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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

The Inactivation of Cytochrome P450 3A5 by 17-Ethynylestradiol Is Cytochrome b₅-Dependent: Metabolic Activation of the Ethynyl Moiety Leads to the Formation of Glutathione Conjugates, a Heme Adduct, and Covalent Binding to the Apoprotein

Department of Pharmacology, University of Michigan, Ann Arbor, Michigan

17-Ethynylestradiol (EE) inactivates cytochrome P450 3A5 (3A5) in the reconstituted system in a mechanism-based manner. The inactivation is dependent on NADPH, and it is irreversible. The inactivation of 3A5 by EE is also dependent on cytochrome b₅ (b₅). The values for the K_I and k_inact of the 7-benzyloxy-4-(trifluoromethyl)coumarin O-debenzylation activity of 3A5 are 26 µM and 0.06 min^–1, respectively. Incubation of 3A5 with EE resulted in a 62% loss of catalytic activity, 60% loss in the reduced CO difference spectrum, and 40% decrease in native heme with the formation of a heme adduct. The partition ratio was 25, and the stoichiometry of binding was 0.3 mol of EE metabolite bound/mol of P450 inactivated. Four major metabolites were formed during the metabolism of EE by 3A5. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [³H]EE was irreversibly bound to 3A5 apoprotein. Liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) revealed that two glutathione (GSH) conjugates with m/z values of 620 were formed only in the presence of b₅. These two conjugates are formed from the reaction of GSH with the ethynyl group with the oxygen being inserted into either the internal or terminal carbon. A heme adduct with the ion at m/z 927 and two dipyrrole adducts with ions at m/z 579 were detected by LC-MS/MS analysis. In conclusion, 3A5 can activate EE to a 17-oxirene-related reactive species that can then partition the oxygen between the internal and terminal carbons of the ethynyl group to form heme and apoprotein adducts, resulting in the inactivation of P450 3A5.

Address correspondence to: Dr. Paul F. Hollenberg, Department of Pharmacology, 2301 MSRB III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail: phollen{at}umich.edu

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