Quantification of 3-Nitrobenzanthrone-DNA Adducts Using Online Column-Switching HPLC-Electrospray Tandem Mass Spectrometry

• Published in: Chemical research in toxicology Vol. 22; no. 11; pp. 1860 - 1868
• Main Authors: Gamboa da Costa, Gonçalo, Singh, Rajinder, Arlt, Volker M, Mirza, Amin, Richards, Meirion, Takamura-Enya, Takeji, Schmeiser, Heinz H, Farmer, Peter B, Phillips, David H
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16-11-2009
• Subjects: Animals; Benz(a)Anthracenes - chemistry; Carcinogens - chemistry; Chromatography, High Pressure Liquid - methods; DNA Adducts - analysis; Female; Hydroxylamine - chemistry; Rats; Rats, Sprague-Dawley; Rats, Wistar; Salmon; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry; Vehicle Emissions
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx900264v

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 32P-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 15N-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 32P-postlabeling. The method required 50 μg 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 32P-postlabeling, and it has sufficient sensitivity and precision to analyze DNA adducts in animals exposed to 3-NBA or its hydroxylamine metabolite.