title: Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries.
abstract: 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.
authors.affiliation: *Oak Ridge Institute for Science Education Postdoctoral Fellow, Oak Ridge, TN, 37831 Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27711 simmons.steve@epa.gov.
authors.forename: Katie
authors.initials: K
authors.lastname: Paul Friedman
authors.affiliation: *Oak Ridge Institute for Science Education Postdoctoral Fellow, Oak Ridge, TN, 37831 National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27711.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Watt
authors.affiliation: Mid-Continent Ecology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Duluth, MN, 55804.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Hornung
authors.affiliation: Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27711.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Hedge
authors.affiliation: National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27711.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Judson
authors.affiliation: National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27711.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Crofton
authors.affiliation: National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27711.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Houck
authors.affiliation: National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27711, simmons.steve@epa.gov.
authors.forename: Steven O
authors.initials: SO
authors.lastname: Simmons
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000633:toxicity*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007453:Iodide Peroxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008297:Male; D020318:Rats, Long-Evans; D018570:Risk Assessment; D054852:Small Molecule Libraries; D034421:Sus scrofa; D013961:Thyroid Gland / Q000187:drug effects* / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D007453:Iodide Peroxidase
keywords: endocrine; predictive toxicology < in vitro and altenatives; thyroid < endocrine toxicology in vitro and altenatives