Grantee Research Project Results
2000 Progress Report: Molecular Mechanisms of Pesticide-Induced Developmental Toxicity
EPA Grant Number: R826886C001Subproject: 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: Center for Air, Climate, and Energy Solutions
Center Director: Robinson, Allen
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, 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:
As stated in the original grant submission, the Molecular Mechanisms Study had two primary objectives for year 2:
- Specific Aim 1: Evaluate the sensitivity of the embryonic midbrain (E12) cells to altered cell cycling and cell viability by model pesticides in vitro.
- Specific Aim 2: Evaluate the sensitivity of the newborn hippocampal (P0) and cerebellar (P7) cells to altered cell viability by model pesticides in vitro.
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. 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 central nervous system (CNS). These specific aims were designed to investigate our stated hypothesis that pesticide-induced effects on CNS development alter the dynamics of cell proliferation, differentiation, and cell viability.
Progress Summary:
Specific Aim 1
A key focus of the year 2 efforts have been to examine the effect of arsenic on gestation day 12 primary rat midbrain neuroepithelial cells. These were exposed to As3+ (0, 2, 4, and 5 μM , 0-48 hours) in vitro. Effects of As3+ on cell cycle kinetics were determined by continuous BrdU labeling and bivariate flow cytometric Hoechst-ethidium bromide analysis. We observed a time- and concentration-dependent inhibition of mitosis as early as 12 hours after exposure. After 24 hours, the fraction of cells in S phase treated with As3+ were significantly higher than untreated controls. In later rounds of cell division, significantly fewer treated cells were present, due to inhibition in earlier phases and/or cell death. Significant inhibition of cell cycle entry from G0/G1 was not seen until 36 hours after treatment. Although cell proliferation was inhibited, cell cycle progression was observed to occur under all exposure conditions.
The present study confirms previous observations of As3+ induced cell cycle inhibition found in other cell types. Taken as a whole, As3+ caused time- and concentration- dependent effects on G1, S, and G2/M phases. These effects likely contribute to its neurodevelopmental toxicity. Further studies investigating changes in gene expression during development will provide a better understanding of mechanisms by which As3+ induces disruption of the cell cycle.
As noted earlier, inorganic arsenic has been shown to be both embryotoxic and teratogenic in animals. Behavioral effects in developing rats have been documented at repeated oral dose levels of 5 mg/kg As5+ (Nagaraja and Desiraju, 1994). How can th ese data be extrapolated to humans? Using a series of assumptions, we conclude that the doses (2-5 μM) used in our experiments represent those that are relevant to possible human exposure levels.
In conclusion, As3+ disrupts many pathways of gene expression and regulation. In our experiment, As3+ treatment altered all phases of the developing rat neuroepithelial cell cycle. Post-treatment, we observed delayed cell cycle exit, delayed entry, and reduced fractions of cells. These effects were cumulative and lasted through three rounds of division. In an organism, coordination of cell cycle progression and cell numbers are critical for normal development. Our findings suggest a wide variety of possible mechanistic hypotheses behind the observed cell cycle effects: As3+ binding to sulfhydryl containing cell components, inducing apoptosis, and/or altering gene expression. Continued analysis of signaling pathways including p21 and ras expression levels would be useful to further characterize observed cell cycle effects.
During year 2, investigators have focused on continuing our evaluation of arsenic. Arsenic distributes throughout the body and is able to pass the placental barrier and reach the developing fetal brain. High-dose inorganic arsenic exposure to animals during development has been associated with neural tube defects, exencephaly, and other disorders, although the molecular mechanisms underlying potential effects from low-dose exposure are uncertain. Although many mechanisms including cell cycle disruption, altered gene expression, and induction of apoptosis have been proposed for As3+ toxicity, the effects on cell proliferation during embryonic/fetal development has not been extensively studied.
To ensure that we are obtaining the information required for cell cycle analysis for each compound, we have decided to stagger the in vitro studies. At this point, dose-range finding studies to define toxicity curves for arsenic have been completed for all cell culture systems (required for both Specific Aim 1 and 2).
Specific Aim 2
To evaluate the contribution of the stress-activated mitogen-activated protein (MAP) kinases in arsenite-induced apoptosis, we measured the kinase activity in cerebellar neurons treated with sodium arsenite. Treatment of cerebellar neurons with sodium arsenite- activated p38 and c-Jun N-terminal kinase 3 (JNK3) but not JNK1 or JNK2. It also induced c-Jun phosphorylation. Furthermore, sodium arsenite induced cerebellar neuron apoptosis. This apoptosis was attenuated by SB203580, an inhibitor of p38, and by CEP-1347, an inhibitor of JNK activation. These data indicate that both JNK and p38 contribute to arsenite-induced apoptosis in primary cerebellar neurons. This is the first evidence that a specific JNK isoform is differentially activated by stress and contributes to neuronal apoptosis. These observations support the overall hypothesis of this proposal that the balance between the extracellular signal-regulated kinases (ERK), JNK, and p38 may regulate neuronal apoptosis and that arsenic’s effects on brain tissue may be to alter the dynamics of cell viability.
Significance
There is current debate in the scientific literature regarding the extent to which arsenic and chlorpyrifos are developmental neurotoxicants. Because we propose mechanistic, neuropathologic, and neurobehavioral experiments, our proposed research has a high probability of providing relevant information that can be used to resolve this debate. Chief among these are whether low-dose, developmental exposures to these model pesticides can result in sub-clinical alterations in neurobehavior and if this altered neurobehavior can be explained by neuropathological alterations associated with impaired cell proliferation and cell death. At present, there are no comprehensive studies being conducted with the goal of resolving these questions.
Studies examining gene expression in flow cytometrically sorted cells have not been successfully conducted before. Our ability to combine flow cytometric sorting with molecular biology analysis has an extremely high potential for improving our understanding of the mechanisms underlying cell death and cell cycle alterations in the developing CNS under normal conditions and following contaminant exposure.
Future Activities:
Initial experiments with chlorpyrifos and chlorpyrifos oxons have been initiated. In this coming year, we expect to complete our examination of cell cycle alterations and apoptosis of arsenic in vitro and will establish the dosing regimen for in vivo studies and initiate in vivo testing prioritizing organophosphate assessments. Inclusion of oxon has resulted from observations from the Paraoxonase Polymorphism Study that have shown oxon is of interest for defining chlorpyrifos neurotoxicity. The studies will be facilitated by the hiring of Dr. Kristina Dam in the Neurobehavioral Assessment Core.
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this subprojectSupplemental 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 developmentRelevant Websites:
http://depts.washington.edu/chc/ Exit
Progress and Final Reports:
Original AbstractMain 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
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.
Project Research Results
4 journal articles for this subproject
Main Center: R826886
89 publications for this center
75 journal articles for this center