In this project, studies are proposed to analyze organophosphate insecticides as toxicants, the consequences of organophosphate exposure, in terms of body burden and inhibition of the target acetylcholinesterase (AChE), and differences in individual susceptibility related to organophosphate target. Fluorescence detection using the AChE target itself will be explored as a means for developing remote sensing and portable detection devices. The disposition of organophosphates in the body will be analyzed by mass spectrometry to assess tissue distribution and conjugation at the active center of AChE. The latter approach enables us to detect inhibited AChE, inhibited and aged AChE and AChE that is spontaneously or oxime reactivated. Hence, a complete kinetic profile of reactions at the active center serine is possible. Because of malathion's prevalent use world-wide and locally, initial studies will involve this organophosphate which must be converted to malaoxon to become an active and toxic agent. Rates of conversion will be studied using animals with enhanced P450 activity which control the conversion of protoxicant to cholinesterase reactive entity. These studies of insecticide exposure will be buttressed by examining transgenic strains where the acetylcholinesterase gene, its alternatively spliced exons and regulatory regions of the gene have been modified. The modified genes enable us to express AChE in select tissues and in discrete cellular locations thereby allowing an analysis of the cellular basis of toxicity in the intact animal. Finally, the investigators have adopted a pharmacogenomic approach to detect single polynucleotide polymorphisms and, in turn, haplotypes to analyze differential sensitivity to cholinesterase inhibitors. Previous studies characterizing the AChE molecule and the single gene encoding the enzyme place the investigators in a strong position to analyze the target molecule of insecticide interaction and determine genetic relationships in individual susceptibility.
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