Yozo Kobayashi

Dose-dependent Induction of 8-Hydroxyguanine and Preneoplastic Foci in Rat Liver by a Food-derived Carcinogen, 2-Amino-3, 8-dimethylimidazo [4,5-f] quinoxaline, at Low Dose Levels

Male F344 rats were administered 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) in the diet at doses of 200, 50, 12.5, 3.2, 0.8, 0.2 and 0.05 ppm for 6 weeks, and partially hepatectomized 1 week after the beginning of MeIQx administration. Quantitative values for glutathione S‐transferase placental form (GST‐P)‐positiye foci in the liver were dose‐dependently increased by the MeIQx treatment. 8‐Hydroxyguanine (8‐OHG) levels assessed after 1 week of dietary MeIQx administration were also dose‐dependently increased, although the effect was no longer observed at the end of the treatment period. The correlation between numbers of GST‐P‐positive foci at week 6 and 8‐OHG levels at week 1 was linear, values for both parameters being higher than the control levels even in the 0.8 ppm dose group. These findings indicate that, in addition to the previously reported MeIQx‐DNA adduct formation, DNA modifications due to oxidative damage may play an important role in MeIQx liver carcinogenesis in rats.

Inhibition by Green Tea Extract of Diethylnitrosamine–initiated but Not Cholinedeficient, L–Amino Acid–defined Diet–associated Development of Putative Preneo–plastic, Glutathione S–Transferase Placental Form–positive Lesions in Rat Liver

The effects of green tea extract (GTE) on exogenous and endogenous models of rat liver carcinogenesis using diethylnitrosamine (DEN) and a choline–deficient, L–amino acid–defined (CDAA) diet were studied. For the exogenous carcinogenesis study, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal dose of 200 mg/Kg body weight of DEN, partially hepatectomized at week 3, and administered GTE at doses of 0, 0.01 and 0.1% in the drinking water from week 2 for 10 weeks. For the endogenous carcinogenesis study, rate were fed the CDAA diet and simultaneously given GTE for 12 weeks. All rats were killed at the end of week 12. After DEN–initiation, the apparent numbers of glutathione S–transferase placental form–positive foci, assayed as putative preneoplastic lesions, were decreased by the administration of GTE, though their sizes were not altered. In contrast, GTE did not significantly reduce the numbers of the lesions induced by the CDAA diet or affect their sizes. While the levels of 8–hydroxyguanine, a parameter of oxidative DNA damage, were reduced by the GTE administration in both experimental models, GTE did not protect against the CDAA–diet–associated liver tissue damage in terms of either histology or plasma marker enzyme levels. We conclude that, while GTE may be a possible chemopreventive agent for nitrosamine–initiated hepato–carcinogenesis in the absence of chronic hepatocyte damage, it does not significantly inhibit lesion development in hepatocarcinogenesis associated with the CDAA diet, a cirrhosis–associated model.

Inhibition of Early‐phase Exogenous and Endogenous Liver Carcinogenesis in Transgenic Rats Harboring a Rat Glutathione S‐Transferase Placental Form Gene

Hepatocarcinogenesis initiated with N‐nitrosodiethylamine (DEN) and that initiated by feeding of a choline‐deficient, l‐amino acid‐defined (CDAA) diet were compared in transgenic male Wistar rats harboring a rat glutathione S‐transferase placental form (GST‐P) gene (GST‐P‐Tg rats) and non‐transgenic (N‐Tg) rats. Eight‐week‐old GST‐P‐Tg and N‐Tg rats were administered DEN intraperitoneally at 100 mg/kg body weight, subjected to a selection procedure with 2‐acetylaminofluorene and CCl4, and killed at the end of weeks 5 and 12. Other groups were fed the CDAA diet for 12 weeks and killed. Five weeks after the DEN treatment, numbers and sizes of γ‐glutamyltransferase (GGT)‐ or GST‐P‐positive lesions and 8‐hydroxyguanine (8‐OHG) levels in the livers were significantly less in GST‐P‐Tg rats than in N‐Tg rats. The lesion numbers were unchanged between the ends of weeks 5 and 12 in GST‐P‐Tg rats, but decreased in N‐Tg rats. The lesion sizes were increased in GST‐P‐Tg rats, but unchanged in N‐Tg rats. While the proliferating cell nuclear antigen labeling indices (PCNA L.I.) in and surrounding the lesions were decreased, more prominently in GST‐P‐Tg rats than in N‐Tg rats, the 8‐OHG levels were also decreased but similarly in both cases. After 12 weeks on the CDAA diet, the lesion incidences, numbers and sizes, 8‐OHG levels, PCNA L.I. in and surrounding the lesions, and liver injury were significantly less in GST‐P‐Tg rats than in N‐Tg rats. These results indicate that insertion of a rat GST‐P transgene alters the early phase of exogenous and endogenous rat hepatocarcinogenesis, presumably due to enhanced detoxification by GST‐P expressed both transiently during the initiation and chronically in the altered hepatocyte populations.

Enhancement of hepatocarcinogenesis initiated with diethylnitrosamine or N-nitrosobis(2-hydroxypropyl)amine by a choline-deficient, L-amino acid-defined diet administered prior to the carcinogen exposure in rats.

Effects of pre-administration of a choline-deficient, L-amino acid-defined (CDAA) diet on hepatocarcinogenesis initiated with diethylnitrosamine (DEN) or N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated. A pre-administrating period was set as 1 week, because CDAA diet induces liver injuries by this time-point. In a time-course study, male Fischer 344 rats, 6 weeks old, received a 1-week pre-administration of choline-supplemented, L-amino acid-defined (CSAA) or CDAA diet, DEN at a dose of 100 mg/kg body weight by a single intraperitoneal injection, then CSAA or CDAA diet for up to 8 weeks, and were sacrificed 4, 6 and 8 weeks after DEN. CDAA diet administered only after DEN significantly increased the numbers of glutathione S-transferase placental form (GST-P)-positive lesions 4, 6 and 8 weeks after DEN and their sizes 6 and 8 weeks after DEN. CDAA diet administered both before and after DEN similarly increased the numbers and sizes of GST-P-positive lesions, but with a significantly greater degree than obtained by the diet administered only after DEN. In a dose response study, rats received vechicle or DEN, at a dose of 0.001, 0.01, 0.1, 1, 10, 20, 50, 100 or 200 mg/kg body weight, 1 week after the commencement of CSAA or CDAA diet, and sacrificed 8 weeks after vehicle or DEN. The significant increases of the numbers of GST-P-positive lesions were obtained after 50-200 mg/kg body weight of DEN under the CSAA diet administration, whereas those were detected after 10-200 mg/kg under CDAA diet administration. Sizes became significantly larger with only 200 mg/kg body weight of DEN in the CSAA case but with 50-200 mg/kg in the CDAA case. Male Wistar rats received a 1-week pre-administration of CSAA or CDAA diet, vehicle or BHP, at a dose of 600 or 1200 mg/kg body weight, by a single intraperitoneal injection, then CSAA or CDAA diet for 8 weeks, and were then sacrificed. The numbers of GST-P-positive lesions demonstrated significant increment with 1200 mg/kg body weight of BHP by CDAA diet administered only after BHP and, to a significantly greater degree, by the diet administered both before and after BHP. While CDAA diet administered only after BHP did not alter the sizes of GST-P-positive lesions, the diet administered both before and after 600 and 1200 mg/kg body weight of BHP significantly increased the sizes of the lesions. These results indicate that the pre- plus post-administration of CDAA diet enhances hepatocarcinogenesis initiated with DEN or BHP, more than the post-administration only, thus providing a sensitive model to detect weak liver carcinogenic potency of environmental chemicals.

Preventive Effects of Various Antioxidants on Endogenous Liver Carcinogenesis in Rats Fed a Choline-Deficient, L-Amino Acid-Defined Diet

A choline-deficient, L-amino acid-defined (CDAA) diet is a unique, semisynthetic diet containing no choline and a limited amount of methionine in the absence of any known carcinogens. Chronic feeding of the CDAA diet to male Fischer 344 rats for 1 year causes a high incidence of hepatocellular carcinomas through the development of putative preneoplastic, enzyme-altered, focal hepatocellular lesions. During this process fat accumulation, hepatocyte necrosis followed by regenerative hepatocyte proliferation, oval cell proliferation, and fibrosis reaching a stage of frank cirrhosis occur sequentially in the liver. Although details of the liver carcinogenic mechanisms have not been fully elucidated, oxidative damage to DNA and extra-DNA components in hepatocytes has been believed to play critical roles independently or together. We introduce chronic feeding of the CDAA diet as a unique and useful model for investigating the mechanisms of endogenous carcinogenesis, which is now appreciated as important. We also present results on the preventive effects of various antioxidants on this endogenous rat liver carcinogenesis.