2002 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, 2001 through July 31,2002
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 study is to determine the role of the serum enzyme paraoxonase (PON1) in the developmental neurotoxicity of organophosphorus (OP) compounds, and whether the two common PON1 isoforms (PON1Q192 and PON1R192) differ in their degree of protection. The specific aims of this research study are to: (1) investigate the acute toxicity of a number of organophosphates and their active oxygen metabolites in PON1 +/+ (wild-type) and PON1-/- (knockout) mice; (2) investigate whether human PON1 offers protection against the toxicity of OPs and whether the PON1Q192 and PON1R192 isoforms differ in their degree of protection against OP toxicity; (3) determine the acute toxicity of OPs in PON1+/+ and PON1-/- mice of different ages and assess whether PON1 offers protection to young animals toward OP toxicity; (4) assess the developmental neurotoxicity of OPs in developing PON1+/+ and PON1-/- mice following repeated exposures on postnatal days 4-20; and (5) determine the PON1 status of children of different ages.

Progress Summary:

The project has continued to pursue its original aims productively.

Specific Aim 1

Into the early part of this year, an abnormally high infant mortality rate had been preventing expansion of the mouse colony to a size large enough to begin the neurobehavioral assessment experiments outlined in specific aim 4. This difficulty has been overcome, and the neurobehavioral assessment experiments are well underway. The first cohort of PON1 knockout mice has been exposed to three different doses of chlorpyrifos oxon (CPO). Neurobehavioral assessment has been completed for this cohort, and timed matings for the second cohort are currently underway.

Specific Aim 2

For examining other possible endpoints to complement the neurobehavioral tests, additional mice were exposed to CPO in parallel to the mice that underwent behavioral testing. These mice were euthanized at various timepoints following exposure for measurement of cholinesterase activity and for histological analyses. As mentioned in the progress report of the Neurobehavioral Assessment C ore, histological analysis of the brains of these mice has yielded interesting new results. After chronic exposure of mice to CPO at a dose that inhibits brain cholinesterase by no more than 20% in PND4 mice, a histological lesion was consistently observed in layers I and II of the motor region of the neocortex, marked most prominently by perinuclear vacuolar structures reminiscent of swollen mitochondria. We are currently following up on this observation. Additional endpoints are being measured to correlate any observed neurobehavioral consequences with either neurochemical changes (detected by immunohistochemistry) or changes in gene expression (detected by hybridization of brain mRNA to microarrays).

Specific Aim 3

We now have robust colonies of both hPONR192 and hPONQ192 mice on the PON1-/- background (i.e., mice whose plasma and liver PON1 have been replaced with either of the human PON1192 isoforms). The two transgenic lines use endogenous human flanking sequences to drive expression of either hPON1Q192 or hPON1R192 faithfully in the liver and not in other tissues. Plasma PON1 levels were equivalent between the two transgenic lines, allowing direct comparison of detoxication efficacy between the two isoforms in vivo. We have completed dose-response and time course experiments examining cholinesterase inhibition and morbidity in these mice (as adults) in response to CPO exposure. These experiments indicated that hPON1Q192 mice were dramatically more sensitive than hPON1R192 mice. Indeed they were almost as sensitive as PON1-/- mice to CPO exposure. These studies corroborated our previous in vivo and in vitro findings that CPO is preferentially metabolized by PON1Arg192, but the magnitude of the difference in detoxication efficacy was unexpected. The two lines of mice are now being tested for sensitivity to diazoxon, as well as to the respective parent compounds, chlorpyrifos and diazinon. Even more importantly, we found that the developmental onset of hPON1 expression is similar in the two transgenic lines, allowing us to now compare the two human PON1 isoforms in vivo for their ability to protect developing animals against the toxicity of OPs.

Specific Aim 4

PON1 status (i.e., PON1Q192R genotype plus plasma PON1 levels) has been measured in 29 babies and children at Children’s Hospital and Regional Medical Center. Sufficient data points are now available to construct developmental curves of plasma PON1 activity from birth to 2 years in children with different PON1Q192R genotypes. The se data indicated that the onset of PON1 expression is variable among individuals, reaching adult levels between 6 and 15 months of age. As mentioned above, the developmental profile of human PON1Arg192 and PON1Gln192 is also being investigated in the transgenic mice.

Specific Aim 5

A collaboration is continuing with the University of California at Berkeley Child Health Center, directed by Brenda Eskenazi. In this study, a cohort of Hispanic pregnant women and their offspring is being tested for correlation among PON1 status, plasma cholinesterase activity, urinary OP metabolites, and behavior. Measurement of PON1 status and plasma cholinesterase activity has been completed in our laboratory for 468 samples from this study, representing samples taken from the women or their offspring at either 26th week of gestation or upon maternal delivery. Additional samples will be processed as they become available at 12 months and 24 months of age.

Significance

The findings from these experiments will provide critical information on the neurobehavioral consequences of OP exposure, which will be integrated with data on cholinesterase inhibition in plasma, brain, and diaphragm and ultimately used to help develop and refine biologically-based risk models for OP compounds. The research will provide new insights into the mechanisms of developmental neurotoxicity related to OP exposure and the role of PON1 in modulating these effects. Experiments with the “humanized” transgenic mice will allow us to assess the importance of the PON1Q192R polymorphism for OP exposures that happen during development. This is especially important, as children already represent a particularly susceptible population due to their lower PON1 levels.

Future Activities:

Work will continue to investigate the developmental effects of CPO in PON1 knockout and wild type mice. Additional endpoints for assessing developmental effects of OP exposure are being pursued that will complement our assessment of cholinesterase inhibition and neurobehavior in mice, including assessment of neurochemical, histopathological, and gene expression end points. We will be following up on the histological observations in the neocortex through the use of electron microscopy, immunohistochemistry for caspases and glial fibrillary acidic protein (GFAP), silver neurodegeneration stains, and TUNEL staining.

Experiments in PON1Arg192 and PON1Gln192 mice will be extended to include the parent compound chlorpyrifos , to develop and refine a pharmacokinetic model in collaboration with Dr. Chuck Timchalk (Pacific Northwest National Laboratories). Additional experiments will be done with diazinon and diazoxon. The two human PON1 isoforms will also be compared in vivo for their ability to protect developing animals against the toxicity of OPs.

A series of investigations will be carried out to substantiate and expand recently published data suggesting that glial cells may represent a primary target for the developmental neurotoxicity of chlorpyrifos and CPO. In vitro experiments with rat cortical astrocytes and a human astrocytomas cell line will be carried out to determine the effects of these OPs on cell cycle and cell viability.


Journal Articles on this Report : 4 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 Costa LG, Richter RJ, Li W-F, Cole T, Guizzetti M, Furlong CE. Paraoxonase (PON1) as a biomarker of susceptibility for organophosphate toxicity. Biomarkers 2003;8(1):1-12. R826886 (2000)
R826886C002 (2002)
R831709 (2005)
R831709 (2007)
R831709C002 (2004)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
    Exit
  • Journal Article Costa LG, Cole TB, Jarvik GP, Furlong CE. Functional genomics of the paraoxonase (PON1) polymorphisms: effects on pesticide sensitivity, cardiovascular disease, and drug metabolism. Annual Review of Medicine 2003;54:371-392. R826886 (2000)
    R826886C002 (2002)
    R831709 (2005)
    R831709 (2007)
    R831709C002 (2004)
  • Abstract from PubMed
  • Abstract: Annual Reviews-Abstract
    Exit
  • Journal Article Furlong CE, Cole TB, Jarvik GP, Costa LG. Pharmacogenomic considerations of the paraoxonase polymorphisms. Pharmacogenomics 2002;3(3):341-348. R826886 (2000)
    R826886C002 (2002)
  • Abstract from PubMed
  • Abstract: FutureMedicine-Abstract
    Exit
  • Journal Article Furlong CE, Li W-F, Shih DM, Lusis AJ, Richter RJ, Costa LG. Genetic factors in susceptibility: serum PON1 variation between individuals and species. Human and Ecological Risk Assessment 2002;8(1):31-43. R826886 (2000)
    R826886C002 (2002)
  • Abstract: Taylor and Francis-Abstract
    Exit
  • 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
  • 2001 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