Our Center currently (in 2004) has two other active research Centers associated with it; the Superfund Basic Research Program Project and The U.S. EPA Health Risks of PM Components Center. We will continue to build on our interdisciplinary collaborations and initiate other funding opportunities in the area of environmental health sciences. Dr. Costa is also the Deputy Director of an NCI-funded Cancer Center, which was just renewed for a five-year period (2003-2008). In that Center, Dr. Costa is the Program Director of the Environmental Carcinogenesis Program, which will also foster collaboration in this important area of environmental health. Many of the members of the Cancer Center are also members of the NIEHS Center. The Department of Environmental Medicine has also initiated an Industrial Associates Program where we will partner with industry in various research ventures. The financial support of prominent industry partners is anticipated to further enhance our interdisciplinary collaboration in the area of environmental health sciences. In recent years we have initiated a very active teaching program at New York University Washington Square campus. With funding from an NIEHS training grant, our graduate program is flourishing and new talent is being brought into the environmental health science field, along with a strong minority recruitment program to bring underrepresented minority students into the environmental health arena. Most, if not all, of these initiatives would not have been possible without the Center grant support from NIEHS and, as we compete for future funding of this core grant, we are grateful for the over 40 years of continuous support we have received from NIEHS that has made our Center highly interactive, scientifically strong, secure and responsive to the evolving needs of the community and the NIEHS.

Future initiatives of the Center build upon the talents of its members and upon its senior leadership, and will continue within the framework of our Center's broad goal of understanding and controlling the adverse impact of environmental factors on human health. The Center leadership continues to evolve to optimize its effectiveness and provide new directions within the Research Cores. In consultation with both the Internal and External Advisory Committees and all Center members, the Center has identified several initiatives for the next 5 years, most of which build upon what would be a natural evolution in our Center as a result of its current research activity.

Initiative 1: To expand the use of novel animal models to assess human toxicity and understand gene environment interactions.

We have obtained exciting results with regard to demonstrating co-carcinogenicity of two important metal carcinogens, arsenic (As) and chromate in a UV-exposed hairless mouse model for skin cancer. A collaboration of various Center members including Drs. Rossman, Burns and Bosland with Biostatistics collaboration (Dr. Nadas) has found that arsenic exposure at levels in the order of those that might be found in drinking water dramatically enhances the incidence of skin cancer following UV exposure. This finding is extremely important since there was evidence that arsenic is a human carcinogen by drinking water exposure, but this had never been demonstrated in any animal model. Dr. Costa, collaborating with the same group of investigators, has extended this model to demonstrate that drinking water exposure to the known human carcinogen chromate (Cr) at concentrations of 2.5 and 5.0 ppm also markedly enhances UV-induced mouse skin cancers. Again, while hexavalent Cr has been shown to be an inhalation carcinogen, there was substantial controversy as to whether it could cause cancers following drinking water exposure because the hexavalent form of chromium could be readily reduced in the stomach to the relatively nontoxic trivalent Cr. Exposure to chromate in the drinking water is a considerable public health concern, but there have been many speculations, especially from industrially funded sources, that drinking hexavalent Cr at levels as high as 10 ppm would be safe even though the drinking water standard is set at 100 ppb. Our animal study adds considerable doubt to the notion that 10 ppm Cr is safe in our drinking water. These studies are examples of how the Center will proceed with developing and using new models of environmentally induced human disease. In addition to these animal studies, Dr. Fred Burns has undertaken a new initiative designed to effectively extrapolate from animals to the human situation. This effort will make the animal models more useful to assessing the human consequences of exposure to toxic agents and will assist in establishing more rigorous methods for risk assessment. The Center has experienced substantial growth in the use of genetically modified mouse models (e.g., Apo/E knockout mice, estrogen receptor-beta knock-out mice, mice expressing human genes for beryllium susceptibility, and mice over expressing metallothionein). Furthermore, there has been a significantly increased focus by Dr. Gordon on the use of genetically defined inbred mice in research on genetic susceptibility to environmental toxicant and carcinogens (see Systemic Toxicology Research Core). These animal model approaches to the study of environmentally-induced human disease, such as environmental asthma, cardiovascular disease, and lung cancers represent a major growth area of the Center, as described in greater detail in the Systemic Toxicology Research Core and in the Molecular Toxicology and Carcinogenesis Research Core.

Drs. Wirgin and Zelikoff have a long history in the use of aquatic models to study the toxicity of chemicals. For example, Dr. Wirgin is using tomcods as sentinels to study the chronic effects of aromatic hydrocarbon contaminants (polychlorinated biphenyls (PCBs) and polychlorinated dibenzodioxins dioxins (PCDDs) on gene expression and early life toxicities, while Dr. Zelikoff studies immunotoxicity in other fish models. Continued and expanded efforts in this area will contribute to the fulfillment of our research initiative on animal models to understand human toxicity.

Initiative 2: To incorporate the use of the array technology to studying mechanisms of respiratory toxicity, cardiovascular disease, chemical carcinogenesis, and molecular epidemiology.

Gene expression array technology offers opportunities to examine changes in gene expression of virtually every gene in the human or murine genomes. These technologies allow a researcher to examine exposed or unexposed animal tissues, human tissues or cultured cells, identify those genes that are increased or decreased in their expression by exposure to chemicals or other environmental agents and determine patterns of coordinated regulation of related genes. Affymetrix GeneChip technology allows for the global screening of expression levels of thousands of genes simultaneously. As a result, GeneChips allow for the identification of clusters of genes whose expression levels are coordinately regulated and the identification of novel genes whose expression levels are altered by toxicant exposure. GeneChips consist of small amounts of thousands of genes [cDNAs, oligonucleotides, expressed sequence tags (ESTs)] printed onto microscope slides in a precise and known pattern. By hybridizing these slides to labeled mRNAs prepared from treated and control organisms, differences in levels of expression of each gene imprinted on the slide can be compared between treatment groups. Microarrays of various types are commercially available for the most common species, including human and mouse, and global gene expression changes can be studied in response to toxicant/stressor exposures. Global gene expression changes are being studied by NYU Center investigators in various in vitro and in vivo models.

Complementary use of custom microarrays will allow for more intensive analysis of these genes in dose or time response studies. The addition of newly developed custom microarray capability to our Center allows investigators to develop and analyze second-generation microarrays that are designed to study alterations in gene expression at target loci previously identified by analysis of global gene expression on Affymetrix GeneChips and/or genes in metabolic/repair/signal transduction pathways known from other studies to be involved in specific toxicities. Use of the custom array instrumentation will allow Center investigators to focus their attention on only those genes that are of demonstrated importance in specific phenotypes. Because of greatly reduced costs compared to Affymetrix GeneChips, the use of custom microarrays will allow investigators to conduct traditional toxicological analyses, such as evaluations of dose-and time-response of specific genes to toxicant exposures. This more intensive analysis will allow for investigations of interactions among responsive genes within individual or among functionally related molecular pathways. It will better enable us to analyze the interactive effects of exposures to mixtures of chemicals, metals and organic pollutants. Also, it will permit dissection of the impacts of toxicants on the disruption of coordinated genetic regulation of developmental pathways such as those induced by metals, dioxins, PCBs, and PAHs, allowing for greater emphasis on the temporal aspects of altered gene expression on phenotypic anchoring. The Molecular and Cell Biology Facility Core will closely coordinate its hands-on microarray activities with the microarray data analysis functions of the Environmental Health Statistics and Bioinformatics Facility Core to ensure that only the highest quality data is passed onto the statistical analysis platform. Thus, custom microarrays can be tailored for each specific research project. Research on alternative animal models utilizing fish species also benefit from the acquisition and development of this technology. Drs. Wirgin and Zelikoff, of the Molecular Toxicology and Carcinogenesis and the Systemic Toxicology Research Cores respectively, are using this approach to understand the mechanistic bases of PCB-induced cardiovascular and immunological impairment in fish models. Also, Dr. Gunnison of the Systemic Toxicology Research Core, in collaboration with the Environmental Epidemiology Research Core, has developed and used custom human microarrays as a tool to screen human populations for genetic polymorphisms in a suite of putative asthma related genes. In collaboration with Dr. Chen, he has also identified changes in genes that control circadiac rhythm following subchronic inhalation exposures to concentrated ambient air particulate matter

Genes that are altered in expression (increased or decreased) in response to an environmental exposure probably have a fundamental relationship to the mechanism of action of the agent tested. Once these genes are identified, further research can focus on the most interesting or relevant gene expression changes using simpler molecular biology techniques (northern blots or RT-PCR) to study the mechanisms by which these chemicals up-regulate or down-regulate specific genes. This approach has been extremely effective in our Center in uncovering new pathways by which toxic agents are acting. For example, using the Affymetrix gene chip, we have been able to determine that exposure of cells to soluble salts of nickel in many cases activates hypoxia-dependent genes, which are regulated by the transcription factor HIF1-alpha. We have recently identified a potential mechanism for this effect in which Ni substitutes for iron (Fe), especially in histidine-chelated Fe enzymes such as proline hydroxylase, that hydroxylates HIF-1 alpha and targets it for degradation. In the presence of soluble Ni, cellular Fe uptake is inhibited and Fe is also displaced from the HIF-proline hydroxylase, resulting in the inhibition of this enzyme and an overall accumulation of HIF protein with subsequent activation of transcription of HIF-dependent genes. This mechanism may also be involved in the way in which Ni transforms normal cells into cancer cells.

To collaborate with us in the design and analysis of these experiments, particularly with regard to custom microarrays, we have added additional statistics and bioinformatics expertise to our new Environmental Health Statistics and Bioinformatics Facility Core. The GeneChip and custom microarray technology is heavily utilized in the Molecular Toxicology and Carcinogenesis Research Core and in the Systemic Toxicology Research Core.

Initiative 3: To develop methods for understanding gene environment interactions by identifying individuals who are exposed to environmental contamination, including mixtures, combined with methodology to understand those that are most genetically susceptible to injury.

It has become increasingly evident that large inter-individual variations in susceptibility to environmental toxicants result from polymorphisms in a variety of genes that metabolize toxicants, repair their damage, or transduce intracellular signals. These differences in susceptibilities to diseases exist between populations and among individuals within populations. Although many of the genes that underlie susceptibility differences have been characterized, many more remain to be identified. The prevalence of alternative alleles needs to be elucidated in human populations, and the association between allelic state and sensitivities to disease needs to be determined. These areas of new research emphasis in the Center are reflected in equipment updates and services provided by the Center. Toward that goal, capillary-based DNA sequencing and PyrosequencingTM technology will enable us to study mutations and DNA methylation patterns in specific human genes. It is very clear that both exposure and genetic predisposition are important factors in determining the prevalence of toxicant-induced disease in human and animal populations. As a result, research and training in genetic susceptibility to toxicant-induced disease has increased within this Center. This is reflected in the themes of our Research Cores and in individual research projects (Drs. Shore, Zeleniuch-Jacquotte, Chen, Wirgin, Gordon, Qu, Rom, Arslan) and in our new course offerings (e.g., Genetic Susceptibility and Toxicogenomics). However, it is becoming increasingly clear, that genes with low penetrance, but high frequencies in populations probably are the most important targets in developing diagnostic, preventive and therapeutic strategies. The identification of susceptibility genes requires the screening in genotyping studies of large cohorts to robustly make the association between genotype and disease outcome. Also, it is becoming increasingly evident that single genetic polymorphisms at single genetic loci only tell a fraction of the story. One of the major goals in post human genome research is to identify and characterize multiple common genetic variants, called haplotypes that may predispose individuals to common, complex diseases. Finally, disease associations and their prevalence in relationship to an environmental exposure will be better understood by common single nucleotide polymorphisms in susceptible genes termed SNPs. Thus, the technology offered by the Center would permit exploration of the association between disease susceptibility and multiple intra-genic and inter-genic genetic polymorphisms. Additional expertise has been added to the Environmental Health Statistics and Bioinformatics Facility Core in statistical methods to assess gene-environment interactions and for the evaluation of the association of SNPs with disease states. The instruments and robotic approaches provided in our Molecular and Cell Biology Facility Core will allow us to analyze the very large number of samples needed to rigorously evaluate the statistical associations between genetic factors and vulnerability to diseases.

In addition to Genetic Susceptibility, research to foster the goals of this initiative involve all four Research Cores, and include: 1) studies on the development and validation of new biomarkers of exposure, effect, and susceptibility, which will aid in assessing the health risks associated with exposure to hazardous substances; 2) studies to validate the efficacy of biomarkers in providing a link between exposure and disease outcome; 3) studies to identify, evaluate, or validate factors in an individual's environment (e.g., nutritional status, home or workplace exposure) or physiological makeup that may lead to an increased likelihood of disease or dysfunction relative to the general population; 4) studies to develop and demonstrate innovative monitoring technologies capable of measuring an individual's incidental or long-term exposure to contaminants at low concentrations; 5) studies to determine how continuous and/or multiple exposures impact health outcomes; and 6) studies to generate biological data and methodologies to develop and validate risk models for translating in vivo and in vitro research findings to affect humans. However, probably the most important aspect of this initiative involves understanding diversity in human genetic susceptibility.