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PubMed2003

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%).

Age Factors↗
PubMed2003

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.

Animals↗
PubMed2003

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.

Animals↗
PubMedDate not supplied

Detection of hypoxia in human brain tumor xenografts using a modified comet assay.

We used the standard comet assay successfully to generate in vitro dose-response curves under oxic and hypoxic conditions. We then made mixtures of cells that had been irradiated with 3 and 9 Gy of X-rays to simulate two subpopulations in a tumor, but efforts to accurately detect and quantify the subpopulations using the standard comet assay were unsuccessful. Therefore, we investigated a modified comet assay to determine whether it could be used for measuring hypoxia in our model systems. U251 MG cells were grown as subcutaneous tumors in athymic mice; U251 MG and U87 MG cells were grown as intracerebral (i.c.) tumors in athymic rats. Animals were injected with RSU 1069, irradiated, and euthanized. Tumors and normal brains were removed, and the cells were analyzed using a modified comet assay. Differences in comet tail moment distributions between tumor and contralateral normal brain, using tail moments at either the 25th or 50th percentile in each distribution, were taken as measures of the degree of tumor hypoxia. For U251 MG tumors, there was a positive relationship between tumor size and the degree of hypoxia, whereas preliminary data from U87 MG i.c. tumors showed less hypoxia and no apparent relationship between tumor size and hypoxia.

Animals↗
PubMed2003

Detection of replicative integrity in small colonic biopsies using the BrdUrd comet assay.

The alkaline single-cell gel electrophoresis or comet assay is a relatively simple method of measuring DNA single-strand breaks and alkali-labile sites in individual cells. Previously, we have used a combination of this with bromodeoxyuridine labelling of DNA and immunolocalisation of the BrdUrd to show that DNA replicative integrity can be assessed in single cultured cells. This study demonstrates the application of the technique to single cells derived from small human colonic biopsies isolated at routine endoscopy. A high level of reproducibility within replicate comet slides and between comet slides prepared from various colonic sites within a single patient is shown. Preliminary results demonstrate that defects in replication can be detected in tumour and premalignant colonic tissue adjacent to the tumour, suggesting that alterations in replicative integrity are an early event in neoplasia, appearing in premalignant mucosal cells. This development deems the BrdUrd comet assay suitable as an ex vivo molecular end point that can be measured easily in tissue collected by biopsy at routine colonic endoscopy. Thus, the BrdUrd comet assay has the potential to facilitate trial investigations of diet- or environment-related factors that may affect replicative integrity in the colon and provides a novel biomarker for colon carcinogenesis.

Aged↗
PubMed2003

Lack of an association between environmental exposure to polychlorinated biphenyls and p,p'-DDE and DNA damage in human sperm measured using the neutral comet assay.

BACKGROUND: Chlorinated organic chemicals, such as polychlorinated biphenyls (PCB), hexachlorobenzene (HCB), dichlorodiphenyl trichloroethane (DDT), and dichlorodiphenyl dichloroethene (DDE, the most stable daughter compound of DDT) are persistent lipophilic compounds found in a large portion of the general population. To explore the hypothesis that environmental exposure to these compounds is associated with altered DNA integrity in human sperm, a study of 212 male partners of a sub-fertile couple who presented to the Massachusetts General Hospital Andrology Laboratory was conducted. METHODS: The neutral single cell microgel electrophoresis assay (comet assay) was used to assess DNA integrity in sperm. VisComet image analysis software was used to measure total comet length, the proportion of DNA present in the comet tail, and tail distributed moment, an integrated measure of length and intensity. RESULTS: In the regression analyses, there were no statistically significant consistent associations between the comet assay parameters and any of the individual PCB congeners, sum of PCB, or p,p'-DDE. CONCLUSION: These results suggest that there are not strong relationships between adult levels of these chlorinated organic compounds and sperm DNA damage as measured by the comet assay.

Adult↗
PubMed2004

Comet-FISH using peptide nucleic acid probes detects telomeric repeats in DNA damaged by bleomycin and mitomycin C proportional to general DNA damage.

For the optimal use of anticancer drugs a knowledge of the whole spectrum of side-effects is required. A potential hazard, so far only scarcely investigated, is uncontrolled effects of drugs such as bleomycin (BLM) and mitomycin C (MMC) on telomere shortening in non-cancerous tissues of the treated person. For the first time, directly labelled telomere-specific peptide nucleic acid (PNA) hybridization probes were applied in comet-FISH to detect DNA fragmentation on an intermediate scale. The effects of BLM and MMC were measured in peripheral blood cells of three human volunteers, following ex vivo incubation. Fragmentation of telomeres and subtelomeric regions was highly specifically detected by the comet-FISH assay, a combination of the comet assay and fluorescence in situ hybridization. As a technical detail, the effects of the hybridization procedure have been studied on the level of single comets. Image analysis before and after the hybridization process reveals a small decrease in the detected fragmented DNA, probably due to diffusion of small fragments. It could not only be shown that both drugs actually induce breaks in telomere-associated DNA, but also that the comet-FISH technique, as a quantitative approach, is a useful tool for the detection and evaluation of the role of sequence-specific DNA damage after mutagenic action. The breakage frequency for DNA of or adjacent to telomeric repeats was found to be proportional to that of the total DNA, which hints at random induction of DNA breaks by BLM and MMC. In terms of therapy, the results indicate that no over- or under-proportional effects on telomeres of BLM or MMC need be expected.

Antibiotics, Antineoplastic↗
PubMed2005

Validation of Drosophila melanogaster as an in vivo model for genotoxicity assessment using modified alkaline Comet assay.

The single cell gel electrophoresis or Comet assay is one of the most popular techniques for genotoxicity assessment. The present study was undertaken to validate our previously modified version of the Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R(+)) with four well-known mutagenic and carcinogenic alkylating agents, i.e. ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and cyclophosphamide (CP). Third instar larvae (74 +/- 2 h) of D.melanogaster were fed different concentrations of EMS, MMS, ENU and CP (0.05, 0.5 and 1.0 mM) mixed standard Drosophila food for 24 h. At 98 +/- 2 h, the anterior midgut from control and treated larvae were dissected out, single-cell suspensions were prepared and Comet assay was performed. Our results show a dose-dependent increase in DNA damage with all the four alkylating agents, in comparison to control. The lower concentration (0.05 mM) of the test chemicals, except MMS, did not induce any DNA damage in the gut cells of the exposed larvae. When comparison of Comet parameters was made among the chemicals, MMS was found to be the most potent genotoxicant and ENU the least. The present study validated our previous observation and shows that D.melanogaster is a sensitive and suitable model for the in vivo assessment of genotoxicity using our modified alkaline Comet assay.

Alkylating Agents↗
PubMed2006

Detection of Alu sequences and mtDNA in comets using padlock probes.

Single cell gel electrophoresis, or the comet assay, is widely used to measure DNA damage and repair. However, the behaviour of the DNA under the conditions used for the comet assay is not fully understood. In developing a method for studying specific gene sequences within comets, using 'padlock probes' (circularizable oligonucleotide probes), we have first applied probes that hybridize to Alu repetitive elements and to mitochondrial DNA (mtDNA). During the sequence of stages in the comet assay, mtDNA progressively disperses into the surrounding agarose gel, showing no tendency to remain with nuclear DNA in the comets. In contrast, Alu probes remain associated with both tail and head DNA.

Alu Elements↗
PubMed2002

Application of the alkaline comet assay in biodosimetry: assessment of in vivo DNA damage in human peripheral leukocytes after a gamma radiation incident.

The alkaline comet assay was employed in the assessment of DNA damage in leukocytes of a worker incidently exposed to gamma radiation (221 mSv, 60Co source). The comet tail lengths and tail moments were studied. By using the alkaline comet assay immediately after accidental exposure a high level of DNA damage was recorded. The highest levels of DNA damage were recorded one day and one week after the radiation incident. Later on, a decrease in both comet parameters was observed. Although the level of DNA damage was diminished during a one year period, it was still elevated compared to normal values recorded in leukocytes of a healthy, unexposed person. The results obtained indicate that the alkaline comet assay is a rapid and sensitive microdosimetric technique and is suitable for in vivo human biomonitoring, especially in cases of incidental exposure to ionising radiation.

Cobalt Radioisotopes↗
PubMed2000

The comet assay of nasal epithelia: measurement of DNA damage for the assessment of genotoxic air pollution.

OBJECTIVES: The alkaline single cell gel electrophoresis or "comet" assay allows measurement of DNA damage in single cells with a high degree of sensitivity, e.g., for investigations of the effect of environmental agents with DNA-damaging potential. This study aimed to adapt this test to respiratory cells of the human nasal mucosa to examine the genotoxic effect of air pollution (cigarette smoke). STUDY DESIGN: In a prospective study, nasal epithelia of 16 cigarette smokers were examined by the adapted comet assay and the results were correlated with the results of the Papanicolaou-stained nasal cytology, carried out in a blinded fashion. The control group comprised 20 non-smoking men. All subjects under investigation were healthy office workers. METHODS: Nasal epithelia were harvested from the maxilloturbinates. One part of cells was Papanicolaou stained and evaluated by cytopathologists. The comet assay was performed on the other part of the cells. The examiners were blinded to the study and control groups. RESULTS: Among cigarette smokers, a significant correlation between cytopathological cell nucleus changes (metaplasia and dysplasia) and the DNA migration (tail lengths) in the comet assay was found as a sign of DNA damage. This was not found in nonsmoking control persons. CONCLUSIONS: These results confirm the sensitivity of the comet assay and the hypothesis that cell nucleus changes in conventional nasal cytology are associated with DNA damage.

Adult↗
PubMed2003

The relationship between cellular radiosensitivity and radiation-induced DNA damage measured by the comet assay.

The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by gamma-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assays. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition for the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells.

Animals↗
PubMed2005

Computerized image analysis software for the comet assay.

Single-cell gel electrophoresis (SCGE) or the comet assay is a powerful tool for the detection of DNA single- and double-strand breaks and base damage and for investigating the kinetics of DNA strand break rejoining in human and animal model systems. It is a versatile technique that can be applied in various areas of biomedical research. This chapter highlights the importance of computerized analysis and data processing for the comet assay and describes the criteria used for manual evaluation of comets and their limitations compared with the computer-based analysis. It describes in detail SCGE-Pro, a semiautomatic software developed in our laboratory for comet evaluation and data processing. For comparison, some of the commercially available software for analysis of data from the comet assay is also described.

Animals↗
PubMed2005

Comet assay with the fish cell line rainbow trout gonad-2 for in vitro genotoxicity testing of xenobiotics and surface waters.

The present study examines the potential of the comet assay using the rainbow trout gonad cell line-2 (RTG-2) as an in vitro indicator test for genotoxicity assessment of aquatic contaminants and native surface waters. Initially, the comet assay protocol was adapted to the RTG-2 cell line. An exposure period of 2 h was found to be optimal, because DNA damage decreased when exposure was prolonged. Then, the sensitivity of the comet assay with RTG-2 cells toward six genotoxic reference substances was evaluated. The lowest-observed-effect concentration values for the directly acting genotoxins, 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine, were in the low nanomolar range. The RTG-2 test system clearly was less sensitive for the indirectly acting genotoxins benzo[a]pyrene, nitrofurantoin, 2-acetylaminofluorene, and dimethylnitrosamine, despite the presence of xenobiotic metabolic capacities in RTG-2 cells. The two effect endpoints used, tail length (TL) and tail moment (TM), did not differ with respect to sensitivity, but the linearity of the concentration-response curve was better with TM than with TL. The overall reproducibility of the assay results was good. Finally, the applicability of the comet assay with RTG-2 cells for genotoxicity screening of native surface water samples was studied. The assay tolerated the use of nonsterile water samples and was able to detect genotoxic potentials in native water samples; that is, extraction and concentration of the samples were not needed. The results of the present study indicate the suitability of the comet assay with the fish cell line, RTG-2, as in vitro screen for detecting genotoxic potencies of xenobiotics and environmental samples.

Animals↗
PubMed2005

Comet-assay in combination with PNA-FISH detects mutagen-induced DNA damage and specific repeat sequences in the damaged DNA of transformed cells.

The Comet-assay was applied to three transformed cell lines (HT1080, CCRF-CEM line and CHO) which were treated with the cytostatics bleomycin (BLM) or mitomycin C (MMC). In addition, PNA probes for the telomere repeat (TTAGGG)(n) were used for detection of telomeric DNA sequences in the damaged DNA. Data were compared with previously obtained results from peripheral leukocytes. The amount of migrating DNA increased in all cell types in a dose-dependent manner after BLM exposure. CHO cells reacted sensitively at low doses of the mutagen, and leukocytes had the highest dose-related effect up to 25 IU/ml which, however, did not further increase. A rather linear dose response characterized the HT1080 cells, the effect was lowest for the CCRF-CEM cells. While MMC at lower doses increased the percentage of migrating DNA in a dose-dependent manner, the higher doses induced shorter comets, on average, than the lower ones in all cell lines. With PNA-Comet-FISH obvious differences were found between the studied cell lines with respect to quantitative head/tail distribution of telomeric signals after BLM exposure. A large number of signal spots of various sizes were found in CHO cells, very small signals could be detected in the comets of both neoplasia cell lines. Dose-dependence of telomeres in the tail was most pro-nounced in CCRF-CEM and normal leukocytes, less in HT1080. The steepest dose-related increase of telomeric signals in the tail was found in CHO cells. The ratio between the migrated DNA and the telomeric signals in the tail varied distinctly between the examined cell types from 3:1 to 1:1. Taken together, Comet-FISH can detect mutagenic effects on specific DNA sequences. This may be of high practical value if amplified DNA sequences will be addressed by those examinations in future.

Animals↗
PubMed1999

Automated comet assay analysis.

BACKGROUND: Recently the "comet assay" or "single-cell gel electrophoresis assay" has been established as a sensitive method for the detection of DNA damage and repair. Most of the software now available to quantify various parameters for DNA damage requires the interaction of a human observer. In this report, we describe an automated analysis system that is based on self-developed software and hardware and needs minimal human interaction. METHODS: The image analysis is divided into two parts: 1) automatic cell recognition and comet classification and 2) quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (Böcker et al.: Phys Med Biol 1997;42:1981-1992) allows for faster analysis. RESULTS: Our recognition software shows a sensitivity of 95.2% and a specificity of 92.7% when tested on test samples from routine work with DNA damage by low-dose radiation (0-2 Gy). The parallel hardware and software concept enables us to analyze 100 comets on one slide in less than 15 min. CONCLUSIONS: A comparison of measurements made on the same samples by manual and automated analysis systems revealed that there are no significant differences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possible.

Algorithms↗
PubMed1999

[Detection of apoptotic cells by comet assay].

Comet assay is a method measuring DNA strand beaks on single cell level, which has been developed for about 10 years. Theoretically, apoptotic cells with extensive DNA internucleosomal fragments can be detected by this method. On the basis that apoptosis in immature mice thymocytes could be induced by dexamethasone(DEX), the detection of apoptotic cells by comet assay was studied and compared with agarose gel electrophoresis, morphological observations with Giemsa staining and TUNEL assay. Apoptotic cells showed a comet feature with a very small head, and a large and wide tail. The optimal conditions for electrophoresis in comet assay was 20 volt in 15 minutes. Comet assay is sensitive, valid, rapid and simple and is highly correlated with Giemsa staining and TUNEL assay in detecting apoptotic cells.

Animals↗
PubMed2005

The comet assay: a sensitive genotoxicity test for the detection of DNA damage.

The comet assay or single-cell gel (SCG) test is a microgel electrophoresis technique that measures DNA damage at the level of single cells. A small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a fluorescent DNA binding dye. Cells with increased DNA damage display increased migration of chromosomal DNA from the nucleus toward the anode, which resembles the shape of a comet. In its alkaline version, which is mainly used, DNA single-strand breaks, DNA double-strand breaks, alkali-labile sites, and single-strand breaks associated with incomplete excision repair sites cause increased DNA migration. On the other hand, crosslinks (DNA-DNA or DNA-protein) can lead to decreased DNA migration. Variations of the comet assay have been established for the detection of specific DNA base modifications. Here we describe the basic methodology of the alkaline comet assay, establishing a sensitive protocol for obtaining reproducible and reliable data. Applications of the comet assay for detecting DNA damage in individual cells are briefly reviewed.

Animals↗