Effect of Halogenated Substituents on the Metabolism and Estrogenic

which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are primarily metabolized to the catechol, 4-hydroxyequile...
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Chem. Res. Toxicol. 2003, 16, 741-749

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Effect of Halogenated Substituents on the Metabolism and Estrogenic Effects of the Equine Estrogen, Equilenin Xuemei Liu, Fagen Zhang, Hong Liu, Joanna E. Burdette, Yan Li, Cassia R. Overk, Emily Pisha, Jiaqin Yao, Richard B. van Breemen, Steven M. Swanson, and Judy L. Bolton* Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street (M/C 877), Chicago, Illinois 60612-7231 Received January 2, 2003

Estrogen replacement therapy has been correlated with an increased risk for developing breast and endometrial cancers. One potential mechanism of estrogen carcinogenesis involves metabolism of estrogens to 2- and 4-hydroxylated catechols, which are further oxidized to electrophilic/redox active o-quinones that have the potential to both initiate and promote the carcinogenic process. Previously, we showed that the equine estrogens, equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are primarily metabolized to the catechol, 4-hydroxyequilenin. This catechol was found to autoxidize to an o-quinone causing oxidation and alkylation of DNA in vitro and in vivo. To block catechol formation from equilenin, 4-halogenated equilenin derivatives were synthesized. These derivatives were tested for their ability to bind to the estrogen receptor, induce estrogen sensitive genes, and their potential to form catechol metabolites. We found that the 4-fluoro derivatives were more estrogenic than the 4-chloro and 4-bromo derivatives as demonstrated by a higher binding affinity for estrogen receptors R and β, an enhanced induction of alkaline phosphatase activity in Ishikawa cells, pS2 expression in S30 cells, and PR expression in Ishikawa cells. Incubation of these compounds with tyrosinase in the presence of GSH showed that the halogenated equilenin compounds formed less catechol GSH conjugates than the parent compounds, equilenin and 17β-hydroxyequilenin. In addition, these halogenated compounds showed less cytotoxicity in the presence of tyrosinase than the parent compounds in S30 cells. Also, as stated above, the 4-fluoro derivatives showed similar estrogenic effects as compared with parent compounds; however, they were less toxic in S30 cells as compared to equilenin and 17β-equilenin. Because 17β-hydroxy-4-halogenated equilenin derivatives showed higher estrogenic effects than the halogenated equilenin derivatives in vitro, we studied the relative ability of the 17β-hydroxy-4-halogenated equilenin derivatives to induce estrogenic effects in the ovariectomized rat model. The 4-fluoro derivative showed higher activity than 4-chloro and 4-bromo derivatives as demonstrated by inducing higher vaginal cellular differentiation, uterine growth, and mammary gland branching. However, 17β-hydroxy-4-fluoroequilenin showed a lower estrogenic activity than 17β-hydroxyequilenin and estradiol, which could be due to alternative pharmacokinetic properties for these compounds. These data suggest that the 4-fluoroequilenin derivatives have promise as alternatives to traditional estrogen replacement therapy due to their similar estrogenic properties with less overall toxicity.

Introduction It has been shown that prolonged exposure to estrogens either through early menarche, late menopause, and/or estrogen replacement therapy will increase the risk of developing breast or endometrial cancer (1-8). In animal models, estrogens have been proven to induce mammary, pituitary, cervical, and uterine tumors (9). Premarin (Wyeth-Ayerst) is the most widely prescribed estrogen replacement formulation, which consists of the equine estrogens, equilin and EN1 and their 17β-hydroxylated analogues, as well as the endogenous estrogens, estrone and 17β-estradiol. Similar to studies with estradiol (10), it has been shown that hamsters treated for 9 months with the equine estrogens, equilin and EN, or sulfatase* To whom correspondence should be addressed. Fax: (312)9967107. E-mail: [email protected].

treated Premarin, resulted in 100% tumor incidence and abundant tumor foci (11). Evidence suggests that metabolism of estrogens to catechols and further oxidation to highly reactive o-quinones could play a major role in 1 Abbreviations: 4-BrEN, 4-bromoequilenin, 4-bromo-3-hydroxyestra1,3,5(10),6,8-pentaen-17-one; 4-ClEN, 4-chloroequilenin, 4-chloro-3hydroxyestra-1,3,5(10),6,8-pentaen-17-one; DCF, dichlorofluoroscein; DCDF-DA, 2′,7′-dichlorodiacylfluoroscein diacetate; ED50, dose of 50% cells killed; EN, equilenin, 3-hydroxyestra-1,3,5(10),6,8-pentaen-17one; 4-FEN, 4-fluoroequilenin, 4-fluoro-3-hydroxyestra-1,3,5(10),6,8pentaen-17-one; 17β-EN, 17β-hydroxyequilenin, estra-1,3,5(10),6,8pentaene-3,17β-diol; 17β-4-FEN, 17β-hydroxy-4-fluoroequilenin, 4-fluoroestra-1,3,5(10),6,8-pentaene-3,17β-diol; 17β-4-ClEN, 17β-hydroxy-4chloroequilenin, 4-chloro-estra-1,3,5(10),6,8-pentaene-3,17β-diol; 17β4-BrEN, 17β-hydroxy-4-bromoequilenin, 4-bromo-estra-1,3,5(10),6,8pentaene-3,17β-diol; LMAgarose, low melting agarose; IC50, dose of half-maximal induction; 4-OHE, 4-hydroxyestrone, 3,4-dihydroxy1,3,5(10)-estratrien-17-one; 4-OHE2, 4-hydroxyestradiol, estra-1,3,5(10)triene-3,4,17β-triol; PSF, penicillin-streptomycin-fungizome; ROS, reactive oxygen species.

10.1021/tx030001f CCC: $25.00 © 2003 American Chemical Society Published on Web 05/06/2003

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Scheme 1. Redox Cycling of 4-OHEN Inducing Damage to DNA

induction of DNA damage leading to initiation of the carcinogenic process (12). Previously, we showed that the major phase I metabolite of both equilin and EN, 4-hydroxyequilenin (4OHEN), could autoxidize to a potent cytotoxic o-quinone and cause a variety of DNA lesions including formation of bulky stable adducts, apurinic sites, oxidation of the phosphate sugar backbone, and purine/pyrimidine bases in vitro and in vivo (Scheme 1) (13-17). This is in contrast to the endogenous catechol estrogen 4-OHE2, which only caused formation of apurinic sites in vivo (18). In addition, our recent data suggest that 4-OHEN was much more effective at transformation of mouse epithelial cells under conditions typically used for tumor promotion or complete carcinogen experiments as compared to similar studies with 4-OHE (19). These studies suggest that 4-OHEN formation from equilin and EN could cause considerably more DNA damage in vivo as compared to endogenous catechol estrogens. Previous studies have shown that estradiol derivatives with halogen substituents at the 4-position maintain estrogenic properties without generating genotoxic species. For example, Liehr and co-workers (9, 20) investigated estrogenic and carcinogenic activities of 4-fluoroestradiol in the Syrian hamster. The compound was presumed to be unable to undergo 4-hydroxylation but was almost as estrogenic as E2. In addition, 4-fluoroestradiol profoundly decreased the induction of renal tumors (9, 20). This was taken as further evidence for the involvement of catechol metabolites in estrogeninduced carcinogenesis. Because 4-OHEN can damage DNA, 4-halogenated EN derivatives were synthesized to decrease the principal route of aromatic A ring hydroxylation of EN. We examined whether similar estrogenic effects and binding activities to estrogen receptors R and β of these 4-halogenated EN derivatives could be observed as compared with the parent compounds. The data showed that 4-fluoro derivatives and the parent compounds had similar estrogenic effects but were more estrogenic than 4-chloro and 4-bromo derivatives. In addition, we investigated whether 4-halogenated EN derivatives formed less catechol metabolites in the presence of tyrosinase and were less toxic in S30 cells as compared with the parent compounds. The results showed that halogenated compounds formed less catechol metabolites and were significantly less toxic in S30 cells than the parent compounds. Finally, we examined the estrogenic activity of 17β-4-halogenated EN derivatives

Liu et al. Scheme 2. Synthesis of 4-Halogenated EN and 17β-4-Halogenated EN Derivatives Where X ) Halogen Atoms

in ovariectomized rats and the in vivo data showed a similar trend as found in vitro. These data suggest that the 4-FEN derivatives have promise as alternatives to traditional estrogen replacement therapy since they retain estrogenic properties with less overall toxicity.

Materials and Methods Caution: The catechol estrogen o-quinones were handled in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens (21). All chemicals were purchased from Aldrich (Milwaukee, WI), Fisher Scientific (Itasca, IL), or Sigma (St. Louis, MO) unless stated otherwise. 17β-EN was synthesized as described previously (22, 23). 4-FEN, 4-ClEN, 4-BrEN, and their 17β-hydroxylated analogues were synthesized as described below (Scheme 2). [3H]Estradiol (83 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA), and Cytoscint was purchased from ICN (Costa Mesa, CA). Human recombinant ERR (estrogen receptor) and ERβ were purchased from Panvera (Madison, WI). Primers of PR, pS2, and β-actin were obtained from Sigma Genosys (Woodlands, TX). Instrumentation. HPLC experiments were performed on a Shimadzu LC-10A gradient HPLC equipped with a SIL-10A auto injector, SPD-M10AV UV/vis photodiode array detector, and SPD-10AV detector. Peaks were integrated with Shimadzu EZ-Chrom software. 1H NMR spectra were obtained with a Bruker Advance DPX300 spectrometer at 300 MHz. Positive and negative ion electrospray mass spectra were obtained using a Thermo Finnigan (San Jose, CA) LCQdeca ion trap mass spectrometer. Tandem MS (MS/MS) spectra were obtained using a Micromass (Manchester, U.K.) Quattro II triple quadrupole mass spectrometer equipped with an electrospray ionization

Halogenated Analogues of Equilenin source. Collision-induced dissociation was carried out using collision energies from 25 to 70 eV and an argon collision gas pressure of 2.7 µBar. HPLC Methodology. Two general methods were used to analyze and separate the various metabolites and GSH conjugates. All retention times reported in the text were obtained using Method A. 1. Method A. Analytical HPLC analysis was performed using a 4.6 mm × 150 mm Ultrasphere C18 column (Beckman). The mobile phase consisted of 5% CH3OH in 0.25% perchloric acid/ 0.25% acetic acid buffer (pH 3.5) at a flow rate of 1.0 mL/min for 5 min, increased to 40% CH3OH over 45 min, isocratic for 5 min, and increased to 90% CH3OH over the next 20 min. 2. Method B. LC-MS/MS analysis of GSH conjugates of o-quinones formed from catechol metabolites was performed using a 4.6 mm × 150 mm Ultrasphere C18 column on a Waters (Milford, MA) 2487 UV detector and a Waters 2690 HPLC system equipped with a Micromass Quattro II triple quadrupole mass spectrometer. The mobile phase consisted of 5% CH3OH in 0.5% ammonium acetate buffer (pH 3.5) at a flow rate of 1.0 mL/min for 5 min, increased to 40% CH3OH over 45 min, isocratic for 5 min, and increased to 90% CH3OH over the next 20 min. Synthesis of 4-FEN. Equilin (500 mg, 1.86 mmol) and N-fluoropyridinium triflate (915 mg, 3.70 mmol) in 1,2-dichloroethane (10 mL) were refluxed under nitrogen for 18 h. The reaction mixture was diluted with 1,2-dichloroethane (100 mL). The resulting mixture was then washed with water (2 × 30 mL) and 10% HCl (30 mL), dried over MgSO4, and filtered, and the solvent was removed in vacuo. The residue obtained was purified by preparative TLC (silica gel, 1 mm, hexane:acetone:MeOH ) 2:1:0.03) to give 4-FEN as a white powder (150 mg, 28% yield). 1H NMR (acetone-d ): δ 0.76 (s, 3H, CH ), 1.77-1.94 (m, 2H), 6 3 2.37-2.40 (m, 1H), 2.58-2.78 (m, 3H), 3.22-3.30 (m, 3H), 7.30 (t, 1H, J ) 8.97 Hz, ArH), 7.41 (d, 1H, J ) 8.37 Hz, ArH), 7.72 (d, 1H, J ) 8.91 Hz, ArH), 7.85 (d, 1H, J ) 8.37 Hz, ArH), 8.85 (b, 1H, OH). 19F NMR (acetone-d6): 152.2 (d, 1F, J ) 9.0 Hz). UV (CH3OH): 238, 270, 328 nm; negative ion electrospray-MS [M - H]- m/z 283 (100%). Synthesis of 4-ClEN. N-Chloroacetamide was prepared by the published procedure (24). Equilin (500 mg, 1.86 mmol) and N-chloroacetamide (174 mg, 1.89 mmol) in anhydrous ethanol (50 mL) were refluxed for 2 h under nitrogen, and the final solution was concentrated in vacuo. The residue obtained was purified by crystallization with MeOH/CHCl3 to give 4-ClEN as a white powder (200 mg, 35% yield). 1H NMR (acetone-d6): δ 0.67 (s, 3H, CH3), 1.83-1.89 (m, 2H), 1.98-2.01 (m, 1H), 2.30-2.36 (m, 1H), 2.59-2.65 (m, 2H), 3.12-3.31 (m, 3H), 7.30 (d, 1H, J ) 9.24 Hz, ArH), 7.41 (d, 1H, J ) 8.64 Hz, ArH), 7.85 (d, 1H, J ) 9.24 Hz, ArH), 7.94 (d, 1H, J ) 8.64 Hz, ArH), 10.21 (b, 1H, OH). UV (CH3OH): 244, 274, 286, 296, 344 nm; negative ion electrospray-MS [M - H]- m/z 299 (100%), m/z 301 (33%). Synthesis of 4-BrEN. 4-Bromoequilin (4-BrEQ) was synthesized according to the published procedure (25). 4-BrEQ was oxidized to 4-BrEN by SeO2 as shown in Scheme 2. Briefly, 4-BrEQ (200 mg, 0.576 mmol) was dissolved in tert-butyl alcohol (40 mL), followed by the addition of pyridine (0.08 mL) and selenium dioxide (100 mg, 0.909 mmol). The mixture was refluxed for 4 h and cooled to room temperature. HCl (10%, 30 mL) was added to the solution, and the solution was stirred for 30 min, followed by extraction with ethyl acetate (50 mL). The final organic layer was washed with water (3 × 40 mL) and dried over Na2SO4. The solvent was removed in vacuo, and the residue was purified by flash chromatography (silica gel) using hexane:ethyl acetate (3:2) as eluent giving 4-BrEN, which was further purified by crystallization from hexane/acetone (180 mg, 90% yield based on 4-BrEQ). 1H NMR (acetone-d6): δ 0.76 (s, 3H, CH3), 1.83-1.88 (m, 2H), 2.35-2.38 (m, 1H), 2.61-2.64 (m, 2H), 2.80-2.83 (m, 1H), 3.28-3.33 (m, 3H), 7.33 (t, 1H, J ) 9.15 Hz, ArH), 7.46 (d, 1H, J ) 8.64 Hz, ArH), 7.96 (d, 1H, J ) 9.15 Hz, ArH), 8.05 (d, 1H, J ) 8.64 Hz, ArH), 8.85 (b, 1H, OH).

Chem. Res. Toxicol., Vol. 16, No. 6, 2003 743 UV (CH3OH): 210, 236, 274, 286, 344 nm; negative ion electrospray-MS [M - H]- m/z 343 (100%), m/z 345 (100%). Synthesis of 17β-Hydroxyequilenin Derivatives. The same reduction procedure was used as described previously for 17β-EN (22, 23). Briefly, the 4-halogenated EN derivatives (800 mg, 3.0 mmol), LiAl(OtBu)3H (2.5 g, 9.25 mmol), and anhydrous THF (60 mL) were refluxed for 14 h under nitrogen and cooled to room temperature. HCl (10%, 100 mL) was added to the mixture on ice and stirred for 10 min. The final solution was extracted with ethyl acetate (2 × 150 mL). The combined organic layers were washed with 2 × 125 mL and dried over Na2SO4. After it was filtered, the solvent was removed in vacuo and the residue was purified by flash chromatography (silica gel) with hexane:ethyl acetate (3:2) as eluent to give the 17β-4-halogenated EN derivatives. 1. 17β-4-ClEN. 1H NMR (acetone-d6): δ 0.67 (s, 3H, CH3), 1.66-1.73 (m, 3H), 2.19-2.25 (m, 3H), 2.77-2.80 (m, 1H), 3.243.36 (m, 2H), 3.74-4.23 (m, 2H), 7.31 (d, 2H, ArH), 7.88 (d, 1H, J ) 9.3 Hz, ArH), 7.96 (d, 2H, J ) 8.7 Hz, ArH), 7.94 (d, 2H, J ) 8.64 Hz, ArH), 10.21 (b, 1H, OH). UV (CH3OH): 232, 284, 296, 344 nm; negative ion electrospray-MS [M - H]- m/z 301 (100%), m/z 303 (33%). 2. 17β-4-FEN. 1H NMR (acetone-d6): δ 0.67 (s, 3H, CH3), 1.63-1.74 (m, 3H), 2.19-2.25 (m, 3H), 3.11-3.31 (m, 2H), 3.773.91 (m, 2H), 7.26 (t, 2H, J ) 8.7 Hz, ArH), 7.68 (d, 1H, J ) 9.0 Hz, ArH), 7.78 (d, 1H, J ) 8.4 Hz, ArH). UV (CH3OH): 238, 270, 280, 340 nm; negative ion electrospray-MS [M - H]- m/z 285 (100%). 3. 17β-4-BrEN. 1H NMR (acetone-d6): δ 0.66 (s, 3H, CH3), 1.30-1.77 (m, 3H), 2.17-2.26 (m, 3H), 2.78-2.90 (m, 1H), 3.273.79 (m, 2H), 7.31 (m, 2H, ArH), 7.98 (m, 2H, ArH). UV (CH3OH): 238, 274, 286, 296, 344 nm; negative ion electrosprayMS [M - H]- m/z 345 (100%), m/z 347 (100%). Oxidation of EN and Its 4-Halogenated EN Derivatives by Tyrosinase. EN or its 4-halogenated EN derivatives were dissolved in DMSO, and GSH was added in phosphate buffer to achieve final concentrations of 0.5 and 5.0 mM, respectively. Reaction solutions containing tyrosinase (0.1 mg/mL, 3620 units/ mL) were conducted for 30 min at 37 °C in 50 mM phosphate buffer (pH 7.4, 1.0 mL total volume). Reactions were terminated by chilling in an ice bath followed by the addition of perchloric acid (50 µL/mL). For control incubations, GSH or tyrosinase was omitted. The reaction mixtures were centrifuged at 18 000g for 6 min, the solution was extracted using solid phase extraction cartridges (Oasis, HLB, Waters), eluted with methanol, and concentrated to 100 µL. Aliquots (25 µL) were analyzed directly using LC-MS (method B). All of these compounds formed the same 4-OHEN GSH conjugates, which were previously described and characterized (26) (Figure 1). Cell Culture Conditions. The S30 cell line, a stable ERR transfectant of MDA-MB-231 cells, was a generous gift from V. C. Jordan (Northwestern University, Chicago, IL). The S30 cells were maintained in phenol red-free MEME supplemented with 1% PSF, 6 µg/L insulin, 1% glutamax (Gibco-BRL, Grand Island, NY), 5% charcoal-dextran-treated FBS (Atlanta Biologicals, Atlanta, GA), and 5% CO2 at 37 °C. Ishikawa cells were provided by Dr. R. B. Hochberg (Yale University, New Haven, CT). Ishikawa cells were maintained in Dulbecco’s modified eagle essential medium (DMEM)/F12 media with 10% heat-inactived FBS, sodium pyruvate (1%), penicillin-streptomycin (1%), and glutamax-1 (1%). One day prior to treating the cells, the medium was replaced with phenol red-free, DMEM/F12 medium containing charcoal/dextran-stripped FBS to remove estrogens. The cells were grown for 24-48 h to maintain logarithmic growth and then treated with various concentrations of estrogens or DMSO in fresh medium. Compounds were dissolved freshly in DMSO, and the final DMSO concentration was 0.05%. ER Competitive Binding Assays (27). The procedure of Obourn et al. (28) was used with minor modifications (29). Briefly, 24 h prior to the assay, a 50% v/v hydroxylapatite slurry was prepared using 10 g of hydroxylapatite in 60 mL of TE buffer (50 mM Tris-Cl, pH 7.4, 1 mM EDTA) and stored at 4

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Figure 1. Formation of 4-halogenated EN GSH adducts in the presence of tyrosinase for 30 min at 37 °C. Aliquots (25 µL) were analyzed directly using LC-MS. (I) 4-OHEN-diSG; (II) 4-OHENSG. (A) EN + tyrosinase; (B) 4-FEN + tyrosinase; (C) 4-ClEN + tyrosinase; (D) 4-BrEN + tyrosinase. °C. The ER binding buffer consisted of 10 mM Tris-Cl (pH 7.5), 10% glycerol, 2 mM dithiothrietol, and 1 mg/mL bovine serum albumin. The ERR wash buffer contained 100 mM KCl, 1 mM EDTA, and 40 mM Tris-Cl (pH 7.5), and the ERβ wash buffer was 40 mM Tris-Cl (pH 7.5). The reaction mixture consisted of 5 µL of test sample in DMSO, 5 µL of pure human recombinant diluted ERR or ERβ (0.5 pmol) in ER binding buffer, 5 µL of “Hot Mix” (400 nM, prepared fresh using 3.2 µL of 25 µM, 83 Ci/mmol 3H-estradiol, 98.4 µL of ethanol, and 98.4 µL of ER binding buffer), and 85 µL of ER binding buffer. The incubations were carried out at room temperature for 2 h, followed by addition of 100 µL of 50% hydroxylapatite slurry, and the tubes were incubated on ice for 15 min with vortexing every 5 min. The appropriate ER wash buffer was added (1 mL), and the tubes were vortexed and then centrifuged at 2000g for 5 min. The supernatant was discarded, and this wash step was repeated three times. The hydroxylapatite pellet containing the ligand-receptor complex was resuspended in 200 µL of ethanol and transferred to scintillation vials. Cytoscint (4 mL/vial) was added, and the tubes were counted using a Beckman (Schaumburg, IL) LS 5801 liquid scintillation counter. The percent inhibition of 3H-estradiol binding to each ER was determined as follows: [1 - (dpmsample - dpmblank)/(dpmDMSO - dpmblank)] × 100. The binding capability (%) of the sample was calculated by comparison to 17β-estradiol (50 nM, 100%). The data represent the average ( SD of three determinations. Induction of Alkaline Phosphatase Activity in Ishikawa Cells. The procedure of Pisha et al. was used as described previously (30). Briefly, Ishikawa cells (5 × 104 cells/well) were incubated overnight with estrogen-free media in 96 well plates. Test samples in DMSO were added to the cells in a total volume of 200 µL media/well and were incubated at 37 °C for 4 days and washed out three times with PBS. Enzyme activity was measured by reading the liberation of p-nitrophenol at 340 nm every 15 s for 16-20 readings with an ELISA reader (Power Wave 200 Microplate Scanning Spectrophotometer, Bio-Tek

Liu et al. Instrument, Winooski, VT). The maximum slope of the lines generated by the kinetic readings was calculated by Table Curve software (SPSS, Chicago, IL). The percent induction for determination of estrogenic activity was calculated as [(slopesample slopecells)/(slopeestrogen - slopecells)] × 100. The data represent the average ( SD of three determinations. RT-PCR Analysis of PR and pS2 mRNA Expression in Ishikawa and S30 Cell Lines, Respectively. Ishikawa cells or S30 cells (4 × 104 cells/wells) were preincubated overnight in estrogen-free media in six well plates. Test samples in DMSO were added and incubated at 37 °C for 4 days (Ishikawa cells) or 48 h (S30 cells). Total RNA from both cell lines was isolated with TRIzol reagent (Gibco, Grand Island, NY) following the manufacturer’s protocol. RT-PCR was carried out using the Super-Script one step RT-PCR system (Gibco) and a DNA thermal cycler 480 (Perkin-Elmer, Foster City, CA). The primers used for PR expression were 5′-CCATGTGGCAGATCCCACAGGAGTT-3′ (sense) and 5′-TGGAAATTCAACACTCAGTGCCCGG-3′ (antisense). The primers used for pS2 expression were 5′-CATGGAGAACAAGGTGATCTG-3′ (sense) and 5′-CAGAAGCGTGTCTGAGGTGTC-3′ (antisense). The PCR products (5 µL) of PR (271 bp) and pS2 (365 bp) were separated by electrophoresis in 1% agarose gels and visualized by staining with ethidium bromide. β-Actin (621 bp) was used as an internal control for both PR and pS2. The sense and antisense primers used for β-actin were 5′-ACACTGTGCCCATCTACGAGG-3′ and 5′-AGGGGCCGGACTCGTCATACT-3′, respectively. The net intensity of the bands was measured using Kodak Digital Science 1D software. The ratio of the intensity of the target gene and the internal control of each sample was calculated as shown in Tables 3 and 4. The data represent the average ( SD of three determinations. Evaluation of the Cytotoxic Potential of the Halogenated EN Derivatives in Breast Cancer Cell Lines. Cell viability was determined by trypan blue exclusion (31, 32). Briefly, the cells (105 cells/mL) were coincubated with various concentrations of the halogenated EN derivatives and tyrosinase (300 µM) or DMSO for 3 h. After treatment, floating cells were collected by centrifugation at 960g for 5 min and attached cells were first trypsinized and then harvested by centrifugation. Floating cells and attached cells were combined, washed with PBS, and stained with 0.4% trypan blue. A drop of cell suspension was placed on a hemocytometer, and the cell number was determined using a light microscope. The LC50 values were obtained by linear regression analysis, and the data represent the average ( SD of four determinations. Analysis of Estrogenic Effects In Vivo. 1. Animals. Guidelines established by our institutional Animal Care and Use Committee and state and federal regulations were followed for all procedures. The protocol complied with the Guidelines for the Care and Use of Laboratory Animals and the facilities are approved by the Association for the Assessment and Accreditation of Laboratory Animal Care. Female ovariectomized Sprague-Dawley rats, 7 weeks old, were purchased from Harlan (Indianapolis, IN). The animals were acclimatized for 3 days prior to experimental manipulations. In this study, eight ovariectomized rats per treatment group were used. Two or three animals per cage were housed with a temperature 22 ( 1 °C, relative humidity 50 ( 10%, and a 12 h light/dark cycle. Rat cages were arranged randomly to limit variations of temperature and light. 2. Diet and Treatment. The rats were fed with AIN-76A (Bethlehem, PA) and kept with unrestricted access to food and water, which was administered through an automatic watering system. The test compounds, 17β-4-halogenated-EN, 17β-EN, and E2, were dissolved in DMSO and suspended in sesame oil to a concentration of 85 µM. Each rat was injected subcutaneously at 73 nmol/(kg d) for 14 days. Controls received DMSO alone. The dose of the test compounds was based on previous studies (33, 34). Estrogenic effects were evaluated based on vaginal cytology, uterine weight, and mammary gland alveolar and ductal structure. The body weight of all animals was

Halogenated Analogues of Equilenin

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Table 1. Estrogen Receptor Competitive Binding Assaya IC50 (nM) compds EN 4-FEN 4-ClEN 4-BrEN 17β-EN 17β-4-FEN 17β-4-ClEN 17β-4-BrEN

ERR 3.6 ( 0.2 3.3 ( 1.8 10.7 ( 2.3 20.6 ( 6.3 1.3 ( 0.3 1.3 ( 1.0 31.6 ( 8.0 51.0 ( 11.0

ERβ 3.2 ( 0.2 4.0 ( 1.4 11.1 ( 3.7 23.3 ( 4.3 0.5 ( 0.1 0.4 ( 0.2 26.4 ( 16.0 44.0 ( 9.7

a The reaction mixture consisted of 5 µL of test samples in DMSO, 5 µL of pure human recombinant diluted ERR in 85 µL of ER binding buffer, and 5 µL of Hot Mix. Samples were incubated for 2 h. Values are expressed as the mean ( SD of three determinations. Experimental details are described in the Materials and Methods.

recorded every other day to monitor toxicity and to adjust the dose accordingly. 3. Vaginal Cellular Differentiation Analysis. Vaginal cytology smears were taken and scored daily to monitor cellular differentiation. A smear was performed before injection to establish a baseline and to confirm that all ovariectomized animal smears showed no cornification. Vaginal smears were taken daily using an eyedropper containing physiological saline, placed on a 24 well plate, and observed under a light microscope using a 10× eyepiece and 10× objective. Cells were identified as leukocytes, nucleated, or cornified epithelial cells. A raw score on a scale of 1-5 was assigned for cell populations ranging from entirely leukocyte (proestrous) to entirely cornified (diestrous). 4. Determination of Uterine Weight and Analysis of the Mammary Glands. At the end of treatment, the animals were killed by CO2 asphyxiation. The uteri were carefully dissected and trimmed of fat and connective tissue. The uteri were then weighed and frozen in 1.5 mL cryogenic vials on dry ice and stored at -80 °C. Mammary gland tissues were collected as a strip of tissues containing the primary duct, the edge of the gland, and part of the mammary tree. Whole mount slides were prepared from the mammary glands as described previously (34, 35). Statistical Analyses. Uterine weights were analyzed using the Student’s t-test. The data were reported as the mean ( SD. Differences among means were considered significant at P < 0.05.

Results Relative Binding Affinity of Halogenated EN Derivatives for ERr and ERβ. To confirm that the halogenated EN derivatives were still bound to estrogen receptors after halogen substitution, the IC50 values of the halogenated ENs for ERR and ERβ as compared to estradiol were measured (Table 1). The parent estrogens, EN and 17β-EN, had similar affinity for both receptors as 4-FEN and 17β-4-FEN, which is likely due to the similar molecular size of the F and H group. However, as the molecular size of the halogenated group was increased from F to Cl to Br, the binding affinity decreased. Induction of Alkaline Phosphatase Activity in Ishikawa Cells. The ER binding assay measures competition with radiolabeled estradiol for ER; however, this assay cannot confirm how the estrogen receptor complex will act within a cell. Therefore, the Ishikawa cell line is used extensively to determine estrogenic activity (30). Ishikawa is an ERR positive endometrial adenocarcinoma cell line derived from a glandular epithelial cell line, which is a stable human endometrial carcinoma cell line

Table 2. Induction of Alkaline Phosphatase Activity in Ishikawa Cellsa compds

ED50 (nM)

compds

ED50 (nM)

EN 4-FEN 4-ClEN 4-BrEN

2.3 ( 0.6 4.2 ( 1.4 19.7 ( 2.4 32.6 ( 4.2

17β-EN 17β-4-FEN 17β-4-ClEN 17β-4-BrEN

2.7 ( 0.3 1.9 ( 0.4 10.8 ( 2.1 26.1 ( 4.9

a Cells (5 × 104 cells/well) were treated with compounds for 4 days. Values are expressed as the mean ( SD of three determinations. Experimental details are described in the Materials and Methods.

Table 3. pS2 Induction in S30 Cellsa compds

ratio to E2

compds

ratio to E2

DMSO E2 E1 EN 4-FEN 4-ClEN

not detected 1.00 0.98 ( 0.10 1.03 ( 0.14 0.78 ( 0.18 0.50 ( 0.07

4-BrEN 17β-EN 17β-4-FEN 17β-4-ClEN 17β-4-BrEN

not detected 0.96 ( 0.04 0.91 ( 0.09 0.71 ( 0.11 not detected

a Cells (4 × 104 cells/well) were treated with compounds for 2 days at 10 nM. Values are expressed as the mean ( SD of three determinations. Experimental details are described in the Materials and Methods.

Table 4. PR Induction in Ishikawa Cellsa compds

ratio to E2

compds

ratio to E2

DMSO E2 E1 EN 4-FEN 4-ClEN

not detected 1.00 1.01 ( 0.09 1.13 ( 0.21 1.02 ( 0.10 0.68 ( 0.17

4-BrEN 17β-EN 17β-4-FEN 17β-4-ClEN 17β-4-BrEN

0.23 ( 0.15 0.96 ( 0.11 1.04 ( 0.02 0.81 ( 0.19 0.28 ( 0.23

a Cells (4 × 104 cells/well) were treated with compounds for 4 days at 10 nM. Values are expressed as the mean ( SD of three determinations. Experimental details are described in the Materials and Methods.

that expresses estrogen inducible alkaline phosphatase (30, 36). This cell line responds to estrogens at concentrations approximating physiological levels (37). Ishikawa cells were used to investigate the estrogenic activity of the halogenated EN derivatives. The results (Table 2) confirmed the findings of the ER binding assay and showed that the halogenated derivatives 4-FEN and 17β4-FEN produced the highest induction of alkaline phosphatase activity. Furthermore, as the size of halogen substituents was increased, the induction of alkaline phosphatase activity decreased. Stimulation of pS2 mRNA Expression in S30 Cells and PR mRNA Expression in Ishikawa Cells. The ERR positive human breast cancer cell line S30 shows pS2 gene induction in the presence of estrogens whereas Ishikawa cells do not (29). In contrast, estradiol-mediated PR expression is not observed in S30 cells but is detectable in Ishikawa cells (29). S30 is a subclone of the MDAMB-231 (ER-) breast cancer cell line that is stably transfected with ERR. We found that 17β-estradiol, estrone, EN, and 17β-EN showed higher stimulated expression of the pS2 gene more than did the halogenated EN derivatives (Table 3). The brominated derivatives did not show any induction. PR induction in Ishikawa cells showed the same trend as pS2 (Table 4). In addition, all of the 17β-hydroxy-4-halogenated EN derivatives induced gene expression more than any of the 17-keto-4-halogenated EN derivatives. These results were consistent with the trends observed for induction of alkaline phosphatase activity.

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Liu et al.

Table 5. Cytotoxic Potential in Breast Cancer Cells with Tyrosinase in 3 ha LC50 compds

tyrosinase

without tyrosinase

EN 4-FEN 4-ClEN 4-BrEN 17β-EN 17β-4-FEN 17β-4-ClEN 17β-4-BrEN

54 ( 9 78 ( 15 >200 >200 69 ( 11 >200 >200 >200

>200 >200 >200 >200 >200 >200 >200 >200

a Cells (105 cells/mL) were incubated with various concentrations or vehicle for 3 h. Values are expressed as the mean ( SD of four determinations. Experimental details are described in the Materials and Methods.

Incubation of 4-Halogenated EN Derivatives with Tyrosinase and GSH. Incubation of EN with tyrosinase in the presence GSH resulted in the formation of only 4-OHEN catechol GSH conjugates, which was confirmed by LC-MS. This experiment showed that the 4-position in the A ring of EN is the most active site for oxidation by tyrosinase. These data further confirm previous results showing that only 4-OHEN GSH conjugates were formed from microsomal incubations of EN in the presence of GSH (26). All of the 4-halogenated EN derivatives formed the same GSH conjugates as EN in the presence of tyrosinase and GSH; however, the total amount of GSH conjugates resulting from each halogenated EN derivatives decreased as the size of the halogen group increased (Figure 1). The ratio of the GSH diadduct formation for EN, 4-FEN, 4-ClEN, and 4-BrEN was 1:0.39:0.21:0.12. 17β-Hydroxy-4-halogenated EN derivatives also showed a similar trend; the ratio of the diadduct formation was 1:0.19:0.07:0.04 (data not shown). These results showed that the introduction of halogens at the 4-position of the A ring decreased hydroxylation at the 4-position. The detection of GSH conjugates from 4-OHEN implied that quinones were formed prior to nucleophilic GSH addition (Figure 1). These o-quinones might produce toxicity through depletion of cellular GSH. Once all of the GSH is depleted, the o-quinones could alkylate Cys residues on cellular proteins resulting in toxic effects (38, 39). Evaluation of the Cytotoxic Potential in Breast Cancer Cells. To confirm that these halogenated compounds formed less catechol estrogens than the corresponding nonhalogenated analogues in the presence of tyrosinase, their cytotoxicity was measured using the trypan blue exclusion assay. In the absence of tyrosinase, no cytotoxic effects were detected from any of the estrogen derivatives in the estrogen receptor positive cells (S30). Coincubation with tyrosinase significantly increased the cytotoxicity of EN and 17β-EN (Table 5); however, inclusion of tyrosinase had no effect on the toxicity of the halogenated derivatives. Analysis of Estrogenic Effects of Halogenated Derivatives In Vivo. 1. Vaginal Cellular Cornification. Vaginal cells showed full cornification in response to E2 and 17β-EN after 3 or 4 days (Figure 2). The administration of 17β-4-FEN exhibited an increase in cell differentiation after 5 days, and at the end of the treatment, cornified epithelial cells were predominant in the smears. These results indicated that the estrogenic effects of 17β-4-FEN were delayed relative to those of 17β-estradiol or 17β-EN. Similar studies with 17β-4-

Figure 2. Vaginal cellular differentiation induced in ovariectomized rats treated with DMSO, E2, 17β-EN, 17β-4-FEN, 17β4-ClEN, and 17β-4-BrEN. Vaginal smears were acquired by aspiration on a daily basis for 14 days from all treated animals. Numbers on the y-axis represent a numeric scale assigned according to predominate cell type with 1 being completely undifferentiated leukocytes, 3 being mostly neucleocytes, and 5 being completely cornified cells. Closed circles, E2; open triangles, 17β-EN; closed triangles, 17β-4-FEN; open squares, 17β-4-ClEN; open circles, 17β-4-BrEN; closed squares, DMSO.

Figure 3. Effect of test compounds on body weight. Closed circles, E2; open triangles, 17β-EN; closed triangles, 17β-4-FEN; open squares, 17β-4-ClEN; open circles, 17β-4-BrEN; closed squares, DMSO.

ClEN and 17β-4-BrEN did not stimulate differentiation as compared to the DMSO control. 2. Body Weight. An ovariectomy has been shown to increase body weight whereas “normal” rats or rats treated with 17β-estradiol do not show significant increases in body weight (40). Over the period of the treatment, the DMSO-treated rats as well as the rats treated with the chloro and bromo derivatives increased their body weight by approximately 25%. On the other hand, the rats treated with the fluoro derivative only increased their body weight by 10% (Figure 3). The rats treated with E2 and 17β-EN maintained a similar body weight over the 14 day treatment. 3. Uterine Weight. Increases in uterine weight provide a biological marker of estrogenic activity in vivo. The chloro and bromo derivatives did not show any induction of uterine weight as compared with DMSO. In contrast, the fluoro derivative showed a significant increase in uterine weight as compared with DMSO (P < 0.05) but

Halogenated Analogues of Equilenin

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Figure 4. Photographs of uteri excised from normal rats or ovariectomized rats treated with DMSO, E2, 17β-EN, 17β-4-FEN, 17β4-ClEN, and 17β-4-BrEN. The photograph demonstrated the changes in uterine thickness in response to test compounds. (A) DMSO; (B) normal rats; (C) E2; (D) 17β-EN; (E) 17β-4-FEN; (F) 17β-4-ClEN; (G) 17β-4-BrEN. Table 6. Weight of Uteri from Ovariectomized Ratsa treatment

n

uterine weight (g)

DMSO E2 17β-EN

8 8 8

0.10 ( 0.02 0.44 ( 0.05b 0.29 ( 0.04b

treatment

n

uterine weight (g)

17β-4-FEN 17β-4-ClEN 17β-4-BrEN

8 8 8

0.18 ( 0.02b 0.10 ( 0.01 0.10 ( 0.01

a The test compounds were dissolved in DMSO and suspended in sesame oil to a concentration of 85 µM. Each rat was injected subcutaneously at 73 nmol/(kg d) for 14 days. b Rats demonstrated a significant increase in uterine weight as compared with DMSO (P < 0.05). Experimental details are described in the Materials and Methods.

less than observed in rats treated with E2 or 17β-EN, indicating a lower potency (Table 6 and Figure 4). These results confirmed the in vitro assays that the fluoro derivatives showed higher estrogenic potency as compared to the chloro and bromo analogues. 4. Mammary Glands. In the mammary gland, proliferation is regulated by the levels of estrogen and progesterone. Therefore, in this study, we examined the duct branching and alveolar structure in the ovariectomized rats. Glands from rats treated with DMSO, 17β4-ClEN, and 17β-4-BrEN displayed thin branches and little alveolar budding. The mammary glands of E2treated rats demonstrated extensive ductal branching and defined buds. Rats that were treated with 17β-4-FEN were comparable to those treated with 17β-EN; slight proliferation of alveolar budding and ductal branching was found as compared to control (data not shown).

Discussion Chemical modification has been used to change both the route and the rate of metabolism of E2 without the loss of estrogenic activity (41). It has been reported that fluorination of the A ring of E2 dramatically alters the route of oxidative metabolism by blocking the formation of potentially toxic catechol estrogens (42) but without loss of estrogenic activity (20). In addition, introduction of fluorine into the C-4 position of E2 profoundly decreases the induction of renal tumors by these compounds (20, 43).

Because we have established that 4-OHEN-o-quinone formation represents the major bioactivation pathway for several equine estrogens, novel 4-halogenated EN derivatives were synthesized and analyzed to determine if cytotoxicity could be reduced without the loss of estrogenic potency. We found that the 4-fluoro derivatives had similar estrogenic potency as compared to their nonhalogenated analogues, which is probably because of the similar molecular size of the H and F groups. In comparison, the 4-chloro and 4-bromo derivatives showed lower binding affinities for estrogen receptors R and β, reduced induction in activity of alkaline phosphatase in Ishikawa cells, and decreased pS2 expression in S30 cells and PR expression in Ishikawa cells. Incubation of the halogenated derivatives with tyrosinase in the presence of GSH showed less formation of catechol GSH conjugates as compared to the parent compounds, EN and 17β-EN. In addition, these halogenated analogues showed lower cytotoxicity in the presence of tyrosinase than the parent compounds in S30 cells. These results are consistent with previous studies, which showed that fluorine, because of its similar size to hydrogen, remains the most feasible chemical alternative to hydrogen for blockade of aromatic hydroxylation and retention of estrogenicity in estrogen analogues (44). Because 17β-hydroxy-4-halogenated EN derivatives showed higher estrogenic activity than the 17-keto-4halogenated EN derivatives in vitro, we then evaluated the potential of the 17β-hydroxy-4-halogenated EN derivatives to induce estrogenic effects in the uterus, vaginal cells, and mammary glands of ovariectomized rats. Ovariectomized rats are often used as in vivo models for estrogenicity of chemicals based on stimulated uterine growth (45). In this study, 4-chloro and 4-bromo derivatives did not show any estrogenic activity as demonstrated by a lower induction of uterine growth, vaginal cell differentiation, and mammary gland branching. On the other hand, 17β-4-FEN exhibited a significant induction in uterine growth as compared with the DMSO control but a lower estrogenic potency than E2 and 17βEN, which could be due to alternative pharmacokinetic properties for these compounds. Although 17β-4-FEN showed lower estrogenic activity than 17β-EN in the

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uterus, 17β-4-FEN exhibited three times higher affinity for ERβ as compared to ERR. This selective effect might offer distinct advantages for estrogen replacement therapy where it would be advantageous to minimize the hyperproliferative action on the endometrium and breast but induce estrogenic activity in the bone and brain where ERβ shows substantial levels of expression (46). Future work will further investigate the tissue selective biological effects of these 4-fluoro derivatives. In summary, fluorine substituents of equine estrogens were found to retain their estrogenic properties in vitro and in vivo while forming lower levels of cytotoxic catechol metabolites. As a result, subsequent autoxidation to an o-quinone would be prevented thereby avoiding DNA oxidation and alkylation in vitro and in vivo. Therefore, 4-fluoro derivatives may represent attractive alternatives for traditional estrogen replacement therapy.

Acknowledgment. This research was supported by NIH Grants CA73638 (J.L.B.) and CA083124 (R.B.v.B.). We are grateful to Dr. V. C. Jordan (Northwestern University) for the gift of the S30 cell line and Dr. R. B. Hochberg (Yale University) for the Ishikawa cells.

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