Mass Spectrometry Based Quantification of 1, 3-Butadiene Induced DNA Adducts: Potential Biomarkers of Cancer Risk

Abstract

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.