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In vitro screening of Xenopus laevis deiodinase 3 for inhibition by ToxCast chemicals
Citation:
Mayasich, S., J. Denny, Joe Korte, S. Degitz, AND M. Hornung. In vitro screening of Xenopus laevis deiodinase 3 for inhibition by ToxCast chemicals. SETAC North America, Sacramento, CA, November 04 - 08, 2018.
Impact/Purpose:
This work helps to address EPA’s need to determine whether chemicals can adversely affect thyroid hormones, which are necessary for amphibian development and metamorphosis. An in vitro assay based on production of amphibian deiodinase enzymes in cultured cell lines has been developed to screen chemicals for their ability to inhibit these enzymes that regulate active thyroid hormone levels in the organism. The results from this effort will provide a basis for making better informed decisions about the predictive potential of screening assays between mammalian and amphibian species.
Description:
Thyroid hormones are necessary for normal sequential development and metamorphosis of amphibian tissues and organs. Deiodinase enzymes in peripheral tissues are critical components of the system that catalyze the removal of an iodine from thyroid hormones to either activate or inactivate the hormone. We conducted a screening study of chemical inhibitory activity toward amphibian (Xenopus laevis) Type 3 iodothyronine deiodinase (DIO 3) enzyme. The chemicals were selected from across the ToxCast Phase 1, Phase 2 and e1k chemical libraries, based on results from previously screening these chemicals against the human DIO 3. Recombinant X. laevis DIO 3 enzyme was produced in cell culture and used in a 96-well plate in an initial single-concentration (200 µM) screening assay. Chemicals were further selected for testing in concentration-response mode if found to inhibit the X. laevis DIO 3 in the single-concentration screening, as well as having low IC50 values for the human DIO 3 enzyme. Some of these chemicals of interest included antimicrobials Triclosan and chlorhexidine diacetate, and the insecticide Fipronil. Xanthohumol was used as a positive control for DIO3 inhibition. The IC50 values for the human DIO 3 enzyme for these four example chemicals were 61.1, 2.6, 1.2 and 0.28 µM, respectively. The IC50 values for X. laevis DIO 3 were 266.3, 9.5, 0.73 and 0.33 µM, respectively. Overall, the data indicate similar activity of the chemicals, likely due to conserved amino acids between these species within the enzymes’ catalytic sites.