Ethynylpyrene functions as a selective suicide substrate of the cytochrome P-450c dependent benzo[a]pyrene hydroxylase activity in rat liver microsomes. Ethynylpyrene can also decrease mammalian cell-mediated mutagenesis by 7,12-dimethylbenz[2]anthracene. Ethynylpyrene may therefore be a representative of a class of compounds which could be used to block the P-450 dependent conversions of polycyclic arylhydrocarbons into their mutagenic and carcinogenic derivatives. Reconstituted monooxygenase systems containing purified cytochrome P-450b or P-450c will be used to study the mechanism responsible for the inhibitory action of ethynylpyrene upon benzo[a]pyrene hydroxylase. The products of the P-450 dependent metabolism of ethynylpyrene will be determine, the partition ratio for product release and enzyme inactivation established, the presence of an ethynylpyrene derivative covalently attached to the P-450 proteins investigated, and a potential mechanism of the inhibition tested. A series of compounds related to ethynylpyrene will be synthesized and structure-activity relationships determined. The effects of modifying both the aryl nucleus and the potential """"""""suicide substituent"""""""" of these structures upon P-450 dependent activities will be established. The effects of the different structural features upon the selectivity of the potential suicide substrates for the benzo[a]pyrene hydroxylase, benzphetamine demethylase, ethylmorphine demethylase, ethoxyresofurin deethylase, and 17-Beta-estradiol 2-hydroxylase activities of microsomes, as well as the effects of these different features upon P-450 destruction will be determined. The effects of selective suicide substrates upon the distribution of metabolites produced from R- and S-warfarin and from 2-acetylaminofluorene by rat liver microsomes, and from benzo[a]pyrene by human epidermal keratinocytes, will be analyzed. The series of compounds related to ethynylpyrene will also be tested as inhibitors of the cell-mediated mutagenesis due to 7,12-dimethylbenz[a]anthracene metabolism. A suicide inhibitor of P-450c will also be tested in vivo as an inhibitor of arylhydrocarbon tumorigenesis in mice. The results of this proposed research could lead ultimately to the design of selective suicide substrates for the P-450 dependent monooxygenases. Such inhibitors could be used to selectively inactivate the P-450 dependent isozymes involved converting procarcinogens such as benzo[a]pyrene into their carcinogenic metabolites.
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