PON1 Status as a Susceptibility Factor in Pesticide Toxicity

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PON1 Status as a Susceptibility Factor in
Pesticide Toxicity Structure and Function of the
Human PON1 PolymorphismsA 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
Davies et al., Nature Genetics 1996 14334-336
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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
L55M Q192R
G-909C G-832A G-162A G-126C T-108C
(CA)n
A/G C/T

• 5 promoter region polymorphisms
• 2 coding region polymorphisms
• 1 intronic polymorphic CA repeat
• 4 3 UTR polymorphisms

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Restriction Enzyme Analysis of the PON1192
Polymorphism
Met55 Gln192
(Low)----5-GACATGGAC---- ----TTACAATCC--------
3-CAGTACCTG---- ----AATGTTAGG---- Cut
with NlaIII No cut with AlwI
Leu55 Arg192
(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 394284-287
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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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Li et al., Pharmacogenetics in press
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Li et al., Pharmacogenetics in press
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Catalytic Efficiency of PON1192 Isoforms
Data from Li et al., Pharmacogenetics in press
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Yuknavage et al., J. Toxicol. Environ. Health
1997 5135-55
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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
L55M Q192R
G-909C G-832A G-162A G-126C T-108C
(CA)n
A/G C/T

• 5 promoter region polymorphisms
• 2 coding region polymorphisms
• 1 intronic polymorphic CA repeat
• 4 3 UTR polymorphisms

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Summary

• SNPs in PON1 contribute to determining PON1
  status
• The Q/R192 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