title: Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry.
abstract: The 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.
authors.affiliation: Institute of Cancer Research, Section of Molecular Carcinogenesis, Sutton, Surrey SM2 5NG, U.K. goncalo.gamboa@fda.hhs.gov
authors.forename: Gonçalo
authors.initials: G
authors.lastname: Gamboa da Costa
authors.forename: Rajinder
authors.initials: R
authors.lastname: Singh
authors.forename: Volker M
authors.initials: VM
authors.lastname: Arlt
authors.forename: Amin
authors.initials: A
authors.lastname: Mirza
authors.forename: Meirion
authors.initials: M
authors.lastname: Richards
authors.forename: Takeji
authors.initials: T
authors.lastname: Takamura-Enya
authors.forename: Heinz H
authors.initials: HH
authors.lastname: Schmeiser
authors.forename: Peter B
authors.initials: PB
authors.lastname: Farmer
authors.forename: David H
authors.initials: DH
authors.lastname: Phillips
mesh_terms: D000818:Animals; D001551:Benz(a)Anthracenes / Q000737:chemistry*; D002273:Carcinogens / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018736:DNA Adducts / Q000032:analysis*; D005260:Female; D019811:Hydroxylamine / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D012474:Salmon; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D001335:Vehicle Emissions
chemical_list: D001551:Benz(a)Anthracenes; D002273:Carcinogens; D018736:DNA Adducts; D001335:Vehicle Emissions; D019811:Hydroxylamine; C117220:3-nitrobenzanthrone