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Determining estrogenic activity in serum from ovariectomized rats treated with environmental compounds using an in vitro estrogen-mediated transcriptional activation assay (T47D-KBluc)
Citation:
WRENCH, N., L. E. GRAY, D. Bermudez, J. R. FURR, AND V. S. WILSON. Determining estrogenic activity in serum from ovariectomized rats treated with environmental compounds using an in vitro estrogen-mediated transcriptional activation assay (T47D-KBluc). Presented at Triangle Consortium for Reproductive Biology Meeting, Durham, NC, February 26, 2011.
Impact/Purpose:
These results demonstrate that the estrogen-mediated T47D-KBluc cell assay can detect circulating estradiol levels with sensitivity comparable to an RIA, and can be used to quantify estrogenic activity in serum from rats treated with BPAF.
Description:
The use of cell-based assays to quantify low levels of estrogen in human serum is an accepted method. These assays are more sensitive but less specific than radioimmunoassays (RIA). Thus, we hypothesized that estrogen responsive T47D-KBluc cells would detect estrogenic activity in rat serum from ovariectomized (ovx) rats dosed with estradiol as well as other estrogenic compounds such as bisphenol A-F (BPAF). Ovx rats were dosed subcutaneously with a negative control (com oil), positive control (1 ug/day estradiol-benzoate; EB) or BPAF (600mg/kg/d) for 3 consecutive days. At necropsy, blood was collected, centrifuged and the serum was stored at 80° C. Cells were exposed to rat serum diluted 100-fold in RPMI media, supplemented with 5% dextran-coated-charcoal stripped fetal bovine serum, at 100ul/well in triplicate. Negative control serum did not induce estrogenic activity. Dilutions ofthe rat serum demonstrated a dose responsive decline in estrogenic activity for both EB and BPAF. Calculated estrogen equivalence (EEQ) values for the EB samples (run in duplicate) averaged: 1.16ng/ml, 0.37ng/ml, 0.22ng/ml. RIA results for the EB samples of: 1.78ng/ml, 0.53ng/ml, 0.30ng/ml, respectively, confirmed the accuracy of the cell assay results. Calculated BPAF equivalents in the rat serum were: 28760ng/ml, 11753ng/ml, 11413ng/ml. These results demonstrate that the estrogen-mediated T47D-KBluc cell assay can detect circulating estradiol levels with sensitivity comparable to an RIA, and can be used to quantify estrogenic activity in serum from rats treated with BPAF. Disclaimer: This abstract does not necessarily reflect EPA policy.