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In vitro screening for potential chemical inhibitors of deiodinase type 1 activity
Citation:
Hornung, M., Joe Korte, J. Olker, J. Denny, C. Knutsen, P. Hartig, M. Cardon, AND S. Degitz. In vitro screening for potential chemical inhibitors of deiodinase type 1 activity. Society of Toxicology, Baltimore, MD, March 12 - 16, 2017.
Impact/Purpose:
An in vitro screening assay developed to identify chemicals with potential for thyroid hormone disruption via inhibition of deiodinase activity will be presented. The results from screening 1800+ chemicals, primarily from the ToxCast phase 1, phase 2 and e1k chemical libraries will be presented. This research helps address the needs of OSCP’s Endocrine Disruptor Screening Program for additional in vitro screening assays covering key molecular initiating events in the thyroid-axis. Results from this assay can support decisions on chemical prioritization for further evaluation and testing for thyroid hormone
Description:
Control of thyroid hormone (TH) signaling in vertebrates is dependent upon 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 19 chemicals was used to establish the assay. Included in this set was the known D1 inhibitor 6-propylthiouracil (used as a positive control). Over 1800 unique chemicals primarily from the EPA’s ToxCast phase 1_v2, phase 2, and e1K chemical libraries 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 nearly 240 chemicals or 13% of the chemicals tested that produced greater than 20% inhibition of D1 activity. Approximately 100 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.