Developmental sensitivity and immunotoxicity of benzo[a]pyrene in the amphibian Xenopus laevis
Gallant, Melanie J 1991-
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The immune system is increasingly recognized as a vulnerable and sensitive target of contaminant exposure. Disruption of this critical system can lead to significant morbidity and/or mortality of organisms in contaminated environments. Amphibians can be simultaneously exposed to both pathogens and contaminants within aquatic habitats, making contaminant-induced immunotoxicity particularly relevant for this taxonomic group. Polycyclic aromatic hydrocarbons (PAHs) are a major group of environmental contaminants that widely occur as mixtures in aquatic environments, including amphibian habitats. Benzo[a]pyrene (B[a]P) is recognized as the most potent toxicant of PAH mixtures and is the most extensively studied PAH with demonstrated adverse effects on the immune system of many vertebrate species. Understanding how aquatic contaminants, such as B[a]P, can impact the immune system of amphibians is imperative since contaminant-induced immunotoxicity is hypothesized to exacerbate disease-driven amphibian population decline. Thus, the overall goal of this thesis was to gain a better understanding of developmental and contaminant-induced changes in the immune system of amphibians, and ultimately how exposure to B[a]P modulates the response to immune stimulation. Specific research questions were addressed through four studies conducted within the broader goal of this thesis. The objective of the first study was to characterize the expression profiles of various pro-inflammatory cytokines in the model amphibian species, Xenopus laevis, throughout larval developmental stages and determine the impacts of thyroidal modulation on their expression. Results suggest that expression of two cytokines, tumor necrosis factor- (TNF) and interleukin-1β (IL-1β) varied over development, increasing with developmental stage, while interferon- (IFN-) remained relatively unchanged. Exogenous manipulation of thyroidal status was successful, as demonstrated by an altered rate of development and thyroid gland histology; however, thyroidal modulation had negligible effects on basal cytokine expression. The dynamic nature of the tadpole immune system suggested that the immunotoxic impacts of environmental contaminants may depend on the stage of development at the time of exposure. Therefore, the second study aimed to identify the sensitive developmental phases for sub-lethal effects of B[a]P exposure in terms of the development (stage, weight and length) and changes in the expression of immune- and detoxification-related genes. Exposure to B[a]P at all life-stages tested (embryo-larval, pre-metamorphic, and pro-metamorphic tadpoles) resulted in CYP1A7 mRNA induction (43, 53, and 47-fold, respectively) as well as stage-specific modulation of pro-inflammatory cytokine expression. Exposure of pre-metamorphic tadpoles to B[a]P demonstrated the greatest effect on weight, length and stage of development. Taken together, these initial studies indicate that the unique development of the tadpole immune system influences their susceptibility to contaminant-induced immune modulation despite having biotransformation capacity across tadpole life-stages. In the subsequent two studies, the ability of B[a]P to alter immune variables and impair a coordinated immune response was evaluated using an immune challenge model both in vivo and in vitro. Juvenile X. laevis were exposed to B[a]P for seven days and then immune stimulated with an injection of lipopolysaccharide (LPS). The highest concentration of B[a]P (350 g/L) impaired the inflammatory response to LPS as indicated by an inability to induce granulocyte:lymphocyte (G:L) ratio or IL-1β mRNA expression, demonstrating that B[a]P can impair the inflammatory immune response to a simulated pathogen. Next, to assess the mechanisms underlying B[a]P-induced effects in amphibians, X. laevis kidney A6 epithelial cell line were used to evaluate the cellular response to B[a]P exposure and identify if B[a]P could directly alter the ability of these cells to respond to an immune stimulation. A time-course of B[a]P exposure showed the progression from up-regulation of biotransformation-related genes, to reactive oxygen species production, and ultimately to cytotoxic effects; pre-exposure to B[a]P did not appear to impair the ability of the A6 cells to mount an inflammatory response to LPS. Taken together, the immune challenge models showed that B[a]P exposure can alter immune variables and impact the ability of juvenile X. laevis to appropriately respond to immune stimulation and that B[a]P-induced biostransformation pathways and production of reactive oxygen species may play a role in this response. Overall, the research presented in this thesis contributes to our understanding of developmental and contaminant-induced changes in the immune system of amphibians. This research provides a strong foundation for further study into the mechanisms underlying chemical-induced immunotoxicity in amphibians. The tools and approaches developed as part of this research could be used in screening chemicals for immunotoxic potential in amphibians and be expanded to evaluate immune responses of ecologically relevant species that are under real risk of compromised immune function and increased disease in contaminated environments.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeJanz, David; Morrissey, Christy; Blakley, Barry; Bollinger, Trent
Copyright DateDecember 2019