A Novel ERβ High Throughput Microscopy Platform for Testing Endocrine Disrupting Chemicals

Authors

Derek A Abbott
Maureen G Mancini
Michael J Bolt
Adam T Szafran
Kaley A Neugebauer
Fabio Stossi
Daniel A Gorelick
Michael A Mancini

Publication Date

1-15-2024

Journal

Heliyon

DOI

10.1016/j.heliyon.2023.e23119

PMID

38169792

PMCID

PMC10758781

PubMedCentral® Posted Date

12-8-2023

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Estrogen receptor beta, High content analysis, High throughput microscopy, Endocrine disruptors, Zebrafish, Estrogen receptor alpha

Abstract

In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERβ), GFP-ERβ:PRL-HeLa, a single-cell-based high throughput (HT) in vitro assay that allows direct visualization and measurement of GFP-tagged ERβ binding to ER-specific DNA response elements (EREs), ERβ-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 ERβ-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 ERβ 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 ERβ and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERβ activity, validating the GFP-ERβ:PRL-HeLa assay as a screening tool for potential ERβ active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERβ:PRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERβ EDCs and improve upon currently available screening tools for this understudied nuclear receptor.