In association with the international validation project to establish an OECD Enhanced Test Guideline 407, we performed a 28-day repeated-dose toxicity study of methoxychlor, a chlorinated hydrocarbon pesticide with pro-estrogenic and anti-androgenic activities. Attention was paid to the sensitivity of certain additional parameters for detecting endocrine related effects of endocrine disrupting chemicals based on the existing TG 407. Seven-week-old Crj:CD(SD)IGS rats were allocated to one of four groups, each consisting often males and ten females, and methoxychlor was administered once daily by gavage at doses of 0 (control), 20, 100 or 500 mg/kg body weight per day. Male rats were killed on the day after the 28th administration. Female rats were killed on the day of the diestrus stage during 4 days after the 28th administration. Male rats receiving methoxychlor showed mainly atrophy of mammary acinus in the 20 mg/ kg and higher groups, together with decreases in prostate and seminal vesicle weights, and atrophy of epididymis, prostate, seminal vesicle and coagulating gland in the 100 and 500 mg/kg groups. In addition, decrease in serum testosterone level, increase in follicle-stimulating hormone level, decrease in testis and epididymis weights, atrophy of semiferous tubules and Leydig cells, decrease in the number of sperm in the caudal epididymis and their motility were observed in the 500 mg/kg group. Female rats receiving methoxychlor showed mainly abnormal estrous cycles, decrease in serum luteinizing hormone level, decrease in ovary weight, proliferation of mammary acinus, atrophy of ovary due to decrease in follicles and corpus luteum in histopathology, hypertrophy of endometrial epithelium of uterus and vagina epithelium in the 100 and 500 mg/kg groups. Among the parameters tested in the present experimental system, effects of methoxychlor on endocrine-related organs were detected with regard to serum hormone, organ weights, histopathological examination in both sexes, estrus cycle in females and sperm examination in males. Based on these results, a no-observed-adverse-effect level (NOAEL) in the present study was estimated to be below 20 mg/kg per day. In particular, the adverse effects were effectively detected in organ weights of accessory sex organs and histopathological examination.

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Dose-dependent promotion effects of combined treatment with sodium nitrite (NaNO 2 ) and ascorbic acid (AsA) on gastric carcinogenesis were examined in rats pretreated with N-methyl-N′ ′ ′ ′-nitro-N-nitrosoguanidine (MNNG). Groups of 15 6-week-old F344 male rats were given 0.01% MNNG in their drinking water for 10 weeks to initiate carcinogenesis in the glandular stomach and a single intragastric administration of 100 mg/kg/ bodyweight of MNNG by stomach tube at week 9 to initiate carcinogenesis in the forestomach. From week 11, they received either drinking water containing 0.05, 0.1 or 0.2% NaNO 2 and a diet supplemented with 0.1 or 0.2% AsA in combination, each individual chemical alone or a basal diet until the end of week 42. In the forestomach, the incidence of hyperplasia was increased dose dependently by the treatment with NaNO 2 alone. Incidences of neoplastic lesions were dramatically increased by the combined treatment with NaNO 2 and AsA in a dosedependent manner, but AsA itself had no effect. In the glandular stomach, only toxicity and regenerative changes were increased by the high-dose combination. In a second short-term experiment conducted for sequential observation, necrosis and strong inflammation were found in the forestomach epithelium shortly after commencing combined treatment with 1.0% AsA and 0.2% NaNO 2 , followed by hyperplasia, whereas there were no obvious effects in the glandular stomach. In addition, after a 4 h treatment with 1.0% AsA and 0.2% NaNO 2 , a slight increase in the 8-hydroxy-deoxyguanosine levels in the forestomach epithelium was observed by high-performance liquid chromatography and an electrochemical detection system, albeit without statistical significance. In vitro, electron spin resonance demonstrated nitric oxide formation during incubation with NaNO 2 and AsA under acidic conditions. Thus, NaNO 2 was demonstrated to exert promoter action in the forestomach, with AsA acting as a strong copromoter through cytotoxicity and regenerative cell proliferation, possibly mediated by oxidative DNA damage, but the combined treatment with NaNO 2 and AsA had little influence on glandular stomach carcinogenesis. (Cancer Sci 2006; 97: 175-182) S odium nitrite is used widely as a food additive to preserve and tinge color-cured meat and fish. We intake nitrate from exogenous sources such as vegetables and water, and this is partly reduced to nitrite in vivo by microflora in the buccal cavity.(1-3) It has been shown that sodium nitrite is a precursor of N-nitroso compounds with strong genotoxic and carcinogenic potential, (4) and an increased incidence of tumors, such as forestomach papillomas and lymphoreticular tumors, has been found in rat carcinogenicity studies of NaNO 2 .(5,6) However, other studies failed to show any effects of nitrite administration on tumor incidence.(7-9) Previously, we reported that when NaNO 2 was administered simultaneously with phenolic antioxidants, such as catechol, 3-methoxycatechol, hydroquinone, tert-butylhydroquinone, gallic acid and pyrogallol...

It is still of importance to investigate renal carcinogenesis by potassium bromate (KBrO 3 ), a by-product of water disinfection by ozonation, for assessment of the risk to man. Five female F344 rats in each group were given KBrO 3 at a dose of 300 mg/kg by single i.g. intubation or at a dose of 80 mg/kg by single i.p. injection, and were killed 48 h after the administration for measurements of thiobarbituric acid-reactive substances (TBARS) and 8-oxodeoxyguanosine (8-oxodG) levels in the kidney. Both levels in the treated animals were significantly elevated as compared with the control values. In a second experiment, 5 male and female F344 rats in each group were administered KBrO 3 at concentrations of 0, 15, 30, 60, 125, 250 and 500 ppm in the drinking water for 4 weeks. KBrO 3 in the drinking water did not elevate TBARS in either sex at any of the doses examined, but 8-oxodG formation in both sexes at 250 ppm and above was significantly higher than in the controls. Additionally, the bromodeoxyuridine-labeling index for proximal convoluted tubules was significantly increased at 30 ppm and above in the males, and at 250 ppm and above in the females. α α α α2u-Globulin accumulation in the kidneys of male rats was increased with statistical significance at 125 ppm and above. These findings suggest that DNA oxidation induced by otassium bromate (KBrO 3 ) was at one time widely used as a maturing agent for flour and as a dough conditioner.1) It was, however, demonstrated to induce renal cell tumors in male and female F344 rats after oral administration for 2 years in the drinking water 2) and the use of KBrO 3 as a food additive is now limited or prohibited, so that exposure of humans via food is very low.3) Nevertheless, there is still concern regarding this chemical in the environment. In order to avoid the formation of trihalomethanes, major by-products in the process of drinking water chlorination 4) that are carcinogenic in rodents, 5) ozone disinfection has been proposed as an alternative method.6) However, it has been shown that ozonation of surface water can generate bromate as one of various by-products in treated drinking water, 7) implying a potential hazard. KBrO 3 has been classified as a genotoxic carcinogen based on positive mutagenicity in the Ames, 8) chromosome aberration 9) and micronucleus tests.10) It has the potential to induce 8-oxodeoxyguanosine (8-oxodG) formation both in vitro and in vivo, [11][12][13][14] and since ribo-and deoxyribonucleosides of 8-oxodG induce sister chromatid exchange in human lymphocytes 15) and 8-oxodG pairs with adenine as well as cytosine, generating GC-to-TA transversion upon replication by DNA polymerases, 16) it has been postulated that this oxidized base is responsible for the mutagenicity and carcinogenicity. 17,18) The formation of oxidized base also indicates that the intra-nuclear redox status is altered in an oxidative direction, and this may lead to the induction of aberrant transcriptional events. However, except for our previous paper, 19) we know of n...

The modification effects of nimesulide, a cyclooxygenase (COX)-2 inhibitor, administration during the postinitiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Male Syrian hamsters were given 4 weekly s.c. injections of BOP at a dose of 10 mg/kg and thereafter administered 0, 100 or 400 ppm nimesulide in the diet for 36 weeks. Additional groups of hamsters were fed 400 ppm nimesulide without prior BOP initiation or nontreated. At week 40, all surviving animals were killed and development of neoplastic and preneoplastic lesions was assessed histopathologically. The incidence of pancreatic adenocarcinomas was significantly (p < 0.05) decreased in the BOP/400 ppm nimesulide group compared to the BOP alone group. The multiplicity of total lesions of pancreatic adenocarcinoma plus atypical hyperplasia was also significantly (p < 0.05) lowered. Immunohistochemically, COX-2 was clearly expressed in pancreatic and lung tumor cells, whereas expression was not remarkably affected by the 400 ppm nimesulide treatment. Proliferating cell nuclear antigen labeling indices of pancreatic ducts were significantly (p < 0.01) reduced by nimesulide. The incidence and multiplicity of neoplastic lesions in other organs did not significantly differ among the BOP-treated groups, though only the multiplicity of lung tumors showed a tendency to decrease. No neoplastic lesions were detected in animals receiving nimesulide alone. Our results clearly indicate that nimesulide protects against BOP-induced pancreatic tumors in hamsters.

Effects of benzyl isothiocyanate (BITC) on urinary bladder carcinogenesis were examined in rats simultaneously treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 20 6-wk-old Fischer 344 male rats were given 10, 100, or 1,000 ppm BITC in the diet or a basal diet with 50 ppm BBN in the drinking water for 40 wk and then killed for autopsy. Additional groups consisting of 10 or 9 rats were similarly given BITC or the basal diet alone without BBN treatment. With BBN treatment, dysplasia, papilloma, and carcinoma incidences and multiplicities were dramatically decreased by simultaneous treatment with BITC in a clear dose-dependent manner. In contrast, epithelial hyperplasia was induced in rats treated with 100 and 1,000 ppm BITC without BBN. These results clearly indicate that although BITC may have weak carcinogenic potency, it is a potent chemopreventive agent against bladder tumor induction by BBN.

In association with the international validation project to establish an OECD Enhanced Test Guideline 407, we performed a 28-day repeated-dose toxicity study of genistein, which is known as a phytoestrogen. Attention was paid to the sensitivity of certain additional parameters, such as histopathology observations and organ weights of endocrine related organs, sperm characteristics, serum hormone levels and estrous cycle, for detecting endocrine-related effects of endocrine-disrupting chemicals based on the existing TG 407. Seven-week-old Crj:CD(SD)IGS rats were assigned to one of four groups, each consisting of ten males and ten females, and genistein was administered once daily by gavage at doses of 0 (control), 120, 400 or 1000 mg/kg body weight per day. Male rats were killed on the day after the 28th administration. Female rats were killed on the day of the diestrus stage during the 4 days after the 28th administration. Endocrine-disrupting effects of genistein were detected in females by histopathology. The changes included vacuolation and mucinification of the vaginal epithelium in the 400 and 1000 mg/kg groups; however, the incidences of the lesion were very low. Although increased serum prolactin levels were recorded in the males of the 1000 mg/kg group, we could not determine whether this was indeed induced by genistein. General toxicological effects of genistein were detected in blood chemistry, such as increased triglycerides and total protein and a decreased albumin/globulin ratio, as well as increased liver weight and glycogen deposition in the periportal hepatocytes. Based on these results, the no-observed-adverse-effect level (NOAEL) in the present study was estimated to be 120 mg/kg per day. In particular, endocrine-related effects were most sensitively detected by histopathology examination of sexual organs. However, the findings indicate that chemicals with weak endocrine-disrupting potential like genistein must be evaluated taking into consideration the results of other test systems.

Combined effects of sodium nitrite (NaNO 2 ) and 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) on liver, colon and Zymbal's gland carcinogenesis were assessed using a rat two-stage carcinogenesis model, with a focus on involvement of oxidative stress. Male 6-week-old F344 rats were given a single intraperitoneal injection of 200 mg/kg of diethylnitrosamine and 4 subcutaneous injections of 40 mg/kg of 1,2-dimethylhydrazine for initiation. Then, they were administered 0 or 300 ppm IQ in the diet or 0, 0.1 or 0.2% NaNO 2 in their drinking water for 27 weeks. The treatment with NaNO 2 1 IQ significantly enhanced colon and Zymbal's gland carcinogenesis and tended to enhance hepatocarcinogenesis. The incidence of lung tumors in the IQ-treated groups was significantly increased as compared with the initiation alone group. In a second experiment, male rats were given IQ or NaNO 2 under the same conditions as before for 1 week, and at sacrifice, their liver and colon tissue or mucosa were collected for analysis of 8-hydroxydeoxyguanosine (8-OHdG), thiobarbituric acid reactive substances (TBARS), acrolein-modified protein and the bromodeoxyuridine-labeling index (BrdU-LI) (in the colon). In the colon, 8-OHdG, acrolein-modified protein levels and BrdU-LI were significantly increased by the combined treatment. These results indicate that the treatment with NaNO 2 enhances IQ-induced colon and Zymbal's gland carcinogenesis in rats and that oxidative DNA damage and lipid peroxidation may partly be involved, especially in the colon. In addition, this experiment showed that IQ can act as a potent lung carcinogen in rats. ' 2005 Wiley-Liss, Inc.Key words: sodium nitrite; 2-amino-3-methylimidazo[4,5-f]quinoline; liver carcinogenesis; colon carcinogenesis; oxidative stress 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), one of the mutagenic and carcinogenic heterocyclic amines (HCA), has been detected in cooked meat and fish 1 and also in cigarette smoke condensate.2 Long-term experiments using rodents have demonstrated that IQ is carcinogenic to the liver, colon, small intestine, Zymbal's gland, clitoral gland and skin in F344 rats 3 and to the liver, forestomach and lung in CDF 1 mice. 4 Metabolically activated IQ forms DNA adducts in liver of rats, mice and monkeys in vivo 1 and in bacteria in vitro.5 In F344 rats, IQ-specific DNA adducts have been detected in the lungs, kidneys, stomach, large intestine, white blood cells and small intestine.6 It was recently suggested that oxidative stress may also participate in HCA carcinogenesis, based on the finding that HCA-induction of preneoplastic or neoplastic lesions was significantly inhibited when antioxidants were given simultaneously 7,8 and that superoxide anion radicals were generated during NADPH/cytochrome P-450 reductase-mediated metabolic activation of HCAs in vitro using a spin-trapping method. 9Sodium nitrite (NaNO 2 ) is used as a color fixative and preservative for meats and fish, and it is also present in vegetables. 10Although it is generally accepted that NaNO 2 is n...