Paraoxonase/arylesterase is a serum enzyme that is associated with HDL particles. It hydrolyzes the toxic oxons of several organophosphorus insecticides and the nerve agents soman and sarin. This enzyme exhibits a substrate dependent polymorphism in humans. One isoform, with arginine at position 192, hydrolyzes paraoxon with a low turnover number. Other substrates such as chlorpyrifos oxon and phenylacetate are hydrolyzed by both isoforms with the same turnover number. The first major objective is to determine the role of human serum paraoxonase in the metabolism of toxic organophosphorus compounds. Purified (natural or recombinant) high and low activity isoforms will be injected into mice and the in vivo protection afforded by each isoform will be determined. Transgenic mice will be constructed which express either the human high or low activity isoform. The transgenic animals will be challenged with organophosphates and the protection provided. by. each isoform determined. The constructs for expression of paraoxonase in the transgenic mice will make use of the cDNAs or subclones from yeast artificial chromosome (YAC) clones already characterized. Epidemiological studies will determine whether serum paraoxonase status affects the response of humans to organophosphorus compounds. Paraoxonase genotype and levels of expression will be determined in individuals with histories of toxicity and compared with values in individuals from the same population who have not experienced episodes of toxicity. PCR, immunological and enzyme assays developed in the first phase of this research will be used to establish the paraoxonase status of each individual. The second major objective is to define the role of paraoxonase in lipid metabolism. Lipase activity will be determined with a number of lipid substrates. Correlations of paraoxonase status with lipoproteins and lipid levels will be examined. The third major objective is to continue the molecular genetic studies on the structure and regulation of the paraoxonase gene. The sequencing and fine structure mapping will be completed and the 5' and 3' noncoding regions from individuals who express very high and low levels of protein win be examined for possible involvement in the regulation of the paraoxonase gene. The completion of the sequencing of the few remaining exon/intron boundries will allow amplification of individual exons from genomic DNA by the polymerase chain reaction (PCR). The amplified paraoxonse coding regions will be examined by single strand conformational polymorphism analysis in non-caucasoid ethnic groups to determine if additional variants exist.
