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PubMedDate not supplied

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.

Biomarkers↗
PubMedDate not supplied

DNA damage (comet assay) and 8-oxodGuo (HPLC-EC) in relation to oxidative stress in the freshwater bivalve Unio tumidus.

The relationships between DNA damage and oxidative stress in the digestive gland, gills and haemocytes of the freshwater bivalve Unio tumidus were investigated. Two markers of genotoxicity were measured: DNA breaks by means of the comet assay, and oxidative DNA lesions by means of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) measured using high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) tissue levels. Effects were studied after exposure of bivalves for 6 days to benzo[a]pyrene (B[a]P) (50 and 100 microg l(-1)) and ferric iron (20 and 40 mg l(-1)), applied alone or in combination. Lipid peroxidation in the digestive gland and gills resulted from exposure to Fe3+ or B[a]P whatever the concentrations tested. DNA oxidatively formed lesions were induced in the two tissues at a higher level after B[a]P exposure than after Fe3+ treatment. No significant dose-response relationship was found with the two compounds and no synergistic effect was observed between Fe3+ and B[a]P. The gills appeared less sensitive than the digestive gland to DNA lesions expressed as 8-oxodGuo and comet results. Good correlations were noted between 8-oxodGuo and comet. MDA and DNA damage did not correlate as well, although it was stronger in the digestive gland than in the gills. Production of mucus by the gills likely served to prevent lesions by reducing the bioavailability of the chemicals tested, which could explain that dose-effect relationships and synergistic effects were not observed.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2002

Basal levels of DNA strand breaks in human leukocytes determined by comet assay.

In order to determine background levels of DNA strand breaks, we examined 80 healthy individuals by comet assay considering age, sex, and smoking as confounding factors. Only age was found to have a significant effect on basal levels. One thousand cells of each donor were graded by eye into 5 categories according to the amount of DNA in the tail: classification group A (no damage) <5%, B (low damage) 5-20%, C (medium damage) 20-40%, D (high damage) 40-95%, and group E (total damage) >95%. The interpretation of the comet assay was modified to achieve a tail factor, which represents the DNA damage of 1000 scored cells as a single number, without the need of an image analysis software package. Hydrogen peroxide and bleomycin used for in vitro exposure of lymphocytes, produced clear dose-related responses in the comet assay. Our data encourage the application of the used classification model for a sensitive and fast quantification of DNA damage. Results in this study are in agreement with most of the earlier investigations.

Adult↗
PubMed2001

In vivo genotoxic effect of zinc sulfate in mouse peripheral blood leukocytes using Comet assay.

Single stranded DNA breaks induced by Zinc sulfate in mice has been studied in vivo using Alkaline Single Cell Gel Electrophoresis (Comet assay). Mice were administered orally with doses of 5.70, 8.55, 11.40, 14.25, 17.10 and 19.95 mg/kg body weight of zinc sulfate respectively. The samples of whole blood were collected at 24, 48, 72, 96 hr and first week post-treatment and the assay was carried out to determine single strand DNA breaks as represented by comet tail-lengths. Results indicated a significant DNA damage at all the doses after treatment with zinc sulfate when compared to controls showing a clear dose-dependent response (p < 0.05). A gradual decrease in the tail-lengths from 48 hr post-treatment onwards was observed indicating a time dependent decrease in the DNA damage. The study confirms that zinc sulfate causes significant DNA damage at the doses used as revealed by comet assay.

Animals↗
PubMed2002

Detection of DNA damage in mouse peripheral blood leukocytes by the comet assay after oral administration of monocrotophos.

The objective of this study was to determine if single/double strand DNA breaks could be induced by monocrotophos (organophosphorus pesticide) in mice in vivo using the comet assay. Mice were dosed orally with 0.046, 0.093, 0.186, 0.373 and 0.746 mg/kg body weight of monocrotophos, and the assay was performed on whole blood after 24, 48 and 72 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24 and 48 h post-treatment with monocrotophos when compared to controls. A decrease in the mean tail length was observed at 72 h post-treatment indicating repair of the damaged DNA. The mean tail length showed a dose-related increase and time dependent decrease. The study reveals that comet assay is a sensitive and rapid method to detect genotoxicity of monocrotophos.

Administration, Oral↗
PubMed2004

Application of the comet assay for investigation of oxidative DNA damage in equine peripheral blood mononuclear cells.

Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and equine recurrent airway obstruction (RAO, a condition similar to human asthma). The images obtained were similar to those previously published for humans, cats, and dogs. The optimum concentration of H(2)O(2) to estimate susceptibility to exogenous damage was 50 microM. Mean intraassay coefficients of variation were 4.7 and 9.7% for endogenous and exogenous tail-DNA quantities, respectively, and 7.3 and 8.3%, respectively, for interassay coefficients. There was no significant difference in either endogenous or exogenous percentages of tail DNA for samples collected from six ponies on three consecutive days. There was no significant difference in endogenous, exogenous, or exogenous (corrected for endogenous) oxidative DNA damage between mature and aged ponies. However, young pony foals had significantly less endogenous DNA damage than mature or aged ponies (P < 0.05). RAO-affected horses without airway inflammation (i.e., in clinical remission) had significantly greater endogenous damage compared with non-RAO-affected control animals (P = 0.009). There was a significant correlation between endogenous percentage of tail DNA in PBMCs and red blood cell hemolysate glutathione concentration (r = 0.720; P < 0.001). In conclusion, the comet assay appears to be suitable for investigating DNA damage in equine PBMCs.

Aging↗
PubMed2001

Chromosomal aberration and single cell gel electrophoresis (Comet) assay in the longitudinal risk assessment of occupational exposure to pesticides.

In recent years the use of pesticides in agriculture has been increasing steadily. At present there are more than 1000 chemicals classified as pesticides. Therefore, the widespread use of pesticides and their potential genetic hazard suggests that evaluation of their genotoxicity should be extended using the newer assays now available. In the present study chromosomal aberration analysis and the alkaline single cell gel electrophoresis (Comet) assay were used to evaluate the extent of DNA damage and DNA repair in peripheral blood lymphocytes of subjects employed in pesticide production. In order to determine possible primary genotoxic effects in workers blood samples were taken after an 8 month long period of exposure to a complex mixture of pesticides. To detect the possible occurrence of DNA repair in lymphocytes of the same subjects the second blood sample was taken after an 8 month long period of absence from the pesticide exposure zone. Regardless of the period of sampling, in the exposed group statistically significantly increased numbers of aberrant cells, chromatid and chromosome breaks, acentric fragments and dicentric chromosomes compared with the controls were found. After the workers had spent 8 months out of the pesticide exposure zone the number of aberrant cells and all types of chromatid and chromosome aberrations decreased significantly compared with sampling after the high exposure period, but it still remained significantly higher in comparison with the control group. After the period of high exposure to a mixture of pesticides statistically significantly increased levels of DNA damage in the Comet assay in terms of tail length and tail moment were found. After the workers were removed from production for 8 months both Comet assay end-points decreased significantly compared with the first sampling point, but they remained increased compared with the control.

Adult↗
PubMed2004

Evaluation of in vivo genotoxicity of cypermethrin in Drosophila melanogaster using the alkaline Comet assay.

The single cell gel electrophoresis (SCGE) assay, also known as the Comet assay, is one of the most promising genotoxicity tests developed in recent years to measure and analyse DNA damage in single cells. The present study was undertaken to assess the in vivo genotoxicity of the synthetic pyrethroid cypermethrin in brain ganglia and anterior mid gut of Drosophila melanogaster. Freshly emerged first instar larvae (22 +/- 2 h) were placed in different concentrations of cypermethrin (0.0004, 0.0008, 0.002, 0.2 and 0.5 p.p.m.) mixed in standard Drosophila food and allowed to grow. At 96 +/- 2 h, brain ganglia and anterior midgut from control and treated larvae were dissected out, single cell suspensions were prepared and a Comet assay was performed. Our results revealed a significant dose-dependent increase in DNA damage in the cells of brain ganglia and anterior midgut of D.melanogaster exposed to cypermethrin as compared with controls (P < 0.05 at 0.002 p.p.m.; P < 0.001 at 0.2 and 0.5 p.p.m.). The present study shows in vivo genotoxicity of cypermethrin even at very low concentrations, which proves D.melanogaster as a model for in vivo genotoxicity assessment using the Comet assay.

Animals↗
PubMed2005

Comparative genotoxic evaluation of 2-furylethylenes and 5-nitrofurans by using the comet assay in TK6 cells.

The genotoxicity of three 2-furylethylene derivatives and four 5-nitrofurans was evaluated by using the comet assay in human lymphoblastoid cultured TK6 cells. The 2-furylethylene derivatives were 2-furyl-1-nitroethene, 1-(5-bromofur-2-yl)-2-nitroethene and 1-(5-bromofur-2-yl)-2-bromo-2-nitroethene, while the 5-nitrofurans were nitrofurantoin, nitrofurazone, furazolidone and 5-nitro-2-furanacrolein. The treatments lasted for 3 h in the absence of metabolic activation. No genotoxic effects were observed for two of the 2-furylethylene compounds, while the derivative 1-(5-bromofur-2-yl)-2-nitroethene showed a statistically significant response mainly at the highest concentration tested; this effect was considered biologically relevant and the compound was classified as slightly genotoxic. On the other hand, for the classical 5-nitrofurans tested there is a tendency towards a dose-related increase of the DNA damage in the comet assay and the observed increases for the parameters analysed (Olive tail moment, tail % DNA and tail length) were significant for all compounds. Then, the four 5-nitrofurans tested were considered genotoxic. These results show that the position of the nitro group influences the genotoxicity of the assayed compounds. Thus, in this comet assay, the 2-furylethylene derivatives having the nitro group attached outside the furan ring appear to be much less genotoxic than the 5-nitrofurans.

Cell Line, Tumor↗
PubMed2005

Genetic polymorphisms and the effect of cigarette smoking in the comet assay.

A potential genotoxic effect of cigarette smoking has repeatedly been investigated with the comet assay (alkaline single cell gel electrophoresis) and conflicting results have been reported. Besides differences in the methodology and the study design used, genetic differences between the subjects investigated might contribute to the variability of test results. Considering genetic polymorphisms of genes involved in metabolism or DNA repair has led to a better discrimination of smoking-related genotoxic effects in some cases but also led to discrepant results. We therefore evaluated our baseline comet assay effects obtained for nonsmokers and smokers in relation to selected genetic polymorphisms. Our study group comprised 52 nonsmokers and 51 smokers who were strictly selected to exclude potential confounding factors. We chose polymorphisms in the genes GSTM1 and CYP1A1 (Ile462Val) because they take part in the metabolism of genotoxins contained in tobacco smoke. In a subgroup of 32 nonsmokers and 31 smokers we also studied polymorphisms in XPD (Lys751Gln), XRCC1 (Arg399Gln) and XRCC3 (Thr241Val) because they are part of DNA repair pathways involved in the repair of tobacco-related DNA damage. Freshly collected peripheral whole blood samples were tested in the alkaline (pH > 13) comet assay. In all experiments a reference standard (untreated V79 cells) was included to correct for assay variability. An independent second evaluation was carried out for all experiments. None of these approaches revealed a significant difference between nonsmokers and smokers.

Comet Assay↗
PubMed2005

Risk assessment of welders using analysis of eight metals by ICP-MS in blood and urine and DNA damage evaluation by the comet and micronucleus assays; influence of XRCC1 and XRCC3 polymorphisms.

The aims of the present study were to assess the occupational risk of welders using analysis of metals in biological fluids, DNA damage evaluation by complementary genotoxic endpoints and the incidence of polymorphisms in DNA repair genes. A biomonitoring study was conducted that included biometrology (blood and urinary concentrations of aluminium, cadmium, chromium, cobalt, lead, manganese, nickel, zinc by ICP-MS), comet and cytokinesis-block micronucleus assays in peripheral lymphocytes and genetic polymorphisms of XRCC1 (p.Arg399Gln) and XRCC3 (p.Thr241Met). This study included 60 male welders divided into two groups: group 1 working without any collective protection device and group 2 equipped with smoke extraction systems. A control group (n = 30) was also included in the study. Higher chromium, lead and nickel blood and urinary concentrations were detected in the two groups of welders compared to controls. Statistically differences between welders of group 1 and group 2 were found for blood concentration of cobalt and urinary concentrations of aluminium, chromium, lead and nickel. The alkaline comet assay revealed that welders had a significant increase of OTMchi2 distribution at the end of a work week compared to the beginning; a significant induction of DNA strand breaks at the end of the week was observed in 20 welders out of 30. The cytokinesis-block micronucleus assay showed that welders of group 1 had a higher frequency of chromosomal damage than controls. The XRCC1 variant allele coding Gln amino acid at position 399 was found to be associated with a higher number of DNA breaks as revealed by the comet assay. Increased metal concentrations in biological fluids, DNA breaks and chromosomal damage in lymphocytes emphasized the need to develop safety programmes for welders.

Adult↗
PubMed2005

The effect of smoking on DNA effects in the comet assay: a meta-analysis.

The comet assay (alkaline single-cell gel electrophoresis, SCG or SCGE) is frequently used in biomonitoring to detect genotoxic effects in humans exposed at the workplace or in their environment. Because of its ready accessibility, blood is most frequently used in such studies. Many studies investigated cigarette smoking either as a genotoxic exposure itself or as a potential confounding factor in occupational studies. However, although smoking is considered to be a relevant exposure towards various genotoxins, conflicting results have been reported in the comet assay studies. The actual reasons for this discrepancy are not known. To further evaluate evidence for smoking-related DNA effects in the comet assay, we now used a meta-analysis approach based on a literature search. We identified 38 studies from 37 publications which were suited for a formal meta-analysis based on the standardized mean difference (SMD) between the study groups. The evaluation of these 38 studies indicated higher levels of DNA damage in smokers than in non-smokers [under a random effects model, SMD = 0.55, 95% confidence interval = (0.16-0.93)]. Subdividing these studies into studies investigating the effect of smoking as a genotoxic exposure (Type A studies, n = 12) and studies investigating smoking as a potential confounder in occupational studies (Type B, n = 26) indicated a significant difference only in Type A studies but not in Type B studies. Furthermore, studies using image analysis or image length measurements (n = 23) only indicated a tendency for a genotoxic effect of smoking, whereas studies using an arbitrary score (n = 15) found a significantly higher level of DNA damage in smokers.

Artifacts↗
PubMed2006

Determination of genotoxicity of classical swine fever vaccine in vitro by cytogenetic and comet tests.

Chromosome damage in lymphocyte cultures induced by live virus vaccine against classical swine fever (CSF) has been observed in previous studies. In vivo cytogenetic tests were made with several doses of vaccines used in Argentina to control the disease. These studies have shown that genotoxic effects increased with dose. In the present study, two different in vitro assays were performed by recording the frequency of cells with chromosome alterations and by assessing the ability of the vaccine to damage DNA, using the single cell gel microelectrophoretic assay (comet test). Frequencies of cells with chromosomal alterations increased significantly when compared with controls and were dose (microl/ml) dependent: 0 = 1.23, 5 = 2.29, 10 = 5.42 and 20 = 11.71%. In the comet assay the variables measured, tail length (TL) and tail moment (TM), also increased. For control cultures TL was 2.32 microm, whereas with concentrations of 20 and 100 microl/ml TL were 12.47 and 42.3 microm, respectively. TM of control cultures was 0.18, whereas with vaccine concentrations of 20 and 100 microl/ml TM were 5.52 and 24.52, respectively. Comet frequency distributions differed significantly among treatments. These results agree with previous in vivo observations. Regarding CSF pathogeny, our results support a direct effect of CSF vaccinal virus on lymphocyte DNA. Genotoxicity of CSF vaccine was corroborated in vitro at the cytogenetic and molecular levels.

Animals↗
PubMed2005

Evaluating the self-esteem of myopic children over a three-year period: The COMET Experience.

PURPOSE: The purposes of this study were to evaluate self-esteem over 3 years in the 469 myopic children participating in the Correction of Myopia Evaluation Trial (COMET), and to examine its relationship with lens assignment (progressive addition lenses [PALs] vs. single-vision lenses [SVLs]), myopia progression, and several other ocular and demographic characteristics. METHODS: Data collection included refractive error measurements, child-reported visual symptoms, attitude toward glasses, adherence, and self-esteem as measured by the Self-Perception Profile for Children (SPPC). A two-way analysis of variance (treatment group x time) was performed to examine whether PAL and SVL wearers differed in self-esteem over time. Multiple regression analyses were used to evaluate associations between self-esteem at follow-up and relevant factors identified by univariate analyses. RESULTS: Regardless of lens assignment or myopia progression, COMET children reported moderate to high levels of self-esteem at follow-up in the areas of scholastic and athletic competence, physical appearance, social acceptance, behavioral conduct, and global self-worth. Mean scores ranged from 2.87 (+/- 0.68) on athletic competence to 3.40 (+/- 0.56) on global self-worth. Self-esteem changed significantly (p < 0.05) over 3 years in the domains of scholastic competence, social acceptance, and physical appearance. Self-esteem at follow-up was associated with visual symptoms, attitude toward glasses, age, gender, and ethnicity. CONCLUSIONS: Lens assignment and myopia progression were not associated with self-esteem in the COMET cohort. These children had high levels of self-esteem, suggesting that having myopia does not negatively impact self-esteem. Follow-up reports will monitor self-esteem and related factors in this cohort of myopic children over the course of adolescence and early adulthood.

Child↗
PubMedDate not supplied

Genotoxicity of nitroso compounds and sodium dichromate in a model combining organ cultures of human nasal epithelia and the comet assay.

Genotoxic effects of xenobiotics are a possible step in tumor initiation in the mucosa of the upper aerodigestive tract. Using the comet assay, detecting genotoxicity in human tissue has been restricted to single incubations in vitro, but in vivo most xenobiotics harm their target in a repetitive or chronic manner. Therefore, we propose a model, which provides repetitive incubations in human upper aerodigestive tract mucosa cultures. Samples of human inferior nasal turbinate mucosa (n = 25) were cultured according to a modified version of a technique originally described by Steinsvåg. On day 1 fresh samples and on days 7, 9 and 11 organ cultures were incubated with N-nitrosodiethylamine (NDEA), sodium dichromate (Na2Cr2O7) and N'-methyl-N-nitro-N-nitrosoguanidine (MNNG). Mucosa samples and organ cultures, respectively, underwent a modified comet assay on days 1, 7 and 11. Genotoxicity could be shown for NDEA, Na2Cr2O7 and MNNG on days 1, 7 and 11. Duration of tissue culture and repetitive incubations did not significantly influence the results for NDEA. Nevertheless, Na2Cr2O7 and MNNG caused higher genotoxic effects on cultures subjected to the comet assay on day 11. This model may help to assess genotoxic hazards posed by environmental pollutants that have a cumulative character in repetitive or chronic exposure in vivo.

Adult↗
PubMed2004

Accommodation and related risk factors associated with myopia progression and their interaction with treatment in COMET children.

PURPOSE: To examine baseline measurements of accommodative lag, phoria, reading distance, amount of near work, and level of myopia as risk factors for progression of myopia and their interaction with treatment over 3 years, in children enrolled in the Correction of Myopia Evaluation Trial (COMET). METHODS: COMET enrolled 469 ethnically diverse children (ages, 6-11 years) with myopia between -1.25 and -4.50 D. They were randomly assigned to either progressive addition lenses (PALs) with a +2.00 addition (n = 235) or single vision lenses (SVLs; n = 234), the conventional spectacle treatment, and were observed for 3 years. The primary outcome measure was progression of myopia by autorefraction after cycloplegia with 2 drops of 1% tropicamide. Other measurements included accommodative response (by an open field of view autorefractor), phoria (by cover test), reading distance, and hours of near work. Independent and interaction analyses were based on the mean of the two eyes. Results were adjusted for important covariates with multiple linear regression. RESULTS: Children with larger accommodative lags (>0.43 D for a 33 cm target) wearing SVLs had the most progression at 3 years. PALs were effective in slowing progression in these children, with statistically significant 3-year treatment effects (mean +/- SE) for those with larger lags in combination with near esophoria (PAL - SVL progression = -1.08 D - [-1.72 D] = 0.64 +/- 0.21 D), shorter reading distances (0.44 +/- 0.20 D), or lower baseline myopia (0.48 +/- 0.15 D). The 3-year treatment effect for larger lags in combination with more hours of near work was 0.42 +/- 0.26 D, which did not reach statistical significance. Statistically significant treatment effects were observed in these four groups at 1 year and became larger from 1 to 3 years. CONCLUSIONS: The results support the COMET rationale (i.e., a role for retinal defocus in myopia progression). In clinical practice in the United States children with large lags of accommodation and near esophoria often are prescribed PALs or bifocals to improve visual performance. Results of this study suggest that such children, if myopic, may have an additional benefit of slowed progression of myopia.

Accommodation, Ocular↗
PubMed2001

Differential staining of DNA strand breaks in dried comet assay slides.

The comet assay involves embedding cells in agarose on microscope slides. After lysis and electrophoresis, staining is usually performed with a fluorescent DNA-binding dye and observation is carried out on fresh wet slides through an epifluorescence microscope. We present here a simple alternative for preservation of the agarose comet slides and a fluorescent staining that allows fine differential analysis of DNA strand breaks under confocal microscopy. Lymphocytes were processed according to previous published methods. Slides were quickly dehydrated in a hot oven at 50C for 20 min. Once the agarose layer was dried and reduced to a thin film, slides were treated with RNase. Image analysis showed higher tail length, total area, and tail moment. Using confocal microscopic optical sectioning, a thickness of approximately 180 microm for wet slides and 12 microm for dehydrated gels was calculated. Acridine orange, used for DNA differential staining, allowed quantitation of metachromasia and orthochromasia with confocal scanning microscopy. Differences between alkaline and neutral comet assay with AO were clear-cut and, in principle, a metachromatic index can be calculated. (J Histochem Cytochem 49:921-922, 2001)

Acridine Orange↗
PubMed2001

Study of N-nitrosomorpholine-induced DNA strand breaks in Caco-2 cells by the classical and modified comet assay: influence of vitamins E and C.

We have studied the genotoxic effects of the well-known heterocyclic liver carcinogen N-nitrosomorpholine (NMOR), an N-nitroso compound, which was prepared in our laboratory by nitrosation of the secondary amine morpholine with NaNO2. NMOR induced DNA strand breaks in human colon carcinoma Caco-2 cells. The concentration-dependent DNA-damaging effects of NMOR were proved by the comet assay. We further characterized DNA strand breaks induced by NMOR as follows: 1) We pretreated cells with vitamins E and C, which are able to eliminate oxidative DNA damage. 2) We varied the pH of the comet assay (12.1 and 13). In general, alkali-labile sites are stable until pH is raised to 12.5. 3) We used the site-specific repair enzymes exonuclease III and formamidopyrimidine-DNA glycosylase in the modified comet assay. Results showed that NMOR-induced DNA strand breaks have their origin exclusively in alkali-labile sites. Nevertheless, vitamins E and C decreased the level of DNA strand breaks. These results showed that antioxidants may have biological activities other than free radical scavenging that relate to their cancer-prevention properties. Our conceptions about reduction of NMOR-induced DNA lesions in Caco-2 cells by vitamins E and C are presented in this work.

Anticarcinogenic Agents↗