Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries

• Published in: Toxicological sciences Vol. 151; no. 1; pp. 160 - 180
• Main Authors: Paul Friedman, Katie, Watt, Eric D, Hornung, Michael W, Hedge, Joan M, Judson, Richard S, Crofton, Kevin M, Houck, Keith A, Simmons, Steven O
• Format: Journal Article
• Language: English
• Published: United States Oxford University Press 01-05-2016
• Subjects: Animal Testing Alternatives; Animals; Dose-Response Relationship, Drug; Enzyme Inhibitors - toxicity; HEK293 Cells; High-Throughput Screening Assays; Humans; Iodide Peroxidase - antagonists & inhibitors; Iodide Peroxidase - metabolism; Male; Rats, Long-Evans; Risk Assessment; Small Molecule Libraries; Sus scrofa; Thyroid Gland - drug effects; Thyroid Gland - enzymology; ToxCast Screening of Thyroperoxidase Inhibitors; predictive toxicology < in vitro and altenatives; thyroid < endocrine toxicology in vitro and altenatives; endocrine
• ISSN: 1096-6080; 1096-0929
• DOI: 10.1093/toxsci/kfw034

High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the U.S. Environmental Protection Agency ToxCast screening assay portfolio. To fill 1 critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast phase I and II chemical libraries, comprised of 1074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single-concentration screen were retested in concentration-response. Due to high false-positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed 2 additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using guaiacol as a substrate to confirm the activity profiles of putative TPO inhibitors. This effort represents the most extensive TPO inhibition screening campaign to date and illustrates a tiered screening approach that focuses resources, maximizes assay throughput, and reduces animal use.