EVALUATING THE SUB-LETHAL TOXICITY OF THE ORGANOPHOSPHATE PESTICIDE, CHLORPYRIFOS, ON THE AMPHIBIAN, XENOPUS LAEVIS

Chlorpyrifos (CPF) is an organophosphate pesticide used extensively in Canada and around the world. Due to its highly conserved mechanism of action involving inhibition of acetylcholinesterase (AChE), CPF has the ability to exert toxicity on non-target species in aquatic systems. In fish species, exposure to CPF has been associated with a range of adverse effects across physiological endpoints including abnormal development, inhibition of AChE, immunomodulation, and molecular level effects such as altered expression of specific genes and global transcriptomes. However, the literature on amphibians exposed to CPF is not as extensive despite the known global declines of amphibian species and the hypothesized links between these declines and anthropogenic pesticide contamination of aquatic systems worldwide. The overall objective of this thesis was to gain a better understanding of the sub-lethal effects of CPF exposure on the model amphibian, Xenopus laevis, across levels of biological organization from molecular to whole animal. \nThe first study (Chapter 2) examined the molecular toxicity pathways and mechanisms of toxicity after short-term exposure of early life-stage (ELS) X. laevis to CPF using whole body transcriptome analyses. The ELS transcriptomic responses were then compared to apical outcomes of chronic exposure to CPF to determine if identified dysregulated pathways could provide early indicators of these adverse outcomes. Post-hatch individuals were exposed to nominal CPF concentrations of 0.4, 2, or 10 μg L-1. A subset of individuals were sampled at 96 hours (h) for whole-body transcriptomic analysis and remaining individuals were transferred to tanks for long-term exposure through to metamorphic climax (~ 75 days). Pathway analysis revealed dysregulated pathways that were related to outcomes known to be associated with exposure to CPF such as altered serine hydrolase activity, impacted metabolic processes, and immune-related outcomes. Other dysregulated pathways with less precedence in the literature included vasculature development and sensory perception of light stimulus. Apical outcomes of chronic CPF exposure included inhibition of AChE activity, increased relative liver weight, and a decrease in percentage of individuals that reached metamorphic climax. Dysregulation of serine hydrolase associated pathways after ELS CPF exposure is in agreeance with the decrease in AChE (a serine hydrolase enzyme) activity observed in the brains of individuals at metamorphic climax. Additionally, an increase in relative liver weight after chronic CPF exposure could be related to dysregulation of ELS pathways associated with metabolic processes and immune function. In fact, several pathways related to immune function were depleted.\nIn Chapter 3, we more closely examined the potential immunotoxicity of sub-lethal CPF exposure. Post-metamorphic individuals were exposed 1 or 10 μg L-1 CPF (nominal) for 7 days (d), then administered a phosphate buffered sodium (PBS) control injection or a lipopolysaccharide (LPS) injection to stimulate an inflammatory response. At 24 h post-injection, morphometric indices were recorded and tissues were sampled for differential leukocyte counts (flow cytometry), liver pro-inflammatory cytokine expression (qPCR), and kidney histopathology. At 1 µg L−1 CPF, there was a decrease in circulating lymphocytes, an increase in circulating granulocytes, and an increase in the granulocyte:lymphocyte (GL) ratio regardless of immune state. Liver expression of pro-inflammatory cytokines TNF-α and CSF-1 was increased in individuals exposed to 10 µg L−1 CPF, independent of immune state. Exposure to 10 μg L-1 CPF increased kidney epithelial cell height (by 18 %) and decreased lumen space in the convoluted tubules of the kidney. This study provided evidence that exposure to CPF can lead to changes in key biomarkers of immune status in amphibians in both immune-rested (PBS-injected) and immune-stimulated (LPS-injected) states. Additionally, we found that LPS was an effective mitogen in our study, capable of inducing a robust and measurable stress response in X. laevis. This response included a decrease in circulating lymphocytes, and increase in circulating monocytes, and an increase in the GL ratio. In addition, increased liver expression of pro-inflammatory cytokines TNF-α, IL-1β, and CSF-1 was induced by LPS injection. We conclude that LPS is an appropriate immunostimulatory agent in an immune challenge assay using X. laevis and that exposure to CPF does not appear to impact the response to LPS exposure. \nOverall, our findings show that exposure to environmentally relevant concentrations of CPF has the ability to impact amphibians at multiple levels of biological organization. A number of affected molecular pathways warrant further study in terms of the underlying mechanisms of CPF-mediated toxicity as well as the associated outcomes of CPF exposure in amphibians. This research provides novel data on the effects of CPF exposure to amphibians, which are generally overlooked and under-represented in the literature despite links between pesticide exposure and globally declining amphibian populations.

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