Date of Award

12-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Committee Member

Lisa J. Bain, Committee Chair

Committee Member

Charles D. Rice

Committee Member

Susan C. Chapman

Committee Member

Peter van den Hurk

Abstract

Arsenic is an environmental contaminant that is commonly found in drinking water, rice, and other food sources around the world. Many epidemiological studies have reported that in utero exposures to arsenic can significantly reduce muscle growth and cause neurodevelopmental abnormalities such as reduced IQ, impaired memory and spatial learning, and sensory functions. However, the mechanisms behind such effects are not well known. Because of this, we wanted to explore the potential mechanisms by which embryonic exposure to environmentally-relevant levels of arsenic are not only impairing growth but also inducing behavioral changes due to altered sensory neuron function. We were also interested in knowing whether these changes are permanent and remain after the exposure is removed. Killifish (Fundulus heteroclitus) are a commonly used model organism for developmental studies, as they can produce a large number of transparent eggs which allows for greater statistical power. They are readily cultured in the laboratory, so we are also able observe changes over the lifetime of the organisms. Further, previous studies have shown that environmentally-relevant levels of arsenic produce adverse effects in killifish similar to those that cause deficits in humans. Other models, such as zebrafish or rodents, require much higher levels of arsenic to produce similar effects. Therefore, we exposed killifish embryos to 0, 50, 200 or 800 parts per billion (ppb) sodium arsenite. After hatching, the juvenile fish were removed from the exposures and raised in clean water until 52 weeks of age. A second study was conducted to determine the effects of arsenic on olfactory development and function, where embryos were exposed to either 0, 10, 50 or 200 ppb arsenite during embryogenesis and raised in clean water until 40 weeks of age. These studies were designed to examine the effects of exposures during critical points of development (embryonic/fetal) and to investigate if deficits persisted well after the exposure was completed. The results of the first arsenic exposure revealed that at 16 weeks post-exposure, condition factors (CF; weight/length3) were significantly reduced by 12-18% in 200 and 800ppb fish compared to controls. At 28 weeks, all three exposure groups had CFs significantly lower than controls. By 52 weeks, however, CFs were similar in all groups. To examine mechanisms responsible for the growth lag in exposed fish, both trunk skeletal muscle fiber density and skeletal muscle expression of insulin-like growth factor-1 (IGF-1) and its receptor, IGF1-R, were examined. Although there were no significant reductions in muscle fiber density at any time point, both IGF-1 and IGF1-R levels were significantly upregulated in exposed fish at 16 weeks of age (2-2.8-fold greater), and these differences persisted through 52 weeks. These results indicate that reductions in growth may be compensated for by upregulation of the IGF growth pathway, and that embryonic arsenic exposure does not permanently impair growth. However, the consequences of constant IGF upregulation over the lifespan of the organism are unknown. Previous studies have shown that in vitro exposure to arsenic can reduce the expression of transcription factors needed for myogenesis, thus inhibiting their differentiation from stem cells. In order to determine if arsenic targets muscle satellite cells as in vivo, we used fish from the previous study to conduct an induced injury experiment. At 28, 40 and 52 weeks of age, three killifish from each exposure replicate were anesthetized, injected with cardiotoxin below the dorsal fin, and allowed to recover until 3, 7, or 10 days post-injury. Baseline PCNA (a marker of proliferation) and collagen levels were also assessed prior to injury. Immunohistochemical analysis revealed consistent increases in both proliferative cells and collagen expression in exposed fish at all time points in uninjured animals. During the repair process, PCNA expression was reduced in exposed fish, and instead, increases in collagen expression were seen. These results indicate that an embryonic-only arsenic exposure impaired proliferation and differentiation of muscle satellite cells and resulted in the enhancement of a fibrotic resolution to injury. Finally, we conducted a second embryonic exposure with slightly lower arsenic levels (0, 10, 50, or 200ppb arsenic) to assess the effects of embryonic-only arsenic exposure on neurogenesis, using the olfactory epithelium and olfactory sensory neurons (OSNs) as models. To test the function of crypt, ciliated, and microvillus OSNs within the epithelium, we performed odorant response tests at 0, 2, 4, 8, 16, 28, and 40 weeks of age using female ovarian extracts, taurocholic acid (TCA), and an amino acid mixture as odorants for each cell type, respectively. Our results showed that exposed killifish took significantly longer to respond to the ovarian extracts and TCA odorants through week 28, and the number or percentage of fish per tank responding to each odorant was also significantly lower in exposed groups compared to controls. Killifish responses to amino acids were not significantly impaired by arsenic exposure. Immunohistochemistry was used to determine if the number of proliferating cells (PCNA), neural stem cells (Sox2), ciliated cells (calretinin), and microvillus cells (Gαi3) in the olfactory epithelium were reduced due to embryonic arsenic exposure. The results of this study show no changes in numbers of PCNA positive cells until week 16, in which their numbers are significantly greater in exposed fish compared to controls. Sox2 expression is decreased through week 16 and calretinin expression (ciliated OSNs) is consistently lower in exposed groups from week 4 to week 28. These changes in proliferation and differentiation of stem cells in the olfactory epithelium are very similar to those seen in muscle satellite cells after embryonic-only exposure, and these results indicate that arsenic impairs the function of stem cells in both neural and muscle tissue, and these deficits continue throughout adulthood. Overall, it appears that arsenic has long-term effects on the presence and function of both muscle and neural stem cells. In spite of attempts to compensate for these deficits, such as upregulating IGF-1 levels in skeletal muscle and increasing proliferation in skeletal muscle and the olfactory epithelium, it is clear that losses in muscle growth are somewhat ameliorated into adulthood while olfactory epithelial functions are not fully recoverable once the exposure has been removed.

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