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Screening the ToxCast Phase 1 chemical library for inhibition of deiodinase type 1 activity
Citation:
Hornung, M., Joe Korte, J. Olker, J. Denny, C. Knutsen, P. Hartig, M. Cardon, AND S. Degitz. Screening the ToxCast Phase 1 chemical library for inhibition of deiodinase type 1 activity. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 162(2):570-581, (2018).
Impact/Purpose:
Deiodinase enzymes perform critical roles in vertebrates by maintaining the balance between having sufficient active thyroid hormone when and where it is needed, and the inactivation of hormone when signaling needs to be reduced. Development of a 96-well plate assay to assess chemical inhibition of deiodinase type 1 activity in vitro and demonstration of its application to screening large chemical libraries is presented in this manuscript. This work supports the US EPA Endocrine Disruptor Screening Program’s need for higher-throughput screening assays to address additional molecular initiating events with the potential to disrupt normal thyroid hormone signaling.
Description:
Thyroid hormone (TH) homeostasis is dependent upon coordination of multiple key events including iodide uptake, hormone synthesis, metabolism and elimination, to maintain proper TH signaling. Deiodinase enzymes catalyze iodide release from THs to interconvert THs between active and inactive forms, and are integral to hormone metabolism. The activity of deiodinases has been identified as an important endpoint to include in the context of screening chemicals for thyroid hormone disruption. To begin to address the potential for chemicals to inhibit these enzymes we utilized an adenovirus expression system to produce human deiodinase type 1 (D1) enzyme, established robust assay parameters for non-radioactive determination of iodide release by the Sandell-Kolthoff method, and employed a 96-well plate format for screening chemical libraries. An initial set of 19 chemicals was used to establish the assay, including the known D1 inhibitor 6-propylthiouracil as a positive control. An additional 292 unique chemicals from the EPA’s ToxCast phase 1_v2 chemical library were screened. Chemicals were initially screened at a single high concentration of 200 µM to identify potential D1 inhibitors. There were 50 chemicals, or 17% of the TCp1_v2 chemicals tested, that produced >20% inhibition of D1 activity. Eighteen of these inhibited D1 activity >50% and were further tested in concentration-response mode to determine IC50s. This work presents an initial effort toward identifying chemicals with potential for affecting thyroid hormones via inhibition of deiodinases and sets the foundation for further testing of large chemical libraries against D1 and the other deiodinase enzymes involved in TH function.
