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The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between ‘desirable’ and ‘essential’ information: ‘essential’ information refers to the precise details that are necessary to assess the quality of the experimental work, whereas ‘desirable’ information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic “DNA ladder” pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.

With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.

The comet assay (single cell gel electrophoresis) is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. The assay depends on the relaxation of supercoiled DNA in agarose-embedded nucleoids (the residual bodies remaining after lysis of cells with detergent and high salt), which allows the DNA to be drawn out towards the anode under electrophoresis, forming comet-like images as seen under fluorescence microscopy. The relative amount of DNA in the comet tail indicates DNA break frequency. The assay has been modified to detect various base alterations, by including digestion of nucleoids with a lesion-specific endonuclease. We describe here recent technical developments, theoretical aspects, limitations as well as advantages of the assay, and modifications to measure cellular antioxidant status and different types of DNA repair. We briefly describe the applications of this method in genotoxicity testing, human biomonitoring, and ecogenotoxicology.

Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The article reviews studies that have used the comet assay to investigate the toxicity of manufactured nanoparticles. It is shown that at least 46 cellular in vitro studies and several in vivo studies have used the comet assay and that the majority of the nanoparticles tested cause DNA strand breaks or oxidative DNA lesions. This is not surprising considering the sensitivity of the method and the reactivity of many nanomaterials. Interactions between the particles and the assay cannot be totally excluded and need further consideration. It is concluded that studies including several particle types, to enable the assessment of their relative potency, are valuable as are studies focusing both on comet assay end points and mutagenicity. Finally, the article discusses the potential future use of the comet assay in human biomonitoring studies, which could provide valuable information for hazard identification of nanoparticles.

Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.

Aquatic species are exposed to a wide spectrum of substances, which can compromise their genomic integrity by inducing DNA damage or oxidative stress. Genotoxicity biomarkers as DNA strand breaks and chromosomal damages developed on sentinel species have already proved to be relevant in aquatic biomonitoring. However, these biomarkers do not reflect DNA oxidative lesions, i.e., the 8-oxodG, recognized as pre-mutagenic lesion if not or mis-repaired in human biomonitoring. The relevance to include the measure of these lesions by using the Fpg-modified comet assay on erythrocytes of the three-spined stickleback was investigated. An optimization step of the Fpg-modified comet assay considering enzyme buffer impact, Fpg concentration, and incubation time has been performed. Then, this measure was integrated in a battery of genotoxicity and cytotoxicity biomarkers (considering DNA strand breaks, DNA content variation, and cell apoptosis/necrosis and density) and applied in a freshwater monitoring program on six stations of the Artois Picardie watershed (3-week caging of control fish). These biomarkers allowed to discriminate the stations regarding the genotoxic potential of water bodies and specifically by the measure of oxidative DNA lesions, which seem to be a promising tool in environmental genotoxicity risk assessment. Graphical abstract

Many efforts have been made in the last 30 years to develop more relevant in vitro models to study genotoxic responses of drugs and environmental contaminants. While 2D HepaRG cells are one of the most promising models for liver toxicology, a switch to 3D cultures that integrate both in vivo architecture and cell-cell interactions has occurred to achieve even more predictive models. Preliminary studies have indicated that 3D HepaRG cells are suitable for liver toxicity screening. Our study aimed to evaluate the response of HepaRG spheroids exposed to various genotoxic compounds using the single cell gel electrophoresis assay. HepaRG spheroids were used at 10 days after seeding and exposed for 24 and 48 hours to certain selected chemical compounds (methylmethansulfonate (MMS), etoposide, benzo[a]pyrene (B[a]P), cyclophosphamide (CPA), 7,12-dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), 4-nitroquinoline (4-NQO), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), acrylamide, and 2-4-diaminotoluene (2,4-DAT)). After treatment, the comet assay was performed on single cell suspensions and cytotoxicity was determined by the ATP assay. Comet formation was observed for all compounds except IQ, etoposide and 2,4-DAT. Treatment of spheroids with rifampicin increased CYP3A4 activity, demonstrating the metabolic capacity of HepaRG spheroids. These data on genotoxicity in 3D HepaRG spheroids are promising, but further experiments are required to prove that this model can improve the predictivity of in vitro models to detect human carcinogens.

The Comet Assay or single cell gel electrophoresis assay is one of the very widely used assays to microscopically detect DNA damage at the level of a single cell. The determination of damage is carried out either through visual scoring of cells (after classification into different categories on the basis of tail length and shape) or by using different commercially available or public domain software (which automatically recognise the extent of damage). In this assay, the shape, size and amount of DNA within the 'comet' play important roles in the determination of the level of damage. The use of a software in particular also provides a range of different parameters, many of which might not be relevant in determining the extent of DNA damage. As a large number of factors could influence the shape, size, identification and determination of induced damage, which includes the scoring criteria, staining techniques, selection of parameters (whilst using the software packages) and appearance of 'hedgehog' or 'clouds', this article aims (a) to provide an overview of evolution of measurements of DNA damage using the Comet Assay and (b) to summarise and critically analyse the advantages and disadvantages of different approaches currently being adopted whilst using this assay. It is suggested that judicious selection of different parameters, staining methods along with inter-laboratory validation and harmonisation of methodologies will further help in making this assay more robust and widely acceptable for scientific as well as regulatory studies.