Bulky adducts detected by 32P-postlabeling in DNA modified by oxidative damage in vitro. Comparison with rat lung I-compounds.

Oxygen free radicals, such as the hydroxyl radical generated by interaction of Fe2+ and H2O2 (Fenton reaction), are produced in mammalian cells as a result of aerobic metabolism and under various pathological conditions and are known to elicit mutations and potentially other adverse effects by reacting with DNA bases. Several products thus formed have recently been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole-ring-opened derivatives of adenine and guanine in DNA. As shown herein by 32P-postlabeling, incubation of DNA under Fenton reaction conditions led to additional products which, by virtue of resistance to nuclease P1 catalyzed 3'-dephosphorylation and chromatographic behavior, appeared to be bulky adducts rather than small polar, hydroxylated or ring-opened nucleotide derivatives. Two major and five minor DNA derivatives were measured after 32P-postlabeling and TLC mapping of DNA oxidized in vitro under conditions known to lead to formation of reactive oxygen species. Amounts of products formed depended on Fe2+ and H2O2 concentrations and increased in the presence of L-ascorbic acid. One of the two major products was also detected in lung DNA of rats where its amount increased with animal age. Thus, at least one I-compound appeared to have its origin in the interaction of DNA with reactive oxygen species.