguang Luo, Li Jiang, Yoshitaka Sekido, Shinya Toyokuni

Pathology international Vol. 72 ( 4 ) page： 242 - 251 2022.4

Language：English Publishing type：Research paper (scientific journal)

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

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

Editorial: Centennial anniversary of vitamin E discovery. Reviewed International journal Open Access

Toyokuni S, Noguchi N, Niki E

Free radical biology & medicine Vol. 183 page： 125 - 126 2022.4

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 Vol. 111 ( 1 ) page： 353 - 353 2022.3

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 Vol. 111 ( 1 ) page： 249 - 249 2022.3

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 Vol. 132 ( 2 ) 2022.1

Language：English Publishing type：Research paper (scientific journal)

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. International journal

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

The Journal of clinical investigation Vol. 132 ( 2 ) 2022.1

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 132 ( 2 ) 2022.1

Language：English Publishing type：Research paper (scientific journal)

Ferroptosis resistance determines high susceptibility of murine A/J strain to iron-induced renal carcinogenesis. Reviewed International journal Open Access

Zhen Cheng, Shinya Akatsuka, Guang Hua Li, Kiyoshi Mori, Takashi Takahashi, Shinya Toyokuni

Cancer science Vol. 113 ( 1 ) page： 65 - 78 2022.1

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed International journal Open Access

Yasumasa Okazaki, Kanako Sasaki, Nanami Ito, Hiromasa Tanaka, Ken-Ichiro Matsumoto, Masaru Hori, Shinya Toyokuni

Free radical research Vol. 56 ( 1 ) page： 17 - 27 2022.1

Language：English Publishing type：Research paper (scientific journal)

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 (1O2), superoxide (O2•-), hydrogen peroxide (H2O2), 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

Enhancement of ethanol production and cell growth in budding yeast by direct irradiation of low-temperature plasma Reviewed International journal Open Access

Hiromasa Tanaka, Shogo Matsumura, Kenji Ishikawa, Hiroshi Hashizume, Masafumi Ito, Kae Nakamura, Hiroaki Kajiyama, Fumitaka Kikkawa, Mikako Ito, Kinji Ohno, Yasumasa Okazaki, Shinya Toyokuni, Masaaki Mizuno, Masaru Hori

Japanese Journal of Applied Physics Vol. 61 ( SA ) 2022.1

Language：English Publishing type：Research paper (scientific journal)

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

Double-edged Sword Role of Iron-loaded Ferritin in Extracellular Vesicles. International journal

Shinya Toyokuni, Yingyi Kong, Hao Zheng, Danyang Mi, Misako Katabuchi, Yashiro Motooka, Fumiya Ito

Journal of cancer prevention Vol. 26 ( 4 ) page： 244 - 249 2021.12

Language：English Publishing type：Research paper (scientific journal)

Human epidemiological and animal studies have demonstrated that excess iron is a risk for cancer. The responsible mechanisms are: 1) increased intracellular iron catalyzes the Fenton reaction to generate hydroxyl radicals, leading to mutagenic oxidative DNA lesions; 2) iron is necessary for cellular proliferation as cofactors of many enzymes. Thus, iron-excess milieu promotes selecting cellular evolution to ferroptosis-resistance, a major basis for carcinogenesis. Ferritin is a 24-subunit nanocage protein required for iron storage under the regulation of the iron-regulatory protein (IRP)/iron-responsive element (IRE) system. Ferritin is a serum marker, representing total body iron storage. However, how ferritin is secreted extracellularly has been unelucidated. We recently discovered that an exosomal marker CD63 is regulated by the IRP/IRE system and that iron-loaded ferritin is secreted as extracellular vesicles under the guidance of nuclear receptor coactivator 4 (NCOA4). On the other hand, we found that macrophages under asbestos-induced ferroptosis emit ferroptosis-dependent extracellular vesicles (FedEVs), which are received by nearby mesothelial cells, resulting in significant mutagenic DNA damage. Therefore, cells, including macrophages, can share excess iron with other cells, via iron-loaded ferritin packaged in extracellular vesicles as safe non-catalytic iron. However, similar process, such as one involving FedEVs, may cause accumulation of excess iron in other specific cells, which may eventually promote carcinogenesis.

DOI： 10.15430/JCP.2021.26.4.244

Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin. Reviewed International journal Open Access

Fumiya Ito, Katsuhiro Kato, Izumi Yanatori, Toyoaki Murohara, Shinya Toyokuni

Redox biology Vol. 47 page： 102174 - 102174 2021.11

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed International journal

Giuseppe Valacchi, Alessandra Pecorelli, Shinya Toyokuni

Archives of biochemistry and biophysics Vol. 711 page： 109006 - 109006 2021.10

Embryonal erythropoiesis and aging exploit ferroptosis. Reviewed International coauthorship International journal Open Access

Hao Zheng, Li Jiang, Tsuyoshi Tsuduki, Marcus Conrad, Shinya Toyokuni

Redox biology Vol. 48 page： 102175 - 102175 2021.10

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

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

CD63 is regulated by iron via the IRE-IRP system and is important for ferritin secretion by extracellular vesicles. Reviewed International journal Open Access

Izumi Yanatori, Des R Richardson, Herschel S Dhekne, Shinya Toyokuni, Fumio Kishi

Blood Vol. 138 ( 16 ) page： 1490 - 1503 2021.10

Language：English Publishing type：Research paper (scientific journal)

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 be also secreted by the exosome pathway (Truman-Rosentsvit M. et al. BLOOD 131 (2018) 342-352), with serum ferritin levels classically reflecting body iron stores. Iron metabolism-associated proteins, such as ferritin, are intricately regulated by cellular iron levels via the iron responsive element (IRE)-iron regulatory protein (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 (UTR) of CD63 mRNA responsible for regulating its expression in response to increased iron. Cellular iron-loading caused a marked increase in CD63 expression and the secretion from cells of CD63 positive (i.e., CD63(+)) EVs, which were shown to contain ferritin-H (FtH) and -L (FtL). 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, poses significant impact 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. Reviewed International journal Open Access

Satomi Yogosawa, Makiko Ohkido, Takuro Horii, Yasumasa Okazaki, Jun Nakayama, Saishu Yoshida, Shinya Toyokuni, Izuho Hatada, Mitsuru Morimoto, Kiyotsugu Yoshida

Communications biology Vol. 4 ( 1 ) page： 1204 - 1204 2021.10

Language：English Publishing type：Research paper (scientific journal)

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 International journal

Toyokuni Shinya, Ito Fumiya, Motooka Yashiro

Metallomics Research Vol. 1 ( 1 ) page： rev-14 - rev-21 2021.10

Language：English Publishing type：Research paper (scientific journal) Publisher：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 2021.10

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Hiromasa Tanaka, Shogo Maeda, Kae Nakamura, Hiroshi Hashizume, Kenji Ishikawa, Mikako Ito, Kinji Ohno, Masaaki Mizuno, Yashiro Motooka, Yasumasa Okazaki, Shinya Toyokuni, Hiroaki Kajiyama, Fumitaka Kikkawa, Masaru Hori

PLASMA PROCESSES AND POLYMERS Vol. 18 ( 10 ) 2021.10

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-V C H VERLAG GMBH

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-H2O2 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

Low temperature plasma irradiation products of sodium lactate solution that induce cell death on U251SP glioblastoma cells were identified. Reviewed International journal Open Access

Hiromasa Tanaka, Yugo Hosoi, Kenji Ishikawa, Jun Yoshitake, Takahiro Shibata, Koji Uchida, Hiroshi Hashizume, Masaaki Mizuno, Yasumasa Okazaki, Shinya Toyokuni, Kae Nakamura, Hiroaki Kajiyama, Fumitaka Kikkawa, Masaru Hori

Scientific reports Vol. 11 ( 1 ) page： 18488 - 18488 2021.9

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 80回 page： [E1 - 6] 2021.9

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 Vol. 80回 page： [S10 - 6] 2021.9

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 Vol. 80回 page： [P2 - 1] 2021.9

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer. Reviewed International journal Open Access

Kenichi Miyata, Yoshinori Imai, Satoshi Hori, Mika Nishio, Tze Mun Loo, Ryo Okada, Liying Yang, Tomoyoshi Nakadai, Reo Maruyama, Risa Fujii, Koji Ueda, Li Jiang, Hao Zheng, Shinya Toyokuni, Toyonori Sakata, Katsuhiko Shirahige, Ryosuke Kojima, Mizuho Nakayama, Masanobu Oshima, Satoshi Nagayama, Hiroyuki Seimiya, Toru Hirota, Hideyuki Saya, Eiji Hara, Akiko Takahashi

Proceedings of the National Academy of Sciences of the United States of America Vol. 118 ( 35 ) 2021.8

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed International journal Open Access

Mayo Miura, Kenji Imai, Hiroyuki Tsuda, Rika Miki, Sho Tano, Yumiko Ito, Shima Hirako-Takamura, Yoshinori Moriyama, Takafumi Ushida, Yukako Iitani, Tomoko Nakano-Kobayashi, Shinya Toyokuni, Hiroaki Kajiyama, Tomomi Kotani

International journal of molecular sciences Vol. 22 ( 17 ) 2021.8

Language：English Publishing type：Research paper (scientific journal)

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 reaction 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 alveolarization 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 protein-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. Reviewed International journal Open Access

Rahul Checker, Deepak Sharma, Santosh K Sandur, Shinya Toyokuni

Free radical research Vol. 55 ( 8 ) page： 755 - 757 2021.8

Non-thermal plasma-induced DMPO-OH yields hydrogen peroxide. International journal

Yasumasa Okazaki, Hiromasa Tanaka, Ken-Ichiro Matsumoto, Masaru Hori, Shinya Toyokuni

Archives of biochemistry and biophysics Vol. 705 page： 108901 - 108901 2021.7

Language：English Publishing type：Research paper (scientific journal)

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 (H2O2), singlet oxygen (1O2), superoxide (O2●-), 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); 1O2 using a fluorescent probe; and O2●- and H2O2 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 O2●- was significantly scavenged by 10 μM DTT and GSH, while 1O2 was not efficiently scavenged by these compounds. GSSG degraded H2O2 more effectively than GSH and DTT, suggesting that the disulfide bonds reacted with H2O2. In the presence of 1-50 mM DMPO, NTP-induced H2O2 quantities were unchanged. The inhibitory effect of tempol concentration (50 and 100 μM) on H2O2 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 O2●-. 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. International journal Open Access

Li Jiang, Hao Zheng, Qinying Lyu, Shotaro Hayashi, Kotaro Sato, Yoshitaka Sekido, Kae Nakamura, Hiromasa Tanaka, Kenji Ishikawa, Hiroaki Kajiyama, Masaaki Mizuno, Masaru Hori, Shinya Toyokuni

Redox biology Vol. 43 page： 101989 - 101989 2021.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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 International journal

Okazaki Y.

Archives of Biochemistry and Biophysics Vol. 700 2021.3

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

Kae Nakamura, Nobuhisa Yoshikawa, Yuko Mizuno, Miwa Ito, Hiromasa Tanaka, Masaaki Mizuno, Shinya Toyokuni, Masaru Hori, Fumitaka Kikkawa, Hiroaki Kajiyama

Cancers Vol. 13 ( 5 ) page： 1 - 15 2021.3

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 165 2021.3

Mechanism of asbestos-induced carcinogenesis via dysregulation of redox-active iron International journal

Ito Fumiya, Toyokuni Shinya

CANCER SCIENCE Vol. 112 page： 998 - 998 2021.2

Mechanism of asbestos-induced carcinogenesis via dysregulation of redox-active iron International journal

Ito Fumiya, Toyokuni Shinya

CANCER SCIENCE Vol. 112 page： 998 - 998 2021.2

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 Vol. 112 page： 610 - 610 2021.2

Asbestos and talc contribute to ovarian carcinogenesis via iron overload

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

CANCER SCIENCE Vol. 112 page： 245 - 245 2021.2

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 Vol. 112 page： 996 - 996 2021.2

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

Homma T.

Free Radical Biology and Medicine Vol. 162 page： 255 - 265 2021.1

Prognostic significance of the MDM2/HMGA2 ratio and histological tumor grade in dedifferentiated liposarcoma International journal

Yamashita K.

Genes Chromosomes and Cancer Vol. 60 ( 1 ) page： 26 - 37 2021.1

Cancer Treatments Using Low-Temperature Plasma. Reviewed International journal

Hiromasa Tanaka, Masaaki Mizuno, Kenji Ishikawa, Shinya Toyokuni, Hiroaki Kajiyama, Fumitaka Kikkawa, Masaru Hori

Current medicinal chemistry Vol. 28 ( 41 ) page： 8549 - 8558 2021

Language：English Publishing type：Research paper (scientific journal)

Low-temperature plasma (LTP) is a partially ionized gas that contains electrons, 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 experiments 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

Mechanisms of toxicity caused by carbon nanotubes

YAMAGUCHI Shin-Ichiro, MORITA Masanobu, ITO Fumiya, XIE Qilin, TOYOKUNI Shinya, NAKAYAMA Masafumi

Annual Meeting of the Japanese Society of Toxicology Vol. 48.1 ( 0 ) page： O-4 - 4 2021

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Toxicology

Multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage cell death and NLRP3 inflammasome activation leading to granuloma and mesothelioma in rodents; however, it remains unknown how macrophages recognize MWCNTs. By a target screening of phagocyte receptors, we here demonstrated that T cell immunoglobulin mucin 4 (Tim4) and Tim1 recognize MWCNTs. Using Tim4-/- Tim1-/- mice, we revealed that Tim4 is involved in MWCNT-associated pathogenesis. Taken together, these results indicate that Tim4 and Tim1 are receptors for MWCNTs.

DOI： 10.14869/toxpt.48.1.0_o-4

The new era for research on polyphenols and food factors International journal

Oteiza P.I.

Archives of Biochemistry and Biophysics Vol. 696 2020.12

The new era for redox research. International journal Open Access

Yuji Naito, Koji Uchida, Shinya Toyokuni

Free radical research Vol. 54 ( 11-12 ) page： 787 - 789 2020.12

Neural stem cell-specific ITPA deficiency causes neural depolarization and epilepsy. International journal Open Access

Yuichiro Koga, Daisuke Tsuchimoto, Yoshinori Hayashi, Nona Abolhassani, Yasuto Yoneshima, Kunihiko Sakumi, Hiroshi Nakanishi, Shinya Toyokuni, Yusaku Nakabeppu

JCI insight Vol. 5 ( 22 ) 2020.11

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Clinical Investigation

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 (Itpa-cKO 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. International journal Open Access

Shinya Toyokuni, Yingyi Kong, Zhen Cheng, Kotaro Sato, Shotaro Hayashi, Fumiya Ito, Li Jiang, Izumi Yanatori, Yasumasa Okazaki, Shinya Akatsuka

Cancers Vol. 12 ( 11 ) page： 1 - 20 2020.11

Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI

Simple Summary: Cancer is a major cause of human mortality worldwide. No life on earth can live without iron. Persistent oxidative stress resulting from continuous use of iron and oxygen may be a fundamental cause of carcinogenesis. Many animal models demonstrated that excess iron may lead to 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 established. We predict that fine use of nanomaterials and non-thermal plasma may be able to reverse this situation.Evolution from the first life on earth to humans took similar to 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

Okazaki Yasumasa, Toyokuni Shinya

JAPANESE JOURNAL OF APPLIED PHYSICS Vol. 59 ( 11 ) 2020.11

Induction of cancer cell-specific ferroptosis by non-thermal plasma exposure International journal

Okazaki Y.

Japanese Journal of Applied Physics Vol. 59 ( 11 ) 2020.11

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

Yanatori I.

Biochimica et Biophysica Acta - General Subjects Vol. 1864 ( 11 ) 2020.11

Adjusted multiple gases in the plasma flow induce differential antitumor potentials of plasma-activated solutions International journal

Nakamura K.

Plasma Processes and Polymers Vol. 17 ( 10 ) 2020.10

Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice International journal

Nishio M.

Cancer Science Vol. 111 ( 10 ) page： 3576 - 3587 2020.10

Novel ovarian endometriosis model causes infertility via iron-mediated oxidative stress in mice International journal

Hayashi S.

Redox Biology Vol. 37 2020.10

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 Vol. 79回 page： OJ1 - 1 2020.10

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

Ito F.

Redox Biology Vol. 36 2020.9

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

Ohara Y.

Oncology Reports Vol. 44 ( 3 ) page： 838 - 848 2020.9

Role of carbonic anhydrases in ferroptosis-resistance International journal

Li Z.

Archives of Biochemistry and Biophysics Vol. 689 2020.8

Ferroptosis at the crossroads of infection, aging and cancer International journal

Toyokuni S.

Cancer Science Vol. 111 ( 8 ) page： 2665 - 2671 2020.8

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

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 Vol. 688 2020.7

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

Okazaki Y.

Cancer Science Vol. 111 ( 6 ) page： 2016 - 2027 2020.6

Frequent homozygous deletion of Cdkn2a/2b in tremolite-induced malignant mesothelioma in rats. Reviewed International journal

Yasumasa Okazaki, Nobuaki Misawa, Shinya Akatsuka, Norihiko Kohyama, Yoshitaka Sekido, Takashi Takahashi, Shinya Toyokuni

Cancer science Vol. 111 ( 4 ) page： 1180 - 1192 2020.4

Publishing type：Research paper (scientific journal)

The onset of malignant mesothelioma (MM) is linked to exposure to asbestos fibers. Asbestos fibers are classified as serpentine (chrysotile) or amphibole, which includes the crocidolite, amosite, anthophyllite, tremolite, and actinolite types. Although few studies have been undertaken, anthophyllite has been shown to be associated with mesothelioma, and tremolite, a contaminant in talc and chrysotile, is a risk factor for carcinogenicity. Here, after characterizing the length and width of these fibers by scanning electron microscopy, we explored the cytotoxicity induced by tremolite and anthophyllite in cells from an immortalized human mesothelial cell line (MeT5A), murine macrophages (RAW264.7), and in a rat model. Tremolite and short anthophyllite fibers were phagocytosed and localized to vacuoles, whereas the long anthophyllite fibers were caught on the pseudopod of the MeT5A and Raw 264.7 cells, according to transmission electron microscopy. The results from a 2-day time-lapse study revealed that tremolite was engulfed and damaged the MeT5A and RAW264.7 cells, but anthophyllite was not cytotoxic to these cells. Intraperitoneal injection of tremolite in rats induced diffuse serosal thickening, whereas anthophyllite formed focal fibrosis and granulomas on peritoneal serosal surfaces. Furthermore, the loss of Cdkn2a/2b, which are the most frequently lost foci in human MM, were observed in 8 cases of rat MM (homozygous deletion [5/8] and loss of heterozygosity [3/8]) by array-based comparative genomic hybridization techniques. These results indicate that tremolite initiates mesothelial injury and persistently frustrates phagocytes, causing subsequent peritoneal fibrosis and MM. The possible mechanisms of carcinogenicity based on fiber diameter/length are discussed.

DOI： 10.1111/cas.14358

A scrutiny of circulating microRNA biomarkers for tubular and glomerular injury in rats

Mth1 deficiency provides longer survival upon intraperitoneal crocidolite injection in female mice Reviewed International journal

Funahashi S.

Free Radical Research Vol. 54 ( 2-3 ) page： 195 - 205 2020.3

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 Reviewed International journal

本岡大社, 伊藤文哉, 豊國伸哉

日本病理学会会誌 Vol. 109 ( 1 ) page： 314 - 314 2020.3

Plasma-activated medium promotes autophagic cell death along with alteration of the mTOR pathway International journal

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

SCIENTIFIC REPORTS Vol. 10 ( 1 ) 2020.1

Augmented oxidative stress increases 8-oxoguanine preferentially in the transcriptionally active genomic regions Reviewed International journal

Akatsuka S.

Free Radical Research 2020

Oxidative stress-dependent and -independent death of glioblastoma cells induced by non-thermal plasma-exposed solutions Reviewed International journal

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 Vol. 9 ( 1 ) page： 13657 2019.9

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. International journal

Yanatori I, Richardson DR, Toyokuni S, Kishi F

Archives of biochemistry and biophysics Vol. 672 page： 108071 2019.9

Carbonic anhydrase 9 confers resistance to ferroptosis/apoptosis in malignant mesothelioma under hypoxia. Reviewed International journal

Zan Li, Li Jiang, Shan Hwu Chew, Tasuku Hirayama, Yoshitaka Sekido, Shinya Toyokuni

Redox biology Vol. 26 page： 101297 - 101297 2019.9

Language：English Publishing type：Research paper (scientific journal)

Hypoxia and acidity provide microenvironment for selection under evolutionary pressure and proliferation in cancer cells. Carbonic anhydrases (CAs) are a superfamily of metalloenzymes present in all life kingdoms, equilibrating the reactions among CO2, bicarbonate and H+. CA9, a membrane-associated α-CA, has been a drug target for various cancers. Whereas iron is essential not only for cancer cells but also for all the lives on earth, little is known on the association among hypoxia, iron metabolism, extracellular acidity and redox regulation. Malignant mesothelioma (MM), an aggressive tumor with poor prognosis, is an intriguing model in that asbestos-associated pathogenesis includes excess iron environment during carcinogenesis. Re-analysis of rat asbestos-induced MM model revealed an inverse association between high CA9 expression and survival. Here we used human MMs to identify the molecular events surrounding CA9 from the viewpoint of iron metabolism. CA9 expression was significantly higher in MM cells than in MeT-5A mesothelial cells, which was further amplified under hypoxia (1%O2) with increased catalytic Fe(II). CA9 suppression by inhibitors (S4 and U104) decreased viability and migration of MM cells, accompanied by overexpression of TFRC, IREB1/2 and FPN1(SLC40A1) and by downregulation of FTH/FTL. This expressional pattern was similar to that of erastin-induced ferroptosis in the same cells. Furthermore, we observed mitochondrial fission and enhanced autophagy with increased catalytic Fe(II) in both mitochondria and lysosomes after CA9 inhibition, accompanied by increased peroxides, mitochondrial O2- and lipid peroxidation. The eventual cell death was significantly inhibited by deferoxamine, ferrostatin-1 and Z-VAD-FMK, suggesting a mixed cell death of ferroptosis and apoptosis. Therefore, CA9 plays a role in equilibrating among hypoxia, iron metabolism and redox regulation in MM cells.

DOI： 10.1016/j.redox.2019.101297

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) Reviewed International journal

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 Vol. 78回 page： E - 1077 2019.9

l-Dehydroascorbic acid recycled by thiols efficiently scavenges non-thermal plasma-induced hydroxyl radicals Reviewed International journal

Yasumasa Okazaki, Hiromasa Tanaka, Masaru Hori, Shinya Toyokuni

Archives of Biochemistry and Biophysics Vol. 669 page： 87 - 95 2019.7

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 Reviewed International journal

水野勇太, 本岡大社, 豊國伸哉

日本病理学会会誌 Vol. 108 ( 1 ) page： 460 - 461 2019.4

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

Toxicology Vol. 415 page： 26-36 2019.3

Non-thermal plasma-activated medium modified metabolomic profiles in the glycolysis of U251SP glioblastoma International journal

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 Vol. 662 page： 83 - 92 2019.2

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 Vol. 32 ( 1 ) page： 19-26 2019.1

Non-thermal plasma specifically kills oral squamous cell carcinoma cells in a catalytic Fe(II)-dependent manner Reviewed International journal

Kotaro Sato, Lei Shi, Fumiya Ito, Yuuki Ohara, Yashiro Motooka, Hiromasa Tanaka, Masaaki Mizuno, Masaru Hori, Tasuku Hirayama, Hideharu Hibi, Shinya Toyokuni

Journal of Clinical Biochemistry and Nutrition Vol. 65 ( 4 ) page： 8 - 15 2019

Iron Metabolism and Ferroptosis Reviewed International journal

Toyokuni Shinya, Yanatori Izumi

FERROPTOSIS IN HEALTH AND DISEASE page： 27 - 41 2019

KAGAWA Takumi, ZÁRYBNICKÝ Tomáš, OMI Takanao, SHIRAI Yuji, TOYOKUNI Shinya, ODA Shingo, YOKOI Tsuyoshi

Annual Meeting of the Japanese Society of Toxicology Vol. 46 ( 0 ) page： P - 233 2019

Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Toxicology

[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

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

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

FREE RADICAL RESEARCH Vol. 52 ( 11-12 ) page： 1424 - 1431 2018.12

Asbestos exposure as a possible cause of ovarian carcinogenesis

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

CANCER SCIENCE Vol. 109 page： 255-255 2018.12

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

Ohara Yuuki, Enomoto Atsushi, Takahashi Masahide, Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 454-454 2018.12

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 Vol. 109 page： 781-781 2018.12

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

Jiang Li, Akatsuka Shinya, Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 830-830 2018.12

Inflammatory microenvironment derived from asbestos increases mutagenesis to repairing mesothelial cell

Ito Fumiya, Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 830 - 830 2018.12

Ferroptosis in Cancer Research

Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 195-195 2018.12

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

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 Vol. 109 page： 781-781 2018.12

Asbestos exposure as a possible cause of ovarian carcinogenesis International journal

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

CANCER SCIENCE Vol. 109 page： 255-255 2018.12

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

Ohara Yuuki, Enomoto Atsushi, Takahashi Masahide, Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 454-454 2018.12

Ferroptosis in Cancer Research International journal

Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 195-195 2018.12

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

Jiang Li, Akatsuka Shinya, Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 830-830 2018.12

Inflammatory microenvironment derived from asbestos increases mutagenesis to repairing mesothelial cell International journal

Ito Fumiya, Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 830-830 - 830 2018.12

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

Fumiya Ito, Hideyuki Hisashi, Shinya Toyokuni

Nagoya journal of medical science Vol. 80 ( 4 ) page： 597 - 604 2018.11

Language：English Publishing type：Research paper (scientific journal) Publisher：NAGOYA UNIV, SCH MED

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 similar to 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 213 carcinogen to humans, which requires considerable regulations for use. In contrast, tangled MWCNTs of similar to 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 page： 1-10 2018.10

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

Toyokuni S

Free radical biology & medicine 2018.10

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 2018.10

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 page： 1-51 2018.9

Plasma Biology for a Novel Cancer Treatment International journal

Toyokuni Shinya

JSAP Annual Meetings Extended Abstracts Vol. 2018.2 ( 0 ) page： 37 - 37 2018.9

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 Vol. 77回 page： 1110 - 1110 2018.9

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 Vol. 77回 page： 118 - 118 2018.9

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. International journal

Koji Uchida, Takahiro Shibata, Shinya Toyokuni, Bareket Daniel, Kamelija Zarkovic, Neven Zarkovic, Shlomo Sasson

Free radical biology & medicine Vol. 124 page： 12 - 20 2018.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier Inc.

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 International journal

Furuta Takahiro, Shi Lei, Toyokuni Shinya

PATHOLOGY INTERNATIONAL Vol. 68 ( 7 ) page： 442 - 443 2018.7

Administration of molecular hydrogen during pregnancy improves behavioral abnormalities of offspring in a maternal immune activation model Reviewed International journal

Kenji Imai, Tomomi Kotani, Hiroyuki Tsuda, Tomoko Nakano, Takafumi Ushida, Akira Iwase, Taku Nagai, Shinya Toyokuni, Akio Suzumura, Fumitaka Kikkawa

Scientific Reports Vol. 8 ( 1 ) page： 9221 2018.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

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 Reviewed International journal

Yuuki Ohara, Takahiko Ito, Makoto Ito, Kyoko Yamashita, Shinya Toyokuni

Medical Mycology Case Reports Vol. 20 page： 39 - 42 2018.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier B.V.

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. Reviewed International journal

Yuuki Ohara, Seiichi Kato, Daisuke Yamashita, Akira Satou, Yoshie Shimoyama, Chie Hamaie, Motoki Sato, Nobutaro Ban, Koji Yamamoto, Takehiro Yamada, Hisashi Kawai, Koichi Ohshima, Shigeo Nakamura, Shinya Toyokuni

Pathology international Vol. 68 ( 6 ) page： 374 - 381 2018.6

Language：English Publishing type：Research paper (scientific journal)

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 18 F-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 Vol. 29 ( 1 ) page： 71 - 71 2018.6

Ferroptosis in carcinogenesis and tumor biology International journal

Toyokuni Shinya

FREE RADICAL BIOLOGY AND MEDICINE Vol. 120 page： S19-S19 - S19 2018.5

Expression of P-REX2a is associated with poor prognosis in endometrial malignancies. International journal

Sho Takeshita, Yoriko Yamashita, Kosuke Shiomi, Nako Suzuki, Jun Yoshida, Aya Naiki-Ito, Shugo Suzuki, Shinya Akatsuka, Shinya Toyokuni, Takashi Takahashi, Shoko Mase, Atsushi Arakawa, Mayumi Sugiura-Ogasawara, Satoru Takahashi

Oncotarget Vol. 9 ( 37 ) page： 24778 - 24786 2018.5

Language：English Publishing type：Research paper (scientific journal)

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 page： 1-5 2018.5

Effect of molecular hydrogen on uterine inflammation during preterm labour Reviewed International journal

Tomoko Nakano, Tomomi Kotani, Kenji Imai, Yukako Iitani, Takafumi Ushida, Hiroyuki Tsuda, Hua Li, Akira Iwase, Shinya Toyokuni, Fumitaka Kikkawa

Biomedical Reports Vol. 8 ( 5 ) page： 454 - 460 2018.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Spandidos Publications

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&lt
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&lt
0.05). The protein levels of Cox2 in the LPS group were also significantly increased compared with those in the control (P&lt
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 Reviewed International journal

Yuuki Ohara, Shan Hwu Chew, Nobuaki Misawa, Shenqi Wang, Daiki Somiya, Kae Nakamura, Hiroaki Kajiyama, Fumitaka Kikkawa, Yuta Tsuyuki, Li Jiang, Kyoko Yamashita, Yoshitaka Sekido, Kenneth E. Lipson, Shinya Toyokuni

Oncotarget Vol. 9 ( 26 ) page： 18494 - 18509 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Impact Journals LLC

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 Reviewed International journal

Kyoko Yamashita, Kenichi Kohashi, Yuichi Yamada, Takeaki Ishii, Yoshihiro Nishida, Hiroshi Urakawa, Ichiro Ito, Mitsuru Takahashi, Takeshi Inoue, Masafumi Ito, Yuuki Ohara, Yoshinao Oda, Shinya Toyokuni

Histopathology Vol. 72 ( 5 ) page： 729 - 738 2018.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Ltd

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 Vol. 107 ( 1 ) page： 291 - 291 2018.4

Phlebotomy as a preventive measure for crocidolite-induced mesothelioma in male rats Reviewed International journal

Yuuki Ohara, Shan-Hwu Chew, Takahiro Shibata, Yasumasa Okazaki, Kyoko Yamashita, Shinya Toyokuni

Cancer Science Vol. 109 ( 2 ) page： 330 - 339 2018.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Ltd

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 Yuuki, Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 781 - 781 2018.1

Molecular mechanisms in oxidative stress-induced carcinogenesis

Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 163 - 163 2018.1

Ferric nitrilotriacetate induced renal tumorigenesis in MUTYH deficient mice

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

CANCER SCIENCE Vol. 109 page： 167 - 167 2018.1

Ferric nitrilotriacetate induced renal tumorigenesis in MUTYH deficient mice International journal

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

CANCER SCIENCE Vol. 109 page： 167-167 2018.1

Molecular mechanisms in oxidative stress-induced carcinogenesis International journal

Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 163-163 2018.1

Phlebotomy attenuates the growth of malignant mesothelioma on rat model International journal

Ohara Yuuki, Toyokuni Shinya

CANCER SCIENCE Vol. 109 page： 781-781 2018.1

Glioblastoma Cell Lines Display Different Sensitivities to Plasma-Activated Medium International journal

Tanaka Hiromasa, Mizuno Masaaki, Ishikawa Kenji, Takeda Keigo, Hashizume Hiroshi, Nakamura Kae, Utsumi Fumi, Kajiyama Hiroaki, Okazaki Yasumasa, Toyokuni Shinya, Akiyama Shinichi, Maruyama Shoichi, Kikkawa Fumitaka, Hori Masaru

IEEE TRANSACTIONS ON RADIATION AND PLASMA MEDICAL SCIENCES Vol. 2 ( 2 ) page： 99 - 102 2018

Language：English Publishing type：Research paper (scientific journal) Publisher：IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC

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

Astaxanthin ameliorates ferric nitrilotriacetate-induced renal oxidative injury in rats.

State of the art in medical applications using non-thermal atmospheric pressure plasma Reviewed International journal

Hiromasa Tanaka, Kenji Ishikawa, Masaaki Mizuno, Shinya Toyokuni, Hiroaki Kajiyama, Fumitaka Kikkawa, Hans-Robert Metelmann, Masaru Hori

Plasma Physics, Association of Asia Pacific Physical Societies 2017 Vol. 1 ( 1 ) 2017.12

Phlebotomy as a preventive measure for crocidolite-induced mesothelioma in male rats. Reviewed International journal

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

Cancer Science 2017.11

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

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 Vol. 67 ( 11 ) page： 564-574 2017.11

Role of catalytic iron and oxidative stress in nitrofen-induced congenital diaphragmatic hernia and its amelioration by Saireito (TJ-114) Reviewed International journal

Shima Hirako, Hiroyuki Tsuda, Fumiya Ito, Yasumasa Okazaki, Tasuku Hirayama, Hideko Nagasawa, Tomoko Nakano, Kenji Imai, Tomomi Kotani, Fumitaka Kikkawa, Shinya Toyokuni

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 61 ( 3 ) page： 176 - 182 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

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 association with nitrofen and Saireito. We observed increased immunostaining 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(II) and pulmonary 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(II). Saireito ameliorated all of these in IMR90-SV cells. In conclusion, catalytic Fe(II)-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 nitrofen-induced CDH.

DOI： 10.3164/jcbn.17-17

Fenton reaction-induced renal carcinogenesis in Mutyh-deficient mice exhibits less chromosomal aberrations than the rat model International journal

Guang Hua Li, Shinya Akatsuka, Shan Hwu Chew, Li Jiang, Takahiro Nishiyama, Akihiko Sakamoto, Takashi Takahashi, Mitsuru Futakuchi, Hiromu Suzuki, Kunihiko Sakumi, Yusaku Nakabeppu, Shinya Toyokuni

PATHOLOGY INTERNATIONAL Vol. 67 ( 11 ) page： 564 - 574 2017.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY

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 p16(INKA) 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. International journal

Brent R Stockwell, José Pedro Friedmann Angeli, Hülya Bayir, Ashley I Bush, Marcus Conrad, Scott J Dixon, Simone Fulda, Sergio Gascón, Stavroula K Hatzios, Valerian E Kagan, Kay Noel, Xuejun Jiang, Andreas Linkermann, Maureen E Murphy, Michael Overholtzer, Atsushi Oyagi, Gabriela C Pagnussat, Jason Park, Qitao Ran, Craig S Rosenfeld, Konstantin Salnikow, Daolin Tang, Frank M Torti, Suzy V Torti, Shinya Toyokuni, K A Woerpel, Donna D Zhang

Cell Vol. 171 ( 2 ) page： 273 - 285 2017.10

Language：English Publishing type：Research paper (scientific journal) Publisher：CELL PRESS

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 Q(10). 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. Reviewed

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 2017.10

Osteogenic differentiation in dedifferentiated liposarcoma: a study of 36 cases in comparison to the cases without ossification. Reviewed International journal

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 2017.10

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. Reviewed

Yanatori I, Richardson DR, Toyokuni S, Kishi F.

Journal of Biological Chemistry Vol. 292 ( 32 ) page： 13205-13229 2017.8

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 International journal

Izumi Yanatori, Des R. Richardson, Shinya Toyokuni, Fumio Kishi

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 292 ( 32 ) page： 13205 - 13229 2017.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

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. Reviewed International journal

Yanatori I, Richardson DR, Toyokuni S, Kishi F

Journal of Biological Chemistry Vol. 292 ( 32 ) page： 13205-13229 2017.8

IRON OVERLOAD ENHANCED CELL DEATH INDUCED BY CARCINOGENIC FIBROUS MATERIALS

Ito Fumiya, Shi Lei, Toyokuni Shinya

AMERICAN JOURNAL OF HEMATOLOGY Vol. 92 ( 8 ) page： E365 - E365 2017.8

IRON OVERLOAD ENHANCED CELL DEATH INDUCED BY CARCINOGENIC FIBROUS MATERIALS International journal

Fumiya Ito, Lei Shi, Shinya Toyokuni

AMERICAN JOURNAL OF HEMATOLOGY Vol. 92 ( 8 ) page： E365 - E365 2017.8

Novel Intraperitoneal Treatment With Non-Thermal Plasma-Activated Medium Inhibits Metastatic Potential of Ovarian Cancer Cells. Reviewed

Nakamura K, Peng Y, Utsumi F, Tanaka H, Mizuno M, Toyokuni S, Hori M, Kikkawa F, Kajiyama H.Sci

Scientific Reports Vol. 7 ( 1 ) page： 6085 2017.7

Novel Intraperitoneal Treatment With Non-Thermal Plasma-Activated Medium Inhibits Metastatic Potential of Ovarian Cancer Cells. International journal

Kae Nakamura, Yang Peng, Fumi Utsumi, Hiromasa Tanaka, Masaaki Mizuno, Shinya Toyokuni, Masaru Hori, Fumitaka Kikkawa, Hiroaki Kajiyama

Scientific reports Vol. 7 ( 1 ) page： 6085 - 6085 2017.7

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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

Non-thermal plasma induces a stress response in mesothelioma cells resulting in increased endocytosis, lysosome biogenesis and autophagy. Reviewed

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 Vol. 108 page： 904-917 2017.7

Astaxanthin ameliorates ferric nitrilotriacetate-induced renal oxidative injury in rats. Reviewed

Okazaki Y, Okada S, Toyokuni S.

Journal of Clinical Biochemistry and Nutrition Vol. 61 ( 1 ) page： 18-24 2017.7

Yasumasa Okazaki, Shigeru Okada, Shinya Toyokuni

Journal of clinical biochemistry and nutrition Vol. 61 ( 1 ) page： 18 - 24 2017.7

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

Daily intake of vegetables can reduce the risk of cancer and lifestyle-related diseases. However, supplementary intake of 13 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 Reviewed International journal

Shinya Toyokuni, Fumiya Ito, Kyoko Yamashita, Yasumasa Okazaki, Shinya Akatsuka

FREE RADICAL BIOLOGY AND MEDICINE Vol. 108 page： 610 - 626 2017.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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 International journal

Lei Shi, Fumiya Ito, Yue Wang, Yasumasa Okazaki, Hiromasa Tanaka, Masaaki Mizuno, Masaru Hori, Tasuku Hirayama, Hideko Nagasawa, Des R. Richardson, Shinya Toyokuni

FREE RADICAL BIOLOGY AND MEDICINE Vol. 108 page： 904 - 917 2017.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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. Reviewed International journal

Okazaki Y, Okada S, Toyokuni S

Journal of Clinical Biochemistry and Nutrition Vol. 61 ( 1 ) page： 18-24 2017.7

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics. Reviewed

Yoshimaru T, Aihara K, Komatsu M, Matsushita Y, Okazaki Y, Toyokuni S, Honda J, Sasa M, Miyoshi Y, Otaka A, Katagiri T.

Scientific Reports Vol. 7 ( 1 ) page： 1821 2017.5

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics International journal

Tetsuro Yoshimaru, Keisuke Aihara, Masato Komatsu, Yosuke Matsushita, Yasumasa Okazaki, Shinya Toyokuni, Junko Honda, Mitsunori Sasa, Yasuo Miyoshi, Akira Otaka, Toyomasa Katagiri

SCIENTIFIC REPORTS Vol. 7 ( 1 ) page： 1821 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Estradiol (E2) and the oestrogen receptor-alpha (ER alpha) 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/ER alpha signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERa activity-regulator synthetic peptide (ERAP: 165-177 amino acids), derived from a-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 a-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. Reviewed

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 Vol. 19 ( 5 ) page： 636-647 2017.5

Significance of low mTORC1 activity in defining the characteristics of brain tumor stem cells International journal

Yi-Peng Han, Atsushi Enomoto, Yukihiro Shiraki, Shen-Qi Wang, Xiaoze Wang, Shinya Toyokuni, Naoya Asai, Kaori Ushida, Hosne Ara, Fumiharu Ohka, Toshihiko Wakabayashi, Jie Ma, Atsushi Natsume, Masahide Takahashi

NEURO-ONCOLOGY Vol. 19 ( 5 ) page： 636 - 647 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS INC

The significance of mammalian target of rapamycin complex 1 (mTORC1) activity in the maintenance of cancer stem cells (CSCs) remains controversial. Previous findings 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 significance of mTORC1 activity in brain tumor stem cells (BTSCs).
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.
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.
Our findings 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 Reviewed International journal

Kyoko Yamashita, Kenichi Kohashi, Yuichi Yamada, Yoshihiro Nishida, Hiroshi Urakawa, Yoshinao Oda, Shinya Toyokuni

Human Pathology Vol. 63 page： 63 - 69 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：W.B. Saunders

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

Cancer and trace metal element

In response to Sharing different perspectives to understand asbestos-induced carcinogenesis: Acomment to Jiang et al. (2016) by Alessandro Francesco Gualtieri (2017) International journal

Shinya Toyokuni, Li Jiang

CANCER SCIENCE Vol. 108 ( 5 ) page： 1089 - 1090 2017.5

Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma. International journal

Shan Hwu Chew, Yasumasa Okazaki, Shinya Akatsuka, Shenqi Wang, Li Jiang, Yuuki Ohara, Fumiya Ito, Hideyuki Saya, Yoshitaka Sekido, Shinya Toyokuni

Free radical biology & medicine Vol. 106 page： 91 - 99 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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 axon 9, with CD44v8-10 among the variant isoforms expressed. The expression of CD44 variable axon 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. Reviewed

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 Vol. 108 ( 4 ) page： 785-794 2017.4

Iron and thiol redox signaling in cancer: An exquisite balance to escape ferroptosis. Reviewed

Toyokuni S, Ito F, Yamashita K, Okazaki Y, Akatsuka S.

Free Radical Biology & Medicine Vol. 108 page： 610-626 2017.4

Iron and thiol redox signaling in cancer: An exquisite balance to escape ferroptosis. Reviewed International journal

Toyokuni S, Ito F, Yamashita K, Okazaki Y, Akatsuka S

Free Radical Biology & Medicine Vol. 108 page： 610-626 2017.4

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor. International journal

Yuki Shikata, Tetsuro Yoshimaru, Masato Komatsu, Hiroto Katoh, Reiko Sato, Shuhei Kanagaki, Yasumasa Okazaki, Shinya Toyokuni, Etsu Tashiro, Shumpei Ishikawa, Toyomasa Katagiri, Masaya Imoto

Cancer science Vol. 108 ( 4 ) page： 785 - 794 2017.4

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY

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. Reviewed

Yamashita K, Kohashi K, Yamada Y, Nishida Y, Urakawa H, Oda Y, Toyokuni S.

Human Pathology Vol. 63 page： 63-69 2017.2

Primary extraskeletal osteosarcoma: a clinicopathological study of 18 cases focusing on MDM2 amplification status. Reviewed International journal

Yamashita K, Kohashi K, Yamada Y, Nishida Y, Urakawa H, Oda Y, Toyokuni S

Human Pathology Vol. 63 page： 63-69 2017.2

Vol. 61 ( 2 ) page： 168 - 173 2017.2

Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma. Reviewed

Chew SH, Okazaki Y, Akatsuka S, Wang S, Jiang L, Ohara Y, Ito F, Saya H, Sekido Y, Toyokuni S.

Free Radical Biology & Medicine Vol. 106 page： 91-99 2017.2

Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma. Reviewed International journal

Chew SH, Okazaki Y, Akatsuka S, Wang S, Jiang L, Ohara Y, Ito F, Saya H, Sekido Y, Toyokuni S

Free Radical Biology & Medicine Vol. 106 page： 91-99 2017.2

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 Vol. 30 page： 27A-27A 2017.2

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 Vol. 97 page： 27A - 27A 2017.2

Clinicopathological and Molecular Features of Dedifferentiated Liposarcoma with Ossification: A Comparative Study with Dedifferentiated Liposarcoma without Ossification and Extraskeletal Osteosarcoma International journal

Kyoko Yamashita, Kenichi Kohashi, Yuichi Yamada, Yoshinao Oda, Shinya Toyokuni

LABORATORY INVESTIGATION Vol. 97 page： 27A - 27A 2017.2

Kyoko Yamashita, Kenichi Kohashi, Yuichi Yamada, Yoshinao Oda, Shinya Toyokuni

MODERN PATHOLOGY Vol. 30 page： 27A - 27A 2017.2

Future perspective of strategic non-thermal plasma therapy for cancer treatment.

Hiroaki Kajiyama, Fumi Utsumi, Kae Nakamura, Hiromasa Tanaka, Shinya Toyokuni, Masaru Hori, Fumitaka Kikkawa

Journal of clinical biochemistry and nutrition Vol. 60 ( 1 ) page： 33 - 38 2017.1

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

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

Primary cause of carcinogenesis: what toxicology can contribute

TOYOKUNI Shinya

Annual Meeting of the Japanese Society of Toxicology Vol. 44 ( 0 ) page： S11 - S11 2017

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 Vol. 61 2017

がんと微量金属元素 (Cancer and trace metal element) International journal

伊藤文哉, 豊國伸哉

臨床検査Journal of clinical laboratory medicine Vol. 61 2017

Future perspective of strategic non-thermal plasma therapy for cancer treatment. Reviewed

Kajiyama H, Utsumi F, Nakamura K, Tanaka H, Toyokuni S, Hori M, Kikkawa F.

Journal of Clinical Biochemistry and Nutrition Vol. 60 ( 1 ) page： 33-38 2016.12

Future perspective of strategic non-thermal plasma therapy for cancer treatment. Reviewed International journal

Kajiyama H, Utsumi F, Nakamura K, Tanaka H, Toyokuni S, Hori M, Kikkawa F

Journal of Clinical Biochemistry and Nutrition Vol. 60 ( 1 ) page： 33-38 2016.12

Editorial: The cutting edge of zinc biology. International journal

Taiho Kambe, Toshiyuki Fukada, Shinya Toyokuni

Archives of biochemistry and biophysics Vol. 611 page： 1 - 2 2016.12

The emerging role of progesterone receptor membrane component 1 (PGRMC1) in cancer biology. International journal

Cahill MA, Jazayeri JA, Catalano SM, Toyokuni S, Kovacevic Z, Richardson DR

Biochimica et biophysica acta Vol. 1866 ( 2 ) page： 339 - 349 2016.12

Molecular hydrogen ameliorates several characteristics of preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model. Reviewed International journal

Takafumi Ushida, Tomomi Kotani, Hiroyuki Tsuda, Kenji Imai, Tomoko Nakano, Shima Hirako, Yumiko Ito, Hua Li, Yukio Mano, Jingwen Wang, Rika Miki, Eiko Yamamoto, Akira Iwase, Yasuko K Bando, Masaaki Hirayama, Kinji Ohno, Shinya Toyokuni, Fumitaka Kikkawa

Free radical biology & medicine Vol. 101 ( 16 ) page： 524 - 533 2016.12

Language：English Publishing type：Research paper (scientific journal)

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 International journal

Ohara, Y; Wang, SQ; Jiang, L; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE Vol. 100 page： S127 - S127 2016.11

Urokinase-type plasminogen activator receptor promotes proliferation and invasion with reduced cisplatin sensitivity in malignant mesothelioma. Reviewed International journal

Shenqi Wang, Li Jiang, Yipeng Han, Shan Hwu Chew, Yuuki Ohara, Shinya Akatsuka, Liang Weng, Koji Kawaguchi, Takayuki Fukui, Yoshitaka Sekido, Kohei Yokoi, Shinya Toyokuni

Oncotarget Vol. 7 ( 43 ) page： 69565 - 69578 2016.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Impact Journals LLC

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. Reviewed

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. Vol. pii: S0891-5849 ( 16 ) page： 30978-9. 2016.10

Molecular hydrogen ameliorates several characteristics of preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model. Reviewed International journal

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. Vol. pii: S0891-5849 ( 16 ) page： 30978-9. 2016.10

Non-thermal plasma prevents progression of endometriosis in mice. Reviewed

Ishida C, Mori M, Nakamura K, Tanaka H, Mizuno M, Hori M, Iwase A, Kikkawa F, Toyokuni S.

Free Radical Research Vol. 50 ( 10 ) page： 1131-1139 2016.10

Non-thermal plasma prevents progression of endometriosis in mice Reviewed International journal

Chiharu Ishida, Masahiko Mori, Kae Nakamura, Hiromasa Tanaka, Masaaki Mizuno, Masaru Hori, Akira Iwase, Fumitaka Kikkawa, Shinya Toyokuni

FREE RADICAL RESEARCH Vol. 50 ( 10 ) page： 1131 - 1139 2016.10

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Endometriosis is observed in approximate to 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). Reviewed International coauthorship International journal

Malyn Ungsurungsie, Young-Joon Surh, Shinya Toyokuni, Michael Jonathan Davies

Free radical research Vol. 50 ( 10 ) page： 1045 - 1046 2016.10

Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450. Reviewed International journal

Cai Zong, C Edwin Garner, Chinyen Huang, Xiao Zhang, Lingyi Zhang, Jie Chang, Shinya Toyokuni, Hidenori Ito, Masashi Kato, Toshihiro Sakurai, Sahoko Ichihara, Gaku Ichihara

Toxicology letters Vol. 258 page： 249 - 258 2016.9

Language：English Publishing type：Research paper (scientific journal)

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 50mg/kg body weight BID (100mg/kg BW/day) for 3days, 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 1200ppm 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(+)/1200ppm 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 Reviewed International coauthorship International journal

Lei Shi, Yue Wang, Fumiya Ito, Yasumasa Okazaki, Hiromasa Tanaka, Masaaki Mizuno, Masaru Hori, Des R. Richardson, Shinya Toyokuni

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 605 page： 109 - 16 2016.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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 mu M) 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 mu M). 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. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2016.05.016

Low-temperature plasma in biology and medicine Reviewed International journal

Hori M.

Archives of Biochemistry and Biophysics Vol. 605 page： 1 - 2 2016.9

Antenatal Saireito (TJ-114) Can Improve Pulmonary Hypoplasia and Pulmonary Vascular Remodeling in Nitrofen-Induced Congenital Diaphragmatic Hernia. Reviewed

Hirako S, Tsuda H, Kotani T, Sumigama S, Mano Y, Nakano T, Imai K, Li H, Toyokuni S, Kikkawa F.

Phytother Res. Vol. 30 ( 9 ) page： 1474-80 2016.9

Urokinase-type plasminogen activator receptor promotes proliferation and invasion with reduced cisplatin sensitivity in malignant mesothelioma. Reviewed

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. page： 11829 2016.9

Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450. Reviewed

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. Vol. 258 page： 249-58 2016.9

Biphasic effects of l-ascorbate on the tumoricidal activity of non-thermal plasma against malignant mesothelioma cells. Reviewed

Shi L, Wang Y, Ito F, Okazaki Y, Tanaka H, Mizuno M, Hori M, Richardson DR, Toyokuni S.

Arch Biochem Biophys. Vol. 605 page： 106-16 2016.9

Antenatal Saireito (TJ-114) Can Improve Pulmonary Hypoplasia and Pulmonary Vascular Remodeling in Nitrofen-Induced Congenital Diaphragmatic Hernia. Reviewed International journal

Shima Hirako, Hiroyuki Tsuda, Tomomi Kotani, Seiji Sumigama, Yukio Mano, Tomoko Nakano, Kenji Imai, Hua Li, Shinya Toyokuni, Fumitaka Kikkawa

Phytotherapy research : PTR Vol. 30 ( 9 ) page： 1474 - 80 2016.9

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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 (C) 2016 John Wiley & Sons, Ltd.

DOI： 10.1002/ptr.5645

Molecular hydrogen suppresses activated Wnt/beta-catenin signaling Reviewed International journal

Yingni Lin, Bisei Ohkawara, Mikako Ito, Nobuaki Misawa, Kentaro Miyamoto, Yasuhiko Takegami, Akio Masuda, Shinya Toyokuni, Kinji Ohno

SCIENTIFIC REPORTS Vol. 6 ( 6 ) page： 31986 2016.8

Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Molecular hydrogen (H-2) is effective for many diseases. However, molecular bases of H-2 have not been fully elucidated. Cumulative evidence indicates that H-2 acts as a gaseous signal modulator. We found that H-2 suppresses activated Wnt/beta-catenin signaling by promoting phosphorylation and degradation of beta-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of beta-catenin abolished the suppressive effect of H-2. H-2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H-2 has no direct effect on GSK3 itself. Knockdown of adenomatous polyposis coli (APC) or Axin1, which form the beta-catenin degradation complex, minimized the suppressive effect of H2 on beta-catenin accumulation. Accordingly, the effect of H-2 requires CK1/GSK3-phosphorylation sites of beta-catenin, as well as the beta-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H-2 reduces the activation of Wnt/beta-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H-2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating beta-catenin accumulation. We first demonstrate that H-2 suppresses abnormally activated Wnt/beta-catenin signaling, which accounts for the protective roles of H-2 in a fraction of diseases.

DOI： 10.1038/srep31986

Contrasting intra- and extracellular distribution of catalytic ferrous iron in ovalbumin-induced peritonitis. Reviewed International journal

Fumiya Ito, Takahiro Nishiyama, Lei Shi, Masahiko Mori, Tasuku Hirayama, Hideko Nagasawa, Hiroyuki Yasui, Shinya Toyokuni

Biochemical and biophysical research communications Vol. 476 ( 4 ) page： 600 - 606 2016.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

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. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2016.06.003

Contrasting intra- and extracellular distribution of catalytic ferrous iron in ovalbumin-induced peritonitis. Reviewed

Ito F, Nishiyama T, Shi L, Mori M, Hirayama T, Nagasawa H, Yasui H, Toyokuni S.

Biochem Biophys Res Commun. Vol. 476 ( 4 ) page： 600-6 2016.8

Molecular hydrogen suppresses activated Wnt/β-catenin signaling. Reviewed

Lin Y, Ohkawara B, Ito M, Misawa N, Miyamoto K, Takegami Y, Masuda A, Toyokuni S, Ohno K.

Sci Rep. Vol. 25 ( 6 ) page： 31986 2016.8

Iron function and carcinogenesis Reviewed International journal

Akatsuka S., Toyokuni S.

Nihon rinsho. Japanese journal of clinical medicine Vol. 74 ( 7 ) page： 1168 - 1175 2016.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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.

Effects of Plasma-Activated Medium on Cells

The emerging role of progesterone receptor membrane component 1 (PGRMC1) in cancer biology. Reviewed

Cahill MA, Jazayeri JA, Catalano SM, Toyokuni S, Kovacevic Z, Richardson DR.

Biochim Biophys Acta. 2016 Jul 22. Vol. pii: S0304-419X ( 16 ) page： 30049-X. 2016.7

Dual preventive benefits of iron elimination by desferal in asbestos-induced mesothelial carcinogenesis Reviewed International journal

Jiang Li, Chew Shan-Hwu, Nakamura Kosuke, Ohara Yuuki, Akatsuka Shinya, Toyokuni Shinya

CANCER SCIENCE Vol. 107 ( 7 ) page： 908 - 915 2016.7

Possible therapeutic option of aqueous plasma for refractory ovarian cancer Reviewed International journal

Kajiyama Hiroaki, Utsumi Fumi, Nakamura Kae, Tanaka Hiromasa, Mizuno Masaaki, Toyokuni Shinya, Hori Masaru, Kikkawa Fumitaka

CLINICAL PLASMA MEDICINE Vol. 4 ( 1 ) page： 14 - 18 2016.7

The emerging role of progesterone receptor membrane component 1 (PGRMC1) in cancer biology. Reviewed International journal

Cahill MA, Jazayeri JA, Catalano SM, Toyokuni S, Kovacevic Z, Richardson DR

Biochim Biophys Acta. 2016 Jul 22. Vol. pii: S0304-419X ( 16 ) page： 30049-X. 2016.7

[Iron function and carcinogenesis].

Akatsuka S, Toyokuni S

Nihon rinsho. Japanese journal of clinical medicine Vol. 74 ( 7 ) page： 1168 - 75 2016.7

[Iron function and carcinogenesis].

Akatsuka S, Toyokuni S

Nihon rinsho. Japanese journal of clinical medicine Vol. 74 ( 7 ) page： 1168 - 75 2016.7

Variable susceptibility of ovarian cancer cells to non-thermal plasma-activated medium. Reviewed

Vol. 35 ( 6 ) page： 3169-77 2016.6

Dual preventive benefits of iron elimination by desferal in asbestos-induced mesothelial carcinogenesis. Reviewed

Jiang L, Chew SH, Nakamura K, Ohara Y, Akatsuka S, Toyokuni S.

Cancer Sci. 2016 Jul;107(7):908-15. Vol. 107 ( 7 ) page： 908-15 2016.6

Dual preventive benefits of iron elimination by desferal in asbestos-induced mesothelial carcinogenesis. Reviewed International journal

Jiang L, Chew SH, Nakamura K, Ohara Y, Akatsuka S, Toyokuni S

Cancer Sci. 2016 Jul;107(7):908-15. Vol. 107 ( 7 ) page： 908-15 2016.6

Variable susceptibility of ovarian cancer cells to non-thermal plasma-activated medium Reviewed International journal

Fumi Utsumi, Hiroaki Kajiyama, Kae Nakamura, Hiromasa Tanaka, Masaaki Mizuno, Shinnya Toyokuni, Masaru Hori, Fumitaka Kikkawa

ONCOLOGY REPORTS Vol. 35 ( 6 ) page： 3169 - 77 2016.6

Language：English Publishing type：Research paper (scientific journal) Publisher：SPANDIDOS PUBL LTD

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 Reviewed International journal

Toyokuni S.

Pathology International Vol. 66 ( 5 ) page： 245 - 259 2016.5

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. Invited Reviewed International journal

Toyokuni S

Pathol Int. Vol. 66 ( 5 ) page： 245-259 2016.5

Oxidative stress as an iceberg in carcinogenesis and cancer biology Reviewed International journal

Toyokuni Shinya

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 595 page： 46 - 49 2016.4

Oxidative stress as an iceberg in carcinogenesis and cancer biology. Reviewed

Toyokuni S.

Arch Biochem Biophys. 2016 Apr 1;595:46-9. Vol. 595 page： 46-9 2016.4

Oxidative stress as an iceberg in carcinogenesis and cancer biology. Reviewed International journal

Toyokuni S

Arch Biochem Biophys. 2016 Apr 1;595:46-9. Vol. 595 page： 46-9 2016.4

Tribute issue: Helmut Sies and oxidative stress: Venit, vidit, vicit Reviewed International journal

Forman H.

Archives of Biochemistry and Biophysics Vol. 595 2016.4

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. Vol. 1863 ( 4 ) page： 727-748 2016.4

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics. International journal

Danuta S Kalinowski, Christian Stefani, Shinya Toyokuni, Tomas Ganz, Gregory J Anderson, Nathan V Subramaniam, Debbie Trinder, John K Olynyk, Anita Chua, Patric J Jansson, Sumit Sahni, Darius J R Lane, Angelica M Merlot, Zaklina Kovacevic, Michael L H Huang, C Soon Lee, Des R Richardson

Biochimica et biophysica acta Vol. 1863 ( 4 ) page： 727 - 48 2016.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

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. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2016.01.026

Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis. Reviewed

Wang Y, Okazaki Y, Shi L, Kohda H, Tanaka M, Taki K, Nishioka T, Hirayama T, Nagasawa H, Yamashita Y, Toyokuni S.

Cancer Sci. Vol. 107 ( 3 ) page： 250-257 2016.3

Pain-reducing anesthesia prevents oxidative stress in human term placenta. Reviewed

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. Vol. 58 ( 2 ) page： 156-60 2016.3

Pain-reducing anesthesia prevents oxidative stress in human term placenta Reviewed International journal

Tsuzuki, Y., Yamashita, Y., Hattori, Y., Li, G.H., Akatsuka, S., Kotani, T., Kikkawa, F., Naiki-Ito, A., Takahashi, S., Nishiwaki, K., Toyokuni, S.

Journal of Clinical Biochemistry and Nutrition Vol. 58 ( 2 ) page： 156 - 60 2016.3

Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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. Reviewed International journal

Yue Wang, Yasumasa Okazaki, Lei Shi, Hiro Kohda, Minoru Tanaka, Kentaro Taki, Tomoki Nishioka, Tasuku Hirayama, Hideko Nagasawa, Yoriko Yamashita, Shinya Toyokuni

Cancer science Vol. 107 ( 3 ) page： 250 - 7 2016.3

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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. Reviewed

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. Vol. 91 page： 154-163 2016.2

Neuroprotective potential of molecular hydrogen against perinatal brain injury via suppression of activated microglia Reviewed International journal

Kenji Imai, Tomomi Kotani, Hiroyuki Tsuda, Yukio Mano, Tomoko Nakano, Takafumi Ushida, Hua Li, Rika Miki, Seiji Sumigama, Akira Iwase, Akihiro Hirakawa, Kinji Ohno, Shinya Toyokuni, Hideyuki Takeuchi, Tetsuya Mizuno, Akio Suzumura, Fumitaka Kikkawa

FREE RADICAL BIOLOGY AND MEDICINE Vol. 91 page： 154 - 63 2016.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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 (H-2), 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. H-2 reduced the LPS-induced expression of pro-inflammatory cytokines, oxidative damage and microglial activation in the fetal brains. Next, we investigated how H-2 contributes to neuroprotection, focusing on microglia, using primary cultured microglia and neurons. H-2 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 H-2, involved in the process of activating microglia. These results suggested that H-2 holds promise for the prevention of inflammation related to perinatal brain injury. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2015.12.015

Plasma with high electron density and plasma-activated medium for cancer treatment Reviewed International journal

Tanaka Hiromasa, Mizuno Masaaki, Ishikawa Kenji, Kondo Hiroki, Takeda Keigo, Hashizume Hiroshi, Nakamura Kae, Utsumi Fumi, Kajiyama Hiroaki, Kano Hiroyuki, Okazaki Yasumasa, Toyokuni Shinya, Akiyama Shin'ichi, Maruyama Shoichi, Yamada Suguru, Kodera Yasuhiro, Kaneko Hiroki, Terasaki Hiroko, Hara Hirokazu, Adachi Tetsuo, Iida Machiko, Yajima Ichiro, Kato Masashi, Kikkawa Fumitaka, Hori Masaru

CLINICAL PLASMA MEDICINE Vol. 3 ( 2 ) page： 72 - 76 2015.12

Ovarian endometriosis-associated stromal cells reveal persistently high affinity for iron. Reviewed

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. Vol. 6 page： 578-586 2015.12

Cancer therapy using non-thermal atmospheric pressure plasma with ultra-highelectron density Reviewed International journal

H. Tanaka, M. Mizuno, S. Toyokuni, S. Maruyama, Y. Kodera, H. Terasaki, T. Adachi, M. Kato, F. Kikkawa, M. Hori

Phys Plasmas Vol. 22 ( 12 ) page： 122004 2015.12

Ovarian endometriosis-associated stromal cells reveal persistently high affinity for iron Reviewed International journal

Masahiko Mori, Fumiya Ito, Lei Shi, Yue Wang, Chiharu Ishida, Yuka Hattori, Masato Niwa, Tasuku Hirayama, Hideko Nagasawa, Akira Iwase, Fumitaka Kikkawa, Shinya Toyokuni

REDOX BIOLOGY Vol. 6 page： 578 - 586 2015.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

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. (C) 2015 The Authors. Published by Elsevier B.V.

DOI： 10.1016/j.redox.2015.10.001

TANAKA Hiromasa, MIZUNO Masaaki, TOYOKUNI Shinya, MARUYAMA Shoichi, KODERA Yasuhiro, ADACHI Tetsuo, TERASAKI Hiroko, KATO Masashi, KIKKAWA Fumitaka, HORI Masaru

Vol. 91 ( 12 ) page： 776 - 779 2015.12

Effects of Plasma-Activated Medium on Cells

TANAKA Hiromasa, MIZUNO Masaaki, TOYOKUNI Shinya, MARUYAMA Shoichi, KODERA Yasuhiro, ADACHI Tetsuo, TERASAKI Hiroko, KATO Masashi, KIKKAWA Fumitaka, HORI Masaru

Vol. 91 ( 12 ) page： 776 - 779 2015.12

Maternal molecular hydrogen administration on lipopolysaccharide-induced mouse fetal brain injury Reviewed International journal

Tomoko Nakano, Tomomi Kotani, Yukio Mano, Hiroyuki Tsuda, Kenji Imai, Takafumi Ushida, Hua Li, Rika Miki, Seiji Sumigama, Yoshiaki Sato, Akira Iwase, Akihiro Hirakawa, Masato Asai, Shinya Toyokuni, Fumitaka Kikkawa

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 57 ( 3 ) page： 178 - 182 2015.11

Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

Fetal brain injury is often related to prenatal inflammation; however, there is a lack of effective therapy. Recently, molecular hydrogen (H-2), a specific antioxidant to hydroxyl radical and peroxynitrite, has been reported to have anti-inflammatory properties. The aim of this study was to investigate whether maternal H-2 administration could protect the fetal brain against inflammation. Pregnant C3H/HeN mice received an intraperitoneal injection of lipopolysaccharide (LPS) on gestational day 15.5 and were provided with H2 water for 24 h prior to LPS injection. Pup brain samples were collected on gestational day 16.5, and the levels of apoptosis and oxidative damage were evaluated using immunohistochemistry. Interleukin-6 (IL-6) levels were examined using real-time PCR. The levels of apoptosis and oxidative damage, as well as the levels of IL-6 mRNA, increased significantly when the mother was injected with LPS than that in the control group. However, these levels were significantly reduced when H2 was administered prior to the LPS-injection. Our results suggest that LPS-induced apoptosis, oxidative damage and inflammation in the fetal brain were ameliorated by maternal H2 administration. Antenatal H2 administration might protect the premature brain against maternal inflammation.

DOI： 10.3164/jcbn.15-90

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. Reviewed

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. Vol. 57 ( 3 ) page： 178-182 2015.11

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 Vol. 57 ( 6 ) page： 548 - 554 2015.11

L. Zhang, C. Zong, S. Ichihara, H. Naito, S. Toyokuni, S. Kumagai, G. Ichihara

TOXICOLOGY LETTERS Vol. 238 ( 2 ) page： S245 - S245 2015.10

Recent information on the outbreak of cholangiocarcinoma in a printing company

Occupational Health Review Vol. 28 ( 2 ) page： 131 - 156 2015.9

Malignant mesothelioma as an oxidative stress-induced cancer: An update. Reviewed International journal

Shan Hwu Chew, Shinya Toyokuni

Free radical biology & medicine Vol. 86 page： 166 - 78 2015.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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-beta pathways are also affected in MM. Recently, analyses of familial MM sorted out BAP] as a novel responsible tumor suppressor gene, whose function is not fully elucidated. Five-year survival of mesothelioma is still similar to 8%, and this cancer is increasing worldwide. Connective tissue growth factor, a secretory protein creating a vicious cycle mediated by beta-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. (C) 2015 Elsevier Inc. All rights reserved.

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 Reviewed International journal

Shinya Toyokuni, Li Jiang, Shenqi Wang, Ayaka Hirao, Tamae Wada, Chieko Soh, Kazumi Toyama, Akira Kawada

PATHOLOGY INTERNATIONAL Vol. 65 ( 8 ) page： 415 - 9 2015.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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 Reviewed International journal

Yamashita Kyoko, Nagai Hirotaka, Toyokuni Shinya

LABORATORY INVESTIGATION Vol. 95 ( 7 ) page： 749 - 764 2015.7

Chemical conversion of human fibroblasts into neuronal cells: dawn of future clinical trials. Open Access

Shinya Toyokuni

Journal of clinical biochemistry and nutrition Vol. 56 ( 3 ) page： 165 - 165 2015.5

Plasma-Activated Solution: Mechanism of Action, Clinical Application, and Industrialization Reviewed International journal

Tanaka H., Mizuno M., Toyokuni S., Maruyama S., Kodera Y., Kikkawa F., Hori M.

Fukuoka igaku zasshi = Hukuoka acta medica Vol. 106 ( 4 ) page： 71 - 76 2015.4

[Plasma-Activated Solution: Mechanism of Action, Clinical Application, and Industrialization].

Hiromasa Tanaka, Masaaki Mizuno, Shinya Toyokuni, Shoichi Maruyama, Yasuhiro Kodera, Fumitaka Kikkawa, Masaru Hori

Fukuoka igaku zasshi = Hukuoka acta medica Vol. 106 ( 4 ) page： 71 - 6 2015.4

Asbestos and multi-walled carbon nanotubes generate distinct oxidative responses in inflammatory cells Reviewed International journal

Funahashi Satomi, Okazaki Yasumasa, Ito Daiki, Asakawa Atsushi, Nagai Hirotaka, Tajima Masafumi, Toyokuni Shinya

Journal of Clinical Biochemistry and Nutrition Vol. 56 ( 2 ) page： 111 - 7 2015.3

Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

Asbestos exposure is considered a social burden by causing mesothelioma. 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-d]pyridazine-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 neutrophils 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 oxidative 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 Vol. 104 ( 1 ) page： 336 - 336 2015.3

Possible involvement of iron-induced oxidative insults in neurodegeneration Reviewed International journal

Takeshi Asano, Masato Koike, Shin-ichi Sakata, Yukiko Takeda, Tomoko Nakagawa, Taku Hatano, Satoshi Ohashi, Manabu Funayama, Kenji Yoshimi, Masato Asanuma, Shinya Toyokuni, Hideki Mochizuki, Yasuo Uchiyama, Nobutaka Hattori, Kazuhiro Iwai

NEUROSCIENCE LETTERS Vol. 588 ( 1 ) page： 29 - 35 2015.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

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. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2014.12.052

MINIMAL INFLAMMOGENICITY OF PRISTINE SINGLE-WALL CARBON NANOTUBES Reviewed International journal

Shinya Toyokuni, Li Jiang, Ryo Kitaura, Hisanori Shinohara

NAGOYA JOURNAL OF MEDICAL SCIENCE Vol. 77 ( 1-2 ) page： 195 - 202 2015.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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.

Catalytic ferrous iron in amniotic fluid as a predictive marker of human maternal-fetal disorders. Reviewed

Minimal inflammogenicity of pristine single-wall carbon nanotubes.

Toyokuni S, Jiang LI, Kitaura R, Shinohara H

Nagoya journal of medical science Vol. 77 ( 1-2 ) page： 195 - 202 2015.2

Minimal inflammogenicity of pristine single-wall carbon nanotubes.

Toyokuni S, Jiang LI, Kitaura R, Shinohara H

Nagoya journal of medical science Vol. 77 ( 1-2 ) page： 195 - 202 2015.2

MINIMAL INFLAMMOGENICITY OF PRISTINE SINGLE-WALL CARBON NANOTUBES

Toyokuni Shinya, Jiang Li, Kitaura Ryo, Shinohara Hisanori

NAGOYA JOURNAL OF MEDICAL SCIENCE Vol. 77 ( 1-2 ) page： 195 - 202 2015.2

Yuka Hattori, Takahiro Mukaide, Li Jiang, Tomomi Kotani, Hiroyuki Tsuda, Yukio Mano, Seiji Sumigama, Tasuku Hirayama, Hideko Nagasawa, Fumitaka Kikkawa, Shinya Toyokuni

Journal of clinical biochemistry and nutrition Vol. 56 ( 1 ) page： 57 - 63 2015.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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 oxidative 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. Reviewed International journal

Yoriko Yamashita, Tetsuro Nagasaka, Aya Naiki-Ito, Shinya Sato, Shugo Suzuki, Shinya Toyokuni, Masafumi Ito, Satoru Takahashi

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc Vol. 28 ( 1 ) page： 111 - 7 2015.1

Language：English Publishing type：Research paper (scientific journal)

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

Maternal molecular hydrogen treatment attenuates lipopolysaccharide-induced rat fetal lung injury. Reviewed

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 Vol. 49 page： 1026-1037 2015

Possible involvement of iron-induced oxidative insults in neurodegeneration. Reviewed

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 Vol. 588 page： 29-35 2015

Receptor role of annexin A2 in the mesothelial endocytosis of crocidolite fibers. Reviewed

Yamashita K, Nagai H and Toyokuni S.

Lab Invest Vol. 95 page： 749-764 2015

Minimal inflammogenicity of pristine single-wall carbon nanotubes. Reviewed

Toyokuni S, Jiang L, Kitaura R and Shinohara S.

Nagoya J Med Sci Vol. 77 page： 195-202 2015

Asbestos and multi-walled carbon nanotubes generate distinct oxidative responses in inflammatory cells. Reviewed

Funahashi F, Okazaki Y, Ito D, Asakawa A, Nagai H, Tajima M and Toyokuni S.

J Clin Biochem Nutr 56: 111-117, 2015. Vol. 56 page： 111-117 2015

Catalytic ferrous iron in amniotic fluid as a predictive marker of human maternal-fetal disorders. Reviewed

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. Vol. 56 page： 57-63 2015

Napsin A is a specific marker for ovarian clear cell adenocarcinoma. Reviewed

Yamashita Y, Nagasaka T, Naiki-Ito A, Sato S, Suzuki S, Toyokuni S, Ito M and Takahashi S.

Mod Pathol Vol. 28 page： 111-117 2015

Malignant mesothelioma as an oxidative stress-induced cancer: An update. Reviewed

Chew SH and Toyokuni S.

Free Radic Biol Med Vol. 86 page： 166-178 2015

Chemical conversion of human fibroblasts into neuronal cells: dawn of future clinical trials. Invited

Toyokuni S.

J Clin Biochem Nutr Vol. 56 page： 165 2015

Application of intermittent microwave irradiation to western blot analysis. Reviewed

Liu YT, Toyokuni S.

Methods Mol Biol Vol. 1314 page： 185-190 2015

Aging rather than sun exposure is a major determining factor for the density of miR-125b-positive epidermal stem cells in human skin. Reviewed

Toyokuni S, Jiang L, Wang S, Hirao A, Wada T, Soh C, Tohyama K and Kawada A.

Pathol Int Vol. 65 page： 415-419 2015

A trial to find appropriate animal models of dichloropropane-induced cholangiocarcinoma based on the hepatic distribution of glutathione S-transferases. Reviewed

Zhang L, Zong C, Ichihara S, Naito H, Toyokuni S, Kumagai S, Ichihara G.

J Occup Health. Vol. 57 ( 6 ) page： 548-554 2015

A trial to find appropriate animal models of dichloropropane-induced cholangiocarcinoma based on the hepatic distribution of glutathione S-transferases International journal

Zhang Lingyi, Zong Cai, Ichihara Sahoko, Naito Hisao, Toyokuni Shinya, Kumagai Shinji, Ichihara Gaku

Journal of Occupational Health Vol. 57 ( 6 ) page： 548 - 554 2015

Language：English Publishing type：Research paper (scientific journal) Publisher：Japan Society for Occupational Health

Objectives: It has been reported that 1,2-Dichloropropane (DCP) induced cholangiocarcinoma (CCA) in offset color proof-printing workers. However, exposure to DCP by inhalation or gavage for 2 year did not induce CCA in mice and rats. The present study mapped the hepatic distribution of GST, which is known to activate dihalogenated alkanes, and proliferative and fibrotic changes in bile ducts in various species to find the most appropriate animal model of DCP-induced CCA. Methods: First, 12 each of C57BL/6J mice, Balb/cA mice, F344 rats, Syrian hamsters, and guinea pigs were divided into four equal groups and exposed to DCP at 0, 300, 1,000, or 3,000 ppm 8 hours/day for 7 days. Second, 32 Balb/cA mice and 32 Syrian hamsters were each divided into four equal groups and exposed to DCP at 0, 200, 400, and 800 ppm 6 hours/day for 14 days. After the last exposure, the animals were decapitated, and the livers were dissected out for histopathological evaluation. Immunostaining was conducted to determine the distribution of GSTT1, GSTM1, and GSTPi, as well as the expression of proliferation marker Ki67. Results: GSTT1, GSTM1, and GSTPi were expressed in both hepatocytes and bile duct cells in all control and exposed animals. There was no clear difference in the expression of Ki67 between the exposed groups and the control. No fibrotic changes were observed in any species or strains examined. Conclusions: Expression of GSTT1 or other GST isozymes might not explain the difference in sensitivity of hepatocytes and the bile duct to DCP between humans and rodents.(J Occup Health 2015; 57: 548–554)

DOI： 10.1539/joh.15-0085-oa

Chemical conversion of human fibroblasts into neuronal cells: dawn of future clinical trials. Invited International journal

Toyokuni S

J Clin Biochem Nutr Vol. 56 page： 165 2015

Application of intermittent microwave irradiation to western blot analysis. Reviewed International coauthorship International journal

Liu YT, Toyokuni S

Methods Mol Biol Vol. 1314 page： 185-190 2015

Maternal molecular hydrogen treatment attenuates lipopolysaccharide-induced rat fetal lung injury Reviewed International journal

Y. Hattori, T. Kotani, H. Tsuda, Y. Mano, L. Tu, H. Li, S. Hirako, T. Ushida, K. Imai, T. Nakano, Y. Sato, R. Miki, S. Sumigama, A. Iwase, S. Toyokuni, F. Kikkawa

FREE RADICAL RESEARCH Vol. 49 ( 8 ) page： 1026 - 37 2015

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Maternal inflammation is associated with spontaneous preterm birth and respiratory impairment among premature infants. Recently, molecular hydrogen (H-2) has been reported to have a suppressive effect on oxidative stress and infl ammation. The aim of this study was to evaluate the effects of H-2 on fetal lung injury caused by maternal infl ammation. 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 H-2-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 H-2 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 H-2 intake. Antenatal H-2 administration may decrease the pulmonary mobility associated with infl ammation in premature infants.

DOI： 10.3109/10715762.2015.1038257

Minimal inflammogenicity of pristine single-wall carbon nanotubes Reviewed International journal

Toyokuni S., Jiang L., Kitaura R., Shinohara H.

Nagoya Journal of Medical Science Vol. 77 ( 1-2 ) page： 195 - 202 2015

Language：English Publishing type：Research paper (scientific journal) Publisher：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.

Elucidation of asbestos-induced mesothelial carcinogenesis and risk assessment of nanomaterials

Application of Intermittent Microwave Irradiation to Western Blot Analysis. Reviewed International journal

Yu-Ting Liu, Shinya Toyokuni

Methods in molecular biology (Clifton, N.J.) Vol. 1314 page： 185 - 90 2015

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

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. Reviewed

Yamashita K, Nagai H, Toyokuni S

Lab Invest Vol. 95 page： 749-764 2015

TOYOKUNI Shinya

Annual Meeting of the Japanese Society of Toxicology Vol. 42 ( 0 ) page： S4 - 2 2015

Establishment of murine model for neurotoxicity of 1-bromopropane, an alternative to ozone-depleting solvents

ZONG Cai, GARNER Edwin, HUANG Chin-Yen, ZHANG Lingyi, ZHANG Xiao, SAKURAI Toshihiro, TOYOKUNI Shinya, ICHIHARA Sahoko, ICHIHARA Gaku

Annual Meeting of the Japanese Society of Toxicology Vol. 42 ( 0 ) page： P - 13 2015

Hepatic distribution of GST cannot explain the gap between human and rodents for induction of cholangiocarcioma following exposure to dichloropropane International journal

ZHANG Lingyi, ZONG Cai, ICHIHARA Sahoko, NAITO Hisao, TOYOKUNI Shinya, KUMAGAI Shinji, ICHIHARA Gaku

Annual Meeting of the Japanese Society of Toxicology Vol. 42 ( 0 ) page： P - 165 2015

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Toxicology

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

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 Vol. 70 page： 337 - 242 2015

Minimal inflammogenicity of pristine single-wall carbon nanotubes Reviewed International journal

Toyokuni S

Nagoya Journal of Medical Science Vol. 77 ( 1-2 ) page： 195 - 202 2015

Genome-wide profiling of 8-oxoguanine reveals its association with spatial positioning in nucleus. Reviewed International journal

Minako Yoshihara, Li Jiang, Shinya Akatsuka, Mikita Suyama, Shinya Toyokuni

DNA research : an international journal for rapid publication of reports on genes and genomes Vol. 21 ( 6 ) page： 603 - 12 2014.12

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

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 Reviewed International journal

Hiromasa Tanaka, Masaaki Mizuno, Kenji Ishikawa, Keigo Takeda, Kae Nakamura, Fumi Utsumi, Hiroaki Kajiyama, Hiroyuki Kano, Yasumasa Okazaki, Shinnya Toyokuni, Shoichi Maruyama, Fumitaka Kikkawa, Masaru Hori

IEEE TRANSACTIONS ON PLASMA SCIENCE Vol. 42 ( 12 ) page： 3760 - 3764 2014.12

Language：English Publishing type：Research paper (scientific journal) Publisher：IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC

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 Reviewed International journal

Yasumasa Okazaki, Yue Wang, Hiromasa Tanaka, Masaaki Mizuno, Kae Nakamura, Hiroaki Kajiyama, Hiroyuki Kano, Koji Uchida, Fumitaka Kikkawa, Masaru Hori, Shinya Toyokuni

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 55 ( 3 ) page： 207 - 15 2014.11

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

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)-O-center dot 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 a-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. Reviewed International journal

T Mukaide, Y Hattori, N Misawa, S Funahashi, L Jiang, T Hirayama, H Nagasawa, S Toyokuni

Free radical research Vol. 48 ( 9 ) page： 990 - 5 2014.9

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.3109/10715762.2014.898844

FREE FERROUS IRON IN AMNIOTIC FLUID AS A PREDICTIVE MARKER OF FETAL DISORDERS IN PREGNANCY Reviewed International journal

Yuka Hattori, Tomomi Kotani, Tomoko Nakano, Yukio Mano, Seiji Sumigama, Hiroyuki Tsuda, Takafumi Usida, Hideko Nagasawa, Shinya Toyokuni, Fumitaka Kikkawa

PLACENTA Vol. 35 ( 9 ) page： A71 - A71 2014.9

Connective tissue growth factor and beta-catenin constitute an autocrine loop for activation in rat sarcomatoid mesothelioma Reviewed International journal

Li Jiang, Yoriko Yamashita, Shan-Hwu Chew, Shinya Akatsuka, Shun Ukai, Shenqi Wang, Hirotaka Nagai, Yasumasa Okazaki, Takashi Takahashi, Shinya Toyokuni

JOURNAL OF PATHOLOGY Vol. 233 ( 4 ) page： 402 - 414 2014.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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 beta-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-GSK3 beta-beta-catenin-TCF-Ctgf autocrine axis and suggest Ctgf as a therapeutic target. Copyright (C) 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. Reviewed International journal

Kanako Shinjo, Yoriko Yamashita, Eiko Yamamoto, Shinya Akatsuka, Nozomi Uno, Akihiro Kamiya, Kaoru Niimi, Yuka Sakaguchi, Tetsuro Nagasaka, Takashi Takahashi, Kiyosumi Shibata, Hiroaki Kajiyama, Fumitaka Kikkawa, Shinya Toyokuni

International journal of cancer Vol. 135 ( 2 ) page： 308 - 18 2014.7

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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.

DOI： 10.1002/ijc.28692

Lack of presence of the human cytomegalovirus in human glioblastoma Reviewed International journal

Yoriko Yamashita, Yoshinori Ito, Hiroki Isomura, Naoaki Takemura, Akira Okamoto, Kazuya Motomura, Takashi Tsujiuchi, Atsushi Natsume, Toshihiko Wakabayashi, Shinya Toyokuni, Tatsuya Tsurumi

MODERN PATHOLOGY Vol. 27 ( 7 ) page： 922 - 9 2014.7

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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.

DOI： 10.1038/modpathol.2013.219

Dexamethasone palmitate ameliorates macrophages-rich graft-versus-host disease by inhibiting macrophage functions. Reviewed International journal

Satoshi Nishiwaki, Takayuki Nakayama, Makoto Murata, Tetsuya Nishida, Seitaro Terakura, Shigeki Saito, Tomonori Kato, Hiroki Mizuno, Nobuhiko Imahashi, Aika Seto, Yukiyasu Ozawa, Koichi Miyamura, Masafumi Ito, Kyosuke Takeshita, Hidefumi Kato, Shinya Toyokuni, Keisuke Nagao, Ryuzo Ueda, Tomoki Naoe

PloS one Vol. 9 ( 5 ) page： e96252 2014.5

Language：English Publishing type：Research paper (scientific journal) Publisher：PUBLIC LIBRARY SCIENCE

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-alpha and IFN-gamma, 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.

DOI： 10.1371/journal.pone.0096252

Maternal molecular hydrogen administration ameliorates rat fetal hippocampal damage caused by in utero ischemia-reperfusion. Reviewed International journal

Yukio Mano, Tomomi Kotani, Mikako Ito, Taku Nagai, Yuko Ichinohashi, Kiyofumi Yamada, Kinji Ohno, Fumitaka Kikkawa, Shinya Toyokuni

Free radical biology & medicine Vol. 69 page： 324 - 30 2014.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Molecular hydrogen (H-2) scavenges hydroxyl radicals. Recently, H-2 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 H-2 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 H-2. Neonatal growth retardation was observed in the IR group, which was alleviated by the H-2 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 '-deoxyguanosine- and 4-hydroxy-2-nonenal-modified proteins. Both oxidative stress markers were significantly increased in the IR group, which was again ameliorated by the H-2 intake. Last, 8-week-old rats were subjected to a Morris water maze test. Maternal H-2 administration improved the reference memory of the offspring to the sham level after IR injury during pregnancy. Overall, the present results support the idea that maternal H-2 intake helps prevent the hippocampal impairment of offspring induced by IR during pregnancy. (C) 2014 Elsevier Inc. All rights reserved.

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 Reviewed International journal

Kaho Fujii, Yoriko Yamashita, Toshimichi Yamamoto, Koji Takahashi, Katsunori Hashimoto, Tomoko Miyata, Kumi Kawai, Fumitaka Kikkawa, Shinya Toyokuni, Tetsuro Nagasaka

HUMAN PATHOLOGY Vol. 45 ( 4 ) page： 717 - 724 2014.4

Language：English Publishing type：Research paper (scientific journal) Publisher：W B SAUNDERS CO-ELSEVIER INC

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. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.humpath.2013.10.031

From Nagoya to the world Reviewed International journal

Toyokuni S.

Nagoya Journal of Medical Science Vol. 76 ( 1-2 ) page： 1 - 2 2014.3

From Nagoya to the world International journal

Toyokuni S

Nagoya Journal of Medical Science Vol. 76 ( 1-2 ) page： 1 - 2 2014.3

RAT MODEL DEMONSTRATES A HIGH RISK OF TREMOLITE BUT A LOW RISK OF ANTHOPHYLLITE FOR MESOTHELIAL CARCINOGENESIS Reviewed

Dilinuer Aierken, Yasumasa Okazaki, Shan Hwu Chew, Akihiro Sakai, Yue Wang, Hirotaka Nagai, Nobuaki Misawa, Norihiko Kohyama, Shinya Toyokuni

NAGOYA JOURNAL OF MEDICAL SCIENCE Vol. 76 ( 1-2 ) page： 149 - 160 2014.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 76 ( 1-2 ) page： 149 - 60 2014.2

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 Vol. 76 ( 1-2 ) page： 149 - 60 2014.2

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 Vol. 76 ( 1-2 ) page： 149 - 160 2014.2

Rat model demonstrates a high risk of tremolite but a low risk of anthophyllite for mesothelial carcinogenesis. Reviewed International journal

Aierken D., Okazaki Y., Chew S.H., Sakai A., Wang Y., Nagai H., Misawa N., Kohyama N., Toyokuni S.

Nagoya journal of medical science Vol. 76 ( 1-2 ) page： 149 - 160 2014.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Shan Hwu Chew, Yasumasa Okazaki, Hirotaka Nagai, Nobuaki Misawa, Shinya Akatsuka, Kyoko Yamashita, Li Jiang, Yoriko Yamashita, Michio Noguchi, Kiminori Hosoda, Yoshitaka Sekido, Takashi Takahashi, Shinya Toyokuni

CARCINOGENESIS Vol. 35 ( 1 ) page： 164 - 72 2014.1

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

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 transcriptionPCR 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.

DOI： 10.1093/carcin/bgt267

Iron overload as a major targetable pathogenesis of asbestos-induced mesothelial carcinogenesis. Reviewed International journal Open Access

Shinya Toyokuni

Redox report : communications in free radical research Vol. 19 ( 1 ) page： 1 - 7 2014.1

Language：English Publishing type：Research paper (scientific journal) Publisher：MANEY PUBLISHING

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

DOI： 10.1179/1351000213Y.0000000075

As a host society to SFRRI 2014 in Kyoto. Open Access

Osamu Handa, Yuji Naito, Shinya Toyokuni

Journal of clinical biochemistry and nutrition Vol. 54 ( 1 ) page： 1 - 1 2014.1

Cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis through dysregulated adipocytokine production. Reviewed

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 Vol. 35 page： 164-172 2014

Preclinical evaluation of an O(6)-methylguanine-DNA methyltransferase-siRNA/liposome complex administered by convection-enhanced delivery to rat and porcine brains. Reviewed

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 Vol. 15 page： 169-178 2014

Dexamethasone palmitate ameliorates macrophages-1 rich graft-versus-host disease by inhibiting macrophage functions. Reviewed

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 Vol. 9 page： e96252 2014

Histological detection of catalytic ferrous iron with the selective turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model. Reviewed

Mukaide T, Hatori Y, Misawa N, Funahashi S, Jiang L, Hirayama T, Nagasawa H and Toyokuni S.

Free Radic Res Vol. 48 page： 990-995 2014

Rat model demonstrates a high risk of tremolite but a low risk of anthophyllite for mesothelial carcinogenesis. Reviewed

Aierken D, Okazaki Y, Chew SH, Sakai A, Wang Y, Nagai H, Misawa N, Kohyama N and Toyokuni S.

Nagoya J Med Sci Vol. 76 page： 149-160 2014

Maternal molecular hydrogen administration ameliorates rat fetal hippocampal damage by in ut ero ischemia-reperfusion. Reviewed

Mano Y, Kotani T, Ito M, Nagai T, Ichinohashi Y, Yamada K, Ohno K, Kikkawa F and Toyokuni S.

Free Radic Biol Med Vol. 69 page： 324-330 2014

Lack of presence of the human cytomegalovirus in human glioblastoma. Modern Pathol 27: 922-929, 2014. Reviewed

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 Vol. 27 page： 922-929 2014

Ovarian mucinous tumors arising from mature cystic teratomas-a molecular genetic approach for understanding the cellular origin. Reviewed

Fujii K, Yamashita Y, Yamamoto T, Takahashi K, Hashimoto K, Miyata T, Kawai K, Kikkawa F, Toyokuni S, Nagasaka T.

Human Pathol Vol. 45 page： 717-724 2014

Iron as a major targetable pathogenesis of asbestos-induced mesothelial carcinogenesis. Reviewed

Toyokuni, S.

Redox Rep. Vol. 19 page： 1-7 2014

Plasma medical science for cancer therapy using non-thermal atomospheric pressure plasma. Reviewed

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 Vol. 42 page： 3760-3764 2014

Iron and thiols as two major players in carcinogenesis: friends or foes? Invited Reviewed

Toyokuni S.

Front Pharmacol Vol. 5 page： 200 2014

Genome-wide profiling of 8-oxoguanine reveals its association with spatial positioning in nucleus. Reviewed

Yoshihara M, Jiang L, Akatsuka S, Suyama M and Toyokuni S.

DNA Res Vol. 21 page： 603-612 2014

Direct exposure of non-equilibrium atmospheric pressure plasma confers simultaneous oxidative and ultraviolet modifications in biomolecules. Reviewed

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 Vol. 55 page： 207-215 2014

Iron and thiols as two major players in carcinogenesis: friends or foes? Reviewed International journal Open Access

Shinya Toyokuni

Frontiers in pharmacology Vol. 5 page： 200 - 200 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：FRONTIERS RESEARCH FOUNDATION

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 Keapl 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.

DOI： 10.3389/fphar.2014.00200

Iron and thiols as two major players in carcinogenesis: friends or foes? Invited Reviewed

Toyokuni S

Front Pharmacol Vol. 5 page： 200 2014

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 Vol. 6 ( 2 ) page： 169 - 78 2014

Preclinical evaluation of an O6-methylguanine-DNA methyltransferase-siRNA/liposome complex administered by convection-enhanced delivery to rat and porcine brains Reviewed International journal

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 Vol. 6 ( 2 ) page： 169 - 178 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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 Vol. 6 ( 2 ) page： 169 - 178 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 2013 ( 0 ) page： 0007 - 0007 2014

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 32 page： 632 - 637 2014

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 Vol. 6 ( 2 ) page： 169 - 178 2014

Genotoxicity and carcinogenicity risk of carbon nanotubes. Reviewed International journal

Shinya Toyokuni

Advanced drug delivery reviews Vol. 65 ( 15 ) page： 2098 - 110 2013.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

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. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.addr.2013.05.011

Deferasirox Induces Mesenchymal-Epithelial Transition in Crocidolite-Induced Mesothelial Carcinogenesis in Rats International journal

Toyokuni, S; Nagai, H; Okazaki, Y; Chew, SH; Yasui, H

FREE RADICAL BIOLOGY AND MEDICINE Vol. 65 page： S25 - S25 2013.11

Deferasirox Induces Mesenchymal-Epithelial Transition in Crocidolite-Induced Mesothelial Carcinogenesis in Rats Reviewed International journal

Hirotaka Nagai, Yasumasa Okazaki, Shan Hwu Chew, Nobuaki Misawa, Hiroyuki Yasui, Shinya Toyokuni

CANCER PREVENTION RESEARCH Vol. 6 ( 11 ) page： 1222 - 30 2013.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER 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.

DOI： 10.1158/1940-6207.CAPR-13-0244

From nagoya to the world Reviewed International journal

Toyokuni S.

Nagoya Journal of Medical Science Vol. 75 ( 1-2 ) page： 1 - 2 2013.9

From nagoya to the world International journal

Toyokuni S

Nagoya Journal of Medical Science Vol. 75 ( 1-2 ) page： 1 - 2 2013.9

Intraperitoneal administration of tangled multiwalled carbon nanotubes of 15nm in diameter does not induce mesothelial carcinogenesis in rats Reviewed International journal

Hirotaka Nagai, Yasumasa Okazaki, Shan Hwu Chew, Nobuaki Misawa, Yasumitsu Miyata, Hisanori Shinohara, Shinya Toyokuni

Pathology International Vol. 63 ( 9 ) page： 457 - 62 2013.9

Language：English Publishing type：Research paper (scientific journal)

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

Erratum to Reduction of diabetes-induced renal oxidative stress by a cantaloupe melon extract/gliadin biopolymers, oxykine, in mice, [International Union of Biochemistry and Molecular Biology, 23, 2, (2005) 85-95] Reviewed International journal

Yuji Naitoa, Satomi Akagiri, Kazuhiko Uchiyama, Satoshi Kokura, Norimasa Yoshida, Goji Hasegawa, Naoto Nakamura, Hiroshi Ichikawa, Shinya Toyokuni, Tetsuo Ijichi, Toshikazu Yoshikawa

BioFactors Vol. 39 ( 5 ) page： 589 - 589 2013.9

CD146 and insulin-like growth factor 2 mRNA-binding protein 3 predict prognosis of asbestos-induced rat mesothelioma Reviewed International journal

Yasumasa Okazaki, Hirotaka Nagai, Shan H. Chew, Jiang Li, Satomi Funahashi, Tohru Tsujimura, Shinya Toyokuni

CANCER SCIENCE Vol. 104 ( 8 ) page： 989 - 95 2013.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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.

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 Reviewed International journal

Hiroshi Hotta, Kazunori Hamamura, Kyoko Yamashita, Hidenobu Shibuya, Noriyo Tokuda, Noboru Hashimoto, Keiko Furukawa, Noriyuki Yamamoto, Hisashi Hattori, Shinya Toyokuni, Minoru Ueda, Koichi Furukawa

GLYCOCONJUGATE JOURNAL Vol. 30 ( 6 ) page： 585 - 97 2013.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

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.

DOI： 10.1007/s10719-012-9458-2

Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells Reviewed International journal

Kyoko Yamashita, Hirotaka Nagai, Yuji Kondo, Nobuaki Misawa, Shinya Toyokuni

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 53 ( 1 ) page： 27 - 35 2013.7

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

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 x1000 magnification (oil-immersion) of the sample with Kernechtrot 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.

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. Reviewed

Michiru Hamakawa, Akimasa Ishida, Keigo Tamakoshi, Haruka Shimada, Hiroki Nakashima, Taiji Noguchi, Shinya Toyokuni, Kazuto Ishida

Journal of clinical biochemistry and nutrition Vol. 53 ( 1 ) page： 8 - 14 2013.7

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

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-mm 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-nonenal-modified 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.

DOI： 10.3164/jcbn.12-72

Multispecificity of Immunoglobulin M Antibodies Raised against Advanced Glycation End Products Reviewed International journal

Miho Chikazawa, Natsuki Otaki, Takahiro Shibata, Hiroaki Miyashita, Yoshichika Kawai, Shoichi Maruyama, Shinya Toyokuni, Yasuyuki Kitaura, Tsukasa Matsuda, Koji Uchida

Journal of Biological Chemistry Vol. 288 ( 19 ) page： 13204 - 14 2013.5

CANCER AS A FERROTOXIC DISEASE: WHAT WE HAVE LEARNED FROM ANIMAL STUDIES TOWARD ITS PREVENTION International journal

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

AMERICAN JOURNAL OF HEMATOLOGY Vol. 88 ( 5 ) page： E168 - E169 2013.5

Met is the most frequently amplified gene in endometriosis-associated ovarian clear cell adenocarcinoma and correlates with worsened prognosis. Reviewed International journal

Yoriko Yamashita, Shinya Akatsuka, Kanako Shinjo, Yasushi Yatabe, Hiroharu Kobayashi, Hiroshi Seko, Hiroaki Kajiyama, Fumitaka Kikkawa, Takashi Takahashi, Shinya Toyokuni

PloS one Vol. 8 ( 3 ) page： e57724 2013.3

Language：English Publishing type：Research paper (scientific journal)

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 (&gt
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&lt
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 International journal

Toyokuni, S

NAGOYA JOURNAL OF MEDICAL SCIENCE Vol. 75 ( 1-2 ) page： 1 - 2 2013.2

Protective effect of cardioplegia with poly (ADP-ribose) polymerase-1 inhibitor against myocardial ischemia-reperfusion injury: in vitro study of isolated rat heart model Reviewed International journal

Kazuhiro Yamazaki, Seigo Tanaka, Ryuzo Sakata, Senri Miwa, Wnimonk Oriyanhan, Kiyoaki Takaba, Kenji Minakata, Akira Marui, Tadashi Ikeda, Shinya Toyokuni, Masashi Komeda, Kunihiro Ueda

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY Vol. 28 ( 1 ) page： 143 - 7 2013.2

Language：English Publishing type：Research paper (scientific journal) Publisher：INFORMA HEALTHCARE

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 mu M) for cardioplegia. During reperfusion, the Group HQ showed significantly greater functional recovery of +dp/dt(max) (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.

DOI： 10.3109/14756366.2011.642373

FROM NAGOYA TO THE WORLD International journal

Toyokuni, S

NAGOYA JOURNAL OF MEDICAL SCIENCE Vol. 75 ( 1-2 ) page： 1 - 2 2013.2

Deferasirox induces mesenchymal-epithelial transition in crocidolite-induced mesothelila carcinogenesis in rats. Reviewed

Nagai H, Okazaki Y, Chew SH, Misawa N, Yasui H and Toyokuni S.

Cancer Prev Res Vol. 6 ( 11 ) page： 1222-1230 2013

Genotoxicity and carcinogenicity risk of carbon nanotubes. Reviewed

Toyokuni S.

Adv Drug Deliver Rev 2013

Repeated short-term daily exercise ameliorates oxidative cerebral damage and the resultant motor dysfunction after transient ischemia in rats. Reviewed

Hamakawa M, Ishida A, Tamakoshi K, Shimada H, Nakashima H, Noguchi T, Toyokuni S and Ishida K.

J Clin Biochem Nutr Vol. 53 page： 8-14 2013

CD146 and IMP3 predict prognosis of asbestos-induced rat mesothelioma. Reviewed

Okazaki Y, Nagai H, Chew SH, Jiang L, Funahashi S, Tsujimura T and Toyokuni S.

Cnacer Sci Vol. 104 page： 989-995 2013

Multi-specificity of IgM antibodies raised against advanced glycation end products: Involvement of electronegative potential of antigens. Reviewed

Chikazawa M, Otaki N, Shibata T, Miyashita H, Kawai Y, Maruyama S, Toyokuni S, Kitaura Y, Matsuda T, Uchida K.

J Biol Chem Vol. 288 page： 13204-13214 2013

Met is the most frequently amplified gene in endometriosis-associated ovarian clear cell adenocarcinoma and correlates with worsened prognosis. Reviewed

Yamashita Y, Akatsuka S, Shinjo K, Yatabe Y, Kobayashi H, Seko H, Kajiyama H, Kikkawa F, Takahashi T and Toyokuni S.

PLoS One Vol. 8 ( 3 ) page： e57724 2013

Evaluation of two distinct methods to quantify the uptake of crocidolite fibers by mesothelial cells. Reviewed

Yamashita K, Nagai H, Kondo Y, Misawa N and Toyokuni S.

J Clin Biochem Nutr Vol. 53 page： 27-35 2013

Metamorphosis of mesothelial cells with active horizontal motility in tissue culture. Reviewed

Nagai H, Chew SH, Okazaki Y, Funahashi S, Namba T, Kato T, Enomoto A, Jiang L, Akatsuka S and Toyokuni S.

Sci Rep Vol. 3 page： 1144 2013

Protective effect of cardioplegia with poly(ADP-ribose)polymerase-1 inhibitor aganst myocardial ischemia-reperfusion injury: in vitro study if isolated rat heart model. Reviewed

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 Vol. 28 page： 143-147 2013

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 Vol. 30 page： 585-597 2013

Intraperitoneal administration of tangled multiwalled carbon nanotubes of 15 nm in diameter does not induce mesothelial carcinogenesis in rats. Reviewed

Nagai H, Okazaki Y, Chew SH, Misawa N, Miyata Y, Shinohara N and Toyokuni S.

Pathol. Int. Vol. 63 page： 457-462 2013

Metamorphosis of mesothelial cells with active horizontal motility in tissue culture Reviewed International journal

Hirotaka Nagai, Shan Hwu Chew, Yasumasa Okazaki, Satomi Funahashi, Takashi Namba, Takuya Kato, Atsushi Enomoto, Li Jiang, Shinya Akatsuka, Shinya Toyokuni

SCIENTIFIC REPORTS Vol. 3 page： 1144 2013

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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. Reviewed

Toyokuni S

Adv Drug Deliver Rev 2013

Multi-specificity of IgM antibodies raised against advanced glycation end products: Involvement of electronegative potential of antigens. Reviewed

Chikazawa M, Otaki N, Shibata T, Miyashita H, Kawai Y, Maruyama S, Toyokuni S, Kitaura Y, Matsuda T, Uchida K

J Biol Chem Vol. 288 page： 13204-13214 2013

Factors regulating the carcinogenicity of multiwalled carbon nanotubes International journal

TOYOKUNI Shinya

Annual Meeting of the Japanese Society of Toxicology Vol. 40 ( 0 ) page： 1044 2013

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〜 International journal

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 Vol. 2012 ( 0 ) page： 48101465 - 48101465 2013

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. Reviewed

Okazaki Y, Nagai H, Chew SH, Jiang L, Funahashi S, Tsujimura T, Toyokuni S

Cnacer Sci Vol. 104 page： 989-995 2013

豊國伸哉

細胞工学 Vol. 32 page： 220 - 224 2013

The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma. Reviewed International journal

Momen Elshazley, Mitsuo Sato, Tetsunari Hase, Ryo Yamashita, Kenya Yoshida, Shinya Toyokuni, Futoshi Ishiguro, Hirotaka Osada, Yoshitaka Sekido, Kohei Yokoi, Noriyasu Usami, David S Shames, Masashi Kondo, Adi F Gazdar, John D Minna, Yoshinori Hasegawa

International journal of cancer Vol. 131 ( 12 ) page： 2820 - 31 2012.12

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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 p21(WAF1/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.

DOI： 10.1002/ijc.27598

YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes Reviewed International journal

T. Mizuno, H. Murakami, M. Fujii, F. Ishiguro, I. Tanaka, Y. Kondo, S. Akatsuka, S. Toyokuni, K. Yokoi, H. Osada, Y. Sekido

ONCOGENE Vol. 31 ( 49 ) page： 5117 - 5122 2012.12

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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. Oncogene (2012) 31, 5117-5122; doi:10.1038/onc.2012.5; published online 30 January 2012

DOI： 10.1038/onc.2012.5

Distinct affinity of nuclear proteins to the surface of chrysotile and crocidolite Reviewed International journal

Yurika Kubo, Hiroyuki Takenaka, Hirotaka Nagai, Shinya Toyokuni

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 51 ( 3 ) page： 221 - 6 2012.11

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

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.

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 Vol. 228 ( 3 ) page： 366 - 377 2012.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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

H. Nagai, Y. Okazaki, L. Jiang, S. Akatsuka, Y. Yamashita, S. Toyokuni

FREE RADICAL BIOLOGY AND MEDICINE Vol. 53 page： S51 - S52 2012.9

Bovine lactoferrin ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats. Reviewed

Yasumasa Okazaki, Isato Kono, Takayoshi Kuriki, Satomi Funahashi, Soichiro Fushimi, Mohammad Iqbal, Shigeru Okada, Shinya Toyokuni

Journal of clinical biochemistry and nutrition Vol. 51 ( 2 ) page： 84 - 90 2012.9

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

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 IF 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.

DOI： 10.3164/jcbn.11-100

Iron overload signature in chrysotile-induced malignant mesothelioma. Reviewed

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 Vol. 228 page： 366-377 2012.8

Differences and similarities between carbon nanotubes and asbestos fibers during mesothelial carcinogenesis: shedding light on fiber entry mechanism. Reviewed International journal Open Access

Hirotaka Nagai, Shinya Toyokuni

Cancer science Vol. 103 ( 8 ) page： 1378 - 90 2012.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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. (Cancer Sci 2012; 103: 13781390)

DOI： 10.1111/j.1349-7006.2012.02326.x

Mitochondria and free radical studies on health, disease and pollution. Invited International journal

Hideyuki J Majima, Shinya Toyokuni

Free radical research Vol. 46 ( 8 ) page： 925 - 6 2012.8

The ferroimmunomodulatory role of ectopic endometriotic stromal cells in ovarian endometriosis. Reviewed International journal

Hiroharu Kobayashi, Yoriko Yamashita, Akira Iwase, Yutaka Yoshikawa, Hiroyuki Yasui, Yoshichika Kawai, Koji Uchida, Nozomi Uno, Shinya Akatsuka, Takashi Takahashi, Fumitaka Kikkawa, Shinya Toyokuni

Fertility and sterility Vol. 98 ( 2 ) page： 415 - 22 2012.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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-1 beta 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. (Fertil Steril (R) 2012;98:415-22. (C) 2012 by American Society for Reproductive Medicine.)

DOI： 10.1016/j.fertnstert.2012.04.047

Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells. Reviewed International journal

Mihoko Horio, Mitsuo Sato, Yoshihiro Takeyama, Momen Elshazley, Ryo Yamashita, Tetsunari Hase, Kenya Yoshida, Noriyasu Usami, Kohei Yokoi, Yoshitaka Sekido, Masashi Kondo, Shinya Toyokuni, Adi F Gazdar, John D Minna, Yoshinori Hasegawa

Annals of surgical oncology Vol. 19 Suppl 3 ( Suppl 3 ) page： S634-45 - S645 2012.7

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

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.

DOI： 10.1245/s10434-011-2142-0

In vitro transformation of mouse testis cells by oncogene transfection. Reviewed

Morimoto H, Lee J, Tanaka T, Ishii K, Toyokuin S, Kanatsu-Shinohara M and Shinohara T.

Biol Reprod Vol. 86 ( 5 ) page： 1-11 2012.5

FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b through MAP2K1 activation. Reviewed International journal

Kei Ishii, Mito Kanatsu-Shinohara, Shinya Toyokuni, Takashi Shinohara

Development (Cambridge, England) Vol. 139 ( 10 ) page： 1734 - 43 2012.5

Language：English Publishing type：Research paper (scientific journal) Publisher：COMPANY OF BIOLOGISTS LTD

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.

DOI： 10.1242/dev.076539

In vitro transformation of mouse testis cells by oncogene transfection. Reviewed International journal

Hiroko Morimoto, Jiyoung Lee, Takashi Tanaka, Kei Ishii, Shinya Toyokuni, Mito Kanatsu-Shinohara, Takashi Shinohara

Biology of reproduction Vol. 86 ( 5 ) page： 148, 1 - 11 2012.5

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC STUDY 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.

DOI： 10.1095/biolreprod.111.095307

Carcinogenic mechanisms through excess iron and reactive oxygen species and its application to cancer prevention and therapy

TOYOKUNI Shinya

Vol. 53 ( 4 ) page： 401 - 408 2012.4

Carcinogenic mechanisms through excess iron and reactive oxygen species and its application to cancer prevention and therapy International journal

TOYOKUNI Shinya

Vol. 53 ( 4 ) page： 401 - 408 2012.4

Genome-wide assessment of oxidatively generated DNA damage. Reviewed International journal

Shinya Akatsuka, Shinya Toyokuni

Free radical research Vol. 46 ( 4 ) page： 523 - 30 2012.4

Language：English Publishing type：Research paper (scientific journal) Publisher：INFORMA HEALTHCARE

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.

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 Vol. 53 ( 4 ) page： 401 - 8 2012.4

[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 Vol. 53 ( 4 ) page： 401 - 8 2012.4

Age- and sun exposure-dependent differences in 8-hydroxy-2 '-deoxyguanosine and N epsilon-(carboxymethyl)lysine in human epidermis Reviewed International journal

Akira Kawada, Akira Date, Shinya Toyokuni

JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY Vol. 66 ( 4 ) page： AB174 - AB174 2012.4

[Carcinogenic mechanisms through excess iron and reactive oxygen species and its application to cancer prevention and therapy]. Reviewed International journal

Toyokuni S.

[Rinshō ketsueki] The Japanese journal of clinical hematology Vol. 53 ( 4 ) page： 401 - 408 2012.4

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 Vol. 101 ( 1 ) page： 280 - 280 2012.3

Array CGH Analysis Reveals Amplification of Met and AKT2 in Clear Cell Carcinoma of the Ovary Reviewed International journal

Y. Yamashita, S. Akatsuka, Y. Yatabe, S. Toyokuni

LABORATORY INVESTIGATION Vol. 92 page： 302A - 302A 2012.2

Array CGH Analysis Reveals Amplification of Met and AKT2 in Clear Cell Carcinoma of the Ovary Reviewed International journal

Y. Yamashita, S. Akatsuka, Y. Yatabe, S. Toyokuni

MODERN PATHOLOGY Vol. 25 page： 302A - 302A 2012.2

FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b genes through MAP2K1 activation. Reviewed

Ishii K, Kanatsu-Shinohara M, Toyokuni S and Shinohara T.

Development Vol. 139 page： 1734-1743 2012

Differences and similarities between carbon nanotubes and asbestos fibers during mesothelial carcinogenesis. Reviewed

Nagai H and Toyokuni S.

Cancer Sci Vol. 103 page： 1378-1390 2012

The ferroimmunomodulatory role of ectopic endometriotic stromal cells in ovarian endometriosis. Reviewed

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 Vol. 98 page： 415-422 2012

Mitochondria and free radical studies on health, disease and pollusion (editorial). Invited

Majima HJ and Toyokuni S.

Free Radic Res Vol. 46 page： 925-926 2012

Distinct affinity of nuclear proteins to the surface of chrysotile and crocidolite. Invited Reviewed

Kubo Y, Takenaka H, Nagai H and Toyokuni S.

J Clin Biochem Nutr Vol. 51 page： 221-226 2012

The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma. Reviewed

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 Vol. 131 page： 2820-2831 2012

YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle promoting genes. Reviewed

Mizuno T, Murakami H, Fujii M, Ishiguro F, Tanaka I, Kondo Y, Akatsuka S, Toyokuni S and Sekido Y.

Oncogene Vol. 31 page： 5117-5122 2012

Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothalioma cells. Reviewed

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 Vol. 19 ( Suppl 3 ) page： 634-645 2012

Genome-wide assessment of oxidatively generated DNA damage. Reviewed

Akatsuka S and Toyokuni S.

Free Radic Res Vol. 46 page： 523-530 2012

Bovine lactoferrin ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats. Reviewed

Okazaki Y, Kono H, Kuriki R, Funahashi S, Fushimi S, Iqbal M, Okada S and Toyokuni S.

J Clin Biochem Nutr Vol. 51 page： 84-90 2012

Differences and similarities between carbon nanotubes and asbestos fibers during mesothelial carcinogenesis. Reviewed

Nagai H, Toyokuni S

Cancer Sci Vol. 103 page： 1378-1390 2012

Fenton Reaction Induced Cancer in Wild Type Rats Recapitulates Genomic Alterations Observed in Human Cancer Reviewed International journal

Shinya Akatsuka, Yoriko Yamashita, Hiroki Ohara, Yu-Ting Liu, Masashi Izumiya, Koichiro Abe, Masako Ochiai, Li Jiang, Hirotaka Nagai, Yasumasa Okazaki, Hideki Murakami, Yoshitaka Sekido, Eri Arai, Yae Kanai, Okio Hino, Takashi Takahashi, Hitoshi Nakagama, Shinya Toyokuni

PLOS ONE Vol. 7 ( 8 ) page： e43403 2012

Language：English Publishing type：Research paper (scientific journal) Publisher：PUBLIC LIBRARY SCIENCE

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.

DOI： 10.1371/journal.pone.0043403

FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bcl6b genes through MAP2K1 activation. Reviewed

Ishii K, Kanatsu-Shinohara M, Toyokuni S, Shinohara T

Development Vol. 139 page： 1734-1743 2012

Carcinogenic mechanisms through excess iron and reactive oxygen species and its application to cancer prevention and therapy

Rinsho Ketsueki Vol. 53 ( 4 ) page： 401 - 408 2012

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. vol.30 page： 2779 - 2785 2012

Asbestos surface provides a niche for oxidative modification

NAGAI Hirotaka, ISHIHARA Toshikazu, LEE Wen-Hua, OHARA Hiroki, OKAZAKI Yasumasa, OKAWA Katsuya, TOYOKUNI Shinya

Cancer Sci. Vol. 102 ( 12 ) page： 2118 - 2125 2011.12

Diameter and rigidity of multiwalled carbon nanotubes are critical factors in mesothelial injury and carcinogenesis. Reviewed International journal

Hirotaka Nagai, Yasumasa Okazaki, Shan Hwu Chew, Nobuaki Misawa, Yoriko Yamashita, Shinya Akatsuka, Toshikazu Ishihara, Kyoko Yamashita, Yutaka Yoshikawa, Hiroyuki Yasui, Li Jiang, Hiroki Ohara, Takashi Takahashi, Gaku Ichihara, Kostas Kostarelos, Yasumitsu Miyata, Hisanori Shinohara, Shinya Toyokuni

Proceedings of the National Academy of Sciences of the United States of America Vol. 108 ( 49 ) page： E1330-8 - 8 2011.12

Language：English Publishing type：Research paper (scientific journal)

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 Reviewed International journal

Hirotaka Nagai, Toshikazu Ishihara, Wen-Hua Lee, Hiroki Ohara, Yasumasa Okazaki, Katsuya Okawa, Shinya Toyokuni

CANCER SCIENCE Vol. 102 ( 12 ) page： 2118 - 25 2011.12

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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. (Cancer Sci 2011; 102: 21182125)

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. Reviewed International journal

Eri Nishikawa, Hirotaka Osada, Yasumasa Okazaki, Chinatsu Arima, Shuta Tomida, Yoshio Tatematsu, Ayumu Taguchi, Yukako Shimada, Kiyoshi Yanagisawa, Yasushi Yatabe, Shinya Toyokuni, Yoshitaka Sekido, Takashi Takahashi

Cancer research Vol. 71 ( 19 ) page： 6165 - 73 2011.10

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC 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. Cancer Res; 71(19); 6165-73. (C)2011 AACR.

DOI： 10.1158/0008-5472.CAN-11-1020

Age- and sun exposure-dependent differences in 8-hydroxy-2'-deoxyguanosine and N-(carboxymethyl)lysine in human epidermis. Reviewed International journal

Shinya Toyokuni, Ayaka Hirao, Tamae Wada, Ryoji Nagai, Akira Date, Takashi Yoshii, Shinya Akatsuka, Yoriko Yamashita, Akira Kawada

Journal of clinical biochemistry and nutrition Vol. 49 ( 2 ) page： 121 - 124 2011.9

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & 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-epsilon-(carboxymethyl)lysine in samples using immunohistochemistry. In the epidermis of the young, negligible amounts of 8-hydroxy-2'-deoxyguanosine and N-epsilon-(carboxymethyl)lysine were detected in unexposed areas, whereas nuclear 8-hydroxy-2'-deoxyguanosine and cytoplasmic N-epsilon-(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-epsilon-(carboxymethyl)lysine in the epidermis of unexposed areas, concomitant with dermal increase in N-epsilon-(carboxymethyl)lysine. However, the immunostaining of 8-hydroxy-2'-deoxyguanosine and N-epsilon-(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.

DOI： 10.3164/jcbn.11-05

BMAL1 depletion represses growth of mesothelioma through induction of mitotic catastrophe Reviewed International coauthorship International journal

Elshazley, MHKA; Hasegawa, Y; Sato, M; Hase, T; Yoshida, K; Toyokuni, S; Seikido, Y; Yokoi, K; Usami, N; Kondo, M

CANCER RESEARCH Vol. 71 2011.9

MET Amplification is a Molecular Hallmark in Endometriosis-associated Ovarian Clear Cell Carcinoma and Correlates With Worse Prognosis Reviewed International journal

Y. Yamashita, Y. Yatabe, S. Akatsuka, H. Kajiyama, F. Kikkawa, T. Takahashi, S. Toyokuni

EUROPEAN JOURNAL OF CANCER Vol. 47 page： S529 - S529 2011.9

Elucidation of Asbestos-induced Mesothelial Carcinogenesis toward Its Prevention International journal Open Access

JIANG Li, TOYOKUNI Shinya

Environmental Mutagen Research Vol. 33 ( 1 ) page： 4 - 9 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：The Japanese Environmental Mutagen Society

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.

DOI： 10.3123/jemsge.33.4

Iron as a target of chemoprevention for longevity in humans. Reviewed International journal

Shinya Toyokuni

Free radical research Vol. 45 ( 8 ) page： 906 - 17 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：INFORMA HEALTHCARE

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.

DOI： 10.3109/10715762.2011.564170

Dietary fish oil promotes colonic apoptosis and mitochondrial proton leak in oxidatively stressed mice. Reviewed International journal

Yang-Yi Fan, Qitao Ran, Shinya Toyokuni, Yasumasa Okazaki, Evelyn S Callaway, Joanne R Lupton, Robert S Chapkin

Cancer prevention research (Philadelphia, Pa.) Vol. 4 ( 8 ) page： 1267 - 74 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER 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 SOD2(Het), Gpx4(Het), and transgenic Gpx4(Tg) 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 (Gpx4(Het) > SOD2(Het) > Wt > Gpx4(Tg)), (ii) dietary n-3 PUFA have an additive effect on the induction of apoptosis in Gpx4(Het) and SOD2(Het) mice; and (iii) dietary n-3 PUFA reverse the phenotype in oxidatively protected Gpx4(Tg) 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. Cancer Prev Res; 4(8); 1267-74. (C) 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 Reviewed International journal

Hiroki Isomura, Mark F. Stinski, Takayuki Murata, Yoriko Yamashita, Teru Kanda, Shinya Toyokuni, Tatsuya Tsurumi

JOURNAL OF VIROLOGY Vol. 85 ( 13 ) page： 6629 - 6644 2011.7

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

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.

DOI： 10.1128/JVI.00384-11

YAP TRANSCRIPTION COACTIVATOR INDUCES MALIGNANT MESOTHELIOMA CELL PROLIFERATION BY UP-REGULATING CELL CYCLE PROGRESSION Reviewed International journal

Tetsuya Mizuno, Hideki Murakami, Makiko Fujii, Futoshi Ishiguro, Yutaka Kondo, Shinya Akatsuka, Shinya Toyokuni, Kohei Yokoi, Hirotaka Osada, Yoshitaka Sekido

JOURNAL OF THORACIC ONCOLOGY Vol. 6 ( 6 ) page： S707 - S708 2011.6

[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]. Reviewed Open Access

Takemi Otsuki, Takashi Nakano, Seiki Hasegawa, Morihito Okada, Tohru Tsujimura, Yoshitaka Sekido, Shinya Toyokuni, Hiroshi Nishimoto, Kazuya Fukuoka, Fumihiro Tanaka, Naoko Kumagai, Megumi Maeda, Yasumitsu Nishimura

Nihon eiseigaku zasshi. Japanese journal of hygiene Vol. 66 ( 3 ) page： 543 - 552 2011.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society for 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 International journal Open Access

TOYOKUNI Shinya, JIANG Li, HU Qian, NAGAI Hirotaka, OKAZAKI Yasumasa, AKATSUKA Shinya, YAMASHITA Yoriko

Nippon Eiseigaku Zasshi (Japanese Journal of Hygiene) Vol. 66 ( 3 ) page： 562 - 7 2011.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society for 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

Osada Hirotaka, Eri Nishikawa, Chinatsu Arima, Yasumasa Okazaki, Shuta Tomida, Yoshio Tatematsu, Ayumu Taguchi, Yukako Shimada, Kiyoshi Yanagisawa, Shinya Toyokuni, Yoshitaka Sekido, Takashi Takahashi

CANCER RESEARCH Vol. 71 2011.4

XCR1 expression and biased VH gene usage are distinct features of diffuse large B-cell lymphoma initially manifesting in the bone marrow. Reviewed International journal

Yoriko Yamashita, Dai Kajiura, Lee Tang, Yuichi Hasegawa, Tomohiro Kinoshita, Shigeo Nakamura, Shinya Akatsuka, Shinya Toyokuni, Naoyoshi Mori

American journal of clinical pathology Vol. 135 ( 4 ) page： 556 - 64 2011.4

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC CLINICAL PATHOLOGY

A total of 29 cases of diffuse large B-cell lymphoma initially manifesting in the bone marrow (BM-DLBCL) were analyzed for V-H 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 V-H gene usage in 6 cases was restricted to V(H)3-7, in 3 cases to V(H)4-34, and in 2 cases to V(H)4-39, which were all previously reported to be autoreactive. In total, 14 of 18 V-H genes were those associated with autoimmune diseases, including V(H)3-21, V(H)3-23, and V(H)3-48. 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 V-H genes seem to be distinct characteristics of BM-DLBCL.

DOI： 10.1309/AJCPCTDC5PY3LXBP

LATS2 is a tumor suppressor gene of malignant mesothelioma

Murakami H.

Cancer Research Vol. 71 ( 3 ) page： 873 - 883 2011.2

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 Vol. 71 ( 3 ) page： 873 - 83 2011.2

Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats. Reviewed International journal

Hiroki Ohara, Shinya Akatsuka, Hirotaka Nagai, Yu-Ting Liu, Li Jiang, Yasumasa Okazaki, Yoriko Yamashita, Tomoyuki Nakamura, Shinya Toyokuni

Free radical research Vol. 45 ( 2 ) page： 211 - 20 2011.2

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.3109/10715762.2010.523702

LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma Reviewed

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 Vol. 71 ( 3 ) page： 873 - 883 2011.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Oxidative DNA Damage and Carcinogenesis

CD8+CD122+ regulatory T cells (Tregs) and CD4+ Tregs cooperatively prevent and cure CD4+ cell-induced colitis. Reviewed International journal Open Access

Agustina Tri Endharti, Yusuke Okuno, Zhe Shi, Nobuaki Misawa, Shinya Toyokuni, Masafumi Ito, Ken-ichi Isobe, Haruhiko Suzuki

Journal of immunology (Baltimore, Md. : 1950) Vol. 186 ( 1 ) page： 41 - 52 2011.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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(+)CD45RB(high) 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(+)CD45RB(low) 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(+)CD45RB(high) 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.

DOI： 10.4049/jimmunol.1000800

3-morpholinosydnonimine participates in the attenuation of neointima formation via inhibition of annexin A2-mediated vascular smooth muscle cell migration. International journal

Kyung-Jong Won, Philyoung Lee, Seung Hyo Jung, Xiaowen Jiang, Chang-Kwon Lee, Hai Yue Lin, Hyun Kang, Hwan Myung Lee, Junghwan Kim, Shinya Toyokuni, Bokyung Kim

Proteomics Vol. 11 ( 2 ) page： 193 - 201 2011.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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.

DOI： 10.1002/pmic.200900834

Iron as a target of chemoprevention for longevity in humans. Reviewed

Toyokuni S.

Free Radic Res Vol. 45 page： 906-917 2011

The human cytomegalovirus gene products essential for late viral gene expression assemble into prereplication complexes before viral DNA replication. Reviewed

Isomura H, Stinski MF, Murata T, Yamashita Y, Kanda T, Toyokuni S and Tsurumi T.

J Virol Vol. 85 page： 6629-6644 2011

Dietary fish oil promotes colonic apoptosis and mitochondrial proton leak in oxidatively stressed mice. Reviewed

Fan YY, Ran Q, Toyokuni S, Okazaki Y, Callaway ES, Lupton JR, and Chapkin RS.

Cancer Prev Res Vol. 4 page： 1267-1274 2011

Age- and sun exposure-dependent differences in 8-hydroxy-2'-deoxyguanosine and Nε-(carboxymethyl)lysine in human epidermis. Reviewed

Toyokuni S, Hirao A, Wada T, Nagai R, Date A, Yoshii T, Akatsuka S, Yamashita Y, and Kawada A.

J Clin Biochem Nutr Vol. 49 page： 1-4 2011

Mysterious link between iron overload and CDKN2A/2B. Reviewed

Toyokuni S.

J Vol. 48 page： 46-49 2011

LATS2 is a tumor suppressor gene of malignancy mesothelioma. Reviewed

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 Vol. 71 page： 873-883 2011

XCR1 expression and biased VH gene usage are distinct features of diffuse large B-cell lymphoma initially manifesting in the bone marrow. Reviewed

Yamashita Y, Kajiura D, Tang L, Hasegawa Y, Kinoshita T, Nakamura S, Akatsuka S, Toyokuni S and Mori N.

Am J Clin Pathol Vol. 135 page： 556-564 2011

Asbetos provides a niche for oxidative modification. Reviewed

Nagai H, Ishihara T, Lee WH, Ohara H, Okazaki Y, Okawa K and Toyokuni S.

Cancer Sci Vol. 102 page： 2118-2125 2011

miR-375 is activated by ASH1 and inhibits YAP1 in a lineage dependent manner in lung cancer. Reviewed

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 Vol. 71 page： 6165-6173 2011

Jiang, L; Toyokuni, S

FREE RADICAL BIOLOGY IN DIGESTIVE DISEASES Vol. 29 page： 55 - 63 2011

Iron as a target of chemoprevention for longevity in humans. Reviewed

Toyokuni S

Free Radic Res Vol. 45 page： 906-917 2011

Mysterious link between iron overload and CDKN2A/2B. Reviewed

Toyokuni S

J Vol. 48 page： 46-49 2011

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 Vol. 71 ( 3 ) 2011

Language：English Publishing type：Research paper (scientific journal)

: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 Vol. 2010 ( 0 ) page： AcOF1030 - AcOF1030 2011

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 29 page： 55 - 63 2011

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 Vol. 22 2011

Oxidative DNA damage and carcinogenesis

Jiang L., Toyokuni S.

Free Radical Biology in Digestive Diseases Vol. 29 page： 55 - 63 2010.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 29 page： 55 - 63 2010.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Frontiers of Gastrointestinal Research

DOI： 10.1159/0003199419

A Beverage Containing Fermented Black Soybean Ameliorates Ferric Nitrilotriacetate-Induced Renal Oxidative Damage in Rats Reviewed

Yasumasa Okazaki, Mohammad Iqbal, Norito Kawakami, Yorihiro Yamamoto, Shinya Toyokuni, Shigeru Okada

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 47 ( 3 ) page： 198 - 207 2010.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Yasumasa, Iqbal Mohammad, Kawakami Norito, Yamamoto Yorihiro, Toyokuni Shinya, Okada Shigeru

Journal of Clinical Biochemistry and Nutrition Vol. 47 ( 3 ) page： 198 - 207 2010.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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 Vol. 47 ( 3 ) page： 198 - 207 2010.11

Biopersistent fiber-induced inflammation and carcinogenesis: lessons learned from asbestos toward safety of fibrous nanomaterials. Reviewed International journal

Hirotaka Nagai, Shinya Toyokuni

Archives of biochemistry and biophysics Vol. 502 ( 1 ) page： 1 - 7 2010.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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 fibeis 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. (C) 2010 Elsevier Inc All rights reserved

DOI： 10.1016/j.abb.2010.06.015

Genome-Scale Approaches to Investigate Oxidative DNA Damage Reviewed International journal

Akatsuka Shinya, Toyokuni Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 47 ( 2 ) page： 91 - 97 2010.9

Genome-Scale Approaches to Investigate Oxidative DNA Damage Reviewed

Shinya Akatsuka, Shinya Toyokuni

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 47 ( 2 ) page： 91 - 97 2010.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Shinya, Toyokuni Shinya

Journal of Clinical Biochemistry and Nutrition Vol. 47 ( 2 ) page： 91 - 97 2010.9

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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. Reviewed International journal Open Access

Tomoyuki Shirase, Kiyoshi Mori, Yasumasa Okazaki, Ken Itoh, Masayuki Yamamoto, Mitsuaki Tabuchi, Fumio Kishi, Li Jiang, Shinya Akatsuka, Kazuwa Nakao, Shinya Toyokuni

The American journal of pathology Vol. 177 ( 2 ) page： 677 - 85 2010.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC

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 beta-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. (Am J Pathol 2010, 177:677-685; DOI: 10.2353/ajpath.2010.090823)

DOI： 10.2353/ajpath.2010.090823

TOWARD A NEW ERA OF THE NAGOYA JOURNAL OF MEDICAL SCIENCE: MESSAGE FROM THE NEW EDITOR-IN-CHIEF International journal

Toyokuni Shinya

NAGOYA JOURNAL OF MEDICAL SCIENCE Vol. 72 ( 3-4 ) page： 107 - 109 2010.8

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 Vol. 72 ( 3-4 ) page： 107 - 9 2010.8

Toward a new era of the Nagoya journal of medical science: message from the new editor-in-chief. Reviewed International journal

Toyokuni S.

Nagoya journal of medical science Vol. 72 ( 3-4 ) page： 107 - 109 2010.8

Lipid Peroxidation Generates Body Odor Component trans-2-Nonenal Covalently Bound to Protein in Vivo Reviewed International journal Open Access

Kousuke Ishino, Chika Wakita, Takahiro Shibata, Shinya Toyokuni, Sachiko Machida, Shun Matsuda, Tomonari Matsuda, Koji Uchida

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 285 ( 20 ) page： 15302 - 15313 2010.5

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

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(epsilon)-3-[(hept-1-enyl)-4-hexyl-pyridinium]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-nonenal-lysine 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.

DOI： 10.1074/jbc.M109.068023

A Novel Method for Efficient Collection of Normal Mesothelial Cells In Vivo Reviewed International journal

Jiang Li, Yamashita Yoriko, Toyokuni Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 46 ( 3 ) page： 265 - 268 2010.5

A Novel Method for Efficient Collection of Normal Mesothelial Cells In Vivo Reviewed

Li Jiang, Yoriko Yamashita, Shinya Toyokuni

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 46 ( 3 ) page： 265 - 268 2010.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Li, Yamashita Yoriko, Toyokuni Shinya

Journal of Clinical Biochemistry and Nutrition Vol. 46 ( 3 ) page： 265 - 268 2010.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

DOI： 10.3164/jcbn.10-01

A Quantitative trait locus responsible for inducing B-cell lymphoblastic lymphoma is a hotspot for microsatellite instability

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 Vol. 64 ( 1 ) page： 54 - 54 2010.5

n-3 polyunsaturated fatty acids promote apoptosis in oxidatively stressed transgenic mouse models International journal

Fan, YY; Toyokuni, S; Callaway, ES; Ran, Q; Lupton, JR; Chapkin, RS

CANCER RESEARCH Vol. 70 2010.4

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

Vol. 101 ( 3 ) page： 800 - 805 2010.3

Homozygous deletion of CDKN2A/2B is a hallmark of iron-induced high-grade rat mesothelioma. Reviewed International journal Open Access

Qian Hu, Shinya Akatsuka, Yoriko Yamashita, Hiroki Ohara, Hirotaka Nagai, Yasumasa Okazaki, Takashi Takahashi, Shinya Toyokuni

Laboratory investigation; a journal of technical methods and pathology Vol. 90 ( 3 ) page： 360 - 73 2010.3

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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 micro-array. 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.

DOI： 10.1038/labinvest.2009.140

A Quantitative trait locus responsible for inducing B-cell lymphoblastic lymphoma is a hotspot for microsatellite instability Reviewed International coauthorship International journal Open Access

Richard H. Kaszynski, Shinya Akatsuka, Takuya Hiratsuka, Guang Jin, Munetaka Ozeki, Tomoko Okuno, Takuro Nakamura, Toshiaki Manabe, Tetsuya Takakuwa, Hiroshi Hiai, Shinya Toyokuni, Keiji Tamaki, Tatsuaki Tsuruyama

CANCER SCIENCE Vol. 101 ( 3 ) page： 800 - 5 2010.3

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL PUBLISHING, INC

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. (Cancer Sci 2010; 101: 800-805)

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 Vol. 99 ( 1 ) page： 217 - 217 2010.3

From the Special Session of the "Special Coordination Funds for Promoting Science and Technology (H18-13-3-1)" Entitled "Malignant Mesothelioma" in the 55^<th> Annual Meeting of the Japanese Society of Occupational Medicine and Traumatology : Brief Overview and Approaches from the Basic Science

OTSUKI Takemi, NAKANO Takashi, HASEGAWA Seiki, OKADA Morihito, TSUJIMURA Tohru, SEKIDO Yoshitaka, TOYOKUNI Shinya, NISHIMOTO Hiroshi, FUKUOKA Kazuya, TANAKA Fumihiro

Japanese journal of occupational medicine and traumatology Vol. 58 ( 1 ) page： 1 - 8 2010.1

*Homozygous deletion of CDKN2A/2B is a hallmark of iron-induced high-grade rat mesothelioma. Reviewed

Hu Q, Akatsuka S, Yamashita Y, Ohara H, Nagai H, Okazaki Y, Takahashi T, Toyokuni S.

Lab. Invest. Vol. 90 page： 360-372 2010.1

Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats. Reviewed

Ohara H, Akatsuka S, Nagai H, Liu YT, Jiang L, Okazaki Y, Yamashita Y, Nakamura T and Toyokuni S.

Free Radic Res page： (in press) 2010

A quantitative trait locus for inducing B-cell lymphoblastic lymphoma is a hot spot for microsatellite instability. Reviewed

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 Vol. 101 page： 800-805 2010

CD8+CD122+ Regulatory T Cells and CD4+ Treg Cooperatively Prevent and Cure CD4+ cell-induced Colitis. Reviewed

Endharti AT, Okuno Y, Shi Z, Misawa N, Toyokuni S, Ito M, Isobe KI and Suzuki H.

Vol. 186 page： 41-52 2010

Toward a new era of the Nagoya Journal of Medical Science: message from the new editor-in-chief. Invited

Toyokuni S.

Nagoy J Med Sci Vol. 72 page： 107-109 2010

A beverage containing fermented black soybean ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats. Reviewed

Okazaki Y, Iqbal M, Kawakami N, Yamamoto Y, Toyokuni S and Okada S.

J Clin Biochem Nutr Vol. 47 page： 198-207 2010

Biopersistent fiber-induced inflammation and carcinogenesis: lessons learned from asbestos toward safety of fibrous nanomaterials. Reviewed

Nagai H and Toyokuni S.

Arch Biochem Biophys Vol. 502 page： 1-7 2010

Genome-scale approaches to investigate oxidative DNA damage. Reviewed

Akatsuka S and Toyokuni S.

J Clin Biochem Nutr Vol. 47 page： 91-97 2010

Lipid peroxidation generates a body odor component trans-2-nonenal covalently bound to protein in vivo. Reviewed

Ishino K, Wakita C, Shibata T, Toyokuni S, Machida S, Matsuda S, Matsuda T and Uchida K.

J Biol Chem Vol. 285 page： 15302-15313 2010

Suppression of SLC11A2 expression is essential to maintain duodenal integrity during dietary iron overload. Reviewed

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 Vol. 177 page： 677-685 2010

Novel method for efficient collection of normal mesothelial cells in vivo. Reviewed

Jiang L, Yamashita Y and Toyokuni S.

J Clin Biochem Nutr Vol. 46 page： 265-268 2010

Homozygous Deletion of CDKN2A/2B is a Hallmark of Iron-induced High-grade Rat Mesothelioma Reviewed International journal

Yasumasa Okazaki, Quan Hu, Shinya Akatsuka, Hiroki Ohara, Hirotaka Nagai, Takashi Takahashi, Shinya Toyokuni

FREE RADICAL BIOLOGY AND MEDICINE Vol. 49 page： S67 - S67 2010

Biopersistent fiber-induced inflammation and carcinogenesis: lessons learned from asbestos toward safety of fibrous nanomaterials. Reviewed

Nagai H, Toyokuni S

Arch Biochem Biophys Vol. 502 page： 1-7 2010

CD8+CD122+ Regulatory T Cells and CD4+ Treg Cooperatively Prevent and Cure CD4+ cell-induced Colitis. Reviewed

Endharti AT, Okuno Y, Shi Z, Misawa N, Toyokuni S, Ito M, Isobe KI, Suzuki H

Vol. 186 page： 41-52 2010

Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats. Reviewed

Ohara H, Akatsuka S, Nagai H, Liu YT, Jiang L, Okazaki Y, Yamashita Y, Nakamura T, Toyokuni S

Free Radic Res page： (in press) 2010

Toward a new era of the Nagoya Journal of Medical Science: message from the new editor-in-chief. Invited

Toyokuni S

Nagoy J Med Sci Vol. 72 page： 107-109 2010

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～ International journal

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 Vol. 26 ( 0 ) page： 35 - 35 2010

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Elucidation of asbestos-induced mesothelial carcinogenesis and its prevention stragegy(Symposium 1: Biomarkers in Environmental Mutagen Research: Lifestyle-related and Environmental Factors)

ToyokuniShinya

( 38 ) page： 75 2009.11

ITPase-deficient mice show growth retardation and die before weaning. Reviewed

Behrmanech M, Sakumi K, Abolhassani N, Toyokuni S, Oka S, Ohnishi YN, Tsuchimoto D, Nakabeppu Y.

Cell Death Differ. Vol. 16 page： 1315-1322 2009.10

ITPase-deficient mice show growth retardation and die before weaning Reviewed International journal Open Access

M. Behmanesh, K. Sakumi, N. Abolhassani, S. Toyokuni, S. Oka, Y. N. Ohnishi, D. Tsuchimoto, Y. Nakabeppu

CELL DEATH AND DIFFERENTIATION Vol. 16 ( 10 ) page： 1315 - 1322 2009.10

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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 alpha-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. Reviewed

Müller C, Ullmann K, Wilkins A, Winterhalter P, Toyokuni S and Steinberg P.

Biosci Biotechnol Biochem Vol. 73 page： 1831-1836 2009.8

Elucidation of Asbestos-induced Carcinogenesis and Its Application to Prevention, Diagnosis and Treatment in Relation to Iron International journal Open Access

Toyokuni Shinya

Haigan Vol. 49 ( 4 ) page： 362 - 367 2009.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japan Lung Cancer Society

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.

DOI： 10.2482/haigan.49.362

Potent Antioxidative Activity of Vineatrol (R) 30 Grapevine-shoot Extract Reviewed International journal Open Access

Carsten Mueller, Kristina Ullmann, Andrea Wilkens, Peter Winterhalter, Shinya Toyokuni, Pablo Steinberg

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Vol. 73 ( 8 ) page： 1831 - 1836 2009.8

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

The health promoting effects of a grapevine-shoot extract named Vineatrol (R) 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 (R) 30. Vineatrol (R) 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 (R) 30 not being superior to Resv alone in both cases. Vineatrol (R) 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 (R) 30 grapevine-shoot extract is a free radical scavenger and potent antioxidant at non-eytotoxic 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 Vol. 68回 page： 163 - 163 2009.8

Genetic Reconstruction of Mouse Spermatogonial Stem Cell Self-Renewal In Vitro by Ras-Cyclin D2 Activation Reviewed International journal Open Access

Jiyoung Lee, Mito Kanatsu-Shinohara, Hiroko Morimoto, Yasuhiro Kazuki, Seiji Takashima, Mitsuo Oshimura, Shinya Toyokuni, Takashi Shinohara

CELL STEM CELL Vol. 5 ( 1 ) page： 76 - 86 2009.7

Language：English Publishing type：Research paper (scientific journal) Publisher：CELL PRESS

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.

DOI： 10.1016/j.stem.2009.04.020

Abnormal DNA methyltransferase expression in mouse germline stem cells results in spermatogenic defects. Reviewed

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.

Vol. 81 page： 155-164 2009.7

Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation. Reviewed

Lee J, Kanatsu-Shinohara M, Morimoto H, Kazuki Y, Takashima S, Oshimura M, Toyokuni S and Shinohara T.

Cell Stem Cell Vol. 5 page： 76-86 2009.7

Abnormal DNA Methyltransferase Expression in Mouse Germline Stem Cells Results in Spermatogenic Defects Reviewed International journal

Seiji Takashima, Masanori Takehashi, Jiyoung Lee, Shinichiro Chuma, Masaki Okano, Kenichiro Hata, Isao Suetake, Norio Nakatsuji, Hiroyuki Miyoshi, Shoji Tajima, Yoriko Tanaka, Shinya Toyokuni, Hiroyuki Sasaki, Mito Kanatsu-Shinohara, Takashi Shinohara

BIOLOGY OF REPRODUCTION Vol. 81 ( 1 ) page： 155 - 64 2009.7

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC STUDY 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.

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. International journal Open Access

Yang-Yi Fan, Yang Zhan, Harold M Aukema, Laurie A Davidson, Lan Zhou, Evelyn Callaway, Yanan Tian, Brad R Weeks, Joanne R Lupton, Shinya Toyokuni, Robert S Chapkin

The Journal of nutrition Vol. 139 ( 7 ) page： 1328 - 1332 2009.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Nutrition

We recently demonstrated that (n-3) PUFA trigger the induction of apoptosis in the colon by enhancing phospholipid oxidation and mitochondrial Ca2+ 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.

DOI： 10.3945/jn.109.106203

The potential role of reactive oxygen species in mammary neoplasm Reviewed International journal

Arafah M., Khabaz M., Toyokuni S.

Australian Journal of Basic and Applied Sciences Vol. 3 ( 3 ) page： 2949 - 2954 2009.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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 International journal

Arafah M, Khabaz M, Toyokuni S

Australian Journal of Basic and Applied Sciences Vol. 3 ( 3 ) page： 2949 - 2954 2009.7

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. Reviewed

Pro-apoptotic effects of dietary (n-3) fatty acids are enhanced in colonocytes of SOD2 knockout mice. Reviewed

Fan YY, Zhan Y, Aukema HM, Davidson LA, Zhou L, Callaway E, Tian Y, Weeks BR, Lupton JR, Toyokuni S, Chapkin RS.

J. Nutr. Vol. 139 page： 1328-1332 2009.5

Effect of edaravone, a novel free radical scavenger, supplemented to cardioplegia on myocardial function after cardioplegic arrest: in vitro study of isolated rat heart. Reviewed

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 Vol. 24 page： 228-235 2009.5

Kazuhiro Yamazaki, Senri Miwa, Shinya Toyokuni, Shintaro Nemoto, Wnimunk Oriyanhan, Kiyoaki Takaba, Yoshiaki Saji, Akira Marui, Takeshi Nishina, Tadashi Ikeda, Masashi Komeda

Heart and vessels Vol. 24 ( 3 ) page： 228 - 235 2009.5

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

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 4A degrees 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 mu M), moderate dose (10 mu M), and high dose (100 mu M) 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/dt (max) (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 mu M) of edaravone.

DOI： 10.1007/s00380-008-1106-1

Pro-apoptotic effects of dietary (n-3) fatty acids are enhanced in colonocytes of SOD2 knockout mice. Reviewed International journal

Fan YY, Zhan Y, Aukema HM, Davidson LA, Zhou L, Callaway E, Tian Y, Weeks BR, Lupton JR, Toyokuni S, Chapkin RS

J. Nutr. Vol. 139 page： 1328-1332 2009.5

Granulocyte colony-stimulating factor protects cardiac mitochondria in the early phase of cardiac injury. Reviewed

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. Vol. 296 page： H823-H832 2009.3

Granulocyte colony-stimulating factor protects cardiac mitochondria in the early phase of cardiac injury Reviewed International journal Open Access

Yoshimi Hiraumi, Eri Iwai-Kanai, Shiro Baba, Yoshihiro Yui, Yuri Kamitsuji, Yasuhiro Mizushima, Hiroshi Matsubara, Motonobu Watanabe, Ken-ichirou Watanabe, Shinya Toyokuni, Hiroaki Matsubara, Tatsutoshi Nakahata, Souichi Adachi

AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Vol. 296 ( 3 ) page： H823 - H832 2009.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

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. Granulocyte colony-stimulating factor protects cardiac mitochondria in the early phase of cardiac injury. Am J Physiol Heart Circ Physiol 296: H823-H832, 2009. First published January 9, 2009; doi:10.1152/ajpheart.00774.2008.-Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardioprotective 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.

DOI： 10.1152/ajpheart.00774.2008

Mechanisms of asbestos-induced carcinogenesis.

Toyokuni S.

Nagoya J. Med. Sci. Vol. 71 page： 1-10 2009.2

MECHANISMS OF ASBESTOS-INDUCED CARCINOGENESIS International journal

Toyokuni Shinya

NAGOYA JOURNAL OF MEDICAL SCIENCE Vol. 71 ( 1-2 ) page： 1 - 10 2009.2

MECHANISMS OF ASBESTOS-INDUCED CARCINOGENESIS

TOYOKUNI S.

Nagoya Journal of Medical Science Vol. 71 ( 1 ) page： 1 - 10 2009.2

Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal

Toyokuni S.

Nagoya journal of medical science Vol. 71 ( 1-2 ) page： 1 - 10 2009.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 71 ( 1-2 ) page： 1 - 10 2009.2

Mechanisms of asbestos-induced carcinogenesis.

Toyokuni S

Nagoya J. Med. Sci. Vol. 71 page： 1-10 2009.2

TOYOKUNI Shinya

Cancer Sci. Vol. 100 ( 1 ) page： 9 - 16 2009.1

Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inihibitor FK228 through AIF translocation. Reviewed International journal Open Access

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 Vol. 124 ( 1 ) page： 55 - 67 2009.1

*Role of iron in carcinogenesis: Cancer as a ferrotoxic disease. Invited Reviewed

Toyokuni S.

Cancer Sci. Vol. 100 page： 6-16 2009.1

Genome-wide analysis identifies a tumor suppressor role for aminoacylase 1 in iron-induced rat renal cell carcinoma. Reviewed

Zhong Y, Onuki J, Yamasaki T. Ogawa O, Akatsuka S and Toyokuni S.

Carcinogenesis Vol. 30 page： 158-164 2009.1

Biased Immunoglobulin VH Recombination and Increased Expression of Chemokine Receptor XCR1 Are Distinct Features of Bone Marrow-Derived Diffuse Large B-Cell Lymphoma International journal

Yamashita, Y; Kajiura, D; Nakamura, S; Toyokuni, S; Mori, N

LABORATORY INVESTIGATION Vol. 89 page： 292A - 292A 2009.1

Biased Immunoglobulin VH Recombination and Increased Expression of Chemokine Receptor XCR1 Are Distinct Features of Bone Marrow-Derived Diffuse Large B-Cell Lymphoma International journal

Yamashita, Y; Kajiura, D; Nakamura, S; Toyokuni, S; Mori, N

MODERN PATHOLOGY Vol. 22 page： 292A - 292A 2009.1

Genome-wide analysis identifies a tumor suppressor role for aminoacylase 1 in iron-induced rat renal cell carcinoma. Reviewed International journal

Yi Zhong, Janice Onuki, Toshinari Yamasaki, Osamu Ogawa, Shinya Akatsuka, Shinya Toyokuni

Carcinogenesis Vol. 30 ( 1 ) page： 158 - 64 2009.1

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

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

Role of iron in carcinogenesis: cancer as a ferrotoxic disease. Reviewed International journal Open Access

Shinya Toyokuni

Cancer science Vol. 100 ( 1 ) page： 9 - 16 2009.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL PUBLISHING, INC

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. (Cancer Sci 2009; 100: 9-16).

DOI： 10.1111/j.1349-7006.2008.01001.x

*Role of iron in carcinogenesis: Cancer as a ferrotoxic disease. Invited Reviewed

Toyokuni S

Cancer Sci. Vol. 100 page： 6-16 2009.1

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. Invited

Liu YT, Toyokuni S.

Methods Mol. Biol. Vol. 536 page： 307-312 2009

Lipid Peroxidation Generates a Body Odor Component Trans-2-Nonenal Covalently Bound to Protein in vivo International journal

Ishino Kousuke, Wakita Chika, Shibata Takahiro, Toyokuni Shinya, Machida Sachiko, Matsuda Shun, Matsuda Tomonari, Uchida Koji

FREE RADICAL BIOLOGY AND MEDICINE Vol. 47 page： S76 - S76 2009

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. Invited

Liu YT, Toyokuni S

Methods Mol. Biol. Vol. 536 page： 307-312 2009

Lipid Peroxidation Generates a Body Odor Component Trans-2-Nonenal Covalently Bound to Protein in vivo Reviewed

Kousuke Ishino, Chika Wakita, Takahiro Shibata, Shinya Toyokuni, Sachiko Machida, Shun Matsuda, Tomonari Matsuda, Koji Uchida

FREE RADICAL BIOLOGY AND MEDICINE Vol. 47 page： S76 - S76 2009

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. Reviewed International journal

Yu-Ting Liu, Shinya Toyokuni

Methods in molecular biology (Clifton, N.J.) Vol. 536 page： 307 - 12 2009

Language：English Publishing type：Research paper (scientific journal)

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 Elucidation of asbestos-induced mesothelial carcinogenesis and its prevention stragegy(Symposium 1: Biomarkers in Environmental Mutagen Research: Lifestyle-related and Environmental Factors)

Toyokuni Shinya

( 38 ) 2009

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 Sci Vol. 99 ( 11 ) page： 2142 - 2151 2008.11

*Characteristics and modifying factors of asbestos-induced oxidative DNA damage. Reviewed

Jiang L, Nagai H; Ohara H, Hara S, Tachibana M, Hirano S, Shinohara Y, Kohyama N, Akatsuka S and Toyokuni S.

Cancer Sci. Vol. 99 ( 11 ) page： 2142-2151 2008.11

Characteristics and modifying factors of asbestos-induced oxidative DNA damage Reviewed International journal Open Access

Li Jiang, Hirotaka Nagai, Hiroki Ohara, Shigeo Hara, Mitsuhiro Tachibana, Seishiro Hirano, Yasushi Shinohara, Norihiko Kohyama, Shinya Akatsuka, Shinya Toyokuni

CANCER SCIENCE Vol. 99 ( 11 ) page： 2142 - 51 2008.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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 H(2)O(2), 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. (Cancer Sci 2008; 99: 2142-2151).

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 Reviewed International journal Open Access

Y. Kamitsuji, J. Kuroda, S. Kimura, S. Toyokuni, K. Watanabe, E. Ashihara, H. Tanaka, Y. Yui, M. Watanabe, H. Matsubara, Y. Mizushima, Y. Hiraumi, E. Kawata, T. Yoshikawa, T. Maekawa, T. Nakahata, S. Adachi

CELL DEATH AND DIFFERENTIATION Vol. 15 ( 11 ) page： 1712 - 22 2008.11

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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. Reviewed International journal Open Access

Jie Liao, Darren N Seril, Gary G Lu, Meng Zhang, Shinya Toyokuni, Allison L Yang, Guang-Yu Yang

Molecular carcinogenesis Vol. 47 ( 8 ) page： 638 - 646 2008.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

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 mm(3). 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 mm(3). 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 mm(3). 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. (C) 2008 Wiley-Liss, Inc.

DOI： 10.1002/mc.20427

Molecular mechanisms of oxidative stress-induced carcinogenesis: from epidemiology to oxygenomics. Reviewed International journal Open Access

Toyokuni S

IUBMB life Vol. 60 ( 7 ) page： 441 - 7 2008.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Protein N-acylation: H2O2-mediated covalent modification of protein by lipid peroxidation-derived saturated aldehydes. Reviewed International journal

Kousuke Ishino, Takahiro Shibata, Takeshi Ishii, Yu-Ting Liu, Shinya Toyokuni, Xiaochun Zhu, Lawrence M Sayre, Koji Uchida

Chemical research in toxicology Vol. 21 ( 6 ) page： 1261 - 70 2008.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

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, N-alpha-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, N-epsilon-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.

DOI： 10.1021/tx800080x

Pluripotency of a single spermatogonial stem cell in mice Reviewed International journal Open Access

Mito Kanatsu-Shinohara, Jiyoung Lee, Kimiko Inoue, Narumi Ogonuki, Hiromi Miki, Shinya Toyokun, Masahito Ikawa, Tomoyuki Nakamura, Atsuo Gura, Takashi Shinohara

BIOLOGY OF REPRODUCTION Vol. 78 ( 4 ) page： 681 - 7 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC STUDY 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 11 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 KIf4) 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.

DOI： 10.1095/biolreprod.107.066068

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model Reviewed International journal

Hakozaki, T; Date, A; Yoshii, T; Toyokuni, S; Yasui, H; Sakurai, H

ARCHIVES OF DERMATOLOGICAL RESEARCH Vol. 300 ( SUPPL. 1 ) page： S51 - S56 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Target Genes of Oxidative Stress-induced Carcinogenesis

TOYOKUNI Shinya

Vol. 63 ( 2 ) page： 282 2008.3

Target Genes of Oxidative Stress-induced Carcinogenesis

TOYOKUNI Shinya

Vol. 63 ( 2 ) page： 282 - 282 2008.3

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 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 Vol. 14 ( 1 ) page： 32 - 40 2008.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Vol. 38 ( 1 ) page： 70 - 78 2008.1

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 Vol. 14 ( 1 ) page： 32 - 40 2008.1

Overexpression of CYP3A aggravates endotoxin-induced liver injury in hypophysectomized female rats Reviewed International journal

Shigekazu Takemura, Yukiko Minamiyama, Shinya Toyokuni, Susumu Imaoka, Seikan Hai, Shoji Kubo, Kazuhiro Hirohashi, Yoshihiko Funae, Shigeru Okada

HEPATOLOGY RESEARCH Vol. 38 ( 1 ) page： 70 - 78 2008.1

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING

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 three days before LPS injection. Liver injury was analyzed 8 h 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.
Conclusions: Overexpression of CYP3A aggravated LPS-induced liver injury in rats, apparently through the formation of reactive oxygen species.

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 Reviewed

Ken Yamaguchi, Masaki Mandai, Shinya Toyokuni, Junzo Hamanishi, Toshihiro Higuchi, Kenji Takakura, Shingo Fujii

CLINICAL CANCER RESEARCH Vol. 14 ( 1 ) page： 32 - 40 2008.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Pluripotency of a single spermatogonial stem cell. Reviewed

Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Yoshida S, Toyokuni S, Lee J, Ogura A and Shinohara T.

Biol. Reprod. Vol. 78 page： 681-687 2008

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. Reviewed

Yamaguchi K, Mandai M, Toyokuni S, Hamanishi J, Higuchi T, Takakura K and Fujii S.

Clin. Cancer Res. Vol. 14 page： 32-40 2008

Molecular mechanisms of oxidative stress-induced carcinogenesis: from epidemiology to oxygenomics. Invited Reviewed

Toyokuni S.

IUBMB Life Vol. 60 page： 441-447 2008

Increased susceptibility of chronic ulcerative colitis-induced carcinoma development in DNA repair enzyme Ogg1 deficient mice. Reviewed

Liao J, Seril DN, Lu GG, Zhang M, Toyokuni S, Yang AL and Yang GY.

Mol. Carcinog. Vol. 47 page： 638-646 2008

Protein N-acylation: H2O2-mediated covalent modification of protein by lipid peroxidation-derived saturated aldehydes. Reviewed

Ishino K, Shibata T, Ishii T, Liu YT, Toyokuni S, Zhu X, Sayre LM and Uchida K.

Chem. Res. Toxicol. Vol. 21 page： 1261-1270 2008

A reporter gene assay to prove the ability of natural compounds to modulate gluthathione peroxidase, superoxide dismutase and catalase gene promoters in V79 cells. Reviewed

Ullmann K, Wiencierz A M, Müller C, Thierbach R, Steege A, Toyokuni S and Steinberg P.

Free Radic. Res. Vol. 42 page： 746-753 2008

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias. Reviewed

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. Vol. 15 page： 1712-1722 2008

Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inihibitor FK228 through AIF translocation. Reviewed

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 Vol. 124 page： 55-67 2008

Genome-wide analysis identifies a tumor suppressor role for aminoacylase 1 in iron-induced rat renal cell carcinoma. Reviewed

Zhong Y, Onuki J, Yamasaki T. Ogawa O, Akatsuka S and Toyokuni S.

Carcinogenesis page： in press 2008

Overexpression of CYP3A aggravates endotoxin-induced liver injury in hypophysectomized female rats. Reviewed

Takemura S, Minamiyama Y, Toyokuni S, Imaoka S, Hai S, Kubo S, Hirohashi K, Funae Y and Okada S.

Hepatol. Res. Vol. 38 page： 70-78 2008

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. Reviewed International journal

Kristina Ullmann, Anne Maria Wiencierz, Carsten Müller, Rene Thierbach, Andreas Steege, Shinya Toyokuni, Pablo Steinberg

Free radical research Vol. 42 ( 8 ) page： 746 - 753 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：INFORMA HEALTHCARE

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. Reviewed

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 Vol. 124 page： 55-67 2008

Molecular mechanisms of oxidative stress-induced carcinogenesis: from epidemiology to oxygenomics. Invited Reviewed

Toyokuni S

IUBMB Life Vol. 60 page： 441-447 - 447 2008

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 Sci. Vol. 98 ( 12 ) page： 1845 - 1852 2007.12

Production of knockout mice by gene targeting in multipotent germline stem cells Reviewed International journal Open Access

Masanori Takehashi, Mito Kanatsu-Shinohara, Hiromi Miki, Jiyoung Lee, Yasuhiro Kazuki, Kimiko Inoue, Narumi Ogonuki, Shinya Toyokuni, Mitsuo Oshimura, Atsuo Ogura, Takashi Shinohara

DEVELOPMENTAL BIOLOGY Vol. 312 ( 1 ) page： 344 - 52 2007.12

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Spermatogonial stein 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. (c) 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 Reviewed International journal Open Access

Khokon Kumar Dutta, Yi Zhong, Yu-Ting Liu, Takuji Yamada, Shinya Akatsuka, Qian Hu, Minako Yoshihara, Hiroki Ohara, Masanori Takehashi, Takashi Shinohara, Hiroshi Masutani, Janice Onuki, Shinya Toyokuni

CANCER SCIENCE Vol. 98 ( 12 ) page： 1845 - 52 2007.12

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING

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.

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 Vol. 171 ( 6 ) page： 1978 - 1988 2007.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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 Vol. 171 ( 6 ) page： 1978 - 1988 2007.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal Open Access

Aya Miyagawa-Hayashino, Tatsuaki Tsuruyama, Hiroto Egawa, Hironori Haga, Hirorni Sakashita, Tomoko Okuno, Shinya Toyolkuni, Keiji Tamaki, Hirohiko Yamabe, Toshialki Manabe, Shinji Uemoto

AMERICAN JOURNAL OF PATHOLOGY Vol. 171 ( 5 ) page： 1499 - 508 2007.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC

Fas-Fas figand (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 (n = 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-gamma 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-gamma(+) T cells, rather than 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 rejection activity in human liver allografts.

DOI： 10.2353/ajpath.2007.070027

Overexpression of a transcription factor LYL1 induces T- and B-cell lymphoma in mice Reviewed International journal

Y. Zhong, L. Jiang, H. Hiai, S. Toyokuni, Y. Yamada

ONCOGENE Vol. 26 ( 48 ) page： 6937 - 47 2007.10

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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 lot 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.

DOI： 10.1038/sj.onc.1210494

Pathological investigation of oxidative stress in the post-genomic era Invited Reviewed International journal

Shinya Toyokuni, Shinya Akatsuka

PATHOLOGY INTERNATIONAL Vol. 57 ( 8 ) page： 461 - 73 2007.8

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING

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 alpha-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.

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 Vol. 66回 page： 343 - 344 2007.8

YOKOO Hideaki, OISHI Takuma, ISODA Koji, NAKAZATO Yoichi, TOYOKUNI Shinya

Vol. 27 ( 3 ) page： 237 - 244 2007.6

Analysis of genes associated with karyomegalic cells in rat kidney after ferric nitilotriacetate treatment

Lee, WH; Toyokuni, S

AMERICAN JOURNAL OF HEMATOLOGY Vol. 82 ( 6 ) page： 602 - 602 2007.6

Novel approach for elucidation of iron-induced carcinogenesis: Oxygenomics and micro-RNA

Toyokuni, S; Dutta, KK; Akatsuka, S

AMERICAN JOURNAL OF HEMATOLOGY Vol. 82 ( 6 ) page： 512 - 512 2007.6

Oxidative stress is related to the formation of Antoni B patterns and eosinophilic hyaline droplets in schwannomas Reviewed International journal

Yokoo, H; Oishi, T; Isoda, K; Nakazato, Y; Toyokuni, S

NEUROPATHOLOGY Vol. 27 ( 3 ) page： 237 - 244 2007.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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

ARMET is a Soluble ER Protein Induced by the Unfolded Protein Response via ERSE-II Element

Novel approach for elucidation of iron-induced carcinogenesis: Oxygenomics and micro-RNA International journal

Toyokuni, S, Dutta, KK, Akatsuka, S

AMERICAN JOURNAL OF HEMATOLOGY Vol. 82 ( 6 ) page： 512 - 512 2007.6

Analysis of genes associated with karyomegalic cells in rat kidney after ferric nitilotriacetate treatment Reviewed International coauthorship International journal

Lee, WH, Toyokuni, S

AMERICAN JOURNAL OF HEMATOLOGY Vol. 82 ( 6 ) page： 602 - 602 2007.6

Akt mediates self-renewal division of mouse spermatogonial stem cells Reviewed International journal Open Access

Jiyoung Lee, Mito Kanatsu-Shinohara, Kimiko Inoue, Narumi Ogonuki, Hiromi Miki, Shinya Toyokuni, Tohru Kimura, Toru Nakano, Atsuo Ogura, Takashi Shinohara

DEVELOPMENT Vol. 134 ( 10 ) page： 1853 - 9 2007.5

Language：English Publishing type：Research paper (scientific journal) Publisher：COMPANY OF BIOLOGISTS LTD

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 (PI3K)-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 PI3K 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 PI3K-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 Reviewed International journal Open Access

Masanori Takehashi, Mito Kanatsu-Shinohara, Kimiko Inoue, Narumi Ogonuki, Hiromi Miki, Shinya Toyokuni, Atsuo Ogura, Takashi Shinohara

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vol. 104 ( 8 ) page： 2596 - 601 2007.2

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

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.

DOI： 10.1073/pnas.0609282104

Adult pancreatic islets require differential pax6 gene dosage Reviewed International journal Open Access

Akihiro Hamasaki, Yuichiro Yamada, Takeshi Kurose, Nobuhiro Ban, Kazuaki Nagashima, Akira Takahashi, Shimpei Fujimoto, Dai Shimono, Michio Fujiwara, Shinya Toyokuni, Yutaka Seino, Nobuya Inagaki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 353 ( 1 ) page： 40 - 6 2007.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

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 (rSey(2)) with a point mutation in the pax6 locus resulting in truncated PAX6 proteins. Analyses of the insulin secretory profile of heterozygous rSey(2)/+ 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 rSey(2)/+. On the other hand, pancreatic insulin and glucagon content and islet architecture in rSey(2)/+ 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. (c) 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 Reviewed International journal

Masaaki Okuno, Kohtaro Minami, Akinori Okumachi, Kazumasa Miyawaki, Norihide Yokoi, Shinya Toyokuni, Susumu Seino

AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Vol. 292 ( 1 ) page： E158 - E165 2007.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

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 diabetes-prone 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.

DOI： 10.1152/ajpendo.00180.2006

Leukemia inhibitory factor enhances formation of germ cell colonies in neonatal mouse testis culture International journal Open Access

Mito Kanatsu-Shinohara, Kimiko Inoue, Narumi Ogonuki, Hiromi Miki, Shosei Yoshida, Shinya Toyokuni, Jiyoung Lee, Atsuo Ogura, Takashi Shinohara

BIOLOGY OF REPRODUCTION Vol. 76 ( 1 ) page： 55 - 62 2007.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC STUDY 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 GS 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.

DOI： 10.1095/biolreprod.106.055863

*Chronic oxidative stress causes amplification and overexpresson of ptprz1 protein tyrosine phosphatase to activate beta-catenin pathway. Reviewed

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. Vol. 171 page： 1978-1988 2007

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model. Reviewed

Hakozaki T, Date A, Yoshii T, Toyokuni S, Yasui H and Sakurai H.

J. Dermatol. Res. Vol. 300 (Supplement 1) page： S51-56 2007

Generation of insulin-secreting cells from pancreatic acinar cells of animal models of type 1 diabetes. Reviewed

Okuno M, Minami K, Okumachi A, Miyawaki K, Yokoi N, Toyokuni S, Seino S.

Am. J. Physiol. Endocrinol. Metab. Vol. 292 page： E158-165 2007

Oxidative stress is related to formation of Antoni B pattern and eosinophilic hyaline droplets in schwannoma. Reviewed

Yokoo H, Oishi T, Isoda, K, Nakazato Y and Toyokuni S.

Neuropathology Vol. 27 page： 237-244 2007

Angiotensin II, oxidative stress, and extracellular matrix degradation during transition to LV failure in rats with hypertension. Reviewed

Takenaka H, Kihara Y, Iwanaga Y, Onozawa Y, Toyokuni S, Kita T.

J. Mol. Cell. Cardiol. Vol. 41 page： 989-997 2007

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. Vol. 76 page： 55-62 2007

Adult pancreatic islets require differential pax6 gene dosage. Reviewed

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. Vol. 353 page： 40-46 2007

Adenovirus-mediated gene delivery into mouse spermatogonial stem cells. Reviewed

Takehashi M, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Ogura A, and Shinohara T.

Proc. Natl. Acad. Sci. USA Vol. 104 page： 2596-2601 2007

Pathological investigation of oxidative stress in the post-genomic era. Invited Reviewed

Toyokuni S and Akatsuka S.

Pathol. Int. Vol. 57 page： 461-473 2007

Overexpressin of a transcription factor LYL-1 induces T- and B-cell lymphoma in mice. Reviewed

Zhong Y, Li J, Hiai H, Toyokuni S* and Yamada Y* (*corresponding author).

Oncogene Vol. 26 page： 6937-6947 2007

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element. Reviewed

Mizobuchi N, Hoseki J, Kubota H, Toyokuni S, Nozaki J, Naitoh M, Koizumi A and Nagata K.

Cell Struct. Funct. Vol. 32 page： 41-50 2007

Akt mediates self-renewal division of mouse spermatogonial stem cells. Reviewed

Lee J, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Kimura T, Nakano T, Ogura A and Shinohara T.

Development Vol. 134 page： 1853-1859 2007

Association of microRNA-34a overexpression with proliferation is cell type-dependent. Reviewed

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. Vol. 98 page： 1845-1852 2007

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. Reviewed

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. Vol. 171 page： 1499-1508 2007

Production of knockout mice by gene targeting in multipotent germline stem cells. Reviewed

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. Vol. 312 page： 344-352 2007

Analysis of genes associated with karyo-megalic cells in rat kidney after ferric nitilotriacetate treatment International journal

Lee, WH; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE Vol. 43 page： S24 - S24 2007

Mizobuchi Naomi, Hoseki Jun, Kubota Hiroshi, Toyokuni Shinya, Nozaki Jun-ichi, Naitoh Motoko, Koizumi Akio, Nagata Kazuhiro

Cell Structure and Function Vol. 32 ( 1 ) page： 41 - 50 2007

Language：English Publishing type：Research paper (scientific journal) Publisher：Japan Society for 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.

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 Vol. 43 page： S154 - S155 2007

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model. Reviewed

Hakozaki T, Date A, Yoshii T, Toyokuni S, Yasui H, Sakurai H

J. Dermatol. Res. Vol. 300 (Supplement 1) page： S51-56 2007

Oxidative stress is related to formation of Antoni B pattern and eosinophilic hyaline droplets in schwannoma. Reviewed

Yokoo H, Oishi T, Isoda, K, Nakazato Y, Toyokuni S

Neuropathology Vol. 27 page： 237-244 2007

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element Reviewed

Naomi Mizobuchi, Jun Hoseki, Hiroshi Kubota, Shinya Toyokuni, Jun-ichi Nozaki, Motoko Naitoh, Akio Koizumi, Kazuhiro Nagata

CELL STRUCTURE AND FUNCTION Vol. 32 ( 1 ) page： 41 - 50 2007

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Angiotensin II, oxidative stress, and extracellular matrix degradation during transition to LV failure in rats with hypertension. Reviewed

Takenaka H, Kihara Y, Iwanaga Y, Onozawa Y, Toyokuni S, Kita T

J. Mol. Cell. Cardiol. Vol. 41 page： 989-997 - 997 2007

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 Vol. 32 ( 1 ) page： 41 - 50 2007

TOYOKUNI Shinya, YASUI Hiroyuki, DATE Akira, HAKOZAKI Tomohiro, AKATSUKA Shinya, KOHDA Haruyasu, YOSHII Takashi, SAKURAI Hiromu

Vol. 56 ( 12 ) page： 760 - 762 2006.12

Angiotensin II, oxidative stress, and extracellular matrix degradation during transition to LV failure in rats with hypertension. Reviewed International journal

Takenaka H, Kihara Y, Iwanaga Y, Onozawa Y, Toyokuni S, Kita T

Journal of molecular and cellular cardiology Vol. 41 ( 6 ) page： 989 - 97 2006.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Shinya Toyokuni, Hiroyuki Yasui, Akira Date, Tomohiro Hakozaki, Shinya Akatsuka, Haruyasu Kohda, Takashi Yoshii, Hiromu Sakurai

PATHOLOGY INTERNATIONAL Vol. 56 ( 12 ) page： 760 - 762 2006.12

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 Sci Vol. 97 ( 11 ) page： 1159 - 1167 2006.11

Deletion and single nucleotide substitution at G:C in the kidney of gpt delta transgenic mice after ferric nitrilotriacetate treatment. Reviewed International journal Open Access

Li Jiang, Yi Zhong, Shinya Akatsuka, Yu-Ting Liu, Khokon Kumar Dutta, Wen-Hua Lee, Janice Onuki, Ken-ichi Masumura, Takehiko Nohmi, Shinya Toyokuni

Cancer science Vol. 97 ( 11 ) page： 1159 - 67 2006.11

Language：English Publishing type：Research paper (scientific journal)

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.

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. Reviewed International journal 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 Vol. 169 ( 4 ) page： 1328 - 42 2006.10

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal Open Access

Wen-Hua Lee, Shinya Akatsuka, Tomoyuki Shirase, Khokon Kumar Dutta, Li Jiang, Yu-Ting Liu, Janice Onuki, Yoshihiro Yamada, Katsuya Okawa, Youichiro Wada, Akira Watanabe, Takahide Kohro, Noriko Noguchi, Shinya Toyokuni

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 453 ( 2 ) page： 168 - 78 2006.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Pre-administration of alpha-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 alpha-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 alpha-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 LLG-PK1 cells, addition of alpha-tocopherol was protective against oxidative stress by H,0, 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 alpha-tocopherol induced N-acetylglucosaminyltransferase 3, our data suggest that alpha-tocopherol modifies glycoprotein metabolism partially by conferring mild ER stress. This adds another molecular mechanism of alpha-tocopherol toward cancer prevention. (c) 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 Vol. 34 ( 9 ) page： 47 - 47 2006.9

Overexpression of LYL1 gene induces T- and B-cell leukemia in mice International journal

Zhong, Y, Jiang, L, Toyokuni, S, Hiai, H, Yamada, Y

EXPERIMENTAL HEMATOLOGY Vol. 34 ( 9 ) page： 47 - 47 2006.9

Historical aspects of free radical chemistry and biology: What's new in oxidative stress-induced carcinogenesis and its prevention? Reviewed International journal

Toyokuni Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 39 ( 1 ) page： 1 - 2 2006.7

Historical Aspects of Free Radical Chemistry and Biology: What’s New in Oxidative Stress-induced Carcinogenesis and Its Prevention?

Toyokuni Shinya

Journal of Clinical Biochemistry and Nutrition Vol. 39 ( 1 ) page： 1 - 2 2006.7

Novel aspects of oxidative stress-associated carcinogenesis Reviewed

Shinya Toyokuni

Antioxidants and Redox Signaling Vol. 8 ( 7-8 ) page： 1373 - 7 2006.7

Language：English Publishing type：Research paper (scientific journal)

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 Shinya, Akatsuka Shinya

Journal of Clinical Biochemistry and Nutrition Vol. 39 ( 1 ) page： 3 - 10 2006.7

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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 Reviewed

Shinya Toyokuni, Shinya Akatsuka

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 39 ( 1 ) page： 3 - 10 2006.7

Language：English Publishing type：Research paper (scientific journal) Publisher：JOURNAL CLINICAL BIOCHEMISTRY & 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.

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 Vol. 39 ( 1 ) page： 3 - 10 2006.7

Historical aspects of free radical chemistry and biology: What's new in oxidative stress-induced carcinogenesis and its prevention? Reviewed International journal

Toyokuni Shinya

JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION Vol. 39 ( 1 ) page： 1 - 2 2006.7

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells Reviewed International journal Open Access

M Kanatsu-Shinohara, M Ikawa, M Takehashi, N Ogonuki, H Miki, K Inoue, Y Kazuki, J Lee, S Toyokuni, M Oshimura, A Ogura, T Shinohara

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vol. 103 ( 21 ) page： 8018 - 23 2006.5

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

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 focus 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.

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 Vol. 95 ( 1 ) page： 213 - 213 2006.4

Anchorage-independent growth of mouse male germline stem cells in vitro Reviewed International journal

M Kanatsu-Shinohara, K Inoue, J Lee, H Miki, N Ogonuki, S Toyokuni, A Ogura, T Shinohara

BIOLOGY OF REPRODUCTION Vol. 74 ( 3 ) page： 522 - 9 2006.3

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC STUDY 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, similar to 4.7 days), they expressed markers of spermatogonia, and grew exponentially for at least 5 months to achieve 1.5 x 10(10)-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.

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 Vol. 12 ( 7 ) page： 1005 - 1012 2006.2

Bancel B, Esteve J, Souquet JC, Toyokuni S, Ohshima H, Pignatelli B

World journal of gastroenterology Vol. 12 ( 7 ) page： 1005 - 12 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal Open Access

K Sakuma, Fujimoto, I, S Hitoshi, F Tanaka, T Ikeda, K Tanabe, S Toyokuni, H Wada, T Mio, M Mishima, K Ikenaka

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 340 ( 3 ) page： 829 - 835 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

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 alpha 1,6-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 of alpha 1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed alpha 1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific alpha 1,6-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. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.12.072

Differences in oxidative stress dependence between gastric adenocarcinoma subtypes Reviewed

Brigitte Bancel, Jacques Esteve, Jean-Christophe Souquet, Shinya Toyokuni, Hiroshi Ohshima, Brigitte Pignatelli

WORLD JOURNAL OF GASTROENTEROLOGY Vol. 12 ( 7 ) page： 1005 - 1012 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed

Toyokuni S, Yasui H, Date A, Hakozaki T, Akatsuka S, Kohda H, Yoshii T and Sakurai H.

Pathol. Int. Vol. 56 page： 760-762 2006

Anchorage-independent growth of mouse male germline stem cells in vitro. Reviewed

Shinohara M, Inoue K, Lee J, Miki H, Ogonuki N, Toyokuni S, Ogura A and Shinohara T.

Biol. Reprod. Vol. 74 page： 522-529 2006

An N-glycan structure correlates with pulmonary metastatic ability of cancer cells. Reviewed

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. Vol. 349 page： 829-835 2006

Novel aspects of oxidative stress-associated carcinogenesis. Reviewed

Toyokuni S.

Antiox. Redox Signal. Vol. 8 page： 1373-1377 2006

Differences in oxidative stress dependence between gastric adenocarcinoma subtypes. Reviewed

Bancel B, Esteve J, Souquet JC, Toyokuni S, Ohshima H, Pignatelli B.

World J. Gastroenterol. Vol. 12 page： 1005-1012 2006

What has been learned from the studies of oxidative stress-induced carcinogenesis: proposal of the concept of oxygenomics. Reviewed

Toyokuni S, Akatsuka S.

J. Clin. Biochem. Nutr. Vol. 39 page： 3-10 2006

Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Reviewed

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 Vol. 103 page： 8018-8023 2006

alfa-Tocopherol induces calnexin in renal tubular cells: Another protective mechanism against free radical-induced cellular damage. Reviewed

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. Vol. 453 page： 168-178 2006

*Contrasting Genome-wide Distribution of 8-Hydroxyguanine and Acrolein-modified Adenine during Oxidative Stress-induced Renal Carcinogenesis. Reviewed

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. Vol. 169 page： 1328-1342 2006

Deletion and single nucleotide substitution at G:C are major mutations in the kidney of gpt delta transgenic mice after the Fenton reaction. Reviewed

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. Vol. 97 page： 1159-1167 2006

Onuki, J; Liu, Y; Lee, W; Jiang, L; Dutta, KK; Akatsuka, S; Toyokuni, S

FREE RADICAL RESEARCH Vol. 40 page： S128 - S128 2006

Iron-Induced Carcinogenesis Reviewed International journal

Toyokuni, S

OXIDATIVE STRESS, DISEASE AND CANCER page： 789 - 805 2006

Novel aspects of oxidative stress-associated carcinogenesis

Toyokuni Shinya

ANTIOXIDANTS & REDOX SIGNALING Vol. 8 ( 7-8 ) page： 1373 - 1377 2006

Oxidative stress and cancer International journal

Toyokuni Shinya

Annual Meeting of the Japanese Society of Toxicology Vol. 33 ( 0 ) page： 15 - 15 2006

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 Vol. 40 page： S128 - S128 2006

Iron-Induced Carcinogenesis

Toyokuni Shinya

OXIDATIVE STRESS, DISEASE AND CANCER page： 789 - 805 2006

Deletion and single nucleotide substitution at G:C are major mutations in the kidney of gpt delta transgenic mice after the Fenton reaction. Reviewed

Jiang L, Zhong Y, Akatsuka S, Liu Y-T, Dutta KK, Lee W-H, Onuki J, Masumura K, Nohmi T, Toyokuni S

Cancer Sci. Vol. 97 page： 1159-1167 2006

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 Vol. 40 page： S129 - S129 2006

Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis Reviewed

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 Vol. 40 page： S129 - S129 2006

Imidapril, an Angiotensin-Converting Enzyme Inhibitor, Reduces Diabetes-Induced Renal Oxidative Damage in Mice

Naito Yuji, Uchiyama Kazuhiko, Akagiri Satomi, Aoi Wataru, Hasegawa Goji, Nakamura Naoto, Kokura Satoshi, Yoshida Norimasa, Ichikawa Hiroshi, Toyokuni Shinya, Yoshikawa Toshikazu

Journal of Clinical Biochemistry and Nutrition Vol. 37 ( 1 ) page： 29 - 37 2005.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SOCIETY FOR FREE RADICAL RESEARCH JAPAN

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. Reviewed International journal

Munetaka Ozeki, Aya Miyagawa-Hayashino, Shinya Akatsuka, Tomoyuki Shirase, Wen-Hua Lee, Koji Uchida, Shinya Toyokuni

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 827 ( 1 ) page： 119 - 26 2005.11

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.1016/j.jchromb.2005.02.025

OZEKI Munetaka, MIYAGAWA HAYASHINO Aya, AKATSUKA Shinya, SHIRASE Tomoyuki, LEE Wen-hua, UCHIDA Koji, TOYOKUNI Shinya

J Chromatogr B Anal Technol Biomed Life Sci Vol. 827 ( 1 ) page： 119 - 126 2005.11

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 Vol. 14 ( 5 ) page： 593 - 604 2005.10

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. International journal

Nobuyuki Yajima, Shu-Ichi Yamada, Takayuki Morisaki, Shinya Toyokuni, Shin Yonehara, Kazuhiro Sakamaki

Transgenic research Vol. 14 ( 5 ) page： 593 - 604 2005.10

Language：English Publishing type：Research paper (scientific journal)

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. International journal Open Access

Kimio Kawasaki, Akiyoshi Nishio, Hajime Nakamura, Kazushige Uchida, Toshiro Fukui, Masaya Ohana, Hazuki Yoshizawa, Shinya Ohashi, Hiroyuki Tamaki, Minoru Matsuura, Masanori Asada, Toshiki Nishi, Hiroshi Nakase, Shinya Toyokuni, Wenrui Liu, Junji Yodoi, Kazuichi Okazaki, Tsutomu Chiba

Laboratory investigation; a journal of technical methods and pathology Vol. 85 ( 9 ) page： 1104 - 17 2005.9

Language：English Publishing type：Research paper (scientific journal)

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 Reviewed International journal

M Kanatsu-Shinohara, N Ogonuki, T Iwano, J Lee, Y Kazuki, K Inoue, H Miki, M Takehashi, S Toyokuni, Y Shinkai, M Oshimura, F Ishino, A Ogura, T Shinohara

DEVELOPMENT Vol. 132 ( 18 ) page： 4155 - 63 2005.9

Language：English Publishing type：Research paper (scientific journal) Publisher：COMPANY OF BIOLOGISTS LTD

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 similar to 10(85)-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 Reviewed International journal Open Access

M Kanatsu-Shinohara, H Miki, K Inoue, N Ogonuki, S Toyokuni, A Ogura, T Shinohara

HUMAN REPRODUCTION Vol. 20 ( 9 ) page： 2376 - 82 2005.9

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

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 (SI) mouse testis that had defective Sertoli cells, spermatogenesis occurred from SI 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.

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 Vol. 31 ( 2 ) page： 126 - 131 2005.8

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 Vol. 31 ( 2 ) page： 126 - 31 2005.8

Language：English Publishing type：Research paper (scientific journal) Publisher：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. International journal

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 Vol. 31 ( 2 ) page： 126 - 31 2005.8

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 167 ( 2 ) page： 327 - 36 2005.8

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 167 ( 2 ) page： 327 - 336 2005.8

Establishment of a diabetic mouse model with progressive diabetic nephropathy Reviewed

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 Vol. 167 ( 2 ) page： 327 - 336 2005.8

Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal Open Access

Hamahata K, Adachi S, Matsubara H, Okada M, Imai T, Watanabe K, Toyokuni S, Ueno M, Akiba S, Katanosaka Y, Wakabayashi S, Kubota M, Nakahata T

European Journal of Pharmacology Vol. 516 ( 3 ) page： 187 - 96 2005.6

Ichikawa, M; Mio, T; Toyokuni, S; Tanaka, F; Wada, H; Mishima, M; Yanagihara, K

JOURNAL OF CLINICAL ONCOLOGY Vol. 23 ( 16 ) page： 682S - 682S 2005.6

Annexin II overexpression correlates with a poor prognosis in surgically resected non-small-cell lung cancer. Reviewed International journal

Ichikawa, M, Mio, T, Toyokuni, S, Tanaka, F, Wada, H, Mishima, M, Yanagihara, K

JOURNAL OF CLINICAL ONCOLOGY Vol. 23 ( 16 ) page： 682S - 682S 2005.6

Two distinct mechanisms for loss of thioredoxin-binding protein-2 in oxidative stress-induced renal carcinogenesis. Reviewed International journal Open Access

Khokon K Dutta, Yumiko Nishinaka, Hiroshi Masutani, Shinya Akatsuka, Than T Aung, Tomoyuki Shirase, Wen-Hua Lee, Yoshihiro Yamada, Hiroshi Hiai, Junji Yodoi, Shinya Toyokuni

Laboratory investigation; a journal of technical methods and pathology Vol. 85 ( 6 ) page： 798 - 807 2005.6

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

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.

DOI： 10.1038/labinvest.3700280

Transcriptional control of fetal liver hematopoiesis: dominant negative effect of the overexpression of the LIM domain mutants of LMO2. Reviewed International journal Open Access

Tomo Terano, Yi Zhong, Shinya Toyokuni, Hiroshi Hiai, Yoshihiro Yamada

Experimental hematology Vol. 33 ( 6 ) page： 641 - 51 2005.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier BV

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.

DOI： 10.1016/j.exphem.2005.01.018

Analysis of rat insulin II promoter-ghrelin transgenic mice and rat glucagon promoter-ghrelin transgenic mice. Reviewed International journal Open Access

Hiroshi Iwakura, Kiminori Hosoda, Choel Son, Junji Fujikura, Tsutomu Tomita, Michio Noguchi, Hiroyuki Ariyasu, Kazuhiko Takaya, Hiroaki Masuzaki, Yoshihiro Ogawa, Tatsuya Hayashi, Gen Inoue, Takashi Akamizu, Hiroshi Hosoda, Masayasu Kojima, Hiroshi Itoh, Shinya Toyokuni, Kenji Kangawa, Kazuwa Nakao

The Journal of biological chemistry Vol. 280 ( 15 ) page： 15247 - 56 2005.4

Language：English Publishing type：Research paper (scientific journal)

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, beta 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.

DOI： 10.1074/jbc.M411358200

Other Link： 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 Vol. 128 ( 4 ) page： A709 - A709 2005.4

Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions. Reviewed International journal

Mito Kanatsu-Shinohara, Hiromi Miki, Kimiko Inoue, Narumi Ogonuki, Shinya Toyokuni, Atsuo Ogura, Takashi Shinohara

Biology of reproduction Vol. 72 ( 4 ) page： 985 - 91 2005.4

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.1095/biolreprod.104.036400

CYP3A induction more impairs endotoxemic liver injury via reactive oxygen species in rats International journal

Minamiyama, Y, Takemura, S, Kodai, S, Toyokuni, S, Yoshikawa, T, Okada, S

GASTROENTEROLOGY Vol. 128 ( 4 ) page： A709 - A709 2005.4

A resected case of multiple early gallbladder carcinomas

URAI Shunji, HAYAKUMO Takanobu, HABU Yasuki, KAWAMURA Takuji, SHIO Seiji, IKEURA Tsukasa, YOSHINO Takuya, HIRAI Kiyoshi, TOYOKUNI Shinya

Nippon Shokakibyo Gakkai Zasshi Vol. 102 ( 3 ) page： 340 - 345 2005.3

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 Vol. 69 ( 0 ) page： 310 2005.3

Circulation journal : official journal of the Japanese Circulation Society Vol. 69 ( 0 ) 2005.3

Genetic selection of mouse male germline stem cells in vitro: offspring from single stem cells. Reviewed International journal Open Access

Mito Kanatsu-Shinohara, Shinya Toyokuni, Takashi Shinohara

Biology of reproduction Vol. 72 ( 1 ) page： 236 - 40 2005.1

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed International journal Open Access

Shinichiro Chuma, Mito Kanatsu-Shinohara, Kimiko Inoue, Narumi Ogonuki, Hiromi Miki, Shinya Toyokuni, Mihoko Hosokawa, Norio Nakatsuji, Atsuo Ogura, Takashi Shinohara

Development (Cambridge, England) Vol. 132 ( 1 ) page： 117 - 22 2005.1

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed

Naito Y, Akagiri S, Uchiyama K, Kokura S, Yoshida N, Hasegawa G, Nakamura N, Ichikawa H, Toyokuni S, Ijichi T, Yoshikawa T.

Biofactors Vol. 23 page： 85-98 2005

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture. Reviewed

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 Vol. 132 page： 4155-4163 2005

Establishment of a diabetic mouse model with progressive diabetic nephropathy. Reviewed

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. Vol. 167 page： 327-336 2005

Free radical-induced carcinogenesis: Target genes and fragile genome sites

Toyokuni, S; Akatsuka, S; Aung, TT; Dutta, KK

FREE RADICAL RESEARCH Vol. 39 page： S30 - S30 2005

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 Vol. 37 ( 1 ) page： 29 - 37 2005

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 Vol. 23 ( 2 ) page： 85 - 95 2005

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) Vol. 23 ( 2 ) page： 85 - 95 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

Y Naito, S Akagiri, K Uchiyama, S Kokura, N Yoshida, G Hasegawa, N Nakamura, H Ichikawa, S Toyokuni, T Ijichi, T Yoshikawa

BIOFACTORS Vol. 23 ( 2 ) page： 85 - 95 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

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 Vol. 37 ( 1 ) page： 29 - 37 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：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.

Control of the peri-operative oxidative stress enables fast recovery from myocardial ischemic damage in cardic surgery. Reviewed

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

Generation of pluripotent stem cells from neonatal mouse testis. Reviewed International journal

Mito Kanatsu-Shinohara, Kimiko Inoue, Jiyoung Lee, Momoko Yoshimoto, Narumi Ogonuki, Hiromi Miki, Shiro Baba, Takeo Kato, Yasuhiro Kazuki, Shinya Toyokuni, Megumi Toyoshima, Ohtsura Niwa, Mitsuo Oshimura, Toshio Heike, Tatsutoshi Nakahata, Fumitoshi Ishino, Atsuo Ogura, Takashi Shinohara

Cell Vol. 119 ( 7 ) page： 1001 - 12 2004.12

Language：English Publishing type：Research paper (scientific journal) Publisher：7

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

Novel mechanism of matrix metalloproteinase inhibitor in the treatment of heart failure:: Direct inhibition of myocardial oxidant stress and NF-κB activation in rats

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 Vol. 119 ( 7 ) page： 1001 - 1012 2004.12

オキシカインによる糖尿病性腎症の進展抑制効果 Reviewed

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 Vol. 7 ( 4 ) page： 375 - 375 2004.11

Takenaka, H; Kihara, Y; Iwanaga, Y; Onozawa, Y; Toyokuni, S; Kita, T

CIRCULATION Vol. 110 ( 17 ) page： 514 - 515 2004.10

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 International journal

Takenaka H, Kihara Y, Iwanaga Y, Onozawa Y, Toyokuni S, Kita T

CIRCULATION Vol. 110 ( 17 ) page： 514 - 515 2004.10

Novel mechanism of matrix metalloproteinase inhibitor in the treatment of heart failure:: Direct inhibition of myocardial oxidant stress and NF-κB activation in rats International journal

Takenaka, H, Kihara, Y, Iwanaga, Y, Onozawa, Y, Toyokuni, S, Kita, T

CIRCULATION Vol. 110 ( 17 ) page： 514 - 515 2004.10

Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo. International journal Open Access

Mito Kanatsu-Shinohara, Shinya Toyokuni, Takashi Shinohara

Biology of reproduction Vol. 71 ( 4 ) page： 1202 - 7 2004.10

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed International journal

Yukiko Minamiyama, Shigekazu Takemura, Shinya Toyokuni, Susumu Imaoka, Yoshihiko Funae, Kazuhiro Hirohashi, Toshikazu Yoshikawa, Shigeru Okada

Free radical biology & medicine Vol. 37 ( 5 ) page： 703 - 712 2004.9

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.1016/j.freeradbiomed.2004.05.022

Gastric inhibitory polypeptide is the major insulinotropic factor in K-ATP null mice Reviewed International journal

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 Vol. 151 ( 3 ) page： 407 - 412 2004.9

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 Vol. 151 ( 3 ) page： 407 - 12 2004.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal

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 Vol. 151 ( 3 ) page： 407 - 12 2004.9

Language：English Publishing type：Research paper (scientific journal)

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. International journal Open Access

Kazuhiro Yamazaki, Senri Miwa, Kunihiro Ueda, Seigo Tanaka, Shinya Toyokuni, Oriyanhan Unimonh, Kazunobu Nishimura, Masashi Komeda

European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery Vol. 26 ( 2 ) page： 270 - 5 2004.8

Language：English Publishing type：Research paper (scientific journal)

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 microM). 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.

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 Reviewed International journal Open Access

Y. Satou, K. Nosaka, Y. Koya, J.-I. Yasunaga, S. Toyokuni, M. Matsuoka

Leukemia Vol. 18 ( 8 ) page： 1357 - 63 2004.8

Regulation of mouse spermatogonial stem cell self-renewing division by the pituitary gland. Reviewed International journal Open Access

Mito Kanatsu-Shinohara, Takeshi Morimoto, Shinya Toyokuni, Takashi Shinohara

Biology of reproduction Vol. 70 ( 6 ) page： 1731 - 7 2004.6

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 53 page： A394 - A394 2004.6

Role of reactive oxygen species in skin carcinogenesis

Nishigori C, Hattori Y, Toyokuni S

ANTIOXIDANTS & REDOX SIGNALING Vol. 6 ( 3 ) page： 561 - 570 2004.6

Redox control of carcinogenesis and tumor biology

Toyokuni S

ANTIOXIDANTS & REDOX SIGNALING Vol. 6 ( 3 ) page： 481 - 482 2004.6

Role of reactive oxygen species in skin carcinogenesis.

Nishigori C, Hattori Y, Toyokuni S

Antioxidants & redox signaling Vol. 6 ( 3 ) page： 561 - 70 2004.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Role of reactive oxygen species in skin carcinogenesis. International journal

Chikako Nishigori, Yukari Hattori, Shinya Toyokuni

Antioxidants & redox signaling Vol. 6 ( 3 ) page： 561 - 70 2004.6

Language：English Publishing type：Research paper (scientific journal)

Redox control of carcinogenesis and tumor biology.

Toyokuni S

Antioxidants & redox signaling Vol. 6 ( 3 ) page： 481 - 2 2004.6

PAX6 regulates islet beta-cell function International journal

Hamasaki A, Yamada Y, Nagashima K, Fujimoto S, Ban N, Toyokuni S, Kurose T, Seino Y

DIABETES Vol. 53 page： A394 - A394 2004.6

Analysis of ghrelin transgenic mice driven by rat insulin II promoter and transgenic mice driven by rat glucagon promoter

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 Vol. 53 page： A357 - A357 2004.6

PAX6 regulates islet β-cell function International journal

Hamasaki, A, Yamada, Y, Nagashima, K, Fujimoto, S, Ban, N, Toyokuni, S, Kurose, T, Seino, Y

DIABETES Vol. 53 page： A394 - A394 2004.6

Redox regulation of annexin 2 and its implications for oxidative stress-induced renal carcinogenesis and metastasis. International journal

Tomoyuki Tanaka, Shinya Akatsuka, Munetaka Ozeki, Tomoyuki Shirase, Hiroshi Hiai, Shinya Toyokuni

Oncogene Vol. 23 ( 22 ) page： 3980 - 9 2004.5

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 23 ( 22 ) page： 3980 - 3989 2004.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Oncogene

DOI： 10.1038/sj.onc.1207555

Neuroprotection by hyperbaric oxygenation after experimental focal cerebral ischemia monitored by MRI. International journal

Wolf-Ruediger Schäbitz, Holger Schade, Sabine Heiland, Rainer Kollmar, Jürgen Bardutzky, Nils Henninger, Harald Müller, Ulrich Carl, Shinya Toyokuni, Clemens Sommer, Stefan Schwab

Stroke Vol. 35 ( 5 ) page： 1175 - 9 2004.5

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 35 ( 5 ) page： 1175 - 1179 2004.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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

Vol. 54 ( 4 ) page： 251 - 255 2004.4

Detection of glyceraldehyde 3-phosphate dehydrogenase messenger RNA using a peptide nucleic acid probe in paraffin-embedded archival specimens. International journal

Makoto Hiroyasu, Shinya Akatsuka, Tomoyuki Shirase, Yoshinobu Toda, Hiroshi Hiai, Shinya Toyokuni

Pathology international Vol. 54 ( 4 ) page： 251 - 5 2004.4

Language：English Publishing type：Research paper (scientific journal)

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. International journal Open Access

Akari Inada, Yoshiyuki Hamamoto, Yoshiyuki Tsuura, Jun-ichi Miyazaki, Shinya Toyokuni, Yu Ihara, Koichiro Nagai, Yuichiro Yamada, Susan Bonner-Weir, Yutaka Seino

Molecular and cellular biology Vol. 24 ( 7 ) page： 2831 - 41 2004.4

Language：English Publishing type：Research paper (scientific journal)

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) Igamma inhibits function of CRE-binding proteins and thus disrupts CRE-mediated transcription in pancreatic beta cells, we generated transgenic mice with beta-cell-directed expression of ICER Igamma, a powerful repressor that is greatly increased in diabetes. Three transgenic lines clearly show that increased ICER Igamma expression in beta cells results in early severe diabetes. From birth islets were severely disorganized with a significantly increased proportion of alpha cells throughout the islet. Diabetes results from the combined effects of impaired insulin expression and a decreased number of beta cells. The decrease in beta 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 beta cells in the postnatal period. These results suggest that CRE and CRE-binding factors have an important role in pancreatic beta-cell physiology not only directly by regulation of gene trans-activation but also indirectly by regulation of beta-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 Vol. 54 ( 4 ) page： 251 - 5 2004.4

Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed

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 Vol. 54 ( 4 ) page： 251 - 255 2004.4

Okada M, Adachi S, Imai T, Watanabe K, Toyokuni S, Ueno M, Zervos AS, Kroemer G, Nakahata T

Blood Vol. 103 ( 6 ) page： 2299 - 2307 2004.3

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 Vol. 103 ( 6 ) page： 2299 - 2307 2004.3

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 Vol. 68 ( 0 ) page： 634 2004.3

Target Genes in Free Radical-Induced Carcinogenesis

TOYOKUNI Shinya

Vol. 59 ( 2 ) page： 105 2004.3

Circulation journal : official journal of the Japanese Circulation Society Vol. 68 ( 0 ) 2004.3

Role of phenobarbital-inducible cytochrome P450s as a source of active oxygen species in DNA-oxidation. Reviewed International journal

Susumu Imaoka, Mayuko Osada, Yukiko Minamiyama, Tokihito Yukimura, Shinya Toyokuni, Shigekazu Takemura, Toyoko Hiroi, Yoshihiko Funae

Cancer letters Vol. 203 ( 2 ) page： 117 - 125 2004.1

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.1016/j.canlet.2003.09.009

Adrenomedullin infusion attenuates myocardial ischemia/reperfusion injury through the phosphatidylinositol 3-kinase/Akt-dependent pathway. International journal

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 Vol. 109 ( 2 ) page： 242 - 8 2004.1

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 109 ( 2 ) page： 242 - 248 2004.1

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal Open Access

Mito Kanatsu-Shinohara, Shinya Toyokuni, Takashi Shinohara

Biology of reproduction Vol. 70 ( 1 ) page： 70 - 5 2004.1

Language：English Publishing type：Research paper (scientific journal)

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 page： 181 - 185 2004

Toyokuni, S; Akatsuka, S; Shirase, T; Than, TA

FREE RADICAL BIOLOGY AND MEDICINE Vol. 36 page： S29 - S29 2004

Target genes in free radical-induced carcinogenesis Reviewed

S Toyokuni

SFRR: PROCEEDINGS OF THE XII BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL page： 181 - 185 2004

Language：English Publishing type：Research paper (international conference proceedings) Publisher：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. Reviewed International journal Open Access

Yoshichika Kawai, Atsunori Furuhata, Shinya Toyokuni, Yasuaki Aratani, Koji Uchida

The Journal of biological chemistry Vol. 278 ( 50 ) page： 50346 - 54 2003.12

Language：English Publishing type：Research paper (scientific journal)

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 Reviewed International journal Open Access

M Kanatsu-Shinohara, N Ogonuki, K Inoue, A Ogura, S Toyokuni, T Shinohara

HUMAN REPRODUCTION Vol. 18 ( 12 ) page： 2660 - 7 2003.12

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

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. Reviewed International journal

Shinya Toyokuni, Tomoyuki Tanaka, Waka Kawaguchi, Neil Ryan Lai Fang, Munetaka Ozeki, Shinya Akatsuka, Hiroshi Hiai, Okezie I Aruoma, Theeshan Bahorun

Free radical research Vol. 37 ( 11 ) page： 1215 - 24 2003.11

Language：English Publishing type：Research paper (scientific journal) Publisher：INFORMA HEALTHCARE

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 mug/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 Vol. 108 ( 17 ) page： 175 - 175 2003.10

Edaravone, a novel free radical scavenger, protects myocardium against oxidative stress after cardioplegic arrest International journal

Yamazaki, K, Miwa, S, Toyokuni, S, Horii, T, Unimonh, O, Sasahashi, N, Takaba, K, Komeda, M

CIRCULATION Vol. 108 ( 17 ) page： 175 - 175 2003.10

Novel aspects in free radical chemistry and biology Reviewed International journal

Toyokuni, S

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 417 ( 1 ) page： 2 - 2 2003.9

Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Reviewed International journal

Mito Kanatsu-Shinohara, Narumi Ogonuki, Kimiko Inoue, Hiromi Miki, Atsuo Ogura, Shinya Toyokuni, Takashi Shinohara

Biology of reproduction Vol. 69 ( 2 ) page： 612 - 6 2003.8

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 69 ( 2 ) page： 612 - 616 2003.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal Open Access

Sakata S, Sakamaki K, Watanabe K, Nakamura N, Toyokuni S, Nishimune Y, Mori C, Yonehara S

Cell death and differentiation Vol. 10 ( 6 ) page： 676 - 86 2003.6

Evidence for oxidative stress in NSAID-induced colitis in IL10-/- mice. International journal

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 Vol. 34 ( 9 ) page： 1153 - 66 2003.5

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 34 ( 9 ) page： 1153 - 1166 2003.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal Open Access

Mito Kanatsu-Shinohara, Shinya Toyokuni, Takeshi Morimoto, Shigeyuki Matsui, Tasuku Honjo, Takashi Shinohara

Biology of reproduction Vol. 68 ( 5 ) page： 1801 - 7 2003.5

Language：English Publishing type：Research paper (scientific journal)

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

Pathol Int Vol. 53 ( 4 ) page： 259 - 261 2003.4

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis

TOYOKUNI Shinya, KAWAGUCHI Waka, AKATSUKA Shinya, HIROYASU Makoto, HIAI Hiroshi

Pathology international Vol. 53 ( 4 ) page： 259 - 261 2003.4

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis

Toyokuni S, Kawaguchi W, Akatsuka S, Hiroyasu M, Hiai H

PATHOLOGY INTERNATIONAL Vol. 53 ( 4 ) page： 259 - 261 2003.4

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis.

Toyokuni S, Kawaguchi W, Akatsuka S, Hiroyasu M, Hiai H

Pathology international Vol. 53 ( 4 ) page： 259 - 61 2003.4

Intermittent microwave irradiation facilitates antigen-antibody reaction in Western blot analysis. International journal

Shinya Toyokuni, Waka Kawaguchi, Shinya Akatsuka, Makoto Hiroyasu, Hiroshi Hiai

Pathology international Vol. 53 ( 4 ) page： 259 - 61 2003.4

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 Vol. 75 ( 5 ) page： 591 - 598 2003.3

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. International journal

Mitsugu Omasa, Tatsuo Fukuse, Shinya Toyokuni, Yoichi Mizutani, Hiroshi Yoshida, Kazuyuki Ikeyama, Seiki Hasegawa, Hiromi Wada

Transplantation Vol. 75 ( 5 ) page： 591 - 8 2003.3

Language：English Publishing type：Research paper (scientific journal)

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. Reviewed International journal

Takeshi Ishikawa, Norimasa Yoshida, Harukuni Tokuda, Eiichiro Ichiishi, Masashi Kuchide, Satoshi Kokura, Yuji Naito, Shinya Toyokuni, Hoyoku Nishino, Toshikazu Yoshikawa

Cancer letters Vol. 191 ( 1 ) page： 41 - 47 2003.2

Language：English Publishing type：Research paper (scientific journal)

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.

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. Reviewed International journal Open Access

Hashimoto M, Sibata T, Wasada H, Toyokuni S, Uchida K

The Journal of biological chemistry Vol. 278 ( 7 ) page： 5044 - 51 2003.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 18 ( 2 ) page： 301 - 12 2003.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 18 ( 2 ) page： 301 - 312 2003.2

Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. International journal

Kazuki Kuroda, Hua Han, Shoichi Tani, Kenji Tanigaki, Tin Tun, Takahisa Furukawa, Yoshihito Taniguchi, Hisanori Kurooka, Yoshio Hamada, Shinya Toyokuni, Tasuku Honjo

Immunity Vol. 18 ( 2 ) page： 301 - 12 2003.2

Language：English Publishing type：Research paper (scientific journal)

Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice. Reviewed International journal

Wataru Aoi, Yuji Naito, Kunihiro Sakuma, Masashi Kuchide, Harukuni Tokuda, Takashi Maoka, Shinya Toyokuni, Shigenori Oka, Masahiro Yasuhara, Toshikazu Yoshikawa

Antioxidants & redox signaling Vol. 5 ( 1 ) page： 139 - 44 2003.2

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 5 ( 1 ) page： 139 - 144 2003.2

Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis. Reviewed International journal Open Access

Mito Kanatsu-Shinohara, Narumi Ogonuki, Kimiko Inoue, Atsuo Ogura, Shinya Toyokuni, Tasuku Honjo, Takashi Shinohara

Biology of reproduction Vol. 68 ( 1 ) page： 167 - 73 2003.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC STUDY 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 Reviewed International journal

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 Vol. 17 ( 12 ) page： 3039 - 3045 2002.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 17 ( 12 ) page： 3039 - 3045 2002.12

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 Vol. 106 ( 19 ) page： 345 - 345 2002.11

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 Vol. 106 ( 19 ) page： 312 - 312 2002.11

Oxydative stress persists in nearby LV area after myocardial infarction (MI) and shows two peaks International journal

Miwa, S, Toyokuni, S, Tanaka, S, Nishina, T, Nomoto, T, Yamazaki, K, Ueda, K, Nishimura, K

CIRCULATION Vol. 106 ( 19 ) page： 312 - 312 2002.11

A novel method of "preparative" myocardial protection using green tea polyphenol in oral uptake Reviewed International journal

Miwa, S, Saito, M, Yamazaki, K, Toyokuni, S, Hyon, SH, Fujita, M, Komeda, M

CIRCULATION Vol. 106 ( 19 ) page： 345 - 345 2002.11

Ex vivo whole-embryo culture of caspase-8-dificient embryos normalize their aberrant phenotypes in the developing neural tube and heart Reviewed International journal

Sakamaki K, Inoue T, Asano M, Sudo K, Kazama H, Sakagami J, Sakata S, Ozaki M, Nakamura S, Toyokuni S, Osumi N, Yonehara S

Cell Death and Differentiation Vol. 9 ( 11 ) page： 1196 - 206 2002.11

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 Vol. 11 ( 8 ) page： 767 - 770 2002.8

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 Vol. 40 ( 8 ) page： 1191 - 1196 2002.8

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 Vol. 36 ( 8 ) 2002.8

Comparison between high-performance liquid chromatography and enzyme-linked immunosorbent assay for the determination of 8-hydroxy-2'-deoxyguanosine in human urine. International journal

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 Vol. 11 ( 8 ) page： 767 - 70 2002.8

Language：English Publishing type：Research paper (scientific journal)

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 Reviewed International journal

Kallay E, Bareis P, Bajna E, Kriwanek S, Bonner E, Toyokuni S, Cross HS

FOOD AND CHEMICAL TOXICOLOGY Vol. 40 ( 8 ) page： 1191 - 1196 2002.8

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)) Reviewed International journal

Toyokuni S., Itani T., Morimitsu Y., Okada K., Ozeki M., Kondo S., Uchida K., Osawa T., Hiai H., Tashiro T.

Free Radical Research Vol. 36 ( 8 ) 2002.8

Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Reviewed International journal

Kazumasa Miyawaki, Yuichiro Yamada, Nobuhiro Ban, Yu Ihara, Katsushi Tsukiyama, Heying Zhou, Shimpei Fujimoto, Akira Oku, Kinsuke Tsuda, Shinya Toyokuni, Hiroshi Hiai, Wataru Mizunoya, Tohru Fushiki, Jens Juul Holst, Mitsuhiro Makino, Akira Tashita, Yukari Kobara, Yoshiharu Tsubamoto, Takayoshi Jinnouchi, Takahito Jomori, Yutaka Seino

Nature medicine Vol. 8 ( 7 ) page： 738 - 42 2002.7

Language：English Publishing type：Research paper (scientific journal)

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(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) 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 Vol. 40 ( 8 ) page： 1191 - 1196 2002.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal 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 Vol. 105 ( 24 ) page： 2867 - 2871 2002.6

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 179 ( 1 ) page： 87 - 93 2002.5

Kondo S, Toyokuni S, Tsuruyama T, Ozeki M, Tachibana T, Echizenya M, Hiai H, Onodera H, Imamura M

Cancer letters Vol. 179 ( 1 ) page： 87 - 93 2002.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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. International journal

Shohei Kondo, Shinya Toyokuni, Tatsuaki Tsuruyama, Munetaka Ozeki, Tsuyoshi Tachibana, Michiko Echizenya, Hiroshi Hiai, Hisashi Onodera, Masayuki Imamura

Cancer letters Vol. 179 ( 1 ) page： 87 - 93 2002.5

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 289 ( 1-2 ) page： 185 - 190 2002.5

Ozeki M, Hoshino S, Hiai H, Toyokuni S

Gene Vol. 289 ( 1-2 ) page： 185 - 90 2002.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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. International journal

Munetaka Ozeki, Shinsuke Hoshino, Hiroshi Hiai, Shinya Toyokuni

Gene Vol. 289 ( 1-2 ) page： 185 - 90 2002.5

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 51 ( 5 ) page： 1409 - 1418 2002.5

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 Vol. 162 ( 1 ) page： 111 - 117 2002.5

Protective effect of colored rice over white rice on Fenton reaction-based renal lipid peroxidation in rats. International journal

Shinya Toyokuni, Tomio Itani, Yasujiro Morimitsu, Kunihiko Okada, Munetaka Ozeki, Shohei Kondo, Koji Uchida, Toshihiko Osawa, Hiroshi Hiai, Toru Tashiro

Free radical research Vol. 36 ( 5 ) page： 583 - 92 2002.5

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 36 ( 5 ) page： 583 - 592 2002.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 162 ( 1 ) page： 111 - 7 2002.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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. International journal

Nobuhiro Ban, Yuichiro Yamada, Yoshimichi Someya, Kazumasa Miyawaki, Yu Ihara, Masaya Hosokawa, Shinya Toyokuni, Kinsuke Tsuda, Yutaka Seino

Diabetes Vol. 51 ( 5 ) page： 1409 - 18 2002.5

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 51 ( 5 ) page： 1409 - 18 2002.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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. International journal

Norimasa Yoshida, Hironobu Murase, Tsutomu Kunieda, Shinya Toyokuni, Tomoyuki Tanaka, Junji Terao, Yuji Naito, Toru Tanigawa, Toshikazu Yoshikawa

Atherosclerosis Vol. 162 ( 1 ) page： 111 - 7 2002.5

Language：English Publishing type：Research paper (scientific journal)

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.

Analysis of suspicious endometrial cytology : based on architectural atypia

Increase in oxidative stress in kidneys of diabetic Akita mice Reviewed International journal

Ueno Y, Horio F, Uchida K, Naito M, Nomura H, Kato Y, Tsuda T, Toyokuni S, Osawa T

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Vol. 66 ( 4 ) page： 869 - 872 2002.4

Increase in Oxidative Stress in Kidneys of Diabetic Akita Mice. International journal

UENO Yuki, HORIO Fumihiko, UCHIDA Koji, NAITO Michitaka, NOMURA Hideki, KATO Yoji, TSUDA Takanori, TOYOKUNI Shinya, OSAWA Toshihiko

Bioscience, Biotechnology, and Biochemistry Vol. 66 ( 4 ) page： 869 - 72 2002.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry

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.

DOI： 10.1271/bbb.66.869

Increase in oxidative stress in kidneys of diabetic Akita mice Reviewed International journal

Ueno Y, Horio F, Uchida K, Naito M, Nomura H, Kato Y, Tsuda T, Toyokuni S, Osawa T

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Vol. 66 ( 4 ) page： 869 - 872 2002.4

SHIMIZU Keiko, OGURA Seiko, KOBAYASHI Hachiro, TOYOKUNI Shinya, NORIMATU Yoshiaki, MORIYA Takuya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 41 ( 2 ) page： 89 - 97 2002.3

Analysis of suspicious endometrial cytology : based on architectural atypia

SHIMIZU Keiko, OGURA Seiko, KOBAYASHI Hachiro, TOYOKUNI Shinya, NORIMATU Yoshiaki, MORIYA Takuya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 41 ( 2 ) page： 89 - 97 2002.3

Adenovirus-mediated gene delivery and in vitro microinsemination produce offspring from infertile male mice. Reviewed International journal

Mito Kanatsu-Shinohara, Atsuo Ogura, Masaya Ikegawa, Kimiko Inoue, Narumi Ogonuki, Kei Tashiro, Shinya Toyokuni, Tasuku Honjo, Takashi Shinohara

Proceedings of the National Academy of Sciences of the United States of America Vol. 99 ( 3 ) page： 1383 - 8 2002.2

Language：English Publishing type：Research paper (scientific journal)

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/Steel(dickie) (Sl/Sl(d)) 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 Vol. 99 ( 3 ) page： 1383 - 1388 2002.2

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 Vol. 160 ( 2 ) page： 419 - 424 2002.2

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 Vol. 38 ( 10 ) page： 765 - 772 2002.2

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 Vol. 38 ( 10 ) page： 765 - 72 2002.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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. International journal

Makoto Hiroyasu, Munetaka Ozeki, Haruyasu Kohda, Michiko Echizenya, Tomoyuki Tanaka, Hiroshi Hiai, Shinya Toyokuni

The American journal of pathology Vol. 160 ( 2 ) page： 419 - 24 2002.2

Language：English Publishing type：Research paper (scientific journal)

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 Vol. 160 ( 2 ) page： 419 - 24 2002.2

Language：English Publishing type：Research paper (scientific journal) Publisher：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. International journal

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 Vol. 38 ( 10 ) page： 765 - 72 2002.2

Language：English Publishing type：Research paper (scientific journal)

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 carcinogenesis: 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. Reviewed International journal

Kazuhiko Yamagami, Yuzo Yamamoto, Shinya Toyokuni, Koichiro Hata, Yoshio Yamaoka

Free radical research Vol. 36 ( 2 ) page： 169 - 76 2002.2

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

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 42degreesC 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 Western-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 Vol. 16 ( 3-4 ) page： 105 - 115 2002

Iron and carcinogenesis: from Fenton reaction to target genes. International journal

Shinya Toyokuni

Redox report : communications in free radical research Vol. 7 ( 4 ) page： 189 - 97 2002

Language：English Publishing type：Research paper (scientific journal)

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 (p16(INK4A) and p15(INK4B) 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

Radiation-induced kidney injury: a role for chronic oxidative stress?

Robbins MEC, Zhao WL, Davis CS, Toyokuni S, Bonsib SM

MICRON Vol. 33 ( 2 ) page： 133 - 141 2002

Toyokuni, S; Hiroyasu, M; Ozeki, M; Hiai, H

XI BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL page： 459 - 462 2002

Target genes in fenton reaction-induced carcinogenisis: A novel concept

Toyokuni, S; Hiroyasu, M; Ozeki, M; Hiai, H

FREE RADICAL BIOLOGY AND MEDICINE Vol. 33 page： S177 - S177 2002

Novel surrogate end-point biomarker to evaluate agents for use in the chemoprevention of reactive oxygen species-associated cancer. Reviewed International journal

Makoto Hiroyasu, Munetaka Ozeki, Aya Miyagawa-Hayashino, Yoshie Fujiwara, Hiroshi Hiai, Shinya Toyokuni

Redox report : communications in free radical research Vol. 7 ( 5 ) page： 335 - 8 2002

Language：English Publishing type：Research paper (scientific journal)

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 p16IK4A 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

Analysis of suspicious endometrial cytology. Based on architectural atypia.:based on architectural atypia

SHIMIZU Keiko, OGURA Seiko, KOBAYASHI Hachiro, TOYOKUNI Shinya, NORIMATSU Yoshiaki, MORIYA Takuya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 41 ( 2 ) page： 89 - 94 2002

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Clinical Cytology

Objective: To increase the accuracy of endometrial cytology, we evaluated the use of architectural atypia as a possible diagnostic criterion and the use of cell culture medium to decrease the frequency of insufficient materials. Study Design: We selected 84 cytologically suspicious cases for which histological diagnoses were available, dividing them into 2 groups-51 in group A diagnosed with cellular atypia, and 33 in group B with architectural atypia and an additional washing smear. Results: Histological findings of group A cases showed 23 (45.1%) that were benign, 21 (41.2%) with endometrial hyperplasia, and 7 (13.7%) with endometrioid adenocarcinoma, grade 1. In group B, 11 (33.3%) cases were benign, 21 (63.7%) were endometrial hyperplasia, and 1 (3.0%) was endometrioid adenocarcinoma, grade 1. The percentage of cases with insufficient material for diagnosis was 32.9% in group A and 20.0% in group B. Conclusion: Focusing on architectural atypia effectively raises the accuracy of endometrial cytological diagnosis for endometrial hyperplasia. The additional washing smear was useful in endometrial cytology.

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 International journal

Toyokuni, S, Hiroyasu, M, Ozeki, M, Hiai, H

FREE RADICAL BIOLOGY AND MEDICINE Vol. 33 page： S177 - S177 2002

Target genes in Fenton reaction-induced carcinogenesis: A novel concept International journal

Toyokuni, S, Hiroyasu, M, Ozeki, M, Hiai, H

XI BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL page： 459 - 462 2002

Robbins ME, Zhao W, Davis CS, Toyokuni S, Bonsib SM

Micron (Oxford, England : 1993) Vol. 33 ( 2 ) page： 133 - 41 2002

Language：English Publishing type：Research paper (scientific journal) Publisher：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? International journal

Mike E Robbins, Weiling Zhao, Charles S Davis, Shinya Toyokuni, Stephen M Bonsib

Micron (Oxford, England : 1993) Vol. 33 ( 2 ) page： 133 - 41 2002

Language：English Publishing type：Research paper (scientific journal)

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 Reviewed International journal

Shimoi K., Kasai H., Yokota N., Toyokuni S., Kinae N.

Cancer Epidemiology Biomarkers and Prevention Vol. 11 ( 8 ) page： 767 - 770 2002

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 17 page： 3039-3045 - 3045 2002

Minamiyama Y, Takemura S, Toyokuni S, Nishino Y, Yamasaki K, Hai S, Yamamoto S, Okada S

BioFactors (Oxford, England) Vol. 16 ( 3-4 ) page： 105 - 15 2002

Language：English Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal

Yukiko Minamiyama, Shigekazu Takemura, Shinya Toyokuni, Yoshihiro Nishino, Keiichi Yamasaki, Seikan Hai, Satoshi Yamamoto, Shigeru Okada

BioFactors (Oxford, England) Vol. 16 ( 3-4 ) page： 105 - 15 2002

Language：English Publishing type：Research paper (scientific journal)

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.

Target genes in oxystress-induced carcinogenesis and its molecular mechanisms

TOYOKUNI Shinya

Environmental mutagen research communications Vol. 23 ( 3 ) page： 177 - 182 2001.12

Language：Japanese Publishing type：Research paper (scientific journal)

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.

Target genes in oxystress-induced carcinogenesis and its molecular mechanisms

TOYOKUNI Shinya

Environmental mutagen research communications Vol. 23 ( 3 ) page： 177 - 182 2001.12

Language：Japanese Publishing type：Research paper (scientific journal)

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 Vol. 104 ( 24 ) page： 2948 - 2954 2001.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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

A case of cervical adenocarcinoma in situ with difficulty in cytological differentiation from recurrent ovarian tumor of borderline malignancy

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 Vol. 104 ( 24 ) page： 2948 - 2954 2001.12

Activation of lectin-like oxidized low-density lipoprotein receptor-1 induces apoptosis in cultured neonatal rat cardiac myocytes Reviewed

Iwai-Kanai E, Hasegawa K, Sawamura T, Fujita M, Araki M, Yanazume T, Toyokuni S, Adachi S, Kihara Y, Sasayama S

Circulation Vol. 104 ( 24 ) page： 2948 - 2954 2001.12

OGURA Seiko, SHIMIZU Keiko, KOBAYASHI Hachiro, TOYOKUNI Shinya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 40 ( 6 ) page： 636 - 640 2001.11

A case of cervical adenocarcinoma in situ with difficulty in cytological differentiation from recurrent ovarian tumor of borderline malignancy

OGURA Seiko, SHIMIZU Keiko, KOBAYASHI Hachiro, TOYOKUNI Shinya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 40 ( 6 ) page： 636 - 640 2001.11

A novel selectin blocker alleviates oxidative stress of lung reperfusion injury. International journal 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 Vol. 101 ( 1 ) page： 91 - 8 2001.11

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 22 ( 9 ) page： 1429 - 1435 2001.9

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 Vol. 22 ( 9 ) page： 1429 - 1435 2001.9

Curcumin and especially tetrahydrocurcumin ameliorate oxidative stress-induced renal injury in mice Reviewed International journal

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

JOURNAL OF NUTRITION Vol. 131 ( 8 ) page： 2090 - 2095 2001.8

Curcumin and especially tetrahydrocurcumin ameliorate oxidative stress-induced renal injury in mice Reviewed International journal

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

JOURNAL OF NUTRITION Vol. 131 ( 8 ) page： 2090 - 2095 2001.8

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 Reviewed International journal

Pignatelli B., Bancel B., Plummer M., Toyokuni S., Patricot L., Ohshima H.

American Journal of Gastroenterology Vol. 96 ( 6 ) page： 1751 - 1757 2001.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 31 ( 1 ) page： 108 - 116 2001.7

Kawanishi S., Inoue S., Oikawa S., Yamashita N., Toyokuni S., Kawanishi M., Nishino K.

Free Radical Biology and Medicine Vol. 31 ( 1 ) page： 108 - 116 2001.7

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

S Kawanishi, S Inoue, S Oikawa, N Yamashita, S Toyokuni, M Kawanishi, K Nishino

FREE RADICAL BIOLOGY AND MEDICINE Vol. 31 ( 1 ) page： 108 - 116 2001.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Ichihashi K, Osawa T, Toyokuni S, Uchida K

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276 ( 26 ) page： 23903 - 23913 2001.6

Ichihashi K., Osawa T., Toyokuni S., Uchida K.

Journal of Biological Chemistry Vol. 276 ( 26 ) page： 23903 - 23913 2001.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 65 ( 6 ) page： 561 - 564 2001.6

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 Vol. 96 ( 6 ) page： 1758 - 1766 2001.6

Difference in thioredoxin expression in viral myocarditis in inbred strains of mice International journal

Miyamoto M, Kishimoto C, Shioji K, Nakamura H, Toyokuni S, Nakayama Y, Kita M, Yodoi J, Sasayama S

JAPANESE CIRCULATION JOURNAL-ENGLISH EDITION Vol. 65 ( 6 ) page： 561 - 564 2001.6

Helicobacter pylori eradication attenuates oxidative stress in human gastric mucosa International journal

Pignatelli B, Bancel B, Plummer M, Toyokuni S, Patricot LM, Ohshima H

AMERICAN JOURNAL OF GASTROENTEROLOGY Vol. 96 ( 6 ) page： 1758 - 1766 2001.6

MIYAMOTO Miki, KISHIMOTO Chiharu, SHIOJI Keisuke, NAKAMURA Hajime, TOYOKUNI Shinya, NAKAYAMA Yukie, KITA Masakazu, YODOI Junji, SASAYAMA Shigetake

Japanese circulation journal Vol. 65 ( 6 ) page： 561 - 564 2001.5

Publishing type：Research paper (scientific journal) Publisher：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 Vol. 65 ( 6 ) page： 561 - 564 2001.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 30 ( 10 ) page： 1130 - 1136 2001.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 30 ( 10 ) page： 1130 - 1136 2001.5

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 Vol. 144 ( 2 ) page： 380 - 383 2001.3

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. Reviewed International journal

Iwakura A, Fujita M, Hasegawa K, Toyokuni S, Sawamura T, Nohara R, Sasayama S, Komeda M

Journal of molecular and cellular cardiology Vol. 33 ( 3 ) page： 419 - 30 2001.3

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 144 ( 2 ) page： 380 - 383 2001.2

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 Vol. 144 ( 2 ) page： 380 - 383 2001.2

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 Vol. 35 ( 3 ) page： 245 - 255 2001

Miyamoto Miki, Kishimoto Chiharu, Shioji Keisuke, Nakamura Hajime, Toyokuni Shinya, Nakayama Yukie, Kita Masakazu, Yodoi Junji, Sasayama Shigetake

Japanese Circulation Journal Vol. 65 ( 6 ) page： 561 - 564 2001

Language：English Publishing type：Research paper (scientific journal) Publisher：The Japanese Circulation Society

DOI： 10.1253/jcj.65.561

A case of cevical adenocarcinoma in situ with difficulty in cytological differentiation from recurrent ovarian tumor of borderline malignancy. International journal

OGURA Seiko, SHIMIZU Keiko, KOBAYASHI Hachiro, TOYOKUNI Shinya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 40 ( 6 ) page： 636 - 640 2001

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Clinical Cytology

Background: The incidence of multiple primary cancers of the female genital tract is increaseing, but these are difficult to correctly diagnose by cytological smear. We report a case of heterochronous double cancer consisting of adenocarcinoma in situ of the uterine cervix and mucinous cystic tumor with borderline malignancy of the ovary. Case: A 55-year-old woman with a ruptured right ovarian tumor had right salping000phorectomy 3 years earlier. The histological diagnosis of her ovarian tumor was mucinous cystic tumor with borderline malignancy. Atypical glandular cells were later found in a uterine cervical smear, but no abnormal findings were seen in clinical examination. Total hysterectomy combined with left salpingooophorectomy was done. The histological examination proved to be adenocarcinoma in situ of the uterine cervix. No malignancy was found in the uterine corpus or left adnexa. Conclusion: Multiple malignancies of the female genital tract should be made combining cytology, clinical data, and diagnostic imaging.

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 Vol. 34 ( 1 ) page： 57 - 68 2001

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

MT Nunez, Tapia, V, S Toyokuni, S Okada

FREE RADICAL RESEARCH Vol. 34 ( 1 ) page： 57 - + 2001

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 34 ( 1 ) page： 57 - + 2001

Helicobacter pylori eradication attenuates oxidative stress in human gastric mucosa International journal

Pignatelli B., Bancel B., Plummer M., Toyokuni S., Patricot L., Ohshima H.

American Journal of Gastroenterology Vol. 96 ( 6 ) page： 1758 - 1766 2001

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 96 ( 6 ) page： 1758 - 1766 2001

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 131 ( 8 ) page： 2090 - 2095 2001

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 22 ( 9 ) page： 1429 - 1435 2001

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 35 ( 3 ) page： 245 - 255 2001

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 nitrilotriacetatetreated rats : the effect of alpha-tocopherol feeding

TOYOKUNI S

Free Radic Res Vol. 35 ( 3 ) page： 245 - 255 2001

Enhanced hepatic lipid peroxidation in patients with primary biliary cirrhosis Reviewed International journal Open Access

Kawamura K., Kobayashi Y., Kageyama F., Kawasaki T., Nagasawa M., Toyokuni S., Uchida K., Nakamura H.

American Journal of Gastroenterology Vol. 95 ( 12 ) page： 3596 - 3601 2000.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 International journal

Kawamura, K; Kobayashi, Y; Kageyama, F; Kawasaki, T; Nagasawa, M; Toyokuni, S; Uchida, K; Nakamura, H

AMERICAN JOURNAL OF GASTROENTEROLOGY Vol. 95 ( 12 ) page： 3596 - 3601 2000.12

Enchanced hepatic lipid peroxidation in patients with primary biliary cirrhosis Reviewed International journal

KAWAMURA K

Am J Gastroenterol Vol. 95 ( 12 ) page： 3596 - 3601 2000.12

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 Vol. 49 ( 7 ) page： 1142 - 1148 2000.7

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 49 ( 7 ) page： 1142 - 1148 2000.7

Activating transcription factor-2 is a positive regulator in CaM kinase IV-induced human insulin gene expression. International journal

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 Vol. 49 ( 7 ) page： 1142 - 8 2000.7

Language：English Publishing type：Research paper (scientific journal)

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 Reviewed International journal

Yamamoto H, Yamamoto Y, Yamagami K, Kume M, Kimoto S, Toyokuni S, Uchida K, Fukumoto M, Yamaoka Y

RESEARCH IN EXPERIMENTAL MEDICINE Vol. 199 ( 6 ) page： 309 - 318 2000.6

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 Vol. 156 ( 6 ) page： 2149 - 2157 2000.6

Heat-shock preconditioning reduces oxidative protein denaturation andameliorates liver injury by carbon tetrachloride in rats Reviewed International journal

YAMAMOTO H.

Res. Exp. Med. Vol. 199 ( 6 ) page： 309 - 318 2000.6

Expression of stress-response and cell proliferation genes in renal cell carcinoma induced by oxidative stress

TANAKA T.

Am. J. Pathol. Vol. 156 ( 6 ) page： 2149 - 2157 2000.6

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 Vol. 135 ( 6 ) page： 465 - 475 2000.6

Itakura K., Oya-Ito T., Osawa T., Yamada S., Toyokuni S., Shibata N., Kobayashi M., Uchida K.

FEBS Letters Vol. 473 ( 2 ) page： 249 - 253 2000.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 473 ( 2 ) page： 249 - 253 2000.5

Antioxidant alpha-tocopherol ameliorates glycemic control of GK rats, a model of type 2 diabetes. Reviewed International journal

Ihara Y, Yamada Y, Toyokuni S, Miyawaki K, Ban N, Adachi T, Kuroe A, Iwakura T, Kubota A, Hiai H, Seino Y

FEBS letters Vol. 473 ( 1 ) page： 24 - 6 2000.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 49 page： A429 - A429 2000.5

Upregulation of thioredoxin (TRX) expression in giant cell myocarditis in rats. Reviewed International journal Open Access

Shioji K, Kishimoto C, Nakamura H, Toyokuni S, Nakayama Y, Yodoi J, Sasayama S

FEBS letters Vol. 472 ( 1 ) page： 109 - 13 2000.4

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Upregulation of thioredoxin (TRX) expression in giant cell myocarditis in rats

SHIOJI K

FEBS Lett Vol. 472 ( 1 ) page： 109 - 113 2000.4

Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C International journal

Kageyama, F; Kobayashi, Y; Kawasaki, T; Toyokuni, S; Uchida, K; Nakamura, H

AMERICAN JOURNAL OF GASTROENTEROLOGY Vol. 95 ( 4 ) page： 1041 - 1050 2000.4

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 Vol. 6 ( 4 ) page： 1394 - 1400 2000.4

Successful interferon therapy reverses enhanced hepatic iron accumulation and lipid peroxidation in chronic hepatitis C Reviewed International journal Open Access

Kageyama F., Kobayashi Y., Kawasaki T., Toyokuni S., Uchida K., Nakamura H.

American Journal of Gastroenterology Vol. 95 ( 4 ) page： 1041 - 1050 2000.4

Language：English Publishing type：Research paper (scientific journal) Publisher：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 International journal

Kageyama, F, Kobayashi, Y, Kawasaki, T, Toyokuni, S, Uchida, K, Nakamura, H

AMERICAN JOURNAL OF GASTROENTEROLOGY Vol. 95 ( 4 ) page： 1041 - 1050 2000.4

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 Reviewed International journal

Kondo S., Toyokuni S., Tanaka T., Hiai H., Onodera H., Kasai H., Imamura M.

Clinical Cancer Research Vol. 6 ( 4 ) page： 1394 - 1400 2000.4

Language：English Publishing type：Research paper (scientific journal) Publisher：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

Clin Cancer Res Vol. 6 ( 4 ) page： 1394 - 1400 2000.4

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 Vol. 6 ( 4 ) page： 1394 - 400 2000.4

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 Vol. 161 ( 3 ) page： 935 - 943 2000.3

YAMAGAMI K

Antioxid Redox Signal Vol. 2 ( 1 ) page： 127 - 136 2000.1

Serum 4-Hydroxy-2-Nonenal-Modified Albumin Is Elevated in Patients with Type 2 Diabetes Mellitus Reviewed International journal

Toyokuni Shinya, Yamada Satoshi, Kashima Minoru, Ihara Yu, Yamada Yuichiro, Tanaka Tomoyuki, Hiai Hiroshi, Seino Yutaka, Uchida Koji

ANTIOXIDANTS & REDOX SIGNALING Vol. 2 ( 4 ) page： 681 - U65 2000

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 Vol. 2 ( 1 ) page： 127 - 136 2000

Serum 4-hydroxy-2-nonenal-modified albumin is elevated in patients with type 2 diabetes mellitus Reviewed International journal

TOYOKUNI S

Antioxid Redox Signal Vol. 2 ( 4 ) page： 681 - 685 2000

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 Vol. 156 ( 6 ) page： 2149 - 2157 2000

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 135 ( 6 ) page： 465 - 475 2000

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 161 ( 3 I ) page： 935 - 943 2000

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 21 ( 6 ) page： 398 - 401 1999.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 21 ( 6 ) page： 398 - 401 1999.12

Fine-needle aspiration cytology of malignant hemangiopericytoma of the salivary gland: A case report Reviewed

K Shimizu, S Ogura, TK Kobayashi, R Kushima, S Toyokuni, Y Iwasa, M Sakurai

DIAGNOSTIC CYTOPATHOLOGY Vol. 21 ( 6 ) page： 398 - 401 1999.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 199 ( 6 ) page： 309 - 318 1999.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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. International journal

Kawamura, K; Kageyama, F; Kobayashi, Y; Kawasaki, T; Toyokuni, S; Uchida, K

HEPATOLOGY Vol. 30 ( 4 ) page： 543A - 543A 1999.10

Enhanced hepatic lipid peroxidation in patients with primary biliary cirrhosis. International journal

Kawamura, K, Kageyama, F, Kobayashi, Y, Kawasaki, T, Toyokuni, S, Uchida, K

HEPATOLOGY Vol. 30 ( 4 ) page： 543A - 543A 1999.10

KOBAYASHI M., NAKAMURA H., YODOI J., TOYOKUNI S., SHIOTA K.

Congenital anomalies Vol. 39 ( 3 ) page： 175 1999.9

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 Vol. 39 ( 3 ) 1999.9

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2'-deoxyguanosine

Toyokuni S., Sagripanti J.

Free Radical Research Vol. 31 ( 2 ) page： 123 - 128 1999.9

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 27 ( 5-6 ) page： 695 - 703 1999.9

Localization of hydroxynonenal protein adducts in normal human kidney and selected human kidney cancers Reviewed

TD Oberley, S Toyokuni, LI Szweda

FREE RADICAL BIOLOGY AND MEDICINE Vol. 27 ( 5-6 ) page： 695 - 703 1999.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 105 ( 1-2 ) page： 179 - 180 1999.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

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

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274 ( 34 ) page： 23787 - 23793 1999.8

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

Journal of Biological Chemistry Vol. 274 ( 34 ) page： 23787 - 23793 1999.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 27 ( 3-4 ) page： 401 - 410 1999.8

Kondo S., Toyokuni S., Iwasa Y., Tanaka T., Onodera H., Hiai H., Imamura M.

Free Radical Biology and Medicine Vol. 27 ( 3-4 ) page： 401 - 410 1999.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 113 ( 1 ) page： 26 - 31 1999.7

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 Vol. 113 ( 1 ) page： 26 - 31 1999.7

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 Vol. 18 ( 25 ) page： 3793 - 3797 1999.6

Tanaka T., Iwasa Y., Kondo S., Hiai H., Toyokuni S.

Oncogene Vol. 18 ( 25 ) page： 3793 - 3797 1999.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 18 ( 25 ) page： 3793 - 3797 1999.6

A 1-hour enzyme-linked immunosorbent assay for quantitation of acrolein- and hydroxynonenal-modified proteins by epitope-bound casein matrix method Reviewed International journal

Satoh K, Yamada S, Koike Y, Igarashi Y, Toyokuni S, Kumano T, Takahata T, Hayakari M, Tsuchida S, Uchida K

ANALYTICAL BIOCHEMISTRY Vol. 270 ( 2 ) page： 323 - 328 1999.6

Satoh K., Yamada S., Koike Y., Igarashi Y., Toyokuni S., Kumano T., Takahata T., Hayakari M., Tsuchida S., Uchida K.

Analytical Biochemistry Vol. 270 ( 2 ) page： 323 - 328 1999.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Satoh K, Yamada S, Koike Y, Igarashi Y, Toyokuni S, Kumano T, Takahata T, Hayakari M, Tsuchida S, Uchida K

ANALYTICAL BIOCHEMISTRY Vol. 270 ( 2 ) page： 323 - 328 1999.6

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 Vol. 47 ( 6 ) page： 833 - 835 1999.6

Toyokuni S., Iwasa Y., Kondo S., Tanaka T., Ochi H., Hiai H.

Journal of Histochemistry and Cytochemistry Vol. 47 ( 6 ) page： 833 - 835 1999.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

J Histochem Cytochem Vol. 47 ( 6 ) page： 833 - 835 1999.6

Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes Reviewed International journal

Ihara Y, Toyokuni S, Uchida K, Odaka H, Tanaka T, Ikeda H, Hiai H, Seino Y, Yamada Y

DIABETES Vol. 48 ( 4 ) page： 927 - 932 1999.4

Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes Reviewed International journal

IHARA Y

Diabetes Vol. 48 ( 4 ) page： 927 - 932 1999.4

Toyokuni S.

Pathology International Vol. 49 ( 2 ) page： 91 - 102 1999.3

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Um SC, Suzuki S, Toyokuni S, Uchida K, Hiai H, Nishimura Y

ANNALS OF PLASTIC SURGERY Vol. 42 ( 3 ) page： 293 - 298 1999.3

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 Vol. 159 ( 3 ) page： A543 - A543 1999.3

Protection of ischemia-reperfusion lung against oxidative stress by nitroglycerin. International journal

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 Vol. 159 ( 3 ) page： A543 - A543 1999.3

Formation of 4-hydroxy-2-nonenal-modified proteins and 3-nitro-L-tyrosine in rat island skin flaps during and after ischemia Reviewed International journal

UM S. C.

Ann. Plast. Surg. Vol. 42 ( 3 ) page： 293 - 298 1999.3

Reactive oxygen species-induced molecular damage and its application in pathology

TOYOKUNI Shinya

Pathology International Vol. 49 ( 2 ) page： 91 - 102 1999.2

Reactive oxygen species-induced molecular damage and its application in pathology

Toyokuni S

PATHOLOGY INTERNATIONAL Vol. 49 ( 2 ) page： 91 - 102 1999.2

The 4-hydroxy-2-nonenal-derived fluorescent pigment International journal

Uchida, K; Itakura, K; Oya-Ito, T; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE Vol. 27 page： S100 - S100 1999

The malondialdehyde-derived fluorescent pigment International journal

Uchida, K; Sakamoto, S; Itakura, K; Kumazawa, S; Nakayama, T; Toyokuni, S; Osawa, T

FREE RADICAL BIOLOGY AND MEDICINE Vol. 27 page： S100 - S100 1999

Detection of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1 and its application

Toyokuni, S

ADVANCES IN DNA DAMAGE AND REPAIR Vol. 302 page： 395 - 405 1999

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 page： 39 - 42 1999

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2 '-deoxyguanosine

Toyokuni S, Sagripanti JL

FREE RADICAL RESEARCH Vol. 31 ( 2 ) page： 123 - 128 1999

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 Vol. 27 page： S149 - S149 1999

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 Vol. 105 ( 1-2 ) page： 179 - 180 1999

Spuamous Cell Carcinoma in Thyroid Papillary Carcinoma International journal Open Access

MINAMINO Masayuki, TSUJI Hiroyuki, NAKAMURA Akihiko, INOUE Toshiya, OHNISHI Sumio, HUJISAWA Takuo, YAMASHITA Toshio, TOYOKUNI Shinya

Practica Oto-Rhino-Laryngologica Vol. 92 ( 3 ) page： 283 - 286 1999

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Society of Practical Otolaryngology

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 Vol. 113 ( 1 ) page： 26 - 31 1999

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 International journal

Uchida, K, Itakura, K, Oya-Ito, T, Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE Vol. 27 page： S100 - S100 1999

The malondialdehyde-derived fluorescent pigment International journal

Uchida, K, Sakamoto, S, Itakura, K, Kumazawa, S, Nakayama, T, Toyokuni, S, Osawa, T

FREE RADICAL BIOLOGY AND MEDICINE Vol. 27 page： S100 - S100 1999

Oxidative stress in the human stomach infected with Helicobacter pylori: effect of bacterial eradication International journal

Pignatelli B, Li CQ, Bancel B, Toyokuni S, Ohshima H

2ND INTERNATIONAL CONGRESS ON GASTROENTEROLOGICAL CARCINOGENESIS page： 39 - 42 1999

Iron chelators modulate the production of DNA strand breaks and 8-hydroxy-2 '-deoxyguanosine

Toyokuni S, Sagripanti JL

FREE RADICAL RESEARCH Vol. 31 ( 2 ) page： 123 - 128 1999

Ihara Y., Toyokuni S., Uchida K., Odaka H., Tanaka T., Ikeda H., Hiai H., Seino Y., Yamada Y.

Diabetes Vol. 48 ( 4 ) page： 927 - 932 1999

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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

A study of imprint smear cytology during surgery of mucinous cystic tumor of ovary : A special reference to borderline malignancy

hOGG1 overexpression and high 8-Oxo-2'-deoxyguanosine lyase activity in human colorectal carcinoma International journal

Toyokuni, S, Kondo, S, Tanaka, T, Hiai, H, Onodera, H, Imamura, M

FREE RADICAL BIOLOGY AND MEDICINE Vol. 27 page： S149 - S149 1999

Detection of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1 and its application International journal

Toyokuni, S

ADVANCES IN DNA DAMAGE AND REPAIR Vol. 302 page： 395 - 405 1999

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 Vol. 105 ( 1-2 ) page： 179 - 180 1999

Oxidative stress in the human stomach infected with Helicobacter pylori:: effect of bacterial eradication International journal

Pignatelli, B, Li, CQ, Bancel, B, Toyokuni, S, Ohshima, H

2ND INTERNATIONAL CONGRESS ON GASTROENTEROLOGICAL CARCINOGENESIS page： 39 - 42 1999

SHIMIZU Keiko, OGURA Seiko, MURATA Masayoshi, TAKAO Yumi, TOYOKUNI Shinya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 37 ( 6 ) page： 583 - 590 1998.11

A study of imprint smear cytology during surgery of mucinous cystic tumor of ovary : A special reference to borderline malignancy

SHIMIZU Keiko, OGURA Seiko, MURATA Masayoshi, TAKAO Yumi, TOYOKUNI Shinya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 37 ( 6 ) page： 583 - 590 1998.11

Improvement in hepatic lipid peroxidation in patients with complete response to interferon-α treatment for chronic hepatitis C. International journal

Kageyama, F; Kobayashi, Y; Kawasaki, T; Toyokuni, S; Uchida, K; Nakamura, H

HEPATOLOGY Vol. 28 ( 4 ) page： 250A - 250A 1998.10

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 Vol. 28 ( 4 ) page： 251A - 251A 1998.10

Role of cytochrome P450 isoforms and free radicals in endotoxemic liver injury. Reviewed

Minamiyama Y, Takemura S, Imaoka S, Toyokuni S, Funae Y, Kinoshita H, Inoue M

HEPATOLOGY Vol. 28 ( 4 ) page： 251A - 251A 1998.10

Improvement in hepatic lipid peroxidation in patients with complete response to interferon-alpha treatment for chronic hepatitis C. Reviewed International journal

Kageyama F, Kobayashi Y, Kawasaki T, Toyokuni S, Uchida K, Nakamura H

HEPATOLOGY Vol. 28 ( 4 ) page： 250A - 250A 1998.10

Improvement in hepatic lipid peroxidation in patients with complete response to interferon-α treatment for chronic hepatitis C. International journal

Kageyama, F, Kobayashi, Y, Kawasaki, T, Toyokuni, S, Uchida, K, Nakamura, H

HEPATOLOGY Vol. 28 ( 4 ) page： 250A - 250A 1998.10

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 Vol. 79 ( 1 ) page： 47 - 53 1998.9

Heat shock preconditioning ameliorates liver injury following normothermic ischemia-reperfusion in steatotic rat livers

YAMAGAMI K

J Surg Res Vol. 79 ( 1 ) page： 47 - 53 1998.9

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

Vol. 89 ( 8 ) page： 814 - 820 1998.8

Development of High-grade Renal Cell Carcinomas in Rats Independently of Somatic Mutations in the Tsc2 and VHL Tumor Suppressor Genes International journal

TOYOKUNI Shinya, OKADA Kunihiko, KONDO Shohei, NISHIOKA Hiroaki, TANAKA Tomoyuki, NISHIYAMA Yasuyuki, HINO Okio, HIAI Hiroshi

Vol. 89 ( 8 ) page： 814 - 820 1998.8

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 Vol. 89 ( 8 ) page： 814 - 820 1998.8

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 Vol. 44 ( 4 ) page： 547 - 559 1998.8

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 Vol. 89 ( 8 ) page： 814 - 820 1998.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 89 ( 8 ) page： 814 - 820 1998.8

Toyokuni S.

Biotherapy Vol. 11 ( 2-3 ) page： 147 - 154 1998.7

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 Reviewed International journal

Tanaka T, Nishiyama Y, Okada K, Satoh K, Fukuda A, Uchida K, Osawa T, Hiai H, Toyokuni S

CARCINOGENESIS Vol. 19 ( 5 ) page： 897 - 903 1998.5

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 Vol. 47 page： A263 - A263 1998.5

Oxidative stress in pancreatic beta-cells of GK rat, a model of non-obese NIDDM International journal

Ihara Y, Yamada Y, Uchida K, Toyokuni S, Seino Y

DIABETES Vol. 47 page： A263 - A263 1998.5

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 Vol. 19 ( 5 ) page： 897 - 903 1998.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 22 ( 3 ) page： 145S - 149S 1998.5

Immunohistochemical detection of 4-hydroxy-2-nonenal-modified-protein adducts in human alcoholic liver diseases Reviewed

M Ohhira, T Ohtake, A Matsumoto, H Saito, K Ikuta, Y Fujimoto, M Ono, S Toyokuni, Y Kohgo

ALCOHOLISM-CLINICAL AND EXPERIMENTAL RESEARCH Vol. 22 ( 3 ) page： 145S - 149S 1998.5

Language：English Publishing type：Research paper (scientific journal) Publisher：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.

The role of oxidative DNA damage in human arsenic carcinogenesis: Detection of 8-OHdG in arsenic related Bowen's disease.

Oxidative stress in pancreatic β-cells of GK rat, a model of non-obese NIDDM International journal

Ihara, Y, Yamada, Y, Uchida, K, Toyokuni, S, Seino, Y

DIABETES Vol. 47 page： A263 - A263 1998.5

Significant deposition of 4-hydroxy-2-nonenal-modified-protein adducts in human alcoholic liver disease; Positive correlation with parenchymal iron. International journal

Ohhira, M; Ohtake, T; Matsumoto, A; Saito, H; Kawashima, T; Ikuta, K; Tanaka, K; Fujimoto, Y; Ono, M; Toyokuni, S; Kohgo, Y

GASTROENTEROLOGY Vol. 114 ( 4 ) page： A1315 - A1315 1998.4

Matsui, M; Nishigori, C; Toyokuni, S; Takada, J; Akaboshi, M; Ochi, H; Ishikawa, M; Imamura, S

JOURNAL OF INVESTIGATIVE DERMATOLOGY Vol. 110 ( 4 ) page： 526 - 526 1998.4

The role of oxidative DNA damage in human arsenic carcinogenesis: Detection of 8-OHdG in arsenic related Bowen's disease. International journal

Matsui, M, Nishigori, C, Toyokuni, S, Takada, J, Akaboshi, M, Ochi, H, Ishikawa, M, Imamura, S

JOURNAL OF INVESTIGATIVE DERMATOLOGY Vol. 110 ( 4 ) page： 526 - 526 1998.4

Enhanced hepatic lipid peroxidation in chronic hepatitis C Reviewed International journal

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 Vol. 26 ( SUPPL. 2 ) page： 149 - 152 1998.4

Enhanced hepatic lipid peroxidation in chronic hepatitis C Reviewed International journal

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 Vol. 26 ( SUPPL. 2 ) page： 149 - 152 1998.4

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 Vol. 101 ( 3 ) page： 785 - 792 1998.3

Involvement of nitric oxide in survival of random pattern skin flap

UM SC

Plast Reconstr Surg Vol. 101 ( 3 ) page： 785 - 792 1998.3

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 Vol. 25 page： S119 - S119 1998

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 page： 349 - 357 1998

A processed grain food inhibits hepatic injury in endotoxemic rats Reviewed

Minamiyama Y, Takemura S, Toyokuni S, Tanimoto Y, Sato EF, Inoue M

JOURNAL OF NUTRITIONAL SCIENCE AND VITAMINOLOGY Vol. 44 ( 4 ) page： 547 - 559 1998

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Center for Academic Publications Japan

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-2nonenal (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

Other Link： https://jlc.jst.go.jp/DN/JALC/00085275768?from=CiNii

Oxidative stress and cancer: The role of redox regulation

Toyokuni S

BIOTHERAPY Vol. 11 ( 2-3 ) page： 147 - 154 1998

A study of imprint smear cytology during surgery of mucinous cystic tumor of ovary. A special reference to borderline malignancy.:A special reference to borderline malignancy International journal

SHIMIZU Keiko, OGURA Seiko, MURATA Masayoshi, TAKAO Yumi, TOYOKUNI Shinya, SAKURAI Masami

The Journal of the Japanese Society of Clinical Cytology Vol. 37 ( 6 ) page： 583 - 590 1998

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Clinical Cytology

Objective: Preoperative diagnosis of ovarian tumors is often difficult. Additional squashed smear cytology during surgery was undertaken for the correct diagnosis of mucinous cystic tumors of the ovary. Study Design: We reviewed the cytological findings of imprint smear during surgery of 34 cases with mucinous cystic tumors of the ovary, especially borderline malignant cases, that we have experienced over an 8 year period (1990-1997). Additional squashed smears were applied in some recent cases and compared with the routine imprint smears. Results: Characteristic cytological findings of mucinous cystic tumors of borderline malignancy were relatively clear background, crowded clumps of cells, and papillary and ball-like architectures. In some cases, an additinal squashed smear technique was useful for the correctdiagnosis, because the structure of cell clusters became more apparent. Conclusion: It was possible to differentiate borderline cases from malignancies, but was difficult to differentiate borderline cases from benign cases by cytological examinations. The additional of the squashed smear technique was useful for correct cytological diagnosis.

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 Reviewed

S Toyokuni

BIOTHERAPY Vol. 11 ( 2-3 ) page： 147 - 154 1998

Ohhira M., Ohtake T., Matsumoto A., Saito H., Ikuta K., Fujimoto Y., Ono M., Toyokuni S., Kohgo Y.

Alcoholism: Clinical and Experimental Research Vol. 22 ( 3 SUPPL. 1 ) 1998

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Minamiyama, Y, Takemura, S, Toyokuni, S, Tanimoto, Y, Sato, EF, Inoue, M

BIOLOGICAL OXIDANTS AND ANTIOXIDANTS: MOLECULAR MECHANISMS AND HEALTH EFFECTS page： 349 - 357 1998

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 Vol. 417 ( 1 ) page： 150 - 152 1997.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 417 ( 1 ) page： 150 - 152 1997.11

Protein modification by lipid peroxidation products: Formation of malondialdehyde-derived N epsilon-(2-propenal)lysine in proteins Reviewed International journal

Uchida K, Sakai K, Itakura K, Osawa T, Toyokuni S

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 346 ( 1 ) page： 45 - 52 1997.10

Uchida K., Sakai K., Itakura K., Osawa T., Toyokuni S.

Archives of Biochemistry and Biophysics Vol. 346 ( 1 ) page： 45 - 52 1997.10

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Uchida K, Sakai K, Itakura K, Osawa T, Toyokuni S

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 346 ( 1 ) page： 45 - 52 1997.10

Enhanced hepatic lipid peroxidation in chronic hepatitis C. International journal

Kageyama, F; Kobayashi, Y; Kawasaki, T; Nakamura, H; Toyokuni, S; Uchida, K

HEPATOLOGY Vol. 26 ( 4 ) page： 79 - 79 1997.10

Yamagami, K; Yamamoto, Y; Kume, M; Kimoto, S; Ozaki, N; Toyokuni, S; Yamaoka, Y

HEPATOLOGY Vol. 26 ( 4 ) page： 844 - 844 1997.10

Impact of the protection from free radical injury in heat shock preconditioning against ischemia-reperfusion injury in steatotic rat livers. International journal

Yamagami, K, Yamamoto, Y, Kume, M, Kimoto, S, Ozaki, N, Toyokuni, S, Yamaoka, Y

HEPATOLOGY Vol. 26 ( 4 ) page： 844 - 844 1997.10

Enhanced hepatic lipid peroxidation in chronic hepatitis C [abstract]. Reviewed International journal

KAGEYAMA F.

Hepatology Vol. 26 ( 4 ) page： 79 - 79 1997.10

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 Vol. 77 ( 2 ) page： 145 - 155 1997.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 77 ( 2 ) page： 145 - 155 1997.8

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 Vol. 77 ( 2 ) page： 145 - 55 1997.8

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 Vol. 409 ( 2 ) page： 287 - 291 1997.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 409 ( 2 ) page： 287 - 291 1997.6

Biomarker evidence of DNA oxidation in lung cancer patients: Association of urinary 8-hydroxy-2'-deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment Reviewed International coauthorship International journal

Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H, Uchida K, Osawa T, Nieminen MM, Alho H, KellokumpuLehtinen P

FEBS LETTERS Vol. 409 ( 2 ) page： 287 - 291 1997.6

SHIMOI Kayoko, SHEN Bingrong, TOYOKUNI Shinya, MOCHIZUKI Rika, FURUGORI Michiyo, KINAE Naohide

Jpn. J. Cancer Res. Vol. 88 ( 5 ) page： 453 - 460 1997.5

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 Vol. 88 ( 5 ) page： 453 - 460 1997.5

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 Vol. 88 ( 5 ) page： 453 - 460 1997.5

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 Vol. 88 ( 5 ) page： 453 - 460 1997.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 88 ( 5 ) page： 453 - 460 1997.5

Copper and iron-induced oxidative damage in non-tumor bearing LEC rats Reviewed International journal

Ma YX, Zhang DX, Kawabata T, Kiriu T, Toyokuni S, Uchida K, Okada S

PATHOLOGY INTERNATIONAL Vol. 47 ( 4 ) page： 203 - 208 1997.4

MA Yuxiang, ZHANG Daxian, KAWABATA Teruyuki, KIRIU Takahiro, TOYOKUNI Shinya, UCHIDA Koji, OKADA Shigeru

Pathology International Vol. 47 ( 4 ) page： 203 - 208 1997.4

Copper and iron-induced oxidative damage in non-tumor bearing LEC rats Reviewed

YX Ma, DX Zhang, T Kawabata, T Kiriu, S Toyokuni, K Uchida, S Okada

PATHOLOGY INTERNATIONAL Vol. 47 ( 4 ) page： 203 - 208 1997.4

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Toyokuni S, Tanaka T, Hatton Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T

LABORATORY INVESTIGATION Vol. 76 ( 3 ) page： 365 - 374 1997.3

Toyokuni S, Tanaka T, Hatton Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T

LABORATORY INVESTIGATION Vol. 76 ( 3 ) page： 365 - 374 1997.3

Quantitative immunohistochemical determination of 8-hydroxy-2'- deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model Reviewed International journal

Toyokuni S., Tanaka T., Hattori Y., Nishiyama Y., Yoshida A., Uchida K., Hiai H., Ochi H., Osawa T.

Laboratory Investigation Vol. 76 ( 3 ) page： 365 - 374 1997.3

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 76 ( 3 ) page： 365 - 74 1997.3

NISHIYAMA Yasuyuki, TANAKA Tomoyuki, NAITOH Hiroshi, MORI Chisato, FUKUMOTO Manabu, HIAI Hiroshi, TOYOKUNI Shinya

Jpn, J. Cancer Res. Vol. 88 ( 2 ) page： 120 - 128 1997.2

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 Vol. 88 ( 2 ) page： 120 - 128 1997.2

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 Vol. 22 ( 6 ) page： 1019 - 1027 1997.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 108 ( 2 ) page： 237 - 237 1997.2

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 Vol. 88 ( 2 ) page： 120 - 128 1997.2

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 Reviewed International journal

Toyokuni S, Luo XP, Tanaka T, Uchida K, Hiai H, Lehotay DC

FREE RADICAL BIOLOGY AND MEDICINE Vol. 22 ( 6 ) page： 1019 - 1027 1997

A case of malignant myoenithelioma in the hard palate

KITA Jun, IWAI Hiroshi, TOYOKUNI Shinya, YANAGIDA Masahiro, KAWASAKI Eiko, MINAMINO Masayuki, NAKASHIMA Yasuaki, YAMASHITA Toshio

OTOLOGIA FUKUOKA Vol. 43 ( 1 ) page： 30 - 33 1997

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：JIBI TO RINSHO KAI

A rare case of low grade malignant myoepithelioma arising from the hard palate is reported. The CT findings indicated a defect of the hard palate due to the tumor. Surgery combined with postoperative irradiation therapy was performed. A histopathological examination indicated that the tumor originated in the myoepithelial cells, invaded the hard palate, and also showed cellular atypia with rare mitoses. Malignant myoepithelioma clinically shows a low sensitivity to both radiation therapy and chemotherapy. Surgery should thus be the first choice of treatment.

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 Vol. 88 ( 2 ) page： 120 - 128 1997

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

S Toyokuni, XP Luo, T Tanaka, K Uchida, H Hiai, DC Lehotay

FREE RADICAL BIOLOGY AND MEDICINE Vol. 22 ( 6 ) page： 1019 - 1027 1997

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

Tanaka T, Nishiyama Y, Okada K, Hirota K, Matsui M, Yodoi J, Hiai H, Toyokuni S

Laboratory Investigation Vol. 77 ( 2 ) page： 145 - 155 1997

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 Vol. 47 ( 4 ) page： 203 - 208 1997

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

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

JOURNAL OF INVESTIGATIVE DERMATOLOGY Vol. 107 ( 5 ) page： 733 - 737 1996.11

8-hydroxy-2'-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure Reviewed International journal

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

JOURNAL OF INVESTIGATIVE DERMATOLOGY Vol. 107 ( 5 ) page： 733 - 737 1996.11

UMEMURA Takashi, HASEGAWA Ryuichi, SAI-KATO Kimie, NISHIKAWA Akiyoshi, FURUKAWA Fumio, TOYOKUNI Shinya, UCHIDA Koji, INOUE Tohru, KUROKAWA Yuji

Jpn J Cancer Res Vol. 87 ( 9 ) page： 882 - 886 1996.9

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 Vol. 87 ( 9 ) page： 882 - 886 1996.9

Prevention by 2-mercaptoethane sulfonate and N-acetylcysteine of renal oxidative damage in rats treated with ferric nitrilotriacetate Reviewed International journal

Umemura T, Hasegawa R, SaiKato K, Nishikawa A, Furukawa F, Toyokuni S, Uchida K, Inoue T, Kurokawa Y

JAPANESE JOURNAL OF CANCER RESEARCH Vol. 87 ( 9 ) page： 882 - 886 1996.9

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 Vol. 87 ( 9 ) page： 882 - 886 1996.9

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Umemura T, Hasegawa R, SaiKato K, Nishikawa A, Furukawa F, Toyokuni S, Uchida K, Inoue T, Kurokawa Y

JAPANESE JOURNAL OF CANCER RESEARCH Vol. 87 ( 9 ) page： 882 - 886 1996.9

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 Vol. 75 ( 2 ) page： 239 - 248 1996.8

Induction of renal cell carcinoma in male Wistar rats treated with cupric nitrilotriacetate Reviewed International journal

Toyokuni S., Tanaka T., Nishiyama Y., Okamoto K., Nakashima Y., Hamazaki S., Okada S., Hiai H.

Laboratory Investigation Vol. 75 ( 2 ) page： 239 - 248 1996.8

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 75 ( 2 ) page： 239 - 248 1996.8

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 Vol. 75 ( 2 ) page： 239 - 48 1996.8

Oxidative stress response in iron-induced renal carcinogenesis: Acute nephrotoxicity mediates the enhanced expression of glutathione S-transferase Yp isozyme Reviewed International journal

Fukuda A, Osawa T, Oda H, Toyokuni S, Satoh K, Uchida K

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 329 ( 1 ) page： 39 - 46 1996.5

Fukuda A., Osawa T., Oda H., Toyokuni S., Satoh K., Uchida K.

Archives of Biochemistry and Biophysics Vol. 329 ( 1 ) page： 39 - 46 1996.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 65 ( 4 ) page： 437 - 441 1996.2

Okamoto K., Toyokuni S., Kim W., Ogawa O., Kakehi Y., Arao S., Hiai H., Yoshida O.

International Journal of Cancer Vol. 65 ( 4 ) page： 437 - 441 1996.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

Int. J. Cancer Vol. 65 ( 4 ) page： 437 - 441 1996.2

Oxidative stress response in iron-induced acute nephrotoxicity: Enhanced expression of heat shock protein 90 Reviewed International journal

Fukuda A, Osawa T, Oda H, Tanaka T, Toyokuni S, Uchida K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 219 ( 1 ) page： 76 - 81 1996.2

Fukuda A., Osawa T., Oda H., Tanaka T., Toyokuni S., Uchida K.

Biochemical and Biophysical Research Communications Vol. 219 ( 1 ) page： 76 - 81 1996.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Fukuda A, Osawa T, Oda H, Tanaka T, Toyokuni S, Uchida K

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 219 ( 1 ) page： 76 - 81 1996.2

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 Vol. 20 ( 6 ) page： 859 - 864 1996

Iron-induced carcinogenesis: The role of redox regulation

Toyokuni S

FREE RADICAL BIOLOGY AND MEDICINE Vol. 20 ( 4 ) page： 553 - 566 1996

Toyokuni S.

Free Radical Biology and Medicine Vol. 20 ( 4 ) page： 553 - 566 1996

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

Free Radic. Biol. Med. Vol. 20 ( 6 ) page： 859 - 864 1996

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 Vol. 107 ( 5 ) page： 733 - 737 1996

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Uchida K, Itakura K, Kawakishi S, Hiai H, Toyokuni S, Stadtman ER

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 324 ( 2 ) page： 241 - 248 1995.12

Uchida K., Itakura K., Kawakishi S., Hiai H., Toyokuni S., Stadtman E.

Archives of Biochemistry and Biophysics Vol. 324 ( 2 ) page： 241 - 248 1995.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Uchida K, Itakura K, Kawakishi S, Hiai H, Toyokuni S, Stadtman ER

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 324 ( 2 ) page： 241 - 248 1995.12

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 Vol. 86 ( 12 ) page： 1150 - 1158 1995.12

NISHIYAMA Yasuyuki, SUWA Hirofumi, OKAMOTO Keisei, FUKUMOTO Manabu, HIAI Hiroshi, TOYOKUNI Shinya

Jpn J Cancer Res Vol. 86 ( 12 ) page： 1150 - 1158 1995.12

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 Reviewed International journal

Nishiyama Y., Suwa H., Okamoto K., Fukumoto M., Hiai H., Toyokuni S.

Japanese Journal of Cancer Research Vol. 86 ( 12 ) page： 1150 - 1158 1995.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 86 ( 12 ) page： 1150 - 1158 1995.12

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 Vol. 62 ( 3 ) page： 309 - 313 1995.7

Toyokuni S., Mori T., Hiai H., Dizdaroglu M.

International Journal of Cancer Vol. 62 ( 3 ) page： 309 - 313 1995.7

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

Int J Cancer. Vol. 62 ( 3 ) page： 309 - 313 1995.7

4-HYDROXY-2-NONENAL-TRAPPING ELISA - DIRECT EVIDENCE FOR THE RELEASE OF A CYTOTOXIC ALDEHYDE FROM OXIDIZED LOW-DENSITY LIPOPROTEINS Reviewed International journal

UCHIDA K, OSAWA T, HIAI H, TOYOKUNI S

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 212 ( 3 ) page： 1068 - 1073 1995.7

DEGRADATION OF DNA IN DRIED TISSUES BY ATMOSPHERIC OXYGEN

MATSUO S, TOYOKUNI S, OSAKA M, HAMAZAKI S, SUGIYAMA T

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 208 ( 3 ) page： 1021 - 1027 1995.3

Matsuo S., Toyokuni S., Osaka M., Hamazaki S., Sugiyama T.

Biochemical and Biophysical Research Communications Vol. 208 ( 3 ) page： 1021 - 1027 1995.3

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

Biochem Biophys Res Commun Vol. 208 ( 3 ) page： 1021 - 1027 1995.3

A RENAL CARCINOGEN FERRIC NITRILOTRIACETATE MEDIATES A TEMPORARY ACCUMULATION OF ALDEHYDE-MODIFIED PROTEINS WITHIN CYTOSOLIC COMPARTMENT OF RAT-KIDNEY Reviewed International journal

UCHIDA K, FUKUDA A, KAWAKISHI S, HIAI H, TOYOKUNI S

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 317 ( 2 ) page： 405 - 411 1995.3

Uchida K., Fukuda A., Kawakishi S., Hiai H., Toyokuni S.

Archives of Biochemistry and Biophysics Vol. 317 ( 2 ) page： 405 - 411 1995.3

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed

K UCHIDA, A FUKUDA, S KAWAKISHI, H HIAI, S TOYOKUNI

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 317 ( 2 ) page： 405 - 411 1995.3

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

TOYOKUNI S, MIYAKE N, HIAI H, HAGIWARA M, KAWAKISHI S, OSAWA T, UCHIDA K

FEBS LETTERS Vol. 359 ( 2-3 ) page： 189 - 191 1995.2

Toyokuni S., Miyake N., Hiai H., Hagiwara M., Kawakishi S., Osawa T., Uchida K.

FEBS Letters Vol. 359 ( 2-3 ) page： 189 - 191 1995.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 88 ( 2 ) page： 157 - 162 1995.1

Cytotoxic and mutagenic effects of ferric nitrilotriacetate on L5178Y mouse lymphoma cells

TOYOKUNI S.

Cancer Lett. Vol. 88 ( 2 ) page： 157 - 162 1995.1

Toyokuni S., Sagripanti J., Hitchins V.

Cancer Letters Vol. 88 ( 2 ) page： 157 - 162 1995.1

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 358 ( 1 ) page： 1 - 3 1995.1

Toyokuni S., Okamoto K., Yodoi J., Hiai H.

FEBS Letters Vol. 358 ( 1 ) page： 1 - 3 1995.1

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

FEBS Lett. Vol. 358 ( 1 ) page： 1 - 3 1995.1

ACUTE NEPHROTOXICITY OF A CARCINOGENIC IRON CHELATE - SELECTIVE-INHIBITION OF A PROTEOLYTIC CONVERSION OF ALPHA(2U)-GLOBULIN TO THE KIDNEY FATTY-ACID-BINDING PROTEIN Reviewed International journal

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

FEBS LETTERS Vol. 357 ( 2 ) page： 165 - 167 1995.1

Uchida K., Fukuda A., Kawakishi S., Toyokuni S., Hiai H., Ikeda S., Horio F.

FEBS Letters Vol. 357 ( 2 ) page： 165 - 167 1995.1

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 212 ( 3 ) page： 1068 - 1073 1995

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

UCHIDA K, TOYOKUNI S, NISHIKAWA K, KAWAKISHI S, ODA H, HIAI H, STADTMAN ER

BIOCHEMISTRY Vol. 33 ( 41 ) page： 12487 - 12494 1994.10

Michael addition type 4-hydroxy-2-nonenal adducts in modified low density lipoproteins : markers for atherosclerosi Reviewed International journal

UCHIDA K.

Biochemistry. Vol. 33 ( 41 ) page： 12487 - 12494 1994.10

Uchida K., Toyokuni S., Nishikawa K., Kawakishi S., Oda H., Hiai H., Stadtman E.

Biochemistry Vol. 33 ( 41 ) page： 12487 - 12494 1994.10

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

OKAMOTO K, TOYOKUNI S, UCHIDA K, OGAWA O, TAKENEWA J, KAKEHI Y, KINOSHITA H, HATTORINAKAKUKI Y, HIAI H, YOSHIDA O

INTERNATIONAL JOURNAL OF CANCER Vol. 58 ( 6 ) page： 825 - 829 1994.9

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 Vol. 58 ( 6 ) page： 825 - 829 1994.9

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

OKAMOTO K, TOYOKUNI S, UCHIDA K, OGAWA O, TAKENEWA J, KAKEHI Y, KINOSHITA H, HATTORINAKAKUKI Y, HIAI H, YOSHIDA O

INTERNATIONAL JOURNAL OF CANCER Vol. 58 ( 6 ) page： 825 - 829 1994.9

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 Vol. 201 ( 3 ) page： 1132 - 1139 1994.6

Hattori-Nakakuki Y., Nishigori C., Okamoto K., Imamura S., Hiai H., Toyokuni S.

Biochemical and Biophysical Research Communications Vol. 201 ( 3 ) page： 1132 - 1139 1994.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 201 ( 3 ) page： 1132 - 1139 1994.6

INCREASED 8-HYDROXYDEOXYGUANOSINE IN KIDNEY AND LIVER OF RATS CONTINUOUSLY EXPOSED TO COPPER

TOYOKUNI S, SAGRIPANTI JL

TOXICOLOGY AND APPLIED PHARMACOLOGY Vol. 126 ( 1 ) page： 91 - 97 1994.5

Toyokuni S., Sagripanti J.

Toxicology and Applied Pharmacology Vol. 126 ( 1 ) page： 91 - 97 1994.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 126 ( 1 ) page： 91 - 97 1994.5

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 Vol. 57 ( 1 ) page： 123 - 128 1994.4

DNA base modification in renal chromatin of wister rats trea, ed with a renal carcinogen, ferric nitrilotriacetate

TOYOKUNI S.

Int. J. Cancer Vol. 57 ( 1 ) page： 123 - 128 1994.4

Toyokuni S., Mori T., Dizdaroglu M.

International Journal of Cancer Vol. 57 ( 1 ) page： 123 - 128 1994.4

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

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 Vol. 91 ( 7 ) page： 2616 - 2620 1994.3

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 Vol. 91 ( 7 ) page： 2616 - 2620 1994.3

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

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 Vol. 91 ( 7 ) page： 2616 - 2620 1994.3

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 Vol. 54 ( 2 ) page： 399 - 402 1994.1

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 Vol. 54 ( 2 ) page： 403 - 407 1994.1

Abnormal bone marrow B-cell differentiation in pre-B lymphoma-prone SL/Kh mice

OKAMOTO K

Cancer Res Vol. 54 ( 2 ) page： 399 - 402 1994.1

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 Vol. 54 ( 2 ) page： 403 - 7 1994.1

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 Vol. 54 ( 2 ) page： 399 - 402 1994.1

Genetic Predisposition to Pre-B Lymphomas in SL/Kh Strain Mice Reviewed International journal

Yamada Y., Matsushiro H., Ogawa M.S., Okamoto K., Nakakuki Y., Toyokuni S., Fukumoto M., Hiai H.

Cancer Research Vol. 54 ( 2 ) page： 403 - 407 1994.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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 International journal

Yamada Y, Matsushiro H, Ogawa M.S, Okamoto K, Nakakuki Y, Toyokuni S, Fukumoto M, Hiai H

Cancer Research Vol. 54 ( 2 ) page： 403 - 407 1994.1

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Cancer Research

Abnormal Bone Marrow B-Cell Differentiation in Pre-B Lymphoma-prone SL/Kh Mice Reviewed International journal

Okamoto K., Yamada Y., Ogawa M.S., Toyokuni S., Nakakuki Y., Ikeda H.

Cancer Research Vol. 54 ( 2 ) page： 399 - 402 1994.1

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 1058 page： 447 - 448 1994

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 Vol. 1058 page： 171 - 172 1994

FORMATION OF 4-HYDROXY-2-NONENAL-MODIFIED PROTEINS IN THE RENAL PROXIMAL TUBULES OF RATS TREATED WITH A RENAL CARCINOGEN, FERRIC NITRILOTRIACETATE International journal

TOYOKUNI, S, UCHIDA, K, OKAMOTO, K, HATTORINAKAKUKI, Y, HIAI, H, STADTMAN, ER

FRONTIERS OF REACTIVE OXYGEN SPECIES IN BIOLOGY AND MEDICINE Vol. 1058 page： 171 - 172 1994

ALTERATION OF THE DEOXYGUANINE NUCLEOTIDES TO 8-HYDROXYDEOXYGUANOSINE IN HUMAN RENAL-CELL CARCINOMA Reviewed International journal

OKAMOTO, K, YOSHIDA, O, HATTORINAKAKUKI, Y, HIAI, H, TOYOKUNI, S

FRONTIERS OF REACTIVE OXYGEN SPECIES IN BIOLOGY AND MEDICINE Vol. 1058 page： 447 - 448 1994

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 Vol. 21 ( 8 ) page： 1126 - 1126 1993.8

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 Vol. 21 ( 8 ) page： 1118 - 1118 1993.8

INDUCTION OF OXIDATIVE SINGLE-STRAND AND DOUBLE-STRAND BREAKS IN DNA BY FERRIC CITRATE

TOYOKUNI S, SAGRIPANTI JL

FREE RADICAL BIOLOGY AND MEDICINE Vol. 15 ( 2 ) page： 117 - 123 1993.8

INDUCTION OF OXIDATIVE SINGLE-STRAND AND DOUBLE-STRAND BREAKS IN DNA BY FERRIC CITRATE Reviewed

S TOYOKUNI, JL SAGRIPANTI

FREE RADICAL BIOLOGY AND MEDICINE Vol. 15 ( 2 ) page： 117 - 123 1993.8

Language：English Publishing type：Research paper (scientific journal) Publisher：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 International journal

YAMADA, Y, SHIMADA, M, OKAMOTO, K, TOYOKUNI, S, NAKAKUKI, Y, FUKUMOTO, M, HIAI, H

EXPERIMENTAL HEMATOLOGY Vol. 21 ( 8 ) page： 1118 - 1118 1993.8

Toyokuni S., Sagripanti J.

Free Radical Biology and Medicine Vol. 15 ( 2 ) page： 117 - 123 1993.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

OKAMOTO, K, YAMADA, Y, SHIMADA, M, TOYOKUNI, S, NAKAKUKI, Y, HIAI, H

EXPERIMENTAL HEMATOLOGY Vol. 21 ( 8 ) page： 1126 - 1126 1993.8

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 Vol. 301 ( 1 ) page： 138 - 142 1993.2

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 Vol. 301 ( 1 ) page： 138 - 142 1993.2

Okada S., Minamiyama Y., Hamazaki S., Toyokuni S., Sotomatsu A.

Archives of Biochemistry and Biophysics Vol. 301 ( 1 ) page： 138 - 142 1993.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 14 ( 2 ) page： 223 - 227 1993.2

DNA single- and double-strand breaks produced by ferric nitrilotriacetate in relation to renal tubular carcinogenesis

TOYOKUNI S.

Carcinogenesis Vol. 14 ( 2 ) page： 223 - 227 1993.2

Toyokuni S., Sagripanti J.

Carcinogenesis Vol. 14 ( 2 ) page： 223 - 227 1993.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 page： 405 - 408 1993

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 page： 835 - 838 1993

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 page： 845 - 848 1993

PATHOLOGICAL EVALUATION OF RABBITS SUBCHRONICALLY EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1 G AND 5 G International journal

NING, JT, TOYOKUNI, S, THOMAS, D, CZERSKA, EM, CASAMENTO, J, SWICORD, ML, DAVIS, CC, ELSON, E

ELECTRICITY AND MAGNETISM IN BIOLOGY AND MEDICINE page： 835 - 838 1993

DOSE-DEPENDENT HEALTH-EFFECTS EXHIBITED IN RABBITS EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1 G AND 5 G International journal

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 page： 845 - 848 1993

CHARACTERIZATION OF IMMORTALIZED CELL-LINES ISOLATED FROM A SOFT-TISSUE TUMOR-DERIVED FROM A RABBIT SUBCHRONICALLY EXPOSED TO 60HZ MAGNETIC-FIELDS AT 1-G Reviewed International coauthorship International journal

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 page： 405 - 408 1993

IRON-MEDIATED DNA DAMAGE - SENSITIVE DETECTION OF DNA STRAND BREAKAGE CATALYZED BY IRON

TOYOKUNI S, SAGRIPANTI JL

JOURNAL OF INORGANIC BIOCHEMISTRY Vol. 47 ( 3-4 ) page： 241 - 248 1992.8

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 Vol. 70 ( 4 ) page： 882 - 886 1992.8

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 Vol. 70 ( 4 ) page： 882 - 886 1992.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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

A case of undifferentiated sarcoma of the liver

Peport of a patient with POEMS/Takatsuki/Crow-Fukase syndrome associated with focal spinal pachymeningeal amyloidsis.

TOYOKUNI S.

Cancer Vol. 70 ( 4 ) page： 882 - 886 1992.8

Iron-mediated DNA damage : sensitive detection of DNA strand breakage catalyzed by iron

TOYOKUNI S

J. Inorg. Biochem. Vol. 47 ( 3-4 ) page： 241 - 248 1992.8

OKUYAMA Takazo, TOYOKUNI Shinya, OKADA Shigeru, NAKASHIMA Yasuaki, YAMABE Hirohiko, KAN Shugen, MORI Keiichiro, OZAWA Kazue

The Journal of the Japanese Society of Clinical Cytology Vol. 31 page： 89 - 90 1992

A case of undifferentiated sarcoma of the liver.

OKUYAMA Takazo, TOYOKUNI Shinya, OKADA Shigeru, NAKASHIMA Yasuaki, YAMABE Hirohiko, KAN Shugen, MORI Keiichiro, OZAWA Kazue

The Journal of the Japanese Society of Clinical Cytology Vol. 31 ( 1 ) page： 89 - 90 1992

A case of undifferentiated sarcoma of the liver

OKUYAMA Takazo, TOYOKUNI Shinya, OKADA Shigeru, NAKASHIMA Yasuaki, YAMABE Hirohiko, KAN Shugen, MORI Keiichiro, OZAWA Kazue

The Journal of the Japanese Society of Clinical Cytology Vol. 31 page： 89 - 90 1992

Iron-mediated DNA damage: Sensitive detection of DNA strand breakage catalyzed by iron

Toyokuni S., Sagripanti J.

Journal of Inorganic Biochemistry Vol. 47 ( 1 ) page： 241 - 248 1992

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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)) Reviewed International journal

Toyokuni

Cancer Research Vol. 52 ( 21 ) 1992

Histochemical detection of burn-induced lipid peroxidation in sebaceous glands of rat skin. International journal

Kitajima T, Hamanaka H, Miyachi Y, Ono Y, Moriguchi T, Toyokuni S, Imamura S

The Journal of dermatology Vol. 18 ( 7 ) page： 393 - 6 1991.7

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 41 ( 3 ) page： 221 - 226 1991.3

Sex differences in the localization and severity of ferric nitrilotriacetate-induced lipid peroxidation in the mouse kidney

OKADA S.

Acta Pathol. Jpn. Vol. 41 ( 3 ) page： 221 - 226 1991.3

Sex Differences in the Localization and Severity of Ferric Nitrilotriacetate‐induced Lipid Peroxidation in the Mouse Kidney International journal

Okada S., Fukunaga Y., Hamazaki S., Yamada Y., Toyokuni S.

Pathology International Vol. 41 ( 3 ) page： 221 - 226 1991.3

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 13 ( 1 ) page： 58 - 60 1991

Congenital myotonic dystrophy with progressive edema and hypoproteinemia

FUJI T

Brain Develop Vol. 13 ( 1 ) page： 58 - 60 1991

Fujii T., Yorifuji T., Okuno T., Toyokuni S., Okada S., Mikawa H.

Brain and Development Vol. 13 ( 1 ) page： 58 - 60 1991

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 13 ( 10 ) page： 732 - 737 1990.10

Matoba Y., Matsumori A., Ohtani H., Tominaga M., Fujiwara H., Kawai C., Toyokuni S., Midorikawa O., Takahashi K.

Clinical Cardiology Vol. 13 ( 10 ) page： 732 - 737 1990.10

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

Clinical Cardiol Vol. 13 ( 10 ) page： 732 - 737 1990.10

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 Vol. 50 ( 17 ) page： 5574 - 5580 1990.9

Combined histochemical and biochemical analysis of sex hormone dependence of ferric nitrilotriacetate-induced renal lipid peroxidation in ddY mice

TOYOKUNI S.

Cancer Res Vol. 50 ( 17 ) page： 5574 - 5580 1990.9

Combined Histochemical and Biochemical Analysis of Sex Hormone Dependence of Ferric Nitrilotriacetate-induced Renal Lipid Peroxidation in ddY Mice Reviewed International journal

Toyokuni S., Okada S., Hamazaki S., Minamiyama Y., Yamada Y., Liang P., Fukunaga Y., Midorikawa O.

Cancer Research Vol. 50 ( 17 ) page： 5574 - 5580 1990.9

Language：English Publishing type：Research paper (scientific journal) Publisher：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 Vol. 50 ( 17 ) page： 5574 - 80 1990.9

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 Vol. 274 ( 2 ) page： 348 - 354 1989.11

Hamazaki S., Okada S., Toyokuni S., Midorikawa O.

Archives of Biochemistry and Biophysics Vol. 274 ( 2 ) page： 348 - 354 1989.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 60 ( 5 ) page： 708 - 711 1989.11

THIOBARBITURIC ACID-REACTIVE SUBSTANCE FORMATION OF RAT-KIDNEY BRUSH-BORDER MEMBRANE-VESICLES INDUCED BY FERRIC NITRILOTRIACETATE Reviewed

S HAMAZAKI, S OKADA, S TOYOKUNI, O MIDORIKAWA

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 274 ( 2 ) page： 348 - 354 1989.11

Okada S., Hamazaki S., Toyokuni S., Midorikawa O.

British Journal of Cancer Vol. 60 ( 5 ) page： 708 - 711 1989.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

Br J Cancer Vol. 60 ( 5 ) page： 708 - 711 1989.11

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 Vol. 272 ( 1 ) page： 10 - 17 1989.7

OXYGEN REDUCTION AND LIPID-PEROXIDATION BY IRON CHELATES WITH SPECIAL REFERENCE TO FERRIC NITRILOTRIACETATE Reviewed

S HAMAZAKI, S OKADA, JL LI, S TOYOKUNI, O MIDORIKAWA

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 272 ( 1 ) page： 10 - 17 1989.7

Hamazaki S., Okada S., Li J., Toyokuni S., Midorikawa O.

Archives of Biochemistry and Biophysics Vol. 272 ( 1 ) page： 10 - 17 1989.7

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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 Vol. 134 ( 6 ) page： 1263 - 1274 1989.6

CIRRHOSIS OF THE LIVER INDUCED BY CUPRIC NITRILOTRIACETATE IN WISTAR RATS - AN EXPERIMENTAL-MODEL OF COPPER TOXICOSIS Reviewed

S TOYOKUNI, S OKADA, S HAMAZAKI, M FUJIOKA, JL LI, O MIDORIKAWA

AMERICAN JOURNAL OF PATHOLOGY Vol. 134 ( 6 ) page： 1263 - 1274 1989.6

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 Vol. 134 ( 6 ) page： 1263 - 74 1989.6

Cirrhosis of the liver induced by cupric nitrilotriacetate in Wistar rats: an experimental model of copper toxicosis. Reviewed

Toyokuni S, Okada S, Hamazaki S, Fujioka M, Li J-L and Midorikawa O.

Am. J. Pathol. Vol. 134 page： 1263-1274 1989

Oxygen reduction and lipid peroxidation by iron chelates with special reference to ferric nitrilotriacetate. Reviewed

Hamazaki S, Okada S, Li J-L, Toyokuni S and Midorikawa O.

Arch. Biochem. Biophys. Vol. 272 page： 10-17 1989

Nasal T-cell lymphoma associated with double gammopathy; A case report. International journal Open Access

Omori Koichi, Kojima Hisayoshi, Nonomura Mitsuharu, Kanaji Myojo, Toyokuno Shinya

Practica Oto-Rhino-Laryngologica Vol. 82 ( 12 ) page： 1739 - 1744 1989

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Society of Practical Otolaryngology

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 immunohistochemical study is indispensable for the diagnosis of nasal non-healing granuloma with necrosis.

DOI： 10.5631/jibirin.82.1739

Renal changes induced by chronic oral administration of potassium bromate of ferric nitrilotriacetate in Wistar rats Reviewed International journal

K. Nakano, S. Okada, S. Toyokuni, O. Midorikawa

Japanese Archives of Internal Medicine Vol. 36 ( 2 ) page： 41 - 47 1989

Cirrhosis of the liver induced by cupric nitrilotriacetate in Wistar rats. An experimental model of copper toxicosis Reviewed International journal

Toyokuni S., Okada S., Hamazaki S., Fujioka M., Li J.L., Midorikawa O.

American Journal of Pathology Vol. 134 ( 6 ) page： 1263 - 1274 1989

Language：English Publishing type：Research paper (scientific journal) Publisher：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.