Mitochondria Mediated Outcomes of Developmental Exposure to Low-level Chemical Mixtures in Zebrafish Danio rerio

Exposure to drinking water contaminants has been linked to developmental outcomes in both epidemiological and model organism studies. However, low level mixture effects, on early development has yet to be explored. It is hypothesized that early chemical exposures may increase disease susceptibility later in life. This work aimed to investigate impacts of a variety of chemicals and concentrated on metals arsenic (As), cadmium (Cd), vanadium (V), and lead (Pb) due to their presence in drinking water and known developmental toxicity. To determine the effects of a metal and organic contaminant co-exposure, the ubiquitously used herbicide glyphosate was also explored. The zebrafish (Danio rerio) model was used to elucidate developmental impacts of lowlevel chemical mixture exposure with a focus on mitochondrial function. An in-depth analysis exploring embryonic oxygen consumption rate (eOCR) in response to all iterations of a 5-part chemical mixture of glyphosate, As, Cd, V, and Pb showed that mitochondria are highly sensitive to mixture toxicity, and that pre-exposure to a metal mixture leave the mitochondria more susceptible to acute chemical stress through depleted reserve capacity. Altered mitochondrial function, along with changes in gene expression and histology suggested that early mixture exposure may contribute to the endemic of chronic kidney disease of unknown etiology (CKDu). To investigate underlying molecular mechanisms that may contribute to CKDu susceptibility, RNA seq data from zebrafish embryos exposed to mixtures of As, Cd, V, and Pb, (+/- glyphosate) and glyphosate alone, suggest that exposure to metal and organic mixtures may be altering the extracellular matrix of kidney tissue. This combined with impaired mitochondrial function, could leave individuals more susceptible to kidney injury CKDu progression. To determine phenotypes associated with mixture exposure, changes in behavior after exposure to a large collection of water samples were explored. A cluster analysis of metals found in drinking water samples were coupled to changes in behavior and revealed that concentrations of Pb, Cd, As, Uranium (U) and Nickel (Ni), should be taken into special consideration when determining drinking water standards. These data suggest that impaired mitochondria, as a result of low-level mixture exposure, may function in the early onset of disease, such as CKDu, and further impair organism development

Effect of Arsenic Exposure on Early Eye Development in Zebrafish (Danio rerio)

Arsenic is a metalloid that contaminates drinking water supplies worldwide. Due to concerns for human health, the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EPA) have established a safe level in drinking water of ≤ 10 ppb. Arsenic has been shown to have carcinogenic effects in humans at high and low doses. Chronic exposure may result in dermal conditions such as hyperkeratosis and hyperpigmentation. Recently, arsenic exposure has also been linked to lower IQ values in children. The effect of arsenic on neurogenesis, specifically eye development, has not been widely explored. This study aimed to examine the effect of environmentally relevant concentrations of arsenic on early eye development by morphological and molecular analysis. The zebrafish (Danio rerio) was chosen to model the impacts of arsenic on retinogenesis because of its similarities to human eye development both structurally and genetically. The effect of arsenic exposure on the gross morphology of the eye and tissue development via histology were examined at three larval stages. It was found that exposure to arsenic has an effect on eye morphology resulting in a significant increase in eye diameter at 14 dpf (days post fertilization) relative to control under all treatment conditions. This was coupled with a trend in thinning of the retinal pigmented epithelium (RPE) layer in fish exposed to 500 ppb arsenic. The impacts of arsenic exposure on gene expression were analyzed following treatments of 10, 50, and 500 ppb from 1 – 72 hpf (hours post fertilization). Molecular analysis of genes associated with eye development was investigated by RT-qPCR at 32 and 48 hpf. RT-qPCR results revealed differential expression of Pax6a, Pax2a, Ngn1, Sox2, and Shha relative to control. Specifically Pax6a, Pax2a, and Sox2 are found to be important in the formation of the RPE. Proper formation of the RPE is necessary for growth of the sclera which, in turn, is responsible for maintaining the shape of the eye. Although we observed a thinning of the RPE we also noted an overall increase in eye size of 14 dpf zebrafish. This could potentially be explained by the disruption of gene expression under arsenic exposure during critical time points in early eye development