No 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.

Reactive oxygen species (ROS: superoxide radical, O2.-; hydrogen peroxide, H2O2; hydroxyl radical, OH.), which arise from the univalent reduction of dioxygen are formed in mitochondria. We summarize here results which indicate that ROS, and also the radical nitrogen monoxide ('nitric oxide', NO), act as physiological modulators of some mitochondrial functions, but may also damage mitochondria. Hydrogen peroxide, which originates in mitochondria predominantly from the dismutation of superoxide, causes oxidation of mitochondrial pyridine nucleotides and thereby stimulates a specific Ca2+ release from intact mitochondria. This release is prevented by cyclosporin A (CSA). Hydrogen peroxide thus contributes to the maintenance of cellular Ca2+ homeostasis. A stimulation of mitochondrial ROS production followed by an enhanced Ca2+ release and re uptake (Ca2+ 'cycling') by mitochondria causes apoptosis and necrosis, and contributes to hypoxia/reperfusion injury. These kinds of cell injury can be attenuated at the mitochondrial level by CSA. When ROS are produced in excessive amounts in mitochondria nucleic acids, proteins, and lipids are extensively modified by oxidation. Physiological (sub-micromolar) concentrations of NO potently and reversibly deenergize mitochondria at oxygen tensions that prevail in cells by transiently binding to cytochrome oxidase. This is paralleled by mitochondrial Ca2+ release and uptake. Higher NO concentrations or prolonged exposure of cells to NO causes their death. It is concluded that ROS and NO are important physiological reactants in mitochondria and become toxic only when present in excessive amounts.

SummaryBlood transfusion therapy is life-saving for patients with b-thalassaemia and sickle cell disease (SCD), but often results in severe iron overload. This pilot study examined whether the biomarkers of tissue injury or inflammation differ in these two diseases. Plasma malondialdehyde (MDA) was significantly increased 1AE8-fold in thalassaemia relative to control patients. In contrast, MDA in SCD was not significantly different from controls. In multivariate analysis, the strongest predictors of elevated MDA were liver iron concentration (P < 0AE001) and specific diagnosis (P ¼ 0AE019). A significant 2-fold elevation of non-transferrin bound iron (NTBI) was observed in thalassaemia relative to SCD. NTBI was not a significant predictor of high MDA in multivariate analysis. SCD patients showed a significant 2AE2-fold elevation of the inflammatory marker interleukin (IL)-6 relative to controls, and a 3AE6-and 1AE7-fold increase in IL-5 and IL-10 relative to thalassaemia. Although a-tocopherol was significantly decreased by at least 32% in both thalassaemia and SCD, indicating ongoing oxidant stress and antioxidant consumption, c-tocopherol, a nitric oxide-selective antioxidant, was increased 36% in SCD relative to thalassaemia. These results demonstrate that thalassaemia patients have increased MDA and circulating NTBI relative to SCD patients and lower levels of some cytokines and c-tocopherol. This supports the hypothesis that the biology of SCD may show increased inflammation and increased levels of protective antioxidants compared with thalassaemia.

Approximately two billion people, mainly women and children, are iron deficient. Two studies examined the effects of iron deficiency and supplementation on rats. In study 1, mitochondrial functional parameters and mitochondrial DNA (mtDNA) damage were assayed in iron-deficient (<5 g͞day) and iron-normal (800 g͞day) rats and in both groups after daily high-iron supplementation (8,000 g͞day) for 34 days. This dose is equivalent to the daily dose commonly given to iron-deficient humans. Iron-deficient rats had lower liver mitochondrial respiratory control ratios and increased levels of oxidants in polymorphonuclear-leukocytes, as assayed by dichlorofluorescein (P < 0.05). Rhodamine 123 fluorescence of polymorphonuclear-leukocytes also increased (P < 0.05). Lowered respiratory control ratios were found in daily high-iron-supplemented rats regardless of the previous iron status (P < 0.05). mtDNA damage was observed in both iron-deficient rats and rats receiving daily high-iron supplementation, compared with ironnormal rats (P < 0.05). Study 2 compared iron-deficient rats given high doses of iron (8,000 g) either daily or every third day and found that rats given iron supplements every third day had less mtDNA damage on the second and third day after the last dose compared to daily high iron doses. Both inadequate and excessive iron (10 ؋ nutritional need) cause significant mitochondrial malfunction. Although excess iron has been known to cause oxidative damage, the observation of oxidant-induced damage to mitochondria from iron deficiency has been unrecognized previously. Untreated iron deficiency, as well as excessive-iron supplementation, are deleterious and emphasize the importance of maintaining optimal iron intake.