Helmut Zarbl

Toxicogenomics: Understanding tissue, strain, species and intra-individual differences in response to environmental toxicants through gene expression analysis
The theme that unifies the components of the FHCRC/UW Toxicogenomics Consortium is the conviction that DNA microarray technologies will provide unprecedented insights into the molecular mechanisms by which of gene-environment interactions determine an individualÂ’s response to exposure to environmental risk factors. How these genes interact with environmental exposure to perturb biological systems studies will ultimately lead to: (1) better classification of toxicants, (2) more facile exposure monitoring based on biological response (biomarkers), (3) the reevaluation of animal models as predictors of human toxicity and/or the replacement of animal testing with mechanism based in vitro assays, (4) better understanding and prediction of individual variation in response to toxicants, and eventually, (5) the development of mechanism based preventative and therapeutic interventions. Intrinsic in this thematic underpinning of our Consortium, are two tenets around which we rally and unify our four diverse research projects. The first is that the intraspecies and interspecies variations in response to stressors and toxicants will be crucial to the interpretation of gene expression profiles induced by these exposures. Among all the genes that respond to an exposure, those that are differentially responsive between differentially sensitive individuals, strains and species will be those linked to the outcome. The second tenet that unifies our research program is the notion that toxicant signatures must first be derived from in vivo experiments or, particularly in the case of human studies, from in vitro experiments that closely mimic the in vivo situation (tissue slices or primary cell cultures). There are numerous reasons to believe that the response to toxicants will be affected by numerous factors that are not necessarily recapitulated by in vitro models such as established cell lines. For example, metabolism of toxicants in vivo may lead to the generation of unidentified metabolic products with increased toxicity. Likewise, the response of an organ to a toxicant that alters a developmental program may be affected by the context of cell-cell and /or cell-matrix interactions. Therefore, a central theme of our program is the need to validate response to toxicants in vitro by comparison to responses seen in vivo, or as close as possible to in vivo. Once toxicant signatures are validated and understood in an in vivo model, the toxicant signatures can be compared to the responses obtained in in vitro models. Nonetheless, we are well aware of the difficulties and limitation associated with performing experiments in these biologically complex settings. It may be necessary to isolate target cells from within organs of exposed animals or primary cultures. In order to obtain sufficient quantities it may be necessary to pool samples of isolated cells or alternatively, to develop methods that allow for representative amplification of mRNA for analysis on microarrays. There are issues pertaining to the selection of dose and the time points after exposure that will yield the most informative data. Standardized experimental protocols, internal standards, microarray platforms and methods for data extraction and analysis will need to be developed to allow for comparison of data among experiments. Standardized methodologies will need to be developed for data collection, normalization, annotation, transmission and storage in a public database. Our combined efforts to address these challenges, within the context of our consortium and within the context of the NIEHS Toxicogeneomics Research Consortium, unifies our proposed research efforts.
Environmental Carcinogenesis: Lung Cancer Research
A major goal of the research in our lab is to understand the molecular mechanisms and genetic regulation of carcinogen action. We have developed mutation assays that permit us to detect specific cancergene mutations in normal tissue samples at a frequency of ~10-6. We use these assays to determine if exposures to carcinogens increase the frequencies of specific cancer gene mutations in the target tissues. For example, we are measuring the frequency andspectrum of cancer gene mutations as a function of anatomical location in the human bronchioepithelial tree. This will allow us to ascertain if exposure to airborne mutagens increases the number of de novo mutations in genes associated with lung cancer.Acomparison of mutation frequencies in the K-ras, p53 and HPRT genes between the normal lung tissue of smokers and non-smokers indicates that the rate of mutation in smokers is only ~1.6 fold higher than in non-smokers. These results suggested that othermechanisms of carcinogenesis, such as promotion and epigenetic changes, also play a major role in smoking associated carcinogenesis.
In order to study cellular resonses to carcinogen exposures in the lung, we have developed a program to generate an integrated picture illustrating the effects of cell exposure to carcinogens by combining data from cell toxicity, mutation fraction, gene expression, genotype, and DNA adduct analyses. In collaboration with the Vouros lab in the Barnett Institute at Northeastern University, we are characterizing global gene expression patterns in human cells exposed to carcinogens using DNA microarrays, and phenotypically anchor the transcriptional responses with assays of cell survival, genotypes of xenobiotic metabolizing enzymes, induced mutation frequencies, and DNA adduct levels as measured by capillary LC-MS. Three well characterized carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-aminobiphenyl (ABP), benzo[a]pyrene (B[a]P), - all of which have been identified as constituents of cigarette smoke and have been implicated as causative agents in lung cancer, will serve as model compounds for this study. 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), an IARC category 2A carcinogen (probable human carcinogen) that is known to be derived primarily from cooked meats will be used to assess the sensitivity of our program to low level carcinogens that can be found in cigarette smoke, but which have not been directly implicated in lung cancer.
It is a widely held hypothesis that formation of DNA adducts due to carcinogen exposure is a potential first step that may lead to the development of tumors and onset of cancer. While there is strong evidence that the occurrence of high levels of DNA adducts is linked to a high rate of cancer incidence, a complete understanding of the chemical and biological effects caused by these toxic compounds is still lacking. This study aims to generate a complete roadmap by developing a correlation between carcinogen exposure, genotype, DNA adduct formation, cellular toxicity, mutation fraction, and variations in gene expression.

This program integrates the technologies of LC/MS for DNA adduct analyses, human cell mutation assays, and DNA microarrays. It is anticipated that the data from these studies will generate comprehensive hypotheses outlining the mechanisms that relate tumor growth with mutations initiated by exposure to smoking or other environmental toxic agents. The combination of expertise in trace level analysis of DNA adducts, microarray technology, cellular biology and toxicogenomics brings unique strength to this program and will very likely lead to a better understanding of the biochemical pathways that mediate cellular responses to DNA damage.

Genetic Susceptibilty to Mammary Cancer
Another application of these sensitive mutation assays tothe study rat mammary carcinogenesis has led to the identification of a novel epigenetic mechanism of carcinogen action. We demonstrated that the activating Hras1 oncogene mutations found in NMU-induced tumors arise as background mutations within cells ofthe developing gland, and that NMU enhanced the phenotypic penetrance of these mutations by initiating alterations in DNA conformation. Moreover we showed that this epigenetic reponse to NMU was present in strains of rats that are genetically predisposed to mammary cancers (e.g. F344), while strains of rats resistant to mammary carcinogenesis (Cop) did not shown this epigenetic respone. In order to identify the putative suppressor of manmmary carcinogenesis responsible for suppressing the response to chemical carcinogenesis, we are using a genetic breeding strategy. Phenotypic analysis of resistant (F344 X Cop)F1 and N2 backcross progeny indicated that the susceptibility is is a polygenic trait that is regulated by multiple quantitative trait loci. Genetic linkage analysis is being used to map novel suppressor(s) of mammary carcinogenesis in the rat in preparation for positional cloning and application to studies of human breast cancer. We are also comparing gene expression profiles before and after carcinogen exposure in mammary tissue from multiple rat strains with differential susceptibilities to mammary carcinogenesis. These studies are providing insights into the biochemical pathways that are regulated by the putative susceptibility genes.

Chemoprevention of Mammary Carcinogenesis.
We are also using the rat tumor model to study mechanisms of chemoprevention. Epidemiological studies have suggested that selenium defficieny is associated with elevated risk of some cancers, and that supplementation of the diet with organic selenium can reduce the risk of some cancers. Selenium-enriched garlic , which contains organic forms of seleium , suppresses tumor formation in the rat mammary tumor model when administered during the promotion/progression phase of mammary carcinogenesis (shortly before or after NMU treatment). Mechanisms through which this dietary chemopreventive agent inhibits mammary carcinogenesis remains to be fully elucidated. The overall goal of our study is to compare gene expression profiles in rat mammary cells following exposure to a carcinogenic dose of NMU, in the presence and absence of chemopreventive regimen of dietary organic selenium. The studies will provide insights into the biochemical processes that mediate selenium chemoprevention.

Development of a Novel Microarray platforms for Genomics and Functional Genomics
In collaboration with Engineering Arts, Inc, we are conducting research funded by the NIH SBIR program that follows two distinct yet complimentary paths. The first involves developing and testing novel piezoelectric pipetting, dispensing, sensing and housing technology and integrating this into a fully automated piezoelectric pipetting system. Innovative piezoelectric based technology enables pipetting of sub-nanoliter volumes of fluid and continuous monitoring to detect clogged tips and other operational states. The second involves using the piezoelectric pipetting system to do cancer related genomics research in the following two areas: 1) Use the proposed piezoelectric pipetting system to develop more robust oligonucleotide or cDNA based microarray platforms for gene expression analyses; 2) Use the proposed piezoelectric pipetting system to develop novel, high-throughput DNA microarray-based assays of cellular function, that will make it possible to assay phenotypic effects of silencing specific genes on a genomic scale. On one hand, the molecular-biology research provides a real-world application to help focus the design of the piezoelctric pipetting system and validate its performance. On the other hand the system provides the enabling technology in terms of reliability, speed, experiment design flexibility, spot reproducibility and density to make the cancer genomics and functional genomics related research feasible. The ultimate goal of the proposed research is to have the two paths come together resulting in a fully functional and tested general-purpose, automated, piezoelectric, fluid pipetting system with the reliability and performance to empower cancer related, genomic and functional genomics research.

DNA microarray technology development, Functional Genomics

1. Application of a novel high throughput method for screening for SNP in populations.
2. Phenotypic anchoring of expression profiles to DNA adducts, mutagenicity and toxicity

1. Development of a novel microarray platform for functional genomic studies in living cells.
2. Determining population frequencies and penetrance of SNPs
3. Gene therapy for cancer, viral infections; gene knockouts in somatic cells