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DEVELOPMENT AND CHARACTERIZATION OF A CELL LINE THAT STABLY EXPRESSES AN ESTROGEN-RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ESTROGEN RECEPTOR AGONIST AND ANTAGONISTS
Citation:
Wilson, V S., K L. Bobseine, AND L E. Gray Jr. DEVELOPMENT AND CHARACTERIZATION OF A CELL LINE THAT STABLY EXPRESSES AN ESTROGEN-RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ESTROGEN RECEPTOR AGONIST AND ANTAGONISTS. TOXICOLOGICAL SCIENCES. Oxford Journals, 81(1):69-77, (2004).
Impact/Purpose:
To describe a highly specific, sensitive assay developed for screening chemicals for estrogenic and antiestrogenic activities.
Description:
Screening for endocrine disrupting chemicals (EDCs) that act as estrogens or antiestrogens relies on the use of in vitro binding and gene expression assays coupled with short-term diagnostic in vivo assays. Although binding assays are useful to identify chemicals that are competitive receptor ligands, estrogen-dependent gene expression assays or some other functional assay is required to identify the nature of the interaction; agonist versus antagonist. While cell proliferation assays have been used to identify environmental estrogens, several advisory committees (EDSTAC, ICCVAM) have recommended that stable estrogen-dependent gene expression assays be developed for screening chemicals for estrogenic activity because of the high degree of specificity of the response and potential for use in a high-throughput mode. In this project we describe a highly specific, sensitive assay developed for screening chemicals for estrogenic and antiestrogenic activities. T47D cells, which naturally express ER alpha and beta, were stably transfected with a triplet ERE-promoter-luciferase reporter gene construct. These cells are sensitive to the potent estrogens, 17 -estradiol, ethynyl estradiol and diethylstibesterol and well characterized weaker environmental estrogens like genistein, HPTE (an estrogenic pesticide metabolite), and nonylphenol. The EC50 for estradiol was about 0.01 nM reaching maximal induction at 0.1 nM. The antiestrogen, ICI-172,780 was able to completely inhibit the induction of luciferase expression by 0.1 nM estradiol at 10 nM, with 1nM being an EC50. In addition, we were able to replicate, in this cell line, the observation that low concentrations of cadmium were able to induce estrogen dependent gene expression, an effect that was completely inhibited by the potent antiestrogen ICI 172,780. The potent glucocorticoid receptor agonist, dexamethasone, was without effect as an ER agonist at concentrations up to 10 nM whereas the potent androgen, dihydrotestosterone (DHT), showed no induction at concentration of 50 M, but was a partial agonist at very high the conc of 0.2 mM and above. In summary, we have developed a highly specific, sensitive estrogen-responsive gene expression assay in a stable cell line that could be used for high throughput screening of large numbers of chemicals for estrogenic and antiestrogenic activity. In addition, herein we also provide key protocol recommendations necessary to identify and eliminate common problems encountered in in vitro screening of estrogenicity.