Developmental neuroendocrine toxicity targeting the kisspeptin pathway

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PROJECT SUMMARY/ABSTRACT Atrazine is the second most common agricultural herbicide used in the US and the most frequent agrichemical contaminant in potable water supplies. Atrazine is regulated by the US EPA at 3 parts per billion (ppb; µg/L) in drinking water, but concentrations above this regulatory limit are often reported. Laboratory and epidemiology studies report various endocrine disrupting impacts with most focused along the hypothalamus-pituitary-gonadal (HPG) axis acting through the suppression of gonadotropin-releasing hormone (GnRH) leading to decreased luteinizing hormone (LH) release. However, there are several reports on atrazine altering additional endocrine axes [including adrenal (HPA) and thyroid (HPT)] and neuronal functions and neurobehavior through multiple neurotransmitter systems (including dopamine and serotonin). A hypothalamic atrazine toxicity target is hypothesized but is yet to be identified. This target would need to be a master regulator of several hormones and neuropeptides. We propose the kisspeptin system as this master regulator and will test the CENTRAL HYPOTHESIS that dysregulation of the kisspeptin pathway by the neuroendocrine toxicant atrazine connects observed impacts on developmental, reproductive, and neurobehavioral outcomes. Kisspeptin is responsible for regulating GnRH neurons and subsequent LH release, which is the primary accepted mechanism of atrazine endocrine disruption along the HPG axis. In addition, kisspeptin modulates the neurotransmitters dopamine and serotonin regulating multiple neurobehaviors including locomotor and anxiety-related behavior, which are also reported to be altered by atrazine exposure. Our studies in the zebrafish model system show that an embryonic atrazine exposure at concentrations around the US EPA regulatory limit in drinking water impacts similar molecular pathways (neuroendocrine, reproductive, neurotransmission, behavior) through transcript, protein, miRNA, and DNA methylation analyses; endocrine axes (HPG, HPA, and HPA); neurotransmission systems (serotonin and dopamine); behavior (locomotor and anxiety-related behavior); and functions (reproductive dysfunction) as identified in humans and rodent models. The kisspeptin system is also conserved in zebrafish with two kiss genes (kiss1 and kiss2) present for the mammalian KISS1 gene. The zebrafish presents as an excellent vertebrate model to address the kisspeptin system, because kiss1 is primarily expressed in the ventral habenula regulating neurotransmission and associated behavioral outcomes, while kiss2 is expressed in the hypothalamus with neurons projecting to the GnRH neurons. As such, we can establish genetic mutants with CRISPR-Cas9 technology to investigate the specificity of these two pathways in aim 1, along with determining the connections with key targets regulated by kisspeptin and altered by atrazine exposure (e.g., GnRH, LH, dopamine, serotonin). In aim 2, we will then test the hypothesis that atrazine developmental neurotoxicity on neuroendocrine and neurotransmission pathways is modulated through the kisspeptin system. This project is novel in significantly advancing our knowledge on the role of kisspeptin as a master regulator in atrazine toxicity pathways.