Dataset Information

Analysis of a malondialdehyde-deoxyguanosine adduct in human leukocyte DNA by liquid chromatography nanoelectrospray-high-resolution tandem mass spectrometry.

ABSTRACT: Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostaglandin biosynthesis, can react with DNA to form stable adducts. These adducts may contribute to the development of such inflammation-mediated diseases as cancer and cardiovascular and neurodegenerative diseases. The predominant MDA-derived DNA adduct formed under physiological conditions is 3-(2-deoxy-?-d-erythro-pentafuranosyl)pyrimido[1,2-?]purin-10(3H)-one deoxyguanosine (M1dG). In this study, we developed a novel liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high-resolution tandem mass spectrometry (HRMS/MS) method for the analysis of M1dG in human leukocyte DNA. After enzymatic hydrolysis of DNA, M1dG and the added internal standard [(13)C3]M1dG were reduced to their 5,6-dihydro derivatives by addition of sodium borohydride to the hydrolysate and purified by solid-phase extraction and column chromatography. The 5,6-dihydro derivatives in the purified samples were analyzed by LC-NSI-HRMS/MS using higher-energy collisional dissociation (HCD) fragmentation, isolation widths of 1 Da for both the analyte and internal standard, and a resolution of 50?000. The detection limit of the developed method is 5 amol on-column, and the limit of quantitation is 0.125 fmol/mg DNA starting with 200 ?g of DNA. Method accuracy and precision were characterized. The developed method was further applied to the analysis of leukocyte DNA from 50 human subjects. M1dG was detected in all samples and ranged from 0.132 to 275 fmol/mg DNA, or 0.004 to 9.15 adducts per 10(8) bases. This unique and highly sensitive HRMS/MS-based method can be used in future studies investigating the pathophysiological role of M1dG in human diseases.

SUBMITTER: Ma B

PROVIDER: S-EPMC4203394 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Analysis of a malondialdehyde-deoxyguanosine adduct in human leukocyte DNA by liquid chromatography nanoelectrospray-high-resolution tandem mass spectrometry.

Ma Bin B Villalta Peter W PW Balbo Silvia S Stepanov Irina I

Chemical research in toxicology 20140915 10

Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostaglandin biosynthesis, can react with DNA to form stable adducts. These adducts may contribute to the development of such inflammation-mediated diseases as cancer and cardiovascular and neurodegenerative diseases. The predominant MDA-derived DNA adduct formed under physiological conditions is 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG). In this study, we ...[more]

PMID: 25181548

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Project description:Cigarette smoking is an important source of human exposure to toxicants and carcinogens and contributes significantly to cancer morbidity and mortality worldwide. Acrolein, a widespread environmental pollutant, is present in relatively high amounts in cigarette smoke and can react directly with DNA to form DNA adducts, which serve as important biomarkers for the assessment of exposure to acrolein and its potential role in smoking related cancer. Etheno-DNA adducts are promutagenic DNA lesions that can derive from exogenous chemicals as well as endogenous sources, including lipid peroxidation. In this study, we developed a combined method for the quantitation of (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8,-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)-one (α-OH-Acr-dGuo), (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8,-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo), 1,N6-etheno-dAdo (εdAdo), and 3,N4-etheno-dCyd (εdCyd) adducts in oral rinse and cytobrush DNA from smokers and nonsmokers by liquid chromatography-nanoelelctrospray ionization-high-resolution tandem mass spectrometry (LC-NSI-HRMS/MS). For oral rinse samples, there was a statistically significant difference between the levels of α-OH-Acr-dGuo, γ-OH-Acr-dGuo, εdAdo, and εdCyd in smokers (12.1 ± 17.9, 163 ± 227, 182 ± 568, and 194 ± 400 adducts/109 nucleotides, respectively) and nonsmokers (1.85 ± 2.08, 5.95 ± 4.23, 7.69 ± 11.7, and 6.07 ± 10.9 adducts/109 nucleotides, respectively). For cytobrush samples, there was a statistically significant difference between the levels of γ-OH-Acr-dGuo and εdAdo in smokers (259 ± 540 and 82.9 ± 271 adducts/109 nucleotides, respectively) and nonsmokers (7.37 ± 5.09 and 16.2 ± 30.2 adducts/109 nucleotides, respectively) but not for α-OH-Acr-dGuo and εdCyd. Our results demonstrate that oral mucosa cells are an excellent source of material for evaluating DNA adducts to be used as biomarkers of tobacco smoke exposure and molecular changes potentially related to cancer.

Project description:We developed a liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method for simultaneous quantitative analysis of 5 oral cell DNA adducts associated with cigarette smoking: (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo, 1) from acrolein; (6S,8S and 6R,8R)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)-one [(6S,8S)γ-OH-Cro-dGuo, 2; and (6R,8R)γ-OH-Cro-dGuo, 3] from crotonaldehyde; 1,N6-etheno-dAdo (4) from acrylonitrile, vinyl chloride, lipid peroxidation, and inflammation; and 8-oxo-dGuo (5) from oxidative damage. Oral cell DNA was isolated in the presence of glutathione to prevent artifact formation. Clear LC-NSI-HRMS/MS chromatograms were obtained allowing quantitation of each adduct using the appropriately labeled internal standards. The accuracy and precision of the method were validated, and the assay limit of quantitation was 5 fmol/μmol dGuo for adducts 1-4 and 20 fmol/μmol for adduct 5. The assay was applied to 80 buccal cell samples selected from those collected in the Shanghai Cohort Study: 40 from current smokers and 40 from never smokers. Significant differences were found in all adduct levels between smokers and nonsmokers. Levels of 8-oxo-dGuo (5) were at least 3000 times greater than those of the other adducts in both smokers and nonsmokers, and the difference between amounts of this adduct in smokers versus nonsmokers, while significant (P = 0.013), was not as great as the differences of the other DNA adducts between smokers and nonsmokers (P-values all less than 0.001). No significant relationship of adduct levels to risk of lung cancer incidence was found. This study provides a new LC-NSI-HRMS/MS methodology for the quantitation of diverse DNA adducts resulting from exposure to the α,β-unsaturated aldehydes acrolein and crotonaldehyde, inflammation, and oxidative damage which are all associated with carcinogenesis. We anticipate application of this assay in ongoing studies of the molecular epidemiology of cancers of the lung and oral cavity related to cigarette smoking.

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Dataset Information

Analysis of a malondialdehyde-deoxyguanosine adduct in human leukocyte DNA by liquid chromatography nanoelectrospray-high-resolution tandem mass spectrometry.

Publications

Analysis of a malondialdehyde-deoxyguanosine adduct in human leukocyte DNA by liquid chromatography nanoelectrospray-high-resolution tandem mass spectrometry.

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