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Validation of T47D-KBluc cell assay for detection of estrogen receptor agonists and antagonists
Citation:
Evans, N., L. E. GRAY, AND V. S. WILSON. Validation of T47D-KBluc cell assay for detection of estrogen receptor agonists and antagonists. Presented at Society of Toxicology (SOT) Annual Meeting, San Francisco, CA, March 11 - 15, 2012.
Impact/Purpose:
There is growing concern of exposure to fish, wildlife, and humans to environmental estrogens and their potential impact on reproductive health. Cell-based assays are useful tools to determine the estrogenic activity of chemicals.
Description:
There is growing concern of exposure to fish, wildlife, and humans to environmental estrogens and their potential impact on reproductive health. Cell-based assays are useful tools to determine the estrogenic activity of chemicals. Confidence in in vitro assay results is strengthened by the use of a well-characterized assay. The T47D-KBluc estrogen receptor transcriptional activation (ERTA) assay utilizes a stable cell line run in a 96-well plate format which contains both human estrogen receptors alpha and beta (ERα and ERß) and an estrogen-responsive luciferase reporter. The cell line was transferred from the EPA lab to CeeTox, Inc for an external validation. Initially about 60 well-characterized estrogenic or anti-estrogenic compounds of varying potency were tested. Testing of this validation set of compounds indicated that the assay produced the expected results and confirmed assay performance. About 50 "unknown" chemicals of interest were then tested. Several compounds were found to be weakly estrogenic such as diethylphthalate and cyclotrisilazane. Only one displayed weak anti-estrogenic effects: N,N-Dimethyltetradecylamine. To further characterize the ERTA assay with respect to ERα and ERß, their respective specific agonists, PPT and DPN were tested. The calculated EC50 of PPT was 4.ge-11M and the EC50 of DPN was 1.5e-8M, compared to that of estradiol which is typically 2.2e-12M. We also tested an ERα specific antagonist, MPP, in competition with either PPT or DPN. Both PPT and DPN-luciferase induced activity were reduced by MPP. Therefore, it cannot be ruled out that DPN, while more specific for ERß is not also activating primarily ERα. Results demonstrate that the T47D-KBluc cell line responds to both ERα and ERß agonists but support the assumption that ERa is inducing more reporter expression than ERß. Future studies will be conducted to further examine the relative proportion of ERα and ERß in reporter gene induction. This abstract does not necessarily reflect EPA policy.