Human serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme. It plays a major role in the detoxication of a number of organophosphate (OP) insecticides in addition to its role in lipid metabolism. PON1 exhibits a substrate dependent polymorphism for the hydrolysis of the toxic metabolites of parathion, chlorpyrifos and diazinon as well as the nerve agents soman and sarin. The available evidence supports the hypothesis that an individual's PON1 status is important in the detoxication of organophosphorus esters (OPs) processed through the cytochrome P450/PON1 pathway. The basis of the polymorphism is an amino acid substitution in PON1 at position 192. Arg192 specifies an isoform with high activity against paraoxon while Gln192 specifies an isoform with low activity against paraoxon. The effect of the polymorphism is reversed for the hydrolysis of diazinon, soman and sarin and leads to the prediction that individuals resistant to parathion will be sensitive to diazinon, soman and sarin, whereas individuals sensitive to parathion will be more resistant to diazinon, soman and sarin. In addition to the activity polymorphism, there is a large variability in the level of enzyme expression between individuals that is stable over time. The combination of genotype and phenotype is referred to as PON1 status. The goals of the proposed research are to: 1) determine the molecular structural basis of the activity polymorphism; 2) refine the mouse model for investigating the functional genomic aspects of the PON1 polymorphism in vivo; and 3) determine the mechanism of regulation of gene expression that results in widely different levels of expression of PON1 between individuals, and is also important in development. To examine the molecular basis of the activity polymorphism, the crystal structure of the Arg192 and Gln192 PON1 isoforms will be determined. Both purified and recombinant PON1 will be used in these studies. For the refinement of the mouse model, we will utilize the PON1 knockout mice, some of which will be given purified human PON1 (Gln192 or Arg192). In addition, we will utilize transgenic mice that express the human Arg192 Or Gln192 isoform as hemizygotes, homozygotes and heterozygotes. The effects of several OP compounds on brain, diaphragm and blood acetylcholinesterases will be assessed as an index of acute toxicity with mice of simulated human PON1 status. To examine the molecular basis of the large individual variability of enzyme levels observed in human populations, the upstream and downstream candidate regulatory sequences will be used to control the expression of the reporter gene luciferase in a liver cell line (HepG2) that expresses paraoxonase message. Sequence cassettes from individuals expressing very high enzyme levels will be compared with those from individuals expressing very low enzyme levels for their ability to regulate the expression of the reporter gene.