Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.
01 Jan 2000-Environmental and Molecular Mutagenesis (ENVIRONMENTAL AND MOLECULAR MUTAGENESIS)-Vol. 35, Iss: 3, pp 206-221
TL;DR: The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) versions of the assay developed by Singh et al.
Abstract: Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.
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TL;DR: This work shows that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity, and suggests that CD133-positive tumour cells could be the source of tumour recurrence after radiation.
Abstract: Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.
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TL;DR: The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells that 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.
Abstract: 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.
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Cites background from "Single cell gel/comet assay: guidel..."
...Genotoxicity is also assessed in cell culture systems, on their own or in conjunction with the microsomal “S9” fraction from liver, that provides enzymes to metabolize chemicals to more reactive forms ( 26 )....
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TL;DR: 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 to measurement of DNA damage in specific genomic sequences.
Abstract: 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.
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TL;DR: Many of the engineered nanomaterials assessed were found to cause genotoxic responses, such as chromosomal fragmentation, DNA strand breakages, point mutations, oxidative DNA adducts and alterations in gene expression profiles.
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Cites background from "Single cell gel/comet assay: guidel..."
...Analysis of the length and fluorescence intensity of the comet tail is directly proportional to the amount of DNA damage [196,197]....
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TL;DR: This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures, and is seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.
Abstract: The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.
968 citations
Cites background or methods from "Single cell gel/comet assay: guidel..."
...Therefore, although a valuable resource with very relevant data, the paper by Sasaki et al. (2000) has areas where technical aspects of the assay differ from the minimal requirements as identified in the present recommendations, as well as from the recommendations given by Tice et al. (2000)....
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...In the present recommendations, minimal standards for obtaining reproducible and reliable Comet assay data were identified which slightly deviate from those concluded following the IWGTP meeting in Washington in 1999 (Tice et al., 2000)....
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...Despite the plethora of data on the Comet assay and the overall agreement on a general protocol (see report of the IGWPT in Tice et al., 2000), it was felt that a more formal document, giving recommendations on the conduct of the in vivo Comet assay for regulatory approval, was required....
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...This paper provides data on more than 200 compounds tested in rodents, all of which were produced before the recommendations by Tice et al. (2000) were available....
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References
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TL;DR: Human lymphocytes were exposed to X-irradiation or treated with H2O2 and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions and this technique appears to be sensitive and useful for detecting damage and repair in single cells.
10,170 citations
"Single cell gel/comet assay: guidel..." refers methods in this paper
...The fluorescent dyes used most frequently are ethidium bromide [e.g., O ̈ stling and Johanson, 1984; Singh et al., 1988], propidium iodide [e....
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...The fluorescent dyes used most frequently are ethidium bromide [e.g., O¨ stling and Johanson, 1984; Singh et al., 1988], propidium iodide [e.g., Olive, 1989], 4,6- diamidino-2-phenylindole (DAPI) [e.g., Gedik et al., 1992], SYBR Green I [e.g., Tice et al., 1998], and YOYO-1…...
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TL;DR: The methods described include the standard plate test, the use and storage of the bacterial tester strains, preparation and use of the liver homogenates, and the methods of inducing the rats for elevated microsomal enzyme activity.
Abstract: We describe in this paper the general methods for using the Salmonella/microsome test as a mutagenesis screening system. The methods described include the standard plate test, the use and storage of the bacterial tester strains, preparation and use of the liver homogenates (S/sub 9/), and the methods of inducing the rats for elevated microsomal enzyme activity. The application of this test system to screening large numbers of compounds, and the interpretation of test results is also discussed.
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"Single cell gel/comet assay: guidel..." refers methods in this paper
...…cofactorsupplemented postmitochondrial fraction (S9) prepared from the livers of male rats treated with enzyme-inducing agents such as Aroclor 1254 [Ames et al., 1975; Natarajan et al., 1976; Matsuoka et al., 1979; Maron and Ames, 1983], or a mixture of phenobarbitone andb-naphthoflavone…...
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...The most commonly used system is a cofactorsupplemented postmitochondrial fraction (S9) prepared from the livers of male rats treated with enzyme-inducing agents such as Aroclor 1254 [Ames et al., 1975; Natarajan et al., 1976; Matsuoka et al., 1979; Maron and Ames, 1983], or a mixture of phenobarbitone and b-naphthoflavone [Matsushima et al....
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TL;DR: Two new tester strains, a frameshift strain and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set and two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine.
Abstract: The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.
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"Single cell gel/comet assay: guidel..." refers methods in this paper
...…from stock cultures, and incubated in appropriate culture medium at 37°C. Lymphocytes isolated from the whole blood of healthy subjects are incubated in appropriate culture medium at 37°C. Rodent hepatocytes are isolated using routine procedures and incubated in appropriate culture medium at 37°C....
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TL;DR: The principles of strand break detection using both the alkaline and neutral versions of the technique are discussed, and a basic methodology with currently used variations is presented.
Abstract: The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. Its use has increased significantly in the past few years. This paper is a review of the studies published to date that have made use of the comet assay. The principles of strand break detection using both the alkaline and neutral versions of the technique are discussed, and a basic methodology with currently used variations is presented. Applications in different fields are reviewed and possible future directions of the technique are briefly explored.
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TL;DR: Mammalian cells were after irradiation suspended in melted agarose, and casted on microscope slides, and DNA had migrated towards the anode and this migration was more pronounced in irradiated than in control cells.
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