Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions

The authors report on the elucidation of DNA-protein cross-links formed in isolated mammalian chromatin upon treatment with H2O2 in the presence of iron or copper ions. Analysis of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization showed the presence of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine (thymine-tyrosine cross-link) on the basis of the gas chromatographic and mass spectrometric characteristics of the trimethylsilylated authentic compound. Other DNA-protein cross-links involving thymine and the aliphatic amino acids and cytosine and tyrosine, which were known to occur in nucleohistone γ-irradiated under anoxic conditions, were not observed. This was due to inhibition by oxygen as clearly shown by experiments that were carried out using ionizing radiation under both oxic and anoxic conditions instead of using H2O2 and metal ions. The yield of the thymine-tyrosine cross-link was higher upon treatment with H2O2/chelated Fe3+ ions than with H2O2/unchelated Fe3+ions. By contrast, h2O2/unchelated Cu2+ ions produced a higher yield than H2O2/chelated Cu2+ions. Superoxide dismutase afforded partial inhibition only when chelated ions were used. The mechanism underlying formation of this DNA-protein cross-link is thought to involve addition of the hydroxyl radical generated allyl radical of thymine to carbon-3 of tyrosine followed by subsequent oxidation of the adduct radical