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.     

Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay

The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.     

Impact of the comet assay in radiobiology

Until the development of single cell gel electrophoresis methods in the 1980s, measurement of radiation-induced DNA strand breaks in individual cells was limited to detection of micronuclei or chromosome breaks that measured the combined effects of exposure and repair. Development of methods to measure the extent of migration of DNA from single cells permitted detection of initial radiation-induced DNA breaks present in each cell. As cells need not be radiolabeled, there were new opportunities for analysis of radiation effects on cells from virtually any tissue, provided a single cell suspension could be prepared. The comet assay (as this method was subsequently named) was able to measure, for the first time, the fraction of radiobiologically hypoxic cells in mouse and human tumors. It was used to determine that the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine "true" DNA strand break rejoining rates. Other examples of applications of the comet assay in radiobiology research include analysis of the inter-individual differences in response to radiation, effect of hypoxia modifying agents on tumor hypoxic fraction, the role of cell cycle position during DNA break induction and rejoining, non-targeted effects on bystander cells, and effects of charged particles on DNA fragmentation patterns.     

Video enhanced laser scanning microscopic analysis of DNA comet formation.

Laser Scanning Microscopy is a sensitive tool that provides a unique method of analyzing biological systems. Coupled with the Single Cell Gel assay, it allows for accurate and reproducible detection of DNA strand breaks. An understanding of the theory of DNA comet formation is lacking. Using dexamethasone induced apoptosis in murine thymocytes as a model for double strand breaks, we used video enhanced laser scanning microscopy to evaluate the leading edge of DNA migration in the single cell gel assay. In this system, comet length increases significantly within the first thirty seconds of electrophoresis, the greatest increase in length is completed within the first minute, and the first two minutes are important in significant increases in DNA migration during DNA comet formation.     

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.     

Measurement of hypoxia using the comet assay correlates with preirradiation microelectrode pO2 histography in R3327-AT rodent tumors

Polarographic determination of tumor oxygenation by Eppendorf histography is currently under investigation as a possible predictor of radiotherapy outcome. Alternatively, the alkaline comet assay has been proposed as a radiobiological approach for the detection of hypoxia in clinical tumor samples. Direct comparisons of these methods are scarce. One earlier study with different murine tumors could not establish a correlation, whereas a weak correlation was reported for a variety of human tumors. Considering the different end points and spatial resolution of the two methods, a direct comparison for a single tumor entity appeared desirable. Anaplastic R3327-AT Dunning prostate tumors were grown on Copenhagen rats to volumes of 1-6 cm(3). Eppendorf histography (100-200 readings in 5 parallel tracks) for 8 different tumors revealed various degrees of oxygenation, with median pO(2) values ranging from 1.1 to 23 mmHg. Within 5 min after an acute exposure to 8 Gy (60)Co gamma rays, tumors were excised from killed animals and rapidly cooled to limit repair, and a single cell suspension was prepared for use with the comet assay. The resulting comet moment distributions did not exhibit two subpopulations (one hypoxic and the other aerobic), and a hypoxic fraction could not be calculated. Instead, the average comet moment distribution was taken as a parameter of overall strand break induction. Corresponding experiments with tumor cells grown in vitro allowed us to derive the relationship between the oxygen enhancement ratio (OER) for the average comet moment and oxygen partial pressure (Howard-Flanders and Alper formula). The validity of this relationship was inferred for cells exposed in situ, and the convolution of a pO(2) distribution with the formula of Howard-Flanders and Alper yielded an array of expected OER values for each tumor. The average expected OER correlated well with the average comet moment (r = 0.89, P < 0.01), and the in situ comet moment distributions could be predicted from the Eppendorf data when 50% repair was taken into account, assuming a 5-min damage half-life. The findings confirm the potential of interstitial polarography to reflect radiobiologically relevant intracellular oxygenation, but also underscore the confounding influence of differences in repair that may occur when cells are prepared from irradiated tissues for use with the comet assay.     

The Liverbeads as a tool for the comet assay

Liverbeads, cryopreserved hepatocytes entrapped within an alginate matrix, were examined for their relevance in the comet assay. It was estimated by their capacity to activate the indirectly acting mutagens, cyclophosphamide (CP), benzo[a]pyrene (BP), dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (2-AAF), into DNA reactive metabolites. The comet assay performed in alkaline condition is a sensitive method for detecting strand breaks at the level of individual cells and allows use of quiescent cells. Experimental conditions as treatment time, cell density, beads dissociation and viability were investigated. Significant statistical positive results assessed by the tail extent moment (TEM) were observed with both human and rat Liverbeads after 12h duration incubation compared to metabolic non-competent cells, HeLa S3. Due to the maintenance of specific functions assessed by the observed capacity to metabolize xenobiotics, Liverbeads represent a suitable tool system, easy to handle, for the detection of promutagens using the comet assay.     

Induction of DNA strand breaks by intermittent exposure to extremely-low-frequency electromagnetic fields in human diploid fibroblasts

Results of epidemiological research show low association of electromagnetic field (EMF) with increased risk of cancerous diseases and missing dose-effect relations. An important component in assessing potential cancer risk is knowledge concerning any genotoxic effects of extremely-low-frequency-EMF (ELF-EMF).Human diploid fibroblasts were exposed to continuous or intermittent ELF-EMF (50Hz, sinusoidal, 24h, 1000microT). For evaluation of genotoxic effects in form of DNA single- (SSB) and double-strand breaks (DSB), the alkaline and the neutral comet assay were used.In contrast to continuous ELF-EMF exposure, the application of intermittent fields reproducibly resulted in a significant increase of DNA strand break levels, mainly DSBs, as compared to non-exposed controls. The conditions of intermittence showed an impact on the induction of DNA strand breaks, producing the highest levels at 5min field-on/10min field-off. We also found individual differences in response to ELF-EMF as well as an evident exposure-response relationship between magnetic flux density and DNA migration in the comet assay.Our data strongly indicate a genotoxic potential of intermittent EMF. This points to the need of further studies in vivo and consideration about environmental threshold values for ELF exposure.     

单细胞凝胶电泳技术及在土壤生态毒理学中的应用

单细胞凝胶电泳技术又称为彗星实验,是最近几年发展起来的一种快速、简单、灵敏、可靠的检测细胞核DNA损伤的技术。总结了近几年来单细胞凝胶电泳技术的发展、原理、方法及其应用,并指出其下一步的发展趋势。彗星实验中,镶嵌于琼脂糖中的细胞核在电场中向正极移动,因细胞核与DNA片段迁移速率不同,而形成类似“彗星”的图像。目前采用的彗星实验有多种,可以检测诸如DNA双链断裂、单链断裂、碱不稳定位点等多种类型的DNA损伤。碱性彗星实验因其高灵敏度而被广泛采用。彗星实验的主要步骤包括细胞核悬浮液的获得、彗星电泳胶板制备、细胞裂解、DNA变性解旋、电泳、中和、染色和观察等。目前彗星实验广泛应用于各个研究领域,近年来开始用于环境污染的基因毒性研究和生物监测,并取得了迅速发展。     

The comet assay for DNA damage and repair

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

Increased levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or X-irradiation of spermatogonia.

To investigate whether DNA damage arising in spermatogenic germ cells can be detected in resultant sperm, we have irradiated murine testis and collected spermatozoa from the vas deferens 45 days later. These cells were derived from spermatogonia present at the time of irradiation. Two forms of irradiation were used, external X-rays (4Gy) and internal auger electrons from contamination of the male mouse with the isotope Indium-114m (1.85MBq), which was localised in the testis. Both forms of irradiation produced a profound fall in vas deferens sperm count and testis weight, Indium-114m being more effective. Using the neutral Comet assay for double strand break detection, significant increases in sperm comet tail length and moment were observed. The levels of damage were similar for both treatments. Care had to be taken during the assay to distinguish between sperm and somatic cells as the proportion of the latter increased after irradiation. We conclude that the comet assay can detect DNA damage in spermatozoa after the in vivo exposure of male germ cells to a known testicular genotoxic agent. The assay may be useful for the assessment of sperm DNA damage (double stranded) associated with male infertility and post-fertilization developmental abnormalities in the offspring.     

Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis.

BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during apoptosis in comparison with standard DNA flow cytometry analysis. METHODS: Staurosporine-induced apoptosis in CHO cells is an adequate model to study a rapid time- and dose-dependent appearance of this process. RESULTS: Nuclear staining with DAPI confirmed the induction of apoptosis with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.     

Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds

An in situ study of the relationship between marine contamination and genotoxic effects was performed on female dab (Limanda limanda) collected from different sites in the eastern English Channel (France) known to be contaminated by polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs). DNA adducts in liver and DNA strand breaks in blood cells were determined respectively by the nuclease P1-enhanced post-labelling technique and an alkaline version of the comet assay. The extent of DNA base oxidation was also assessed for three of the six sampling sites in the study, using a comet assay in combination with a specific DNA repair enzyme, formamidopyrimidine glycosylase (Fpg).With Comet data, two groups of sites that seem in accordance with the pollution level have been distinguished. The extent of DNA strand breaks was higher in adult than juvenile female dab. From a technical point of view, comet assay sensitivity was affected by high intra-individual variability that accounted for nearly 70% of total variance (the site factor represented no more than 26%). The combined use of the comet assay and Fpg showed the presence of DNA oxidised bases in environmentally exposed dab.Although qualitative differences between the sampling sites were observed in DNA adduct profiles, no significant differences were found for total DNA adduct levels. DNA adducts did not appear to be associated with PAH exposure.Histopathological studies showed hepatic steatosis in most of the animals examined. Only one pre-cancerous lesion (an early stage of hyperplasia) was detected (associated frequency of 0.8%).     

The comet assay: a method to measure DNA damage in individual cells

We present a procedure for the comet assay, a gel electrophoresis-based method that can be used to measure DNA damage in individual eukaryotic cells. It is versatile, relatively simple to perform and sensitive. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-links, base damage and apoptotic nuclei. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell. DNA damage and its repair in single-cell suspensions prepared from yeast, protozoa, plants, invertebrates and mammals can also be studied using this assay. Originally developed to measure variation in DNA damage and repair capacity within a population of mammalian cells, applications of the comet assay now range from human and sentinel animal biomonitoring (e.g., DNA damage in earthworms crawling through toxic waste sites) to measurement of DNA damage in specific genomic sequences. This protocol can be completed in fewer than 24 h.     

Comet assay: rapid processing of multiple samples

The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets.     

Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 λunits/g liver at 37°C for 20 λmin) for isolation of intact cells for subsequent comet assay. We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation ( -85°C for 72 λh). To minimize this damage, minced liver (1.0 λg) and kidney (0.5 λg) tissues were added to 20 λml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO ( -85°C for 72 λh and 3 weeks), and to a slightly lesser extent with glycerol (72 λh), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150 λunits/g liver and 300 λunits/g kidney for 10 λmin produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at -85°C for 72 λh. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.     

Assessment of cytogenetic damage in lymphocytes and in exfoliated nasal cells of dental laboratory technicians exposed to chromium,cobalt, and nickel

The alkaline comet assay is able to identify in individual cells DNA strand breaks associated with different processes. Topoisomerase inhibitors, some of which are used as chemotherapeutic agents, stabilise topoisomerase-DNA cleavable complexes by stimulating DNA strand cleavage and inhibiting religation. This can result in the activation of stress-associated signalling pathways, inducing cell cycle arrest and activation of the biochemical cascade of apoptosis. The aim of our study was to assess the ability of the comet assay to detect stabilisation of cleavable complexes and induction of apoptosis by two topoisomerase II inhibitors, etoposide and ellipticine, and two topoisomerase I inhibitors, camptothecin and topotecan. The study was carried out on Chinese hamster ovary (CHO) cells, DC3F cells and DC3F/C-10, its camptothecin-resistant counterpart. The comet assay was able to identify stabilised cleavable complexes through the presence of DNA strand breaks after 1h treatment that disappeared within 24h after drug removal. Kinetics studies allowed to discriminate between these early DNA damages and DNA fragmentation related to apoptosis characterised by reappearance of DNA strand breaks 48h after treatment.     

Comet assay with nuclear extract incubation

Alkaline comet assay is a simple sensitive method for detecting DNA strand breaks. However, at the time of cell lysis, only a fraction of the entire DNA damage appears as DNA strand breaks, while some DNA strand breaks may have been rejoined and some DNA lesions may still remain unexcised. We showed that nuclear extract (NE) prepared from human cells could excise the DNA adducts induced by UVC, X-ray, and methyl methanesulfonate (MMS). Thus, the comet assay with NE incubation allows a closer estimation of total DNA damage. Among the human urothelial carcinoma cell lines we tested, the NE of NTUB1 cells showed higher activity in excising the DNA adducts induced by UVC, but with a lower activity in excising the DNA adducts induced by MMS than the NE of BFTC905 cells. Moreover, under the same dose of X-ray irradiation, a larger difference in total DNA damage between two cell lines was revealed in comet assay incubated with NE than without NE. Therefore, the comet assay with NE incubation may be useful in the research of cancer risk, drug resistance, and DNA repair proteins.     

Ultrasensitive detection of hydrogen peroxide-mediated DNA damage after alkaline single cell gel electrophoresis using occultation microscopy and TUNEL labeling.

DNA damage at the level of individual cells can be detected using the single cell gel electrophoresis (SCGE) or 'comet' assay. In the present study, we report novel variations on the conventional comet assay that can be used to enhance the microscopic detection of DNA damage. Hydrogen peroxide-treated peripheral blood leukocytes were used as a DNA damage model system. Cells were embedded in agarose, treated, and electrophoresed according to the procedure of Singh et al. [N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988), p. 184-191]. However, sites of strand breaks were directly labeled with the TUNEL (TdT-mediated fluorescein-dUTP nick end labeling) method. This labeling protocol revealed clumps and/or a series of stripes in the comet tail perpendicular to the direction of electrophoresis; these sites may account for the substructure seen in conventional comet assays. In a second comet variation, we passed an opaque disk into a field-conjugated plane of the microscope near the lamp, thus occluding the nucleus' image. Nuclear occultation allows the intensified charge-coupled device (ICCD) camera gain to increase to a single photon detection level thus revealing low levels of DNA damage in the tail. These methods offer a substantial improvement in sensitivity.     

Rejoining of DNA strand breaks by T4 DNA ligase in mammalian cells

We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks.