Estrogenic activity assessment of environmental chemicals using in vitro assays: Identification of two new estrogenic compounds

Environmental chemicals with estrogenic activities have been suggested to b e associated with deleterious effects in animals and humans. To characteriz e estrogenic chemicals and their mechanisms of action, we established in vi tro and cell culture assays that detect human estrogen receptor alpha (hER alpha)-mediated estrogenicity. First, we assayed chemicals to determine the ir ability to modulate direct interaction between the hER alpha and the ste roid receptor coactivator-1 (SRC-1) and in a competition binding assay to d isplace 17 beta-estradiol (E-2). Second, we tested the chemicals for estrog en-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the enviro nment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophen yl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estroge nic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of the se xenobiotics seemed to be able to induce ER/SRC-1 interactions, most like ly because the conformation of the activated receptor would not allow direc t contacts with this coactivator. However, these compounds were able to inh ibit [H-3]-E-2 binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natu ral hormone as revealed by the receptor/coactivator interaction analysis.