title: Metabolism of the antimammary cancer antiestrogenic agent tamoxifen. I. Cytochrome P-450-catalyzed N-demethylation and 4-hydroxylation.
abstract: Previous studies suggested that the therapeutic effect of the antimammary cancer agent tamoxifen might be related to its metabolism. This study examined the cytochrome P-450 enzymes in rat and human liver catalyzing the metabolism of tamoxifen. Incubations of tamoxifen with rat liver microsomes yielded three major polar metabolites identified as the N-oxide, N-desmethyl, and 4-hydroxy derivatives. N-Oxide formation was catalyzed by the flavin-containing monooxygenase (see part II). Carbon monoxide, SKF-525A, metyrapone, and benzylimidazole strongly inhibited N-demethylation and 4-hydroxylation, indicating the participation of P-450 monooxygenase in these reactions. Antibodies to NADPH-P450 reductase inhibited N-demethylation and 4-hydroxylation. Comparison of the metabolism of tamoxifen in untreated male and female rats demonstrated some sexual dimorphism. N-Demethylation was higher in the male rat and 4-hydroxylation was higher in the female. Treatment of rats with phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), and methylcholanthrene (MC) enhanced N-demethylation, demonstrating the potential participation of multiple P-450s in N-demethylation. Evidence strongly indicates that CYP3A enzyme(s) catalyzes N-demethylation in liver microsomes of PB- and PCN-treated rats (PB and PCN microsomes, respectively): i) N-demethylation was inhibited by cortisol and erythromycin (alternate substrates) and a time-dependent inhibition was observed with troleandomycin (TAO) in vitro; ii) treatment of female rats with TAO, followed by dissociation of the microsomal TAO-P-450 complex, elevated N-demethylation; iii) treatment of PCN-induced female rats with chloramphenicol inhibited N-demethylation; and iv) polyclonal antibodies (PAbs) to CYP3A1 inhibited N-demethylation in PCN- and PB-treated female rats. Although we were unable to reconstitute the N-demethylation activity with purified CYP3A1, which is difficult to reconstitute, collectively the evidence demonstrated that CYP3A enzymes catalyze N-demethylation in PB and PCN microsomes. By contrast, antibodies against CYP2B1/B2 did not inhibit N-demethylation and reconstituted 2B1 did not catalyze N-demethylation of tamoxifen, indicating that 2B1 was not involved. The increase in N-demethylation by MC treatment appears to be due to elevation of CYP1A1/1A2 (P-450c/d). Alternate substrates of CYP1A1/1A2 inhibited N-demethylation and reconstituted rat CYP 1A1-catalyzed N-demethylation. Surprisingly, monoclonal antibodies (MAbs) against CYP1A1/1A2 only partially inhibited, and PAbs against CYP1A1 did not inhibit N-demethylation in MC microsomes, indicating that in MC microsomes, 1A1 does not contribute significantly to that reaction. Mab anti-CYP2C11/2C6 (P-450h/k) inhibited N-demethylation in PB, PCN, and control male rat liver microsomes, suggesting that CYP2C11 and/or CYP2C6 catalyze this reaction to some extent.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
authors.forename: C
authors.initials: C
authors.lastname: Mani
authors.forename: H V
authors.initials: HV
authors.lastname: Gelboin
authors.forename: S S
authors.initials: SS
authors.lastname: Park
authors.forename: R
authors.initials: R
authors.lastname: Pearce
authors.forename: A
authors.initials: A
authors.lastname: Parkinson
authors.forename: D
authors.initials: D
authors.lastname: Kupfer
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000627:therapeutic use; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D004790:Enzyme Induction; D005260:Female; D006801:Humans; D006900:Hydroxylation; D008297:Male; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy*; D008745:Methylation; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012737:Sex Factors; D013379:Substrate Specificity; D013629:Tamoxifen / Q000378:metabolism* / Q000627:therapeutic use
chemical_list: D000970:Antineoplastic Agents; D013629:Tamoxifen; D003577:Cytochrome P-450 Enzyme System