Date of Award

Spring 5-11-2016

Document Type


Degree Name

Bachelor of Arts


Biochemistry & Molecular Biology

First Advisor

Wade H. Powell


The aryl hydrocarbon receptor (AHR) mediates the toxic effects of halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs) in vertebrates. Frogs and salamanders are unusually insensitive to the toxicity of these xenobiotics because amphibian AHRs typically bind these agonists with lower affinity than more sensitive mammalian and teleost receptors. The AHR signaling pathway in the African clawed frog, Xenopus laevis, displays another unusual characteristic: the presence of two closely related AHRs, AHR1α and AHR1β. These proteins are encoded by paralogous genes that resulted from genome duplication in a cross-species hybridization event 25-40 million years ago. Previous studies of frog AHR signaling in our lab made extensive use of XLK-WG, an epithelial cell line derived from adult X. laevis kidney. In this study, we launched the characterization of AHR function in the X. laevis tadpole cell line, XTC-2. XTC-2 cells are of particular interest because the cell line functions as a model for gene expression during metamorphosis, the development of a froglet from a tadpole. AHR function was assessed by measuring the transcriptional responsiveness of XTC-2 cells to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the most potent xenobiotic AHR agonist, and 6-formylindolo[3,2-b]carbazole (FICZ), a well- characterized endogenous AHR ligand. Surprisingly, XTC-2 cells were largely refractory to induction of cytochrome P450 1A6 (CYP1A6) and cytochrome P450 1A7 (CYP1A7), the primary AHR target genes, by TCDD. Additional AHR target genes, gap junction gamma 1 (GJC1) and spectrin repeat-containing nuclear envelope protein 1 (SYNE-1), were also minimally induced in by TCDD. The only AHR target gene altered by TCDD was aryl hydrocarbon receptor repressor (AHRR), which was induced ~10-fold. In contrast, FICZ induced CYP1A6 ~80-fold, CYP1A7 ~250-fold, and AHRR ~12-fold.While TCDD-induced target gene expression in XTC-2 cells is blunted compared to XLK-WG cells, FICZ responsiveness is comparable between the two cell lines.

These data indicate that AHR activity in XTC-2 cells is distinct from that in XLK- WG cells. To elucidate the underlying mechanisms distinctive AHR function, we sought to characterize the transcriptional role of each AHR paralog in isolation in knockout cell lines, comparing gene expression with similar mutants derived from XLK-WG. We successfully employed TALENs to generate AHR1β mutations in XTC-2 cells. Further genomic characterization of mutant cell lines is needed to confirm the homozygosity of the mutation and the absence of off target mutations. Future work should be directed towards the generation of a homozygous AHR1α knockout derived from XTC-2.

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