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Identifying Environmental Chemicals as Agonists of the Androgen Receptor by Applying a Quantitative High-throughput Screening Platform
Lynch, C., S. Sakamuru, R. Huang, D. Stavea, L. Varticovski, G. Hager, Richard S. Judson, Keith A. Houck, N. Kleinstreuer, W. Casey, R. Paules, A. Simeonov, AND M. Xia. Identifying Environmental Chemicals as Agonists of the Androgen Receptor by Applying a Quantitative High-throughput Screening Platform. TOXICOLOGY. Elsevier Science Ltd, New York, NY, 385:48-58, (2017).
• Agency Research Drivers - The US EPA needs to prioritize thousands of chemicals within the Endocrine Disruptor Screening Program (EDSP). For many of these chemicals, little-to-no toxicity data exist and hazard identification methods are needed to prioritize chemicals for screening purposes. • Science Challenge – Strict regulation of androgen signaling through the androgen receptor (AR) is required for reproductive organ development and function. The current EDSP screening tests program are too time-consuming and costly to screen the tens-of-thousands of compounds currently in use. Alternative methods are necessary to screen and prioritize compounds for androgen disruption potential. • Research Approach – This research used cellular, biochemical, and in vitro methods to evaluate the effects of chemical exposure on AR activity. • Results – This study screened 10,000 chemical compounds for their ability to activate AR. Over 100 compounds were identified from the primary screens based on their activity to be studied in follow-up assays. Nineteen compounds identified were found to bind to AR, and eighteen of them translocated AR into the nucleus. Seven potentially novel AR activators were discovered. • Anticipated Impact/Expected use – The information gained from the current study can be directly used to prioritize compounds for further in-depth toxicological evaluations, as well as their potential to disrupt the endocrine system via AR activation.
Background: The androgen receptor (AR, NR3C4) is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function. It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation. Pathological processes involving AR include Kennedy’s disease and Klinefelter’s syndrome, as well as prostate, ovarian, and testicular cancer. Strict regulation of sex hormone signaling is required for normal reproductive organ development and function. Objectives: This study was conducted to screen the Tox21 10K compound library for their ability to activate AR.Methods: We performed a quantitative high-throughput screen using two reporter gene cell lines, AR ß-lactamase (AR-bla) and AR-luciferase (AR-luc). Over 100 compounds were identified from the primary screens based on their activity to be studied in follow-up assays. Biochemical binding and AR nuclear translocation assays were performed to confirm direct binding and activation of AR from these compounds.Results: The compound confirmation screen rates were 97.8% and 92.2% in the AR-luc and AR-bla assays, respectively. The top nineteen compounds identified were found to bind to AR, and eighteen of them translocated AR from the cytoplasm into the nucleus. Seven potentially novel AR agonists were discovered through primary and follow-up studies.Conclusions: We have identified multiple AR activators, including known AR agonists such as testosterone, as well as novel compounds prulifloxacin and GSK232420A. The information gained from the current study can be directly used to prioritize compounds for further in-depth toxicological evaluations, as well as their potential to disrupt the endocrine system via AR activation.