Date of Award
12-2010
Document Type
Thesis
Degree Name
Master of Science (MS)
Legacy Department
Environmental Toxicology
Committee Chair/Advisor
Rice, Charles D.
Committee Member
Scott , Thomas R
Committee Member
Hurk , Peter Van den
Abstract
The aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor normally found in the cytoplasmic compartment of cells held by chaperones and immunophilin-like proteins. Ligand binding dissociates the AhR/ligand from chaperone proteins, allowing translocation to the nucleus with subsequent transcription of a suite of responsive genes, most notably Phase I, II, and III drug metabolism genes. Select environmental contaminants such as co-planar PCBs, planar polyaromatic hydrocarbons (PAHs), and halogenated aromatic hydrocarbons (HAHs) are potent AhR agonists, with 2,3,7,8 -tetrachlorodibenzodioxin (TCDD) being one of the most potent. Adverse effects of exposure to these potent environmental AhR ligands include immune suppression, reproductive, and developmental disorders. Mammals express a single AhR protein, while fish express both AhR1 and AhR2. However, to date only the AhR2 protein appears to be involved in mediating the toxic effects of known xenobiotic AhR ligands.
Using bacterial expression plasmid systems, a recombinant Atlantic killifish, Fundulus heteroclitus , AhR2 protein was expressed and used to produce a monoclonal antibody (mAb 5B6) that does not cross-react with AhR1. AhR2 expression can be detected with abundance in the cytosol of individual cells and in select organs. By testing the antibody against paraffin-embedded tissues, it was found that mAb 5B6 requires microwaving tissues under high pH conditions to properly recognize its epitope. . High levels of AhR2 protein are detected in the liver, spleen, intestine, and anterior kidney. Experimental exposures to the potent AhR2 ligand PCB-126 induce
expression of both AhR2 and CYP1A proteins in most tissues, especially the intestine and liver. Basal expression of AhR2 protein was determined in livers of Atlantic killifish collected at the US-EPA Superfund site in Portsmouth VA, a site heavily contaminated with creosote and containing very high levels of PAHs. Several livers from the Superfund site harbored aggressive tumors and other hepatic lesions. AhR2 protein expression was high in normal tissue, but not cells within lesions. Overall, CYP1A protein expression patterns mirror those of AhR2 protein. This is the first study to examine AhR2 expression in tissues isolated from fish collected in the field, and like CYP1A, this protein may be a sentinel biomarker in future studies.
Recommended Citation
Wojdylo, Josephine, "Development, characterization, and technical applications of an atlantic killifish AhR-2 specific monoclonal antibody (mAb 5B6)" (2010). All Theses. 1034.
https://open.clemson.edu/all_theses/1034