Characterization of flavonoids with potent and subtype-selective actions on estrogen receptors alpha and beta.

The initial step in estrogen-regulated transcription is the binding of a ligand to its cognate receptors, named estrogen receptors (ERα and ERß). Phytochemicals present in foods and environment can compete with endogenous hormones to alter physiological responses. We screened 224 flavonoids in our engineered biosensor ERα and ERß PRL-array cell lines to characterize their activity on several steps of the estrogen signaling pathway. We identified 83 and 96 flavonoids that can activate ERα or ERß, respectively. While most act on both receptors, many appear to be subtype-selective, including potent flavonoids that activate ER at sub-micromolar concentrations. We employed an orthogonal assay using a transgenic zebrafish in vivo model that validated the estrogenic potential of these compounds. To our knowledge, this is the largest study thus far on flavonoids and the ER pathway, facilitating the identification of a new set of potential endocrine disruptors acting on both ERα and ERß.

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

In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERß), GFP-ERß:PRL-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-ERß:PRL-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-ERß:PRL-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.