The aim of the proposed research is to elucidate the mechanisms involved in the induction of hepatocellular carcinomas in hamsters and rats induced by synthetic estrogens and to examine the role which Alpha-naphthoflavone (ANF) plays in the tumorigenic process. Other natural and synthetic sex steroids will also be evaluated to determine if this new liver tumor model imitates the clinical causal relationships shown between hepatic tumors and certain anabolic androgens and synthetic progestins. Our general hypothesis is that estrogen intermediates, particularly those involved in the catechol pathway and whose metabolism is modified by 17Alpha-alkyl substitution, are more directly involved in the carcinogenic process in the hamster liver than the parent compound. This implies that steroidal and stilbene estrogens undergo activation in order to elicit tumorigenesis in these rodent livers. The following specific studies are proposed: 1. To determine the affect of ANF on the metabolism of ethinyl estradiol (EE) in liver microsomal incubations. Such studies should indicate any changes in EE metabolite formation that occurs in the presence or absence of ANF treatment. 2. Since a direct role for ANF in the carcinogenic process cannot definitely be ruled out as yet, we propose to evaluate the effect of ANF and synthetic estrogen pretreatment on ANF metabolism using radiolabeled ANF in liver microsomal and hepatocyte incubations. It has been suggested that the inhibitory and stimulatory activity of ANF on P-450 monooxygenases may reside in particular metabolic products of ANF. The proposed studies should provide important insights in our understanding of how a flavonoid and synthetic estrogen effect hepatic cell transformation in these rodent species. 3. An important criteria for demonstrating enhanced formation of potential reactive intermediates of EE in liver microsomes and isolated hepatocytes as a consequence of in-vivo ANF exposure would be to assess the amount of irreversible binding of radiolabeled EE metabolites to hamster liver DNA. These findings will be compared to similar hepatic incubations in non-ANF exposed animals. 4. Oxidative metabolism of estrogens appears pertinent to the carcinogenic activity of these hormones as initial data in our laboratory indicate. Therefore, modulation of epoxide hydrolase, catechol-O-methyltransferase, and estrogen 2-/4-hydroxylase activities will be assessed in livers of ANF treated and deprived hamsters using various estrogen derivatives. 5. We propose to determine if natural estrogens such as equine estrogens are capable of transforming hepatic cells in the presence of ANF and a select number of anabolic androgens and progestins.
