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Development of a tiered screening strategy for a molecular-initiating event: thyroperoxidase inhibition (SOT)
Citation:
Paul Friedman, K., Eric Watt, J. Hedge, K. Crofton, M. Hornung, AND Steve Simmons. Development of a tiered screening strategy for a molecular-initiating event: thyroperoxidase inhibition (SOT). Presented at SOT Annual Meeting, San Diego, CA, March 22 - 26, 2015. https://doi.org/10.23645/epacomptox.5178835
Impact/Purpose:
Present poster at SOT annual meeting in San Diego, CA on March 24, 2015
Description:
Adverse outcome pathway (AOP) analyses illustrate that some molecular-initiating events (MIEs) for thyroid disruption, including thyroperoxidase (TPO) inhibition, are not evaluated by current ToxCast/Tox21 high-throughput screening (HTS) assays. A novel HTS assay for TPO inhibition was developed by adaptation of the guaiacol oxidation assay with a fluorescent peroxidase substrate (Amplex UltraRed, AUR, LifeTech) in a rat thyroid microsome-based assay optimized to 384-well format. Initial testing of the AUR-TPO assay was conducted using a 21-chemical training set that included a reference chemical, methimazole (MMI), known TPO inhibitors, and negative controls. The AUR-TPO assay signal was stable (30-120 min), the dynamic range and Z’ score with MMI were 11-fold and 0.93, and the IC50 for MMI was 0.025 µM (compared to 2.20 µM in a guaiacol-based 96-well assay). The ToxCast Phase I & II libraries (1060 chemicals) were tested with the AUR-TPO assay using an initial screen of one high concentration to identify candidate inhibitors. Chemicals that exceeded 20% inhibition were then screened in concentration-response (6 or 8 concentrations for Phase I & II). In addition, 2 parallel screens were conducted: ATP availability in HEK293T cells (24 hr exposure) to indicate cytotoxicity; and, luciferase inhibition to identify nonspecific protein inhibitors. Results demonstrated known and novel chemicals that inhibited TPO at concentrations below cytotoxicity and that did not inhibit luciferase. AOP-based analyses for thyroid screening enabled development of a new HTS assay that allowed screening of ToxCast chemicals for TPO inhibition. This assay provides an additional data stream to support prioritization of chemicals within the Endocrine Disruptor Screening Program. This abstract does not necessarily reflect the policy of the US EPA.
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DOI: Development of a tiered screening strategy for a molecular-initiating event: thyroperoxidase inhibition (SOT)