Project 1: Determination of endogenous apurinic/apyrimidinic sites in genomic DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine in brain and nasal respiratory and olfactory tissues of healthy canines exposed to air pollutants
Project 2: Fluorescence in situ hybridization assays for Non-Hodgkin’s Lymphoma translocations using tissue microarrays: a validation study
Project 3: Selenium status and viral mutations: measurement of oxidative stress
Project 4: Binding network of Pol Eta in DNA damage responses
Project 5: Toxicokinetic modeling using space/time mapping of exposure fields with existing health outcome data in a measurement error model
Project 6: Base excision repair polymorphisms and oxidative stress
Project: Determination of endogenous apurinic/apyrimidinic sites in genomic DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine in brain and nasal respiratory and olfactory tissues of healthy canines exposed to air pollutants
Exposure to complex mixtures of air pollutants produces inflammation in the upper and lower respiratory tract. Because the nasal cavity is a common portal of entry, respiratory and olfactory epithelia are vulnerable targets for toxicological DNA damage. The purpose of this study- using a highly sensitive slot blot assay- is to determine the number of endogenous apurinic/apyrimidinic (AP) sites in genomic DNA and to quantitate 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by HPLC in olfactory and respiratory nasal mucosae, olfactory bulb, entorrhinal, frontal, medial temporal, hippocampal, parietal, and cerebellar tissues from 16 healthy well nourished mongrel canine residents in Southwest Metropolitan Mexico City (SWMMC), a highly polluted urban region. Findings will be compared to those in 14 age-matched dogs from Tlaxcala, a less polluted control city. We hypothesize that AP sites and 8-oxodG will be increased in animals with a life exposure to air pollutants, particularly in the olfactory-limbic associated structures, and nasal tissues. We will also attempt to demonstrate an association between the DNA damage and the neuropathological findings, specifically the expression of NF-kB and iNOS. Persistent respiratory inflammation and deteriorating olfactory and respiratory barriers, the production of proinflammatory cytokines, the damage to the blood-brain barrier(BBB),and the microcirculation by the iNOS may play a role in the increased DNA damage to the involved structures and the neuropathology observed in the brains of these highly exposed canines. The innovation of this project is not only to study naturally exposed animals and use highly sensitive assays, but also to establish an association between the DNA damage endpoints and the neuropathological and immunohistochemistry findings representing critical information regarding neurodegenerative diseases. Neurodegenerative disorders such as Alzheimer’s may begin early in life with air pollutants playing a crucial role.
Project: Fluorescence in situ hybridization assays for Non-Hodgkin’s Lymphoma translocations using tissue microarrays: a validation study
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.
Project: Selenium status and viral mutations: measurement of oxidative stress
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.
Project: Binding network of Pol Eta in DNA damage responses
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.
Project: Toxicokinetic modeling using space/time mapping of exposure fields with existing health outcome data in a measurement error model
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.
Project: Base excision repair polymorphisms and oxidative stress
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.
Funded by NIEHS Grant # P30 ES010126