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Screening the ToxCast Phase I, II, and e1K Chemical Libraries for Inhibition of Deiodinase Type 1,2 and 3 Enzyme Activity
Citation:
Denny, J., Joe Korte, C. Knutsen, J. Olker, J. Christensen, P. Kent, P. Hartig, M. Cardon, S. Degitz, AND M. Hornung. Screening the ToxCast Phase I, II, and e1K Chemical Libraries for Inhibition of Deiodinase Type 1,2 and 3 Enzyme Activity. SETAC North America, Minneapolis, MN, November 12 - 16, 2017.
Impact/Purpose:
This is a presentation of the development of an in vitro screening assay to identify chemicals with potential for thyroid hormone disruption via inhibition of deiodinase activity. The results from screening approximately 1800 chemicals at a single concentration from the ToxCast phase 1, phase 2, and e1k chemical libraries will be presented. This work within the CSS 16.01 High Throughput Toxicology Project addresses needs of OSCP’s Endocrine Disruptor Screening Program for additional thyroid-axis in vitro screening assays help prioritize chemicals for further evaluation for thyroid hormone disruption.
Description:
Thyroid hormone (TH) signaling and homeostasis is dependent upon coordination of multiple key events including thyroidal iodide uptake and hormone synthesis, and peripheral metabolism and elimination. Deiodinase enzymes play an essential role in converting the pro-hormone thyroxine to the active hormone triiodothyronine. They also convert these THs to inactive forms via further removal of iodide from the substrate hormones. The activity of the deiodinases has been recognized as an important endpoint to include in the development of screening assays to identify thyroid hormone disrupting chemicals. To address the lack of data regarding the potential for chemicals to inhibit these enzymes a research effort was initially focused on human deiodinase type 1 (D1). We utilized an adenovirus expression system for production of 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 chemicals. An initial set of 19 chemicals was used to establish the performance of the assays. This successful approach was extended to include human deiodinase types 2 and 3. Over 1800 unique chemicals, primarily from the EPA’s ToxCast phase 1_v2, phase 2, and e1K chemical libraries, were tested in each of the three screening assays. Chemicals were initially screened at a single high concentration of 200 µM to identify potential enzyme inhibitors. The majority of the chemicals did not inhibit deiodinase activity in this initial screen. Across the three enzymes, only 15-20% of the chemicals produced enzyme inhibition of 20% or greater. Approximately half of these inhibited the enzymes by greater than 50% compared to control enzyme activity. Chemicals that inhibited deiodinase activity by greater than 50% were further tested in concentration-response mode to determine relative potency. This work presents an initial effort toward screening chemicals with the potential for affecting thyroid hormone status via inhibition of deiodinase activity. It sets the groundwork for development and evaluation of structure-activity relationships for deiodinase inhibition, and provides a dataset for the targeted selection of chemicals for testing to identify deiodinase inhibition adverse outcomes. This abstract does not necessarily reflect US EPA policy.