17β-Estradiol Metabolism by Hamster Hepatic Microsomes

metabolites of 17β-estradiol arising from both these metabolic pathways have ... Thus, the usual method (COMT plus ascorbate) of determining 17β-est...
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Chem. Res. Toxicol. 1996, 9, 793-799

793

17β-Estradiol Metabolism by Hamster Hepatic Microsomes: Comparison of Catechol Estrogen O-Methylation with Catechol Estrogen Oxidation and Glutathione Conjugation Michael Butterworth,† Serrine S. Lau, and Terrence J. Monks* Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712 Received November 20, 1995X

Catechol estrogens, the cytochromes P450 mediated metabolites of 17β-estradiol, undergo further metabolism either via catechol O-methyltransferase (COMT) catalyzed methylation, or by oxidation and subsequent thioether formation with glutathione (GSH). Secondary metabolites of 17β-estradiol arising from both these metabolic pathways have been identified in vivo. However, the relative contribution of catechol O-methylation, and catechol oxidation followed by GSH conjugation, to the disposition of the catechol estrogens is unclear. We have therefore quantified both pathways of catechol estrogen disposition, generated in situ from 17β-estradiol, in hamster hepatic microsomes. 17β-Estradiol was readily converted to 2- and 4-hydroxy-17β-estradiol, both of which were effectively methylated in the presence of COMT (300 units/mL). Addition of GSH (50 µM-1 mM) to microsomal incubations resulted in the formation of four catechol estrogen-derived GSH conjugates. Three conjugates of 2-hydroxy17β-estradiol were identified: 2-hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy-1glutathion-S-yl-17β-estradiol, and 2-hydroxy-4-glutathion-S-yl-17β-estradiol. In contrast, just one GSH conjugate of 4-hydroxy-17β-estradiol was identified: 4-hydroxy-1-glutathion-S-yl17β-estradiol. When a combination of COMT and GSH were simultaneously added to microsomal incubations, both metabolic pathways competed for the same pool of catechol estrogens, and ascorbate dramatically influenced which of these two pathways predominate. In the presence of ascorbate, catechol estrogen methylation predominated over catechol estrogen oxidation and GSH conjugation. In the absence of ascorbic acid, catechol estrogen methylation, and catechol estrogen oxidation linked to GSH conjugation, contributed equally to the disposition of the catechol estrogens. 17β-Estradiol 2- and 4-hydroxylase activity was always higher in the absence of ascorbate, irrespective of whether GSH or COMT was used as the trapping agent. Thus, the usual method (COMT plus ascorbate) of determining 17β-estradiol 2- and 4-hydroxylase activity underestimates enzyme activity by ∼50% when compared to the value obtained when GSH is used to trap the o-quinones in the absence of ascorbate. A reassessment of 17β-estradiol 2- and 4-hydroxylase activity in different species and tissues is required to permit a more informed evaluation of the role of catechol estrogens in estrogeninduced carcinogenesis.

Introduction Exposure to estrogens has been associated with neoplastic changes in both humans and laboratory animals (1). For example, in an established model of estrogenmediated carcinogenesis, male Syrian golden hamsters develop renal carcinoma following prolonged exposure to estrogens (2-4). Although the underlying mechanism of this, and other estrogen-induced carcinoma, remains to be fully elucidated, a number of mechanisms have been proposed to explain estrogen-mediated carcinogenesis. For example, because the receptor-binding affinity for a series of estrogens did not correlate to their carcinogenic index (5), their carcinogenicity was linked to metabolism to reactive catechols or quinones (1). Aromatic hydroxylation of 17β-estradiol at the C-2 or C-4 position generates catechol estrogens, 2-hydroxy-17β-estradiol and 4-hy* Address correspondence to this author at the above address. Tel: (512) 471-6699; FAX: (512) 471-5002; EMail: [email protected]. † Present address: MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Rd., Leicester LE1 9HN, U.K. X Abstract published in Advance ACS Abstracts, May 1, 1996.

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droxy-17β-estradiol, which have limited affinity for estrogen receptors (6). However, 4-hydroxy-17β-estradiol, but not 2-hydroxy-17β-estradiol, causes renal tumors upon prolonged administration to hamsters (7). Catechol estrogens are readily oxidized to reactive semiquinones and o-quinones (1), both of which may have pathological effects through their ability to interact with genetic material, either directly or indirectly via the generation of reactive oxygen species, and initiate tumor formation (1). While the oxidative metabolism of estrogens occurs in a number of organs, the liver is the major site of catechol estrogen formation (8). Thus, the generation of catechol estrogens in hamster hepatic microsomes is greater than in kidney microsomes, and reactive electrophilic metabolites of [4-14C-]-17β-estradiol covalently bind to hamster hepatic microsomal protein substantially more than to renal microsomal protein (9). However, in male Syrian hamsters, the kidney is the target of estrogen-mediated carcinogenicity. It has therefore been proposed that the in situ generation of catechol estrogens in hamster kidney, which expresses relatively high levels © 1996 American Chemical Society

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of the P450 catalyzing the 4-hydroxylation of 17βestradiol, or the delivery of liver-derived catechol estrogens to the kidney, which should be greater in the hamster because of the low hepatic and blood levels of catechol O-methyltransferase (COMT),1 contributes to the species and tissue selectivity (10, 11). Indeed, the species specificity of estrogen-mediated renal carcinogenesis has to some extent been rationalized on differences in the ability of COMT to detoxify catechol estrogens. In addition to the methylation of catechol estrogens, they may also undergo oxidation coupled to conjugation with glutathione (GSH). Whereas the ability of catechol estrogens to redox cycle is quenched by COMT-mediated monomethylation due to the masking of the catechol moiety, conjugation of quinones with GSH does not eliminate their reactivity (12). Although thioether metabolites of the catechol estrogens have been identified in vivo and in vitro (13-20), their quantitative contribution to the metabolism of catechol estrogen is unknown. The aim of the present work was therefore to determine the relative importance of catechol O-methylation, and oxidation followed by GSH conjugation, in the disposition of catechol estrogens generated in situ from 17β-estradiol.

Material and Methods Materials. 17β-Estradiol, 16R-hydroxyestrone, estrone, estriol, 2-hydroxyestrone, 2-methoxyestradiol, GSH, S-adenosylmethionine, magnesium chloride, ascorbic acid, and COMT (porcine liver, 1680 units of activity/mg of protein) were purchased from Sigma Chemical Co. (St. Louis, MO). 4-Methoxyestradiol was obtained from Steraloids (Wilton, NH). Reagents for the NADPH generating system (NADP+, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase) were purchased from Boehringer Mannheim (Indianapolis, IN). [4-14C]17β-Estradiol (98% radiochemical purity, 55.5 mCi/mmol) was obtained from Dupont Co. (Boston, MA). Animals. Male Syrian golden hamsters, weighing 90-110 g, were purchased from Sasco (Omaha, NE). Animals were housed on a 12h dark-12 h light cycle and allowed food and water ad libitum. Synthesis and Purification of Catechol Estrogens. 2and 4-hydroxy-17β-estradiol were prepared from 17β-estradiol by the method of Gelbke et al. (21). Briefly, 17β-estradiol (0.75 g) was dissolved in acetone (250 mL) and acidified with 10% acetic acid (v/v in water, 400 mL). The mixture was stirred, and potassium nitrosodisulfonate (2.5 g) was added at 0 and 15 min. The o-quinones were extracted into chloroform (50 mL × 3) and, following acidification with acetic acid (50 mL), were reduced to catechol estrogens by the addition of excess potassium iodide (∼2 g). Iodine formed in this reaction was reduced with 0.1 N sodium thiosulfate (∼20 mL). Water (100 mL) was added, and the chloroform layers were removed. The aqueous portion was further extracted with ethyl acetate/dichloromethane (1:1 v/v). The organic layers were pooled, and their volume was reduced to approximately 5 mL by rotary evaporation under vacuum. The crude catechol estrogen mixture was finally dried under a stream of nitrogen, and 2- and 4-hydroxy-17β-estradiols were isolated by semipreparative HPLC. A mixture of 2- and 1 Abbreviations: COMT, catechol O-methyltransferase; SAM, Sadenosyl methionine; GSH, glutathione; estriol, 1,3,5[10]-estratriene3,16R,17β-triol; 17β-estradiol, 1,3,5[10]-estratriene-3,17β-diol; 2-hydroxy17β-estradiol, 1,3,5[10]-estratriene-2,3,17β-triol; 2OH-1,4-bis-(GS)E2, 2-hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol; 2OH-4-(GS)E2, 2-hydroxy-4-glutathion-S-yl-17β-estradiol; 2OH-1-(GS)E2, 2-hydroxy-1-glutathion-S-yl-17β-estradiol; 4-hydroxy-17β-estradiol, 1,3,5[10]-estratriene3,4,17β-triol; 4OH-1-(GS)E2, 4-hydroxy-1-glutathion-S-yl-17β-estradiol; 4-hydroxy-17β-estradiolestrone, 3-hydroxy-1,3,5[10]-estratrien-17-one; 2-hydroxyestrone, 2,3-dihydroxy-1,3,5[10]-estratrien-17-one; 2-methoxy-17β-estradiol, 1,3,5[10]-estratriene-2,3,17β-triol 2-methyl ether; 4-methoxy-17β-estradiol, 1,3,5[10]-estratriene-3,4,17β-triol 4-methyl ether; NADPH, reduced β-nicotinamide adenine dinucleotide phosphate; NADP+, β-nicotinamide adenine dinucleotide phosphate.

Butterworth et al. 4-hydroxy-17β-estradiol (20 mg) was dissolved in methanol/1% acetic acid (3:1, 4 mL) and centrifuged, and the supernatant purified by HPLC (Shimadzu LC-6A). Aliquots (1 mL) were injected onto a 5 µm Ultrasphere ODS (Beckman) reverse phase semipreparative column (25 cm × 10 mm) and eluted at a flow rate of 3 mL/min with a linear gradient of methanol/water/acetic acid (54:45:1 v/v) to methanol/water/acetic acid (55.5:43.5:1 v/v) over 30 min, followed by a 6 min column wash with methanol (100%) and a further 10 min wash with 54% methanol to equilibrate to the initial conditions. The eluate was monitored at 284 nm with a Shimadzu UV spectrophotometric detector (SPD-6A). Under these conditions, two major UV absorbing peaks were eluted from the column with retention times of 24.0 and 28.2 min, which corresponded to authentic 4- and 2-OHE2, respectively. The catechol estrogen fractions from several injections were collected, and the methanol was eliminated by rotary evaporation. The aqueous component was frozen in dry ice/acetone and lyophilized. The resultant powders were reanalyzed by analytical HPLC, and each gave rise to a single UV absorbing peak. Synthesis and Purification of Catechol Estrogen GSH Conjugates. GSH conjugates of both 2- and 4-hydroxy-17βestradiol were prepared using a modified version of the method of Jellinck and Elce (22). Purified 2- or 4-hydroxy-17β-estradiol (50 mg) were dissolved in 50% acetic acid (40 mL,v/v) and stirred gently at room temperature. The catechol estrogens were oxidized with sodium periodate (1.4 mmol), producing a deep red solution. The quinones were quickly extracted into dichloromethane (10 mL × 2). The organic layer was acidified by the addition of 50% acetic acid (v/v, 8mL) and the mixture shaken with GSH (∼0.5-1.0 g) until decoloration of the solution. The organic and aqueous phases were separated, and the lower organic layer was washed with water (5 mL × 2). The aqueous phase and the washings were pooled and the volume was reduced by rotary evaporation. The GSH conjugates of 2- or 4-hydroxy-17β-estradiol were purified by reverse phase semipreparative HPLC using a 5 µm Ultrasphere ODS (Beckman) column. The samples were eluted with a linear gradient of methanol/water/acetic acid (46:53:1 v/v) to methanol/water/acetic acid (49:50:1 v/v) over 32 min at a flow rate of 3 mL/min, and the eluate was monitored at 284 nm. Oxidation of 2-hydroxy17β-estradiol, in the presence of GSH, gave rise to 3 major UV absorbing peaks with retention times of 9.7 min (product 1), 19.3 min (product 2), and 31.3 min (product 3). Oxidation of 4-hydroxy-17β-estradiol, in the presence of GSH, gave rise to only one major UV absorbing peak with a retention time of 28.2 min (product 4). The fractions from several injections were collected, and the methanol was removed by rotary evaporation. The residual aqueous fractions were frozen in a mixture of dry ice and acetone and lyophilized overnight. The resulting white powders were further analyzed by analytical HPLC and found to be greater than 95% pure. The structural identity of each product was characterized by 1H NMR spectroscopy (Bruker AM-500). The yield of product 1 could be increased by oxidizing either products 2 or 3 with sodium periodate and subsequently reducing with GSH, suggesting that products 2 and 3 were monosubstituted GSH conjugates and that product 1 was a bissubstituted GSH conjugate of 2-hydroxy-17β-estradiol. Characterization of 2-Hydroxy-17β-estradiol and Its GSH Conjugates. 2-Hydroxy-17β-estradiol was obtained as a beige powder, and in 1% acetic acid/methanol (4:1) exhibited a UV spectrum with λmax, nm (log max, M-1 cm-1) at 231 (3.51) and 285 (3.57). 1H NMR (DMSO-d6), chemical shifts (δ) in ppm, δ 6.62 (s, 1H, aromatic proton at C-4), δ 6.37 (s, 1H, aromatic proton at C-1), δ 4.46 (s, 1H, hydroxyl at C-17), δ 3.50 (t, 1H, proton at C-17), δ 1.0-2.0 (m,18H), δ 0.66 (s, 3H, methyl at C-18). 2-Hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol (product 1) was obtained as a white powder and in 1% acetic acid/methanol (9:1) gave a UV spectrum with λmax, nm (log max, M-1 cm-1) at 233 (4.08), 279 (3.83), and 313 (3.46). 1H NMR (D2O) δ 4.46 (s, 1H, hydroxyl at C-17), δ 4.23 (dd, 2H, cysteine R), δ 3.62 (m, 4H, glycine R), δ 3.50 (t, 1H, proton at C-17), δ 2.67 and 2.58 (dd, 2H, cysteine β), δ 2.27 (m, 4H, glutamate γ), δ 1.94 (m, 4H glutamate β), δ 1.0-2.0 (m, 18H), δ 0.68 (s, 3H, methyl at C18). 2-Hydroxy-4-glutathion-S-yl-17β-estradiol

Catechol Estrogen Methylation and Oxidation (product 2) was obtained as a white powder and in 1% acetic acid/methanol (9:1) exhibited a UV spectrum with λmax, nm (log max, M-1 cm-1) at 232 (3.93), 260 (3.55), and 302 (3.58). 1H NMR (DMSO-d6) δ 6.44 (s, 1H, aromatic proton at C-1), δ 4.46 (s, 1H, hydroxyl at C-17), δ 4.02 (dd, 1H, cysteine R), δ 3.63 (m, 2H, glycine R), δ 3.55 (t, 1H, proton at C-17), δ 2.67 and 2.58 (dd, 1H, cysteine β), δ 2.27 (m, 2H, glutamate γ), δ 1.94 (m, 2H, glutamate β), δ 1.0-2.0 (m, 18H), δ 0.68 (s, 3H, methyl at C-18). 2-Hydroxy-1-glutathion-S-yl-17β-estradiol (product 3) was obtained as a white powder. In 1% acetic acid/methanol (4:1), this conjugate gave a UV spectrum with λmax, nm (log max, M-1 cm-1) at 231 (3.80), 260 (3.36), and 301 (3.40). 1H NMR (DMSOd6) δ 6.73 (s, 1H, aromatic proton at position 4), δ 4.47 (s,1H, hydroxyl at C-17), δ 4.23 (dd, 1H, cysteine R), δ 3.62 (m, 2H, glycine R), δ 3.49 (t, 1H, proton at C-17), δ 3.03 and 2.93 (dd, 1H, cysteine β), δ 2.27 (m, 2H, glutamate γ), δ 1.94 (m, 2H, glutamate β), δ 0.686 (s, 3H, methyl at C-18). The NMR spectra of the GSH conjugates of 2-hydroxy-17β-estradiol were in agreement with previously published data (16-18). The glutamate R proton in each of the spectra was obscured by a strong water signal. Characterization of 4-Hydroxy-17β-estradiol and Its GSH Conjugate. 4-Hydroxy-17β-estradiol was obtained as an off-white powder and in 1% acetic acid/methanol (4:1) exhibited a UV spectrum with λmax, nm (log max, M-1 cm-1) at 231 (3.54) and 277 (3.23). 1H NMR (DMSO-d6) δ 6.53 (S, 2H, aromatic protons at C1 and C-2), δ 3.5 (t, 1H, proton at C-17), δ 1.0-2.0 (m, 17H), δ 0.64 (s, 3H, methyl at C-18). 4-Hydroxy-1glutathion-S-yl-17β-estradiol (product 4) was obtained as a white powder and in 1% acetic acid/methanol (9:1) exhibited a UV spectrum with λmax, nm (log max, M-1 cm-1) at 232 (3.92), 254 (3.63), and 291 (3.30). 1H NMR (DMSO-d6) δ 6.73 (s, 1H, aromatic proton at C-2), δ 4.47 (s, 1H, hydroxyl at C-17), δ 4.32 (dd, 1H, cysteine R), δ 3.67 (m, 1H, glutamate R), δ 3.5 (t, 1H, proton at C-17), δ 2.85 and 2.67 (dd, 1H, cysteine β), δ 2.37 (m, 2H, glutamate γ), δ 1.95 (m, 2H, glutamate β), δ 1.0-2.0 (m, 18H), δ 0.66 (s, 3H, methyl at C-18). Preparation of Hepatic Microsomes. Hamsters were euthanized by cervical dislocation and their livers rapidly removed, weighed, and minced on ice. The minced tissue was homogenized with HEPES-KCl buffer (pH 7.4; 20 mM HEPES; 1.15% KCl) (w/v, 1:10) at 4 °C. The homogenates were centrifuged at 9000g for 20 min, and the resulting supernatant removed and centrifuged at 100000g for 1 h. The microsomal pellet was washed and gently resuspended in HEPES-KCl buffer prior to a further centrifugation at 100000g for 1 h. The final microsomal pellet was resuspended in HEPES-KCl buffer and the protein content determined by the method of Lowry et al. (23) using bovine serum albumin as a standard. The microsomal suspension was further diluted to give a final protein content of 0.5 mg/mL. Microsomes were always freshly prepared on the day of use. Microsomal Incubations. Optimal conditions for the methylation of the catechol estrogens generated in situ by hamster hepatic microsomes from 17β-estradiol have been previously established (24). Incubations (final volume 1 mL) were carried out at 37 °C in 25 mL glass tubes, with 75 µM 17β-estradiol and 300 units/mL COMT. All incubation mixtures contained magnesium chloride (1 mM), ascorbate (1 mM, unless indicated otherwise), and S-adenosyl-L-methionine (0.3 mM) and were initiated by the addition of an NADPH generating system [NADP+ (0.52 mM), glucose 6-phosphate (8 mM), and glucose6-phosphate dehydrogenase (300 units/mL)]. Subsequent incubations examined the microsomal oxidation and GSH conjugation of catechol estrogens generated from 17β-estradiol. The effect of GSH (1 mM) on the methylation of catechol estrogens derived from [4-14C]-17β-estradiol (75 µM) by hamster hepatic microsomes was determined in the presence and absence of ascorbate (1 mM). The reaction was stopped by adding 10% perchloric acid (100 µL). An aliquot (500 µL) was stored on ice and following centrifugation used to determine the water-soluble metabolite fraction (estriol, 2-hydroxy-1-glutathion-S-yl-17βestradiol, 4-hydroxy-1-glutathion-S-yl-17β-estradiol, 2-hydroxy4-glutathion-S-yl-17β-estradiol, and 2-hydroxy-1,4-bis(glutathionS-yl)-17β-estradiol). A second aliquot (500 µL) was extracted

Chem. Res. Toxicol., Vol. 9, No. 4, 1996 795

Figure 1. HPLC elution profile of 17β-estradiol and some authenticated metabolites: 1, 2-hydroxy-1,4-bis(glutathion-Syl)-17β-estradiol (11.3 min); 2, 2-hydroxy-4-glutathion-S-yl-17βestradiol (15.1 min); 3, estriol (17.9 min); 4, 4-hydroxy-1glutathion-S-yl-17β-estradiol (23.1 min); 5, 2-hydroxy-1-glutathion-S-yl-17β-estradiol (25.3 min); 6, 4-hydroxy-17β-estradiol (29.2 min); 7, 2-hydroxy-17β-estradiol (31.0 min); 8, estrone (36.7 min); 9, 17β-estradiol (41.2 min), 10, 2-methoxy-17β-estradiol (44.8 min). 4-Methoxy-17β-estradiol (not shown) elutes with a retention time of 43.0 min. Conditions for the HPLC assay are described in the Materials and Methods. with ethyl acetate/dichloromethane (1:1 v/v) (1.5 mL × 3). The organic extracts were pooled to form the lipophilic metabolite fraction (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxy-17βestradiol, 4-hydroxy-17β-estradiol, estrone, 17β-estradiol, 2-methoxy-17β-estradiol, and 4-methoxy-17β-estradiol). With the exception of estrone (93%), recovery of the organic metabolites was 100%. The organic solvent was removed under a stream of N2 and the dried residue dissolved in methanol/1% acetic acid (1:1 v/v) (500 µL) prior to HPLC analysis. The metabolism of [4-14C]17β-estradiol was assessed by determining the covalent binding of reactive electrophilic [4-14C]-17β-estradiol metabolites to microsomal protein, and by the profile of 17β-estradiol metabolites in the resultant supernatant, as assessed by analytical HPLC. Measurement of Covalent Binding of Reactive Electrophilic [4-14C]-17β-Estradiol Metabolites to Hepatic Microsomal Protein. Microsomal incubations were terminated by the addition of 100 µL of 10% perchloric acid. The microsomal protein pellet was then extracted extensively with 4 mL aliquots of methanol/ethyl acetate (1:1v/v) until no further radioactivity could be removed from the pellet. The pellet was solubilized by boiling in 0.5 M sodium hydroxide (1.5 mL) for 15 min. An aliquot (0.5 mL) was transferred to a miniscintillation vial (Poly-Q-vial, Beckman) and radioactivity assessed after the addition of liquid scintillation cocktail (Ready Value, Beckman). Samples were analyzed on a Beckman LS1800 liquid scintillation spectrometer. Radioactivity was corrected for background quenching and expressed as nanomoles covalently bound [4-14C]-17β-estradiol equivalents per milligram of microsomal protein. Measurement of 17β-Estradiol and Its Metabolites by HPLC. Separation of 17β-estradiol and its metabolites was achieved using analytical HPLC. Aliquots (100 µL) of either the organic extractable metabolites from the microsomal incubations or the residual aqueous supernatant, containing the water-soluble metabolites, were injected onto a 5 µm Partisil ODS3 analytical cartridge column (25 cm × 4.6 mm) (Whatman) and eluted with an initial solvent of methanol/water/acetic acid (42:67:1 v/v) for 6 min, followed by a linear increase to 55% methanol over 30 min, followed by a linear gradient to methanol/ water/acetic acid (68:31:1 v/v) over 18 min, at a flow rate of 1 mL/min. The eluate was monitored at 280 nm. A typical HPLC profile for authentic 17β-estradiol metabolites is shown in Figure 1.

Results Formation of Thioether Conjugates of Catechol Estrogens by Hamster Hepatic Microsomes. The

796 Chem. Res. Toxicol., Vol. 9, No. 4, 1996

Figure 2. The effect of glutathione concentration on the formation of catechol estrogen glutathione conjugates in hamster hepatic microsomes. Microsomes were incubated with 17βestradiol (75 µM) in the presence of an NADPH generating system, ascorbic acid (1 mM), and glutathione (0-1 mM). The reactions were terminated after 30 min, and the water-soluble fraction was analyzed by HPLC with UV detection, as described in the Materials and Methods. Each point represents the mean ( SEM (n ) 3). Symbols represent (0) 2-hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol; (9) 2-hydroxy-1-glutathion-S-yl17β-estradiol; (O) 2-hydroxy-4-glutathion-S-yl-17β-estradiol; and (b) 4-hydroxy-1-glutathion-S-yl-17β-estradiol. Statistical significance was determined by one-way analysis of variance with a post hoc Student Newman-Keul’s test. Data were significantly different from experiments carried out in the absence of exogenously added glutathione at *p < 0.05.

conversion of 17β-estradiol to catechol estrogens is concentration dependent (24). C2 and C4 hydroxylation of 17β-estradiol is maximal at 75 µM 17β-estradiol, and monomethylation at the C2 position is 3-fold greater than at C4 after 30 min incubation with 300 units/mL COMT (24). The addition of GSH (50-1000 µM) to hamster hepatic microsomes containing 17β-estradiol, in the presence or absence of ascorbate (1 mM), gave rise to four catechol estrogen thioether conjugates (Figure 2) which were identified by comparison with the HPLC retention times and UV spectra of authentic standards. Three GSH conjugates of 2-hydroxy-17β-estradiol were identified: 2-hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy-1-glutathion-S-yl-17β-estradiol, and 2-hydroxy-4glutathion-S-yl-17β-estradiol. In contrast, only one GSH conjugate of 4-hydroxy-17β-estradiol was identified, 4-hydroxy-1-glutathion-S-yl-17β-estradiol. Significant increases (p < 0.05) in the formation of each of the GSH conjugates, in the absence of ascorbate, occurred at 50 µM GSH. In contrast, a concentration of 500 µM GSH was required to produce significant (p < 0.05) increases in the recovery of the conjugates (Figure 2) in the presence of ascorbate. Thus, under conditions optimized for catechol estrogen O-methylation substantial oxidation of the catechols occurs, and the resulting o-quinones are effectively trapped with GSH. The formation of the GSH conjugates in the absence of exogenously added GSH is due to the presence of residual GSH (32 ( 4 nmol/0.5 mg of microsomal protein, equivalent to a concentration of 16 µM). The ability of GSH to trap the o-quinones was maximal when experiments were carried out in the absence of ascorbate (Figures 2 and 3), presumably because the 1,4Michael addition of GSH competes with the reduction of the o-quinones by ascorbate, thereby reducing the pool available for conjugation. The enhanced (8-fold) formation of 2-hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol in the absence of ascorbate was especially striking. By comparison, the recovery of 2-hydroxy-4-glutathion-S-yl17β-estradiol, 2-hydroxy-1-glutathion-S-yl-17β-estradiol, and 4-hydroxy-1-glutathion-S-yl-17β-estradiol was en-

Butterworth et al.

Figure 3. The effect of ascorbic acid on the formation of catechol estrogen glutathione conjugates in hamster hepatic microsomes. Microsomes were incubated with 17β-estradiol (75 µM) in the presence of an NADPH generating system, ascorbic acid (0 or 1 mM), and glutathione (1 mM). The reactions were terminated after 30 min, and the water-soluble fraction was analyzed by HPLC with UV detection, as described in the Materials and Methods. Each bar represents the mean ( SEM (n ) 9). Statistical significance was determined by one-way analysis of variance with a post hoc Student Newman-Keul’s test. Data were significantly different from experiments carried out in the presence of ascorbate at *p < 0.01, †p < 0.05.

Figure 4. Effect of COMT and glutathione on the recovery of catechol estrogen metabolites. Hamster hepatic microsomes were incubated with 17β-estradiol (75 µM) in the presence of an NADPH generating system, ascorbic acid (1 mM), glutathione (0 or 1 mM), and COMT (0 or 300 units/mL). The reactions were terminated after 30 min and the methoxy (0) and GSH conjugates (9) of 2- and 4-hydroxy-17β-estradiol analyzed by HPLC with UV detection, as described in the Materials and Methods. Each bar represents the mean ( SEM (n ) 3). Statistical significance was determined by one-way analysis of variance with a post hoc Student Newman-Keul’s test (p < 0.05). *Significantly different from control incubations and those carried out in the presence of COMT, and COMT in combination with GSH; §significantly different from control incubations and those carried out in the presence of COMT in combination with GSH; †significantly different from control incubations; ¶significantly different from incubations carried out in the presence of either COMT, or COMT in combination with GSH.

hanced 2.6-, 2.7-, and 1.3-fold, respectively, in the absence of ascorbate. The increased yield of conjugates was accompanied by a substantial diminution of catechol estrogens, whereas the formation of estriol remained unaffected (24), indicating that neither ascorbate nor GSH influences 16R-hydroxylase activity. Effect of GSH on the Methylation of Catechol Estrogens by COMT. O-Methylation of the catechol estrogens was greater in the absence of ascorbate (29.9 ( 2.9 nmol/(mg‚30 min), mean ( SEM) than in its presence (18.3 ( 1.7 nmol/(mg‚30 min), mean ( SEM) (Figure 4). In the presence of 1 mM GSH, O-methylation and oxidation coupled to GSH conjugation contribute

Catechol Estrogen Methylation and Oxidation

Figure 5. Effect of glutathione on the covalent binding of reactive electrophilic metabolites of [4-14C]-17β-estradiol to microsomal protein. Hamster hepatic microsomes were incubated with [4-14C]-17β-estradiol (75 µM, 100 µCi) in the presence (O) or absence (b) of 1 mM ascorbic acid, in the presence of an NADPH generating system and varying concentrations of glutathione (0, 0.25, 0.50, 0.75, 1.0, and 5.0 mM). The reactions were terminated after 30 min, and covalent binding was determined as described in the Materials and Methods. Each point represents the mean ( SEM (n ) 3). The inset shows the same data expressed as a % of control values. Statistical significance was determined by one-way analysis of variance, with a post hoc Student Newman-Keul’s test. Data were significantly different from experiments carried out in the absence of ascorbate at *p < 0.01, †p < 0.05.

equally to the disposition of the catechol estrogens (13.5 ( 1.1 and 13.2 ( 1.5 nmol/(mg‚30 min), mean ( SEM). When both GSH and ascorbate are present, catechol O-methylation exceeds oxidation and GSH conjugation by about 2:1 (16.6 ( 1.7 vs 6.5 ( 0.1 nmol/(mg‚30 min), mean ( SEM). Thus, depending upon either the presence or absence of ascorbate, oxidation coupled to GSH conjugation represents between 28% and 50% of the fate of the catechol estrogens in hamster liver microsomal incubations. Interestingly, COMT quenches GSH conjugate formation in the presence or absence of ascorbate (Figure 4) without increasing the recovery of methylated metabolites. We speculate that a fraction of the catechol estrogen GSH conjugates may undergo enzymatic Omethylation, in a manner similar to that reported for the O-methylation of the corresponding cysteine conjugates (25, 26). We are currently addressing this possibility experimentally. Effect of GSH, Ascorbate, and COMT on the Covalent Binding of Electrophilic Metabolites of [4-14C]-17β-Estradiol to Microsomal Protein. Covalent binding of reactive electrophilic metabolites of [4-14C]-17β-estradiol to microsomal protein was substantially reduced by the addition of GSH, even in the presence of ascorbate (Figure 5). Thus, 1 mM GSH decreased covalent binding by 50.3 ( 4.6% in the presence of ascorbate, and by 74.8 ( 2.2% in the absence of ascorbate. The inhibition of covalent binding by 5 mM GSH was identical in the presence or absence of ascorbate (82.7 ( 3.1% and 83.3 ( 2.6%, respectively). Because there are differences in the fraction of covalently bound metabolites in the presence and absence of ascorbate, the residual fraction of reactive electrophilic metabolites available for trapping when GSH is added to the incubations differs substantially. The inset to Figure 5 clearly shows that the ability of GSH to trap the “ascorbate insensitive” fraction of electrophilic metabolites is less efficient than its ability to trap the “ascorbate sensitive” pool, consistent with the finding that higher concentra-

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Figure 6. Effect of COMT and glutathione on the covalent binding of reactive electrophilic metabolites of [4-14C]-17βestradiol to microsomal protein. Hamster hepatic microsomes were incubated with [4-14C]-17β-estradiol (75 µM, 100 µCi) in the presence of an NADPH generating system, glutathione (0 or 1 mM), COMT (0 or 300 units/mL), and ascorbate (1 mM). The reactions were terminated after 30 min, and covalent binding was determined as described in the Materials and Methods. Each bar represents the mean ( SEM (n ) 6). Statistical significance was determined by one-way analysis of variance with a post hoc Student Newman-Keul’s test (p < 0.01). *Significantly different from control incubations and those carried out in the presence of COMT. Table 1. The Effects of Glutathione and Catechol O-Methyltransferase on the Determination of the 2- and 4-Hydroxylation of 17β-Estradiol in Hamster Liver Microsomesa total 2- and 4-hydroxylated metabolites [nmol/(mg‚30 min)] no additions COMT (300 units/mL) glutathione (1 mM) COMT (300 units/mL) and glutathione (1 mM)

w/o ascorbate

ascorbate

8.2 ( 0.3 32.8 ( 2.9 39.5 ( 2.2 26.7 ( 2.4

9.3 ( 1.7 21.1 ( 1.5 16.4 ( 2.1 23.1 ( 1.8

a Hamster hepatic microsomes were incubated with 17β-estradiol (75 µM) in the presence of an NADPH generating system, ascorbic acid (0 or 1 mM), glutathione (0 or 1 mM), and COMT (0 or 300 units/mL). The reactions were terminated after 30 min and the methoxy- and thiol-derived metabolites of 2- and 4-hydroxy-17β-estradiol analyzed by HPLC with UV detection, as described in the Materials and Methods. Data represent the mean ( SEM (n ) 3).

tions of GSH are required to produce significant increases in the yield of GSH conjugates in the presence of ascorbate (Figure 2). In contrast to GSH, COMT had no effect on the covalent binding of [4-14C]-17β-estradiol to microsomal protein, and the combination of COMT and GSH was just as effective as GSH alone (Figure 6). Changes in covalent binding were accompanied by alterations in the profile of [4-14C]-17β-estradiol-derived metabolites (Figure 4). Thus, the process of catechol estrogen oxidation and GSH conjugation competes with catechol O-methylation for the same pool of catechol estrogens, as reflected by changes in the levels of 2- and 4-methoxy17β-estradiol, and differences in the formation of secondary thioether metabolites. It is of interest to note that the recovery of thioether metabolites in the presence of GSH (Figure 4) approximates the amount of covalent binding to microsomal protein in the absence of GSH (Figure 5). Thus, when total microsomal 2- and 4-hydroxylase activity is determined, the estimate of metabolism is higher when GSH is used to trap the o-quinones than when COMT is used to trap the corresponding catechols (Table 1). Moreover, 2- and 4-hydroxylase activity is always higher in the absence of ascorbate,

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irrespective of whether GSH or COMT are used as the trapping agent (Table 1).

Discussion The secondary metabolism of 17β-estradiol involves both catechol estrogen methylation and catechol estrogen oxidation coupled to GSH conjugation. One of the aims of the present work was to quantitate the oxidation and GSH conjugation of 2- and 4-hydroxy-17β-estradiol in hamster hepatic microsomes. 2-Hydroxy-1,4-bis(glutathion-S-yl)-17β-estradiol, 2-hydroxy-1-glutathion-S-yl17β-estradiol, 2-hydroxy-4-glutathion-S-yl-17β-estradiol, and 4-hydroxy-1-glutathion-S-yl-17β-estradiol were identified as metabolites of 17β-estradiol (75 µM) in hamster hepatic microsomes supplemented with 1 mM GSH (Figure 2). Thus, under conditions optimized for catechol estrogen O-methylation (24), catechol estrogen oxidation occurs readily, and in the presence of GSH the o-quinones are trapped as GSH conjugates. The competition between catechol O-methylation and catechol oxidation coupled to GSH conjugation may have important implications for the in vivo disposition of 17βestradiol. Intracellular concentrations of GSH are relatively high in mammalian tissue, typically in the range 0.4-6 mM. GSH thioethers are formed in rat or human liver slices incubated with either 17β-estradiol or 2-hydroxy-17β-estradiol (27). In addition, 17β-estradiol depletes GSH in both isolated rat hepatocytes and microsomal incubations (28). Although the relative importance of catechol estrogen thioether formation to the overall metabolism of estrogens in vivo is unclear, Ball et al. (27) noted that in liver, the most active of the peripheral tissues that metabolize estrogens, the major metabolites of 17β-estradiol were present mainly in the water-soluble fraction, and emphasized that “the overall importance of catechol estrogen monomethyl ethers should not be overestimated...the major importance of conjugates and minor importance of methyl ethers in vivo should be stressed.” Moreover, COMT activity in hamster tissue is lower than in several other rodent species (10). In particular, COMT activity in hamster liver is 10-fold less than in mouse liver, and 100-fold less than in rat liver. Thus the fraction of catechol estrogen available for oxidation and conjugation with GSH is likely to be greater in the hamster than in other rodent species. Preliminary experiments on the in vivo disposition of 17β-estradiol support this view. In addition, Stalford et al. (29) recently reported that only a small fraction (4-7%) of 2-hydroxy-17β-estradiol excreted in hamster bile was methylated, and no 2-methoxy-17β-estradiol was found in urine, consistent with the view that catechol estrogen O-methylation plays a minor role in the disposition of catechol estrogens in hamsters in vivo. The important modulatory role attributed to COMT in this model of estrogen-mediated carcinogenesis is therefore incompatible with both our data and that of Stalford et al. (29). The balance of O-methylation, and oxidation followed by GSH conjugation, is substantially influenced by the availability of ascorbic acid (Figures 3 and 4). Interestingly, ascorbate inhibited catechol O-methylation by about 40% (Figure 4). Catechol estrogen oxidation coupled to GSH conjugation was also inhibited by ascorbate (Figures 3 and 4) presumably because ascorbate effectively reduces the o-quinones to catechols, thereby reducing the fraction of the o-quinone available for the addition of GSH. Thus, depending upon the concentration of ascorbate, oxidation coupled to GSH conjugation

Butterworth et al.

represents between 28% and 50% of the fate of the catechol estrogens in hamster liver microsomal incubations. Ascorbate has also been shown to diminish the formation of catechol estrogens by rat microsomes, and their further metabolism to GSH conjugates (30) reduces the covalent binding of reactive electrophilic [14C]-17βestradiol metabolites to rat microsomal protein (13, 31) and reduces the proportion of 17β-estradiol converted to aqueous metabolites in isolated rat hepatocytes (31). Both GSH and cysteine conjugates are also formed in incubations of 17β-estradiol with human liver homogenates, although no estrogen mercapturic acid has yet been demonstrated in human urine (32). It has therefore been suggested that, in contrast to the rat, humans do not synthesize estrogen-GSH conjugates (33). An alternative, and equally plausible, possibility is that catecholestrogen-thioether conjugates are formed in the human (as evidenced by the in vitro data), but that they are metabolized to products which are either not readily excreted or which become bound to tissue macromolecules. Indeed, following the administration of radiolabeled 2-hydroxy-1-glutathion-S-yl-17β-estradiol to rats, only 15% of the dose was recovered in the urine and 5% in the feces after several days (19) and only the Nacetylcysteine derivative could be identified in the urine. The authors of this paper noted that “Oestrogen-glutathione conjugates formed in the intact rat may be excreted in an apparently non-steroidal, possibly proteinbound form”. Consistent with these views is our finding that, in the absence of ascorbate, the pattern of thioether metabolites shifts toward formation of 2-hydroxy-bis(glutathion-S-yl)-17β-estradiol (Figure 3), indicating that 2-hydroxy-1-glutathion-S-yl-17β-estradiol and 2-hydroxy4-glutathion-S-yl-17β-estradiol are also capable of undergoing oxidation. Thus, conjugation of the o-quinones with GSH does not eliminate their ability to undergo further oxidation and/or alkylation reactions. Since hepatic GSH competes with COMT for the same pool of catechol estrogens, it is pertinent to consider the possible subcellular distribution of 17β-estradiol and its metabolites, and how this may affect its disposition. The hydrophobicity of 17β-estradiol permits its preferential partitioning into lipid membranes such as the endoplasmic reticulum, which facilitates access to P450, and metabolism to catechol estrogens. The resultant catechols are also lipophilic in nature and may be retained in the hydrophobic environment of the endoplasmic reticulum, where they may undergo oxidation to the corresponding o-quinones. In support of this view, both 2- and 4-hydroxyestrones are rapidly oxidized by P450 (34). However, the fraction of 2- and 4-hydroxy-17βestradiol that escapes the endoplasmic reticulum may then undergo either catechol O-methylation or oxidation followed by GSH conjugation. The ability of COMT, primarily located in the cytosol (35), to interact with catechol estrogens generated from 17β-estradiol will be limited if they are preferentially associated with intracellular lipid, and will be dependent upon the relative distribution of ascorbic acid and GSH. The finding that the recovery of thioether metabolites in the presence of GSH (Figure 4) approximates the amount of covalent binding to microsomal protein in the absence of GSH (Figure 5) indicates that GSH gains access to the microsomal compartment where it is an efficient trapper of reactive electrophilic metabolites. The presence of UDPGA glucuronyl transferases within the endoplasmic reticulum may further limit the fraction of catechol estrogens reaching the cytosol.

Catechol Estrogen Methylation and Oxidation

It is important to note that the apparent microsomal 2- and 4-hydroxylase activity is higher when GSH is used to trap the o-quinones than when COMT is used to trap the corresponding catechols (Table 1). Moreover, 17βestradiol 2- and 4-hydroxylase activity is usually determined in the COMT-linked assay, in the presence of ascorbate. However, our data indicate that the apparent activity of 17β-estradiol 2- and 4-hydroxylase is always higher in the absence of ascorbate, irrespective of whether GSH or COMT is used as the trapping agent (Table 1). Thus, utilizing the usual method (COMT plus ascorbate) to determine 17β-estradiol 2- and 4-hydroxylase activity (21.1 ( 1.5 nmol/(mg‚30 min)) underestimates enzyme activity by ∼50% when compared to the value obtained when GSH is used to trap the o-quinones in the absence of ascorbate (39.5 ( 2.2 nmol/(mg‚30 min)). A reassessment of 17β-estradiol 2- and 4-hydroxylase activity in different species and tissue is required to permit a more informed evaluation of the role of catechol estrogens in estrogen-induced carcinogenesis. In summary, we have shown that, under in vitro conditions optimized for catechol estrogen methylation, 2- and 4-hydroxy-17β-estradiols undergo oxidation coupled to GSH conjugation. Although our data and that of Stalford et al. (29) are incompatible with the proposition that COMT plays an important modulatory role in the Syrian hamster model of estrogen-mediated carcinogenesis, the mechanistic involvement of catechol estrogens in this process remains to be elucidated.

Acknowledgment. We would like to thank Dr. Judy Bolton (University of Illinois at Chicago) for providing us with copies of manuscripts prior to their publication. The project described in this paper was supported by Grant CA 58036 from the National Cancer Institute, NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCI.

References (1) Liehr, J. G., and Roy, D. (1990) Free radical generation of cycling estrogens. Free Radical Biol. Med. 8, 415-423. (2) Matthews, V. S., Kirkman, H., and Bacon, R. L. (1947) Kidney damage in the golden hamster following chronic administration of diethylstilbestrol and sesame oil. Proc. Soc. Exp. Biol. Med. 66, 195-196. (3) Kirkman, H., and Bacon, R. L. (1952) Estrogen-induced tumors of the kidney. 1. Incidence of renal tumors in intact and gonadectomized male golden hamster treated with diethylstilbestrol. J. Natl. Cancer Inst. 13, 745-755. (4) Kirkman, H. (1959) Estrogen-induced tumors of the kidney III. Growth characteristics in the Syrian hamster. Natl. Cancer Inst. Monogr. 1, 1-57. (5) Liehr, J. G. (1983) 2-Fluoroestradiol; separation of estrogenicity from carcinogenicity. Mol. Pharmacol. 23, 278-281. (6) Martucci, C. P., and Fishman, J. (1993) P450 enzymes of estrogen metabolism. Pharmacol. Ther. 57, 237-257. (7) Liehr, J. G., Fang, W.-F., Sirbasku, D. A., and Ari-Ulubelen, A. (1986) Carcinogenicity of catechol estrogens in Syrian hamsters. J. Steroid Biochem. 24, 353-356. (8) Ball, P., Haupt, M., and Knuppen, R. (1972) Comparative studies on the metabolism of estradiol in the brain, the pituitary and the liver of the rat. Acta Endocrinol. 87, 1-11. (9) Haaf, H., Li, S. A., and Li, J. J. (1987) Covalent binding of estrogen metabolites to hamster liver microsomal proteins: inhibition by ascorbic acid and catechol O-methyltransferase. Carcinogenesis 8, 209-215. (10) Li, S. A., Purdy, R. H., and Li, J. J. (1989) Variations in catechol O-methyltransferase activity in rodent tissues: possible role in estrogen carcinogenicity. Carcinogenesis 10, 63-67. (11) Weisz, J., Bui, Q. D., Roy, D., and Liehr, J. G. (1992) Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a

Chem. Res. Toxicol., Vol. 9, No. 4, 1996 799

(12) (13)

(14)

(15)

(16)

(17) (18) (19) (20) (21) (22) (23) (24)

(25) (26) (27)

(28) (29)

(30) (31)

(32)

(33) (34)

(35)

potential pathway of metabolic activation of estrogens. Endocrinology 131, 655-661. Monks, T. J., and Lau, S. S. (1992) Toxicology of quinonethioethers. Crit. Rev. Toxicol. 22, 243-270. Nelson, S. D., Mitchell, J. R., Dybing, E., and Sasame, H. A. (1976) Cytochrome P-450-mediated oxidation of 2-hydroxyestrogens to reactive intermediates. Biochem. Biophys. Res. Commun. 70, 1157-1165. Marks, F., and Hecker, E. (1969) Metabolism and mechanism of action of estrogens. XII. Structure and mechanism of formation of water-soluble and protein-bound metabolites of estrone in rat liver microsomes in vitro and in vivo. Biochim. Biophys. Acta 187, 250-265. Kuss, E. (1969) Water-soluble metabolites of oestradiol-17β. III. Separation and identification of 1- and 4-glutathione thioethers of 2,3-dihydroxy-oestratrienes. Hoppe-Seyler’s Z. Physiol. Chem. 350, 95-97. Kuss, E. (1971) Microsomal oxidation of estradiol-17β. 2-Hydroxylation and 1- or 4-thioether formation with and without 17hydroxyl dehydrogenation. Hoppe-Seylers, Z. Physiol. Chem. 352, 817-836. Elce, J. S. (1970) Metabolism of a glutathione conjugate of 2-hydroxyoestradiol by rat liver and kidney preparations in vitro. Biochem. J. 116, 913-917. Elce, J. S., and Harris, J. (1971) Conjugation of 2-hydroxyestradiol-17β [1,3,5,(10)-estratriene-2,3,17β-triol] with glutathione in the rat. Steroids 18, 583-591. Elce, J. S. (1972) Metabolism of a glutathione conjugate of 2-hydroxyoestradiol-17β in the adult male rat. Biochem. J. 126, 1067-1071. Elce, J. S., and Chandra, J. (1973) Estrogen mercapturic acids in the adult male rat. Steroids 22, 699-705. Gelbke, H. P., Haupt, O., and Knuppen, R. (1973) A simple method for the synthesis of catechol estrogens. Steroids 21, 205. Jellinck, P. H., and Elce, J. S. (1969) Synthesis of estrogen glutathione and cysteine derivatives. Steroids 13, 711-718. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-275. Butterworth, M., Lau, S. S., and Monks, T. J. (1996) 17β-Estradiol metabolism in hamster liver microsomes. Implications for the catechol O-methyltransferase mediated detoxication of catechol estrogens. Drug Metab. Dispos. In press. Knuppen, R., Ball, P., Haupt, O., and Breuer, H. (1972) Enzymic methylation of thioethers of 2-hydroxyoestradiol by human and rat liver in vitro. Hoppe-Seyler’s Z. Physiol. Chem. 353, 565-568. Ball, P., Hoppen, H. O., and Knuppen, R. (1974). Metabolism of oestradiol-17β and 2-hydroxyoestradiol-17β in rat liver slices. Hoppe-Seyler’s Z. Physiol. Chem. 355, 1451-1462. Ball, P., Haupt, M., and Knuppen, R. (1983) Biogenesis and metabolism of catechol estrogens in vitro. In Catechol Estrogens (Merriam, G. R., and Lipsett, M. B., Eds.) pp 91-103, Raven Press, New York. Ruiz-Larrea, M. B., Garrido, M. J., and Lacort, M. (1993) Estradiol-induced effects on glutathione metabolism in rat hepatocytes. J. Biochem. 113, 563-567. Stalford, A. C., Maggs, J. L., Gilchrist, T. L., and Park, B. K. (1994) Catecholestrogens as mediators of carcinogenesis: Correlation of aromatic hydroxylation of estradiol and its fluorinated analogs with tumor induction in Syrian hamsters. Mol. Pharmacol. 45, 1259-1267. Jellinck, P. H., Hahn, E. F., and Fishman, J. (1986) Absence of reactive intermediates in the formation of catechol estrogens by rat liver microsomes. J. Biol. Chem. 261, 7729-7732. Brueggemeier, R. W., Kimball, J. G., and Kraft, F. (1984) Estrogen metabolism in rat liver microsomal and isolated hepatocyte preparationssII inhibition studies. J. Steroid Biochem. 21, 709716. Ball, P., Favthmann, E., and Knuppen, R. (1976) Comparative studes on the metabolism of oestradiol-17β and 2-hydroxy oestradiol-17β in man in vitro and in vivo. J. Steroid Biochem. 1, 139143. Elce, J. S., Bird, C. F., and Chandra, J. (1973) Water soluble products of the estrogens. Bile metabolites of 2-hydroxyestradiol17β in man. J. Clin. Endocrinol. Metab. 36, 1027-1030. Iverson, S. L., Shen, L., Anlar, N., and Bolton, J. L. (1996) Bioactivation of estrone and its catechol metabolites to quinoidglutathione conjugates in rat liver microsomes. Chem. Res. Toxicol. 9, 492-499. Thakker, D. R., and Creveling, C. R. (1990) O-Methylation. In Conjugation Reactions in Drug Metabolism (Mulder, G. J., Ed.) pp 193-232, Taylor and Francis, London.

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