In vitro synthesis of 1,N6-etheno-2'-deoxyadenosine and 1,N2-etheno-2'-deoxyguanosine by 2,4-dinitrophenol and 1,3-dinitropyrene in presence of a bacterial nitroreductase.

The formation of covalent nitro-PAH DNA adducts and nitro-PAH mediated oxidative lesions are two possible mechanisms for the initiation of nitro-PAH carcinogenesis. Sixty-minute incubation of 1,3-dinitropyrene (100 microM) or 1,4-dinitrophenol (100 microM) with a mixture of 150 microM NADH, 0.5 units of E. coli nitroreductase, 100 microM linoleic acid, 0.5 mM ferrous iron, and 100 microM 2'-deoxyadenosine (2'-dA) or 100 microM 2'-deoxyguanosine (2'-dG) were analyzed by liquid chromatography multistage mass spectrometry. Mixtures of 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) plus 4-oxo-2-nonenal (4-ONE) and 1,N(2)-etheno-2'-deoxyguanosine (epsilondG) plus 4-ONE could be detected from 2'-dA and 2'-dG, respectively. Addition of 2% propanol inhibited the formation of etheno adducts. Analyses of disappearance kinetics of dA and dG showed that dG was more rapidly eliminated than does dA (t[1/2] = 23.3 min and 98.3 min for dG and dA, respectively). Curves of formation kinetics revealed that the peak of epsilondG was at 55.6 min while that of epsilondA was at 186.9 min. These peaks represented 1.43% and 1.25% of the original dG and dA, respectively. In both cases, the peaks were followed by rapid degradations of etheno adducts. The results, obtained in this system, do not allow any extrapolation to realistic cellular responses; nevertheless, these data questioned the validity of the use of unsubstituted etheno adducts as reliable oxidative stress and nitro-PAH exposure biomarkers.