ascorbic-acid and 8-hydroxyguanine

Antioxidants are believed to prevent many types of disease. Some previous studies suggest that dietary supplementation with vitamin C results in a decrease in the level of one of the markers of oxidative damage-8-oxoguanine in the DNA of peripheral blood mononuclear cells (PBMC). AIM OF TRIAL: To investigate the effect of different dose levels of dietary supplementation with vitamin C on oxidative DNA damage.. A randomised double-blind placebo-controlled trial was carried out using three different levels (80, 200 and 400 mg) of dietary vitamin C supplementation in a healthy population of 160 volunteers; supplementation was for a period of 15 weeks followed by a 10 week washout period. Peripheral blood samples were obtained every 5 weeks from baseline to 25 weeks.. An increase in PBMC vitamin C levels was not observed following supplementation in healthy volunteers. There was no effect found on 8-oxoguanine measured using HPLC with electrochemical detection for any of the three supplemented groups compared to placebo. 8-oxoadenine levels were below the limit of detection of the HPLC system used here.. Supplementation with vitamin C had little effect on cellular levels in this group of healthy individuals, suggesting their diets were replete in vitamin C. The dose range of vitamin C used did not affect oxidative damage in PBMC DNA.

Topics: Adolescent; Adult; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Cross-Over Studies; Dietary Supplements; DNA Damage; Dose-Response Relationship, Drug; Double-Blind Method; Female; Guanine; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Oxidation-Reduction; Oxidative Stress

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods. The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10(6) dG were respectively 7.85 and 5.87 (p=0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Ascorbic Acid; Deoxyguanosine; DNA; DNA Damage; Fertility; Guanine; Humans; Infertility, Male; Leukocytes; Male; Oxidative Stress; Semen; Smoking; Spermatozoa

The aim of the present study was to evaluate the oxidative status in healthy full-term children and piglets. Urinary excretion of 8-oxoGua (8-oxoguanine) and 8-oxodG (8-oxo-2'-deoxyguanosine) were determined using HPLC/GS/MS methodology and concentrations of vitamins A, C and E with HPLC technique. The levels of 8-oxoGua in urine samples were about 7-8 times higher in newborn children and piglets when compared with the level of adult subjects, while in the case of 8-oxodG the difference was about 2.5 times. The levels of vitamin C and E in umbilical cord blood of newborn children significantly depend on the concentration of these compounds in their mother's blood. However, the values of vitamin C in human's cord blood were about 2-times higher than in respective mother blood, while the level of vitamin E showed an opposite trend. The results suggest that: (i) healthy, full-term newborns are under potential oxidative stress; (ii) urinary excretion of 8-oxoGua and 8-oxodG may be a good marker of oxidative stress in newborns; and (iii) antioxidant vitamins, especially vitamin C, play an important role in protecting newborns against oxidative stress.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Animals, Newborn; Antioxidants; Ascorbic Acid; Biomarkers; Chromatography, High Pressure Liquid; Deoxyguanosine; Female; Guanine; Humans; Infant, Newborn; Male; Mass Spectrometry; Oxidative Stress; Swine; Vitamin E

Animal studies have demonstrated that ozone exposure can induce lung tumors. Recent epidemiological studies have also shown that increased ozone exposure is associated with a greater risk of lung cancer. This study used single-cell gel electrophoresis (the Comet assay) and flow cytometry to investigate DNA damage in A549 cells exposed to ozone levels below the current ambient standard. Cells were exposed to ozone at levels of 0, 60, 80, and 120 ppb, and then DNA single-strand breaks and 8-oxoguanine levels were measured. Additionally, the formamidopyrimidine glycosylase (Fpg) repair enzyme was added to the Comet assay to enhance detection of oxidative damage. Vitamins C and E were also added to determine their inhibitory effects on ozone-induced 8-oxoguanine. Measurements of tail length, tail intensity, and tail moment of the Comet assay were shown to correlate with each other. However, tail moment appeared to be more sensitive than the other two indicators in detecting DNA single-strand breaks. Tail moments of cells exposed to 80 and 120 ppb of ozone were significantly higher than those exposed to 0 ppb (P<0.05). These three indicators of DNA single-strand breaks with Fpg were shown to be increased and more sensitive than those without Fpg. After Fpg was introduced, the tail moments at ozone levels of 60, 80, and 120 ppb were significantly higher than those at 0 ppb (P<0.05). Furthermore, 8-oxoguanine levels, determined by fluorescence intensity, at 80 and 120 ppb of ozone exposure were significantly higher than the level at 0 ppb. Pretreatment with vitamins C and E reduced the 8-oxoguanine levels caused by ozone. We conclude that ozone levels below current ambient standards may induce DNA breaks and oxidative DNA damage. Moreover, the Fpg repair enzyme in the Comet assay can increase the sensitivity of oxidative damage detection in vitro.

Topics: Antioxidants; Ascorbic Acid; Comet Assay; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Dose-Response Relationship, Drug; Guanine; Humans; Lung Neoplasms; Oxidants, Photochemical; Oxidative Stress; Ozone; Tumor Cells, Cultured; Vitamin E

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.

Topics: Alleles; Ascorbic Acid; Cell Line; Chloramphenicol O-Acetyltransferase; Dehydroascorbic Acid; DNA; DNA, Complementary; Dose-Response Relationship, Drug; Glutathione; Guanine; HL-60 Cells; Humans; Hydrogen Peroxide; Models, Biological; Mutation; Oxidative Stress; Oxygen; Plasmids; Tyrosine

The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA repair enzymes.

Topics: Ascorbic Acid; DNA; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Edetic Acid; Escherichia coli; Escherichia coli Proteins; Ferric Compounds; Gamma Rays; Guanine; Hydrogen Peroxide; Lysine; Mutagenesis, Site-Directed; N-Glycosyl Hydrolases; Proline; Structure-Activity Relationship

The effects of co-supplementing healthy volunteers with iron (14 mg/day ferrous sulphate) and vitamin C (either 60 mg/day or 260 mg/day as ascorbic acid) on levels of oxidative DNA damage in white blood cells were studied. The subjects were divided into two groups: one group of 20 volunteers with a higher mean initial level of plasma vitamin C (71.9 +/- 14.0 mumol/l) and a second group of 18 volunteers with a lower mean level (50.4 +/- 25.8 mumol/l). In the first group there was a significant rise in several oxidative DNA base damage products and in total oxidative DNA damage in DNA extracted from white blood cells, but not in 8-hydroxyguanine, after 6 weeks of supplementation. However, after 12 weeks levels returned approximately to normal. In the group with the lower initial level of plasma ascorbate, presupplemental levels of oxidative DNA damage were higher and decreased on supplementation with iron and ascorbate. Since oxidative DNA damage has been suggested as a risk factor for the development of cancer, the implications of increased levels in well-nourished subjects after iron/ascorbate supplementation are disturbing in view of the frequent use of dietary supplements containing both iron salts and ascorbate.

Topics: Adult; Antioxidants; Ascorbic Acid; DNA Damage; Drug Interactions; Female; Guanine; Humans; Iron; Iron, Dietary; Leukocytes; Male; Middle Aged; Oxidative Stress; Time Factors

Topics: Ascorbic Acid; DNA; DNA Adducts; Guanine; Humans; Lymphocytes; Oxidation-Reduction; Reactive Oxygen Species

Topics: Ascorbic Acid; Clinical Trials as Topic; DNA; DNA Adducts; Guanine; Humans; Lymphocytes; Oxidation-Reduction; Reactive Oxygen Species; Research Design