Title: A Mechanistic High-Content Analysis Assay Using a Chimeric Androgen Receptor That Rapidly Characterizes Androgenic Chemicals.

Authors: Szafran, Adam T; Bolt, Michael J; Obkirchner, Caroline E; Mancini, Maureen G; Helsen, Christine; Claessens, Frank; Stossi, Fabio; Mancini, Michael A

Published In SLAS Discov, (2020 08)

Abstract: Human health is at risk from environmental exposures to a wide range of chemical toxicants and endocrine-disrupting chemicals (EDCs). As part of understanding this risk, the U.S. Environmental Protection Agency (EPA) has been pursuing new high-throughput in vitro assays and computational models to characterize EDCs. EPA models have incorporated our high-content analysis-based green fluorescent protein estrogen receptor (GFP-ER): PRL-HeLa assay, which allows direct visualization of ER binding to DNA regulatory elements. Here, we characterize a modified functional assay based on the stable expression of a chimeric androgen receptor (ARER), wherein a region containing the native AR DNA-binding domain (DBD) was replaced with the ERα DBD (amino acids 183-254). We demonstrate that the AR agonist dihydrotestosterone induces GFP-ARER nuclear translocation, PRL promoter binding, and transcriptional activity at physiologically relevant concentrations (<1 nM). In contrast, the AR antagonist bicalutamide induces only nuclear translocation of the GFP-ARER receptor (at μM concentrations). Estradiol also fails to induce visible chromatin binding, indicating androgen specificity. In a screen of reference chemicals from the EPA and the Agency for Toxic Substances and Disease Registry, the GFP-ARER cell model identified and mechanistically grouped activity by known (anti-)androgens based on the ability to induce nuclear translocation and/or chromatin binding. Finally, the cell model was used to identify potential (anti-)androgens in environmental samples in collaboration with the Houston Ship Channel/Galveston Bay Texas A&M University EPA Superfund Research Program. Based on these data, the chromatin-binding, in vitro assay-based GFP-ARER model represents a selective tool for rapidly identifying androgenic activity associated with drugs, chemicals, and environmental samples.

PubMed ID: 32392092

MeSH Terms: DNA-Binding Proteins/genetics; Dihydrotestosterone/pharmacology; Endocrine Disruptors/adverse effects; Endocrine Disruptors/pharmacology*; Environmental Exposure/adverse effects; Estrogen Receptor alpha/genetics*; Green Fluorescent Proteins/pharmacology; HeLa Cells; High-Throughput Screening Assays; Humans; Receptors, Androgen/genetics*; Recombinant Fusion Proteins/genetics*; United States