Optimization of an estrogen receptor-alpha transcriptional activation assay for testing a diverse spectrum of chemicals.
Estrogen receptor (ER) transactivation assays were initially designed to study endogenous mechanisms of steroid hormone action, but more recently have been used to assess industrial chemicals for potential estrogenic activity. Given the diverse spectrum of physicochemical properties of these chemicals, we examined the effects of pH (a weak organic and strong inorganic acid and base), hyperosmolality (NaCl, mannitol) and two different vehicles (DMSO, Triton X-100) on responses to estradiol-17beta (E2) in an ER transactivation assay. MCF-7 human breast cancer cells were transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc), treated with E2 under various test conditions, and then assessed for ER-mediated luciferase activity. Maximal E2-induced reporter activity was observed at pH 7.8 (pre-incubation), but was markedly reduced at pH < or =7.5, or > or =8.0 (P < 0.001), even though there was no evidence of cytotoxicity. Hyperosmolality induced by addition of mannitol (> or =25 mM) resulted in significant decreases in overall assay responsiveness, whereas NaCl (> or =80 mM) decreased the sensitivity of the assay by increasing the no-observed-effect level for E2 compared to control cultures (330 mOsm). The maximal DMSO concentration that resulted in consistently high E(2)-induced reporter activity was 0.1%, whereas concentrations of Triton X-100 above 1 ppm inhibited E2-induced reporter responses and were cytotoxic above 10 ppm. These results indicate that various physicochemical factors have the potential to confound assay data if not kept within predefined operational limits.