Linking a developmental origin of atrazine-induced reproductive system alterations in Zebrafish: Identifying changes in the genetic and epigenetic landscape
An emerging body of research into genetic and epigenetic alterations observed in adult diseases points to a developmental origin through reprogramming of cellular processes that occur during development and influences by the surrounding environment. As a corollary, recent research concerning the effects of endocrine disrupting chemicals (EDCs) on human health has increased substantially and heightens the need for further research into the mechanistic effects of exposure to these compounds. Atrazine is a commonly used herbicide predominately applied as a pre-emergent herbicide to control broadleaf and grassy weeds in a variety of crops and is implicated as an EDC and potential carcinogen. The mechanisms of atrazine toxicity and health risks associated with atrazine exposure, especially at low levels likely to be encountered in the environment, are not distinctly understood. The studies in this dissertation were designed to investigate the immediate, later-in-life, and transgenerational effects from exposure to environmentally-relevant concentrations of atrazine using the zebrafish vertebrate model. Embryonic zebrafish were exposed to 0, 0.3, 3 or 30 ppb of atrazine, concentrations very likely to occur in environmental exposures to the general population including pregnant mothers and their fetuses (the exposure regimen this study design targets). Transcriptomic profiling of zebrafish immediately following the exposure period resulted in enrichment in genes associated with reproductive system function and development, cell cycle regulation, and cancer (Chapter 2). A significant decrease in mating success was observed in adults developmentally exposed to 30 ppb atrazine with 4% of females presenting with severe swelling of the abdomen and an inability to spawn eggs. Transcriptomic profiling of brain tissue of adult females revealed 31 common genes with known human homologs with altered expression in all three treatments. Furthermore, pathway analysis was similar in all three atrazine treatments indicating an enrichment of expression alterations of genes associated with organismal, tissue, and nervous system development and behavior (Chapter 3). To investigate if effects were inherited in an unexposed generation, control and 30 ppb atrazine exposed adults were bred within treatment groups as well as crossed between groups to determine if the effects were passed on to a future generation through the male or female germline. The greatest impacts were observed in the offspring of the 30 ppb male x control female cross (the male was developmentally exposed to atrazine and the female was from the control group) with RNA sequencing at 72 hpf indicating an enrichment in expression alterations of genes associated with embryonic and tissue development, including development of head and body axis, development of sensory organs, eye development and eye morphology as well as genes associated with neurological and ophthalmic diseases. In addition, when the F2 generation was raised to maturity adult females in this same cross presented with the same swelling of the abdomen which points to a paternal inheritance of this effect (Chapter 4). Furthermore, to identify changes in epigenetic mechanisms that may link the observed phenotypic trait observed in the adults to a developmental origin, embryonic zebrafish were exposed to the same atrazine concentrations as stated above and microRNA (miRNA) expression assessed. Analysis revealed alterations to miRNAs targeting genes previously identified to be altered during developmental exposure to atrazine to downstream functional pathways including synaptic long term depression and axonal guidance (Chapter 5). Overall, data obtained in these studies provides the framework for future analyses into the genetic and epigenetic mechanisms that are altered during atrazine exposure and are linked to later-in-life and transgenerational effects.
Sepulveda, Purdue University.
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