The extensive use of pesticides in California's agricultural industry, the disappearance of dry corridors which separate agricultural areas and the public perception of every date contact with pesticides in foodstuffs create unusual and growing problems for this class of chemicals. However, a major subclass of these agents, the organophosphates and carbamates are unique among superfund chemicals in that their target site for acute toxicity, acetylcholinesterase (AChE) is encoded by a single gene in mammals. Moreover, the chemical mechanism of ACHE inhibition has been examined in great detail and recent crystallographic studies provide an atomic resolution template for understanding the intimate details of specificity of these agents. Our proposal relies heavily on these mechanistic studies of inhibition and our ability to manipulate the AChE gene to regulate AChE inhibition to the accumulation of phosphorylated enzyme. Through the use of unique surrogate cholinesterase inhibitors of known absolute stereochemistry and Sp- and Rp-enantiomeric selectivity, inhibition of AChE will be distinguished from other serine hydrolase as targets for chronic toxicity. With other AChE inhibitors having unique dispositions in the body, we propose to inhibit selectivity peripheral and central AChE and correlate the cumulative inhibition through phosphorylation in regional CNS and tissues of the peripheral nervous system with alterations in gene expression. Similar studies will be extended to the carbamate insecticides and pesticide combinations of AChE inhibitors and organochlorines. Particular synergisms and antagonistic relations in production of toxicity are predicted. Finally, transgenic animals with compromised AChE gene expression (total gene knockout, conditional knockout, expression of selected splice variants) and mice with diminished sensitivity to oxidative stress will be examined as candidate strains unusually susceptible to AChE inhibition. Not only should these studies shed light on changes in gene expression associated with chronic AChE inhibition, but may yield mice strains and cell lines with unusual sensitivity to AChE inhibition. The animals should provide models for genetic variations and developmental stages in the human population (newborns and children) where enhanced toxicity may be manifest.
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