2001 Progress Report: Genetic Susceptibility to Pesticides (Paraoxonase Polymorphism or PON1 Study)

EPA Grant Number: R826886C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R826886
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: University of Washington
Center Director: Faustman, Elaine
Title: Genetic Susceptibility to Pesticides (Paraoxonase Polymorphism or PON1 Study)
Investigators: Faustman, Elaine
Institution: University of Washington
EPA Project Officer: Callan, Richard
Project Period: August 1, 1998 through December 31, 2003
Project Period Covered by this Report: August 1, 2000 through July 31,2001
Project Amount: Refer to main center abstract for funding details.
RFA: Centers for Children's Environmental Health and Disease Prevention Research (1998) RFA Text |  Recipients Lists
Research Category: Children's Health , Health Effects , Health

Objective:

The general aim of this project remains that of determining the role of the enzyme paraoxonase (PON1) in the developmental toxicity and neurotoxicity of organophosphorus (OP) insecticides. The specific aims also remain as follows: (1) investigate the acute toxicity of a number of OPs in wild type and PON1 knockout mice; (2) investigate whether human PON1, either the Arg192 or Gln192 isozyme, would offer protection against the toxicity of OP s in PON1-/- mice; (3) determine the acute toxicity of OPs in developing mice; (4) assess the developmental neurotoxicity of OPs in wild type and PON1 knockout mice; and (5) determine the PON1 status in infants and children.

Progress Summary:

We have focused on a series of pilot experiments aimed at defining the dose-regimen of chlorpyrifos oxon (CPO) to be utilized in the developmental neurotoxicity studies. CPO was dissolved in dimethyl sulfoxide (DMSO) and administered by s.c. injection to 4- day-old wild type (PON1+/+) mice. CPO caused a dose-dependent inhibition of brain and serum cholinesterase (ChE) in the 0.25–1.0 mg/kg dose range. The lowest dose (0.25) mg/kg), causing a 20% inhibition of brain ChE, was used in subsequent repeated exposure studies, where CPO was given daily from postnatal day 4 to 21. Two control groups were used, one treated with the vehicle DMSO and another that was left untreated. There were no differences among groups in body weight gain, and no clinical symptoms of toxicity were observed. When measured 24 hours after the last CPO injection, brain ChE activity did not differ among groups.

Groups of PON1 knockout (PON1 -/-) mice were also utilized in acute CPO experiments. Administration of various doses of CPO (0.1-0.25 mg/kg, s.c. in DMSO) caused a dose-dependent inhibition of brain and plasma ChE. As the 0.16 mg/kg and the 0.20 mg/kg dose caused 6% and 40% inhibition of brain ChE activity, an intermediate dose, 0.18 mg/kg was also tested, which caused an approximate 20% inhibition of this enzyme. This dose will be used in the repeated treatment (day 4-21).

We previously identified three polymorphisms in the PON1 regulatory region that affect expression levels in cultured human hepatocytes. We then determined the genotypes of three regulatory-region polymorphisms for 376 white individuals and examined their effect on plasma-PON1 levels, determined by rates of phenylacetate hydrolysis. The –108 polymorphism had a significant effect on PON1-activity level, whereas the –162 polymorphism had a lesser effect. The –909 polymorphisms, which is in linkage disequilibrium with the other sites, appears to have little or no independent effect on PON1-activity level in vivo. Other studies have found that the L55M polymorphism in the PON1-coding region is associated with differences in both PON1 mRNA and PON1 activity levels. Our results indicate that the L55M effect of lowered activity is not due to the amino acid change but is, rather, largely due to linkage disequilibrium with the –108 regulatory-region polymorphism. The codon 55 polymorphism marginally appeared to account for 15.3% of the variance in PON1 activity, but this dropped to 5% after adjustments for the effects of the –108 and Q192R polymorphisms were made. The –108C/T polymorphism accounted for 22.8% of the observed variability in PON1 expression levels, which was much greater than that attributable to the other PON1 polymorphisms.

Finally, taking advantage of a study on children diagnosed with phenylketonuria, a number of serum samples are being collected from infants and children. These samples are analyzed for PON1 activity using different substrates. Preliminary results show an increase of serum PON1 are activity with age.

Significance

Animal studies done so far are progressing toward the final goal of the project (i.e., the role of PON1 in the developmental neurotoxicity of OPs). The human study will provide unique information on the developmental profile of serum PON1 in children. Overall, the significance of the project lies in the understanding of whether the combined genetic background and developmental expression of PON1 may render children more at risk for OP toxicity and neurotoxicity.

Future Activities:

This project was originally proposed as a 3-year study. However, the project will continue for a fourth year. Funds for the continuation were approved using a portion of the restored year 3 funds. In the fourth year of the PON-1 project, we plan to continue pursuing the Specific Aims. In particular, we will continue the collection of samples and the assays of human serum from infants and children. Furthermore, we will conduct studies toward the goals of Aims 2 and 4. We have obtained from Drs. Lusis and Shih at the University of California at Los Angeles (UCLA) breeding pairs of PON1 Arg192 and PON1 Gln192 mice. These are PON1 knockout mice that only express the Q or R allele of the human gene. Once we have obtained a sufficiently large colony of these animals, we will conduct a number of in vivo studies, as done in the past, to assess the acute effects of various organophosphates (chlorpyrifos, parathion, diaxinon) and their oxon on brain and diaphragm acetylcholinesterase levels. These experiments will expand our previous studies and contribute to Aim 2 of this proposal with a unique in vivo transgenic model.

We will also conduct in vivo experiments on developing PON1 -/- mice. We identified 0.18 mg/kg as the dose of CPO causing 20% of brain ChE inhibition. This dose will be used in the repeated exposure treatment (day 4-21).

Behavioral experiments will also be started in collaboration with the Neurobehavioral Core. First, we will conduct a pilot study to assess behavioral phenotypes of PON1 knockout mice. In addition to developmental landmarks, a battery of behavioral tests will be used to assess motor activity, prepulse inhibition, and learning and memory. After the se initial studies, experiments will be initiated to evaluate the effect of CPO, given from postnatal day 4 to 21 to wild type and PON1 knockout mice at doses causing 10, 20, and 40% inhibition of brain cholinesterase.


Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other subproject views: All 14 publications 14 publications in selected types All 11 journal articles
Other center views: All 87 publications 77 publications in selected types All 73 journal articles
Type Citation Sub Project Document Sources
Journal Article Brophy VH, Hastings MD, Clendenning JB, Richter RJ, Jarvik GP, Furlong CE. Polymorphisms in the human paraoxonase (PON1) promoter. Pharmacogenetics 2001;11(1):77-84. R826886 (2000)
R826886C002 (2001)
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  • Journal Article Brophy VH, Jampsa RL, Clendenning JB, McKinstry LA, Jarvik GP, Furlong CE. Effects of 5' regulatory-region polymorphisms on paraoxonase-gene (PON1) expression. American Journal of Human Genetics 2001;68(6):1428-1436. R826886 (2000)
    R826886C002 (2001)
    R831709 (2007)
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  • Journal Article Furlong CE, Li WF, Brophy VH, Jarvik GP, Richter RJ, Shih DM, Lusis AJ, Costa LG. The PON1 gene and detoxication. NeuroToxicology 2000;21(4):581-587. R826886 (2000)
    R826886C002 (2001)
  • Abstract from PubMed
  • Journal Article Furlong CE, Li WF, Richter RJ, Shih DM, Lusis AJ, Alleva E, Costa LG. Genetic and temporal determinants of pesticide sensitivity: role of paraoxonase (PON1). NeuroToxicology 2000;21(1-2):91-100. R826886 (2000)
    R826886C002 (2000)
    R826886C002 (2001)
  • Abstract from PubMed
  • Journal Article Li WF, Costa LG, Richter RJ, Hagen T, Shih DM, Tward A, Lusis AJ, Furlong CE. Catalytic efficiency determines the in-vivo efficacy of PON1 for detoxifying organophosphorus compounds. Pharmacogenetics 2000;10(9):767-779. R826886 (2000)
    R826886C002 (2000)
    R826886C002 (2001)
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  • Supplemental Keywords:

     , RFA, Health, Scientific Discipline, Toxics, Geographic Area, Environmental Chemistry, Health Risk Assessment, State, pesticides, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Children's Health, genetic susceptability, farmworkers, health effects, pesticide exposure, dermal exposure, sensitive populations, community-based intervention, biological response, environmental risks, exposure, Washington (WA), children, Human Health Risk Assessment, assessment of exposure, children's vulnerablity, public health, human exposure, insecticides, pesticide residues, environmental health hazard, environmental toxicant, exposure pathways, harmful environmental agents, take home exposure, agricultural community, exposure assessment, intervention, environmental hazard exposures

    Relevant Websites:

    http://depts.washington.edu/chc/ Exit

    Progress and Final Reports:

    Original Abstract
  • 1999
  • 2000 Progress Report
  • 2002 Progress Report
  • 2003
  • Final

  • Main Center Abstract and Reports:

    R826886    University of Washington

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R826886C001 Molecular Mechanisms of Pesticide-Induced Developmental Toxicity
    R826886C002 Genetic Susceptibility to Pesticides (Paraoxonase Polymorphism or PON1 Study)
    R826886C003 Community-Based Participatory Research Project
    R826886C004 Pesticide Exposure Pathways Research Project