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Metabolism of 2-Acetylaminofluorene by Two 3-Methylcholanthrene-inducible Forms of Rat Liver Cytochrome P-450

Toxicology Research and Testing Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

The present study examines the contribution of two major 3-methylcholanthrene (3-MC)-inducible forms of rat liver cytochrome P-450 (P-448_MC and P-448_HCB) to the metabolism of 2-acetylaminofluorene (AAF). In a reconstituted enzyme system, purified rat liver P-448_MC metabolized AAF at a 10-fold greater rate than P-448_HCB. The major metabolites produced by cytochomre P-448_MC were 3-hydroxy (OH) (30%), 5-OH (24%), 7-OH (22%), and 9-OH (10%). N-OH-AAF (3%) was a minor metabolite. In contrast, P-448_HCB catalyzed the N-hydroxylation of AAF preferentially (15% of total metabolites). The other primary metabolites formed by this isozyme were 7-OH-AAF (30%), 5-OH-AAF (29%), and 9-OH-AAF (25%). Cytochrome P-448_HCB catalyzed the formation of less 3-OH-AAF (7%) than did P-448_MC (30%). Since cytochrome P-448_HCB is immunochemically related to P-448_MC, specific antisera to both isozymes were made by immunoabsorption with the appropriate antigen bound covalently to Sepharose. Anti-P-448_MC inhibited AAF metabolism approximately 43% in microsomes from 3-MC-induced male rats and 30% in microsomes from rats treated with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), another 3-MC-type inducer. Anti-P-448_HCB inhibited total metabolism of AAF by only 22 and 38% in microsomes from 3-MC- and HCB-induced rats. However, anti-P-448_HCB inhibited N-hydroxylation by 60% in both 3-MC- and HCB-induced microsomes. Anti-P-448_MC did not inhibit N-hydroxylation. Neither antibody affected AAF metabolism in control microsomes. These data suggest that, in rat liver, two 3-MC-inducible isozymes of cytochrome P-450 metabolize AAF; however, N-hydroxylation is catalyzed primarily by one of these isozymes, cytochrome P-448_HCB.

¹ To whom requests for reprints should be addressed, at Toxicology Research and Testing Program, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709.

Received 2/15/84. Accepted 5/29/84.