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Screening the ToxCast Phase I Chemical Library for inhibition of Deiodinase Type I enzyme activity
Citation:
Denny, J., Joe Korte, C. Knutsen, J. Olker, S. Degitz, M. Hornung, P. Hartig, AND M. Cardon. Screening the ToxCast Phase I Chemical Library for inhibition of Deiodinase Type I enzyme activity. SETAC Midwest Chapter, Minneapolis, MN, July 20 - 22, 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 in vertebrates is dependent upon coordination of multiple key events including iodide uptake, hormone synthesis, metabolism and elimination, to maintain proper homeostasis of the hormones. Deiodinase enzymes interconvert THs between less active and more active forms via release of iodide from the substrate hormones. The activity of deiodinases has been identified as an important endpoint to include in the context of screening chemicals for thyroid hormone disruption. 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 chemical libraries. An initial set of 13 chemicals was used to establish the assay. Included in this set was the known D1 inhibitor 6-propylthiouracil (used as a positive control). Over 300 unique chemicals primarily from the EPA’s ToxCast phase 1_v2 chemical library were tested in the screening assay. Chemicals were initially screened at a single high concentration of 200 µM to identify potential D1 inhibitors. The majority of the chemicals did not inhibit D1 activity in this initial screen as defined as a response of less than 20% inhibition compared to control D1 activity. There were 50 chemicals or 17 % of the chemicals tested that produced greater than 20% inhibition of D1 activity. Approximately 18 of these inhibited D1 activity by greater than 50% and were further tested in concentration-response mode to determine IC50s. This work presents an initial effort toward screening chemicals with the potential for affecting thyroid hormone status via inhibition of D1 activity. Further work to determine whether this in vitro activity translates to effects in vivo is needed.