Abstract
Exposure to estrogens increases the risk of breast and endometrial cancer. It is proposed that the estrogen receptor (ER) may contribute to estrogen carcinogenesis by transduction of the hormonal signal and as a "Trojan horse" concentrating genotoxic estrogen metabolites in the nucleus to complex with DNA, enhancing DNA damage. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in estrogen replacement formulations, autoxidizes to a redox-cycling quinone that has been shown to cause DNA damage. 4-OHEN was found to be an estrogen of nanomolar potency in cell culture using a luciferase reporter assay and, using a chromatin immunoprecipitation assay, was found to activate ERalpha binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells by comparing ERalpha(+) versus ERalpha(-) cells and 4-OHEN versus menadione, a reactive oxygen species (ROS)-generating, but non-estrogenic, quinone. 4-OHEN selectively induced DNA damage in ERalpha(+) cells, whereas menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ERalpha(+) cells, whereas ER status had no effect on the rate of menadione-induced damage. Imaging of ROS induced by 4-OHEN showed accumulation selective for the nucleus of ERalpha(+) cells within 5 min, whereas in ERalpha(-) or menadione-treated cells, no selectivity was observed. These data support ERalpha acting as a Trojan horse concentrating 4-OHEN in the nucleus to accelerate the rate of ROS generation and thereby amplify DNA damage. The Trojan horse mechanism may be of general importance beyond estrogen genotoxins.
Highlights
The collective evidence supports contributions to estrogensensitive breast cancer from both the proliferative and antiapoptotic hormonal effects of estrogen itself [12,13,14,15,16] and the genotoxic and mutagenic effects of estrogen metabolites [17, 18]
The hormonal estrogenicity of 4-OHEN was assayed using cellular reporters; a comparison was made between estrogen receptor (ER)␣-positive and -negative cells testing 4-OHEN-induced DNA damage; a comparison was made with the reactive oxygen species (ROS)-generating, but non-estrogenic, quinone, menadione; and nuclear localization of 4-OHEN and generation of ROS was determined by chromatin immunoprecipitation (ChIP) assay and fluorescence confocal microscopy
No PCR amplification product was detected in the chromatin immunoprecipitation pulled down with IgG. These results showed that 4-OHEN activates ER␣ to bind to the promoter region of estrogen-responsive genes in cells
Summary
The catechol estrogens were handled in accordance with NIH Guidelines for the Laboratory Use of Chemical Carcinogens [35]. The S30 cells were maintained in phenol red-free MEM supplemented with the same solution as the MDA-MB-231 cells except for the addition of 5% charcoal-dextran-treated fetal bovine serum and 500 g/ml Geneticin. Cells were treated with DMSO, E2, or 4-OHEN for 45 min, washed with phosphate-buffered saline (PBS), and cross-linked with 1.5% formaldehyde at room temperature for 15 min. Cells were rinsed twice with ice-cold PBS, collected into lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8, and 1ϫ protease inhibitor tablet (Roche Applied Science)), and sonicated 20 times for 10 s at 10% strength (Fisher Model 300 Sonic Dismembrator) followed by centrifugation at 21,000 ϫ g for 10 min at 4 °C. Tukey’s multiple comparison tests using GraphPad Prism version 4 for Windows
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