The ability of four catechol estrogens of 17ß-estradiol and estrone to induce DNA adducts in Syrian hamster embryo fibroblasts

Eiichi Yagi, J.Carl Barrett1,2 and Takeki Tsutsui

Department of Pharmacology, The Nippon Dental University, School of Dentistry at Tokyo, Tokyo 102-8159, Japan and
1 National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA and National Cancer Institute, Bethesda, MD 20892, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Catechol estrogens are considered critical intermediates in estrogen-induced carcinogenesis. We demonstrated previously that 17ß-estradiol (E2), estrone (E1) and four of their catechol estrogens, 2- and 4-hydroxyestradiols (2- and 4-OHE2), and 2- and 4-hydroxyestrones (2- and 4-OHE1) induce morphological transformation in Syrian hamster embryo (SHE) fibroblasts, and the transforming abilities vary as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 E2, E1. To examine the involvement of catechol estrogens in the initiation of hormonal carcinogenesis, we studied the ability of E2, E1 and their catechol estrogens to induce DNA adducts in SHE cells by using a 32P-post-labeling assay. DNA adducts were detected in cells treated with each of all the catechol estrogens at concentrations of 10 µg/ml for 1 h and more. 2- or 4-OHE2 formed a single DNA adduct, which was chromatographically distinct from each other. In contrast, 2- or 4-OHE1 produced one major and one minor adduct, and the two adducts formed by each catechol estrogen exhibited identical mobilities on the chromatograms. Neither E2 nor E1 at concentrations up to 30 µg/ml induced DNA adducts. The abilities of the estrogens to induce DNA adducts were ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 > > E2, E1, which corresponds well to the transforming and carcinogenic abilities of the estrogens. In addition, the level of DNA adducts induced by the catechol estrogens was markedly decreased by co-treatment of cells with the antioxidant L-ascorbic acid. The results indicate the possible involvement of oxidative metabolites of catechol estrogens of E2 and E1 in the initiation of endogenous estrogen-induced carcinogenesis.

Abbreviations: E2, 17ß-estradiol; E1, estrone; 4-OHE2, 4-hydroxyestradiol; 4-OHE1, 4-hydroxyestrone; 2-OHE2, 2-hydroxyestradiol; SHE, Syrian hamster embryo; DES, diethylstilbestrol; 2-OHE1, 2-hydroxyestrone; DMSO, dimethyl sulfoxide; B[a]P, benzo[a]pyrene; CFE, colony-forming efficiency


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Estrogens are carcinogenic in humans and rodents (1,2), but the mechanisms by which these hormones induce cancer are not fully elucidated (3). Epigenetic mechanisms of estrogens, related to stimulation of cell proliferation, mediated through the estrogen receptor are not sufficient to explain the carcinogenic activity of estrogens in vivo and in vitro (4). Accumulating evidence has suggested that another mechanism related to genetic alterations is involved in the initiation of estrogen-induced carcinogenesis (4,5). The mutagenic mechanism of estrogens, in conjunction with the epigenetic mechanism, may contribute to hormonal carcinogenesis.

Endogenous estrogens are implicated as a possible etiological factor in the causation of certain types of human cancers such as breast, endometrium, ovary, prostate and, possibly, brain cancers (6). Catechol estrogens, particularly the 4-hydroxy metabolites of 17ß-estradiol (E2) and estrone (E1), are possible critical intermediates in estrogen-induced cancer. Exposure for 6–10 months by implantation of 4-hydroxyestradiol (4-OHE2) or 4-hydroxyestrone (4-OHE1) induces renal carcinomas in male Syrian hamsters (7,8). Administration of E2, 2-OHE2 or 4-OHE2 for 12–18 months also induces uterine adenocarcinomas in mice, and the carcinogenic abilities of these estrogens are ranked as follows: 4-OHE2 > 2-OHE2 > E2 (9). The oxidative metabolites of 2- or 4-OHE1(E2), i.e. E1(E2)-2,3 or 3,4-quinones, covalently bind to calf thymus DNA and form DNA adducts (10). Cavalieri et al. (11) found that intramammillary injection of 4-OHE2 or E2-3,4-quinone results in DNA adducts in mammary glands of female Sprague–Dawley rats. Treatment of Syrian hamster embryo (SHE) fibroblasts, which have endogenous oxidative and peroxidative metabolizing enzymes, with 2-OHE2 or 4-OHE2 induces DNA adducts, in parallel with the induction of cellular transformation (12). These results suggest that oxidative metabolites of catechol estrogens play an important role in the initiation of endogenous estrogen-induced cancers.

Chemically induced DNA damage that may initiate various cancers can be examined by detection of DNA adducts through covalent modification of the DNA. Liehr et al. (13,14) demonstrated the presence of covalent DNA adducts in the premalignant lesion in the kidneys of male Syrian hamsters treated with E2 or diethylstilbestrol (DES) by using a 32P-post-labeling assay. Adduct formation in the kidney DNA is detected at 1–3 months but not at 2 weeks after the beginning of treatment. DNA adducts are also detected in kidney, liver and other organs of hamsters 4 h after treatment with an i.p. injection of a single dose of DES (15). Although these data support a genotoxic mechanism of E2 or DES, the correlation between DNA adduct formation and the initiation of the estrogen-induced carcinogenesis still remains to be clarified.

We have used SHE fibroblast cell cultures as a model system to study the ability of estrogens to transform cells directly (4). An advantage of the SHE cell model for studies of carcinogenesis is that cellular transformation and genetic effects can be measured in the same target cells (16). SHE cells do not express measurable levels of estrogen receptor, and estrogen treatment is not mitogenic to the cells (4). Thus, estrogenic stimulation of cell proliferation can be excluded as the mechanism of action for cellular transformation in this in vitro assay. The cells do, however, have the ability to metabolize estrogens (4). We have shown that treatment of SHE cells with E2, E1 or four of their catechol estrogens induces cellular transformation. The transforming abilities of the estrogens vary with the following rank order: 4-OHE1 > 2-hydroxyestrone (2-OHE1) > 4-OHE2 > 2-OHE2 >= E2, E1 (17). In the present study, to examine a direct involvement of catechol estrogens in the initiation of estrogen-induced cell transformation, we studied the abilities of these estrogens to induce DNA adducts in SHE cells by using a 32P-post-labeling assay, and compared these findings with the transforming abilities.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cells and chemicals
SHE cell cultures were established from 13-day gestation Syrian hamster fetuses and grown as described previously (16). E2, 2-OHE2, 4-OHE2, E1, 2-OHE1 and 4-OHE1 were purchased from Sigma (St Louis, MO). E1 and E2 were dissolved with dimethyl sulfoxide (DMSO; Sigma) at 10 mg/ml. Their catechol estrogens and benzo[a]pyrene (B[a]P; Sigma) were dissolved with DMSO at 3 and 1 mg/ml, respectively. These solutions were diluted with complete medium to the final concentrations. L-Ascorbic acid phosphate magnesium salt n-hydrate (L-ascorbic acid; Wako Pure Chemical, Osaka, Japan) was dissolved with complete medium and filter-sterilized.

Cytotoxicity
The cytotoxicity of test estrogens on SHE cells was determined from the colony-forming efficiency (CFE) of cells treated with the estrogens. Cells (5x105) were plated into 75-cm2 flasks (Costar, Cambridge, MA), incubated for 2 days, and treated with various test estrogens at 1–10 µg/ml (~3.5– 37 µM) for 6 h. Control cultures were incubated with medium containing 0.33% DMSO (DMSO medium); this concentration did not affect the CFE as compared with untreated cells. After cells were trypsinized, 2000 cells were replated, in triplicate, onto 100-mm dishes (Costar) and incubated for 7 days for colony formation. Cells were fixed with absolute methanol and stained with a 10% aqueous Giemsa solution. The number of surviving colonies with >50 cells was then counted and the percent cell survival expressed as the number of colonies in the treated dishes divided by the number in control dishes x100. Statistical analysis was performed by Student's t-test.

Isolation of DNA and 32P-post-labeling
Cells (5x105) were plated into 75-cm2 flasks, incubated for 2 days and treated with test estrogens at varying concentrations for 1–6 h. Exposure of cells to B[a]P with the same treatment regimen was used as a positive control. Control cultures were incubated with DMSO medium, which did not affect DNA adduct formation as compared with untreated cultures. DNA from the treated cells was isolated as described previously (12).

DNA adducts were analyzed by the nuclease P1 enhancement version of the 32P-post-labeling assay (12,18). Briefly, 20 µg of isolated DNA was digested to deoxyribonucleoside 3'-monophosphates with 6 U of micrococcal nuclease (Worthington, Freehold, NJ) and 0.06 U of spleen phosphodiesterase (Worthington). The digest of DNA (10 µg) was then incubated with 4 mg nuclease P1 per ml (Sigma) for 1 h at 37°C and subsequently 32P-labeled with a mixture of 5 U of T4 polynucleotide kinase (USB, Cleveland, OH) and 21 pmol of [{gamma}-32P]ATP (259 TBq/mmol, ICN, Irvine, CA) for 1 h at 37°C. Unreacted [{gamma}-32P]ATP was digested with 3.3 U/ml apyrase for 45 min at 37°C. Labeled DNA adducts were mapped by chromatography on polyethyleneimine (PEI) cellulose sheets (Polygram CEL 300 PEI, Machery-Nagel, Düren, Germany) and developed with 2.3 M sodium phosphate buffer (pH 6.0) (D1). Adducts remaining at the original spot were contact-transferred to a new PEI cellulose sheet and developed in 3.8 M lithium formate/6.8 M urea (pH 3.5) (D3) and 0.8 M lithium chloride/0.5 M Tris–HCl/8.5 M urea (pH 6.8) (D4). Adducts were located by autoradiography on X-ray film (Fuji Photo Film, Tokyo, Japan) with intensifying screens (Konica, Tokyo, Japan) for 24 h at –80°C. For quantitative estimation of adduct levels, located adducts were excised and counted by the Cerenkov counting assay using comparable background areas for control counts as described by Reddy and Randerath (18). All experiments were performed two or three times, and the results obtained were reproducible.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytotoxicity
Relative cell survivals following treatment of SHE cells with E2, E1 or their catechol estrogens at 1–10 µg/ml for 6 h are shown in Figure 1Go. Cell survivals were not decreased by treatment with E2 at any concentration (Fig. 1AGo). However, statistically significant decreases in cell survivals were elicited by treatment with 2- or 4-OHE2 in a concentration-dependent manner (Figure 1B and CGo). When 2- or 4-OHE2 was administered to cells at 1 or 3 µg/ml, the cytotoxicities, as determined by cell survivals, were similar with the both catechol estrogens. However, 2-OHE2 was more cytotoxic than 4-OHE2 when administered at 10 µg/ml (Figure 1B and CGo). Similar results were observed in cells treated with E1 or its catechol estrogens (Figure 1D–FGo).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Relative cell survival of SHE cells following treatment with various estrogens for 6 h. (A) E2; (B) 2-OHE2; (C) 4-OHE2; (D) E1; (E) 2-OHE1; (F) 4-OHE1. The actual CFE of control cells was 9.3 ± 1.3 (SD)%. Each symbol represents the mean of three determinations. Bars denote SD. When not indicated, SD are within the symbol. *Significantly different from control (P<0.05, t-test). **Significantly different from control (P<0.01, t-test).

 
DNA adduct formation
When SHE cells were treated for 6 h with 1 µg/ml B[a]P as a positive control, DNA from the cells exhibited a number of extra spots on the chromatogram not seen in the control cells (Figure 2A and BGo). In contrast, DNA adducts were not detected in SHE cells treated with E2 at 10 and 30 µg/ml for 6 h (Figure 2C and DGo). No detectable adducts were formed in DNA from cells treated with E1 at 10 and 30 µg/ml for 6 h (Figure 2E and FGo). DNA adducts were not induced even in cells treated with either E1 or E2 at 10 µg/ml for 24 h (data not shown). Treatment with E2 or E1 at 30 µg/ml for 24 h was too toxic to analyze DNA adduct formation.



View larger version (111K):
[in this window]
[in a new window]
 
Fig. 2. 32P-post-labeling analysis of DNA from SHE cells treated with either E2 or E1 at 10 or 30 µg/ml for 6 h. Autoradiography was performed for 24 h at –80°C. (A) Control cells; (B) cells treated with B[a]P at 1 µg/ml for 6 h; (C) and (D) cells treated with E2 at 10 and 30 µg/ml, respectively; (E) and (F) cells treated with E1 at 10 and 30 µg/ml, respectively.

 
On the other hand, SHE cells treated with 2- or 4-OHE2 at 10 µg/ml for 6 h exhibited a single DNA adduct (Figure 3A and BGo). The adduct spot produced by 2-OHE2 was observed at a different location on the chromatogram from that produced by 4-OHE2. To clarify the difference in the location, DNA from SHE cells treated with 2-OHE2 was analyzed by co-chromatography with DNA from cells treated with 4-OHE2. As shown in Figure 3CGo, the DNA adduct produced by 2-OHE2 (spot a) migrated faster than that induced by 4-OHE2 (spot b), indicating that both adducts can be resolved separately in co-chromatography experiments.



View larger version (140K):
[in this window]
[in a new window]
 
Fig. 3. 32P-post-labeling analysis of DNA from SHE cells treated with either 2- or 4-OHE2 at 10 µg/ml for 6 h. Autoradiography was performed for 24 h at –80°C. (A) DNA from cells treated with 2-OHE2; (B) DNA from cells treated with 4-OHE2; (C) a mixture of DNA samples shown in (A) and (B); (D) control cells. Three separate experiments were performed with similar results.

 
When cells were treated with 2- or 4-OHE1 at 10 µg/ml for 6 h, one major and one minor adduct were detected on the chromatograms (Figure 4Go). The levels of adducts produced by 4-OHE1 were greater than those induced by 2-OHE1. There were, however, no differences in the location of the two adduct spots between the cells treated with either 2- or 4-OHE1. These results were confirmed by co-chromatography with the mixture of DNAs from both cells treated with 2- or 4-OHE1 (data not shown).



View larger version (51K):
[in this window]
[in a new window]
 
Fig. 4. 32P-post-labeling analysis of DNA from SHE cells treated with either 2- or 4-OHE1 at 10 µg/ml for 6 h. Autoradiography was performed for 24 h at –80°C. (A) Control cells; (B) cells treated with 2-OHE1; (C) cells treated with 4-OHE1. Two separate experiments were performed with similar results.

 
We next examined the treatment time and concentration dependencies of DNA adduct formation. When cells were treated with 10 µg/ml 4-OHE, for 1, 3 or 6 h, two adduct spots were detected as described above. Quantitative estimation by Cerenkov counting was performed to compare the levels of DNA adducts. The total number of two DNA adducts formed in the 1, 3 or 6 h-treatment group was estimated to be 1.89, 3.81 or 4.00 adducts in 108 nucleotides, respectively. Treatment of cells with 4-OHE1 at 0.3–10 µg/ml for 6 h induced DNA adducts in a concentration-dependent manner as shown in Figure 5Go. The same results were obtained with 2-OHE1. Although DNA adducts were not detected in cells treated with 2-OHE1 at 0.3 or 1 µg/ml for 6 h, treatment of cells with 2-OHE1 at 3 or 10 µg/ml elicited DNA adducts in a concentration-dependent manner. To compare the ability of estrogens to induce DNA adducts in SHE cells, the levels of DNA adducts induced were quantitatively estimated by Cerenkov counting (Table IGo). When compared, the total number of DNA adducts in 108 nucleotides in cells treated with various estrogens at 10 µg/ml for 6 h, the capability of inducing DNA adducts was ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 > > E2, E1.



View larger version (110K):
[in this window]
[in a new window]
 
Fig. 5. 32P-post-labeling analysis of DNA from SHE cells treated with varying concentrations of 4-OHE1 for 6 h. Autoradiography was performed for 24 h at –80°C. (A) Control cells; (B) 0.3 µg/ml; (C) 1 µg/ml; (D) 3 µg/ml; (E) 10 µg/ml. Two separate experiments were performed with similar results.

 

View this table:
[in this window]
[in a new window]
 
Table I. The levels of DNA adducts induced by E2, E1 or its catechol metabolites. SHE cells were treated with varying concentrations of E2 or its metabolites for 6 h
 
Effect of L-ascorbic acid on the levels of catechol estrogen-induced DNA adducts
When SHE cells were treated with 0.25 mM L-ascorbic acid for 6 h, no DNA adducts were detected as compared with the control cells (Figure 6BGo). DNA adducts induced by treatment of cells with 10 µg/ml 2-OHE2 for 6 h were markedly decreased by co-treatment with 0.25 mM L-ascorbic acid (Figure 6C and DGo). The co-treatment with 2-OHE2 and L-ascorbic acid decreased the level of DNA adducts by 23% compared with that of adducts induced by 2-OHE2 alone. A slight level of DNA adducts was observed even when cells were co-treated with 2.5 mM L-ascorbic acid (data not shown). The same results were observed in cells treated with 10 µg/ml 4-OHE2 in the presence of 0.25 mM L-ascorbic acid (Figure 6E and FGo). Co-treatment of cells with L-ascorbic acid also profoundly decreased DNA adducts induced by 2- or 4-OHE1 at 10 µg/ml for 6 h (data not shown).



View larger version (110K):
[in this window]
[in a new window]
 
Fig. 6. 32P-post-labeling analysis of DNA from SHE cells treated with either 2- or 4-OHE2 at 10 µg/ml for 6 h in the absence or presence of 0.25 mM L-ascorbic acid. Autoradiography was performed for 24 h at –80°C. (A) Control cells; (B) cells treated with L-ascorbic acid alone; (C) cells treated with 2-OHE2 alone; (D) cells treated with 2-OHE2 in the presence of L-ascorbic acid; (E) cells treated with 4-OHE2 alone; (F) cells treated with 4-OHE2 in the presence of L-ascorbic acid. Two separate experiments were performed with similar results.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we examined the abilities of E2, E1 and four of their catechol metabolites to induce DNA adducts in SHE cells to investigate a direct involvement of DNA damage in the initiation of estrogen-induced cell transformation. DNA adducts were induced by treatment of cells with each of the catechol estrogens for 1 h and more, but, not by treatment with E2 or E1. The adduct-inducing abilities of the estrogens were ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2, indicating that the catechol estrogens of E1 have higher abilities than the catechol metabolites of E2 in SHE cells. Moreover, 4-OHE1 had a high ability compared with 2-OHE1. The same difference was observed between 4- and 2-OHE2. The transforming abilities of these estrogens in SHE cells (17) treated for 48 h at the concentrations that induced DNA adducts are ranked in the same order as the DNA adduct-inducing abilities. In addition, the adduct-inducing abilities correspond with the abilities of these catechol estrogens to induce chromosome aberrations and gene mutations at the Na+/K+ ATPase and/or hprt loci in SHE cells (17). These results indicate that genetic damage as detected by 32P-post-labeling can act as the initiator in estrogen-induced carcinogenesis. However, it is not clear whether the difference in the abilities to induce the genetic effects among the catechol estrogens is attributed to the difference in the intrinsic feature or the metabolic fate of the chemicals. In the hamster kidney tumor model, 4-hydroxy catechol estrogens are carcinogenic, whereas 2-hydroxy catechol estrogens are not (7,8). Liehr and his collaborators demonstrated that hamster kidney contains high estrogen 4-hydroxylase activity (19) and that the methylation of 4-OHE2 by catechol-O-methyltransferase is inhibited by 2-OHE2 in vitro (20). This may facilitate accumulation of 4-OHE2 in the hamster kidney. However, the lack of carcinogenic activity of 2-OHE2 in the hamster model can also be due to rapid methylation and rapid metabolic clearance of 2-OHE2 itself (21). Cavalieri et al. (11) showed that 2- or 4-hydroxy catechol estrogens of E2 and E1 are oxidized to E2(E1)-2,3- or 3,4-quinones and form DNA adducts in vitro and in vivo. Recently, Newbold and Liehr (9) have demonstrated that although the incidence is lower than that of 4-OHE2, 2-OHE2 also induces uterine adenocarcinoma in mice. In addition, our previous results showed that direct treatment of SHE cells with 2-hydroxy catechol estrogens (2-OHE2 and 2-OHE1) induces morphological transformation, chromosome aberrations and aneuploidy in the cells (17). These findings suggest an intrinsic carcinogenic and mutagenic activity of 2-hydroxy catechol estrogens.

The molecular characteristic of DNA adducts induced by 2- or 4-OHE2(E1) in SHE cells is unknown. The depurinating adduct 4-OHE2(E1)-1({alpha},ß)-N7 guanine is formed in vitro by reaction of E2(E1)-3,4-quinones with DNA or by activation of 4-OHE2(E1) with horseradish peroxidase, lactoperoxidase or cytochrome P450 in the presence of DNA (11). The same depurinating adduct is detected in rat mammary glands following exposure by intramammillary injection of 4-OHE2 or E2-3,4-quinone (11). When E1-2,3-quinone is reacted with deoxyguanosine or deoxyadenosine in vitro, 2-OHE1-6-N2 deoxyguanosine or 2-OHE1-6-N6 deoxyadenosine is formed, respectively (10). In SHE cells, a single DNA adduct was formed by 2- or 4-OHE2, whereas one major and one minor adduct were produced by 2- or 4-OHE1. Studies on the molecular characterization of the adducts formed in SHE cells are currently in progress.

The levels of DNA adducts induced by 4-OHE2(E1) are higher than those by 2-OHE2(E1). The differential levels of DNA adducts induced by 2- and 4-OHE2(E1) may come from dissociation of 2-OHE2(E1)-induced adducts from DNA because of high sensitivity to enzyme digestion during a 32P-post-labeling assay. This is, however, unlikely because the DNA adduct-inducing abilities of both catechol estrogens correlate well with the abilities of these estrogens to induce cellular transformation, chromosome aberrations and gene mutations in SHE cells as described above.

Although E2 and E1 are carcinogenic, they failed to induce DNA adducts in SHE cells. Administration by implantation of E2 for up to 9 months induces renal carcinoma in 100% of male Syrian hamsters (14). DNA adducts are detected in the kidney. The adduct levels are maximum at 5 months after initial E2 implantation but weak at 1 month (22), indicating that DNA adduction by E2 becomes manifest only after prolonged chronic exposure of animals to the estrogen. In SHE cells, E2 induces morphological transformation, but fails to induce any detectable gene mutations or chromosome aberrations over the concentration range that induces cellular transformation (17). The failure of E2 to induce gene mutations and chromosome aberrations is consistent with the lack of DNA adduct-inducing ability of E2, which suggests that SHE cells do not retain some metabolizing enzymes or that the exposure times are too short to induce the changes seen in vivo. E2 induces a specific type of genetic change, i.e. aneuploidy (17). Because aneuploidy and cellular transformation in SHE cells are mechanistically related (4), the ability of E2 to induce aneuploidy could participate in SHE cell transformation by E2. As far as we know, there is no report on the induction of DNA adducts by E1. E1 induces cellular transformation, chromosome aberrations and aneuploidy in SHE cells. However, gene mutations are not elicited in SHE cells by E1 (17). No mutagenic activity of E1 in Chinese hamster V79 cells is also noted by Drevon et al. (23). Clastogenic and/or aneugenic activities could play a role in the carcinogenic activity of E1.

Epidemiological studies have shown that L-ascorbic acid intake has the statistically significant inverse association with breast cancer risk (24). Additionally, L-ascorbic acid is not carcinogenic to rats or mice given 5% with diet for 2 years (25). Co-administration of L-ascorbic acid to E2- or DES-treated male Syrian hamsters reduces the incidence of renal carcinoma by ~50% (26). Concomitant administration of L-ascorbic acid and DES decreases the levels of DNA adducts by 70% in the kidney of hamsters and the in vitro concentrations of DES-4',4''-quinone, which is the reactive metabolic intermediate of DES (27). The inhibitory effect of L-ascorbic acid on hormonal carcinogenesis and DNA adduct formation may be a result of preventing oxidation of catechol estrogens or DES to their respective quinones and/or scavenging free radicals. In the present study, L-ascorbic acid decreased DNA adduct levels induced by 2- or 4-OHE2(E1) in SHE cells, indicating that oxidative intermediates of the catechol estrogens may contribute to DNA adduct formation in SHE cells. However, it is not clear whether oxidative products of the catechol estrogens bind to DNA directly, or whether free radicals generated by redox cycling between the catechol estrogens and the quinone intermediates participate in the adduct formation.

No growth-stimulating activities are observed in SHE cells treated with any of the estrogens examined in the present study (17), which is consistent with the findings that SHE cells do not express measurable levels of estrogen receptor (4). Newbold and Liehr (9) compared estrogenic potency and carcinogenic activity of catechol estrogens in neonatal mouse uteri. 2-OHE2 was less estrogenic but more carcinogenic than E2. The estrogenic activity of 4-OHE2 was 60% higher but its carcinogenic activity was 9-fold higher than E2. Their findings indicate that the estrogenicity is not sufficient to explain the carcinogenic activity of these catechol estrogens, and the involvement of genetic alterations in the catechol estrogen-induced carcinogenesis is also suggested.

In summary, we have demonstrated that catechol metabolites of E2 and E1 induce DNA adducts in SHE cells, but neither E2 nor E1 are active in inducing DNA adducts. The ability to induce DNA adducts correlated well with the transforming ability of these estrogens described previously (17). The levels of DNA adducts induced by the catechol estrogens were decreased by co-treatment of cells with L-ascorbic acid. The data indicate the involvement of oxidative metabolites of catechol estrogens of E2 and E1 in the initiation of endogenous estrogen-induced carcinogenesis.


    Notes
 
2 To whom correspondence should be addressed

Email: barrett{at}mail.nih.gov Back


    Acknowledgments
 
The authors are grateful to Ms Yukiko Tamura for her excellent technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. IARC (1979) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Sex Hormones(II), Vol 21. International Agency for Research on Cancer, Lyon.
  2. IARC (1999) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Hormonal Contraception and Post-menopausal Hormonal Therapy, Vol 72, International Agency for Research on Cancer, Lyon.
  3. Huff,J., Boyd,J. and Barrett,J.C. (1996) Cellular and Molecular Mechanisms of Hormone Carcinogenesis: Environmental Influences. Wiley-Liss, New York, NY.
  4. Tsutsui,T. and Barrett,J.C. (1997) Neoplastic transformation of cultured mammalian cells by estrogens and estrogenlike chemicals. Environ. Health Perspect., 105 (Suppl. 3), 619–624.[Medline]
  5. Liehr,J.G. (1997) Dual role of oestrogens as hormones and pro-carcinogens: tumour initiation by metabolic activation of oestrogens. Eur. J. Cancer Prev., 6, 3–10.[Medline]
  6. Zhu,B.T. and Conney,A.H. (1998) Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis, 19, 1–27.[Abstract]
  7. Liehr,J.G., Fang,W.-F., Sirbasku,D.A. and Ari-Ulubelen,A. (1986) Carcinogenicity of catechol estrogens in Syrian hamsters. J. Steroid Biochem., 24, 353–356.[Medline]
  8. Li,J.J. and Li,S.A. (1987) Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism. Fed. Proc., 46, 1858–1863.[Medline]
  9. Newbold,R.R. and Liehr,J.G. (2000) Induction of uterine adenocarcinoma in CD-1 mice by catechol estrogens. Cancer Res., 60, 235–237.[Abstract/Full Text]
  10. Stack,D.E., Byun,J., Gross,M.L., Rogan,E.G. and Cavalieri,E.L. (1996) Molecular characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides. Chem. Res. Toxicol., 9, 851–859.[Medline]
  11. Cavalieri,E.L.,Stack,D.E., Devanesan,P.D. et al. (1997) Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc. Natl Acad. Sci. USA, 94, 10937–10942.[Abstract/Full Text]
  12. Hayashi,N., Hasegawa,K., Komine,A., Tanaka,Y., McLachlan,J.A., Barrett,J.C. and Tsutsui,T. (1996) Estrogen-induced cell transformation and DNA adduct formation in cultured Syrian hamster embryo cells. Mol. Carcinogenesis, 16, 149–156.[Medline]
  13. Liehr,J.G., Randerath,K. and Randerath,E. (1985) Target organ-specific covalent DNA damage preceding diethylstilbestrol-induced carcinogenesis. Carcinogenesis, 6, 1067–1069.[Abstract]
  14. Liehr,J.G., Gladek,A., Macatee,T., Randerath,E. and Randerath,K. (1991) DNA adduct formation in liver and kidney of male Syrian hamsters treated with estrogen and/or {alpha}-naphthoflavone. Carcinogenesis, 12, 385–389.[Abstract]
  15. Gladek,A. and Liehr,J.G. (1989) Mechanism of genotoxicity of diethylstilbestrol in vivo. J. Biol. Chem., 264, 16847–16852.[Abstract]
  16. Barrett,J.C., Bias,N.E. and TS'O,P.O.P. (1978) A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation. Mutat. Res., 5, 121–136.
  17. Tsutsui,T., Tamura,Y., Yagi,E. and Barrett,J.C. (2000) Involvement of genotoxic effects in the initiation of estrogen-induced cellular transformation: studies using Syrian hamster embryo cells treated with 17ß-estradiol and eight of its metabolites. Int. J. Cancer, 86, 8–14.[Medline]
  18. Reddy,M.V. and Randerath,K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-post-labeling test for structurally diverse DNA adducts. Carcinogenesis, 7, 1543–1551.[Abstract]
  19. Weisz,J., Bui,Q.D., Roy,D. and Liehr,J.G. (1992) Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a potential pathway of metabolic activation of estrogens. Endocrinology, 131, 655–661.[Abstract]
  20. Roy,D., Weisz,J. and Liehr,J.G. (1990) The O-methylation of 4-hydroxyestradiol is inhibited by 2-hydroxyestradiol: implications for estrogen-induced carcinogenesis. Carcinogenesis, 11, 459–462.[Abstract]
  21. Roy,D. and Liehr,J.G. (1999) Estrogen, DNA damage and mutations. Mutat. Res., 42, 107–115.
  22. Liehr,J.G., Avitts,T.A., Randerath,E. and Randerath,K. (1986) Estrogen-induced endogenous DNA adduction: Possible mechanism of hormonal cancer. Proc. Natl Acad. Sci. USA, 83, 5301–5305.[Medline]
  23. Drevon,C., Piccoli,C. and Montesano,R. (1981) Mutagenicity assays of estrogenic hormones in mammalian cells. Mutat. Res., 89, 83–90.[Medline]
  24. Howe,G.R., Hirohata,T., Hislop,T.G., et al. (1990) Dietary factors and risk of breast cancer: combined analysis of 12 case-control studies. J. Natl Cancer Inst., 82, 561–569.[Abstract]
  25. Douglas,J.F., Huff,J. and Peters,A.C. (1984) No evidence of carcinogenicity for L-ascorbic acid (vitamin C) in rodents. J. Toxicol. Environ. Health, 14, 605–609.[Medline]
  26. Liehr,J.G. and Wheeler,W.J. (1983) Inhibition of estrogen-induced renal carcinoma in Syrian hamsters by vitamin C. Cancer Res., 43, 4638–4642.[Abstract]
  27. Liehr,J.G., Roy,D. and Gladek,A. (1989) Mechanism of inhibition of estrogen-induced renal carcinogenesis in male Syrian hamsters by vitamin C. Carcinogenesis, 10, 1983–1988.[Abstract]
Received January 8, 2001; revised May 14, 2001; accepted June 1, 2001.