ASSESSMENT OF ENVIRONMENTAL AND THERAPEUTIC ESTROGEN MIMICS USING RECOMBINANT HUMAN ESTROGEN-a . Elisabeth Jisa*, Agnes Thalhammer, James L. Wittliff. ¹Biochemistry & Brown Cancer Center, University of Louisville, Louisville, KY.

The gene for human estrogen receptor-a (hER) was expressed in Saccheromyces cerevisiae as a ubiquitin fusion under the control of a CUP1 promoter. hER preparations exhibited 6-8 pmol/mg extract protein (Kd = 0.05-0.6 nM for estradiol-17b ). Wild-type ER from calf uterus was used for comparison. Ligand recognition by hER was determined from an array of titration curves generated with tritium-labeled estradiol-17b in the absence (control) and presence of an increasing concentration of a candidate estrogen mimic. Ligand binding assessed in the titration mode was analyzed by Lundon OneSiteâ software, while inhibition of binding was determined using Lundon Competeâ software. hER recognition of wild-type ERE was analyzed using gel mobility shift assays in the presence and absence of candidate mimics. Supershift assays were also performed using monoclonal antibodies (MAb) recognizing epitopes in functional domains of hER. Estrone & equilin, naturally-occurring estrogens, bound hER with Kd values = 1.4 nM, and supershifted with AER 314 to a position identical to that observed with hER either unliganded or associated with estradiol-17b . Zearalanone from Fusarium exhibited similar properties indicating its estrogen mimicry. The phytoestrogen, diadzein, bound hER (Kd = 0.1-0.3 m M) and supershifted with MAb. Evaluation of various pesticides and herbicides by this approach gave the following results: 1) chlordecone exhibited high affinity (Kd = 0.2 m M) while retaining supershift properties of hER, 2) DDT, its metabolite DDE, heptachlor & methoxychlor associated with hER with Kd values = 10-50 m M and supershifted with AER 314, 3) DDD, DEET, atrazine & simazine exhibited considerably lower affinities. The therapeutic estrogens, tamoxifen & [4-OH]tamoxifen (4-HT), bound hER with Kd values of 0.1-0.3 m M & 0.1-0.5 nM respectively and supershifted with AER 314. However, when hER was occupied by tamoxifen and supershifted with MAb to hsp 27, it migrated differently than hER-estradiol-17b complexes supershifted with the same antibody. Diethylstilbestrol gave similar results to those observed with 4-HT. These results indicate the candidate estrogen mimics studied inhibit hER binding of estradiol-17b to various extents without altering recognition of ERE either alone or in the presence of monoclonal antibodies to hER. Supported in part by a grant from Department of Veterans Affairs (#Hendler 0006). EJ & AT are research fellows in the International Scholars Program.