title: Oxidative DNA damage induced by equine estrogen metabolites: role of estrogen receptor alpha.
abstract: Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in 8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (NADH) or deplete GSH (diethyl maleate). The role of the estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist tamoxifen, where decreases in 8-oxo-deoxyguanosine were observed. Another equine estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of estrogen-dependent alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine estrogen metabolites could involve oxidative stress and that the estrogen receptor may play a role in this process.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, The University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, USA.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Liu
authors.forename: Jiaqin
authors.initials: J
authors.lastname: Yao
authors.forename: Emily
authors.initials: E
authors.lastname: Pisha
authors.forename: Yanan
authors.initials: Y
authors.lastname: Yang
authors.forename: Yousheng
authors.initials: Y
authors.lastname: Hua
authors.forename: Richard B
authors.initials: RB
authors.lastname: van Breemen
authors.forename: Judy L
authors.initials: JL
authors.lastname: Bolton
mesh_terms: D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004855:Equilenin / Q000031:analogs & derivatives* / Q000633:toxicity*; D045166:Estradiol Congeners / Q000633:toxicity*; D047628:Estrogen Receptor alpha; D005260:Female; D006801:Humans; D017382:Reactive Oxygen Species / Q000378:metabolism; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
chemical_list: D004273:DNA, Neoplasm; D045166:Estradiol Congeners; D047628:Estrogen Receptor alpha; D017382:Reactive Oxygen Species; D011960:Receptors, Estrogen; D020845:Selective Estrogen Receptor Modulators; D013629:Tamoxifen; C065250:4-hydroxy-equilenin; D004855:Equilenin