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Evaluation of the Functionality of XRCC1 Polymorphisms Using Aldehydic Deoxyribose Lesions
EPA Grant Number: FP916436Title: Evaluation of the Functionality of XRCC1 Polymorphisms Using Aldehydic Deoxyribose Lesions
Investigators: Pachkowski, Brian F.
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Manty, Dale
Project Period: January 1, 2004 through December 31, 2006
Project Amount: $93,570
RFA: STAR Graduate Fellowships (2004)
Research Category: Academic Fellowships , Fellowship - Toxicology , Health Effects
Description:
Objective:Abatement of DNA damage by repair proteins is critical for maintaining genomic stability and preventing adverse health effects. The base excision repair (BER) pathway is one of the protective mechanisms against the constant generation of endogenous mutagens as well as environmental chemicals. It is believed that the orchestrated events of BER are coordinated by the protein interactions of the non-enzymatic, scaffold protein XRCC1. Single nucleotide polymorphisms (SNPs) at codons 399, 280, and 194 of XRCC1 have been detected in the human population and have been suspected of potentially increasing susceptibility to chronic diseases. Although not definitive, population-based studies have shown a link between specific XRCC1 SNPs and cancer. To get a more transparent understanding of the molecular relationship between XRCC1 variants and cancer susceptibility, mechanistic data are needed to clarify the biological significance of polymorphic gene products. The objective of this research is to monitor the persistence of DNA lesions and their repair intermediates in mammalian cells expressing variant forms of XRCC1 after exposure to genotoxic agents.
Approach:To evaluate the functionality of XRCC1 SNPs, mammalian cells will be exposed to alkylative or oxidative conditions with subsequent comparisons of endpoint measurements. Isogenic Chinese hamster ovary (CHO) cell lines expressing various forms of XRCC1 protein will be the model for characterizing the influence of XRCC1 variants on DNA repair. Among these cell lines are DNA repair proficient AA8 cells and repair deficient EM9 cells that produce a truncated XRCC1 protein, a theoretical knock-out model. In addition, EM9 cells transfected with human wild-type XRCC1 (EM9-HX) will be compared to EM9 cells expressing either the 399 (EM9-HX399), 280 (EM9-HX280), or the 194 (EM9-HX194) human variant protein as well as to an EM9 cell line expressing an empty vector (EM9-V). Cell lines will be challenged by methyl methanesulfonate or hydrogen peroxide to induce DNA damage that is repaired by XRCC1 mediated pathways. In addition, a set of environmentally relevant chemicals will be investigated to demonstrate their effect on mammalian cells expressing XRCC1 variants. A battery of assays will be employed to measure endpoints including cytotoxicity, DNA strand break formation, and damage to DNA bases and deoxyribose. A fluorometric, propidium iodide based cytotoxicity assay will be used to establish appropriate dose ranges for genotoxicity experiments. A novel, real-time single strand break (SSB) assay to monitor intracellular NAD(P)H levels, an indicator of poly(ADP-ribose)polymerase (PARP) activation and SSB repair, will be used to evaluate the affects of XRCC1 variants on DNA repair. Doses that generate significant differences in SSB formation between repair proficient and deficient or variant cell lines will be selected for additional exposures. DNA from the CHO cell lines that have been exposed to optimized doses of genotoxicants will be analyzed for a spectrum of DNA lesions and their repair intermediates. A general measure of DNA damage in the form of aldehydic DNA lesions, which include abasic sites, deoxyribose lesions, and potentially aldehydic base lesions, will be determined by an ELISA-like slot blot assay. Glycosylase coupled slot-blot and immuno-slot-blot assays will be used to measure oxidized and alkylated DNA bases. The DNA lesion data for repair proficient cells will be compared to values from deficient or variant cell lines to determine if XRCC1 genotype influenced the persistence of these lesions. These exposure scenarios will characterize the functionality of XRCC1 variants as reflected by DNA repair proficiency in mammalian cells and identify potential modes of action for environmental contaminants.
Supplemental Keywords:fellowship, DNA damage, DNA repair, XRCC1, XRCC1 polymorphisms, cancer susceptibility, base excision repair, polymorphisms, mammalian cells, cytotoxicity, ELISA,
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ENVIRONMENTAL MANAGEMENT, Scientific Discipline, Health, RFA, Susceptibility/Sensitive Population/Genetic Susceptibility, Risk Assessment, genetic susceptability, Health Risk Assessment, Biochemistry, Genetics, carcinogenesis, genetic polymorphism, genetic research, DNA analysis, DNA damage, DNA repair, aldehydic deoxyribose lesions, chemical exposure, human susceptibility, biological contol agents, cancer risks, health risks