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PON1 Status as a Susceptibility Factor in Pesticide Toxicity Structure and Function of the Human PON1 Polymorphisms: A Detailed Look at One Set of Insecticides Clement E. Furlong Departments of Genome Sciences & Medicine, Division of Medical Genetics University of Washington, Seattle, WA

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CYTOCHROME P450-PARAOXONASE PATHWAY FOR O.P. DETOXICATION CYTOCHROME P450-PARAOXONASE PATHWAY FOR O.P. DETOXICATION Davies et al., Nature Genetics 1996; 14:334-336

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T.L. Diepgen & M. Geldmacher-v. Mallinckrodt. Arch. Toxicol. Suppl. 9, 154-158 (1986)

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Determining the molecular basis of the PON1 polymorphism required: Purifying rabbit and human plasma PON1s to homogeneity Gel purifying active rabbit PON1 Sequencing the purified rabbit PON1 Designing probes and screening a rabbit liver cDNA library Isolating and characterizing the rabbit PON1 cDNA Using rabbit PON1 cDNA to isolate the human cDNA Characterizing the human PON1 cDNAs to reveal polymorphisms (M55L & Q192R) Determining which polymorphism governed activity (Q192R)

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PON1 Polymorphisms 5 promoter region polymorphisms 2 coding region polymorphisms 1 intronic polymorphic CA repeat 4 3 UTR polymorphisms L55M Q192RG-909C G-832A G-162A G-126C T-108C (CA) n A/G C/T

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Restriction Enzyme Analysis of the PON1 192 Polymorphism Met 55 Gln 192 (Low) ----5-GACATGGAC--------TTACAATCC---- ----3-CAGTACCTG--------AATGTTAGG---- Cut with NlaIII No cut with AlwI Leu 55 Arg 192 (High) ----5-GACTTGGAC--------TTACGATCC---- ----3-CTGAACCTC--------AATGCTAGG---- No cut with NlaIII Cut with AlwI

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PON1 Status Q,Q Q,R R,R

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Understanding the Role of PON1 in OP sensitivity: development of an animal model Correlation of PON1 levels with OP resistance Injection of purified PON1 into mice Sensitivity of PON1 knockout mice to OPs Reconstitution of resistance in KO mice with PON1 Conclusions from the animal model studies

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Half Life of Injected PON1

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Chlorpyrifos-oxon Toxicity in PON1 -/- Mice Shih et al., Nature 1998; 394:284-287

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Diazoxon Toxicity in PON1 -/- Mice Li et al., Pharmacogenetics, in press

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Paraoxon Toxicity in PON1 -/- Mice Li et al., Pharmacogenetics in press

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Catalytic Efficiency of PON1 192 Isoforms Data from Li et al., Pharmacogenetics in press

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Yuknavage et al., J. Toxicol. Environ. Health 1997; 51:35-55

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Time Course of PON1 Appearance in Infants Richter and Furlong, unpublished data

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Analysis of PON1 Promoter Haplotypes

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PON1 Polymorphisms 5 promoter region polymorphisms 2 coding region polymorphisms 1 intronic polymorphic CA repeat 4 3 UTR polymorphisms L55M Q192RG-909C G-832A G-162A G-126C T-108C (CA) n A/G C/T

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Summary SNPs in PON1 contribute to determining PON1 status The Q/R 192 polymorphism determines PON1 catalytic efficiency Promoter SNPs contribute to determining PON1 levels PON1 status affects both the normal physiological function of PON1 as well as OP metabolism by PON1