Single cell gel electrophoresis assay: methodology and applications

The single cell gel electrophoresis or Comet assay is a sensitive, reliable, and rapid method for DNA double- and single-strand breaks, alkali-labile sites and delayed repair site detection, in eukariotic individual cells. Given its overall characteristics, this method has been widely used over the past few years in several different areas. In this paper we review the studies published to date about the principles, the basic methodology with currently used variations. We also explore the applications of this assay in: genotoxicology, clinical area, DNA repair studies, environmental biomonitoring and human monitoring.     

Single cell gel electrophoresis assay: a new technique for human biomonitoring studies

Human biomonitoring using the single cell gel electrophoresis (SCGE) or comet assay is a novel approach for the assessment of genetic damage in exposed populations. This assay enables the detection of various forms of DNA damage in individual cells with ease and speed and is, therefore, well suited to the analysis of a large group in a population. Here, application of SCGE assay in the identification of dietary protective factors, in clinical studies and in monitoring the risk of DNA damage resulting from occupational, environmental or lifestyle exposures is reviewed. Also, the comparative sensitivity of SCGE assay and conventional cytogenetic tests to detect genetic damage is discussed. Finally, strengths and shortcomings of the SCGE assay are addressed.     

Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay

The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76+/-1.21 (arbitrary units) for males as compared to 3.37+/-1.47 for females (P<0.05)], tail DNA (%) [10.2+/-2.96 for males as compared to 9.40+/-2.83 for females (P<0.05)] and tail length (microm) [59.65+/-9.23 for males and 49.57+/-14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.     

Germ cell and dose-dependent DNA damage measured by the comet assay in murine spermatozoaa after testicular X-irradiation

The single-cell gel electrophoresis (Comet) assay has been widely used to measure DNA damage in human sperm in a variety of physiological and pathological conditions. We investigated the effects of in vivo radiation, a known genotoxin, on spermatogenic cells of the mouse testis and examined sperm collected from the vas deferens using the neutral Comet assay. Irradiation of differentiating spermatogonia with 0.25-4 Gy X-rays produced a dose-related increase in DNA damage in sperm collected 45 days later. Increases were found when measuring Comet tail length and percentage of tail DNA, but the greatest changes were in tail moment (a product of tail length and tail DNA). Spermatids, spermatocytes, differentiating spermatogonia, and stem cell spermatogonia were also irradiated in vivo with 4 Gy X-rays. DNA damage was indirectly deduced to occur at all stages. The maximum increase was seen in differentiating spermatogonia. DNA damaged cells were, surprisingly, still detected 120 days after stem cell spermatogonia had been irradiated. The distribution of DNA damage among individual sperm cells after irradiation was heterogeneous. This was seen most clearly when changes in the Comet tail length were measured when there were discrete undamaged and damaged populations. After increasing doses of irradiation, an increasing proportion of cells were found in the damaged population. Because a proportion of undamaged sperm cells remains after all but the highest dose, the possibility of normal fertility remains. However, fertilization with a spermatozoa carrying high amounts of DNA damage could lead to effects as diverse as embryonic death and cancer susceptibility in the offspring.     

Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay

The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76±1.21 (arbitrary units) for males as compared to 3.37±1.47 for females (P<0.05)], tail DNA (%) [10.2±2.96 for males as compared to 9.40±2.83 for females (P<0.05)] and tail length (μm) [59.65±9.23 for males and 49.57±14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.     

Association of GSTM1 and GSTT1 with ageing in auto repair shop workers

We evaluated the possible influence of glutathione S-transferase mu (GSTM1) and glutathione S-transferase theta (GSTT1) genes on genetic damage due to occupational exposure, which contributes to accelerate ageing. This study was conducted on 120 car auto repair workshop workers exposed to occupational hazards and 120 controls without this kind of exposure. The null and non-null genotypes of GSTM1 and GSTT1 genes were determined by multiplex PCR. Micronucleus frequency, Comet tail length and relative telomere length differences between the null and non-null genotypes of the GSTM1 gene were significantly greater in the exposed group. Lack of GSTT1 did not affect the damage biomarkers significantly (P > 0.05), while lack of GSTM1 was associated with greater susceptibility to genomic damage due to occupational exposure. It was concluded that early ageing is under the influence of these genes and the environmental and socio-demographic factors. Duration of working time was significantly associated with micronucleus frequency, Comet tail length and relative telomere length.     

Evaluation of DNA damage in flight personnel by Comet assay

There have been some suggestions that air-crew are at a higher-than-normal risk of developing cancer, since they are exposed to potential genotoxic factors. These include cosmic radiations, airborne pollutants such as the combustion products of jet propulsion, ozone, and electromagnetic fields. We used the Comet assay to investigate DNA damage in flight personnel with the aim of assessing potential health hazards in this occupational category. We studied 40 civil air-crew members who had been flying long-haul routes for at least 5 years, and compared them with a homogeneous control group of 40 healthy male ground staff. The Comet assay, or single-cell gel electrophoresis (SCGE), detects DNA single- and double-strand breaks (DSBs) and alkali-labile lesions in individual cells, and is a powerful and sensitive technique for detecting genetic damage induced by different genotoxic agents. Taking into consideration occupational risk and possible confounding factors, this assay showed a small increase, that did not reach statistical significance, of DNA damage in long-haul crew members compared to controls, indicating a lack of evident genotoxic effects. An association, although again not statistically significant, was found between reduced DNA damage and use of protective drugs (antioxidants).     

Proteomic analysis of plasma proteins of workers exposed to benzene

In this study, we analyzed the proteins in plasma of workers exposed to benzene by two-dimensional gel electrophoresis, in the hope of finding a specific protein suitable for the biomonitoring of benzene exposure. Comet assays were also carried out to evaluate lymphocytes DNA damage. Fifty workers from a printing company and 38 matched unexposed healthy subjects were enrolled in the study. DNA damage was found to be significantly higher in the exposed workers than in the controls. The tail moments of the two groups were 2.07 +/- 0.35 and 1.48 +/- 0.41, respectively (P < 0.0001). The mean values of trans, trans-muconic acid (t,t-MA) in workers exposed to benzene and in unexposed subjects were 1.011 +/- 0.249 and 0.026 +/- 0.028 mg/g creatinine, respectively. Protein profiles were significantly different (P < 0.05) in the two groups, as identified by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry and confirmed by Western blot. T cell receptor beta chain (TCR beta), FK506-binding protein (FKBP51) and matrix metalloproteinase-13 (MMP13) were found to be up-regulated in the benzene-exposed workers. In addition, the correlation between TCR beta and the tail moments of lymphocytes was statistically significant (r-value, 0.428). We conclude that TCR beta in plasma could be used for the early detection of exposure to benzene.     

Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females

The single-cell gel electrophoresis or Comet assay measures qualitative and quantitative DNA damage in single cells. Its simplicity and non-invasive nature has made it widely accepted for the monitoring of human genotoxicity, employing peripheral blood lymphocytes. Factors, such as gender, age, and dietary and smoking habits are known to affect the Comet assay responses in lymphocytes. However, there is no information regarding the influence of the menstrual cycle on the results of the assay in lymphocytes of females. A study was therefore undertaken among 18 healthy Indian female volunteers to assess the effect of the menstrual cycle on Comet assay responses. During a complete menstrual cycle, only minor changes were observed in the basal levels of DNA damage in the lymphocytes as evident by Comet parameters, such as tail length (microm), tail DNA (%) and Olive tail moment (arbitrary units). To assess the effect of the estrogen 17beta-estradiol (at physiological concentrations of 0.5, 1.0 and 2.0 nM) on the Comet assay responses, an in vitro study was conducted in the human lymphocyte cell line JM-1 and the breast cancer cell line MCF-7. As was evident from the Comet parameters, a significant (p < 0.01) concentration-dependent increase in the level of DNA damage was observed in the MCF-7 cells while no significant change was found in the JM-1 cells. The results indicate that the menstrual cycle does not influence the Comet assay responses in lymphocytes; hence, these can serve as a model for monitoring genotoxicity in females.     

Evaluation of DNA damage by the Comet assay in shoe workers exposed to toluene and other organic solvents

The alkaline single-cell gel electrophoresis (or Comet) assay was applied to evaluate DNA damage in cryopreserved peripheral blood mononuclear leukocytes from 34 female shoe workers exposed to organic solvents and a group of 19 non-exposed women. We also investigated whether the polymorphisms of glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genes affect individual level of DNA damage possibly induced by the solvent exposure. Chemical measurements of workplace air in the two factories studied showed that the workers were exposed to acetone, gasoline, and toluene in both factories and to ethylacetate and diisocyanate in one factory. In the exposed workers, the average level of blood hemoglobin was lower and that of urinary hippuric acid higher than in the non-exposed individuals. However, the occupational exposure to organic solvents did not affect the Comet values. Neither did age, smoking, or the GSTM1 genotype have any effect on the outcome of this assay. The low prevalence of the GSTT1-null genotype precluded conclusions on the influence of GSTT1 polymorphism.     

Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females

The single-cell gel electrophoresis or Comet assay measures qualitative and quantitative DNA damage in single cells. Its simplicity and non-invasive nature has made it widely accepted for the monitoring of human genotoxicity, employing peripheral blood lymphocytes. Factors, such as gender, age, and dietary and smoking habits are known to affect the Comet assay responses in lymphocytes. However, there is no information regarding the influence of the menstrual cycle on the results of the assay in lymphocytes of females.A study was therefore undertaken among 18 healthy Indian female volunteers to assess the effect of the menstrual cycle on Comet assay responses. During a complete menstrual cycle, only minor changes were observed in the basal levels of DNA damage in the lymphocytes as evident by Comet parameters, such as tail length (μm), tail DNA (%) and Olive tail moment (arbitrary units).To assess the effect of the estrogen 17β-estradiol (at physiological concentrations of 0.5, 1.0 and 2.0 nM) on the Comet assay responses, an in vitro study was conducted in the human lymphocyte cell line JM-1 and the breast cancer cell line MCF-7. As was evident from the Comet parameters, a significant (p < 0.01) concentration-dependent increase in the level of DNA damage was observed in the MCF-7 cells while no significant change was found in the JM-1 cells.The results indicate that the menstrual cycle does not influence the Comet assay responses in lymphocytes; hence, these can serve as a model for monitoring genotoxicity in females.     

Environmental genotoxicity assessment of an urban stream using freshwater planarians

Pollution is a major concern in urban areas. Due to its biological significance, genotoxicity should be a main focus for pollution biomonitoring, due mainly to the increasing complexity of the chemical environment in which organisms are exposed. Diluvio's Basin (Porto Alegre, RS, Brazil) is a heavily polluted urban ecosystem impacted by urban wastewater. Planarians are useful organism for evaluating environmental genotoxicity because of their high sensitivity, low cost, high proliferative rate and also because of their basal evolutionary position in relation to complex metazoans. Comet assay is a powerful and highly sensitive method of evaluating primary DNA lesions. Based on the unique features of planarians and the current environmental state of Diluvio's Basin, the aim of this work was to evaluate the genotoxic potential of this body of water using comet assay in planarians. Planarians were exposed to the water for 13 days in a laboratory and comet assay was performed in order to screen possible DNA damages. The results indicated an increasing gradient of damage towards basin's mouth. Such a gradient could be related to the gradual increase of pollutants among the different sample sites. Moreover, there seems to be a correlation between the urbanization gradient that exists within the watershed and the genotoxicity. Historical physical-chemical data was also gathered and examined for possible correlations with genotoxicity. Comet assay in planarians is a very promising test for environmental monitoring studies. Its application should be expanded.     

Evaluation of DNA damage in exfoliated tear duct epithelial cells from individuals exposed to air pollution assessed by single cell gel electrophoresis assay

The search for relevant target cells for human monitoring purposes has increased during the last few years. Cells such as sperm, buccal or nasal and gastric epithelium are being used. In this study, we report the use of exfoliated tear duct epithelial cells as a potential material for human biomonitoring studies, since these cells are a target for environmental pollutants. We employed the alkaline single cell gel electrophoresis (SCGE) assay to evaluate for differences in the basal level of DNA damage between young adults from the south (exposed mainly to high levels of ozone) and from the north (exposed principally to hydrocarbons) regions of Mexico City. We found an increase in DNA migration in tear duct epithelial cells from individuals who live in the southern part of the city compared to those living in the northern part. Moreover, young people who live in the southwest part of the city with the highest values of ozone presented the highest values of DNA damage. These results show the feasibility of using exfoliated tear duct epithelial cells in human biomonitoring studies.     

Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals

The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements.     

Children's exposure to environmental pollutants and biomarkers of genetic damage. II. Results of a comprehensive literature search and meta-analysis

The present review is based on findings from 178 publications retrieved through an extensive search of the MedLine/PubMed database for a 25 years time period (1980-2004) and 10 manually identified papers. Among the cytogenetic biomarkers that are frequently used in field studies, chromosome aberrations (CA) and micronuclei (MN) but not sister chromatid exchanges (SCE) were found consistently increased in children exposed to environmental pollutants. Meta-analysis of the studies reporting SCE in cord blood showed similar levels of SCE in exposed and in non-exposed newborns. Exposure to airborne pollutants, soil and drinking water contaminants, mostly increased CA and, to a lesser extent, MN levels in children. The effect of exposure to airborne urban pollutants was consistently reported by field studies measuring DNA, albumin and hemoglobin adducts. Prenatal (in utero) and postnatal exposure (environmental tobacco smoke, ETS) to tobacco smoke compounds were associated with increased frequencies of DNA and hemoglobin adducts and CA. The limited number of field studies measuring DNA fragmentation (Comet assay), hypoxanthine-guanine phosphoribosyltransferase (HPRT) and the glycophorinA (GPA) mutation frequency in environmentally exposed children precluded a meaningful evaluation of the usefulness of these assays. Meta-analyses performed in children exposed to ETS and in newborns exposed in utero to their mothers' smoke showed 1.3 and 7 times higher levels of hemoglobin adducts compared to referent subjects, respectively. These increases are consistent with the epidemiological evidence of higher lung cancer risks reported in adults who had never smoked and were exposed to ETS during childhood and with 7-15 times higher lung cancer risks reported in smokers than in non-smokers. Higher levels of PAH-DNA adducts were found in fetal than in maternal tissue, suggesting a specific susceptibility of the fetus to this class of ubiquitous environmental pollutants. According to these findings, future research and biomonitoring programs on children would greatly benefit from the inclusion of selected biomarkers that could provide biologically based evidence for the identification of intervention priorities in environmental health.     

Exposure to environmental polycyclic aromatic hydrocarbons: influences on cellular susceptibility to DNA damage (sampling Kosice and Sofia)

The aim of this study was to investigate a possible influence of occupational exposure to carcinogenic environmental polycyclic aromatic hydrocarbons (c-PAHs) on cellular susceptibility to the induction of the DNA damage. Monitoring was performed and blood samples were collected from two groups of male subjects: occupationally exposed and matched controls. The group exposed to c-PAHs (average age of 35.1 years) consisted of 52 policemen from Košice and 26 policemen and 25 bus drivers (51 altogether) from Sofia. The control group (average age of 36.4 years) consisted of 54 unexposed subjects from Košice and 24 from Sofia. In the investigated groups 52.5% of exposed subjects and 45.3% of control were current smokers. A challenging dose of X-rays (3 Gy) and an alkaline version of the single cell gel electrophoresis (SCGE) assay, known as Comet assay, were used to evaluate levels of induced DNA damage and repair kinetics in isolated human blood lymphocytes. DNA damage detected in lymphocytes prior to or after irradiation did not differ significantly between exposed and unexposed subjects. A significant decrease in repair efficiency due to exposure to PAHs was observed in the exposed individuals from Košice and Sofia, when analysed separately or together. A negative influence of tobacco smoking on the efficiency of DNA repair was observed. Statistically significant differences were found between subgroups stratified according to education level in Sofia: the half times for DNA repair declined with the increasing level of education. These results confirm that environmental exposure to c-PAHs can alter the ability of blood lymphocytes to repair DNA damage and, as a result could potentially lead to effects that are hazardous to human health.     

Response to challenging dose of X-rays as a predictive assay for molecular epidemiology

Human biomonitoring, as a tool to identify health risk from environmental exposures, has gained increasing interest especially in the areas of cancer risk assessment and response to therapy. Chromosome aberrations resulting from direct DNA breakage or from inhibition of DNA repair or synthesis, as measured in peripheral blood lymphocytes, have been used successfully in the assessment of environmental health. Susceptibility to the induction of genotoxicity has been evaluated by the use of an in vitro challenge dose of UV or X-rays. In this report, DNA damage was analyzed with the use of single cell gel electrophoresis (SCGE) assay in healthy donors and cancer patients. Studies have shown a good correlation between DNA damage induced in vivo or in vitro and cytogenetic measures. Results from studies on susceptibilities and repair competence in 475 controls, exposed workers and cancer patients are discussed. The possible effects of exposures and influence of the diet and other confounding factors are shown. The prospective use of a challenging dose of radiation combined with the SCGE assay as a predictive assay is suggested and the limitations are discussed.