2002 Progress Report: Molecular Mechanisms of Pesticide-Induced Developmental ToxicityEPA 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, 2001 through July 31,2002
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
As stated in the original grant submission, the Molecular Mechanisms study has seven specific aims. These are to:
- Evaluate the sensitivity of the embryonic midbrain (E12) cells to altered cell cycling and cell viability by model pesticides in vitro;
- Evaluate the sensitivity of the new born hippocampal (P0) and cerebellar (P7) cells to altered cell viability by model pesticides in vitro;
- 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;
- Determine whether pesticide-induced cell cycle inhibition observed in vitro or in vivo is associated with alterations in cell cycle regulatory protein levels or gene expression;
- Determine whether toxicant-induced alterations in cell death relate to underlying alterations in the apoptotic pathway;
- Determine whether pesticide -induced apoptosis perturbs the activities of stress-activated mitogen-activated protein (MAP) kinases; and
- 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 include arsenic (historically used as a pesticide), benomyl, and chlorpyrifos (CP). We are continuing our activities for each of these aims in Year 5. 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.
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 Cole (Neurobehavioral Assessment Core), 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. Due to delays in the establishment of this core, our joint activity with this core will now primarily occur in Year 5 as a priority project to address Specific Aim 7.
Arsenic/In Vitro Studies/Midbrain, Cortical, and Cerebellar Neuronal Investigations
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 that were designed to address Specific Aims 1, 2, 3, 5, and 6 for arsenic and 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 have been determined to be mediated 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/Hippocampal Cortical Neurons
CP- increased lactate dehydrogenase release reduced cortical neuron viability and caused nuclear fragmentation and condensation. These effects were detectable at 15 – 30 μM CP, concentrations that are lower or comparable to those used by other investigators to study CP toxicity. Because many of the toxic effects of CP have been attributed to chlorpyrifos-oxon (CPO) inhibition of the brain acetylcholinesterase activity, we examined the effect of CPO on neuronal apoptosis. CPO is about three orders of magnitude more potent than the parent compound in inhibition of brain acetylcholinesterase activity. Therefore, if CP-induced apoptosis is mediated by acetylcholinesterase inhibition, the oxon form should induce apoptosis at concentrations two to three orders of magnitude lower than CP. However, CPO was only slightly more potent than CP in inducing cell death. 3,5,6-trichloro-2-pyridinol (TCP) is the major breakdown product of the detoxification of CP and CPO. It is generally thought to be non-toxic. Our results showed that TCP does not induce cortical neuron cell death at concentrations up to 130 μM. These data suggest that CP causes cytotoxicity in primary cortical neurons via a mechanism that is independent of acetylcholinesterase inhibition.
CP activates the JNK signaling pathway in cortical neurons. Because JNK signaling has been implicated in several forms of neuronal apoptosis including that induced by taxol and arsenite, we investigated if CP also activates this pathway. Western analysis using an antibody that recognizes phosphorylated and activated JNK (anti-p-JNK) showed no increase in total JNK phosphorylation after treatment with CP. However, CP caused an increase in total c-Jun phosphorylation at serine 73 for at least 24 hours, indicating c-Jun activation. An analysis of subcellular compartments showed an increase in p-JNK in the nucleus of neurons and a decrease in p-JNK in neuronal processes. This was accompanied by an increase in c-Jun phosphorylation in the nucleus. The increased nuclear JNK and c-Jun phosphorylation was detected up to 24 hours later. These data suggest that a sub-pool of JNK is activated for a sustained period of time in the nucleus, which causes c-Jun phosphorylation, and presumably AP-1-mediated transcription. The discrepancy between p-JNK Western and p-JNK immunostaining analysis is probably due to the high basal JNK activity in neurons that masks specific pools of JNK activation when total JNK was analyzed by Western blotting. These results provide supporting evidence for our hypothesis that activation of JNK and c-Jun mediated de nova gene expression may be required for neuronal apoptosis induced by CP.
CP activates the p38 signaling pathway in cortical neurons. Because p38 signaling has been implicated in several forms of neuronal apoptosis including that induced by nitric oxide and arsenite, we investigated if CP also activates p38. Our data show that p38 was activated by CP in cortical neurons, providing supporting evidence for our hypothesis that activation of p38 may be required for CP-induced apoptosis.
Chlorpyrifos/In Vitro Studies/Midbrain
Investigators examined the effects of CP and 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, and 50 μg/mL) or CPO (0.1, 1, 10, and 50 μg/mL ) for 24 hours under serum-free conditions. Medium 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 added additional time points in our evaluation. 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.
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 have made this task difficult. Specific patterns of cell death produced by CP and CPO under these conditions are being 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 using pregnancy specific pharmacokinetic models.
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 : 2 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|
||Namgung U, Xia Z. Arsenic induces apoptosis in rat cerebellar neurons via activation of JNK3 and p38 MAP kinases. Toxicology and Applied Pharmacology 2001;174(2):130-138.||
||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.||
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
Progress and Final Reports:Original Abstract
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