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Extramural Research

2006 Progress Report: Molecular Mechanisms of Pesticide-Induced Developmental Toxicity

EPA Grant Number: R831709C001
Subproject: this is subproject number 001 , established and managed by the Center Director under grant R831709
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: University of Washington Center for Child Environmental Health Risks Research
Center Director: Faustman, Elaine
Title: Molecular Mechanisms of Pesticide-Induced Developmental Toxicity
Investigators: Faustman, Elaine
Institution: University of Washington
EPA Project Officer: Callan, Richard
Project Period: November 1, 2003 through October 31, 2008 (Extended to October 31, 2010)
Project Period Covered by this Report: November 1, 2006 through October 31,2007
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2003)
Research Category: Children's Health , Health Effects

Description:

Objective:

The objective of this research project is to identify cellular, biochemical, and molecular mechanisms for the adverse developmental neurotoxicity of pesticides.

Approach:

During the previous cycle of this research project, we conducted studies evaluating three classes of pesticides in both in vitro and in vivo assessments. A final manuscript has resulted from Dr. Xia’s studies comparing the impact of chlorpyrifos (CP) in embryonic and newborn cortical neurons.  Her investigations into the proposed mechanisms of toxicity associated with CP and its two major metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TCP; the breakdown product of both CP and CP-oxon) were reported recently in a publication (Caughlan, et al., 2004) and are summarized in this progress report.  We focused our experiments to address arsenic and methylmercury neurotoxicity and expanded significantly our understanding of the molecular mechanisms of toxicity associated with CP and its two major metabolites in embryonic midbrain cultures.

Progress Summary:

FAUSTMAN LABORATORY: During the report period, the Faustman laboratory research group conducted  pilot  studies in order to find appropriate dose-response doses for the in vivo mouse neurobehavioral study (Neurobehavioral Assessment facility core). Based on available literature of CPS exposure studies in rat, the first pilot study  for the CPS doses in mice were chosen as 0, 1, 2, and 5 mg/kg/d, administered by sc injection from GD6-17.  Maternal toxicity and acetylcholinesterase (AchE) activity in maternal brain and fetal tissues were used to determine the appropriateness of the doses selected. We found there was no measurable ChE inhibition at the doses chosen in either maternal brain or fetal tissues. Another pilot study was performed using 0, 5, 10, and 15 mg/kg CPS. We found that maternal brain AchE activity significantly decreased in the 10 and 15mg/kg dose with a decrease about 28% and 68%, respectively at 6h and decreased about 13% and 64% at 24h.  At the 10 and 15mg/kg dose, fetal brain AchE activity decreased around 12% and 15%, respectively at 6h and around 13% and 16% at 24h. The repeated dosing of CP resulted in a steep decrease in maternal AchE activity between the 10 and 15mg/kg dose at both 6h and 24h. The characterization of the AchE activity provides important evidence to verify the dose-response relationship in maternal and fetus. In addition, these pilot studies provide important information on plug rates and pregnancy rates for use in the experimental design of the gestational CPS exposure study.  Based on above results from pilot studies, final doses for the in vivo neurobehavioral study were chosen as 0, 4, 8, and 12 mg/kg CPS. The highest dose was expected to produce 50% inhibition of maternal AchE activity and the middle dose to produce 20% inhibition and low dose to produce no inhibition.
 
Neurobehavioral Assessment batteries require n=12 mice of each sex per dose, the total number of litters required for completion of the study is 48 litters. Therefore,  the experiment will be completed in four experimental cohorts, each consisting of 12 litters.We have finished gestational treatment  of three cohorts, and neurobehavioral assessment are underway in the Neurobehavioral Assessment facility core.
 
The Faustman lab continues experiments and explores advanced tools to address specific aims 1, 2 and 3 in regards to arsenic. These studies are designed to significantly expand our understanding of the molecular mechanisms of toxicity associated with chlorpyrifos and its two major metabolites. First, we successfully developed and characterized a mouse micromass primary culture system. The developmental dynamics of cell cycling and differentiation were characterized over nine days in culture and compared with rodent in vitro development. We also applied this culture system to investigate the key cell cycle regulatory gene p53 on neurogenesis (proliferation and differentiation) under normal and treatment conditions. In addition, we applied genomic approaches to investigate the gene pathways involved in the toxic responses to metals. Furthermore, we developed a system based approach to interpret dose- and time-dependent microarray data. We developed an extended program (GO-Quant) to quantitatively evaluate the gene expression values and to calculate the average intensity or ratio for those significantly altered by functional gene category based on MAPPFinder results. To demonstrate its application, we applied this approach to a previously published dose- and time-dependent toxicogenomic data set (J. F. Dillman et al., 2005, Chem. Res. Toxicol. 18, 28–34). Our results indicate that the above systems approach can describe quantitatively the degree to which functional gene systems change across dose or time. Additionally, this approach provides a robust measurement to illustrate results compared to single-gene assessments and enables the user to calculate the corresponding ED50 for each specific functional GO term, important for risk assessment. This system-based approach will greatly help to both qualitatively and quantitatively identify the key important gene pathways in pesticides exposures.
 
COSTA LABORATORY:  In recent years, several reports have suggested that organophosphates (OPs) may cause oxidative stress, characterized by exposure to excessive reactive oxygen species (ROS). Oxidative stress has been described in acute and chronic exposure to OPs, in both animals and humans, as well in some in vitro studies.  Of particular interest was the suggestion that oxidative stress may be involved in the developmental neurotoxicity of OPs. We have investigated the ability of OPs to induce oxidative stress in a genetic model of glutathione deficiency. For this purpose we utilized an in vitro system consisting of cerebellar granule neurons isolated from wild-type mice (GCLM (+/+) or from mice lacking the modifier subunit of glutamate cysteine ligase (GCLM -/-), the first and limiting step in the synthesis of glutathione. These neurons display very low levels of glutathione and are more susceptible to the toxicity of agents that increase oxidative stress. The cytotoxicity of chlorpyrifos (CPF) and diazinon (DZ), their active metabolites chlorpyrifos oxon (CPO) and diazoxon (DZO), and their “inactive” metabolites tricloropyridinol (TCP) and  2-isopropyl-6-methyl-4-pyrimidol (IMP) was assessed by the MTT assay. All compounds caused a concentration-dependent cytotoxicity; the oxons were the most toxic (IC50s = 2.5-4.9 uM),  while TCP and IMP were the least toxic. Cytotoxicity of CPF, CPO, DZ and DZO was greatly enhanced in neurons from GCLM (-/-) mice (IC50s increased by 10-25-fold). Two antioxidants, phenyl-N-butylnitrone and catalase protected neurons from the cytotoxicity of these compounds. Incubation of neurons with GSH ethyl ester, which significantly increased GSH levels, also conferred protection. When neurons from GCLM (+/+) mice were exposed to buthionine sulfoximine (BSO), a GSH synthase inhibitor, GSH levels decreased from 12.5 to 3.7  nmol/mg protein. Under this condition, the toxicity of CPF, CPO, DZ, DZO was significantly increased, and GCLM (+/+) neurons were as sensitive as GCLM(-/-) cells, not treated with  BSO.
 
CPF, DZ and their oxygen analogs increased intracellular ROS, measured using 2,7’-dichlorofluorescein acetate, in a time-dependent manner. ROS production was higher in neurons from GCLM (-/-) mice. Lipid peroxidation, assessed by measurement of malonyldialdehyde, was also increased by these compounds, with a greater effect in neurons from GCLM(-/-) mice.
 
SIGNIFICANCE: The focus of the Faustman and Costa laboratories is to understand the potential for, and magnitude of, impacts of pesticides on neurogenesis and gliogenesis. In both these essential neurodevelopmental pathways the balance between initial proliferation and subsequent specific differentiation is integral for proper neurodevelopment. In this project critical molecular pathways facilitating proliferation and toxicant response are being investigated. Knowledge about the timing and sensitivity of these critical pathways will directly translate into information relevant for establishing conditions promoting environmental and public health safety.

Future Activities:

FAUSTMAN LABORATORY:  During the next year, the Faustman lab will continue their investigations of specific cell cycle control check point pathways in defining susceptility of neurons to pesticide-induced effects in neurogenesis by using newly established mouse micromass cell cultures and using precursor neuronal cells from transgenic mouse models for checkpoint response  (proliferation) (Specific Aim 2). They will also apply genomic (microarray) and proteomic approaches to indentify the critical molecular pathways of neurogenesis disrupted by CPS gestation exposures.  
 
In collaboration with the Neurobehavioral Facility Core, we will finish the in vivo assessment study of prenatal mice exposed to pesticides in assesing the molecular, biochemical and neurobehavioral alterations (Specific Aim 7). The Faustman lab will work closely with with Risk Characterization Facility Core to incorporate the molecular research findings into biologically-based dose response models for assessing potential for neurodevelopmental toxicity.
 
COSTA LABORATORY: In the coming year we will continue our work on OPs and oxidative stress as a potential mechanism involved in the developmental neurotoxicity of these compounds. Specifically, we will investigate the cellular/molecular mechanism(s) by which OPs may induce oxidative stress. The role of acetylcholinesterase, muscarinic and nicotinic cholinergic receptors, the homeostasis of reduced and oxidized glutathione, the role of calcium (both extracellular and intracellular), and the involvement of mitochondria, will be investigated in the same in vitro system (cerebellar granule neurons from GCLM (+/+) and   GCLM (-/-) mice).


Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other subproject views: All 34 publications 17 publications in selected types All 16 journal articles
Other center views: All 175 publications 127 publications in selected types All 107 journal articles

Type Citation Sub Project Document Sources
Journal Article Costa LG. Current issues in organophosphate toxicology. Clinica Chimica Acta 2006;366(1-2):1-13. R831709 (2005)
R831709 (2006)
R831709 (2007)
R831709C001 (2006)
  • Abstract from PubMed
  • Abstract: Science Direct-Abstract
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  • Journal Article Faustman EM, Gohlke J, Judd NL, Lewandowski TA, Bartell SM, Griffith WC. Modeling developmental processes in animals: applications in neurodevelopmental toxicology. Environmental Toxicology and Pharmacology 2005;19(3):615-624. R831709 (2005)
    R831709 (2006)
    R831709 (2007)
    R831709C001 (2004)
    R831709C001 (2005)
    R831709C001 (2006)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
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  • Abstract: Science Direct-Abstract
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  • Other: ScienceDirect-Full Text PDF
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  • Journal Article Giordano G, Afsharinejad Z, Guizzetti M, Vitalone A, Kavanagh TJ, Costa LG. Organophosphorus insecticides chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency. Toxicology and Applied Pharmacology 2007;219(2-3):181-189. R831709 (2005)
    R831709 (2006)
    R831709 (2007)
    R831709C001 (2006)
    R831709C001 (2007)
    R831709C002 (2007)
  • Abstract from PubMed
  • Full-text: ScienceDirect - Full Text HTML
    Exit
  • Abstract: ScienceDirect - Abstract
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  • Other: ScienceDirect - Full Text PDF
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  • Journal Article Guizzetti M, Pathak S, Giordano G, Costa LG. Effect of organophosphorus insecticides and their metabolites on astroglial cell proliferation. Toxicology 2005;215(3):182-190. R831709 (2005)
    R831709 (2006)
    R831709 (2007)
    R831709C001 (2005)
    R831709C001 (2006)
    R831709C002 (2006)
  • Abstract from PubMed
  • Full-text: Science Direct-Full Text HTML
    Exit
  • Abstract: Science Direct-Abstract
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  • Other: Science Direct- Full Text PDF
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  • Journal Article Sidhu JS, Ponce RA, Vredevoogd MA, Yu X, Gribble E, Hong S-W, Schneider E, Faustman EM. Cell cycle inhibition by sodium arsenite in primary embryonic rat midbrain neuroepithelial cells. Toxicological Sciences 2006;89(2):475-484. R831709 (2005)
    R831709 (2006)
    R831709 (2007)
    R831709C001 (2005)
    R831709C001 (2006)
    R832733 (Final)
  • Abstract from PubMed
  • Full-text: Oxford Journals-Full Text HTML
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  • Abstract: Oxford Journals-Abstract
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  • Other: Oxford Journals-Full Text PDF
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  • Journal Article Yu X, Griffith WC, Hanspers K, Dillman III JF, Ong H, Vredevoogd MA, Faustman EM. A system-based approach to interpret dose-and time-dependent microarray data:quantitative integration of gene ontology analysis for risk assessment. Toxicological Sciences 2006;92(2):560-577. R831709 (2005)
    R831709 (2006)
    R831709 (2007)
    R831709C001 (2006)
    R832733 (2007)
    R832733 (Final)
  • Abstract from PubMed
  • Full-text: Oxford Journals-Full Text HTML
    Exit
  • Abstract: Oxford Journals-Abstract
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  • Other: Oxford Journals-Full Text PDF
    Exit
  • Supplemental Keywords:

    children’s health, epidemiology, genetics, health risk assessment, risk assessment, assessment of exposure, asthma, children’s environmental health, diesel exhaust, environmental risks, exposure assessment, genetic mechanisms, genetic risk factors, genetic susceptibility, maternal exposure, nutritional risk factors, Environmental Management, Scientific Discipline, Health, RFA, Risk Assessment, Health Risk Assessment, Children's Health, Biochemistry, Environmental Chemistry, health effects, children's environmental health, assessment of exposure, developmental neurotoxicity, agricultural community, community-based intervention, pesticide exposure, genetic polymorphisms, biological response, environmental health, environmental risks, children's vulnerability
    , RFA, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Biochemistry, Children's Health, Risk Assessment, health effects, pesticide exposure, environmental health, community-based intervention, developmental neurotoxicity, environmental risks, biological response, Human Health Risk Assessment, children's vulnerablity, assessment of exposure, children's environmental health

    Progress and Final Reports:
    Original Abstract
    2004 Progress Report
    2005 Progress Report
    2007 Progress Report


    Main Center Abstract and Reports:
    R831709    University of Washington Center for Child Environmental Health Risks Research

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R831709C001 Molecular Mechanisms of Pesticide-Induced Developmental Toxicity
    R831709C002 Genetic Susceptibility to Pesticides
    R831709C003 Community-Based Participatory Research Project
    R831709C004 Pesticide Exposure Pathways Research Project

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    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.

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