Mamoru Mutai

Effects of carbon tetrachloride administration on initiation of liver cell foci by the non-hepatocarcinogens N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (B(a)P)

In a development trial for an initiation bioassay system, cell proliferation kinetics after partial hepatectomy (PH) or CCl4 administration (1 ml/kg b.w., i.g.) and the effect of administration time after PH or CCl4 treatment on liver cell foci induction by the direct and indirect non-hepatocarcinogens, N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (B(a)P) were investigated. Male F344 rats were killed 12, 18, 24, 36, 48, 72 or 96 h after PH or CCl4 treatment and liver cell proliferation was examined with the bromodeoxiuridine (BrdU) labeling method. Appreciable increase in the BrdU labeling index was observed 18-36 h after PH with a peak at 24 h, and 18-72 h following treatment with CCl4 with a peak at 48 h. MNNG (80 mg/kg i.g.) or B(a)P (100 mg/kg i.g.) were administered to 7-week-old male F344 rats at various times after PH or CCl4 treatment and lesion induction was assessed using the resistant hepatocyte model. MNNG caused significant numbers of glutathione S-transferase placental form (GST-P)-positive liver cell foci in rats when given 12-36 h after PH, with a peak at 24 h. In contrast, the numbers of foci induced by B(a)P were maximal with exposure at 12 h after PH. In the CCl4 study, both MNNG and B(a)P induced significant increase in GST-P-positive liver cell foci when given 12-72 h after CCl4 treatment, with a peak at 48 h, the results being directly in line with the changes in BrdU labeling. From these findings, it is concluded that initiation assay protocols with a CCl4 proliferative stimulus to hepatocytes may prolong the appropriate administration period for effective detection of the initiation potential of both direct and indirect carcinogens targeting sites other than the liver.

Greater Expression of Transforming Growth Factor α and Proliferating Cell Nuclear Antigen Staining in Mouse Hepatoblastomas Than Hepatocellular Carcinomas Induced by a Diethylnitrosamine-Sodium Phenobarbital Regimen

Transforming growth factor α (TGF-α) is a potent stimulator of normal hepatocyte proliferation, considered to have relationship to the liver regeneration or carcinogenesis. In this study, we investigated immunohistochemically the association between expression of TGF-α and cell proliferation activity in mouse hepatoblastoma s (HBs) and hepatocellular carcinomas (HCCs) induced in B6C3F1 mice by diethylnitrosamine and sodium phenobarbital. The TGF-α-positive rate in HBs (29.2%) was significantly higher than that in HCCs (12.7%). Likewise, the proliferating cell nuclear antigen-positive rate (22.2%) was higher than the HCC value (14.5%). On the individual data for both TGF-α and PCNA, most of the HBs showed higher positive rates than HCCs. In HBs, TGF-α was localized only in the nuclei, whereas some HCC cells stained positive both in their nuclei and cytoplasm (0.6%). These results suggest expression of TGF-α and its localization might be linked to cell proliferation and play a role in malignant progression of mouse HBs.

Dose-dependent promoting effects of catechol on glandular stomach carcinogenesis in BALB/c mice initiated with N-methyl-N-nitrosourea

The effects of cateehol administration in the diet on stomach carcinogenesis in mice after initiation with N‐methyl‐N‐nitrosourea (MNU) in the drinking water were investigated in a development trial for a new experimental protocol. Male 6‐week‐old BALB/c mice were given MNU in the drinking water intermittently for a total of three one‐week periods, with one‐week intervals, at the concentration of 120 ppm (groups 1 and 2). Groups 3 and 4 served as non initiated controls. From week 7, groups 1 and 3 were divided into three subgroups and the mice were fed on diet containing 0.05% (groups la and 3a), 0.2% (groups Ib and 3b), 0.8% (groups 1c and 3c) or 0% (groups 2 and 4) cateehol for 29 weeks. At week 20, appreciably enhanced development of pepsinogen 1‐altered pyloric glands was noted in all catechol‐treated groups, in a partially dose‐dependent manner (12.8±12.5, 13.8±11.7, and 24.0±12.7/100 pyloric glands respectively, for groups 1, 2 and 3). The incidences of adenomas (groups 1, 2 and 3) were also increased. At week 35, dose‐dependent induction of adenocarcinomas in groups 1 (3/19), 2 (3/19) and 3 (14/20) was evident. In addition, the depth of invasion of the adenocarcinomas was enhanced by cateehol in a dose‐dependent manner, though the histological type was not influenced. Thus, the administration of cateehol in the diet strongly enhanced the pre‐neoplastic and neoplastic lesions in mouse glandular stomach induced by MNU in the drinking water, in a dose‐dependent manner

Decreased Expression of Bcl-x Protein during Hepatocarcinogenesis Induced Exogenously and Endogenously in Rats

Dysregulations of apoptosis have been widely recognized as important events in multi‐stage carcinogenesis. Bcl‐x, a member of the Bcl‐2 family, is known to act as a regulator of apoptosis. The present study was conducted to assess the role of altered Bcl‐x protein expression in exogenous and endogenous hepatocarcinogenesis in rats. In the short‐term exogenous models, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 200 mg/kg body weight, partially hepatectomized at the end of week 3, administered phenobarbital at a concentration of 0.05% from the end of week 2 for 6 weeks, and sacrificed. In the livers, glutathione S‐transferase (GST‐P)‐positive, putative preneoplastic lesions were induced, and Bcl‐x protein expression was decreased in 24.7% of such lesions. The incidence of GST‐P‐positive lesions with decreased Bcl‐x increased depending on the size of the lesions; 18.9%, 32.4% and 86.5% in the lesions smaller than 0.03, between 0.03 and 0.3, and larger than 0.3 mm2, respectively. In GST‐P‐positive lesions larger than 0.3 mm2, both apoptosis induction and cell proliferation activity were enhanced when Bcl‐x protein expression was decreased. In the long‐term exogenous models, rats were given 10 mg/kg of DEN, partially hepatectomized 4 h after treatment, administered 0.5 mg/kg of colchicine at the end of days 1 and 3, subjected to a selection procedure, and sacrificed at the end of week 45. Hepatocellular carcinomas were induced with the decreased Bcl‐x protein expression. In the endogenous model, rats were fed a choline‐deficient, l‐amino acid‐defined diet for 16 or 80 weeks and sacrificed. Bcl‐x protein expression was decreased both in GST‐P‐positive lesions and hepatocellular carcinoma. These results suggest that this decrease of Bcl‐x protein might serve as an indicator of the advanced form of preneoplastic lesions, and that this decrease could also be associated with a potential to progress into carcinoma in both exogenous and endogenous hepatocarcinogenesis of rats.