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Evidence That the Catechol 3,4-Dihydroxytamoxifen Is a Proximate Intermediate to the Reactive Species Binding Covalently to Proteins^1,2,

Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545

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: 6052–6058, 1991). The current study examines the nature of the tamoxifen metabolite that is proximate to the reactive intermediate(s). The rate of covalent binding of tamoxifen metabolites, tamoxifen N-oxide, N-desmethyltamoxifen, and tamoxifen N-oxide-epoxide was approximately equal to or less than that of tamoxifen. By contrast, covalent binding of 4-hydroxytamoxifen (4-OH-tam) was 3–5-fold higher than that of tamoxifen, indicating that among the metabolites examined, 4-OH-tam or its metabolite(s) is most proximate to the reactive intermediate(s). Incubation of 4-OH-tam with liver microsomes from PCN-treated rat yielded three detectable metabolites. One was identified as 4-OH-tam N-oxide via its facile reduction back to 4-OH-tam by titanium(III) chloride. Another metabolite of 4-OH-tam, assumed to be 3,4-dihydroxytamoxifen (3,4-di-OH-tam) catechol, was demonstrated by its monomethylation with [³H]S-adenosyl-L-methionine ([³H]SAM) in the presence of endogenous catechol-O-methyltransferase. Monomethylated catechol from 4-OH-tam was formed at a 3–4-fold higher rate than from tamoxifen. It was reasoned that if the catechol is the most proximate metabolite to the reactive intermediate, then its methylation would reduce the formation of the reactive intermediate and result in lower rate of covalent binding. In fact, addition of radioinert SAM to incubations of tamoxifen inhibited covalent binding by 17–23%. By contrast, inclusion of 1.0 mM S-adenosyl-L-homocysteine, 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 glutathione, 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 results in covalent binding to microsomal proteins.

¹ A preliminary account of a portion of this study was presented at the Sixth International Society for the Study of Xenobiotics Meeting (1994) in Raleigh, North Carolina.

² Financial support provided by United States Public Health Service Grant ES00834 from the National Institute for Environmental Health Sciences is gratefully acknowledged.

³ To whom requests for reprints should be addressed, at Worcester Foundation for Biomedical Research, 222 Maple Avenue, Shrewsbury, MA 01545. Phone: (508) 842-8921; Fax: (508) 842-9632.

Received 10/11/95. Accepted 1/12/96.

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