Yuji Kurokawa

Preventive effects of green tea against liver oxidative DNA damage and hepatotoxicity in rats treated with 2-nitropropane

Male rats were given 2% green tea as their drinking water for 2 wk before a single ip injection of the carcinogen 2-nitropropane (2NP) (100 mg/kg body weight) and liver nuclear 8-hydroxydeoxyguanosine (8-OHdG) levels and hepatotoxicity parameters were determined 6 or 15 hr thereafter. The increase of 8-OHdG adducts in liver nuclear DNA caused by 2NP was depressed 50% at both time points with the green tea pretreatment. The time-dependent elevations of serum aminotransferases and lactate dehydrogenase values by 2NP were also effectively prevented. However, green tea had no obvious effects on the falls in serum lipid peroxide and triglyceride levels associated with carcinogen exposure. Increases of hepatic lipid peroxide levels with 2NP were depressed 100 and 30%, at 6 and 15 hr, respectively, by green tea and the decrease in hepatic glycogen content at 6 hr was clearly alleviated. Histopathological examination revealed effective protection against induction of hepatic degenerative changes by 2NP at 15 hr. Drinking crude catechin extract solution with the same concentration of (-)epigallocatechin gallate as green tea provided protection at 6 hr, but with only half the effectiveness. These findings demonstrate that green tea can effectively block oxidative DNA damage to the liver as well as hepatotoxicity in rats treated with 2NP.

The protective role of glutathione, cysteine and vitamin C against oxidative DNA damage induced in rat kidney by potassium bromate

The roles of glutathione (GSH), cysteine, vitamin C., liposome‐encapsulated superoxide dismutase (L‐SOD) and vitamin E in preventing oxidative DNA damage and cytotoxicity in the rat kidney after administration of potassium bromate (KBrO3) to male F344 rats were investigated by measuring 8‐hydroxydeoxyguanosine (8‐OH‐dG), an oxidative DNA product, lipid peroxidation (LPO) levels and relative kidney weight (RKW). Combined pre‐ and posttreatment of animals with 2 × 800 mg/kg GSH i.p. inhibited the increase of 8‐OH‐dG, LPO levels and RKW caused by 80 mg/kg KBrO3 i.p. administration. In contrast, pretreatment with 0.3 ml/kg diethylmaleate (DEM) i.p., a depletor of tissue GSH, was associated with elevation of 8‐OH‐dG, LPO levels and RKW after a 20 mg/kg KBrO3 i.p. treatment, which itself caused no change. Administration of KBrO3 itself reduced renal non‐protein thiol levels, but this was inhibited by the two doses of exogenous GSH. Combined treatment with DEM and KBrO3 lowered the non‐protein thiol level in the kidney more than did DEM treatment alone. Protective effects against the oxidative damage caused by KBrO3 were also observed for pre‐ and posttreatment with 400 mg/kg cysteine i.p., another sulfhydryl compound, and daily i.g. application of 200 mg/kg vitamin C for 5 days. However, no influence was evident after pre‐ and posttreatment with 18,000 U/kg L‐SOD i.p. or daily i.g. 100 mg/kg of vitamin E for 5 days. The results suggest that intracellular GSH plays an essential protective role against renal oxidative DNA damage and nephrotoxicity caused by KBrO3.

A Collaborative Study in Japan on Optimal Treatment Period and Parameters for Detection of Male Fertility Disorders Induced by Drugs in Rats

The tripartite-harmonized International Conference on Harmonization reproductive guideline (1993) recommends administration of test substances for 4 weeks to male rats before mating. However, scientific or experimental rationale for this recommendation is not firmly based, and the most appropriate parameters have not been established in experimental models. Therefore, a team consisting of 16 Japanese pharmaceutical companies and the National Institute of Health Sciences performed a collaborative study to determine the optimal period and parameters for detection of male fertility disorders in rats. Sixteen compounds, including four anticancer drugs, two psychotropic drugs, two nootropic drugs, two vitamins, two hormones, one antihypertensive agent, one diuretic drug, and two general chemicals were administered to male rats for 4 or 9 weeks before mating. Parameters used to examine effects on the male reproductive system were organ weights, sper-matogenic endpoints, mating behavior, cesarean section findings, and histopathology. From the results, treatment for 4 weeks before mating was concluded to be sufficient to detect adverse effects on male fertility, with the histopathology of the testis being the most sensitive index for the drugs used. Sperm parameters, especially number, and genital organ weight determination provided information confirming toxicity. Tests of reproductive activity were generally found to be insensitive, except where the drugs affected sperm maturation. Based on this study, it is concluded that a 4-week treatment period is appropriate for detection of drug effects on male fertility, and that histopath-ological examination of the testis is the most sensitive approach.

Long-term toxicity/carcinogenicity of musk xylol in B6C3F1 mice

The long-term toxicity/carcinogenicity of musk xylol, a synthetic nitro musk, was examined in B6C3F1 mice of both sexes. Musk xylol was administered at dietary levels of 0 (control), 0.075 or 0.15% for 80 wk. The overall tumour incidences in all treated groups of both sexes were significantly higher than those in the corresponding controls. Combined malignant and benign liver cell tumours were clearly increased in both sexes, and in males a positive significant trend was also noted for the occurrence of hepatocellular carcinomas. In males the incidence of Harderian gland tumours was also significantly greater in treated groups than in controls. Some other neoplasms, such as lung tumours in both sexes and Harderian gland tumours and lymphomas in females, occurred in greater numbers in the treated groups, although the differences were not statistically significant in comparison with the controls. In addition, the incidences and total numbers of malignant tumours were significantly increased in treated groups of both sexes, although the increases were not dose dependent. The results demonstrated that musk xylol is carcinogenic in B6C3F1 mice when given at dose levels of 0.075 or 0.15% in the diet for 80 wk.

Oxidative DNA damage, lipid peroxidation and nephrotoxicity induced in the rat kidney after ferric nitrilotriacetate administration

8-Hydroxydeoxyguanosine (8-OH-dG) was examined in the kidneys of rats after single i.p. administration of ferric nitrilotriacetate (Fe-NTA) for variable periods of time at various doses along with the measurement of lipid peroxidation and serum biochemical parameters and histopathological examination. Though lipid peroxide level increased rapidly and decreased sharply after reaching a much higher peak 1 h after treatment, significant higher levels of 8-OH-dG were observed at 1, 6 and 24 h after injection. On the other hand, the increase of 8-OH-dG formation was observed in a similar dose-dependent manner to the appearance of nephrotoxic responses in terms of serum biochemical and histopathological changes.

Relationship between hepatic peroxisome proliferation and 8-hydroxydeoxyguanosine formation in liver DNA of rats following long-term exposure to three peroxisome proliferators; di(2-ethylhexyl) phthalate, aluminium clofibrate and simfibrate

To elucidate the relationship between hepatic peroxisome proliferation and oxidative DNA damage induced by hepatocarcinogenic peroxisome proliferators, 3 agents, namely, di(2-ethylhexyl) phthalate (DEHP, aluminium clofibrate and simfibrate were fed at doses of 1.2%, aluminium clofibrate 0.5% and 0.5% in the diet, respectively, to male F-344 rats for up to 1 year. Evidence of hepatic peroxisome proliferation and 8-hydroxydeoxyguanosine (8-OH-dG) formation in liver and kidney DNA were assessed at 1, 2, 3, 6, 9 and 12 months. Peroxisomal beta-oxidation enzyme activities were increased 3- to 8-fold and catalase was elevated to 1.4- to 2.2-fold the control level by DEHP, aluminium clofibrate and simfibrate from months 1 to 12 of the treatment. 8-OH-dG levels in liver DNA of DEHP-, aluminium clofibrate- and simfibrate-fed rats were increased approximately 2-fold after 1 month, the tendency for elevation also being observed in the liver DNA at 2, 3, 9 and 12 months. The results thus clearly demonstrate that persistent peroxisome proliferation in the liver leads to continued specific oxidative DNA damage.

Studies on the promoting and complete carcinogenic activities of some oxidizing chemicals in skin carcinogenesis

Six oxidizing chemicals were tested for promoting and complete carcinogenic activities in skin carcinogenesis using female Sencar mice. In the promotion tests, the chemicals were applied twice a week for 51 weeks after initiation with dimethylbenzanthracene (DMBA). In the tests for complete carcinogenic activities, the chemicals alone were applied for 51 weeks. Benzoyl peroxide was found to be a potent promoter as reported previously. Moreover, possible complete carcinogenic action of this chemical was found in this study. Potential promoting effect was suspected in sodium chlorite. Potassium bromate, ammonium persulphate, hydrogen peroxide and sodium hypochlorite were inactive either as a promoter or a complete carcinogen.

Short-term exposure to the peroxisome proliferators, perfluorooctanoic acid and perfluorodecanoic acid, causes significant increase of 8-hydroxydeoxyguanosine in liver DNA of rats

To elucidate the relationship between peroxisome proliferation by perfluorinated compounds and oxidative DNA damage, perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), perfluorobutyric acid (PFBA) and perfluorooctane (PFO) were administered to 6-week-old F-344 male rats. After a single intraperitoneal (i.p.) injection of PFOA, PFBA or PFO in corn oil at a dose of 100 mg/kg, significant increases of liver weight and 8-hydroxydeoxyguanosine (8-OH-dG) levels in liver DNA were observed in PFOA-treated rats. Oral administration of powdered diet containing 0.02% PFOA or 0.01% PFDA for 2 weeks resulted in significant increases of liver weight and 8-OH-dG levels in liver DNA in rats given both chemicals. On the other hand, no increase in 8-OH-dG levels in kidney DNA was found in either of the studies. Our results demonstrate that, as with other peroxisome proliferators (phthalic ester plasticizers and hypolipidemic drugs), PFOA and PFDA induced peroxisome proliferation also leads to organ specific oxidative DNA damage.

Protective Effects of Green Tea on Hepatotoxicity, Oxidative DNA Damage and Cell Proliferation in the Rat Liver Induced by Repeated Oral Administration of 2-Nitropropane

To evaluate the benefit of green tea in mitigating hazards caused by repeated exposure of 2-nitropropane (2NP), we examined the effects of the tea on toxic indices, oxidative DNA damage and cell proliferation in the liver of 2NP-treated rats. Male Fischer 344 rats were administered, by gastric intubation, a total of six doses of 60 mg/kg 2NP(L), or alternatively two doses of 90 mg/kg and then four doses of 120 mg/kg 2NP(H) during 2 weeks. Green tea infusion was given to the rats as drinking water 1 week before the 2NP treatments and throughout the experiment. Significant elevation of hepatotoxic indices was evident in the 2NP(H)-treated group, such as an increase of serum glutamic-oxaloacetic transaminase (GOT) activity and of hepatic lipid peroxidation, together with a decrease in hepatic glycogen and serum triglyceride, and degenerative changes in the hepatocytes. A dose-related increase was observed in oxidative DNA damage and cell proliferation in the liver. Green tea effectively inhibited all of above changes induced by 2NP treatment, suggesting that tea intake may be effective for preventing the hepatic injuries after chronic exposure to 2NP.

Chronic toxicity study on formaldehyde administered orally to rats

Groups of 20 male and 20 female Wistar rats were given formaldehyde solution in their drinking water at concentrations of 0.50, 0.10, 0.02 and 0% for 24 months. Significant decreases in body weight and food and water intake were observed in the 0.50% group of both sexes and all rats in this group died by 24 months. Various non-neoplastic lesions were observed in rats, mostly in the 0.50% group. In this group, erosions and/or ulcers were evident in both the forestomach and glandular stomach. In the forestomach, squamous cell hyperplasia with or without hyperkeratosis and downward growth of basal cells were observed. Glandular hyperplasia of the fundic mucosa was noted along the limiting ridge. A few of such changes of the upper GI tract were seen in the 0.10% group. No toxicological abnormalities were found in 0.02% group of both sexes. There were no significant differences in the incidences of any tumors among groups of both sexes. Based on these findings, the no observable effect level of formaldehyde was 0.02% in the drinking water (10 mg/kg body wt./day).