2001 Progress Report: Molecular Mechanisms of Pesticide-Induced Developmental Toxicity

EPA Grant Number: R826886C001
Subproject: this is subproject number 001 , 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: Molecular Mechanisms of Pesticide-Induced Developmental Toxicity
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, 2000 through July 31,2001
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:

As stated in the original grant submission, the Molecular Mechanisms study has seven specific aims. These are:

  1. Evaluate the sensitivity of the embryonic midbrain (E12) cells to altered cell cycling and cell viability by model pesticides in vitro.
  2. Evaluate the sensitivity of the new born hippocampal (P0) and cerebellar (P7) cells to altered cell viability by model pesticides in vitro.
  3. Evaluate the in vivo sensitivity of the embryonic midbrain (E12), fetal hippocampus (P0), and post natal cerebellum (P7) to altered cell cycling, apoptosis, and necrosis by model pesticides.
  4. Determine whether pesticide-induced cell cycle inhibition observed in vitro (Specific Aim 1) or in vivo (Specific Aim 3) is associated with alterations in cell cycle regulatory protein levels or gene expression.
  5. Determine whether toxicant-induced alterations in cell death relate to underlying alterations in the apoptotic pathway.
  6. Determine whether pesticide -induced apoptosis perturbs the activities of stress-activated mitogen-activated protein (MAP) kinases.
  7. Determine whether there is a correlation between the level of pesticide-induced alteration in cell proliferation and/or death at critical times in the developing central nervous system (CNS) and quantifiable alterations in postnatal behavior.

As defined in the original grant submission, the model pesticides used in the overall project are to include arsenic (historically used as a pesticide), benomyl, and chlorpyrifos (CP). We are continuing our activities for each of these aims in Year 4. No changes in either compounds of interest or in our specifications are foreseen. These pesticides were chosen because of existing information suggesting plausible mechanisms through which they may interfere with neuronal cell production and loss in the developing CNS.

Progress Summary:

Molecular Mechanisms study researchers investigate the relationship between pesticide-induced alterations in cell proliferation and cell death during embryonic/postnatal neurodevelopment and deficits in learning and development. Our hypothesis is that certain pesticides affect learning, growth, and development by altering the balance between cell proliferation and cell death. Four laboratories are involved in this research core: the Faustman laboratory studies the role of regulators of cell proliferation, the Mirkes laboratory investigates the role of apoptotic factors, and the Xia laboratory addresses the role of cell death-signaling pathways and survival pathways. Finally, the rodent neurobehavioral laboratory, lead by Drs. Burbacher and Dam (Neurobehavioral Assessment C ore), studies the relationship between toxicant-induced alteration in cell proliferation and/or death in the prenatal/postnatal CNS and subsequent postnatal alterations in learning and development.

Arsenic/In Vitro Studies/Midbrain, Cortical, and Cerebellar Neuronal Investigations

As reported in a previous progress report , dose-response relationships have been established for As3+ in midbrain, cortical, and cerebellar neuronal tissues, and research evaluating the effects on cell viability, proliferation, and cell signaling have been conducted or are in progress. This research represents investigations designed to address specific aims 1, 2, 3, 5 and 6 for arsenic. These investigations have focused on evaluating arsenic concentrations (2–5 μM) that represent those of relevance to possible human exposure levels. Toxic effects in cortical and cerebellar neurons appear to be via stress activated cell signaling pathways including specific c-Jun N-terminal protein kinases (JNK) and p38 MAP kinase pathways. For example, arsenite induced cerebellar neuron apoptosis requires new gene expression and caspase activation, and sodium arsenite selectively activated p38 and JNK3 but not JNK1 or 2 pathways in cerebellar neurons.

Chlorpyrifos/In Vitro Studies/Midbrain

Recently, concern has begun to grow about the effects of CP on the developing nervous system, where there is some evidence to suggest that it may have unique targets. For example, immature animals are more sensitive to CP than their adult counterparts even though they recover acetylcholinesterase activity more quickly. In addition, it has been shown that CP can reduce macromolecule and protein synthesis as well as inhibit neuronal differentiation in PC12 cells. The effects of CP on neuronal apoptosis, however, have not been previously characterized.

With the help of Dr. Kristina Dam, whose work bridges the Molecular Mechanisms study and the Neurobehavioral Assessment C ore, studies have commenced examining the effects of CP and chlorpyrifos-oxon (CPO) on gestational day 12 primary rat midbrain epithelial cells in vitro. Cells were cultured under serum-free conditions and were exposed to CP (0.5, 5, 50, and 500 μg/mL ) or CPO (0.1, 1, 10 and 100 μg/mL ) for 24 hours under serum-free conditions. Medi um was then replaced with serum-containing media (standard micromass media), with no pesticide present, for a total of 5 days in culture. Cells were completely eliminated by the highest doses of CP and CPO with a less robust decrease in viability with 10 μg/mL CPO at 24 hours after the initial exposure. By 5 days after initial plating no changes in viability could be seen with any of the concentrations, except 500 and 100 μg/mL CP and CPO, respectively, in which there were no cells present to recover. Data from the lower concentrations suggest that the cells are capable of completely recovering from the initial toxicant insult.

Studies in PC-12 cells indicate that differentiated cells are more susceptible to pesticide insults than proliferating cells. Using this information, we changed the time course of pesticide exposure for our in vitro work. By 5 days after plating, primary rat midbrain epithelial cells have differentiated into neurons, exhibiting multiple neurites. For this reason we chose to expose the cells to CP and CPO for a 24-hour period beginning on day 5. As suspected, the differentiated cells were more sensitive to the pesticides, at least for CP. In this case 50 μg/mL CP (10-fold lower than used in the studies above) decreased viability by about 90 percent. Although there was a trend towards decreased viability with CPO, the effects were not statistically significant.

In Year 4, we will measure the degree of cholinesterase inhibition produced by exposure to CP and CPO under these same dose response conditions. However, very low levels of cholinesterase in the embryo make this task difficult. Specific patterns of cell death produced by CP and CPO under these conditions will be evaluated. Finally, we are working with the Risk Characterization C ore (Crispin Pierce and Bill Griffith) to evaluate the in vitro dose response with our projected in vivo CP concentration.

CP/In Vitro Studies/Cerebellar/Cortical

We have started investigating the neurotoxicity of CP. Our data show: (1) h igh doses (150 mM) of CP induce apoptosis in postnatal cortical neurons cultured in the presence of serum; (2) h igh doses (150 mM) of CP induce apoptosis in embryonic cortical neurons cultured in the presence of serum; (3) CPO at concentrations as high as 150 mM does not induce cell death in postnatal cortical neurons cultured in the presence of serum; (4) 3,5,6-trichloro-2-pyridinol (TCP) does not induce apoptosis in embryonic cortical neurons cultured in the presence of serum; (5) CP (50 mM) induces apoptosis in postnatal cortical neurons cultured in the absence of serum; (6) CP (30 and 50 mM) induces apoptosis in embryonic cortical neurons cultured in the absence of serum; and (7) CPO (30 and 50 mM) induces apoptosis in postnatal cortical neurons cultured in the absence of serum.

Significance

In summary, we have data demonstrating that: CP, at concentrations similar to or lower than those used by other investigators, induces apoptosis in young neurons cultured in serum-free conditions. Furthermore, CPO is not much more toxic than the parent compound in inducing cell death, suggesting that CP-induced apoptosis does not involve choline sterase inhibition. Moreover, CP and its metabolites, at concentrations lower than those needed to induce apoptosis, potently inhibit mitochondria function (measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide [MTT] metabolism). This may provide another biomarker for CP toxicity.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other subproject views: All 4 publications 4 publications in selected types All 4 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 Mirkes PE, Little SA. Cytochrome c release from mitochondria of early postimplantation murine embryos exposed to 4-hydroperoxycyclophosphamide, heat shock, and staurosporine. Toxicology and Applied Pharmacology 2000;162(3):197-206. R826886 (2000)
R826886C001 (2001)
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  • Journal Article Mirkes PE, Wilson KL, Cornel LM. Teratogen-induced activation of ERK, JNK, and p38 MAP kinases in early postimplantation murine embryos. Teratology 2000;62(1):14-25. R826886 (2000)
    R826886C001 (2001)
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  • Journal Article Umpierre CC, Little SA, Mirkes PE. Co-localization of active caspase-3 and DNA fragmentation (TUNEL) in normal and hyperthermia-induced abnormal mouse development. Teratology 2001;63(3):134-143. R826886 (2000)
    R826886C001 (2001)
    R826886C001 (2002)
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  • Supplemental Keywords:

     , RFA, Health, Scientific Discipline, Toxics, Environmental Chemistry, Health Risk Assessment, pesticides, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Children's Health, genetic susceptability, health effects, pesticide exposure, sensitive populations, biological response, developmental toxicity, environmental risks, neurodevelopment, exposure, children, Human Health Risk Assessment, neurotoxicity, neurodevelopmental, assessment of exposure, children's vulnerablity, polychlorinated biphenyls, susceptibility, neurodevelopmental toxicity, human exposure, growth and development, environmental health hazard, environmental toxicant, exposure pathways, environmentally caused disease, growth & development, windows of sensitivity, sensitivity, developmental disorders, exposure assessment, neurological development

    Relevant Websites:

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

    Progress and Final Reports:

    Original Abstract
  • 1999
  • 2000 Progress Report
  • 2002 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