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dc.contributor.authorGamboa da Costa, Goncalo
dc.contributor.authorSingh, Rajinder
dc.contributor.authorArlt, Volker M.
dc.contributor.authorMirza, Amin
dc.contributor.authorRichards, Meirion
dc.contributor.authorTakamura-Enya, Takeji
dc.contributor.authorSchmeiser, Heinz H.
dc.contributor.authorFarmer, Peter B.
dc.contributor.authorPhillips, David H.
dc.date.accessioned2010-11-05T11:29:28Z
dc.date.available2010-11-05T11:29:28Z
dc.date.issued2009-11
dc.identifier.citationChem. Res. Toxicol. 2009, 22 (11):1860-1868en
dc.identifier.issn1520-5010
dc.identifier.pmid19916526
dc.identifier.doi10.1021/tx900264v
dc.identifier.urihttp://hdl.handle.net/10146/114771
dc.description.abstractThe aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on (32)P-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of (15)N-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by (32)P-postlabeling. The method required 50 microg of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N(2)-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R(2) = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more automated analytical approach that also provides structural confirmation of the adducts detected by (32)P-postlabeling, and it has sufficient sensitivity and precision to analyze DNA adducts in animals exposed to 3-NBA or its hydroxylamine metabolite.
dc.description.sponsorshipThe authors acknowledge financial support from Cancer Research UK and from the European Union Network of Excellence ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility, www.ecnis.org, Contract No. FOOD-CT-2005-513943), and from the Medical Research Council (Grant No. G0100873).en
dc.language.isoenen
dc.relation.urlhttp://pubs.acs.org/doi/abs/10.1021/tx900264ven
dc.subjectCarcinogensen
dc.subjectBenz(a)Anthracenesen
dc.subjectHydroxylamineen
dc.subjectDNA Adductsen
dc.subjectRatsen
dc.subjectChromatography, High Pressure Liquiden
dc.subjectTandem Mass Spectrometryen
dc.subject.meshAnimals
dc.subject.meshBenz(a)Anthracenes
dc.subject.meshCarcinogens
dc.subject.meshChromatography, High Pressure Liquid
dc.subject.meshDNA Adducts
dc.subject.meshFemale
dc.subject.meshHydroxylamine
dc.subject.meshRats
dc.subject.meshRats, Sprague-Dawley
dc.subject.meshRats, Wistar
dc.subject.meshSalmon
dc.subject.meshSpectrometry, Mass, Electrospray Ionization
dc.subject.meshTandem Mass Spectrometry
dc.subject.meshVehicle Emissions
dc.titleQuantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry.en
dc.typeArticleen
dc.identifier.journalChemical Research in Toxicologyen
html.description.abstractThe aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on (32)P-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of (15)N-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by (32)P-postlabeling. The method required 50 microg of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N(2)-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R(2) = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more automated analytical approach that also provides structural confirmation of the adducts detected by (32)P-postlabeling, and it has sufficient sensitivity and precision to analyze DNA adducts in animals exposed to 3-NBA or its hydroxylamine metabolite.


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