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In vitro screening assays for chemical inhibition of mammalian and amphibian iodotyrosine deiodinase
Citation:
Olker, J., Joe Korte, J. Denny, P. Hartig, M. Cardon, J. O'Flanagan, M. Hornung, AND S. Degitz. In vitro screening assays for chemical inhibition of mammalian and amphibian iodotyrosine deiodinase. Society of Toxicology, Baltimore, MD, March 10 - 14, 2019.
Impact/Purpose:
Iodotyrosine deiodinase (IYD) has an important role in vertebrate thyroid hormone homeostasis through catalyzing iodide recycling and promoting retention of iodide in thyroid follicular cells. This poster presents the development of assays for inhibition of human and amphibian IYD in a 96-well plate format and the application of the human IYD inhibition assay to screen a large set of chemicals. Included are the results from the test set of 10 chemicals with a cross-species comparison and the results from screening the ~300 chemicals in the ToxCast phase 1_v2 chemical library for inhibition of human IYD. These assays can be used for future screening of large chemical libraries for inhibition of IYD activity. This work supports the U.S. 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:
Iodotyrosine deiodinase (IYD, dehalogenase) catalyzes the recycling of iodide from monoiodotyrosine (MIT) and diiodotyrosine (DIT) and thus has an important role in retention of iodide for synthesis of thyroid hormone. In an effort to understand the susceptibility of IYD to chemical perturbation, a 96-well plate in vitro assay was developed to screen chemicals for inhibition of mammalian and amphibian IYD enzyme activity. With recombinant human IYD enzyme and Xenopus laevis liver microsomal fractions, robust assays were established using MIT as the substrate and 3-nitro-L-tyrosine (MNT) as the positive control, and tested against an initial set of ten chemicals (7 known/suspected IYD inhibitors and 3 non-inhibitors). Performance metrics support use of these assays for screening, with high Z’ factor (0.6 or greater) and low variability in the control chemicals (DMSO, NaOH, and MNT). There was strong cross-species agreement between human and Xenopus IYD in this initial test set of chemicals, with similar maximum inhibition and rank order potencies; however, a larger set of chemicals will be tested to complete this species comparison. The ToxCast phase 1_v2 chemical library was then screened with recombinant human IYD to identify chemicals that have the potential to disrupt the thyroid axis via this pathway. The majority of these chemicals did not inhibit IYD enzyme activity at a single, high concentration (target of 200 µM). Of the 293 unique ToxCast chemicals tested, 28 chemicals (9.6%) inhibited human IYD activity by 20% or greater, including 3 chemicals previously reported to inhibit IYD. Of these 28 chemicals, 7 produced inhibition of 50% or greater and were further tested in concentration-response mode to determine IC50s and rank order potency. These IYD inhibition assays can be used for future screening of large chemical libraries and expand the limited number of in vitro assays used to identify chemicals having potential to interfere with thyroid hormone homeostasis. Additionally, these results suggest that IYD response to potential chemical inhibitors is conserved across vertebrates.