DNA base damage in chromatin of gamma-irradiated cultured human cells

We report on the chemical characterization of DNA base damage in chromatin of gamma-irradiated cultured human cells. Chromatin was isolated from unirradiated and irradiated cells and analyzed by gas chromatography/mass spectrometry with selected-ion monitoring after acidic hydrolysis of chromatin and trimethylsilylation of hydrolysates. Prior to analysis of chromatin samples, experimental conditions for acidic hydrolysis were optimized by determining the relative molar response factors of modified bases under non-acidic and acidic conditions, and their release from DNA under various acidic conditions. A number of modified bases in chromatin isolated from irradiated cells were identified and quantitated. These were 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin, 5-(hydroxymethyl)uracil, cytosine glycol, thymine glycol, 5,6-dihydroxycytosine, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine. Radiation doses ranging from 42 to 420 Gy (J.kg-1) were used. Background levels of all modified bases were observed in chromatin isolated from unirradiated cells. The radiation yields of a number of modified bases were increased significantly over their background levels at a dose as low as 42 Gy. In most cases, linear dose-yield relationships were obtained up to approximately 200 Gy. At radiation doses higher than 420 Gy, no additional increase in the yields of modified bases was observed. The yields of guanine-derived bases amounted to approximately 45% of the total net yield of modified bases measured, followed by almost equal yields of adenine-, cytosine- and thymine-derived bases. Modified bases identified were typical products of hydroxyl radical attack on DNA bases, indicating the involvement of hydroxyl radical, although their induction in part by the direct effect of ionizing radiation through ionization of DNA bases cannot be excluded. The yields of modified bases were lower than those previously measured after gamma-irradiation of fully expanded chromatin in aqueous buffer solutions.