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

University of Minnesota Ph.D. dissertation. November 2014. Major: Medicinal Chemistry. Advisor: Natalia Tretyakova. 1 computer file (PDF); xxiii, 300 pages.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

NanoHPLC-nanoESI + -MS/MS Quantitation of Bis -N7-Guanine DNA–DNA Cross-Links in Tissues of B6C3F1 Mice Exposed to subppm Levels of 1,3-Butadiene

1,3-butadiene (BD) is an important industrial chemical and a common environmental pollutant present in urban air. BD is classified as a human carcinogen based on epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its potent carcinogenicity in laboratory mice. A diepoxide metabolite of BD, 1,2,3,4-diepoxybutane (DEB), is considered the ultimate carcinogenic species of BD due to its ability to form genotoxic DNA-DNA cross-links. We have previously employed capillary HPLC-ESI+-MS/MS methods to quantify DEB-induced DNA-DNA conjugates, e.g. 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD), 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and 1,N6-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (1,N6-HMHP-dA), in tissues of laboratory mice exposed to 6.25 – 625 ppm BD (Goggin et al. Cancer Research 69(6), 2479–2486, 2009). However, typical BD human exposure levels are 0.01 to 3.2 ppb in urban air and 1– 2.0 ppm in an occupational setting, requiring greater detection sensitivity for these critical lesions. In the present study, a nanoHPLC-nanoESI+-MS/MS method was developed for ultra-sensitive, accurate, and precise quantitation of bis-N7G-BD in tissues of laboratory mice treated with low ppm and sub-ppm concentrations of BD. The LOD value of the new method is 0.5 fmol/100 μg DNA, and the LOQ is 1.0 fmol/100 μg DNA, making it possible to quantify bis-N7G-BD adducts present at concentrations of 3 per 109 nucleotides. Bis-N7G-BD adduct amounts in liver tissues of mice exposed to 0.5, 1.0, 1.5 ppm BD for 2 weeks were 5.7 ± 3.3, 9.2 ± 1.5, and 18.6 ± 6.9 adducts per 109 nucleotides, respectively, suggesting that N7G-BD adduct formation is more efficient under low exposure conditions. To our knowledge, this is the first quantitative analysis of DEB specific DNA adducts following low ppm and sub-ppm exposure to BD

NanoLC/ESI+ HRMS3 Quantitation of DNA Adducts Induced by 1,3-Butadiene

Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI(+)-HRMS(3) analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5-1.5 ppm). EB-GII concentrations increased linearly from 1.15 ± 0.23 to 10.11 ± 0.45 adducts per 10(8) nucleotides in HT1080 cells treated with 0.5-10 μM EB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17 ± 0.05, 0.33 ± 0.08, and 0.50 ± 0.04 adducts per 10(8) nucleotides, respectively [corrected]. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20 ± 0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI(+)-HRMS(3) Orbitrap methodology to quantitative analysis of DNA adducts in vivo

Persistence and Repair of Bifunctional DNA Adducts in Tissues of Laboratory Animals Exposed to 1,3-Butadiene by Inhalation

1,3-butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen. The mechanism of BD-mediated cancer is of significant interest because of the widespread exposure of humans to BD from cigarette smoke and urban air. BD is metabolically activated to 1,2,3,4-diepoxybutane (DEB), which is a highly genotoxic and mutagenic bis-alkylating agent believed to be the ultimate carcinogenic species of BD. We have previously identified several types of DEB-specific DNA adducts, including bis-N7-guanine cross-links (bis-N7-BD), N6-adenine-N7-guanine cross-links (N6A-N7G-BD), and 1,N6-dA exocyclic adducts. These lesions were detected in tissues of laboratory rodents exposed to BD by inhalation (Goggin et al. Cancer Res. 2009;69:2479–2486). In the present work, persistence and repair of bifunctional DEB-DNA adducts in tissues of mice and rats exposed to BD by inhalation were investigated. The half-lives of the most abundant cross-links, bis-N7G-BD, in mouse liver, kidney, and lungs were 2.3–2.4 days, 4.6–5.7 days, and 4.9 days, respectively. The in vitro half-lives of bis-N7G-BD were 3.5 days (S,S isomer) and 4.0 days (meso isomer) due to their spontaneous depurination. In contrast, tissue concentrations of the minor DEB adducts, N7G-N1A-BD and 1,N6-HMHP-dA, remained essentially unchanged during the course of the experiment, with an estimated t 1/2 of 36–42 days. No differences were observed between DEB-DNA adduct levels in BD-treated wild type mice and the corresponding animals deficient in methyl purine glycosylase or the Xpa gene. Our results indicate that DEB-induced N7G-N1A-BD and 1,N6-HMHP-dA adducts persist in vivo, potentially contributing to mutations and cancer observed as a result of BD exposure

Bis-butanediol-mercapturic acid (bis-BDMA) as a urinary biomarker of metabolic activation of butadiene to its ultimate carcinogenic species

Human carcinogen 1,3-butadiene (BD) undergoes metabolic activation to 3,4-epoxy-1-butene (EB), hydroxymethylvinyl ketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD) and 1,2,3,4-diepoxybutane (DEB). Among these, DEB is by far the most genotoxic metabolite and is considered the ultimate carcinogenic species of BD. We have shown previously that BD-exposed laboratory mice form 8- to 10-fold more DEB–DNA adducts than rats exposed at the same conditions, which may be responsible for the enhanced sensitivity of mice to BD-mediated cancer. In the present study, we have identified 1,4-bis-(N-acetyl-l-cystein-S-yl)butane-2,3-diol (bis-BDMA) as a novel DEB-specific urinary biomarker. Isotope dilution high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was employed to quantify bis-BDMA and three other BD-mercapturic acids, 2-(N-acetyl-l-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-l-cystein-S-yl)-2-hydroxy-but-3-ene (MHBMA, from EB), 4-(N-acetyl-l-cystein-S-yl)-1,2-dihydroxybutane (DHBMA, from HMVK) and 4-(N-acetyl-l-cystein-S-yl)-1,2,3-trihydroxybutane (THBMA, from EBD), in urine of confirmed smokers, occupationally exposed workers and BD-exposed laboratory rats. Bis-BDMA was formed in a dose-dependent manner in urine of rats exposed to 0–200 p.p.m. BD by inhalation, although it was a minor metabolite (1%) as compared with DHBMA (47%) and THBMA (37%). In humans, DHBMA was the most abundant BD-mercapturic acid excreted (93%), followed by THBMA (5%) and MHBMA (2%), whereas no bis-BDMA was detected. These results reveal significant differences in metabolism of BD between rats and humans

Epigenetic Events Determine Tissue-Specific Toxicity of Inhalational Exposure to the Genotoxic Chemical 1,3-Butadiene in Male C57BL/6J Mice

1,3-Butadiene (BD), a widely used industrial chemical and a ubiquitous environmental pollutant, is a known human carcinogen. Although genotoxicity is an established mechanism of the tumorigenicity of BD, epigenetic effects have also been observed in livers of mice exposed to the chemical. To better characterize the diverse molecular mechanisms of BD tumorigenicity, we evaluated genotoxic and epigenotoxic effects of BD exposure in mouse tissues that are target (lung and liver) and non-target (kidney) for BD-induced tumors. We hypothesized that epigenetic alterations may explain, at least in part, the tissue-specific differences in BD tumorigenicity in mice. We evaluated the level of N-7-(2,3,4-trihydroxybut-1-yl)guanine adducts and 1,4-bis-(guan-7-yl)-2,3-butanediol crosslinks, DNA methylation, and histone modifications in male C57BL/6 mice exposed to filtered air or 425 ppm of BD by inhalation (6 h/day, 5 days/week) for 2 weeks. Although DNA damage was observed in all three tissues of BD-exposed mice, variation in epigenetic effects clearly existed between the kidneys, liver, and lungs. Epigenetic alterations indicative of genomic instability, including demethylation of repetitive DNA sequences and alterations in histone-lysine acetylation, were evident in the liver and lung tissues of BD-exposed mice. Changes in DNA methylation were insignificant in the kidneys of treated mice, whereas marks of condensed heterochromatin and transcriptional silencing (histone-lysine trimethylation) were increased. These modifications may represent a potential mechanistic explanation for the lack of tumorigenesis in the kidney. Our results indicate that differential tissue susceptibility to chemical-induced tumorigenesis may be attributed to tissue-specific epigenetic alterations

Cellular Repair of DNA–DNA Cross-Links Induced by 1,2,3,4-Diepoxybutane

Xenobiotic-induced interstrand DNA–DNA cross-links (ICL) interfere with transcription and replication and can be converted to toxic DNA double strand breaks. In this work, we investigated cellular responses to 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) cross-links induced by 1,2,3,4-diepoxybutane (DEB). High pressure liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS) assays were used to quantify the formation and repair of bis-N7G-BD cross-links in wild-type Chinese hamster lung fibroblasts (V79) and the corresponding isogenic clones V-H1 and V-H4, deficient in the XPD and FANCA genes, respectively. Both V-H1 and V-H4 cells exhibited enhanced sensitivity to DEB-induced cell death and elevated bis-N7G-BD cross-links. However, relatively modest increases of bis-N7G-BD adduct levels in V-H4 clones did not correlate with their hypersensitivity to DEB. Further, bis-N7G-BD levels were not elevated in DEB-treated human clones with defects in the XPA or FANCD2 genes. Comet assays and γ-H2AX focus analyses conducted with hamster cells revealed that ICL removal was associated with chromosomal double strand break formation, and that these breaks persisted in V-H4 cells as compared to control cells. Our findings suggest that ICL repair in cells with defects in the Fanconi anemia repair pathway is associated with aberrant re-joining of repair-induced double strand breaks, potentially resulting in lethal chromosome rearrangements

NanoHPLC-nanoESI⁺-MS/MS Quantitation of <i>Bis</i>-N7-Guanine DNA–DNA Cross-Links in Tissues of B6C3F1 Mice Exposed to subppm Levels of 1,3-Butadiene

1,3-Butadiene (BD) is an important industrial chemical and a common environmental pollutant present in urban air. BD is classified as a human carcinogen based on epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its potent carcinogenicity in laboratory mice. A diepoxide metabolite of BD, 1,2,3,4-diepoxybutane (DEB), is considered the ultimate carcinogenic species of BD due to its ability to form genotoxic DNA–DNA cross-links. We have previously employed capillary HPLC-ESI⁺-MS/MS (liquid chromatography-electrospray ionization tandem mass spectrometry) methods to quantify DEB-induced DNA–DNA conjugates, e.g. 1,4-<i>bis</i>-(guan-7-yl)-2,3-butanediol (<i>bis</i>-N7G-BD), 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and <i>1,N</i>^<i>6</i>-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (<i>1,N</i>^<i>6</i>-HMHP-dA), in tissues of laboratory mice exposed to 6.25–625 ppm BD (Goggin et al. <i>Cancer Res.</i> <b>2009</b>, <i>69</i>(6), 2479–2486). However, typical BD human exposure levels are 0.01 to 3.2 ppb in urban air and 1–2.0 ppm in an occupational setting, requiring greater detection sensitivity for these critical lesions. In the present study, a nanoHPLC-nanoESI⁺-MS/MS method was developed for ultrasensitive, accurate, and precise quantitation of <i>bis</i>-N7G-BD in tissues of laboratory mice treated with low ppm and subppm concentrations of BD. The LOD value of the new method is 0.5 fmol/100 μg DNA, and the LOQ is 1.0 fmol/100 μg DNA, making it possible to quantify <i>bis</i>-N7G-BD adducts present at concentrations of 3 per 10⁹ nucleotides. <i>Bis</i>-N7G-BD adduct amounts in liver tissues of mice exposed to 0.5, 1.0, and 1.5 ppm BD for 2 weeks were 5.7 ± 3.3, 9.2 ± 1.5, and 18.6 ± 6.9 adducts per 10⁹ nucleotides, respectively, suggesting that <i>bis</i>-N7G-BD adduct formation is more efficient under low exposure conditions. To our knowledge, this is the first quantitative analysis of DEB specific DNA adducts following low ppm and subppm exposure to BD