Fred Hutchinson Cancer Research Center / University of Washington

The FHCRC/UW Toxicogenomics Consortium

David Eaton, Ph.D.
deaton@u.washington.edu

Project Description

A central tenet of toxicology is that with the possible exception of acute cell necrosis, every toxic exposure leads to an alteration in the pattern of gene expression. This altered pattern of gene expression reflects the cell's attempt to cope with the toxic insult, and can range from induction of xenobiotic metabolism to the extreme of cell suicide or apoptosis. While numerous studies have looked at changes in the expression of a limited number of genes thought to play a role in these adaptive responses, the power of array technology is the ability to obtain a comprehensive survey of thousands of genes simultaneously. This global analysis of gene expression affords researchers the ability to discern specific patterns or signatures of expression that are associated with particular classes of toxicants. These signatures are likely to include classes of genes not previously implicated in response to specific toxic insult. As such the applications of array technologies to toxicology will undoubtedly enhance our understanding of the cellular response to toxicants, which should by inference also provide insight into the mechanism of drug responses, toxicity, development effects and induction of disease. The unifying theme of the present proposal is that the comparison of gene expression profiles induced by stressors or toxicants in cells that are differentially sensitive to their effects will be particularly useful in dissecting the biochemical pathways underlying a toxic response.

We have assembled a team experts in the areas of biotransformations, neurodevelopmental toxicology, and carcinogenesis with the common goal of using DNA microarray technologies to compare gene expression profiles among mouse and rat, and human cells that are differentially sensitive to a variety of environmental agents. A series of four projects and a Toxicology Research Core Project, all of which make use of genetically defined rats or mice, transgeneic and knockout mice and primary human and rodent cell cultures are proposed. The projects will be supported by an administrative core and essential three facility cores. The DNA microarray facility core will provide project researchers access to the state of the art facility established at the Fred Hutchinson Cancer Research Center by Dr. Zarbl. The tissue acquisition core will project researchers access to the Transgeneic /Knockout Mice facility at the University of Washington, providing researchers access to genetically defined mice and their tissues. Recognizing the need to look at individual cell types comprising organs, we also included within this core access to cell enrichment technologies. The tissue acquisition core will provide researchers access to high speed cell sorting and laser capture microdissection capabilities. The final facilities core will provide Bioinformatic and Biostatistics support to the project researchers. These latter will be required for coordinating the acquisition, processing, storing and analyzing the large volumes of data that will be generated by the project researchers. Each of the cores will also interact with other consortium members and the central contractor through the Toxicicology Research Core Project to perform cross species and cross platform comparisons of the, and to develop standards for data standardization. The latter will be essential for the generation of a public database for data generated by all members of the consortium.

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Project 1: Toxicogenomic Pathway Analysis of Sensitive and Resistant Mammalian Models for Neurodevelopmental Toxicology

Principal Investigator: Elaine Faustman
austman@u.washington.edu

The long-term objective of this project is to utilize microarray analysis to facilitate the evaluation of mechanisms of mammalian susceptibility to neurodevelopmental toxicants. Adverse neurodevelopment is a common congenital defect, with prevalence rates ranging from as high as 1 per 1,000. These neurodevelopmental defects (NDDs) can encompass abnormal neural tube closure resulting in anencephaly, exencephaly and spina bifida as well as microcephaly and other Central Nervous System (CNS) abnormalities including adverse functional and behavioral changes. Various agents (such as heat, methyl mercury, cadmium, valproic acid, etc) have been identified as mammalian neurodevelopmental toxicants; however, even across and within various mammalian species, differential responses have been identified. For example, SWV mice are sensitive and C57BL/6J mice are resistant to hyperthermia-induced neurodevelopmental defects. Both human and animal studies suggest that the genetic component of neurodevelopmental abnormalities is complex and likely to involve multiple loci. The etiology of such effects remains poorly understood, however, recent developments in gene expression profiling using DNA microarrays, offers the promise to screen thousands of genes simultaneously and to begin the dissection of subsets of genes that are associated with susceptibility to particular disease states. Using this technology, we propose to use mouse strains differentially sensitive to teratogen induced NDDs (SWV- sensitive mice versus C57B1/6J resistant mice) to identify genes and their associated pathways that are modified by two well characterized neurodevelopmentally toxic agents, hyperthermia and methyl mercury (MeHg).

We will evaluate exposure and temporal characteristics of gene responses under conditions of sensitivity and resistance to NDDs. These initial studies will begin to test our ultimate hypothesis that neurodevelopmental toxicants share a common pattern of gene expression that is pathognomic for sensitivity to neurodevelopmental toxicity.

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Project 2: Effect of the Human Paraoxonase (PON1) Polymorphisms on the Consequences of Developmental Exposure to the Organophosphorus (OP) Insecticide Chlorpyrifos Oxon (CPO)

Principal Investigator: Clement Furlong
clem@u.washington.edu

The major aim of the proposed research is to examine the effect of the human paraoxonase (PON1) polymorphisms on the consequences of developmental exposure to the organophosphorus (OP) compound chlorpyrifos oxon (CPO). The HDL-associated PON1 enzyme plays a major role in detoxifying chlorpyrifos oxon (CPO) and diazoxon (DZO), the highly toxic oxon forms of the organophosphorus (OP) insecticides chlorpyrifos and diazinon. This enzyme is polymorphically distributed in human populations. A Gln/Arg polymorphism at position 192 of this protein determines catalytic efficiency for hydrolysis of specific substrates, while promoter polymorphisms determine the level of PON 1 expressed in a given individual. Animal model experiments have provided convincing evidence that high PON1 levels are protective against acute exposures to chlorpyrifos oxon and diazoxon while low levels of PON1 most likely render an individual highly sensitive to exposure to these compounds. It is also known that newborns have very low PON1 levels, which do not reach adult levels until about a year of age in humans and 20 days in rats and mice. Very little is known about the relationship of PON 1 status in modulating effects of OP exposure during development. The proposed research aims to explore this relationship.

The proposed studies compare patterns of gene expression in the mouse brain at different times of development and assess changes in these patterns elicited by developmental exposure to CPO. For this purpose, mice will be treated with CPO during postnatal days 4-21 and gene expression will be analyzed by gene microarrays at days p5, p21 and p90 to determine acute, short term and long term effects of CPO exposure on gene expression. To determine how PON1 status modulates the effects of developmental CPO exposure, PON1 knockout mice will be exposed to CPO and gene expression profiles will be analyzed by mouse gene microarrays. Analysis of arrays from untreated PON1 knockout mice and wild type mice will provide information on any changes in brain gene expression profiles due simply to knocking out the PON1 gene and will also serve as controls for the exposure experiments. Wild type and PON1 knockout mice will be exposed to increasing doses of CPO in order to determine a minimal effective or no observed effect level of GPO in eliciting changes in gene expression. Results from these studies will be compared with those from a parallel study funded from the NIEHS UW Children's Health Center, which is investigating the behavioral effects of CPO exposure on cognitive, motor and sensory functions in developing wild type and PON1 knockout mice. Based on the findings of these initial studies, gene expression patterns will be examined in subregions of the brain suggested by behavioral studies to be particularly affected by OP exposure to determine if some subregions are more susceptible than others. Examination of the brain subregions will be done with assistance from the Tissue Acquisition Core in collaboration with Dr. Kavanagh. The proposed studies will also investigate the role of the human PON1Q/R192 polymorphism on any observed developmental effects of CPO exposure on gene expression profiles. For this purpose, the study will make use of "humanized mice", where either the PONlQio2 allele or the PONlRjg2 allele replaces the mouse PON1 gene. Mice will be used that express levels of PON1 equivalent to the average levels observed in human population studies for each PON1,92 genotype (Q/Q homozygotes and R/R homozygotes). This "cross-species" comparison should provide some insights into the developmental consequences of OP exposure on global gene expression in the brain and generate testable hypotheses about the mechanisms responsible for differential sensitivity in humans.

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Project 3: Species and Zone-Specific Hepatic Gene Expression

Principal Investigator: Curtis Omiecinski
cjo10@psu.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 crossspecies 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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Project 4: Response of Mammary Cells From Sensitive and Resistant Rat Strains to Carcinogens

Principal Investigator: Helmut Zarbl (FHCRC)
Zarbl@eohsi.rutgers.edu

The overall goal of the proposed studies is to understand the mechanistic basis for differential sensitivities to the induction mammary carcinomas following exposure of pubescent female rats to environmental toxicants. By studying the differential sensitivities among rat strains, we will gain insight into mechanistic basis for differential sensitivities in humans and generate testable hypotheses about gene-environment interactions in human breast cancer. Genetic breeding experiments in rats suggested that resistance to mammary carcinogenesis is a polygenic trait. Our recent results suggest that resistance to mammary carcinogenesis is controlled by Quantitative Trait Loci (QTL) that affect both tumor multiplicity and latency. To complicate matters even further, the comparison of linkage data from different genetic crosses suggest that resistance to different chemical or physical carcinogens is mediated by different tumor suppressors and includes epigentic mechanisms. Together, these results suggest a very complex picture of genetic and epigenetic regulation of cellular responses following exposure to different mammary carcinogens. Thus, even after the cloning and characterization of all these tumor suppressors, understanding the mechanism by which they interact with each other and with different carcinogens will remain a major challenge. Here we propose the utilization of DNA microarray technologies to compare the patterns of gene expression induced in mammary cells in response to different toxicants in strains of rats that are sensitive or resistant to mammary carcinogenesis. Gene expression profiling of mammary cells from differentially sensitive strains as a function of the inducing agent, dose and time after exposure will greatly facilitate the dissection of the interactions among biochemical pathways that underlie the observed differences in gene-environment interactions.

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