The goal of this research is to develop a pharmacological screen for drugs that inhibit cytochrome P450 isoforms responsible for chlorpyrifos oxidation in developing zebrafish, using the neurotoxic behavioral effects of chlorpyrifos as a screening phenotype. Developing fetuses, infants, and children may be particularly vulnerable to the effects of organophosphate pesticides such as chlorpyrifos. These high priority chemical threat agents act after being metabolized by cytochrome P450 oxidases (CYPs) to oxon forms, which are active as acetylcholinesterase inhibitors. We hypothesize that oxon formation can be pharmacologically altered in developing vertebrates by inhibiting P450-mediated oxon generation to reduce the individual effects of thion pesticide exposure on these sensitive stages. We will initially examine drugs previously approved for pediatric use that exhibit CYP inhibitory activity in humans. In animal models, developmental chlorpyrifos exposure leads to altered locomotor behaviors and learning deficits at doses that do not elicit acute cholinergic toxicity, but that depend on the activation of chlorpyrifos. These altered behaviors form the basis of a pharmacological screen, in conjunction with an effort to determine the specific zebrafish CYP isoforms responsible for chlorpyrifos bioactivation. Inhibition of oxon formation will be reflected in decreased behavior toxicity, guiding the development of a screen for organophosphate therapeutics. We propose also to determine which zebrafish CYP isoforms are involved in chlorpyrifos-oxon formation, using a combination of molecular modeling and in vitro studies. These results would be used to validate the behavioral screen, as isoform-specific CYP knockdown in zebrafish would demonstrate the specificity of positive hits from the initial screen. The proposed studies provide the 'mouth' of a drug-development pipeline focused on organophosphate threat agents.
Developing fetuses, infants, and children are particularly vulnerable to the effects of organophosphate pesticides such as chlorpyrifos. The goal of this research is to develop a screen for drugs that inhibit cytochrome P450 isoforms responsible for chlorpyrifos activation in developmental stages, in the zebrafish model. Neurotoxic behavioral effects of chlorpyrifos will be the sensitive phenotype screened. We will determine which zebrafish cytochrome P450 isoforms are primarily involved in chlorpyrifos metabolism, using a combination of molecular modeling and in vitro studies, to validate the behavioral screen. The studies lay a foundation for use of zebrafish for assessing therapeutic intervention in organophosphate exposure.
