Disruption of thyroid hormone action by environmental contaminants in vertebrates




Hinther, Ashley

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Thyroid hormones (THs) are important hormones involved in developmental processes, including foetal brain maturation. THs are also involved in the maintenance of homeostasis. One in three people in Canada are considered to have some form of thyroid disorder. One reason for the high level of thyroid disorders may be the increasing amount of anthropogenic chemicals released into the environment that affect normal hormone action. Amphibian metamorphosis is completely dependent on TH and provides a model to study such chemicals. This thesis uses the Rana catesbeiana tadpole as a model to study potential TH disrupting chemicals by developing a novel screening assay called the cultured tail fin biopsy assay, or the “C-fin” assay. The C-fin assay uses tail biopsies from premetamorphic tadpoles, Taylor-Kollros stage VI-VIII. The biopsies are cultured in serum-free media along with the test chemical for 48 hours. QPCR is used to measure the mRNA steady-state levels of selected gene transcripts. Two TH-responsive gene transcripts were measured: the up-regulated gene transcript, thyroid hormone receptor β (TRβ) and the down-regulated gene transcript, Rana larval keratin type I (RLKI). Heat-shock protein 30 (HSP30) and catalase (CAT) were used as indicators of cellular stress. Another model system used in this thesis is rat pituitary cells, or GH3 cells. QPCR was used to measure the mRNA steady-state levels of three TH-responsive genes growth hormone (GH), deiodinase I (DIOI), and prolactin (PRL); heat-shock protein 70 (HSP70) was used as an indicator of cellular stress. Nanoparticles, used in various consumer products, were one class of chemicals examined. Using the C-fin assay, nanosilver and quantum dots (QDs) caused perturbations in TH-signalling and also showed signs of cellular stress. There was no overt toxicity observed as was determined by the normalizer, house-keeping gene transcript, ribosomal protein L8. The GH3 cells also detected TH disrupting effects by both nanosilver and QDs; however, nanosilver did not appear to cause cellular stress whereas QDs did. Nitrate and nitrite, major waterway contaminants, were also examined and there were no TH-perturbations observed using the C-fin assay. Finally, two antimicrobials used in many consumer products, triclocarban (TCC), triclosan (TCS) and its metabolite, methyl-TCS (mTCS) were examined using both the C-fin assay and GH3 cells. Both the C-fin assay and the GH3 cells determined mTCS to be more potent than TCS in disrupting TH action. TCC also caused perturbations in TH-signalling as well as causing a significant amount of cellular stress. Overall the C-fin assay and the GH3 cells proved to be excellent models in studying the potential disruptors of the TH axis. The C-fin assay and GH3 cells detected novel TH disruptors and gave further insight into already known disruptors of the TH axis.



thyroid hormone, Rana catesbeiana, mRNA expression, QPCR, nanometals, antimicrobials