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.

Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is formed by the near diffusion-limited reaction of ⅐NO and O 2 . during activation of phagocytes.Chronic inf lammation induced by phagocytes is a major contributor to cancer and other degenerative diseases. We examined how ␥-tocopherol (␥T), the principal form of vitamin E in the United States diet, and ␣-tocopherol (␣T), the major form in supplements, protect against peroxynitriteinduced lipid oxidation. Lipid hydroperoxide formation in liposomes (but not isolated low-density lipoprotein) exposed to peroxynitrite or the ⅐NO and O 2 . generator SIN-1 (3-morpholinosydnonimine) was inhibited more effectively by ␥T than ␣T. More importantly, nitration of ␥T at the nucleophilic 5-position, which proceeded in both liposomes and human low density lipoprotein at yields of Ϸ50% and Ϸ75%, respectively, was not affected by the presence of ␣T. These results suggest that despite ␣T's action as an antioxidant ␥T is required to effectively remove the peroxynitrite-derived nitrating species. We postulate that ␥T acts in vivo as a trap for membranesoluble electrophilic nitrogen oxides and other electrophilic mutagens, forming stable carbon-centered adducts through the nucleophilic 5-position, which is blocked in ␣T. Because large doses of dietary ␣T displace ␥T in plasma and other tissues, the current wisdom of vitamin E supplementation with primarily ␣T should be reconsidered.

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