Dataset Information

Isotope Dilution nanoLC/ESI+-HRMS3 Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene.

ABSTRACT: 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.

SUBMITTER: Sangaraju D

PROVIDER: S-EPMC5515386 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Isotope Dilution nanoLC/ESI<sup>+</sup>-HRMS<sup>3</sup> Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene.

Sangaraju Dewakar D Boldry Emily J EJ Patel Yesha M YM Walker Vernon V Stepanov Irina I Stram Daniel D Hatsukami Dorothy D Tretyakova Natalia N

Chemical research in toxicology 20170117 2

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 capa ...[more]

PMID: 27997139

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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 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:DNA alkylation or adduct formation occurs at nucleophilic sites in DNA, mainly the N7-position of guanine. Ever since identification of the first N7-guanine adduct, several hundred studies on DNA adducts have been reported. Major issues addressed include the relationships between N7-guanine adducts and exposure, mutagenesis, and other biological endpoints. It became quickly apparent that N7-guanine adducts are frequently formed, but may have minimal biological relevance, since they are chemically unstable and do not participate in Watson Crick base pairing. However, N7-guanine adducts have been shown to be excellent biomarkers for internal exposure to direct acting and metabolically activated carcinogens. Questions arise, however, regarding the biological significance of N7-guanine adducts that are readily formed, do not persist, and are not likely to be mutagenic. Thus, we set out to review the current literature to evaluate their formation and the mechanistic evidence for the involvement of N7-guanine adducts in mutagenesis or other biological processes. It was concluded that there is insufficient evidence that N7-guanine adducts can be used beyond confirmation of exposure to the target tissue and demonstration of the molecular dose. There is little to no evidence that N7-guanine adducts or their depurination product, apurinic sites, are the cause of mutations in cells and tissues, since increases in AP sites have not been shown unless toxicity is extant. However, more research is needed to define the extent of chemical depurination versus removal by DNA repair proteins. Interestingly, N7-guanine adducts are clearly present as endogenous background adducts and the endogenous background amounts appear to increase with age. Furthermore, the N7-guanine adducts have been shown to convert to ring opened lesions (FAPy), which are much more persistent and have higher mutagenic potency. Studies in humans are limited in sample size and differences between controls and study groups are small. Future investigations should involve human studies with larger numbers of individuals and analysis should include the corresponding ring opened FAPy derivatives.

Project description: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.

Project description:Dietary exposure to aflatoxin B1 (AFB1) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB1 exposure leads to the formation of AFB1-N7-guanine (AFB1-N7-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) in DNA. These adducts lead to G ? T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB1 as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB1-N7-Gua and the two diastereomers of AFB1-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB1-treated mice. Levels varying from 1.5 to 45 lesions/106 DNA bases in AFB1-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB1 treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB1 treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB1-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB1-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB1 exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

Dataset Information

Isotope Dilution nanoLC/ESI+-HRMS3 Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene.

Publications

Isotope Dilution nanoLC/ESI<sup>+</sup>-HRMS<sup>3</sup> Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene.

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