Chaga mushroom extract inhibits oxidative DNA damage in lymphocytes of patients with inflammatory bowel disease

Inflammatory Bowel Disease (IBD) is partly caused by oxidative stress from free radicals and reduced antioxidant levels. Using hydrogen peroxide to induce oxidative stress in vitro in peripheral lymphocytes we investigated the induction of DNA damage supplemented with ethanolic extract of Chaga mushroom as a protective antioxidant. Lymphocytes were obtained from 20 IBD patients and 20 healthy volunteers. For treatment, a constant H_{2}O_{2 } dose (50 microg/ml) was used with variable doses of Chaga extract (10-500 microg/ml). DNA damage was evaluated in 50 cells per individual and dose using the Comet assay (making 1000 observations per experimental point ensuring appropriate statistical power). Chaga supplementation resulted in a 54.9% (p < 0.001) reduction of H_{2}O_{2 } induced DNA damage within the patient group and 34.9% (p < 0.001) within the control group. Lymphocytes from Crohn's disease (CD) patients had a greater basic DNA damage than Ulcerative Colitis (UC) patients (p < 0.001). Conclusively, Chaga extract reduces oxidative stress in lymphocytes from IBD patients and also healthy individuals when challenged in vitro. Thus, Chaga extract could be a possible and valuable supplement to inhibit oxidative stress in general.     

Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress

The present study aimed to evaluate the association between the environmental tobacco smoke (ETS) and DNA damage in relation to oxidative stress (OS) in children. Sixty-four children of age 1-8 years, selected from the outpatient clinic of Mansoura University Children Hospital were divided into two groups (23 children/group) based on high (>20 cigarettes/day) or low (<20 cigarettes/day) exposure to ETS at home. Twenty symptom-free children with normal cotinine level and with no exposure to ETS were recruited as controls. The comet assay was used to quantify the level of DNA damage in lymphocytes isolated from all children. Spectrophotometric methods were used to assess the serum level of malondialdehyde (MDA) and activity of glutathione peroxidase (GSH-Px) in erythrocytes. Also, serum level of tocopherol fractions (alpha, gamma, delta) was assessed by high performance liquid chromatography (HPLC). Children exposed to ETS exhibited retarded growth, more chest problems, and gastroenteritis than the control. A significant increase in mean comet tail length indicating DNA damage was observed in ETS-exposed children (P<0.001) compared to controls. ETS-exposed children had significantly (P<0.001) higher MDA level paralleled with significant (P<0.001) decrease in the level of GSH-Px and tocopherol fractions compared with controls. The GSH-Px activity and tocopherol levels were inversely correlated with the increase of ETS exposure. These results show that inhalation of ETS is associated with an increase in the level of oxidants and a simultaneous decrease in the level of antioxidants in the children's blood. This status of oxidant-antioxidant imbalance (OS) may be one of the mechanisms leading to DNA damage detected in lymphocytes of ETS-exposed children. In conclusion, the present study gives an indication of an association between DNA damage and ETS exposure in children.     

SIN-1-induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay)

Human lymphocytes were exposed to increasing concentrations of SIN-1, which generates superoxide and nitric oxide, and the formation of single-strand breaks (SSB) in individual cells was determined by the single-cell gel electrophoresis assay (comet assay). A dose- and time-dependent increase in SSB formation was observed rapidly after the addition of SIN-1 (0.1-15 mM). Exposure of the cells to SIN-1 (5 mM) in the presence of excess of superoxide dismutase (0.375 mM) increased the formation of SSB significantly, whereas 1000 U/ml catalase significantly decreased the quantity of SSB. The simultaneous presence of both superoxide dismutase and catalase before the addition of SIN-1 brought the level of SSB to that of the untreated cells. Moreover, pretreatment of the cells with the intracellular Ca(2+)-chelator BAPTA/AM inhibited SIN-1-induced DNA damage, indicating the involvement of intracellular Ca(2+) changes in this process. On the other hand, pretreatment of the same cells with ascorbate or dehydroascorbate did not offer any significant protection in this system. The data suggest that H2O2-induced changes in Ca(2+) homeostasis are the predominant pathway for the induction of SSB in human lymphocytes exposed to oxidants.     

Effect of phytoestrogen and antioxidant supplementation on oxidative DNA damage assessed using the comet assay

Antioxidant species may act in vivo to decrease oxidative damage to DNA, protein and lipids thus reducing the risk of coronary heart disease and cancer. Phytoestrogens are plant compounds which are a major component of traditional Asian diets and which may be protective against certain hormone-dependent cancers (breast and prostate) and against coronary heart disease. They may also be able to function as antioxidants, scavenging potentially harmful free radicals. In this study, the effects of the isoflavonoids (a class of phytoestrogen) genistein and equol on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide significantly increased the levels of DNA strand breaks. Pre-treatment of the cells with both genistein and equol offered protection against this damage at concentrations within the physiological range. This protection was greater than that offered by addition of the known antioxidant vitamins ascorbic acid and alpha-tocopherol, or the compounds 17beta-oestradiol and Tamoxifen which have similar structures to isoflavonoids and are known to have weak antioxidant properties. These findings are consistent with the hypothesis that phytoestrogens can, under certain conditions, function as antioxidants and protect against oxidatively-induced DNA damage.     

Investigating oxidative DNA damage and its repair using the comet assay

The comet assay is not the only way to measure oxidative DNA damage, but it is one of the most sensitive and accurate, being relatively free of artefacts. It is a valuable tool in population monitoring, for example in assessing the role of oxidative stress in human disease, and in monitoring the effects of dietary antioxidants. A simple modification allows the measurement of DNA repair. In combination with the analysis of polymorphisms in relevant genes, the comet assay - especially when adapted for analysis of large numbers of samples - can provide important information on the interactions between genetic variation and environmental factors in maintaining genome stability.     

Endurance exercise results in DNA damage as detected by the comet assay

To determine if 6 weeks of supplementation with antioxidants could alleviate exercise-induced DNA damage, we studied 21 runners during a 50 km ultramarathon. Subjects were randomly assigned to one of two groups: (1) placebos (PL) or (2) antioxidants (AO) (1000 mg vitamin C and 400 IU RRR-alpha-tocopheryl acetate). The comet assay was used to assess DNA damage in circulating leukocytes at selected time points: pre-, mid-, and 2 h postrace and daily for 6 days postrace. All subjects completed the race: run time 7.1 +/- 0.1 h, energy expenditure 5008 +/- 80 kcal for women (n = 10) and 6932 +/- 206 kcal for men (n = 11). Overall, the percentage DNA damage increased at midrace (p <.02), but returned to baseline by 2 h postrace, indicating that the exercise bout induced nonpersistent DNA damage. There was a gender x treatment x time interaction (p <.01). One day postrace, women taking AO had 62% less DNA damage than women taking PL (p <.0008). In contrast, there were no statistically significant differences between the two treatment groups of men at any time point. Thus, endurance exercise resulted in DNA damage as shown by the comet assay and AO seemed to enhance recovery in women but not in men.     

DNA damage induced by paclitaxel and DNA repair capability of peripheral blood lymphocytes as evaluated by the alkaline comet assay

This study was designed to assess whether the chemotherapeutic drug paclitaxel can induce DNA damage in peripheral blood lymphocytes of human healthy donors, and to evaluate if such damage could be repaired. Venous blood was collected by routine venipuncture, the lymphocytes were isolated and cultured and then treated with 100nM, 500nM, 10microM, and 30microM of taxol for 4h. The alkaline comet assay technique was used to quantify the level of DNA damage and the DNA repair in lymphocytes. A significant increase in DNA damage was achieved when the cells were incubated with paclitaxel concentrations of 10microM or above. To test the DNA repair capability, the lymphocytes were allowed to recover for 2, 4, 6, and 24h. The DNA damage was almost completely repaired after 24h of incubation demonstrating a time-dependent repair capability. In conclusion, we demonstrate that paclitaxel induces DNA damage in peripheral blood lymphocytes and that this damage can be repaired.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25+/-8.45 microm; non-smokers, 21.6+/-2.06 microm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5+/-20.34 microm; non-smokers, 79.2+/-11.59 microm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13+/-10.73 microm; non-smokers, (27.2+/-4.13 microm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

Measuring oxidative damage to DNA; HPLC and the comet assay compared

Depending on the analytical method employed estimates of background levels of base oxidation in human DNA vary over orders of magnitude. It is now realised that oxidation of guanine in vitro can result in serious overestimation of the nucleoside by HPLC (with electrochemical detection). We have modified procedures of isolation, hydrolysis and storage of DNA with the aim of eliminating this artefact. Vacuum- or freeze-drying, and dialysis, tend to encourage oxidation. We compare results obtained with HPLC and with the comet assay, which employs lesion-specific enzymes to introduce breaks in DNA at sites of oxidative damage. Although estimates of background levels of DNA oxidation using the comet assay are several-fold lower than the estimates by HPLC, both approaches have been used successfully to detect differences between human subjects or population groups that seem to relate to human disease and nutritional factors.     

Measuring oxidative damage to DNA and its repair with the comet assay

Background

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases.

Scope of review

With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells.

Major conclusions

There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay.

General significance

In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Measuring oxidative damage to DNA; HPLC and the comet assay compared

Depending on the analytical method employed estimates of background levels of base oxidation in human DNA vary over orders of magnitude. It is now realised that oxidation of guanine in vitro can result in serious overestimation of the nucleoside by HPLC (with electrochemical detection). We have modified procedures of isolation, hydrolysis and storage of DNA with the aim of eliminating this artefact. Vacuum- or freeze-drying, and dialysis, tend to encourage oxidation. We compare results obtained with HPLC and with the comet assay, which employs lesion-specific enzymes to introduce breaks in DNA at sites of oxidative damage. Although estimates of background levels of DNA oxidation using the comet assay are several-fold lower than the estimates by HPLC, both approaches have been used successfully to detect differences between human subjects or population groups that seem to relate to human disease and nutritional factors.     

Basal damage and oxidative DNA damage in children with chronic kidney disease measured by use of the comet assay

One consequence of chronic kidney disease (CKD) is an elevated risk for cancer. There is sufficient evidence to conclude that there is an increased incidence of at least some cancers in kidney-dialysis patients. Cancer risk after kidney transplantation has mainly been attributed to immunosuppressive therapy. There are no data evaluating DNA damage in children with CKD, in dialysis patients, or following kidney transplantation. In this study, the comet assay and the enzyme-modified comet assay - with the use of endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) enzymes - were conducted to investigate the basal damage and the oxidative DNA damage as a result of treatment in peripheral blood lymphocytes of children. Children at various stages of treatment for kidney disease, including pre-dialysis patients (PreD) (n=17), regular hemodialysis patients (HD) (n=15), and those that received kidney transplants (Tx) (n=17), comprised the study group. They were compared with age- and gender-matched healthy children (n=20) as a control group. Our results show that the %DNA intensity, a measure of basal damage, was significantly increased in children with CKD (mean ± SD) (5.22 ± 1.57) and also in each of the PreD, HD, and Tx groups [(4.92 ± 1.23), (4.91 ± 1.35), and (5.79 ± 1.94), respectively, vs the healthy children (2.74 ± 2.91) (p<0.001). Significant increases in oxidative DNA damage were only found in the FPG-sensitive sites for the PreD and Tx groups, compared with control and HD groups (p<0.05), suggesting that basal DNA damage was more evident for the PreD, HD, and Tx groups. The findings of the present study indicate a critical need for further research on genomic damage with different endpoints and also for preventive measures and improvements in treatment of pediatric patients, in order to improve their life expectancy.     

Detection of alkylation damage in human lymphocyte DNA with the comet assay

The enzyme 3-methyladenine DNA glycosylase II (AlkA) is a bacterial repair enzyme that acts preferentially at 3-methyladenine residues in DNA, releasing the damaged base. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay (single cell gel electrophoresis) they appear as DNA strand breaks. AlkA is no t lesion-specific, but has a low activity even w ith undamagedbases. We have tested the enzyme at different concentrations to find conditions that maximise detection of alkylated bases with minimal attack on normal, undamaged DNA. AlkA detects damage in the DNA of cells treated with low concentrations of methyl methanesulphonate. We also find low background levels of alkylated bases in normal human lymphocytes.     

Modified comet assay as a biomarker of sodium dichromate-induced oxidative DNA damage: optimization and reproducibility.

Hexavalent chromium (Cr[VI]) is a genotoxic carcinogen that has been associated with an increased risk of nasal and respiratory tract cancers following occupational exposure. Although the precise mechanism(s) remain to be elucidated, there is evidence for a role of oxidative DNA damage in the genotoxicity of Cr(VI). In the current study, human white blood cells were treated in vitro with non-cytotoxic concentrations of sodium dichromate (1-100 microM) for 1 h. Analysis by immunocytochemistry indicated the presence of elevated levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine at concentrations of sodium dichromate greater than 10 microM. In contrast, the lowest concentration of dichromate that resulted in a statistically significant increase in levels of formamidopyrimidine DNA glycosylase (FPG)-dependent DNA strand breaks was 100 nM (p<0.05). In addition, levels of both control and dichromate-induced FPG-dependent strand breaks from blood samples taken from the same individuals over 10 months proved remarkably reproducible in the individuals studied. The coefficients of variation over three different times of the year in control and dichromate-induced oxidative DNA damage for the four individuals were 54, 1, 37 and 4, and 45, 6, 21 and 18%, respectively. In summary, these results indicate that physiologically relevant, nanomolar concentrations of sodium dichromate cause DNA base oxidation in human white blood cells in vitro as assessed by the FPG-modified comet assay. Furthermore, comet assay data from an individual are reproducible over an extended period. This consistency is sufficient to suggest that the modified comet assay might prove to be a useful and sensitive biomonitoring tool for individuals occupationally exposed to hexavalent chromium.     

DNA damage induced by mutagens in plant and human cell nuclei in acellular comet assay

Higher plant cells have a long tradition of use in the studies on environmental mutagenesis in situ, especially in relation to human health risk determination. The studies on the response of plant and human cells to physical and chemical mutagens showed differences in their sensitivity. The differences in the presence of cell components in plants and humans could influence such response. Additionally, the level of the organization of the employed material could influence DNA-damaging effect: leukocytes are isolated cells and plant--an intact organism. To preclude these obstacles, the effects of direct treatment of isolated nuclei with genotoxic agents were determined to compare the sensitivity of plant and human cells. In the present study, we have determined the DNA-damaging effects of two chemical mutagens: maleic acid hydrazide (MH) and N-methyl-N-nitroso-urea (MNU) applied to isolated nuclei of both plant and human cells. In order to compare the sensitivity of the nuclei of Nicotiana tabacum var. xanthi and the nuclei of leukocytes, the acellular Comet assay was carried out. The results showed higher sensitivity of the nuclei of leukocytes as compared to the nuclei of plant cells to mutagenic treatment with the applied doses of MH and MNU.     

Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay

The correlation of oxidative stress on development and DNA damage in bovine embryos was investigated by the comet assay (single-cell microgel electrophoresis), an effective technique for detecting single-strand DNA breakage. After in vitro maturation and fertilization, one-cell stage embryos without cumulus cells were cultured for 8 days in SOF medium containing amino acids plus 5% FCS under low (5%) and atmospheric (20% ) oxygen concentration. After 8 days of culture, the extent of blastocyst formation was significantly decreased (P<0.001) when embryos were cultured under 20% oxygen concentration (5.8 +/- 2.4%) when compared to embryos cultured under 5% oxygen concentration (35.1 +/- 6.7%). At the day 3 of development, DNA damage of individual embryos cultured under 5% or 20% oxygen concentration was measured by the comet assay, which entails microgel electrophoresis that can readily detect damaged DNA. After measuring the DNA damage in individual embryos by the comet assay, the length (149.9 +/- 15.3 microm) of the migrating DNA fragment that is indicative of damaged DNA was significantly increased (P<0.001) in the embryos cultured under 20% oxygen concentration when compared to embryos cultured in 5% oxygen concentration (42.3 +/- 7 microm). The length of damaged DNA in more than 50% of embryos was less than 50 microm. when embryos were cultured under 5% oxygen concentration. In contrast, the distribution of damaged DNA shifted to the more damaged extent when embryos were cultured under 20% oxygen concentration. These results demonstrate that the retardation in bovine embryo development than in likely due oxidative stress as a consequence of the higher atmospheric oxygen concentration is positively correlated with an increase in the extent of DNA damage. Moreover, these results demonstrate that the comet assay is a useful method to evaluate embryo culture conditions.     

Assessment of reference values for DNA damage detected by the comet assay in human blood cell DNA

Genotoxicity measured by the comet assay is expressed by different researchers using parameters that are not easy to conceptualize, except for percent tail DNA (%T) or visual score (arbitrary units). A total of 125 publications have reported genotoxicity as DNA damage (representing strand breaks, alkaline labile sites, and transient repair sites), endonuclease III (ENDOIII), or formamidopyrimidine DNA glycosylase (FPG) sensitive sites. I have recalculated the visual score so that it is expressed in the range of 0-100, similar to that of %T. Similar values were obtained for DNA damage and ENDOIII sites, regardless of whether of the data were reported as %T or visual score. Thus, these endpoints can be used interchangeably, assuming that the visual score is expressed in the 0-100 range. Pooled analysis of %T and visual score data showed that the median (25-75%) values of DNA damage, ENDOIII, and FPG sites were 8.6 (4.4-14.5), 11.0 (4.2-19.5), 7.6 (3.2-14.2), respectively. The duration of alkaline treatment and electrophoresis had no significant effect on the level of DNA damage. There was a positive correlation between age and the level of DNA damage. A sub-analysis of DNA damage obtained from European countries showed a negative correlation with latitude. In conclusion, reference values for DNA lesions measured by the comet assay are around 7-11 %T or arbitrary units.     

The comet assay for DNA damage and repair

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.