Grantee Research Project Results

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.