Bioactivation of Estrone and Its Catechol Metabolites to Quinoid

(2-OHE) and 4-hydroxyestrone (4-OHE) as well as from estrone in rat liver ... observed from the 2-OHE-o-quinone as well as a conjugate where GSH had a...
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Chem. Res. Toxicol. 1996, 9, 492-499

Bioactivation of Estrone and Its Catechol Metabolites to Quinoid-Glutathione Conjugates in Rat Liver Microsomes Suzanne L. Iverson, Li Shen, Nilgun Anlar, 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 October 18, 1995X

Although the carcinogenic effects of estrogens have been mainly attributed to hormonal properties, there is interest in estrogens acting as chemical carcinogens by binding to cellular macromolecules. In the present study, we explored factors which influence the rate of P450catalyzed formation of the o-quinones (3,5-cyclohexadiene-1,2-diones) from 2-hydroxyestrone (2-OHE) and 4-hydroxyestrone (4-OHE) as well as from estrone in rat liver microsomes. The initially formed o-quinones were trapped as their GSH conjugates which were separated and characterized by HPLC with electrospray-MS detection. Two mono-GSH conjugates were observed from the 2-OHE-o-quinone as well as a conjugate where GSH had added twice to the molecule producing a di-GSH conjugate. 4-OHE-o-quinone gave only one mono-GSH adduct as well as a di-GSH adduct. Both 2-OHE and 4-OHE were excellent substrates for P450, generating o-quinone GSH adducts at 94 and 40 times, respectively, the rate of estrone. 2-OHE but not 4-OHE saturated P450 at unusually low concentrations (0.2 nmol of P450/mL) perhaps due to differences in the stability of the o-quinones formed in the active site of the enzyme. Preliminary data suggest that the o-quinones of both 2-OHE and 4-OHE could isomerize to quinone methides (4-alkyl-2,5-cyclohexadien-1-ones, QMs). The o-quinones of the catechol estrogens were incubated at 37 °C (pH 7.4) in the absence of GSH. Aliquots were removed at various times and combined with GSH. From the pseudo-first-order rate of disappearance of the o-quinone GSH adducts, the half-lives of the o-quinones were determined. The o-quinone from 2-OHE has a half-life of 42 ( 3 s at 37 °C (pH 7.4), and the o-quinone from 4-OHE has a half-life of 12.2 ( 0.4 min under identical conditions. The o-quinones of the AB ring analogs of the catechol estrogens (3,4-dihydroxy-5,6,7,8-tetrahydronaphthalene and 1,2-dihydroxy5,6,7,8-tetrahydronaphthalene) isomerize to QMs, suggesting that a similar reaction pathway could occur with the o-quinones from catechol estrogens. In support of this, oxidation of 4-OHE and quenching with GSH after 70 min produced 9-dehydro-4-hydroxyestrone (3-hydroxy-1,3,5(10),9(11)-estratetraen-17-one), a product which could result from either the QM hydrolysis product or the QM-glutathione conjugate, both of which could eliminate to give the conjugated alkene of 4-OHE. The implications of the o-quinone/QM pathway to the in vivo effects of catechol estrogens are not known; however, given the direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.

Introduction There is a clear association between excessive exposure to synthetic and endogenous estrogens and the development of cancer in several tissues including breast, endometrium, liver, and kidney (1, 2). Although the carcinogenic effects of estrogens have been mainly attributed to hormonal properties (1), there is interest in estrogens acting as chemical carcinogens by binding to cellular macromolecules. The endogenous steroids estrone and 17β-estradiol are hydroxylated by various cytochrome P450 (P450)1 enzymes at several positions including the 2- and 4-carbons to form the 2- and 4-catechol metabolites (Figure 1) (3, 4). The catechol * Address correspondence to this author at the Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, The University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612-7231. FAX (312) 996-7107; E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, February 1, 1996.

0893-228x/96/2709-0492$12.00/0

metabolites are oxidized to o-quinones that undergo redox cycling mediated through cytochrome P450/P450 reductase and thus could contribute to the carcinogenicity through the induction of oxidative damage to DNA (5, 6). In addition to generating reactive oxygen species, o-quinones are Michael acceptors, and covalent modification of DNA has been observed by the 32Ppostlabeling method (7, 8). The structures of the DNA adducts have not yet been determined; however, a recent 1 Abbreviations: 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; estrone, 3-hydroxy-1,2,5(10)-estratrien-17-one; 9-dehydro-4-OHE, 9-dehydro-4-hydroxyestrone, 3,4-dihydroxy-1,3,5(10),9(11)estratetraen-17-one; 2-THNC, 3,4-dihydroxy-5,6,7,8-tetrahydronaphthalene; 4-THNC, 1,2-dihydroxy-5,6,7,8-tetrahydronaphthalene; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; P450, cytochrome P450; QM, quinone methide, 4-alkyl-2,5-cyclohexadien-1-one; o-quinone, 3,5cyclohexadiene-1,2-dione; CI-MS, chemical ionization mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; electrosprayMS, electrospray mass spectrometry.

© 1996 American Chemical Society

Quinoids from Catechol Estrogens

Chem. Res. Toxicol., Vol. 9, No. 2, 1996 493

Figure 1. Bioactivation of estrone to catechol estrogens, o-quinones, and quinone methides.

Figure 2. Structures of the quinoids and GSH conjugates produced from 2-THNC.

report has shown that the o-quinone of 4-hydroxyestrone (4-OHE) modifies adenine at the C8 position on the purine ring under reductive conditions (9). Chemical or enzymatic activation of estrone or 17β-estradiol and their catechol metabolites also leads to protein alkylation (10, 11). In rat hepatocytes, 17β-estradiol produced a dosedependent depletion of cellular GSH without formation of GSSG (12). Previous work has shown that both o-quinones of the catechol estrogen metabolites readily react with GSH, suggesting GSH depletion is due to conjugate formation. GSH adds at the 1- and 4-positions in the case of the o-quinone from 2-hydroxyestrone (2OHE) giving two mono-GSH adducts in a ratio of 3.5:1 (13, 14) and only at the 2-position for the 4-OHE-oquinone (15). As both studies relied on TLC separation of the adducts, additional GSH conjugates, especially diadducts, could have been overlooked. We have previously shown that the AB ring analog of 2-OHE (2-THNC) can be oxidized by cytochrome P450 in the presence of [3H]GSH to give two o-quinone GSH conjugates, as shown in Figure 2 (structures 1 and 2) (16). In addition, the bile of male hamsters treated with 17β-estradiol contained the di-GSH adduct of 2-hydroxy-17β-estradiol as well as the above-mentioned mono-GSH adducts (17). Di-GSH adducts of both catechol estrogens have been reported in two early studies (18, 19) which may indicate that they contribute to the cytotoxic properties of the mono-GSH conjugates as reported for other polyhydroxylated glutathionyl ethers (20). Hydroxylation of estrone or estradiol at the 2-position

predominates over 4-hydroxylation in rat liver (21, 22) by about a factor of 10. Although qualitatively the metabolic pathways for estrogen metabolism are the same in humans, quantitatively aromatic hydroxylation is much more significant in male rats. Studies have shown that the particular P450 enzyme responsible for catechol formation is a constitutive enzyme found in adult male rat liver (23). The subsequent two-electron oxidation of the catechol estrogens to o-quinones is believed to be catalyzed by the peroxidase activity of P450 1A1 (24); however, the rates of formation of the o-quinones were determined spectrophotometrically, which, depending on the stability of these reactive intermediates, could give erroneous results. The present investigation concerns factors that influence the rate of P450-catalyzed formation of the oquinones from 2- and 4-OHE as well as from estrone. The initially formed o-quinones were trapped as their GSH conjugates which were separated and characterized by HPLC with electrospray-MS detection. The quantities of o-quinones formed from 2-OHE, 4-OHE, and estrone in untreated rat liver microsomes were determined as their [3H]GSH conjugates. The lifetimes of the estrogen o-quinones under physiological conditions were estimated by time-dependent trapping experiments with GSH. The results of this investigation confirm previous work which showed the 4-OHE-o-quinone is considerably more stable than the 2-OHE-o-quinone (8). Finally, the hypothesis that catechol estrogen o-quinones could isomerize to highly electrophilic quinone methides (QMs) was studied with the AB ring catechol analog of 4-OHE (4-THNC) since we had previously shown that the o-quinone from the 2-OHE model compound (2-THNC) isomerizes to the QM shown in Figure 2 (16). 4-THNC has the potential to form two QMs, as shown in Figure 3. These results provided evidence that formation of redox-active oquinones followed by isomerization to the more electrophilic QMs may be a general bioactivation pathway for catechols (16, 25), including catechol estrogens. In fact, QM formation from the o-quinone of 4-OHE was confirmed through isolation of a dehydration product. These results suggest that the o-quinone/QM isomerization pathway may contribute to the deleterious effects of catechol estrogens.

494 Chem. Res. Toxicol., Vol. 9, No. 2, 1996

Figure 3. Structures of the quinoids and GSH conjugates produced from 4-THNC.

Figure 4. HPLC analysis of adducts produced from (A) 4-OHE and (B) 2-OHE by rat liver microsomes (0.4 nmol of P450/mL) in the presence of an NADPH-generating system and 1.0 mM [3H]GSH. Radioactivity eluting from the HPLC column was measured in fractions collected at 18-s intervals. The data for the di-SG adducts have been divided by 2 to correct for the additional [3H]GSH moiety. The question marks refer to unidentified [3H]GSH adducts.

Materials and Methods Materials. All chemicals were purchased from Aldrich (Milwaukee, WI), Fisher Scientific (Itasca, IL) or Sigma (St. Louis, MO) unless stated otherwise. [3H]GSH (glycine-2-3H) was obtained from Dupont (Boston, MA) and diluted to a specific activity of 10 nCi/nmol. 2-OHE and 4-OHE were either purchased from Sigma (St. Louis, MO) or synthesized as described previously (26). 9-Dehydroestrone was obtained from Steraloids (Wilton, NH). 2,3-Dihydroxy-5,6,7,8-tetrahydronaphthalene (2THNC) and 1,2-dihydroxy-5,6,7,8-tetrahydronaphthalene (4THNC) were synthesized from 5,6,7,8-tetrahydro-2-naphthol according to literature procedures (27). The spectral data for 2-THNC have previously been reported (16). 4-THNC: 1H-NMR (CDCl3) δ 1.76 (m, 4H, 2 × CH2), 2.66 (m, 4H, 2 × benzyl CH2), 5.15 (bs, 2H, 2 × OH), 6.54 (d, J ) 8 Hz, 1H, ArH), 6.65 (d, J ) 8 Hz, 1H, ArH); UV (CH3OH) 224, 282 nm; GC-MS, CI (positive mode, methane carrier gas) m/z 165 (100) (MH+). GSH Conjugates of o-Quinones. The structures of the o-quinone GSH conjugates of 2-OHE and 4-OHE are shown in Figure 4. The GSH conjugates of 2-THNC were previously synthesized and characterized (5, 6). The adducts of 4-THNC

Iverson et al. (Figure 3) were prepared by oxidizing the catechol (5.0 mM) with silver oxide (250 mg) in 5 mL of acetonitrile and combining 205 µL of the acetonitrile solution with 5.0 mM GSH in 5 mL of 50 mM sodium phosphate buffer (pH 6.0) at 25 °C (16, 25). 2-OHE and 4-OHE were oxidized with peroxidase using a previously described procedure (28) in the presence of GSH. Briefly, a mixture of each catechol (0.5 mM), GSH (1.0 mM), peroxidase (100 µg/mL horseradish peroxidase type VI), and 0.5 mM H2O2 in 5 mL of sodium phosphate buffer (50 mM, pH 7.4) was incubated at 37 °C for 60 min. The adducts were isolated from the aqueous phase on C-18 extraction cartridges (J. T. Baker) 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 Hewlett-Packard (Palo Alto, CA) 1090L gradient HPLC equipped with an 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-THNC Conjugates: 5, 6: UV (CH3OH) 228, 276, 306 nm; positive ion electrosprayMS, m/z 470 (100) (MH+); retention time 49 and 51 min. 7: UV (CH3OH) 208, 278, 308 nm; positive ion electrospray-MS, m/z 775 (100) (MH+); retention time 33 min. 2-OHE Conjugates: 2-OHE-1-SG and 2-OHE-4-SG: UV (CH3OH) 260, 300 nm similar to the reported values of 261, 302 nm (19); positive ion electrospray-MS, m/z 592 (100) (MH+); retention time 63 and 65 min. 2-OHE-diSG: UV (CH3OH) 280, 317 nm, literature value 315 nm (18); positive ion electrospray-MS, m/z 449 (100) (M + 2H+)2+, 896 (14) (M+); retention time 54 min. 4-OHE Conjugates: 4-OHE-2-SG: UV (CH3OH) 256, 290 nm; positive ion electrospray-MS, m/z 592 (100) (MH+); retention time 65 min. 4-OHE-diSG: UV (CH3OH) 236, 260, 310 nm; positive ion electrospray-MS, m/z 448 (45) (M + H+)2+, 897 (100) (MH+); retention time 43 min. GSH Conjugates of 4-THNC-QMs. The 2-THNC-QM GSH adducts were previously synthesized and characterized (Figure 2). The structures of the 4-THNC-QM GSH conjugates are shown in Figure 3. The QMs were prepared using a modification of the procedure described for vinyl QMs (16, 25, 29). Silver oxide (5 g, 21.6 mmol) was added to 100 mg (0.7 mmol) of 4-THNC in 100 mL of acetonitrile and the mixture stirred for 30 min at 60 °C. The solution was filtered and added in aliquots (4 × 25 mL) to 100 mL of potassium phosphate buffer (pH 7.4). The CH3CN was removed after each addition under vacuum. The final concentration of the QMs in the aqueous solution was 25 mM. The solution was incubated for 80 min at 25 °C to allow for the initially formed o-quinone to isomerize to the QMs. After 80 min, GSH was added to give a final concentration of 10 mM. The aqueous solution was washed with ether. The adducts were isolated from the aqueous phase on C-18 extraction cartridges (J. T. Baker) and eluted with methanol. The eluates were concentrated and subjected to LCMS with electrospray detection as described above. Addition of GSH to the prochiral p-benzyl center gives two diasteriomeric QM conjugates in approximately equal amounts. 8, 9: UV (CH3OH) 220, 306 nm; positive ion electrospray-MS, m/z 470 (100) (MH+); retention time 32 and 33 min. o-QM formation from this analog is also possible, and a probable diastereomeric pair of GSH adducts (10, 11) were observed at 27 and 28 min; however, the amount formed was below the limit of electrospray-MS detection. Small peaks were observed at the appropriate retention times when only those ions at 470 ( 2 were monitored. Kinetic Experiments. Various oxidation methods had to be used to oxidize the catechols to o-quinones because of differences in enzymatic and chemical specificity. The oquinones of the catechol estrogens were generated by 2,3dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation in the absence of GSH. The catechol estrogen (2 mg) was dissolved in 3 mL of acetonitrile containing 2 mg of DDQ. The solution was heated at 60 °C for 10 min, and then an aliquot (750 µL) was combined with 14.25 mL of potassium phosphate buffer (pH 7.4).

Quinoids from Catechol Estrogens The o-quinone of 2-THNC was synthesized by incubating 0.5 mM 2-THNC with mushroom tyrosinase (0.009 mg/mL) in 50 mM sodium phosphate buffer (5 mL, pH 6.0) at 25 °C. After 5 min incubation time, the solution was extracted twice with 10 mL of ether, concentrated under a stream of N2, and exchanged with acetonitrile (0.5 mL final volume). The o-quinone of 4-THNC was generated by silver oxide oxidation in acetonitrile as described above. Aliquots of the acetonitrile o-quinone solutions (0.5 mM final concentration) were added to 10 mL of pH 7.4 phosphate buffer (50 mM) and incubated at 37 °C. The remainder of the time course was the same for all o-quinones examined. At variable time increments, 500 µL aliquots were removed and combined with 25 µL of 100 mM GSH. After the addition of perchloric acid (25 µL), the incubates were centrifuged at 13 000 rpm for 6 min. Aliquots of the supernatant (100 µL) were analyzed directly by HPLC with a 4.6 × 150 mm Ultrasphere C-18 column (Beckman) on a Shimadzu LC-10A gradient HPLC equipped with an SIL-10A auto injector and 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 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. The rate of o-quinone disappearance was determined from the decay of the di-SG conjugates. Isomerization of 4-OHE-o-Quinone. The o-quinone of 4-OHE (10 mg) was prepared as described above on a larger scale (15 mL). The acetonitrile solution was added in aliquots (3 × 5 mL) to 50 mL of potassium phosphate buffer (pH 7.4). The CH3CN was removed after each addition under vacuum. After 70 min incubation at 37 °C, 2.5 mL of 0.2 M GSH was added. The products were isolated from the aqueous phase on C-18 extraction cartridges (J. T. Baker) and eluted with methanol. The eluates were concentrated and subjected to semipreparative HPLC with an Ultrasphere ODS column (10 × 250 mm, Beckman) with a flow rate of 3.5 mL/min. The mobile phase consisted of 5% methanol in 0.25% perchloric acid/ 0.25% acetic acid (pH 3.5) for 5 min, increased to 40% CH3OH over 5 min, isocratic for 10 min, increased to 60% CH3OH over 10 min, isocratic for 7 min, and increased to 99% CH3OH over the remaining 6 min of the run. Under these conditions, the dehydration product 9-dehydro-4-OHE elutes at 43 min. Using the analytical HPLC conditions, 9-dehydro-4-OHE elutes at 68 min. 9-Dehydro-4-OHE: 1H-NMR (CD3OD) δ 0.93 (s, 3H, CH3), 1.28 (m, 2H), 1.70 (m, 2H), 2.24 (m, 5H), 2.54 (m, 2H), 3.03 (dd, J ) 6, 12 Hz, 1H, R-C6 H), 6.08 (d, J ) 6 Hz, 1H, vinyl H), 6.63 (d, J ) 9 Hz, 1H, ArH), 7.00 (d, J ) 9 Hz, 1H, ArH); UV (CH3OH) 220, 272 nm; positive ion CI-MS, m/z 285 (100) (MH+). Incubations. Male Sprague-Dawley rats (180-200 g) were obtained from Sasco Inc. (Omaha, NE). Microsomes were prepared from rat liver, and protein and P450 concentrations were determined as described previously (30). Incubations containing microsomal protein were conducted for 10 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 10 mCi/mmol) was added in phosphate buffer, to achieve final concentrations of 0.5 and 1.0 mM, respectively. An NADPH-generating system consisting of 0.4 mM NADP+, 7.5 mM glucose 6-phosphate, and 1 unit/mL glucose-6-phosphate 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 incubates were centrifuged at 13 000 rpm for 6 min to precipitate microsomal protein. Aliquots of the supernatant (100 µL) were analyzed directly by HPLC as described above (kinetic experiments). 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

Chem. Res. Toxicol., Vol. 9, No. 2, 1996 495 converting the data to nanomolar amounts using the specific activity of the [3H]GSH. 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, and 1H-NMR spectra were obtained with a Varian XL-300 spectrometer at 300 MHz. Electrospray mass spectra were obtained using a Hewlett-Packard 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 350 °C and a flow rate of 50 mL/min were used for evaporation of solvent from the electrospray. The range m/z 200-900 was scanned over approximately 2 s during LC-MS.

Results and Discussion GSH Adducts of Catechol Estrogen o-Quinones. Previous work has shown that catechol estrogens can readily be oxidized by a variety of chemical and enzymatic agents including sodium metaperiodate (31), manganese dioxide (13, 32), tyrosinase (33), and peroxidase (8). These model oxidizing agents are more efficient than liver microsomes or purified P450s in generating sufficient quantities of o-quinones for spectroscopic characterization. In the present study, we generated the o-quinones using peroxidase-catalyzed oxidation of the 2-OHE and 4-OHE in the presence of GSH. An HPLC method was developed to separate and characterize the GSH conjugates by UV and electrospray-MS (Figure 4). Similar to studies with the catechols from 17β-estradiol (17), the data indicate that three GSH conjugates are produced from 2-OHE, including two mono-GSH adducts as well as the di-SG product. Only two GSH adducts were formed from 4-OHE, a mono-GSH conjugate resulting from attack at the C2 position and the 4-OHE diGSH adduct. This result was surprising since 4-THNC, the AB ring analog of 4-OHE, gives both mono-GSH conjugates as well as the di-GSH adduct. GSH would be expected to react more selectively at C2 since the resulting anion enjoys greater resonance stabilization (34). It is not clear whether formation of mono-GSH conjugates is a detoxification route for catechol estrogen o-quinones in vivo, because these conjugates may autoxidize at higher rates than the corresponding catechol (20, 35). However, it may be argued that GSH conjugation decreases the number of electrophilic sites in the oquinone and, by increasing the hydrophilicity, facilitates excretion. Oxidation of Catechol Estrogens and Estrone by Cytochrome P450. We examined the oxidation of the 2-OHE, 4-OHE, and estrone to quinoid metabolites in rat liver microsomes by trapping these reactive species with [3H]GSH (Figures 4 and 5, Table 1). The trapping reaction should be very efficient due to the high concentration of GSH in the medium (1.0 mM), and the relatively fast rate of addition of thiols to quinoids relative to amino or hydroxyl groups (36, 37). For example, the pseudo-first-order rate of addition of GSH to 4-methyl-o-quinone is 1.2 × 102 s-1, which was 3-4 orders of magnitude greater than non-sulfhydryl-containing nucleophiles (38). Nevertheless, a small amount of binding to microsomal protein is possible, so conjugate

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Figure 5. HPLC analysis of adducts produced from estrone by rat liver microsomes (0.4 nmol of P450/mL) in the presence of an NADPH-generating system and 1.0 mM [3H]GSH. Radioactivity eluting from the HPLC column was measured in fractions collected at 18-s intervals. The data for the di-SG adducts have been divided by 2 to correct for the additional [3H]GSH moiety. Adducts eluted 3-5 min earlier relative to Figure 4. Adducts were identified by their electrospray mass spectrum and by coinjection of the catechol estrone incubations.

formation shown in Table 1 is a lower limit for the generation of quinoids. The radiochromatograms (Figures 4 and 5) gave peaks with retention times and UV spectra identical to those derived from addition of GSH to the o-quinones (data not shown); QM-derived GSH adducts were not observed under these experimental conditions as the rate of trapping of the o-quinone by GSH is much faster than the rate of isomerization of the o-quinone to the QM (16, 25). Quantitative data for metabolism of the catechols showed that the rate of formation of the 2-OHE-o-quinone GSH conjugates are significantly faster (2.5-fold) than the 4-OHE-o-quinone GSH adducts (Table 1) under these incubation conditions. Considerably less o-quinone derived GSH adducts were produced in incubations with estrone, supporting the intermediacy of the catechols on the bioactivation pathway. The 2-OHE adducts predominated over 4-OHE conjugates only by a factor of 6 even though hydroxylation at the C2 position is favored over C4 hydroxylation by a factor of 10 in hepatic microsomes from male rats (3). This discrepancy may result from differences in the dependence of the catechol oxidation on P450 concentration (see below). In addition to the o-quinone GSH products listed in Table 1, three additional GSH conjugates were detected in the estrone incubations that were not present in experiments with the catechol estrogens. Their exact structures are unknown at present; however, they may result from GSH conjugation with the QM formed from direct two-electron oxidation of estrone. Precedence for formation of these highly substituted QMs from steroids has been established during oxidation of 11-oxoestrones by dichlorodicyanobenzoquinone (39). Reaction of GSH with arene oxides of estrone is also possible (40). o-Quinone GSH conjugates were also observed in incubations in the absence of NADPH. These results indicate that the majority of two-electron oxidation is mediated by P450; however, the reduced amount of o-quinone-derived GSH products in the control incubations suggests that an additional oxidation mechanism(s) not involving P450s may be involved in the metabolism of these catechols. The dependence of catechol oxidation on incubation time and protein concentration was also determined in

Iverson et al.

rat liver microsomes. Formation of o-quinone GSH adducts from both catechols increased in a linear fashion up to 20 min incubation time (data not shown). In contrast, dramatic differences were observed when the concentration of microsomal protein was varied (Figure 6). Incubations with 2-OHE showed a much faster initial rate of o-quinone GSH conjugate formation compared to 4-OHE; however, it also saturated P450 at an unusually low concentration (0.2 nmol of P450/mL). With 4-OHE, saturation of the enzyme was not observed at any concentration examined (maximum 1.5 nmol of P450/ mL); however, its rate of formation appeared to slow at higher P450 concentrations. The observed differences in o-quinone formation from catechol estrogens may be due to variable o-quinone-mediated inhibition of further P450-catalyzed π-oxidation, as quinones can interfere with the electron supply to the hemoprotein by accepting electrons directly from P450 reductase (41). Alternatively, the higher reactivity of the 2-OHE-o-quinone compared to the 4-OHE-o-quinone (see below) may better explain the P450 saturation effect. These data suggest that, in cells with low levels of P450, oxidation of 2-OHE will be favored over 4-OHE metabolism. In contrast, oxidation of equal amounts of the catechol estrogens in cells with relatively high concentration of P450, such as hepatocytes, should show enhanced formation of 4-OHEo-quinone GSH conjugates. Reactivity of the Catechol Estrogen o-Quinones. Preliminary data suggests that the o-quinones of both catechol estrogens (Figure 1) isomerize to QMs. 2-OHEo-quinone has the potential to form two QMs (Figure 1), a QM stabilized by two alkyl substituents on the methylene group in the C ring and a QM with only one alkyl substituent in the B ring. In contrast, 4-OHE-o-quinone can isomerize to the potentially more stable C ring p-QM as well as the B ring o-QM as described above for 4-THNC. The o-quinones of the catechol estrogens were incubated at pH 7.4, 37 °C in the absence of GSH. Aliquots were removed at various times and combined with GSH. Peroxidase-catalyzed or DDQ oxidation gives the di-GSH conjugate as the major product for both 2-OHE and 4-OHE. From the pseudo-first-order rate of disappearance of the o-quinone di-GSH adducts (Figure 7), the half-lives of the o-quinones were determined (Table 2). The o-quinone from 2-OHE was found to be 17-fold more reactive in aqueous solution compared to the 4-OHE-o-quinone, confirming previous qualitative reports of reactivity differences between the two oquinones (8). Exogenous 4-OHE, although not 2-OHE, is carcinogenic in hamsters (42), possibly because of the slower rate of isomerization of the o-quinone to highly electrophilic QMs in addition to its relatively slow rate of metabolic inactivation via O-methylation of the catechol function (43). In both GSH trapping studies, new GSH conjugates appear over the course of the experiment which may be QM GSH adducts; however, the adducts were unstable, and isolation and spectral characterization have not been successful. Isomerization of the o-Quinone of 4-THNC to QMs. The isomerization of o-quinones to QMs is unlikely to occur in cells with high levels of GSH since the rate of GSH trapping of most o-quinone is orders of magnitude higher than the rate of isomerization. Once the stores of intracellular GSH have been depleted, however, isomerization of o-quinones to QMs could readily occur, resulting in potent electrophiles which could potentially alkylate numerous biopolymers, including proteins (44) and

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Table 1. Formation of o-Quinone-Glutathione Conjugates in Rat Liver Microsomesa substrate [nmol/(nmol of P450‚min)] metabolite

2-OHE

(-NADPH)

2-OHE-Di-SG 2-OHE-C1-SG 2-OHE-C4-SG 4-OHE-di-SG 4-OHE-C2-SG

0.92 ( 0.13 12.5 ( 2.0 28.8 ( 4.0

0.65 4.0 5.4

4-OHEb

0.50 ( 0.13 16.33 ( 2

(-NADPH)

estronec

(-NADPH)

ND 2.96

0.07 ( 0.01 0.46 ( 0.05 0.55 ( 0.06 ND 0.18 ( 0.03

0.06 NDd 0.06 ND ND

a Incubations were conducted for 10 min with 0.5 mM substrate, 1.0 mM [3H]GSH (sp act. ) 10 nCi/nmol), untreated rat liver microsomes (0.1 nmol of P450/mL), and a NADPH-generating system. Product formation was linear with time for at least 20 min. Results are the means ( SD of 3 determinations. b 0.2 nmol of P450/mL. c 0.4 nmol of P450/mL. d Not detectable.

Figure 8. HPLC chromatogram of the GSH adducts observed after incubating the 4-THNC o-quinone in pH 7.4 phosphate buffer for 2 h at 25 °C followed by addition of 5 mM GSH. Numbers correspond to structures shown in Figure 3. Figure 6. Effect of changing P450 concentration on the metabolism of 4-OHE and 2-OHE in rat liver microsomes. Incubations were conducted for 10 min with 0.5 mM substrate, 1.0 mM GSH, untreated rat liver microsomes, and an NADPHgenerating system: Mono-GSH adducts, squares; di-GSH adducts, circles; 2-OHE, closed symbols; 4-OHE, open symbols.

Figure 7. Time course for incubation of the o-quinones of 2-OHE and 4-OHE at 37 °C, pH 7.4. 500 µL aliquots were combined with 5.0 mM GSH at various times. (b) Peak area ratio of 2-OHE-diSG, (×) peak area ratio of 4-OHE-diSG. Table 2. Rates of o-Quinone Disappearance (pH 7.4, 37 °C) catechol

half-life (min)a

2-THNC 2-OHE 4-THNCb 4-OHE

5.4 ( 0.4 0.7 ( 0.05 17 ( 0.5 12.2 ( 0.4

a The rate of disappearance of the di-SG adduct for each catechol as described in Materials and Methods. b The rate of disappearance of the sum of the mono-SG adducts due to coelution of the di-SG adduct and DDQ peaks in the chromatogram.

nucleic acids (45, 46). It is not clear if generation of QMs from o-quinones is a bioactivation process or a detoxification mechanism in vivo as several factors need to be considered, including the redox and addition chemistry of the o-quinone as well as the reactivity of the QM. Previously we showed that the o-quinone of 2-THNC was the initially formed kinetic product, which isomerized to the thermodynamically more stable QM intermediate over time (Figure 2; 16). Thus, incubation of 2-THNC with tyrosinase, ether extraction to separate the incubate from the enzyme, addition to pH 6.0 buffer, and quench-

ing with GSH at various times showed a time-dependent decrease of o-quinone GSH adducts along with the appearance of the GSH conjugates resulting from trapping of the 2-THNC-QM. The first-order rate constant of isomerization was estimated from the decrease in o-quinone GSH adducts to be 5.8 × 10-4 s-1 (t1/2 ) 20 min, 25 °C). In the present study, we have shown that a similar process occurs with 4-THNC; oxidation with either tyrosinase or silver oxide followed by quenching with GSH at various times also yielded a time-dependent decrease in o-quinone GSH adducts (Table 2, Figure 8). Two sets of diastereoisomeric QM GSH conjugates appeared during the course of the incubation; the major set (8, 9) is predicted to result from reaction of GSH with the p-QM, and the minor GSH adducts (10, 11) could result from trapping the o-QM. The o-quinone from 2-THNC disappeared at a faster rate compared to that from 4-THNC (Table 2); however, the differences were not as dramatic as compared to the catechol estrogen o-quinone data. Isomerization of the o-Quinone of 4-OHE to p-QM. Although the GSH adducts of the suspected catechol estrogen QMs were unstable, we have obtained evidence that the 4-OHE o-quinone isomerizes to a QM by isolating and characterizing 9-dehydro-4-OHE (Figure 9). The 1HNMR spectrum of 9-dehydro-4-OHE revealed a signal corresponding to one proton at 6.08 ppm similar to the chemical shift of the vinylic proton in 9-dehydroestrone (6.10 ppm) consistent with a vinylic proton at C11. Further support for the double bond assignment at C9C11 was obtained by comparison with the 1H-NMR spectrum of 4-OHE (Figure 9B). The benzyl proton at C9 in 4-OHE has a resonance at 2.7 ppm which is absent from the spectrum of 9-dehydro-4-OHE (Figure 9A). Finally, the CI mass spectra gave a protonated molecular ion at 285 daltons expected for a molecule with an additional site of unsaturation relative to 4-OHE. This product could be formed by elimination of water or GSH as shown in Figure 9. Alternatively, direct rearomatization of the 4-OHE-QM could occur (47). A similar reaction would be expected with the 2-OHE o-quinone as two p-QMs could form. Future work will concentrate on searching for the elimination products and stabilizing the suspected QM GSH conjugates in order to unequivo-

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Iverson et al.

Figure 9. Mechanism of formation of 9-dehydro-4-OHE from 4-OHE-o-quinone. (A) Partial 1H-NMR spectrum (CD3OD) of 9-dehydro4-OHE showing the vinylic proton resonance at 6.09 ppm and the absence of the 4-OHE C9 benzylic resonance at 2.7 ppm. (B) Partial 1H-NMR spectrum 4-OHE showing the analogous regions.

cally confirm QM formation from both catechol estrogen o-quinones. In conclusion, data have been presented on the bioactivation of catechol estrogens to o-quinone GSH conjugates in rat liver microsomes. Dramatic differences were observed between 2-OHE and 4-OHE with the former saturating P450 at unusually low concentrations. The enhanced reactivity of the 2-OHE-o-quinone may explain the observed effect. Studies are in progress with other catechols that form o-quinones of known lifetimes to determine if this is a general phenomenon for P450catalyzed metabolism of catechols. The o-quinones of the AB ring analogs of the catechols isomerize to QMs which implies that a similar reaction pathway could occur with the o-quinones from catechol estrogens. Finally, we isolated and characterized an elimination product consistent with the isomerization of 4-OHE o-quinone to an C ring p-QM. The implications of the o-quinone/QM pathway to the in vivo effects of catechol estrogens are not known; however, given the direct link between excessive exposure to endogenous estrogens and the enhanced risk of breast cancer, the potential for formation of additional reactive intermediates needs to be explored.

Acknowledgment. This research was supported by NIH Grant ES06216, NSERC Grant WFA0122931, and the University of Illinois at Chicago. We thank Mr. Zhiwen Huang for the synthesis of 4-THNC. The electrospray-MS expertise provided by Dr. Richard B. van Breemen (Liquid Chromatography-Mass Spectrometry Laboratory, University of Illinois at Chicago) is gratefully appreciated.

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