Dataset Information

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.

ABSTRACT: 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-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,N(6)-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-HMHP-dA), in tissues of laboratory mice exposed to 6.25-625 ppm BD (Goggin et al. Cancer Res. 2009, 69(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 bis-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 ?g DNA, and the LOQ is 1.0 fmol/100 ?g DNA, making it possible to quantify bis-N7G-BD adducts present at concentrations of 3 per 10(9) nucleotides. Bis-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 10(9) nucleotides, respectively, suggesting that bis-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.

SUBMITTER: Sangaraju D

PROVIDER: S-EPMC3298759 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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.

Sangaraju Dewakar D Goggin Melissa M Walker Vernon V Swenberg James J Tretyakova Natalia N

Analytical chemistry 20120126 3

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 prev ...[more]

PMID: 22220765

Similar Datasets

Project description:1,3-Butadiene (BD) is a high volume industrial chemical commonly used in polymer and rubber production. It is also present in cigarette smoke, automobile exhaust, and urban air, leading to widespread exposure of human populations. Upon entering the body, BD is metabolized to electrophilic epoxides, 3,4-epoxy-1-butene (EB), diepoxybutane (DEB), and 3,4-epoxy-1,2-diol (EBD), which can alkylate DNA nucleobases. The most abundant BD epoxide, EBD, modifies the N7-guanine positions in DNA to form N7-(2, 3, 4-trihydroxybut-1-yl) guanine (N7-THBG) adducts, which can be useful as biomarkers of BD exposure and metabolic activation to DNA-reactive epoxides. In the present work, a capillary HPLC-high resolution ESI?-MS/MS (HPLC-ESI?-HRMS/MS) methodology was developed for accurate, sensitive, and reproducible quantification of N7-THBG in cell culture and in human white blood cells. In our approach, DNA is subjected to neutral thermal hydrolysis to release N7-guanine adducts from the DNA backbone, followed by ultrafiltration, solid-phase extraction, and isotope dilution HPLC-ESI?-HRMS/MS analysis on an Orbitrap Velos mass spectrometer. Following method validation, N7-THBG was quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of DEB and in DNA isolated from blood of smokers, nonsmokers, individuals participating in a smoking cessation program, and occupationally exposed workers. N7-THBG concentrations increased linearly from 31.4 ± 4.84 to 966.55 ± 128.05 adducts per 10? nucleotides in HT1080 cells treated with 1-100 ?M DEB. N7-THBG amounts in leukocyte DNA of nonsmokers, smokers, and occupationally exposed workers were 7.08 ± 5.29, 8.20 ± 5.12, and 9.72 ± 3.80 adducts per 10? nucleotides, respectively, suggesting the presence of an endogenous or environmental source for this adduct. The availability of sensitive HPLC-ESI?-HRMS/MS methodology for BD-induced DNA adducts in humans will enable future population studies of interindividual and ethnic differences in BD bioactivation to DNA-reactive epoxides.

Project description:1,3-Butadiene (BD) is a common environmental and industrial chemical widely used in plastic and rubber manufacturing and also present in cigarette smoke and automobile exhaust. BD is classified as a known human carcinogen based on evidence of carcinogenicity in laboratory animals treated with BD by inhalation and epidemiological studies revealing an increased risk of leukemia and lymphohematopoietic cancers in workers occupationally exposed to BD. Upon exposure via inhalation, BD is bioactivated to several toxic epoxides including 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB); these are conjugated with glutathione and excreted as 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA), and 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA). Exposure to DEB generates monoalkylated DNA adducts, DNA-DNA crosslinks, and DNA-protein crosslinks, which can cause base substitutions, genomic rearrangements, and large genomic deletions. In this study, we developed a quantitative nanoLC/NSI+-HRMS methodology for 1,4-bis-(gua-7-yl)-2,3-butanediol (bis-N7G-BD) adducts in urine (LOD: 0.1 fmol/mL urine, LOQ: 1.0 fmol/mL urine). This novel method was used to quantify bis-N7G-BD in urine of mice treated with 590 ± 150 ppm BD for 2 weeks (6 h/day, 5 days/week). Bis-N7G-BD was detected in urine of male and female BD-exposed mice (574.6 ± 206.0 and 571.1 ± 163.4 pg/mg of creatinine, respectively). In addition, major urinary metabolites of BD, bis-BDMA, MHBMA and DHBMA, were measured in the same samples. Urinary bis-N7G-BD adduct levels correlated with DEB-derived metabolite bis-BDMA (r = 0.80, Pearson correlation), but not with the EB-derived DNA adducts (EB-GII) or EB-derived metabolites MHBMA and DHBMA (r = 0.24, r = 0.14, r = 0.18, respectively, Pearson correlations). Urinary bis-N7G-BD could be employed as a novel non-invasive biomarker of exposure to BD and bioactivation to its most mutagenic metabolite, DEB. This method will be useful for future studies of 1,3-butadiene exposure and metabolism.

Project description:1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a known human carcinogen. Occupational exposure to BD in the polymer and monomer industries is associated with an increased incidence of lymphoma. BD is present in automobile exhaust, cigarette smoke, and forest fires, raising concern about potential exposure of the general population to this carcinogen. Following inhalation exposure, BD is bioactivated to 3,4-epoxy-1-butene (EB). If not detoxified, EB is capable of modifying guanine and adenine bases of DNA to form nucleobase adducts, which interfere with accurate DNA replication and cause cancer-initiating mutations. We have developed a nanoLC/ESI+-HRMS3 methodology for N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human urine (limit of detection: 0.25 fmol/mL urine; limit of quantitation: 1.0 fmol/mL urine). This new method was successfully used to quantify EB-GII in urine of F344 rats treated with 0-200 ppm of BD, occupationally exposed workers, and smokers belonging to two different ethnic groups. EB-GII amounts increased in a dose-dependent manner in urine of laboratory rats exposed to 0, 62.5, or 200 ppm of BD. Urinary EB-GII levels were significantly increased in workers occupationally exposed to 0.1-2.2 ppm of BD (1.25 ± 0.51 pg/mg of creatinine) as compared to administrative controls exposed to <0.01 ppm of BD (0.22 ± 0.08 and pg/mg of creatinine) (p = 0.0024), validating the use of EB-GII as a biomarker of human exposure to BD. EB-GII was also detected in smokers' urine with European American smokers excreting significantly higher amounts of EB-GII than African American smokers (0.48 ± 0.09 vs 0.12 ± 0.02 pg/mg of creatinine, p = 3.1 × 10-7). Interestingly, small amounts of EB-GII were observed in animals and humans with no known exposure to BD, providing preliminary evidence for its endogenous formation. Urinary EB-GII adduct levels and urinary mercapturic acids of BD (MHBMA, DHBMA) were compared in a genotyped multiethnic smoker cohort.

Project description:1,3-Butadiene is a known carcinogen primarily targeting lymphoid tissues, lung, and liver. Cytochrome P450 activates butadiene to epoxides which form covalent DNA adducts that are thought to be a key mechanistic event in cancer. Previous studies suggested that inter-species, -tissue, and -individual susceptibility to adverse health effects of butadiene exposure may be due to differences in metabolism and other mechanisms. In this study, we aimed to examine the extent of inter-individual and inter-species variability in the urinary N7-(1-hydroxy-3-buten-2-yl)guanine (EB-GII) DNA adduct, a well-known biomarker of exposure to butadiene. For a population variability study in mice, we used the collaborative cross model. Female and male mice from five strains were exposed to filtered air or butadiene (590 ppm, 6 h/day, 5 days/week for 2 weeks) by inhalation. Urine samples were collected, and the metabolic activation of butadiene by DNA-reactive species was quantified as urinary EB-GII adducts. We quantified the degree of EB-GII variation across mouse strains and sexes; then, we compared this variation with the data from rats (exposed to 62.5 or 200 ppm butadiene) and humans (0.004-2.2 ppm butadiene). We show that sex and strain are significant contributors to the variability in urinary EB-GII levels in mice. In addition, we find that the degree of variability in urinary EB-GII in collaborative cross mice, when expressed as an uncertainty factor for the inter-individual variability (UFH), is relatively modest (≤threefold) possibly due to metabolic saturation. By contrast, the variability in urinary EB-GII (adjusted for exposure) observed in humans, while larger than the default value of 10-fold, is largely consistent with UFH estimates for other chemicals based on human data for non-cancer endpoints. Overall, these data demonstrate that urinary EB-GII levels, particularly from human studies, may be useful for quantitative characterization of human variability in cancer risks to butadiene.

Project description:1,3-Butadiene (BD) is an important industrial chemical used in the manufacture of rubber and plastics as well as an environmental pollutant present in automobile exhaust and cigarette smoke. It is classified as a known human carcinogen based on the epidemiological evidence in occupationally exposed workers and its ability to induce tumors in laboratory animals. BD is metabolically activated to several reactive species, including 1,2,3,4-diepoxybutane (DEB), which is hypothesized to be the ultimate carcinogenic species due to its bifunctional electrophilic nature and its ability to form DNA-DNA and DNA-protein cross-links. While 1,4- bis-(guan-7-yl)-2,3,-butanediol ( bis-N7G-BD) is the only type of DEB-specific DNA adduct previously quantified in vivo, four regioisomeric guanine-adenine (G-A) cross-links have been observed in vitro: 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), 1-(guan-7-yl)-4-(aden-3-yl)-2,3-butanediol (N7G-N3A-BD), 1-(guan-7-yl)-4-(aden-7-yl)-2,3-butanediol (N7G-N7A-BD), and 1-(guan-7-yl)-4-(aden-6-yl)-2,3-butanediol (N7G-N (6)A-BD) ( Park ( 2004) Chem. Res. Toxicol. 17, 1638- 1651 ). The goal of the present work was to develop an isotope dilution HPLC-positive mode electrospray ionization-tandem mass spectrometry (HPLC-ESI (+)-MS/MS) method for the quantitative analysis of G-A DEB cross-links in DNA extracted from BD-exposed laboratory animals. In our approach, G-A butanediol conjugates are released from the DNA backbone by thermal or mild acid hydrolysis. Following solid-phase extraction, samples are subjected to capillary HPLC-ESI (+)-MS/MS analysis with (15)N 3, (13)C 1-labeled internal standards. The detection limit of our current method is 0.6-1.5 adducts per 10 (8) normal nucleotides. The new method was validated by spiking G-A cross-link standards (10 fmol each) into control mouse DNA (0.1 mg), followed by sample processing and HPLC-ESI (+)-MS/MS analysis. The accuracy and precision were calculated as 105 +/- 17% for N7G-N3A-BD, 102 +/- 25% for N7G-N7A-BD, and 79 +/- 11% for N7G-N (6)A-BD. The regioisomeric G-A DEB adducts were formed in a concentration-dependent manner in DEB-treated calf thymus DNA, with N7G-N1A-BD found in the highest amounts. Under physiological conditions, N7G-N1A-BD underwent Dimroth rearrangement to N7G-N (6)A-BD ( t 1/2 = 114 h), while hydrolytic deamination of N7G-N1A-BD to the corresponding hypoxanthine lesion was insignificant. We found that for in vivo samples, a greater sensitivity could be achieved if N7G-N1A-BD adducts were converted to the corresponding N7G-N (6)A-BD lesions by forced Dimroth rearrangement. Liver DNA extracted from female B6C3F1 mice that underwent inhalation exposure to 625 ppm BD for 2 weeks contained 3.1 +/- 0.6 N7G-N1A-BD adducts per 10 (8) nucleotides ( n = 5) (quantified as N7G-N (6)A-BD following base-induced Dimroth rearrangement), while the amounts of N7G-N3A-BD and N7G-N7A-BD were below the detection limit of our method. None of the G-A cross-links was present in control animals. The formation of N7G-N1A-BD cross-links may contribute to the induction of AT base pair mutations following exposure to BD. Quantitative methods presented here may be used not only for studies of biological significance in animal models but potentially to predict risk associated with human exposure to BD.

Dataset Information

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.

Publications

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.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets