You are here:
SCIENTIFIC AND TECHNOLOGICAL SUPPORT ON IN VITRO ASSAYS FOR THE AGENCY'S ENDOCRINE DISRUPTOR SCREENING PROGRAM
Citation:
Hartig, P C., V S. Wilson, C R. Lambright, K L. Bobseine, M C. Cardon, S C. Laws, R L. Cooper, AND L E. Gray Jr. SCIENTIFIC AND TECHNOLOGICAL SUPPORT ON IN VITRO ASSAYS FOR THE AGENCY'S ENDOCRINE DISRUPTOR SCREENING PROGRAM. Presented at Endocrine Disruptors Research Program Review, Research Triangle Park, NC, October 29-31, 2003.
Description:
In response to the 1996 legislative mandate for an endocrine screening and testing program, we are helping develop, standardize and validate relatively sensitive, robust and relatively simple methods for in vitro screening of chemicals that affect estrogen, and androgen function (Table below). The following types of assays are considered in this project: ER alpha binding with the Pan Vera Assay, ER alpha ,and beta transcriptional activation assays, AR binding assays and AR transcriptional activation assays (with transient, transduced, and stable cells). Androgen receptor binding assays. There are several in vitro binding assays for the AR. These include using AR isolated from rat ventral prostate or COS whole cell and a newer, and unvalidated assay using a chimeric rat AR (Pan Vera). If some of the newer AR binding assays could be standardized and validated then they could replace the standard assay which uses AR from animal tissues (3, 4) AR-dependent gene expression assays with transiently transfected or transduced cells. Androgen-dependent gene expression can be assessed in vitro using mammalian cells that are transiently transfected with a luciferase reporter gene and hAR. The binding assays, cannot distinguish agonists from antagonists. Gene expression assays using transiently transfected, or transduced cell, or stably transfected cell lines are required to make this distinction. In our laboratory, we have used CV-1 cells that were transiently cotransfected with MMTV-luc and hAR or hGR. We also have used Adenovirus to transduce CV-1 cells with MMTV-luc and hAR or MDA-453 cells with MMTV-luc. (2,3,4,5) AR-dependent gene expression assays. K Bobseine has developed two stable cells lines to screen for androgenic and antiandrogenic chemicals. One of the assays uses a cell line that has endogenous levels hAR (MDA-KB2) and has been stably transfected with and androgen-responsive reporter gene while the other stable expresses both hAR and reporter genes. These assays offer several advantages over the transiently transfected cell lines (interassay variability, transfection of cells with genes not necessary). These assays, and those above, have all been evaluated with a common suite of AR agonists and antagonists. (2,6,7). In vitro assessment of hER binding. The Fluorescence Polarization System (Pan Vera) is used to assess binding of chemicals to ER using the new ES2 kit. This is a competitive binding assay using purified receptor, fluorescent ligand. We have demonstrated that this assay provides reproducible data that are consistent with ER binding affinities collected from more established assays using about 20 chemicals (including 17 beta-estradiol, estrone, ethinyl estradiol, genistein, DES, phenols, DDT/DDEs). In contrast to the literature, we found that the ES1 kit was not useful and recommend that it not be used and it was subsequently removed from the market. Development of Cell lines that stably express hER alpha and/or beta for screening EDCs. We are developing cells that stably express ER for research and screening purposes. The ZR-75-1 and T47D cells are being evaluated with chemicals that display known estrogenic activities. T47D breast cancer cell line has both ERs while the ZR-75-1 parental cell line only has ER alpha. Both estrogen responsive cell lines demonstrate luciferase induction by E2 exposure while ICI consistently inhibits this response.