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

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Abstract
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 μM) or 1,4‐dinitrophenol (100 μM) with a mixture of 150 μM NADH, 0.5 units of E. coli nitroreductase, 100 μM linoleic acid, 0.5 mM ferrous iron, and 100 μM 2′‐deoxyadenosine (2′‐dA) or 100 μM 2′‐deoxyguanosine (2′‐dG) were analyzed by liquid chromatography multistage mass spectrometry. Mixtures of 1,N6‐etheno‐2′‐deoxyadenosine (εdA) plus 4‐oxo‐2‐nonenal (4‐ONE) and 1,N2‐etheno‐2′‐deoxyguanosine (εdG) 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 εdG was at 55.6 min while that of εdA 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. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 222–227, 2007.

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