DNA oxidation matters: The HPLC–electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine

Abstract

Oxidative DNA damage is important in aging and the degenerative diseases 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 difficulties 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 successful nonphenol method. A more significant reduction in the baseline level was achieved with a modification of the recently introduced chaotropic NaI method, reducing our estimate of the level of steady-state oxidative adducts by an order of magnitude to 24,000 adducts per cell in young rats and 66,000 adducts per cell in old rats. Of several alternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo 8 dG values. In further studies we show that human urinary 8-oxo-guanine (oxo 8 Gua) excretion is not affected by the administration of allopurinol, suggesting that, unlike some methylated adducts, oxo 8 Gua is not derived enzymatically from xanthine oxidase. Lastly, we discuss remaining uncertainties inherent both in steady-state oxo 8 dG measurements and in estimates of endogenous oxidation (“hit rates”) based on urinary excretion of oxo 8 dG and oxo 8 Gua.