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.     

Could a strong alkali deproteinization replace the standard lysis step in alkaline single cell gel electrophoresis (comet) assay (pH > 13)?

The alkaline version of single cell gel electrophoresis (comet) assay is widely used for evaluating DNA damage at the individual cell level. The standard alkaline method of the comet assay involves deproteinization of cells embedded in agarose gel using a high salt–detergent lysis buffer, followed by denaturation of DNA and electrophoresis using a strong alkali at pH > 13 [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) 184–191]. However, a recent report showed that a strong alkali treatment results in simultaneous deproteinization of cells and denaturation of genomic DNA [P. Sestili, C. Martinelli, V. Stocchi, The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single cell-level, Mutat. Res. 607 (2006) 205–214]. This study was carried out to test whether the strong alkali deproteinization of cells could replace the high salt–detergent lysis step used in the standard method of the alkaline comet assay. Peripheral blood lymphocytes from 3 healthy individuals were irradiated with gamma rays at doses varying between 0 and 10 Gy. Following irradiation, the comet assay was performed according to the standard alkaline method (pH > 13) and a modified method. In the modified method, agarose embedded cells were treated with a strong alkali (0.3 M NaOH, 0.02 M Trizma and 1 mM EDTA, pH > 13) for 20 min to allow deproteinization of cells and denaturation of DNA. This was followed by electrophoresis using the same alkali solution to obtain comets. DNA damage expressed in terms of comet tail length, percentage of DNA in comet tail and tail moment obtained by the standard alkaline method and the modified method were compared. In both methods, DNA damage showed a good correlation with the dose of gamma ray. The results indicate a satisfactory sensitivity of the modified method in detecting radiation-induced DNA damage in human peripheral blood lymphocytes.     

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.     

Single strand DNA breaks in rat brain cells exposed to microwave radiation

This investigation concerns with the effect of low intensity microwave (2.45 and 16.5 GHz, SAR 1.0 and 2.01 W/kg, respectively) radiation on developing rat brain. Wistar rats (35 days old, male, six rats in each group) were selected for this study. These animals were exposed for 35 days at the above mentioned frequencies separately in two different exposure systems. After the exposure period, the rats were sacrificed and the whole brain tissue was dissected and used for study of single strand DNA breaks by micro gel electrophoresis (comet assay). Single strand DNA breaks were measured as tail length of comet. Fifty cells from each slide and two slides per animal were observed. One-way ANOVA method was adopted for statistical analysis. This study shows that the chronic exposure to these radiations cause statistically significant (p<0.001) increase in DNA single strand breaks in brain cells of rat.     

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.     

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.     

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.     

DBD-fish on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells

Humanblood leukocytes exposed to X-rays were immersed in an agarose microgel on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characteristics of DNA migration (i.e., area of the comet) are related to the DNA double-strand breaks (dsbs) yield. After electrophoresis, comets were briefly incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. These motifs behave as target sites for hybridization with a whole genome probe, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is produced in the comet by the alkali and more DNA probe hybridizes, resulting in an increase in the mean fluorescence intensity. Since radiation-induced DNA single-strand breaks (ssbs) are far more frequent than dsbs, the mean fluorescence intensity of the DBD-FISH signal from the comet is related to the ssb level, whereas the surface area of the same comet signal is indicative of the dsb yield. Thus, both DNA break types may be simultaneously analyzed in the same cell. This was confirmed in a repair assay performing the DBD-FISH on neutral comets from a human cell line defective in the repair of dsbs. Otherwise, treatment with hydrogen peroxide, a main inducer of ssbs, increased the mean fluorescence intensity, but not the surface, of X-ray-exposed human leukocytes.     

Assessment of DNA sensitivity in peripheral blood leukocytes after occupational exposure to microwave radiation: the alkaline comet assay and chromatid breakage assay

DNA sensitivity in peripheral blood leukocytes of radar-facility workers daily exposed to microwave radiation and an unexposed control subjects was investigated. The study was carried out on clinically healthy male workers employed on radar equipment and antenna system service within a microwave field of 10 μW/cm²–20 mW/cm² with frequency range of 1,250–1,350 MHz. The control group consisted of subjects of similar age. The evaluation of DNA damage and sensitivity was performed using alkaline comet assay and chromatid breakage assay (bleomycin-sensitivity assay). The levels of DNA damage in exposed subjects determined by alkaline comet assay were increased compared to control group and showed inter-individual variations. After short exposure of cultured lymphocytes to bleomycin cells of subjects occupationally exposed to microwave (MW) radiation responded with high numbers of chromatid breaks. Almost three times higher number of bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes were determined in comparison with control group. The difference in break per cell (b/c) values recorded between smokers and non-smokers was statistically significant in the exposed group. Regression analyses showed significant positive correlation between the results obtained with two different methods. Considering the correlation coefficients, the number of metaphase with breaks was a better predictor of the comet assay parameters compared to b/c ratio. The best correlation was found between tail moment and number of chromatid with breaks. Our results indicate that MW radiation represents a potential DNA-damaging hazard using the alkaline comet assay and chromatid breakage assay as sensitive biomarkers of individual cancer susceptibility.     

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell ^1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.     

Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid

Cadmium (Cd) is one of the important pollutants of soil and the genotoxicity of Cd-contaminated soil was studied in combination with imidacloprid. The single cell gel electrophoresis or comet assay was used to quantify DNA strand breaks as a measure of DNA damage induced by Cd and imidacloprid contamination in soil. The soil was artificially contaminated by Cd 2 h at 25℃ and were used in the comet assay. DNA damage was measured as the values of percentage of nuclei with tails, tail length, tail DNA, tail moment (TM), and Olive tail moment (OTM). DNA damages of root tips of Vicia faba increased after Cd treatment and there were dose-related increases in DNA damage measured as these parameters. However, the addition of imidacloprid further increased the DNA damage. These data confirmed the genotoxic effect of Cd to plants, and that the combined pollution with imidacloprid can enhance the genotoxicity of Cd.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Tail profile: a more accurate system for analyzing DNA damage using the Comet assay

The Comet assay (single cell gel electrophoresis assay) measures DNA strand breaks in individual cells. In the assay cells are embedded in agarose, lysed, and electrophoresed under low voltage, allowing migration of damaged DNA. The DNA is stained and subsequently viewed with an epifluorescent microscope. If DNA damage has occurred the electrophoresed DNA fragments appear as a diffuse tail behind the nucleus known as a "comet". Many computer-aided analysis systems are currently in use to quantify the amount of DNA damage that is represented by a comet image. Here, we present a novel method of analysis known as "tail profile". This method of analysis provides several advantages over currently employed methods, which rely primarily on the "tail moment" method of analysis. We compared the amount of DNA damage reported from both the tail profile and tail moment methods of analysis and observed a 26% (P<0.0001) increase in damage detected by tail profile across the 10-25 microm range of tail length, where the majority of the relevant comet data is concentrated. We further report that this increase in sensitivity is not only limited to assessing DNA damage, but also to gathering data from DNA repair assays. Furthermore, we demonstrate increased functionality and extended data analysis capabilities with the use of a compressed collection of images called a "comet chip" and through a visual representation of data called a "profile plot". Use of the custom macros enabled us to detect an unexpected characteristic of the electrophoretic profile, giving us novel insight into the nature of comet analysis. In addition to the increased analytical sensitivity proffered by this system, the tail profile macros are upgradeable and platform independent.     

Hepatic iron accumulation is not directly associated with induction of DNA strand breaks in the liver cells of Long-Evans Cinnamon (LEC) rats.

Effects of accumulation of copper and iron on induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant hepatitis. Copper and iron accumulated in the liver of LEC rats in an age-dependent manner from 4 to 15 weeks. Low-iron diet prevented the accumulation of iron in the liver, but did not prevent accumulation of copper. The amounts of DNA strand breaks that were estimated by comet assay in the liver cells of rats fed standard diet increased with age from 4 to 15 weeks. No significant differences were observed in the proportions of LEC rat liver cells without tail and the average lengths of tail momentum in the comet images between LEC rats that had been fed standard MF diet and low-iron diet. These results support the idea that accumulation of iron is not directly associated with the induction of DNA damage in the liver cells of LEC rats.     

In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses

A global DNA methylation and proteomics approach was used to investigate somatic embryo maturation in hybrid larch. Each developmental step during somatic embryogenesis was associated with a distinct and significantly different global DNA methylation level: from 45.8% mC for undifferentiated somatic embryos (1‐week proliferation) to 61.5% mC for immature somatic embryos (1‐week maturation), while maturation was associated with a decrease in DNA methylation to 53.4% for mature cotyledonary somatic embryos (8‐weeks maturation). The presence of 5‐azacytidine (hypo‐methylating agent) or hydroxyurea (hyper‐methylating agent) in the maturation medium altered the global DNA methylation status of the embryogenic cultures, and significantly reduced both their relative growth rate and embryogenic potential, suggesting an important role for DNA methylation in embryogenesis. Maturation was also assessed by examining changes in the total protein profile. Storage proteins, identified as legumin‐ and vicilin‐like, appeared at the precotyledonary stage. In the proteomic study, total soluble proteins were extracted from embryos after 1 and 8 weeks of maturation, and separated by two‐dimensional gel electrophoresis. There were 147 spots which showed significant differences between the stages of maturation; they were found to be involved mainly in primary metabolism and the stabilization of the resulting metabolites. This indicated that the somatic embryo was still metabolically active at 8 weeks of maturation. This is the first report of analyses of global DNA methylation (including the effects of hyper‐ and hypo‐treatments) and proteome during somatic embryogenesis in hybrid larch, and thus provides novel insights into maturation of conifer somatic embryos.     

Effect of vinblastine sulfate on gamma-radiation-induced DNA single-strand breaks in murine tissues

The effect of vinblastine sulfate on gamma-radiation-induced DNA strand breaks in different tissues of tumour bearing mice, was studied by single-cell gel electrophoresis. Intraperitonial administration of different doses (0.25-2.0mg/kg body weight) of vinblastine sulfate 30 min prior to 4 Gy gamma-radiation exposure showed a dose-dependent decrease in the yield of DNA strand breaks in murine fibrosarcoma, blood leukocytes and bone marrow cells. The dose-dependent protection of cellular DNA against radiation-induced strand breaks as evidenced from comet tail length, tail moment and percent DNA in the tail, was more pronounced in bone marrow cells than in the cells of the tumor fibrosarcoma. In fibrosarcoma cells, the decrease in comet tail length, tail moment and percent DNA in the tail was detected at lower doses of vinblastine sulfate administration and these parameters were not significantly altered at higher doses, from that of the control irradiated. From this study, it appears that in addition to anticancer activity, vinblastine sulfate could offer protection to the normal tissues against gamma-radiation-induced DNA strand breaks.     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa

The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances.     

Recovery and regeneration of embryogenic cultures from female flowers of False Horn Plantain

Somatic embryogenesis from immature male flowers in Musa is only suitable for genotypes with a male bud. Six friable embryogenic cultures were obtained from 28 cultured buds of female flowers of the AAB False Horn Plantains, ‘Curraré’ and ‘Curraré Enano’. Embryogenic suspensions were established from these embryogenic cultures. Somatic embryogenesis was demonstrated histologicaly. Regeneration of plants was obtained either from somatic embryos directly isolated from embryogenic cultures or from suspensions after plating on a semi-solid medium. This study demonstrates that somatic embryogenesis from immature flowers is suitable for genotypes of Musa with or without male buds. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.