TY - JOUR T1 - Formation of Both Heme and Apoprotein Adducts Contributes to the Mechanism-Based Inactivation of Human CYP2J2 by 17<em>α</em>-Ethynylestradiol JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 813 LP - 822 DO - 10.1124/dmd.118.080903 VL - 46 IS - 6 AU - Hsia-lien Lin AU - Haoming Zhang AU - Paul F. Hollenberg Y1 - 2018/06/01 UR - http://dmd.aspetjournals.org/content/46/6/813.abstract N2 - 17α-Ethynylestradiol (EE), a major component of many oral contraceptives, affects the activities of a number of the human cytochrome P450 (P450) enzymes. Here, we characterized the effect of EE on CYP2J2, a major human P450 isoform that participates in metabolism of arachidonic acid. EE inactivated the hydroxyebastine carboxylation activity of CYP2J2 in a reconstituted system. The loss of activity is time and concentration dependent and requires NADPH. The KI and kinact values for the inactivation were 3.6 μM and 0.08 minute−1, respectively. Inactivation of CYP2J2 by EE was due to formation of a heme adduct as well as an apoprotein adduct. Mass spectral analysis of CYP2J2 partially inactivated by EE showed two distinct protein masses in the deconvoluted spectrum that exhibited a mass difference of approximately 312 Da, which is equivalent to the sum of the mass of EE and one oxygen atom. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis revealed a heme adduct with MH+ ion at m/z 875.5, corresponding to alkylation of an iron-depleted prosthetic heme by EE plus one oxygen atom. The reactive intermediate responsible for covalently modifying both the prosthetic heme and apoprotein was characterized by trapping with glutathione (GSH). LC-MS/MS analysis revealed two GSH conjugate isomers with MH+ ions at m/z 620, which were formed by reaction between GSH and EE with the oxygen being added to either the internal or terminal carbon of the ethynyl moiety. High-pressure liquid chromatography analysis revealed that three other major metabolites were formed during EE metabolism by CYP2J2. ER -