EVIDENCE THAT THE CATECHOL 3,4-DIHYDROXYTAMOXIFEN IS A PROXIMATE INTERMEDIATE TO THE REACTIVE SPECIES BINDING COVALENTLY TO PROTEINS

Metabolism of tamoxifen by rat and human hepatic microsomal cytochrome P450s (CYPs) forms a reactive intermediate that irreversibly binds to microsomal proteins (C. Mani and D, Kupfer, Cancer Res., 51: 6052-605 8, 1991). The current study examines the nature of the tamoxifen metab olite that is proximate to the reactive intermediate(s). The rate of c ovalent binding of tamoxifen metabolites, tamoxifen N-oxide, N-desmeth yltamoxifen, and tamoxifen N-oxide-epoxide was approximately equal to or less than that of tamoxifen. By contrast, covalent binding of 4-hyd roxytamoxifen (4-OH-tam) was 3-5-fold higher than that of tamoxifen, i ndicating that among the metabolites examined, 4-OH-tam or its metabol ite(s) is most proximate to the reactive intermediate(s), Incubation o f 4-OH-tam with liver microsomes from PCN-treated rat yielded three de tectable metabolites. One was identified as 4-OH-tam N-oxide via its f acile reduction back to 4-OH-tam by titanium(III) chloride. Another me tabolite of 4-OH-tam, assumed to be 3,4-dihydroxytamoxifen (3,4-di-OH- tam) catechol, was demonstrated by its monomethylation with [H-3]S-ade nosyl-L-methionine ([H-3]SAM) in the presence of endogenous catechol-O -methyltransferase. Monomethylated catechol from 4-OH-tam was formed a t a 3-4-fold higher rate than from tamoxifen. It was reasoned that if the catechol is the most proximate metabolite to the reactive intermed iate, then its methylation would reduce the formation of the reactive intermediate and result in lower rate of covalent binding. In fact, ad dition of radioinert SAM to incubations of tamoxifen inhibited covalen t binding by 17-23%. By contrast, inclusion of 1.0 mM S-adenosyl-L-hom ocysteine, a potent inhibitor of catechol-O-methyltransferase-mediated methylation of 3,4-di-OH-tam, essentially overcame the inhibition of the covalent binding by SAM. Additionally, ascorbic acid and glutathio ne, inhibitors of covalent binding of tamoxifen, produced an elevation of methylated catechol. These findings collectively indicate that 3,4 -di-OH-tam is proximate to the ultimate reactive intermediate that res ults in covalent binding to microsomal proteins.