Comparison of amphibian and mammalian thyroperoxidase inhibition by xenobiotics
Knutsen, C. AND M. Hornung. Comparison of amphibian and mammalian thyroperoxidase inhibition by xenobiotics. SETAC Midwest Chapter, Minneapolis, MN, March 20 - 22, 2017.
An in vitro assay developed to assess thyroperoxidase (TPO) inhibition in an amphibian model (Xenopus laevis) will be presented. The activity of a subset of chemicals tested previously for their capacity to inhibit this enzyme that catalyzes thyroid hormone synthesis, are compared between this amphibian model and the activity determined previously in a rat-based TPO inhibition assay. This information can help support program office risk assessments for potential thyroid hormone disruptors by addressing the conservation of this response and potential differences in species sensitivity based on this endpoint.
Thyroperoxidase (TPO) catalyzes the production of thyroid hormones in the vertebrate thyroid gland by oxidizing iodide (I- ) to produce iodinated tyrosines on thyroglobulin, and further coupling of specific mono- or di-iodinated tyrosines to generate the triiodo- and tetra-iodothyronine, precursors to thyroid hormone. This enzyme is a target for thyroid disrupting chemicals. TPO-inhibition by xenobiotics is a molecular initiating event that is known to perturb the thyroid axis by preventing synthesis of thyroid hormone. Previous work on TPO-inhibition has been focused on mammalian TPO; specifically, the rat and pig. A primary objective of this experiment was to directly measure TPO activity in a non-mammalian system, in this case a thyroid gland homogenate from Xenopus laevis; as well as compare chemical inhibition from past mammalian studies to the amphibian data generated. Thyroid glands obtained from X. laevis tadpoles at NF stages 58-60, were pooled and homogenized by sonication in phosphate buffer. This homogenate was then used to test 24 chemicals for inhibition of TPO as measured by conversion of Amplex UltraRed (AUR) substrate to its fluorescent product. The test chemicals were selected based upon previous results from rat in vitro TPO assays, and X. laevis in vitro and in vivo studies for thyroid disrupting endpoints, and included both positive and negative chemicals in these assays. An initial screening of the chemicals was done at a single high concentration, 1 mM. From this data, 2-aminobenzothiazole, benzothiazole, dibutylphthalate, diethylhexylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, phenol, and sodium perchlorate showed little to no TPO inhibition. The remaining 16 chemicals were tested to produce concentration response curves, determine rank-order potencies, and verification of activity. There was strong agreement between chemical between the rat TPO-AUR assay and the X laevis TPO-AUR assay. The results from this relatively small set of chemicals suggests that TPO functionality and response to potential chemical inhibitors is well-conserved across vertebrates.