INVESTIGATION OF ARSENIC (AS) AND LEAD (PB) MIXTURE DEVELOPMENTAL TOXICITY

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. 

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., vegfaa, wnt7aa, and lrp1aa) and genes associated with tight junctions (i.e., cldn5a and cldn5b) were assessed at 72 hpf. Increased cldn5b expression was detected in all treatment groups, while cldn5a was increased in only the 10 ppb Pb treatment group. In addition, wnt7aa was only decreased in the 10 ppb Pb treatment group. Alternatively, vegfaa was increased in the 100 ppb As and 100 ppb mixture treatment groups and no changes were detected for lrp1aa. 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. 

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 in silico 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.