Date of Award

5-16-2013

Document Type

Thesis

Degree Name

Bachelor of Arts

First Advisor

Powell, Wade

Abstract

Amphibian metamorphosis is a postembryonic developmental process driven by thyroid hormone (TH) and mediated by the thyroid hormone receptor (TR). Toxicity of planar halogenated aromatic hydrocarbons, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is mediated by the aryl hydrocarbon receptor (AHR). TCDD can disrupt TH function in humans and other species via AHR-mediated uridine 5'-diphospho-glucuronosyltransferase (UDP-GT) induction. Here we used the African Clawed Frog (Xenopus laevis) as a model to probe the interaction of TR and AHR signaling. We quantified relative mRNA expression of both TR and AHR target genes in XLK-WG cells and in prometamorphic tadpoles following exposure to both TR and AHR agonists. XLK-WG were exposed to 50 nM T3 and/or 100 nM TCDD for 24 hrs. TR target genes KLF-9, TRë_a, and TRë_b were not strongly induced by TCDD alone, and AHR target genes CYP1A6 and UDP-GT1a were not responsive to T3. However, exposure to both compounds resulted in enhanced expression of both transcript classes. Compared with T3-exposed cultures, KLF-9, TRë_a, and TRë_b mRNAs were 78-128% more abundant following co-exposure with TCDD. Similarly, CYP1A6 and UDP-GT1a mRNA was 77% and 55% more abundant, respectively, in XLK-WG cells following T3/TCDD co-treatment than with exposure to TCDD alone. Comparable patterns were observed in tadpoles (NF 51-54) exposed to 10 nM T3 and/or 5 nM TCDD for 18 hours. The effect of TCDD on circulating TH varies with species and can differ for T4 and T3. While the superinduction of TH-responsive genes could involve TCDD-related alteration of TH levels, the requirement for co-treatment and the effect in cultured cells in the absence of serum suggests the possibility of a transcriptional mechanism. We examined the 5 promoter regions of KLF-9, TRë_a, TRë_b, and CYP1A6, finding potential cognate binding elements for each receptor. Overall, our results suggest that AHR and TR co-regulate expression of their respective targets. Frogs display great insensitivity to TCDD toxicity resulting from key differences in the ligand binding domain of the AHR that lower its affinity for TCDD. However, TCDD-like chemicals have not been environmentally relevant until recent decades and did not likely contribute to the evolution of this phenomenon. Therefore, we examined the effects of a candidate endogenous AHR ligand, 6-formylindolo[3,2-b]carbazole (FICZ), in co-treatments with T3, and observed similar trends to those produced by TCDD. We hypothesize that the induction of TR-regulated genes by AHR may represent an additional mechanism by which metamorphosis is protected from disruption by endogenous or xenobiotic AHR agonists, and nominate FICZ as a possible selective pressure in the evolution of AHR/TR synergy.

Comments

Includes bibliographical references (p. 46-52)

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