Evaluation of androgen assay results using a curated Hershberger database

Abstract

A set of 39 reference chemicals with reproducible androgen pathway effects in vivo, identified in the companion manuscript [1], were used to interrogate the performance of the ToxCast/Tox 21 androgen receptor (AR) model based on 11 high throughput assays. Cytotoxicity data and specificity confirmation assays were used to distinguish assay loss-of-function from true antagonistic signaling suppression. Overall agreement was 66% (19/29), with ten additional inconclusive chemicals. Most discrepancies were explained using in vitro to in vivo extrapolation to estimate equivalent administered doses. The AR model had 100% positive predictive value for the in vivo response, i.e. there were no false positives, and chemicals with conclusive AR model results (agonist or antagonist) were consistently positive in vivo. Considering the lack of reproducibility of the in vivo Hershberger assay, the in vitro AR model may better predict specific AR interaction and can rapidly and cost-effectively screen thousands of chemicals without using animals.

CoMPARA: Collaborative Modeling Project for Androgen Receptor Activity

“…First, a multiassay AR pathway model was developed based on the results of 11 assays covering the androgen signaling pathway and combining the in vitro results into an AUC score representing the whole AR activity to mimic the in vivo results (Kleinstreuer et al 2017). These assays were run on the same initial library of 1,855 ToxCast™ chemicals that the ER assays were run on, and the developed pathway model was validated using reference chemicals with known in vitro results from the literature (Kleinstreuer et al 2017) and further compared with a set of chemicals with reproducible results in vivo (Browne et al 2018;Kleinstreuer et al 2018a). Note that the goal of this project is to predict in vitro AR activity.…”

“…There is significant discrepancy between in vitro AR activity and the results of the in vivo Hershberger assay, especially for antagonist mode. However, as demonstrated by Kleinstreuer et al (Kleinstreuer et al 2018a), most of these discrepancies are due to the in vivo activity occurring at internal concentrations well above the upper limit of testing in the in vitro assays (100 lM). The resulting AR pathway activity AUC scores were used as a training set in a large AR modeling consortium called the Collaborative Modeling Project for Androgen Receptor (CoMPARA).…”

Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders

“…To put this issue into perspective, a recent study reported on the performance of the ToxCast/Tox 21 AR in vitro model, based on 11 high-throughput assays for predicting a positive antiandrogenic response in the Hershberger rodent model (Kleinstreuer et al 2018). The AR in vitro model had 100% positive predictive value for the in vivo response, and chemicals with conclusive in vitro results were consistently positive in vivo, indicating that few false positives existed.…”

Single-Cell Distribution Analysis of AR Levels by High-Throughput Microscopy in Cell Models: Application for Testing Endocrine-Disrupting Chemicals

“…One of the largest efforts to date has been the U.S. Environmental Protection Agency's (EPA) Endocrine Disruptor Screening Program (EDSP), which aimed to combine HTS with computational analyses to characterize chemicals that interfere with endocrine hormones, focusing on three nuclear receptors pathways: estrogen (ER), androgen (AR), and thyroid (TR) receptors. 1 Through the extensive efforts of the Tox21/ToxCast initiatives, via testing hundreds of chemicals in more than a hundred high-throughput (HT) assays, the National Institute of Environmental Health Sciences (NIEHS) and EPA curated sets of reference chemicals for the ER, AR, and TR pathways [2][3][4] that are relevant to in vivo animal studies, which have included the development of computational models for quantifying activity on ER and AR. 3,5,6 Many studies, including several from our group, 7-11 have described the development of HT high-content assays (HCAs) using the AR as a model system (reviewed in Campana et al 12 ).…”

“…1 Through the extensive efforts of the Tox21/ToxCast initiatives, via testing hundreds of chemicals in more than a hundred high-throughput (HT) assays, the National Institute of Environmental Health Sciences (NIEHS) and EPA curated sets of reference chemicals for the ER, AR, and TR pathways [2][3][4] that are relevant to in vivo animal studies, which have included the development of computational models for quantifying activity on ER and AR. 3,5,6 Many studies, including several from our group, 7-11 have described the development of HT high-content assays (HCAs) using the AR as a model system (reviewed in Campana et al 12 ). However, the vast majority of efforts have been concentrated on nonnative and/or cell-free assays that measured several characteristics of the AR pathway, including ligand binding (e.g., NVS_NR_hAR in ToxCast), coregulator recruitment (via protein complementation assay, OT_AR_ARSRC1), reporter gene activation (Tox21_AR_Luc_MDAKB2, etc.…”