DNA OXIDATION MATTERS - THE HPLC-ELECTROCHEMICAL DETECTION ASSAY OF 8-OXO-DEOXYGUANOSINE AND 8-OXO-GUANINE

Oxidative DNA damage is important in aging and the degenerative diseas es of aging such as cancer, Estimates commonly rely on measurements of 8-oxo-2'-deoxyguanosine (oxo(8)dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair, Here we examine difficulti es inherent in the analysis of oxo(8)dG, identify sources of artifacts , and provide solutions to some of the common methodological problems, A frequent criticism has been that phenol in DNA extraction solutions artificially increases the measured level of oxo(8)dG, We found that phenol extraction of DNA contributes a real but minor increase in the level of oxo(8)dG when compared, under equivalent conditions, with a s uccessful nonphenol method, A more significant reduction in the baseli ne level was achieved with a modification of the recently introduced c haotropic NaI method, reducing our estimate of the level of steady-sta te oxidative adducts by an order of magnitude to 24,000 adducts per ce ll in young rats and 66,000 adducts per cell in old rats, Of several a lternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo(8)d G values. In further studies we show that human urinary 8-oxo-guanine (oxo(8)Gua) excretion is not affected by the administration of allopur inol, suggesting that, unlike some methylated adducts, oxo(8)Gua is no t derived enzymatically from xanthine oxidase, Lastly, we discuss rema ining uncertainties inherent both in steady-state oxo(8)dG measurement s and in estimates of endogenous oxidation (''hit rates'') based on ur inary excretion of oxo(8)dG and oxo(8)Gua.