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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES009883-13
Application #
8461516
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Mcallister, Kimberly A
Project Start
1999-09-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
13
Fiscal Year
2013
Total Cost
$555,898
Indirect Cost
$196,575
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Marsillach, Judit; Costa, Lucio G; Furlong, Clement E (2016) Paraoxonase-1 and Early-Life Environmental Exposures. Ann Glob Health 82:100-10
Furlong, Clement E; Marsillach, Judit; Jarvik, Gail P et al. (2016) Paraoxonases-1, -2 and -3: What are their functions? Chem Biol Interact :
Burton, Casey; Marsillach, Judit; Zhang, Yu Shrike et al. (2015) Announcing our finalists. Bioanalysis 7:1667-73
Marsillach, Judit; Becker, Jessica O; Vaisar, Tomas et al. (2015) Paraoxonase-3 is depleted from the high-density lipoproteins of autoimmune disease patients with subclinical atherosclerosis. J Proteome Res 14:2046-54
Marsillach, Judit; Suzuki, Stephanie M; Richter, Rebecca J et al. (2014) Human valacyclovir hydrolase/biphenyl hydrolase-like protein is a highly efficient homocysteine thiolactonase. PLoS One 9:e110054
Kim, Daniel Seung; Crosslin, David R; Auer, Paul L et al. (2014) Rare coding variation in paraoxonase-1 is associated with ischemic stroke in the NHLBI Exome Sequencing Project. J Lipid Res 55:1173-8
Cole, Toby B; Li, Wan-Fen; Co, Aila L et al. (2014) Repeated gestational exposure of mice to chlorpyrifos oxon is associated with paraoxonase 1 (PON1) modulated effects in maternal and fetal tissues. Toxicol Sci 141:409-22
Strelitz, Jean; Engel, Lawrence S; Keifer, Matthew C (2014) Blood acetylcholinesterase and butyrylcholinesterase as biomarkers of cholinesterase depression among pesticide handlers. Occup Environ Med 71:842-7
Costa, Lucio G; de Laat, Rian; Dao, Khoi et al. (2014) Paraoxonase-2 (PON2) in brain and its potential role in neuroprotection. Neurotoxicology 43:3-9
Marsillach, Judit; Hsieh, Edward J; Richter, Rebecca J et al. (2013) Proteomic analysis of adducted butyrylcholinesterase for biomonitoring organophosphorus exposures. Chem Biol Interact 203:85-90

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