Grantee Research Project Results
2011 Progress Report: Molecular Mechanisms
EPA Grant Number: R834514C003Subproject: this is subproject number 003 , established and managed by the Center Director under grant R834514
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials
Center Director: Faustman, Elaine
Title: Molecular Mechanisms
Investigators: Faustman, Elaine
Current Investigators: Faustman, Elaine , Fenske, Richard , Griffith, William C. , Yost, Michael , Costa, Lucio G , Furlong, Clement , Thompson, Engelberta , Vigoren, Eric M. , Carr, Catherine J
Institution: University of Washington
EPA Project Officer: Callan, Richard
Project Period: September 25, 2008 through September 24, 2016
Project Period Covered by this Report: August 1, 2010 through July 31,2011
RFA: Children's Environmental Health and Disease Prevention Research Centers (with NIEHS) (2009) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
Pesticide-induced alterations in the regulatory dynamics of normal cell proliferation, differentiation, and cell death result in altered central nervous system (CNS) morphogenesis. These alterations are correlated with subsequent deficits in learning and development. Thorough understanding of mechanisms of toxicity at the molecular, cellular, and organism level is therefore important as these mechanisms may define “windows of susceptibility” for many neurodevelopmental toxicants as well as for pesticides. The Molecular Mechanism Research Project evaluates these mechanistic processes across several pesticide classes in order to understand the commonality and uniqueness of pesticide toxicity across dose, lifestage, cell type, and behavioral domain. These efforts bring together the expertise from different laboratories and focus this expertise on the relationship between pesticide-induced alterations in cell proliferation and death during embryonic/postnatal development and postnatal alterations in behavior. The laboratory of Dr. Elaine Faustman addresses the impacts of pesticides and model neurotoxicants on prenatal neurogenesis (gestational day 12-17) and the laboratory of Dr. Lucio Costa evaluates the postnatal effects on gliogenesis.Progress Summary:
In the last 2 years, the Faustman lab initiated studies to explore new in vitro models for the evaluation of neurogenesis including proliferation and differentiation. The highlights of this year include the following:
- Establishing human neuronal stem cell cultures. Direct application of this culture system has provided critical information on chemical impacts on human cell proliferation and differentiation, which is relevant for neurogenesis in human tissues. We investigated the effects of chlorpyrifos (CP) and arsenic on proliferation and differentiation. Human neural stem cells (hNPCs) were expanded in serum free proliferation expansion medium (MEM) or differentiated in neural differentiation medium (HDM). Cells were incubated for 72 hours in either MEM or HDM with CP. Dose-dependent decreases in cell viability were observed in both cells cultured in proliferation MEM and differentiation HDM medium, with significantly greater effects of CP on cells in HDM.
- The Faustman lab further investigated whether CP differentially altered epigenetic markers during proliferation and differentiation. We examined protein expression of epigenetic markers. These initial results suggest that CP exposure in differentiating hNPCs resulted in significant alteration in expression of several epigenetic markers associated with transcriptional activation while exposure of cells to CP during proliferation showed fewer changes in these epigenetic factors. Differential alterations of epigenetic markers during proliferation and differentiation may provide cell stage specific mechanistic clues for examining effects on neural development. These examinations of the effect of pesticides on human cells should provide valuable information and help to interpret in vivo animal studies and more directly inform prediction for impacts in human populations.
- In addition, the Faustman lab is invested in optimizing biomarker methods to allow for analysis from human samples to profile the potential impacts of pesticides on exposed populations. Due to their non-invasive nature and low cost to collect, buccal epithelial cells have been used for the isolation of DNA and, in limited cases, for obtaining RNA for gene expression analyses. We have tested several types of DNA and RNA isolation methods. For DNA, an ethanol precipitation method following a proteinase K digestion (Gentra Puregene kit) was found to provide superior yields compared to a phenol extraction method and to a protocol using magnetic beads (Invitrogen Chargeswitch). DNA isolated with the optimal method is of high quality and able to be amplified using whole genome amplification methods to provide ample material for multiplexed genotyping arrays. With regards to RNA, a column-based isolation (Qiagen RNeasy) paired with a short proteinase K digestion step outperformed a phenol-chloroform (Invitrogen Trizol) extraction method. A comparison of whole transcriptome amplification methods showed that amplification products generated with the Nugen WT Ovation kit provided greater signals in RT-PCR assays than did those produced by the Ambion WT Expression kit.
- We characterized field samples collected by the CBPR project to identify potential differences in farmworker and non-farmworker families in metabolite profiles with the Affymetrix Drug Metabolizing Enzyme and Transporter (DMET) Plus platform. We investigated if genotypes for key CYP450 genes might modify the urinary identifications of OP metabolites and this relationship and our analysis has identified genetic variation in the CYP3A5 gene (6986A>G, rs776746) (ANOVA, p < 0.05). Post-hoc testing showed that individuals with the G/G genotype had higher AChE inhibition per unit AZ or DMTP compared to A/A individuals. Because the G allele produces a splice defect that is associated with lower enzyme activity, future PBPK modeling will determine if genotypic variation at this locus could be used to identify individuals who may be more sensitive to OP-associated effects.
During the past year, work in the Costa lab has progressed according to plans, addressing the proposed specific aims. The Costa lab had previously shown that the organophosphorus (OP) insecticide diazinon and its active oxygen analog diazoxon induced oxidative stress, by measuring levels of reactive oxygen species (ROS) in rat cortical astrocytes. Both compounds were effective at concentrations ranging from 0.1 to 10 uM. The highlight of this year is that we now have found that exposure of astrocytes for 24 h to diazinon or diazoxon (both at 10 uM), followed by complete wash-out, and by co-culture of astrocytes with rat hippocampal neurons for an additional 48 h, significantly inhibits the ability of astrocytes to foster neuronal differentiation. Control astrocytes, when incubated with hippocampal neurons, promote neurite outgrowth. When astrocytes were pre-exposed to either diazinon or diazoxon, the length of the longest neurite (previously determined by tau staining to be the axon [Guizzetti, et al., 2008]) decreased by 50%, while length of minor neurites decreased by 40%. There were no changes in the number of neurites/cell. Incubation of astrocytes with a known oxidant, H2O2, which was used as a positive control, induced oxidative stress in astrocytes, and reduced neuritogenesis in hippocampal neurons to an extent similar to the OPs. Astrocyte-mediated neuritogenesis has been shown to be mediated by permissive neuritogenic factors, which are expressed and secreted by astrocytes. We had previously shown that two extracellular matrix proteins, fibronectin and laminin, play a major role in astrocyte-mediated neuritogenesis. In a preliminary experiment, we found that diazinon and diazoxon decrease fibronectin protein level in the astrocyte lysate, as does H2O2. In contrast, neither OPs nor H2O2 had any effect on laminin levels.
Journal Articles:
No journal articles submitted with this report: View all 55 publications for this subprojectSupplemental Keywords:
RFA, Scientific Discipline, Health, INTERNATIONAL COOPERATION, ENVIRONMENTAL MANAGEMENT, Biochemistry, Environmental Monitoring, Children's Health, Environmental Policy, Biology, Risk Assessment, pesticide exposure, age-related differences, pesticides, children's vulnerablity, molecular research, biological markers, agricultural communityRelevant Websites:
Center for Child Environmental Health Risks Research | Department of Environmental & Occupational Health Science | University of Washington School of Public Health ExitProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R834514 Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R834514C001 Community-Based Participatory Research
R834514C002 Pesticide Exposure Pathways
R834514C003 Molecular Mechanisms
R834514C004 Genetic Susceptibility
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
16 journal articles for this subproject
Main Center: R834514
510 publications for this center
178 journal articles for this center