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In Vitro screening for chemical inhibitors of human and amphibian iodotyrosine deiodinase activity
Citation:
Olker, J., Joe Korte, P. Hartig, M. Cardon, M. Hornung, AND S. Degitz. In Vitro screening for chemical inhibitors of human and amphibian iodotyrosine deiodinase activity. SETAC North America, Sacramento, CA, November 04 - 08, 2018.
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 with a non-radioactive detection method. Included are the results from a test set of 10 chemicals and a cross-species comparison. These assays can be used for future screening of large chemical libraries for inhibition of IYD activity. This work supports the US 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:
odotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling from monoiodotyrosine (MIT) and diiodotyrosine (DIT) and promotes retention of iodide in thyroid follicular cells. IYD has an important role in maintaining appropriate levels of thyroid hormones through conservation of this scarce micronutrient and is especially critical for organisms in low iodide environments, including most freshwater ecosystems. Loss of function or chemical inhibition of IYD reduces thyroid hormone synthesis, which leads to insufficiency in tissues and subsequent negative developmental consequences. Our objective was to develop a 96-well plate in vitro assay that could be used to screen chemicals for inhibition of IYD and to compare inhibition between mammals and amphibians. We utilized recombinant human IYD enzyme and Xenopus laevis liver microsomal fractions to establish robust assays using MIT as the substrate, 3-nitro-L-tyrosine (MNT) as the positive control, and a non-radioactive detection method. A set of ten chemicals was used to establish the assay, including seven known or suspected inhibitors of IYD (MNT, 3,5-dinitrotyrosine, 3,5-dibromotyrosine, triclosan, phloxine-B, L-mimosine, and bromoxynil) and three suspected non-inhibitors (genistein, dibutyl phthalate, and bisphenol A). Chemicals were tested at a maximum concentration of 200 µM, with seven graded concentrations to produce an inhibition concentration-response curve. Assay performance metrics support use of this assay for screening, with high Z’ factor (0.7 or greater) and low variability in the control chemicals (DMSO and MNT). With the recombinant human IYD, five chemicals produced inhibition of IYD ranging from 60-100% (compared to DMSO control), with IC50 ranging from 0.05 µM to >200 µM. There was strong cross-species agreement between human and Xenopus, and all chemicals produced comparable maximum inhibition and IC50s, with minor shifts in rank order potency. These assays for inhibition of IYD activity in a 96-well plate format can be used for future screening of large chemical libraries. Additionally, these results indicate that IYD response to potential chemical inhibitors is conserved across vertebrates.