Effect of 17- -Ethynylestradiol on Activities of Cytochrome P450 2B (P450 2B) Enzymes: Characterization of Inactivation of P450s 2B1 and 2B6 and Identification of Metabolites

17-Ethynylestradiol (17EE) inactivated purified, reconstituted rat hepatic cytochrome P450 (P450) 2B1 and human P450 2B6 in a mechanism-based manner. Little or no inactivation was observed when P450s 2B2 or 2B4 were incubated with 17EE. The inactivation of P450s 2B1 and 2B6 was entirely dependent on both NADPH and 17EE and followed pseudo-first order kinetics. The maximal rate constants for the inactivation of P450s 2B1 and 2B6 at 30°C were 0.2 and 0.03 min , respectively. For P450s 2B1 and 2B6 the apparent KI was 11 and 0.8 M, respectively. Incubation of P450 2B1 with 17EE and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 20 to 25% loss of the enzyme’s ability to form a reduced CO complex. With P450 2B6, an 83% loss in enzymatic activity and a 5 to 10% loss in the CO reduced spectrum were observed. The extrapolated partition ratios for 17EE with P450 2B1 and 2B6 were 21 and 13, respectively. Simultaneous incubation of an alternate substrate together with 17EE protected both enzymes from inactivation. A 1.3:1 stoichiometry of labeling for binding of the radiolabeled 17EE to P450 2B1 and 2B6 was seen. These results indicate that 17EE inactivates P450s 2B1 and 2B6 in a mechanism-based manner, primarily by the binding of a reactive intermediate of 17EE to the apoprotein. Analysis of the 17EE metabolites showed that 2B enzymes that become inactivated differ primarily by their ability to generate two metabolites that were not produced by P450s 2B2 or 2B4. Liver microsomal cytochromes P450 are involved in the metabolism of many drugs and carcinogens. P450 enzymes catalyze the metabolism of numerous structurally distinct substrates (Porter and Coon, 1991; Rendic and Di Carlo, 1997). The catalytic mechanism appears to be common to all P450s and involves a two-electron reduction of molecular oxygen to form a reactive oxygen intermediate and water (Porter and Coon, 1991). Information about the critical active site amino acid residues involved in substrate binding and catalysis has come primarily from site-directed mutagenesis studies or from observations with naturally occurring mutants (Kedzie et al., 1991; Halpert, 1995). Additional insight into the active site structure has been gained from examining the crystal structures of a number of bacterial P450s (Ravichandran et al., 1993; Cupp-Vickery and Poulos, 1995) as well as a lowresolution crystal structure of mammalian P450 2C5 (Williams et al., 2000). Mechanism-based inactivators that undergo catalytic conversion to reactive intermediates that covalently bind to amino acid side chains have been used to identify peptides or critical amino acid residues within the active sites that are involved in substrate metabolism. Studies with the 2B rat and rabbit enzymes, using acetylenic compounds such as 2-ethynylnaphtalene and 9-ethynylphenanthrene (for review, see Kent et al., 2001) or secobarbital (He et al., 1996), were particularly successful in identifying such critical residues. Relatively little is known about the physiological role of P450 2B6, the human 2B homolog, although some studies suggest that P450 2B6 was expressed at elevated levels in human breast tumor samples compared with nontumor tissue (Hellmold et al., 1998). The recent interest in 2B enzymes This study was supported by National Institutes of Health Grant CA 16954 from the National Cancer Institute. Portions of this work were presented at the Ninth Annual Meeting of the International Society for the Study of Xenobiotics in Nashville, TN, Oct. 24–28, 1999. ABBREVIATIONS: P450, cytochrome P450; 17EE, 17-ethynylestradiol; HPLC, high-performance liquid chromatography; ; DLPC, dilauroyl-L-phosphatidylcholine; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; GC-MS, gas chromatographymass spectrometry; GSH, glutathione; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; TMCS, trimethylchlorosilane; LC-MS, liquid chromatography-gas chromatography; TIC, total ion chromatogram; TMS, trimethylsilane. RP73401, 3-cyclopentyloxy-N-(3,5-clichloro-4-pyridyl)-4methoxybenzamide. 0022-3565/02/3002-549–558$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 300, No. 2 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4473/960590 JPET 300:549–558, 2002 Printed in U.S.A. 549 at A PE T Jornals on Sptem er 8, 2017 jpet.asjournals.org D ow nladed from stems from observations that they may play a role in the activation of procarcinogens (Osborne et al., 1993). P450 2B6 comprises about 2 to 10% of the total P450s in human liver microsomes and may not be expressed in all human livers (Shimada et al., 1994). However, a recent study indicated that P450 2B6 could be induced by phenobarbital in all the human livers that were screened with a polyclonal anti-2B6 antibody (Madan et al., 1996). Recombinant P450 2B6 isolated from vacciniaor baculovirus expression systems has been shown to metabolize a number of different substrates such as nicotine (McCracken et al., 1992), aminochrysene and 3-methoxy-4-aminoazobenzene (Mimura et al., 1993), tamoxifen (Styles et al., 1994), 7-ethoxycoumarin (Yamazaki et al., 1996), testosterone (Ono et al., 1996), diazepam (Ono et al., 1996), and antiretroviral drugs (Hesse et al., 2001). P450 2B6 also appears to be inactivated by mechanism-based inactivators such as 9-ethynylphenathrene, n-propylxanthate, and 2-phenyl-2-(piperidinyl)propane that have also been shown to inactivate rat P450 2B1 (for review, see Kent et al., 2001). Recently, several substrates such as bupropion, RP 73401, and ketamine have been identified that appear to be metabolized exclusively by P450 2B6 (Stevens et al., 1997; Faucette et al., 2000; Yanagihara et al., 2001). 17EE, developed in 1938, is the major synthetic steroid component of many oral contraceptives (Innhoffen and Holweg, 1938). Although the acetylenic moiety increased the oral availability of 17EE, incorporation of this group into a compound metabolized by P450 enzymes can also lead to the inactivation of these enzymes (Ortiz de Montellano and Reich, 1986). It was shown that 17EE, when incubated with human liver microsomes, abolished the NADPH-dependent activity of P450 3A4 (Guengerich, 1988). Concurrently, a loss in the spectrally detectable P450 was observed (Guengerich, 1988). At least 10 metabolites of 17EE have been isolated from human urine, with the 2-hydroxy species being the major metabolite (Williams et al., 1975; Guengerich, 1990). P450 enzymes and estrogens have also been implicated in the development of certain cancers. Elevated levels of P450 1B1 and of 4-hydroxyestradiol have been linked to the occurrence of breast cancer in humans (Osborne et al., 1993). Studies by Osborne et al. (1993) also suggested that an increase in breast tissue levels of C16 -hydroxylation of 17 -estradiol might be a biomarker of breast cancer risk. Steroids such as testosterone also are good substrates for P450 2B enzymes (Code et al., 1997). For these reasons it was of interest to determine whether 17EE would be metabolized by 2B enzymes and to study the effects of the 17EE metabolism on the activity of some of the known P450 2B isoforms. In this study 17EE was found to inactivate the major phenobarbital-inducible rat liver P450 2B1 and the human 2B homolog P450 2B6 by a classical mechanism-based mechanism (Silverman, 1996). Loss of enzymatic activity of P450 2B1 and 2B6 was primarily due to the binding of a reactive intermediate of 17EE to the apoprotein. P450s 2B2 and 2B4 were not significantly inactivated by 17EE. 2-Hydroxy-17ethynylestradiol was the major metabolite generated by all four isoforms. HPLC analysis of the 17EE metabolites revealed two peaks, C and E, that were primarily produced by P450s 2B1 and 2B6, suggesting the possibility that either or both may have been derived from a reactive intermediate of 17EE involved in the inactivation. Experimental Procedures Materials. Dilauroyl-L-phosphatidylcholine (DLPC), NADPH, catalase, 17EE, estradiol, and estrone were purchased form Sigma Chemical (St. Louis, MO). 4-Hydroxy-estradiol, 2-hydroxy-estradiol, 16-hydroxy-estrone, 4-hydroxyestrone, and 2-hydroxyestrone were obtained from Steraloids (Newport, RI). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) was from Molecular Probes (Eugene, OR) and 7-hydroxy-4-(trifluoromethyl)coumarin (HFC) was purchased from Enzyme Systems Products (Livermore, CA). Bicinchoninic acid reagent and Slide-A-Lyzer cassettes were from Pierce Chemical (Rockford, IL). Ultima Gold liquid scintillation cocktail was obtained from Packard (Meridien, CT). 2-Hydroxy-ethynylestradiol was a generous gift from Dr. William Slikker (Department of Health and Human Services, Food and Drug Administration, Jefferson, AR). Synthesis of 17-Carboxy-estradiol. A solution of ethylbromoacetate in sodium-dried benzene (2.16 g, 13 mmol in 20 ml) was added dropwise to a stirring mixture containing 1 g (15.3 mmol) of activated zinc and 1 g of estrone (3.7 mmol) in 15 ml of dry ether over the course of 30 min. The resulting mixture was warmed gently for 1 h and then refluxed for 5 h. After cooling to room temperature, 50 ml of ice-cold 10% sulfuric acid was added to the mixture. After transferring the mixture to a separatory funnel, the aqueous layer was removed. The benzene layer was washed twice with 50 ml of 5% sulfuric acid, once with 50 ml of 10% sodium bicarbonate followed by two 25-ml washes with water. The combined acid washes were extracted with ether. The organic phases were pooled and dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure. The crude product was purified by silica gel flash chromatography by using 25% ether in hexane as the eluent. The yield of estra-3,17-diol-17-ethyl acetate was 0.76 g (57%, melting point 66–68°C). GC-MS analysis with direct probe insertion yielded m/z (%) fragments of 330.30 (25.1), 312.25 (7.29), 270.20 (20.83), and 213.15 (78.30). Estra-3,17-diol-17-ethyl acetate (0.7 g, 1.96 mmol) was added to a stirring mixture composed of 20 ml of 10% aqueous sodium hydroxide and 50 ml of ethanol. The mixture was stirred at room temperature for 2 days. The progress of the reaction was monitored using thin layer chromatography with chloroform as the solvent. Ethanol was evaporated under reduced pressure. The residue was diluted with 50 ml of water and cooled in an ice bath. The solution was acidified by slowly adding dilute sulfuric acid from a dropping funnel until the solution was acidic to Congo red paper. The solution was extracted three times with 50 ml of ether. The ether extracts were combined and dried over anhydrous sodium sulfate, filtered, and then the solvent was evaporated. The product was purified using preparative thin layer chromatography with chloroform as the solvent. The yield of the purified acid was 0.41 g (63%). The white solid had a melting point of 142 to 144°C. GC-MS analysis with direct probe insertion yielded the following m/z (%) species: 358.30 (32.0), 340.30 (7.25), 252.20 (47.14), and 213.20 (100). Proton NMR spectra were recorded from samples dissolved in CDCl3 by using a GE omega 400-MHz FT-NMR spectrophotometer. The observed values for the estradiol 17ester were 6.97 ppm (d, 1H, H-AR), 6.40 to 6.50 ppm (m, 2H, H-AR), 4.13 ppm (q, 2H, -CH2-CH3), 2.81 to 2.85 ppm (m, 3 H), 2.52 ppm (d, 1H), 2.24 to 2.31 ppm (m, 1H), 2.03 to 2.10 ppm (m, 1H), 1.28 to 1.76 ppm (m, 14H), and 0.95 ppm (s, 3H, CH3). For the estradiol 17-a acetic acid the values were 6.98 ppm (d, 1H, H-AR), 6.50 to 6.60 ppm (m, 2H, H-AR), 2.80 to 2.83 ppm (m, 2H), 2.60 to 2.71 ppm (m, 2H), 2.28 to 2.32 ppm (m, 1H), 1.30 to 2.01 (m, 12H), and 0.95 ppm (s, 3H, CH3). Purification of P450 and Reductase. P450 2B1 was purified from microsomes isolated from livers of fasted male Long Evans rats (175–190 g; Harlan Bioproducts for Science, Indianapolis, IN) given 0.1% phenobarbital in the drinking water for 12 days according to published procedures (Saito and Strobel, 1981). Reductase was purified after expression in Escherichia coli as previously described (Hanna et al., 1998b). P450s 2B2 and 2B6 were expressed in E. coli 550 Kent et al. at A PE T Jornals on Sptem er 8, 2017 jpet.asjournals.org D ow nladed from MV1304 cells and purified as previously described (Hanna et al., 1998a, 2000). P450 2B4 was purified from livers of phenobarbitalinduced rabbits as described by Coon et al. (1978). Enzyme Activity Assays and Inactivation. Purified P450 2B1 and reductase were reconstituted with lipid for 45 min at 4°C. Incubation mixtures contained 0.5 M P450 2B1 or 0.67 M P450 2B6, 1 M reductase, 200 g of DLPC/ml, 110 units of catalase/ml, 17EE, or dimethyl sulfoxide in 50 mM potassium phosphate buffer, pH 7.4. In some instances, the P450 2B6-reconstituted system also contained equimolar amounts of cytochrome b5. P450s 2B2 and 2B4 were reconstituted as described for P450 2B1 except that equimolar amounts of cytochrome b5 were also added to these isoforms. After equilibrating the reaction mixture at 30°C for 3 min, the reactions were initiated by adding NADPH to a final concentration of 1.2 mM (primary reaction mixture). The 7-EFC O-deethylation activity was measured spectrofluorometrically as described by Buters et al. (1993). At the indicated times, duplicate 10l samples (5 pmol of P450 2B1) of the primary reaction mixture were removed and mixed with 990 l of a secondary reaction containing 0.2 mM NADPH, 100 M 7-EFC, and 40 g bovine serum albumin/ml in 50 mM potassium phosphate buffer, pH 7.4, and incubated at 30°C for 5 min. For P450 2B6, duplicate 12l samples (8 pmol of P450 2B6) of the primary reaction mixture were mixed with 988 l of the secondary reaction mixture and incubated for 10 min at 30°C. Enzyme activity was stopped by adding ice-cold acetonitrile to a final concentration of 25%. Fluorescence of the samples was measured directly at room temperature on an SLM-Aminco model SPF-500 C spectrofluorometer (SLM-Aminco, Urbana, IL) with excitation at 410 nm and emission at 510 nm. Substrate Protection. Substrate protection from 17EE-dependent inactivation of P450 2B1 was assayed by including 10 M 17EE together with 7-EFC at molar ratios of 1:0.25, 1:0.5, 1:1, and 1:2 of 17EE/7-EFC in the primary reaction. At the indicated times duplicate 10l aliquots were removed and assayed for activity remaining as described above. For P450 2B6, substrate protection was assayed by including a 5-fold molar excess of ethoxycoumarin over 5 M 17EE in the primary incubation mixture. Partition Ratio. To estimate the partition ratio, P450 2B1 samples were incubated in the presence of 2.5 to 300 M 17EE for 20 min to ensure the assay had proceeded to completion. Duplicate aliquots were removed and assayed for 7-EFC activity as described above. P450 2B6 was incubated with 5 to 350 M 17EE, incubated for 20 min, and assayed for residual activity with 7-EFC. Irreversibility of Inactivation of P450 2B1 and 2B6 by 17EE. Cytochromes P450 2B1 or 2B6 (0.5 nmol) were reconstituted and inactivated with 20 M or 50 M 17EE in a total volume of 138 l as described above. Control samples were incubated with 17EE but without NADPH. After 10 min at 30°C, the samples (0.13 ml) were dialyzed overnight at 4°C against 2 500 ml of 50 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol and 0.1 mM EDTA. The dialyzed samples were reconstituted with 10 g of lipid for 30 min on ice. Some samples also received fresh reductase. Enzymatic activity was assayed with 7-EFC as described above. Stoichiometry and Specificity of Binding. The stoichiometry of binding was determined by extensively dialyzing 500l samples containing 1 nmol of P450 2B1 reconstituted with reductase and lipid as described above that had been incubated with 40 M radiolabeled [H]17EE, 10 mM GSH, and with or without NADPH for 10 min at 30°C. Aliquots were removed to measure the extent of inactivation of P450 2B1 based on the residual 7-EFC O-deethylation activity and to determine the amount of heme loss by reduced CO difference spectroscopy before dialysis (Omura and Saito, 1964). Samples were dialyzed in Slide-A-Lyzer cassettes against 4 500 ml of 50 mM potassium phosphate, pH 7.4, containing 20% glycerol, 10 mM sodium cholate, and 0.1 mM EDTA. Aliquots were removed and the radioactivity remaining after dialysis was measured by liquid scintillation counting. Cytochrome P450 2B1 recovery was determined spectrophotometrically by measuring the reduced CO difference spectra. The stoichiometry of binding was calculated after subtracting the background counts from dialyzed samples incubated with 17EE in the absence of NADPH. P450 2B6 was incubated with 10 M 17EE, 10 mM GSH, and with or without NADPH for 20 min. The samples were assayed for residual activity and P450 content by reduced CO difference spectroscopy before dialysis as described for P450 2B1. Spectrophotometric Quantitation of P450 2B1 and P450 2B6. At the times indicated, 200l aliquots of the primary reaction incubation were removed and diluted with 800 l of ice-cold 50 mM potassium phosphate, pH 7.4, containing 40% glycerol and 0.6% Tergitol Nonidet P-40. The sample was gently bubbled with CO for 60 s and the spectrum was recorded from 400 to 500 nm on a DW2 UV/Vis spectrophotometer (SLM-Aminco) equipped with an OLIS spectroscopy operating system (On-Line Instrument Systems, Bogart, GA). Dithionite was added and the reduced carbonyl spectrum was recorded (Omura and Saito, 1964). For absolute spectral determinations, P450 2B1 and reductase were reconstituted at a 1:1 ratio. The final concentration was 1 M P450 2B1, 1 M reductase, 200 g of DLPC/ml, and 110 U of catalase/ml in 50 mM potassium phosphate, pH 7.4. The reference contained catalase and lipid in 50 mM potassium phosphate, pH 7.4. Spectra were recorded by scanning from 375 to 500 nm. Isolation of 17EE Metabolites. P450s were reconstituted with reductase and lipid as previously described. For assays with P450 2B2, 2B4, and 2B6, cytochrome b5 was also included in equimolar amounts. Each assay contained 1 M P450, 1 M reductase, 1 M cytochrome b5, 25 g/ml DLPC, 200 g/ml ascorbate, 110 units of catalase, 40 M 17EE, and 50 mM potassium phosphate buffer, pH 7.4, in a total volume of 500 l. In some instances, 17EE contained trace amounts of [H]17EE. Reactions were initiated with 1.2 mM NADPH. P450 2B1 samples were incubated at 30°C for 30 min, whereas P450s 2B2, 2B4, and 2B6 were incubated for 60 min. The reaction mixtures were quenched with 2 ml of N2-saturated methylene chloride. 17EE and its metabolites were extracted into the organic phase. Each sample received 3 l of dimethyl sulfoxide and the methylene chloride was evaporated under N2. The samples were dissolved in 100 l of 50% solvent B (49.9% CH3OH, 50% CH3CN, 0.1% acetic acid) before HPLC analysis. Metabolites were chromatographed on a C18 reverse phase column (25 cm, 5 m, 100 Å; Microsorb MV; Rainin Instruments, Woburn, MA) equilibrated with 70% solvent A (0.1% acetic acid in H2O) and 30% solvent B at a flow rate of 1.2 ml/min. After 5 min, the concentration of solvent B was raised to 50% over 3 min, followed by a linear increase to 60% over 12 min, and then to 95% over 10 min. After 10 min at 95% solvent B, the column was brought back to initial equilibration conditions. Under these conditions 17EE and its metabolites eluted between 10 and 25 min. The retention times of the metabolites were compared with authentic standards. Metabolites were quantified by integrating the area under the peak by using the Millenium program (Waters, Milford, MA). Identification of 17EE Metabolites. Fractions containing the 17EE metabolites were collected, dried, derivatized with BSTFA/ TMCS, and analyzed by GC-MS essentially as described by Suchar et al. (1995). Each sample was incubated with 5 l of redistilled pyridine and 20 l of BSTFA, 1% TMCS for 30 min at 70°C. Each sample (4 l) was chromatographed on a 30-m DB1 fused silica capillary column (0.32-mm i.d., 0.25m film coating; J&W Scientific, Folsom, CA) with a temperature gradient of 10°C/min from 80 to 320°C and analyzed over an m/z range from 45 to 750 on a JEOL JMS AX-505H double focusing mass spectrometer coupled to a Hewlett Packard 5890J gas chromatograph via a heated interface.