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Chem. Res. Toxicol. 2001, 14, 1654-1659
Equine Estrogen Metabolite 4-Hydroxyequilenin Induces DNA Damage in the Rat Mammary Tissues: Formation of Single-Strand Breaks, Apurinic Sites, Stable Adducts, and Oxidized Bases Fagen Zhang, Steven M. Swanson, Richard B. van Breemen, Xuemei Liu, Yanan Yang, Chungang Gu, and Judy L. Bolton* Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231 Received September 28, 2001
Epidemiological data strongly suggest that a woman’s risk of developing breast cancer is directly related to her lifetime estrogen exposure. Estrogen replacement therapy in particular has been correlated with an increased cancer risk. Previously we showed that the equine estrogens equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are metabolized to the catechol, 4-hydroxyequilenin which autoxidizes to an o-quinone causing oxidation and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1227]. In the present study, we injected 4-hydroxyequilenin into the mammary fat pads of Sprague-Dawley rats. Analysis of cells isolated from the mammary tissue for DNA single-strand breaks and oxidized bases using the comet assay showed a dose-dependent increase in both types of lesions. In addition, LC-MS-MS analysis of extracted mammary tissue showed the formation of an alkylated depurinating guanine adduct. Finally, extraction of mammary tissue DNA, hydrolysis to deoxynucleosides, and analysis by LC-MS-MS showed the formation of stable cyclic deoxyguanosine and deoxyadenosine adducts as well as oxidized bases. This is the first report showing that 4-hydroxyequilenin is capable of causing DNA damage in vivo. In addition, the data showed that 4-hydroxyequilenin induced four different types of DNA damage that must be repaired by different mechanisms. This is in contrast to the endogenous estrogen 4-hydroxyestrone where only depurinating guanine adducts have been detected in vivo. These results suggest that 4-hydroxyequilenin has the potential to be a potent carcinogen through the formation of variety of DNA lesions in vivo.
Introduction Despite the well-documented effects of estrogen replacement therapy on reducing menopausal symptoms (1), the risk of osteoporosis (2), and a potentially beneficial effect on the prevention of coronary heart disease (3, 4), concerns about developing hormone-dependent cancers cause many women to avoid taking estrogens (5). The association between female endocrine factors and increased cancer risk in hormone-sensitive tissues, particularly the breast and endometrium, has been thoroughly established by numerous epidemiological studies (6-9). However, the molecular mechanism(s) involved in the carcinogenic action of estrogens remains both controversial and elusive (10). One potential mechanism could involve modification of critical cellular macromolecules by biotransformation of estrogens to either redox active and/or electrophilic o-quinones (11-13). Redox cycling between the catechol metabolites of estrogens and their o-quinones generates reactive hydroxyl radicals that could cause DNA single-strand breaks (14-16) and/or oxidation of the DNA bases (17). * To whom correspondence should be addressed. Phone: (312) 9965280. Fax: (312) 996-7107. E-mail:
[email protected].
The most widely prescribed estrogen replacement formulation in North America is Premarin (WyethAyerst), which contains the equine estrogens equilenin and equilin, as well as the endogenous estrogens estrone and 17β-estradiol. However, there is little information on the metabolism of the equine estrogens in animal models (18-21) let alone in women. Li et al. reported that exposing hamsters for 9 months to estrone, equilin plus equilenin, or sulfatase-treated Premarin resulted in 100% tumor incidence and abundant tumor foci (19). We have shown previously that the major metabolite of both equilin and equilenin was 4-hydroxyequilenin (4-OHEN,1 Figure 1) (11, 22, 23). This catechol estrogen was found to autoxidize to an o-quinone that caused depletion of reducing equivalents and molecular oxygen, alkylation of drug metabolism enzymes, and a variety of DNA lesions in vitro, including single-stand cleavage, oxidation 1 Abbreviations: 4-OHEN, 4-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8-estrapentaen-17-one; 4-OHE, 4-hydroxyestrone, 3,4-dihydroxy1,3,5(10)-oestratrien-17-one; DMSO, dimethyl sulfoxide; Fpg, E. coli formamidopyrimidine-DNA glycosylase; equilenin, 1,3,5(10),6,8-estrapentaen-3-ol-17-one; equilin, 1,3,5(10),7-estratetraen-3-ol-17-one; LCMS, liquid chromatography-mass spectrometry; MRM, multiple reaction monitoring; MCP, microchannel plate, ROS, reactive oxygen species.
10.1021/tx010158c CCC: $20.00 © 2001 American Chemical Society Published on Web 11/27/2001
4-OHEN Induces DNA Damage in Vivo
Figure 1. DNA damage induced by 4-OHEN quinoids in vitro.
of DNA bases, formation of apurinic sites, and bulky stable adducts (11). In addition, we recently reported that 4-OHEN-induced DNA damage and apoptosis in breast cancer cell lines (24) and cellular transformation in vitro (25). In the present study, we examined whether 4-OHENo-quinone could produce DNA damage in vivo by injecting the catechol precursor into the mammary glands of Sprague-Dawley rats and analyzing for DNA damage using the comet assay, LC-MS, and LC-MS-MS. In addition, the potential formation of depurinating adducts was studied by extraction of the mammary tissue and analysis of the tissue extracts by LC-MS-MS. The data showed that 4-OHEN is capable of inducing a variety of DNA lesions including stable bulky adducts, apurinic sites, single-strand cleavage, and oxidation of DNA bases all of which could contribute to the carcinogenic effects of equine estrogens.
Materials and Methods The catechol estrogen o-quinones were handled in accordance with NIH guidelines for the Laboratory Use of Chemical Carcinogens (26). All chemicals were purchased from Aldrich (Milwaukee, WI), Fisher Scientific (Pittsburgh, PA), or Sigma Chemical Co. (St. Louis, MO) unless stated otherwise. 4-OHEN was synthesized as described previously (21). The standard cyclic deoxynucleoside 4-OHEN adducts (4-OHEN-dA, 4-OHENdG) were prepared as previously described (27, 28). Mass Spectrometry. A Micromass (Manchester, U.K.) QTOF 2 hybrid tandem mass spectrometer equipped with electrospray and a Waters (Milford, MA) 2690 HPLC was used for LC-MS-MS. In all experiments, the electrospray capillary voltage was held at +3.1 kV, and the cone voltage was at 30 V. Source block and drying gas temperatures were maintained at 110 and 330 °C, respectively. Nitrogen was used as both nebulizing gas and drying gas. Following the mass selection for [M + H]+ and [M + Na]+ ions of the possible adducts by the quadrupole mass analyzer, collision-induced dissociation (CID) was carried out with argon at the collision energy of 30 eV. The acceleration voltage of TOF was 9.1 kV and the end plate of the reflectron was 11.2 kV. The TOF cycle time was 43 µs for the mass range of m/z
