An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands

Oxidative cell damage is involved in the pathogenesis of atherosclerosis, cancer, diabetes and other diseases. Uptake of fruit juice with especially high content of antioxidant flavonoids/polyphenols, might reduce oxidative cell damage. Therefore, an intervention study was performed with a red mixed berry juice [trolox equivalent antioxidative capacity (TEAC): 19.1 mmol/L trolox] and a corresponding polyphenol-depleted juice (polyphenols largely removed, TEAC 2.4 mmol/L trolox), serving as control. After a 3-week run-in period, 18 male probands daily consumed 700 mL juice, and 9 consumed control juice, in a 4-week intervention, followed by a 3-week wash-out. Samples were collected weekly to analyze DNA damage (comet assay), lipid peroxidation (plasma malondialdehyde: HPLC/fluorescence; urinary isoprostanes: GC-MS), blood glutathione (photometrically), DNA-binding activity of nuclear factor-kappaB (ELISA) and plasma carotenoid/alpha-tocopherol levels (HPLC-DAD). During intervention with the fruit juice, a decrease of oxidative DNA damage (p<5x10(-4)) and an increase of reduced glutathione (p<5x10(-4)) and of glutathione status (p<0.05) were observed, which returned to the run-in levels in the subsequent wash-out phase. The other biomarkers were not significantly modulated by the juice supplement. Intervention with the control juice did not result in reduction of oxidative damage. In conclusion, the fruit juice clearly reduces oxidative cell damage in healthy probands.     

Carbohydrate restriction does not change mitochondrial free radical generation and oxidative DNA damage

Many previous investigations have consistently reported that caloric restriction (40%), which increases maximum longevity, decreases mitochondrial reactive species (ROS) generation and oxidative damage to mitochondrial DNA (mtDNA) in laboratory rodents. These decreases take place in rat liver after only seven weeks of caloric restriction. Moreover, it has been found that seven weeks of 40% protein restriction, independently of caloric restriction, also decrease these two parameters, whereas they are not changed after seven weeks of 40% lipid restriction. This is interesting since it is known that protein restriction can extend longevity in rodents, whereas lipid restriction does not have such effect. However, before concluding that the ameliorating effects of caloric restriction on mitochondrial oxidative stress are due to restriction in protein intake, studies on the third energetic component of the diet, carbohydrates, are needed. In the present study, using semipurified diets, the carbohydrate ingestion of male Wistar rats was decreased by 40% below controls without changing the level of intake of the other dietary components. After seven weeks of treatment the liver mitochondria of the carbohydrate restricted animals did not show changes in the rate of mitochondrial ROS production, mitochondrial oxygen consumption or percent free radical leak with any substrate (complex I- or complex II-linked) studied. In agreement with this, the levels of oxidative damage in hepatic mtDNA and nuclear DNA were not modified in carbohydrate restricted animals. Oxidative damage in mtDNA was one order of magnitude higher than that in nuclear DNA in both dietary groups. These results, together with previous ones, discard lipids and carbohydrates, and indicate that the lowered ingestion of dietary proteins is responsible for the decrease in mitochondrial ROS production and oxidative damage in mtDNA that occurs during caloric restriction.     

Gender differences in steady-state levels of oxidative damage to DNA in healthy individuals

Oxidative damage to DNA has often been used as a biomarker for oxidative stress and more specifically for cancer risk. Indeed, the measurement of oxidative damage to DNA, particularly of 8-hydroxyguanine (8OHG) and 8-hydroxy-2'-deoxyguanosine (8OHdG), has been adopted as a method for establishing the effects of antioxidant supplementation towards protection from certain cancers, cardiovascular and neuro-degenerative diseases, both in patients and healthy individuals. However, reported levels of 8OHdG or 8OHG vary considerably, possibly due to the different methodologies used, and only few data are available for the non-smoking and the female population. In this paper, steady-state levels of oxidative damage to DNA measured in a group of 20 males and 19 females are reported. Significant gender differences in levels of modified DNA bases such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine), 8-hydroxyadenine (8OHA) and 5-hydroxycytosine (5OHC), measured by gas chromatography-mass spectrometry (GC/MS), were observed. The results are discussed in relation to the Vitamin C and iron status of the subjects and to the existing, yet limited, literature data. The role of gender in predisposition to oxidative damage to DNA needs to be addressed in future studies.     

Iron supplementation and oxidative damage to DNA in healthy individuals with high plasma ascorbate

Previously, we have investigated the potential for a pro-oxidant interaction of iron and ascorbate in vivo in iron and ascorbate cosupplementation or ascorbate supplementation studies. In this study, for the first time, the effects of iron supplementation on oxidative damage to DNA in healthy individuals with plasma ascorbate levels at the upper end of the normal range were examined. Forty female and male volunteers (mean plasma ascorbate approximately equal to 70 micromol/L) were supplemented with a daily dose of syrup (ferrous glycine sulphate equivalent to 12.5 mg iron) for 6 weeks. Serum ferritin, transferrin bound iron, % saturation of transferrin and plasma ascorbate were assessed and the mean dietary intakes of all subjects were estimated through food frequency questionnaires. Oxidative damage to DNA bases from white blood cells was measured by gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM), using isotope-labelled standards for quantification. Iron supplementation did not affect any of the iron status parameters. There were also no detrimental effects, over the period under investigation, in terms of oxidative damage to DNA. However, the effects of larger doses or of longer supplementation periods should also be investigated.     

Losartan reduces oxidative damage to renal DNA and conserves plasma antioxidant capacity in diabetic rats

Increased reactive oxygen species (ROS) levels produced by hyperglycemia and angiotensin-II (AT-II) are considered among the pathogenic factors in the malignant transformation of diabetic renal cells. We aimed to investigate the potential role of AT-II in the increased cancer risk seen in diabetes; measuring oxidative damage to renal DNA and protective antioxidant defenses, including adiponectin (Adp) and plasma antioxidant capacity by the Ferric Reducing Ability of Plasma (FRAP) method. In the kidney of streptozotocin (STZ)-induced (55 mg/kg) diabetic rats either treated or not treated for 3 weeks with losartan, an AT-II type 1 receptor antagonist (20 mg/kg/day); we measured 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) levels, as an index of oxidative DNA damage, circulating Adp and FRAP. Diabetic rats showed significantly higher 8-oxodGuo levels in renal DNA (8.48 ± 0.98 × 10⁻⁶ dG, mean ± SEM n = 11) than normoglycemic ones (1.18 ± 0.04 × 10⁻⁶ dG, mean ± SEM, n=7) and lower plasma Adp and FRAP levels in comparison to normoglycemics. The treatment of diabetic rats with losartan significantly (P < 0.01) reduced 8-oxodGuo levels (5.4 ± 0.58 × 10⁻⁶ dG, mean ± SEM n=9) in renal DNA and conserved FRAP values. Moreover, an inverse correlation was found between 8-oxodGuo in kidney DNA and circulating Adp levels in normoglycemic and diabetic rats. Losartan treatment preserves FRAP levels, reduces DNA oxidative injury and thus the carcinogenesis risk. Furthermore, our results indicate that Adp plasma levels are a further marker of oxidative injury to the kidney and confirm that it is an important part of the plasma antioxidant defense.     

Tomato consumption modulates oxidative DNA damage in humans.

Consumption of a single serving of tomatoes by healthy human volunteers was sufficient to alter levels of oxidative DNA base damage in white cell DNA within 24 h. Levels of the mutagenic oxidized purine base 8-hydroxyguanine decreased, especially in those subjects whose initial levels of this base were higher than the mean. However, total DNA base damage remained unchanged since levels of 8-hydroxyadenine rose. The ability of tomato consumption to modulate oxidative DNA damage in the short term may indicate why daily consumption of fruits and vegetables is beneficial in decreasing cancer incidence.     

Diet modification affects DNA oxidative damage in healthy humans

DNA 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a promising biomarker for oxidative damage. We assessed its responsiveness to diet in 32 nonsmoking, healthy subjects (12 male, 20 female) aged 31+/-7.6 years. They consumed two liquid formula diets (Ensures) as the sole source of nutrition for 10-d in a randomized crossover design, with 5-d control solid food diets as washout before each liquid diet period. Reformulated Ensure (Re-En) had a vitamin E/ PUFA of 3.5 compared to standard Ensure (En) of 1.1. We hypothesized that subjects would have lower leukocyte 8-OHdG/deoxyguanosine (dG) ratios while consuming Re-En compared to En. But 8-OHdG/dG ratios did not change with the consumption of either Re-En or En. The mean ratios of 8-OHdG/dG after 10 days of Re-En and En consumption were (2.12+/-0.68)x10(-5) and (2.16+/-0.63)x10(-5), respectively. However, there was a 22% decrease in 8-OHdG/dG by the end of the study and a significant downward trend of leukocyte 8-OHdG among all subjects throughout all nutrient-rich diet phases as the study progressed (Test for trend: p = .04; paired t-test: p = .07). Because all the experimental diets provided antioxidant nutrients at higher quantities than typically consumed by a U.S. age-matched population, this study adds to the few in vivo studies that show a decrease in DNA damage in healthy nonsmoking subjects through dietary intervention.     

Dietary fish oil reduces oxidative DNA damage in rat colonocytes

Prolonged generation of reactive oxygen species by inflammatory mediators can induce oxidative DNA damage (8-oxodG formation), potentially resulting in intestinal tumorigenesis. Fish oil (FO), compared to corn oil (CO), has been shown to downregulate inflammation and upregulate apoptosis targeted at damaged cells. We hypothesized FO could protect the intestine against 8-oxodG formation during dextran sodium sulfate- (DSS-) induced inflammation. We provided 60 rats with FO- or CO-supplemented diets for 2 weeks with or without 3% DSS in drinking water for 48 h. Half the treated rats received 48 additional h of untreated water before termination. Due to DSS treatment, the intestinal epithelium had higher levels of 8-oxodG (p =.04), induction of repair enzyme OGG1 mRNA (p =.02), and higher levels of apoptosis at the top of colonic crypts (p =.01) and in surface cells (p <.0001). FO-fed rats, compared to CO, had lower levels of 8-oxodG (p =.05) and increased apoptosis (p =.04) in the upper crypt region; however, FO had no significant effect on OGG1 mRNA. We conclude that FO protects intestinal cells against oxidative DNA damage in part via deletion mechanisms.     

Fruit juice consumption modulates antioxidative status,immune status and DNA damage

Polyphenolic compounds exert a variety of physiological effects in vitro including antioxidative, immunomodulatory and antigenotoxic effects. In a randomized crossover study in healthy men on a low-polyphenol diet, we determined the effects of 2 polyphenol-rich juices (330 ml/d) supplemented for 2 weeks on bioavailability of polyphenols, markers of antioxidative and immune status, and reduction of DNA damage. Juices provided 236 mg (A) and 226 mg (B) polyphenols with cyanidin glycosides (A) and epigallocatechin gallate (B) as major polyphenolic ingredients. There was no accumulation of plasma polyphenols after two weeks of juice supplementation. In contrast, plasma malondialdehyde decreased with time during juice interventions. Moreover, juice consumption also increased lymphocyte proliferative responsiveness, with no difference between the two juices. Interleukin-2 secretion by activated lymphocytes and the lytic activity of natural killer cells were significantly increased by both juices. Juice intervention had no effect on single DNA strand breaks, but significantly reduced oxidative DNA damage in lymphocytes. A time-delay was observed between the intake of fruit juice and the reduction of oxidative DNA damage and the increase in interleukin-2 secretion. We conclude that consumption of either juice enhanced antioxidant status, reduced oxidative DNA damage and stimulated immune cell functions. However, fruit juice consumption for 2 weeks did not result in elevated plasma polyphenols in subjects after overnight fasting. Further studies should focus on the time-delay between juice intake and changes in measured physiological functions, as well as on active polyphenolic metabolites mediating the observed effects.     

Monitoring of oxidative free radical damage in vivo: analytical aspects.

Free radical damage is an important factor in many pathological and toxicological processes. During the last decade a wide range of methods has been developed to determine free radical damage in various biological fluids and at various stages of development. This review offers an overview of the state of the art of monitoring free radical damage in vivo, with special emphasis on the analytical aspects of non-invasive methods.     

Effects of polyphenols from grape seeds on oxidative damage to cellular DNA

Grape seed polyphenols have been reported to exhibit a broad spectrum of biological properties. In this study, eleven phenolic phytochemicals from grape seeds were purified by gel chromatography and high performance liquid chromatography (HPLC). The antioxidant activities of five representative compounds with different structure type were assessed by the free radical-scavenging tests and the effects of the more potent phytochemicals on oxidative damage to DNA in mice spleen cells were investigated. Procyanidin B4, catechin, epicatechin and gallic acid reduced ferricyanide ion and scavenged the stable free radical, alpha, alpha-diphenyl-beta-picrylhydrazyl (DPPH) much more effectively than the known antioxidant vitamin ascorbic acid, while epicatechin lactone A, an oxidative derivative of epicatechin, did not reduce ferricyanide ion appreciably at concentrations used and was only about half as effective on free radical-scavenging as epicatechin. Mice spleen cells, when pre-incubated with relatively low concentration of procyanidin B4, catechin or gallic acid, were less susceptible to DNA damage induced by hydrogen peroxide (H2O2), as evaluated by the comet assay. In contrast, noticeable DNA damage was induced in mice spleen cells by incubating with higher concentration (150 microM) of catechin. Collectively, these data suggest that procyanidin B4, catechin, gallic acid were good antioxidants, at low concentration they could prevent oxidative damage to cellular DNA. But at higher concentration, these compounds may induce cellular DNA damage, taking catechin for example, which explained the irregularity of dose-effect relationship.     

Protective effects of grape seed extract against oxidative and nitrative damage of plasma proteins

Oxidative stress, vascular inflammation, endothelial dysfunction plays a crucial role in the pathogenesis of cardiovascular diseases. The aim of our in vitro study was to examine the antioxidative properties of grape seed extract, and its potential protective effect on the haemostatic function of human fibrinogen under oxidative stress conditions, induced by peroxynitrite (100 μM). The preincubation of plasma with the tested extract (0.5-50 μg/ml or 0.5-300 μg/ml) reduced the formation of 3-nitrotyrosine and diminished oxidation of thiol groups in plasma proteins. The low concentrations (0.5-50 μg/ml) of grape seed extract also decreased the level of carbonyl groups, however at higher concentrations (100-300 μg/ml) this effect was not observed. Furthermore, grape seed extract counteracted the inhibitory effect of peroxynitrite on human plasma clotting. The results obtained in this study indicate that components of the grape seed extract posses antioxidative properties and may be promising substances for the creation of new dietary supplements.     

Blue light induces mitochondrial DNA damage and free radical production in epithelial cells

Exposure of biological chromophores to ultraviolet radiation can lead to photochemical damage. However, the role of visible light, particularly in the blue region of the spectrum, has been largely ignored. To test the hypothesis that blue light is toxic to non-pigmented epithelial cells, confluent cultures of human primary retinal epithelial cells were exposed to visible light (390-550 nm at 2.8 milliwatts/cm2) for up to 6 h. A small loss of mitochondrial respiratory activity was observed at 6 h compared with dark-maintained cells, and this loss became greater with increasing time. To investigate the mechanism of cell loss, the damage to mitochondrial and nuclear genes was assessed using the quantitative PCR. Light exposure significantly damaged mitochondrial DNA at 3 h (0.7 lesion/10 kb DNA) compared with dark-maintained controls. However, by 6 h of light exposure, the number of lesions was decreased in the surviving cells, indicating DNA repair. Isolated mitochondria exposed to light generated singlet oxygen, superoxide anion, and the hydroxyl radical. Antioxidants confirmed the superoxide anion to be the primary species responsible for the mitochondrial DNA lesions. The effect of lipofuscin, a photoinducible intracellular generator of reactive oxygen intermediates, was investigated for comparison. Exposure of lipofuscin-containing cells to visible light caused an increase in both mitochondrial and nuclear DNA lesions compared with non-pigmented cells. We conclude that visible light can cause cell dysfunction through the action of reactive oxygen species on DNA and that this may contribute to cellular aging, age-related pathologies, and tumorigenesis.     

The effect of consumption of purple grape juice in the gestational period on oxidative stress parameters in Wistar rat foetuses

This study aimed to evaluate the effects of purple grape juice consumption in pregnancy on oxidative stress parameters in Wistar rat fetuses. Twenty-four pregnant rats were divided into five groups: control group, indomethacin group (received a single dose of indomethacin in DG20), group grape juice DG14 (received an amount for 14 days/first and second gestational trim), group grape juice DG20 (received a dose throughout the gestational period), group grape juice two doses (received two doses, at morning and afternoon). On the 20th day of pregnancy (DG20), rats were anesthetized, and a cesarean section was performed to obtain the fetuses. A sample of liver, heart, and total brain of fetuses was collected for oxidative stress analyses. Values P<0.05 were considered significant. In fetuses’ heart, we observed that the grape juice two dose group decreased sulfhydryl and increased SOD. In the liver, the grape juice decreased TBARS and SOD. There was a decrease in carbonyl and sulfhydryl in the indomethacin and grape juice one dose groups in the brain. We conclude that indomethacin altered oxidative stress parameters only in the fetal brain, and grape juice was presented as an important modulator of antioxidant capacity when consumed in a dose.     

Cyclosporine treatment reduces oxygen free radical generation and oxidative stress in the brain of hypoxia-reoxygenated newborn piglets

Oxygen free radicals have been implicated in the pathogenesis of hypoxic-ischemic encephalopathy. It has previously been shown in traumatic brain injury animal models that treatment with cyclosporine reduces brain injury. However, the potential neuroprotective effect of cyclosporine in asphyxiated neonates has yet to be fully studied. Using an acute newborn swine model of hypoxia-reoxygenation, we evaluated the effects of cyclosporine on the brain, focusing on hydrogen peroxide (H₂O₂) production and markers of oxidative stress. Piglets (1–4 d, 1.4–2.5 kg) were block-randomized into three hypoxia-reoxygenation experimental groups (2 h hypoxia followed by 4 h reoxygenation)(n = 8/group). At 5 min after reoxygenation, piglets were given either i.v. saline (placebo, controls) or cyclosporine (2.5 or 10 mg/kg i.v. bolus) in a blinded-randomized fashion. An additional sham-operated group (n = 4) underwent no hypoxia-reoxygenation. Systemic hemodynamics, carotid arterial blood flow (transit-time ultrasonic probe), cerebral cortical H₂O₂ production (electrochemical sensor), cerebral tissue glutathione (ELISA) and cytosolic cytochrome-c (western blot) levels were examined. Hypoxic piglets had cardiogenic shock (cardiac output 40–48% of baseline), hypotension (mean arterial pressure 27–31 mmHg) and acidosis (pH 7.04) at the end of 2 h of hypoxia. Post-resuscitation cyclosporine treatment, particularly the higher dose (10 mg/kg), significantly attenuated the increase in cortical H₂O₂ concentration during reoxygenation, and was associated with lower cerebral oxidized glutathione levels. Furthermore, cyclosporine treatment significantly attenuated the increase in cortical cytochrome-c and lactate levels. Carotid blood arterial flow was similar among groups during reoxygenation. Conclusively, post-resuscitation administration of cyclosporine significantly attenuates H₂O₂ production and minimizes oxidative stress in newborn piglets following hypoxia-reoxygenation.     

Generation of superoxide free radical by neocarzinostatin and its possible role in DNA damage

Spectroscopic analysis of the reduction of both nitro blue tetrazolium and ferricytochrome c induced by neocarzinostatin shows that superoxide free radical is produced during the spontaneous degradation of the antibiotic. The amount of superoxide free radical produced from neocarzinostatin is not affected by the presence of thiol, although earlier work has shown that DNA damage is stimulated at least 1000-fold by thiol. Transition metals are not involved in this reaction. Although superoxide dismutase inhibits the reduction of nitro blue tetrazolium and cytochrome c induced by neocarzinostatin, neither it nor catalase interferes with the action of neocarzinostatin on DNA, whether or not drug has been activated by thiol. The pH profiles for spontaneous base release and alkali-labile base release (a measure of nucleoside 5'-aldehyde formation at a strand break) do not correspond with that for the generation of superoxide free radical from neocarzinostatin. The same holds for supercoiled DNA cutting by neocarzinostatin chromophore in the absence of a thiol, which is an acid-favored reaction. These results indicate that the generation of superoxide free radical by the drug does not correlate with DNA damage activity, whether or not thiol is present. Furthermore, the failure of hydroxyl free-radical scavengers to inhibit drug-induced single-strand breaks in supercoiled DNA in the absence of thiol also indicates that a diffusible hydroxyl free radical is most probably not involved in this reaction.     

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O6-guanine and to form interstrand cross-links (ISC) has been studied using Mer+ and Mer- human embryonic cells. Mer+ (IMR-90) cells have been reported to contain an O6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer- (VA-13) cell line, which is deficient in the repair of O6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.