Abstract
Oxidized bases in DNA can be measured directly by high-performance liquid chromatography (HPLC). 7,8-Dihydro-8-oxo-guanine (8-OHgua), as the most abundant oxidation product, is often regarded as an indicator of oxidative stress. Estimates of endogenous 8-OHgua levels in human lymphocyte DNA are between 2 and 8 for every 10(5) unaltered bases--a high frequency in view of the potential mutagenicity of this base alteration and of the presence of an effective base excision repair pathway in eukaryotic cells. An alternative approach to the measurement of oxidized bases makes use of repair endonucleases with appropriate lesion specificities--endonuclease III, for oxidized pyrimidines and formamidopyrimidine glycosylase for 8-OHgua. These enzymes introduce breaks at sites of damage in DNA. The comet assay (single cell gel electrophoresis) can then be used to detect the DNA breaks. This modified comet assay, like other enzyme-linked DNA breakage assays, gives a value for endogenous oxidative base damage that is more than 10-fold lower than most estimated from HPLC. It is possible that HPLC-based estimates are artificially high because oxidation of guanine occurs during isolation, storage, or hydrolysis of DNA. Using a revised DNA isolation procedure designed to decrease in vitro oxidation, we have obtained results for 8-OHgua concentrations in human lymphocytes that are closure to the figures obtained by the comet assay. It is still an open question whether 8-OHgua, measured by HPLC or by the comet assay, is a valid marker for oxidative damage.