Induction of mammary cytochromes P-450: an essential first step in the metabolism of 7,12-dimethylbenz[a]anthracene by rat mammary epithelial cells.
Rat mammary epithelial cells (RMEC) in culture have been shown to activate polycyclic aromatic hydrocarbon (PAH) carcinogens. This study investigates the role of mammary cytochrome P-450 monooxygenases in these metabolic processes. Monooxygenation of 7,12-dimethylbenz[a]anthracene (DMBA) by RMEC in culture exhibited a 6-h lag period before reaching a constant rate. The mechanism for this time-dependent expression of DMBA monooxygenase activity was investigated in lysed cells, where both conjugation and in situ induction of P-450 are prevented. Although metabolism of DMBA by untreated RMEC lysates was undetectable (less than 1 pmol/mg cell protein/h), prior exposure of cultured cells to benz[a]anthracene (BA) induced DMBA metabolism, (approximately 100 pmol/mg cell protein/h). BA pretreatment also eliminated the lag period for metabolism of DMBA by cultured RMEC but did not prevent additional induction of DMBA monoxygenase activity by the substrate. The distribution of monooxygenated DMBA metabolites formed by BA-induced cell lysates was clearly different from that obtained with purified P-450c, the predominant PAH-inducible isozyme in rat liver. For example, the carcinogen precursor DMBA 3,4-dihydrodiol, which is not formed by P-450c, was a clearly detectable product in RMEC. The low epoxide hydratase activity of BA-induced lysate (approximately 400-fold lower compared to that in the liver) limited formation of all DMBA dihydrodiols. The formation of DMBA 3,4-dihydrodiol increased by 5-fold following addition of exogenous purified epoxide hydratase. The DMBA monooxygenase activity of BA-induced RMEC lysates was completely inhibited by alpha-naphthoflavone but was only partially inhibited (50%) by a polyclonal antibody raised against cytochrome P-450c. Anti P-450c completely inhibited formation of some of the metabolites, partially inhibited formation of others and notably stimulated formation of DMBA 3,4-dihydrodiol by 60%. A polyclonal antibody that recognized both rat hepatic P-450a and a group of P-450 isozymes related to P-450h, and which totally inhibited DMBA 3,4-dihydrodiol formation by rat liver microsomes, did not inhibit formation of any DMBA metabolite in RMEC, including DMBA 3,4-dihydrodiol. Western blot analyses of RMEC homogenates demonstrated that BA pretreatment induces P-450c, but not P-450a or any of the P-450h-related isozymes. We conclude that metabolism of DMBA by RMEC depends on induction of P-450c and at least one additional form of cytochrome P-450 which is immunochemically distinct from rat hepatic P-450a and P-450h related isozymes, but is sensitive to alpha-naphthoflavone.
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000031:analogs & derivatives / Q000378:metabolism*; D000085:Acetates; D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004848:Epithelium / Q000378:metabolism; D005260:Female; D007527:Isoenzymes / Q000096:biosynthesis*; D008321:Mammary Glands, Animal / Q000166:cytology / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D014867:Water
D000085:Acetates; D000906:Antibodies; D007527:Isoenzymes; D014867:Water; C018970:7-hydroxymethyl-12-methylbenz(a)anthracene; D015127:9,10-Dimethyl-1,2-benzanthracene; C007650:ethyl acetate; D003577:Cytochrome P-450 Enzyme System