The development and validation of EpiComet‐Chip,a modified high‐throughput comet assay for the assessment of DNA methylation status

DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher‐throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation‐dependent endonuclease, McrBC, to develop a modified alkaline comet assay, “EpiComet,” which allows single platform evaluation of genotoxicity and global DNA methylation [5‐methylcytosine (5‐mC)] status of single‐cell populations under user‐defined conditions. Further, we leveraged the CometChip platform to create an EpiComet‐Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet‐Chip on 63 matched samples, we correctly identified single‐sample hypermethylation (≥1.5‐fold) at 87% (20/23), hypomethylation (≥1.25‐fold) at 100% (9/9), with a 4% (2/54) false‐negative rate (FNR), and 10% (4/40) false‐positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75‐fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA‐regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. Environ. Mol. Mutagen. 58:508–521, 2017. © 2017 Wiley Periodicals, Inc.     

Convenient,multi‐well plate‐based DNA damage response analysis using DT40 mutants is applicable to a high‐throughput genotoxicity assay with characterization of modes of action

Chemists continually synthesize myriad new chemicals (～2,000/year), some of which make their way into the environment or otherwise pose possible threats to humans who potentially become exposed to the compounds. Regulators must determine whether these, along with the glut (～80,000) of existing, chemicals are toxic and at what exposure levels. An important component of this determination is to ascertain the mode of action (MOA) of each compound as it relates to the pathway the compound uses to induce genotoxicity. Several assays have traditionally been used to reveal these effects to the genome: the Ames test, tests with yeast and mammalian cell lines, and animal studies. Previously, we described a new multi‐well plate‐based method which makes use of the DT40 isogenic cell line and its dozens of available mutants knocked out in DNA repair and cell cycle pathways and we now provide a detailed protocol of the further improvement of the assay. Although the DT40 line has existed for some time and has been used in numerous studies of DNA repair pathways, little use has been made of this valuable resource for toxicological investigations. Our method introduces the 2,3‐bis (2‐methoxy‐4‐nitro‐5‐sulfophenyl)?5‐[(phenylamino) carbonyl]?2H‐tetrazolium hydroxide dye scheme determination of cell survival in a manner that greatly increases throughput and reduces cost while maintaining reasonable sensitivity. Although this new genotoxicity assay requires validation with many more mutagens before becoming an established, regulatory decision‐making analysis tool, we believe that this method will be very advantageous if eventually added to the repertoire of those investigating MOAs of potentially genotoxic substances. Environ. Mol. Mutagen. 52:153–160, 2011. © 2010 Wiley‐Liss, Inc.     

Influence of experimental conditions on data variability in the liver comet assay

The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post‐mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time‐dependent increase in comet parameters. Environ. Mol. Mutagen. 55:114–121, 2014. © 2013 Wiley Periodicals, Inc.     

Genotoxicity of a polluted river system measured using the alkaline comet assay on fish and earthworm tissues

Monitoring genotoxicity in the environment by using endemic organisms as sentinels requires sensitive assays. In this study the genotoxic properties of water and sediment collected from the Noyyal River, which is polluted with industrial effluent and sewage, was determined in fish (Cyprinus carpio) and earthworms (Eisenia foetida) using the alkaline comet assay. Upon electrophoresis, extensive DNA damage, measured as the DNA length:width ratio of the DNA mass, was observed in erythrocytes, liver, and kidney cells of fish exposed to polluted water samples and the amount of damage increased with the duration of exposure. Similarly, the mean DNA length:width ratio was significantly higher in the coelomocytes of earthworms placed in sediment samples. The highest levels of DNA damage were obtained with samples taken at and immediately downstream of urban centers. The results of this study indicate that the Noyyal River system is contaminated with substances that are genotoxic to fish and earthworms and that the comet assay has sufficient sensitivity to detect the genotoxicity.     

Comparison of sensitivity to methyl methanesulphonate among tadpole developmental stages using the alkaline single-cell gel electrophoresis (comet) assay

In a previous study, we demonstrated that tadpoles are suitable organisms for monitoring small bodies of water (e.g., creeks, ponds, and drainage ditches) for genotoxicity using the alkaline single-cell gel DNA electrophoresis (SCG) or “comet” assay [Ralph and Petras, 1997]. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a “comet with tail” formation. In this initial study, most of the tadpoles collected were in the early stages of larval development, but this is not always possible. The present study evaluated the sensitivity of tadpoles, at different stages of larval development, to a range of concentrations of the genotoxicant methyl methane-sulphonate (MMS). Four specific phases of Rana clamitans (green frog) larval development were examined: first-year limbless tadpoles (Stage I as defined by Taylor and Kollros [1946]), second-year limbless tadpoles (Stages II–III), second-year tadpoles with only hindlimbs (Stages X–XVIII), and second-year tadpoles with all four limbs evident and a tail undergoing resorption (Stages XXII–XXIII). Twenty-four hour exposures to MMS of tadpoles in the three earliest phases produced a significant (P < 0.01) added variance component among tadpoles for DNA damage and there were significant increases (P < 0.05) in the length:width ratios of the DNA patterns at concentrations as low as 1.56 mg/l. However, tadpoles in the last phase studied (both pairs of limbs present) showed no significant (P > 0.05) added variance component and no significant increases (P > 0.05) in DNA damage upon exposure to any of the MMS doses tested. A nested ANOVA indicated that, for each of the tested concentrations of MMS, but not the dechlorinated water control, there was significant heterogeneity (P < 0.05) in DNA damage when tadpoles of all four phases studied were compared. However, when tadpoles of the last phase of development were removed from the comparison, there was no significant heterogeneity (P > 0.05) among tadpoles of the remaining three phases. Possible reasons for this insensitivity to MMS as animals enter the metamorphic climax were considered. The results indicate that pooling of the early tadpole phases of R. clamitans for SCG environmental genotoxicity biomonitoring is acceptable. Environ. Mol. Mutagen. 31:374–382, 1998. © 1998 Wiley-Liss, Inc.     

Proposal of an in vivo comet assay using haemocytes of Drosophila melanogaster

This study presents the first application of an in vivo alkaline comet assay using haemocytes of Drosophila melanogaster larvae. These cells, which play a role similar to that of mammalian blood, can be easily obtained and represent an overall exposure of the treated larvae. To validate the assay, we evaluated the response of these cells to three well‐known mutagenic agents: ethyl methanesulfonate (EMS), potassium dichromate (PD), and gamma radiation (γ‐irradiation). Third‐instar Drosophila larvae were exposed to different concentrations of EMS (1, 2, and 4 mM) and PD (0.5, 1, and 2.5 mM) and to different doses of γ‐irradiation (2, 4, and 8 Gγ). Subsequently, haemolymph was extracted from the larvae, and haemocytes were isolated by centrifugation and used in the comet assay. Haemocytes exhibited a significant dose‐related increase in DNA damage, indicating that these cells are clearly sensitive to the treatments. These results suggest that the proposed in vivo comet test, using larvae haemocytes of D. melanogaster, may be a useful in vivo assay for genotoxicity assessment. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss, Inc.     

Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared with carcinogenicity studies in rodents. Current in vitro genotoxicity tests are also often limited by relatively low throughput. The aim of this study was to validate an in vitro genotoxic assay in a 96‐well plate format that allows the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (γH2AX), which reflects a global genotoxic insult, using the In‐Cell Western technique. The assay was evaluated on HepG2 cells by testing a list of 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), whose genotoxic potential has already been characterized. The γH2AX assay on HepG2 cell line was highly sensitive: 75% of the genotoxic compounds gave a positive result, and specific: 90–100% of nongenotoxic compounds gave negative results. Compared with the micronucleus genotoxicity assay using the same cell line and test compounds, the γH2AX assay was more sensitive and specific. In sum, the high‐throughput γH2AX assay described here can accurately detect simultaneously the genotoxic and the cytotoxic potential of compounds with different modes of mutagenic action, notably those who required metabolic activation. The use of this assay in the early discovery phase of drug development may prove to be a valuable way to assess the genotoxic potential of xenobiotics. Environ. Mol. Mutagen. 54:737–746, 2013. © 2013 Wiley Periodicals, Inc.     

Biomonitoring with the comet assay of Greek greenhouse workers exposed to pesticides

The pesticides in use in Greek greenhouses include a number of agents known to be mutagens and carcinogens. In the present study, we evaluated whether occupational exposure of agricultural workers to a complex mixture of pesticides resulted in a significant increase in DNA damage in human peripheral blood lymphocytes (PBLs). A total of 116 healthy individuals were divided into groups based on exposure to pesticides, smoking status, and gender. Alkaline comet assays performed on PBLs from these individuals indicated no statistically significant differences in basal DNA damage between the study groups. In addition, exposure of PBLs to a dose of hydrogen peroxide led to a similar degree of DNA damage and subsequent repair for all the study populations. The results of the study indicate that the agricultural workers who participated in this study had no detectable increase in DNA damage or alteration in the cellular response to DNA damage.     

Alkaline single cell gel (comet) assay and genotoxicity monitoring using bullheads and carp

Monitoring genotoxicity of the environment using endemic organisms as sentinels requires the development of sensitive assays. Toward this end, we explored the feasibility of applying the alkaline single cell gel (SCG) or “comet” assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which, on electrophoresis, migrate from the nuclear core, resulting in a “comet with tail” formation. Tail length has been correlated with level of genotoxicant exposure in a number of organisms. The fish used in this study were benthic feeding bullheads (Ameiurus nebulosus) and carp (Cyprinus carpio). On electrophoresis of erythrocyte DNA under alkaline conditions, we found a linear increase in the tail length/core width ratio over a broad range of cyclophosphamide doses. Freshly caught bullheads from seven different sites showed a wide range of DNA damage. Bullheads from Big Creek (western Lake Erie), Hamilton Harbour (western Lake Ontario), and the Detroit River gave ratios of 3.81 to 4.65. Based on polycyclic aromatic hydrocarbon (PAH) and polychlorinated biphenyl (PCB) levels, the sediment at these three sites is considered to be heavily polluted. Bullheads from southern Lake Huron, which is relatively clean, and from a fish hatchery in Brockport, New York, gave ratios between 1.30 and 1.40. Bullheads from Big Creek, maintained in the laboratory for 3 months, gave ratios which approached those seen in hatchery-bred fish. Results for carp were similar. Carp from Big Creek gave ratios of about 4.50, while carp from Lake Huron and laboratory-maintained carp gave values of 1.23 and 1.36, respectively. The results of the SCG procedure in bullheads and carp indicate that this assay is extremely sensitive and should be useful in detecting DNA damage caused by environmental contaminants. © 1995 Wiley-Liss, Inc.     

Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (comet) assay

To monitor genotoxicity in small bodies of water (e.g., creeks, ponds, and drainage ditches) we examined tadpole erythrocytes of two species: Rana clamitans and Rana pipiens, using the alkaline single cell gel DNA electrophoresis (SCG) or “comet” assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a “comet with tail” formation. Fifty-six samples, a total of 606 tadpoles, from 18 sites in southern Ontario, collected between 1993 and 1995, were examined. Samples of R. clamitans tadpoles collected in 1994 and 1995, from regions with heavy agricultural activity, gave significantly higher (P < 0.001) DNA length to width ratios than samples of R. clamitans tadpoles collected from sites in the Bruce Peninsula and near the French River, which have little or no agriculture. Samples of R. pipiens tadpoles collected in 1994 from sites on the outskirts of Windsor, Ontario, sites which receive genotoxic inputs from nearby industries, gave significantly higher (P < 0.001) DNA ratios than samples from agricultural areas and the Bruce Peninsula. R. clamitans tadpoles showed significant annual variation in DNA damage which was greater in samples of tadpoles collected from agricultural areas than from the Bruce Peninsula. The higher levels of DNA damage in tadpoles collected from agricultural areas may be due to the pesticides used, and the increased variation in DNA damage in the same areas is likely due to the impact of crop rotation, including leaving fields fallow, the timing of rainfall, and/or the application of pesticides. R. clamitans tadpoles, especially those collected from agricultural areas, also showed significant seasonal variation in DNA damage. There was no significant (P > 0.05) seasonal or annual variation in the levels of DNA damage in R. pipiens tadpoles collected from the Tallgrass Prairie. This study indicates that both species are suitable for use in the alkaline SCG assay and as in situ sentinel organisms for environmental biomonitoring. Environ. Mol. Mutagen. 29:418–430, 1997. © 1997 Wiley-Liss, Inc.     

Alkaline single-cell gel (comet) assay and genotoxicity monitoring using two species of tadpoles

Small bodies of water (e.g., creeks, ponds, and drainage ditches) have received very little attention in genotoxicity studies, yet these areas are important because they are often the first to be affected by industrial effluents, sewage contaminants, accidental spills, internal combustion engine emissions, landfill runoffs, and pesticide uses. To address this deficiency, we examined erythrocytes in two species of tadpoles, Rana clamitans and Bufo americanus, using the alkaline single-cell gel (SCG) (“comet”) assay. This approach involves detection, under alkaline conditions, of cell DNA fragments, which on electrophoresis migrate from the nuclear core, resulting in a “comet-with-tail” formation. Exposure of R. clamitans tadpoles to a range of concentrations of methyl methanesulfonate (MMS) produced a linear increase in DNA length to DNA core width ratios. This is consistent with findings in a number of other species. Time-dose experiments using MMS suggest that the peak level of DNA damage in R. clamitans tadpoles occurred 42 hr after exposure. B. americanus tadpoles exposed to 6.25 mg/l of MMS for 12 hours had a significant increase in DNA damage over that seen in the controls. Freshly caught R. clamitans tadpoles from Highgate and B. americanus tadpoles from Duart, both on the north shore of Lake Erie, gave ratios of 2.78 and 2.07, respectively. This region of Ontario is a prime agricultural area and pesticide use is extensive. Tadpoles from Highgate and Duart, maintained in the laboratory for 4 months and 6 weeks, respectively, gave ratios of 1.29 and 1.44. The results of the SCG procedure in tadpoles indicate that this assay is extremely sensitive and suitable for detecting genotoxicity in the environment. © 1996 Wiley-Liss, Inc.     

Lack of an association between environmental exposure to polychlorinated biphenyls and p,p'-DDE and DNA damage in human sperm measured using the neutral comet assay

BACKGROUND: Chlorinated organic chemicals, such as polychlorinated biphenyls (PCB), hexachlorobenzene (HCB), dichlorodiphenyl trichloroethane (DDT), and dichlorodiphenyl dichloroethene (DDE, the most stable daughter compound of DDT) are persistent lipophilic compounds found in a large portion of the general population. To explore the hypothesis that environmental exposure to these compounds is associated with altered DNA integrity in human sperm, a study of 212 male partners of a sub-fertile couple who presented to the Massachusetts General Hospital Andrology Laboratory was conducted. METHODS: The neutral single cell microgel electrophoresis assay (comet assay) was used to assess DNA integrity in sperm. VisComet image analysis software was used to measure total comet length, the proportion of DNA present in the comet tail, and tail distributed moment, an integrated measure of length and intensity. RESULTS: In the regression analyses, there were no statistically significant consistent associations between the comet assay parameters and any of the individual PCB congeners, sum of PCB, or p,p'-DDE. CONCLUSION: These results suggest that there are not strong relationships between adult levels of these chlorinated organic compounds and sperm DNA damage as measured by the comet assay.     

DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuels

Exposure to jet fuel damages DNA and results in a number of physiological changes in liver, lung, immune, and neurological tissue. In this study the single-cell gel electrophoresis assay or comet assay was used to compare the DNA damage in human peripheral lymphocytes produced by three jet propulsion fuels: JP-8, JP-5, and JP-8+100. These fuels consist of complex mixtures of aliphatic, aromatic, and substituted naphthalene hydrocarbons. Two exposure times were investigated which correspond to estimated occupational exposure times and concentrations of fuels were used that were based on previous fuel toxicity studies. Analysis of samples for the extent of DNA damage as determined by tail moment and percent tail DNA was performed on exposed cells following a brief recovery time. All fuels produced significant increases in DNA damage; however, only JP-8+100 was genotoxic at the lowest exposure concentration (1:500). At the highest exposure concentration (1:75), the mean tail moments for JP-8 and JP-8+100 (32.041 +/- 2.599 and 45.774 +/- 4.743, respectively) were significantly greater than for JP-5 (1.314 +/- 0.474). These results indicate that JP-8+100 is the most potent inducer of DNA damage in human peripheral lymphocytes and that both JP-8+100 and JP-8 are capable of damaging lymphocyte DNA to a greater extent than JP-5.     

Genotoxicity biomonitoring in regions exposed to vehicle emissions using the comet assay and the micronucleus test in native rodent Ctenomys minutus

Exposure to motor vehicle emissions represents an important concern for possible long-term health effects. The present report describes: 1) the application and verification of the alkaline comet assay in Ctenomys minutus to detect the possible genotoxicity of automobile emissions; 2) a comparison of the comet assay results with peripheral blood micronucleus (MN) assay results performed in the same animals; and 3) the identification of agents involved in the responses and in the seasonal variation of the effects. Ctenomys minutus (Octodontidae-Rodentia) were captured in two different fields from both sides of RS/030, a highway on the coastal plain of the Brazilian state of Rio Grande do Sul. Reference animals were obtained from a nearby field that was about 3 km distant from any road. By the end of this study, 123 rodents (73 females and 50 males) were live-trapped. Our results indicate that there was an increase in cells with DNA damage for C. minutus environmentally exposed to automobile emissions, as demonstrated by the alkaline comet assay, but there was no increase in micronucleated cells. The alkaline comet assay showed age and gender differences in the response. The comet assay results suggest that adult females are the principal population affected by air pollutants from vehicle emissions. Chemical data were also collected from areas exposed to automobile exhaust and these indicated that elevated levels of hydrocarbons, metals, and NO(2) were associated with the elevated levels of damaged cells observed in the wild rodent C. minutus. Our results agree with previous data on engine and fuel components, where weak increases in damage for native rodents exposed to emissions have been observed. Other larger, controlled studies are needed to better understand how the metabolism of C. minutus affects its response to emission exposure.     

Development of a high‐throughput genotoxicity assay using Umu test strains expressing human cytochrome P450s and NADPH‐P450 reductase and bacterial O‐acetyltransferase

Umu test is one of the in vitro genotoxicity test that has been used widely. It was developed as a high‐throughput test system using the 96‐well microplate. We have previously constructed new umu test strains for the evaluation of genotoxicity of procarcinogenic metabolic products formed by cytochrome P450 (CYP) enzymes. In this study, a highly sensitive high‐throughput genotoxicity test was developed using four umu test strains (OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4) that express human CYPs and NADPH‐P450 reductase. We found that the modified umu‐microplate method was more sensitive than the conventional microplate method using strain OY1002/1A2. In addition, the new microplate method was better able to detect genotoxicity than the test tube method when the strain OY1002/1A2 was used and had similar sensitivity for the remaining three strains. When the microplate method was used, OY1002/1A2 showed stronger umuC gene expression in the presence of 2‐amino‐6‐methyldipyrido[1,2‐a:3',2'‐d]imidazole, 2‐amino‐3‐methylimidazo[4,5‐f]quinoline, 2‐amino‐3,4‐dimethylimidazo[4,5‐f]quinoline, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline, 2‐aminofluorene, and 2‐aminoanthracene compared to other strains. We also confirmed CYP1A2 expression in OY1002/1A2 in this condition. These results indicate that the microplate version of this test system can detect the genotoxicity of heterocyclic and aromatic amines with high sensitivity and can be used for high‐throughput screening of potentially genotoxic compounds. Environ. Mol. Mutagen. 58:209–216, 2017. © 2017 Wiley Periodicals, Inc.     

Evaluation of the Pig‐a,micronucleus, and comet assay endpoints in a 28‐day study with ethyl methanesulfonate

Ethyl methanesulfonate (EMS) was evaluated as part of the validation effort for the rat Pig‐a mutation assay and compared with other well‐established in vivo genotoxicity endpoints. Male Sprague‐Dawley (SD) rats were given a daily dose of 0, 6.25, 12.5, 25, 50, or 100 mg/kg/day EMS for 28 days, and evaluated for a variety of genotoxicity endpoints in peripheral blood, liver, and colon. Blood was sampled pre‐dose (Day 1) and at various time points up to Day 105. Pig‐a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC^CD59? and RET^CD59? frequencies. The first statistically significant increases in mutant frequencies were seen in RETs on Day 15 and in RBCs on Day 29 with the maximum RET^CD59? on Day 29 and of RBC^CD59? on Day 55. The lowest dose producing a statistically significant increase of RET^CD59? was 12.5 mg/kg on Day 55 and 25 mg/kg for RBC^CD59? on Day 55. EMS also induced significant increases in % micronucleated RETs (MN‐RETs) in peripheral blood on Days 3, 15, and 28. No statistically significant increases in micronuclei were seen in liver or colon. Results from the in vivo Comet assay on Day 29 showed generally weak increases in DNA damage in all tissues evaluated with little evidence for accumulation of damage seen over time. The results with EMS indicate that the assessment of RBC^CD59? and/or RET^CD59? in the Pig‐a assay could be a useful and sensitive endpoint for a repeat dose protocol and complements other genotoxicity endpoints. Environ. Mol. Mutagen. 55:492–499, 2014. © 2014 Wiley Periodicals, Inc.     

Vitamin C modulates DNA damage induced by hydrogen peroxide in human colorectal adenocarcinoma cell lines (HT29) estimated by comet assay in vitro

Introduction

Cancer cells, compared to normal cells, are under increased oxidative stress associated with oncogenic transformation, alterations in metabolic activity, and increased generation of reactive oxygen species.

Material and methods

We investigated the ability of vitamin C to reduce the damage induced by hydrogen peroxide, in human colorectal adenocarcinoma cells in vitro by the comet assay. Additionally, we measured the kinetics and efficacy of the repair of DNA damage after incubation with vitamin C in the presence of H₂O₂.

Results

The obtained results showed that 1 h pre-incubation with vitamin C and exposure to H₂O₂ for the last 10 min of incubation caused a statistically significant (p < 0.05) increase in DNA migration in comet tails in all experimental series. For the 10 µM, 25 µM, 50 µM, 100 µM vitamin C concentrations the levels of DNA damage were as follows: 18.6%, 21.1%, 25.3% and 27.2%, respectively, as compared to the untreated cells (3.26%). However, in comparison with H₂O₂ alone (29.1%), we observed a statistically significant (p < 0.05) decrease of the genotoxic effect in HT29 cells induced by H₂O₂ for the two lowest of concentrations of vitamin C: 10 µM and 25 µM. The HT29 cells were able to achieve effective repair of the damaged DNA within 60 and 120 min after incubation with the tested compounds. All the values obtained in the test were statistically significant (p < 0.05).

Conclusions

Vitamin C caused a weaker DNA damaging effect of hydrogen peroxide and positively influences the level of oxidative DNA damage in HT29 cells (decrease ∼ 30%). We noted that DNA damage was effectively repaired during 120 min postincubation in the tested cells and that oxidative damage was the major type of damage.