1,3-Butadiene-Induced DNA Damage: Ethnic Differences and Sources of Formation

Abstract

In the United States, lung cancer is the leading cause of cancer-related deaths. Cigarette smoking is a major risk factor for lung cancer development, with approximately 80% of lung cancer cases directly related to smoking. Up to 1 in 4 smokers will develop lung cancer over the course of their lifetime. This risk varies by racial/ethnic group, with African Americans and Native Hawaiians at greater risk as compared to whites, and Japanese and Latinos at a relatively lower risk than whites. This racial/ethnic difference in risk is not explained by differences in smoking dose, diet, occupation, or socioeconomic status. Cigarette smoke is comprised of over 7000 chemical compounds, more than 70 of which are known human carcinogens. These carcinogens undergo metabolic activation to reactive species which can form adducts with DNA, leading to mutations and eventually lung cancer development. Polymorphisms in metabolic genes responsible for the bioactivation and detoxification of tobacco smoke carcinogens have been hypothesized to play a role in the racial/ethnic differences in lung cancer risk. Among tobacco smoke carcinogens, 1,3-butadiene (butadiene, BD) is one of the most abundant and has the highest cancer risk index. BD is metabolically activated to reactive epoxides 3,4-epoxy-1-butene (EB), 1,2-dihydroxy-3,4-epoxybutane (EBD), and 1,2,3,4-diepoxybutane (DEB) by CYP2E1 and 2A6. These epoxides can be detoxified through glutathione conjugation by GSTT1 to form 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene and 1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA) from EB, N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA) from EB-derived hydroxymethylvinyl ketone (HMVK), 4-(N-acetyl-L-cystein-S-yl)-1,2,3-trihydroxybutane (THBMA) from EBD, and bis-butanediol mercapturic acid (bis-BDMA) from DEB. If not detoxified, these epoxide species can form covalent adducts with DNA such as N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) from EB, N7-(2,3,4-trihydroxybut-1-yl) guanine (N7-THBG) from EBD, and 1,4-bis-(guan7-yl)-2,3-butanediol (bis-N7G-BD) from DEB. The first goal of this thesis work was to investigate urinary levels of EB-GII as a biomarker of lung cancer risk in smokers. In Chapter 2 of this thesis, we report the temporal stability and association with smoking of urinary EB-GII. Urinary EB-GII levels were stable over time in smokers, indicating that single adduct measurements provide reliable levels of EB-GII. Additionally, we observed a 34% decrease in the levels of urinary EB-GII upon smoking cessation, indicating that it is associated with smoking status but may also have other sources of formation. In Chapter 3 we quantified urinary EB-GII adducts in smokers and non-smokers belonging to three racial/ethnic groups with differing risks of lung cancer development: Native Hawaiian, white, and Japanese American. We observed higher levels of urinary EB-GII excretion in Japanese Americans as compared to whites and Native Hawaiians and these differences could not be explained by GSTT1 gene deletion or CYP2A6 activity. In Chapter 4 we directly examined the association between urinary EB-GII and lung cancer incidence, revealing that EB-GII levels are elevated in lung cancer cases as compared to smokers without lung cancer (OR = 1.91). In Chapters 2 and 3, we observed that there were low but detectable levels of urinary EB-GII in smokers following smoking cessation and in non-smokers, suggesting additional sources of EB-GII adduct formation. In Chapter 5, we utilized stable isotope tracing to investigate the formation of BD DNA adducts and metabolites from endogenous sources. Laboratory rats were treated with low ppm (0.3-3.0 ppm) concentrations of BD-d6 to approximate occupational exposure to BD (~1 ppm). Levels of exogenous (deuterated) EB-GII, MHBMA, and DHBMA increased in a dose-dependent manner following BD exposure, while endogenous (unlabeled) adducts and metabolites were unaffected by BD dose. While urinary EB-GII and MHBMA were formed primarily by exogenous exposure, significant amounts of endogenous DHBMA were observed. Additionally, urinary exogenous EB-GII was associated with butadiene-induced genomic EB-GII, suggesting that urinary EB-GII can be used as a non-invasive surrogate measurement for genomic BD-DNA damage. In Chapter 6, formation of a novel BD-DNA adduct, N6-[2-deoxy-D-erythro-pentofuranosyl]-2,6-diamino-3,4-dihydro-4-oxo-5-N-1-(oxiran-2-yl)propan-1-ol-formamidopyrimidine (DEB-FAPy-dG) was investigated. A sensitive isotope dilution nanoLC-ESI+-HRMS/MS methodology was developed and applied to quantitation of DEB-FAPy-dG formation in DEB treated calf thymus DNA. DEB-FAPy-dG formation was dependent on DEB concentration and pH, with higher adduct levels observed at higher pH. Detection of DEB-FAPy-dG in mouse embryonic fibroblast cells and nuclei treated with DEB was unsuccessful, likely due to the adduct forming in low quantities at physiological pH.