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Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II
Katie Paul Friedman1, Eric D Watt2, Michael W Hornung3
1*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.
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.
Insights
This study developed a new assay to screen for thyroid-disrupting chemicals by measuring inhibition of thyroperoxidase (TPO). A tiered approach using multiple assays identified selective TPO inhibitors, reducing false positives and animal use.
Area of Science:
- Environmental Toxicology
- Endocrinology
- Assay Development
Background:
- Thyroid hormone (TH) homeostasis is crucial and can be disrupted by environmental chemicals.
- Existing screening assays in the U.S. EPA ToxCast portfolio have limitations in identifying TH-disrupting chemicals.
- Thyroperoxidase (TPO) plays a key role in TH synthesis, making its inhibition a critical molecular-initiating event (MIE).
Purpose of the Study:
- To develop and implement a high-throughput screening assay for identifying chemicals that inhibit TPO.
- To fill a critical gap in screening for MIEs related to TH disruption.
- To establish a tiered screening strategy for efficient and accurate identification of TPO inhibitors.
Main Methods:
- Screened 1074 chemicals from ToxCast libraries using the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay at a single high concentration.
- Retested positive compounds in concentration-response and employed parallel cell-free luciferase inhibition and cytotoxicity assays to identify nonspecific inhibition and cytotoxicity.
- Validated findings by comparing AUR-TPO results with an orthogonal guaiacol peroxidase assay for 150 chemicals.
Main Results:
- Identified a subset of chemicals exhibiting selective AUR-TPO inhibition after accounting for nonspecific effects.
- Stratified approximately 300 putative TPO inhibitors based on selective activity, cytotoxicity, and nonspecific enzyme inhibition.
- Confirmed activity profiles of putative TPO inhibitors through orthogonal assay comparison.
Conclusions:
- The developed AUR-TPO assay and tiered screening approach effectively identify selective TPO inhibitors.
- This strategy enhances the efficiency and accuracy of screening for thyroid-disrupting chemicals.
- The approach minimizes resource use and reduces the need for animal testing in chemical safety assessments.
