4-hydroxytamoxifen-induced cytotoxicity and bisphenol A: Competition for estrogen receptors in human breast cancer cell lines

Increasing concerns over the effects of environmental estrogens on wildlife and humans have highlighted the need for screening systems to assess poten tially estrogenic effects of test compounds. As a result, in vitro screenin g methods such as cell proliferation assays using the estrogen-responsive h uman breast cancer cell line, MCF-7, have been developed. The present study describes an alternative in vitro approach for the assessment of such xeno estrogens, based on estrogenic rescue of MCF-7 cells from antiestrogen-indu ced cytotoxicity. This method measures the ability of various estrogenic co mpounds to compete with a known estrogen-receptor-mediated antihormonal dru g, 4-hydroxytomoxifen, using the 1-[4,5-dimethylthiazol-2-yl]-3,5-diphenylf ormazan (MTT) assay to assess mitochondrial activity. Because 4-hydroxytamo xifen treatment of cells results in a dramatic decrease in mitochondrial de hydrogenase activity which is directly related to their estrogen-receptor c ontent, inhibition of this effect with estrogenic compounds represents an e strogen-receptor interaction, or estrogenic rescue. The estrogenic compound s tested include a weak xenoestrogen, bisphenol A (BPA), and two biological estrogens, 17 alpha- and 17 beta-estradiol. Competitive inhibition of 4-hy droxytamoxifen-induced cytotoxicity by BPA was compared to that of the biol ogical estrogens. The results indicate that the biological estrogens can su ccessfully compete with the antiestrogen in a dose-dependent manner. In add ition, the assay is sensitive enough to detect estrogenic rescue by even th e very weak xenoestrogen, BPA, albeit at high BPA concentrations. This simp le in vitro method could be used as an alternative or second-line screen fo r potential xenoestrogens.