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Xenopus laevis and human type 3 iodothyronine deiodinase enzyme cross-species sensitivity to inhibition by ToxCast chemicals
Citation:
Mayasich, S., J. Korte, J. Denny, Philip Hartig, J. Olker, P. Degoey, J. O'Flanagan, S. Degitz, AND M. Hornung. Xenopus laevis and human type 3 iodothyronine deiodinase enzyme cross-species sensitivity to inhibition by ToxCast chemicals. TOXICOLOGY IN VITRO. Elsevier Science Ltd, New York, NY, 73:105141, (2021). https://doi.org/10.1016/j.tiv.2021.105141
Impact/Purpose:
This manuscript describes optimization of activity and expression of the amphibian Xenopus laevis type 3 iodothyronine deiodinase enzyme (Xldio3) and its use in screening assays to identify specific chemical inhibitors and to evaluate cross-species sensitivity of Xldio3 compared to human DIO3. The Xldio3 assays were conducted similarly to the human DIO3 assays in a 96-well plate format. Single concentration screening of 356 chemicals was conducted, with further concentration-response testing for 79 of these chemicals to calculate IC50s and to compare rank order potency for the purpose of determining whether human screening results can be predictive for those of the amphibian. This work supports the U.S. EPA Endocrine Disruptor Screening Program’s need for higher-throughput in vitro screening assays to address additional molecular initiating events, expanding the ToxCast suite of assays to include non-mammalian vertebrate orthologous protein targets.
Description:
Deiodinase enzymes are critical for tissue specific and temporal control of activation or inactivation of thyroid hormones during vertebrate development, as well as in amphibian metamorphosis. From the results of an in vitro screening study of the ToxCast Phase 1, Phase 2 and e1k chemical libraries for inhibitory activity toward human Type 3 iodothyronine deiodinase (hDIO3) enzyme we identified a subset of 356 chemicals to test for amphibian (Xenopus laevis) dio3 (Xldio3) enzyme inhibition. Our goals were to identify specific chemical inhibitors of Xldio3 and to evaluate cross-species sensitivity. Recombinant Xldio3 was produced in cell culture and used in 96-well plate screening assays at a single test chemical concentration (200 µM). From the initial Xldio3 single concentration screening results, 79 chemicals were tested in concentration-response mode. A general relationship between steep Hill slopes (a potential indication of non-specific inhibition) and LogP (octanol:water) values >4.5 emerged. Of the 43 chemicals with a LogP > 4.5, 33 had steep Hill slopes (much less than -1) for one or both species, with most Xldio3 IC50 values much higher than for hDIO3. This may be because the activity level of the enzyme in the Xldio3-transfected cell lysate was lower than that of hDIO3, requiring 25 times more protein in the Xldio3 assays. However, eight of the most potent chemicals with IC50 of 14 µM or less had Hill slopes near -1 and were not significantly different between species. These 8 chemicals may be specific, competitive inhibitors. The similar responses of hDIO3 and Xldio3 to these chemicals are likely due to conservation of the catalytically active amino acids between these species. More work is needed to distinguish between specific and non-specific inhibitors, and to improve expression of recombinant non-mammalian proteins for HTS studies of cross-species sensitivity to potential endocrine-disrupting chemicals.
URLs/Downloads:
DOI: Xenopus laevis and human type 3 iodothyronine deiodinase enzyme cross-species sensitivity to inhibition by ToxCast chemicals