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Cytochrome P-450-mediated activation and irreversible binding of the antiestrogen tamoxifen to proteins in rat and human liver: possible involvement of flavin-containing monooxygenases in tamoxifen activation.
Cancer Res. 1991 Nov 15; 51(22):6052-8.CR

Abstract

Tamoxifen (TXF), a triphenylethylene antiestrogen, is the major therapeutic agent for breast cancer. In rare cases, TXF treatment appears to increase incidence of endometrial cancer. Also in rats, TXF was found to induce hepatocellular carcinoma. Previous studies suggested that metabolism of TXF may contribute to its antiestrogenic and anticancer activity. The current study demonstrates a novel route of TXF metabolism. TXF is metabolized by rat and human liver microsomes into a reactive intermediate (txf*) which binds irreversibly to microsomal proteins. The binding requires NADPH and O2 and is inhibited by CO, inhibitors of P-450, and antibodies to rat NADPH-P450 reductase, indicating catalysis by P450. Phenobarbital treatment of rats markedly increases binding, suggesting the involvement of induced P450s. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from incubation of [14C] TXF with phenobarbital-treated microsomes exhibits a major radiolabeled zone which corresponds to a molecular weight of approximately 54,000, suggesting binding to a P-450. Cysteine and glutathione inhibited the binding of TXF without significantly affecting P-450-mediated metabolism of TXF, possibly by reacting with txf* or by competing for the same binding sites. Exposure of phenobarbital-treated microsomes and control-microsomes to 50 degrees C for 90 s, which inactivates the flavin-containing monooxygenase (FMO), diminished binding and pH 8.6 enhanced binding. Also, alternate FMO substrates inhibited binding. These findings indicate that P-450 and possibly FMO catalyze the reactions leading to the formation of txf*. However, incubations with single-labeled and dual-radiolabeled tamoxifen or with [14C]TXF-N-oxide demonstrated that monodesmethyl-TXF and TXF-N-oxide, the principal P-450 and FMO-mediated metabolites, respectively, are not on the major route of txf* formation, indicating that txf* could not be an aldehyde derived from tamoxifen nitrone. Thus, though the structure of txf* was not characterized, certain possibilities were excluded. Speculations on the structure of txf* and on its possible pharmacological and toxicological activity are presented.

Authors+Show Affiliations

Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.No affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

1933868

Citation

Mani, C, and D Kupfer. "Cytochrome P-450-mediated Activation and Irreversible Binding of the Antiestrogen Tamoxifen to Proteins in Rat and Human Liver: Possible Involvement of Flavin-containing Monooxygenases in Tamoxifen Activation." Cancer Research, vol. 51, no. 22, 1991, pp. 6052-8.
Mani C, Kupfer D. Cytochrome P-450-mediated activation and irreversible binding of the antiestrogen tamoxifen to proteins in rat and human liver: possible involvement of flavin-containing monooxygenases in tamoxifen activation. Cancer Res. 1991;51(22):6052-8.
Mani, C., & Kupfer, D. (1991). Cytochrome P-450-mediated activation and irreversible binding of the antiestrogen tamoxifen to proteins in rat and human liver: possible involvement of flavin-containing monooxygenases in tamoxifen activation. Cancer Research, 51(22), 6052-8.
Mani C, Kupfer D. Cytochrome P-450-mediated Activation and Irreversible Binding of the Antiestrogen Tamoxifen to Proteins in Rat and Human Liver: Possible Involvement of Flavin-containing Monooxygenases in Tamoxifen Activation. Cancer Res. 1991 Nov 15;51(22):6052-8. PubMed PMID: 1933868.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cytochrome P-450-mediated activation and irreversible binding of the antiestrogen tamoxifen to proteins in rat and human liver: possible involvement of flavin-containing monooxygenases in tamoxifen activation. AU - Mani,C, AU - Kupfer,D, PY - 1991/11/15/pubmed PY - 1991/11/15/medline PY - 1991/11/15/entrez SP - 6052 EP - 8 JF - Cancer research JO - Cancer Res VL - 51 IS - 22 N2 - Tamoxifen (TXF), a triphenylethylene antiestrogen, is the major therapeutic agent for breast cancer. In rare cases, TXF treatment appears to increase incidence of endometrial cancer. Also in rats, TXF was found to induce hepatocellular carcinoma. Previous studies suggested that metabolism of TXF may contribute to its antiestrogenic and anticancer activity. The current study demonstrates a novel route of TXF metabolism. TXF is metabolized by rat and human liver microsomes into a reactive intermediate (txf*) which binds irreversibly to microsomal proteins. The binding requires NADPH and O2 and is inhibited by CO, inhibitors of P-450, and antibodies to rat NADPH-P450 reductase, indicating catalysis by P450. Phenobarbital treatment of rats markedly increases binding, suggesting the involvement of induced P450s. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from incubation of [14C] TXF with phenobarbital-treated microsomes exhibits a major radiolabeled zone which corresponds to a molecular weight of approximately 54,000, suggesting binding to a P-450. Cysteine and glutathione inhibited the binding of TXF without significantly affecting P-450-mediated metabolism of TXF, possibly by reacting with txf* or by competing for the same binding sites. Exposure of phenobarbital-treated microsomes and control-microsomes to 50 degrees C for 90 s, which inactivates the flavin-containing monooxygenase (FMO), diminished binding and pH 8.6 enhanced binding. Also, alternate FMO substrates inhibited binding. These findings indicate that P-450 and possibly FMO catalyze the reactions leading to the formation of txf*. However, incubations with single-labeled and dual-radiolabeled tamoxifen or with [14C]TXF-N-oxide demonstrated that monodesmethyl-TXF and TXF-N-oxide, the principal P-450 and FMO-mediated metabolites, respectively, are not on the major route of txf* formation, indicating that txf* could not be an aldehyde derived from tamoxifen nitrone. Thus, though the structure of txf* was not characterized, certain possibilities were excluded. Speculations on the structure of txf* and on its possible pharmacological and toxicological activity are presented. SN - 0008-5472 UR - https://www.unboundmedicine.com/medline/citation/1933868/Cytochrome_P_450_mediated_activation_and_irreversible_binding_of_the_antiestrogen_tamoxifen_to_proteins_in_rat_and_human_liver:_possible_involvement_of_flavin_containing_monooxygenases_in_tamoxifen_activation_ L2 - http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=1933868 DB - PRIME DP - Unbound Medicine ER -