James A. Swenberg, DVM - US grants

The theme of this program project is to develop ultrasensitive biomarkers of exposure and genetic effects to carcinogens, as an index of individual exposure, and to investigate the hydrogeological and ecological factors which contribute to variability in human population exposure. The program consists of six integrated research projects, research cores on chemistry and epidemiology, and an administrative/training core. Vinyl chloride (VC), pentachlorophenol (PCP) and polynuclear aromatic hydrocarbons (PAH) have been selected for investigation due to their widespread potential for human exposure. Three biomedical projects will concentrate on developing biomarkers of exposure and effect in order to examine their utility in future epidemiology studies. Project 1 will develop ultrasensitive methods for quantifying the DNA adducts of VC and PCP and conduct molecular dosimetry studies. Project 2 will determine the mutational spectra of VC and PCP in human cells and animals as an early biomarker of genetic effect. Project 3 will utilize new techniques for identifying and quantifying hemoglobin and albumin adducts of VC and PCP. Three hydrogeologic projects will examine the fate and transport of chemicals to identify critical factors that determine the extent of human exposure. Project 4 will examine microbial transformation of chemicals in the subsurface environment that can affect both toxicity and mobility. Project 5 will investigate multicomponent, multiphase flow and transport processes in subsurface systems. These data will be useful in developing stochastic modeling in Project 6, to better predict the spatial variability of contaminant transport. These models will be of immense value, both in selecting populations for investigation of exposure-response relationships and, ultimately, in predicting human exposure based on the hydrogeologic factors identified. The biomarker and engineering components of this program project are designed so that their data and methods will become interactive during the second half of the project. The biomarkers will be evaluated over a large exposure range to define their limits of detection. The engineering components will identify likely environmental exposure scenarios. By frequent interactions between these two groups, we will develop much greater insight into the feasibility of future epidemiology studies and requirements for additional methods development and modeling. This program project will also provide strong interdisciplinary training for 19 pre- and post-doctoral fellows in environmental sciences by combining coursework, seminars and research in environmental health sciences, hydrogeological engineering, and microbial ecology.

DESCRIPTION (provided by applicant) This training grant application requests support for 8 pre-doctoral and 4 postdoctoral trainees in the Curriculum in Toxicology program at the University of North Carolina-Chapel Hill. The proposed training program brings together a highly interactive and productive faculty of basic scientists, physician scientists and public health researchers from the Schools of Medicine, Public Health, and Pharmacy at UNC, plus outstanding researchers and mentors from the U.S. EPA and NIEHS in Research Triangle Park. The training program faculty includes 44 basic researchers and 11 DVM/MDs with proven research records in environmental health. The research interests of the faculty can be broadly divided into a) Mechanisms, b.) Systems Biology, Biomarkers, Metabolism, c) Organ-Specific Toxicology, and d) Carcinogenesis. This unique blend of environmental health researchers has worked together for the past 30 years to produce 134 Ph.D. graduates and 73 postdoctoral fellows who have gone on to productive careers in academia, government, and industry. The investigators' research training focuses on providing pre-doctoral and postdoctoral trainees the environment, infrastructure, and resources to conduct interdisciplinary cross-cutting research in environmental toxicology, systems toxicology/biomarkers, research translation, and animal models of human diseases. In order to maintain this successful training program, the investigators continuously update their training approaches, incorporating feedback from their External Advisory Committee and their own internal evaluation. During the previous funding period, the investigators implemented measures to enhance program cohesiveness, increased co-mentoring, and focused on interdisciplinary and translational research with critical clinical and public health impact. In the current application, they will add further improvements including a mentor led ethics seminar and opportunities to acquire professional skills. The Curriculum in Toxicology at UNC-CH has outstanding didactic instruction, excellent oversight, unparalleled resources, and a superb environment to support the proposed training. As outlined in this application, the investigators' previous record demonstrates that the outstanding new scientists they train will excel at interdisciplinary approaches that result in the mechanistic understanding and translation of how the environment influences human disease.

The theme of this program project is to develop ultrasensitive biomarkers of exposure and genetic effects to carcinogens, as an index of individual exposure, and to investigate the hydrogeological and ecological factors which contribute to variability in human population exposure. The program consists of six integrated research projects, research cores on chemistry and epidemiology, and an administrative/training core. Vinyl chloride (VC), pentachlorophenol (PCP) and polynuclear aromatic hydrocarbons (PAH) have been selected for investigation due to their widespread potential for human exposure. Three biomedical projects will concentrate on developing biomarkers of exposure and effect in order to examine their utility in future epidemiology studies. Project 1 will develop ultrasensitive methods for quantifying the DNA adducts of VC and PCP and conduct molecular dosimetry studies. Project 2 will determine the mutational spectra of VC and PCP in human cells and animals as an early biomarker of genetic effect. Project 3 will utilize new techniques for identifying and quantifying hemoglobin and albumin adducts of VC and PCP. Three hydrogeologic projects will examine the fate and transport of chemicals to identify critical factors that determine the extent of human exposure. Project 4 will examine microbial transformation of chemicals in the subsurface environment that can affect both toxicity and mobility. Project 5 will investigate multicomponent, multiphase flow and transport processes in subsurface systems. These data will be useful in developing stochastic modeling in Project 6, to better predict the spatial variability of contaminant transport. These models will be of immense value, both in selecting populations for investigation of exposure-response relationships and, ultimately, in predicting human exposure based on the hydrogeologic factors identified. The biomarker and engineering components of this program project are designed so that their data and methods will become interactive during the second half of the project. The biomarkers will be evaluated over a large exposure range to define their limits of detection. The engineering components will identify likely environmental exposure scenarios. By frequent interactions between these two groups, we will develop much greater insight into the feasibility of future epidemiology studies and requirements for additional methods development and modeling. This program project will also provide strong interdisciplinary training for 19 pre- and post-doctoral fellows in environmental sciences by combining coursework, seminars and research in environmental health sciences, hydrogeological engineering, and microbial ecology.

Advances in analytical methods have made it possible to quantitate DNA adducts in a variety of tissues and cells of experimental animals and humans and to begin to relate these biomarkers to extent of exposure and potential risk for cancer. These DNA adducts arise from exogenous exposure to chemicals as well as from endogenous processes. Modulation of the repair pathways that exist for both exogenous and endogenous DNA adducts by environmental exposure to xenobiotics could play an important role in the induction of neoplasia in experimental animals exposed to Maximum Tolerated Doses, and in individuals exposed to hazardous wastes. The long range goal of this project is to examine the formation and repair of both endogenous and exogenous DNA adducts associated with environmental chemical exposures in order to identify mechanisms that are of critical importance for the accurate prediction of human risk for cancer. We will determine if exposure to environmental carcinogens (1,1,2-trichloroethylene, 1,1,2,2- tetrachloroethylene, 1,1,2-trichloroethane, chloroform, and carbon tetrachloride) induces or modulates the formation and/or repair of cyclic DNA adducts. These studies will quantitate adducts that are formed by direct alkylation of DNA by the test chemical, as well as similar adducts that arise from oxidative stress such as lipid peroxidation. We will examine dose-response relationships for adduct formation and repair to determine if important nonlinearities exist that could affect cancer risk assessments. We will continue to develop ultrasensitive and highly specific methods for quantitating DNA adducts. In collaboration with the Chemistry Core, we will further develop applications of mass spectrometry to DNA adducts. Differences in DNA repair could greatly affect age, tissue and cell type susceptibilities to mutagenesis and carcinogenesis. We will measure the expression of N-methylpurine-DNA-glycosylase (MPG) mRNA in a variety of tissues and cells from rodents and humans using quantitative RT/PCR. The effect of exposure to environmental carcinogens on MPG activity will be examined and correlated with DNA adducts. Since MPG excises adducted bases, the utility of monitoring urinary excretion of etheno adducts as an index of exposure will be investigated. We will determine the molecular dose of the dinitrotoluene DNA adducts and the extent of cell proliferation, and relate these effects to hepatocyte initiation. This effort will provide critical information on dose- response, ranging from the bioassay doses and extending down two orders of magnitude. Data from this research project will greatly improve the scientific bases needed to improve the accuracy of biologically-based low dose risk estimates for these important Superfund chemicals.

Peroxisome proliferators are a group of structurally diverse compounds that cause an increase in both the number and size of peroxisomes, elevate rates of cell proliferation and cause liver cancer in rodents. Although the mechanism of carcinogenesis is not known, two main hypotheses have been proposed. One views oxidative stress as a critical event in the carcinogenic process, while the other contends that elevated and sustained cell replication is responsible for the induction of tumors. The latter is considered to be the main mechanism of promotion, although, the role of oxidative stress in initiation is controversial. However, these two hypotheses are not mutually exclusive. In exciting new experiments, we showed that at least one of the base excision repair enzymes is upregulated by chronic administration of WY-14,643, a model peroxisome proliferator. This led us to the hypothesis that peroxisome proliferators induce both increased formation of oxidative DNA adducts and repair of these lesions. Furthermore, we hypothesize that oxidative stress is involved in signaling pathways for cell proliferation leading to higher probabilities of mutation, promotion and progression. We will utilize frozen tissues from the NTP study to investigate following questions: 1) Do peroxisome proliferators induce the formation of oxidative and/or etheno DNA adducts in rodent liver? 2) Does DNA repair play a role in peroxisome proliferator- induced carcinogenesis? 3) Are increases in free radicals following chronic exposure to peroxisome proliferators consistent with oxidants as a signaling mechanism for cell proliferation? We predict that the poor correlation between hepatic oxidative DNA lesions and carcinogenic potency of peroxisome proliferators is due to the limited number of DNA adducts that has been studied and the induction of DNA repair. State-of-the-art equipment and techniques developed in this laboratory will allow us to look at a broad spectra of DNA adducts, the activity of multiple DNA repair enzymes, markers of oxidative stress, and a signal transduction pathway for cell proliferation. We expect to find a good correlation between hepatocarcinogenic potency of peroxisome proliferators and their ability to produce oxidants using a responsive species (rat) and a nonresponsive species (hamster). The unique design of the NTP study will allow us to examine our hypotheses using tissues from WY-14,643- exposed rates and hamsters by comparing different doses and time points. The strengths of this novel application lie in the evaluation of specific hypotheses designed to fill critical gaps in our knowledge regarding the mechanism(s) of action of this important but poorly understood class of compounds. This research also will have important implications for mechanistically based risk assessment.

In exciting new studies, we showed that phthalic acid esters and lipid lowering drugs (e.g., diethylhexylphthalate (DEHP) and WY-14,643) activate phagocytosis by the resident hepatic macrophages, Kupffer cells. Further, increased cell proliferation was blocked in vivo by both an antibody to TNFalpha and methyl palmitate, an inactivator of Kupffer cells. Based on this new information, we hypothesize that phthalates activate Kupffer cells to produce mitogenic cytokines that stimulate cell proliferation and cause tumors. To test this hypothesis, three questions will be posed. First, are Kupffer cells responsible for increased cell proliferation due to plasticizers? Kupffer cells will be treated with DEHP and the lipid lowering drug WY-14,643 in vitro or isolated from rats treated in vivo and cultured. Conditioned media from Kupffer cells will be added to cultured hepatocytes and cell proliferation will be assessed. Similar experiments will be performed with human macrophages and hepatocytes since whether or not these chemicals cause cancer in humans remains controversial. Nimodipine, methyl palmitate and dietary glycine will be used to prevent or minimize activation of Kupffer cells. We expect these experiments to demonstrate conclusively that TNFalpha produced by Kupffer cells is responsible for enhanced cell proliferation caused by plasticizers. Second, experiments will be performed to understand how phthalates activate Kupffer cells to produce mitogens. We propose that phthalates will enter Kupffer cells membranes based on their lipophilicity, increase intracellular calcium, and activate PKC by altering second messenger systems. Accordingly, uptake of radiolabelled drugs of different lipid solubility will be compared. Further, the effect of phthalate treatment in vivo on intracellular calcium and PKC will be measured in isolated Kupffer cells. To determine if PKC activates NADPH oxidase and increases NF/kappa/B-mediated TNFalpha production, superoxide and NF/kappa/B will be monitored and correlated with changes in TNFalpha mRNA. Third, can phthalate-stimulated increases in cell proliferation and tumors be prevented in vivo by modulation of Kupffer cells? Dietary glycine, which inhibits Kupffer cell activation, will be used to prevent cell proliferation, changes in apoptosis, and the formation of preneoplastic foci and tumors, which is the "gold-standard." By pursuing these new hypotheses, we will fill important gaps in our knowledge regarding mechanisms. Another important impact of this work will be to provide governmental regulators with mechanistic information which will allow them to shift emphasis away from the fact that these chemicals induce peroxisomes and focus on cell proliferation.

Disinfection of drinking water generates halogenated by-products, whose potential biological effects have important public health implications. Several disinfection by-products are carcinogenic when administered to experimental animals. The extrapolation of such findings to human health, however, is unclear, since these tests generally involved exposures that were several orders of magnitude greater than likely human exposures. In addition, the mechanisms underlying the carcinogenic effects are poorly understood. Carcinogenesis may involve covalent modification of DNA, either by direct chemical reaction or by modulation of endogenous processes to produce reactive electrophiles. We will test this hypothesis by the following specific aims: Specific Aim 1. Using a battery of well-characterized and ultrasensitive endpoints, we will investigate whether disinfection by-products enhance endogenous DNA damage (i.e., DNA modification derived from reactive oxygen species). Specific Aim 2. The formation of direct DNA adducts that derive from the disinfection by-product, and 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) will be compared with those derived from reactive oxygen species. Specific Aim 3. Dose-response relationships of DNA modification will be determined, and those findings will be related to other mechanistic data, such as cell proliferation and to the outcomes of 2-year bioassays (tumor incidence). We propose to investigate three disinfection by-products, bromodichloromethane, dibromoacetic acid, and MX in Specific Aims 1 and 3, and MX in Specific Aim 2. A strength of this proposal will be the ability to relate DNA adducts to other mechanistic data and to tumors that occur under the same exposure conditions. Analysis of different types of DNA damage will result in a better understanding of the mechanisms underlying the toxicity and carcinogenicity of disinfection by-products. In addition, DNA adducts will provide a mechanistic basis for conducting extrapolations across doses and between species, which will improve the prediction of human health risks from animal studies.

DESCRIPTION (provided by applicant) The primary objective of the NIEHS Superfund Basic Research Program (SBRP) is to understand the human health and environmental risks associated with hazardous waste sites, with the goal of more accurately assessing risks and more efficiently remediating hazardous waste sites. Since its inception, the UNC SBRP has focused its multidisciplinary research on addressing scientific issues that underpin the assessment of human risk and the development of improved methods for remediation of hazardous waste sites, in the proposed research, we will further advance this goal through the following objectives: 1) apply new molecular and analytical tools in a systems biology framework to understand critical pathways for environmental disease and bioremediation;2) develop biomarkers of exposure and effect for humans and experimental models of environmental disease;3) use this knowledge to elucidate the modes of action for major chlorinated hydrocarbons and polycyclic aromatic hydrocarbons;4) examine the effects of dose-response on such modes of action;5) evaluate and understand genetic susceptibility to hazardous chemicals;6) determine the factors controlling dermal absorption of polycyclic aromatic hydrocarbons and the relationship of internal dose to both skin and systemic exposure;and 7) develop and evaluate remediation methods in complex, field-relevant systems for their potential to reduce overall human exposures and risks. These objectives will be accomplished through six diverse, but highly interdigitated research projects, two research support cores, a research translation core, a training core and an administrative core.

Carcinogenicity is the major human health risk associated with the regulation of chemicals on the National Priority List. In the past, clean up standards for most of the Superfund sites used linear risk assessment based on animal cancer bioassay data. The 1996 Revised Cancer Risk Assessment Guidelines contain many important changes in how future cancer risk assessments will be performed. Paramount to these changes is the incorporation of more science regarding the mode of action and dose response of the chemical in question. The proposed research focuses on developing a sound scientific understanding of the mode of action and the observed and expected dose response relationship of several of the major hazardous chemicals on the National Priority List. These data will be suitable for use with the revised guidelines and will improve the accuracy of the risk assessments driving site remediation. We hypothesize that several aliphatic and aromatic chlorinated hydrocarbons share an important mode of action referred to as "oxidative stress." Through this mechanism, Reactive Oxygen Species (ROS) cause damage to DNA and activated signal transduction pathways involved in gene regulation and cell proliferation. Little is known about the dose response relationship of oxidative stress induced by these chemicals. The proposed research will identify the most useful biomarkers for oxidative stress and use these to examine the extent and type of oxidative stress on tissues from rodents to determine if linear or non-linear models best fit the data. In addition, these markers of this research are: (1) To develop and validate a comprehensive battery of biomarkers of oxidative stress; (2) To evaluate the role of oxidative stress in the toxicity and carcinogenicity of chlorinated aliphatic hydrocarbons; (3) to evaluate the role of ROS in damage to DNA and plasma proteins of individuals with known plasma PCB characterization; and (5) to collaborate with Projects 2 and 3 in the measurement of oxidative stress and other DNA adducts.

The Chemistry and Analytical Core will provide support for the individual research projects. The Core consists of two components: the Synthesis Laboratory and the Mass Spectrometry Facility. The Synthesis Laboratory will be responsible for providing, on a continuing basis, specific compounds for which need has been established by the Program Projects. Compounds synthesized by the Core will be used as standards in quantitation, for in vitro formation of DNA and protein adducts for in vivo dosing protocols. The availability of standards multiply labeled with stable isotopes from the Synthesis Laboratory will crucial to the development of isotope dilution methods for ultra-trace analysis of metabolites and DNA and protein adducts by the Mass Spectrometry Facility. Specific classes of compounds for which synthetic needs have been established are: Project 1, tricyclic nucleobases, deoxynucleosides and deoxynucleotides; protein adducts of malondialdehyde and 4-hydroxynon-2-enal; cysteine contual basis for dosing protocols; Projects 3 and 8, S-phenyl-d5-cysteine and mercapturic acid; Project 4, fungal metabolites of PAH and Project 5, 13C-labeled PAH phenoles. Synthesis Laboratory will also be available for consultation on questions involving structural characterizations, and application of spectroscopic techniques to problems of quantitation and characterization. Other areas where Core expertise may be useful to Program Projects are in the application of structure-activity relationships to direct efforts in isolation and characterization of products of metabolism or non-enzymatic decay. The Core has access to 500 and 500 MHz NMR spectrometers with multinuclear and variable temperature capability. Core personnel are trained operators, proficient in all aspects of data acquisition, work-up and interpretation, and will fill the NMR needs of program projects. Additional techniques of physical characterization accessible through the Core are circular dichroism X-ray crystallography, EPR and FTIR. The Mass Spectrometry Facility will provide support for Program Projects when mass spectrometry requirements are beyond the capacity of equipment belonging to Program Projects or when extensive methods development is necessary. The Mass Spectrometry Facility performs characterization and ultra-trace analysis on a routine basis by a variety of mass spectrometric techniques, and is also involved in developmental work. Major objectives of the Mass Spectrometry Facility will be to develop on-line sample clean-up and analyte pre-concentration procedures and to apply HPLC/MS/MS techniques to characterization of DNA adducts, protein adducts and PAH metabolites.

DNA adducts are believed to be a major source of the mutations involved in carcinogenesis. The availability of innovative technologies for investigating the presence of these adducts will greatly aid basic research epidemiological and chemoprevention studies, risk assessment and occupational health. We propose to develop technologies for the routine analysis of DNA adducts that arise from medical, environmental and occupational exposures, as well as from endogenous processes. We have previously developed highly sensitive gas chromatography/mass spectroscopy (GC-MS) methods for DNA adducts, but these methods are time consuming and labor intensive. The primary focus of this Phase I application will be the development of ultrasensitive techniques for monitoring DNA adduct formation using liquid- chromatography/electrospray mass spectroscopy (LC-MS). Specifically, we will develop LC-MS methods for 7-hydroxyethylguanine, 7- methylguanine, O6- methylguanine, N2,3-ethenodeoxyguanosine, l ,N2- ethenodeoxyguanosine, l ,N6-ethenodeoxyadenosine, 3,N4-ethenodeoxy- cytosine, 7-(2-hydroxy-2-phenylethyl)guanine, and 7-(2-hydroxy- l - phenyl-ethyl)guanine. These adducts are formed in DNA from animals and humans exposed to vinyl chloride, simple alkylating agents and styrene oxide, and most can be demonstrated in DNA of unexposed animals and humans. Following LC-MS methods development, the methods will be compared with our GC-MS methods to determine the sensitivity, specificity and feasibility of LC-MS for routine use. Triangle Laboratories, with three LC-MS/MS instruments, will be the primary site for the development of the new technology. This will be facilitated by the expertise and stable isotope internal standards that exist in Dr. Swenberg's laboratory. Since the equipment, standards and expertise required for these analyses are extensive, it will be more cost and time effective for many investigators in academia, industry and government to have such assays run by a specialized biotechnology laboratory. PROPOSED COMMERCIAL APPLICATIONS: The availability of innovative technologies for investigating the presence of these adducts will greatly aid basic research, epidemiological and chemoprevention studies, risk assessment and occupational health. Since the equipment, standards and expertise required for these analyses are extensive, it will be more cost and time effective for many investigators in academia, industry and government to have such assays run by a specialized biotechnology laboratory.

Description: (Taken from the Investigator's Abstract) The principal objective of the Biomarkers Facilities Core is to provide facilities and expertise to generate highly specific and ultra sensitive measurements of selected biomarkers for funded and proposed new research by members of the NIEHS Center. The biomarkers to be measured include assays of DNA and protein adducts, abasic sites in DNA, oxidative stress, and GSH, as well as methods for immunoaffinity chromatography and immunohistochemistry. Such assays are critical for assessing specific hypotheses regarding basic mechanisms of carcinogenesis and other diseases, the roles of causal agents and covariates (e.g., nutrition), bioactivation and detoxication, DNA repair and biomarkers of exposure in occupational and environmental epidemiology. By providing this newest technology, the Biomarkers Core will provide members of the NIEHS Center a major opportunity for ground breaking studies. The aims of the Core are to: 1) to make available to Center members state of the art assays for biomarkers of cancer and other diseases; 2) to assist Center members with experimental design, tissue collection and assays of various biomarkers; and, 3) to foster applications of biomarkers in pilot projects and new collaborative research projects.

This application proposes to establish a Center under the NIEHS Environmental Health Sciences Center Grants (P30) program in the School of Public Health, at the University of North Carolina (UNC), Chapel Hill. The focus of this "UNC-CH Center on Environmental Health and Susceptibility" is in the area of environmental epidemiology and toxicology. Three research cores will form the intellectual heart of the Center, organized around the following areas of concentration: Genetic Susceptibility, bringing together laboratory and molecular epidemiologic research on genomic determinants of susceptibility; Developmental Susceptibility, addressing the role of different stages in the life cycle and how these influence susceptibility to exposure, with a particular concentration on exposures received from conception through childhood; and Toxicokinetic Susceptibility, reexamining inter-individual variability in physiologic and metabolic factors that are responsible for the wide ranges in response to an exogenous agent. Four facility cores will provide critical services and will result in cost-efficiency for Center investigators: High Throughput Genotyping, Biostatistics and Epidemiologic Methods, Biomarkers, and Nutrient Assessment. The Administrative Core will have responsibility for coordination of Center activities, strategic planning and evaluation: the Pilot Projects Program; membership decisions; financial matters; leadership and visibility for UNC-CH's environmental health research. The Community Education and Outreach Program will assist the Administrative Core in dissemination and education about Center- related themes on environmental health to professionals, the media, and the public at large, with a focus on the state of North Carolina, and will promote two-way scientist/citizen interactions. This Center is designed to maximize cross-disciplinary integration to promote new research collaborations with exciting scientific potential, understand the mechanistic basis of chemical toxicity, and effectively reduce the burden of environmentally-related disease.

Description (provided by applicant): The goal of the Biomarker Facility Core is to provide access to state of the art facilities for mass spectrometry, microarray, proteomics, metabolomics, mouse genotyping, DNA damage and immunohistochemistry for members of the CEHS to facilitate and enhance research on environmentally related disease. This will be accomplished by the following objectives: 1) to make available to Center members well equipped laboratories for analytical, molecular, genomic and histochemical biomarkers; 2) to assist Center members with experimental design, tissue collection and assays of various biomarkers; 3) to foster the application of biomarkers in pilot projects and new collaborative research projects; 4) to provide education and training in methods related to biomarkers.

DESCRIPTION (provided by applicant): The Biomarker Mass Spectrometry Facility supports research on the UNC-Chapel Hill Campus by providing mass spectrometric identification, characterization and quantitation of biomarkers of exposure. The Biomarker Mass Spectrometry Facility seeks funding to acquire an ultra-high performance bench top triple quadrupole LC-MS/MS system. Our need for the requested instrument is two-fold. Currently the instruments operated by the Biomarker MS Facility are operating around the clock (except for instrument down-time and maintenance) and analyses require four or more weeks advance scheduling. The current volume of demand for the Facility's analytical services exceeds our capacity, resulting in delays in scheduling analyses and leaving no leeway for new projects that are being initiated as assays for DNA damage which we have developed are being transferred to other users on campus. Our other pressing need is for higher-sensitivity analysis than can be provided with the current instrumentation. Because we are measuring analytes which are present at low levels in samples such as human tissue which are available in very limited quantity only, we need sensitivity of detection well beyond what can be achieved with the instruments currently operating in the facility. We therefore seek an instrument with LC-MS/MS, high-throughput capability and high sensitivity. We also need this instrument to accept nano and capillary LC input. This instrument will support research into cisplatin DNA adducts, lipid composition, molecular dosimetry of DNA adducts derived from butadiene, ethylene oxide, propylene oxide and isoprene, and studies on choline metabolism, cancer causality and susceptibility, DNA damage and repair, molecular epidemiology, exposure assessment, and other healthrelated topics. The instrument of choice is an Applied Biosystems/MDS Sciex API 4000 interfaces with an LC Packings (Dionex) capillary/nanoflow high pressure liquid chromatography system. This instrument will be installed in an established and productive shared-use facility staffed by experienced personnel, where it will support the research of ten holders of eleven current or pending R01 and U01 NIH grants.

DESCRIPTION (provided by applicant): 1,3-butadiene (BD) is a known carcinogen. However, the DNA adducts responsible for mutations remain unknown. The overall goals of the proposed research are to examine the molecular dose of previously unexplored DNA adducts in rodents exposed to BD and 3-butene-1, 2-diol (BD-diol), comparing the data with mutation frequencies and mutational spectra to determine if a particular adduct could be used as a quantitative indicator of mutagenesis, and to evaluate effects of exposure on gene expression. The first hypothesis to be tested is that hydroxymethylvinyl ketone (HMVK) is formed in vivo during exposure to BD and BD-diol in a sex, species, and exposure concentration dependent manner resulting in important differences in mutagenicity. The second hypothesis is that promutagenic N1 adenine adducts convert to the more stable inosine adducts which are poorly repaired and accumulate during chronic exposure to BD. Several specific aims will be accomplished while addressing these hypotheses. Specific Aim 1 is to examine the formation of potentially mutagenic DNA adducts (specifically 1, N2-propanodeoxyguanosine) by HMVK in vivo. The second aim is to determine the utility of the N-terminal valine adduct of HMVK (HMVK-Val) as a biomarker of HMVK formation by BD and BD-diol. Specific Aim 3 is to develop methods for detecting N1- inosine, N1 - and N6 adenine adducts derived from BD metabolites in vivo. Specific Aim 4 is to determine the mutagenic responses induced by BD exposures and characterize the impact of BD-diol derived metabolites on the spectra of mutations induced by BD exposure in the B6C3F1 mouse and F344 rat to identify which adducts studied in Aims 1 and 3 are quantitative indicators of mutagenesis. Specific Aim 5 will examine the effects of exposure to BD and BD-diol on gene expression and DNA repair pathways. Collectively, these experiments have been designed to look at adduct formation, DNA repair, mutagenicity, and genomic alterations in rodents exposed BD and BD-diol, as well as the impact of glutathione depletion and DNA repair deficiency. Finally, HMVK-Val adducts will be measured in samples from BD exposed humans. Our research will identify critical metabolites and adducts that are responsible for BD mutagenicity, as well as develop biomarkers suitable for future molecular epidemiology studies. Ultimately these data will improve our understanding of critical mechanisms of toxicity and ability to accurately assess the risk of BD to humans.

[unreadable] DESCRIPTION [unreadable] [unreadable] Researchers from the University of North Carolina School of Public Health seek partial support for a conference entitled, "Current and Future Challenges in Environmental Health & Protection in Eastern & Central Europe" to be held in Kiev, Ukraine, April 28-May 1, 2006. [unreadable] [unreadable] The objectives are: 1) To bring together scientists, students and regulators from the US and Europe; 2) To present and share the best science; 3) To expose students and young investigators to this area. [unreadable] [unreadable] [unreadable] [unreadable]

The research of this project will utilize newly developed biomarkers of oxidative stress to evaluate the mode of action and dose response of hazardous chemicals of importance to Superfund sites. Our studies will primarily address effects of polyhalogenated and polycyclic aromatic hydrocarbons including dibenzo(a,l)pyrene (DBF), RGBs and dioxin. These studies will analyze snap frozen tissues from two of the largest and best characterized carcinogenicity bioassays: the NTP Toxic Equivalency Factor studies on PCBs and dioxin in rats; and the ED(0.1) DBF study in rainbow trout. We hypothesize that oxidative stress is an important mode of action for toxicity and carcinogenesis. These studies will be accomplished by comparing a comprehensive series of biomarkers for oxidative DNA damage that includes lesions removed by long and short patch base excision repair and utilizes different glycosylases, as well as by nucleotide excision repair. The studies will examine dose-response relationships for oxidative DNA damage and compare this with P450 induction, cell proliferation, and in the case of DBF, 32P-postlabeling studies of bulky DNA adducts. In addition, it will conduct similar research on fish from the Hudson River and less polluted waterways that have exposures to different mixtures and amounts of PAHs, PCBs and dioxins. In collaboration with the NYU SBRP, laboratory exposures to similar mixtures of hazardous chemicals will be conducted to investigate the development of resistance to PCBs toxicity and differences in life stage susceptibility to these agents. Finally, we will continue our basic research on oxidative DNA damage and repair to further our understanding of the biology of DNA damage and repair, and its implications for environmental health and individual susceptibility. The long term goals will be to develop an in-depth understanding of how these chemicals cause toxicity and under what conditions they elicit responses. These data will fill critical gaps in knowledge that will impact the basis of low dose extrapolation and improve the scientific basis of risk assessment and the setting of remediation standards. We will interact with each of the other scientific projects of this program by performing biomarker analyses, providing data or determining the ability of various environ-mental chemicals or their metabolites to cause oxidative DNA damage. The research will also make heavy use of both the Chemistry and Analytical Core and the Mathematical and Statistical Analysis and Modeling Core.

[unreadable] DESCRIPTION (provided by applicant) [unreadable] [unreadable] The proposal is requesting support for a conference to investigate bioavailability from the chemical partitioning behavior in the environment through transport, exposure, intake and distribution in the body, to a disease outcome. The stress of the workshop is on this picture of bioavailability as a whole, not on one of these separate areas or boxes. The conference will consist of one session, rather than concurrent sessions, and will devote a fair amount of time to discussion and synthesis of the current state of knowledge in order to identify gaps in our knowledge and define research needs. The product of the conference will be a paper to be submitted to Environmental Health Perspectives that will "synthesize the recommendations for research addressed to bioavailability." [unreadable] [unreadable] [unreadable] [unreadable]

Pilot Projects; programs

The goal of the Systems Biology Facility Core is to bring together the wide array of advanced technologies available to CEHS researchers and provide the resources to assist investigators in the design and implementation of new studies to take maximal advantage of these powerful tools. The previous Biomarkers Facility Core has facilitated access to a wide array of the modern 'omics' technologies. The University has continued to support these efforts and has committed extensive resources to insuring that state-of-the-art instrumentation is available in areas such as genomics, proteomics and metabolomics. These high-throughput methods have been extensively used by CEHS investigators to generate and test new hypotheses in the areas of environmental exposure and toxicology. In order to provide the most efficient access to the expanded capabilities at UNC and integrate new resources in bioinformatics, the Biomarkers Facility Core is being upgraded to the Systems Biology Facility Core (SBFC) which will be closely integrated with the Biostatistics and Bioinformatics Facility Core and the Integrated Health Sciences Facility Core. The SBFC is comprised of the six existing core facilities listed in Table 1. Support from CEHS gives our investigators priority access to these capabilities for their research in the form of reduced rates for services and support for pilot projects. Table 1. Core Facilities within the Systems Biology Facility Core. Sub-Core Facility Genomics Core DNA Damage Biomarkers Mass Spectrometry Proteomics Core Metabolomics Anatomic Pathology Core A systems biology investigation can generate a staggering amount of data. To address the need to analyze and integrate these data, the SBFC will work with the BBFC to develop the Computational Biology Resource (CBR). This will be comprised of individuals working with the BBFC who will have direct expertise in the high level analysis of multiple omics datasets. The individual sub-core facilities will do the initial processing of the raw data, typically using the software provided with the specific instrumentation. These may generate data such as lists of perturbed genes, proteins or metabolites, but transforming these datasets into testable biological hypotheses requires a tremendous amount of manual intervention. By using the computational modeling tools of systems biology, a more automated, objective means of generating high level biological knowledge is enabled. Computational biology tools work with massive databases of genetic and protein interactions along with metabolic pathways to uncover new relationships between genes and their downstream products. The CBR will provide advanced biostatistics analyses to identify the most critical alterations in the data, but will also integrate the data to generate biological pathways and networks. By applying the tools of the individual sub-core facilities and utilizing the Computational Biology Resource, CEHS investigators will be able to develop a systems level understanding of problems related to environmental exposures and toxicology leading to new hypotheses on how these risk factors can be mitigated to improve human health. A long-term goal of the CBR is to be able to apply similar approaches to the data generated in human studies conducted in the Interdisciplinary Health Sciences Facility Core. New investigators are easily overwhelmed by the rapidly advancing technologies in the systems biology world. To facilitate the translation of important biological questions into feasible systems biology research projects, the Systems Biology Research Network (SBRN) is being developed. The SBRN will provide a single point of contact to direct new investigators on how to initiate new projects and guide in the progression of existing projects toward the best resources. The director of the SBFC will serve as the head of the SBRN and will work closely with the Research Navigator to optimize the applications of systems biology. A biweekly seminar/journal club will be developed focusing on various techniques and applications to provide the non-expert with a forum to gain a better understanding of systems biology.

DESCRIPTION (provided by applicant): Specifically, the current and proposed Career Development Program includes: 1) a revised CEHS Pilot Project Program, with pilot project award opportunities that specifically target junior faculty, physician-scientists, and large interdisciplinary projects led by senior faculty;2) a seminar series focused on bringing outside speakers to enhance the CEHS scientific discourse;3) CEHS scientific retreats that address a specific theme and include poster presentations of postdoctoral fellows, graduate students and Facility Cores;(4) availability of the CEHS core facilities, each with clear training and consultation services;5) a mentoring program, led by senior CEHS members, directed towards junior faculty and other junior investigators, which includes a grant writing course for Assistant Professors and advanced postdoctoral trainees;6) training grants, led by senior Center members, or other opportunities that will support students engaged in environmental health research;the latter include providing additional classroom training opportunities by utilizing the expertise of the members of the new Integrative Health Sciences Facility Core and the Biostatistics and Bioinformatics Facility Core;and 7) a return to providing partial salary support, targeted to recruiting new junior faculty focused on environmental health research.

The primary mission of the Integrative Health Sciences Facility Core (IHSFC) is to support CEHS investigators who conduct research using human study participants. IHSFC addresses four broad areas of interest. First, the IHSFC provides scientific consultation for clinical and population-based studies. These services promote collaborations and enhance scientific productivity. Second, biospecimen processing and genotyping sub-core laboratories provide platforms for short-term pilot projects and long-term epidemiologic studies for Center members. These sub-core laboratories provide access to state of the art equipment and a high level of quality control and maximal throughput that could not be achieved by individual investigators. The genotyping sub-core also provides typing of laboratory rodent strains in order to promote cross-disciplinary collaborations in functional and comparative genomics for Center members. Third, the IHSFC provides expertise in spatial analysis and geographic information systems, in order to integrate human epidemiologic studies with environmental exposure measurement. Fourth, the IHSFC serves as a liaison to a variety of core laboratories, resources and facilities at UNC that are of use to specific CEHS investigators. The IHSFC is closely integrated with the Biostatistics and Bioinformatics Facility Core (BBFC) and the Systems Biology Facility Core (SBFC) through the Research Navigator and the Flexible Interdisciplinary Research Groups (FIRG).

Dr. Kaufmann will serve as the Research Navigator and direct the Interdisciplinary Flexible Research Group. He will be responsible for coordinating the regular research group meetings, motivating the group to develop new research initiatives, and fostering an atmosphere wherein collaborations will become the norm. He will oversee the development and progress of the group's pilot projects. He will participate in the Internal Advisory Committee meetings, act as the liaison between the Administration of the Center and the research group members, and will provide input about priorities for specific pilot projects in his area of expertise.

The Administrative Core has primary responsibility for oversight and guidance of the University of North Carolina at Chapel Hill Superfund Research Program (UNC-CH SRP). This includes the overall planning and coordination of UNC-SRP research activities, fostering interdisciplinary interaction, enriching the program through seminars and retreats, scheduling meetings of Program researchers and trainees, the Executive Committee and the External Advisory Committee, as well as fiscal and resource management and planning. The proposed UNC-SRP includes five research projects, two research support cores, a Research Translation Core, and the Administrative Core. The Administrative Core will continue to be led by Professor Swenberg, Director of the UNC SRP. He will be assisted by Professor Rusyn, Core Co-Leader, and Ms. Nataliya Vanchosovych, Program Coordinator. The goals of the core are to: (1) foster strong interdisciplinary interactions and high productivity of the research projects and cores; (2) manage the fiscal resources of the program; (3) organize and schedule monthly meetings of all UNC-SRP researchers, including students and post-docs during the academic year, monthly meetings of the Executive Committee (immediately before or after monthly Program meetings), enrichment seminars, and annual meetings of the External Advisory Committee (in conjunction with an annual retreat); and (4) facilitate interactions of our scientists and trainees with the Research Translation Core to maximize communication with the National Institute of Environmental Health Sciences, the U.S. Environmental Protection Agency, the Agency for Toxic Substances and Disease Registry, the North Carolina Department of Environment and Natural Resources, other government agencies, communities, the public, and potential users of the technology developed by our SRP, in translating research concepts developed in our Program.

The CEHS Community Outreach and Education Core's efforts focus on improving public understanding of how susceptibilities and environmental factors interact to cause disease, with a goal of enabling people to make informed decisions about reducing disease risk and hazard exposure. In our work, we rely on CEHS and other peer-reviewed environmental health science research, and we focus on reaching populations most susceptible to the diseases studied by CEHS and those who provide services to these populations. The COEC's specific aims are to: 1. Increase CEHS members'understanding of stakeholder needs through interactions with a Community Advisory Committee. 2. Enhance the capacity of susceptible populations and public health practitioners to understand environmental health risks and to reduce disease risk and hazard exposure in homes and communities. 3. Raise awareness of emerging research and environmental health concepts among children participating in summer camps and among broad community-based audiences through our web site and other media. Target Audiences We strive to engage the populations most susceptible to the diseases being studied by CEHS. Certain vulnerable populations (e.g., children, specific minority groups, and people living in poverty) are not only susceptible to these diseases but also have more serious health outcomes, making them logical target audiences. For instance, in North Carolina, childhood lifetime asthma prevalence and current asthma prevalence exceed the national median, and African- and Native-Americans have significantly greater mortality rates due to asthma than Caucasians (Jensen, 2006). Similar mortality trends have been documented for African-American women with breast cancer, and recent studies by CEHS investigators have found more lethal cancer subtypes appearing in younger African-American women (Carey, 2006). Although melanoma is largely a disease of older Caucasians, five-year survival rates are significantly higher for Caucasians than African-Americans (ACS, 2005), and incidence in children, adolescents, and young adults increased by almost 3% per year from 1973-2001 (Strouse, 2005). Further, evidence suggests that sunburn during childhood may raise the risk of adult melanoma (Noonan, 2001). To reach a cross-section of these susceptible populations, we propose to work with faith- and community-based organizations, particularly in African- and Native-American communities, in several North Carolina counties. Church-based health promotion interventions have been shown to be effective at impacting health behavior, especially in addressing some of the specific health issues reflected in the CEHS research foci (such as breast cancer and obesity) (Campbell, 2007). Examples of successful strategies include family programs, lay health advisors, pastor leadership, church-sponsored education events, and increasing access to health care and low-cost screening and follow-up. The COEC already employs some of these strategies with predominantly black churches in the rural counties surrounding the Triangle area. For instance, the COEC partnership with local Breast and Cervical Cancer Control Program (BCCCP) coordinators has enabled low income women who attend our church-hosted Breast Cancer, Genes, and the Environment workshops to enroll in the BCCCP to acquire free mammograms and other services associated with diagnosing and treating breast cancer. This initial breast cancer outreach to a small group of churches also served as a catalyst for relationships with multiple congregations and interfaith councils and provided opportunities to introduce other environmental health topics to these groups, in response to their requests. One of the specific areas of interest of these congregations was youth environmental health outreach. For this reason, we are partnering with a local science center to develop a summer camp program that introduces CEHS science to middle school students and can serve as a template for youth outreach in our target communities. The middle school years are a time of tremendous change and development for students and also a pivotal time in their understanding of and enthusiasm for science. Research has shown that if educators do not capture student interest in science by middle school, students may lose interest altogether (NSTA, 2003). We also propose to work with public health practitioners in a set of target counties and across the state to prepare them to educate their clients/patients on environmental health and healthy homes issues. A number of recent studies underscore the need for health promotion involvement in environmental health practice (Howze, EH, 2004;Kreuter, MW, 2004;Freudenberg, 2004). At least one has shown that public health professionals can strengthen community capacity by increasing access to accurate science and building strong relationships between communities and local health departments, among other factors (Freudenberg, 2004). Furthermore, in COEC outreach to health practitioners in the previous grant cycle we heard a strong desire for environmental health education. Participating professional associations stressed the need for such education among their members, and the health departments that participated in an evaluation of educational materials on effectively engaging communities in environmental health/hazard issues requested follow-up training for larger groups of their employees. Initially, our efforts will be focused in Chatham, Wake, and Craven Counties and, to a lesser extent, the counties that border them. In later years, we hope to add a county in eastern North Carolina with a substantial Native American population (e.g., Robeson Co.). In addition to population demographics, our choices were influenced by existing relationships with community partners, a desire to work in multiple regions of the state, and the relative proximity of these counties to UNC-Chapel Hill, enabling us to have more active and sustained relationships.

Environmental health research, whether involving experiments at the cellular level, investigations using laboratory animals, or studies of human populations, is becoming increasingly complex. There are several reasons for this added complexity. The valid quantification of human exposures to environmental hazards requires consideration of complicated time-dependent individual-specific multiple exposure profiles. Assessment of early biologic effects (e.g., biomarkers) requires elucidation of how these exposure profiles relate to multiple, intermediate, and often correlated endpoints. Assessment of exposure-disease relationships requires knowledge of the connections between exposure, early biological effects, and multiple correlated health outcomes. The further necessity of simultaneously considering the impact of gene-environment interactions and molecular-level mechanisms argues strongly for the use of innovative, well-designed, and appropriately analyzed environmental health research studies to insure the validity and precision of scientific conclusions. Such validity and precision requirements mandate that biostatisticians and bioinformaticians be involved as active research collaborators from the planning stages of environmental health studies through final analyses and dissemination of research conclusions via presentations and publications. Without state-of-the-art input from biostatisticians on study design and data analysis, there is no question that environmental health research studies can be severely biased and can suffer from a lack of sufficient precision to detect important exposure-response relationships. The Biostatistics and Bioinformatics Facility Core (BBFC) provides the CEHS with the needed expertise to apply state-of-the-art statistical methods to the design and analysis of all types of environmental health research studies, ranging from laboratory study design, analysis of multidimensional gene by environment interactions, and quantification of complex time-dependent exposures. BBFC personnel have expertise in essentially all state-of-the-art statistical methods required to address the most complex of study design and data analysis issues. It is fair to say that the CEHS cannot function effectively without making continued use of the BBFC, from study planning through data analysis and interpretation. It is an efficient and cost-effective allocation of resources to assign these important statistical design and data analysis activities to the BBFC. In the sections to follow, detailed descriptions of BBFC (formerly the Biostatitics and Epidemiological Methods Facility Core, or BEMFC) activities since April 2006 will be given. In particular, important functional changes will be highlighted, BBFC members and their unique expertise will be detailed, involvements of BBFC members in CEHS pilot and research projects will be described, and BBFC enrichment activities will be mentioned.

DESCRIPTION (provided by applicant): The UNC-CH Program's multidisciplinary research will address scientific issues that underpin the assessment and reduction of risks to human health associated with high priority chemicals at Superfund sites. The overall specific aims are to improve the scientific foundation for risk assessment, elucidate mechanisms responsible for inter-individual susceptibility, advance approaches to assessing exposure to hazardous chemicals, and more efficiently reduce risks by remediation of hazardous waste sites. In this competing continuation, we will focus on three major classes of chemicals ~ polycyclic aromatic hydrocarbons (PAHs), halogenated hydrocarbons, and heavy metals ~ in three biomedical and two non-biomedical projects, two Research Support Cores, a Research Translation Core (RTC), and an Administrative Core. Research themes that cross multiple projects and cores include: (1) developing biomarkers of exposure and effect for human and experimental models of environmental disease over a range of exposure levels to improve low-dose quantitative risk assessment;(2) applying new molecular tools in a systems biology framework to understand metabolic pathways critical for environmental disease, predict in vivo inter-individual differences in susceptibility and risk, and evaluate complex microbial communities in bioremediation systems;(3) using advanced analytical tools to identify mechanisms of genotoxicity;(4) using advanced statistical and bioinformatics methods to evaluate gene-environment interactions;and (5) quantifying the chronic exposure and bioavailability of toxic compounds in environmental systems. This work will also be integrated by sharing methods and resources across projects and cores, by regular meetings of all researchers, and by co-advising of trainees by faculty in different projects and cores. Working with investigators, the RTC will enhance the capacity of government agencies to provide technical assistance to communities, develop improved decision-support tools, and promote the commercialization of our research products. This Program is highly relevant to Superfund by addressing high-priority chemicals and by focusing on mechanisms underlying health effects, exposure assessment, and remediation to mitigate exposure and toxicity.

This project will utilize newly developed assays for a battery of oxidative DNA adducts to better define dose and time responses for oxidative stress induced by exposure to PCBs and TCDD using liver and lung samples from 13, 30 and 52-week tissues from Sprague-Dawley rats exposed to pentachlorodibenzofuran, PCB 118, or mixtures of TCDD, PeCDF and PCB 126 from the NTP Toxic Equivalency Factor (TEF) studies. These studies will demonstrate the relationships between endogenous DNA adducts and carcinogenesis and determine if the oxidative stress Mode of Action for this important set of nongenotoxic chemicals is supported. Endogenous DNA adducts can be converted to mutations if DNA replication takes place before repair. Unlike DNA adducts, mutations cannot be repaired and are heritable in the progeny of the originally mutated cell. We have shown that endogenous DNA lesions are always present in genomic DNA, attaining >40,000 adducts per cell. Since most of these lesions are potentially mutagenic, this nonzero background of endogenous DNA damage is a likely cause of the nonzero spontaneous background mutation rate. We will develop new methods for studying mutations using the PIG gene in DT-40 cells and will employ this system to evaluate the dose-response for mutations resulting from chemicals that form DNA adducts identical to endogenously formed adducts in cells and tissues. This research will provide highly informative scientific data to be used in future cancer risk assessments, rather than relying on linear default science policy decisions that may not provide additional protection to public health, but pose expensive and often non-attainable clean-up levels for Superfund sites. Finally, we will collaborate with Projects 2, 3, 4 and 5 respectively, to determine the role of oxidative stress in the toxicity of trichloroethylene; identify critical DNA response pathways for cadmium; evaluate CAFLUX and CALUX cell biological responses to samples of environmental contaminants; and examine toxicity and DNA damage response pathways of original extracts and their fractionated and purified samples of PAH mixtures that have undergone bioremediation.

Our previous research has established that the genetic makeup of the host plays a key role in metabolism and its biological effects of trichloroethylene (TCE) in mouse liver. Genetic polymorphisms have a profound effect on differences between individuals who may have developed disease after exposure to environmental agents, yet these factors are not being fully considered in risk assessment. Indeed, the need to account for differences among humans in cancer susceptibility other than from possible early-life susceptibility is becoming ever more evident to both the scientific community and the regulatory agencies. A hypothesis that apparent species- and organ-specific metabolism and toxicity of TCE are genetically controlled and that the mechanisms of susceptibility can be successfully elucidated using a panel of inbred mice will be addressed here. First, we will elucidate genetic determinants of inter-individual differences in TCE metabolism by collecting time course, dose-response, and repeat dose data on TCE metabolites in blood and tissues from a large panel of genetically diverse inbred mouse strains. The data will be used to investigate the genetic causes of variation in the metabolism of TCE, a step crucial for understanding the potential for TCE-induced adverse health effects in a heterogeneous human population. Second, we will build population-wide pharmacokinetic models for TCE metabolism, which will account for inter-individual variability in metabolism from the genetics point of view by using the time-course and dose-response data obtained on the genetically-diverse animals. Third, we will determine the effects of inter-individual genetic variability on strain-specific responses to TCE through dose-response modeling of gene expression and metabolomic data. Collectively, this project is timely in proposing a paradigm that will not only offer valuable insights into the molecular basis for genetically-determined variability in response to TCE, and develop PBPK and statistical models, but also will provide necessary science-based underpinnings and tools for the new paradigms being incorporated into the risk assessment and decision-making on TCE and related chlorinated solvents, as well as other environmental agents.

The toxic heavy metal cadmium is a high priority contaminant identified at more than one third of all Superfund Sites. While cadmium is a known and well-studied carcinogen, here we will address cadmium's effects as a developmental toxicant. Newborns exposed to cadmium during the prenatal period have increased risk for poor birth outcomes, including low birthweight. In addition to the immediate postnatal concerns, low birthweight is also associated with increased risk for chronic diseases later in life, such as diabetes, hypertension, and cardiovascular disease. While poor birth outcomes have been associated with environmental exposure to cadmium and other metals, the underlying biological mechanisms remain under studied. Integrating our preliminary findings and our interest in understanding how metal exposure influences pregnancy outcome in the United States, we will examine gene-environment interactions that influence cadmium-induced signaling of inflammatory response genes and will determine the association of pathway modulation with birthweight. We hypothesize that gene-environment interactions influence cadmium's effects on signaling of inflammatory response genes and that this signaling is associated with newborn birth outcomes. This study will use complementary in vitro and in vivo approaches to test our hypothesis. To identify genes that influence cadmium-induced toxicity, we will use a panel of cell lines derived from a genetically diverse human population. The integrated in vivo aims will assess the impact of fetal genotypes of inflammatory response genes on newborn birthweight and the interaction effects between fetal genotypes and cadmium exposure. The modulation of the expression levels of members of inflammatory response genes in newborns and association with maternal cadmium exposure will be determined. The role of DNA methylation in controlling the gene expression alterations will be established. The results obtained from the proposed research will help to elucidate molecular pathways associated with cadmium-induced toxicity and disease.

The proposed research addresses the broad SRP theme of Detection Research and more specifically the development of passive samplers for multi-chemical detection and determination of the degree of bioavailability in water and sediment. Aim 1 will develop a universal passive sampling device (PSD) for measuring the time-weighted-average chronic exposure to hundreds of organic chemicals in water. We will advance the theory, design, and application of PSDs to a very broad range of physico-chemical properties (e.g., KOW = 0-9) so that nearly every organic chemical on the Superfund Priority List of Chemicals, and many of their metabolites, will be sampled by a single PSD. We hypothesize that a mixed-polymer sorptive phase contained within a non-selective and highly porous membrane will allow linear uptake of nearly all organic chemicals. Aim 2 will establish the use of PSDs to measure the bioavailable fraction of PCBs and PCB metabolites in water, sediment, and soil. We will conduct laboratory bioavailability experiments with PCB-contaminated soil, sediment and water to advance our understanding of the mechanisms controlling PCB bioavailability and perform field verification at NPL sites. In collaboration with Project 1, we will use extracts of our samples to determine the relationship of our bioavailability measure to the dioxin toxic equivalency factor response in Project 1 cell assays. Aim 3 will establish the use of PSDs to measure the bioavailable fraction of PAHs and PAH metabolites in water, sediment, and soil. In collaboration with Project 5, we will perform studies very similar to those in Aim 2 to develop the use of PSDs to measure PAH and PAH metabolite bioavailability under both controlled laboratory conditions and at NPL sites. This work will advance our understanding of and ability to measure the partitioning of PAHs and metabolites among dissolved organic carbon, soft and hard (soot) particulate carbon, weathered oil/oil product phases, and biota. We hypothesize that our novel PSD design will provide an accurate measure of bioavailable chronic exposure under a broad range of conditions and that PSD-derived data will overcome a critical barrier to more accurate estimates of bioavailability and risk.

Bioremediation is an established technology for removing PAHs from contaminated soil, but previous studies have shown that it does not always lead to a reduction in toxicity. The causes of toxicity and the mechanisms by which toxicity might be avoided or diminished are not well-understood. We hypothesize that metabolites produced by PAH-degrading bacteria, which have been observed to accumulate in field-contaminated soil and sediment, are responsible at least in part for the toxicity that can result from bioremediation. We also hypothesize that bioremediation conditions influence the community of PAH-degrading microorganisms in contaminated soil, which in turn influences both PAH removal and the extent to which metabolites might accumulate. We propose to explore the effects of bioremediation conditions on PAH removal and soil toxicity, using a slurry-phase bioreactor as the experimental platform. Among the conditions we will evaluate is the addition of a hydrophobic surfactant at a low dose, which we recently demonstrated can improve the bioavailability and biodegradation of PAHs that remained in a field-contaminated soil after conventional bioremediation. This approach will be developed further by demonstrating its efficacy in a semi-continuous process. We will combine our recent work on stable-isotope probing with a high-throughput DNA sequencing method to identify the PAH-degrading bacteria most likely to influence PAH removal, metabolite accumulation, and toxicity in the treated soil. Genomes of these organisms will be sequenced to identify genes associated with PAH metabolism, and the key genes will be expressed. Differences in the ability to metabolize various PAHs will be correlated to sequence differences in these genes so that genetic determinants of metabolite accumulation can be identified. In parallel, we will use fractionation techniques and advanced analytical tools to identify compounds responsible for toxicity of the treated soil. Finally, variables that can be controlled during bioremediation will be evaluated for their ability to preclude or mitigate toxicity. Our overall goal is to fill key gaps in knowledge that will inform and improve field applications of bioremediation that lead to true reductions in risk.

The Biostatistics Core is designed to enhance the UNC Superfund Research Program's assessment and reduction of risks to human health associated with Superfund high priority chemicals. The five projects in the SRP present considerable biostatistical issues and bioinformatics challenges that are central to the success of the projects. Our overall aims are to (i) provide state-of-the-art biostatistics and bioinformatics expertise, end-user analytical support, and tool development; (ii) to develop new methods and tools as necessary to address project objectives; (iii) to foster a unique training environment for students, postdoctoral fellows, and faculty to prepare them for the new and complex challenges presented by modern datasets. The Core faculty and staff have a range of complementary skills, which will lead to a unified approaches to data interpretation, integration and cross-platform analysis. Collectively, the Core is highly relevant to the Superfund Program, as it will: ¿ support interdisciplinary (toxicology, genetics, biostatistics, pharmacokinetic modeling) research to elucidate the genetic basis of dose-response and susceptibility; ¿ develop and use state-of-the-art statistical techniques and analysis tools for systems biology approaches; ¿ identify potential biomarkers linked to genetic differences in toxicant metabolism and/or response; and ¿ generate knowledge that is directly applicable to quantitative elucidation of risk. The Core will be involved at the earliest stages of each project, assisting investigators in each step of planning and executing the projects. Each Core faculty member is assigned a role to two projects. A staff statistician with considerable bioinformatics experience will serve as a dedicated analyst, under the guidance of Core faculty members. The Core director will report to the SRP Director, and members, who already collaborate extensively, will meet regularly with the investigators and with each other to plan and exchange ideas.

The Chemistry and Analytical Core of the UNC-SRP is a research support core that provides analytical support to the research projects and synthesis, purification and characterization of chemicals that are not available commercially or that can be cost-effectively prepared in-house. The Chemistry Core offers particular expertise in preparation of standards and internal standards labeled with stable isotopes ([15]N, [13]C, [2]H) that allow development of assays using mass spectrometric techniques for highly specific and highly sensitive identification and quantitation. The specific goals of the Chemistry Core are based on the aims and accomplishments of the individual research projects, and thus will evolve in response to research needs. The overall aims of the Chemistry and Analytical Core include: (1) provide advice and consultation on analytical methodology and chemical handling, including analytical method development, appropriate choice of analytes, instrumentation, and standards (Projects 1, 2, 3, 4 and 5); (2) develop analytical methodology, including gas and liquid chromatography, for detection and quantitation of parent compounds, degradation products, metabolites, and macromolecular adducts (Projects 1, 2, and 5); (3) provide service in carrying out routine assays for detection and measurement by quantitative spectroscopy and mass spectrometry of PAH, PAH metabolites, PCBs, oxidative damage, and macromolecular adducts (Projects 1, 2, 3 and 5); (4) prepare novel and rare chemicals, including isotope-labeled chemicals, devise synthetic routes and offer advice and guidance for UNC-SRP researchers wishing to undertake syntheses and product purification with their own personnel, or carry out the entire preparation as needed (Projects 1, 2, 4 and 5); (5) analysis and structural identification of unknown degradation products, metabolites, and macromolecular adducts and provide expertise in interpretation of spectral data in support of structural elucidation (Projects 1, 2, 4 and 5). Specific major tasks include preparation of [[13]C]-labeled vinyl carbamate and its epoxide for Project 1, assisting Project 2 with assay of trichloroethylene metabolites, assisting Project 4 with assays of environmental complex mixtures, assisting Project 5 with fractionation of extracts of bioremediated soil and identification of genotoxically-active constituents of the extracts and preparation of [U-[13]C] PAH quinones, and providing assays of biomarkers of oxidative stress as needed for Projects 1-5.

The Research Translation Core (RTC) focuses on improving scientific and public understanding of how Superfund chemicals harm human health and how to reduce exposure to those chemicals, enabling government officials and the public to make informed decisions about reducing risk. We emphasize the high priority Superfund chemicals studied within the UNC SRP. The RTC aims to: (1) foster and coordinate research translation efforts within UNC SRP and share our results with NIEHS and other SRPs, (2) raise awareness of UNC SRP research among federal, state and local government agencies and build their capacity to protect health and the environment around contaminated sites; (3) advance the practical contributions of our research through development of decision support tools and innovative environmental technology; (4) raise awareness among teachers and other broad audiences of UNC SRP research findings and general environmental health concepts related to hazardous chemical exposure; and (5) increase our students' knowledge of research translation concepts. The RTC will continue current efforts to raise awareness of UNC SRP research and extend them to new state agency partners. We will also implement new initiatives designed to respond to research needs identified by state agencies and local health departments in communities with hazardous waste sites. The RTC will continue to assist NCDENR and NCDHHS in creating decision making tools that index cumulative exposure to pollutants and health outcomes, refining beta versions of these tools and implementing new efforts to train agency staff in their use. We will also work with the UNC Office of Technology Development to assist researchers in the commercialization process. In addition, the RTC will continue and expand its teacher professional development activities, enabling us to effectively share Superfund related science with broad audiences in appropriate ways. We will also conduct short courses on research translation topics for Environmental Sciences and Engineering students.

DESCRIPTION (provided by applicant): We are requesting funding to purchase a versatile, high sensitivity high-throughput system consisting of an HPLC-chip interfaced to a triple quadrupole mass spectrometer. This instrument will be located in the Biomarker Mass Spectrometry Facility Core in the Department of Environmental Sciences and Engineering of the University of North Carolina at Chapel Hill (UNC-CH), which is a shared facility that serves researchers from the entire university and beyond. This instrumentation will provide us with at least an order of magnitude higher sensitivity than the existing instrumentation. This will allow detection and quantitation of analytes that are present at levels too low for our current instrumentation, and/or where the total amount of sample available precludes analysis with our current instrumentation. The equipment of choice is an Agilent 1200 series nanoflow HPLC interfaced via a 1260 Infinity Chip Cube with an Agilent 6490 LCMS Triple Quadupole mass spectrometry system. This combination is the most sensitive LC-MS/MS on the market, and will allow quantitation of biomarkers of interest with the smallest mass and volume of sample. The Chip Cube interface is also very well suited for use in a shared instrument facility where trace-level analysis is very easily compromised by cross-contamination. This equipment will support nine NIH- funded researchers. PUBLIC HEALTH RELEVANCE: The Biomarker Mass Spectrometry Facility provides accessible routine service for measuring small molecules and biomarkers to the University of North Carolina at Chapel Hill biomedical research community. The LCMS/MS system requested for the Biomarker Mass Spectrometry Facility will enable the Facility to extend by at least an order of magnitude the sensitivity of assays for small molecules, DNA adducts, DNA-protein crosslinks, and other biomarkers in complex biological matrices. This will enhance studies by nine NIH-funded researchers at the UNC-CH School of Public Health and School of Medicine, including three physicianscientists, who address critical health problems related to cancer, cystic fibrosis and other respiratory disease and drug abuse.

?DESCRIPTION (provided by applicant) The theme of the UNC Center for Environmental Health and Susceptibility (UNC-CEHS) is translating interdisciplinary research on environmental health threats to improve public health in North Carolina. The Center encourages and facilitates collaborations among basic researchers, public health scientists, and clinicians to generate high impact discoveries that improve public health. The strategic vision is implemented by the Administrative Core which manages budgets, oversees the Facility Cores, evaluates and funds innovative pilot projects, sponsors seminars and symposia, supports developing careers, communicates with Center membership, prepares reports and newsletters, and tracks publications and grant proposals. The Center has three Facility Cores that facilitate biospecimen acquisition, molecular analyses, and data analysis and experimental design. These cores ensure dissemination of proven and innovative new technologies to environmental disease research. The Integrated Health Sciences Facility Core provides expertise in biospecimen processing and helps encourage new collaborations using clinical and population-based studies. The Biostatistics and Bioinformatics Facility Core provides expertise in study design, data analysis, training and methodology development that especially supports the application of powerful omics technologies. The Molecular Analysis Facility Core provides expertise and instrumentation for mass spectrometry, assessment of DNA damage and its responses, translational histopathology and the full complement of genotyping and genomics technology. Each of the cores is designed to enhance productivity of UNC-CEHS investigators. Three Translational Research focus areas are supported by the Center: environmental cancer, cardiopulmonary disease, and developmental disease. North Carolina exposures often are associated with multiple outcomes, and thus research on similar environmental health threats unifies center investigators, provides connectivity, and ensures integration of UNC-CEHS. Scientific studies benefit from multi-directional communication, led by the UNC-CEHS Community Outreach and Engagement Core, which provides a means to disseminate research discoveries to public health professionals, community health workers, susceptible populations, families and lifelong learners. The science and outreach projects are supported by a competitive Pilot Projects program that prioritizes junior faculty. UNC-CEHS also actively mentors and cultivates leadership of junior and mid-level career investigators to train the next generation of environmental health researchers. Through the Stakeholder Advisory Board and External Advisory Board the UNC-CEHS receives input on community concerns and feedback on Center effectiveness. UNC-CEHS serves North Carolina, and ultimately the southeastern United States and the nation with innovative and strategic scientific activities and community engagement.