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 (0.0, 0.2, 0.5, 1.0, 2.0 mg· kg^?1 dry soil) or Cd (0.0, 0.2, 0.5, 1.0, 2.0 mg · kg^?1 dry soil) and imidacloprid (0.5 mg · kg^?1 dry soil). Roots ofVicia faba were exposed to the contaminated soil for 2 h at 25°C 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 ofVicia 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.     

Genotoxicity of imidacloprid in relation to metabolic activation and composition of the commercial product

Imidacloprid is a neonicotinoid insecticide combining excellent efficiency against parasites with low toxicity for mammals. Commercially, it is co-formulated with dimethyl sulfoxide, methylpyrrolidone, propylene carbonate and mineral oil, which can modify its bioavailability and toxicological profile for humans following occupational exposure. A combined in vitro approach employing the comet assay and the micronucleus test was used to assess the genotoxicity of imidacloprid in relation to formulation, metabolic activation and exposure level. Human peripheral blood lymphocytes from unexposed healthy volunteers were treated with imidacloprid (0.2, 2 and 20 μM) and with equimolar concentrations of a commercial product, with and without addition of S9 fraction. Imidacloprid significantly increased the comet score and the frequency of micronuclei only at the highest concentration tested. DNA damage was slightly more severe with the commercial product, and was increased, though not significantly, by metabolic activation. Formation of reactive oxygen species (ROS) does not seem to be involved as a mechanism of genotoxicity, but this result may be explained by the insufficient sensitivity of the 2',7'-dichlorofluorescein diacetate assay at the test concentrations of imidacloprid. These results suggest that at concentrations<20 μM imidacloprid is not genotoxic to human lymphocytes in vitro. Nonetheless, the presence of co-formulants in the commercial product and occupational exposure, along with poor safety procedures, may present an increased risk for DNA fragmentation and chromosomal aberrations.     

Application of Euglena gracilis cells to comet assay: evaluation of DNA damage and repair

Alkaline single-cell gel electrophoresis (comet assay) enables sensitive detection of DNA damage in eukaryotic cells induced by genotoxic agents. We performed a comet assay of unicellular green alga Euglena gracilis that was exposed to genotoxic chemicals, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), benzo[a]pyrene (BAP), mitomycin C (MMC) and actinomycin D (AMD). Tail length and tail moment in migrated DNA were measured as indications of DNA damage. MNNG and BAP were found to cause concentration-dependent increases in DNA damage. The responses were more sensitive than those of human lymphocytes under the same treatment conditions. MMC and AMD showed no positive response, as reported elsewhere. The comet assays performed at specified times after treatment revealed that the DNA damaged by MNNG and gamma-ray irradiation was repaired during the initial 1h. The results clearly show that the comet assay is useful for evaluating chemically-induced DNA damage and repair in E. gracilis. Given the ease of culturing and handling E. gracilis as well as its sensitivity, the comet assay of this alga would undoubtedly prove to be a useful tool for testing the genotoxicity of chemicals and monitoring of environmental pollution.     

Enhanced prediction of potential rodent carcinogenicity by utilizing comet assay and apoptotic assay in combination

The comet assay has been recently validated as a sensitive and specific test system for the quantification of DNA damage. The objectives of this study are to investigate the utility of comet assay for detecting mutagens with 11 substances that demonstrated positive results in at least one test among four standard short-term genotoxicity tests, and to evaluate its ability to predict rodent carcinogenicity. Out of 11 test substances, positive comet results were obtained for colchicine, hydroxyurea and actinomycin D. No effect on DNA migration, determined as the tail moment, was found with theophylline or 2,4-dinitrophenol. Bisphenol A, vinblastine, paclitaxel and p-anisidine appeared cytotoxic clastogens because these induced tail moment at concentrations showing 60% or less cell survival. In addition, among three test substances showing the bimodal distribution of DNA damage, which is a characteristic of apoptosis, true apoptosis result was obtained for camptothecin and dexamethasone with the Annexin V affinity assay. With this limited data-set, an investigation into the predictive value of these short-term genotoxicity tests for determining the carcinogenicity showed that comet assay has relatively high sensitivity and superior specificity to other four short-term genotoxicity assay. Therefore, our data suggest that comet assay, especially in combination with apoptotic assay, would be a good predictive test to minimize false-positives in evaluation of the potential rodent carcinogenicity.     

Detection of DNA damage in workers exposed to JP-8 jet fuel

The genotoxicity of jet propulsion fuel 8 (JP-8) was assessed in the leukocytes of archived blood specimens from U.S. Air Force personnel using the comet assay. No differences in mean comet assay measurements were found between low, moderate, and high exposure groups before or after a 4h work shift. Before the work shift, mean tail DNA and mean tail (Olive) moment increased as the concentration of benzene measured in end-exhaled breath increased, indicating that prior environmental or work-related exposures to benzene produced DNA damage. The number of cells with highly damaged DNA decreased as the pre-shift benzene concentration in breath increased. It is not clear why the decrease is occurring. Mean tail DNA and mean tail (Olive) moment decreased as the concentrations of benzene and naphthalene measured in breath immediately after the work shift increased. These inverse relationships may reflect a slower rate of absorption or a faster rate of expiration of benzene in the lung. The number of cells with highly damaged DNA increased as the concentration of urinary (2-methoxyethoxy)acetic acid (MEAA) increased. This relationship was not seen in urinary MEAA adjusted for creatinine. MEAA is a metabolite of the deicing agent 2-(2-methoxyethoxy)ethanol contained in JP-8. MEAA or a component of JP-8 correlated with MEAA may have a toxic effect on DNA.     

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.     

DNA damage and repair in human peripheral blood lymphocytes following treatment with microcystin-LR

The purpose of this study was to find a possible explanation of the inconsistency of data regarding the genotoxicity of microcystin-LR (MC-LR). We compared the results of the comet assay with the results of the analysis of chromosome aberrations and apoptosis. In order to investigate the influence of MC-LR on DNA damage in human lymphocytes, cells were treated with MC-LR at different concentrations (1, 10 and 25 microg/ml) for 6, 12, 18 and 24 h. Analyses of Olive Tail Moment (OTM) as an indicator of DNA damage showed that MC-LR treatment induced DNA damage in a time-dependent manner, reaching its maximum after 18 h. The lowest values of OTM were observed after 24 h. MC-LR had no effect on the frequency of chromosome aberrations in lymphocytes. Since some data available in the literature indicate that apoptosis may lead to overestimated or false positive results regarding the genotoxicity of mutagens in the comet assay, we measured the frequency of late apoptotic cells by use of the comet assay and the frequency of early apoptotic cells with the TUNEL method. The comet assay results revealed that the highest level of apoptosis was observed after 24 h and the lowest after 18 h. The comparison of the frequency of apoptotic cells determined by the comet assay with DNA damage (OTM) examined by the comet assay revealed a statistically significant, negative correlation. The TUNEL results showed that the frequency of apoptotic cells progressively increased in a dose- and time-dependent manner. The comparison of the frequency of apoptotic cells determined by TUNEL method with DNA damage (OTM) examined by the comet assay showed a significant positive correlation for lymphocytes treated with MC-LR for 6, 12 and 18 h. Therefore, our findings indicate that microcystin-LR-induced DNA damage observed in the comet assay may be related to the early stages of apoptosis due to cytotoxicity but not genotoxicity. In addition, we examined the DNA repair kinetics in lymphocytes following treatment with microcystin-LR and ionizing radiation. Our results indicate that MC-LR has an inhibiting effect on the repair of radiation-induced damage.     

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.     

JP-8 jet fuel-induced DNA damage in H4IIE rat hepatoma cells

We investigated the genotoxicity of middle distillate jet fuel, Jet Propulsion 8 (JP-8), on H4IIE rat hepatoma cells in vitro. DNA damage was evaluated using the comet (single cell gel electrophoresis) assay. Cells were exposed for 4h to JP-8 (solubilized in ethanol (EtOH) at 0.1% (v/v)) to concentrations ranging from 1 to 20microg/ml. Exposure to JP-8 resulted in an overall increase in mean comet tail moments ranging from 0.74+/-0.065 (0.1% EtOH control) to 3.13+/-0.018,4.36+/-0.32,5.40+/-0.29,7.70+/-0.52 and 11.23+/-0.77 for JP-8 concentrations 3, 5, 10, 15 and 20microg/ml, respectively. Addition of DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C) to cell culture with JP-8 resulted in accumulation of DNA damage strand breaks and increase in comet tail length. Inclusion of 4mM HU and 40microM Ara-C with 3, 5, 10 and 20microg/ml JP-8 concentrations resulted in increased mean tail moments to 5.94+/-0.43,10.12+/-0.72,17.03+/-0.96,and29.25+/-1.55. JP-8, in the concentrations used in this study, did not result in cytotoxicity or significant apoptosis, as measured using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-X nick end labeling (TUNEL) assay. These results demonstrate that relevant exposures to JP-8 result in DNA damage to H4IIE cells, and suggest that DNA repair is involved in mitigating these effects.     

A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells

Cadmium (Cd) is a toxic heavy metal of continuing occupational and environmental concern with a wide variety of adverse effects. Several studies have shown that cadmium produces DNA strand breaks, DNA-protein cross-links, oxidative DNA damage, chromosomal aberrations, dysregulation of gene expression resulting in enhanced proliferation, depressed apoptosis and/or altered DNA repair. This study was undertaken to investigate the ability of cadmium chloride (CdCl(2)) and cadmium sulphate (CdSO(4)) to induce point mutations in codon 12 of the K-ras protooncogene assessed by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP) and RFLP-enriched PCR methods. Also their genotoxic effects were analyzed by the comet assay and sister chromatid exchanges test. The human lung fibroblast cell line MRC-5 was used for the experiments. Sister chromatid exchanges assay (SCEs) frequencies were significantly increased in cells exposed to cadmium salts in relation to controls (p<0.001). Despite the slow increment observed in the three comet parameters considered when cells were treated with cadmium chloride, significant differences between groups were only found in the variable comet moment (CM) (p<0.005). On the other hand, when cells were exposed to cadmium sulphate, the Kruskal-Wallis test showed highly significant differences between groups for migration, tail moment and comet moment parameters (p<0.001). Nevertheless, a null or weak point mutation induction in K-ras protooncogene was detected using polymerase chain reaction-low ionic strength-single strand conformation polymorphisms (PCR-LIS-SSCP) and RFLP-enriched PCR methods when cells were treated with cadmium salts. Thus, inorganic cadmium produces genotoxicity in human lung fibroblast MRC-5 cells, in the absence of significant point mutation of the K-ras gene.     

Assessment of reference values for DNA damage detected by the comet assay in human blood cell DNA

Genotoxicity measured by the comet assay is expressed by different researchers using parameters that are not easy to conceptualize, except for percent tail DNA (%T) or visual score (arbitrary units). A total of 125 publications have reported genotoxicity as DNA damage (representing strand breaks, alkaline labile sites, and transient repair sites), endonuclease III (ENDOIII), or formamidopyrimidine DNA glycosylase (FPG) sensitive sites. I have recalculated the visual score so that it is expressed in the range of 0-100, similar to that of %T. Similar values were obtained for DNA damage and ENDOIII sites, regardless of whether of the data were reported as %T or visual score. Thus, these endpoints can be used interchangeably, assuming that the visual score is expressed in the 0-100 range. Pooled analysis of %T and visual score data showed that the median (25-75%) values of DNA damage, ENDOIII, and FPG sites were 8.6 (4.4-14.5), 11.0 (4.2-19.5), 7.6 (3.2-14.2), respectively. The duration of alkaline treatment and electrophoresis had no significant effect on the level of DNA damage. There was a positive correlation between age and the level of DNA damage. A sub-analysis of DNA damage obtained from European countries showed a negative correlation with latitude. In conclusion, reference values for DNA lesions measured by the comet assay are around 7-11 %T or arbitrary units.     

Does toxoplasmosis cause DNA damage? An evaluation in isogenic mice under normal diet or dietary restriction

Toxoplasmosis is an anthropozoonotic widespread disease, caused by the coccidian protozoan parasite Toxoplasma gondii. Since there are no data regarding the genotoxicity of the parasite in vivo, this study was designed to evaluate the genotoxic potential of the toxoplasmosis on isogenic mice with normal diet or under dietary restriction and submitted to a treatment with sulfonamide (375 microg/kg per day). DNA damage was assessed in peripheral blood, liver and brain cells using the comet assay (tail moment). The results for leucocytes showed increases in the mean tail moment in mice under dietary restriction; in infected mice under normal diet; in infected, sulfonamide-treated mice under normal diet; in infected mice under dietary restriction and in infected sulfonamide-treated mice under dietary restriction. In liver and brain cells, no statistically significant difference was observed for the tail moment. These results indicated that dietary restriction and T. gondii were able to induce DNA damage in peripheral blood cells, as detected by the comet assay.     

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.     

Increased genotoxic susceptibility of breast epithelial cells to ethylene oxide

This study was carried out with the aim of elucidating the organ-specific effects of ethylene oxide in comparison with the sensitivity of cells from different tissues. An increased incidence of leukemia and lymphoma has been observed in workers exposed to ethylene oxide. However, contradictory findings exist regarding its ability to induce other tumor types, such as breast cancer. We characterized the genotoxicity of ethylene oxide by means of the alkaline version of comet assay in in vitro systems, in order to investigate the hypothesized role of this substance in the development of breast cancer. For this study, we used primary and secondary cultures of lymphoblasts (well-known target cells of the genotoxicity of ethylene oxide), breast epithelial cells (hypothesized target), peripheral blood lymphocytes (cells commonly used in biomonitoring), and of keratinocytes and cervical epithelial cells. DNA damage was measured and expressed as tail DNA, tail length, and tail moment. In the concentration range 0-100 microM, ethylene oxide induced a dose-dependent increase of DNA damage in the investigated cell types without notable cytotoxicity. A statistically significant increase of DNA damage could be observed after treatment with 20 microM ethylene oxide in lymphoblasts (51% increase of tail moment over the background), breast epithelial cells (26% increase) and peripheral lymphocytes (71% increase). In keratinocytes (5% increase) and cervical epithelial cells (5% increase) significant DNA damage could not be detected at this dose, but at higher concentrations (50-100 microM), such an increase was observed. These results are indicative of an increased sensitivity of breast epithelial cells towards genotoxic insults of ethylene oxide. Our observations provide additional data to evaluate the hypothesis that exposure to ethylene oxide may play a role in breast cancer, and the findings may contribute to the development of screening tests for monitoring an early response to genotoxic insults in occupational settings.     

Free radicals-mediated induction of oxidized DNA bases and DNA-protein cross-links by nickel chloride

Using the comet assay, we showed that nickel chloride at 250-1000 microM induced DNA damage in human lymphocytes, measured as the change in comet tail moment, which increased with nickel concentration up to 500 microM and then decreased. Observed increase might follow from the induction of strand breaks or/and alkali-labile sites (ALS) by nickel, whereas decrease from its induction of DNA-DNA and/or DNA-protein cross-links. Proteinase K caused an increase in the tail moment, suggesting that nickel chloride at 1000 microM might cross-link DNA with nuclear proteins. Lymphocytes exposed to NiCl(2) and treated with enzymes recognizing oxidized and alkylated bases: endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), displayed greater extent of DNA damage than those not treated with these enzymes, indicating the induction of oxidized and alkylated bases by nickel. The incubation of lymphocytes with spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by nickel. The pre-treatment with Vitamin C at 10 microM and Vitamin E at 25 microM decreased the tail moment of the cells exposed to NiCl(2) at the concentrations of the metal causing strand breaks or/and ALS. The results obtained suggest that free radicals may be involved in the formation of strand breaks or/and ALS in DNA as well as DNA-protein cross-links induced by NiCl(2). Nickel chloride can also alkylate DNA bases. Our results support thesis on multiple, free radicals-based genotoxicity pathways of nickel.     

In-vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia (HL-60) cells using the MTT and alkaline single cell gel electrophoresis (Comet) assays

Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD₅₀ value of 6.4 ± 0.6 μg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.     

利用彗星试验检测Cu2+对驯化蚯蚓的基因损伤

为研究Cu2+对驯化蚯蚓的损伤影响,将赤子爱胜蚓(Eisenia fetida)在非致死浓度(100mgCu2+·kg-1)下驯化培养2周,以未驯化的蚯蚓为对照,测定Cu2+对驯化及未驯化蚯蚓的急性毒性,并通过彗星试验(cometassay)观察铜胁迫下(400mg·kg-1)驯化后蚯蚓基因损伤的动态变化。结果显示:14d时,Cu2+对驯化蚯蚓和未驯化蚯蚓的半致死浓度(LC50)分别为321.83～542.45和230.83～342.91mg·kg-1,驯化后蚯蚓的存活率得到显著提高。彗星试验结果表示:蚯蚓体腔细胞的尾长、尾部DNA含量以及尾矩呈非正态分布,在11和14d时,驯化后的蚯蚓基因损伤程度明显比未驯化蚯蚓低。彗星试验是检测Cu2+对蚯蚓活体基因损伤的有效手段,蚯蚓体的DNA损伤可以作为指示重金属污染物影响的生物标志物。     

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.     

Laser scanning cytometry for comet assay analysis

BACKGROUND: The comet assay (single-cell gel electrophoresis) is a sensitive method for evaluating nuclear DNA damage. Previously used evaluation methods for the comet assay are time consuming and have an inherent risk of biased selection of comets due to manual selection and categorization of comet images. Laser scanning cytometry (LSC), the principle of which is equivalent to flow cytometry, enables quantification of fluorescence emitted from the cells on a microscope slide. In the present study, we explored whether LSC could be used to determine the degree of DNA damage demonstrated by the comet assay. METHODS: DNA damage was induced by ultraviolet A irradiation of keratinocytes and visualized by the comet assay. The evaluation included (a) LSC determination of DNA-specific fluorescence in 1,000 comet heads (undamaged DNA), (b) image acquisition of comets by rescanning of the microscope slide, and (c) digital image analysis and computation of tail moment and DNA content in the comet tails. RESULTS: Cells with damaged DNA were observed in a sub-G(1) area because the comet head loses DNA to the tail. We found a strong inverse correlation between tail moment and DNA content per nucleus. CONCLUSIONS: LSC enables an automated method for cell recognition and evaluation of the comets, thus providing quantitative information about nuclear DNA damage without subjective selection of analyzed comets.     

Comet assay to sense neutron 'fingerprint'

The suitability of comet assay to identify DNA damage induced by neutrons of varying energy was tested. For this purpose, monoenergetic neutrons from Hiroshima University Radiobiological Research Accelerator (HIRRAC) were used to induce DNA damage in irradiated human peripheral blood lymphocytes. The level of damage was computed as tail moment for different doses (0.125-1 Gy) and compared with the effects resulting from irradiation with (60)Co gamma. The neutron-irradiated cells exhibited longer comet tails consisting of tiny pieces of broken DNA in contrast to the streaking tails generated by (60)Co gamma. The peak biological effectiveness occurred at 0.37 and 0.57 MeV; a further increase or decrease in neutron energy led to a reduced RBE value. The RBE values, as measured by the comet assay, were 6.3, 5.4, 4.7, 4.3, 2.6, and 1.7 for 0.37, 0.57, 0.79, 0.186, 1, and 2.3 MeV neutrons. The lower RBE value obtained by the comet assay when compared to that for other biological end points is discussed. This study reports the usefulness of the alkaline comet assay for identifying DNA damage induced by neutrons of the same radiation weighting factor. The comet assay is a potential tool for use in neutron therapy, as well as a method for the rapid screening of samples from individuals accidentally exposed to radiation.