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In vitro chemical screening assays to identify thyroid hormone disruptors.
Citation:
Hornung, M., S. Degitz, J. Olker, Joe Korte, J. Denny, S. Laws, T. Stoker, P. Hartig, M. Cardon, D. Hallinger, A. Murr, A. Buckalew, J. Wang, K. Paul-Friedman, AND Steve Simmons. In vitro chemical screening assays to identify thyroid hormone disruptors. SETAC North America Focused Topic Meeting, Durham, NC, April 16 - 18, 2018.
Impact/Purpose:
Identification of chemicals with potential to disrupt thyroid hormone function is a priority of the US EPA. In vitro screening assays can be used to significantly reduce the number of chemicals that need to be selected for testing in higher tiered test protocols. Three assays were developed to more rapidly assess the interaction of chemicals with the thyroid-axis. These included two assays covering activities important for thyroid hormone production and one assay addressing chemical inhibition of hormone activation and inactivation. Over 1,800 chemicals were screened and the data generated from this screening effort will help the EPA make prioritization decisions for chemicals of concern to the Agency.
Description:
Identification of chemicals with potential to impact thyroid hormone function is a priority of the US EPA’s Endocrine Disruptor Screening Program (EDSP). In vitro screening assays can be used to significantly reduce the number of chemicals that need to be considered for testing in higher-tiered test protocols by identifying chemicals that can affect critical key events in pathways necessary for normal thyroid hormone function, and more importantly, by identifying chemicals that are not likely to directly affect the thyroid-axis. Three molecular initiating events deemed of greatest priority were addressed first to develop, adapt, and implement assays to more rapidly assess the interaction of chemicals with these targets within the thyroid-axis. These included two assays covering activities integral to thyroid hormone production: a thyroperoxidase (TPO) inhibition assay and an iodide uptake inhibition assay. Deiodinase (DIO) enzyme activity assays were also developed to address chemical inhibition of hormone activation and catabolism. A TPO inhibition assay was optimized for use in a 384-well plate format, utilizing rat thyroid gland microsomes as the source of enzyme, and a fluorescent substrate, Amplex UltraRed, to measure enzyme activity. To determine chemical inhibition of iodide uptake, a new cell line was engineered to express the human sodium iodide symporter (NIS; hNIS-HEK293T-EPA). Uptake of radioactive iodide into these cells was measured in a 96-well format. DIO enzymes were produced via an adenoviral expression system and cell homogenates were used as source material for the 96-well plate format DIO inhibition assays. Chemical inhibition of iodide released from thyroid hormone substrates was determined by colorimetric assay. To date, these assays have been used to screen over 1,800 ToxCast chemicals for inhibitions of TPO activity, iodide uptake via NIS, and DIO enzyme activity. Data generated from this screening effort will help the EDSP incorporate potential impacts on thyroid-axis endpoints in prioritization decisions for chemicals of concern to the Agency, and inform thyroid-axis Adverse Outcome Pathway development.