Browsing by Subject "DNA adducts"

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    Biological Consequences Of Complex Dna Lesions Induced By Bis-Electrophiles
    (2014-08) Wickramaratne, Susith
    Genomic DNA is constantly modified by bis–electrophiles which induce a wide array of DNA adducts. DNA adducts can threaten cell viability and genomic integrity by interfering with DNA replication and transcription. Unless repaired, they can be misread by DNA polymerases, leading to heritable mutations and cancer. Amongst known human carcinogens is 1,3–butadiene (BD), an important industrial chemical and environmental pollutant. BD is metabolized to reactive epoxides that induce a range of DNA adducts. In the first part of this thesis, we investigated the ability of human cells to repair three potentially mutagenic BD–adenine lesions: N 6–(2–hydroxy–3–buten–1–yl)–2 ’–deoxyadenosine, 1,N 6–(2–hydroxy–3–hydroxymethylpropan–1,3–diyl)–2 ’–deoxyadenosine and N 6–,N 6–(2,3–dihydroxybutan–1,4–diyl)–2 ’–deoxyadenosine. Repair assays using site– and stereo–specifically adducted oligodeoxynucleotides revealed that all three BD–dA adducts were recognized by base excision repair (BER) pathway. Repair inhibition by a BER inhibitor, and 5’ excision products characterized by HPLC–ESI––MS/MS analysis confirmed the involvement of BER. Exposure to bis–electrophiles or reactive oxygen species can irreversibly trap cellular proteins on DNA, forming super–bulky lesions known as DNA–protein cross–links (DPCs). The complexity and diversity of DPCs have prevented researchers from investigating their biological effects. In the second part of this thesis, we have developed bioconjugation methods to generate site–specific DPCs and examined their effects on DNA replication. Hydrolytically stable 7–deazaguanine adducted DPCs, which are structurally analogous to cellular DPCs induced by antitumor nitrogen mustards, were prepared by post–synthetic reductive amination. Further, Huisgen cycloaddition was used to generate DPCs involving the C5–thymidine of DNA. Replication bypass using human translesion synthesis polymerases η and κ revealed that large polypeptides cross–linked to either 7–deazaguanine or C5–thymidine completely blocked replication, while the corresponding decapeptide cross–links were bypassed, suggesting that bypass of DPCs in cells is mediated by proteolytic degradation of the cross–linked proteins. Steady–state kinetic studies provided evidence for the highly error–prone bypass of C5–thymidine peptide cross–links and error–free bypass of N7–guanine cross–linked peptides. Further, HPLC–ESI––MS/MS characterization of replication products of C5–thymidine peptide cross–links revealed large numbers of deletion and substitution mutations. Taken together, our data suggest that the efficiency and the fidelity of DNA replication past DPCs are dependent on the lesion size, the cross–linking site and DNA polymerase identity.
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    Mass Spectrometry Based Quantification of 1, 3-Butadiene Induced DNA Adducts: Potential Biomarkers of Cancer Risk
    (2014-11) Sangaraju, Dewakar
    Chemical carcinogenesis involves metabolic activation of carcinogens to electrophilic species which can react with important cellular biomolecules including DNA to form covalent adducts. Covalent carcinogen-DNA adducts which are not removed by DNA repair mechanisms can induce transforming mutations, ultimately leading to cancer. Hence, carcinogen-DNA adducts are deemed the ultimate biomarkers of carcinogen exposure, metabolic activation, and possibly of cancer risk. 1,3-Butadiene (BD) is a recognized human and animal carcinogen present in cigarette smoke, automobile exhaust, wood fires, and also in some occupational settings such as BD monomer and polymer plants. BD is metabolically activated by CYP2E1 to form three electrophilic epoxides: 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). EB, EBD, and DEB can modify DNA bases to form covalent DNA adducts such as N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII), N7-(2, 3, 4-trihydroxybut-1-yl)-guanine (N7-THBG) and 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD). Although BD-DNA adducts had been successfully detected and quantified in tissues of laboratory animals exposed to relatively high concentrations of BD ( ≥ 6.25 ppm), they had not been previously quantified in humans, preventing their use as biomarkers of BD exposure, metabolic activation, and cancer risk. The main purpose of this research was to develop ultra-sensitive bioanalytical methodologies based on mass spectrometry to enable the detection and quantitation of BD-DNA adducts in animals treated with sup-ppm levels of BD and in exposed human populations. In Chapter 2 of the thesis, a novel nanoHPLC-nanoESI+-MS/MS method was developed for sensitive, accurate, and precise quantitation of BD-induced guanine-guanine cross-links (1,4-bis-(guan-7-yl)-2,3,-butanediol, bis-N7G-BD) in tissues of laboratory mice treated with low - sub-ppm concentrations of BD (0.5-1.5 ppm) which approximate human occupational exposure to BD (1 ppm). Bis-N7G-BD concentrations increased in a concentration-dependent manner in mouse liver DNA as a function of BD exposure. In Chapter 3 of this Thesis, we investigated DNA repair mechanisms responsible for bis-N7G-BD repair using isogenic Chinese hamster lung fibroblasts proficient or deficient in nucleotide excision repair (NER) and Fanconi Anemia (FA) repair pathways. We found that while both pathways contributed to bis-N7G-BD removal, FA pathway was most effective at alleviating the toxicity and replication blockage imposed by bis-N7G-BD cross-links. To enable BD-DNA adduct detection in humans, we developed an isotope dilution capillary HPLC-ESI+-HRMS/MS methodology for the most abundant BD-DNA adducts identified in vivo: N7-(2,3,4-trihydroxybut-1-yl)-guanine (N7-THBG) (Chapter 4). This method was successfully applied to quantify N7-THBG adducts in blood leukocyte DNA of smokers, nonsmokers, and occupationally exposed workers. In addition, we have developed an isotope dilution nanoLC/ESI+-HRMS3 methodology for the quantitation of BD-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human blood leukocyte DNA and human urine (Chapters 5 and 6). This method was applied to quantify EB-GII adducts in blood and urine of BD-exposed populations such as smokers, nonsmokers, and occupationally exposed workers. Overall, during the course of the studies described in this Thesis, we have developed a range of novel mass spectrometry-based quantitative methods which have excellent sensitivity, accuracy, and precision, and can be used for future human BD exposure biomonitoring studies. Furthermore, these methodologies are now being employed in epidemiological studies to identify any ethnic/racial differences in BD bioactivation and to help understand the origins of ethnic/racial differences in lung cancer risk in smokers.