A major objective of the proposed research is to understand the physiological consequences of genetic variability in the human PON1 gene and to identify factors that contribute to the large differences in plasma PON1 levels among individuals. Development of E. coli PON1 expression and purification protocols provides a rapid means for characterizing the effects of sequence variants on the catalytic efficiency of PON1 and at the same time provides sufficient recombinant PON1 protein for toxicology experiments and structure/function studies. Demonstration of the therapeutic potential of recombinant human PON1 for treating cases of acute OP exposure has provided the impetus for Specific Aims 1 and 2 of the proposed research. The research proposed in this application has 5 specific aims. The goal of Specific Aim 1 is to improve the expression and purification protocols for generating native and variant human PON1s in the E. coli expression system. The goal of Specific Aim 2 is to generate PON1 variants that have sufficient catalytic efficiency to protect against exposures to tricresyl phosphate and paraoxon. The goal of Specific Aim 3 is to identify and validate biomarkers for exposure to organophosphorus (OP) compounds. There is a pressing need for biomarkers of exposure that extend the time-frame of exposure analyses beyond the few day window that measuring urinary or blood metabolites provides.
Specific Aim 4 addresses the importance of maternal PON1 status in protecting the fetus from OP (diazoxon) exposure during gestation. The data generated to date indicate that PON1 status is important in determining sensitivity to diazinon/diazoxon and chlorpyrifos/chlorpyrifos oxon.
Specific Aim 5 examines the influence of promoter region epigenetic methylation on PON1 plasma levels. Our research to date has characterized promoter polymorphisms that significantly influence plasma PON1 levels. Identification of approximately 200 new polymorphisms has shown that only 30% of the variability of plasma PON1 levels appears to be dependent on promoter-region polymorphisms. Preliminary examination of methylation of CpGs in the PON1 promoter region revealed variability that may explain another significant source of regulation. Data from transgenic PON1 mouse pedigrees are consistent with this additional level of regulation.
Data generated from this project has assisted the EPA in negotiating a reduction in diazinon and chlorpyrifos exposure. The PON1 status analyses have identified low PON1 status as a risk factor for carotid artery disease and have also identified differences between male Parkinson's patients and controls. Engineering recombinant human PON1 will provide therapeutics for treating nerve agent and insecticide exposures.
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