2007 Progress Report: Molecular Mechanisms of Pesticide-Induced Developmental Toxicity
EPA Grant Number:
Subproject: this is subproject number 001 , established and managed by the Center Director under
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
University of Washington Center for Child Environmental Health Risks Research
Molecular Mechanisms of Pesticide-Induced Developmental Toxicity
University of Washington
EPA Project Officer:
November 1, 2003 through
October 31, 2008
(Extended to October 31, 2010)
Project Period Covered by this Report:
November 1, 2006 through October 31,2007
Centers for Children's Environmental Health and Disease Prevention Research (2003)
The objective of this research project is to identify cellular, biochemical, and molecular mechanisms for the adverse developmental neurotoxicity of pesticides.
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.
FAUSTMAN LABORATORY: Exposure to pesticides such as chlorpyrifos (CPF) during critical “windows of susceptibility” can alter behavior and development of the central nervous system (CNS) in rodents by interfering with the regulatory pathways that control the production and selective cell loss of neurons. Specifically, pesticide-induced alterations in the regulatory dynamics of normal cell proliferation, differentiation, and cell death result in altered CNS morphogenesis and these alterations are correlated with subsequent deficits in learning and development. Microarray technology is a powerful tool in its ability to simultaneously monitor thousands of genes at same time. In addition, it has been proved to be an efficient method for identifying gene pathways involved in the process of abnormal development. During the reported period, the Faustman laboratory has applied a genomic (Affymetrix mouse whole genome array) approach to characterize the gene expression alteration resulting from CPF exposure. Furthermore, we have started to explore a non-labeling quantitative proteomic approach to examine proteomic change following CPF exposure during neurodevelopment.
Pregnant mice (GD6) were administered by sc injection doses of 0, 2, 4,10, 12, 15 mg/kg/d CP. 17-Acetylcholinesterase (AchE) activity in maternal brain and fetal tissues were measured. Total RNA was extracted from maternal brain and fetal tissues using Trizol reagent. RNA quality was checked by Agilent Bioanalyzer. Samples were prepared for hybridization to Affymetrix mouse whole genome 430 2.0 oligonucleotide arrays (39,000 transcripts). BRB Array Tools were used for normalization and statistical comparison analysis (ANOVA). Hierarchical clustering analysis was conducted in MeV. To establish the associations between the treatment and the affected gene ontology (GO) term and pathways, we used DAVID 2006 and the GenMAPP program. Furthermore, we applied our recently developed system-based GO-Quant approach to quantitatively evaluate the dose-dependent response in functional gene pathway. We found CPF exposure resulted in significant gene expression changes; 322 and 1372 genes changed in maternal and fetal brains, respectively (ANOVA, p≤ 0.005). Hierarchical clustering analysis show distinct differential gene expression pattern in maternal brain and fetus brain. Gene ontology (GO) and pathway analysis showed that CPF exposure affected multiple functional targets. For the maternal brain, the primary functional category changes included cell communication, protein modification, nervous system development, and synaptic transmission. In the fetal brain, the main primary functional category changes included sterol biosynthesis, RNA metabolism (processing, spicing), and regulation of cell growth. Our gene expression analysis demonstrated that different gene targets are affected following CPF exposure in maternal brains versus fetal brains.
The Faustman lab continues to explore advanced proteomic tools to examine pesticide-induced effects on neurodevelopment at the protein level. We collaborated with Dave Goodlett, Director of Mass Spectrometry Facility in the Department of Medicinal Chemistry, University of Washington, to explore non-labeling quantitative proteomic approaches to examine proteomic changes following CPF exposure during neurodevelopment. We have finished the first pilot study which investigated the best protein sample preparation for the MASS spectrometry. We found that our protein sample preparation protocol enabled us to identify approximately 500 protein peptides in the fetal brain under four different conditions by shotgun MASS analysis. We believe that the comparative genomic and proteomic analysis will enable us to identify the critical molecular pathways of neurogenesis disrupted by CPF gestation exposures.
COSTA LABORATORY: In the previous year we investigated the ability of organophosphates (OPs) to induce oxidative stress in a genetic model of glutathione (GSH) deficiency. For this purpose we utilized an in vitro system consisting of cerebellar granule neurons (CGNs) isolated from wild-type mice (Gclm +/+) or from mice lacking the modifier subunit of glutamate cysteine ligase (Gclm -/-), the first and limiting step in the sysnthesis of glutathione. These neurons display very low levels of glutathione and are more susceptible to the toxicity of agents that increase oxidative stress. The cytotoxicity of CPF and diazinon (DZ), their active metabolites chlorpyrifos oxon (CPO) and diazoxon (DZO), and their “inactive” metabolites tricloropyridinol (TCP) and 2-isopropyl-6-methyl-4-pyrimidol (IMP), was assessed by the MTT assay. All compounds caused a concentration-dependent cytotoxicity; the oxons were the most toxic, while TCP and IMP were the least toxic. Cytotoxicity of CPF, CPO, DZ and DZO was greatly enhanced in neurons from Gclm (-/-) mice (IC50s increased by 10-25-fold). Two antioxidants, phenyl-N-butylnitrone and catalase, protected neurons from the cytotoxicity of these compounds. Incubation of neurons with GSH ethyl ester, which significantly increased GSH levels, also conferred protection. When neurons from Glcm (+/+) mice were exposed to buthionine sulfoximine (BSO), a GSH synthase inhibitor, GSH levels decreased from 12.5 to 3.7 nmol/mg protein. Under this condition, the toxicity of CPF, CPO, DZ, and DZO was significantly increased, and Gclm (+/+) neurons were as sensitive as Gclm (-/-) cells not treated with BSO. CPF, DZ and their oxygen analogs increased intracellular ROS, measured using 2,7’-dichlorofluorescein acetate, in a time-dependent manner. ROS production was higher in neurons from Gclm (-/-) mice. Lipid peroxidation, assessed by measurement of malonyldialdehyde, was also increased by these compounds, with a greater effect in neurons from Gclm (-/-) mice.
More recently we also found that the calcium chelator BAPTA -AM partially but significantly antagonized cytotoxicity induced by all tested compounds (CPF, CPO, DZ, and DZO). The protection afforded by treating both Gclm (+/+) and Gclm (-/-) neurons with BAPTA -AM suggests that the OPs toxicity might be mediated by an increase in intracellular calcium.
To investigate this hypothesis we measured the intracellular calcium level [Ca2+]i using the Ca2+ -sensitive fluorescent dye fluo-3/AM (3μM). CPO and CPF at 1μM caused an increase in [Ca2+]i after a 15 min incubation. Fig. 1 shows the results in CGNs from Gclm (+/+) mice, but the results were identical in both genotypes. This increase was not abolished by using free-calcium medium, indicating that such increase was due to a release from intracellular calcium stores. Dantrolene (100µM), a ryanodine receptor antagonist that blocks the release of calcium from the endoplasmic reticulum (ER) into the cytosol, partially prevented CPO-induced cell death in both Gclm (+/+) and Gclm (-/-) neurons (Fig.2). This finding suggests that CPO may induce calcium release from ER through a ryanodine receptor-mediated mechanism, and that this is involved in cell death.
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 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 directly translate into information relevant for establishing conditions promoting environmental and public health safety.
FAUSTMAN LABORATORY: During the next year, the Faustman lab will continue their bioinformatic data mining for gene expression analysis to indentify the critical molecular pathways of neurogenesis disrupted by CP gestation exposures. We will apply our recently developed system-based GO-Quant approach to quantitatively evaluate the dose- dependent response in functional gene pathway. We will also further explore and characterize the criticl gene pathways by using realtime RT-PCR. In addition, in collaboration with the Mass Spectrometry Facility, we will develop a quantitative proteomic approach to examine the protein changes resulting from pesticide exposure (Specific Aim 7).
COSTA LABORATORY: In the coming year we will continue our work on OPs and oxidative stress as a potential mechanism involved in the developmental neurotoxicity of these compounds. Specifically, we will investigate the cellular/molecular mechanism(s) by which OPs may induce oxidative stress, following-up on our findings with CPF and CPO on intracellular calcium. Intracellular calcium measurements will be carried out for DZ and DZO, and the effect of dantrolene on DZ and DZO-induced neurotoxicity will be determined. The effect of dantrolene on CPO,CPS, DZ, and DZO-induced increase in ROS levels will then be measured. We will then investigate the possible role of inositol 1,4,5-trisphosphate receptors (IP3R) in OP-induced intracellular calcium release. For this purpose we will carry out experiments similar to those described above with the IP3R antagonist xestospongin C. These experiments will start providing a clearer picture on the role of intracellular calcium in OP-induced oxidative stress and cell death in mouse CGNs.
on this Report
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|| Costa LG, Cole TB, Furlong CE. Gene-environment interactions: paraoxonase (PON1) and sensitivity to organophosphate toxicity. LabMedicine 2006;37(2):109-113.
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|| Costa LG, Giordano G, Guizzetti M, Vitalone A. Neurotoxicity of pesticides: a brief review. Frontiers in Bioscience 2008;13(4):1240-1249.
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|| Giordano G, Afsharinejad Z, Guizzetti M, Vitalone A, Kavanagh TJ, Costa LG. Organophosphorus insecticides chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency. Toxicology and Applied Pharmacology 2007;219(2-3):181-189.
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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, Environmental Management, Scientific Discipline, Health, RFA, Risk Assessment, Health Risk Assessment, Children's Health, Biochemistry, Environmental Chemistry, health effects, children's environmental health, assessment of exposure, developmental neurotoxicity, agricultural community, community-based intervention, pesticide exposure, genetic polymorphisms, biological response, environmental health, environmental risks, children's vulnerability
, RFA, Health, Scientific Discipline, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Biochemistry, Children's Health, Risk Assessment, environmental health, health effects, pesticide exposure, community-based intervention, developmental neurotoxicity, biological response, environmental risks, Human Health Risk Assessment, assessment of exposure, children's vulnerablity, children's environmental health
Progress and Final Reports:
2004 Progress Report
2005 Progress Report
2006 Progress Report
Main Center Abstract and Reports:
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