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Chem. Res. Toxicol. 1998, 11, 917-924

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Covalent Binding of Catechol Estrogens to Glutathione Catalyzed by Horseradish Peroxidase, Lactoperoxidase, or Rat Liver Microsomes Kai Cao,† Prabu D. Devanesan,† Ragulan Ramanathan,‡ Michael L. Gross,‡ Eleanor G. Rogan,† and Ercole L. Cavalieri*,† Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, and Department of Chemistry, Washington University, One Brookings Drive, St. Louis, Missouri 63130-4899 Received December 22, 1997

Oxidation of catechol estrogens (CE) leads to the reactive electrophilic CE quinones. Reaction of CE-3,4-quinones with DNA has been implicated in tumor initiation. One pathway to prevent this reaction is conjugation of CE quinones with glutathione (GSH). Four CE, 4-hydroxy estrone (4-OHE1), 4-hydroxyestradiol (4-OHE2), 2-OHE1, and 2-OHE2, were conjugated with GSH after oxidation catalyzed by horseradish peroxidase (HRP), lactoperoxidase (LP), or rat liver microsomal cytochrome P450. This reaction is a free-radical chain autoxidation that produces very high yields of products. Six mono-GSH conjugates, 4-OHE1(E2)-2-SG, 2-OHE1(E2)-1-SG, and 2-OHE1(E2)-4-SG, and four di-GSH conjugates, 4-OHE1(E2)-1,2-bisSG and 2-OHE1(E2)1,4-bisSG, were identified and quantified. These di-GSH conjugates were also obtained quantitatively from oxidation of mono-GSH conjugates by the same enzymes. HRP and LP gave very similar product profiles. Phenobarbital- and 3-methylcholanthrene-induced microsomes with either NADPH or cumene hydroperoxide as cofactor oxidized 4-OHE2 to form similar amounts of GSH conjugates. Enzymatic oxidation of 2-OHE1(E2) in the presence of GSH produced more 2-OHE1(E2)-4-SG than the 1-isomer. This contrasts with the direct reaction of E1(E2)-2,3-Q and GSH, in which the 1-isomer is formed more abundantly than the 4-isomer (Cao, K., Devanesan, P. D., Ramanathan, R., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1998) Chem. Res. Toxicol. 11, 909-916). Competitive enzymatic oxidation of equimolar 4-OHE2 and 2-OHE2 in the presence of an equimolar amount of GSH yielded more 2-OHE2 conjugates than 4-OHE2 conjugates, despite E2-3,4-Q being more reactive with GSH than E2-2,3-Q. These results suggest that 2-OHE2 is a better substrate than 4-OHE2 in the catalytic oxidation to quinones, despite the greater reactivity of E2-3,4-Q, compared to E2-2,3-Q, with GSH.

Introduction Reaction of endogenous catechol estrogen-3,4-quinones (CE-3,4-Q)1 with DNA has been implicated in the initiation of many types of cancer (1, 2). This hypothesis is based on two major lines of evidence. One is related to the carcinogenicity of the 4-catechol estrogens, namely, 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4OHE2), in the kidney of Syrian golden hamsters (3, 4). The second is suggested by the formation of depurinating DNA adducts by CE-3,4-Q (1, 2). This type of DNA adduct has been correlated with oncogenic mutations in mouse skin papillomas induced by carcinogenic polycyclic aromatic hydrocarbons (5). Conjugation of CE-Q with glutathione (GSH) may represent a preventive pathway that competes with * To whom correspondence should be addressed. † University of Nebraska Medical Center. ‡ Washington University. 1 Abbreviations: CAD, collisionally activated dissociation; CE-Q, catechol estrogen quinone(s); COSY, homonuclear two-dimensional chemical shift correlation spectroscopy; CuOOH, cumene hydroperoxide; E1, estrone; E2, 17β-estradiol; ESI, electrospray ionization; GSH, glutathione; HRP, horseradish peroxidase; LP, lactoperoxidase; MC, 3-methylcholanthrene; OHE1, hydroxyestrone; OHE2, hydroxyestradiol; PB, phenobarbital; -SG, glutathione moiety bound at the sulfur atom; TFA, trifluoroacetic acid.

reaction of CE-Q and DNA. GSH, the most abundant intracellular nonprotein thiol, can react nonenzymatically with electrophiles and free radicals, as well as detoxify a wide variety of compounds via the catalytic action of GSH S-transferase (6). The resultant GSH conjugates are generally less reactive and more readily excreted. GSH conjugates of bioactivated CE have been identified both in vitro and in vivo (7-14). In addition to the monoGSH conjugates, di-GSH conjugates were reported (1214). Efficient, quantitative conversion of CE-Q to CEGSH conjugates was obtained by direct nonenzymatic coupling of CE-Q with GSH (15). In this paper, we report the oxidation of CE catalyzed by peroxidases and cytochrome P450s in the presence of GSH. Cytochrome P450s induced by phenobarbital (PB) or 3-methylcholanthrene (MC) were investigated along with the two cofactors NADPH and cumene hydroperoxide (CuOOH). We also studied the competitive reactivity of E2-3,4-Q vs E2-2,3-Q and enzymatically oxidized 4-OHE2 vs 2-OHE2 with GSH.

Experimental Section Caution: CE-Q are toxic and were handled according to NIH guidelines (16).

S0893-228x(97)00230-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/21/1998

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Scheme 1. Enzymatic Oxidation of 4-OHE1(E2) and 2-OHE1(E2) and Formation of Mono-GSH and Di-GSH Conjugates

Chemicals. The 2-OHE1, 2-OHE2, 4-OHE1, 4-OHE2, and the corresponding quinones were synthesized by two different methods, as previously described (17-19). GSH (reduced form) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Horseradish peroxidase (HRP), lactoperoxidase (LP), and CuOOH were obtained from Sigma Chemical Co. (St. Louis, MO). PBand MC-induced rat liver microsomes were prepared from MRC Wistar rats (Eppley Colony) as previously described (20). HPLC Analysis. HPLC was conducted on a Waters (Milford, MA) 600E system equipped with a Waters 990 photodiode array detector interfaced with an APC-IV Powermate computer. Analyses were conducted by using a reversed-phase YMC (Morris Plains, NJ) 5-µm, 120-Å ODS-AQ column (6.0 × 250 mm) at a flow rate of 1 mL/min. The analytical gradient started with 20% acetonitrile [0.4% trifluoroacetic acid (TFA)] in water (0.4% TFA) for 5 min, and then a 95-min linear gradient to 100% acetonitrile (0.4% TFA). Preparative HPLC was conducted by using a YMC ODS-AQ 5-µm, 120-Å column (20 × 250 mm) at a flow rate of 6 mL/min. The preparative gradient started with 30% methanol (0.4% acetic acid) in water (0.4% acetic acid) for 10 min, followed by a 55-min linear gradient to 100% methanol (0.4% acetic acid). The analyses were monitored at 302 nm for reactions with 2-OHE1 and 2-OHE2 and at 292 nm for reactions with 4-OHE1 and 4-OHE2. The product yields (% of starting CE) were calculated from the peak areas determined by the photodiode array detector at the absorbance maximum for each compound (Table 1) and an extinction coefficient determined for each compound. Characterization of Compounds. The UV spectra were obtained during HPLC with the acetonitrile/water/TFA gradient as described above using the photodiode array detector. Proton and homonuclear two-dimensional chemical shift correlation spectroscopy (COSY) NMR spectra were recorded in Me2SO-d6 on a Varian Unity 500 at 499.835 MHz at 25 °C. Chemical shifts are reported relative to Me2SO (2.49 ppm). Mass Spectrometry. Electrospray ionization (ESI) tandem (product-ion) mass spectra of the CE-mono- and CE-di-GSH conjugates were obtained by using a VG ZAB-T four-sector mass spectrometer (Manchester, UK) that was described in the previous paper in this issue (15). The spray needle of the VG

Table 1. Comparison of UV Maxima of CE, CE-Mono-GSH, and CE-Di-GSH Conjugates CE

λmax, λmax, nm CE-mono-GSH nm

4-OHE1 280 4-OHE1-2-SG 4-OHE2 280 4-OHE2-2-SG 2-OHE1 289 2-OHE1-1-SG 2-OHE1-4-SG 2-OHE2 289 2-OHE2-1-SG 2-OHE2-4-SG

292 292 304 302 304 302

CE-di-GSH

λmax, nm

4-OHE1-1,2-bisSG 311 4-OHE2-1,2-bisSG 311 2-OHE1-1,4-bisSG 314 2-OHE2-1,4-bisSG 314

ESI source was maintained at 8000 V, the counter electrode (pepper pot) was at 5000 V, and the sampling cone, skimmer lens, skimmer, hexapole, and ring electrode were at 4417, 4125, 4119, 4117, and 4116 V, respectively. Nitrogen was used as both bath and nebulizer gases (at 80 °C) with a flow rate of approximately 300 and 12 L/h, respectively. A Harvard model 22 syringe pump (South Natick, MA) was used to infuse a solution of 50:49:1 methanol/water/acetic acid to the spray needle at a rate of 10 µL/min. The elemental composition of the ESI-produced [M + 2H]2+ ions of di-SG conjugates was confirmed by measurements of exact mass in a voltage scan at a mass resolving power of 5000. A solution mixture of PEG 400 and 600 was used to generate two reference ions whose m/z values bracketed that of the analyte. Peroxidase-Catalyzed Oxidation of CE and Conjugation with GSH. CE were bound to GSH in 5-mL reaction mixtures containing 0.067 M sodium-potassium phosphate (pH 7.0), 200 µg of enzyme (HRP, type VI, or LP), 0.5 mM H2O2, and indicated amounts of CE in Me2SO and GSH. The mixtures were incubated at 37 °C for 1 h. An equal volume of ethanol was added to stop the reaction, the precipitated protein was removed by centrifugation, and the mixtures were evaporated under vacuum to dryness. The residues were redissolved in 2 mL of Me2SO (10% acetic acid), filtered, and analyzed by HPLC. Control reactions without enzyme or H2O2 were included. Cytochrome P450-Catalyzed Oxidation of CE and Conjugation with GSH. PB- or MC-induced rat liver microsomal cytochrome P450 was used to catalyze the oxidation of CE with either CuOOH or NADPH as the cofactor. Reaction mixtures containing 150 mM Tris-HCl, pH 7.5, 150 mM KCl, 5 mM

Enzymic Formation of Catechol Estrogen-GSH Adducts

Chem. Res. Toxicol., Vol. 11, No. 8, 1998 919

Scheme 2. Pathway of Formation of Mono-GSH and Di-GSH Conjugates from 2-OHE1(E2)

MgCl2, 1 mg/mL microsomal protein, and indicated amounts of GSH, CE in Me2SO, and CuOOH or NADPH were incubated at 37 °C for 1 h. An equal volume of ethanol was added to stop the reaction, the precipitated protein was removed by centrifugation, and the mixtures were evaporated under vacuum to dryness. The residues were redissolved in 2 mL of Me2SO (10% acetic acid), filtered, and analyzed by HPLC. Control reactions without microsomes or cofactor were included.

Results and Discussion Enzymatic Oxidation of CE and Conjugation with GSH. Oxidation of 4-OHE1(E2) with HRP, LP, or cytochrome P450 in the presence of GSH yielded the mono-GSH conjugate 4-OHE1(E2)-2-SG, as expected, and the 4-OHE1(E2)-1,2-bisSG conjugate (Scheme 1). Enzymatic oxidation of 2-OHE1(E2) yielded 2-OHE1(E2)-1-SG, 2-OHE1(E2)-4-SG, and 2-OHE1(E2)-1,4-bisSG. The diGSH adducts were formed by oxidation of the mono-GSH adducts and subsequent conjugation with GSH (see below), as shown for the 2-hydroxy adducts in Scheme 2. Unless otherwise noted, the concentration of GSH in the reaction mixtures was three times that of the CE. Identification of GSH Conjugates. GSH conjugates were separated by HPLC, and a typical chromatogram for the enzymatic oxidation of 2-OHE2 shows the monoGSH and di-GSH conjugates (Figure 1). The mono-GSH conjugates were identified by comparison of their retention times and mass spectra with those of the authentic standards prepared and characterized as described in the previous paper in this issue (15). Structural information on the mono-GSH conjugates was obtained by tandem mass spectrometry by using the [M + H]+ ions that were produced by ESI. The spectra of product ions formed upon high-energy collisional activation are nearly identical to those of the synthetic reference conjugates described in the previous paper in this issue (15). We were able to confirm the various isomers on the basis of type-4 ions. These product ions are not formed when GSH is linked at C-2 of the A-ring of the estrogen, but they are moderately abundant when the attachment is at C-4 and most abundant when the attachment is at C-1.

Figure 1. HPLC separation of GSH conjugates formed by HRPcatalyzed oxidation of 2-OHE2 in the presence of GSH. Conditions are given in the Experimental Section.

To elucidate the structures of the di-GSH conjugates, large-scale enzymatic preparations were carried out. The structures of the di-GSH adducts were elucidated by a combination of UV, NMR, and mass spectrometry. The UV spectra of the di-GSH adducts have characteristic red shifts of approximately 10 nm compared to the absorb-

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Figure 2. Tandem mass spectrum of ESI-produced [M + H]+ ions from 4-OHE2-1,2-bisSG, obtained by using a four-sector mass spectrometer.

ance maxima of the mono-GSH conjugates (Table 1), which in turn have 10 nm red shifts compared to the corresponding CE (Table 1) (15). The NMR spectra of the four di-GSH conjugates from 2-OHE1(E2) and 4-OHE1(E2) show none of the aromatic proton resonance signals present in the spectra of the mono-GSH adducts (15); this is consistent with two SG groups bonded to the aromatic ring. The di-GSH conjugates give abundant [M + 2H]2+ ions of m/z 449.2 for 2-OHE1-1,4-bisSG and 4-OHE1-1,2-bisSG and of m/z 450.2 for 2-OHE2-1,4-bisSG and 4-OHE2-1,2bisSG. They also produce less abundant [M + H]+ ions of m/z 897 and 899 for the E1 and E2 conjugates, respectively. Exact mass measurements are 449.1545 and 450.1625 for the [M + 2H]+ ions for the E1 and E2 conjugates, respectively, confirming the formulas C38H54N6O15S2 and C38H56N6O15S2 to within 2 ppm of the calculated molecular weight. Collisionally activated dissociation (CAD) experiments were conducted only with the [M + H]+ ions because the software for the tandem four-sector mass spectrometer does not accommodate scanning to m/z values that are greater than that of the precursor ion. Both E1 and E2 conjugates dissociated to give similar tandem mass spectra, except that fragment ions containing the CE moiety are shifted higher by 2 u for the E2 conjugates. We discuss only the CAD spectra of the E2 diconjugates because they were not reported previously. Upon collisional activation, decompositions of [M + H]+ (Figure 2) occurred to give [M + H - H2O]+, **b2 (modified b2 formed by loss of one Gly; the double asterisk is used to denote the GSH conjugated with an estrogen that is substituted with the second GSH, whereas the single asterisk refers to a GSH conjugated with the estrogen only), **a2, [M + H - GSH]+, *b2 (modified b2, loss of a second Gly), and * y2 of m/z 881, 824, 771, 695, 594, 519, and 465, respectively. Other fragment ions observed with the mono-GSH conjugates (15) were also observed at m/z 362, 345, 319, 317, 177, and 130. Isomer distinction based on tandem mass spectrometry is difficult, but confirmation was achieved that di-GSH conjugates were produced. Comparison of Enzyme-Catalyzed Formation of CE-GSH Conjugates. Direct coupling between equi-

Table 2. HRP- and LP-Catalyzed Oxidation of CE and Conjugation with GSHa product (% yield) substrate

HRP

4-OHE1

4-OHE1-2-SG (11%) 4-OHE1-1,2-bisSG (70%) 4-OHE2-2-SG (21%) 4-OHE2-1,2-bisSG (5%) 2-OHE1-1-SG (17%) 2-OHE1-4-SG (20%) 2-OHE1-1,4-bisSG (25%) 2-OHE2-1-SG (20%) 2-OHE2-4-SG (32%) 2-OHE2-1,4-bisSG (18%)

4-OHE2 2-OHE1 2-OHE2

LP

4-OHE2-2-SG (15%) 4-OHE2-1,2-bisSG (5%)

2-OHE2-1-SG (18%) 2-OHE2-4-SG (33%) 2-OHE2-1,4-bisSG (18%)

a 3.5 mM CE (5 mg/250 µL of Me SO, except for 4-OHE , 5 mg/ 2 2 500 µL of Me2SO), 200 µg of enzyme (HRP or LP), 10.5 mM GSH, and 0.5 mM H2O2 in 0.067 M sodium-potassium phosphate (pH 7.0, total volume 5 mL) were incubated at 37 oC for 1 h. The yield of product is expressed as percent of starting CE.

molar amounts of E1(E2)-3,4-Q and GSH produced 4-OHE1(E2)-2-SG as the sole product, whereas E1(E2)2,3-Q plus GSH yielded 2-OHE1(E2)-1-SG and 2-OHE1(E2)4-SG in a ratio of 2.5:1, respectively (15). (1) Peroxidase-Catalyzed Oxidation. Oxidation of 4-OHE1(E2) catalyzed by HRP or LP produced 4-OHE1(E2)2-SG and 4-OHE1(E2)-1,2-bisSG (Table 2). In this reaction, the formation of CE-Q occurs via a one-electron oxidation of the CE to form a semiquinone radical anion that, by disproportionation, yields CE and CE-Q (21). The concentration of cofactor, H2O2, in this reaction was 1/7 that of the substrate, and 2 equiv of H2O2 are necessary to oxidize a molecule of CE to CE-Q. If the reaction occurred stoichiometrically, one would expect a maximum amount of primary product, mono-GSH, equal to 7% of the starting CE. Much higher product yields were obtained not only in the reactions catalyzed by HRP and LP (Table 2) but also in those catalyzed by cytochrome P450 (Tables 3-5). The high product yields can be explained by a freeradical chain reaction that occurs during autoxidation of the CE, as previously observed for catecholamines (2224). The initiating step of the autoxidation involves transfer of one electron from the CE to molecular oxygen to form superoxide radical in a metal-catalyzed process

Enzymic Formation of Catechol Estrogen-GSH Adducts

Chem. Res. Toxicol., Vol. 11, No. 8, 1998 921

Table 3. PB-Induced Rat Liver Microsome-Catalyzed Oxidation of CE and Conjugation with GSHa substrate

product (% yield)

4-OHE1

4-OHE1-2-SG (30%) 4-OHE1-1,2-bisSG (44%) 4-OHE2-2-SG (23%) 4-OHE2-1,2-bisSG (41%) 2-OHE1-1-SG (12%) 2-OHE1-4-SG (15%) 2-OHE1-1,4-bisSG (20%) 2-OHE2-1-SG (16%) 2-OHE2-4-SG (59%) 2-OHE2-1,4-bisSG (16%)

4-OHE2 2-OHE1 2-OHE2

a 0.437 mM CE (0.25 mg/12.5 µL of Me SO), 2 mg of PB-induced 2 microsomal protein, 1.31 mM GSH with 0.085 mM CuOOH as cofactor in Tris-HCl, pH 7.5, 150 mM KCl, and 5 mM MgCl2 (total volume 2 mL) were incubated at 37 °C for 1 h. The yield of product is expressed as percent of starting CE.

Table 4. Formation of 4-OHE2 Conjugates by Different Cytochrome P450 Isoforms with Two Cofactorsa products, % yield enzyme PB-microsomes

cofactor

CuOOH NADPH MC-microsomes CuOOH NADPH

4-OHE2-2-SG 4-OHE2-1,2-bisSG 25 21 24 20

7 4 3 4

a Reaction mixtures (total volume 4 mL) containing 4 mg of protein from either PB- or MC-induced microsomes, 3.15 mM GSH, 1.05 mM 4-OHE2 (0.6 mg/30 µL of Me2SO), 0.085 mM CuOOH, or 0.06 mM NADPH as cofactor, in 150 mM Tris-HCl, pH 7.5, 150 mM KCl, and 5 mM MgCl2 were incubated at 37 °C for 1 h. The yield of product is expressed as percent of starting CE.

(23). In turn, the superoxide radical reacts with the CE to form a semiquinone radical anion and H2O2. Thus, the free-radical chain autoxidation leads to the high product yields observed (Tables 2-5). With HRP, 4-OHE1 appeared to be a better substrate than 4-OHE2 in the formation of both the mono-GSH and di-GSH adducts. The lower product yield with 4-OHE2 (Table 2), in fact, results from the larger amount of the radical-quenching Me2SO required to dissolve 4-OHE2. Activation of 4-OHE2 by HRP and LP yielded similar amounts of adducts. Oxidation of 2-OHE1(E2) catalyzed by HRP or LP yielded similar profiles of the three conjugates, 2-OHE1(E2)-1-SG, 2-OHE1(E2)-4-SG, and 2-OHE1(E2)-1,4-bisSG (Table 2). Among the mono-GSH conjugates, the 4-isomer was consistently more abundant than the 1-isomer. This is in contrast to the result obtained by direct reaction of E1(E2)-2,3-Q with GSH (see above) (15). This result has two possible explanations. Either the 4-isomer is formed more abundantly in the enzymecatalyzed reaction than the 1-isomer or 2-OHE1(E2)-1SG might be more abundantly formed but is more rapidly converted to 2-OHE1(E2)-1,4-bisSG than is 2-OHE1(E2)4-SG. To test these two alternative hypotheses, equimolar amounts of 2-OHE2-1-SG, 2-OHE2-4-SG, and GSH were incubated together with HRP. Ninety-five percent of the GSH was converted to 2-OHE2-1,4-bisSG, leaving 79% unreacted 2-OHE2-1-SG and 20% unreacted 2-OHE24-SG. An LP-mediated reaction produced a similar profile of adducts. These results indicate that under the conditions employed the 4-isomer was not only more abundantly formed but was also more easily converted to the di-GSH conjugate. The 2-OHE1(E2) is a better

Figure 3. HPLC separation of GSH conjugates formed by reaction of equimolar E2-2,3-Q, E2-3,4-Q, and GSH.

substrate for the formation of the 4-SG conjugate, and the 4-SG is a better substrate than the 1-SG in the formation of the di-GSH adduct. (2) Cytochrome P450-Catalyzed Oxidation. Cytochrome P450 also catalyzes the formation of CE-GSH conjugates. When each of the four CE was incubated with PB-induced rat liver microsomes in the presence of CuOOH and GSH, both mono-GSH and di-GSH conjugates were formed (Table 3). The 4-OHE1 yielded the two conjugates in amounts similar to those produced with 4-OHE2. The 2-OHE2 produced much more (59% vs 15%) 4-SG conjugate than 2-OHE1, suggesting higher substrate specificity for 2-OHE2 in the conversion to the 4-SG conjugate. Both the cytochrome P450 isoforms and the cofactor employed are important parameters in the oxidation of CE and their conjugation with GSH. Redox cycling of CE is closely related to lipid peroxidation, and lipid hydroperoxides can serve as cofactors for cytochrome P450 (25-28). NADPH has also been used as a cofactor in the P450-catalyzed oxidation of CE (12, 14). To investigate the role of the above parameters, the ability of PB-induced microsomes to form GSH conjugates was compared to that of MC-induced microsomes in the presence of CuOOH or NADPH (Table 4). Under the conditions used, no significant differences were observed with the two types of microsomes and the two cofactors, suggesting that this oxidation and conjugation can be

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Table 5. HRP-, LP-, and Cytochrome P450-Catalyzed Oxidation and Conjugation of a Mixture of Equimolar 4-OHE2, 2-OHE2, and GSH product, % yield

total conjugates, %

enzyme

2-OHE21-SG

2-OHE24-SG

2-OHE21,4-bisSG

4-OHE22-SG

4-OHE21,2-bisSG

from 2-OHE2

from 4-OHE2

ratio of conjugates, 2-OHE2 vs 4-OHE2

HRPa LP a PB-microsomesb MC-microsomesb

8 7 18 17

9 14 30 31

9 2 2 3

11 6 40 34

2 trace 3 6

26 23 50 51

13 6 43 40

2:1 3.8:1 1.2:1 1.3:1

a HRP- or LP-catalyzed reactions: 1.75 mM 4-OHE (2.5 mg/250 µL of Me SO), 1.75 mM 2-OHE (2.5 mg/250 µL of Me SO), 1.75 mM 2 2 2 2 GSH, 2 mg of enzyme (HRP or LP), and 0.5 mM H2O2 in 0.067 M sodium-potassium phosphate (pH 7.0, total volume 5 mL) were incubated at 37 °C for 1 h. The yield of product is expressed as percent of starting CE. b Microsome-catalyzed reactions: 0.31 mM 4-OHE2 (0.45 mg/45 µL of Me2SO), 0.31 mM 2-OHE2 (0.45 mg/45 µL of Me2SO), 0.31 mM GSH, 5 mg PB- or MC-induced microsomal protein, with 0.085 mM CuOOH as cofactor, in 150 mM Tris-HCl, pH 7.5, 150 mM KCl, and 5 mM MgCl2 (total volume 5 mL) were incubated at 37 °C for 1 h. The yield of product is expressed as percent of starting CE.

Figure 4. HPLC separation of GSH conjugates formed by oxidation of equimolar 2-OHE2 and 4-OHE2 by PB-induced microsomes in the presence of an equimolar amount of GSH.

efficiently catalyzed by several P450 isoforms with either cofactor. Competitive Formation of GSH Conjugates. To compare the relative reactivity of E2-3,4-Q and E2-2,3-

Q, equimolar amounts (35 µmol) of E2-2,3-Q and E2-3,4-Q were prepared in 2 mL of acetonitrile by oxidation of their respective CE; GSH (35 µmol) in 2 mL of water was added dropwise to the mixture of the two quinones. The

Enzymic Formation of Catechol Estrogen-GSH Adducts

reaction was stopped after 10 min by addition of excess N-acetylcysteine, which rapidly consumed any unreacted quinone, and an aliquot of the reaction mixture was analyzed by HPLC. The analysis showed that 80% of the GSH was converted to 4-OHE2-2-SG and less than 10% reacted to give 2-OHE2-1-SG plus 2-OHE2-4-SG (Figure 3). Thus, the reactivity of E2-3,4-Q with GSH is greater than that of E2-2,3-Q. Similar results were obtained with the quinones of E1. The relative ability of 2-OHE2 and 4-OHE2 to be enzymatically activated and conjugated with GSH was compared (Table 5 and Figure 4). With HRP or LP, 2-OHE2 formed two to four times more conjugates than 4-OHE2, despite the greater reactivity of E2-3,4-Q compared to E2-2,3-Q. With PB- or MC-induced microsomes supported by CuOOH, only slightly more conjugates were formed with 2-OHE2 than with 4-OHE2. 2-OHE2 consistently formed more 4-SG than 1-SG conjugate. These results indicate either that 2-OHE2 is a better substrate than 4-OHE2 in the catalytic oxidation to quinones or that catechol estrogen semiquinones are also involved in the enzymatically catalyzed coupling reaction.

Conclusions Cytochrome P450 and the peroxidases HRP and LP efficiently catalyze the oxidation of CE to form GSH conjugates. This reaction is a free-radical chain autoxidation that affords high yields of products. Both monoand di-GSH conjugates are formed, indicating that monoGSH conjugates are very good substrates for further conjugation. Both PB- and MC-induced rat liver microsomes are capable of oxidizing 4-OHE2 with either NADPH or CuOOH as cofactor. In the catalytic oxidation of 2-OHE1(E2) in the presence of GSH, the 2-OHE1(E2)-4-SG isomer is not only more abundantly formed with respect to the 1-isomer but also more easily converted to the di-GSH conjugate. These results suggest that 2-OHE1(E2) is a better substrate for formation of the 4-SG conjugate. This contrasts with the direct reaction of E1(E2)-2,3-Q with GSH, in which the 1-isomer is 2.5 times more abundantly formed than the 4-isomer (15). E2-3,4-Q is more reactive than E2-2,3-Q, as was determined by competitive reaction experiments. Competitive enzymatic oxidation of equimolar amounts of 4-OHE2 and 2-OHE2 in the presence of GSH yields more 2-OHE2 conjugates than 4-OHE2 conjugates (Table 5). These results suggest that 2-OHE2 is a better substrate than 4-OHE2 in the catalytic oxidation, even though E2-3,4-Q is more reactive than E2-2,3-Q with GSH. Alternatively, catechol estrogen semiquinones are also involved in the enzymatically catalyzed coupling reaction.

Acknowledgment. We acknowledge support from U.S. PHS Grant No. P01 CA49210. Core support at the Eppley Institute was provided by Grant No. CA 36727 from the National Cancer Institute and at Washington University by Grant No. P41 RR00954. K.C. received fellowship support from the University of Nebraska Center for Environmental Toxicology.

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