Yuzo Hayashi

Inhibitory Effects of Antioxidants on N‐Bis(2‐hydroxypropyl)nitrosamine‐induced Lung Carcinogenesis in Rats

Potential second‐stage modifying effects of 8 antioxidants on lung tumorigenesis initiated by N‐bis(2‐hydroxypropyl)nitrosamine (DHPN) were examined in male F344 rats. After an initial 2‐week treatment with DHPN (0.1% in drinking water), rats were administered one of the antioxidants supplemented in the diet for 30 weeks. Although the incidences of lung adenomas were not affected, those of carcinomas were lowered by 2% butylated hydroxyanisole (BHA, 2 rats/20 rats), 1% butylated hydroxytoluene (BHT, 1/20), 0.8% ethoxyquin (EQ, 3/20) and 1%α‐tocopherol (α‐TP, 2/20) treatments as compared to the control level (9/20), while 5% sodium l‐ascorbate (SA), 0.8% catechol (CC), 0.8% resorcinol (RN), and 0.8% hydroquinone (HQ) did not exert any significant effect on incidence. Quantitative analysis of adenomas and carcinomas (numbers and areas of lesions per unit area of lung section) revealed obvious inhibitory effects of SA, CC, and RN as well as BHA, BHT, EQ, and α‐TP. Among the antioxidants, BHT exerted the strongest inhibitory activity. In contrast, DHPN‐induced thyroid tumorigenesis was significantly enhanced by BHT (14/20) and EQ (20/20) treatments (control=5/20). Thus the antioxidants showed opposite effects on lung and thyroid carcinogenesis in the rat.

Overview of genotoxic carcinogens and non-genotoxic carcinogens

It is known that carcinogens designated on the basis of longterm animal test results are extremely diverse in character, both in terms of potencies and the mechanism of action, which leads to complexity in their assessment for cancer risk to humans. The classification of carcin0ogens into two categories, namely, genotoxic and non-genotoxic varieties has been proposed to give a longical foundation on which cancer risk assessment can be reasonably based. The term genotoxic carcinogen indicates a chemical capable of producing cancer by directly altering the genetic material of target cells, while non-genotoxic carcinogen represents a chemical capable of producing cancer by some secondary mechanism not related to direct gene damage. This classification has contributed to the exclusion of various rodent-specific carcinogens from the group of chemicals with potential cancer risk to humans. However, the term, nongenotoxic carcinogen tends to give the mistaken impression that carcinogens shown to be negative for mutagenicity in a series of test systems might be harmless to humans. It should be realized that clear-cut criteria for this classification have not been established because of insufficiencies in the available information concerning mechanisms of action of non-genotoxic carcinogens. Future scientific advances leading to elucidation of the subcellular mechanisms of carcinogenesis are necessary for establishment of the unified, more realistic and mechanism-based approach to cancer risk estimation form exposure to chemicals.

Promoting effect of peroxisome proliferators in two-stage rat renal tumorigenesis.

A two-stage rat renal tumorigenesis model was employed to examine the promoting effects of peroxisome proliferators in the kidney. Groups of 20 male F344 rats were given 0.05% N-ethyl-N-hydroxyethylnitrosamine (EHEN) orally for the first 2 weeks as the initiator. Subsequently they were treated with clofibrate, simfibrate or di(2-ethylhexyl)phthalate (DEHP), respectively, at dietary concentrations of 0.35%, 0.35% and 1.2% for 24 weeks and killed for microscopical examination of the kidney. The incidences of renal cell tumors (RCT) and the numbers of RCT/kidney in rats given DEHP after initiation were significantly increased as compared to the controls. On the other hand, renal promoting effects were not evident in groups given clofibrate or simfibrate. Under the condition of this study, DEHP was found to act as a renal promoter.

Effect of dibutyltin dichloride on incidence of pancreatic adenocarcinoma induced in hamsters by a single dose of N-nitrosobis(2-oxopropyl)amine

The effects of timing of a single intragastric application of dibutyltin dichloride, at a dosage of 30 mg/kg body wt, on N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic carcinomas have been studied in female Syrian golden hamsters. Dibutyltin dichloride, which has been shown to produce selected bile duct injury, was administered either 1 week before or 1 week after a single injection of BOP (20 mg/kg body wt). Control hamsters were injected with BOP alone or were given dibutyltin dichloride alone. The incidence of ductal adenocarcinomas strikingly decreased in hamsters when dibutyltin dichloride was ingested after BOP treatment, but remained unaffected when dibutyltin dichloride was given before carcinogen treatment. Two cases of sarcoma were observed in the group treated with dibutyltin dichloride before BOP injection. The incidence of insulomas, which were considered as spontaneous tumors, was not influenced by dibutyltin dichloride. These results showed that intragastric application of dibutyltin dichloride after BOP treatment significantly reduced the induction of pancreatic cancer. These findings do not support speculations based on epidemiologic studies as to a promoting effect of cholestasis.

Enhancing effect of oxymetholone, an anabolic steroid, on development of liver cell foci in rats initiated with N-diethylnitrosamine

The promoting potential of oxymetholone (OXM) administration on development of liver cell foci was investigated in male F344 rats previously treated with N-diethylnitrosamine (DEN). One week after a single injection of DEN (100 mg/kg, i.p.), rats were given OXM at a dietary level of 0.2% for the first 4 weeks and then at a concentration of 0.1% for an additional 35 weeks. All rats were killed at week 40 for histopathological and immunohistopathological examination of liver tissue. The numbers and areas of both clear cell and glutathione S-transferase placental form (GST-P) positive foci were significantly increased in the group treated with DEN and OXM as compared with the respective values for the DEN alone group. The results thus suggested that OXM possesses promoting potential for rat liver carcinogenesis.

Effects of 2-phenyl-1,4-benzoquinone and 2,5-dihydroxybiphenyl on two-stage mouse skin carcinogenesis

Two metabolites of sodium o-phenylphenate (OPP-Na), 2-phenyl-1,4-benzoquinone (PBQ) and 2,5-dihydroxybiphenyl (5-OH), were examined for initiating, promoting or complete carcinogenic activity for skin carcinogenesis in female CD-1 mice. While PBQ treatment (1 mg per mouse, twice a week for 34 weeks) did cause sustained hyperplasia like 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment (2.5 micrograms, twice/week for 34 weeks), it showed weak, but not statistically significant, tumor promoting potential for skin tumor development initiated with 7,12-dimethylbenz[a]anthracene (DMBA, 10 micrograms x 10 in 5 weeks). On the other hand, 5-OH applied at a dose of 10 mg/mouse using a similar protocol did not exert any promoting influence, and neither of these chemicals administered continually for 40 weeks without prior DMBA initiation treatment induced any skin tumors. Furthermore, both chemicals applied 10 times in 5 weeks at higher doses (2 mg for PBQ and 20 mg for 5-OH) in association with subsequent TPA treatment did not initiate any skin tumor development. Thus, neither of the OPP-Na metabolites demonstrated any capacity to influence skin tumor development in any manner, despite the fact that OPP-Na itself was previously found to exert skin tumor promoting potential in the mouse.

Immunohistochemical and Biochemical Identification of Pepsinogen Isozymes in the Hamster Lungs: Induction by Polychlorinated Biphenyls

Pesinogens are acid protease enzymes of pepsin usually found in gastric mucosa. In the present study, we demonstrated the presence of pepsinogen isozymes in male Syrian golden hamster lung tissues by a combined immunohistochemical and biochemical approach. Immunohistochemically, using rat pepsinogen 1 antibody, pepsinogen positive cells were observed mainly in the epithelia of the terminal bronchioles. They demonstrated morphological features of Clara cells. The pepsinogen isozyme pattern of lung tissue determined by polyacrylamide gel electrophoresis was similar to that of stomach mucosa. Treatment of hamsters with polychlorinated biphenyls at a dose of 500 mg/kg body weight ip caused a 2.8-fold increase in pepsinogen content (p < 0.01) as well as increase in numbers of pepsinogen positive cells in the lung.

Initiation/Promotion Designs in Carcinogenicity Bioassays

Routine carcinogenicity testings have been designed and conducted primarily for providing data to show whether or not the test compounds have a potential of inducing tumors in animals. Therefore, in the case of safety assessment or risk assessment of test compounds in humans, additional data are needed, at times, to learn how intensely the test compound can induce tumors in animals or by which mechanism the test compound can induce tumors in animals. The initiation/promotion experiment is performed as a proceeding for such requests. This paper describes the updating principle and procedures to evaluate initiation effects and promotion effects separately. However, it must be realized that our present knowledge about the initiation/promotion is still limited to some qualitative evidences. Actually, we know very little about mechanistic background and quantitative aspect of the initiation/promotion such as the site or mode of action of promoter action, the organ-specificity of promoter action, or threshold of the initiator action and promoter action. All these problems are necessary to be studied systematically in order that the initiation/promotion design can make a more important contribution to the evaluation of carcinogenicity of chemicals.

Peroxisome proliferation of hepatocytes in rats by a microbial degradation product of cholic acid, 4-(Decahydro-6-methyl-3-oxocyclopenta(f)quinoline-7-yl)valeric acid

Three-week oral administration of 4-(decahydro-6-methyl-3-oxo-cyclopenta(f)quinoline-7-yl)valeric acid (32-1328) in the diet supplemented at concentrations of 0.1% or 0.3% was associated with hepatomegaly and hypotriglyceridemia in male F344 rats. Electron microscopic examination of the liver revealed a remarkable increase of peroxisomes in hepatocytes both in number and size. Biochemically, there were increased activities of peroxisomal marker enzymes including the heat-labile enoyl-CoA hydratase and catalase while the mitochondrial enoyl-CoA hydratase activity was unchanged after feeding of 32-1328. These findings indicate that 32-1328 can exert peroxisome-proliferating activity to rat liver in a manner similar to typical peroxisome proliferators such as clofibrate or di(2-ethylhexyl)phthalate.

Relevance of cellular autonomy to regulatory decisions.

DR. KING: This is the panel discussion. Hopefully, all kinds of ideas and concepts will be coming forward for this discussion. Again, the idea and the objectives are to take the concept that we can have a standard carcinogenicity protocol, and to be able to constructively suggest changes which may, at the end, assist pathologists in the interpretation of lesions which will be used for carcinogenesis evaluation, and more importantly, will be ultimately used for extrapolation to man. We would like to address these questions with the gentlemen before you now. The members of this panel are or have been associated with regulatory agencies. I would like to make it clear that any statements or opinions made by the panelists are based on their own experience and may not represent an official position of the agencies they represent. Our goal is, as it was last year, to attempt to integrate the academic, the indus-