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Extramural Research

2004 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 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, 2003 through October 31, 2004
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:

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.

Arsenic Effects in Midbrain (E12) Neurons

Since the last progress report, we have completed the analysis of experiments conducted in E12 midbrain cultures examining the impact of arsenite on proliferation, cytotoxicity, apoptosis, and stress-activated mitogen-activated signaling pathways.  A manuscript examining impacts on proliferation, apoptosis, and cytotoxicity is in the process of being submitted for publication.  Briefly, our findings demonstrate that arsenite mediates toxicity in developing cells specifically via alterations to the progression of the cell cycle.  Although our studies also provide morphological evidence suggestive of cell stress associated with apoptotic events, we were unable to demonstrate significant molecular evidence for either mechanism at an arsenite concentration below 4 μM, whereas significant impacts on cell cycle progression were evident between 0.5-1 μM.  We assessed stress-activated (SAPK) mitogen-activated protein kinases (MAPK) by a combination of Western analysis and phosphospecific antibodies directed against various members of these pathways (p38 MAPK, SAPK/JNK, and ERK1/2 MAPK).  We also assessed apoptosis by detection of cleaved caspase-3 expression, as well as functional activity assessment of caspase-3/7 and caspase-8 activities.  The mechanism underlying arsenite-mediated impacts on cell cycle progression and resulting toxicity at lower concentrations currently is under investigation and may reside in specific impacts to the ubiquitin proteasome system and altered cell cycle regulatory protein expression.

Methylmercury Effects on Cell Cycle in Embryonic Fibroblasts

The Faustman laboratory has been investigating the use of embryonic fibroblasts from transgenic animal models to determine the importance of specific cell cycle control checkpoint pathways in mediating methylmercury’s effects on cell cycle.  We have shown that methylmercury (historically used as pesticide) particularly mediated its effects via the p21 cell cycle checkpoint pathways.  In Gribble, et al. (2005), we show that p53 signaling pathways play a significant (but not exclusive) role in mediating methylmercury’s effects on cell cycle progression.  These new mechanistic observations should expand our understanding of upstream control processes responsible for mediating the cell cycle inhibition seen with many neurodevelopmental toxicants.

Chlorpyrifos Studies Summary

In our last progress report, we proposed to examine four general types of molecular responses, proliferation, cytotoxicity, apoptosis, and SAPKs/MAPKs associated with CP-mediated developmental toxicity in midbrain neurons during early organogenesis and in neurons in later critical development.  We extended our assessments to include both CP and its two major metabolites, TCP and CPO.  Dr. Xia’s group recently has published a manuscript demonstrating differential toxicity of CP and its metabolites in primary cortical neuronal cultures derived from embryonic (E17), as well as from newborn (P0) brain.  Their findings conclude that only a marginal difference is observed between CP and CPO with regard to induction of apoptosis.  This result is somewhat surprising because it is agreed generally that the oxon metabolite is considered three orders of magnitude more potent than CP in inhibition of brain acetylcholinesterase activity.  The results would indicate, therefore, that CP-mediated apoptosis occurs independent of cholinesterase inhibition.  

In contrast to results for both CP and CPO, Dr. Xia’s study concludes that TCP exerts only marginal toxicity and, even at 130 μM, does not induce apoptosis in postnatal cortical neurons.  They report that embryonic neurons are more sensitive to CP than newborn cortical neurons.  The Faustman laboratory has examined CP treatments in midbrain micromass cells between E17-18, during which time the cells are just completing their active proliferation and are starting to differentiate into neurons; therefore, they are likely to exhibit a greater degree of susceptibility to pesticide-induced toxicity than cells that are no longer proliferating and are more advanced as differentiated neural cells.  The results of our findings support those of the Xia group in that both CP and CPO induced a dose-dependent cytotoxicity between 0-75 μM in midbrain micromass cells, whereas TCP exerted only a minimal response even at the highest dose examined (126 μM).  As with the Xia group’s findings, most effects were observed at doses ranging between 15-50 μM; this included impact on cell morphology and cell viability.  The results also included an increased disposition to apoptosis, as evidenced by increased cleaved caspase-3 expression and caspase-3/7 and caspase-8 activities.  In contrast to the studies in newborn cortical neurons (P0), however, micromass cells (E17) exhibited a pronounced and dose-dependent elevation in cleaved caspase-3 expression and caspase-3/7 activity, as well as caspase-8 activity induced by TCP.  These findings are intriguing because they highlight the commonality and potential variability between cell types and the corresponding windows of susceptibility to pesticide exposure.

Significance

In summary, we provide data showing the differential impact of CP, CPO, and TCP on cytotoxicity, differentiation, and apoptosis in embryonic midbrain neuronal cells.  We demonstrate that CPO and TCP actually are more robust in inducing apoptosis than the parent compound, but for all three agents the mechanism appears independent of activation of stress-signaling pathways.  We will continue to incorporate our molecular findings in the biologically based dose response models for developmental toxicity that are being developed by the Risk Characterization Facility Core.  During this project period, we developed toxicokinetic and dynamic models for neurodevelopmental toxicity (Faustman, et al., 2004).  We also have listed our biologically based toxicodynamic model for midbrain and neocortical development with ethanol (Gohlke, et al., 2002, 2004) and methylmercury (Lewandowski, et al., 2003), respectively.

Future Activities:

We plan to examine the correlation between the level of CP-induced alteration in cell proliferation and/or death at critical times in the developing central nervous system (CNS) and quantifiable alterations in postnatal development/behavior.  These studies have been delayed because of the longer than expected time needed to establish the Neurobehavioral Core facility and the larger timeline for our initial postnatal CP neurodevelopmental studies.

Astrocyte proliferation has been suggested to be a target for organophosphorus pesticides (OPs) and be responsible, at least in part, for their possible developmental neurotoxicity.  As a followup to our preliminary results, Dr. Costa’s laboratory will measure [3H]thymidine incorporation in proliferating astrocytes and 1321N1 astrocytoma cells (a validated model for astrocytes) exposed to different concentrations (10-200 µM) of CP, CPO, and TCP.  Under the same conditions, they also will measure 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.


Journal Articles on this Report : 8 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 Caughlan A, Newhouse K, Namgung U, Xia Z. Chlorpyrifos induces apoptosis in rat cortical neurons that is regulated by a balance between p38 and ERK/JNK MAP kinases. Toxicological Sciences 2004;78(1):125-134.
abstract available  
R831709 (2007)
R831709C001 (2004)
  • Abstract from PubMed
  • Full-text: Toxicological Sciences-Full Text HTML
    Exit
  • Other: Toxicological Sciences-Full Text PDF
    Exit
  • Journal Article Daston G, Faustman E, Ginsberg G, Fenner-Crisp P, Olin S, Sonawane B, Bruckner J, Breslin W, McLaughlin TJ. A framework for assessing risks to children from exposure to environmental agents. Environmental Health Perspectives 2004;112(2):238-256.
    abstract available  
    R831709 (2007)
    R831709C001 (2004)
  • Full-text from PubMed
  • Abstract from PubMed
  • Journal Article Eskenazi B, Gladstone EA, Berkowitz GS, Drew CH, Faustman EM, Holland NT, Lanphear B, Meisel SJ, Perera FP, Rauh VA, Sweeney A, Whyatt RM, Yolton K. Methodologic and logistic issues in conducting longitudinal birth cohort studies: lessons learned from the Centers for Children's Environmental Health and Disease Prevention Research. Environmental Health Perspectives 2005;113(10):1419-1429.
    abstract available   full text available
    R831709 (2005)
    R831709C001 (2004)
    R827027 (2002)
    R829389 (2003)
    R829389 (2004)
    R829389 (2005)
    R829389 (Final)
    R829390 (2005)
    R829390 (Final)
    R829390C002 (2005)
    R831710 (2005)
    R831710 (Final)
    R831710C001 (2006)
    R831710C002 (2006)
    R831711 (2005)
    R831711 (2006)
    R831711 (2007)
    R831711 (Final)
    R831711C001 (2006)
    R831711C002 (2004)
    R831711C002 (2006)
    R831711C003 (2006)
    R832141 (2005)
    R832141 (2007)
    R832141 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • 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.
    abstract available   full text available
    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. The role of cell death during neocortical neurogenesis and synaptogenesis: implications from a computational model for the rat and mouse. Developmental Brain Research 2004;151(1-2):43-54.
    abstract available   full text available
    R831709 (2005)
    R831709 (2007)
    R831709C001 (2004)
  • 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 Gribble EJ, Hong SW, Faustman EM. The magnitude of methylmercury-induced cytotoxicity and cell cycle arrest is p53-dependent. Birth Defects Research Part A: Clinical and Molecular Teratology 2005;73(1):29-38.
    abstract available  
    R831709 (2005)
    R831709 (2007)
    R831709C001 (2004)
  • Abstract from PubMed
  • Abstract: Wiley Online-Abstract
    Exit
  • Journal Article Lewandowski TA, Ponce RA, Charleston JS, Hong S, Faustman EM. Changes in cell cycle parameters and cell number in the rat midbrain during organogenesis. Developmental Brain Research 2003;141(1-2):117-128.
    abstract available   full text available
    R831709 (2005)
    R831709 (2007)
    R831709C001 (2004)
  • 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 Lewandowski TA, Ponce RA, Charleston JS, Hong S, Faustman EM. Effect of methylmercury on midbrain cell proliferation during organogenesis:potential cross-species differences and implications for risk assessment. Toxicological Sciences 2003;75(1):124-133.
    abstract available   full text available
    R831709 (2005)
    R831709 (2007)
    R831709C001 (2004)
  • 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
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  • 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
    2005 Progress Report
    2006 Progress Report
    2007 Progress Report


    Main Center Abstract and Reports:
    R831709    University of Washington

    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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