The Major Metabolite of Equilin, 4-Hydroxyequilin ... - ACS Publications

Equine estrogens constitute approximately 50% of the estrogens in the widely prescribed estrogen replacement therapy marketed under the name of Premar...
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Chem. Res. Toxicol. 1999, 12, 204-213

The Major Metabolite of Equilin, 4-Hydroxyequilin, Autoxidizes to an o-Quinone Which Isomerizes to the Potent Cytotoxin 4-Hydroxyequilenin-o-quinone Fagen Zhang, Yumei Chen, Emily Pisha, Li Shen, Yansan Xiong, 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 September 15, 1998

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 a slight increased cancer risk. Previously, we showed that equilenin, a minor component of Premarin (Wyeth-Ayerst), was 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. (1998) Chem. Res. Toxicol. 11, 1113-1127]. In this study, we have compared the chemistry of the major catechol metabolite of equilin (4-hydroxyequilin), which is found in several estrogen replacement formulations, to the equilenin catechol (4-hydroxyequilenin). Unlike endogenous catechol estrogens, both equilin and equilenin were primarily converted by rat liver microsomes to 4-hydroxylated rather than 2-hydroxylated o-quinone GSH conjugates. With equilin, a small amount of 2-hydroxyequilin GSH quinoids were detected (4-hydroxyequilin:2-hydroxyequilin ratio of 6:1); however, no peaks corresponding to 2-hydroxyequilenin were observed in incubations with equilenin. These data suggest that unsaturation in the B ring alters the regiochemistry of P450-catalyzed hydroxylation from primarily 2-hydroxylation for endogenous estrogens to 4-hydroxylation for equine estrogens. 4-Hydroxyequilenin-o-quinone reacts with GSH to give two mono-GSH conjugates and one di-adduct. The behavior of 4-hydroxyequilin was found to be more complex than 4-hydroxyequilenin as conjugates resulting from 4-hydroxyequilenin were detected in addition to the 4-hydroxyequilin-GSH adducts. The mechanism of decomposition of 4-hydroxyequilin likely involves isomerization to a quinone methide which readily aromatizes to 4-hydroxyequilenin followed by autoxidation to 4-hydroxyequilenin-o-quinone. Similar results were obtained with 2-hydroxyequilin, although, in contrast to 4-hydroxyequilenin, 2-hydroxyequilenin does not autoxidize and the reaction stops at the catechol. Since 4-hydroxyequilin is converted to 4-hydroxyequilenin and 4-hydroxyequilenin-o-quinone, similar effects were observed for this equine catechol, including consumption of NAD(P)H likely by the 4-hydroxyequilenin-o-quinone, depletion of molecular oxygen by 4-hydroxyequilenin or its semiquinone radical, and alkylation of deoxynucleosides and DNA by 4-hydroxyequilenin quinoids. Finally, preliminary studies conducted with the human breast tumor cell line MCF-7 demonstrated that the cytotoxic effects of the catechol estrogens from estrone, equilin, and 2-hydroxyequilenin were similar, whereas 4-hydroxyequilenin was a much more potent cytotoxin (∼30-fold). These results suggest that the catechol metabolites of equine estrogens have the ability to cause alkylation/redox damage in vivo primarily through formation of 4-hydroxyequilenin quinoids.

Introduction Equine estrogens constitute approximately 50% of the estrogens in the widely prescribed estrogen replacement therapy marketed under the name of Premarin (WyethAyrest). The most abundant of the equine estrogens in this formulation is equilin which differs from endogenous human estrogens in that it contains a 7,8-double bond in the B ring. Equilin is also present as a minor component in other estrogen replacement formulations, including Estratab (Solvay Pharmaceuticals) and Menest * To whom correspondence should be addressed: Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612-7231. Fax: (312) 996-7107. E-mail: [email protected].

(SmithKline Beecham). Although estrogen replacement therapy is considered a safe and effective pharmaceutical, with a wide variety of beneficial effects (1), a slight increase in breast and endometrial cancers has been reported especially in long-term, high-dose studies (2-4). The exact mechanism(s) by which estrogens cause these hormone-dependent cancers is unknown. One pathway might involve metabolism of estrogens to catechols which are then oxidized to redox active/electrophilic o-quinones and/or electrophilic quinone methides. It is well established that the endogenous estrogens, estrone and 17β-estradiol, are metabolized by cytochrome P450 isozymes to 2- and 4-hydroxylated catechols (510). The o-quinones formed from peroxidase/P450catalyzed oxidation of these catechols have previously

10.1021/tx980217v CCC: $18.00 © 1999 American Chemical Society Published on Web 01/09/1999

Quinoids from Equine Estrogens

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 205

Scheme 1. Metabolism of Equilenin to 4-Hydroxyequilenin-o-quinone-GSH Conjugates

Scheme 2. Metabolism of Equilin to 4-Hydroxyequilin- and 2-Hydroxyequilin-o-quinone-GSH Conjugates

been implicated as the ultimate carcinogens. Redox cycling between the catechols and their o-quinones generates reactive hydroxyl radicals which causes oxidation of the purine/pyrimidine residues of DNA (7). In addition, o-quinones are Michael acceptors that could alkylate DNA to form adducts that have been detected both in vitro (11-15) and in vivo (16-19). There have been few studies on the potential for equine estrogens to be metabolized to reactive intermediates (20-23). It has been demonstrated that an increasing level of unsaturation in the B ring leads to a change in product distribution from primarily 2-hydroxylation as observed for endogenous estrogens to exclusively 4-hydroxylation for equilenin (Scheme 1). This is of some concern since only 4-hydroxylated estrogens have been found to be carcinogenic in the hamster kidney, which is the established animal model for hormonal carcinogen-

esis (24). In addition, we have shown that 4-hydroxyequilenin autoxidizes to quinoids which can consume reducing equivalents and molecular oxygen (25). These quinoids are potent inactivators of the phase II enzyme, glutathione S-transferase (26), and can cause a variety of damage to DNA, including formation of bulky stable adducts and apurinic sites (10, 27, 28) and oxidation of the phosphate-sugar backbone and purine/pyrimidine bases (29). All of these deleterious effects could contribute to the cytotoxic/genotoxic effects of equilenin in vivo. In this study, we have examined the metabolism of equilenin (Scheme 1) and equilin (Scheme 2) in rat liver microsomes. The data show that both were primarily metabolized to 4-hydroxylated o-quinone GSH conjugates. A small amount of GSH conjugates corresponding to reaction with the 2-hydroxyequilin-o-quinone1 were detected with equilin, although no 2-hydroxyequilenin-

206 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

o-quinone GSH conjugates were observed in incubations with equilenin. In a manner similar to that of 4-hydroxyequilenin, 4-hydroxyequilin also autoxidizes to an o-quinone, which isomerizes to 4-hydroxyequilenin quinoids through a mechanism likely involving formation of unstable equilin quinone methides. As a result, comparable cytotoxic effects were observed with 4-hydroxyequilin, including consumption of reducing equivalents and molecular oxygen and formation of DNA adducts.

Materials and Methods Caution: The catechol estrogen o-quinones were handled in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens (30). All chemicals were purchased from Aldrich (Milwaukee, WI), Fisher Scientific (Itasca, IL), or Sigma (St. Louis, MO) unless stated otherwise. [3H]GSH ([2-3H]glycine) was obtained from Dupont (Boston, MA) and diluted to a specific activity of 20 mCi/mmol. 4-Hydroxyequilenin was synthesized by treating equilin with Fremy’s salt as described previously (31) with minor modifications (25). 4-Hydroxyequilin was synthesized by demethylation of 4-methoxyequilin as described previously (26). Similarly, 2-hydroxyequilin and 2-hydroxyequilenin were synthesized by demethylation of 2-methoxyequilin followed by aromatization of the B ring in the case of 2-hydroxyequilenin (32). The GSH adducts of both 4-hydroxyequilenin and 4-hydroxyequilin-o-quinones were previously synthesized and characterized (26). GSH Conjugates of 2-Hydroxyequilin-o-quinone. The o-quinone GSH conjugates of 2-hydroxyequilin were prepared by incubating the catechol (0.5 mM) with 5.0 mM GSH and tyrosinase (0.4 µg/mL) in 2 mL of 50 mM sodium phosphate buffer (pH 7.4) at 37 °C for 30 min. The adducts were isolated from the aqueous phase using solid phase extraction cartridges (Oasis; Waters Corp., Milford, MA) and eluted with methanol. The methanol was concentrated to 150 µL, and aliquots (25 µL) were analyzed directly by HPLC with a 4.6 mm × 150 mm Ultrasphere C-18 column (Beckman) on a Hewlett-Packard (Palo Alto, CA) 1090L gradient HPLC system 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% CH3OH in 0.5% ammonium acetate (pH 3.5) at a flow rate of 1.0 mL/min for 5 min, which was increased to 40% CH3OH over the course of 45 min, isocratic for 5 min, and increased to 90% CH3OH over the last 20 min. 2-Hydroxyequilin-diSG: UV (CH3OH/H2O) 280, 330 nm; positive ion electrospray MS m/z 895 (100% relative intensity) [M + H]+, with a retention time of 50 min. 2-Hydroxyequilin-SG 1: UV (CH3OH) 270, 318 nm; positive ion electrospray MS m/z 589 (100% relative intensity) [M + H]+, with a retention time of 69 min. 2-Hydroxyequilin-SG 2: UV (CH3OH) 227, 290 nm; positive ion electrospray MS m/z 589 (100% relative intensity) [M + H]+, with a retention time of 70 min. Similar experiments with 2-hydroxyequilenin did not show any GSH conjugates using a variety of oxidation conditions. Kinetic Studies. The rate of formation or disappearance of the conjugates was determined by monitoring product formation and disappearance with HPLC. 4-Hydroxyequilin (0.5 mM) was oxidized to the o-quinone with tyrosinase (0.1 mg) in 5 mL of potassium phosphate buffer (pH 7.4, 37 °C). Aliquots were 1 Abbreviations: 4-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8estrapentaen-17-one; 2-hydroxyequilenin, 2,3-dihydroxy-1,3,5(10),6,8estrapentaen-17-one; 4-hydroxyequilin, 3,4-dihydroxy-1,3,5(10),7(8)estratetraen-17-one; 2-hydroxyequilin, 2,3-dihydroxy-1,3,5(10),7(8)estratetraen-17-one; 4-hydroxyestrone, 3,4-dihydroxy-1,3,5(10)-estratrien17-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-estratetraen3-ol-17-one; dG, 2′-deoxyguanosine; dA, 2′-deoxyadenosine; dC, 2′deoxycytidine; DT-diaphorase, NAD(P)H-quinone oxidoreductase; LC/ MS, liquid chromatography/mass spectrometry; electrospray MS, electrospray mass spectrometry.

Zhang et al. removed at various times (250 µL) and the reactions quenched with perchloric acid (50 µL/mL) and GSH (5.0 mM). Aliquots of the supernatant (100 µL) were analyzed directly by HPLC with a 4.6 mm × 150 mm Ultrasphere C-18 column (Beckman) on a Shimadzu LC-10A gradient HPLC system equipped with an SIL-10A auto injector, an SPD-M10AV UV/vis photodiode array detector, and an SPD-10AV UV detector set at 280 nm. The mobile phase consisted of 5% methanol in 0.25% perchloric acid/ 0.25% acetic acid (pH 3.5) at a flow rate of 1.0 mL/min for 5 min, which was increased to 40% CH3OH over the course of 45 min, isocratic for 5 min, and increased to 90% CH3OH over the last 20 min. The rate of conversion of 2-hydroxyequilin to 2-hydroxyequilenin was monitored spectrophotometrically at 342 nm, pH 8.5, and 37 °C. After 40 min, the pH was adjusted to 4 and the solution was extracted with CHCl3 (2 mL). The organic layer was concentrated and analyzed by positive ion CI-MS (methane). The MH+ ion of 2-hydroxyequilin (MH+ at m/z 285) was reduced 70% compared to the starting material, and the MH+ ion of 2-hydroxyequilenin (MH+ at m/z 283) was the base peak in the spectrum. Incubations. Female Sprague-Dawley rats (180-200 g) were obtained from Sasco Inc. (Omaha, NE). The rats were pretreated with dexamethasone to induce P450 3A4 (33). Dexamethasone was administered by ip injections of 100 mg/kg in corn oil daily for 4 days, and the animals were sacrificed on day 5. Microsomes were prepared from rat liver, and protein and P450 concentrations were determined as described previously (34). Incubations containing microsomal protein were conducted 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 dimethyl sulfoxide, and [3H]GSH (specific activity of 20 mCi/mmol) was added in phosphate buffer, to achieve final concentrations of 0.5 and 1.0 mM, respectively. A NADPH-generating system consisting of 1.0 mM NADP+, 5 mM isocitric acid, and 0.2 unit/ mL isocitric dehydrogenase was used together with 5.0 mM MgCl2. For control incubations, NADP+ was omitted. The reactions were initiated by the addition of NADP+ and terminated by chilling in an ice bath followed by the addition of perchloric acid (25 µL). Adduct Quantification. The reaction mixtures were centrifuged at 13 000 rpm for 6 min to remove precipitated microsomal protein. Aliquots of the supernatant (100 µL) were analyzed directly by HPLC as described above for the GSH conjugates. For quantification of GSH conjugates, 0.3 mL aliquots of the column effluent were collected during each run, and radioactivity was measured with a Beckman model LS 5801 liquid scintillation counter. Concentrations of the GSH conjugates were calculated by summing the radioactivity associated with each peak and converting the data to nanomolar amounts using the specific activity of the [3H]GSH. Each analysis was performed immediately after the incubation to limit degradation of the o-quinone-GSH adducts. Oxidation of NAD(P)H. Reduction of o-quinones was monitored by UV/visible spectrophotometry using a Hewlett-Packard HP8452 diode array spectrophotometer. 4-Hydroxyequilin (30 µM) was added to 50 mM potassium phosphate buffer (pH 7.4) containing 0.7 mg/mL bovine serum albumin and the mixture allowed to autoxidize to the o-quinone. NADH (0.2 mM) was added, and the oxidation of NADH was monitored at 340 nm with and without DT-diaphorase (1.67 µg/mL) at 25 °C. Concentrations of NADH were determined on the basis of literature molar absorptivities (35). Reactions were carried out in the presence and absence of dicumarol (20 µM). Reductions of o-quinones with purified P450 reductase were assessed in a similar fashion by monitoring NADPH oxidation spectrophotometrically at pH 7.4 and 25 °C. With rat liver microsomes, incubations containing microsomal protein were conducted at 37 °C in 50 mM phosphate buffer (pH 7.4, 1 mL total volume; 36). 4-Hydroxyequilin was also added in DMSO (1 µL of a 100 mM solution) and the mixture allowed to autoxidize to the o-quinone prior to addition of microsomes and

Quinoids from Equine Estrogens NADPH. For control incubations, heat-inactivated microsomes were used. The reactions were initiated by the addition of NADPH, and the rate of NADPH oxidation was monitored at 340 nm. Oxygen Uptake. Oxygen uptake was measured in 600 µL reaction mixtures using a Clark-type polarographic micro oxygen probe (Yellow Springs Instrument Co., Yellow Springs, OH) at 25 or 37 °C. Air-saturated potassium phosphate buffer (50 mM, pH 7.4) was used to represent 100% oxygen. Substrates were introduced as described above. Oxygen uptake was monitored over the course of 20 min and converted to micromoles of oxygen per minute. Reaction of 4-Hydroxyequilin with DNA. Calf thymus DNA (Sigma) (10 mg) was dissolved in 5 mL of 50 mM phosphate buffer containing 1 mM EDTA and the mixture repeatedly precipitated with 70% ethanol/water to remove all impurities. A solution of DNA (2 mg/mL) was incubated with 4-hydroxyequilin (2.0 mM) and ampicillin (100 µg/mL) for 4 h at 37 °C in phosphate buffer (pH 7.4). At the conclusion of the incubation, the DNA was precipitated with 70% ethanol/water and washed with three 5 mL portions of 70% ethanol. The ethanol washings were pooled; the solvent was removed in vacuo and the residue redissolved in 1.0 mL of methanol for LC/MS analysis of alkylated deoxynucleosides. The precipitated DNA was hydrolyzed to deoxynucleosides using the following literature procedures with minor modifications (28, 37, 38). Briefly, the DNA was redissolved in 1 mL of water, and the DNA was denatured in boiling water for 2 min. The solution was cooled in an ice bath and diluted with 4 mL of 30 mM sodium acetate (pH 5.3) and 20 mM zinc sulfate (500 µL) and incubated with nuclease P1 (80 units/mg DNA) and alkaline phosphatase (60 units/mg DNA) for 3 h at 37 °C. The pH was adjusted to 8.5 using 0.5 M Tris buffer and the solution incubated for an additional 3 h at 37 °C. Adducts were isolated as described above and analyzed by electrospray LC/MS. Cytotoxicity Studies in MCF-7 Cells. Assays were performed by procedures developed by the National Cancer Institute (39) as described previously (40, 41). Briefly, MCF-7 cells were maintained in MEME supplemented with 1% penicillin, streptomycin, fungizome, 10 mg/L insulin, 1% nonessential amino acids, and 10% fetal bovine serum. The medium was changed 24 h before beginning cytotoxicity assays to maintain logarithmic growth. The cells were harvested by trypsinization, counted, diluted in media to 3-5 × 104 cells/mL, and added to 96-well plates containing the test compound dissolved in DMSO (0.5% final concentration). The test samples were assayed in triplicate, and final concentrations ranged from 1.6 to 100 µM. For each catechol estrogen, attempts were made to treat the cells with doses that were 2.5-6.25-fold above and below the estimated ED50 value. Each assay included negative controls (cells treated with DMSO only) that were used to define 100% cell viability. The plates were incubated for 3 days. Following incubation, the cells were fixed with trichloroacetic acid and stained with 0.4% sulforhodamine B in 1% acetic acid. The bound dye was liberated with 0.1 M Tris base, and the absorbance at 515 nm was measured with a microtiter plate reader. The ED50 values were obtained by regression and linear estimation analysis. The data represent the mean ( SD of triplicate determinations. Instrumentation. HPLC experiments were performed on the above-mentioned Shimadzu HPLC system. Peaks were integrated with Shimadzu EZ-Chrom software and a 486-33 computer. UV spectra were measured with a Hewlett-Packard model 8452 diode array UV/vis spectrophotometer. 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

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 207 Table 1. Conversion of Equine Estrogens to o-Quinone-Derived GSH Conjugates by Rat Liver Microsomesa

conjugate

retention time (min)

4-hydroxyequilenin-diSG 4-hydroxyequilin-diSG 2-hydroxyequilin-diSG 4-hydroxyequilenin-SG 4-hydroxyequilenin-SG 4-hydroxyequilin-SG 4-hydroxyequilin-SG 2-hydroxyequilin-SG

40 47 53 58 60 64 66 69

equileninb 4-hydroxyequilenin-diSG 4-hydroxyequilenin-SG 4-hydroxyequilenin-SG

40 58 60

substrate equilin

rate of formation (nmol/nmol of P450 per 10 min) 0.10 ( 0.01 0.08 ( 0.01 0.024 ( 0.001 0.03 ( 0.01 0.016 ( 0.004 0.05 ( 0.01 0.13 ( 0.02 0.04 ( 0.01 total of 0.47 0.45 ( 0.05 0.47 ( 0.04 0.09 ( 0.04 total of 1.01

a Incubations were conducted for 30 min with 0.5 mM substrate and rat liver microsomes (1.0 nmol of P450/mL) in the presence of an NADPH-generating system and 1.0 mM [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 samples without NADP+ have been subtracted from each peak. b Incubation time was 10 min.

for nebulization of the HPLC effluent, and nitrogen bath gas at 250 °C and a flow rate of 10 L/min was used for evaporation of solvent from the electrospray. The range of m/z 200-900 was scanned every 2 s during LC/MS.

Results Oxidation of Equine Estrogens by Cytochrome P450. Microsomal incubations with equilenin or equilin showed the formation of o-quinone-trapped GSH adducts (Table 1). For equilenin, only products corresponding to 4-hydroxylated metabolites were detected during HPLC analysis of the incubation mixture, confirming previous studies (20, 23) which showed that aromatization of the B ring causes a change in metabolism from primarily 2-hydroxylation for estrone and 17β-estradiol to exclusively 4-hydroxylation for equilenin. It has been proposed that the lack of 2-hydroxylase activity for equilenin in microsomes has more to do with unsaturation in the B ring of the estrogen than the regiospecificity of the P450 isozyme responsible for aromatic hydroxylation (23). The first step in the P450-catalyzed oxidation mechanism likely involves abstraction of the hydrogen atom from the phenolic OH group, generating a phenoxy radical. Carboncentered radical resonance structures can be drawn which put the radical on the C2 or C4 position for subsequent hydroxylation by P450; however, the C4 radical resonance structure could be expected to have much more radical character than the C2 structure since this resonance structure is not stabilized by an aromatic B ring (23). Similar experiments with equilin did show some 2-hydroxylated products, although 4-hydroxyequilin-o-quinone-GSH conjugates predominated by a ratio of 6:1 (Table 1). In addition, 4-hydroxyequilenin-o-quinone-GSH conjugates were detected which presumably result from isomerization of 4-hydroxyequilin-o-quinone (see below). These data differ from what has been reported for incubations of equilin in baboon liver microsomes where the catechols were quantified as their methyl ether

208 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

Figure 1. Time course for incubation of 4-hydroxyequilin-oquinone at 37 °C and pH 7.4. Aliquots (250 µL) were combined with 5.0 mM GSH and 12.5 µL of PCA at various times. The data represent the peak area ratios of the di-GSH conjugates.

metabolites (20). In this earlier study, the ratio of 4-hydroxyequilin:2-hydroxyequilin was found to be 0.4: 1. Species differences in metabolism may account for the change in regioselectivity. Alternatively, differences in the products analyzed [i.e., catechol methyl ethers (20) vs GSH conjugates (this study)] may explain the change in product distribution. Finally, in terms of the total amount of o-quinone-GSH conjugates produced in rat liver microsomes, equilenin seems to be a slightly better substrate (2-fold, Table 1) than equilin which is analogous to what was observed in the experiments with baboon liver microsomes (20). Isomerization of 4-Hydroxyequilin-o-quinone to 4-Hydroxyequilenin-o-quinone. In the absence of a trapping agent such as GSH, we have found that 4-hydroxyequilin-o-quinone will isomerize to 4-hydroxyequilenin-o-quinone. We took advantage of the fact that GSH reacts very rapidly with o-quinones (42) and that the resulting GSH conjugates do not appear to autoxidize or isomerize under acidic conditions. Figure 1 shows the effect of incubating 4-hydroxyequilin-o-quinone under physiological conditions and adding GSH at various times. Qualitatively, the rate of formation of 4-hydroxyequilenin-diSG eluting at 40 min corresponds to the rate of disappearance of 4-hydroxyequilin-diSG (47 min retention time). Similar behavior was observed with the mono-GSH conjugates. From these studies, we determined that the half-life of the 4-hydroxyequilin-o-quinone was much shorter (12 min) than that of the 4-hydroxyequilenin-o-quinone (138 min; 25). A proposed mechanism of isomerization of 4-hydroxyequilin-o-quinone to 4-hydroxyequilenin-o-quinone is shown in Scheme 3. Initially, 4-hydroxyequilin autoxidizes to 4-hydroxyequilin-o-quinone. Because of the relatively acidic hydrogens in the 6 position, this o-quinone readily isomerizes to an unstable quinone methide. The return to aromaticity is probably the driving force which rearranges the quinone methide to 4-hydroxyequilenin which then readily autoxidizes to 4-hydroxyequilenin-o-quinone. Chemistry of 2-Hydroxylated Equine Estrogens. 2-Hydroxyequilin autoxidizes very slowly at physiological pH and temperature. Unlike the 4-hydroxylated estrogens where the o-quinone chromophore could be clearly observed, little absorbance was detected at 420 nm. Instead, a new species was observed after 4 h under physiological conditions. Figure 2 shows the change in the UV spectrum of 2-hydroxyequilin after incubation under basic conditions (pH 8.5) to enhance the rate of conversion. The new species formed at the end of the reaction had the same chromophore as 2-hydroxyequilenin which is very similar to that of equilenin (43). In

Zhang et al.

addition, extraction of the aqueous solution with CHCl3 and analysis by CI-MS revealed that the MH+ ion at m/z 283 corresponding to the protonated molecular ion of 2-hydroxyequilenin was the base peak. It is likely that 2-hydroxyequilin isomerizes to 2-hydroxyequilenin through a mechanism analogous to that shown in Scheme 3 for 4-hydroxyequilin. The rate of conversion of 2-hydroxyequilin to 2-hydroxyequilenin was determined to be (3.4 ( 0.01) × 10-4 s-1 at pH 8.5 and 37 °C which is 2.4-fold slower than the rate of formation of 4-hydroxyequilenino-quinone from 4-hydroxyequilin under the same conditions. Finally, it should be noted that 2-hydroxyequilenin does not autoxidize. After 15 h at 37 °C [0.15 mM 2-hydroxyequilenin (pH 7.4) and phosphate buffer, with scans from 200 to 600 nm every 20 min], no change was observed in the UV spectra and the characteristic yellow color attributed to o-quinones was absent. Also, even in the presence of oxidative enzymes or chemical oxidizing conditions, we have not been able to detect any GSH conjugates from reaction of the 2-hydroxyequilenin-oquinone with GSH. These data suggest that although 2-hydroxyequilenin does not appear to be formed from equilenin in rat liver microsomes, it is possible that 2-hydroxyequilenin is a metabolite of equilin in vivo. o-Quinone-Mediated Oxidation of NAD(P)H. Previously, we have shown that 4-hydroxyequilenin-oquinone can consume NAD(P)H and the rate of NAD(P)H consumption can be increased by including reductive enzymes such as P450 reductase or DT-diaphorase (25). Similarly, in this study, we incubated 4-hydroxyequilin with and without DT-diaphorase, P450 reductase, or rat liver microsomes and followed the disappearance of the NAD(P)H chromophore at 340 nm. As observed previously with 4-hydroxyestrone-o-quinone (44), naphthalene1,2-dione (45), and 4-hydroxyequilenin-o-quinone (25), 4-hydroxyequilin-o-quinone was spontaneously reduced by NAD(P)H. However, unlike 4-hydroxyestrone-o-quinone, which is not a substrate for DT-diaphorase (44), the rate of reduction for 4-hydroxyequilin-o-quinone was increased when DT-diaphorase, P450 reductase, or rat liver microsomes were included in the incubation (Figure 3A). In the presence of dicumarol, a potent inhibitor of DT-diaphorase (35), the rate enhancement for 4-hydroxyequilin-o-quinone was abolished. Compared to 4-hydroxyequilenin-o-quinone, 4-hydroxyequilin-o-quinone was 3-9fold less effective at consuming NAD(P)H which may imply that 4-hydroxyequilin-o-quinone must isomerize to 4-hydroxyequilenin-o-quinone prior to NAD(P)H consumption. Catechol- and/or Semiquinone Radical-Mediated Oxygen Consumption. In addition to NAD(P)H consumption, we also examined the relative ability of 4-hydroxyequilin and/or the 4-hydroxyequilin semiquinone radical to consume molecular oxygen using the Clarktype oxygen electrode (Figure 3B). DT-diaphorase, P450 reductase, and rat liver microsomes enhanced the rate of oxygen consumption, like what was observed with 4-hydroxyequilenin (25). With DT-diaphorase, 4-hydroxyequilenin was 6-fold more effective at oxygen consumption; however, both 4-hydroxyequilin and 4-hydroxyequilenin were equally effective with P450 reductase and rat liver microsomes. Competitive Reaction of Deoxynucleosides and DNA with 4-Hydroxyequilin. 4-Hydroxyequilin and 4-hydroxyequilenin were incubated with the four deoxynucleosides and analyzed by HPLC. The adducts pro-

Quinoids from Equine Estrogens

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 209

Scheme 3. Mechanism of Isomerization of 4-Hydroxyequilin to 4-Hydroxyequilenin-o-quinone

Figure 2. UV/vis spectral analysis of the conversion of 2-hydroxyequilin to 2-hydroxyequilenin. Incubations contained 2-hydroxyequilin (0.15 mM) in potassium phosphate buffer (50 mM, pH 8.5, 37 °C). Scans were taken every 5 min for 1 h.

duced from either catechol estrogen had the same retention time, UV spectra, and electrospray mass spectra which suggests that 4-hydroxyequilin-o-quinone does not react directly with deoxynucleosides. Instead, 4-hydroxyequilin-o-quinone isomerizes to 4-hydroxyequilenin-oquinone which then alkylates dG, dA, and dC, forming the same unusual cyclic adducts as 4-hydroxyequilenin (Scheme 4; 28). Previous work established that no reaction occurs between 4-hydroxyequilenin-o-quinone and thymidine (28), likely because of the lack of an exocyclic amino group in the latter. Although the adducts formed with 4-hydroxyequilin are the same as those produced from 4-hydroxyequilenin, the rate of adduct formation is considerably slower (Figure 4). We then incubated solutions of calf thymus DNA with 4-hydroxyequilin and isolated, washed, and hydrolyzed the DNA. As observed with 4-hydroxyequilenin (28), adenine adducts were detected in the ethanol wash prior to DNA hydrolysis since these adducts in particular are very unstable and readily depurinate. In addition, the DNA hydrolysate showed two stable adducts resulting from alkylation of dG and dC which we also detected using 4-hydroxy-

equilenin as the substrate (28). As observed with the deoxynucleoside experiments, the extent of alkylation by 4-hydroxyequilin was less than that observed with 4-hydroxyequilenin (data not shown). Cytotoxicity Experiments in MCF-7 Cells. Preliminary studies conducted with the human breast tumor cell line MCF-7 demonstrated that the cytotoxic effects of the catechol estrogens from estrone, equilin, and 2-hydroxyequilenin were similar whereas 4-hydroxyequilenin was a much more potent cytotoxin. For example, the ED50 values for the estrone catechols (10), the equilin catechols, and 2-hydroxyequilenin (data not shown) ranged from 33 to 44 µM, whereas the ED50 value for 4-hydroxyequilenin was consistently 20-30-fold lower (ED50 ) 1.4 µM; 10). The estrone catechol estrogens require oxidative enzymes to form their o-quinones, and these enzymes also dramatically enhance the rate of formation of the o-quinones from 2-hydroxyequilin and 4-hydroxyequilin. In addition, it is quite possible that quinone methide formation from 2-hydroxyestrone in particular could contribute to the cytotoxic effects of this catechol (10, 46, 47). Unstable quinone methides can also form from the equilin catechols as discussed above; however, we have found that these quinone methides readily aromatize to the corresponding equilenin catechols which could contribute to the toxic effects through formation of 4-hydroxyequilenin in particular. This catechol autoxidizes very rapidly without the need for enzymatic catalysis, and it also forms a very long-lived redox active/electrophilic o-quinone likely responsible for the damage in these cells.

Discussion Most epidemiology studies about the link between estrogen replacement therapy and increased risk for

210 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

Figure 3. Rates of (A) NAD(P)H oxidation and (B) oxygen consumption by 4-hydroxyequilenin compared with those of 4-hydroxyequilin. The 4-hydroxyequilenin data are from ref 25. Reaction mixtures contained 1.67 µg of DT-diaphorase/mL [specific activity of 320 µmol of 2,6-dichlorophenolindophenol reduced min-1 (mg of protein)-1], 0.7 mg/mL BSA, 0.2 mM NADH, 0.03 mM catechol, and phosphate buffer (pH 7.4) at 25 °C. The concentration of dicumarol was 20 µM. For P450 reductase, reaction mixtures contained 8.8 µg of P450 reductase/ mL [specific activity of 32 µmol of cytochrome c reduced min-1 (mg of protein)-1], phosphate buffer (pH 7.4), 0.03 mM catechol, and 0.2 mM NADPH at 25 °C. For microsomal incubations, reaction mixtures contained 0.4 mg/mL microsomal protein, 0.3 mM NADPH, 0.1 mM catechol, and phosphate buffer (pH 7.4) at 37 °C. The values represent averages ( SD of three determinations. The numbers on top of the black bars represent the fold increase in the rate of NAD(P)H or oxygen consumption for 4-hydroxyequilenin compared with that of 4-hydroxyequilin.

breast and endometrial cancers have been conducted using Premarin. In this study, we have shown that two of the equine estrogen components of Premarin, equilin and equilenin, can be converted by rat liver microsomes to quinoid-GSH conjugates. Equilenin is only converted to 4-hydroxylated products, whereas equilin forms both catechols. It is likely that 2-hydroxylation of equilenin is unfavorable since the oxidation mechanism should require disruption of the aromaticity of the B ring (23). With equilin, it is less clear why 4-hydroxylation would predominate over 2-hydroxylation especially since the latter is the dominant hepatic pathway for estrone and 17β-estradiol (5, 46). Previous studies have shown that in the absence of GSH the endogenous catechol estrogen o-quinones, 4-hydroxyestrone-o-quinone, and 2-hydroxyestrone-o-quinone can isomerize to electrophilic p-quinone methides (46, 47). The relative importance of these p-quinone methides to the toxicity of estrogens is not known; however, quinone methides have very different chemistry compared to quinones, and as a result, they could alkylate a variety of functional groups on cellular macromolecules, including amino, hydroxyl, and phenolic groups as well as

Zhang et al.

sulfhydryl sites which are the primary reaction site of o-quinones. We have previously shown that the relative importance of this o-quinone/p-quinone methide isomerization mechanism increases with increasing acidity of the benzyl hydrogens on the carbon which forms the “methide” functional group of the quinone methide (48, 49). For example, 4-allylcatechol (hydroxychavicol)-oquinone quantitatively isomerizes to the p-quinone methide, whereas for a structural analogue, which does not contain an allylic alkene group R to the benzylic hydrogens (4-propylcatechol), the o-quinone/p-quinone methide isomerization pathway is a very minor one at physiological pH and temperature. With 4-hydroxyequilin-oquinone, which could be regarded as a cyclic version of 4-allylcatechol, it is not surprising that it readily isomerizes to the p-quinone methide shown in Scheme 3. Unlike the endogenous estrogens, this quinone methide is very unstable because of the driving force generating an aromatic B ring, and the quinone methide rearranges, forming 4-hydroxyequilenin which autoxidizes to 4-hydroxyequilenin-o-quinone. As a result, the cytotoxic/ genotoxic effects attributed to this catechol estrogen could be due to both 4-hydroxyequilin quinoids and 4-hydroxyequilenin quinoids. 2-Hydroxyequilin also autoxidizes to an o-quinone which isomerizes and aromatizes to 2-hydroxyequilenin. 2-Hydroxyequilenin does not autoxidize which suggests that if this metabolite were formed in vivo from metabolism of equilin, it would be unlikely to make a significant contribution to the toxicity of equilin. In support of this, an analogue of 2-hydroxyequilenin-o-quinone, 2,3-dihydroxynaphthene-o-quinone, has never been synthesized to our knowledge, although it has been trapped as a Diels-Alder adduct (50). Calculations indicated that this o-quinone exhibits distinct destabilization relative to its structural isomer 1,2-dihydroxynaphthene-o-quinone (51). This instability seems to be the result of constraining the molecule in a completely quinoid structure. Calculations also show that 2,3-dihydroxynaphthene-o-quinone prefers a significantly nonplanar structure, contributing to the high energy of this o-quinone (52). These data support the conclusion that 2-hydroxyequilenin is a remarkably stable catechol and oxidation to the o-quinone is unlikely to occur except under harsh chemical reaction conditions. We have not detected any 2-hydroxyequilenin-GSH conjugates in incubations with equilenin; however, the 2-hydroxyequilenin quinoids do not appear to react with GSH, and it is possible that the products of 2-hydroxylation of equilenin were not detected. As literature studies also report a lack of 2-hydroxylase activity for equilenin in microsomes, it seems more likely that 2-hydroxyequilenin is not a metabolite of equilenin. Finally, 2-hydroxyequilenin was significantly less toxic in MCF-7 cells compared to 4-hydroxyequilenin (29-fold), supporting the conclusion that even if 2-hydroxyequilenin was formed from equilenin, it would not significantly contribute to the toxicity of equilenin. Since 4-hydroxyequilin is readily converted to 4-hydroxyequilenin, it is not surprising that similar qualitative effects are detected in vitro, although most of the biological effects are diminished relative to those of 4-hydroxyequilenin. For example, 4-hydroxyequilin-oquinone will consume NAD(P)H and the corresponding catechol and/or semiquinone radical can deplete molecular oxygen directly. The rate of consumption is enhanced in the presence of DT-diaphorase, P450 reductase, and

Quinoids from Equine Estrogens

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 211

Scheme 4. Adducts Formed from Reaction of 4-Hydroxyequilin with Deoxynucleosides

Figure 4. Rate of formation of 4-hydroxyequilenin deoxynucleoside adducts from 4-hydroxyequilenin compared with that of 4-hydroxyequilin. The catechol estrogens (1.5 mM) were incubated at pH 7.4 and 37 °C with 5.0 mM dG, dA, dC, and thymidine. At various time points, aliquots (60 µL) were analyzed by HPLC using a 4.6 mm × 150 mm Ultrasphere C-18 column (Beckman) with a flow rate of 1.0 mL/min. The mobile phase consisted of 5% methanol in 0.25% perchloric acid/0.25% acetic acid (pH 3.5) for 2 min, which was increased to 27% methanol over the course of 18 min, isocratic for 25 min, and increased to 60% methanol over the course of 15 min. The data represent the sum of the peak area ratios of the adducts.

rat liver microsomes; however, with one or two exceptions, 4-hydroxyequilin was found to be 2-6-fold less effective than 4-hydroxyequilenin. These data suggest that 4-hydroxyequilin must be converted to 4-hydroxyequilenin quinoids to cause any damage in vivo. In support of this, only adducts resulting from reaction of 4-hydroxyequilenin quinoids with deoxynucleosides or DNA were detected in samples treated with 4-hydroxyequilin. In summary, data have been presented about the metabolism of equilin and equilenin to o-quinone-GSH conjugates in rat liver microsomes. Equilenin undergoes

exclusively 4-hydroxylation, whereas trace amounts of products corresponding to 2-hydroxylated quinoids were detected with equilin. Both 4-hydroxyequilin and 2-hydroxyequilin autoxidize to o-quinones which in the absence of GSH readily isomerize to unstable quinone methides and then aromatize to 4-hydroxyequilenin and 2-hydroxyequilenin, respectively. Unlike 4-hydroxyequilenin, which readily autoxidizes to an o-quinone, 2-hydroxyequilenin does not autoxidize and is unlikely to contribute to the toxic effects of equilin. Since 4-hydroxyequilin is readily converted to 4-hydroxyequilenin, similar deleterious effects were observed with this equine catechol estrogen, including consumption of reducing equivalents and molecular oxygen as well as formation of the same DNA adducts observed with 4-hydroxyequilenin. These results suggest that the catechol metabolites of these equine estrogens have the ability to cause alkylation/ redox damage in vivo primarily through formation of 4-hydroxyequilenin quinoids.

Acknowledgment. This research was supported by NIH Grant CA73638-01. We thank Drs. David Ross and David Siegel from the University of Colorado Health Sciences Center for supplying us with DT-diaphorase and for helpful discussions.

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