Final Report: Molecular Mechanisms

EPA Grant Number: R834514C003
Subproject: 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: University of Washington Center for Child Environmental Health Risks Research (2010)
Center Director: Faustman, Elaine
Title: Molecular Mechanisms
Investigators: Faustman, Elaine , Carr, Catherine J , Costa, Lucio G , Fenske, Richard , Furlong, Clement , Griffith, William C. , Thompson, Engelberta , Vigoren, Eric M. , Yost, Michael
Institution: University of Washington
EPA Project Officer: Callan, Richard
Project Period: September 25, 2008 through September 24, 2016
RFA: Children's Environmental Health and Disease Prevention Research Centers (with NIEHS) (2009) RFA Text |  Recipients Lists
Research Category: Children's Health , Health

Objective:

The project had four initial aims:
  1. To investigate the direct effects of organophosphates (OPs) on neurite outgrowth, neuronal proliferation and viability in neurodevelopmental-stage specific in vitro models.
  2. To elucidate the impact of OP-induced oxidative stress and its effects on neuritogenesis and neurogenesis in neurodevelopmental-stage specific in vitro models.
  3. To examine developmental stage dependent impacts of chlorpyrifos (CP) on proliferation and differentiation gene expression pathways.
  4. To investigate whether OP exposure results in impairment of glial-neuronal interactions affecting the ability of astrocytes to promote neuritogenesis.

Work in Dr. Costa’s laboratory has focused on the effects of OPs on neuritogenesis and possible underlying mechanisms, thus encompassing Aims 1, 2, and 4. Work in Dr. Faustman’s lab encompasses Aims 1, 2, and 3.

Summary/Accomplishments (Outputs/Outcomes):

Aims of this research project are carried out in a collaborative manner by two laboratories, the Faustman Laboratory and the Costa Laboratory. Studies and results are reported herein for these joint efforts.
 
In the years preceding this latest funding cycle, Dr. Costa’s laboratory had been exploring novel mechanisms by which OP insecticides may adversely influence brain development. Two important findings were that OPs were able to inhibit proliferation of astrocytes, particularly in the presence of mitogens such as carbachol (an analog of acetylcholine) (Guizzetti, et al., 2005), and that the cytotoxicity of OPs on neurons was due to their ability to induce oxidative stress (Giordano, et al., 2007). For this latter study, 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 synthesis of glutathione. These neurons display very low levels of glutathione and are more susceptible to the toxicity of agents that increase oxidative stress.
 
During the same period, the Costa Laboratory studied the interactions of astrocytes and neurons in mediating chemical toxicity. Various in vitro systems were implemented to study such interactions, and a preliminary characterization of a number of parameters was carried out. Rat cortical or hippocampal astrocytes, when co-cultured with hippocampal neurons, increase their differentiation, and stimulation of astrocytes with carbachol greatly enhances their ability to induce neuritogenesis. This effect is mediated by an effect of carbachol on the expression and release of at least three neuritogenic factors: fibronectin, laminin, and PAI-1 (Guizzetti, et al., 2008). Ethanol can interfere with muscarinic receptor signaling in astrocytes and inhibit their ability to foster neuritogenesis in hippocampal neurons (Guizzetti, et al., 2010). More germane to this project is the finding that manganese, by accumulating and causing oxidative stress in astrocytes, inhibits their ability to induce neuritogenesis in hippocampal neurons (Giordano, et al., 2009).
 
These initial findings obtained with OPs or with other compounds served as the basis for some of the hypotheses and the specific aims indicated above.
 
The first series of studies sought to investigate whether the widely-used OP diazinon (DZ) and its oxygen metabolite diazoxon (DZO) would affect glial-neuronal interactions as a potential mechanism of developmental neurotoxicity. Specifically, the effects of DZ and DZO on the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons were investigated (Pizzurro, et al., 2014a). The results showed that both DZ and DZO adversely affect astrocyte function, resulting in inhibited neurite outgrowth in hippocampal neurons. This effect appears to be mediated by oxidative stress, as indicated by OP-induced increased reactive oxygen species production in astrocytes and prevention of neurite outgrowth inhibition by antioxidants. The concentrations of OPs were devoid of cytotoxicity and cause limited acetylcholinesterase inhibition in astrocytes (18 and 25% for DZ and DZO, respectively). Among astrocytic neuritogenic factors, a most important one is the extracellular matrix protein fibronectin. DZ and DZO decreased levels of fibronectin in astrocytes, and this effect also was attenuated by antioxidants. Underscoring the importance of fibronectin in this context, adding exogenous fibronectin to the co-culture system successfully prevented inhibition of neurite outgrowth caused by DZ and DZO. These results indicate that DZ and DZO increase oxidative stress in astrocytes, and this in turn modulates astrocytic fibronectin, leading to impaired neurite outgrowth in hippocampal neurons.
 
A second series of studies focused again on DZ and DZO and explored their ability to directly impair neurite outgrowth in rat primary hippocampal neurons as a mechanism of developmental neurotoxicity (Pizzurro, et al., 2014b). Both DZ and DZO (0.5–10 μM) significantly inhibited neurite outgrowth in hippocampal neurons at concentrations devoid of any cyototoxicity. These effects appeared to be mediated by oxidative stress, as they were prevented by antioxidants (melatonin, N-t-butyl-alpha-phenylnitrone and glutathione ethyl ester). Inhibition of neurite outgrowth was observed at concentrations below those required to inhibit the catalytic activity of acetylcholinesterase. The presence of astrocytes in the culture was able to provide protection against inhibition of neurite outgrowth by DZ and DZO. Astrocytes increased neuronal glutathione (GSH) in neurons, to levels comparable to those of GSH ethyl ester. Astrocytes depleted of GSH by L-buthionine-(S,R)-sulfoximine no longer conferred protection against DZ- and DZO-induced inhibition of neurite outgrowth. The findings indicate that DZ and DZO inhibit neurite outgrowth in hippocampal neurons by mechanisms involving oxidative stress, and that these effects can be modulated by astrocytes and astrocyte-derived GSH. Oxidative stress from other chemical exposures, as well as genetic abnormalities that result in deficiencies in GSH synthesis and regulation, may render individuals more susceptible to these developmental neurotoxic effects of OPs.
 
In conjunction with Dr. Costa, Dr. Faustman’s laboratory also has been examining developmental neurotoxic effects of OPs. In vitro models of neuronal differentiation are emerging as an important tool for high throughput and high content screening in neurodevelopmental toxicology. Understanding when, how, and at what doses neurotoxicants exposures affect normal development is critical for our ability to predict impacts of exposures before populationwide exposures occur. To further explore the importance of developmental context and timing in neurotoxicity, we exposed human neuronal progenitor cells (hNPCs) grown in proliferating and differentiating conditions to CP and arsenic (As), two well established neurotoxicants. The effects of CP or As treatment on hNPC morphology and cell viability were measured 24 hours and 72 hours post-treatment; at 72 hours post-treatment, changes in protein expression levels of neural differentiation and cell stress markers, cell viability and histone H3 modifications were observed (Figures 1 and 2). Cell viability, differentiation status, and epigenetic results suggest that hNPC cultures respond to CP and As treatment with different degrees of sensitivity, dependent on differentiation/proliferation status and on the toxicant concentration and length of exposure. Toxicant-related responses in sensitivity and protein expression patterns of neuronal markers that occurred 72 hours post-treatment were dependent on the cell growth conditions. Histone modifications, as measured by changes in histone H3 phosphorylation, acetylation, and methylation, varied for each toxicant and growth condition, suggesting that differentiation status can influence the epigenetic effects of CP and As exposures (Figure 2).
 
 
Figure 1. Changes in neuronal and stage-specific protein marker expressions following treatment of hNPC cultured under proliferation and differentiation conditions. Protein expression was quantified from western blotting following 72 hours treatment with CP or As under proliferating or differentiating conditions. Plots show means and 95 percent confidence intervals. * = p < 0.05; ** = p < 0.01
 
 
 
Figure 2. Changes of histone H3 acetylation and methylation following CP and As treatment of hNPC cultured under proliferation and differentiation conditions at 72 hours post-treatment. Histone modifications at specific sites were quantified by western blotting following 72 hours treatment with CP or As under proliferating or differentiating conditions. Plots show means and 95 percent confidence intervals. * = p < 0.05; ** = p < 0.01.

In addition, Dr. Faustman’s laboratory has characterized pathway dynamics throughout neuronal differentiation of the hNPC line that provides a particularly promising, scalable, and reproducible model for high-throughput and high-content neurodevelopmental toxicity screening. The laboratory cultured hNPCs up to 21 days in differentiation conditions and used Western blotting and immunofluorescence to measure changes in protein expression though time (Figures 3 and 4). Global gene expression dynamics were measured using Affymetrix Human gene 2.0 ST microarrays. Over time in differentiation conditions, hNPCs acquired morphological characteristics of mature neuronal networks and increased expression of neuronal differentiation markers, including beta tubulin III, MAP2 and synaptophysin. Significantly changed genes were organized according to temporal expression patterns using K-means clustering, revealing three phases of gene expression. Quantitative pathway analysis identified gene ontology (GO) terms enriched among genes expressed in each of these phases and created a quantitative summary or temporal pathway trends in vitro. GO terms enriched among genes significantly decreased over time are largely associated with proliferation and stem cell maintenance. GO terms enriched among genes with significantly increasing expression over time are dominated by key developmental processes, including neuronal differentiation, migration, and synaptogenesis. Enrichment of several GO terms associated with forebrain development indicates that these culture conditions promote differentiation towards a forebrain identity.

 
Figure 3. Morphological development of differentiating hNPCs.  Differentiating hNPCs were fixed and β-tubulin III expression was visualized with a fluorescent tag (green). Nuclei are countered stained with Hoechst 33342 (blue). Cells were visualized with a fluorescent microscope at 400x magnification. The increased expression of β-tubulin III expression and morphological development indicates a growing population of differentiating neurons.
 
 
 
Figure 4. Protein expression in differentiating hNPC cultures through time. Protein was harvested from differentiating hNPCs and expression of specific markers was evaluated by western blotting, with equal amounts of protein loaded in each sample. Data are normalized to Actin expression and presented as fold change expression intensity at each time point over expression average across time and reflects results of three independent experiments. Error bars indicate standard error. β-tubulin III, MAP2, α-synuclein, and nestin expression all increase significantly over time (one-way ANOVA p < 0.05).
 

We compared gene expression in vitro with publicly available gene expression data from developing human brain tissue in vivo and found substantial concordance in relative gene expression intensity (Figure 5). Genes highly expressed in both samples were enriched for key processes of brain development, including proliferation, migration, differentiation, synapse formation, and neurotransmission (Table 1). Conversely, GO terms enriched among genes highly expressed only in vivo or only in vitro reveal important differences between systems. For example, genes highly expressed in vitro are enriched for more stress and apoptosis pathways. This analysis provides a timeline of progression through differentiation, facilitating identification of key phases of sensitivity in vitro. Key processes important for the identification of Adverse Outcome Pathways (AOPs) of proliferation, differentiation, and functional maturation matched in vivo patterns (Table 1). Given the heightened sensitivity of the brain to toxicant perturbation during critical windows of development, it is important that we understand which sensitive developmental pathways are captured in vitro and which are not so that in vitro assays can be interpreted appropriately. These observations of morphology, protein, and gene expression provide a timeline of progression through differentiation, facilitating identification of key phases of sensitivity. By anchoring in vitro dynamics to in vivo reference points, this work clarifies the extent to which fundamental processes of brain development are captured in our model.

 
 
Figure 5. Methods for comparison of gene expression in actively differentiating tissues (in vivo period 2 vs. in vitro day 14)
 
 
Table 1. Summary of Pathways Related to Brain Development Enriched Among Genes Highly Expressed (>75th Percentile of Relative Expression Intensity) In Vivo Period 2 versus in Vitro Day 14
 

 
 
GO Terms Enriched Among Genes Above the 75th Percentile of Expression In Vivo (Period 2) and In Vitro (Day 14): Processes of Brain Development
Enriched Both In Vivo and In Vitro
Enriched In Vivo Only
Enriched In Vitro Only
Enriched GO Biological Processes
Z-Score
Enriched GO Biological Processes
Z-Score
Enriched GO Biological Processes
Z-Score
Stem Cell Maintenance
Glial cell proliferation
2.34
Neural precursor cell proliferation
2.29
None
 
Regulation of stem cell maintenance
2.24
 
 
 
 
 
 
 
 
 
 
Neurogenesis
Regulation of neurogenesis
4.81
Neurogenesis
2.95
None
 
 
 
 
 
 
 
 
 
 
 
 
 
Neural Migration
Cerebral cortex cell migration
3.17
Neuron migration
2.98
None
 
 
 
 
 
 
 
 
 
 
 
 
 
Neural Differentiation
Forebrain radial glial cell differentiation
4.14
Cerebral cortex neuron differentiation
4.23
None
 
 
 
Forebrain neuron differentiation
3.24
 
 
 
 
Glial cell differentiation
2.87
 
 
Neurite Outgrowth and Synapse Formation
Axon guidance
4.94
Axon guidance
6.06
Positive regulation of axonogenesis
2.19
Peripheral nervous system axon ensheathment
2.98
Neuron cell-cell adhesion
5.62
 
 
Myelin assembly
2.98
Neuron recognition
4.98
 
 
Neurotransmission
Vesicle-mediated transport
7.78
Positive regulation of excitatory postsynaptic membrane potential
4.48
None
 
Regulation of synaptic vesicle exocytosis
2.86
Neurotransmitter uptake
3.42
 
 
 
 
Regulation of respiratory gaseous exchange by neurological system process
2.72
 
 
Brain Structures and Functions
Corpus callosum development
3.48
Regulation of nervous system development
4.85
Neural tube development
2.30
Forebrain development
3.39
Brain development
4.25
 
 
Brain development
3.37
Dentate gyrus development
3.92
 
 
                 

 

Conclusions:

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.
 
By anchoring in vitro gene expression dynamics to in vivo dynamics, the analysis above begins to define the appropriate applications of Dr. Costa’s in vitro model of neuronal differentiation for developmental neurotoxicology. The neuronal differentiation model evaluated here captures several essential processes of early brain development in vivo, including neuronal differentiation and development, neuronal migration, synapse formation, and neurotransmission. This model also captures several generic developmental pathways important throughout development. For example, pathway analysis revealed activity in signal transduction pathways and general differentiation and morphogenesis processes that are ubiquitous in development. Detection of perturbation of generic developmental pathways in this model may be able to predict perturbation in a broader set of developmental contexts.
 
Taken together, Dr. Faustman’s results support the idea that dose, time, and biological context are important factors that need to be considered when developing in vitro models for toxicity testing, interpreting results, and comparing findings across endpoints and platforms.
 
Key Center Findings Related to the Molecular Mechanisms Project:
  • Both DZ and DZO adversely affect astrocyte function, resulting in inhibited neurite outgrowth in hippocampal neurons. Inhibited outgrowth is associated with neurodegenerative diseases.
  • DZ and DZO increase oxidative stress in astrocytes, and this in turn modulates astrocytic fibronectin, leading to impaired neurite outgrowth in hippocampal neurons.
  • DZ and DZO significantly inhibited neurite outgrowth in hippocampal neurons at concentrations devoid of cyototoxicity. These effects appeared to be mediated by oxidative stress, as they were prevented by antioxidants (melatonin, N-t-butyl-alpha-phenylnitrone, and glutathione ethyl ester).
  • Inhibition of neurite outgrowth was observed at concentrations below those required to inhibit the catalytic activity of acetylcholinesterase. DZ and DZO inhibit neurite outgrowth in hippocampal neurons by mechanisms involving oxidative stress, and these effects can be modulated by astrocytes and astrocyte-derived GSH. These responses are similar to those observed following OP exposure.
  • Differentiation status (or developmental context) modifies the epigenetic effects of CP and As exposure.
  • Characterization of neurodevelopmental toxicants using human neuroprogenitor cells provides a particularly promising, scalable, and reproducible model for high-throughput and high content neurodevelopmental toxicity screening.
  • We have identified common mechanisms of actions for OP pesticides that have informed our analysis of adverse outcome pathways, which link OPs to adverse neurodevelopmental outcomes.
Plans
 
During the no-cost extension, the Faustman and Costa laboratories continued with their investigations to support the aims of the Molecular Mechanisms project and publish drafted manuscripts.

 

References:

  1. Giordano G, Afsharinejad Z, Guizzetti M, Vitalone A, Kavanagh T, Costa L. Organophosphorus insecticides chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency. Toxicology and Applied Pharmacology 2007;219:181-189.
  2. Giordano G, Pizzuro D, VanDeMark K, Guizzetto M, Costa LG. Manganeze inhibits the ability of astrocytes to promote neuronal differentiation. Toxicology and Applied Pharmacology 2009;240:226-235.
  3. Guizzetti M, Pathak S, Giordano D, Costa L. Effect of organophosporus insecticides and their metabolites on astroglial cell proliferation. Toxicology 2005;215:182-190.
  4. Guizzetti M, Moore NH, Giordano G, Costa LG. Modulation of neuritogenesis by astrocyte muscarinic receptors. Journal of Biological Chemistry 2008;283:1884-1897.
  5. Guizzetti M, More NH, Giordano G, VanDeMark KL, Costa LG. Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors. Glia 2010;58:1395-1406.
  6. Pizzurro DM, Dao K, Costa LG. Diazinon and diazoxon impair the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons. Toxicology and Applied Pharmacology 2014a;274(3):372-382.
  7. Pizzurro DM, Dao K, Costa LG. Astrocytes protect against diazinon- and diazoxon-induced inhibition of neurite outgrowth by regulating neuronal glutathione. Toxicology 2014b;318:59-68.


Journal Articles on this Report : 16 Displayed | Download in RIS Format

Other subproject views: All 55 publications 38 publications in selected types All 16 journal articles
Other center views: All 507 publications 224 publications in selected types All 175 journal articles
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Journal Article Costa LG, Pellacani C, Dao K, Kavanagh TJ, Roque PJ. The brominated flame retardant BDE-47 causes oxidative stress and apoptotic cell death in vitro and in vivo in mice. NeuroToxicology 2015;48:68-76. R834514 (2015)
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  • Journal Article Harris S, Hermsen SA, Yu X, Hong SW, Faustman EM. Comparison of toxicogenomic responses to phthalate ester exposure in an organotypic testis co-culture model and responses observed in vivo. Reproductive Toxicology 2015;58:149-159. R834514 (Final)
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  • Journal Article Harris S, Wegner S, Hong SW, Faustman EM. Phthalate metabolism and kinetics in an in vitro model of testis development. Toxicology in Vitro 2016;32:123-131. R834514 (Final)
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  • Journal Article Harris S, Shubin SP, Wegner S, Van Ness K, Green F, Hong SW, Faustman EM. The presence of macrophages and inflammatory responses in an in vitro testicular co-culture model of male reproductive development enhance relevance to in vivo conditions. Toxicology In Vitro 2016;36:210-215. R834514 (Final)
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  • Journal Article Kim HY, Wegner SH, Van Ness KP, Park JJ, Pacheco SE, Workman T, Hong S, Griffith W, Faustman EM. Differential epigenetic effects of chlorpyrifos and arsenic in proliferating and differentiating human neural progenitor cells. Reproductive Toxicology 2016;65:212-223. R834514 (Final)
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  • Journal Article Moreira EG, Yu X., Robinson JF, Griffith W, Hong SW, Beyer RP, Bammler TK, Faustman EM. Toxicogenomic profiling in maternal and fetal rodent brains following gestational exposure to chlorpyrifos. Toxicology and Applied Pharmacology 2010;245(3):310-325. R834514 (2011)
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  • Journal Article Pizzurro DM, Dao K, Costa LG. Astrocytes protect against diazinon- and diazoxon-induced inhibition of neurite outgrowth by regulating neuronal glutathione. Toxicology 2014;318:59-68. R834514C003 (Final)
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  • Journal Article Pizzurro DM, Dao K, Costa LG. Diazinon and diazoxon impair the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons. Toxicology and Applied Pharmacology 2014;274(3):372-382. R834514C003 (Final)
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  • Journal Article Robinson JF, Guerrette Z, Yu X, Hong S, Faustman EM. A systems-based approach to investigate dose-and time-dependent methylmercury-induced gene expression response in C57BL/6 mouse embryos undergoing neurulation. Birth Defects Research. Part B: Developmental and Reproductive Toxicology 2010;89(3):188-200. R834514 (2011)
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  • Journal Article Robinson JF, Griffith WC, Yu X, Hong S, Kim E, Faustman EM. Methylmercury induced toxicogenomic response in C57 and SWV mouse embryos undergoing neural tube closure. Reproductive Toxicology 2010;30(2):284-291. R834514 (2011)
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  • Journal Article Robinson JF, Yu X, Moreira EG, Hong S, Faustman EM. Arsenic-and cadmium-induced toxicogenomic response in mouse embryos undergoing neurulation. Toxicology and Applied Pharmacology 2011;250(2):117-129. R834514 (2011)
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  • Journal Article Robinson JF, Theunissen PT, van Dartel DA, Pennings JL, Faustman EM, Piersma AH. Comparison of MeHg-induced toxicogenomic responses across in vivo and in vitro models used in developmental toxicology. Reproductive Toxicology 2011;32(2):180-188. R834514 (Final)
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  • Journal Article Wegner SH, Yu X, Pacheco Shubin S, Griffith WC, Faustman EM. Stage-specific signaling pathways during murine testis development and spermatogenesis: a pathway-based analysis to quantify developmental dynamics. Reproductive Toxicology 2015;51:31-39. R834514 (2015)
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  • Journal Article Wegner S, Yu X, Kim HY, Harris S, Griffith WC, Hong S, Faustman EM. Effect of dipentyl phthalate in 3-dimensional in vitro testis co-culture is attenuated by cyclooxygenase-2 inhibition. Journal of Toxicology and Environmental Health Sciences 2014;6(8):161-169. R834514 (2015)
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  • Journal Article Yu X, Robinson JF, Sidhu JS, Hong S, Faustman EM. A system-based comparison of gene expression reveals alterations in oxidative stress, disruption of ubiquitin-proteasome system and altered cell cycle regulation after exposure to cadmium and ethylmercury in mouse embryonic fibroblast. Toxicological Sciences 2010;114(2):356-377. R834514 (2011)
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  • Journal Article Zhou C, Chen J, Zhang X, Costa LG, Guizzetti M. Prenatal ethanol exposure up-regulates the cholesterol transporters ATP-binding cassette A1 and G1 and reduces cholesterol levels in the developing rat brain. Alcohol and Alcoholism 2014;49(6):626-634. R834514 (2015)
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  • Supplemental Keywords:

    RFA, Health, Scientific Discipline, 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 community

    Progress and Final Reports:

    Original Abstract
  • 2009
  • 2010
  • 2011 Progress Report
  • 2012
  • 2013 Progress Report
  • 2014
  • 2015 Progress Report

  • Main Center Abstract and Reports:

    R834514    University of Washington Center for Child Environmental Health Risks Research (2010)

    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