Oregon Health & Science University

Center for Research on Occupational & Environmental Toxicology (CROET)

NeuroToxicogenomics and Child Health
Peter Spencer, Ph.D.
spencer@ohsu.edu

Project Description

This is an application to become a Member of the NIEHS Toxicogenomics Research Consortium. The application originates from the synergistic fusion of two components of the Oregon Health Sciences University (OHSU), the resulting entity working in close cooperation with the Massachusetts General Hospital (MGH) at Harvard University. Leadership of this research program and the Toxicology Research Core Project (TRCP) is located in the OHSU Center for Research on Occupational and Environmental Toxicology (CROET), a dedicated research institute with strength in cellular, animal and human toxicology, with an emphasis on neurotoxicology. Co-leadership of this proposal is located in the nearby OHSU School of Medicine Department of Pediatrics which, together with participation from the Division of Medical Informatics and the Department of Medical and Molecular Genetics, brings strength in genomics, proteomics and bioinformatics, with emphasis on the developing brain and child health. Core Cl combines these components to support the toxicogenomics research needs of the TRCP and the Research Projects P1-P3. Project PI examines growth factor signaling in neural cells and its modulation by the neurotoxic anti-cancer drug taxol. Project P2 investigates early and late effects of genotoxic agents on neural development. Project P3, a subcontract with MGH, determines if neural and glial cells, as well as cells from different genetic strains, have overlapping or distinct patterns of toxicant-induced changes in gene expression that affect specific cellular functions, such as migration and division. The OHSU-MGH collaboration further strengthens this application by pooling our respective ongoing toxicogenomics studies and building an interdependent relationship for the benefit of the Consortium. Additionally, the participation in this proposal of scientific leaders of the NIEHS-funded Superfund Basic Research Center (SBRC) at OHSU (located in CROET) will ensure that the benefits of Toxicogenomics Research Consortium Membership are rapidly spread to other NTEHS-supported investigators at CROET and its collaborating SBRC institutions in the Pacific Northwest.

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Project 1: Shc Modulation of IGF Action in Neuronal Cells

Principal Investigator: Charles Roberts
robertsc@ohsu.edu

Normal neural development requires the careful orchestration of responses to an array of neurotrophic factors and other agents that exert a mixture of proliferative, differentiative and pro and anti-apoptotic signals. The accurate integration of these signals is crucial to the maturation of the nervous system, as well as its response to injury. A key player in the regulation of CNS development is the insulin-like growth factor (IGF) signaling system. The IGF ligands act through the IGF-I receptor to elicit proliferative, differentiative, and anti-apoptotic responses in many cell types, including neural-derived cells. The IGF-I receptor is a transmembrane tyrosine kinase that is linked to the MAP kinase and PI3 kinase cascades through scaffolding/adapter proteins of the Shc and IRS families. The chemotherapeutic agent taxol has recently been demonstrated to trigger the tyrosine and serine phosphorylation of specific Shc isoforms, modifications that would be expected to modulate their function in signal transduction. Based upon these findings, we hypothesize that differential expression of Shc isoforms modulates the differentiative and neuroprotective effects of IGF in neural cells, and that taxol modulates IGF signaling in neural-derived cells through effects on Shc phosphorylation. To evaluate these hypotheses, we propose the following specific aims: Specific aim #1. Characterize Shc expression patterns and IGF signaling and target genes in neural differentiation. Specific aim #2. Ascertain the requirement for Shc function in IGF action. Specific aim #3. Determine the effect of taxol on Shc-mediated IGF action. With the support of the Genomics (Cl) and Technical (C2) cores, we will employ molecular biological and gene profiling approaches to define IGF target genes that control differentiation and sensitivity to apoptosis, as well as the molecular mechanisms and gene expression patterns that may underlie the potential neurodevelopmental toxicity of taxol and other microtubule-targeted agents. The data obtained will be correlated with the results of Projects 2 and 3, which also address issues of neural development.

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Project 2: Role of Gene Expression in Brain Injury

Principal Investigator: Sri Nagalla
nagallas@ohsu.edu

The developing central nervous system is an especially vulnerable organ and susceptible to influence by endogenous or environmental factors that may produce acute or chronic brain injury. The objective of the present studies is to characterize gene expression profiles of the developing nervous system, using neuronal and glial cell cultures in two different species as model systems. These models will serve as the foundation to characterize the role of gene expression to assess the short-term and long-term effects of environmental agents.

Oxidative stress and DNA damage are two mechanisms that have been proposed to mediate short-term and long-term brain injury, respectively. We will model toxicant-induced oxidative stress using buthionine sulfoxamine (BSO) and toxicant-induced DNA damage using the environmental genotoxin methylazoxymethanol (MAM) that is strongly linked to a progressive neurodegenerative disorder.

We propose that the developing CNS is particularly vulnerable to environmental agents that produce either acute or a chronic progressive neurotoxicity through transient or persistent changes in gene expression. We will test this hypothesis by comparing the gene expression profiles of developing and mature murine and human neuronal and glial cells in the presence or absence of environmental agents that induce oxidative stress (e.g., BSO) or DNA damage (e.g., MAM). The findings from these studies will increase our understanding of the short- and long-term impact of environmental agents on the developing nervous system and will address whether the observed effects of environmental agents on the developing nervous system is species-dependent.

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Project 3: Gene Expression in Developmental Neurotoxicology

Principal Investigator: Jeffrey Miller
miller@helix.mgh.harvard.edu

Cellular stress can result from exposure to a range of environmental agents as well as pharmaceutical substances. Effects of these toxicants are determined largely by their hepatic interactions, including stimulation of various genes encoding detoxification and bioactivation enzymes, as well as activation of key signaling pathways that mediate cellular stress responses. We have developed a unique and highly defined primary hepatocyte culture model that faithfully displays a broad range of differentiation markers, and accurately reproduces hormone responsiveness, signaling patterns, as well as responses to chemical stressors and inducers that occur in the liver. The studies proposed in this project will test the principle hypothesis that responses to chemical stress can be accurately modeled and predicted through the use of well-defined primary hepatocyte culturing methodologies. Employing a selected battery of real-time, quantitative RT-PCR fluorescence assays as well as DNA microarray methodologies, this hypothesis will be initially tested using in vivo and in vitro rodent models, with exposure to either of two important environmental toxicants [Aroclor 1254 and Bis(2-ethylhexyl) Phthalate]. To extend cross species comparisons, primary human hepatocytes will be similarly characterized for gene expression signatures subsequent to chemical exposures. The power of DNA microarray technology will also be applied to distinguish chemically-altered, gene-specific profiles in hepatocytes enriched from either the pericentral or periportal regions of the liver acinus, providing a framework for understanding the basis for profoundly different response capacities of these cells. In collaboration with the Toxicology and Facility Cores associated with this proposal, a robust model will emerge detailing hepatic gene expression signatures across species, with the capacity to predict toxicological impact following chemical exposures.

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