Chem. Res. Toxicol. 1992,5, 828-833
828
Synthesis and Characterization of Estrogen 2,3- and 3,4-Quinones. Comparison of DNA Adducts Formed by the Quinones versus Horseradish Peroxidase-Activated Catechol Estrogens I. Dwivedy, P. Devanesan, P. Cremonesi, E. Rogan, and E. Cavalieri' Eppley Institute for Research in Cancer and Allied Diseases, Uniuersity of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805 Received June 1. 1992
Catechol estrogens (CE) are among the major metabolites of estrone (El) and 178-estradiol (E2). Oxidation of these metabolites to semiquinones and quinones could generate ultimate carcinogenic forms of E1 and E2. The 2,3- and 3,4-quinones of E1 and E2 were synthesized by MnO2 oxidation of the corresponding CE, following the method for synthesizing El-3,4-quinone [Abul-Hajj (1984) J.Steroid Biochem. 21,621-6221. Characterization of these compounds was accomplished by UV, nuclear magnetic resonance, and mass spectrometry. The relative stability of these compounds was determined in DMSO/H20 (2:l) a t room temperature, and the 3,4quinones were more stable than the 2,3-quinones. The four quinones directly reacted with calf thymus DNA to form DNA adducts analyzed by the 32P-postlabelingmethod. The adducts were compared to those formed when the corresponding CE were activated by horseradish peroxidase (HRP) to bind to DNA. The El- and E2-2,3-quinones formed much higher levels of DNA adducts than the corresponding 3,4-quinones. In addition, many of the adducts (7090%) formed by the El- and E2-2,3-quinones appeared to be the same as those formed by activation of 2-OHE1 or 2-OHE2 by HRP to bind to DNA. Little overlap was observed between the adducts formed by El- and E2-3,4-quinonesand HRP-activated 4-OHE1and 4-OHE2. These results suggest that semiquinones and/or quinones are ultimate reactive intermediates in the peroxidatic activation of catechol estrogens.
Introduction Although the carcinogeniceffectsof estrogens (1-4) have been mainly attributed to hormonal properties, namely, promoting effects resulting in cellular proliferation and differentiation, there is interest in estrogens acting as chemical carcinogens by binding covalently to cellular macromolecules. Covalent binding of activated estrogen metabolites to DNA is thought to be an initiating event in chemical carcinogenesis by these compounds (5). Chemical, peroxidatic, or microsomal activation of diethylstilbestrol and steroid estrogensleads to their covalent binding to DNA and proteins (6-14). Catechol estrogens (CE)' are among the major metabolites of estrone (El) and l7g-estradiol (E21 (15),and their formation is presumably catalyzed by different cytochrome P-450 isoforms (16). These metabolites are rapidly stabilized by monomethylation at the 2-, 3-, or 4-hydroxy group, catalyzed by catechol 0-methyltransferase (15). With elevated rates of CE synthesis or deficient monomethylation of CE, these metabolites can be easily oxidized to semiquinones and quinones. Autoxidation of CE to semiquinones and subsequently to quinones is possible in neutral and alkaline solutions (17). Horseradish peroxidase (HRP)-catalyzed oxidation of CE forms semiquinones, which by disproportionation produce quinones and CE, while tyrosinase produces quinones, which in the
* Author to whom correspondence should be addressed.
Abbreviations: CE,catecholestrogen(&;DMSO, dimethylsulfoxide; El, estrone;E*,l7g-eetradiol;HPLC,high-pressureliquid chromatography; HRP, horseradish peroxidase; MS, mass spectrometry; NMR, nuclear magnetic resonance; PEI, poly(ethy1enimine).
presence of CE by reverse disproportionation produce semiquinones (17). Formation of quinones from CE is also catalyzed by cytochrome P-450 supported by organic hydroperoxides (18). Reactive semiquinones and/or quinones could be the ultimate carcinogenic forms of E1 and E2. Results obtained by induction of renal carcinomas in castrated male hamsters treated with estrogens suggest that the CE pathway may be involved in the tumor initiation process (19-22). We report in this paper the synthesis and characterization of the four quinones of the CE of E1 and E2 following the method of Abul-Hajj (23)adopted for the preparation of El-3,4-quinone by oxidation of its CE with activated MnO2. When incubated with DNA, these quinones produce adducts, as determined by the 32P-postlabeling method. Many of these adducts are the same as those obtained by HRP activation of the corresponding CE.
Materials and Methods Chemicals. El and Ez were purchased from Sigma Chemical Co. (St. Louis, MO), and all other reagents used in the syntheses were obtained from Aldrich Chemical Co. (Milwaukee,WI). The quinones of El and Ez are extremely toxic and are handled according to NIH guidelines (24). [+P]ATP (sp act. 5000 Ci/ mmol)was purchased from AmershamCorp. (ArlingtonHeights, IL);calf thymus DNA was obtained from Pharmacia (Piscataway, NJ); and HRP, apyrase, nuclease P1, dithiothreitol, and bicine were from Sigma Chemical Co. Micrococcal nuclease and spleen phosphodiesterase were from Worthington Biochemical Corp. (Freehold,NJ), and T4 polynucleotide kinase was from Bethesda ResearchLaboratories (Gaithersburg,MD). Poly(ethy1enimine) (PE1)-cellulose thin-layerchromatographyplates were purchased from Brinkmann Instruments (Des Plaines, IL).
0893-228x/92/2705-0828$03.00/00 1992 American Chemical Society
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 829
DNA Adducts of Catechol Estrogens and Estrogen Quinones
AcO
J-$p yofl 1: R = O 4:
5:
R
7: R
10: R
9:R=O 12:
z : R = O
R = H, 6-OAC
-0
Md& AcO
on
OMc
R = H.
E-OAC
13:
= 0 R = H.
15: R
H. E-OAC
E-OAC
= H. E-OH
= H. R-OH
* @-++A
3: R = O
-6:
R = H, E-OAC
8:
R = H, 6-OH
11:
R = 0
14: R
O&l
16:
0
H, 8-OAC
R = H. 8-OH
Figure 1. Reaction scheme for the synthesis of the 3,4-quinones of E1 and EO.
Figure 2. Reaction scheme for the synthesis of the 2,3-quinones of E1 and Ez.
Analysis of HPLC. All of the compounds were analyzed by high-pressure liquid chromatography (HPLC) using a Waters 600E system controller coupled with a Waters 994 programmable photodiode array detector (Millipore Corp., Waters Division, Milford, MA) and a reverse-phase YMC-Pack 5 - ~ m ODS-AQ313 column (6.0 X 250 mm) (YMC, Morris Plains, NJ). The column was eluted with a l b m i n linear gradient from 50 % CH3CN in HzO to 100% CH3CN at a flow rate of 1 mL/min. The eluant was monitored for UV absorbance at 254 nm. The purity of the compounds by HPLC was greater than 99%. Characterization of Compounds. All of the compoundswere characterized by nuclear magnetic resonance (NMR), UV, and mass spectrometry (MS). The UV spectra were obtained during HPLC with the CH3CN-HzO gradient as described above by using the Waters 994 photodiode array detector. Melting points were taken on a Melt-Temp I1 apparatus and are uncorrected. NMR spectra were recorded in CDCl3 on a Varian XL-300 at 299.938 MHz. Chemical shifts are reported relative to tetramethylsilane, which was employed as an internal reference. Mass spectra were recorded on a Kratos MS-50 instrument at the Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska-Lincoln, and an AEI MS-9 modified mass spectrometer at the Eppley Institute, University of Nebraska Medical Center. Syntheses. (A) 4-Hydroxyestrone (2). A mixture of 4-methoxyestrone 3-acetate (1, Figure 1) (100 mg, 0.33 mmol) and freshly distilled pyridine hydrochloride (300 mg, 1.46 mmol) was heated to reflux under a dry Nz atmosphere for 15 min. The mixture was cooled to room temperature, added to a saturated aqueous solution of ascorbic acid (15 mL), and extracted three times with an equal volume of CHCl3. The CHC13 layer was washed with a saturated solution of ascorbic acid, dried over anhydrous NazS04, and concentrated at reduced pressure to give 4-OHE1(2),70 mg (73.4% yield), mp 260 "C dec [lit. mp 260-265 "C (25)l. (B)Estrone 3,4-Quinone [ 1,5(lO)-Estradiene-3,4,17-trione] (3). This compound was synthesized following the procedure of Abul-Hajj (23).To a stirred solution of 4-OHE1(2) (20 mg, 0.069 mmol) in CDCl3 (20 mL) at -30 "C under Nz atmosphere was added activated MnOz (10 mg, 0.11 mmol). The reaction was complete in 3 min. The suspension was passed very quickly through a syringe equipped with a filter to remove MnOz and give a dark yellow solution. The NMR spectrum was then recorded. UV: ,A, (nm) 275,430. lH NMR 0.85 (s,3 H, 18-CH3),6.21 (d, 1 H, 2-H, J = 10.5 Hz), 7.16 (d, 1 H, 1-H, J = 10.5 Hz). (C) 4-Hydroxyestradiol 178-Acetate (5). A mixture of 4-methoxyestradiol3,17P-diacetate (4) (200mg,0.518 mmol) and freshly distilled pyridine hydrochloride (600mg, 2.91 mmol) was heated to reflux for 20 min under Nz. The reaction mixture was cooled to room temperature and treated with a saturated aqueous
solution of ascorbic acid (25 mL). The solution was extracted three times with an equal volume of CHCls, washed with a saturated solution of ascorbic acid, and dried over anhydrous NazS04. After evaporation of CHCl3 under reduced pressure, the residue obtained was 4-OHEz-17B-acetate(5) (120 mg, 70.6% yield). It was crystallized from CHCb-hexane, mp 185 "C dec. 'H NMR 0.82 (9, 3 H, 18-CH3). 2.06 (8, 3 H, COCHs), 4.68 (t, 1 H, 17-H, J = 7.8 Hz), 6.69 (d, 1H, 1-H, J = 8.4 Hz), 6.75 (d, 1H, 2-H, J = 8.4 Hz). MS m/z: 330 (100, M), in agreement with C20H2604; 288 (18.9, M - COCH3). (D)Estradiol 178-Acetate3,a-Quinone [ 17,9-Acetoxy-1,5(lO)-estradiene-3,4-dione] (6). Toastirred solution of4-OHEz17D-acetate (5) (10 mg, 0.030 mmol) in CDCl3 (20 mL) at -40 "C under a Nz atmosphere was added activated MnOz (5mg, 0.055 mmol). The mixture was further stirred for 3 min, and then the suspensionwas filtered to remove MnOz and give a yellow solution of the quinone (6). U V A, (nm) 275,430. lH NMR 0.85 (s,3 H, l&CH3), 2.1 ( ~ ,H, 3 COCHs), 4.7 (t, 1H, 17-H, J = 7.5 Hz), 6.25 (d, 1H, 2-H, J=10.5Hz),7.17(d,1H,l-H,J=10.5Hz). MSm/z: 328(13.1, M), in agreement with C20H2404; 330 (100, M + 2); 288 (14.9, M 2 - COCH3). (E) Estradiol 3,4-Quinone [ 178-Hydroxy-1,5( 10)-estradiene-3,l-dioneI (8). To a stirred solution of 5 (200 mg, 0.60 mmol) and ascorbic acid (500 mg, 2.8 mmol) in methanol (30 mL) waa added 5 N HzS04 (6 mL) under an argon atmosphere. The reaction mixture was kept in the dark for 3 days at room temperature. It was then poured into a saturated aqueous solution of ascorbic acid (60 mL) and extracted with ether (4 X 50 mL). The combined organic extracts were washed with a saturated solution of ascorbic acid (2 X 50 mL) and dried over anhydrous NazS04. The solvent was evaporated under reduced pressure, and the residue was dried in vacuum to give 4-OHEz (7) (145 mg, 83.3% yield). To a stirred solution of 7 (10 mg, 0.034 mmol) in CDCl3 at -40 "C under Nz was added activated MnOn (5mg, 0.055 mmol), and the mixture was stirred for 3 min. The suspension was filtered to remove MnOz, and a yellow solution of the quinone (8) was obtained. UV: A,, (nm) 275,431. 'H NMR 0.72 (s,3 H, 18-CHd, 4.09 (t, 1 H, 17-H, J = 7.5 Hz), 6.18 (d, 1H, 2-H, J 10.8 Hz), 7.11 (d, 1H, 1-H,J = 10.5 Hz). MS m/z: 286 (13.36, M), in agreement with ClaHzz03; 288 (100, M + 2). (F)2-Hydroxyestrone (10). A mixture of 2-methoxyestrone 3-acetate (9, Figure 2) (100 mg, 0.33 mmol) and freshly distilled pyridine hydrochloride (300 mg, 1.46 mmol) was heated to reflux for 30 min under an argon atmosphere. The workup procedure used for 4-OHE1 (2, Figure 1)was followed to give 2-OHE1(10) (70 mg, 73.4% yield), mp 190 "C [lit. mp 192-194 "C (2511. (G) Estrone 2,3-Quinone [1(10),4(5)-Estradiene-2,3,17trione] (11). To a stirred solution of 2-OHE1(10) (2 mg, 0.0069
+
Dwivedy et al.
830 Chem. Res. Toxicol., Vol. 5, No. 6, 1992
mmol) in 5 mL of CDC13 at -60 O C under an argon atmosphere was added activated MnO2 (1mg, 0.003 mmol), and the mixture was further stirred for 2 min. The suspension was passed through a syringe equipped with a filter to remove MnOz and give a yellow solution of El-2,3-quinone (11). UV: A,, (nm) 277,412. lH NMR: 0.89 (s,3 H, 18-CH3),6.17 (8, 1H, 1-H),6.24 (d, 1H, 4-H, J = 2.4 Hz). MS m/z: 284 (68.2, M) in agreement with CleHzoO3; 286 (100, M + 2). (H) 2-Hydroxyestradiol178-Acetate(13). Synthesis of this compound was carried out starting from 2-methoxyestradiol3,17g-diacetate (12),following the procedure for the synthesis of 4-OHEz-17j3-acetate(5). The yield obtained was 85.5%, mp 95 O C [lit. mp 100-109 O C (25)l. UV: ,A, (nm) 285. lH NMR 0.85 (s, 3 H, l8-CH3), 2.08 (s, 3 H, COCHs),4.72 (t, 1H, 17-H, J = 7.86 Hz), 6.59 ( ~ , H, 1 1-H), 6.80 (8, 1 H, 4-H). MS m/z: 330 (100, M), in agreement with C2oHzaO4; 288 (11.3, M - COCH3). (I) Estradiol 17B-Acetate 2,3-Quinone [ 178-Acetoxy-l(10),4(5)-estradiene-2,3-dione] (14). Oxidation of 2-OHE2-17@acetate (13) by MnOz was carried out following the procedure described for the synthesis of quinone 11. UV: ,A, (nm) 273,311,398. lH NMR: 0185 (s,3 H, l8-CH3), 2.2 (8, 3 H, COCH,), 4.64 (t, 1H, 17-H,J = 8.4 Hz), 6.14 (9, 1H, 1-H), 6.22 (d, 1 H, 4-H, J = 2.4 Hz). (J)Estradiol 2,3-Quinone [178-Hydroxy-l(10),4(5)-estradiene-2,j-dioneI (16). 2-OHE2 (15) was obtained by acid hydrolysis of 2-OHE2-178-acetate (13) following the same procedure as described for the synthesis of 4-OHE2 (7). Oxidation of 2-OHE2 (15) by MnO2 under the reaction conditions described for the synthesis of quinone 11 gave E2-2,3-quinone (16). UV: A,, (nm) 278,419. lH NMR: 0.78 (s,3H, 18-CH3),6.14 (s, 1H, 1-H), 6.24 (d, 1H, 4-H, J = 2.4 Hz). MS m/z: 286 (62.9, M) in agreement with C18H2203; 288 (100, M + 2). Stability of Quinones in DMSO-H20. Quinones were freshly prepared in CHC13 (1mg/mL of El- and E2-2,3-quinone and 2 mg/mL of El- and Ez-3,4-quinone). A 600-pL aliquot of the quinone solution was mixed with dimethyl sulfoxide (DMSO) (200pL), and the CHC13was quickly evaporated under Nz. Tripledistilled Hz0 (100 pL) was added at room temperature (22 f 1 OC), and at various time intervals 25-pL aliquots were analyzed by HPLC as described above. The DMSO-H20 solution had a pH of 6.7. The retention times of the four quinones were the following: Ez-2,3-quinone,9.7 min; El-2,3-quinone, 12.0 min; E23,4-quinone, 13.0 min; and E1-3,4-quinone, 16.0 min. Covalent Binding of Quinones to DNA. A CHCl3 solution of quinone (200 pL, 1 mg/mL) was mixed with DMSO (50 pL), and the CHC13 was evaporated under a stream of argon. The DMSO solution of quinone was quickly mixed with calf thymus DNA (7.0 mM, 1mL in HzO) and incubated at 37 OC for 2 h. Two volumes of ethanol (2 mL) were added; the solution was evaporated under argon, and the residue was dissolved in 0.015 NaC1-0.0015 M sodium citrate (1 mL). The concentration of DNA was measured by UV absorbance at 260 nm, and the DNA was used for 32P-postlabelinganalysis. Extraction of the incubation mixture with CHC13 after the reaction did not affect the adduct profile; therefore, this step was subsequently omitted. Covalent Binding of Catechol Estrogens to DNA. Covalent binding of CE to DNA was carried out by activation with HRP in the presence of Hz02. Reaction mixtures (1 mL) containing 2.8 mM calf thymus DNA in 0.067 M sodiumpotassium phosphate (pH 7.0), CE (1 mg/50 p L of DMSO), 100 pg HRP (type VI), and 0.5 mM H202 were incubated for 24 h at 37 OC. H202 was added at five intervals during the course of the incubation. The first two additions were made at 1-h intervals and the last three at 2-h intervals. DNA was purified by extraction with phenol and chloroform and precipitation with ethanol, as previously published (26). The concentration was measured and the DNA used for 32P-postlabelinganalysis. In the absence of enzyme, no adducts were obtained. 32P-Postlabeling Method. The 32P-postlabeling method described by Reddy and Randerath (26) was used for detection of DNA adducts of quinones and CE. The complete postlabeling
method has been described previously (26,27). The postlabeled mixtures were applied to 10-cm X 10-cm PEI-cellulose plates. The chromatographic conditions and the solvents used were the following: (1)1 M NaHzP04, pH 6.8 D1 direction overnight with paper wick; (2) 0.15 M ammonium formate, pH 4.0 for washing of plates; (3) 2.5 M lithium formate, 7.0 M urea, pH 4.0 DZdirection; (4) 0.7 M sodium phosphate, 7 M urea, pH 6.4 D3 14 h with paper wick; (5) 1.7 M NaHzP04, pH 6.0 D4 direction 6 h with paper wick. The adduct spots were detected by autoradiography with exposure at room temperature. The amounts of adducts were determined by scraping each spot off the plate and liquid scintillation counting of the 32P.
-
--
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Results and Discussion Synthesis and Characterization of Quinones. Quinones were synthesized by MnO2 oxidation of the corresponding CE, following the method described by Abul-Hajj for the preparation of E1-3,Cquinone (23).The 2,3-quinones from 2-hydroxyestrogens were previously characterized as derivatives (28,291. More recently, EO2,3-quinone was isolated and characterized by UV and Fourier transform infrared analysis (30). 4-Methoxy-El-3-acetate (1, Figure 1) and 2-methoxyEl-3-acetate (9, Figure 2) in pyridine hydrochloride at reflux quantitatively yielded 4-OHE1 and 2-OHE1, respectively, wtih contemporaneous deacetylation and demethylation. MnO2 oxidation of 4-OHE1(2) in CDCl3 at -30 "Cafforded El-3,Cquinone (3). The NMR spectrum shows two characteristic doublets in the aromatic region (J= 10.5 Hz), corresponding to 1-H and 2-H at 7.16 and 6.21 ppm, respectively. Evaporation of CDC13 at room temperature led to formation of adark brown gum. HPLC and NMR analysis of this gum showed a complicated mixture with no trace of quinone. However, when CDCl3 was evaporated after addition of DMSO, the quinone was not decomposed, as evidenced by its characteristic retention time on HPLC and its UV spectrum. When stored at -80 "C in CHC13, this quinone is stable for at least 1 month. Synthesis of E1-2,3-quinone (11) was achieved under the same conditions as that of E1-3,4-quinonee The structure of the product was determined by a combination of UV, NMR, and MS. The UV shows two broad maxima at 277 and 412 nm. The NMR shows a broad singlet at 6.17 ppm, corresponding to the aromatic 1-H,andadoublet at 6.25 ppm (J = 2.4 Hz), corresponding to the 4-H. The small coupling constant of the 4-H is due to long-range coupling with 6-H. The mass spectrum shows an M+ ion of mlz 284 and a basic (M + 2)+ ion of mlz 286, characteristic of the quinone structure (31). This compound was even less stable than El-3,4-quinone (see below). On heating to reflux with pyridine hydrochloride, 4-methoxy-E2-3,178-diacetate (4, Figure 1)and 2-methoxyE2-3,178-diacetate (12, Figure 2) afforded the 178-acetoxy of 4-OHE2(5) and 2-OHE2 (13), respectively. With these compounds, only deacetylation at C-3 occurred. Characterization of the 17j3-acetoxyderivatives arose from the characteristic triplet signal with resonance at 4.67 ppm (J = 7.5 Hz) in their NMR spectra. Hydrolysis of the 178acetoxy derivatives was obtained with 5 N H2S04 in the presence of ascorbic acid in methanol to yield 4-OHE2 (7, Figure 1)and 2-OHE2 (15, Figure 2). Oxidation of the CE with MnO2 in CDC13yielded the corresponding quinones (8, Figure 1, and 16, Figure 2). The structure of the E23,4-quinone was evidenced by the two characteristic aromatic doublets (J= 10.8 Hz) at 6.18 and 7.11 ppm, 2-H
Chem. Res. Toxicol., Vol. 5, No. 6, 1992 831
DNA Adducts of Catechol Estrogens and Estrogen Quinones
and l-H, respectively. Its UV spectrum with,,A at 275 and 431 nm is characteristic of o-quinones. The mass spectrum shows an M+ ion of mlz 286 and an (M + 2)+ ion at mlz 288 as a base peak, which is characteristic of o-quinones (30).The structure of the E2-2,3-quinonewas confirmed by the characteristic aromatic singlet at 6.14 ppm and the doublet (J = 2.4 Hz) at 6.25 ppm, corresponding to the l-H and 4-H, respectively. The UV and MS were similar to those of the other quinones. E2-17@-acetate-3,4-quinone(6, Figure 1) and E2-178acetate-2,3-quinone (14, Figure 2) were also synthesized and characterized similarly to the quinones described above. Stability of the Quinones. Upon evaporation of CHC13, the quinones totally decomposed, even a t -30 OC, as determined by HPLC and NMR analysis. The nature of the decomposition products is unknown, but we suspect that Diels-Alder reactions occur, as logically suggested by Abul-Hajj (23). To avoid decomposition, various organic solvents such as DMSO and trioctanoin were added to the quinones in CHCL solution and the latter solvent was removed by evaporation. In these organic solvents, the quinones can be kept for several days at -80 "C; at room temperature, however,the quinones decompose in a matter of hours. The stability of the 2,3- and 3,4-quinones of E1 and E2 was determined in a mixture of triple-distilled H2O and DMSO (pH 6.7) (1:2) at room temperature. The pH was checked periodically and was found to remain constant. The 2,3-quinones (Figure 3A) are less stable than the 3,4quinones (Figure 3B). In fact, after 90 min, the 2,3quinones were 70 % decomposed, whereas at this time the 3,4-quinones are only 15-20% decomposed. Total decomposition of 2,3-quinonesrequired less than 3 h, whereas after 7 h about half of the 3,4-quinones remains intact. Covalent Binding to DNA. The 2,3- and 3,4-quinones of E1 and E2 were covalently bound to DNA by direct reaction. The resulting DNA adducts were compared to those formed when 2-OHE1,2-OHE2,4-OHE1,and 4-OHEz were activated by HRP to bind to DNA. The DNA adducts were analyzed by the 32P-postlabelingmethod (Table I, Figures 4 and 5). 2-OHE1,2-OHE2,and the 2,3-quinones yielded larger quantities of DNA adducts, as determined by 32P-postlabeling, than 4-OHE1, 4-OHE2, or the 3,4quinones. Under the conditions of this assay, DNA incubated alone or in the presence of HRP and H202 yields no adduct spots (Figure 6, panels A and B, respectively). 2-OHE1 (Figure 4A) yielded 7 different adduct spots adductslnucleotide, whereas the totaling 30.0 X corresponding E1-2,3-quinone(Figure4B) yielded 8adduct adductslnucleotide (Table I). spots totaling 85.9 X Five of the spots (90% of total adducts), spots 2,4,6, and 7 and part of spot 5, appear to be common between the DNA adducts of 2-OHE1 and E1-2,3-quinone. 2-OHE2 (Figure 5A) yielded significantly more DNA adducts, with 8 adduct spots totaling 92.6 X lo-' adducts/nucleotide, while the corresponding E2-2,3-quinone (Figure 5B) also adducts/ yielded 8 adduct spots to total 11.4 X nucleotide. Adduct spots 3,4,7, and 8 and part of spot 5 (70% of total adducts) appear to be in common between the two compounds. Interestingly, 2-OHE2 was activated to a greater extent by HRP than 2-OHE1, whereas the reaction of their corresponding quinones with DNA in the absence of enzyme showed the opposite effect, with El-
I
0 El-2,3-quinone
0 E2-2,3-quinone 80
c
-1
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TIME (min)
I
I
I
100
I
I
Ih
0 El-3,4-quinone 0 E,-3,4-quinone
80
Y z3
60
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4o
4
20
0 0
I
I
I
I
I
90
180
270
360
450
IF
24(h)
TIME (min)
Figure 3. Stability of (A) El- and E2-2,3-quinoneend (B)Eland E2-3,4-quinone. Table I. 32P-Postlabeling Analysis of DNA Adducts adduct level adduct level compound x 107 compound x 107 2-OHE1 30.0 (7)" 4-OHE1 1.9 (8) El-2,3-quinone 85.9 (8) quinone 2.4 (6) 2-OHE2 92.6 (8) 4-OHEz 2.1 (5) E2-2,3-quinone 11.4 (8) Ez-3,4-quinone 1.1 (10) (1
Number of adduct spots.
2,3-quinone affording more DNA adducts than E2-2,3quinone. Both 4-OHE1 (Figure 4C) and 4-OHE2 (Figure 5C) yielded relatively smaller quantities of adducts than their 2-OH counterparts. A total of 8 adducts were detected with 4-OHE1 and 6 adducts for the corresponding El3,4-quinone (Figure 4D) a t relative adduct levels of 1.9 X lW7 and 2.4 X adductslnucleotide, respectively. Upon comparison, adduct spots 7 and 8 appear to be common and El-SP-quinone; they among the adducts of constitute 44% of the 4-OHE1 adducts and 64% of the E1-3,Cquinoneadducts. exhibited 5DNA adduct spots on 32P-postlabelinganalysis, while E2-3,4-quinone (Figure 5D) yielded 10 spots at relative adduct levels of
Dwivedy et al.
832 Chem. Res. Toxicol., Vol. 5, No. 6, 1992
Figure 6. Autoradiogramsof 3T-poastlabeledDNA (A) incubated alone or (B) incubated with HRP and H202. Autoradiograms were exposed for 18 h.
Fund, Inc., Chicago, IL, the Lincoln Family Foundation, Lincoln, NE, and USPHS Grant R01 CA44686 from the National Cancer Institute.
References Figure 4. Autoradiograms of 32P-postlabeled DNA containing adducts formed from (A) 2-OHE1 (2-h exposure); (B) E1-2,3quinone (1.75-h exposure);(C) 4-OHE1 (15-h exposure);and (D) E1-3,rl-quinone (15-h exposure).
Figure 5. Autoradiograms of 32P-postlabeled DNA containing adducts formed from (A) 2-OHEz (0.67-h exposure); (B) E2-2,3quinone (7.5-h exposure); (C) 4-OHE2 (10-h exposure); and (D) E2-3,4-quinone (10-h exposure).
2.1 X 10-7and 1.1 X 10-7adducts/nucleotide, respectively. Of these, the major adduct, spot 3, appears to be common between the two compounds; it constitutes 60% of the adducts formed by the CE, but only 9 % of those from the quinone. In conclusion, the quinones covalently bind to DNA, and many of the adducts obtained appear to be the same as those formed by peroxidatic activation of the corresponding CE. These results suggest that semiquinones and/or quinones are ultimate reactive intermediates in the peroxidatic activation of catechol estrogens.
Acknowledgment. We acknowledge with gratitude support of this research by the Wendy Will Case Cancer
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