Characterization of free radical-induced base damage in DNA at biologically relevant levels

Abstract

DNA damage induced by oxygen radicals, e.g., hydroxyl radicals generated in living cells either by cellular metabolism or external agents such as ionizing radiations, appears to play an important role in mutagenesis, carcinogenesis, and aging. Elucidation of the chemical nature of such DNA lesions at biologically significant quantities is required for the assessment of their biological consequences at biologically significant quantities is required for the assessment of their biological consequences and repair. For this purpose, a sensitive method using gas chromatography-mass spectrometry with the selected-ion-monitoring technique ( GC-MS SIM ) was developed in the present work. DNA was exposed to hydroxyl radicals and hydrogen atoms produced by ionizing radiation in N 2O-saturated aqueous solution. DNA samples were subsequently hydrolyzed with formic acid, trimethylsilylated, and analyzed by GC-MS SIM . Characteristic ions from previously known mass spectra of DNA base products as their trimethylsilyl derivatives were recorded and the area counts of each ion were integrated. From these acquired data, a partial mass spectrum of each product was generated and then compared with those of authentic materials. This technique permitted the detection and characterization of a large number of free radical-induced based products of DNA, i.e., 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-hydroxycytosine, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine, simultaneously in a single sample after radiation doses from 0.1 to 10 Gy. Detectable amounts of the base products were found to be as low as ∼10 fmol per injection. Because the GC-MS SIM technique provides rapid and absolute characterization of a large number of free radical-induced base products simultaneously and does so with a high degree of sensitivity, it is suggested as an ideal analytical tool for the identification of such base lesions in cellular DNA, for their detection in biological fluid, and for the study of their repair and biological consequences.