Modification of DNA bases in mammalian chromatin upon treatment with hydrogen peroxide in the presence of ferric and cupric ions was studied. Ten DNA base products in mammalian chromatin were identified and quantitated by the use of gas chromatography-mass spectrometry with selected-ion monitoring after hydrolysis of chromatin and trimethylsilylation of hydrolysates. This technique permitted the analysis of modified DNA bases in chromatin without the necessity of isolation of DNA from chromatin first. Modified bases identified were typical hydroxyl radical-induced products of DNA, indicating the involvement of hydroxyl radical in their formation. This was also confirmed by inhibition of product formation by typical scavengers of hydroxyl radical. The inhibition of product formation was much more prominent in the presence of chelated ions than unchelated ions, indicating a possible site-specific formation of hydroxyl radical when metal ions are bound to chromatin. Hydrogen peroxide in the presence of cupric ions caused more DNA damage than in the presence of ferric ions. Chelation of cupric ions caused a marked inhibition in product formation. By contrast, DNA was damaged more extensively in the presence of chelated ferric ions than in the presence of unchelated ferric ions. The presence of ascorbic acid generally increased the yields of the products, indicating increased production of hydroxyl radical by reduction of metal ions by ascorbic acid. Superoxide dismutase afforded partial inhibition of product formation only in the case of chelated iron ions. The yields of the modified bases in chromatin were lower than those observed with calf thymus DNA under the same conditions.
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