Synthesis and Reactivity of the Catechol Metabolites from the Equine Estrogen, 8,9-Dehydroestrone

Home , Estriol phosphate, Estriol sulfamate

754 Chem. Res. Toxicol. 2001, 14, 754-763

Fagen Zhang, Dan Yao, Yousheng Hua, Richard B. van Breemen, and Judy L. Bolton* Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois, 60612-7231

Received March 1, 2001

The risk factors for women developing breast and endometrial cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy in particular has been correlated with an increased cancer risk. Previously, we showed that the equine estrogens equilin and equilenin, which are major components of the widely prescribed estrogen replacement formulation Premarin, are metabolized to highly cytotoxic quinoids which caused oxidative stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. Chem. Res. Toxicol. 1998, 11, 1113-1127]. In this study, we have synthesized 8,9-dehydroestrone (a third equine estrogen component of Premarin) and its potential catechol metabolites, 4-hydroxy-8,9-dehydroestrone and 2-hydroxy-8,9-dehydroestrone. Both 2-hydroxy- 8,9-dehydroestrone and 4-hydroxy-8,9-dehydroestrone were oxidized by tyrosinase or rat liver microsomes to o-quinones which reacted with GSH to give one mono-GSH conjugate and two di-GSH conjugates. Like endogenous estrogens, 8,9-dehydroestrone was primarily converted by rat liver microsomes to the 2-hydroxylated rather than the 4-hydroxylated o-quinone GSH conjugates; the ratio of 2-hydroxy-8,9-dehydroestrone versus 4-hydroxy-8,9-dehydroestrone was 6:1. Also in contrast to experiments with equilin, 4-hydroxyequilenin was not observed in microsomal incubations with 8,9-dehydroestrone or its catechols. The behavior of 2-hydroxy- 8,9-dehydroestrone was found to be more complex than 4-hydroxy-8,9-dehydroestrone as GSH conjugates resulting from 2-hydroxy-8,9-dehydroestrone were detected even without oxidative enzyme catalysis. Under physiological conditions, 2-hydroxy-8,9-dehydroestrone isomerized to 2-hydroxyequilenin to form the very stable 2-hydroxyequilenin catechol; however, 4-hydroxy- 8,9-dehydroestrone was found to be stable under similar conditions. Finally, preliminary studies conducted with the human breast tumor S-30 cell lines demonstrated that the catechol metabolites of 8,9-dehydroestrone were much less toxic than 4-hydroxyequilenin (20-40-fold). These results suggest that the catechol metabolites of 8,9-dehydroestrone may have the ability to cause cytotoxicity in vivo primarily through formation of o-quinones; however, most of the adverse effects of Premarin estrogens are likely due to formation of 4-hydroxyequilenin o-quinone from equilin and equilenin.

Introduction tissue selectivity in postmenopausal women, and a distinct pharmacological profile that results in significant There are many benefits of estrogen replacement clinical activity in vasomotor, neuroendocrine, and bone therapy including relief of menopausal symptoms, pre- preservation parameters (10, 11). vention of osteoporosis, and cardiovascular disease (1). Despite the numerous benefits of estrogen replacement In addition, there is some evidence that estrogen replace- therapy, there have been troubling, controversial reports ment therapy can protect women from Alzheimer’s concerning the increase in risk of developing breast and - disease (2 5) and stroke (6, 7), and improve motor endometrial cancers, particularly for women on long-term disability in Parkinsonian postmenopausal women with high dose estrogen replacement therapy (12-18). The motor fluctuations (8). Equine estrogens make up ap- exact mechanism(s) by which estrogens cause these proximately 50% of the estrogens in the widely prescribed hormone-dependent cancers is unknown. One mechanism estrogen replacement therapy marketed under the name of Premarin (Wyeth-Ayerst). One of these equine estro- 1 Abbreviations: 8,9-dehydroestrone, 3-hydroxy-1,3,5(10),8,9-es- gens, 8,9-dehydroestrone differs from endogenous human tratetraen-17-one; 2-hydroxy-8,9-dehydroestrone, 2,3-dihydroxy-1,3,5- (10),8,9-estratetraen-17-one; 4-hydroxy-8,9-dehydroestrone, 3,4-dihy- estrogens in that it contains a 8,9-double bond in the B droxy-1,3,5(10),8,9-estratetraen-17-one;equilenin,3-hydroxy-1,3,5(10),6,8- 1 ring. The presence of 8,9-dehydroestrone in Premarin estrapentaen-17-one; 4-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8- is the primary reason that there has never been a generic estrapentaen-17-one; 2-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8- estrapentaen-17-one; 4-hydroxyequilin, 3,4-dihydroxy-1,3,5(10),7(8)- replacement for Premarin (9). Recent studies have shown estratetraen-17-one; 2-hydroxyequilin, 2,3-dihydroxy-1,3,5(10),7(8)- that 8,9-dehydroestrone has estrogenic activity, novel estratetraen-17-one; 4-hydroxyestrone, 3,4-dihydroxy-1,3,5(10)-estratrien- 17-one; estrone, 3-hydroxy-1,3,5(10)-estratrien-17-one; equilenin, 1,3,5(10),6,8-estrapentaen-3-ol-17-one; equilin, 1,3,5(10),7-estratetraen- * To whom correspondence should be addressed. Fax: (312) 996- 3-ol-17-one; LC-MS, liquid chromatography-mass spectrometry; ER, 7107. E-mail: [email protected]. estrogen receptor.

Scheme 1. Synthesis of 8,9-Dehydroestrone

Scheme 2. Synthesis of 2-Hydroxy-8,9-dehydroestrone

might involve metabolism of estrogens to catechols, which 2-hydroxylation as observed for endogenous estrogens to are then oxidized to redox active/electrophilic o-quinones mainly 4-hydroxylation for equilenin. Recently, we have that could initiate the carcinogenic process by binding found that the equine catechol estrogens, 4-hydrox- to cellular macromolecules. For example, it is well yequilenin and 4-hydroxyequilin, form unusual cyclic established that the endogenous estrogens, estrone and adducts with DNA in vitro (32-34). If the similar adducts 17â-estradiol, are metabolized by P450 enzymes to 2- and are formed in vivo, which are not repaired efficiently, 4-hydroxylated catechols (19, 20). The o-quinones formed mutations could result leading to initiation of the carci- from peroxidase/P450-catalyzed oxidation of these cat- nogenic process in hormone sensitive tissues. echols have previously been implicated as the ultimate There has only been one report on the metabolism of carcinogens. Redox cycling between the catechols and 8,9-dehydroestrone which suggested that the only me- their quinones generates reactive hydroxyl radicals which tabolite was 17â-hydroxy-8,9-dehydroestradiol (35). To cause oxidation of the purine/pyrimidine residues of DNA accurately study the potential for catechol formation from (21). The o-quinones could also alkylate DNA to form 8,9-dehydroestrone, we synthesized 8,9-dehydroestrone adducts which have been detected both in vitro (22-24) (Scheme 1), 4-hydroxy-8,9-dehydroestrone (Scheme 2), and in vivo (25-27). There have also been a few studies and 2-hydroxy-8,9-dehydroestrone (Scheme 3). We then on the potential for equine estrogens to be metabolized examined the metabolism of 8,9-dehydroestrone in rat to reactive catechol intermediates (28-31). It has been liver microsomes and showed that both 2-hydroxylated demonstrated that increasing unsaturation in the B ring and 4-hydroxylated GSH conjugates were formed (Scheme leads to a change in product distribution from primarily 4). Finally, we tested the relative toxicity of 8,9-dehy- 756 Chem. Res. Toxicol., Vol. 14, No. 6, 2001 Zhang et al.

Scheme 3. Synthesis of 4-Hydroxy-8,9-dehydroestrone

Scheme 4. Metabolism of 8,9-Dehydroestrone to o-Quinones and Reaction with GSH

droestrone and its catechol metabolites in breast cancer on Hewlett-Packard (Palo Alto, CA) UV-vis spectrophotometer. cells. The data suggest that although catechols were 1H NMR spectra were obtained with a Bruker (Billerica, MA) formed from 8,9-dehydroestrone, they were considerably Avance DPX300 spectrometer at 300 MHz. Positive ion electro- less toxic then 4-hydroxyequilenin which implies that spray mass spectra were obtained using a Hewlett-Packard quinoids formed from equilin and equilenin are more 5989B MS Engine quadrupole mass spectrometer equipped with likely to contribute to the adverse effects of Premarin. a ChemStation data system and high-flow pneumatic nebulizer- assisted electrospray LC-MS interface. The mass spectrometer was interfaced to a Hewlett-Packard 1050 gradient HPLC Materials and Methods system. The quadrupole analyzer was maintained at 120 °C and unit resolution was used for all measurements. Nitrogen at a Caution: The catechol estrogen o-quinones were handled in pressure of 80 psi was used for nebulization of the HPLC accordance with NIH guidelines for the Laboratory Use of effluent, and nitrogen bath gas at 300 °C and a flow rate of 10 Chemical Carcinogens (36). All chemicals were purchased from Aldrich (Milwaukee, WI), Fisher Scientific (Itasca, IL), or Sigma L/min were used for evaporation of solvent from the electro- (St. Louis, MO) unless stated otherwise. (3H)-GSH (glycine-2- spray. A mass range from 300 to 1050 mass units were scanned 3H) was obtained from NEN Life Science Products, Inc. (Boston, every 1.5 s (1.5 s/scan). Tandem MS (MS-MS) spectra were MA) and diluted to a specific activity of 40 mCi/mmol. 2-Hy- obtained using a Micromass (Manchester, U.K.) Quattro II triple droxyequilenin was synthesized as described previously (37). quadrupole mass spectrometer equipped with an electrospray Instrumentation. HPLC experiments were performed on a ionization source. Collision-induced dissociation was carried out Shimadzu (Columbia, MD) LC-10A gradient HPLC equipped using a range of collision energy from 25 to 70 eV and argon with a SIL-10A auto injector, SPD-M10AV UV-vis photodiode collision gas pressure of 2.7 µbar. array detector, and SPD-10AV detector. Peaks were integrated HPLC Methodology. Two general methods were used to with Shimadzu EZ-Chrom software. UV spectra were measured analyze and separate the various metabolites and GSH conju- Catechol Metabolites from 8,9-Dehydroestrone Chem. Res. Toxicol., Vol. 14, No. 6, 2001 757

Scheme 5. Mechanism of Isomerization of 2-Hydroxy-8,9-dehydroestrone to 2-Hydroxyequilenin

gates. All retention times reported in the text were obtained and the solvent removed in vacuo to give a yellow liquid which using Method A. was purified by column chromatography (silica gel) using ethyl Method A. Analytical HPLC analysis was performed using a acetate/hexane (1:8 v/v) as eluent to give 3 (2.5 g, 52% yield): 1 4.6 × 250 mm Ultrasphere C-18 column (Beckman) on the H NMR (CDCl3) δ 0.23 (s, 6H, Si(CH3)2), 0.99 (s, 9H, C(CH3)3), Shimadzu HPLC system described above. The mobile phase 2.30 (m, 2H, CH2), 2.69 (t, 2H, CH2), 5.15 (dd, 1H, Jcis ) 10.6 consisted of 20% acetonitrile in 0.25% perchloric acid/0.25% Hz, Jgem ) 1.8 Hz, Hcis of CH2dCH), 5.50 (dd, 1H, Jtrans ) 10.6 acetic acid (pH 3.5) at a flow rate of 1.0 mL/min for 5 min, Hz, Jgem ) 1.8 Hz, Htrans of CH2dCH), 6.08 [m, 1H, H-C(2)], increased to 18% acetonitrile over 5 min, isocratic for 50 min, 6.56 (m, 1H, CH2dCH), 6.66 (m, 2H, ArH), 7.20 (d, 1H, J ) 9 increased to 45% over the next 10 min, and increased to 90% Hz, ArH); CI-MS m/z 287 (100%) [M + H]+. acetonitrile over the last 5 min of the run. (3) 3-tert-Butyldimethylsiloxy-8,9-dehydroestrone,4. The cy- Method B. For LC-MS-MS analysis of GSH conjugates of cloaddition reaction procedure described in the literature was catechol metabolites from 8,9-dehydroestrone, analytical HPLC followed (39). To a 100 mL, three-necked, round-bottom flask, analysis was performed using a 4.6 × 250 mm Ultrasphere C-18 a solution of TiCl4 (1 mL, 9.15 mmol) in dry CH2Cl2 (15 mL) column (Beckman) on the above-mentioned Hewlett-Packard was added to a solution of 2-methylcyclopent-2-en-1-one (0.32 HPLC system, and tandem mass spectra were obtained using g, 3.33 mmol) at -78 °C. The yellow mixture was stirred for 15 the Micromass system interfaced with the Hewlett-Packard min and a solution of 6-tert-butyldimethylsiloxy-1-vinyl-3,4- HPLC system. The mobile phase consisted of 20% acetonitrile dihydronaphathalene 3 (2.32 g, 8.40 mmol) in dry CH2Cl2 (15 in 0.5% ammonium acetate (pH 3.5) at a flow rate of 1.0 mL/ mL) was added dropwise over 1 h. The mixture was stirred for min for 5 min, increased to 18% acetonitrile over 5 min, isocratic another5hat-78 °C until 2-methylcyclopent-2-en-1-one could for 50 min, increased to 45% over the next 10 min, and increased not be detected by TLC (silica gel, hexane/ethyl acetate ) 8:1). to 90% acetonitrile over the last 5 min of the run. The mixture was warmed to 0 °C and 1 M HCl (70 mL) was Synthesis of 8,9-Dehydroestrone (Scheme 1). (1) 6-tert- added. The final mixture was extracted with ethyl ether (2 × Butyldimethylsiloxytetralone, 2. 6-tert-Butyldimethylsiloxyte- 250 mL), washed with brine (2 × 250 mL), dried over sodium tralone was synthesized as described in the literature (38). sulfate, filtered, and evaporated to give a deep yellow liquid Briefly, to a stirred mixture of 6-hydroxy-1-tetralone 1 (2.5 g, which was further purified by flash column chromatography 15.4 mmol) and imidazole (2.6 g, 38.5 mmol) in DMF (14 mL) (silica gel) using ethyl acetate/hexane (1:8 v/v) as eluent to give 1 under a stream of nitrogen at room temperature was added tert- 4 as a yellow power (0.66 g, 7.2% yield): H NMR (acetone-d6) butyldimethylsilyl chloride (2.9 g, 18.7 mmol). After stirring for δ 0.21 (s, 6H, Si(CH3)2), 0.99 (s, 9H, C(CH3)3), 1.02 (s, 3H, CH3), 18 h, the reaction mixture was diluted with diethyl ether (280 1.41 (m, 1H), 1.77 (m, 2H), 2.12 (m, 1H), 2.36 (m, 5H), 2.47 (m, mL). The resulting mixture was then washed with 1 M HCl (200 1H), 2.70 (m, 2H), 2.80 (m, 1H), 6.63 (s, 1H), 6.68 (d, 1H, J ) ) 13 mL) and brine (2 × 50 mL), dried over MgSO4, filtered, and the 8.3 Hz, ArH), 6.88 (d, 1H, J 8.3 Hz, ArH); C NMR (acetone- solvent removed en vacuo. The residue obtained was purified d6) δ -4.25 [Si(CH3)2], 18.7 [C(CH3)3], 20.7, 22.5, 26.2, 26.4, 27.4, by flash chromatography (silica gel, hexane/ethyl acetate, 3:1) 28.1 29.5, 37.0, 47.4, 49.2, 118.2, 119.8, 123.9, 126.8, 130.6, to give 2 as a pale yellow liquid (4.6 g, 96% yield): 1H NMR 133.4, 137.9, 154.8, 221.7 (C17); CI-MS (positive ion, methane) + + (CDCl3) δ 0.23 (s, 6H, Si(CH3)2), 0.97 (s, 9H, C(CH3)3), 2.09 (m, m/z 383 (100%) [M H] . 2H, CH2), 2.61 (t, 2H, J ) 6.3 Hz, CH2), 2.90 (t, 2H, J ) 6.3 Hz, (4) 8,9-Dehydroestrone, 5. To a flame dried flask, 6-tert- CH2), 6.65 (s, 1H, ArH), 6.74 (d, 1H, J ) 8.4 Hz, ArH), 7.96 (d, butyldimethylsiloxy-8,9-dehydroestrone 4 (300 mg, 0.785 mmol), ) 13 - 2H, J 8.4 Hz, ArH); C NMR (CDCl3) δ 4.09, 18.4, 23.6, dry THF (15 mL), and Bu4NF (1.5 mL,1MinTHF) were added. 25.8, 30.1, 39.1, 118.9, 119.3, 127.1, 129.7, 147.0, 160.5, 197.5; The mixture was stirred for 20 min at room temperature, CI-MS m/z 277 (100%) [M + H]+. followed by addition of THF (50 mL). This solution was washed (2) 6-tert-Butyldimethylsiloxy-1-vinyl-3,4-dihydronaph- with brine (2 × 50 mL), dried over Na2SO4, filtered, and the athalene, 3. To a flame dried flask, 6-tert-butyldimethylsilox- solvent was removed en vacuo to give a yellow residue which ytetralone 2 (4.6 g, 16.6 mmol) and anhydrous THF (50 mL) was further purified by flash chromatography (silica gel, hexane: were added. Vinylmagnesium bromide (50 mL,1MinTHF) was acetone ) 2:1 + 1% MeOH) to give 8,9-dehydroestrone 5 (160 added to this mixture through a dropping funnel at room mg, 76% yield). Complete characterization was accomplished temperature. After stirring for 16 h, the mixture was refluxed by 1D and 2D NMR experiments including 1H, 13C, HMQC, 1 for 40 min, cooled to room temperature, and HCl (60 mL, 1 M) HMBC, and COSY: H NMR (acetone-d6) δ 1.01 (s, 3H, 18-CH3), was added through a dropping funnel. The final reaction 1.41 (m, 1H, H11), 1.74 (m, 2H, H12), 2.12 (m, 1H, H12), 2.27 (m, mixture was extracted with diethyl ether (2 × 100 mL). The 5H, 2 × H15), 2.49 (m, 1H, H16), 2.4 (m, 2H, H11), 2.67 (m, 1H, 14 4 1 combined organic layers were washed with saturated NaHCO3 H ), 6.63 (m, 2H, H ), 7.10 (s, 1H, J ) 8.3 Hz, H ), 8.18 (bs, 3 13 (2 × 60 mL) and brine (2 × 60 mL), dried over Na2SO4, filtered, 1H, OH ); C NMR (acetone-d6) δ 20.7 (C18), 22.6 (C15), 26.2 758 Chem. Res. Toxicol., Vol. 14, No. 6, 2001 Zhang et al.

(C6), 27.5 (C11,), 28.1 (C12), 29.4 (C16), 37.0 (C9), 47.4 (C13), 49.2 4-(2′,3′-dimethoxyphenyl)butanoic acid 14 (42). Compound 14 (C14), 113.4 (OCH3), 115.1 (C1), 124.1 (C4), 126.9 (C7), 128.8 (C5), was cyclized with polyphosphoric acid (PPA) to produce 5,6- 132.2 (C10), 137.9 (C8), 146.3 (C2), 156.7(C3), 221.8 (C17); UV dimethoxy-1-tetralone 15 (42). Demethylation of 5,6-dimethoxy- +• (CH3OH), 220, 286 nm; EI-MS m/z 268 (100%) [M] . 1-tetralone 15 using anhydrous AlCl3 /benzene gave 5,6- 1 Synthesis of 2-Hydroxy-8,9-dehydroestrone (Scheme 2). dihydroxy-1-tetralone 16: H NMR (acetone-d6) δ 2.07 (m, 2H, (1) 6,7-Dihydroxy-1-tetralone, 7. To a flame dried flask, 6,7- CH2), 2.49 (t, 2H, J ) 6.3 Hz, CH2), 2.97 (t, 2H, J ) 6.3 Hz, ) ) dimethoxy-1-tetralone 6 (5.0 g, 24 mmol), anhydrous AlCl3 (25.5 CH2), 6.81 (d, 1H, J 8.4 Hz, ArH), 7.46 (d, 1H, J 8.4 Hz, 13 g, 192 mmol), and benzene (100 mL) were added under a stream ArH), 9.17 (b, 2H, OH); C NMR (CDCl3) δ 23.5, 23.7, 39.1, of nitrogen. The mixture was refluxed for 2.5 h, cooled to room 113.8, 120.2, 126.8, 132.9, 142.3, 149.9, 197.1; CI-MS m/z 179 temperature, and 3 N HCl (100 mL) was added at 0 °C. After (100%) [M + H]+. filtration, the deep yellow solid was collected and recrystallized (2) 5,6-Di-tert-butyldimethylsiloxytetralone, 17. 5,6-Di-tert- from hot water to gave 6,7-dihydroxy-1-tetralone 7 (1.5 g, 35% butyldimethylsiloxytetralone 17 was prepared by reacting 5,6- 1 yield): H NMR (acetone-d6) δ 2.04 (m, 2H, CH2), 2.44 (t, 2H, J dihydroxy-1-tetralone 16 (2.6 g, 14.5 mmol) with tert-butyldim- ) 6.3 Hz, CH2), 3.11 (t, 2H, J ) 6.3 Hz, CH2), 6.71 (s, 1H, ArH), ethylsilyl chloride (7.2 g, 46.44 mmol) in DMF (50 mL) and 13 7.35 (s, 1H, ArH), 8.44 (b, 2H, OH); C NMR (CDCl3) δ 23.8, imidazole (6.8 g, 100.8 mmol) according to the same procedure 1 25.8, 39.1, 119.1, 119.7, 127.3, 129.9, 147.2, 152.5, 196.5; CI- described above (6.4 g, 94% yield): H NMR (acetone-d6) δ 2.05 + MS m/z 179 (100%) [M + H] . (m, 2H, CH2), 2.52 (m, 2H, J ) 7.0 Hz, CH2), 2.93 (m, 2H, CH2), (2) 6,7-Di-tert-butyldimethylsiloxytetralone, 8. 6,7-Di-tert- 6.92 (d, 1H, J ) 8.4 Hz, ArH), 7.58 (d, 1H, J ) 8.4 Hz, ArH); butyldimethylsiloxytetralone 8 (3.2 g, 94% yield) was prepared CI-MS m/z 407 (100%) [M + H]+. by reacting 6,7-dihydroxy-1-tetralone 7 (1.5 g, 8.42 mmol) with (3) 5,6-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphath- tert-butyldimethylsilyl chloride (3.17 g, 20.5 mmol) in DMF (20 alene, 18. 5,6-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaph- mL) and imidazole (2.84 g, 42.1 mmol) according to the same athalene 18 (1.1 g) was obtained by reacting 5.6-di-tert-bu- 1 procedure described above: H NMR (acetone-d6) δ 0.24 (s, 6H, tyldimethylsiloxytetralone 17 (4.21 g, 10.4 mmol) with vinyl- Si(CH3)2), 0.27 (s, 6H, Si(CH3)2), 1.01 (s, 18H, C(CH3)3), 2.05 magnesium bromide (70 mL,1MinTHF) following the same (m, 2H, CH2), 2.50 (t, 2H, J ) 7.0 Hz, CH2), 2.84 (t, 2H, J ) 7.0 procedure as described above for 6-tert-butyldimethylsiloxy-1- 13 Hz, CH2), 6.81 (s, 1H, ArH), 7.46 (d, 1H, ArH); C NMR vinyl-3,4-dihydronaphathalene. - (acetone-d6) δ 4.50, 18.3, 18.4, 23.6, 25.5, 25.6, 118.2, 120.3, (4) 3,4-Di-tert-butyldimethylsiloxy-8,9-dehydroestrone, 19. Ac- + 139.3, 145.7, 151.8,195.6; CI-MS m/z 407 (100%) [M + H] . cording to the same cycloaddition reaction procedure described (3) 6,7-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphath- above, 5,6-di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphathalene, 9. 6,7-Di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaph- alene 18 (1.1 g, 2.64 mmol) was converted into 3,4-di-tert- athalene 9 (1.08 g, 31% yield) was obtained by reacting 6,7-di- butyldimethylsiloxy-8,9-dehydroestrone 19 (30 mg, 2.5% yield) tert-butyldimethylsiloxytetralone 8 (3.5 g, 8.6 mmol) with vi- by reaction with 2-methylcyclopent-2-en-1-one (0.16 g, 1.66 1 nylmagnesium bromide (50 mL,1MinTHF) following the same mmol) in the presence of TiCl4 (6.4 mmol): H NMR (ace- procedure as described above for 6-tert-butyldimethylsiloxy-1- tone-d6) δ 6.71 (d, 1H, J ) 8.4 Hz, ArH), 6.76 (d, 1H, J ) 1 vinyl-3,4-dihydronaphathalene 3: H NMR (CDCl3) δ 0.23 (s, 8.4 Hz, ArH); APCI-MS (positive ion) m/z 513 (100%) [M + + 12H, Si(CH3)2), 0.99 (s, 18H, C(CH3)3), 2.21 (m, 2H, CH2), 2.62 H] . ) ) (m, 2H, CH2), 5.15 (dd, 1H, Jcis 10.6 Hz, Jgem 1.8 Hz, Hcis of (5) 4-Hydroxy-8,9-dehydroestrone, 20. 3,4-Di-tert-butyldi- d ) ) CH2 CH), 5.51 (dd, 1H, Jtrans 10.6 Hz, Jgem 1.8 Hz, Htrans methylsiloxy-8,9-dehydroestrone 19 (25 mg, 1.95 mmol) was d - d of CH2 CH), 6.07 [m, 1H, H C(2)], 6.56 (m, 1H, CH2 CH), 6.64 deprotected using Bu4NF to give 4-hydroxy-8,9-dehydroestrone + 1 (s, 1H, ArH), 6.89 (d, 1H, ArH); CI-MS m/z 417 (100%) [M 20 (7 mg, 51% yield): H NMR (acetone-d6) δ 1.02 (s, 3H, CH3), + H] . 1.42 (m, 1H), 1.80 (m, 2H), 2.10 (m, 1H), 2.30 (m, 5H), 2.45 (m, (4) 2,3-Di-tert-butyldimethylsiloxy-8,9-dehydroestrone, 10. Ac- 1H), 2.55 (m, 2H), 2.80 (m, 1H), 6.61 (d, 1H, J ) 8.4 Hz, ArH), cording to the same cycloaddition reaction procedure described 6.66 (d, 1H, J ) 8.4 Hz, ArH), 7.11 (b, 1H, OH), 8.24 (b, 1H, above, 6,7-di-tert-butyldimethylsiloxy-1-vinyl-3,4-dihydronaphath- OH), Negative APCI-MS m/z 283 (100%) [M - H]-. alene 9 (1.8 g, 4.3 mmol) was converted into 2,3-di-tert- GSH Conjugates of 2-Hydroxy-8,9-dehydroestrone-o- butyldimethylsiloxy-8,9-dehydroestrone 10 (250 mg, 11% yield) quinone and 4-Hydroxy-8,9-dehydroestrone-o-quinone. by reaction with 2-methylcyclopent-2-en-1-one (0.21 g, 2.18 The o-quinone GSH conjugates of 2-hydroxy-8,9-dehydroestrone 1 mmol) in the presence of TiCl4 (6.4 mmol): H NMR (acetone- and 4-hydroxy-8,9-dehydroestrone were prepared by incubating d6) δ 0.22 (s, 12H, Si(CH3)2), 0.99 (s, 18H, C(CH3)3), 1.02 (s, 3H, the corresponding catechol (0.5 mM) with 5.0 mM GSH and CH3), 1.41 (m, 1H), 1.73 (m, 2H), 2.15 (m, 1H), 2.30 (m, 5H), tyrosinase (0.4 µg/mL) in 2 mL of 50 mM sodium phosphate 2.40 (m, 1H), 2.65 (m, 2H), 2.81 (m, 1H), 6.67 (s, 1H), 6.74 (d, buffer (pH 7.4), 37 °C for 30 min. The conjugates were isolated 13 - 1H, ArH); C NMR (acetone-d6) δ 3.69, 19.14, 20.8, 22.6, 26.3, from the aqueous phase using solid-phase extraction cartridges 27.4, 28.1, 28.4, 29.5, 37.0, 47.4, 49.4, 116.3, 121.0, 126.7, 129.6, (Oasis; Waters Corporation, Milford, MA) and eluted with 130.6, 133.8,145.6, 147.5, 221.7 (C17); APCI-MS (positive ion, methanol. The methanol extract was concentrated to 150 µL + + methane) m/z 513 (100%) [M H] . and aliquots (25 µL) were analyzed directly by HPLC. Two di- (5) 2-Hydroxy-8,9-dehydroestrone, 11. 2-Hydroxy-8,9-dehy- GSH conjugates and one mono-GSH conjugates (Scheme 4) were droestrone 11 (8 mg, 37% yield) was obtained by deprotection obtained with both 2-OHDHES and 4-OHDHES. Since the of 2,3-di-tert-butyldimethylsiloxy-8,9-dehydroestrone 10 (50 mg, tandem mass spectrometry of the di-SG and mono-SG conju- 0.097 mmol) with Bu4NF (0.5 mL, 1.0 M in THF) according to gates showed a similar pattern for each GSH conjugate, MS- 1 the same procedure described above: H NMR (acetone-d6) δ MS data are only reported for the 4-OHDHES-diSG and 1.01 (s, 3H, CH3), 1.41 (m, 1H), 1.74 (m, 2H), 2.15 (m, 1H), 2.26 4-OHDHES-SG (method A): 4-OHDHES-diSG1,UV(CH3OH/ + (m, 5H), 2.40 (m, 1H), 2.55 (m, 2H), 2.91 (m, 1H), 6.61 (s, 1H, H2O) 256 nm, positive ion electrospray MS m/z 895 [M + H] + ArH), 6.72 (s, 1H, ArH), 7.60 (b, 2H, OH); CI-MS m/z 285 (100%) (100%); MS-MS with CID of m/z 895 gave m/z 877 [MH - H2O] [M + H]+. (10%), 820 [MH - Gly]+ (5%), 766 [MH - Glu]+ (30%), 637 [MH Synthesis of 4-Hydroxy-8,9-Dehydroestrone (20) (Scheme - 2Glu]+ (20%), retention time 20 min; 4-OHDHES-diSG2,UV 3). (1) 5,6-Dihydroxy-1-tetralone, 16. 2-Carboxyethyltriphe- (CH3OH/H2O) 262 nm, positive ion electrospray MS m/z 895 [M nylphosphonium bromide was obtained by refluxing triph- + H]+ (100%); CID MS-MS pattern same as 4-OHDHES-diSG1, enylphosphine with 3-bromopropanoic acid in the presence of retention time 21 min; 4-OHDHES-SG,UV(CH3OH/H2O) 255 benzene (40). 2,3-Dimethoxybenzaldehyde 12 was treated with nm, positive ion electrospray MS m/z 590 [M + H]+ (100%); MS- 2-carboxyethyltriphenylphosphonium bromide in the presence MS with CID of m/z 590 gave m/z 515 [MH - Gly]+ (20%), 461 of NaH and DMSO to give 4-(3′,5′-dimethoxyphenyl)-3-butenoic [MH - Glu]+ (40%), 358 [MH - 2Glu]+ (60%), 315 [MH - SG + + acid bromide 13 (41). Hydrogenation of 13 using 5% Pd/C gave 32] , retention time 55 min; 2-OHDHES-diSG1,UV(CH3OH/ Catechol Metabolites from 8,9-Dehydroestrone Chem. Res. Toxicol., Vol. 14, No. 6, 2001 759

H2O) 260, 334 nm; positive ion electrospray MS m/z 895 [M + obtained by regression and linear estimation analysis. The data H]+ (100%); MS-MS pattern same as 4-OHDHES-diSG1, reten- represent the mean ( SD of triplicate determinations. tion time 24 min; 2-OHDHES-diSG2,UV(CH3OH/H2O) 257, + + 335 nm, positive ion electrospray MS m/z 895 [M H] (100%), Results and Discussion retention time 25 min; 2-OHDHES-SG,UV(CH3OH/H2O) 272, + + 337 nm, positive ion electrospray MS m/z 590 [M H] (100%); Synthesis of 8,9-Dehydroestrone. There are reports retention time 51 min. in the literature on the synthesis of 8,9-dehydroestrone Kinetic Studies. The rate of formation 2-hydroxyequilenin and 3-methoxy-8,9-dehydroestrone (39, 47-49). One from the autoxidation of 2-hydroxy-8,9-dehydroestrone (0.5 mM) method involved isomerization of equilin to 8,9-dehy- in potassium phosphate buffer (pH 7.4, 37 °C) was determined droestrone in the presence of glacial acetic acid and by monitoring product formation by UV-visible spectroscopy hydrochloric acid (47). However, this study did not report using a Hewlett-Packard HP8452 diode array spectrophotometer. After 1.5 h, the pH was adjusted to 4 and the solution was sufficient spectral data in order to unequivocally confirm the structure of 8,9-dehydroestrone. When this method extracted with CHCl3 (2 mL). The organic layer was concentrated and analyzed by positive ion CI-MS (methane). The [M was attempted, only a mixture of products resulted which + H]+ ion of 2-hydroxy-8,9-dehydroestrone (285) was reduced could not be separated by chromatography. Other litera- 95% compared to the starting material and the [M + H]+ ion of ture synthetic methods included multistep reactions (39, 2-hydroxyequilenin (283) was the base peak in the spectrum. 48, 49). However, all of these methods concentrated on Incubations. Female Sprague-Dawley rats (180-200 g) the synthesis of 3-methoxy-8,9-dehydroestrone which were obtained from Sasco Inc. (Omaha, NE). The rats were could not be easily converted into 8,9-dehydroestrone. pretreated with dexamethasone to induce P450 3A isozymes Demethylation of 3-methoxy-8,9-dehydroestrone was at- (43). Dexamethasone was administered by i.p. injections of 100 tempted by using mild demethylation agents, such as mg/kg in corn oil daily for 4 days and the animals were sacrificed BBr3 (50), BBr3‚SMe2 (51), AlCl3/C2H5SH (52), and BCl3 on day 5. Microsomes were prepared from rat liver, and protein (53); however, these experiments were not successful due and P450 concentrations determined as described previously to the instability of the conjugated 8,9-double bond in the (44). Incubations containing microsomal protein were conducted B ring. Therefore, an alternative method for the synthesis for 10-30 min at 37 °C in 50 mM phosphate buffer (pH 7.4, 500 µL total volume). Substrates were added as solutions in of 8,9-dehydroestrone was developed (Scheme 1). In this dimethyl sulfoxide, and (3H)-GSH (specific activity of 40 mCi/ methodology, the commercially available 6-hydroxy-1- mmol) was added in phosphate buffer to achieve final concen- tetralone 1 was used as starting material. After protec- trations of 0.5 and 1.0 mM, respectively. A NADPH generating tion of the 6-hydroxy group with tert-butyldimethylsilyl system consisting of 1.0 mM NADP+, 5 mM isocitric acid, and chloride, 6-tert-butyldimethylsiloxy-1-tetralone 2 was 0.2 units of isocitric dehydrogenase/mL were used together with obtained. The protected tetralone was converted into + 5.0 mM MgCl2. For control incubations NADP was omitted. 1-vinyl-6-tert-butyldimethylsiloxy-3,4-dihydronaphatha- + The reactions were initiated by the addition of NADP , and lene 3 by reacting compound 2 with commercially avail- terminated by chilling in an ice bath followed by the addition able vinylmagnesium bromide. Catalytic cycloaddition of of perchloric acid (25 µL). 1-vinyl-6-tert-butyldimethylsiloxy-3,4-dihydronaphatha- Adduct Quantification. The reaction mixtures were cen- lene with 2-methylcyclopent-2-en-1-one in the presence trifuged at 13 000 rpm for 6 min to remove precipitated of TiCl4 gave 3-tert-butyldimethylsiloxy-8,9-dehydroestro- microsomal protein. Aliquots of the supernatant (100 µL) were ne 4. Removal of the protecting group under mild analyzed directly by HPLC as described above for the GSH conditions with Bu NF gave 8,9-dehydroestrone 5.Onthe conjugates. For quantification of GSH conjugates, 0.3 mL 4 aliquots of the column effluent were collected every 18 s during basis of this methodology, the potential catechol metabo- each run, and radioactivity was measured with a Beckman lites of 8,9-dehydroestrone, 4-hydroxy-8,9-dehydroestrone model LS 5801 liquid scintillation counter. Concentrations of (Scheme 3) and 2-hydroxy-8,9-dehydroestrone (Scheme the GSH conjugates were calculated by summing the radioactiv- 2), were also synthesized. ity associated with each peak and converting the data to GSH Conjugates of 2-Hydroxy-8,9-dehydroestro- nanomolar amounts using the specific activity of the (3H)-GSH. ne- and 4-Hydroxy-8,9-dehydroestrone-o-quinone. Each analysis was performed immediately after the incubation Previous studies have shown that enzymatic oxidation in order to limit degradation of the o-quinone GSH conjugates. of endogenous catechol estrogens generated o-quinones Cytotoxicity Experiments in S-30 Breast Cancer Cells. which could be trapped in situ by GSH (54, 55). In Cell viability was determined by Trypan blue exclusion (45, 46). addition, we have shown that 4-hydroxyequilenin and The S-30 cell line was a generous gift from V. C. Jordan 4-hydroxyequilin readily autoxidized to o-quinones which (Northwestern University, Evanston, IL). Briefly, S-30 cells were maintained in MEME supplemented with 1% penicillin, strep- reacted with GSH to form the corresponding GSH tomycin, fungizome, 10 mM nonessential amino acids, and 5% conjugates at physiological pH and temperature (50, 56). 2× stripped bovine serum. The medium was changed 24 h before In contrast, 4-hydroxy-8,9-dehydroestrone was found to beginning cytotoxicity assays to maintain logarithmic growth. be very stable under physiological conditions; however, The cells were treated with 2-OHDHES, 4-OHDHES, 4-OHEN, it could be oxidized to an o-quinone by oxidative enzymes. 8,9-dehydroestrone, and equilenin for 18 h. The test samples This o-quinone was trapped with GSH to form one mono- were assayed in triplicate, and final concentrations ranged from GSH conjugate (retention time 55 min) and two di-GSH 1.6 to 500 µM. Each assay included negative controls (cells conjugates eluting at 20 and 21 min (HPLC method A). treated with DMSO only) that were used to define 100% cell Similar to other equine catechol estrogen GSH conju- viability. After treatment, floating cells were collected by gates, these GSH conjugates were unstable and we could centrifugation at 3000 rpm for 5 min, and attached cells were not obtain sufficient material for NMR characterization, first trypsinized and then harvested by centrifugation. Floating and attached cells were combined, washed with PBS, and and as a result their structures could not be determined stained with 0.4% Trypan blue. A drop of cell suspension was unambiguously. However, based on the LC-MS and MS- placed on a hemocytometer and the cell numbers were deter- MS data, the structures of the GSH conjugates (mono or mined under a microscope. The dead cells were stained blue di) are consistent with reaction of the o-quinone of while viable cells remained unstained. The LC50 values were 4-hydroxy-8,9-dehydroestrone with one or two GSH 760 Chem. Res. Toxicol., Vol. 14, No. 6, 2001 Zhang et al.

MS pattern as that of the 4-hydroxy-8,9-dehydroestrone GSH conjugates. However, incubation of 2-hydroxy-8,9- dehydroestrone in phosphate buffer (pH 7.4) gave a distinctive time-dependent UV absorbance change (Fig- ure 3). The new species formed at the end of the incubation had the same UV absorbance pattern as that of 2-hydroxyequilenin (Figure 3). This transformation was further confirmed by HPLC with a co-injection of an authentic sample 2-hydroxyequilenin. In addition, extraction of the final incubation solution with CHCl3 and analysis by CI-MS showed an ion at 283 corresponding to the protonated molecule of 2-hydroxyequilenin. It is likely that the transformation of 2-hydroxy-8,9-dehydroestrone to 2-hydroxyequilenin under these conditions was accomplished through a analogous o-quinone me- thide mechanism (Scheme 5) by which 4-hydroxyequilin Figure 1. Positive ion electrospray MS/MS with CID of the isomerized to 4-hydroxyequilenin (34). The GSH conju- protonated molecule (m/z 590) of 4-hydroxy-8,9-dehydroestrone gates of 2-hydroxy-8,9-dehydroestrone and 4-hydroxy-8,9- mono-GSH conjugate. dehydroestrone imply that quinones were formed prior to nucleophilic GSH addition. These o-quinones might produce toxicity through depletion of cellular GSH. Once GSH is depleted, the o-quinones could alkylate cysteine residues on cellular proteins, resulting in toxic effects (59, 60). Oxidation of Catechol Estrogens and 8,9-Dehy- droestrone by Rat Liver Microsomes. We examined the oxidation of 2-hydroxy-8,9-dehydroestrone, 4-hydroxy-8,9-dehydroestrone, and 8,9-dehydroestrone to quinoid metabolites in rat liver microsomes by trapping these reactive species with [3H]GSH (Table 1). The trapping reaction should be very efficient because of the high concentration of GSH in the incubation medium and the relatively fast rate of GSH addition as compared to amino and hydroxyl groups (61, 62). However, a small amount of binding to the microsomal protein by the quinoids is possible and as a result conjugate formation Figure 2. Positive ion electrospray MS/MS with CID of the shown in Table 1 should be considered a lower limit for protonated molecule (m/z 895) of 4-hydroxy-8,9-dehydroestrone the generation of quinoids. Microsomal incubations with di-GSH conjugate. the catechols showed that 2-hydroxy-8,9-dehydroestrone molecules (Figure 1, ref 2). The molecule at m/z 590 was was a slightly better substrate than 4-hydroxy-8,9- consistent with the protonated molecule of the mono- dehydroestrone in terms of o-quinone formation (Table GSH conjugate (Figure 1). The product ion spectrum of 1). Incubations with 8,9-dehydrodroestrone also showed this ion of m/z 590 gave abundant fragment ions at m/z production of mono-GSH conjugates from both catechols 515 [MH - 75]+ (loss of glycine), m/z 461 [MH - 129]+ although considerably less o-quinone derived GSH con- (loss of pyroglutamic acid), and m/z 315. The two GSH jugates were produced in incubations with the parent conjugates of 4-hydroxy-8,9-dehydroestrone had similar phenol as compared to similar experiments with the MS-MS patterns. The MS-MS pattern of 4-hydroxy-8,9- catechols. These data differ from what has been reported dehydroestrone-diSG 1 is shown in Figure 2. The product for the metabolism of 8,9-dehydroestrone in female dogs ion spectrum of the ion at m/z 895 gave abundant where only the 17â-hydroxy-8,9-dehydroestradiol was fragment ions at m/z 820 [MH - 75]+ (loss of glycine), detected (35). It is possible that these catechols were m/z 766 [MH - 129]+ (loss of pyroglutamic acid), and m/z formed in the female dogs which were not detected 637 [MH - 258]+ (loss of two pyroglutamic acid moieties). because of the instability of the catechols and o-quinones. Loss of 129 units (pyroglutamic acid) from the protonated Regioselectivity was observed in the P-450-catalyzed molecule is a characteristic fragment ion of GSH conju- hydroxylation of 8,9-dehydroestrone since 2-hydroxy-8,9- gates formed during MS-MS (57, 58). The behavior of dehydroestrone GSH conjugates predominated over 4-hy- 2-hydroxy-8,9-dehydroestrone was found to be different droxy-8,9-dehydroestrone GSH conjugates by a factor of from 4-hydroxy-8,9-dehydroestrone under similar condi- 6. Previously, we studied the metabolism of estrone, tions. This catechol formed one mono-GSH conjugate at equilin, and equilenin to catechol o-quinone GSH conju- a retention time of 51 min and two di-GSH conjugates gates in rat liver microsomes (50, 54). It was shown that at 24 and 25 min. These conjugates were formed either estrone was primarily metabolized to 2-hydroxyestrone in the absence or in the presence of oxidative enzymes (2:4 hydroxylation ratio ) 6:1), equilin was primarily (P450 or tyrosinase). Like the GSH conjugates of 4-hy- metabolized to 4-hydroxyequilin (2:4 hydroxylation ratio droxy-8,9-dehydroestrone, these GSH conjugates were ) 1:6), and equilenin was exclusively metabolized to also unstable and only LC-MS-MS spectra were obtained. 4-hydroxyequilenin. The present data showed that the The mono- and di-GSH conjugates showed the same MS- ratio of 2:4 hydroxylation of 8,9-dehydroestrone was 6:1 Catechol Metabolites from 8,9-Dehydroestrone Chem. Res. Toxicol., Vol. 14, No. 6, 2001 761

Table 1. Conversion of 8,9-Dehydroestrone and Its Catechol Metabolites to o-Quinone-Derived GSH Conjugates by Rat Liver Microsomesa retention rate of formation substrate conjugate time (min) (nmol/nmol of P450‚10 min) 8,9-dehydroestrone 2-OHDHES-SG 51 0.037 ( 0.003 4-OHDHES-SG 55 0.007 ( 0.001 total ) 0.044 ( 0.004 2-hydroxy-8,9-dehydroestrone 2-OHDHES-diSG 1 24 0.17 ( 0.02 2-OHDHES-diSG 2 25 0.23 ( 0.01 2-OHDHES-SG 51 4.50 ( 0.03 total ) 4.90 ( 0.06 4-hydroxy-8,9-dehydroestrone 4-OHDHES-diSG 1 20 0.31 ( 0.01 4-OHDHES-diSG 2 21 0.48 ( 0.02 4-OHDHES-SG 55 2.07 ( 0.03 total ) 2.86 ( 0.06 a Incubations were conducted for 30 min with 0.5 mM substrate, rat liver microsomes (1.0 nmol P450/mL) in the presence of an NADPH- generating system and [3H]GSH, at 37 °C. Radioactivity eluting from the HPLC column was measured in fractions collected at 18-s intervals. Results are the average ( SD of three incubations. Background radioactivity in the control (-NADP+) samples have been subtracted from each peak.

Table 2. Cytotoxicity of Equine Estrogens and Their Catechol Metabolites in S-30 Breast Cancer Cellsa

substrate LC50 (µM) 2-hydroxy-8,9-dehydroestrone 84 ( 7 4-hydroxy-8,9-dehydroestrone 147 ( 7 4-hydroxyestrone 251 ( 9 4-hydroxyequilenin 4.0 ( 0.1b 8,9-dehydroestrone 126 ( 7 equilenin 155 ( 8 a Cell viability was assessed by Trypan blue exclusion as described in Materials and Methods. Values are expressed as the mean ( SD of at least three determinations. b From ref 63.

8,9-dehydroestrone in rat liver microsomes and the relative cytotoxicity of these catechols in breast cancer cells. We have found that 8,9-dehydroestrone was primarily metabolized to 2-hydroxy-8,9-dehydroestrone in rat liver microsomes and that this catechol can autoxidize, isomerize, and aromatize giving 2-hydroxyequilenin. In contrast, 4-hydroxy-8,9-dehydroestrone is a stable catechol under physiological conditions. These data might - Figure 3. (A) UV vis spectral analysis of the conversion of explain the enhanced toxicity of 2-hydroxy-8,9-dehy- 2-hydoxy-8,9-dehydroestrone to 2-hydroxyequilenin. Incubations contained 2-hydroxy-8,9-dehydroestrone (0.15 mM) in potassium droestrone compared to 4-hydroxy-8,9-dehydroestrone in phosphate buffer (50 mM, pH 7.4, 37 °C). Scans were taken breast cancer cells. In should be noted that both catechol every 5 min for 1.5 h. (B) UV spectrum of 2-hydroxyequilenin. metabolites of 8,9-dehydroestrone were much less toxic (20-40-fold) than 4-hydroxyequilenin which is the major which was quite similar to that of the endogenous metabolite of both equilin and equilenin (34, 50). These estrogen, estrone. results may suggest that the catechol metabolites of 8,9- Cytotoxicity of 8,9-Dehydroestrone and Catechol dehydroestrone might have the ability to cause cytotox- Metabolites in Breast Cancer Cells (S-30). Prelimi- icity in vivo primarily through formation of catechol R nary studies conducted with the ER stably transfected quinoids; however, most of the adverse effects of Pre- human breast tumor cell line S-30, demonstrated that marin estrogens are likely due to formation of quinoids the cytotoxicity of the catechol metabolites from 8,9- from equilin and equilenin. dehrdroestrone were much higher than that of 4-hydroxyestrone, but less than that of 4-hydroxyequilenin (63). Acknowledgment. This research was supported by The cytotoxicity of 2-hydroxy-8,9-dehydroestrone is al- NIH Grant CA73638 (J.L.B.) and CA83124 (R.B.v.B.). We most double that of 4-hydroxy-8,9-dehydroestrone which are grateful to Dr. V. C. Jordan (Northwestern Univer- had similar cytotoxic effects as 8,9-dehrydroestrone and sity) for the gift of the S-30 cell line. equilenin (Table 2). The higher cytotoxicity of 2-hydroxy- 8,9-dehydroestrone might result from the fact that this catechol can autoxidize to 2-hydroxy-8,9-dehydroestrone References o-quinone under physiological conditions without oxida- (1) Grodstein, F., Stampfer, M. J., Colditz, G. A., Willett, W. C., tive enzymatic catalysis (see above). In addition, the Manson, J. E., Joffe, M., Rosner, B., Fuchs, C., Hankinson, S. E., microsomal incubations suggest that the amount of Hunter, D. J., Hennekens, C. H., and Speizer, F. E. (1997) quinoids formed in the S30 cells is likely higher for Postmenopausal hormone therapy and mortality. New Eng. J. Med. 336, 1769-1775. 2-hydroxy-8,9-dehydroestrone as compared to 4-hydroxy- (2) Wickelgren, I. (1997) Estrogen: A new weapon against Alzhe- 8,9-dehydroestrone. imer’s. Science 276, 676-677. In conclusion, data have been presented on the forma- (3) Henderson, V. W. (1997) The epidemiology of estrogen replace- tion of GSH conjugates from the catechol metabolites of ment therapy and Alzheimer’s disease. Neurology 48, S27-S35. 762 Chem. Res. Toxicol., Vol. 14, No. 6, 2001 Zhang et al.

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