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Screening the ToxCast Phase 1, 2, and e1k Chemical Libraries for Inhibition of Deiodinase Type 1, 2 and 3 Enzyme Activity
Citation:
Olker, J., Joe Korte, J. Denny, J. 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 Type 1, 2 and 3 Enzyme Activity. Society of Toxicology, San Antonio, TX, March 11 - 15, 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 manuscript presents the development of 96-well plate assays to assess chemical inhibition of deiodinase types 2 and 3 and the results from screening large chemical libraries for chemical inhibition of deiodinase types 1, 2, and 3. 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 converting the pro-hormone thyroxine (T4) to the active hormone triiodothyronine (T3) and converting 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 established robust in vitro assays and screened over 1800 unique chemicals for inhibition of human deiodinase types 1 (D1), 2 (D2), and 3 (D3). The activity of the deiodinase enzymes was determined utilizing the Sandell-Kolthoff reaction for non-radioactive determination of iodide in a 96-well plate format. The majority of the chemicals did not inhibit deiodinase activity in the initial screen with a single high concentration (target concentration of 200 µM). Across the three enzymes, only 12-17% of the chemicals produced enzyme inhibition of 20% or greater.The EPA’s ToxCast phase 1_v2, phase 2, and e1k chemical libraries each included multiple chemicals that produced enzyme inhibition of 50% or greater in the three deiodinase assays; these chemicals were further tested in concentration-response mode to determine relative potency. 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 toward expanding the limited number of in vitro assays used to identify chemicals having the potential to interfere with thyroid hormone homeostasis. Further work to determine whether this in vitro activity translates to effects in vivo is needed. 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.