Nadph-dependent Formation of Polar and Nonpolar Estrogen Metabolites following Incubations of 17 -estradiol with Human Liver Microsomes

By using a versatile high-pressure liquid chromatography method (total elution time 135 min) developed in the present study, we detected the formation of some 20 nonpolar radioactive metabolite peaks (designated as M1 through M20), in addition to a large number of polar hydroxylated or keto metabolites, following incubations of [H]17 -estradiol with human liver microsomes or cytochrome P450 3A4 in the presence of NADPH as a cofactor. The formation of most of the nonpolar estrogen metabolite peaks (except M9) was dependent on the presence of human liver microsomal proteins, and could be selectively inhibited by the presence of carbon monoxide. Among the four cofactors (NAD, NADH, NADP, NADPH) tested, NADPH was the optimum cofactor for the metabolic formation of polar and nonpolar estrogen metabolites in vitro, although NADH also had a weak ability to support the reactions. These observations suggest that the formation of most of the nonpolar estrogen metabolite peaks requires the presence of liver microsomal enzymes and NADPH. Chromatographic analyses showed that these nonpolar estrogen metabolites were not the monomethyl ethers of catechol estrogens or the fatty acid esters of 17 -estradiol. Analyses using liquid chromatography/mass spectrometry and NMR showed that M15 and M16, two representative major nonpolar estrogen metabolites, are diaryl ether dimers of 17 -estradiol. The data of our present study suggest a new metabolic pathway for the NADPH-dependent, microsomal enzyme-mediated formation of estrogen diaryl ether dimers, along with other nonpolar estrogen metabolites. The endogenous estrogens undergo extensive metabolism in humans (Martucci and Fishman, 1993; Zhu and Conney, 1998a), such as oxidation (largely mediated by P450 enzymes), interconversion between 17 -estradiol and estrone, and various conjugation-deconjugation reactions. In addition, the catechol-O-methyltransferase-mediated O-methylation of endogenous catechol estrogens to monomethyl ethers (Zhu and Conney, 1998b) and the acyltransferase-mediated esterification of estrogens with fatty acyl-CoAs (Mellon-Nussbaum et al., 1982; Hochberg, 1998) result in the formation of lipophilic estrogen derivatives. These multiple metabolic pathways not only determine the pharmacokinetic features of the endogenous estrogens in the body and in various target tissues, but they also diversify the biological actions of endogenous estrogens in certain target sites through metabolic formation of biologically active estrogen metabolites, such as 4-hydroxyestradiol, 15 -hydroxyestradiol, 16 -hydroxyestrone, and 2-methoxyestradiol (a predominant O-methylation product of 2-hydroxyestradiol). During our recent analysis of the NADPH-dependent metabolism of [H]E2 and [ H]estrone to various hydroxylated or keto metabolites by human liver microsomes (Lee et al., 2001, 2002), we consistently detected a cluster of coeluted radioactive peaks at the end of a 60-min HPLC run, with their chromatographic polarities less than estrone. Notably, similar nonpolar radioactive peaks were also noted earlier when radioactive E2 or estrone was incubated with rat or mouse liver microsomes (Aoyama et al., 1990; Haaf et al., 1992; Suchar et al., 1995, 1996; Zhu et al., 1997, 1998). Further characterization of these nonpolar metabolite peaks has never been pursued, largely because there was no evidence in the literature that would provide a rationale for the suggestion that highly lipophilic metabolites would be formed from steroid hormones or xenobiotics by microsomal enzymes using NADPH as a cofactor. However, in our recent study to characterize the NADPH-dependent metabolism of [H]E2 and [ H]estrone by 15 selectively expressed human P450 isozymes, we noted that this cluster of radioactive nonpolar metabolite peaks appeared to be selectively formed in large quantities only with certain human P450 isozymes, most notably, CYP3A4 and CYP3A5 (Lee et al., 2003). This interesting observation has prompted us to investigate further the possibility that the formation of some of these nonpolar radioactive E2 metabolite peaks might be dependent on the presence of specific drug-metabolizing enzymes, such as certain P450 isoforms. In the present study, we first developed a versatile HPLC method for the detection and quantification of various polar and nonpolar This study was supported, in part, by a Department of Defense Breast Cancer Research Program Predoctoral Training Award (DAMD17-02-1-0566, to A.J.L.), a National Institutes of Health grant (RO1 CA 92391, to B.T.Z.), and an American Cancer Society research grant (RSG-02-143-01-CNE). ABBREVIATIONS: P450, cytochrome P450; E2, 17 -estradiol; NADP and NADPH, nicotinamide dinucleotide phosphate and its reduced form, respectively; NAD and NADH, nicotinamide dinucleotide and its reduced form, respectively; HPLC, high-pressure liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; CO, carbon monoxide; NMR, nuclear magnetic resonance. 0090-9556/04/3208-876–883$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 8 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 1353/1163922 DMD 32:876–883, 2004 Printed in U.S.A. 876 at A PE T Jornals on Sptem er 8, 2017 dm d.aspurnals.org D ow nladed from metabolites formed by human liver microsomes using [H]E2 as a substrate. By using this new method, we have studied the formation of these nonpolar estrogen metabolite peaks by human liver microsomal enzymes and NADPH. Two of the major nonpolar metabolites (M15 and M16) formed were identified to be the diaryl ether dimers of E2. Materials and Methods Chemicals and Human Liver Microsomes. E2, NADPH, and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO). [2,4,6,7,16,17-H]E2 (specific activity of 123.0 Ci/mmol) was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). The scintillation cocktail (ScintiVerse LC) and all the solvents (HPLC grade or better) were purchased from Fisher Scientific (Atlanta, GA). Human liver microsomes used in this study were obtained from Human Biologics International (Scottsdale, AZ) and BD Gentest (Woburn, MA). Assay of the NADPH-Dependent Metabolism of [H]E2 by Human Liver Microsomes. It is of note that the conditions used for the in vitro metabolic formation of the nonpolar E2 metabolites were exactly the same as those used in our recent studies of the NADPH-dependent oxidative metabolism of estrogens (Lee et al., 2001, 2002). Specifically, the reaction mixture consisted of human liver microsomes (1 mg of protein/ml), a desired concentration of E2 (containing 2 Ci of [ H]E2) in 5 l of ethanol, 2 mM NADPH, and 5 mM ascorbic acid in a final volume of 0.5 ml of 0.1 M Tris-HCl/0.05 M HEPES buffer, pH 7.4. The enzymatic reactions were initiated by the addition of liver microsomes, and the incubations were carried out at 37°C for 20 min with periodic mild shaking. The reactions were arrested by placing the reaction tubes on ice followed by addition of 10 l of 10 mM nonradioactive E2 substrate to reduce nonspecific adsorption of estrogens to the test tubes. The mixture was then immediately extracted with 8 ml of ethyl acetate, and the supernatants were transferred to another set of test tubes and dried under a stream of nitrogen. The resulting residues were redissolved in 100 l of methanol, and an aliquot (50 l) was injected into the HPLC apparatus for analysis of estrogen metabolite composition. Notably, all the test tubes used in our present study were silanized with 5% (v/v) dimethyldichlorosilane in toluene as described in our recent studies (Lee et al., 2001, 2002, 2003). The H-labeled E2 was repurified by HPLC a day before it was used as a substrate in the in vitro metabolism experiments. HPLC Analytical System. Analysis of radioactive E2 metabolites was performed with an HPLC system coupled with in-line radioactivity and UV detection. The HPLC system consisted of a Waters 2690 separation module (Waters, Milford, MA), a radioactivity detector ( -RAM; IN/US Systems, Inc., Tampa, FL), a Waters UV detector (model 484), and an Ultracarb 5 ODS column (150 4.60 mm; Phenomenex, Torrance, CA). The solvent system for the separation of E2, estrone, and their metabolites consisted of acetonitrile (solvent A), water (solvent B), and methanol (solvent C). The solvent gradient (solvent A/solvent B/solvent C) used for the selective separation of various polar estrogen metabolites (method I) was as follows: 8 min of an isocratic elution at 16:68:16, 7 min of a concave gradient (curve number 9) to 18:64:18, 13 min of a concave gradient (curve number 8) to 20:59:21, 10 min of a convex gradient (curve number 2) to 22:57:21, 13 min of a concave gradient (curve number 8) to 58:21:21, followed by a 0.1-min step to 92:5:3 and an 8.9-min isocratic period at 92:5:3. The gradient was then FIG. 1. Representative HPLC traces showing the separation and detection of both polar and nonpolar [H]E2 metabolites formed by human liver microsomes and NADPH. The incubation mixtures consisted of human liver microsomes (1 mg protein/ml), 50 M E2 (containing 2 Ci [ H]E2), 2 mM NADPH, and 5 mM ascorbic acid in a final volume of 0.5 ml of Tris-HCl (0.1 M) and HEPES (50 mM) buffer, pH 7.4. The human liver microsomes (HBI226, upper inset; HBI217, lower panel) were obtained from Human Biologics International. The method I mobile phase gradient (described under Materials and Methods) was used to generate the HPLC trace in the upper small inset, and the method II gradient was used to generate the main HPLC trace in the lower panel. The polar estrogen metabolites were identified by comparing their HPLC and gas chromatography retention times as well as the gas chromatography/MS mass fragmentation pattern of their trimethylsilyl derivatives against those of authentic reference compounds as described in our recent study (Lee et al., 2001). 877 FORMATION OF POLAR AND NONPOLAR ESTROGEN METABOLITES at A PE T Jornals on Sptem er 8, 2017 dm d.aspurnals.org D ow nladed from returned to the initial condition (16:68:16) and held for 10 min before analysis of the next sample. The solvent gradient (solvent A/solvent B/solvent C) used for simultaneous separation of both polar and nonpolar estrogen metabolites (method II) was as follows: 8 min of an isocratic elution at 16:68:16, 7 min of a concave gradient (curve number 9) to 18:64:18, 13 min of a concave gradient (curve number 8) to 20:59:21, 10 min of a convex gradient (curve number 2) to 22:57:21, 32 min of a linear gradient (curve number 6) to 30:40:30, 10 min of a concave gradient (curve number 9) to 35:35:30, 10 min of a concave gradient (curve number 9) to 40:30:30, 10 min of a concave gradient (curve number 9) to 45:25:30, 10 min of a concave gradient (curve number 9) to 50:20:30, 10 min of a concave gradient (curve number 9) to 55:15:30, 5 min of a concave gradient (curve number 9) to 60:10:30, and 5 min of a concave gradient (curve number 9) to 65:5:30. The gradient was then returned to the initial condition (16:68:16) and held for 15 min before analysis of the next sample. The solvent gradient (solvent A/solvent B/solvent C) used to selectively separate nonpolar estrogen metabolites (method III) was as follows: 20 min of an isocratic elution at 30:40:30, 10 min of a convex gradient (curve number 3) to 35:30:35; 10 min of a convex gradient (curve number 3) to 40:25:35, 10 min of a convex gradient (curve number 3) to 50:20:30, 10 min of a convex gradient (curve number 3) to 60:10:30, followed by 10 min of convex gradient (curve number 3) to 65:5:30. The gradient was then returned to the initial condition (30:40:30) and held for 5 min before analysis of the next sample. LC/MS Analysis of the Dansyl Derivative of M15 and M16. M15 and M16 were formed following large-scale incubations of E2 with selectively expressed human CYP3A4 in the presence of NADPH, and then were isolated by using the HPLC (methods I, II, and III). After evaporation to dryness of the collected HPLC fraction containing M15 or M16, the residues were then redissolved in 100 l of sodium bicarbonate buffer (100 mM, pH adjusted to 10.5 with 1 N NaOH), and followed by vortex-mixing for 1 min. Dansyl chloride solution (100 l, at 1.0 mg/ml in acetone) was then added and followed by vortex-mixing for another 1 min. The samples were incubated at 60°C for 2 h in a heat block and then cooled to room temperature. An aliquot (10 l) was injected into the LC/MS for determination of the mass. The LC/MS system consisted of an Agilent 1100 HPLC apparatus (Palo Alto, CA), an AquaSep column (50 2.0 mm; ES Industries, West Berlin, NJ), and a Micromass Quattro LC triple quadruple MS detector (Waters). The solvent system consisted of water with 0.1% formic acid (A) and 95% acetonitrile with 0.1% formic acid (B) at a 0.2 ml/min flow rate. The mobile phase gradient (A/B) was as follows: 4 min of an isocratic elution at 80:20, 26 min of a linear gradient to 0:100, and 30 min of an isocratic elution at 0:100. The mass was detected using an electrospray mode at a 3.0-kV capillary voltage and a 32-V cone voltage. The source block temperature and desolvation temperature were set at 100 and 320°C, respectively. Results and Discussion Establishment of an HPLC Method for the Separation of Nonpolar Estrogen Metabolite Peaks. In several of our recent studies (Lee et al., 2001, 2002, 2003), we had used an HPLC method that could selectively separate a large number of polar metabolites (mostly hydroxylated and keto metabolites) of E2 and estrone in a 60-min HPLC run. A representative HPLC trace is shown in Fig. 1 (as a small upper inset). With this HPLC method (as well as some other similar HPLC methods used by others), the nonpolar estrogen metabolite peaks were always eluted together at the end of a chromatogram. To develop an HPLC method that could separate both polar and nonpolar estrogen metabolites in a single run, we focused on selectively modifying the last 20 min of the HPLC mobile phase gradient while still using the original column. We chose this strategy because we desire to retain the versatile ability of our original HPLC method for the separation of all polar estrogen metabolites but, simultaneously, to also to acquire additional ability to resolve the nonpolar estrogen metabolite peaks. Experimentally, we modified each component of the mobile phase gradient in a systematic and incremental manner and then tested each modified gradient for the separation of polar and nonpolar [H]E2 metabolites formed with a representative human FIG. 2. Detection of various polar and/or nonpolar metabolites of [H]E2 formed by human liver microsomes using three different HPLC methods. The composition of the incubation mixtures was the same as that described in the legend to Fig. 1, except that 20 M E2 was used as substrate. Human liver microsomes (HG42, obtained from BD Gentest) were used in this study. Details of the three HPLC mobile phase gradients (methods I, II, and II) were described under Materials and Methods. 878 LEE AND ZHU at A PE T Jornals on Sptem er 8, 2017 dm d.aspurnals.org D ow nladed from