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

• Published in: SLAS discovery Vol. 25; no. 7; pp. 695 - 708
• Main Authors: Szafran, Adam T., Bolt, Michael J., Obkirchner, Caroline E., Mancini, Maureen G., Helsen, Christine, Claessens, Frank, Stossi, Fabio, Mancini, Michael A.
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA Elsevier Inc 01.08.2020; SAGE Publications
• Subjects: androgen receptor; androgens; chromatin binding; endocrine-disrupting chemicals; high-content analysis; androgen receptor; androgens; endocrine-disrupting chemicals; chromatin binding; high-content analysis
• ISSN: 2472-5552; 2472-5560
• DOI: 10.1177/2472555220922917

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.