Jump to main content or area navigation.

Contact Us

Extramural Research

2005 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
Current 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, 2004 through October 31, 2005
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.

Progress Summary:

Faustman Laboratory

The Faustman laboratory and the Neurobehavioral Assessment Facility Core recently initiated pilot studies for the in vivo neurobehavioral study.  During the reporting period they met 12 times to discuss the experiments and finalize the study design.  These efforts have been supported by the Risk Characterization Facility Core to ensure a robust study design and to build quality assurance/quality control (QA/QC) methods into the procedures.  For this study, initial doses of chlorpyrifos (CPS) were chosen based on a review of the available literature.  Initial dose range-finding studies have begun, using multiple measures of maternal and fetal toxicity.  CPS doses were chosen initially as 0, 1, 2, and 5 mg/kg/day, administered by subcutaneous injection from gestation day (GD) 6 to 17.  The appropriateness of these CPS doses is being assessed currently in a pilot study examining maternal toxicity and various biochemical measurements in maternal and fetal tissues, including measurement of cholinesterase inhibition.  

Because of limitations of time and equipment, the maximum number of mice that can undergo neurobehavioral assessment is 96, obtained from 24 litters (one male and one female from each litter is used for each of the two test batteries).  Based on typical pregnancy rates in the mouse colony, it was decided to monitor the estrus cycle, to set up timed matings with 48 females and 12 males, and to monitor plug-positive females for weight gain before the onset of dosing at GD 6.  Pregnant mice will be exposed daily to CPS from GD 6 to 17, and litters will be culled to six mice at postnatal day 4, followed by one of two neurobehavioral assessment batteries as in the postnatal study reported previously.

The Faustman laboratory also continues experiments to address arsenic and methylmercury neurotoxicity.  We have expanded significantly our understanding of the molecular mechanisms of toxicity associated with chlorpyrifos (CP) and its two major metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TCP).  A key focus of these studies has been the successful development and characterization of a mouse micromass primary culture system.  This will allow for evaluation of specific cell signaling response pathways in the etiology of pesticide-induced effects on neurogenesis (proliferation and differentiation).  The developmental dynamics of cell cycling and differentiation were characterized over 9 days in culture and compared with rat in vitro development.

The Faustman laboratory continues to work closely with the Risk Characterization Facility Core to model their molecular and cellular research findings on neocortex neurogenesis and synaptogenesis.  These efforts especially have been important as they have identified (using model neurodevelopmental toxicants) the potential for significant impacts at low levels on early proliferative processes in neurogenesis.  Thus, this research has directly addressed questions posed in the literature regarding the relative importance of environmental impacts on proliferative versus apoptotic processes of normal neurodevelopment.  The results also have shown the proportionally greater impact on necortical development of impacts on proliferation versus the less dramatic impacts for comparable exposures during synaptogenesis.

Costa Laboratory

Astrocyte proliferation has been suggested to be a target for organophosphorus pesticides (OPs) and to be responsible, at least in part, for their possible developmental neurotoxicity.  As a followup to their preliminary results, Dr. Costa’s laboratory has measured [3H]thymidine incorporation in proliferating astrocytes and 1321N1 astrocytoma cells (a validated model for astrocytes) exposed to different concentrations of CP, CPO, and TCP.  Under the same conditions, they measured the percentage of astrocytoma cells incorporating trypan blue (as an index of cell viability) to discriminate between a specific effect of OPs on DNA synthesis and possible cytotoxic effects, which may lead, ultimately, to inhibition of cell proliferation.  

Similar experiments also were carried out with diazinon (DZ) and its active metabolite, diazinon oxon; its other metabolite; as well as with patathion (PS), paraoxon (PO), and p-nitrophenol (PNP).  The OPs and their oxons inhibited DNA synthesis in rat cortical astrocytes and astrocytoma cells with IC50s of around 50 μM, whereas the other metabolites were less effective.  The effects were similar in both cell types.  These effects were caused, in minimal part, by cytotoxicity.  OPs and their oxons also inhibited DNA synthesis stimulated by muscarinic agonists and by epidermal growth factor in synchronized astrocytoma cells, with a higher potency (IC50s in the low micromolar range).  Additional experiments with flow cytometry indicated that these compounds increased the number of cells in the G0/G1 phase of the cell cycle.  These experiments indicate that several OPs and their active metabolites interfere with DNA synthesis in astroglial cells and that the G0/G1-S progression especially may be sensitive to their action.  Though effects were seen at concentrations that exceed those necessary to inhibit acetylcholinesterase, they appear independent of this action as they also were caused by the parent compounds.

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 of 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 translate directly into information relevant for establishing conditions promoting environmental and public health safety.

Future Activities:

Faustman Laboratory 

During the next year, the Faustman laboratory will extend their investigations of specific cell cycle control checkpoint pathways in defining susceptibility of neurons to pesticide-induced effects in neurogenesis (proliferation).  This will be accomplished using their newly established mouse micromass cell cultures, as well as using precursor neuronal cells from transgenic mouse models, for checkpoint response.

Building on their pilot in vivo studies, they will, in collaboration with the Neurobehavioral Assessment Facility Core, establish the in vivo assessment study of prenatal mice exposed to pesticides.  The developing mice will be assessed for molecular, biochemical, and neurobehavioral alterations.

The Faustman laboratory will continue to work closely with the Risk Characterization Facility Core to incorporate the molecular research findings into biologically based dose-response models for assessing potential for neurodevelopmental toxicity.

Costa Laboratory 

During next year, the Costa laboratory is planning to address another issue related to the mechanism of developmental neurotoxicity of OPs.  There is a growing body of literature suggesting that OPs may exert toxic effects mediated by oxidative stress and independent of acetylcholinesterase inhibition.  To address this issue, cerebellar granule neurons will be used.  They will be prepared from wild-type mice and from mice whose GCLM gene has been knocked out.  These mice display very low levels of glutathione, a major cellular antioxidant.  Utilizing this in vitro model, three OPs (CP, DZ, and PS) and their oxons will be tested.  Endpoints will be cytotoxicity, generation of reactive oxygen species, and lipid peroxidation.  Protection by antioxidants also will be evaluated.  The hypothesis is that cells derived from GCLM-knockout mice will be more sensitive to OP toxicity because of reduced ability to counteract oxidative stress.  Mechanisms by which OPs may increase oxidative stress also will be investigated.


Journal Articles on this Report : 4 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 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
    Exit
  • Abstract: Science Direct-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • Journal Article Gohlke JM, Griffith WC, Faustman EM. A systems-based computational model for dose-response comparisons of two mode of action hypotheses for ethanol-induced neurodevelopmental toxicity. Toxicological Sciences 2005;86(2):470-484. R831709 (2004)
    R831709 (2005)
    R831709 (2007)
    R831709C001 (2005)
  • Abstract from PubMed
  • Full-text: Oxford Journals-Full Text HTML
    Exit
  • Abstract: Oxford Journals-Abstract
    Exit
  • Other: Oxford Journals-Full Text PDF
    Exit
  • 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
    Exit
  • Other: Science Direct- Full Text PDF
    Exit
  • 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
    Exit
  • Abstract: Oxford Journals-Abstract
    Exit
  • 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,, 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

    Relevant Websites:

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

    Progress and Final Reports:
    Original Abstract
    2004 Progress Report
    2006 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

    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.

    Jump to main content.