Oxidative DNA damage: biological significance and methods of analysis.

All forms of aerobic life are subjected constantly to oxidant pressure from molecular oxygen and also reactive oxygen species (ROS), produced during the biochemical utilization of O2 and prooxidant stimulation of O2 metabolism. ROS are thought to influence the development of human cancer and more than 50 other human diseases. To prevent oxidative DNA damage (protection) or to reverse damage, thereby preventing mutagenesis and cancer (repair), the aerobic cell possesses antioxidant defense systems and DNA repair mechanisms. During the last 20 years, many analytical techniques have been developed to monitor oxidative DNA base damage. High-performance liquid chromatography-electrochemical detection and gas chromatography-mass spectrometry are the two pioneering contributions to the field. Currently, the arsenal of methods available include the promising high-performance liquid chromatography-tandem mass spectrometry technique, capillary electrophoresis, 32P-postlabeling, fluorescence postlabeling, 3H-postlabeling, antibody-base immunoassays, and assays involving the use of DNA repair glycosylases such as the comet assay, the alkaline elution assay, and the alkaline unwinding method. Recently, the use of liquid chromatography-mass spectrometry has been introduced for the measurement of a number of modified nucleosides in oxidatively damaged DNA. The bulk of available chromatographic methods aimed at measuring individual DNA base lesions require either chemical hydrolysis or enzymatic digestion of oxidized DNA, following extraction from cells or tissues. The effect of experimental conditions (DNA isolation, hydrolysis, and/or derivatization) on the levels of oxidatively modified bases in DNA is enormous and has been studied intensively in the last 10 years.