A novel ERß high throughput microscopy platform for testing endocrine disrupting chemicals.

ABSTRACT

In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERß), GFP-ERßPRL-HeLa, a single-cell-based high throughput (HT) in vitro assay that allows direct visualization and measurement of GFP-tagged ERß binding to ER-specific DNA response elements (EREs), ERß-induced chromatin remodeling, and monitor transcriptional alterations via mRNA fluorescence in situ hybridization for a prolactin (PRL)-dsRED2 reporter gene. The model was used to accurately (Z' = 0.58-0.8) differentiate ERß-selective ligands from ERα ligands when treated with a panel of selective agonists and antagonists. Next, we tested an Environmental Protection Agency (EPA)-provided set of 45 estrogenic reference chemicals with known ERα in vivo activity and identified several that activated ERß as well, with varying sensitivity, including a subset that is completely novel. We then used an orthogonal ERE-containing transgenic zebrafish (ZF) model to cross validate ERß and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERß activity, validating the GFP-ERßPRL-HeLa assay as a screening tool for potential ERß active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERßPRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERß EDCs and improve upon currently available screening tools for this understudied nuclear receptor.