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Premarin (Wyeth-Ayerst). In this study we have synthesized the catechol metabolites of equilenin [4-hydroxyequilenin (4-OHEN)] and equilin [4-hydroxye...
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Chem. Res. Toxicol. 1998, 11, 758-765

Inhibition of Glutathione S-Transferase Activity by the Quinoid Metabolites of Equine Estrogens Minsun Chang, Fagen Zhang, Li Shen, Nikkole Pauss, Iram Alam, Richard B. van Breemen, Sylvie Y. Blond, 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 December 10, 1997

The risk factors for women developing breast and endometrium cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy (ERT) in particular has been correlated with a slight increased cancer risk, although the numerous benefits of ERT may negate this harmful side effect. Equilenin and equilin are equine estrogens which make up between 30% and 45% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst). In this study we have synthesized the catechol metabolites of equilenin [4-hydroxyequilenin (4-OHEN)] and equilin [4-hydroxyequilin (4-OHEQ)] and examined how changing unsaturation in the B ring affects the formation of o-quinone GSH conjugates and the ability of the o-quinones and/or GSH conjugates to inhibit glutathione S-transferase (GST). Interestingly, both 4-OHEN and 4-OHEQ autoxidized to o-quinones without the need of oxidative enzyme catalysis. 4-OHEN-o-quinone reacts with GSH to give two mono-GSH conjugates and one diadduct. The behavior of 4-OHEQ was found to be more complex than 4-OHEN as conjugates resulting from 4-OHEN were detected in addition to the 4-OHEQ GSH adducts. Both 4-OHEN and 4-OHEQ were found to be potent inhibitors of GSTcatalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene. In contrast, the endogenous catechol estrogens, 4-hydroxyestrone (4-OHE) and 2-hydroxyestrone (2-OHE), were without effect unless tyrosinase was present to convert the catechols to o-quinones. Scavengers of reactive oxygen species and metal chelators had no effect on GST inhibition by catechol estrogens with the exception of the catalase which protected GST activity. Kinetic studies showed that 4-OHEN was a potent irreversible inactivator of GST. Preincubation of the enzyme with 4-OHEN showed a time-dependent increase in inhibitory effect, and gel filtration did not restore GST activity confirming the irreversible nature of the enzyme inactivation. Analysis of the Kitz-Wilson plot gave a dissociation constant of the reversible enzyme-inhibitor complex (Ki ) 620 µM) and a rate constant of conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2 ) 7.3 × 10-3 s-1). These data suggest that 4-OHEN is an irreversible inactivator with relatively low affinity for GST; however, once formed the 4-OHEN enzyme complex is rapidly converted to the irreversibly inhibited enzyme. The inhibition mechanism likely involves oxidation of the catechol estrogens to o-quinones and covalent modification and/or oxidation of critical amino acid residues on GST. In addition, hydrogen peroxide generated through redox cycling of the o-quinone and/or semiquinone radical and GSH could cause oxidative damage to GST.

Introduction Excessive exposure to estrogens either through early menarche and late menopause and/or through estrogen replacement therapy (ERT)1 increases the risk of women developing breast or endometrial cancer (1-5). Premarin (Wyeth-Ayerst) is the most widely prescribed estrogen replacement formulation, and yet there is very little * Address correspondence to: Dr. Judy L. Bolton. Fax: (312) 9967107. E-mail: [email protected]. 1 Abbreviations: GST, glutathione S-transferase; CDNB, 1-chloro2,4-dinitrobenzene; 2-OHE, 2-hydroxyestrone, 2,3-dihydroxy-1,3,5(10)estratrien-17-one; 4-OHE, 4-hydroxyestrone, 3,4-dihydroxy-1,3,5(10)estratrien-17-one; 4-OHEN, 4-hydroxyequilenin, 3,4-dihydroxy-1,3, 5(10),6,8-estrapentaen-17-one; 4-OHEQ, 4-hydroxyequilin, 3,4-dihydroxy-1,3,5(10),7-estrabutaen-17-one; estrone, 3-hydroxy-1,2,5-(10)estratrien-17-one; equilenin, 1,3,5(10),6,8-estrapentaen-3-ol-17-one; equilin, 1,3,5(10),7-estratetraen-3-ol-17-one; ERT, estrogen replacement therapy; P450, cytochrome P450; o-quinone, 3,5-cyclohexadiene1,2-dione; PCA, perchloric acid.

information on the potential cytotoxic/genotoxic effects of the 10 different estrogens present in Premarin. It is known that treating hamsters for 9 months with either estrone, equilin plus equilenin, or sulfatase-treated Premarin resulted in 100% tumor incidences and abundant tumor foci (6). One potential carcinogenic pathway could involve metabolism of the Premarin estrogens to catechols (6-9) which could be enzymatically or spontaneously converted to o-quinones (10). As o-quinones are Michael acceptors as well as potent redox cycling agents, damage in cells could occur to DNA, lipids, critical structural proteins, and enzymes such as the phase II enzyme glutathione S-transferase (GST). GSTs are a family of enzymes which play an extremely important role in the detoxification of electrophiles including Michael acceptors by catalyzing conjugation with GSH (11, 12). It has been shown that several

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Catechol Estrogen-Mediated GST Inhibition

Figure 1. Structures of the catechol estrogens investigated.

naturally occurring as well as synthetic quinones can inhibit GST activity (13-16). As catechol estrogens are readily oxidized to o-quinones, it is quite possible that the estrogen o-quinones formed from Premarin estrogens could inhibit GST activity in vivo. Such an event could indirectly contribute to the cytotoxic effects of estrogens through decreases in the GST-mediated detoxification of xenobiotic as well as endogenous carcinogens. It is unlikely that the estrogen o-quinones are substrates for GST since quinones are known to react very rapidly with sulfur nucleophiles including GSH without the need for enzyme catalysis (17-19). We previously synthesized the major metabolite of equilenin, 4-hydroxyequilenin (4-OHEN; Figure 1), and examined how aromatization of the B ring affects the formation and reactivity of the equilenin quinoids (10). Unlike the endogenous catechol estrogens, 4-OHEN rapidly autoxidized to 4-OHEN-o-quinone which readily entered into a redox couple with the semiquinone radical catalyzed by NAD(P)H, P450 reductase, or quinone reductase (10). Significant oxygen consumption was also detected consistent with in vitro models that have shown that 4-OHEN-o-quinone increases the amount of oxidative damage to DNA by 50% compared to control levels (20). Finally, we showed that 4-OHEN-semiquinone radical forms very unusual cyclic adducts with deoxynucleosides and DNA which may represent one mechanism for equilenin carcinogenesis (21, 22). In the present study, we examined the relative ability of four of the Premarin catechol estrogen metabolites shown in Figure 1 to inhibit the GST-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB). These data suggest that without oxidative enzyme catalysis the endogenous estrogen metabolites 4-hydroxyestrone (4-OHE) and 2-hydroxyestrone (2-OHE) would have little effect on GST activity; however, the equine estrogen metabolites 4-hydroxyequilin (4-OHEQ) and 4-hydroxyequilenin (4-OHEN), both of which autoxidize to o-quinones, were found to be potent inhibitors of GST.

Materials and Methods Caution: All quinones used in this study and the catechol estrogen o-quinones in particular were handled in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens (23). Materials. All chemicals were purchased from Aldrich (Milwaukee, WI), Fisher Scientific (Itasca, IL), or Sigma (St. Louis, MO) unless stated otherwise. Recombinant human GST Μ1-1 was purchased from Pan Vera (Madison, WI). The specific activity was 221 µmol of CDNB GSH conjugate formed/min/mg of protein. 2-OHE and 4-OHE were either purchased from

Chem. Res. Toxicol., Vol. 11, No. 7, 1998 759 Sigma or synthesized as described previously (24). 4-OHEN was synthesized by treating equilin with Fremy’s salt as described previously (20, 25) with minor modifications (10). 4-OHEQ was synthesized by demethylation of 4-methoxyequilin which was synthesized according to the published procedure (26) as shown in Figure 2. Briefly, equilin was brominated to 4-bromoequilin (2) with N-bromoacetamide. The 17-keto group in 4-bromoequilin was protected by reaction with ethylene glycol to give 3. Treatment of 3 with sodium methoxide produced 4, and acid hydrolysis gave 4-methoxyequilin (5): 1H NMR (acetone-d6) δ 0.75 (s, 3H, 18-CH3), 3.78 (s, 3H, OCH3), 5.57 (m, 1H, H7), 6.78 (d, J ) 8.40 Hz, 1H, ArH), 6.90 (d, J ) 8.40 Hz, ArH); 13C NMR (CDCl3) δ 13.7, 19.6, 24.5, 32.0, 32.1, 35.7, 40.3, 49.5, 50.5, 60.3, 114.0, 115.0, 124.2, 126.9, 130.3, 135.5, 143.9, 146.6, 205.1; CIMS (positive ion, methane) m/z 299 (MH+, 100%). 4-OHEQ. Compound 5 (32 mg, 0.11 mmol) was dissolved in anhydrous ClCH2CH2Cl (6 mL), and the solution was cooled to -15 °C. BBr3 (1.2 mL of 1 M in ClCH2CH2Cl) was added to the cooled solution, and the mixture was stirred for 9 h. It should be noted that solvent and reaction temperature are critical to the demethylation of 5. Water (8 mL) was added to quench the reaction, and the solution was extracted with ethyl acetate (3 × 20 mL). The organic layer was washed with 10% sodium bicarbonate and water and dried over sodium sulfate. After filtration, the solvent was removed in vacuo, and the final residue was purified by preparative TLC (silica gel) with hexane/ acetone/methanol (2:1:0.03) as eluent. 4-OHEQ readily dimerizes in solution within 24 h, and it should be stored as a solid at -70 °C under argon. Complete characterization was accomplished by 1D and 2D NMR experiments including 1H, 13C, HMQC, HMBC, and DQF-COSY: 1H NMR (acetone-d6) δ 0.76 (s, 3H, CH3), 1.50 (m, 1H, H11), 1.64 (m, 1H, H12), 1.83 (m, 1H, H12), 2.05 (m, 2H, 2 H15), 2.20 (m, 1H, H11), 2.27 (dd, 1H, H14), 2.49 (m, 2H, 2H16), 3.15 (m, 1H, H9), 3.35 (m, 2H 2H6), 5.58 (m, 1H, H7), 6.62 (d, J ) 9.0 Hz, 1H, H1), 6.72 (d, J ) 9.0 Hz, 1H, H2), 7.12 (s, D2O exchangeable 1H, OH4), 8.07 (s, D2O exchangeable, 1H, OH3); 13C NMR (acetone-d6) δ 14.0 (C18), 20.3 (C15), 25.3 (C6), 33.2 (C11, C12), 36.1 (C16), 41.3 (C9), 49.9 (C13), 51.2 (C14), 114.2 (C2), 116.1 (C7), 119.2 (C1), 121.7 (C5), 130.5 (C10), 136.6 (C8), 142.6 (C4), 142.7 (C3), 219.1 (C17); CI-MS (positive ion, methane) m/z 285 (MH+, 100%). GSH Conjugates of 4-OHEN- and 4-OHEQ-o-quinones. The o-quinone GSH conjugates of 4-OHEN were prepared by incubating the catechol (0.5 mM) with 5.0 mM GSH in 5 mL of sodium phosphate buffer (50 mM) (pH 7.4) at 25 °C, 5 min. The adducts were isolated from the aqueous phase on Waters Oasis extraction cartridges (Waters Corp. Milford, MA) and eluted with methanol. The methanol was concentrated to 500 µL, and aliquots (25 µL) were analyzed directly by HPLC with a 4.6- × 150-mm Ultrasphere C-18 column (Beckman) on a HewlettPackard (Palo Alto, CA) 1090L gradient HPLC equipped with a photodiode array UV/vis absorbance detector set at 230-350 nm and a 5989B MS Engine quadrupole mass spectrometer. The mobile phase consisted of 5% methanol in 0.5% ammonium acetate (pH 3.5) at 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 last 20 min. 4-OHEN GSH Conjugates. 4-OHEN-DiSG: UV (CH3OH) 274, 324 nm; positive ion electrospray-MS m/z 893 (100%) (MH+), retention time 39 min. 4-OHEN-SG 1: UV (CH3OH) 276, 324 nm; positive ion electrospray-MS m/z 586 (100%) (M+ - 1), retention time 54 min. 4-OHEN-SG 2: UV (CH3OH) 270, 318 nm; positive ion electrospray-MS, m/z 586 (100%) (M+ 1), retention time 60 min. 4-OHEQ GSH Conjugates. 4-OHEQ GSH conjugates were synthesized by incubating 4-OHEQ (0.5 mM) with GSH (5.0 mM) in pH 7.4 phosphate buffer for 5 min at 37 °C. The adducts were isolated and characterized as described above for 4-OHEN. In addition, to the 4-OHEN conjugates the following conjugates were also detected. 4-OHEQ-DiSG: UV (CH3OH) 308 nm; positive ion electrospray-MS m/z 896 (25%) (MH+), 587 (30%) (MH+ - GSH), 448 (MH2+), retention time 46 min. 4-OHEQ-

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Figure 2. Synthesis of 4-OHEQ. Reactions are described in the text. SG 1: UV (CH3OH) 250, 280 nm; positive ion electrospray-MS m/z 590 (100%) (M+), retention time 63 min. 4-OHEQ-SG 2: UV (CH3OH) 270, 318 nm; positive ion electrospray-MS m/z 591 (100%) (MH+), retention time 65 min. GST Conjugation of CDNB. The initial rates of GSTcatalyzed conjugation of GSH with CDNB were determined spectrophotometrically according to the method of Habig et al. (27). Reactions contained GST M1-1 (0.5 µg/mL), 2.5 mM GSH, and 1.5 mM CDNB in a total volume of 1.0 mL of potassium phosphate buffer (50 mM, pH 6.5), 25 °C. In inhibition studies, concentrated solutions of the catechol estrogens in DMSO were included, and the percent inhibition was calculated from the difference in rates compared to samples with DMSO only. The absorbance differences were recorded on a Hewlett-Packard model 8452 diode array UV/vis spectrophotometer at 340 nm. Kinetics of Irreversible Inhibition. GST (50 µg/mL) was preincubated at 25 °C with different concentrations of 4-OHEN or 4-OHEQ for various times. Aliquots were removed and diluted 100-fold into incubation buffer containing 2.5 mM GSH and 1.5 mM CDNB in a total volume of 1.0 mL of potassium phosphate buffer (50 mM, pH 6.5). Initial rates were determined spectrophotometrically as described above. Irreversible kinetic parameters were obtained according to refs 28 and 29 using the following equations where it is assumed that [I] . [Eo]. [I] is the inhibitor concentration, [Eo] is the concentration of GST at time 0, EI is the enzyme inhibitor complex, and E* is the inactivated enzyme. Ki

k2

I + E y\z EI 98 E* kapp )

k2 1+

Ki

(1)

[I]

Ki 1 1 ) + kapp k2 k2[I]

(2)

Ki and k2 were obtained from double-reciprocal plots (i.e., eq 2) of the apparent first-order rate constant versus inhibitor concentration. Gel Filtration Experiments. GST (2 mg/mL, equine liver; Sigma) was dissolved in potassium phosphate buffer (pH 6.5), and the enzyme was treated with either DMSO (2 µL), 4-OHEN (0.8 mM), or 4-OHEQ (0.8 mM). Size-exclusion chromatography was carried out at 4 °C using a Pharmacia LKB FPLC system on a semipreparative grade Superose 12 HR 10/30 column (Pharmacia Biotech) with a mobile phase of 50 mM potassium phosphate buffer (pH 7.5) containing 5 mM MgCl2 at a flow rate of 0.5 mL/min. Fractions were collected every minute, and the absorbance was measured at 280 nm. Under these conditions, GST eluted from 26 to 30 min. Protein concentrations were determined using the Bradford assay (Bio-Rad), and activity was measured spectrophotometrically as described above.

Instrumentation. HPLC experiments were performed on a Shimadzu LC-10A gradient HPLC equipped with an SIL-10A autoinjector, SPD-M10AV UV/vis photodiode array detector, and SPD-10AV UV detector. Peaks were integrated with Shimadzu EZ-Chrom software and a 486-33 computer. Gel filtration experiments were performed on a Pharmacia LKB FPLC system equipped with a P-500 pump, UV-1 UV detector, FRAC-100 fraction collector, and LCC-501 Plus system controller. 1H NMR spectra were obtained with a Bruker Avance DPX300 spectrometer at 300 MHz, and CI mass spectra were obtained with a Finnigan MAT 90 magnetic sector mass spectrometer. Positive ion electrospray mass spectra were obtained using a HewlettPackard 5989B MS Engine quadrupole mass spectrometer equipped with a ChemStation data system and high-flow pneumatic nebulizer-assisted electrospray LC/MS interface. The mass spectrometer was interfaced to the above-mentioned Hewlett-Packard gradient HPLC system. The quadrupole analyzer was maintained at 120 °C, and unit resolution was used for all measurements. Nitrogen at a pressure of 80 psi was used for nebulization of the HPLC effluent, and nitrogen bath gas at 250 °C and a flow rate of 10 L/min were used for evaporation of solvent from the electrospray. The range m/z 200-900 was scanned every 2 s during LC/MS.

Results GSH Conjugates of 4-OHEN- and 4-OHEQ-oquinones. Previous studies have shown that enzymatic oxidation of endogenous catechol estrogens generates o-quinones which can be trapped in situ by GSH (17, 30). With 4-OHEN, chemical or enzymatic oxidation is not required to generate the o-quinone as this catechol readily autoxidizes under physiological conditions (10). Once formed 4-OHEN-o-quinone reacts with GSH to give two mono-GSH conjugates at retention times of 54 and 60 min and one diadduct at 39 min (Figure 3B). Electrospray-MS analysis of the conjugates revealed that some had been oxidized to the corresponding o-quinone adducts during electrospray ionization; instead of observing the expected MH+ ion at 588 mass units, 586 mass units was detected, representing MH+ of the oxidized adduct. We previously observed similar behavior with 4-OHEN (i.e., MH+ at 281 mass units instead of 283 mass units, ref 10). There are reports in the literature of the electrospray needle acting as an electrochemical cell (31), and it is possible that a similar phenomenon occurs with these highly redox-active catechol GSH conjugates. The exact position of GSH on the A or B ring of 4-OHEN is not known since these conjugates could not be stabilized sufficiently for 1H NMR analysis. Some stability is achieved by including perchloric acid in the HPLC

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Figure 4. Concentration-dependent inactivation of GST M1-1 activity by 4-hydroxyestrogens. Incubations contained GST (0.5 µg/mL), GSH (2.5 mM), CDNB (1.5 mM), and various concentrations of 4-hydroxyestrogens at 25 °C, pH 6.5: b, 4-OHEN; 0, 4-OHEQ; 9, 4-OHE. The results represent duplicate determinations ( SD.

Figure 3. HPLC analysis of GSH conjugates produced from (A) 4-OHEQ and (B) 4-OHEN. The catechols (0.5 mM) were incubated with 5.0 mM GSH, pH 7.4 phosphate buffer, 37 °C for 30 min. PCA (50 µL/mL) was added, and aliquots (100 µL) were analyzed by HPLC using the Shimadzu HPLC system. The mobile phase was changed to 0.25% PCA/0.25% acetic acid (pH 3.5). All other conditions were as described for LC/MS experiments in Materials and Methods.

samples; however, even under these conditions the conjugates only have a half-life of 2-3 h. In this investigation, we have shown that 4-OHEQ also autoxidizes to an o-quinone. Starting from the catechol, the first-order autoxidation rate was determined by monitoring the appearance of 4-OHEQ-o-quinone spectrophotometrically (λmax ) 392 nm). At 37 °C (pH 8.0) the rate constant for o-quinone formation is (8.1 ( 0.6) × 10-4 s-1, which is 230-fold slower than that for 4-OHEN [(1.9 ( 0.09) × 10-1 s-1] under similar reaction conditions. Attempts were made to compare the autoxidation rates at pH 7.4; however, the rate of oxidation of 4-OHEQ under these conditions was found to be too slow to obtain accurate kinetic rate constants by monitoring formation of the o-quinone chromophore. However, it should be noted that GSH conjugates were detected in incubations with either 4-OHEN or 4-OHEQ in buffers ranging from pH 6.5 to 8.0 (see below). The behavior of 4-OHEQ was found to be more complex than that of 4-OHEN as GSH conjugates resulting from 4-OHEN were detected in addition to the 4-OHEQ GSH adducts (Figure 3A). A 4-OHEQ di-GSH conjugate was observed at 46 min as well as two mono-GSH conjugates at retention times of 63 and 65 min. The 4-OHEN conjugates formed from 4-OHEQ have identical retention times (Figure 3A, 39 min for 4-OHEN-DiSG, 54 min for 4-OHEN-SG) and mass spectra as those obtained from incubations with 4-OHEN (Figure 3B). The amount of 4-OHEN GSH conjugates produced from 4-OHEQ was 21-fold less than

Figure 5. Inactivation of human GST M1-1 activity by 4-hydroxyestrogens. Incubations contained GST (0.5 µg/mL), GSH (2.5 mM), CDNB (1.5 mM), and 0.1 mM 4-hydroxyestrogens at 25 °C, pH 6.5. The concentration of tyrosinase was 50 µg/mL. The results represent triplicate determinations ( SD. *Represents values significantly different from no tyrosinase, p < 0.05.

those obtained from 4-OHEN directly, and the total amount of GSH conjugates was also reduced (12-fold). As discussed above for 4-OHEN GSH adducts, the conjugates formed from 4-OHEQ are also very unstable, and they could not be isolated for NMR analysis. Finally, we have determined that GSH conjugates can be produced from 4-OHEQ under the GST assay conditions (i.e., pH 6.5, data not shown), although at the lower pH, the yield of GSH conjugates was 2.7- and 1.8-fold reduced for 4-OHEQ and 4-OHEN, respectively. Inhibition of GSTs by Catechol Estrogens. It was of interest to determine whether the Premarin catechol estrogens could inhibit GSTs since we have shown that they readily oxidize to o-quinones and form GSH conjugates. Either the o-quinones or their GSH conjugates have the potential to inhibit GSTs through mechanisms involving alkylation of the enzyme, oxidation of the enzyme, and/or competitive binding of the GSH conjugate

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Figure 6. Effect of scavengers of reactive oxygen species on the inactivation of GST activity by 4-hydroxyestrogens. Incubations contained GST (0.5 µg/mL), GSH (2.5 mM), CDNB (1.5 mM), and 4-hydroxyestrogens (0.1 mM) at 25 °C, pH 6.5. For experiments with 4-OHE, 50 µg/mL tyrosinase was included. The concentrations of scavengers were as follows: SOD (210 units/mL), catalase (880 units/mL), mannitol (10 mM), sodium benzoate (10 mM), bathocuprone (200 µM). The results represent the average ( SD of duplicate determinations. *Represents values significantly different from no additions, p < 0.05.

in the active site of the enzyme. The inhibition of GST M1-1 is shown in Figure 4. Both 4-OHEQ and 4-OHEN caused concentration-dependent inhibition of GST activity, although 4-OHEN was considerably more effective than 4-OHEQ. The difference in potency between these catechol estrogens is likely due to lower concentrations of 4-OHEN- and 4-OHEQ-o-quinones formed from 4-OHEQ under the assay conditions (see above). In contrast, 4-OHE (Figure 4) or 2-OHE (data not shown) was without effect under these conditions. If an oxidative enzyme (tyrosinase) was included in the incubation (Figure 5), 4-OHE and 2-OHE did inhibit GST M1-1 presumably through formation of o-quinones. Similar studies with 4-OHEN and tyrosinase did not increase the inhibitory effect since oxidative enzymes are not required to form the 4-OHEN-o-quinone (10). In contrast, tyrosinase increased the inhibitor potency of all of the catechol estrogens studied to give an inhibitory effect equal to that of 4-OHEN. Since reactive oxygen species have been shown to be involved in the cytotoxic as well as genotoxic effect of catechol estrogens (32, 33), we investigated if the inhibitory effect of 4-hydroxyestrogens on GST activity could be modulated with scavengers of reactive oxygen species (Figure 6). The data show that superoxide dismutase, mannitol or sodium benzoate, or bathocuprone had no significant effect on catechol estrogen inhibitory activity toward GST which suggests that superoxide, free hydroxyl radical, and copper ion, respectively, do not play a significant role in the inhibitory process. In contrast, catalase protected GST from inhibition by catechol estrogens which may indicate a role for hydrogen peroxide in the mechanism of inhibition of the enzyme. Since 4-OHEQ- or 4-OHEN-o-quinones or their oquinone GSH conjugates could cause irreversible inactivation of GST, through either oxidation and/or alkylation of the enzyme, we examined whether treating GST with these catechol estrogens followed by gel filtration restored enzyme activity. Before gel filtration 79% of the 4-OHEN-treated enzyme activity was inhibited and 72%

Figure 7. Kinetics of GST inhibition by 4-OHEN. (A) GST (50 µg/mL) was incubated with 0.3 mM (O), 0.75 mM (b), 1.25 mM (0), or 1.5 mM (9) 4-OHEN in pH 6.5 phosphate buffer at 25 °C. Aliquots were removed at various times, diluted 100-fold, and combined with GSH (2.5 mM) and CDNB (1.5 mM), and GST activity was determined as described in Materials and Methods. The ln of the ratio of the remaining activity (E) and the initial activity (Eo) was plotted against the time of incubation. The slopes of the lines represent the apparent rate constants of inhibition (kapp) determined from linear regression analysis. Values represent the means ( SD of duplicate determinations. (B) Kitz-Wilson double-reciprocal plot of GST inhibition by 4-OHEN. Double-reciprocal plot of kapp (A) versus 4-OHEN concentration. The rate constant for irreversible inhibition (k2) was obtained from the y-intercept, and the dissociation constant (Ki) was obtained from the slope of the line multiplied by k2 as described in eq 2 (Materials and Methods).

inhibition of GST was observed with 4-OHEQ; after gel filtration similar inhibitory activity was detected (85% for 4-OHEN and 76% for 4-OHEQ). These data show that both 4-OHEN and 4-OHEQ are irreversible inactivators of GST. Kinetics of Enzyme Inactivation. To study the kinetics of GST inhibition by 4-OHEN, the enzyme was incubated with 0.3, 0.5, 0.75, 1.25, and 1.5 mM 4-OHEN in assay buffer at 25 °C. Aliquots were removed, diluted 100-fold, and assayed for enzyme activity at various time points. The time course of inhibition showed first-order kinetics. The natural logarithm of the ratio of the remaining activity (E) and the initial activity (Eo) was plotted as a function of incubation time giving linear relationships as shown in Figure 7A. The slope of the lines represents the apparent rate constant (kapp) for inhibition by the corresponding 4-OHEN concentration. The Kitz-Wilson plot (Figure 7B, 1/kapp versus 1/[I]) gave a linear relationship which was used to calculate the kinetic parameters. The constant for the conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2) was (7.3 ( 0.9) × 10-3 s-1, and the dissociation constant for the reversible complex (Ki) was 620 ( 80 µM. Similar kinetic studies were attempted with 4-OHEQ; however, unlike 4-OHEN firstorder kinetic behavior was not observed likely due to the formation of numerous inhibitory species (i.e., 4-OHEQ

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Figure 8. Proposed model for inhibition of GST using 4-OHEN as an example. [H] refers to any reducing agent.

and 4-OHEN quinoids). These data suggest that both 4-OHEN and 4-OHEQ are irrevesible inactivators likely acting through oxidation to o-quinones and covalent modification and/or oxidation of critical amino acid residues on GST. In addition, hydrogen peroxide generated through redox cycling of the o-quinone and/or semiquinone radical and GSH could cause oxidative damage to GST.

Discussion In the present investigation, we have synthesized the major catechol metabolites of equilenin and equilin and examined how changing unsaturation in the B ring affects the formation of o-quinone GSH conjugates and the ability of the o-quinones and/or GSH conjugates to inhibit GST. Interestingly, both 4-OHEN and 4-OHEQ autoxidized to o-quinones without oxidative enzyme catalysis. 4-OHEN-o-quinone reacts with GSH to give two mono-GSH conjugates and one diconjugate. The behavior of 4-OHEQ was found to be more complex than that of 4-OHEN as conjugates resulting from 4-OHEN were detected in addition to the 4-OHEQ GSH conjugates. As a result, it is quite possible that 4-OHEN quinoids and/or 4-OHEN GSH conjugates formed from 4-OHEQ are responsible for some of the abberant biological effects of 4-OHEQ in addition to contributions from 4-OHEQ quinoids and GSH conjugates. It should be noted that equilenin or 17-dihydroequilenin is the major urinary (34) and biliary (35) metabolites of equilin, and it is quite reasonable that the B ring of 4-OHEQ could aromatize to 4-OHEN under physiological conditions. Since 4-OHEN and 4-OHEQ autoxidized to electrophilic/redox-active quinoids, we examined whether these

catechol metabolites of the Premarin estrogens could inhibit the phase II enzyme GST. Both 4-OHEN and 4-OHEQ were found to be potent inhibitors of the human GST isoform M1-1, although 4-OHEQ was less potent than 4-OHEN. The inhibition occurs in the absence of oxidative enzymes since both of these catechols only require molecular oxygen to form semiquinone radicals and o-quinones. This is in direct contrast to the endogenous catechol estrogens, 4-OHE and 2-OHE, which do not autoxidize. As a result, they do not inhibit GST unless an oxidative enzyme (tyrosinase) is present to catalyze quinoid formation (Figures 4, 5). The experiments with scavengers of reactive oxygen species (Figure 6) suggest that redox reactions play a role in inhibition of GSTs. Although no effect was observed with scavengers of free hydroxyl radical, superoxide, or copper chelators, catalase which scavenges hydrogen peroxide did protect GSTs from catechol estrogen-mediated inhibition. These data suggest that hydrogen peroxide, produced from redox cycling between the semiquinone radical and the quinone and/or the corresponding GSH conjugates, is directly or indirectly involved in the inhibition mechanism. Inhibition kinetics was studied according to Kitz and Wilson (28), since GST activity could not be restored after gel filtration, inhibition of GST was not reversible by dilution (100-fold), and the inhibition progressed exponentially with time (Figure 7). Analysis of the KitzWilson plot gave a dissociation constant of the reversible enzyme-inhibitor complex (Ki ) 620 µM) and a rate constant of conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2 ) 7.3 × 10-3 s-1). These data suggest that 4-OHEN is an

764 Chem. Res. Toxicol., Vol. 11, No. 7, 1998

irreversible inactivator with relatively low affinity for GST; however, once formed the 4-OHEN enzyme complex is rapidly converted to the irreversibly inhibited enzyme. A scheme summarizing these results is shown in Figure 8. o-Quinones and/or o-quinone GSH conjugates may be involved in two types of irreversible interactions with cellular enzymes. They could cause oxidation of cysteine residues leading to disulfide bond formation and disruption of the protein tertiary structure. Alternatively, the quinoids could alkylate critical amino acid residues leading to inactivation of the enzyme. At present the relative contribution of each reactive species to the inhibition mechanism is not known; however, it has been shown with chlorinated benzoquinones that the corresponding GSH conjugates are much more potent irreversible inactivators of the enzyme than the unsubstituted quinones (15, 16). This is likely the result of an increase in redox activity with GSH substitution (36, 37) leading to an increase in production of reactive oxygen species and a corresponding increase in inhibition potency. Also, several GSH conjugates can act as competitive inhibitors of GST, a fact which has been exploited in the design of chemotherapeutic agents to overcome multidrug resistance (12). As GSH conjugates are readily formed from both 4-OHEN and 4-OHEQ under physiological conditions, it seems likely that the conjugates also contribute to GST inhibition. Finally, cellular reducing agents [i.e., NAD(P)H, P450 reductase, quinone reductase, etc.] could act to promote redox cycling by reducing the o-quinone and semiquinone radical back to the catechol which then autoxidizes generating superoxide and hydrogen peroxide, contributing to GST inhibition. In summary, on the basis of these data, it may be concluded that the involvement of quinoids in catechol estrogen-mediated inhibition of GST depends on a combination of the rate of formation of the o-quinone, the rate of disappearance of the o-quinone, and the electrophilic/redox reactivity of the quinoids. The formation of reactive oxygen species especially hydrogen peroxide likely plays a role for the highly redox-active estrogens, 4-OHEN and 4-OHEQ. In addition, we have shown that 4-OHEQ is readily converted to 4-OHEN quinoid GSH conjugates under physiological conditions which suggests that GST inhibition could be mediated by either quinoid and/or GSH conjugate. For the endogenous catechol estrogens, oxidative enzymes are necessary to form the quinoids prior to any inhibitory effects. All of these critical factors are closely linked to structure as well as microenvironment, and the details are just beginning to be elucidated. Given the direct link between excessive exposure to estrogens, metabolism of estrogens, and increased risk of hormone-dependent cancer, it is crucial that factors which affect the formation, reactivity, and cellular targets of estrogen quinoids be explored.

Acknowledgment. This research was supported by NIH Grant CA73638-01.

Chang et al.

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