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Screening the ToxCast Phase 1, 2, and e1k chemical libraries for inhibition of Deiodinase Types 1, 2 and 3 enzyme activity
Citation:
Olker, J., Joe Korte, J. Denny, P. Hartig, M. Cardon, C. Knutsen, P. Kent, J. Christensen, S. Degitz, AND M. Hornung. Screening the ToxCast Phase 1, 2, and e1k chemical libraries for inhibition of Deiodinase Types 1, 2 and 3 enzyme activity. SETAC Focused Topic Meeting, Durham, NC, April 16 - 18, 2018.
Impact/Purpose:
Deiodinase enzymes perform critical roles in vertebrate thyroid hormone homeostasis by maintaining the balance between having sufficient active thyroid hormone when and where it is needed, and the catabolism of hormone when signaling needs to be reduced.This poster presents the results from screening large chemical libraries for inhibition of deiodinase types 1, 2, and 3 using recently developed assays in 96-well plate format with a non-radioactive detection method. This work supports the US EPA Endocrine Disruptor Screening Program’s need for higher-throughput screening assays to address additional molecular initiating events, beyond those currently in the ToxCast suite of assays, with the potential to disrupt normal thyroid hormone signaling.
Description:
Thyroid hormone (TH) homeostasis is dependent on multiple proteins for TH synthesis, transport, and peripheral metabolism and elimination. Deiodinase enzymes play an essential role in converting THs between active and inactive forms by deiodinating the pro-hormone thyroxine (T4) to the active hormone triiodothyronine (T3) and modifying both T4 and T3 to inactive forms. Chemical inhibition of deiodinase activity has been identified as an important endpoint to include in screening chemicals for thyroid hormone disruption. To address the lack of data regarding the potential of chemicals to inhibit the deiodinase enzymes, we developed robust 96-well in vitro assays for screening chemicals for inhibition of human deiodinase types 1, 2, and 3 enzyme activity using a non-radioactive detection method. With these assays, we screened over 1800 unique chemicals from the EPA’s ToxCast phase 1_v2, phase 2, and e1k libraries to identify those that have the potential to disrupt the thyroid axis via deiodinase inhibition. The majority of the chemicals did not inhibit deiodinase activity in the initial screen with a single high concentration (target concentration of 200 µM). There were 411 chemicals that produced enzyme inhibition of 20% or greater in at least one of the three deiodinase assays. Of these, 228 chemicals produced enzyme inhibition of 50% or greater; these chemicals were further tested in concentration-response mode to determine relative potency. The chemicals identified as ?‘active’ were from all three chemical libraries and a range of chemical types (e.g., pesticides, industrial products, food additives, pharmaceuticals). Comparison across the three deiodinase isoforms shows similar results for over 90% of the tested chemicals; however, there are over 50 chemicals that show greater than 50% inhibition of only one of the three deiodinases. This set of three deiodinase inhibition assays is a significant contribution to the effort towards expanding the limited number of in vitro assays used to identify chemicals having the potential to interfere with thyroid hormone homeostasis. Additionally, this study sets the groundwork for development and evaluation of structure-activity relationships for deiodinase inhibition, and informs targeted selection of chemicals for further testing to identify adverse outcomes of deiodinase inhibition.