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Evidence that the catechol 3,4-Dihydroxytamoxifen is a proximate intermediate to the reactive species binding covalently to proteins.
Cancer Res. 1996 Mar 15; 56(6):1283-90.CR

Abstract

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 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 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.

Authors+Show Affiliations

Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8640815

Citation

Dehal, S S., and D Kupfer. "Evidence That the Catechol 3,4-Dihydroxytamoxifen Is a Proximate Intermediate to the Reactive Species Binding Covalently to Proteins." Cancer Research, vol. 56, no. 6, 1996, pp. 1283-90.
Dehal SS, Kupfer D. Evidence that the catechol 3,4-Dihydroxytamoxifen is a proximate intermediate to the reactive species binding covalently to proteins. Cancer Res. 1996;56(6):1283-90.
Dehal, S. S., & Kupfer, D. (1996). Evidence that the catechol 3,4-Dihydroxytamoxifen is a proximate intermediate to the reactive species binding covalently to proteins. Cancer Research, 56(6), 1283-90.
Dehal SS, Kupfer D. Evidence That the Catechol 3,4-Dihydroxytamoxifen Is a Proximate Intermediate to the Reactive Species Binding Covalently to Proteins. Cancer Res. 1996 Mar 15;56(6):1283-90. PubMed PMID: 8640815.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Evidence that the catechol 3,4-Dihydroxytamoxifen is a proximate intermediate to the reactive species binding covalently to proteins. AU - Dehal,S S, AU - Kupfer,D, PY - 1996/3/15/pubmed PY - 1996/3/15/medline PY - 1996/3/15/entrez SP - 1283 EP - 90 JF - Cancer research JO - Cancer Res VL - 56 IS - 6 N2 - 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 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 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. SN - 0008-5472 UR - https://www.unboundmedicine.com/medline/citation/8640815/Evidence_that_the_catechol_34_Dihydroxytamoxifen_is_a_proximate_intermediate_to_the_reactive_species_binding_covalently_to_proteins_ L2 - http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=8640815 DB - PRIME DP - Unbound Medicine ER -