Date of Award

5-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Health Sciences

Committee Chair

Jennifer L. Freeman

Committee Member 1

Jason R. Cannon

Committee Member 2

Wei Zheng

Committee Member 3

Maria S. Sepulveda

Abstract

Environmental toxicants are chemical or physical agents that enter the environment and can have adverse health effects on populations of humans. Chemical contamination of rivers, lakes, and ground water is a significant public health concern; 23% of private wells are contaminated with at least one significant environmental toxicant and chemical spill incidences can have severe consequences to human health and the environment. Even low doses of chemicals in the environment can have an effect on human health, and it is suggested by the Developmental Origins of Health and Disease (DOHaD) hypothesis that embryonic exposure to toxicants alters health status and disease prevalence later in life. Therefore, it is important to study the legacy, emergent, and current-use chemicals that are found in low levels in drinking water sources to assess the risk of early exposure. The first chapter of this dissertation introduces the problem of drinking water contamination and provides a background on the zebrafish biomedical model used in this dissertation as well as the zebrafish neurotransmitter systems and methods used to evaluate neurotoxicity. The second chapter is focused on the legacy toxicant, trichloroethylene (TCE). TCE, an industrial solvent and degreaser, is an environmental toxicant that contaminates over half of Superfund sites. In the study described in Chapter 2, the zebrafish model was used to evaluate the acute developmental toxicity of near regulatory concentrations of TCE by monitoring survivability, percent hatching, morphological measurements, and neurobehavior. The percent survival was not significantly different between treatments, but the 500 ppb treatment had increased hatching at 48 hpf. Morphological measurements indicated that the 500 ppb treatment had significantly larger head length to body length ratios and significantly shorter brains compared to the controls. No significant differences were observed during the evaluation of neurobehavior. The expression of four cytochrome p450 enzymes was also evaluated and the expression of cyp24a1 was significantly downregulated, suggesting altered vitamin D3 metabolism. The reported alterations suggest that developmental TCE toxicity is still

a concern near regulatory concentrations and that TCE should remain a priority environmental toxicant. The third chapter describes the acute toxicity of the emergent chemical mixtures in the methylcyclohexanemethanol (MCHM) family. In January 2014, a chemical mixture containing crude MCHM contaminated the water supply of Charleston, West Virginia. Although the mixture was later identified as a mix of crude MCHM and stripped propylene glycol phenyl ethers, initial risk assessment focused on 4-MCHM, the predominant component of crude MCHM. The mixture’s exact composition and the toxicity differences between 4-MCHM, crude MCHM, and the tank mixture were unknown. In the studies described in Chapter 3, the chemical compositions of crude MCHM and the tank mixture were analyzed via GC/MS and found to be different from each other and the reported composition of crude MCHM. The percent mortality and percent hatch, larval morphology alterations, and larval visual motor response test were used to establish toxicity profiles for each of the chemicals or mixtures. The acute toxicity differed between 4-MCHM, crude MCHM and the tank mixture with significant differences in survival, hatching, morphology, and locomotion. The acute toxicity increased as the complexity of the mixture increased, but the developmental toxicity was greatest with the 4-MCHM parent compound. Additionally, behavior was changed at levels as low as the short-term screening level of 1 ppm, suggesting a need for further research into human health risks. The final toxicant of interest is the current use herbicide, atrazine (ATZ). ATZ is the second most commonly used pesticide and frequently contaminates rural and urban water sources. Exposure to ATZ is linked to endocrine disruption, cancer, changes in genome methylation, and alterations in neurochemistry and behavior. The studies described in Chapter 4 focus on ATZ related developmental neurotoxicity while the studies described in Chapter 5 examine the later life effects on the nervous system of embryonic ATZ exposure. In Chapter 4, the relative expression of six genes was monitored throughout a developmental time course to determine normal expression and to determine if developmental exposure to 0, 0.3, 3, or 30 ppb ATZ altered expression. One gene, cyp17a1, had dynamic expression during development that was not related to ATZ exposure, but ttc3 and tpd52l1 both had ATZ related changes in relative gene expression. The brain lengths were measured and larval behavior was monitored to determine physical and functional outcomes of developmental ATZ exposure. Increased brain length was observed in larvae exposed to 0.3 ppb ATZ, while hypoactivity was observed larvae with developmental exposure to 30 ppb ATZ. Proteomic analysis identified 28 proteins with a significant label-free quantification intensity value and the proteins were associated with pathways related to organism and organ system development, intracellular signaling, protein handling and degradation, epigenetic regulation of gene expression, and cancer. The results suggest that the effects of developmental ATZ exposure are broad, time sensitive, and involve the interaction of many pathways. In Chapter 5, the studies use adult zebrafish aged 9 to 14 months to tests the hypothesis that embryonic exposure to low levels of ATZ results in age and sex-specific changes in behavior, the adult brain transcriptome, and adult body and brain pathology, according to the DOHaD hypothesis. At age 9 months, a novel tank test, a light-dark box, and an open field test evaluated adult behavior; microarray analysis investigated ATZ related differences in gene expression; and brain histopathology investigated neuropathology. At 14 months, the body length, weight, and brain weight was measured to evaluate effects of ATZ on mature body and brain size. The 9 month adult behavioral tests found nonmonotonic, sex-specific behavior changes, with male zebrafish having decreased activity and female zebrafish having increased signs of anxiety. Microarray analysis identified sex-specific transcriptomic alterations, with females having altered expression of genes in pathways related to cancer and organismal injury and males having altered gene expression in organismal development and reproductive system development and function pathways. Genes with persistant altered expression were also identified. Adult zebrafish also had nonmonotonic, sex-specific alterations in body length, body weight, and brain weight. This study suggests that developmental exposure to ATZ does cause sex-specific alterations in adult neural function later in life. In summary, developmental exposure to legacy, emergent, and current use environmental toxicants can alter neurodevelopment and later life neural function in zebrafish, suggesting a greater need for human health risk assessments.

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