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<p>   </p> <p>Environmental toxicants such arsenic (As) and lead (Pb) are chemicals that enter the environment and can result in adverse health effects humans, especially during development. This dissertation work evaluated As and Pb to determine if developmental toxicity significantly changes at lethal and sub-lethal mixture concentrations using the zebrafish model. Joint action models were applied to survival data to determine the type of interaction. Metal exposures were from 1-120 hours post fertilization (hpf). As concentrations were 0-7,500 ppb. Pb concentrations were 0–100,000 ppb. The LC25, LC50, and LC75 values at 120 hpf from single metal exposures were used to select mixture concentrations for modeling. The survival data indicated an additive effect occurred at lethal concentrations. </p> <p>The impact of the mixture on behavior, morphology, and gene expression was then evaluated at sub-lethal concentrations of 10 and 100 ppb As and Pb individually or in mixtures. Data was analyzed with a repeated measures ANOVA (behavior) or an ANOVA (morphology and qPCR) with the least significant difference test (α=0.05). Zebrafish larvae exposed to 10 ppb As exhibited hyperactivity in all dark phases for the distance moved, time moving, and velocity, while those exposed to 10 ppb Pb only showed an increase in distance moved and velocity in the first dark phase. The 10 ppb mixture was found to have an intermediate impact with increased time moving in all dark phases and increased distance moved and velocity only in the first dark phase. In contrast, hyperactivity was observed only in the 100 ppb As and 100 ppb mixture treatment in the last two dark phases for time spent moving. No significant behavioral alterations occurred in the 100 ppb Pb treatment. A decrease in mean brain length and brain length ratio to the total length in the 10 ppb mixture was observed with no significant morphology changes observed for head length, head width, or total length. Alternatively, measurements of cerebral vasculature morphology in the mesencephalon (midbrain) and cerebellum (hindbrain) uncovered decreased total vascularization at 72 hpf (exposure 1-72 hpf) in both brain regions. This decrease occurred in all treatment groups in the mesencephalon and in all treatment groups, except the 100 ppb Pb and 10 ppb As treatment groups in the cerebellum. In addition, decreased sprouting and branching occurred in the mesencephalon, while only decreased branching was measured in the cerebellum. The 10 ppb Pb treatment group showed unique perturbations in several cerebral vasculature endpoints evaluated, which was also observed in a specific gene expression alteration pattern different from the other treatment groups. To identify molecular changes associated with these changes, expression of genes related to angiogenesis and vasculogenesis (i.e., <em>vegfaa, wnt7aa,</em> and<em> lrp1aa</em>) and genes associated with tight junctions (i.e., <em>cldn5a</em> and <em>cldn5b</em>) were assessed at 72 hpf. Increased <em>cldn5b</em> expression was detected in all treatment groups, while <em>cldn5a </em>was increased in only the 10 ppb Pb treatment group. In addition, <em>wnt7aa</em> was only decreased in the 10 ppb Pb treatment group. Alternatively, <em>vegfaa</em> was increased in the 100 ppb As and 100 ppb mixture treatment groups and no changes were detected for <em>lrp1aa</em>. In summary, cerebral vascular toxicity outcomes in the 10 ppb mixture treatment were primarily driven by changes in the 10 ppb Pb treatment group, while perturbations in the 100 ppb mixture treatment group aligned with the 100 ppb As alterations. In addition, the non-linear dose response for 10 and 100 ppb Pb treatment groups agree with observations in prior studies. qPCR results indicate that both metals together and separately alter cerebral vasculature development at environmental regulatory levels. </p> <p>Lastly, with the increase in the prevalence of neurodegenerative diseases increasing globally, there is a need to evaluate more therapeutics at a high through-put pace and to pinpoint the cause of the sporadic cases. CRISPR-Cas9 technology offers a relatively inexpensive, reliable, and precise advantages over its predecessors when it comes to producing mutant disease models of neurodegeneration. A method to create the expression vector needed for creation of a CRISPR Cas9 knock-in model is detailed in this dissertation. The methodology to insert a chimeric DNA sequence contain human DNA has been created, and the <em>in silico</em> assays used to produce the reactant for this methodology were successful. It has been determined that the efficiency of this knock-in method is limited to the success of producing the chimeric model which is limited itself by the number of molecules included into the chimeric sequence. Overall, the results show that the overlap primers designed in silico need to be re-designed to improve efficiency of the initial reactions required to produce the first plasmid containing the required machinery for a successful knock in of exogenous human DNA.</p>

<p>Per- and polyfluoroalkyl substances (PFAS) are a group of emerging global contaminants used in a variety of industrial processes and consumer products, such as personal care products and fast-food wrappers. However, due to their carbon-fluorine bonds, these chemicals resist degradation and persist in the environment. PFAS toxicity is driven by a compound’s functional group and chain length with perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide dimer acid (GenX) being of focal concern due to their toxicity to wildlife and presence in the environment. Despite growing concern regarding these contaminants, inadequate attention has been given to evaluating what organismal characteristics influence uptake and depuration of these chemicals, such as life stage and sex. <em>Xenopus laevis</em> tadpoles are a useful model to assess the influence of sex on PFAS kinetics since they have a life history that includes a gill to lung transition. Previous studies have shown that air-breathing organisms depurate PFAS more slowly than water-breathing organisms, but this relationship has never been directly tested. Sex has been shown to be an important factor in the depuration of PFOA for rats, with female rats depurating PFOA in four hours while males depurate in four days. The early portion of bioaccumulation curves are also understudied even though tadpoles accumulate PFAS rapidly, reaching steady state within 48 hours of exposure. <em>Xenopus laevis</em> are used to study multiple endpoints for endocrine disrupting chemicals including PFAS. Despite this, toxicity reference values (TRVs) have not been described for the uptake and elimination of PFAS using <em>X. laevis</em>. To address these gaps in knowledge, I first developed TRVs for <em>X. laevis</em> tadpoles exposed to PFOA throughout metamorphosis and evaluated the influence of sex on phenotypic endpoints. Results showed a no observed effect concentration (NOEC) of 11.1 ppm for body mass at day 14 and no effect of sex on apical endpoints. Next, I described the early bioaccumulation of four PFAS with differing structure (chain lengths and functional groups). PFOS was the only chemical to bioaccumulate with a log bioconcentration factor (BCF) at 10 and 1,000 ppm of 1.33 and 1.18, respectively. PFHxS, PFOA, and GenX had BCFs less than 0. Finally, I examined the impact of life-stage and sex on <em>X. laevis</em> tadpole and juvenile depuration rates. Larval tadpoles depurated four times faster than juveniles, indicating a significant effect of life stage on elimination rates. Sex had no influence on elimination rates. These are the first studies conducted evaluating the significance of life stage and sex in toxicokinetics of PFAS in amphibians.</p>

<p dir="ltr">Atrazine is an herbicide used to control broadleaf and grassy weeds on agricultural fields in the US and other global regions, but this herbicide has been banned from use in the European Union since 2003, based mainly on risk of contamination of surface and groundwater. Atrazine is categorized as an endocrine disrupting chemical (EDC) but the specific mechanism that leads to this disruption is not yet clearly defined. This study investigated the hypothesis that the main target of neuroendocrine atrazine toxicity is the kisspeptin system, which then leads to multiple adverse health outcomes that are seen affecting multiple endocrine axes. Furthermore, an embryonic atrazine exposure was expected to result in adverse effects on neurohormones and associated gene expression along the endocrine axes into adulthood. Using the zebrafish model, concentrations of estradiol, dopamine, kisspeptin, and luteinizing hormone were measured in atrazine exposed larvae as well as male and female adult brains using ELISA. Adult wild type zebrafish were bred to obtain embryos, collected at 1 hour post fertilization (1 hpf), and randomly assigned to 0, 0.3, 3, or 30 ppb (µg/L) atrazine treatment, surrounding the current US EPA regulatory level in drinking water of 3 ppb. Exposure ceased at the end of embryogenesis (72 hpf) and fish placed in filtered aquaria water for continued development. Zebrafish were either collected for larval evaluation [72, 120, 144, or 168 hpf] or grown to different timepoints in adulthood for collection of brains [6 months post fertilization (mpf), 2 years post fertilization (ypf), or 2.5 ypf]. Gene expression of neuroendocrine molecular targets was examined to determine if an embryonic atrazine exposure perturbed neuroendocrine development using qPCR. Behavior analysis was conducted on larvae and in adults to assess downstream functional changes related to dopaminergic signaling. Significant findings in gene expression, neurohormone and protein concentration, and behavior were observed and a CRISPR-Cas9 knockdown model was designed to further investigate the kisspeptin system as a viable target of atrazine toxicity in connection with the multitude of adverse effects. Studies to further examine perturbations along the pathways associated with these biomarkers are necessary to elucidate the mechanism of atrazine and further characterize the role of the kisspeptin system in atrazine toxicity.</p>

Ionized calcium (Ca2+), acting as an intracellular messenger, controls numerous biological processes that are essential for life. However, it is also able to convey signals that result in cell death. The fidelity of Ca2+ as a universal second messenger therefore depends on mechanisms that specifically and dynamically regulate its levels within a cell, as well as maintain resting intracellular Ca2+ concentration ([Ca2+]i) very low. One such mechanism for Ca2+ signaling and homeostasis is the plasma membrane Ca2+-ATPase (PMCA), which is a primary active Ca2+ transporter that translocates Ca2+ from a low intracellular Ca2+ environment to a high extracellular environment. There are four mammalian PMCA isoforms (PMCA1−4), which are differentially expressed depending on tissue or cell type. PMCA isoforms possess different sensitivities to biochemical regulation of Ca2+ efflux activity and are also able to subtly alter the dynamics of Ca2+ signals. These properties suggest that the PMCA is not merely a trivial mechanism for Ca2+ extrusion but is influential in contributing to the Ca2+ signaling requirements and unique physiology of different cells. The indispensable nature of Ca2+ signaling in organs such as the brain, heart and skeletal muscle has been the studied extensively but little is known about the roles and regulation of Ca2+ in the mammary gland. This is despite the fact that the mammary gland is a site of extensive Ca2+ flux during lactation. However, cumulating evidence indicates that upregulation of PMCA2 expression in the mammary gland is a major mechanism for milk Ca2+ enrichment. Therefore, the PMCA is likely to be an important mediator of bulk Ca2+ homeostasis in the mammary gland. Studies in other model systems also suggest that PMCAs may regulate other cellular processes such as cell proliferation, differentiation and apoptosis that are required for normal mammary gland physiology. These basic cellular processes are also disturbed in breast cancer and hence deregulation of PMCA expression in the mammary gland may have pathophysiological consequences. Previous studies show that PMCA1 expression is greater in tumorigenic MCF-7 and MDA-MB-231 human breast cancer cells compared to non-tumorigenic MCF-10A human breast epithelial cells. Furthermore, the expression of PMCA1b and PMCA4b is lower in human skin and lung fibroblasts neoplastically transformed by simian virus 40, compared to non-transformed counterparts. It is therefore hypothesized that regulation of PMCA isoform expression is disrupted in breast cancer and that inhibition of PMCA expression in an in vitro model of breast cancer has important effects in modulating intracellular Ca2+ homeostasis, cell proliferation, differentiation and apoptosis. This thesis describes the use of real time RT-PCR to compare PMCA isoform mRNA expression in tumorigenic and non-tumorigenic mammary gland epithelial cells. It demonstrates that particular breast cancer cell lines overexpress PMCA2, an isoform with restricted tissue distribution and which is present in abundant amounts in the lactating rat mammary gland. Thus, some breast cancers may be characterized by the overexpression of Ca2+ transporters that are normally upregulated during the physiological course of lactation. The pathophysiological significance of PMCA2 overexpression in breast cancer is uncertain and future investigations should look at whether levels of PMCA isoform expression correlate with malignancy, prognosis or survival. To address the second hypothesis of this thesis, a stable MCF-7 Tet-off human breast cancer cell line able to conditionally express PMCA antisense was generated. This strategy was necessary due to the current lack of specific pharmacological inhibitors of the PMCA. This thesis shows that PMCA antisense expression significantly inhibits PMCA protein expression, while subtly affecting PMCA-mediated Ca2+ efflux without causing cell death. However, it also reveals that inhibition of PMCA expression has major effects in mediating cell proliferation and cell cycle progression. Moderate changes in PMCA expression and PMCA-mediated Ca2+ transport result in dramatic consequences in MCF-7 cell proliferation. These studies not only support the supposition that modulation of Ca2+ signaling is a viable therapeutic approach for breast cancer but also suggest that PMCAs are possible drug targets. Alternatively, inhibitors of the PMCA may act as adjuvants to augment the efficacy of other anti-neoplastic agents like tamoxifen that have been shown to modulate Ca2+ signaling. Since the discovery of a new family of primary active Ca2+ transporters, which are related to PMCAs, the opportunities in this field of research are very promising.

Ionized calcium (Ca2+), acting as an intracellular messenger, controls numerous biological processes that are essential for life. However, it is also able to convey signals that result in cell death. The fidelity of Ca2+ as a universal second messenger therefore depends on mechanisms that specifically and dynamically regulate its levels within a cell, as well as maintain resting intracellular Ca2+ concentration ([Ca2+]i) very low. One such mechanism for Ca2+ signaling and homeostasis is the plasma membrane Ca2+-ATPase (PMCA), which is a primary active Ca2+ transporter that translocates Ca2+ from a low intracellular Ca2+ environment to a high extracellular environment. There are four mammalian PMCA isoforms (PMCA1−4), which are differentially expressed depending on tissue or cell type. PMCA isoforms possess different sensitivities to biochemical regulation of Ca2+ efflux activity and are also able to subtly alter the dynamics of Ca2+ signals. These properties suggest that the PMCA is not merely a trivial mechanism for Ca2+ extrusion but is influential in contributing to the Ca2+ signaling requirements and unique physiology of different cells. The indispensable nature of Ca2+ signaling in organs such as the brain, heart and skeletal muscle has been the studied extensively but little is known about the roles and regulation of Ca2+ in the mammary gland. This is despite the fact that the mammary gland is a site of extensive Ca2+ flux during lactation. However, cumulating evidence indicates that upregulation of PMCA2 expression in the mammary gland is a major mechanism for milk Ca2+ enrichment. Therefore, the PMCA is likely to be an important mediator of bulk Ca2+ homeostasis in the mammary gland. Studies in other model systems also suggest that PMCAs may regulate other cellular processes such as cell proliferation, differentiation and apoptosis that are required for normal mammary gland physiology. These basic cellular processes are also disturbed in breast cancer and hence deregulation of PMCA expression in the mammary gland may have pathophysiological consequences. Previous studies show that PMCA1 expression is greater in tumorigenic MCF-7 and MDA-MB-231 human breast cancer cells compared to non-tumorigenic MCF-10A human breast epithelial cells. Furthermore, the expression of PMCA1b and PMCA4b is lower in human skin and lung fibroblasts neoplastically transformed by simian virus 40, compared to non-transformed counterparts. It is therefore hypothesized that regulation of PMCA isoform expression is disrupted in breast cancer and that inhibition of PMCA expression in an in vitro model of breast cancer has important effects in modulating intracellular Ca2+ homeostasis, cell proliferation, differentiation and apoptosis. This thesis describes the use of real time RT-PCR to compare PMCA isoform mRNA expression in tumorigenic and non-tumorigenic mammary gland epithelial cells. It demonstrates that particular breast cancer cell lines overexpress PMCA2, an isoform with restricted tissue distribution and which is present in abundant amounts in the lactating rat mammary gland. Thus, some breast cancers may be characterized by the overexpression of Ca2+ transporters that are normally upregulated during the physiological course of lactation. The pathophysiological significance of PMCA2 overexpression in breast cancer is uncertain and future investigations should look at whether levels of PMCA isoform expression correlate with malignancy, prognosis or survival. To address the second hypothesis of this thesis, a stable MCF-7 Tet-off human breast cancer cell line able to conditionally express PMCA antisense was generated. This strategy was necessary due to the current lack of specific pharmacological inhibitors of the PMCA. This thesis shows that PMCA antisense expression significantly inhibits PMCA protein expression, while subtly affecting PMCA-mediated Ca2+ efflux without causing cell death. However, it also reveals that inhibition of PMCA expression has major effects in mediating cell proliferation and cell cycle progression. Moderate changes in PMCA expression and PMCA-mediated Ca2+ transport result in dramatic consequences in MCF-7 cell proliferation. These studies not only support the supposition that modulation of Ca2+ signaling is a viable therapeutic approach for breast cancer but also suggest that PMCAs are possible drug targets. Alternatively, inhibitors of the PMCA may act as adjuvants to augment the efficacy of other anti-neoplastic agents like tamoxifen that have been shown to modulate Ca2+ signaling. Since the discovery of a new family of primary active Ca2+ transporters, which are related to PMCAs, the opportunities in this field of research are very promising.

<p>Neurological disorders are a major public health concern due to prevalence, severity of symptoms, and impact on caregivers and economic losses. While genetic susceptibility likely has a role in most cases, exposure to toxicants can lead to neurotoxicity, including potentially developmental origins of adult disease or increased risk of disease onset. These exposures are not necessarily large, acute exposures, but could accumulate, with a chronic low-dose exposure, causing toxicity. This research focuses on the potential neurotoxicity of two classes of dietary toxins/toxicants, heterocyclic aromatic amines (HAAs) and per- and polyfluoroalkyl substances (PFAS). HAAs, such as PhIP, harmane, and harmine, are formed in charred or overcooked meat, coffee, tobacco, and other foods. PFAS are largely used in making household materials, but are found in small amounts in eggs and dairy products and largely in contaminated water. While these two classes are diverse in terms of structure, common neurotoxic targets and mechanisms often exist. Therefore, we tested the effects of these chemicals on cell viability and neurotoxicity. In the first aim, we aimed to elucidate the mechanism of toxicity of harmane and harmine, focusing on their ability to cause mitochondrial dysfunction. The second aim was to determine the effects of either harmane or PhIP on the nigrostriatal motor systems and motor function of rats and mice, respectively. The third aim determined the effects of PFAS on neurodevelopment of Northern leopard frogs, focusing on changes in neurotransmitter levels and accumulation in the brain. Harmane did not cause motor dysfunction, but potentially affected the nigro-striatal motor system in an age- or sex-dependent manner. PhIP had differential effects on dopamine levels over time and caused motor dysfunction after subchronic exposure in mice. Perfluorooctane sulfonate (PFOS) accumulated in the brains of frogs and PFAS caused changes in neurotransmitter levels that were dose- and time-dependent. Overall, this research shows that toxins/toxicants humans are exposed to over their whole lives through their diet and contaminated water can cause neurotoxicity, potentially leading to or increasing risk of disease states. </p>

<p>  </p> <p>Perfluoroalkyl substances (PFAS) are synthetic chemicals that are composed of fluorinated aliphatic chains and are widely used in industrial and consumer products. These chemicals are very stable and persist in the environment. Due to concerns linked with longer chain PFAS, shorter chain chemicals are being used as replacements. There are limited human health data regarding the shorter chain chemicals. In addition, these alternatives are persistent in the environment similar to the longer chain PFAS. The main objective of this dissertation was to assess developmental toxicity of the shorter chain PFAS or shorter chain PFAS with chemical modifications represented by perfluorobutanoic acid (PFBA, C4), perfluorohexanoic acid (PFHxA, C6), perfluorobutane sulfonate (PFBS, C4), and perfluoro-2-proxypropanoic acid (GenX, C6). Overall, the results showed that chain length and functional group are determinants of toxicity of PFAS. All tested PFAS induced one or more developmental adverse outcome, but the effects of each chemical are unique, warranting further studies to address the toxicity of the replacement PFAS. </p>

<p dir="ltr">Neurological and neuropsychological dysfunctions resulting from manganese (Mn) accumulation in the human brain are well acknowledged; however, the underlying mechanisms are not yet fully understood. Amongst currently proposed Mn neurotoxicity mechanisms, some were only detectable at concentrations that can lead to over 50% viability loss in acute insult which cannot represent human brain exposure scenarios. Meanwhile, epidemiological reports suggest that exposures over a timeframe of years to decades at Mn levels near or even lower than the regulatory workspace threshold can still lead to adverse outcomes in the central nervous system. Therefore, how to model environmentally relevant chronic Mn exposures at near-threshold levels in <i>in vitro</i> experimental settings and how neurotoxicity is developed under this exposure paradigm are central questions awaiting to be answered.</p><p dir="ltr">Considering the essentiality of Mn as a critical metallic co-factor of multiple enzymes, this study aims to test the hypothesis that interrupted homeostasis of cellular functions that utilize Mn under physiological conditions are the most sensitive respondents to Mn overload. To test this hypothesis, a wide range of Mn concentrations were exposed in cell-based neuronal models across multiple durations. The responses of Mn-dependent biological processes were evaluated by protein phosphorylation quantifications, transcriptomic analyses, and functional measurements. Findings from these assessments highlighted the sensitivity of insulin/PI3K/AKT/mTOR signaling, cAMP/PKA/CREB signaling, cell adhesion, axonal guidance, and homeostatic regulation of divalent metals to near-physiological-threshold Mn overload. Alterations of these pathways illustrate a network of cellular functions that relies on optimal intracellular Mn content and vitally contributes to the neurodegeneration risks induced by chronic Mn exposures.</p><p dir="ltr">In conclusion, this work contributes to a more nuanced understanding of Mn neurotoxicity mechanisms, emphasizing the importance of both concentration and duration of exposure in the context of neurodegenerative risk, and paving the way for future research into constructing adverse outcome pathways of Mn via advanced <i>in vitro</i> modeling.</p>

<p dir="ltr">The Northwest Atlantic Ocean, which surrounds the US eastern coastline, is an area rich in marine life. The US eastern coastline is also highly urbanized, resulting in a lot of pollutants (like heavy metals) entering the marine environment. This is of concern for long-lived marine species like sea turtles. Since sea turtles are long-lived and highly migratory, their tissues can often incorporate these pollutants through environmental and dietary exposure. I collected tissue samples from 5 different sea turtle populations in the Northwest Atlantic and analyzed them for concentrations of silver (Ag), aluminum (Al), arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), selenium (Se) and zinc (Zn) using an Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The first chapter looks at skin (reflects exposure ~1 year ago) and scute (reflects exposure from 4-6 years ago) samples collected during necropsies of juvenile green (<i>Chelonia mydas</i>) (n=8), Kemp’s ridley (<i>Lepidochelys kempii</i>) (n=30) and loggerhead (<i>Caretta caretta</i>) (n=17) turtles that were found cold-stunned in Cape Cod Bay, Massachusetts. In scute samples, the heavy metal with the highest concentration for green turtles was iron, zinc for loggerhead turtles, and arsenic for Kemp’s ridley turtles. In skin samples, the heavy metal with the highest concentration for green turtles was iron, arsenic for loggerhead turtles, and aluminum for Kemp’s ridley turtles. Overall, I found scute samples to have higher heavy metal concentrations than skin samples. The second chapter looks at scute samples collected from loggerhead turtles of different life stages. These samples were collected during necropsies of cold-stunned loggerhead turtles from Cape Cod Bay, Massachusetts (CCB; n=17), as well as from live loggerhead turtles in the Mid-Atlantic Bight (MAB; n=37) and off the coast of North Carolina (NC; n=9). We also collected commonly known loggerhead turtle prey items including whelk (<i>Buccinum undatum</i>) (n=12), Atlantic scallop (<i>Placopecten magellanicus)</i> (n=10) and Jonah crab (<i>Cancer borealis</i>) (n=5) from the Mid-Atlantic Bight region to study the occurrence of biomagnification through trophic pathways. NC loggerhead turtles had higher heavy metal concentrations than other locations except for cadmium and zinc, where CCB loggerhead turtles were higher. I found that all heavy metals except silver, cadmium, and lead appear to be biomagnified (TTF>1) in loggerhead turtles. These two chapters provided baseline information on heavy metal concentrations in sea turtles in east coast US.</p>