[Show abstract][Hide abstract] ABSTRACT: Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a constituent of plant derived foods, beverages and herbal remedies. We investigated its DNA protective properties in a placebo controlled human intervention trial in single cell gel electrophoresis experiments. Supplementation of drinking water with GA (12.8 mg/person/d) for three days led to a significant reduction of DNA migration attributable to oxidised pyrimidines (endonuclease III sensitive sites) and oxidised purines (formamidopyrimidine glycosylase sensitive sites) in lymphocytes of healthy individuals by 75% and 64% respectively. Also DNA damage caused by treatment of the cells with reactive oxygen species (ROS) was reduced after GA consumption (by 41%). These effects were paralleled by an increase of the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase and glutathion-S-transferase-π) and a decrease of intracellular ROS concentrations in lymphocytes, while no alterations of the total antioxidant capacity (TAC), of malondialdehyde levels in serum and of the urinary excretion of isoprostanes were found. Experiments with rats showed that GA reduces oxidatively damaged DNA in lymphocytes, liver, colon and lungs and protects these organs against γ-irradiation-induced strand breaks and formation of oxidatively damaged DNA-bases. Furthermore, the number of radiation-induced preneoplastic hepatic foci was decreased by 43% after oral administration of the phenolic. Since we did not find alterations of the TAC in plasma and lipid peroxidation of cell membranes but intracellular effects it is likely that the antioxidant properties of GA seen in vivo are not due to direct scavenging of radicals but rather to indirect mechanisms (e.g. protection against ROS via activation of transcription factors). As the amount of GA used in the intervention trial is similar to the daily intake in Middle Europe (18 mg/person/day), our findings indicate that it may contribute to prevention of formation of oxidatively damaged DNA in humans.
Full-text · Article · Jul 2011 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
[Show abstract][Hide abstract] ABSTRACT: A controlled intervention trial was conducted to assess the impact of spinach consumption on DNA stability in lymphocytes and on health-related biochemical parameters.
The participants (n = 8) consumed homogenised spinach (225 g/day/person) over a period of 16 days. DNA migration was monitored in single cell gel electrophoresis-comet assays under standard conditions, which reflect single- and double-strand breaks, after treatment of nuclei with lesion-specific enzymes (formamidopyrimidine glycosylase, FPG and endonuclease III, ENDO III) and after treatment of intact cells with H(2)O(2) before, during and after intervention.
While no reduction in DNA damage was observed under standard conditions after different time intervals of spinach intake, other endpoints, namely ROS sensitivity and DNA migration attributable to the formation of oxidatively damaged DNA bases (i.e. pyrimidines-ENDO III-sensitive sites and purines-FPG sensitive sites) were reduced 6 h after consumption of the first portion and after 11 days of continuous consumption. In the case of ENDO III-sensitive sites, also after 16 days, a decrease in comet formation was observed. At the end of a 40 days washout period, the DNA stability parameters were not significantly different from the background values. Other biochemical parameters which were significantly altered by spinach intake were the folate (+27%) and homocysteine (-16%) concentrations in blood, and it was found in an earlier human study that folate may prevent oxidative damage to DNA bases.
Taken together, our results show that moderate consumption of spinach causes protection against oxidative DNA damage in humans and that this phenomenon is paralleled by alterations of health-related biochemical parameters.
Full-text · Article · Mar 2011 · European Journal of Nutrition
[Show abstract][Hide abstract] ABSTRACT: Coffee is among the most frequently consumed beverages. Its consumption is inversely associated to the incidence of diseases related to reactive oxygen species; the phenomenon may be due to its antioxidant properties. Our primary objective was to investigate the impact of consumption of a coffee containing high levels of chlorogenic acids on the oxidation of proteins, DNA and membrane lipids; additionally, other redox biomarkers were monitored in an intervention trial.
The treatment group (n=36) consumed instant coffee co-extracted from green and roasted beans, whereas the control consumed water (800 mL/P/day, 5 days). A global statistical analysis of four main biomarkers selected as primary outcomes showed that the overall changes are significant. 8-Isoprostaglandin F2α in urine declined by 15.3%, 3-nitrotyrosine was decreased by 16.1%, DNA migration due to oxidized purines and pyrimidines was (not significantly) reduced in lymphocytes by 12.5 and 14.1%. Other markers such as the total antioxidant capacity were moderately increased; e.g. LDL and malondialdehyde were shifted towards a non-significant reduction.
The oxidation of DNA, lipids and proteins associated with the incidence of various diseases and the protection against their oxidative damage may be indicative for beneficial health effects of coffee.
Full-text · Article · Dec 2010 · Molecular Nutrition & Food Research
[Show abstract][Hide abstract] ABSTRACT: Coffee is among the most frequently consumed beverages worldwide and epidemiological studies indicate that its consumption is inversely related to the incidence of diseases in which reactive oxygen species (ROS) are involved (liver cirrhosis, certain forms of cancer and neurodegenerative disorders). It has been postulated that antioxidant properties of coffee may account for this phenomenon. To find out if consumption of paper filtered coffee which is the most widely consumed form in Central Europe and the US protects humans against oxidative DNA-damage, a controlled intervention trial with a cross-over design was conducted in which the participants (n=38) consumed 800ml coffee or water daily over 5 days. DNA-damage was measured in peripheral lymphocytes in single cell gel electrophoresis assays. The extent of DNA-migration attributable to formation of oxidised purines (formamidopyrimidine glycosylase sensitive sites) was decreased after coffee intake by 12.3% (p=0.006). Biochemical parameters of the redox status (malondialdehyde, 3-nitrotyrosine and the total antioxidant levels in plasma, glutathione concentrations in blood, intracellular ROS levels and the activities of superoxide dismutase and glutathione peroxidase in lymphocytes) were not markedly altered at the end of the trial, also the urinary 8-isoprostaglandine F2α concentrations were not affected. Overall, the results indicate that coffee consumption prevents endogenous formation of oxidative DNA-damage in human, this observation may be causally related to beneficial health effects of coffee seen in earlier studies.
Full-text · Article · Oct 2010 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
[Show abstract][Hide abstract] ABSTRACT: Antioxidant requirements have neither been defined for endurance nor been defined for ultra-endurance athletes. To verify whether an acute bout of ultra-endurance exercise modifies the need for nutritive antioxidants, we aimed (1) to investigate the changes of endogenous and exogenous antioxidants in response to an Ironman triathlon; (2) to particularise the relevance of antioxidant responses to the indices of oxidatively damaged blood lipids, blood cell compounds and lymphocyte DNA and (3) to examine whether potential time-points of increased susceptibility to oxidative damage are associated with alterations in the antioxidant status. Blood that was collected from forty-two well-trained male athletes 2 d pre-race, immediately post-race, and 1, 5 and 19 d later was sampled. The key findings of the present study are as follows: (1) Immediately post-race, vitamin C, α-tocopherol, and levels of the Trolox equivalent antioxidant capacity, the ferric reducing ability of plasma and the oxygen radical absorbance capacity (ORAC) assays increased significantly. Exercise-induced changes in the plasma antioxidant capacity were associated with changes in uric acid, bilirubin and vitamin C. (2) Significant inverse correlations between ORAC levels and indices of oxidatively damaged DNA immediately and 1 d post-race suggest a protective role of the acute antioxidant responses in DNA stability. (3) Significant decreases in carotenoids and γ-tocopherol 1 d post-race indicate that the antioxidant intake during the first 24 h of recovery following an acute ultra-endurance exercise requires specific attention. Furthermore, the present study illustrates the importance of a diversified and well-balanced diet to maintain a physiological antioxidant status in ultra-endurance athletes in reference to recommendations.
Full-text · Article · Oct 2010 · The British journal of nutrition
[Show abstract][Hide abstract] ABSTRACT: Berberis lycium Royle (Berberidacea) from Pakistan and its alkaloids berberine and palmatine have been reported to possess beneficial pharmacological properties. In the present study, the anti-neoplastic activities of different B. lycium root extracts and the major constituting alkaloids, berberine and palmatine were investigated in p53-deficient HL-60 cells. The strongest growth inhibitory and pro-apoptotic effects were found in the n-butanol (BuOH) extract followed by the ethyl acetate (EtOAc)-, and the water (H(2)O) extract. The chemical composition of the BuOH extract was analyzed by TLC and quantified by HPLC. 11.1 microg BuOH extract (that was gained from 1mg dried root) contained 2.0 microg berberine and 0.3 microg/ml palmatine. 1.2 microg/ml berberine inhibited cell proliferation significantly, while 0.5 microg/ml palmatine had no effect. Berberine and the BuOH extract caused accumulation of HL-60 cells in S-phase. This was preceded by a strong activation of Chk2, phosphorylation and degradation of Cdc25A, and the subsequent inactivation of Cdc2 (CDK1). Furthermore, berberine and the extract inhibited the expression of the proto-oncogene cyclin D1. Berberine and the BuOH extract induced the acetylation of alpha-tubulin and this correlated with the induction of apoptosis. The data demonstrate that berberine is a potent anti-neoplastic compound that acts via anti-proliferative and pro-apoptotic mechanisms independent of genotoxicity.
No preview · Article · Nov 2009 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
[Show abstract][Hide abstract] ABSTRACT: Also physical exercise in general is accepted to be protective, acute and strenuous exercise has been shown to induce oxidative stress. Enhanced formation of free radicals leads to oxidation of macromolecules and to DNA damage. On the other hand ultra-endurance events which require strenuous exercise are very popular and the number of participants is continuously increasing worldwide. Since only few data exists on Ironman triathletes, who are prototypes of ultra-endurance athletes, this study was aimed at assessing the risk of oxidative stress and DNA damage after finishing a triathlon and to predict a possible health risk. Blood samples of 42 male athletes were taken 2 days before, within 20 min after the race, 1, 5 and 19 days post-race. Oxidative stress marker increased only moderately after the race and returned to baseline after 5 days. Marker of DNA damage measured by the SCGE assay with and without restriction enzymes as well as by the sister chromatid exchange assay did either show no change or deceased within the first day after the race. Due to intake during the race and the release by the cells plasma concentrations of vitamin C and α-tocopherol increased after the event and returned to baseline 1 day after. This study indicates that despite a temporary increase in some oxidative stress markers, there is no persistent oxidative stress and no DNA damage in response to an Ironman triathlon in trained athletes, mainly due to an appropriate antioxidant intake and general protective alterations in the antioxidant defence system.
[Show abstract][Hide abstract] ABSTRACT: The major aims of this study were to investigate the effect of an Ironman triathlon on DNA migration in the single cell gel electrophoresis assay, apoptosis and necrosis in the cytokinesis-block micronucleus cytome assay with lymphocytes and on changes of total antioxidant capacity in plasma. Blood samples were taken 2 days (d) before, within 20 min, 1 d, 5 d and 19 d post-race. The level of strand breaks decreased (p<0.05) immediately after the race, then increased (p<0.01) 1 d post-race and declined (p<0.01) until 19 d post-race. Apoptotic and necrotic cells decreased (p<0.01) and the total antioxidant status increased (p<0.01) immediately after the race. The results indicate that ultra-endurance exercise does not cause prolonged DNA damage in well-trained male athletes.
Full-text · Article · Aug 2009 · Free Radical Research
[Show abstract][Hide abstract] ABSTRACT: Inhalative exposure to vanadium pentoxide (V(2)O(5)) causes lung cancer in rodents.
The aim of the study was to investigate the impact of V(2)O(5) on DNA stability in workers from a V(2)O(5) factory.
We determined DNA strand breaks in leukocytes of 52 workers and controls using the alkaline comet assay. We also investigated different parameters of chromosomal instability in lymphocytes of 23 workers and 24 controls using the cytokinesis-block micronucleus (MN) cytome method.
Seven of eight biomarkers were increased in blood cells of the workers, and vanadium plasma concentrations in plasma were 7-fold higher than in the controls (0.31 microg/L). We observed no difference in DNA migration under standard conditions, but we found increased tail lengths due to formation of oxidized purines (7%) and pyrimidines (30%) with lesion-specific enzymes (formamidopyrimidine glycosylase and endonuclease III) in the workers. Bleomycin-induced DNA migration was higher in the exposed group (25%), whereas the repair of bleomycin-induced lesions was reduced. Workers had a 2.5-fold higher MN frequency, and nucleoplasmic bridges (NPBs) and nuclear buds (Nbuds) were increased 7-fold and 3-fold, respectively. Also, apoptosis and necrosis rates were higher, but only the latter parameter reached statistical significance.
V(2)O(5) causes oxidation of DNA bases, affects DNA repair, and induces formation of MNs, NPBs, and Nbuds in blood cells, suggesting that the workers are at increased risk for cancer and other diseases that are related to DNA instability.
Full-text · Article · Jan 2009 · Environmental Health Perspectives
[Show abstract][Hide abstract] ABSTRACT: Sumach (Rhus coriaria L.) is widely used as a spice. The aim of this study was the investigation of its DNA-protective effects in humans and animals. Prevention of the formation of strand breaks and oxidized DNA bases as well as the protection against H(2)O(2)- and (+/-)-anti-benzo[a]pyrene-7,8-dihydro-diol-9,10-epoxide (BPDE)-induced DNA-damage were monitored in human lymphocytes in a placebo controlled trial (N=8/group) with ethanolic extract of sumach (3.0g/day, 3 days) in single cell gel electrophoresis assays. Furthermore, DNA-protective effects of sumach were monitored in different inner organs of rats under identical conditions. No alteration of DNA-migration was detectable in human lymphocytes under standard conditions, but a decrease of the tail-lengths due to formation of oxidized purines and pyrimidines (52% and 36%) was found with lesion-specific enzymes. Also damage caused by H(2)O(2) and BPDE was significantly reduced by 30% and 69%, respectively. The later effect may be due to induction of glutathione S-transferase (GST). After the intervention, the overall GST (CDNB) activity in plasma was increased by 40%, GST-alpha by 52% and GST-pi by 26% (ELISA). The antioxidant effects of extract are probably due to scavenging which was observed in in vitro experiments, which also indicated that gallic acid is the active principle of sumach. The animal experiments showed that sumach also causes protection in inner organs. Supplementation of the drinking water (0.02g/kg per animal) decreased the formation of oxidized DNA bases in colon, liver, lung and lymphocytes; also after gamma-irradiation pronounced effects were seen.
Full-text · Article · Nov 2008 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
[Show abstract][Hide abstract] ABSTRACT: Single cell gel electrophoresis (SCGE- or comet-) assay is based on the determination of DNA-migration in an electric field and can be used in human intervention studies to investigate the impact of dietary factors on DNA stability. The use of this technique has yielded new and unexpected results concerning the antioxidant and chemoprotective properties of food components.
No preview · Article · Sep 2008 · ACS Symposium Series
[Show abstract][Hide abstract] ABSTRACT: This article summarises the results of human dietary intervention trials employing the comet assay (single cell gel electrophoresis, SCGE), which have been published in the last few years (i.e., between 2005 and 2008) and describes new trends and developments as well as current problems concerning the design of intervention trials and the interpretation of the results. Most new studies were carried out with complex plant derived foods and juices; only a few were conducted with individual food constituents. With specific vegetables, for example with water cress and Brussels sprouts, potent antioxidant effects were observed; also coffee caused a protective effect and it is notable that it was more effective than consumption of a diet containing increased levels of fruits and vegetables. Interesting recent developments include the development of protocols which enable us to monitor protection towards genotoxic chemicals contained in the human diet, and it was shown in preliminary studies that alterations of the activities of drug metabolising enzymes by dietary factors lead to altered sensitivity of lymphocytes against DNA damage caused by certain dietary carcinogens. Another novel approach is the development of methods to monitor the effects of dietary factors on DNA repair. The development of protocols for experiments with exfoliated buccal cells is another potentially valuable innovation. The adequate experimental design of SCGE trials is still a matter of debate and the evaluation of the available data shows that there is an urgent need to develop guidelines concerning the number of participants, sampling periods, duration of trials, use of placebos, and definition of adequate run-in and wash-out phases. Recent studies showed that the results of dietary studies could be biased by factors such as age, sex, body mass index and life style habits and by seasonal effects. Another still unsolved problem is the interpretation of the results of SCGE trials in regard to potential beneficial health effects. The use of -omics techniques may contribute to provide mechanistic explanations in addition to conventional approaches (such as enzyme measurements). Information on health effects of dietary factors and on prevention of diseases related to DNA damage can also be obtained in experiments with animals, using SCGE to detect decreases in DNA damage in inner organs.
Full-text · Article · Sep 2008 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
[Show abstract][Hide abstract] ABSTRACT: This article describes the principles and limitations of methods used to investigate reactive oxygen species (ROS) protective properties of dietary constituents and is aimed at providing a better understanding of the requirements for science based health claims of antioxidant (AO) effects of foods. A number of currently used biochemical measurements aimed of determining the total antioxidant capacity and oxidised lipids and proteins are carried out under unphysiological conditions and are prone to artefact formation. Probably the most reliable approaches are measurements of isoprostanes as a parameter of lipid peroxidation and determination of oxidative DNA damage. Also the design of the experimental models has a strong impact on the reliability of AO studies: the common strategy is the identification of AO by in vitro screening with cell lines. This approach is based on the assumption that protection towards ROS is due to scavenging, but recent findings indicate that activation of transcription factors which regulate genes involved in antioxidant defence plays a key role in the mode of action of AO. These processes are not adequately represented in cell lines. Another shortcoming of in vitro experiments is that AO are metabolised in vivo and that most cell lines are lacking enzymes which catalyse these reactions. Compounds with large molecular configurations (chlorophylls, anthocyans and polyphenolics) are potent AO in vitro, but weak or no effects were observed in animal/human studies with realistic doses as they are poorly absorbed. The development of -omics approaches will improve the scientific basis for health claims. The evaluation of results from microarray and proteomics studies shows that it is not possible to establish a general signature of alterations of transcription and protein patterns by AO. However, it was shown that alterations of gene expression and protein levels caused by experimentally induced oxidative stress and ROS related diseases can be normalised by dietary AO.
Full-text · Article · Jun 2008 · The British journal of nutrition
[Show abstract][Hide abstract] ABSTRACT: To find out if the cancer protective effects of Brussels sprouts seen in epidemiological studies are due to protection against DNA-damage, an intervention trial was conducted in which the impact of vegetable consumption on DNA-stability was monitored in lymphocytes with the comet assay. After consumption of the sprouts (300 g/p/d, n = 8), a reduction of DNA-migration (97%) induced by the heterocyclic aromatic amine 2-amino-1-methyl-6-phenyl-imidazo-[4,5-b]pyridine (PhIP) was observed whereas no effect was seen with 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2). This effect protection may be due to inhibition of sulfotransferase 1A1, which plays a key role in the activation of PhIP. In addition, a decrease of the endogenous formation of oxidized bases was observed and DNA-damage caused by hydrogen peroxide was significantly (39%) lower after the intervention. These effects could not be explained by induction of antioxidant enzymes glutathione peroxidase and superoxide dismutase, but in vitro experiments indicate that sprouts contain compounds, which act as direct scavengers of reactive oxygen species. Serum vitamin C levels were increased by 37% after sprout consumption but no correlations were seen between prevention of DNA-damage and individual alterations of the vitamin levels. Our study shows for the first time that sprout consumption leads to inhibition of sulfotransferases in humans and to protection against PhIP and oxidative DNA-damage.
Full-text · Article · Mar 2008 · Molecular Nutrition & Food Research
[Show abstract][Hide abstract] ABSTRACT: Epidemiological studies indicate a correlation of cruciferous vegetables consumption with reduced incidence of cancer. This study was designed to investigate molecular mechanisms, which may help to understand the beneficial effects of Brussels sprout consumption. In order to avoid the limitations of in vitro model systems, we performed a dietary intervention study with five participants. We investigated, whether sprout consumption affects the proteome profile of primary white blood cells. In order to achieve maximal sensitivity in detecting specific adaptive proteome alterations, we metabolically labelled freshly isolated cells in the presence of (35) S-methionine/cysteine and performed autoradiographic quantification of protein synthesis. Proteins were separated by 2-DE and spots of interest were cut out, digested and identified by MS. After the intervention, we found a significant up-regulation of the synthesis of manganese superoxide dismutase (1.56-fold) and significant down-regulation of the synthesis of heat shock 70 kDa protein (hsp70; 2.27-fold). Both proteins play a role in malignant transformation of cells. Hsp-70 is involved in the regulation of apoptosis, which leads to elimination of cancer cells, while SOD plays a key role in protection against reactive oxygen species mediated effects. Our findings indicate that the alteration of the synthesis of these proteins may be involved in the anticarcinogenic effects of cruciferous vegetables, which was observed in earlier laboratory studies with animals.
No preview · Article · Jan 2008 · Proteomics. Clinical applications
[Show abstract][Hide abstract] ABSTRACT: Quaternary ammonium compounds (QACs) are cationic surfactants that are widely used as disinfectants. In the present study, we tested two important representatives, namely, benzalkonium chloride (BAC) and dimethyldioctadecyl-ammonium bromide (DDAB) in four genotoxicity tests, namely, in the Salmonella/microsome assay with strains TA 98, TA 100 and TA 102, in the single-cell gel electrophoresis (SCGE) assay with primary rat hepatocytes and in micronucleus (MN) assays with peripheral human lymphocytes and with root tip cells of Vicia faba. In the bacterial experiments, consistently negative results were obtained in the dose range between 0.001 and 110 microg per plate in the presence and absence of metabolic activation while significant induction of DNA migration was detected in the liver cells. With BAC, a moderate but significant effect was found with an exposure concentration of 1.0 mg/l while DDAB caused damage at lower doses (0.3 mg/l). The effects were not altered when the nuclei were treated with formamidopyridine glycosylase, indicating that they are not due to formation of oxidized purines. The MN assays with blood cells were carried out under identical conditions to the SCGE experiments and a significant increase was seen at the highest dose levels (BAC: 1.0 and 3.0 mg/l; DDAB: 1 mg/l). Both compounds also caused significant induction of MN as well as inhibition of cell division in plant cells, the lowest effective levels were 1.0 and 10 mg/l for DDAB and BAC, respectively. Our findings show that both chemicals induce moderate but significant genotoxic effects in eukaryotic cells at concentrations which are found in wastewaters and indicate that their release into the environment may cause genetic damage in exposed organisms. Furthermore, the direct contact of humans to QAC-containing detergents and pharmaceuticals that contain substantially higher concentrations than those which were required to cause effects in eukaryotic cells in the present study should be studied further in regard to potential DNA-damaging effects in man.
[Show abstract][Hide abstract] ABSTRACT: Aim of the study was to investigate the impact of coffee on DNA-stability in humans. DNA-damage was monitored in lymphocytes of eight individuals with single cell gel electrophoresis assays before and after consumption of 600 ml coffee (400 ml paper filtered and 200 ml metal filtered/d) for five days. Under standard conditions, no alteration of DNA-migration was seen, but a strong reduction of DNA-migration attributable to endogenous formation of oxidised purines and pyrimidines was detected with restriction enzymes; furthermore DNA-damage caused by reactive oxygen radicals (H2O2 treatment) and by the heterocyclic aromatic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole-acetate was significantly reduced after coffee consumption by 17% and 35%, respectively. Also in in vitro experiments, inhibition of H2O2 induced DNA-damage was observed with coffee at low concentrations (<or= 25 microl/ml) whereas the diterpenoids cafestol and kahweol caused only marginal effects indicating that the effects of coffee are due to scavenging effects of other constituents. Enzyme measurements showed that additionally induction of antioxidant enzymes may play a role: while the activity of glutathione peroxidase was only marginally increased after coffee consumption, a significant (38%) increase of superoxide dismutase activity was detected. Comparisons with results of earlier studies suggest that coffee consumption may prevent oxidative DNA-damage to a higher extent as diets enriched in fruits and vegetables.
Full-text · Article · Sep 2007 · Food and Chemical Toxicology