Mechanism-Based Inactivation of Cytochrome P450 3A4 by 17 -Ethynylestradiol: Evidence for Heme Destruction and Covalent Binding to Protein

17 -Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6 -hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanismbased manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the KI and kinact were 18 M and 0.04 min , respectively, and the t1/2 was 16 min. Incubation of 50 M EE with P450 3A4 at 37°C for 30 min resulted in a 67% loss of testosterone 6 -hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EEdependent inactivation. The partition ratio was 50. The stoichiometry of binding was approximately 1.3 nmol of an EE metabolite bound per nmol of P450 3A4 inactivated. SDSpolyacrylamide gel electrophoresis analysis demonstrated that [H]EE was irreversibly bound to the P450 3A4 apoprotein. After extensive dialysis of the [H]EE inactivated samples, high-pressure liquid chromatography (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M H) of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site. The cytochromes P450 comprise a large family of microsomal heme-containing monooxygenases that are involved in the metabolism of a wide variety of xenobiotics including drugs, pesticides, environmental pollutants, and carcinogens, as well as endogenous compounds such as steroids, retinoids, and fatty acids. The catalytic mechanism appears to be common to all P450s and involves a two-electron reduction of molecular oxygen to form a reactive oxygen species and water (Porter and Coon, 1991; Rendic and Di Carlo, 1997). 17 -Ethynylestradiol (EE), the major estrogenic component of many oral contraceptives, can be metabolized by P450 enzymes in various animal species and humans (Bolt, 1979). Drugs, hormones, insecticide synergists, carcinogens, or dietary constituents that influence the expression and activity of various P450s can modulate the efficacy and side effects of EE (Bolt and Kassel, 1976; Guengerich, 1990a,b; He et al., 1998b). Studies using microsomes from phenobarbital-induced rats have demonstrated that the metabolism of EE results in a decrease in the cytochrome P450 content determined by the reduced CO difference spectrum and leads to the formation of the green pigments obtained from N-alkylated porphyrins (White, 1978; Ortiz de Montellano et al., 1979; Blakey and White, 1986). In addition, the mechanismbased inactivation of human liver microsomal P450s by EE with the loss of the P450-CO spectrum during incubation with NADPH has been shown (Guengerich, 1988). The EEmediated inactivation in both rat and human liver microsomal P450s was postulated to be due to the metabolism of the acetylenic moiety of EE and the ensuing modification of the This work was supported in part by National Institutes of Health Grant CA-16954 (P.F.H.) and by a grant (DRR-00480) from the Biotechnology Research Technology Program, National Center for Research Resources, National Institutes of Health (Michigan State University). ABBREVIATIONS: P450, cytochrome P450; EE, 17 -ethynylestradiol; 3A4, cytochrome P450 3A4; reductase, NADPH-cytochrome P450 reductase; HPLC, high-pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; 2-OH-EE, 2-hydroxyethynylestradiol; TIC, total ion chromatogram; GC-MS, gas chromatography-mass spectrometry. 0022-3565/02/3011-160–167$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4667/973873 JPET 301:160–167, 2002 Printed in U.S.A. 160 at A PE T Jornals on O cber 7, 2017 jpet.asjournals.org D ow nladed from heme moiety of the P450s. However, the details of the kinetic parameters for the inactivation and modification of the apoprotein by an EE-derived metabolite were not reported (Guengerich, 1990a). Cytochrome P450 3A4 (3A4) is the most abundant P450 isoform in human liver, has very broad substrate specificity, and is believed to be responsible for the metabolism of more than 60% of all clinically relevant drugs including contraceptive steroids (Guengerich, 1995). The substrates for 3A4 can be inactivators as well as inducers; thus during therapy with multiple drugs, drug-drug interactions may result in problems of clinical significance. 3A4 has been shown to be the principal catalyst involved in the oxidation of EE. In a series of human liver microsomes, the rate of the 2-hydroxylation of EE correlated well with both the rate of nifedipine oxidation and immunochemically determined 3A4 (Guengerich, 1988). We have used purified 3A4 reconstituted with phospholipids, NADPH-cytochrome P450 reductase (reductase), and catalase to characterize the EE-dependent inactivation of the testosterone 6 -hydroxylase activity of 3A4. The following parameters were measured: the values for the concentration of EE required to give the half-maximal rate of inactivation (KI); the maximal rate constant of inactivation at saturating concentrations of EE (kinact); the time required for half the P450 to be inactivated at saturating concentrations of EE (t1/2); the partition ratio; the UV-visible spectrum; the reduced CO difference spectrum; the effect of an alternate substrate on the loss of catalytic activity; and the stoichiometry and specificity of EE binding. The reactive intermediates of acetylenic compounds formed by several isoforms of P450 have been known to alkylate the prosthetic heme group as well as to bind covalently to the protein (Ortiz de Montellano and Correia, 1995). Studies with P450 2B1 demonstrated that 2-ethylnylnaphthalene predominantly inactivates P450 2B1 through modification of the apoprotein, whereas phenylacetylene inactivates P450 2B1 via N-alkylation of heme (Ortiz de Montellano and Komives, 1985; Roberts et al., 1993). In this report, radiolabeled EE was employed in studies using SDSPAGE and reversed-phase HPLC analysis to investigate the targets for EE-mediated 3A4 modification. To elucidate the identity of the reactive metabolite(s) involved in the heme and apoprotein modification and the mechanism(s) for inactivation by EE, the metabolites of EE were resolved by HPLC and further characterized by GC-MS. Materials and Methods Chemicals. Cholic acid, catalase, NADPH, glutathione, testosterone, 6 -hydroxytestosterone, 6 -hydroxytestosterone, estradiol, estrone, and EE were purchased from Sigma-Aldrich (St. Louis, MO). 2 -, 4 -, and 16 -hydroxyestradiol were obtained from Steraloids Inc. (Newport, RI). Synthesis of 17 -carboxyestradiol was described in the previous study (Kent et al., 2002). 2-Hydroxyethynylestradiol (2-OH-EE) was a generous gift from Dr. William Slikker (Department of Health and Human Services, Food and Drug Administration, Jefferson, AR). 17 -[6,7-H]Ethynylestradiol (46.2 Ci/mmol) with radiochemical purity of 99% was obtained from Amersham Biosciences (Piscataway, NJ). All other chemicals and solvents were of the highest purity from commercial sources. Purification of Enzyme. Both 3A4 and reductase were expressed in Escherichia coli and purified to homogeneity as described (Hanna et al., 1998; He et al., 1999). Enzyme Assay and Inactivation. For the primary reaction mixture, 0.5 nmol of 3A4 was reconstituted with 60 g of a mixture (1:1:1) of L-dilauroyl-phosphocholine, L-dioleyl-sn-glycero-3phosphocholine, and L-phosphatidylserine, 200 g of recrystallized sodium cholate, 1 nmol of reductase, 100 U of catalase, and 2 mM glutathione in 1 ml of assay buffer containing 50 mM HEPES (pH 7.5), 20% glycerol, 30 mM MgCl2, and 0.5 mM EDTA. In studies of the timeand concentration-dependent inactivation by EE, the reactions were initiated by the addition of 1 mM NADPH to the primary reaction mixture, or the same volume of water was added as a control and the reaction mixtures were incubated at 37°C for the times indicated. The secondary reactions were started by transferring 50 l of the primary reaction mixtures to 950 l of assay buffer containing 200 M testosterone and 200 M NADPH. Incubations were carried out at 37°C for 20 min and then the reactions were terminated by the addition of 2 ml of ethyl acetate. The internal standard, 6 -hydroxytestosterone, was added and the products were extracted into the organic phase. The major metabolite, 6 -hydroxytestosterone, and the internal standard were quantified after separation by HPLC as described previously (He et al., 1999). The activity of 3A4 in the reconstituted system was 14.8 1.2 nmol of 6 -hydroxytestosterone formed per minute per nanomole of P450. Spectral Analysis. After incubating the primary reaction mixtures with 50 M EE in the presence (inactivated sample) or absence of NADPH (control sample), the absolute spectra and reduced CO difference spectra of 0.25 nmol of 3A4 were determined by scanning from 400 to 600 nm on an SLM-AMINCO 3000 spectrophotometer (Omura and Sato, 1964). In addition, 50 l aliquots of the reaction mixtures were removed for the determination of the testosterone 6 -hydroxylation activity. To test the irreversibility of inactivation, the control and inactivated samples were dialyzed overnight at 4°C against 1 liter of assay buffer and then reanalyzed both for enzymatic activity and reduced CO difference spectra. Substrate Protection. The inactivation of 3A4 by EE in the presence or absence of substrate was investigated by adding an 8and 16-fold molar excess of testosterone over EE to the primary reaction mixture. At the end of the incubation time, aliquots were removed for the determination of 3A4 activity remaining as de-