Department of Pharmacology, University of Michigan, Ann Arbor, Michigan.
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan
Drug Metab Dispos. 2018 Jun;46(6):813-822. doi: 10.1124/dmd.118.080903. Epub 2018 Mar 30.
17-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 and values for the inactivation were 3.6 M and 0.08 minute, 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 / 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 / 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.
17-乙炔基雌二醇（EE）是许多口服避孕药的主要成分，它会影响许多人类细胞色素 P450（CYP）酶的活性。在这里，我们研究了 EE 对 CYP2J2 的影响，CYP2J2 是一种主要的人类 P450 同工酶，参与花生四烯酸的代谢。EE 在重组体系中使 CYP2J2 的羟基依巴斯汀羧化活性失活。这种失活作用具有时间和浓度依赖性，需要 NADPH。失活的 和 值分别为 3.6 M 和 0.08 分钟。EE 通过形成血红素加合物和脱辅基蛋白加合物使 CYP2J2 失活。用 EE 部分失活的 CYP2J2 的质谱分析显示，在去卷积图谱中出现了两个明显不同的蛋白质量，其质量差约为 312 Da，相当于 EE 和一个氧原子的质量总和。液相色谱-串联质谱（LC-MS/MS）分析显示 MH 离子为 / 875.5 的血红素加合物，对应于 EE 加一个氧原子对缺铁血红素辅基的烷基化。用谷胱甘肽（GSH）捕获鉴定出导致辅基血红素和脱辅基蛋白共价修饰的反应性中间产物。LC-MS/MS 分析显示，MH 离子为 / 620 的两种 GSH 结合异构体，是由 GSH 与 EE 反应形成的，氧原子分别加到乙炔基的内部或末端碳原子上。高效液相色谱分析显示，CYP2J2 代谢 EE 时还形成了另外三种主要代谢物。
