Evidence for the DNA binding and adduct formation of estrone and 17β-estradiol after dimethyldioxirane activation

Chemico-Biological Interactions 110 (1998) 173 – 187

Evidence for the DNA binding and adduct formation of estrone and 17b-estradiol after dimethyldioxirane activation Fu-Li Yu a,*, Mian-Ying Wang a, Dong-Hui Li b, Wanda Bender a, Wei-Yun Zheng a a

Department of Biomedical Sciences, Uni6ersity of Illinois, College of Medicine at Rockford, 1601 Park6iew A6enue, Rockford, IL 61107, USA b Department of Clinical In6estigation, Uni6ersity of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA Received 19 September 1997; received in revised form 9 January 1998; accepted 13 January 1998

Abstract Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E1) and 17b-estradiol (E2) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957 – 1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E1 and E2 after activation are able to bind DNA. This paper reports that after DMDO activation, [3H]E1 and [3H]E2 were able to bind to both A-T and G-C containing DNAs. Furthermore, the formation of E1 –DNA and E2 – DNA adducts was detected by 32P-postlabeling analysis. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

* Corresponding author. Tel.: +1 815 3955680; fax: + 1 815 3955666; e-mail: [email protected] 0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-2797(98)00007-6

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Keywords: Dimethyldioxirane epoxidation; DNA binding; 17b-Estradiol; Estrone; postlabeling

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P-

1. Introduction Breast cancer leads all cancer incidence among US women, accounting for 30% of the 1997 estimated new cases in the USA, and is the second leading cause of cancer deaths, estimated at 44000 per year [1]. Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine [2 – 4] and breast cancers [5–10]. Since estrogens are required for the growth and development of the target tissues, it has been assumed that they are promoters for carcinogenesis [11 –13]. Many chemical carcinogens with diversified structures, including the polycyclic aromatic hydrocarbons [14–19], aflatoxin B1 (AFB1) [20 – 25], vinyl carbamate [26], etc., require metabolic activation to epoxide in order to initiate carcinogenesis. Dimethyldioxirane (DMDO) is a very versatile epoxide-forming oxidant [27]. It has been used for the activation by epoxidation of a variety of chemical carcinogens, including AFB1 [24,25,28], vinyl carbamate [26], and phenyl and 4-nitrophenyl vinyl ethers [29]. Recently, we found that estrone (E1) and 17b-estradiol (E2) could be activated by DMDO, resulting in the inhibition of liver nuclear and nucleolar RNA synthesis [30]. Since it has been shown that the AFB1 epoxides produced by DMDO in vitro are the same as the microsome P450 enzymes produced in vivo [31,32], and the epoxides so produced are mutagenic in bacteria and carcinogenic in animal tests [26,29], we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis [30]. Chemical carcinogenesis is a multistage process including initiation, promotion and progression [14 – 19]. Initiation involves the covalent binding of a chemical carcinogen to DNA, which is believed to be the critical first step in carcinogenesis [14– 19]. It is clear that, in order to further support the proposed hypothesis, evidence showing that estrogens after activation are able to bind to DNA is critical. Using several synthetic DNAs of different base content and sequence, this paper reports that [3H]E1 and [3H]E2 after DMDO activation are able to bind both A-T and G-C containing DNAs. Additionally, the E1 –DNA and E2 –DNA adducts are clearly detected by 32P-postlabeling analysis.

2. Materials and methods

2.1. Materials Estrone, 17b-estradiol, poly[d(I-C)], poly[d(A-T)], poly[d(G-C)], poly(dG) · poly(dC), poly(dC) and calf thymus DNA were all purchased from Sigma (St. Louis, MO). [2,4,6,7-3H]Estrone (80–120 Ci/mmol), [2,4,6,7-3H]estradiol (70– 120 Ci/mmol), guanosine 5%-[a-32P]triphosphate (3000 Ci/mmol), uridine 5%-

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[a-32P]triphosphate (3000 Ci/mmol) were purchased from Amersham Life Science (Arlington Heights, IL).

2.2. Acti6ation of E1 and E2 by DMDO The procedure using the specific epoxide-forming oxidant DMDO for the activation of E1 and E2 was essentially the same as described previously by Yu and Bender [30]. DMDO was prepared by the procedure of Adam et al. [27]. Briefly, aliquots of 1 ml of the freshly prepared DMDO were mixed with 1 mg E1 (3.70 mmol) or E2 (3.67 mmol) at room temperature for 60 min with gentle shaking. After activation, the residual DMDO in the reaction mixture was completely removed by vacuum and the activated E1 or E2 (the E1 or E2 epoxide group) was redissolved in the solvent dimethylsulfoxide (DMSO) before use. In parallel, tubes containing 1 mg of E1 or E2 were mixed with 1 ml of acetone and reacted the same way as DMDO at room temperature for 60 min and then vacuum dried. Again, it was redissolved in DMSO before use (the E1 or E2 control group).

2.3. Assay of E1 and the DMDO-acti6ated E1 on DNA-dependent RNA synthesis For the assay of E1 and the DMDO-activated E1 on DNA-dependent RNA synthesis, aliquots containing 200 mg (0.74 mmol) or an otherwise indicated amount of either E1 (control) or DMDO-activated E1 (E1 epoxide group) in 40 ml DMSO was reacted with 0.1 A260 unit DNA, i.e. poly[d(I-C)], poly[d(A-T)], poly(dG) · poly(dC) or poly(dC) in 20 ml H2O at room temperature for 30 min. Then, 0.5 ml of the RNA polymerase assay medium [22–25] containing 0.1 mCi [a-32P]GTP or [a-32P]UTP as required was added to the reaction mixture. RNA synthesis was initiated by the addition of 0.1 ml of free RNA polymerase [33,34], and incubated at 37°C with shaking for 30 min. The reaction was terminated by the addition of 3 ml of 10% trichloroacetic acid containing 1% pyrophosphate. The radioactive RNA was collected onto Whatman GF/C filters, which were washed and counted as before [22 – 25]. RNA synthesis was measured in pmol [32P]GMP or [32P]UMP incorporated per A260 unit DNA.

2.4. Binding of [ 3H]E1 and [ 3H]E2 epoxides to DNA One milligram of E1 (3.70 mmol) or E2 (3.67 mmol) containing 10 mCi [3H]E1 or [ H]E2 was reacted with either 1 ml of acetone (control group) or 1 ml of DMDO (epoxide group) at room temperature for 1 h. Aliquots of 0.2 ml (200 mg) of the reaction mixture from both the control and the epoxide groups were dried. It was then reacted with 10 mg DNA as indicated (Table 2), i.e. calf thymus DNA, poly[d(A-T)], poly[d(G-C)], poly(dG) · poly(dC) and poly[d(I-C)], in 20 ml H2O plus 40 ml DMSO (i.e. 66% DMSO final concentration) at room temperature for 30 min. In order to make the DNA recovery and subsequent optical density measurement easier, two reaction tubes were combined, and the DNA was precipitated with 2 ml of absolute alcohol in the presence of 0.1 volume of 30% sodium acetate (pH 5) for 3

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1 h at −20°C. The DNA precipitate was collected by centrifugation, and the pellet was then washed twice with 75% ethanol. Finally, it was dissolved in 0.5 ml of H2O for optical density measurement and scintillation counting. The binding of [3H]E1 or [3H]E2 to DNA was expressed in pmol per A260 unit or mg DNA as indicated.

2.5. Detection of E1 – DNA and E2 –DNA adducts by

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The preparations of the DMDO-activated E1 and E2 and the binding of them to poly[d(A-T)] or poly[d(G-C)] were the same as described in the preceding sections. Except for the present studies, E1 and E2 were not labeled. The poly[d(A-T)] and poly[d(G-C)] controls were prepared by reacting with acetone-treated E1 or E2 under identical conditions as the DMDO-activated E1 or E2 group. A total of eight samples, i.e. poly[d(A-T)] and poly[d(G-C)] treated with E1 controls, poly[d(A-T)] and poly[d(G-C)] treated with the DMDO-activated E1 groups, poly[d(A-T)] and poly[d(G-C)] treated with E2 controls, and poly[d(A-T)] and poly[d(G-C)] treated with the DMDO-activated E2 groups, were used for DNA adduct analyses using the nuclease P1-enhanced version of 32P-postlabeling technique [35]. Briefly, 1–2 mg sample and 0.25 mg internal standard were digested with a mixture of micrococcal nuclease and spleen phosphodiesterase at 37°C for 3.5 h to produce the normal and adducted 3%-nucleotides [35]. The normal 3%-nucleotides were then selectively dephosphorylated by nuclease P1 to nucleosides which were not substrates for the subsequent polynucleotide kinase (PNK) labeling [35]. The adducted 3%-nucleotides, which were resistant to nuclease P1 digestion, were then labeled with [g-32P]ATP by PNK to 5%-32P-labeled deoxyribonucleoside 3%,5%-bisphosphates [35]. The excess [g-32P]ATP was then removed by hydrolysis with potato apyrase. These labeled adducts were separated by anion-exchange thin-layer chromatography (TLC) on a polyethyleneimine – cellulose plate as described previously [36]. An internal standard adducted DNA, which was derived from DNA treated with a weak carcinogen dibenzo(a,j)acridine, was included in each sample to monitor the adequacy of enzyme digestion, efficiency of labeling, completeness of contact transfer and other experimental conditions. Before the two-dimensional mapping of adducts, normal nucleotides and residual [g-32P]ATP were removed by chromatography with 2.3 M sodium phosphate, pH 5.75, for overnight in direction 1 (D1). After autoradiography, a 2.4 cm strip was cut from each lane of the D1 chromatogram. The labeled nucleotides were contact-transferred to a 16.5 cm×13 cm TLC plate by a magnet transfer technique and separated by two-dimensional chromatography [37]. Kodak XAR-5 or Du Pont Cronex 4X-ray films and Du Pont Lightning Plus intensifying screens were used for detection of DNA adducts [36,37].

3. Results In order to further test our hypothesis, several synthetic DNAs with different base content and sequence were used to study the transcriptional effects of E1 before and after DMDO activation. The results, as shown in Table 1, indicate that

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DMDO activated E1 at a dose of 200 mg per 0.1 A260 unit DNA produced 68% inhibition of poly[d(I-C)], 50% of poly(dG) · poly(dC), 46% of poly[d(A-T)] and 47% of poly(dC) directed RNA synthesis under the experimental conditions used. To expand these studies, dose – response inhibition curves were obtained. Fig. 1 shows that the template function of poly[d(I-C)] was inhibited by 27, 37 and 56%, respectively, when 50, 100 and 200 mg DMDO-activated E1 per 0.1 A260 unit DNA were used. It is also clear from Fig. 1 that E1 without DMDO activation had no inhibitory effect. Fig. 2 shows that the template function of poly[d(A-T)] was inhibited 26, 30 and 51%, respectively, when 50, 100 and 200 mg DMDO-activated E1 per 0.1 A260 unit DNA were used. Again, E1 per se had little effect on the template function of poly[d(A-T)]. Fig. 3 shows the dose–response inhibition curve of poly(dG) · poly(dC). The results show that the template function was inhibited by 26, 31 and 50%, respectively when 50, 100 and 200 mg DMDO-activated E1 were used. Again, E1 without DMDO treatment had no inhibitory effect. The first step in the initiation of chemical carcinogenesis is believed to be the covalent binding of an ultimate carcinogen to DNA [14–19]. Although we have shown from correlation studies that the inhibition of DNA-dependent RNA synthesis is a direct reflection of the binding of a carcinogen to DNA [38,39], it is clearly necessary, in order to further support our hypothesis, to directly demonstrate that estrogens after DMDO activation are able to bind DNA. Table 2 summarizes the binding of DMDO-activated [3H]E1 to several DNAs with different base content and sequence. It is clear from these data that E1 after DMDO activation was able to bind both A-T and G-C containing DNAs. However, the data also indicate that, although the best substrate for the binding was calf thymus DNA, there was a preferential binding of DMDO-activated E1 to A-T over G-C Table 1 Transcriptional effect of DMDO activated estrone (E1) on several single- and double-stranded DNA templates Template

Activated E1

RNA polymerase activitya (pmol [a-32P]XMP incorporated/A260)

%

Poly[d(I-C)

– + – + – + – +

11491 91507 3673 9498 784 9 18 427 9 24 5795 9 797 2866 9578 1441 9154 767 9165

100 32 100 54 100 50 100 53

Poly[d(A-T)] Poly(dG) · poly(dC) Poly(dC)

DMDO-activated E1 (200 mg) in 40 ml DMSO was reacted with 0.1 A260 unit DNA in 20 ml H2O at room temperature for 30 min. Control contained 200 mg E1. RNA synthesis was assayed with 0.5 ml assay medium containing 0.1 mCi [a-32P]GTP or [a-32P]UTP as required with 0.1 ml free enzyme for 30 min at 37°C. Values given are means 9S.E. of 2 – 5 separate experiments.

a

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Fig. 1. Effect of dimethyldioxirane (DMDO)-activated estrone (E1) on the template function of poly[d(I-C)]. E1, 1 mg, was reacted with 1 ml of acetone (control group), or 1 ml of the freshly prepared DMDO (DMDO-activated E1 group) at room temperature for 1 h. Then, 50 ml (50 mg), 100 ml (1OO mg) and 200 ml (200 mg) aliquots in duplicate were taken from each group and dried. To each tube, a mixture containing 20 ml (0.1 A260) poly[d(I-C)] in H2O and 40 ml of dimethylsulfoxide (DMSO) was added and reacted at room temperature for 30 min, followed with 0.5 ml of the RNA assay medium containing 1 mCi [a-32P]GTP. The RNA synthesis was started by the addition of 0.1 ml of the rat liver nuclear free RNA polymerase, and the synthesis was carried out at 37°C for 30 min with shaking. E1 control, ; DMDOactivated E1, “. Values given are the average of 2 – 3 separate experiments.

bases in DNA. With the dose used at 200 mg per 10 mg DNA, the binding affinity to poly[d(A-T)] was about two to three times higher than to G-C containing DNAs, e.g. poly[d(G-C)] and poly(G) · poly(dC). It should be mentioned that the binding values of E1 without activation were about 2–5% of the corresponding values of the activated E1, and these values were subtracted from the calculations as the unspecific binding background. Fig. 4 shows the dose-dependent binding curves of [3H]E1 after DMDO activation to calf thymus DNA, poly[d(A-T)] and poly[d(G-C)]. In good agreement with the results obtained in Table 2, E1 bound preferentially to calf thymus DNA over the poly[d(A-T)] and poly[d(G-C)]. When 50, 100 and 200 mg DMDO-activated E1 per 10 mg DNA were used, there were respectively 10000, 15300 and 26900 pmol E1 bound per mg calf thymus DNA. The corresponding binding values were 800, 7100 and 24000 for poly[d(A-T)], and 1400, 3100 and 5500 for poly[dG-C)]. Also, it is clear from these binding curves that the dramatic effect of the preferential binding to A-T over G-C bases in DNA by E1 after DMDO activation is seen only at the higher doses. This is because that, while the binding of DMDO-activated E1 to poly[d(G-C)] is almost linear throughout, the binding to poly[d(A-T)] is exponential with increasing doses. In this regard, the shape of the dose-dependent binding curve of poly[d(A-T)] is quite similar to that of calf thymus DNA.

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Fig. 5 shows the dose-dependent binding curves of DMDO-activated E2 to the above-mentioned three DNAs. As E1 in Fig. 4, calf thymus DNA again was the preferred DNA for the binding. At 50, 100 and 200 mg E2 after DMDO activation, the binding values were 4500, 7500 and 23100 pmol E2 bound per mg. The corresponding binding values for poly[d(A-T)] were 400, 2900 and 15000 and for poly[d(G-C)] were 1600, 4200 and 6300 pmol bound per mg, respectively. Again, the preferential binding to poly[d(A-T)] over poly[d(G-C)] was observed only at the high dose. In order to further confirm that E1 and E2, after activation, are able to bind DNA and form E1 – DNA and E2 –DNA adducts, 32P-postlabeling was used to detect these adducts. Fig. 6 shows the 32P-postlabeling maps of the E1-treated poly[d(A-T)] control (panel A), DMDO-activated E1-treated poly[d(A-T)] (panel B), E1-treated poly[d(G-C)] control (panel C) and poly[d(G-C)] reacted with DMDO-activated E1 (panel D). Since the intensities of the radioactive spots in the control groups were very low, the films for the control groups (i.e. panels A and C) were exposed for 16 h at −80°C. The films for the epoxide groups (i.e. panels B and D) were exposed only for 6 h. As shown in panel B, there were eight detectable DNA adducts (marked as dotted line cycles) from the DMDO-activated E1 -treated poly[d(A-T)] sample. Among these, spots 4, 5 and 6 were the major adducts. There

Fig. 2. Effect of dimethyldioxirane (DMDO)-activated estrone (E1) on the template function of poly[d(A-T)]. The conditions to activate E1 by DMDO and the subsequent assays of the DNA template function for RNA synthesis were the same as described in Fig. 1, except in this study poly[d(A-T)l and [a-32P]UTP were used. E1, ; DMDO-activated E2, “. Values given are the average of 2 – 3 separate experiments.

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Fig. 3. Effect of dimethyldioxirane (DMDO)-activated estrone (E1) on the template function of poly(dG) · poly(dC). The conditions to activate E2 by DMDO and the subsequent assays of the DNA template function were the same as described in Fig. 1, except in this study poly(dG) · poly(dC) and [a-32P]GTP were used. E1, ; DMDO-activated E1, “. Values given are the average of 2 – 4 separate experiments.

were 11 detectable DNA adducts from the DMDO-activated E1-treated poly[d(GC)] sample. Spots 1, 2, 5, 6 and 10 were the major adducts of this group. The spot marked ‘IS’ was the internal standard DNA adduct marker. Fig. 7 shows the 32P-postlabeling patterns of E2-treated poly[d(A-T)] control (panel A), DMDO-activated E2-treated poly[d(A-T)] (panel B), E2-treated poly[d(G-C)] control (panel C) and DMDO-activated E2-treated poly[d(G-C)] (panel D). Again, the films for the control groups were exposed for 16 h at −80°C, and the DMDO-activated groups were exposed for 6 h. As shown in panel B, there Table 2 Binding specificities of ([3H]E1) after dimethyldioxirane activation to several DNAs with different base content and sequence Template

pmol/A260

%

pmol/mg

%

Calf thymus DNA Poly[d(A-T)] Poly[d(G-C)] Poly(dG) · poly(dC) Poly[d(I-C)]

9469133 7949 35 2299 8 345959 4399 55

100 84 24 36 46

21709 93114 14438 9814 4033 9202 6456 91075 5698 9863

100 67 19 30 26

a

Values given are mean 9 S.E. of three separate experiments.

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Fig. 4. Binding of dimethyldioxirane (DMDO)-activated 3H-labeled estrone ([3H]E1) to calf thymus DNA, poly[d(A-T)] and poly[d(G-C)]. [3H]E1 control and DMDO-activated E1 groups were prepared the same way as described in Fig. 1. DNA, 10 mg as indicated, was reacted with 50, 100 or 200 mg [3H]E1 or DMDO-activated E1 in 66% DMSO at room temperature for 30 min. The DNA was then precipitated and washed with alcohol. The DNA precipitate was finally dissolved in H2O for radioactivity counting. The control values, ranging from 2 to 5% of the DMDO-activated groups, were subtracted as the background counts. Calf thymus DNA, ; poly[d(A-T)], “; poly[d(G-C)], ×. Values given are the average of 2–4 separate experiments.

were 11 detectable DNA adducts from the activated E2-treated poly[d(A-T)] sample. Spots 5, 7, 8 and 11 were the major adducts. There were nine detectable DNA adducts from the activated E2-treated poly[d(G-C)] sample. Spots 1, 5, 6 and 7 were the major adducts.

4. Discussion In animal studies, it has been clearly demonstrated that estrogens can induce mammary [12,40] and renal tumors [12,41]. Epidemiological evidence suggests that estrogen is a risk factor in endometrial [2–4] and breast cancers [5–10]. There are three working hypotheses to explain the carcinogenic properties of estrogens. Since estrogens are required for the growth and development of the target tissues, they are believed to be the promoters for carcinogenesis [11–13]. The second hypothesis believes that estrogens go through the metabolic redox cycles between the semiquinone and quinone forms and the free radicals generated can cause oxidative damage to DNA. Moreover, it is believed that the semiquinone and quinone forms

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of estrogens can covalently bind and form DNA adducts [42,43]. The third hypothesis proposed by this laboratory 2 years ago [30] suggests that the aromatic A ring of E1 and E2, like many of the well-known chemical carcinogens including AFB1 [20 – 25], and the polycyclic aromatic hydrocarbons [14–19], can be activated to epoxide and to bind DNA and to initiate carcinogenesis (Fig. 8). Three independent lines of experimental evidence supporting our hypothesis are presented in this report. The results presented in Table 1 and Figs. 1–3 indicate clearly that E1 per se is not able to inhibit RNA synthesis. However, after DMDO activation, E1 is able to inhibit in a dose-dependent manner of DNA-dependent RNA synthesis of both A-T and G-C containing DNAs. Using [3H]E1 and after DMDO activation, the data in Table 2 show that the activated E1 is able to bind DNA. The data also indicate that, although calf thymus DNA is the best substrate for the binding, the activated E1 binds preferentially to A-T over G-C containing DNAs. However, the results presented in Figs. 4 and 5 indicate that there is no clear binding preference of the DMDO-activated [3H]E1 or [3H]E2 to the DNA bases at lower doses. In this regard, it should be mentioned that this property of the DMDO-activated E1 and E2 is quite different from the DMDO-activated AFB1 ,

Fig. 5. Binding of dimethyldioxirane (DMDO)-activated 3H-labeled 17b-estradiol ([3H]E2) to calf thymus DNA, poly[d(A-T)] and poly[d(G-C)]. Control and DMDO-activated [3H]E2 groups were prepared the same way as described in Fig. 1. DNA, 10 mg as indicated, was reacted with 50, 100 and 200 mg [3H]E2 or DMDO-activated E2 in 66% DMSO at room temperature for 30 min. The DNA was then precipitated and washed with alcohol. The DNA precipitate was finally dissolved in H2O for radioactivaty counting. The control values, ranging from 2 to 5% of the epoxide groups, were substracted as the background counts. Calf thymus DNA, ; poly[d(A-T)], “; poly[d(G-C)], × . Values given are the average of 2–3 separate experiments.

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Fig. 6. 32P-postlabeling maps of the estrone (E1) and DMDO-activated E1-treated poly[d(A-T)] and poly[d(G-C)]. The conditions used for the initial binding of E1 and DMDO activated E1 were the same as described in Figs. 4 and 5, except E1 in this case was not labeled. The nuclease P1-enchanced version of the 32P-postlabeling procedure was used to detect DNA adducts. (A) E1-treated poly[d(A-T)]; (B) DMDO-activated E1-treated poly[d(A-T)]; (C) E1-treated poly[d(G-C)]; (D) DMDO-activated E1-treated poly[d(G-C)]. Since the intensities of the radioactive spots from the control groups were very low, the films of A and C were exposed for 16 h at − 80°C. In contrast, the films of B and D were exposed for only 6 h. The spot marked ‘IS’ was the internal standard DNA adduct marker.

which binds preferentially to G-C containing DNAs at all dose levels studied [22– 24]. Also, it should be mentioned that, from our earlier studies, we found the binding values for DMDO-activated AFB1 to poly[d(G-C)], poly(dG) · poly(dC) and poly[d(I-C)] were respectively 12117, 9903 and 5509 pmol/A260 unit DNA [24]. And the binding values, as shown in Table 2, for DMDO-activated E1 to poly[d(G-C)], poly(dG) · poly(dC) and poly[d(I-C)] were 229, 345, 439 pmol/A260 unit DNA, respectively. Furthermore, for the DMDO-activated AFB1 binding studies, a ratio of 8 mg (0.026 mmol) of the activated AFB1 per mg DNA was used [24], compared

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to a ratio of 20 mg (0.074 mmol) of the activated E1 per mg DNA for the present study. Taking all the above evidence into consideration, it can be estimated that the binding ability of the DMDO-activated E1 to DNA is one or two orders of magnitude less than the DMDO-activated AFB1. A similar conclusion has been reached based on the ability in the inhibition of DNA-dependent RNA synthesis after DMDO activation [30]. Since AFB1 is the most potent chemical carcinogen known [44], and if the ability of binding to DNA after activation is an indicator of the potency of a carcinogen, these results suggest that E1 and E2 are relatively weak carcinogens. Finally, the results from 32P-postlabeling studies have firmly established the fact that E1 and E2 after DMDO activation are able to bind covalently to DNA and to

Fig. 7. 32P-postlabeling maps of the 17b-estradiol (E2) and DMDO-activated E2-treated poly[d(A-T)] and poly[d(G-C)]. The conditions used for the initial binding of E2 and DMDO-activated E2 were the same as described in Figs. 4 and 5, except E2 was not labeled. For 32P-postlabeling studies, the conditions used were the same as described in Fig. 6. (A) E2-treated poly[d(A-T)]; (B) DMDO-activated E2-treated poly[d(A-T)]; (C) E2-treated poly[d(G-C)]; (D) DMDO-activated E2-treated poly[d(G-C)].

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Fig. 8. Estrogen epoxidation as the initiation mechanism of carcinogenesis.

form DNA adducts. These results have therefore provided the strongest evidence so far in the support of our hypothesis [30]. It is not surprising to see, as shown in Figs. 6 and 7, that multiple DNA adduct spots are detected from both of the activated E1- and E2-treated poly[d(A-T)] and poly[d(G-C)] templates. There are at least two possible reasons to explain these results. (1) Previous studies on AFB1 have established that epoxidation by either microsomal P450 enzymes or DMDO is not exclusively stereospecific [31,32]. After AFB1 epoxidation both exo- and endoepoxides are formed [31,32]. If what we believe is true—i.e. DMDO activation of E1 and E2 is in reality the conversion of E1 and E2 to epoxides—it is conceivable that the stereoisomers of E1 and E2 epoxides may also be formed after DMDO activation and these stereoisomers after reacting with DNA should produce various isomeric DNA adducts. (2) It is known that there are multiple target sites on each of the four DNA bases vulnerable to electrophilic attack by carcinogen epoxides [19]. This may be another reason why multiple E1 –DNA and E2 –DNA adducts are seen. Electron-density surrounding the target bases and the steric configurations between the target sites and the attacking E1 and E2 epoxides may be the important determining factors for the formation of major and minor DNA adducts. In conclusion, a hypothesis has been proposed suggesting that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis [30]. We are encouraged by the fact that the experimental evidence accumulated so far has been strongly supportive. Realizing the complexity of tumorigenesis in general, and breast cancer in particular, it is our hope that this hypothesis will stimulate further study on the potential initiation role of these estrogens in carcinogenesis and in the induction of breast cancer.

Acknowledgements This investigation was supported by PHS Grant Number CA-70466 awarded by the National Cancer Institute DHHS (F.L.Y.), and a grant from the University of Texas, M.D. Anderson Cancer Center Breast Cancer Research Program (D.H.L.).

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