Toyohiko Aoki

Enhancing potential of 6 different carcinogens on multi-organ tumorigenesis after initial treatment with N-methyl-N-nitrosourea in rats

The advantages of applying a whole‐body concept to the assessment of carcinogenic potential of compounds in a two‐stage model after initiation by N‐methyl‐N‐nitrosourea (MNU) were investigated. Male, 6‐week‐old F344 rats were injected with MNU (20 mg/kg, i. p.) twice a week for 4 weeks and they then received 3,2′‐dimethyl‐4‐aminobiphenyl (DMAB) (50 mg/kg, s.c., once a week), N,N′‐dibutylnitrosaraine (DBN) (0.05%, in drinking water), N‐bis(2‐hydroxypropyl)nitrosamine (DHPN) (0.1%, in drinking water), diethylstilbestrol (DES) (2.5 ppm, in diet), sodium o‐phenylphenate (S. OPP) (2%, in diet) or captafol (0.15%, in diet) for 20 weeks. All six carcinogens enhanced the incidences of preneoplastic and neoplastic lesions in their respective target organs: liver, pancreas, small intestine and urinary bladder with DMAB; liver, esophagus, forestomach and urinary bladder with DBN; thyroid, lung, liver, esophagus, forestomach, small intestine and urinary bladder with DHPN; liver and forestomach with DES; and thyroid, forestomach, kidney and urinary bladder with S. OPP; liver and forestomach with captafol. The results suggested that prior treatment with MNU sensitized the tissues to the organotropic carcinogenic potential of chemicals given thereafter for as short a period as 20 weeks. Thus, this system could be utilized as a whole‐body medium‐term bioassay system for the screening of environmental carcinogens, bridging the gap between in vitro mutagenicity and long‐term carcinogenicity tests.

Effects of Modifying Agents on Conformity of Enzyme Phenotype and Proliferative Potential in Focal Preneoplastic and Neoplastic Liver Cell Lesions in Rats

Development of preneoplastic lesions in the rat liver under the influence of various modifiers was investigated with particular attention to changes in simultaneous expression of altered enzyme phenotype within the lesions (conformity) and proliferation potential. Degree of conformity of marker enzymes such as glutathione S‐transferase placental form (GST‐P), glucose‐6‐phosphate dehydrogenase (G6PD), glucose‐6‐phosphatase, adenosine triphosphatase and γ‐glutamyltranspeptidase was compared with levels of S‐bromo‐2‐deoxyuridine labeling. After initiation with diethylnitrosamine, rats were administered the hepatopromoter sodium phenobarbital (PB, 0.05%), the antioxidant ethoxyquin (EQ, 0.5%), or a peroxisome proliferator, clofibrate (CF, 1.0%) or di(2‐ethylhexyl)‐phthalate (0.3%) and killed at week 16 or 32. The PB promoting regimen was clearly associated with increase in the numbers of high conformity class lesions simultaneously expressing three to five enzymes, and elevated proliferation potential. The inhibitor, EQ, in contrast, brought about a time‐dependent decrease in conformity so that only 1 or 2 alterations were most commonly observed at week 32. Lesion populations in the peroxisome proliferator‐ and especially CF‐treated cases were characterized by obvious dissociation between degree of conformity and proliferative status. Such treatment‐dependent differences were not always correlated with the size of the lesion. The results thus suggested that the conformity and proliferation potential of preneoplastic lesions are dependent on modification treatment. Overall, GST‐P was found to be the most reliable marker, although G6PD was less influenced in the peroxisome proliferator cases.

Modifying Effects of Various Chemicals on Tumor Development in a Rat Wide-spectrum Organ Carcinogenesis Model

The efficacy of a wide‐spectrum organ carcinogenesis model for detection of modification potential of exogenous agents was investigated in F344 male rats. Groups of animals were sequentially injected with N‐bis(2‐hydroxypropyl)nitrosamine (1000 mg/kg body weight, i.p., in saline, twice in week 1), N‐ethyl‐N‐hydroxyethylnitrosamine (1500 mg/kg body weight, i.g., in distilled water, twice in week 2) and 3,2′‐dimethyl‐4‐aminobiphenyl (75 mg/kg body weight, s.c., in corn oil, twice in week 3) for wide‐spectrum initiation of target organs and then given one of 10 test chemicals, comprising 6 hepatocarcinogens and 4 non‐hepatocarcinogens, for 12 weeks. All 10 chemicals exerted modifying effects in their respective target organs. Enhancing influence could be detected in the liver and urinary bladder with 2‐acetylaminofluorene, ethionine, and 3′‐methyl‐4‐dimethylaminoazobenzene; in the liver and thyroid with 4,4′‐diaminodiphenylmethane and phenobarbital; in the esophagus and urinary bladder with N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine; in the forestomach and urinary bladder with butylated hydroxyanisole; in the liver with 7,12‐dimethylbenz[a]anthracene and in the liver and lung with 3‐methylcholanthrene. Inhibitory effects on development of glutathione S‐transferase placental form‐positive liver cell foci were observed with clofibrate. The results indicate that the present model can be reliably utilized as a whole body medium‐term bioassay system for assessment of environmental cancer modifiers.

Modulatory interaction between initial clofibrate treatment and subsequent administration of 2-acetylaminofluorene or sodium phenobarbital on glutathione S-transferase positive lesion development

The effects of 2-acetylaminofluorene (2-AFF) or sodium phenobarbital (PB) treatment subsequent to clofibrate (CF) administration in terms of preneoplastic lesion development and induction of hepatocellular carcinomas (HCC) were studied using Fischer 344 rats. Animals received CF (0.3% in diet) for the initial 30 weeks, and then either 2-AAF (0.01% in diet), PB (0.05% in diet) or basal diet until week 78. Further groups were initially given basal diet, and then treated with 2-AAF or PB week 30. Two-thirds partial hepatectomy was carried out on all animals at week 3, sacrifice of representative groups being performed at weeks 30, 48 and 78. No glutathione S-transferase placental form positive (GST-P+) or negative focal or nodular lesions were apparent at the cessation of CF administration. The induction of GST-P+ focal lesions by 2-AAF was markedly decreased at week 48 in the group previously given CF (P less than 0.05) and furthermore, the respective incidences of HCC at week 78 were 4/17 (23.5%) in the CF----2-AAF group and 7/17 (41.2%) in the 2-AAF alone case. No significant differences between CF----PB and PB alone groups were evident with regard to either GST-P+ lesions and HCC at weeks 48 and 78. No CF-specific GST-P negative neoplastic nodules or HCC were observed in any of the experimental groups. These results suggest that pretreatment with CF may inhibit the induction of GST-P+ focal lesions and HCC by subsequently administrated 2-AAF and that CF demonstrates no initiating activity for liver carcinogenesis under the present condition.

Analysis of the Effects of Modifying Agents on Six Different Phenotypes in Preneoplastic Foci in the Liver in Medium-Term Bioassay Model in Rats

Recently a great deal of interest has been expressed in characterizing the altered enzyme phenotype of putative preneoplastic rat liver lesions. In particular, attention has been given to the changes in drug metabolizing potential, conferring physiological advantage to initiated cells, and their usefulness as marker lesions for the analysis of the development of neoplasia1–2.

Spontaneous carcinosarcoma originating from the renal pelvis ina rat.

Carcinosarcoma is a rare neoplasm composed of malignant epithelial and stromal elements, and, for rats, carcinosarcomas in the kidney have not been reported. In a long-term study to gather background data, we encountered a spontaneous carcinosarcoma originating from the renal pelvis with metastasis to the lung. At necropsy, a mass was observed in the abdominal cavity, and white nodules were scattered in lung lobes. Microscopically, there was polypoid hyperplasia of the urothelium accompanied by hyperplasia of spindle stromal cells in the pelvis. The intra-abdominal tumor was composed of epithelial and stromal elements; in the lung, the tumor cells invaded along alveoli/bronchi and occasionally invaded the parenchyma from the blood vessels. Immunohistochemical and electron microscopic examinations revealed that the epithelial element consisted of transitional epithelial cells and that the stromal element consisted of lipoblasts. The tumor was diagnosed as a carcinosarcoma originating from the renal pelvis, and this is the first report of a carcinosarcoma originating from the renal pelvis in a rat.