RT Journal Article
SR Electronic
T1 Formation of Both Heme and Apoprotein Adducts Contributes to the Mechanism-Based Inactivation of Human CYP2J2 by 17α-Ethynylestradiol
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP dmd.118.080903
DO 10.1124/dmd.118.080903
A1 Hsia-lien Lin
A1 Haoming Zhang
A1 Paul F. Hollenberg
YR 2018
UL http://dmd.aspetjournals.org/content/early/2018/03/30/dmd.118.080903.abstract
AB 17α-Ethynylestradiol (EE), a major component of many oral contraceptives, affects the activities of a number of the human cytochrome P450 (CYP) enzymes. Here, we characterized the effect of EE on CYP2J2, a major human CYP isoform which 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. KI and kinact values for the inactivation were 3.6 μM and 0.08 min-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 in 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.