Research methods pilot projects

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Molecular Tags for high sensitivity quantification of mutations in amplicon and DNA capture protocols

Principal Investigator: Piotr Mieczkowski, PhD, Research Assistant Professor, Genetics

Abstract

Targeted sequencing gives the promise of sensitive and quantative measurement and monitoring of mutations in several assays including the free circulation DNA (fc DNA) in peripheral blood. From a diagnostic perspective, the fc DNA can carry information about tumor burden12-17, virus load (EBV19-27 and HPV28-33) as well as mutation loads in cells which underwent apoptosis, necrosis, or were eliminated by the Immune system. To acquire all this information from the fc DNA sequencing data, we need to improve our strategy for Next Generation Sequencing assays to increase the sensitivity of variant detection. Currently, a PCR artifacts and an error rate produced from the DNA sequencing technology (103) affects the sensitivity of Amplicon Sequencing assays. Additionally, the Whole Genome Sequencing or the DNA capture technology requires a high coverage for variant discovery to reduce false discovery rate sbased on sequencing data. Both of these applications are unable to confidently call variants lower than those of a frequency level of 0.1%. Introducing the Molecular Tags (Duplex tags), used for low DNA input samples like the fc DNA, finally allow for high sensitivity variant detection for both Amplicon and DNA capture assays.

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Discovery of New Detoxification Pathways Using System Biology of Liver Cultures

Principal Investigator: Jeffrey M. Macdonald, PhD, Associate Professor of Biomedical Engineering, SoM

Abstract

A novel system biology approach combining fluxomics with targeted kinomics will be applied to test the hypothesis that substrate cycles, aka futile cycles, are detoxification mechanisms designed to protect the cell from excess redox energy and subsequent generation of reactive oxygen species. Substrate cycling can dissipate an overloaded mitochondrial proton motive force generated by the electron transport chain by consuming NADH or shuttling it to the cytosol. This action regulates redox balance and maintains constant energy flow, similar to how a linear regulator maintains constant voltage in an electrical circuit. A unique 13C-labeling strategy encodes metabolic memory in specific 13C-metabolites enabling quantification of substrate cycles. The response of 2D and 3D cultures of hepatocytes to two toxicants, ethanol, and acetaldehyde, will test the hypothesis. A multidisciplinary team consisting of Directors from four CEHS Cores of Drs. Macdonald (Systems Biology; Metabolomics), Graves (Proteomics), and Bodnar (Biomarkers), will supervise the acquisition of fluxomics/metabolomics, targeted kinomics and targeted metabolite data, respectively, from 2D and 3D hepatocytes cultures (Dr. LeCluyse, Hamner Institute). Dr. Gomez (UNC, SOM), will develop a mechanistic metabolic flux analysis (MFA) model to interpret the dataset.

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Pathways for human uptake of emerging chemicals of concern in land-applied sewage sludge

Principal Investigator:  Steve Wing, Associate Professor of Epidemiology
Award: $25,000

Abstract

Annually, an estimated 4 million dry tons of U.S. sewage sludge are disposed of by application to land. Sludges, especially those from waste water treatment plants with household and industrial inputs, also contain complex mixtures of chemical contaminants. Although human exposures to these compounds are likely as many are persistent and bioaccumulative in the environment and wildlife, little is known about pathways of human exposure. Our objective is to identify important routes of exposure to sludge contaminants, specifically perfluorinated compounds (PFCs) and polybrominated diphenyl ethers (PBDEs), and identify the population of individuals exposed. We will assess multiple pathways of exposure to sludge contaminants, including airborne dispersion of contaminants, local fish consumption and drinking water contamination. Combining these data with previously assessed pathways, we will use probabilistic models to assess the contribution of each pathway to human internal contaminant dose. This research will provide the first comprehensive assessment of human exposure pathways to land-applied sludge contaminants and will be used to inform an assessment of the impacts of sludge contaminant mixtures on human health.

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Genome-wide analysis of DNA replication fork stalling due to environmentally-induced DNA damage

Principal Investigator: Cyrus Vaziri, Ph.D.; Associate Professor of Pathology and Laboratory Medicine
Award: $25,000

Abstract

Benzo[a]pyrene (B[a]P) and Ultraviolet (UV) radiation are ubiquitous environmental agents that damage DNA and cause cancer. During S-phase DNA replication forks are particularly vulnerable to the detrimental consequences of DNA damage. Failure to accurately replicate and repair DNA leads to many hallmarks of cancer cells including mutagenesis and gross chromosomal rearrangements. This project will test the hypothesis that DNA replication defects arising from environmental DNA-damaging agents are over-represented at specific loci in the human genome. We have devised an innovative protocol for isolating chromatin at sites where replication fork stalling occurs specifically in response to B[a]P- or UV-induced DNA damage. We will perform next-generation sequencing and Microarray-based Comarative Genomic Hybridization (CGH) to perform genome-wide analysis of replication events in occurring in the absence and presence of genotoxins. The CEHS Biostatistics and Bioinformatics core facility will provide expertise and assistance for alignment of sequence reads on the human genome and identification of sites of significant over-representation. We anticipate we will be able to identify novel DNA sequences and epigenetic marks associated with replication fork stalling due to environmental carcinogens.

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High throughput analysis of environmental and endogenous agents

Principal Investigator: Paul Chastain, Ph.D.; Research Associate of Pathology and Lab Medicine
Award: $25,000

Abstract

The rapid assessment of cellular responses to different biological, environmental, and endogenous challenges is critical for understanding how these agents may increase genomic instability, disease formation and ultimately affect quality of life. One way to assess this response is to determine their influence on DNA replication and how this change in replication dynamics is dealt with by the cell. Recently, we have adopted the technique of replication analysis of DNA on extended single DNA fibers spread onto glass slides to expand our understanding of how DNA damaging agents influence DNA replication. This CEHS pilot project is designed to allow us to develop automated techniques to perform immunostaining and computer-assisted analysis of the slides, thereby decreasing analysis time and eliminating bias associated with analyzing the fibers manually. In addition, this methodology would accelerate the study of the effect of DNA damaging agents on isogenic cell lines with different defects in damage recognition, repair, and checkpoint pathways, thus increasing the depth of our understanding of the role of the various proteins involved in DNA replication and repair.

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A mathematical model describing both the normal transition of cells from G2 into mitosis and the process that occurs following DNA damage

Principal Investigator: Kevin J. Kesseler, Postdoctoral Fellow, Dept. of Pathology and Laboratory Medicine, School of Medicine
Award: $25,000

Abstract

In order to study the G2 DNA damage checkpoint we created a mathematical model of the protein interactions involved in the G2 to M transition and the G2 DNA damage arrest mechanism. This proposal calls for the addition of DNA damage signaling pathways and additional compartments to the model as well as the modeling of G2 checkpoint function in various cell lines.

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Repair of formaldehyde-induced DNA-protein crosslinks in Drosophila

Principal Investigator: Jeff Sekelsky, PhD, Associate Professor of Biology and Program in Molecular Biology & Biotechnology
Award: $25,000

Abstract

DNA-protein crosslinks (DPCs) arise from both exogenous and endogenous sources. One important source the environmental carcinogen formaldehyde. Cells can repair DPCs, but the repair mechanisms are poorly understood. Previous studies in E. coli and mammalian cells suggests that repair involves components of two well-studied repair pathways: nucleotide excision repair (NER) and homologous recombination (HR). We propose to study repair of DPCs in vivo in the model organism Drosophila melanogaster. We will first determine whether the NER endonucleases XPG and XPF-ERCC1 (the excinucleases) are required to excise DPCs caused by exogenous formaldehyde. We will then investigate the role of HR in the response to DPCs induced by exogenous formaldehyde. To explore the contribution of endogenous formaldehyde to DNA damage, we will attempt to block spontaneous mitotic crossing, which may arise during repair of DPCs, with a formaldehyde quencher. These experiments will further our understanding of in vivo repair of DPCs in animals.

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PCaP-GIS: A Geospatial Analysis of Environmental Risk Factors for Prostate Cancer Severity in North Carolina

Principal Investigator: Jane C. Schroeder DVM, PhD, Assistant Professor of Epidemiology, SPH

Abstract

African Americans are at greater risk of dying of prostate cancer than whites, in part because of racial differences in tumor characteristics that may reflect differences in etiologic mechanisms. Prior studies suggest that pesticide exposures may increase the risk or severity of prostate cancer, and it has been hypothesized that African Americans may be more highly exposed or susceptible to pesticide-mediated carcinogenesis than whites. The PCaP Geographic Information Study (PCaP-GIS) study will be based on existing data from approximately 450 African American and 450 white North Carolina prostate cancer patients enrolled in the North Carolina Louisiana Prostate Cancer Project (PCaP), a population-based study of racial disparities in prostate cancer aggressiveness. Community-level markers of pesticide exposures and healthcare availability will be derived using spatial statistics, and will be analyzed in combination with PCaP interview data concerning race, farming, pesticide-associated occupations and healthcare access to estimate race-specific associations between pesticides and prostate cancer aggressiveness. The PCaP-GIS will provide a unique and cost-efficient opportunity to evaluate environmental factors that may contribute to racial differences in prostate cancer severity among North Carolina men.

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New Statistical Approaches for Combining Multiple-Source Environmental Data

Principal Investigator: Amy H. Herring, PhD, Associate Professor of Biostatistics, SPH

Abstract

We propose to develop statistical methodology that allows researchers to incorporate latent variables, useful in summarizing high-dimensional exposures, and complex spatial correlation structures in one common framework. This work is challenging methodologically due to non-normal measured variables, spatial misalignment, and the need for spatial change of support techniques. In addition, we will incorporate methods for measurement error and uncertainty analysis. We will apply these methods to a study of physical activity in women after pregnancy, exploring whether air pollution levels, meteorological variables, and the built and perceived environment (e.g., conduciveness of neighborhood to physical activity) affect physical activity levels. These methods are widely applicable to other settings, including the National Children’s Study, e.g. in the planned analysis of indoor exposures and environment, air pollution, and childhood asthma incidence and exacerbations. These methods will be applicable when researchers wish to combine spatial methods with hierarchical or random effects models and thus have far-reaching potential applications.

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Environmental Agents and Early Onset of Aging and Age-Related Diseases

Principal Investigator: Paul Chastain II, PhD, postdoctoral research associate, Department of Pathology and Laboratory Medicine

Abstract

Cells are continuously exposed to environmental agents that directly or indirectly create reactive oxygen species (ROS). When the cell’s capacity to eliminate ROS is overwhelmed, oxidative stress occurs and results in widespread oxidation of proteins and DNA. This macromolecular oxidation process has been implicated in the developments of cancer, degenerative diseases, and aging. However, the mechanism by which ROS promote the onset of disease and aging has not been identified. Recently, we found that a large proportion of oxidative damage in the form of abasic (AP) sites occurs non-randomly in cellular DNA. In this pilot project we propose to determine the location of non-random AP sites in low-passage and high-passage/near senescence cells using microarray technology and bioinformatics; establish whether environmentally-induced oxidative stress increases the number of non-random sites; and search for common and/or unique sequence features among genomic regions that contain non-random sites. These findings may allow us to correlate exposure of young cells to environmental agent promoting oxidative damage to premature aging and disease, and exposure of old cells to the same agents to accelerated induction of disease.

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Dietary a-tocopherol Supplementation as a Way to Decrease Neuroinflammation Following Exposure to Environmental Agents

Principal Investigator: Patricia A. Sheridan, PhD, postdoctoral fellow, Department of Nutrition

Abstract

Herpes simplex virus encephalitis (HSE) is the most common fatal sporadic encephalitis in humans. Treatment with anti-viral medication such as acyclovir, decreases HSE associated mortality from 70% to 20%, however, only 38% of HSE patients recover to normal function. HSE, primarily caused by herpes simplex virus (HSV)-1 infection of the brain, results in increased levels of oxidative stress including production of reactive oxygen species, free radicals and nitric oxide and significant neuroinflammation. The most biologically active and most abundant form of vitamin E (VE) is a-tocopherol. In cellular membranes, VE prevents lipid peroxidation by scavenging free radicals and functions as an anti-oxidant. Supplementation with VE has been shown to decrease immunosenescence, improve immune function and may be neuroprotective. In this proposal we will test the hypothesis that dietary VE supplementation: 1) will decrease symptoms of HSE, viral load and spread, and decrease the production of pro-inflammatory cytokines in the brain of experimentally infected mice and 2) decrease HSE symptoms and inflammation by decreasing oxidative stress, lipid peroxidation and cyclooxygenase (COX)-2 levels and subsequent prostaglandin (PG) E2 production.

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Comparison of Two Measures of Oxidative DNA Damage, the Comet Assay and 7-hydroxy-8-oxo-2-deoxyguanosine, in Healthy African American and White Adults

Principal Investigator: Jessie Satia, PhD, MPH, Assistant Professor of Epidemiology and Nutrition

Abstract

Development and optimization of intermediate biomarkers of cancer is crucial for identifying individuals at elevated risk. Oxidative DNA damage, a significant contributor to the development of many cancers, is commonly measured by direct measurements of DNA fragmentation (e.g., alkaline Comet assay) and indirect measurements based on biomarkers of DNA damage (e.g., 7-hydroxy-8-oxo-2′-deoxyguanosine (8-oxo-dG)). However, there is limited data comparing the concordance of these methods. Using data from a population-based study of associations between antioxidant nutrients and oxidative DNA damage in 165 African American and White adults in North Carolina, we are requesting funds to perform 8-oxo-dG assays as a second measure of oxidative DNA damage (along with the Comet assay) and compare the degree of agreement between these methods, as current funding only permits the Comet assays. Given the absence of solid data comparing these methods and their frequent use in research investigations, the larger sample and diverse population in this study would be useful in potentially identifying or confirming the absence of an association. Therefore, this project would represent a major methodological advance in this area of research.

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Measuring protein adducts in dried blood spots

Principal Investigator: S. M. Rappaport, Ph.D., Professor of Environmental Sciences and Engineering

Abstract

The purpose of this pilot project is to develop an assay for protein adducts of environmental toxicants in dried blood spots (DBS). Because DBS are considerably simpler to obtain and store than venous blood samples and are routinely collected from live births throughout the U.S., the ability to measure protein adducts in DBS would generate opportunities for molecular epidemiology studies. While the ultimate goal of our work will be to measure protein adducts from different environmental toxicants, here we seek proof of concept by focusing upon hemoglobin (Hb) and albumin adducts of 1,4-benzoquinone (1,4-BQ), a metabolite thought to be responsible for the leukemogenicity and hematotoxicity of benzene. We have measured these adducts (designated 1,4-BQ-Hb and 1,4-BQ-Alb) in human blood from benzene exposed workers, from control workers and from human volunteers. In order to extend this method to DBS, we must develop new procedures for isolating small amounts of Hb and albumin and for quantifying 1,4-BQ-Hb and 1,4-BQ-Alb at trace levels. We will develop these procedures using GC- or LC-MS/MS in collaboration with the Biomarkers Facility Core.

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A novel function of a translesion DNA polymerase against DNA-protein crosslinks

Principal Investigator: Jun Nakamura, D.V.M., Ph.D., Research Assistant Professor of Environmental Sciences and Engineering

Abstract

Cellular DNA is continuously exposed to endogenous and exogenous agents that produce DNA-protein crosslinks (DPCs). While the biological significance of DPCs have not been fully investigated, DPCs likely interrupt DNA replication, repair, recombination, transcription, and chromatin remodeling. Interestingly, epidemiological studies have reported a positive association between basal levels of DPCs and the incidence of breast cancer. Many low fidelity translesion DNA polymerases involved in the bypass of DNA lesions have been recently discovered in eukaryotic cells. The various insertive functions of these polymerases allow them to bypass DNA lesions but cause the main replicative polymerase to stall. Strikingly, our preliminary results showed that cells deficient in one of the translesion DNA polymerases are hypersensitive to physiological levels of endogenous bifunctional aldehydic compounds. Based on this evidence, we hypothesize that a single specific polymerase is involved in the removal of DPCs during the course of DNA repair or replicative bypass of these DNA lesions. This hypothesis will be extensively tested using a variety of genetic and functional approaches. The project will provide critical information regarding the mechanisms by which cells tolerate DPCs and the roles that translesion synthesis DNA polymerases have in counteracting DPCs induced under physiological conditions in order to maintain genomic integrity.

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Health effects of air pollution on lupus (HEAPL): a nested pilot study on a community-based panel of lupus patients and controls

Principal Investigator: Jiu-Chiuan (JC) Chen, MD, MPH, Sc.D., Assistant Professor of Epidemiology

Abstract

In the proposed pilot panel of 265 patients with systemic lupus erytheomatosus (SLE) and 302 community-based controls from the Carolina Lupus Study, we will use geocoded residential information, air pollution data recorded in EPA’s Aerometric Information Retrieval System, and other relevant spatiotemporal data to construct Bayesian Maximum Entropy (BME) models and estimate personal exposures to ambient PM10, PM2.5 (particulate matter with aerodynamic diameter =10 and 2.5µm respectively), and ozone. Inflammatory responses to air pollution (as assessed by increases in C-reactive protein [CRP]) will be compared between SLE patients and controls. Among SLE patients, clinical markers of disease severity and activity will be examined for their associations with exposures to air pollution. We will employ generalized linear models to estimate effects of air pollution on CRP and indicators of disease severity and activity, adjusting for relevant confounders. By demonstrating a sophisticated modeling approach applicable to studying other environmental determinants of health outcomes in North Carolina, our pilot study will generate preliminary but essential data to evaluate the susceptibility of SLE patients and the potential effects of air pollution on rheumatoid disorders.

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Regulation of neuronal apoptosis: focus on identifying novel functions of MicroRNAs

Principal Investigator: Mohanish Deshmukh, Ph.D., Assistant Professor of Cell and Developmental Biology, School of Medicine and Neuroscience Center

Abstract

Exposure to environmental neurotoxins or other pathological situations can induce neurodevelopmental and neurological deficits by triggering excessive neuronal death by apoptosis. Therefore, understanding how neurons undergo apoptosis is essential for developing therapeutic strategies aimed at preventing neuronal death.

Recently, microRNAs (miRNAs) have been discovered as important regulators of many biological processes. MiRNAs are short, noncoding RNA molecules that negatively regulate protein expression. The focus of this pilot proposal is to determine whether miRNAs regulate apoptosis in mammalian neurons (using primary mouse sympathetic and cerebellar granule neurons). In Specific Aim 1, we will examine whether two known antiapoptotic Drosophila miRNAs and the cancer-associated mammalian miRNAs inhibit apoptosis in mammalian neurons. If these miRNAs are found to be neuroprotective, we will examine the mechanism by which they inhibit neuronal apoptosis. In Specific Aim 2 we will take an unbiased approach to discover novel miRNAs that regulate neuronal apoptosis by using a custom miRNA microarray to identify miRNAs that are transcriptionally induced in neurons undergoing apoptosis. Subsequent experiments will focus on identifying the mechanism by which these miRNAs promote neuronal apoptosis.

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Effects of posttranslational modifications of P450 2E1 on butadiene metabolism in vitro

Principal Investigator: Gunnar Boysen, Ph.D., Postdoctoral Fellow, Environmental Sciences and Engineering

Abstract

Butadiene (BD) is a multi-species, multi-site carcinogen in rodents, with mice being a much more sensitive species than rats. This proposal evaluates whether phosphorylation or covalent binding of activated metabolites alter the catalytic activity of P450 2E1 and are responsible for the differences in BD metabolism and tumorigenesis. State of the art mass spectrometry approaches will be used to identify covalent binding sites of 1,2-epoxybutene (EB) and 1,2;3,4-diepoxybutane (DEB) and phosphorylation sites. Preliminary data have already demonstrated distinct binding sites of EB. After these posttranslational modifications have been identified, their effects on catalytic activity will be determined. Therefore, it is proposed to exam the kinetics for the conversions of BD to EB and EB to DEB. To normalize and verify these results, the catalytic activities will also be determined for p-Nitrophenol hydroxylation and Chlorzoxazone 6-hydroxylation. If posttranslational modifications alter the catalytic activity of P450 2E1 in vitro, these studies will establish a novel level of P450 2E1 regulation and it is proposed to extent this project to in vivo samples from tissues of BD treated mice and rats.

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Development of an infectivity assay for noroviruses using tissue engineering

Principal Investigator: Jan Vinjé, Ph.D., Research Assistant Professor, Environmental Sciences and Engineering

Abstract

Noroviruses (NoVs) are the leading causes of acute gastroenteritis in humans. The inability to replicate NoVs in cell culture has been a serious barrier to understanding their basic virology. Recently, it has been shown that humans possessing certain ABH histo-blood group antigens are susceptible to norovirus infection and that humans lacking these antigens cannot be infected. In addition to the need for the presence of an appropriate ABH histo-blood group antigen on cells two additional restrictions could be: 1) the need to use authentic gastrointestinal cells and culture conditions as hosts, since NoVs are observed in vivo in gastrointestinal tissue (small intestine), and 2) a possible need for the viruses to replicate in a stem/progenitor cell compartment and then be able to mature along with the host cells to an appropriate adult cell stages to yield the mature virus. We propose to test the relevance of ABH histo-blood group antigen, the use of gastrointestinal-derived cell types, and/or a lineage-stage culture model system to see if any (or all) of the variables are critical to enable NoVs to replicate ex vivo.

If our approach is successful, this research will become a cornerstone for NoV research. A culture system for human NoV would represent a major advance for the field, and therefore should lead to competitive applications and funding opportunities.

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Modeling complex exposures and time-to-event data

Principal Investigator: Amy H. Herring, Assistant Professor, Biostatistics

Abstract

Investigators often go to great lengths to obtain careful, detailed measures of exposure, which may have multiple dimensions and may change over time, e.g. diet, stress, or blood pressure. In assessing the association between an individual’s exposure history and the time or rate of occurrence of a health event, it is important to reduce the dimensionality of this multivariate exposure history data in order to increase statistical power. Although replacing the multivariate exposure information with a simple summary, as is typically done in practice, can sometimes improve interpretability and statistical power, it is typically not clear how best to summarize the information at hand. In addition, reducing detailed data into naive summaries often runs counter to the study goals of obtaining the most accurate assessment of exposure possible. We are interested in developing and applying statistical methods, allowing evidence-based summaries to be constructed objectively in a manner that maximizes information about the outcomes of interest.

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Specific fatty acids alter cellular responses to environmental agents

Principal Investigator: Rosalind A. Coleman, Nutrition; Co-P.I.: Melinda Beck, PhD

Abstract

Lipid rafts are specialized detergent-insoluble regions on cell plasma membrane that are entry points for viruses including influenza and HIV, and a variety of toxic proteins including bacterial toxins, prions, and plant toxins such as the potential bioterrorism agent ricin. Lipid rafts are comprised of lipids and proteins that differ from the bulk components of the plasma membrane. They contain more cholesterol and sphingomyelin as well as specific proteins, many of which are anchored by saturated fatty acids. Polyunsaturated fatty acids (PUFA) modify lipid rafts and alter their ability to regulate the entry of environmental agents, including viruses and toxins. Recently reported studies suggest that exposure to polyunsaturated fatty acids can alter the protein composition of these regions. This proposal uses influenza virus infection to model the changes in the lipid and protein composition of cell membranes and their lipid raft domains.

We will use a knockout mouse with aberrant lipid composition and cultured cells exposed to different fatty acids in order to delineate alterations in lipid raft function that may affect the entry of environmental toxins. We develop methods for phospholipid fatty acid analysis. These methods will be used to analyze the fatty acid composition of phospholipids from a variety of samples: influenza virus that have been isolated from GPAT KO and WT mice, lung tissue and macrophages from these mice, PUFA-treated cells infected with influenza virus, and virus itself after replication in cells exposed to different fatty acids that alter viral replication. Proteins present in lipid rafts will be identified before and after the lipid modifications, as will the viral coat proteins hemaglutinin and neuraminidase. We will also analyze macrophage eicosanoids from KO and WT mice. These studies will delineate the changes that occur in lipid rafts that restrict entry of environmental agents and toxins into cells.

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Genetic analysis of transcriptional regulation in liver

Principal Investigator: Ivan Rusyn, Assistant Professor, Environmental Sciences and Engineering

Abstract

Exposure to environmental agents is inevitable and all individuals encounter various chemicals, doses and times of exposure throughout the lifetime. The end-effects usually vary greatly across a population of similarly exposed individuals. Genetic variation in commonly used animal models, such as mice, is as complex as in man, but the availability of genetically controlled “inbred” animal populations provides a significant benefit impossible to achieve in humans. These genetic tools become even more powerful as the complete haplotype maps of many commonly used mouse inbred strains become available. This application aims to combine microarray-based assays of mRNA level with gene mapping methods to detect polymorphic loci that co-regulate extensive molecular networks in mouse liver. Our specific experimental approach is to analyze the variation in basal gene expression among genetically controlled mice by using a panel of BXD recombinant inbred (RI) strains derived from C57BL/6J and DBA/2J. Steady-state mRNA levels will be measured and used as quantitative traits to map potential susceptibility and resistance genes and, more importantly, networks of gene interactions related to liver injury susceptibility. The latter will be achieved by combining the data from this project with an already established web interface containing genotypes, phenotypic data and already collected gene expression measurements from other organs (e.g., brain) of BXD mice. This will allow exploration of networks of correlated molecular and phenotypic traits, and to uncover shared upstream modulation of expression in several diverse tissue types targeting genes known to be potential targets for toxicants. Collectively, this project will serve as a springboard to future studies on the genetic dissection of novel genotype-phenotype correlations associated with in vivo responses to toxic agents that affect the liver. This project will allow us to collect pilot data necessary to infer genetic causes for phenotypic variation across RI strains. Furthermore, this approach will serve as an example of how genetics, animal models, genomics and traditional mechanistic toxicology can be combined to bring new discoveries and technical advances into the mainstream of environmental science.

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Human responses to UV-induced DNA damage

Principal Investigator: Marila Cordeiro-Stone, Ph.D., Professor of Pathology and Toxicology

Abstract

This pilot project seeks to acquire preliminary data in support of a competitive application for a program project grant on “Human responses to UV-induced DNA damage.” Four independent researchers and a junior investigator with expertise in biochemistry, proteomics, molecular biology and radiobiology will pursue interdisciplinary research projects focused on the responses of human cells to UV-induced DNA damage. Project investigators independently have established strong records of productivity and accomplishment in the fields of DNA repair and cell cycle checkpoints. The joint effort by these investigators will be directed toward developing in vitro systems that are amenable to biochemical manipulations and responsive to DNA damage. The primary goal will be to demonstrate that these model systems could support mechanistic studies on the primary components of DNA damage checkpoints, mainly damage sensors, mediators, signal transducers, and effectors of specific responses. The focus of this pilot project will be on the intra-S phase checkpoint and the signal transduction reactions that underlie the inhibition of replicon initiation. Synergistic approaches fostered by this pilot project will lead to a new program of study to investigate the multiple interactions among cell cycle checkpoint and DNA repair proteins in human keratinocytes and fibroblasts damaged by UV. Solar UV radiation is a ubiquitous environmental carcinogen accounting for over a million new cases of skin cancer each year. Information gained by study of human responses to UV will facilitate understanding of responses to other environmental toxins that damage DNA and induce cancer at internal sites.

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Fluorescence in situ hybridization assays for Non-Hodgkin’s Lymphoma translocations using tissue microarrays: a validation study

Principal Investigator: Jane C. Schroeder, D.V.M., Ph.D., Assistant Professor of Epidemiology

Abstract

Defining non-Hodgkin’s lymphoma (NHL) case-subtypes by acquired chromosomal translocations has been advocated to increase the etiologic specificity of NHL outcomes for epidemiologic research. To realize the full potential of this approach, cases need to be reliably subtyped according to multiple translocations, in studies large enough to allow associations to be estimated with reasonable precision. New fluorescence in situ hybridization (FISH) assays can detect common NHL translocations, including t(14;18), t(8:14), t(11;14), BCL6 and ALK translocations, with sensitivity as good or better than polymerase chain reaction assays or karyotyping. Using new tissue microarray techniques (TMA), over 100 individual tumor samples can be arrayed on a single slide and assayed using the quantity of reagents normally required for two individual samples. TMAs may greatly improve efficiency and reduce the cost of FISH for NHL translocations, but these specific assays have not been validated for use on TMAs. Our goal is to validate FISH NHL translocation assays for TMAs, using anonymous NHL tumor blocks obtained from the UNC Hospitals archive. Cases will be selected from histologic subtypes likely to be positive for each translocation of interest, and full diameter sections will be used to identify positive cases. A TMA will be constructed using a minimum of two cores per case, taken from representative areas identified by a collaborating hematopathologist. The proportion of positive cases identified on TMA will be used to determine the sensitivity of TMA FISH for each translocation. This work will increase the investigators’ experience with FISH assays and TMA construction techniques relevant to a proposed study of subtype-specific associations among cases enrolled in a large population-based study conducted by the National Cancer Institute. TMA and standard sections are available for these cases, which have not been subtyped on a molecular level. Of particular interest will be associations with pesticides that were related to t(14;18) NHL in a previous study. In addition, we hope to evaluate relations between translocation subtypes and gene polymorphisms that may affect immunoglobulin gene rearrangement and double strand DNA repair.

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Selenium status and viral mutations: measurement of oxidative stress

Principal Investigator: Patricia Gallagher, Research Assistant Professor of Nutrition

Abstract

Previous work in our laboratory has demonstrated that a host deficiency in the trace mineral selenium (Se) can influence the genome of a viral pathogen. A normally avirulent coxsackievirus becomes virulent and a normally mild strain of influenza virus becomes highly virulent in Se-deficient animals. Once the mutations in the viral pathogen has occurred, even animals with normal Se status were now vulnerable to the newly pathogenic strains. Se is an essential component of glutathione peroxidase, an antioxidant enzyme. Thus, a deficiency in Se leads to decreased function of this enzyme. We hypothesize that oxidative stress is increased in Se-deficient hosts, due to a deficiency in glutathione peroxidase activity. One difficulty in understanding the mechanism by which oxidative stress affects the viral genome is the ability to measure the oxidative stress in tissues at the site of infection. The specific aim of this proposal is to develop and validate a measure of oxidative damage in virally infected tissue in order to directly correlate increased oxidative stress with viral mutations. F2-isoprostanes are novel biomarkers of in vivo lipid peroxidation. F2-isoprostanes are formed in situ on membrane phospholipids by free radical mediated, non-enzymatic peroxidation of membrane bound polyunsaturated fatty acids. Several studies have shown that F2-isoprostanes are increased during oxidative stress. Morrow et al. developed a reliable and accurate gas chromatographic mass spectrometric (GC/MS) assay for F-2 isoprostanes, however it requires extensive purification and derivitization and costly specialized equipment not available in many laboratories. Enzyme immunoassay kits (EIA) for 8-iso-PGF2a are commercially available, however the accuracy and reliability of these kits has not been completely validated by simultaneous GC/ MS assay. Our aim is to develop a precise EIA assay for measuring tissue levels of F2-isoprostanes by improving the extraction procedure. The ability to reproducibly and accurately measure F2-isoprostanes will allow us to directly relate the oxidative stress status of the host with an increase in viral mutations. In addition, this method can be used for future studies examining the effects of other oxidative stressors, including environmental factors.

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Binding network of Pol Eta in DNA damage responses

Principal Investigator: Marila Cordeiro-Stone, Ph.D., Professor of Pathology and Toxicology

Abstract

Maintenance of genetic stability depends on an integrated system of biochemical pathways that prevent DNA damage, remove DNA lesions, coordinate cell cycle checkpoints and DNA repair, reduce the probability of fixing errors during bypass replication, or repair secondary lesions in DNA synthesized from damaged templates. Inactivation of any of these pathways increases disease susceptibility. This pilot project is focused on post-replication repair, which includes mechanisms of DNA lesion bypass and elimination of daughter strand gaps in nascent DNA. DNA polymerase h plays an important role in mutation avoidance by catalyzing efficient and accurate replication past UV-induced cyclobutane pyrimidine dimers. Absence of such activity underlies the cancer-prone syndrome xeroderma pigmentosum variant. Although this is a rare autosomal recessive disease, mutations in other gene products that regulate or interact with pol h might also increase cancer risk. This project will use a novel proteomic approach to identify proteins that bind to pol h. These proteins could represent functional partners of pol h during catalysis of translesion synthesis, and/or negative regulators that block access of this bypass polymerase to undamaged DNA, which is replicated by higher fidelity DNA polymerases a and d. Recombinant pol h, tagged with six histidines and a c-myc epitope, will be modified with chemical groups that can be photo-activated to cross-link binding proteins. We will demonstrate that the modified pol h retains DNA polymerization and lesion bypass activities by using primer extension assays (single enzyme catalysis) and SV40 origin-dependent in vitro replication of closed circular duplexes (requiring interaction of pol h with other replication factors). Pol h partners will be identified by mass spectrometry of tryptic fragments of the proteins covalently bound to it by the photo cross-linking reaction. Once the proteins making direct contact with pol h are identified, these primary partners can be used to search for other proteins that might participate in a pol h translesion synthesis complex. Starting with pol h as the initial bait, this pilot project has potential to develop into a major research initiative leading to the characterization of complex modules of proteins involved in post-replication repair and other DNA damage responses.

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Toxicokinetic modeling using space/time mapping of exposure fields with existing health outcome data in a measurement error model

Principal Investigator: Marc Serre, Ph.D., Research Assistant Professor, Department of Environmental Science and Engineering

Abstract

We are interested in using the Bayesian Maximum Entropy (BME) space/time modeling framework and it’s numerical library, BMElib, to obtain a space/time model providing a representation of the distribution of the exposure to an environmental contaminant across space and time. Using the BME model we then obtain estimates of the exposure to the contaminant at the location where existing health outcome data is available. For each of the estimated exposure value we also obtain the associated mapping uncertainty (i.e. interpolation error). This provides the dataset of exposure-health outcomes from which associations can be analysed. The research question that we are interested in is to account for the estimation uncertainty of the estimated exposure data in the analysis of association between exposure and health outcome. The estimation uncertainty is provided by the BME method and it can be treated as a measurement error for the risk factor (exposure to contaminant), which will be incorporated in the analysis of association through a Measurement Error Model (MEM). The quantification for the association between the error prone risk factor (the estimated exposure to dust) and the health outcome will be attenuated if the measurement is not taken in consideration. Therefore accounting for the measurement error allows correcting for this attenuation. This research will provide a novel and rigorous framework for linking existing health outcome data with exposure information collected in a neighbouring monitoring network, and analysing the association between exposure and health outcome. This new framework will combine areas of excellence of the toxicokinetic susceptibility research core, namely in space/time modelling and in analysis of exposure-health effect association with measurement errors, and should lead to applications and funding opportunities in exposure studies and toxicokinetic modelling.

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Base excision repair polymorphisms and oxidative stress

Principal Investigator: Jun Nakamura, D.V.M., Ph.D., Research Assistant Professor, Department of Environmental Science and Engineering

Abstract

The purpose of this project is to test the functionality of base excision repair SNPs in human cultured cells under oxidative conditions. Oxidative DNA lesions induced by oxygen free radicals such as superoxide and hydroxyl radicals appear to be repaired predominantly by base excision repair pathway. While genotype analysis of single nucleotide polymorphisms (SNPs) has identified base excision repair polymorphisms, the phenotypic significance of these polymorphisms has not been fully characterized. Since X-ray repair cross-complementing group 1 (XRCC1) variants at 399 are associated with increased cancer susceptibility and interact with other base excision repair enzymes such as apurinic/apyrimidinic (AP) endonuclease 1 (APE1), we hypothesize that XRCC1 (Arg->Gln) variants will reduce the repair efficiency of DNA lesions induced by oxidative stress. To test this hypothesis, we propose to examine the incidence of major base excision repair SNPs (8-hydroxyguanine DNA glycosylase: Ser326Cys; APE1: Asp148Glu; DNA polymerase: Arg228Leu; XRCC1: Arg399Gln) in peripheral lymphocytes collected from 200 women from a currently on-going breast cancer-control study. We will then quantitate the number of AP sites and oxidative base lesions in these lymphocytes to test the association between the number of oxidative DNA lesions in lymphocytes and XRCC1 399 SNP. We will further test if a reduced capacity for base excision repair pathways towards DNA lesions induced by oxidative stress exist in human lymphoblastoid cell lines established from lymphocytes with either XRCC1 399Gln or 399Arg homozygotes. Using our novel AP site assays combined with oxidized base measurements, the repair kinetics of oxidative DNA lesions and their repair intermediates will be compared between wild type and variant groups. The information on base excision repair obtained from this project will provide critical evidence regarding cancer-susceptible populations who repair oxidized DNA lesions less efficiently.

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Functional ATM Polymorphisms

Principal Investigator: William K. Kaufmann
Biostatistics Consultant: Fei Zou

Abstract

This project will determine whether certain missense mutations in ATM produce a trait of G2-irradiation chromosomal hypersensitivity. A case-control study is underway at UNC-CH in which peripheral blood lymphocytes are tested for mutations in ATM and chromosomal hypersensitivity following treatment with ionizing radiation in G2. Cases represent women with newly diagnosed breast cancer and controls represent hospital patients matched for age and race but without cancer. To date ATM mutations and chromosomal hypersensitivity have been detected in both cases and controls. The frequency of the traits is increased about three-fold in cases relative to controls suggesting that ATM mutations and chromosomal hypersensitivity predispose to development of breast cancer. Several patient samples have been identified which display both mutations in ATM and chromosomal hypersensitivity. To prove that the trait of chromosomal hypersensitivity seen in these samples was a consequence of the mutation in ATM, a functional assay must be developed. We propose to clone ATM cDNA into a retroviral vector for efficient, stable transduction and expression of ATM in ATM-null cell lines. Expression of ATM in AT cells should restore DNA damage checkpoint functions and reduce chromosomal sensitivity to irradiation. Having a system in which ATM function can be demonstrated by restoration of checkpoint function and chromosomal repair, we will test the effect of introducing specific mutations in the ATM cDNA by site-directed mutagenesis. Missense mutations that were seen in lymphocytes with chromosomal hypersensitivity will be transduced alone or in combination with wildtype ATM. We will determine whether the missense mutations reduce the ability of ATM to enforce cell cycle checkpoints and enhance repair of chromatid breaks, and whether the mutations can override the function of wildtype ATM (dominant negative). This effort will help to define functional polymorphisms in a DNA repair gene.

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The Sea Urchin Embryo as a Model for Developmental Susceptibility to Environmental Neurotoxins

Principal Investigator: Jean Lauder
Biostatistics Consultant: Donglin Zeng

Abstract

The purpose of the proposed project is to develop an animal model for in vivo screening of environmental neurotoxicants, using the early sea urchin embryo. The proposed studies will investigate mechanisms underlying embryotoxic effects of the organophosphate insecticide, chlorpyrifos (cpf). We aim to demonstrate that this invertebrate model allows identification of cellular targets and cellular/molecular mechanisms of cpf embryotoxicity that are relevant to neurotoxic effects of cpf on mammalian brain development. The Specific Aims are designed to test the hypothesis that embryotoxic effects of cpf on early sea urchin embryos are due to its ability to significantly increase intracellular calcium (Ca2+) by activation of nACh receptors (nAChR) and Ca2+ channels. We will determine whether the protective actions of lipophilic acetylcholine analogs, Ca2+ channel blockers or nAChR antagonists against embryotoxic effects of cpf involve prevention of increased intracellular Ca2+. Specifc Aim 1 will determine whether cellular substrates for chlopyrifos (cpf) embryotoxicity during vulnerable periods of early sea urchin development involve increased intracellular Ca2+. Specific Aim 2 will determine whether protective actions of lipophilic acetylcholine analogs, Ca2+ channel blockers or nAChR antagonists are mediated by their ability to block cpf-induced increases in intracellular Ca2+. These studies will provide clues as to possible cellular/molecular mechanisms of cpf embryotoxicity and provide evidence regarding the relevance of these mechanisms to cpf actions on mammalian brain development Future studies will utilize this information to delve deeper into cellular/molecular mechanisms. Ultimately, this simple animal model could provide the opportunity for development of pharmacologic agents useful for prevention of neurotoxicant effects on pregnancy outcome and brain development.

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Dermal Exposure to 1,6-Hexamethylene Diisocyanate Spray Painting Operations

Principal Investigator: Leena A. Nylander-French
Biostatistics Consultant: Lawrence Kupper

Abstract

Development and application of monitoring methods for measurement of 1,6-hexamethylene diisocyanate (HDI) exposure to spray painters are required to estimate the risk for dermal sensitization and development of HDI induced occupational asthma. The importance of this problem and the availability of a well-defined “at-risk” study population warrant the comprehensive investigation of multiple routes of HDI exposure. We will recruit 15 spray painters from an U.S. Marine/Navy base located in North Carolina for this pilot project. Personal breathing-zone exposure to HDI will be measured using the Iso-CheckT sampler. Dermal exposure will be measured using a non-invasive method for tape-stripping skin (stratum corneum) and quantification by analytical chemistry. Urine samples will be collected for analysis of 1,6-hexamethylene diamine (HDA) as a marker for exposure and systemic absorption. Together, these data will allow us to correlate dermal exposure to systemic exposure and to determine the significance of dermal exposure. In addition, we will synthesize the S-cysteinyl-(6-aminohexamethylene)carbamyl conjugate of the keratin-1 (KRT1) and keratin-10 (KRT10) fragments with the structures Gly3ArgPheSer2CysGly2 and Gly4CysGly5, respectively. If successful, these conjugates will be used to develop an enzyme-linked immunosorbent assay (ELISA) to determine cysteinyl-KRT1 and -KRT10 protein adducts as biomarkers of exposure to HDI in a future study. ELISA will allow sensitive detection of HDI-adducted keratin as a biomarker to measure HDI absorption and the potential systemic bioavailability from the skin. Correlation between the biologically available dose and the dose measured in the biological specimen is critical to developing an understanding of the role of isocyanate exposure through the skin. The results obtained with this study will provide us essential preliminary data for a development of comprehensive research proposal to investigate the relationship between dermal exposure, sensitization, and potential development of occupational asthma in this “at-risk” population.