Catechol estrogen metabolites and conjugates in mammary tumors and hyperplastic tissue from estrogen receptor-
knock-out (ERKO)/Wnt-1 mice: implications for initiation of mammary tumors
Prabu Devanesan1,
Richard J. Santen2,
Wayne P. Bocchinfuso3,
Kenneth S. Korach3,
Eleanor G. Rogan1 and
Ercole Cavalieri1,4
1 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6085,
2 Division of Hematology, Oncology and Endocrinology, Cancer Center, University of Virginia Health Science Center, Charlottesville, VA 22908 and
3 Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA
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Abstract
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A novel model of breast cancer was established by crossing mice carrying the Wnt-1 transgene (100% of adult females develop spontaneous mammary tumors) with the ERKO mouse line, in which mammary tumors develop despite a lack of functional estrogen receptor-
. To begin investigating whether metabolite-mediated genotoxicity of estrogens may play an important role in the initiation of mammary tumors, the pattern of estrogen metabolites and conjugates was examined in ERKO/Wnt-1 mice. Extracts of hyperplastic mammary tissue and mammary tumors were analyzed by HPLC with identification and quantification of compounds by multichannel electrochemical detection. Picomole amounts of the 4-catechol estrogens (CE) were detected, but their methoxy conjugates, as well as the 2-CE and their methoxy conjugates, were not. 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These preliminary findings show that the estrogen metabolite profile in the mammary tissue is unbalanced, in that the normally minor 4-CE metabolites were detected in the mammary tissue and not the normally predominant 2-CE. These results are consistent with the hypothesis that the mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations.
Abbreviations: CE, catechol estrogen(s); CE-Q, catechol estrogen quinone(s); Cys, cysteine; E1, estrone; E2, estradiol; ER, estrogen receptor; ERKO, estrogen receptor-
knock-out; GSH, glutathione; NAcCys, N-acetylcysteine; OHE1, hydroxyestrone; OHE2, hydroxyestradiol; -SG, glutathione moiety
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Introduction
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Breast cancer has been associated with exposure to estrogens. The standard paradigm states that estrogens, through receptor-mediated processes, stimulate proliferation of breast epithelial cells, rendering them more susceptible to genetic errors during DNA replication, with less time for DNA repair before cell division. If uncorrected, these mutations can ultimately lead to malignancy (1). While receptor-mediated processes undoubtedly play a role in the development and growth of tumors, specific oxidative metabolites of estrogens can be endogenous carcinogens that react with DNA to generate mutations with the potential to initiate cancer (2).
The biological effects of estrogenic hormones are mediated through estrogen receptor-
(ER-
) (3), although a second receptor, ER-ß, may mediate estrogen signaling in some tissues (47). The estrogen/ER-
signaling pathway may act via both autocrine and paracrine mechanisms. Thus, ER-
positive epithelial or stromal cells may respond to estradiol (E2) and transmit growth factor signals for proliferation to surrounding ER-
negative cells (8). To investigate the estrogen/ER-
signaling pathway, an ER-
knock-out (ERKO) mouse was developed (9).
The MMTVWnt-1 transgene is known to induce mammary hyperplasia and carcinomas in mice (1012). Almost all female mice with the MMTVWnt-1 transgene develop mammary hyperplasia and tumors, with about half developing tumors within 6 months (10). The MMTV-Wnt-1 transgene was introduced into ERKO mice to investigate the role of ER-
in mammary carcinogenesis (13). ER-ß was not detected in the breast tissue of these animals. The ERKO/Wnt-1 female mice developed mammary hyperplasia and tumors, with an average latency of 48 weeks, twice that of the Wnt-1 mice that produce ER-
(13). These results indicate that estrogen/ER-
-mediated signaling is not required for induction of mammary hyperplasia and tumors by Wnt-1 in ERKO/Wnt-1 mice. Preliminary data suggest that reduction of circulating estrogens by oophorectomy delays the onset of tumor development to 60 ± 11 weeks and reduces the incidence in ERKO/Wnt-1 mice (13). These observations suggest a role for E2 acting independently of its receptor. The mammary tumors observed in ERKO/Wnt-1 mice may be initiated by endogenous carcinogenic estrogen metabolites that react with DNA to generate oncogenic mutations (2).
The pathway leading to endogenous carcinogenic estrogen metabolites has been hypothesized (2,14) to involve metabolism of estrone (E1) and/or E2 to the carcinogenic catechol estrogens (CE) (15,16), 4-hydroxyestrone (4-OHE1) and/or 4-hydroxyestradiol (4-OHE2), as well as the non-carcinogenic CE (15,16), 2-OHE1 and/or 2-OHE2. The CE are further oxidized to quinones (CE-Q), which may react with DNA to form stable (by CE-2,3-Q) and depurinating (by CE-3,4-Q) adducts of adenine and guanine (2,14). Oncogenic mutations are thought to arise primarily from depurinating adducts (1719). In extrahepatic tissues, oxidation of CE to CE-Q is typically prevented by methylation of the CE, catalyzed by catechol-O-methyltransferase (20). If the CE-Q are formed, they can be neutralized by reaction with glutathione (GSH) (21) or be reduced back to CE, catalyzed by quinone oxidoreductase (22), protective mechanisms that reduce the possibility of the CE-Q reacting with DNA.
To examine the possibility that ERKO mice carrying the Wnt-1 transgene have abnormal metabolism of estrogens, leading to metabolites that induce oncogenic mutations in the mammary epithelium, the profile of estrogen metabolites and GSH conjugates was determined. Evidence supporting the hypothesis that mammary tumors in ERKO/Wnt-1 mice are initiated by endogenous carcinogenic CE-3,4-Q could include one, two or all of the following points: (i) relatively high levels of 4-CE and low levels of 2-CE, as in women with breast cancer (laboratory animals and humans, including women without breast cancer, normally have significantly higher levels of 2-CE than those of 4-CE) (23,24); (ii) little or no methylated CE, indicating a lack of protection of CE from further oxidation to CE-Q; and (iii) detection of CE-glutathione (-SG) conjugates, plus their products of hydrolysis and acetylation, namely, CE-cysteine (Cys) and CE-N-acetylcysteine (NAcCys) conjugates, respectively, which are obtained from the mercapturic acid biosynthesis pathway for the GSH conjugates (25); these conjugates demonstrate the formation of CE-Q in mammary tissue. In contrast, balanced estrogen metabolism can be broadly defined as relatively high levels of 2-CE compared with 4-CE, relatively high levels of methylated CE, and little or no CE-SG conjugates. Under these conditions, DNA damage by CE-3,4-Q is not expected.
Mammary hyperplastic tissue or tumor tissue from one female ERKO/Wnt-1 mouse or ovariectomized ERKO/Wnt-1 mouse was analyzed, or tissue from two mice was combined to give ~1 g of tissue. Because of the limited number of mice available, the mice ranged from 4 to 12 months of age. The samples were minced, ground to a fine powder in liquid nitrogen, suspended in 3 ml of 50 mM ammonium acetate, pH 5.0, and incubated for 6 h at 37°C with 10 000 U of ß-glucuronidase (Helix pomatia, Sigma, St Louis, MO, also containing 900 U of arylsulfatase). At the end of the incubation, methanol was added to achieve a final concentration of 60%, and the mixture was extracted with 8 ml of hexane to remove lipids. The aqueous phase was then diluted with 50 mM ammonium acetate, pH 4.0, containing 2 mg/ml ascorbic acid (to minimize oxidation of CE and CE conjugates), to an approximate final concentration of 25% methanol. It was applied to a Certify II Sep-Pak (200 mg cartridge, Varian, Palo Alto, CA). The cartridge was first eluted with 3 ml of pH 4.0 buffer, followed by elution with 2 ml each of 20, 40 and 70% methanol in buffer, and the three fractions were collected and analyzed by HPLC.
Analyses were carried out by using a Luna(2) C18 reverse-phase column (250x4.6 mm, 5 µm, Phenomenex, Torrance, CA) on an HPLC system equipped with dual ESA Model 580 solvent delivery modules, an ESA Model 540 autosampler and an 8-channel ESA CoulArray electrochemical detector (ESA, Chelmsford, MA). The oxidation potentials of the electrochemical cells were set at 20, 30, 70, 150, 280, 380, 470 and 540 mV for channels 18, respectively. A linear gradient from 100% acetonitrile/methanol/water/1 M ammonium acetate, pH 4.4 (15:5:70:10) to 90% acetonitrile/methanol/water/1M ammonium acetate, pH 4.4 (50:20:20:10) over 50 min at a flow rate of 1 ml/min was employed to separate the compounds. Estrogen metabolites and conjugates from the mice were identified and quantified by comparison with authentic standards, based on their retention time, oxidation potential and peak height ratios between the dominant peak and the preceding and trailing peaks in adjacent channels in the chromatogram (26). Data analyses were carried out by using ESA CoulArray software. This method is sufficiently sensitive to detect routinely 1 pmol of metabolites and conjugates injected in the column. The 2-CE and 4-CE, as well as their methoxy conjugates (27), and the 4-CE-2-SG, 4-CE-2-Cys and 4-CE-2-NAcCys conjugates (28) were synthesized for use as standards. The 2-CE-(1&4)-SG, 2-CE-(1&4)-Cys and 2-CE-(1&4)-NAcCys conjugates were not available for use in these analyses.
In the first experiment, hyperplastic tissue and tumor from the mammary glands of ERKO/Wnt-1 mice and ovariectomized ERKO/Wnt-1 mice were compared (Table I
). The 4-CE and their conjugates were detected in three of the groups, but not in a mammary tumor from an ovariectomized mouse, as anticipated. Observation of these compounds in hyperplastic tissue from an ovariectomized mouse was surprising, and analysis of such tissue must be explored in a future study. Both the hyperplastic tissue and tumor from ERKO/Wnt-1 mice showed the presence of the carcinogenic 4-CE, but not the noncarcinogenic 2-CE. Furthermore, the 4-methoxyCE conjugates were not detected, suggesting that the amount of catechol-O-methyltransferase, which catalyzes the methylation of CE, is low or it is absent. Small amounts of the GSH, Cys and NAcCys conjugates were detected, suggesting that the CE-3,4-Q were formed and reacted with GSH. The presence of only the 4-CE and some of their conjugates, coupled with the absence of any detectable 4-methoxyCE or 2-CE metabolites, suggests that these mice contain only the carcinogenic 4-CE metabolites. As no or minimal O-methylation occurs, the 4-CE metabolites can be oxidized to the ultimate carcinogenic metabolites, CE-3,4-Q, as evidenced by formation of the GSH conjugates.
In a second experiment with tissue from additional mice, we corroborated the results of the first experiment (Table I
), namely, the levels of 4-CE were higher than those of 2-CE, no methoxyCE were found, and GSH conjugates of the 4-CE were formed (Table II
). In addition, we investigated whether some of the metabolites were present as glucuronide and/or sulfate conjugates (Table II
), as previously found in hamsters (26,29). Indeed, from the few data obtained, the CE metabolites did not appear to be conjugated with glucuronic or sulfuric acid. The results from this experiment once again substantiate the presence of the carcinogenic 4-CE, the absence of methylated CE, and the presence of GSH conjugates that demonstrate formation of the ultimate carcinogenic CE-3,4-Q.
In conclusion, the normally abundant 2-CE were present in small amounts in only one sample of mammary tissue and were not detected in the others. The normally minor 4-CE were present in relatively large amounts in most samples. The 2-methoxyCE and 4-methoxyCE were not detected, suggesting poor protection of the CE from being oxidized to their quinones. The formation of CE-3,4-Q was demonstrated by detection of CE conjugates with GSH or their hydrolysis products, CE-Cys and CE-NAcCys.
The results from this preliminary study suggest that ERKO/Wnt-1 mice display an estrogen metabolite profile that is unbalanced toward the formation of 4-CE and their quinones and against inactivation through methylation. Further studies will be required to distinguish among three possible consequences of these metabolic alterations: (i) that this metabolic alteration is the mechanism for Wnt-1 induced carcinogenesis; (ii) that Wnt-1 induces tumors independently of this metabolic process; or (iii) that specific estrogen metabolites, CE-3,4-Q, initiate and Wnt-1 promotes tumor development through stimulation of c-myc or other mechanisms. To examine these possibilities, future experiments will be conducted with Wnt-1, ERKO and ERKO/Wnt-1 mice.
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Notes
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4 To whom correspondence should be addressed:
Email: ecavalie{at}unmc.edu 
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Acknowledgments
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This research was supported by US Public Health Service grant P01 CA49210 and a supplemental grant awarded under the Activities to Promote Research Collaborations program of the National Cancer Institute. Core support in the Eppley Institute is provided by grant P30 CA36727 from the National Cancer Institute.
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Received February 20, 2001;
revised May 15, 2001;
accepted June 1, 2001.