Grantee Research Project Results

2000 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: Center for Air, Climate, and Energy Solutions
Center Director: Robinson, Allen
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, 1999 through July 31,2000
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 , Human 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:

During the second year studies were carried out to further understand the in vivo function of PON1 for detoxifying OP insecticides utilizing transgenic mice. We had previously found that PON1 -/- mice were extremely sensitive to the toxicity of diazoxon but not of paraoxon. In vitro assays indicated that the PON1_R192 isoform hydrolyzed diazoxon less rapidly than did the PON_Q₁₉₂ isoform. In vivo experiments, where PON1-knockout mice received the same amount of either PON1₁₉₂ isoform via i.p. injection 4 hours prior to exposure, showed that both isoforms provided a similar degree of protection against diazoxon, while PON1_R192 conferred better protection against chlorpyrifos-oxon than PON1_Q₁₉₂. Injection of purified rabbit PON1 or either human PON1₁₉₂ isoform did not protect PON1-knockout mice from paraoxon toxicity nor did over-expression of the human PON1_R192 transgene in wild type mice. Kinetic analysis of the two PON1₁₉₂ isoforms revealed that the catalytic efficiency (V_max/K_m) determines the in vivo efficacy of PON1 for organophosphate detoxication. The results indicate that PON1 plays a major role in the detoxication of diazoxon and chlorpyrifos oxon but not paraoxon.

Since a wide range of expression levels of PON1 among individuals has been observed, we examined the promoter region of PON1 for genetic factors that might affect PON1 activity levels. We conducted a deletion analysis of the PON1 promoter region in transient transfection assays and found that cell-type promoter elements for liver and kidney are present in the first 200 bases upstream of the coding sequence. Sequence analysis of DNA from several individuals identified five polymorphisms in the first 1000 bases upstream of the coding region at positions –108, –126, –162, –832, and –909. The two polymorphisms at –126 and –832 have no apparent effect on expression level in the reporter gene assay. The polymorphisms at position –909, –162, and –108 each have approximately a two-fold effect on expression level. The expression level effects of the three polymorphisms does not appear to be additive and may depend on context effects.

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 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:

In the third year of this 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 Aim 3. For this purpose, we will assess the developmental profile of serum and liver PON1 activity with different substrates and of liver PON1 mRNA in C57 mice, which are of the same strain as the PON1-/- mice we utilize. We will then conduct a series of studies to determine the acute effects of various doses of OPs in mice of different ages (postnatal days 4, 7, 10, 15, 21, and 28), comparing wild type and PON1- knockout mice. The end-point in these experiments will be, as always, inhibition of brain and diaphragm acetylcholinesterase activity. These experiments will also provide important information (e.g., doses of OPs) for the studies of developmental neurotoxicity.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Publications Views
Other subproject views:	All 14 publications	14 publications in selected types	All 11 journal articles
Other center views:	All 89 publications	79 publications in selected types	All 75 journal articles

Publications
Type	Citation	Sub Project	Document Sources
Journal Article	Costa LG, Li WF, Richter RJ, Shih DM, Lusis A, Furlong CE. The role of paraoxonase (PON1) in the detoxication of organophosphates and its human polymorphism. Chemico–Biological Interactions 1999;119-120:429-438.	R826886 (2000) R826886C002 (2000)	Abstract from PubMed Full-text: ScienceDirect-Full Text HTML Exit Other: ScienceDirect-Full Text PDF Exit
Journal Article	Costa LG. The emerging field of ecogenetics. Neurotoxicology 2000;21(1-2):85-89.	R826886 (2000) R826886C002 (2000)	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)	Abstract from PubMed Abstract: Ovid-Abstract Exit

Supplemental Keywords:

children, health, pesticide, exposure., 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

Main Center Abstract and Reports:

R826886 Center for Air, Climate, and Energy Solutions

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

Top of Page

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.