研究者詳細 - 豊國　伸哉

Thioredoxin as a biomarker for oxidative stress in patients with rheumatoid arthritis. 国際誌

Takumi Jikimoto, Yuko Nishikubo, Masahiro Koshiba, Sugayo Kanagawa, Sahoko Morinobu, Akio Morinobu, Ryuichi Saura, Kosaku Mizuno, Shohei Kondo, Shinya Toyokuni, Hajime Nakamura, Junji Yodoi, Shunichi Kumagai

Molecular immunology 38 巻 ( 10 ) 頁： 765 - 72 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

There is no doubt that oxidative stress occurs in patients with rheumatoid arthritis (RA) and play an important role in both inflammation and destruction of RA joints. Thioredoxin (TRX) is a ubiquitous redox-active protein and is known to be induced in several cells against oxidative stress and to be secreted extracellularly. To clarify whether plasma thioredoxin levels could be a marker for oxidative stress in patients with RA, we measured plasma TRX levels in patients with RA using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and investigated its relationship to TRX concentrations in the inflammatory joints. We have found that the plasma TRX levels of RA patients were significantly higher than those of normal subjects (86.8 +/-54.1 ng/ml versus 38.6 +/-18.5 ng/ml, P<0.0001). The plasma levels were correlated with the disease activity of RA and also with serum C-reactive protein (CRP) values (P<0.01). The concentration of TRX in synovial fluid (SF) from RA was 353.3 +/- 220.1 ng/ml (mean +/- S.D.) which was significantly higher than that in SF from osteoarthritis patients (70.6 +/- 31.0 ng/ml, P<0.0001). The SF TRX concentration was significantly correlated with the number of leukocytes infiltrating in SF and with the serum CRP levels. The serum TRX levels were significantly positively correlated with the SF TRX concentrations in RA patients (P<0.05). By the histological examination for synovial tissue of RA patients, TRX was shown to be present on the surface of synovial lining layer as well as in the leukocytes.Moreover, urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage by endogenously generated oxygen radicals, was significantly higher in RA patients than in healthy subjects (11.55 +/- 4.71 versus 7.76 +/- 2.26 ng/mg creatinine, P<0.0001). Plasma TRX levels were significantly correlated with urinary excretion of 8-OHdG (P<0.005). We concluded that plasma TRX level is a new biomarker for the disease activity of RA and may reflect higher levels of oxidative stress in RA patients.

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. Open Access

Hiroyasu M, Ozeki M, Kohda H, Echizenya M, Tanaka T, Hiai H, Toyokuni S

The American journal of pathology 160 巻 ( 2 ) 頁： 419 - 24 2002年2月

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

Oxidative tissue damage has been shown to be associated with carcinogenesis. In human cancers p16INK4A is one of the most frequently mutated tumor suppressor genes. The present study used the ferric nitrilotriacetate (Fe-NTA)-induced rat renal carcinogenesis model to determine whether oxidative damage can cause specific allelic loss of p16INK4A. By the use of fluorescent in situ hybridization in combination with imprint cytology at single-cell resolution, we found that the number of renal tubular cells with aneuploidy (1 or 3 signals) at the p16INK4A locus was significantly and specifically increased (1 week, 37.2 ± 2.3%; 3 weeks, 37.8 ± 1.3% vs control, 22.5 ± 1.9%; mean ± SE, N = 8; P < 0.001 and P < 0.0001, respectively) after repeated intraperitoneal administration of 5 to10 mg of iron/kg in the form of Fe-NTA for 3 weeks. No increase in aneuploidy was observed at the loci of either the p53 or vhl tumor suppressor gene. Furthermore, the increase in the cells with 3 signals was followed by a continuous increase in those with 1 signal. Therefore, the p16INK4A locus is specifically vulnerable to oxidative damage, leading to its allelic loss within weeks, presumably due to a deficiency in the replication of both the alleles.

DOI： 10.1016/S0002-9440(10)64860-2

Specific allelic loss of p16 (INK4A) tumor suppressor gene after weeks of iron-mediated oxidative damage during rat renal carcinogenesis. 国際誌 Open Access

Makoto Hiroyasu, Munetaka Ozeki, Haruyasu Kohda, Michiko Echizenya, Tomoyuki Tanaka, Hiroshi Hiai, Shinya Toyokuni

The American journal of pathology 160 巻 ( 2 ) 頁： 419 - 24 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Oxidative tissue damage has been shown to be associated with carcinogenesis. In human cancers p16(INK4A) is one of the most frequently mutated tumor suppressor genes. The present study used the ferric nitrilotriacetate (Fe-NTA)-induced rat renal carcinogenesis model to determine whether oxidative damage can cause specific allelic loss of p16 (INK4A). By the use of fluorescent in situ hybridization in combination with imprint cytology at single-cell resolution, we found that the number of renal tubular cells with aneuploidy (1 or 3 signals) at the p16(INK4A) locus was significantly and specifically increased (1 week, 37.2 +/- 2.3%; 3 weeks, 37.8 +/- 1.3% vs control, 22.5 +/- 1.9%; mean +/- SE, N = 8; P < 0.001 and P < 0.0001, respectively) after repeated intraperitoneal administration of 5 to10 mg of iron/kg in the form of Fe-NTA for 3 weeks. No increase in aneuploidy was observed at the loci of either the p53 or vhl tumor suppressor gene. Furthermore, the increase in the cells with 3 signals was followed by a continuous increase in those with 1 signal. Therefore, the p16 (INK4A) locus is specifically vulnerable to oxidative damage, leading to its allelic loss within weeks, presumably due to a deficiency in the replication of both the alleles.

DOI： 10.1016/S0002-9440(10)64860-2

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. 査読有り国際誌

Yamagami K, Yamamoto Y, Toyokuni S, Hata K, Yamaoka Y

Free radical research 36 巻 ( 2 ) 頁： 169 - 76 2002年2月

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

Heat shock preconditioning (HSPC) is a promising strategy for providing ischemic tolerance. The objective of this study is to investigate the effectiveness of HSPC in preventing oxidative damage of cellular proteins and DNA during ischemia-reperfusion of the liver. Male Wistar rats were divided into a heat shock group (group HS) and control (group C). Forty-eight hours prior to ischemia, rats in group HS received HSPC at 42°C for 15 min. All rats received hepatic warm ischemia for 30 min and subsequent reperfusion. The formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) modified proteins in liver tissue, survival rate of the animals, and changes in biochemical and histological parameters were compared between groups. Heat shock protein 72 was produced only in group HS. The 7-day survival of rats was significantly better in group HS (10/10) than in group C (5/10) (p <0.01). The serum release of alanine aminotransferase (n =10, p < 0.01) and the concentration of adenosine triphosphate in liver tissue (n = 10, p < 0.01) 40 min after reperfusion was significantly better in group HS than in group C. The formation of 8-OHdG in liver tissue measured by high-performance liquid chromatography was suppressed in group HS (p < 0.01). The production of HNE-modified proteins as determined by Westem-blot analysis was also decreased in group HS. These results were also confirmed by immunohistochemical analysis. As determined by levels of 8-OHdG and HNE-modified proteins produced during ischemia-reperfusion of the liver, HSPC reduced the oxidative injury of cellular proteins and DNA in the liver tissue.

DOI： 10.1080/10715760290006385

Amelioration of cisplatin toxicity by a fermented grain food product

Minamiyama Y, Takemura S, Toyokuni S, Nishino Y, Yamasaki K, Hai S, Yamamoto S, Okada S

BIOFACTORS 16 巻 ( 3-4 ) 頁： 105 - 115 2002年

Iron and carcinogenesis: from Fenton reaction to target genes. 国際誌

Toyokuni S

Redox report : communications in free radical research 7 巻 ( 4 ) 頁： 189 - 97 2002年

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

Reactive oxygen species (ROS) have been shown to be associated with a wide variety of pathological phenomena such as carcinogenesis, inflammation, radiation and reperfusion injury. Iron, the most abundant transition metal ion in our body, may work as a catalyst for the generation of ROS in pathological conditions. In the past few years, there have been great advances in the understanding of iron metabolism. These include the discoveries of iron transporters and the gene responsible for hereditary hemochromatosis. Iron overload has been shown to be associated with carcinogenesis. We recently identified the major target genes (p16INK4A and p15INK4B tumor suppressor genes, which encode cyclin-dependent kinase inhibitors) in a ferric nitrilotriacetate-induced rat renal carcinogenesis model, in which the Fenton reaction is induced in the renal proximal tubules. Allelic loss of the p16 gene occurs early in carcinogenesis and specifically at the p16 loci as compared with other tumor suppressor genes. This led to the novel concept of 'genomic sites vulnerable to the Fenton reaction'. Here, recent new findings on iron metabolism are reviewed and the concept of the vulnerable sites explored. More effort to link iron metabolism with human carcinogenesis is anticipated.

DOI： 10.1179/135100002125000596

Radiation-induced kidney injury: a role for chronic oxidative stress?

Robbins MEC, Zhao WL, Davis CS, Toyokuni S, Bonsib SM

MICRON 33 巻 ( 2 ) 頁： 133 - 141 2002年

Toyokuni, S; Hiroyasu, M; Ozeki, M; Hiai, H

XI BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 頁： 459 - 462 2002年

Target genes in fenton reaction-induced carcinogenisis: A novel concept

Toyokuni, S; Hiroyasu, M; Ozeki, M; Hiai, H

FREE RADICAL BIOLOGY AND MEDICINE 33 巻頁： S177 - S177 2002年

Novel surrogate end-point biomarker to evaluate agents for use in the chemoprevention of reactive oxygen species-associated cancer. 査読有り国際誌

Hiroyasu M, Ozeki M, Miyagawa-Hayashino A, Fujiwara Y, Hiai H, Toyokuni S

Redox report : communications in free radical research 7 巻 ( 5 ) 頁： 335 - 8 2002年

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

Cancer chemoprevention is the use of chemical agents to inhibit, delay or reverse carcinogenesis. We established a novel method to evaluate agents for use in the chemoprevention of reactive oxygen species (ROS)-associated cancer. Induction of renal cell carcinoma in rats by ferric nitrilotriacetate (Fe-NTA) is an established model of ROS-associated cancer. We recently identified the p16INK4A tumor suppressor gene as one of the major target genes in this model, and showed by the use of in situ hybridization that allelic loss of p16INK4A occurs in the increased fraction of renal tubular cells within a few weeks. In the present study, we tested whether diets including green tea powder or a processed grain food are effective chemopreventive agents in this animal model. Consumption of these modified diets led to a significant decrease in the fraction of aneuploid cells after 1 week of repeated Fe-NTA administration. A decrease in renal lipid peroxidation after a single administration of Fe-NTA was also observed. Therefore, intake of green tea or processed grain foods stabilizes p16INK4A in the genome, at least in this model, and might be helpful for the prevention of ROS-associated cancer. This novel method is versatile, and may work as a surrogate end-point biomarker for screening the usefulness of agents for cancer chemoprevention.

DOI： 10.1179/135100002125000938

子宮内膜細胞診疑陽性例の検討構造異型を加味した判定基準を主体に:構造異型を加味した判定基準を主体に

清水恵子, 小椋聖子, 小林八郎, 豊國伸哉, 則松良明, 森谷卓也, 桜井幹己

日本臨床細胞学会雑誌 41 巻 ( 2 ) 頁： 89 - 94 2002年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：特定非営利活動法人日本臨床細胞学会

目的:子宮内膜細胞診は判定が困難な分野として認識されており, さらに各施設により採取法, 検体処理法, 疑陽性とする細胞診断基準が異なっている.今回われわれは当院に於ける細胞診疑陽性例の判定基準を主体にした検討 (特に構造異型を加味した判定基準に対する検討) を行い若干の知見を得たので報告する. 方法:1995年5月から2000年9月までの疑陽性例のうち組織診が施行された84件を対象とし, 細胞異型を中心に判定した期間 (期間A) と細胞異型に構造異型を加味して判定した期問 (期間B) の成績の差異について検討した.標本作製法はエンドサイト直接塗抹法で期間BではRPMI液による採取器具洗浄を加えた. 成績:検討症例84件の組織診断の内訳は期間Aでは良性23件, 増殖症21件, 腺癌7件, 期間Bでは良性11件, 増殖症21件, 腺癌1件であった.検体不良率は, 期間Aでは32.9%, 期間Bでは20%であった. 結論:細胞異型に構造異型を加味して判定した期間では, 細胞異型を中心に判定した期間に比べて増殖症の正診率が向上し腺癌の疑陽性率は減少した.また, 直接塗抹標本に器具洗浄標本を加えることにより検体不良率の減少がみられた.

DOI： 10.5795/jjscc.41.89

CiNii Research

Target genes in fenton reaction-induced carcinogenisis: A novel concept 国際誌

Toyokuni, S, Hiroyasu, M, Ozeki, M, Hiai, H

FREE RADICAL BIOLOGY AND MEDICINE 33 巻頁： S177 - S177 2002年

Radiation-induced kidney injury: a role for chronic oxidative stress? 国際誌

Mike E Robbins, Weiling Zhao, Charles S Davis, Shinya Toyokuni, Stephen M Bonsib

Micron (Oxford, England : 1993) 33 巻 ( 2 ) 頁： 133 - 41 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）

Kidney irradiation clearly leads to a progressive reduction in function associated with concomitant glomerulosclerosis and/or tubulointerstitial fibrosis. However, the particular cell types, mediators and/or mechanisms involved in the development and progression of radiation nephropathy remain ill defined. Angiotensin II (Ang II) plays a major pathogenic role; administration of Ang II blockers markedly abrogates the severity of radiation nephropathy in experimental models. Both ionizing radiation and Ang II signal via generation of reactive oxygen species (ROS). Thus, we hypothesized that localized kidney irradiation might lead to a chronic oxidative stress. In view of the difficulty in measuring ROS in vivo we adopted an indirect immunohistochemical approach in which we used a monoclonal antibody specific for 8-hydroxy-2'-deoxyguanosine (8-OHdG), one of the most commonly used markers of DNA oxidation. The right kidney of 7-8 week-old male Sprague-Dawley rats was removed. Five to 6 weeks later the remaining hypertrophied kidney was irradiated with single doses of 0-20.0 Gy X-rays. Groups of rats, three per dose, were killed at 4, 8, 16 and 24 weeks post-irradiation, their kidneys fixed, and sections stained with the 8-OHdG-specific antibody N45.1. For quantitation of glomerular DNA oxidation with the N45.1 antibody stained sections, 50 glomeruli/animal were counted. The presence of any intensely stained nuclei within the glomerular tuft was scored as positive. Quantitation of tubular DNA oxidation employed a 10 x 10 point ocular grid. Sections were examined at 400 magnification; 250 tubular profiles were counted. All tubules with any nuclear staining were scored as positive.Sham-irradiated kidneys showed little evidence of DNA oxidation over the experimental period. In contrast, localized kidney irradiation led to a marked, dose-independent increase in glomerular and tubular cell nuclear DNA oxidation. This increase was evident at the first time point studied, i.e. 4 weeks after irradiation, and persisted for up to 24 weeks postirradiation. DNA oxidation in the irradiated kidney was only seen in apparently viable glomerular and tubular cells. Thus, while from 16 to 24 weeks post-irradiation structural alterations had progressed to glomerular sclerosis and tubular atrophy, positive staining for 8-OHdG was not observed in severely atrophic tubules. Similarly, fewer positive staining cells were noted in glomeruli undergoing sclerosis, while none were seen in totally sclerotic glomeruli. These data support the hypothesis that renal irradiation is associated with a chronic and persistent oxidative stress.

Radiation-induced kidney injury: a role for chronic oxidative stress?

Robbins ME, Zhao W, Davis CS, Toyokuni S, Bonsib SM

Micron (Oxford, England : 1993) 33 巻 ( 2 ) 頁： 133 - 41 2002年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Micron

Kidney irradiation clearly leads to a progressive reduction in function associated with concomitant glomerulosclerosis and/or tubulointerstitial fibrosis. However, the particular cell types, mediators and/or mechanisms involved in the development and progression of radiation nephropathy remain ill defined. Angiotensin II (Ang II) plays a major pathogenic role; administration of Ang II blockers markedly abrogates the severity of radiation nephropathy in experimental models. Both ionizing radiation and Ang II signal via generation of reactive oxygen species (ROS). Thus, we hypothesized that localized kidney irradiation might lead to a chronic oxidative stress. In view of the difficulty in measuring ROS in vivo we adopted an indirect immunohistochemical approach in which we used a monoclonal antibody specific for 8hydroxy-2′-deoxyguanosine (8-OHdG), one of the most commonly used markers of DNA oxidation. The right kidney of 7-8 week-old male Sprague-Dawley rats was removed. Five to 6 weeks later the remaining hypertrophied kidney was irradiated with single doses of 0-20.0 Gy X-rays. Groups of rats, three per dose, were killed at 4, 8, 16 and 24 weeks post-irradiation, their kidneys fixed, and sections stained with the 8-OHdG-specific antibody N45.1. For quantitation of glomerular DNA oxidation with the N45.1 antibody stained sections, 50 glomeruli/animal were counted. The presence of any intensely stained nuclei within the glomerular tuft was scored as positive. Quantitation of tubular DNA oxidation employed a 10 × 10 point ocular grid. Sections were examined at 400 magnification; 250 tubular profiles were counted. All tubules with any nuclear staining were scored as positive. Sham-irradiated kidneys showed little evidence of DNA oxidation over the experimental period. In contrast, localized kidney irradiation led to a marked, dose-independent increase in glomerular and tubular cell nuclear DNA oxidation. This increase was evident at the first time point studied, i.e. 4 weeks after irradiation, and persisted for up to 24 weeks postirradiation. DNA oxidation in the irradiated kidney was only seen in apparently viable glomerular and tubular cells. Thus, while from 16 to 24 weeks post-irradiation structural alterations had progressed to glomerular sclerosis and tubular atrophy, positive staining for 8-OHdG was not observed in severely atrophic tubules. Similarly, fewer positive staining cells were noted in glomeruli undergoing sclerosis, while none were seen in totally sclerotic glomeruli. These data support the hypothesis that renal irradiation is associated with a chronic and persistent oxidative stress. © 2001 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/s0968-4328(01)00006-3

Target genes in Fenton reaction-induced carcinogenesis: A novel concept 国際誌

Toyokuni, S, Hiroyasu, M, Ozeki, M, Hiai, H

XI BIENNIAL MEETING OF THE SOCIETY FOR FREE RADICAL RESEARCH INTERNATIONAL 頁： 459 - 462 2002年

Comparison between high-performance liquid chromatography and enzyme-linked immunosorbent assay for the determination of 8-hydroxy-2′-deoxyguanosine in human urine 査読有り国際誌

Shimoi K., Kasai H., Yokota N., Toyokuni S., Kinae N.

Cancer Epidemiology Biomarkers and Prevention 11 巻 ( 8 ) 頁： 767 - 770 2002年

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

8-Hydroxy-2′-deoxyguanosine (8-OH-dG), which has been regarded as a potential marker of oxidative DNA damage induced by reactive oxygen species, was measured in human urine by a commercial ELISA using a monoclonal antibody N45.1 and by high-performance liquid chromatography (HPLC) coupled to an electrochemical detector (HPLCECD) to evaluate whether the ELISA system is applicable to human monitoring studies. The urine samples were collected from 120 healthy men ages 18-58 in a steel-manufacturing company. A good correlation (r = 0.833; P < 0.0001) was observed between the two methods on HPLC-purified 8- OH-dG fractions from 23 urine samples. The mean value (±SD) of 8-OH-dG (μg/g creatinine) was 5.47 (±2.97) by HPLC-ECD assay and 5.50 (±2.36) by ELISA. However, the correlation (r) between the two methods on 120 original urine samples was 0.460 [P < 0.001; mean value (±SD) of 8-OH-dG (μg/g creatinine) was 4.46 (±2.03) by the HPLC assay and 9.33 (±3.23) by ELISA]. ELISA estimates were about 2-fold higher than the HPLC estimates on original urine. For an unknown reason, 10% of the urine samples showed more than a 4-fold increase in value by ELISA. It is suggested that the ELISA system is applicable for comparative human monitoring studies. Prepurification of 'samples is required to determine the absolute value of 8-OH-dG in individual urine samples by ELISA.

Amelioration of cisplatin toxicity by a fermented grain food product.

Birth of offspring transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination

Shinohara, T, Inoue, K, Ogonuki, N, Kanatsu-Shinohara, M, Miki, H, Nakata, K, Kurome, M, Nagashima, H, Toyokuni, S, Kogishi, K, Honjo, T, Ogura, A

Human Reproduction 17 巻頁： 3039-3045 - 3045 2002年

Minamiyama Y, Takemura S, Toyokuni S, Nishino Y, Yamasaki K, Hai S, Yamamoto S, Okada S

BioFactors (Oxford, England) 16 巻 ( 3-4 ) 頁： 105 - 15 2002年

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

The most noticeable hypothesis regarding the pathogenesis of cisplatin toxicity, seen mainly in kidney and intestine, is oxidative stress, an imbalance between free-radical generating cisplatin and radical scavenging systems. This paper describes the role of the antioxidant system in cisplatin-induced toxicity and the protective effect by a processed grain food (Antioxidant Biofactor: AOB®), which has been shown to exhibit strong antioxidant activity. Male Fischer 344 rats were used. They were pre-fed either a basal diet (control, 15 g/day) or the diet supplemented with AOB® to provide 6.5% or 20% of total diet throughout the experiment. Cisplatin (5 mg/kg, i.v.) was administered at the start of the experiment, and the animals were sacrificed 5 days later. Blood urea nitrogen (BUN) and plasma creatinine, NO2- and NO3- (NOx) were determined from the plasma. The levels of 4-hydroxy-2-nonenal (a lipid peroxidation product), 8-hydroxy-deoxyguanosine (8-OHdG, an oxidatively modified DNA adduct) and nitrotyrosine were histologically analyzed. The cisplatin administration resulted in a loss of body weight and elevations of BUN, serum creatinine and NOx levels, whereas AOB supplement reversed these effects. The severe morphological damages induced in the kidney and intestine by the cisplatin administration were markedly improved in the AOB group. The levels of lipid peroxidation, 8-OHdG, and nitrotyrosine all paralleled the morphological damage. The AOB effect was dose dependent. In conclusion, the present study suggests that certain food additives like AOB may be of benefit against the side effects of cisplatin.

DOI： 10.1002/biof.5520160306