Vitamin E prevents increase in oxidative damage to lipids and DNA in liver of ODS rats given total body X-ray irradiation

We examined the effects of dietary vitamin E (VE) on oxidative damage to DNA and lipids in the liver a few days after total body irradiation (TBI). ODS rats, which lack vitamin C synthesis, were fed either a low VE diet (4.3 λmg λVE/kg) or a basal VE diet (75.6 λmg λVE/kg) for 5 weeks while vitamin C was supplied in the drinking water. The VE level in the liver of the low VE group was lower and the levels of lipid peroxides were higher compared to those of the basal VE group: the relative levels in the two groups were 1:30 for VE, 18:1 for 4-hydroxynonenal (HNE), and 10:1 for hexanal (HA). The level of 8-hydroxydeoxyguanosine (8OHdG), a marker of oxidative DNA damage, did not differ between the low VE and the basal VE groups. When the rats received TBI at the dose of 3 λGy and were killed on day 6, the levels of HNE, HA and 8OHdG increased by 2.2-, 2-, and 1.5-times, respectively, in the low VE group, but TBI did not cause such increases in the basal VE group. Changes in antioxidative enzymes (glutathione peroxidase, catalase, and Cu/Zn-SOD) in the liver could not explain the different responses of the two diet groups to TBI-induced oxidative damage. The concentrations of vitamin C and glutathione in the liver did not differ between the two groups. These results suggest that dietary VE can prevent the oxidative damage to DNA and lipids in the liver which appear a few days after TBI at dose of 3 λGy.     

Vitamin C prevents cigarette smoke-induced oxidative damage in vivo

Our recent in vitro results [4] indicate that cigarette smoke induces oxidation of human plasma proteins and extensive oxidative degradation of the guinea pig lung, heart, and liver microsomal proteins, which is almost completely prevented by ascorbic acid. In this paper, we substantiate the in vitro results with in vivo observations. We demonstrate that exposure of subclinical or marginal vitamin C-deficient guinea pigs to cigarette smoke causes oxidation of plasma proteins as well as extensive oxidative degradation of the lung microsomal proteins. Cigarette smoke exposure also results in some discernible damage of the heart microsomal proteins. The oxidative damage has been manifested by SDS-PAGE, accumulation of carbonyl and bityrosine, as well as loss of tryptophan and protein thiols. Cigarette smoke exposure also induces peroxidation of microsomal lipids as evidenced by the formation of conjugated dienes, malondialdehyde, and fluorescent pigment. Cigarette smoke-induced oxidative damage of proteins and peroxidation of lipids are accompanied by marked drop in the tissue ascorbate levels. Protein damage and lipid peroxidation are also observed in cigarette smoke-exposed pair-fed guinea pigs receiving 5 mg vitamin C/animal/day. However, complete protection against protein damage and lipid peroxidation occurs when the guinea pigs are fed 15 mg vitamin C/animal/day. Also, the cigarette smoke-induced oxidative damage of proteins and lipid is reversed after discontinuation of cigarette smoke exposure accompanied by ascorbate therapy. The results, if extrapolated to humans, indicate that comparatively large doses of vitamin C may protect the smokers from cigarette smoke-induced oxidative damage and associated degenerative diseases.     

Vitamin E protects DNA from oxidative damage in human hepatocellular carcinoma cell lines

Expression of multiple drug resistant (MDR) phenotype and over-expression of P-glycoprotein (P-gp) in the human hepatocellular carcinoma (HCC) cell clone P1(0.5), derived from the PLC/PRF/5 cell line (P5), are associated with strong resistance to oxidative stress and a significant (p < 0.01) increase in intracellular vitamin E content as compared with the parental cell line. This study evaluates the role of vitamin E in conferring resistance to drugs and oxidative stress in P1(0.5) cells. Parental drug-sensitive cells, P5, were incubated in alpha-tocopherol succinate (alpha-TS, 5 microM for 24 h) enriched medium to increase intracellular vitamin E content to levels comparable to those observed in P1(0.5) cells at basal conditions. Susceptibility to lipid peroxidation and oxidative DNA damage were assessed by measuring the concentration of thiobarbituric-reactive substances (TBARS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) at basal and after experimental conditions. Cell capacity to form colonies and resistance to doxorubicin were also studied. P5 cells, treated with alpha-TS, became resistant to ADP-Fe3+ and to ionizing radiation-induced lipid peroxidation as P1(0.5) cells. Exposure to ADP-Fe3+ or ionizing radiation increased TBARS and the 8-OHdG content in the P5 cells, while vitamin E enrichment abolished these effects. Irradiation doses at 5 cGy increased TBARS and 8-OHdG. They also inhibited cell capacity to form colonies in the untreated P5 cells. Incubation with alpha-TS fully reverted this effect and significantly (p < 0.01) reduced the inhibitory effect of cell proliferation induced by irradiation doses at >500 cGy. Resistance to doxorubicin was not affected by alpha-TS. These observations demonstrate the role of vitamin E in conferring protection from lipid peroxidation, ionizing radiation and oxidative DNA damage on the human HCC cell line. They also rule out any role of P-gp over-expression as being responsible for these observations in cells with MDR phenotype expression.     

Vitamin C for DNA damage prevention

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels>50μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels>50μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels>50μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.     

Vitamin E protects against methyl ethyl ketone peroxide-induced peroxidative damage to rat brain DNA

Peroxidative damage to DNA initiated by methyl ethyl ketone peroxide, a potent initiator of lipid peroxidation, and protection against this damage by vitamin E were studied in rats. Groups of rats were fed a casein-based diet that contained 10% tocopherol-stripped corn oil and either 0, 3, 5, or 10 IU of DL-alpha-tocopherol acetate/kg; the groups were named 0, 3, 5, or 10, respectively. DNA isolated from the brains of these rats was analyzed for template activity, bound tryptophan, and malondialdehyde-type DNA-protein and interstrand DNA crosslinks. The DNA of groups 5 and 10 had significantly higher template activity, less bound tryptophan, and fewer crosslinks than that of groups 3 and 5, respectively. The DNA of group 3 had significantly fewer interstrand DNA crosslinks than that of group 0, and the most significant differences were between groups 3 and 5. Loss of template activity correlated best with interstrand DNA crosslinks, and bound tryptophan correlated best with DNA-protein crosslinks. Electrophoresis of the RNA transcribed from the isolated DNA showed that a significantly higher percentage of longer RNA was made from the DNA of groups 5 and 10 than from that of group 0. The apparent molecular weight of the DNA of group 0 was less than that of group 10 and was more heterogeneous, which suggests fragmentation and/or crosslinking. The DNA from group 10 had maximum observed template activity; therefore, 10 IU of vitamin E/kg of diet appeared to be adequate to protect brain DNA against the damage measured in this study.     

Vitamin C prevents DNA mutation induced by oxidative stress

The precise role of vitamin C in the prevention of DNA mutations is controversial. Although ascorbic acid has strong antioxidant properties, it also has pro-oxidant effects in the presence of free transition metals. Vitamin C was recently reported to induce the decomposition of lipid hydroperoxides independent of metal interactions, suggesting that it may cause DNA damage. To directly address the role of vitamin C in maintaining genomic integrity we developed a genetic system for quantifying guanine base mutations induced in human cells under oxidative stress. The assay utilized a plasmid construct encoding the cDNA for chloramphenicol acetyl transferase modified to contain an amber stop codon, which was restored to wild type by G to T transversion induced by oxidative stress. The mutation frequency was determined from the number of plasmids containing the wild type chloramphenicol acetyl transferase gene rescued from oxidatively stressed cells. Cells were loaded with vitamin C by exposing them to dehydroascorbic acid, thereby avoiding transition metal-related pro-oxidant effects of ascorbic acid. We found that vitamin C loading resulted in substantially decreased mutations induced by H(2)O(2). Depletion of glutathione led to cytotoxicity and an increase in H(2)O(2)-induced mutation frequency; however, mutation frequency was prominently decreased in depleted cells preloaded with vitamin C. The mutation results correlated with a decrease in total 8-oxo-guanine measured in genomic DNA of cells loaded with vitamin C and oxidatively stressed. These findings directly support the concept that high intracellular concentrations of vitamin C can prevent oxidation-induced mutations in human cells.     

Dietary supplementation of Vitamin E protects heart tissue from exercise-induced oxidant stress

Exhaustive endurance exercise in adult female albino rats (C-Ex) increased the generation of free radicals (R ·) in the myocardium, probably through enhanced oxidative mechanisms. Free radical mediated lipid peroxidation measured in the form of tissue MDA content also increased in C-Ex animals, suggesting the exercise-induced oxidative stress in these animals. Dietary supplementation of Vit E, for a period of 60 days significantly increased Vit E incorporation into the serum and myocardium, more so in the myocardium. Vit E supplementation to exercising animals completely abolished the radical production. The protection of Vit E against oxidative stress appears to be not mediated through the improvement of antioxidant mechanisms by enzymes like SOD, catalase and Se-GSH Px. However the non Se-GSH Px, the enzyme involved in the reduction of endoperoxides increased significantly in control and Vit E fed animals in response to exercise. The protection of Vit E against exercise-induced oxidative stress was correlated with its multivarious activities like a) scavenger of free radicals; b) inhibition of lipoxygenases; and c) reduction of peroxides in association with lipoxygenases. These studies indicate that dietary supplementation of Vit E protects the animals from the possible oxidative damages of endurance exercise.     

Vitamin E prevents oxidation of antiapoptotic proteins in neuronal cells

Oxidative damage to neuronal proteins appears to be central to the toxicity associated with a number of neuropathologies, including Alzheimer's disease. We have examined this by using oxidative stress to induce apoptosis in a mouse hippocampal neuronal cell line (HT-22). Oxidatively modified proteins were measured by high-resolution two-dimensional gel electrophoresis coupled with oxidation-specific immunostains. Under these conditions the oxidatively stressed cells undergo apoptosis, and specific proteins are oxidized. The three proteins that appeared to be most susceptible to oxidation were identified by mass spectrometry. Those oxidized proteins are heat shock protein 60 and vimentin, both believed to function as antiapoptotic proteins, and a third protein with sequence homology to hemoglobin alpha-chain. When the cells were pretreated with vitamin E, these proteins were not oxidized and the cells did not undergo apoptosis.     

Vitamin E prevents nonylphenol-induced oxidative stress in testis of rats

In the present study we have investigated if administration of nonylphenol-induced oxidative stress in various subcellular fractions of adult rat testis and the effect of vitamin E on reactive oxygen species mediated nonylphenol toxicity. Male rats were administered orally with nonylphenol at 1, 10 and 100 microg/kg body weight per day for 45 days with and without supplementation of vitamin E (20 mg/kg body weight). In nonylphenol-treated rats the activities of antioxidant enzymes superoxide dismutase and glutathione reductase decreased significantly while the levels of lipid peroxidation increased significantly in the crude homogenate and in the mitochondrial and microsome-rich fractions of testis. Co-administration of nonylphenol and vitamin E did not cause changes in the activities of antioxidant enzymes in various subcellular fractions of rat testis. The results suggest that graded doses of nonylphenol elicit depletion of antioxidant defence system in rat testis, indicating nonylphenol induced oxidative stress in the testis of rats which could be reversed by the administration of vitamin E.     

Hemimethylation prevents DNA replication in E. coli

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.     

Garlic supplementation prevents oxidative DNA damage in essential hypertension

Oxygen-free radicals and other oxygen/nitrogen species are constantly generated in the human body. Most are intercepted by antioxidant defences and perform useful metabolic roles, whereas others escape to damage biomolecules like DNA, lipids and proteins. Garlic has been shown to contain antioxidant phytochemicals that prevent oxidative damage. These include unique water-soluble organosulphur compounds, lipid-soluble organosulphur compounds and flavonoids. Therefore, in the present study, we have tried to explore the antioxidant effect of garlic supplementation on oxidative stress-induced DNA damage, nitric oxide (NO) and superoxide generation and on the total antioxidant status (TAS) in patients of essential hypertension (EH). Twenty patients of EH as diagnosed by JNC VI criteria (Group I) and 20 age and sex-matched normotensive controls (Group II) were enrolled in the study. Both groups were given garlic pearls (GP) in a dose of 250 mg per day for 2 months. Baseline samples were taken at the start of the study, i.e. 0 day, and thereafter 2 months follow-up. 8-Hydroxy-2′-deoxyguanosine (8-OHdG), lipids, lipid peroxidation (MDA), NO and antioxidant vitamins A, E and C were determined. A moderate decline in blood pressure (BP) and a significant reduction in 8-OHdG, NO levels and lipid peroxidation were observed in Group I subjects with GP supplementation. Further, a significant increase in vitamin levels and TAS was also observed in this group as compared to the control subjects. These findings point out the beneficial effects of garlic supplementation in reducing blood pressure and counteracting oxidative stress, and thereby, offering cardioprotection in essential hypertensives.     

Dietary supplementation of vitamin A, C and E prevents p-dimethylaminoazobenzene induced hepatic DNA damage in rats

The preventive effect of antioxidant vitamins A, C, E and their analogues against DNA damage induced by a hepatocarcinogen p-dimethylaminoazobenzene (DAB) was assessed by comet assay. For genotoxicity (DNA damage) study, male albino rats were divided into 11 groups, consisting of four rats each. Group I served as control. Group II to VII received 1, 10, 100, 200, 300 and 400 mg per kg body wt of DAB respectively; group VIII to XI received 500 mg/kg body wt of DAB. They were sacrificed by cervical decapitation 3, 6, 12 and 24 h after treatment; livers were excised immediately and subjected to comet assay to measure DNA damage. To study the effect of vitamins, experiments were conducted on a group of 275 rats divided into 3 sets of 25 rats each. First set served as control; second set received 0.06% DAB and third set received 0.06% DAB, along with analogues of vitamins A, C and E. Rats fed with 0.06% DAB were provided water ad libitum for a period of 4 months, followed by a normal (basal) diet for further 2 months. Vitamins A (10,000-50,000 IU), C (75-1000 mg) and E (50-500 mg) and their analogues were given (per kg body wt) to the third set of rats by gavage route once in a week for a period of 6 months. The DAB induced DNA damage only at the highest tested dose of 500 mg/kg body wt. Administration of high doses of vitamin A acid, L-ascorbic acid and vit. E succinate individually prevented the DNA damage. However, administration of a mixture of these vitamins at low doses prevented the DAB-induced DNA damage, which may be due to their synergistic effect. The results indicate that there is a significant advantage in mixed vitamins therapy at low dose over the treatment with individual vitamins.     

Vitamin C prevents cigarette smoke induced oxidative damage of proteins and increased proteolysis

Aqueous extract of cigarette smoke (CS) contains some stable oxidants, which oxidize human plasma proteins, bovine serum albumin, amino acid homopolymers, and also cause extensive oxidative degradation of microsomal proteins. Similar observations are made when the aqueous extract of cigarette smoke is replaced by whole phase CS solution or whole phase cigarette smoke. CS-induced microsomal protein degradation is a two step process: (i) oxidation of proteins by the oxidants present in the CS and (ii) rapid proteolytic degradation of the oxidized proteins by proteases present in the microsomes. Using aqueous extract of CS equivalent to that produced from one-twentieth of a cigarette, the observed initial and postcigarette smoke treated values of different parameters of oxidative damage per milligram of microsomal proteins are respectively: 0.24 and 1.74 nmoles for carbonyl formation, 125.4 and 62.8 fluorescence units for tryptophan loss, 10.2 and 33.4 fluorescence units for bityrosine formation, and 58.3 and 12.2 nmoles for loss of protein thiols. When compared with sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of untreated microsomal proteins, the extent of microsomal protein degradation after treatment with whole phase CS solution or aqueous extract of CS is above 90%. Ascorbate (100 microM) almost completely prevents cigarette smoke-induced protein oxidation and thereby protects the microsomes from subsequent proteolytic degradation. Glutathione is partially effective, but other antioxidants including superoxide dismutase, catalase, vitamin E, probucol, beta-carotene, mannitol, thiourea, and histidine are ineffective. The gas phase cigarette smoke contains unstable reactive oxygen species such as superoxide (O2*-) and hydrogen peroxide (H2O2) that can cause substantial oxidation of pure protein like albumin but is unable to produce significant oxidative damage of microsomal proteins. Gas phase cigarette smoke-induced albumin oxidation is not only inhibited by ascorbate and glutathione but also by superoxide dismutase, catalase and mannitol. The stable oxidants in the cigarette smoke are not present in the tobacco and are apparently produced by the interaction of O2*-/H2O2/OH* of the gas phase with some components of the tar phase during/following the burning of tobacco.     

Vitamin E isoform-specific inhibition of the exercise-induced heat shock protein 72 expression in humans.

Increased levels of reactive oxygen and nitrogen species, as seen in response to exercise, challenge the cellular integrity. Important protective adaptive changes include induction of heat shock proteins (HSPs). We hypothesized that supplementation with antioxidant vitamins C (ascorbic acid) and E (tocopherol) would attenuate the exercise-induced increase of HSP72 in the skeletal muscle and in the circulation. Using randomization, we allocated 21 young men into three groups receiving one of the following oral supplementations: RRR-alpha-tocopherol 400 IU/day + ascorbic acid (AA) 500 mg/day (CEalpha), RRR-alpha-tocopherol 290 IU/day + RRR-gamma-tocopherol 130 IU/day + AA 500 mg/day (CEalphagamma), or placebo (Control). After 28 days of supplementation, the subjects performed 3 h of knee extensor exercise at 50% of the maximal power output. HSP72 mRNA and protein content was determined in muscle biopsies obtained from vastus lateralis at rest (0 h), postexercise (3 h), and after a 3-h recovery (6 h). In addition, blood was sampled for measurements of HSP72, alpha-tocopherol, gamma-tocopherol, AA, and 8-iso-prostaglandin-F2alpha (8-PGF2alpha). Postsupplementation, the groups differed with respect to plasma vitamin levels. The marker of lipid peroxidation, 8-iso-PGF2alpha, increased from 0 h to 3 h in all groups, however, markedly less (P < 0.05) in CEalpha. In Control, skeletal muscle HSP72 mRNA content increased 2.5-fold (P < 0.05) and serum HSP72 protein increased 4-fold (P < 0.05) in response to exercise, whereas a significant increase of skeletal muscle HSP72 protein content was not observed (P = 0.07). In CEalpha, skeletal muscle HSP72 mRNA, HSP72 protein, and serum HSP72 were not different from Control in response to exercise. In contrast, the effect of exercise on skeletal muscle HSP72 mRNA and protein, as well as circulating HSP72, was completely blunted in CEalphagamma. The results indicate that gamma-tocopherol comprises a potent inhibitor of the exercise-induced increase of HSP72 in skeletal muscle as well as in the circulation.     

Tomato-oleoresin supplement prevents doxorubicin-induced cardiac myocyte oxidative DNA damage in rats

Doxorubicin (DOX) is an efficient chemotherapeutic agent used against several types of tumors; however, its use is limited due to severe cardiotoxicity. Since it is accepted that reactive oxygen species are involved in DOX-induced cardiotoxicity, antioxidant agents have been used to attenuate its side effects. To determine tomato-oleoresin protection against cardiac oxidative DNA damage induced by DOX, we distributed Wistar male rats in control (C), lycopene (L), DOX (D) and DOX+lycopene (DL) groups. They received corn oil (C, D) or tomato-oleoresin (5mg/kg body wt. day) (L, DL) by gavage for a 7-week period. They also received saline (C, L) or DOX (4mg/kg body wt.) (D, DL) intraperitoneally at the 3rd, 4th, 5th, and at 6th week. Lycopene absorption was checked by HPLC. Cardiac oxidative DNA damage was evaluated by the alkaline Comet assay using formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (endo III). Cardiomyocyte levels of SBs, SBs FPG and SBs Endo III were higher in rats from D when compared to other groups. DNA damage levels in cardiomyocytes from DL were not different when compared to C and L groups. The viability of cardiomyocytes from D or DL was lower than C or L groups (p<0.01). Lycopene levels (mean+/-S.D.nmol/kg) in saponified hearts were similar between L (47.43+/-11.78) and DL (49.85+/-16.24) groups. Our results showed: (1) lycopene absorption was confirmed by its cardiac levels; (2) DOX-induced oxidative DNA damage in cardiomyocyte; (3) tomato-oleoresin supplementation protected against cardiomyocyte oxidative DNA damage.     

Non-erythroid alpha spectrin prevents telomere dysfunction after DNA interstrand cross-link damage

Telomere integrity is critical for telomere function and genomic stability. We previously demonstrated that non-erythroid α-spectrin (αIISp) is present in mammalian cell nuclei where it is important in repair of DNA interstrand cross-links (ICLs) and chromosome stability. We now demonstrate that αIISp is also important for telomere maintenance after ICL damage. It localizes to telomeres in S phase after ICL damage where it has enhanced association with TRF1 and TRF2 and is required for recruitment of the ICL repair protein, XPF, to damage-induced foci at telomeres. In telomerase-positive normal cells depleted of αIISp by siRNA or in Fanconi anemia, complementation group A (FA-A) cells, where αIISp levels are 35–40% of normal, ICL damage results in failure of XPF to localize to telomeres, markedly increased telomere dysfunction-induced foci, followed by catastrophic loss of telomeres. Restoration of αIISp levels to normal in FA-A cells corrects these deficiencies. Our studies demonstrate that αIISp is critical for repair of DNA ICLs at telomeres, likely by facilitating the recruitment of repair proteins similar, but not identical, to its proposed role in repair of DNA ICLs in genomic DNA and that this function in turn is critical for telomere maintenance after DNA ICL damage.     

Vitamin E protects proteins against free radical damage in lipid environments

The fragmentation of the membrane protein monoamine oxidase in submitochondrial particles was induced by defined free radicals during radiolysis and by a system dependent on hydrogen peroxide and a transition metal. By injection of alpha-tocopherol in vivo, the levels of this physiological antioxidant in the mitochondrial preparations could be elevated more than ten-fold. In both radical-generating systems the presence of high levels of alpha-tocopherol in the membrane substantially retarded the protein fragmentation, in parallel with lipid peroxidation. It is suggested that membrane-bound proteins are damaged during lipid peroxidation and that alpha-tocopherol protects cells against both types of damage.