Extent Of DNA Migration Research Articles

GENOPROTECTIVE EFFECTS OF ETHANOLIC STEM EXTRACTS OF TINOSPORA CORDIFOLIA ON SODIUM ARSENITE-INDUCED DNA DAMAGE IN SWISS MICE LYMPHOCYTES BY COMET ASSAY

Objective: The study was aimed to evaluate the DNA protective effects of ethanolic extracts of Tinospora cordifolia stem on Swiss albino mice lymphocytes against the sodium arsenite-induced genotoxicity.
 Methods: In this experiment, 25 animals of nearly the same age were equally divided. Group 1 (control) fed on tap water while rest four (2, 3, 4, and 5) groups were treated with 4 mg/kg body weight, per orally sodium arsenite for 12 weeks. Sodium arsenite pretreated groups (4 and 5) were followed by administration of 80 mg/kg body weight, per orally T. cordifolia extracts for the next 12 weeks. Animals were sacrificed at the 8th week and 12th week, respectively, for the assessment of sodium arsenite-induced genotoxicity and comparative genoprotective effects of experimental plant extracts. The extent of DNA migration is directly proportional to the magnitude of DNA damage. Percentage tail DNA content and olive tail moment parameters were used in the comet to relate DNA damage.
 Results: The findings suggested that the ethanolic stem extracts of T. cordifolia significantly inhibited the sodium arsenite-induced oxidative genotoxicity. The phytoconstituents of T. cordifolia shown to retard genetic damage associated with arsenic exposure.
 Conclusion: T. cordifolia may be used as a preventive herbal preparation against chemical or arsenical toxicity.

Antigenotoxic Effect of Trametes spp. Extracts against DNA Damage on Human Peripheral White Blood Cells.

Trametes species have been used for thousands of years in traditional and conventional medicine for the treatment of various types of diseases. The goal was to evaluate possible antigenotoxic effects of mycelium and basidiocarp extracts of selected Trametes species and to assess dependence on their antioxidant potential. Trametes versicolor, T. hirsuta, and T. gibbosa were the species studied. Antigenotoxic potentials of extracts were assessed on human peripheral white blood cells with basidiocarp and mycelium extracts of the species. The alkaline comet test was used for detection of DNA strand breaks and alkali-labile sites, as well as the extent of DNA migration. DPPH assay was used to estimate antioxidative properties of extracts. Fruiting body extracts of T. versicolor and T. gibbosa as well as T. hirsuta extracts, except that at 20.0 mg/mL, were not genotoxic agents. T. versicolor extract had at 5.0 mg/mL the greatest antigenotoxic effect in both pre- and posttreatment of leukocytes. The mycelium extracts of the three species had no genotoxic activity and significant antigenotoxic effect against H2O2-induced DNA damage, both in pre- and posttreatment. The results suggest that extracts of these three species could be considered as strong antigenotoxic agents able to stimulate genoprotective response of cells.

In vitro genoprotective and genotoxic effect of nicotine on human leukocytes evaluated by the comet assay

The comet assay was used to measure the DNA damage induced in vitro by nicotine in human leukocytes as the extent of DNA migration in the comet head area, tail length, percent DNA in the tail, and Olive tail moment. Samples of whole blood were collected and blood cells were challenged with acute doses of 0.1, 1 and 10 µM of (−)-nicotine for 60 minutes. We found that nicotine treatment had dose-dependent effects on the level of DNA damage. At 1 and 10 µM of nicotine, both Olive tail moment and percent DNA in the tail significantly increased (p < 0.001), compared to the control. In the presence of 10 µM of nicotine, the shortest tail length and the smallest head area were detected. At a concentration of 0.1 µM, surprisingly, DNA damage detected by the comet assay was lower than in the control, which was proved by the observed significantly (p < 0.001) lower Olive tail moment and percent DNA in the tail as well as larger head area. The results suggest that nicotine, at a reasonably low concentration (0.1 µM), comparable to those found in the blood of habitual smokers, may have a protective effect, whereas higher doses of nicotine (1 and 10 µM) are genotoxic. The possible participation of reactive oxygen species in the DNA-damaging potential of nicotine is discussed.

Evaluation of Oxidative DNA Damage Using an Alkaline Single Cell Gel Electrophoresis (SCGE) Comet Assay, and the Protective Effects of N-Acetylcysteine Amide on Zearalenone-induced Cytotoxicity in Chang Liver Cells

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium that are found in cereals and agricultural products. ZEN has been implicated in mycotoxicosis in farm animals and in humans. The toxic effects of ZEN are well known, but the ability of an alkaline Comet assay to assess ZEN-induced oxidative DNA damage in Chang liver cells has not been established. The first aim of this study was to evaluate the Comet assay for the determination of cytotoxicity and extent of DNA damage induced by ZEN toxin, and the second aim was to investigate the ability of N-acetylcysteine amide (NACA) to protect cells from ZEN-induced toxicity. In the Comet assay, DNA damage was assessed by quantifying the tail extent moment (TEM; arbitrary unit) and tail length (TL; arbitrary unit), which are used as indicators of DNA strand breaks in SCGE. The cytotoxic effects of ZEN in Chang liver cells were mediated by inhibition of cell proliferation and induction of oxidative DNA damage. Increasing the concentration of ZEN increased the extent of DNA damage. The extent of DNA migration, and percentage of cells with tails were significantly increased in a concentration-dependent manner following treatment with ZEN toxin (p < 0.05). Treatment with a low concentration of ZEN toxin (25 μM) induced a relatively low level of DNA damage, compared to treatment of cells with a high concentration of ZEN toxin (250 μM). Oxidative DNA damage appeared to be a key determinant of ZEN-induced toxicity in Chang liver cells. Significant reductions in cytolethality and oxidative DNA damage were observed when cells were pretreated with NACA prior to exposure to any concentration of ZEN. Our data suggest that ZEN induces DNA damage in Chang liver cells, and that the antioxidant activity of NACA may contribute to the reduction of ZEN-induced DNA damage and cytotoxicity via elimination of oxidative stress.

Exposure to 1800 MHz radiofrequency electromagnetic radiation induces oxidative DNA base damage in a mouse spermatocyte-derived cell line

Whether exposure to radiofrequency electromagnetic radiation (RF-EMR) emitted from mobile phones can induce DNA damage in male germ cells remains unclear. In this study, we conducted a 24h intermittent exposure (5min on and 10min off) of a mouse spermatocyte-derived GC-2 cell line to 1800MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rates (SAR) of 1W/kg, 2W/kg or 4W/kg. Subsequently, through the use of formamidopyrimidine DNA glycosylase (FPG) in a modified comet assay, we determined that the extent of DNA migration was significantly increased at a SAR of 4W/kg. Flow cytometry analysis demonstrated that levels of the DNA adduct 8-oxoguanine (8-oxoG) were also increased at a SAR of 4W/kg. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS); these phenomena were mitigated by co-treatment with the antioxidant α-tocopherol. However, no detectable DNA strand breakage was observed by the alkaline comet assay. Taking together, these findings may imply the novel possibility that RF-EMR with insufficient energy for the direct induction of DNA strand breaks may produce genotoxicity through oxidative DNA base damage in male germ cells.

Pre-cancerous (DNA and chromosomal) lesions in professional sports

Exhaustive exercises may become detrimental, causing disturbance of intracellular oxidant-antioxidant balance and damage to macromolecules, leading to genomic instability when DNA/chromosomes get damaged. As these are precancerous lesions, cancer occurrence is probable. Because professional sports requires high-intensity training and increasing physical demand, there may occur cellular genomic instability. To evaluate genetic damage at DNA and chromosomal levels in hockey and baseball-soft ball players and compare with levels in age- and sex-matched sedentary controls. Players professionally active in their sport from 3 to 11 years on a daily training session of 4h/day were contacted during their camps, and the study was approved by the Institutional Ethics Committee. All participants were healthy individuals, not on any medication and were not taking any supplements. Genetic damage using the single cell gel electrophoresis assay and buccal micronucleus cytome assay of 56 individuals (36 players and 20 controls) was evaluated. Student's t -test, ANOVA, Pearson correlation and linear regression and Chi-square analysis were performed. Players had significantly elevated levels of genetic damage. There were no gender differences and also no significant difference in the genetic damage incurred in both sports types. However, the extent of DNA migration in hockey players was higher. Significantly increased genomic instability in players of both sports was observed. Both repaired and repairable genetic damage cells were observed in different tissues of the same subject. The presence of such genetic damage implies that these players are at an individual risk from cancer- and age-related diseases.

Protective Effects of Aqueous and Ethanolic Extracts of Portulaca oleracea L. Aerial Parts on H 2O 2- Induced DNA Damage in Lymphocytes by Comet Assay

The comet assay is a standard method for measuring DNA damage. In this study, the protective effects of ethanolic and aqueous extracts of Portulaca oleracea L. ( P. oleracea) on human lymphocyte DNA lesions were evaluated with the comet assay. Lymphocytes were isolated from blood samples taken from healthy volunteers. Human lymphocytes were incubated in H 2O 2 (50,100, and 200 μM), aqueous extract (0.05, 0.1, 0.5, 1, and 2.5 mg/ml), and ethanolic extracts (0.05, 0.1, 0.5, 1, and 2.5 mg/ml) of P. oleraceae aerial parts alone with a combination of H 2O 2 (100 μM) with either 1 or 2.5 mg/ml of both extracts at 4°C for 30 minutes. The extent of DNA migration was measured using the alkaline single cell gel electrophoresis approach assay, and DNA damage was expressed as percentage tail DNA. We found that the aqueous extract of P. oleracea significantly inhibited DNA damage, while there was no effect of the ethanolic extract. These data suggest that the aqueous extract of P. oleracea can prevent oxidative DNA damage to human lymphocytes, which is likely due to antioxidant constituents in the extract.

DNA damage in barn swallows ( Hirundo rustica) from the Chernobyl region detected by use of the comet assay

We investigated levels of DNA damage in blood cells of barn swallows ( Hirundo rustica) inhabiting the Chernobyl region to evaluate whether chronic exposure to low-level radioactive contamination continues to induce genetic damage in free-living populations of animals. Blood samples were obtained from barn swallows collected at sites with different background levels of radiation, including a relatively uncontaminated area. The extent of DNA damage was evaluated using the alkaline (pH = 12.1) comet assay, a robust and sensitive electrophoresis-based technique widely employed in research ranging from biomonitoring to clinical studies. We found that levels of DNA damage, as indexed by the extent of DNA migration, were increased in barn swallows living in areas surrounding Chernobyl when compared to swallows sampled at low-level sites. The results we obtained are consistent with previous findings on this same species, which showed that swallows breeding in areas heavily contaminated with radionuclides have increased mutation rates, higher oxidative stress and incidence of morphological aberrations and tumors. Overall, these results indicate that chronic exposure to radioactive contaminants, even 20 years after the accident at the Chernobyl nuclear power plant, continues to induce DNA damage in cells of free-living animals.

DNA instability in low-risk myelodysplastic syndromes: refractory anemia with or without ring sideroblasts

We tested genomic instability in patients with myelodysplastic syndrome (MDS) by the comet assay and verified the suitability of this approach as a tool for analysis of ineffective hematopoiesis in refractory anemia (RA) and RA with ring sideroblasts (RARS). Erythroid and myeloid cell populations from bone marrow aspirates of 20 RA, 14 RARS and 15 control subjects were separated by differential expression of glycophorin A and subjected to comet assay. The extent of DNA migration was measured in single cells (200 cells/bone marrow fraction/subject). The results were in agreement with the concept of increased apoptosis in low-risk MDS subtypes. The RA samples had a significantly higher DNA instability than controls in glycophorin A positive cells, and the extent of DNA breakage correlated with the degree of cytopenia. Although RARS had an even higher rate of genomic instability in bone marrow cells than RA, there was no clear relationship to peripheral cytopenia. This suggests an additional DNA instability of non-apoptotic origin. Whether this increase is associated with an increased repair of oxidative damage in DNA arising due to iron deposits in ring sideroblasts remains to be formally proven. Comet assay provides a promising tool for the investigation of difference between RA and RARS pathobiology.

Investigation of genotoxic effects of inhalative occupational exposure to vanadium: results of a multiple endpoint study

Animal experiments showed that inhalative exposure to vanadate causes lung cancer. To assess DNA damage in exposed workers (EW, n = 52) of a metal factory, we monitored DNA migration and formation of micronuclei in blood cells of EW, who are exposed to vanadium pentoxide via inhalation and in a matched control group (CG, n = 54). Median level of vanadium serum concentrations of EW (2.23 μg/l) was found to be 7-fold increased compared to CG (0.31 μg/l). In the standard single cell gel electrophoresis (SCGE) assay with leucocytes, no differences were detected between EW and CG, but increased levels of oxidised DNA bases (detected with FPG and ENDO III) were found (p < 0.05). Pretreatment of leukocytes with bleomycin resulted in a higher extent of DNA migration (27%, p < 0.001) and reduced capacity of DNA repair (by 42%, p < 0.001) in EW. Furthermore, the cytochalasin blocked micronucleus (CBMN) assay was carried out with peripheral lymphocytes (n = 24 per group). EW showed a 2.5-fold higher MN frequency as CG and a 7fold increase of nucleoplasmic bridges (which reflect formation of dicentric chromosomes) was seen. Nuclear buds (attributable to gene amplification events) were 3fold higher in EW (p < 0.001 for all events). Apoptosis rates did not differ significantly between the two groups, whereas necrosis rates doubled in EW (p < 0.001). Taken together, our results show increased genetic damage in individuals which inhale vanadium dust, possibly indicating increased risk for cancer.

Antigenotoxic effects of Satureja hortensis L on rat lymphocytes exposed to oxidative stress

DNA damage and oxidative stress are widely recognized as major factors in many degenerative diseases and aging [1, 2]. Numerous reports have demonstrated that plant products possess a variety of in vitro antioxidant properties and, that the consumption of food or beverages rich in antioxidant phytochemicals resulted in positive effects on human health and aging process [3]. The protective properties of Satureja hortensis L. on the rat lymphocytes DNA lesions were tested. Lymphocytes were isolated from blood samples taken from healthy rats. DNA breaks and resistance to H2O2-induced damage were measured with the comet assay [4]. Rat lymphocytes were incubated in S. hortensis ethanolic extract (SHE) (0.05, 0.1, 0.5, 1 and 2.5mg/mL), essential oil (SHEO)(0.05, 0.1, 0.5, 1 and 2.5µL/mL), H2O2 (50, 100 and 200µM), a combination of H2O2 (200 M) with either SHE (1, 2.5mg/mL) or SHEO (1, 2.5µL/mL) at 4 oC for 30min, and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Treatment of rat lymphocytes with SHE or SHEO resulted in significant reduction of H2O2-induced DNA damage compared to controls. SHE exhibited a significant (P<0.01) inhibitory effect on oxidative DNA damage at 2.5mg/mL. SHEO (1 and 2.5µL/mL) also showed significant inhibitory effects (P<0.01) on H2O2 induced chromosomal damage. In conclusion both the ethanolic extract and the essential oil of the plant were able to reverse the oxidative damage on rat lymphocytes induced by hydrogen peroxide.

Antigenotoxic effects ofSatureja hortensis L. on rat lymphocytes exposed to oxidative stress

The protective properties of Satureja hortensis L. on the rat lymphocytes DNA lesions were tested. Lymphocytes were isolated from blood samples taken from healthy rats. DNA breaks and resistance to H2O2-induced damage were measured with the comet assay. Rat lymphocytes were incubated in S. hortensis ethanolic extract (SHE) (0.05, 0.1, 0.5, 1.0, and 2.5 mg/ mL), essential oil (SHEO)(0.05, 0.1, 0.5, 1.0, and 2.5 microL/mL), H2O2 (50, 100, and 200 microM), a combination of H2O2 (200 mM) with either SHE (1.0, 2.5 mg/mL) or SHEO (1.0, 2.5 microL/mL) at 4 degrees C for 30 min, and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Treatment of rat lymphocytes with SHE or SHEO resulted in significant reduction of H2O2-induced DNA damage compared to controls. SHE exhibited a significant (P < 0.01) inhibitory effect on oxidative DNA damage at 2.5 mg/mL. SHEO (1.0 and 2.5 microL/mL) also showed significant inhibitory effects (P < 0.01) on H2O2 induced chromosomal damage. In conclusion both the ethanolic extract and the essential oil of the plant reversed the oxidative damage to rat lymphocytes induced by hydrogen peroxide.

Investigation into possible DNA damaging effects of ultrasound in occupationally exposed medical personnel — the alkaline comet assay study

In the present paper the possible DNA damaging effects of ultrasound in occupationally exposed medical personnel were investigated using the alkaline comet assay. The extent of DNA migration in peripheral blood leucocytes was measured. Parameters of the comet assay were studied in 30 medical workers occupationally exposed to ultrasound and in 30 corresponding unexposed control subjects. It was found that the subjects who were occupationally exposed to ultrasound for various periods of time showed a highly significant increase in levels of DNA damage compared with the control. The results obtained have confirmed the usefulness of the alkaline comet assay as a sensitive biodosimetric method, reflecting the current level of DNA damage and/or repair in peripheral blood leucocytes of ultrasound-exposed subjects. In spite of their limitations, the results of the present investigation indicate that individuals occupationally exposed to ultrasound may experience an increased genotoxic risk, emphasizing the need for more research into the nature and extent of the biological consequences to medical personnel working with ultrasonic equipment.

DNA lesions and cytogenetic changes induced by N-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E

Introduction: N -Nitrosomorpholine (NMOR), present in the workplace of tyre chemical factories, is a known hepatocarcinogen. This compound belongs to the group of N -nitrosamines, which are indirect-acting and require metabolic activation. However, the mechanism of its carcinogenic effect is not completely clear. Aims: The objective of this study was (i) to compare the DNA-damaging and clastogenic effects of NMOR in three cell lines (HepG2, V79 and VH10) with different levels of metabolizing enzymes and (ii) to determine the protective effects of Vitamins A, C and E against deleterious effects of NMOR. Methods: The exponentially growing cells were pre-treated with Vitamins A, C and E and treated with NMOR. Genotoxic effects of NMOR were evaluated by single-cell gel electrophoresis (SCGE, comet assay), while the chromosomal aberration assay was used for the study of clastogenic effects. Key results: NMOR-induced a significant dose-dependent increase of DNA damage as analyzed by SCGE, but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by SCGE confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pre-treated with Vitamins A, C and E we found a significant decrease of the percentage of tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E; Vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. Conclusions: The fat-soluble Vitamins A and E, which are dietary constituents, reduce the harmful effects of N -nitrosomorpholine in human hepatoma cells HepG2, which are endowed with the maximal capacity for metabolic activation of several drugs.

DNA lesions and cytogenetic changes induced by N-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E

Introduction: N-Nitrosomorpholine (NMOR), present in the workplace of tyre chemical factories, is a known hepatocarcinogen. This compound belongs to the group of N-nitrosamines, which are indirect-acting and require metabolic activation. However, the mechanism of its carcinogenic effect is not completely clear. Aims: The objective of this study was (i) to compare the DNA-damaging and clastogenic effects of NMOR in three cell lines (HepG2, V79 and VH10) with different levels of metabolizing enzymes and (ii) to determine the protective effects of Vitamins A, C and E against deleterious effects of NMOR. Methods: The exponentially growing cells were pre-treated with Vitamins A, C and E and treated with NMOR. Genotoxic effects of NMOR were evaluated by single-cell gel electrophoresis (SCGE, comet assay), while the chromosomal aberration assay was used for the study of clastogenic effects. Key results: NMOR-induced a significant dose-dependent increase of DNA damage as analyzed by SCGE, but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by SCGE confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pre-treated with Vitamins A, C and E we found a significant decrease of the percentage of tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E; Vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. Conclusions: The fat-soluble Vitamins A and E, which are dietary constituents, reduce the harmful effects of N-nitrosomorpholine in human hepatoma cells HepG2, which are endowed with the maximal capacity for metabolic activation of several drugs.

Evaluation of DNA Damage in Lymphocytes of Cardiologists Exposed to Radiation During Cardiac Catheterization by the COMET ASSAY

This study was designed to evaluate the possible in vivo induction of DNA damage by exposure to radiation in cardiologists. The alkaline comet assay (single cell gel electrophoresis, SCGE), which appears to be a promising tool with which to estimate DNA damage at the single cell level, has been used. The assay was carried out on 30 cardiologists currently employed in a busy cardiac service and 30 healthy unexposed controls. Venous blood samples were obtained from the exposed and control subjects and SCGE was examined in 100 cells graded as undamaged, intermediate, and tailed nuclei. The number of undamaged nuclei was almost the same in control and exposed subjects. The extent of DNA migration (SCGE assay) did not distinguish between the samples in either the nonsmoker exposed or nonsmoker control subjects, which leads one to wonder whether a difference in DNA damage really exists. Previous studies reported increased DNA damage in blood lymphocytes of smokers. In our study, the percentage of damaged cells increased either with the frequency of smoking or exposure to radiation. A statistically significant difference was observed both in smokers and exposed subjects. In conclusion, the elevated grade of DNA damage in cardiologists exposed to radiation indicates a possible genotoxic hazard, therefore, careful measures and full cooperation between cardiologists and radiologists should be undertaken to reduce the exposure to radiation.

Prevention of heterocyclic amine-induced DNA damage in colon and liver of rats by different lactobacillus strains

The aim of the present study was to investigate the impact of four different lactobacillus (LB) strains, namely Lactobacillus bulgaricus 291, Streptococcus thermophilus F4, S.thermophilus V3 and Bifidobacterium longum BB536, which are used for the production of yogurt, on the DNA-damaging effects of heterocyclic aromatic amines (HCAs). Male F344 rats were treated orally with HCA mixtures containing 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline, 2-amino-9H-pyrido[2,3-b]indole and 2-amino-3-methyl-3H- imidazo[4,5-f]quinoline, which were representative of the HCA contents found in fried beef ('beef mix') and chicken ('chicken mix'). Suspensions of LB were given by gavage to the animals simultaneously with and at different time periods before administration of the HCAs. Subsequently, the extent of DNA migration was measured in colon and liver cells in single cell gel electrophoresis (SCGE) assays. All four strains caused complete inhibition of DNA damage induced with beef mix after administration of 1 x 1010 LB cells/animal, whereas with chicken mix only marginal (non-significant) effects were seen. The inhibition of beef-induced DNA damage was dose dependent and was still significant when 1 x 107 cells/animal were administered. Kinetics studies showed that the protective effects were still significant when LB was given 12 h before the beef mix. A comparison of the present results with chemical analytical data from in vitro experiments suggests that the strong reduction in DNA migration seen in the animals can be only partly explained by direct binding effects. The results of the present study show that LB are highly protective against the genotoxic effects of HCAs under conditions which are relevant for humans and provide a possible explanation for the reduced colon cancer rates observed in some studies in individuals with either high LB counts in their feces or with a high consumption of LB-containing foods.

Genotoxicity of Fecal Water in a Free-Living Irish Population

The alkaline single-cell gel electrophoresis (comet) assay was used to investigate the genotoxicity of fecal water (FW) isolated from 47 Irish subjects using Caco-2 colonocytes as target cells. Two methods of comet assay analysis were compared to determine the extent of DNA damage and to categorize the samples as having no, low-to-moderate, or high genotoxicity. FW was isolated from stool samples by centrifugation and tested for its ability to induce DNA damage in Caco-2 cells. DNA damage was assessed using the comet assay by measuring the extent of DNA migration from the nucleus (μm, tail length) or by classifying the nuclei into five different categories depending on their morphology. Data collected from the two methods were used to categorize the FW samples on the basis of their genotoxic activity. Both methods showed good agreement. There was an approximately 50:50 split, with half the samples having some level of genotoxic activity and half having no genotoxicity. About one-third of the samples were considered to be highly genotoxic. There was a trend for low pH of the FW to be associated with increased DNA damage, but this was not significant. The results presented in this report show a relatively high incidence of genotoxic FW in samples derived from a free-living Irish population. Our data demonstrate the suitability of classifying nuclei on the basis of their morphology as a means of determining DNA damage. This procedure is very rapid and, therefore, advantageous in analyzing a large number of slides in the absence of an image analysis system.

DNA damage by cadmium and arsenic salts assessed by the single cell gel electrophoresis assay

Human exposure to metals is frequent due to their ubiquity, wide use in industry, and environmental persistence. Direct and indirect genotoxic effects of cadmium (Cd) and arsenic (As) were reported. However, the mechanisms of induction of genetic damage are not well known. The aim of the present work was to evaluate the degree of damage induced by Cd and As salts in a human lung fibroblasts cell line using the single cell gel electrophoresis assay (SCGE). MRC-5 cells were treated with cadmium chloride (CdCl 2), cadmium sulfate (CdSO 4), sodium arsenite (NaAsO 2) and cacodylic acid (C 2H 7AsO 2). A significant dose-dependent increment in the extent of DNA migration as well as in the percentage of cells with tails was observed ( P<0.001) after treatment with CdSO 4 and NaAsO 2. Treatment with CdCl 2 induced a relatively low level of DNA strand breaks in comparison with that induced by CdSO 4. The increase migration observed with the three compounds could be originated either by the direct induction of DNA lesions or by the inhibition of excision repair mechanisms. On the other hand, cells treated with C 2H 7AsO 2 showed a decrease in the migration length with the three doses employed ( P<0.001). The decrease in the rate of DNA migration could be a consequence of the induction of DNA cross-links by organic arsenicals. Cd and As salts induced DNA damage in fibroblast cells, detected as DNA migration in the single cell gel (SCG) assay. The distribution of DNA migration among cells as a function of dose revealed that the majority of exposed cells showed more DNA damage than cells obtained from control cultures. The potential for human exposure to both metals has been increased over the years due to the increment in their use. For this reason, elucidation of carcinogenic mechanisms is very important.

Mutagen sensitivity of nasopharyngeal cancer patients

Primary nasopharyngeal carcinomas (NPCs) may be of various types, including squamous cell carcinomas, undifferentiated carcinomas, and lymphoepitheliomas. Tumor initiation has been linked to the Epstein-Barr virus and, in some geographical regions, to alimentary factors. Possible hereditary components for the appearance of NPCs have not yet been clearly identified. In this study, genetic sensitivity to the genotoxic effects of carcinogenic xenobiotics as an endogenous risk factor of tumor initiation was investigated. The single cell microgel electrophoresis assay was used to quantify chemically-induced DNA damage in lymphocytes of 30 NPC patients and 30 non-tumor donors. The xenobiotics investigated were N′-nitrosodiethylamine, sodium dichromate, and nickel sulphate, with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) as positive and negative controls, respectively. The extent of DNA migration in the solvent control cultures was not significantly different between the two groups (1.2±0.5 mean Olive tail moment and standard deviation of 30 individuals for NPC patients; 1.1±0.4 for non-tumor donors). With constant exposure and electrophoretic conditions, genotoxic effects of varying degrees were induced by the different xenobiotics in tumor and non-tumor patients (nickel sulphate: 7.1±2.5 for NPC patients and 5.9±1.6 for non-tumor donors; sodium dichromate: 18.1±5.3 for NPC patients and 16.2±5.4 for non-tumor donors; MNNG: 47.8±13.3 for NPC patients and 52.7±13.6 for non-tumor donors). Only N′-nitrosodiethylamine proved to induce significantly more DNA migration in lymphocytes of tumor patients (9.8±3.1) as compared to non-tumor patients (8.2±2.3). Although for sodium dichromate the degree of DNA migration did not significantly differ, variability in migration patterns proved to be lower in the tumor group. Mutagen sensitivity of NPC patients was shown to be elevated for a selected xenobiotic, whereas a general elevation of DNA fragility was not present. Further studies on mutagen sensitivity as an endogenous risk factor influencing the susceptibility of patients at the time of first diagnosis of nasopharyngeal carcinomas are warranted.