The comet assay: ready for 30 more years.

During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the comet assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the comet assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the comet assay and they hold promise that the assay is ready for the next 30 years.

New developments in comet-FISH.

The comet assay combined with fluorescence in-situ hybridisation (FISH) is a powerful technique for comparative analyses of damage induction and repair in genomes and in specific DNA sequences within single cells. Recent advances in the methodology of comet-FISH will be considered here, with particular attention to the design and generation of fluorescent probes. In general, all the approaches must fulfil a few basic requirements: the probes should be no longer than ~300 nucleotides in length (single or double stranded) to be able to penetrate the gel in which the target genomic DNA is embedded, they should be sequence-specific, and their signal should be detectable and distinct from the background fluorescence and the dye used to stain the DNA.

Experiences with the in vivo and in vitro comet assay in regulatory testing.

The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages.

Worldwide interest in the comet assay: a bibliometric study.

The comet assay is a rapid, sensitive and relatively simple method for measuring DNA damage. A bibliometric study was performed to evaluate temporal and geographical trends, research quality and main areas of interest in scientific production in this field. A PubMed search strategy was developed and 7674 citations were retrieved in the period 1990-2013. Notably, the MeSH (Medical Subject Headings) term 'comet assay', officially introduced in 2000, is used by indexers only in two thirds of papers retrieved. Articles on the comet assay were published in 78 countries, spread over the 5 continents. The EU contributed the greatest output, producing >2900 articles with IF (42.0%) and totalling almost 10000 IF points, and was followed by USA. In the new millennium, research with this assay reached a plateau or slow decline in the most industrialised areas (USA, Germany, UK, Italy), while its use has boomed in emerging countries, with increases of 5- to 7-fold in the last 10 years in China, India and Brazil, for instance. This transition resulted in a slow decrease of scientific production quality, as the countries that increased their relative weight typically had lower mIFs. The most common MeSH terms used in papers using the comet assay referred to wide areas of interest, such as DNA damage and repair, cell survival and apoptosis, cancer and oxidative stress, occupational and environmental health. Keywords related to humans, rodents and cell culture were also frequently used. The top journal for the comet assay articles was found to be Mutation Research, followed by Mutagenesis. Most papers using the comet assay as a biomarker were published in genetic and toxicology journals, with a stress on environmental and occupational disciplines.

Application of the comet assay method in clinical studies.

The comet assay or single-cell gel electrophoresis (SCGE) assay is now widely accepted as a standard method for assessing DNA damage in individual cells. It finds use in a broad variety of applications including human biomonitoring, genotoxicology, ecological monitoring and as a tool for investigation of DNA damage and repair in different cell types in response to a range of DNA-damaging agents. The comet assay should be eminently suitable for use in clinical practice since it is a relatively simple and inexpensive technique which requires only a few cells, and results can be obtained within a matter of hours. This method can be used in the study of cancer as well as in lifestyle and dietary studies. In cancer it is useful for measuring DNA damage before, throughout and after therapy (either radiotherapy or chemotherapy). Another use of this method is in lifestyle study, such as investigation of the effect on DNA of common human activities (e.g. smoking, or working with a potentially genotoxic agent). The final use of comet assay in this paper is dietary study. In this type of study we observe the effects of consumption of specific foods or supplements which may be protective for DNA against damage.

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood.

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~250 µl volumes, at -80°C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at -80°C, unless a cryopreservative is present. Our "small volume" approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.

The comet assay in nanotoxicology research.

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.

Use of single cell gel electrophoresis assays for the detection of DNA-protective effects of dietary factors in humans: recent results and trends.

This article summarises the results of human dietary intervention trials employing the comet assay (single cell gel electrophoresis, SCGE), which have been published in the last few years (i.e., between 2005 and 2008) and describes new trends and developments as well as current problems concerning the design of intervention trials and the interpretation of the results. Most new studies were carried out with complex plant derived foods and juices; only a few were conducted with individual food constituents. With specific vegetables, for example with water cress and Brussels sprouts, potent antioxidant effects were observed; also coffee caused a protective effect and it is notable that it was more effective than consumption of a diet containing increased levels of fruits and vegetables. Interesting recent developments include the development of protocols which enable us to monitor protection towards genotoxic chemicals contained in the human diet, and it was shown in preliminary studies that alterations of the activities of drug metabolising enzymes by dietary factors lead to altered sensitivity of lymphocytes against DNA damage caused by certain dietary carcinogens. Another novel approach is the development of methods to monitor the effects of dietary factors on DNA repair. The development of protocols for experiments with exfoliated buccal cells is another potentially valuable innovation. The adequate experimental design of SCGE trials is still a matter of debate and the evaluation of the available data shows that there is an urgent need to develop guidelines concerning the number of participants, sampling periods, duration of trials, use of placebos, and definition of adequate run-in and wash-out phases. Recent studies showed that the results of dietary studies could be biased by factors such as age, sex, body mass index and life style habits and by seasonal effects. Another still unsolved problem is the interpretation of the results of SCGE trials in regard to potential beneficial health effects. The use of -omics techniques may contribute to provide mechanistic explanations in addition to conventional approaches (such as enzyme measurements). Information on health effects of dietary factors and on prevention of diseases related to DNA damage can also be obtained in experiments with animals, using SCGE to detect decreases in DNA damage in inner organs.

Viable human buccal mucosa cells do not yield typical nucleoids: impacts on the single-cell gel electrophoresis/Comet assay.

Buccal mucosa (BM) cells have been used in human biomonitoring studies for detecting DNA adducts and chromosomal damage in an epithelial cell population. In the present study, we have investigated if human BM cells are suitable for use in the single-cell gel electrophoresis (SCGE)/Comet assay as an approach for estimating the exposure of epithelial cells to DNA-damaging agents. Our results indicate that only a few cells from BM cell samples yield comets that can be analyzed by current methods, and that the yield of cells with comets is independent of the percentage of viable BM cells in the sample. Data generated after enzymatic enrichment of viable cells and immunomagnetic separation of epithelial cells suggest that most of the BM cells that do form comets are probably leukocytes. Moreover, by reevaluating specific cells after running the Comet assay, we found that viable epithelial BM cells give rise to atypical comets that are not included in the analysis. Comparing DNA migration patterns between small groups of smokers and nonsmokers indicated that long-term smoking had no effect on the subpopulation of cells that yield typical comets. Our results indicate that the SCGE assay, as it is commonly performed, may not be useful for genotoxicity monitoring in human epithelial BM cells.