Characterization of free radical-induced damage to DNA by the combined use of enzymatic hydrolysis and gas chromatography—mass spectrometry

Abstract

The use of gas chromatography—mass spectrometry (GC—MS) for characterization of free radical-induced based damage to DNA is presented. Damage introduced to DNA by reactive oxygen species such as hydroxyl radicals appears to play an important role in mutagenesis, carcinogenesis and aging. Elucidation of the chemical nature of such DNA lesions is necessary for the assessment of their biology consequences and enzymatic repair. DNA exposed to radiation-generated hydroxyl radicals in aqueous solution was hydrolyzed to 2′-deoxyribonucleosides with a mixture of DNase I, venom and spleen exonucleases and alkaline phosphatase. The hydrolysate was subsequently trimethylsilylated and analyzed by GC-MS. A large number of DNA lesions were separated and identified. Mass spectra obtained were interpreted on the basis of the typical fragmentation pathways of trimethylsilylated nucleosides. The use of GC—MS with selected-ion monitoring facilitated the detection of these lesions at the very low quantities and radiation doses (below 10 Gray) that might be relevant to those in biological systems.