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Xenopus laevis deiodinase 3 expression for in vitro screening of potential chemical inhibitors
Citation:
Mayasich, S., P. Kent, C. Knutsen, S. Degitz, AND M. Hornung. Xenopus laevis deiodinase 3 expression for in vitro screening of potential chemical inhibitors. SETAC North America, Minneapolis, MN, November 12 - 16, 2017.
Impact/Purpose:
This work helps to address EPA’s need to determine whether chemicals can adversely affect thyroid hormones, which are necessary for vertebrate development and metamorphosis. An in vitro assay based on production of amphibian deiodianase enzymes in cultured cell lines is being 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 essential for normal sequential development and metamorphosis of amphibian tissues and organs. Critical to this process are the deiodinase (DIO) enzymes which catalyze the removal of an iodine from thyroid hormones to either activate or inactivate the hormone. As part of an effort to evaluate chemicals for thyroid hormone disrupting potential, work at our laboratory is ongoing using recombinant human deiodinase enzymes in a 96-well screening assay to identify chemicals that can inhibit deiodinase activity. However, it is uncertain whether the human enzyme screening results can be a surrogate for activity in other species. Therefore, we initiated a cross-species comparison of chemical inhibitory activity between human and amphibian (Xenopus laevis) deiodinases. Deiodinases are membrane-bound selenoproteins containing the rare amino acid selenocysteine. Expression of functional enzyme in vitro requires not only the protein-coding region of the gene but also the selenocysteine insertion sequence (SECIS) within the 3′-untranslated region (UTR). Initial efforts to express the X. laevis DIO Type 3 in a human cell line (HEK293) produced low activity suggesting potential compatibility issues when expressing amphibian enzyme in mammalian cells. Constructing the gene with the X. laevis protein-coding sequence fused to the 3’ UTR from the human DIO 3 gene resulted in 70 % increased production of functional X. laevis enzyme compared to the native X. laevis sequence. Additionally, we found substrate turnover rate for human DIO 3 to be more than 20 fold higher than for X. laevis DIO 3, and the pH optimum also differed between these species. An initial screening of X. laevis DIO 3 with 25 chemicals was conducted at a single concentration of 200 μM. The results for the X. laevis DIO 3 were in agreement with the human results for most chemicals, however amphiphilic compounds including PFDA that fully inhibited human DIO 3 showed less than 10 % inhibition of X. laevis DIO 3. The low activity of the X. laevis DIO 3 required the use of mg amounts of the HEK293 cell lysates in the assays, which was found to reduce the apparent PFDA inhibitory activity. Further steps to enhance X. laevis protein production and enrich enzyme activity to allow using less lysate may improve the sensitivity of the current assay and improve its utility as a screening tool. This abstract does not necessarily reflect U.S. EPA policy.