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

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 KI and kinact of the 7-benzyloxy-4-(trifluoromethyl)coumarin O-debenzylation activity of 3A5 are 26 M and 0.06 min , 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 [H]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. Cytochrome P450 3A4 (3A4) and cytochrome P450 3A5 (3A5), the two most abundant P450 enzymes in human liver and intestine, and which exhibit approximately 85% sequence identity, are estimated to metabolize more than 60% of clinically important drugs and to exhibit significant overlaps in substrate specificity. Because the overall metabolic rate of 3A5 for many substrates is much lower than that of 3A4, the contribution of 3A5 to drug and xenobiotic metabolism has generally been considered to be not particularly significant (Wrighton et al., 1990). However, because 3A5 exhibits significant genetic polymorphisms, wide tissue distribution, and high interindividual/interracial variability in expression, 3A5 may, in fact, be a much more important contributor to variability in human metabolism of drugs, endogenous substrates, and carcinogens than had been reported previously (Kuehl et al., 2001; Koch et al., 2002; Huang et al., 2004). The metabolism of 17 -ethynylestradiol (EE), the major estrogenic component in most oral contraceptives, has been extensively investigated for more than half-a century. Although previous studies had reported that 3A5 cannot metabolize EE (Wrighton et al., 1990), the ability of 3A5 to catalyze the metabolism of EE to 2-OH-EE has been shown recently (Wang et al., 2004). Some of the differences in these metabolic capabilities observed between 3A4 and 3A5 may have resulted from the experimental conditions used (Huang et al., 2004). Because EE has been proven previously to be an effective mechanism-based inactivator of 3A4 (Lin et This work was supported in part by National Institutes of Health Grant CA-16954. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.117861. ABBREVIATIONS: 3A4, cytochrome P450 3A4; 3A5, cytochrome P450 3A5; EE, 17 -ethynylestradiol; b5, cytochrome b5; GSH, glutathione; LC-MS/MS, liquid chromatography-tandem mass spectrometry; BFC, 7-benzyloxy-4-(trifluoromethyl)-coumarin; HPLC, high-pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Lys C, lysyl endopeptidase; Ni-NTA, nickel-nitrilotriacetic acid; TFA, trifluoroacetic acid; ESI, electrospray ionization; M, metabolite; XIC, extracted ion chromatogram; G, glutathione adduct. 0022-3565/07/3211-276–287$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 321, No. 1 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 117861/3193586 JPET 321:276–287, 2007 Printed in U.S.A. 276 at A PE T Jornals on Sptem er 7, 2017 jpet.asjournals.org D ow nladed from al., 2002), it is interesting to elucidate in detail the pathways for the metabolism of EE by 3A5 and the mechanism(s) by which it inactivates this P450. The importance of the acetylenic group in EE for the inactivation and destruction of the heme in rat liver cytochrome P450 was first noted almost three decades ago (White and Muller-Eberhard, 1977). The initial studies in liver microsomes from phenobarbital-induced rats clearly demonstrated the covalent binding of ethynylsterols to the prosthetic heme moiety (Ortiz de Montellano et al., 1979; Ortiz de Montellano and Kunze, 1980). Later, the covalent binding of other acetylenic compounds to the apoprotein was demonstrated (Gan et al., 1984; CaJacob et al., 1988; Hammons et al., 1989; Chan et al., 1993; Roberts et al., 1993). It was proposed that P450 inactivation by the ethynyl compounds involves the initial oxygenation of the ethynyl groups (Kunze et al., 1983; Ortiz de Montellano and Komives, 1985; CaJacob et al., 1988; Chan et al., 1993). Whether the inactivation was due to alkylation of prosthetic heme or to modification of the apoprotein was determined by the site of addition of the oxygen to either the internal or terminal carbon of the carbon–carbon triple bond, respectively. However, the factors determining to which carbon of the triple bond the oxygen is transferred, and whether heme or protein modification or both occur, are not clear. An inactivation mechanism involving both heme destruction and covalent binding of inactivator to apoprotein was first characterized for the inactivation of P450 2B1 by secobarbital (He et al., 1996). Subsequently, similar phenomena involving heme and apoprotein modification were found during EE inactivation of 3A4, t-butyl acetylene-inactivation of P450 2E1, and the inactivation of P450 3A and 2B subfamilies by bergamottin (Lin et al., 2002; Blobaum et al., 2002; Lin et al., 2005). In some cases, the masses of the heme and the apoprotein adducts have been determined. Interestingly, it was found that cytochrome b5 (b5) was required for the inactivation of 3A5 but not 3A4 by EE. Because both heme and protein modifications occur during the inactivation of 3A4 and 3A5, the possibility that two different forms of reactive species were formed during the inactivations was investigated. To address this issue, glutathione (GSH) was used to trap electrophilic reactive intermediates of EE formed during the metabolism and inactivation of both P450 3A4 and 3A5 in the reconstituted system. The resulting GSH conjugates were then characterized using standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches (Baillie and Davis, 1993; Samuel et al., 2003). That inactivation of 3A5 by EE is b5-dependent has led to some very interesting observations and some valuable conclusions. We report here that 3A5 not only metabolizes EE to four metabolites but also catalyzes the bioactivation of the carbon–carbon triple bond of EE, leading to heme destruction and covalent binding of EE to the apoprotein. In addition, the structures of two GSH conjugates formed during the metabolic activation of the ethynyl moiety by 3A5 in the presence of b5 have been determined. Materials and Methods Chemicals. NADPH, EE, and GSH were purchased from SigmaAldrich (St. Louis, MO). 2-OH-EE was a generous gift from Dr. William Slikker (Department of Health and Human Services, Food and Drug Administration, Jefferson, AR). 7-Benzyloxy-4-trifluoromethyl(coumarin) (BFC) and 7-hydroxy-(trifluoromethyl)coumarin were obtained from Invitrogen (Eugene, OR) and Indofine Chemical Co., Inc. (Hillsborough, NJ), respectively. [6,7-H]EE (46.2 Ci/mmol) was obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All other chemicals and solvents were of the highest purity available from commercial sources. Purification of Enzymes. The plasmid for P450 3A5 was a generous gift from Dr. J. R. Halpert (University of Texas Medical Branch, Galveston, TX) and was expressed as an His-tagged protein in Escherichia coli Topp 3 cells (Domanski et al., 2001). P450 3A4 and NADPH-cytochrome P450 reductase (reductase) were expressed in E. coli DH5 and Topp 3 cells, respectively. Cytochrome b5 was purified from liver microsomes of phenobarbital-treated Long-Evans rats. The purification procedures for all of the proteins were described previously (Lin et al., 2005). Enzyme Assay and Inactivation. The primary reaction mixture contained 60 g of a mixture (1:1:1) of L-dilauroyl-phosphocholine, L-dioleyl-sn-glycero-3-phosphocholine, and L-phosphatidylserine, 1 nmol of 3A5, 2 nmol of reductase, 1 nmol of b5, 100 units of catalase, and 2 mM GSH in 1 ml of 50 mM HEPES buffer, pH 7.5, containing 20% glycerol, 30 mM MgCl2, and 0.5 mM EDTA. The reconstituted system was incubated at room temperature for 30 min and then kept on ice until used for the experiments. For the studies on the concentrationand time-dependent inactivation of 3A5 by EE, the reactions were initiated by adding 1 mM ADPH to the primary reaction mixture containing various concentrations of EE at 37°C. At the time points indicated, 10l aliquots were transferred into 990 l of a secondary reaction mixture containing 50 M BFC and 200 M NADPH in 200 mM potassium phosphate buffer, pH 7.7. Incubations were carried out for 15 min, and the reactions were terminated by the addition of 365 l of 0.1 M Tris in 80% acetonitrile. The formation of the 7-hydroxy-(trifluoromethyl)coumarin product was determined by fluorescence measurement (excitation, 409 nm; emission, 530 nm) using an RF-5301PC spectrofluorophotometer (Shimadzu, Kyoto, Japan). All samples were done in duplicate. Partition Ratio. EE at concentrations ranging from 2.5 to 300 M was added to the primary reaction mixture containing the reconstituted system with 1 M 3A5. The reaction mixtures were initiated by the addition of 1 mM NADPH and incubated at 37°C for 1 h, allowing the inactivation to go to completion (Silverman, 1996). Aliquots were removed and assayed for residual BFC activity as