title: Bioactivation of the cancer chemopreventive agent tamoxifen to quinone methides by cytochrome P4502B6 and identification of the modified residue on the apoprotein.
abstract: The nonsteroidal antiestrogen tamoxifen was introduced as a treatment for breast cancer 3 decades ago. It has also been approved as a chemopreventive agent and is prescribed to women at high risk for this disease. However, several studies have shown that use of tamoxifen leads to increased risk of endometrial cancer in humans. One potential pathway of tamoxifen toxicity could involve metabolism via hydroxylation to give 4-hydroxytamoxifen (4OHtam), which may be further oxidized to form a quinone methide. CYP2B6 is a highly polymorphic drug-metabolizing enzyme, and it metabolizes a number of clinically important drugs. Earlier studies from our laboratory have shown that tamoxifen is a mechanism-based inactivator of CYP2B6. The aim of the current study was to investigate the possible formation of reactive intermediates through detection of protein covalent binding and glutathione ethyl ester adduct (GSHEE) formation. The incubation of tamoxifen with 2B6 gave rise to an adduct of 4OHtam with glutathione, which was characterized as the 4OHtam quinone methide + GSHEE with an m/z value of 719, and the structure was characterized by liquid chromatography-tandem mass spectrometry. The metabolic activation of tamoxifen in the CYP2B6 reconstituted system also resulted in the formation of an adduct to the P4502B6 apoprotein, which was identified using liquid chromatography mass spectrometry. The site responsible for the inactivation of CYP2B6 was determined by proteolytic digestion and identification of the labeled peptide. This revealed a tryptic peptide ¹⁸⁸FHYQDQE¹⁹⁴ with the site of adduct formation localized to Gln193 as the site modified by the reactive metabolite formed during tamoxifen metabolism.
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Chitra
authors.initials: C
authors.lastname: Sridar
authors.forename: Jaime
authors.initials: J
authors.lastname: D'Agostino
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
mesh_terms: D016588:Anticarcinogenic Agents / Q000493:pharmacokinetics* / Q000494:pharmacology; D018931:Antineoplastic Agents, Hormonal / Q000493:pharmacokinetics / Q000494:pharmacology; D001059:Apoproteins / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001711:Biotransformation; D065702:Cytochrome P-450 CYP2B6; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D045563:Indolequinones / Q000493:pharmacokinetics*; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology
chemical_list: D016588:Anticarcinogenic Agents; D018931:Antineoplastic Agents, Hormonal; D001059:Apoproteins; D045563:Indolequinones; D013629:Tamoxifen; C068040:quinone methide; C016601:afimoxifene; C042431:S-ethyl glutathione; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D005978:Glutathione