Identification and characterization of the major DNA adduct formed chemically and in vitro from the environmental genotoxin 3-nitrofluoranthene

The genotoxic environmental pollutant 3-nitrofluoranthene (3-NFA)was reduced chemically and allowed to react with calf thymusDNA, yielding one major adduct which was determined to be N-deoxyguanosin-8-yl)-3-amino-fluoran-thenebased on Fast Atom Bombardment Mass Spectrometry (FAB-MS), protonnuclear magnetic resonance, ultra-violet-visible wavelengthlight spectroscopy (UV-VIS), and fluorescence data. Extensivecharacterization of the isolated adduct by tandem mass spectrometry(MS/MS) was necessary to demonstrate definitively that the adductisolated was the dG: C8 adduct, and not the isomeric dG: N2adduct. The extent of modification of the initial calf thymusDNA by chemically reduced 3-NFA was 0.12% (1.2 adducts/10P nucleosides),which was sufficient to allow several hundred micrograms ofthe adduct to be isolated and purified. The chemically synthesizedadduct was utilized as a reference standard for comparison tothe major adduct isolated from xanthine-oxidase-catalyzed reductionof 3-NFA in vitro. The yield from the in vitro biological systemwas 2.4 adducts/10⁵ nucleosides; the adduct isolated possessedthe same mass spectrometric. UV-VIS, and fluorescence characteristicsas the purified standard, and co-eluted with the standard onHPLC. No evidence for other adducts was found, either in vitroor in the chemical synthesis, based on FAB-MS examination ofwhole extracts of the reaction mixture for the presence of ionsrelated to other possible adducts. Therefore, if minor adductswere present they were formed in substantially lesser amountsthan N-(deoxyguanosin-8-yl)-3-aminoflouranthene.