Variation in the cytochrome P4501A response to dioxin-like compounds in avian species
Creators: name: Head, Jessica
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2006
Subjects: subject: Biology, Molecular.

description: Toxicological risk assessment for dioxin-like compounds in avian wildlife is currently limited by a lack of species sensitivity data. This gap in the literature can be partially attributed to difficulties associated with performing in vivo dosing studies with wild birds. For this reason, in vitro approaches for estimating dioxin sensitivity are required. One such bioassay measures the cytochrome P4501A (CYP1A) response to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) as ethoxyresorufin-O-deethylase (EROD) activity in cultured embryo hepatocytes. Application of the EROD bioassay to risk assessment in avian wildlife is complicated by the extreme variability of the CYP1A response. The experiments presented in this thesis characterize factors contributing to variation in the CYP1A response to TCDD among individuals, populations, and species. These experiments have the dual purpose of investigating mechanisms underlying variation in dioxin sensitivity in avian species, and improving in vitro methods for obtaining species sensitivity estimates. Basal and TCDD-induced CYP1A mRNA expression was found to be extremely variable in hepatocyte cultures prepared from 55 individual herring gull embryos. Significant differences in basal CYP1A expression between populations suggested that there may be a genetic component to this variation. Possible effects of exposure to contaminants throughout development were investigated using a chicken model. Embryonic exposure to dioxin-like compounds at current environmental levels was determined to be unlikely to contribute to the CYP1A response to TCDD in cultured hepatocytes for all but the most contaminated individuals. Regardless of the source, variation between individuals was shown to be a confounding factor for obtaining reproducible sensitivity estimates from the CYP1A bioassay. Characterization of mechanisms underlying species differences in the CYP1A response to TCDD led to the development of novel in vitro approaches for assessing dioxin sensitivity in birds. A comparison of previously published toxicity data and aryl hydrocarbon receptor (AHR) sequences cloned from 18 avian species revealed that key amino acids in the AHR ligand binding domain predict sensitivity to dioxin-like compounds in diverse avian orders. This finding suggests that a simple genetic test could be used to identify species that are particularly sensitive to dioxin-like compounds within contaminated environments. A second series of experiments led to the development of a novel method for measuring CYP1A enzymatic activity (EROD) and mRNA expression in the same sample. This technique increases the versatility of the EROD bioassay and maximizes the amount of data that can be obtained from scarce tissues. Together the results presented in this thesis provide important insight into mechanisms underlying variation in dioxin sensitivity in birds, and enhance our ability to estimate risk to avian wildlife through the use of in vitro bioassays.