Background & Principle of the Comet Assay
The comet assay is a versatile and sensitive method that detects, in its standard version, DNA strand breaks (SBs) and alkali-labile sites. The first paper on this single-cell gel electrophoresis assay was published in 1984 by Ostling and Johanson. This was a neutral assay in which the lysis and electrophoresis were done under neutral conditions. Staining was done with acridine orange. The image obtained looked like a comet with a distinct head (comprising of intact DNA) and a tail (consisting of damaged or broken pieces of DNA), hence the name "comet assay".
The more versatile alkaline method of the comet assay was developed by N.P. Singh and co workers in 1988. This method was developed to measure low levels of strand breaks with high sensitivity. Several reviews have been published in recent years to highlight the procedures, advantages and limitations of this assay in genotoxicological, ecotoxicological and biomonitoring studies (Collins, 2004; Dixon et al., 2002; Fairbairn et al., 1995; Lee and Steinert, 2003).
The protocol is simple: briefly, cells embedded in agarose on a microscope slide are lysed to remove membranes and soluble components (including histones) leaving nucleoids (i.e., supercoiled DNA attached at intervals to a nuclear matrix forming loops). Next, nucleoids undergo alkaline unwinding and electrophoresis. The presence of SBs in the DNA relaxes the supercoiled loops and enables the DNA to migrate towards the anode. The resulting comet-shaped figures, called comet(s), are visualised with a DNA fluorescent dye and fluorescence microscopy.
Representative pictures of comets visualized with fluorescence microscopy:
Modifications of the Comet Assay
The comet assay can be used to measure not just SBs but also specific DNA lesions. For instance, the enzyme is formamidopyrimidine DNA glycosylase (FPG), which recognises 8- oxoguanine (and some other altered guanines).
This enzyme-modified comet assay includes an extra step between lysis and alkaline treatment; i.e. incubation with DNA repair enzymes from bacteria or human cells to gain further information on specific classes of DNA lesions.
Examples of enzymes commonly used in the enzyme-modified comet assay:
formamidopyrimidine DNA glycosylase (Fpg) detects oxidized purines, formamidopyrimidines (ring-opened adenine or guanine) and ring-opened N7 guanine adducts;
human 8-oxo-guanine (8-oxoG) DNA glycosylase (hOGG1) detects oxidized purines and formamidopyrimidines; and
T4 endonuclease V (T4endoV) detects dimerised pyrimidines.
The comet assay (with and without the inclusion of lesion-specific enzymes) is widely used as a biomarker assay in human population studies and genotoxicity testing (including regulatory toxicology) - primarily to measure DNA damage, but increasingly also to assess the activity of cells for DNA repair.
In the original publication, Ostling and Johanson also reported the first experiments to measure DNA repair by simply following the decrease of ionising radiation-induced SBs over time – referred to as a challenge assay or cellular repair assay. However, this approach merely measures the final step in the repair process (i.e., ligation). Still, useful information on the kinetics of NER and BER has been gained by following the removal of pyrimidine dimers or oxidised bases, respectively, using appropriate DNA repair enzymes. Then again, this approach is time-consuming and laborious, and therefore not optimal for biomonitoring or intervention studies, which typically require high-throughput processing of many samples.
An alternative in vitro approach called the comet-based in vitro DNA repair assay is based on assessing the ability of repair proteins in a sample extract to recognize and incise substrate DNA that contains induced lesions. The whole-cell extract can be prepared from blood cells, ground tissues or cultured cells, by ‘snap-freezing’ and subsequent lysis with Triton® X-100. The comet-based in vitro DNA repair assay was first devised in 1994 to measure NER and BER activity in a human cell extract. However, over the past two decades, it has been modified and improved, as well as being applied to tissue samples in addition to cell suspensions.
The table gives the comparison between the four main versions of the comet assay, i.e. the standard comet assay, the enzyme-modified comet assay, the cellular repair assay and the comet-based in vitro DNA repair assay.
Applications of the Method
Human biomonitoring in environmental and occupational exposure scenarios
The comet assay has been employed in around 140 studies of occupational exposure to xenobiotics, and approximately 40 investigations of environmental exposure of humans. Levels of DNA damage have been measured in populations exposed to environmental pollution, including polycyclic aromatic hydrocarbons, particulate matter, benzene and heavy metals, and in addition environmental tobacco smoke. Effects of exposure to ionising radiation following the Chernobyl accident, indoor radon, and high natural radiation levels have also been investigated.
Occupational exposure studies have focused on volatile organic compounds such as styrene, benzene, toluene, vinyl chloride; metals such as welding fumes, chromium, lead, mercury; pesticides; asbestos and mineral fibres; and anaesthetic gases, chemotherapy drugs and ionising radiation in medical personnel. (It is worth noting that the assay is also used in ecological biomonitoring, and much of the methodological discussion here applies equally to that field).
The influence of nutritional factors on DNA damage levels (and by inference on disease incidence, notably cancer) has been studied with the comet assay using standard epidemiological approaches, in particular observational or ecological studies, and dietary interventions. Analyses of DNA damage levels in combination with information about dietary intake or measurements of micronutrients in the blood have shown, for instance, a negative correlation between carotenoids and DNA base oxidation, and a reduction of DNA damage in subjects on vegetarian or high vitamin content diets. Such correlations do not necessarily indicate causal relationships, but might simply be associations, brought about by other, undefined common factors. Intervention studies give more conclusive evidence of cause and effect. Many such studies have been carried out, with antioxidant or other phytochemical supplements, addition of specific foods (usually fruits) to the diet, or major changes in the diet (e.g. including 600 grams per day of vegetables or fruits).
To be reliable, trials should be placebo-controlled, though 'placebo' is a misnomer where real foods are concerned. Crossover studies are popular, in which each subject takes placebo or supplement in two phases separated by a washout period.
With DNA damage as an endpoint, roughly half the studies have shown a reduction in SBs or oxidised bases and half have shown no effect. Individual antioxidant status can be assessed by treating white blood cells in vitro with an oxidising chemical (H2O2); a low yield of SBs indicates a high antioxidant status. The value of decreasing oxidative base damage in healthy individuals is debatable, since a certain amount of oxidative stress is inevitable and even desirable; for example, the inflammatory response deploys ROS as a defence mechanism, and ROS are involved in crucial cell signalling pathways.
Other endpoints are also amenable to study, notably DNA repair - shown in several studies to be enhanced by nutritional factors (Collins et al., 2012).
DNA damage as an indicator of disease
Oxidised DNA bases and/or SBs are detected at elevated levels in PBMN cells from patients with diabetes, inflammatory disorders (arthritis, ankylosing spondylitis), various cancers (breast, cervix, lung, oesophagus, prostate), neurodegenerative disorders (Alzheimer’s and Parkinson’s diseases), and cardiovascular diseases; the DNA oxidation damage is often accompanied by depressed antioxidant status.
To determine whether DNA damage is a cause or an effect of disease, prospective studies are necessary, but have not yet been carried out; a large cohort of healthy individuals would have their DNA damage analysed, and would be monitored for years so that disease incidence and mortality could be retrospectively linked to the earlier burden of DNA damage.
A similar approach is needed to establish whether intrinsic ability to repair DNA is a factor in determining susceptibility to disease (particularly cancer).
DNA damage and aging
It is commonly believed that a major player in the process of aging is the accumulation of oxidative damage to biomolecules, including DNA. Such an accumulation might result from increased exposure to ROS (for instance, if mitochondria become 'leaky') or from depressed defenses, such as a decline in DNA repair capacity.
Most studies to date have been carried out in PBMN cells, and results are not consistent, with only a weak positive correlation overall. DNA repair capacity, also, has been variously reported as not affected, decreased or even increased with age.
Ecogenotoxicological studies and environmental monitoring
The use of plants as well as a wide range of terrestrial and aquatic species in the comet assay has dramatically increased in the last decade (Costa et al., 2014; de Lapuente et al., 2015; Santos et al., 2015), particularly in environmental risk assessment (ERA). A recent validation study has indicated that the in vitro comet assay combined with FPG may be an effective complementary line-of-evidence in ERA even in particularly challenging natural scenarios such as estuarine environments (Costa et al., 2014).
Regulatory genotoxicological studies
The importance of this assay has also being realized in regulatory genotoxicological studies (Tice et al., 2000, Hartmann et al., 2003, Burlinson et al., 2007) .Because of the versatility of the assay, it is widely used in genotoxicity testing of novel chemicals and nanomaterials as screening tool for pharmaceuticals and nanomaterials (NMs). An OECD test guideline exists for using the comet assay in in vivo genotoxicity studies (OECD TG 489) , but despite the wide variety of applications, no OECD guidelines exist for in vitro genotoxicity testing or for biomonitoring applications of the comet assay. One reason for the absence of OECD guidelines for the in vitro comet assay is poor comprehension of the molecular mechanisms of comet formation after single cell gel electrophoresis, which can easily lead to poorly conducted experiments, complicating the possibilities to identify the underlying MoA of genotoxic compounds. Also detecting direct versus indirect genotoxicity is a challenge with the standard in vitro comet assay. On the other hand, modifications of the assay may provide new insights and need further attention.
DNA repair as a biomarker
The comet-based in vitro DNA repair assay has been used in some cell culture and animal studies – studying the effect of nutrition and ageing – but it is mostly used in human biomonitoring and intervention studies. We previously reviewed the different in vitro, in vivo animal and human studies where this technique has been applied to measure DNA repair activity (Azqueta et al. 2014). In the near future, we also plan to use the assay in genotoxicity testing to unravel the role of DNA repair in the Mode-of-Action (MoA) of potential (non)genotoxic carcinogens. In addition, DNA repair has recently been defined as a key event (KE) in an adverse outcome pathway (AOP) that was submitted to the OECD Extended Advisory Group for Molecular Screening and Toxicogenomics (EAGMST) for internal review – which may also promote the use of the assay.
High throughput versions
A medium throughput format for the comet assay was introduced in 2009, comprising 12 minigels (each of 5 µL of agarose) per slide (Shaposhnikov et al., 2010). Each minigel can be incubated separately with different extract/enzyme/buffer when placed into the 12-gel comet assay unit produced by Severn Biotech (www.severnbiotech.com). This unit allows analysis of many more extracts in one experiment, and uses fewer substrate cells and less extract volume per reaction. Several reports describe the use of this format in the enzyme-modified comet assay (Muruzabal et al., 2019) and the in vitro DNA repair assay (Azqueta et al., 2014).
Example of 12 gel comet assay unit and minigels pipetted on a microscope slide:
Alternatively, 48 up to 96 well formats can be used for genotoxicity testing purposes. Various high throughput modifications of the assay were previously reviewed (Brunborg et al., 2014). Both in vivo and in vitro applications would gain great advantage from further improvements in efficiency, standardization of protocol, and throughput.
In addition, a CometChip platform has been described (Ge et al., 2014) that allows comet assay detection of DNA damage in mammalian cells patterned into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.
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> Azqueta A, Langie SA, Slyskova J, Collins AR. Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay--a methodological overview. DNA Repair (Amst). 2013 Nov;12(11):1007-10. doi: 10.1016/j.dnarep.2013.07.011
> L. María Sierra and Isabel Gaivão (eds.), Genotoxicity and DNA Repair: A Practical Approach, Methods in Pharmacology and Toxicology, DOI 10.1007/978-1-4939-1068-7_1, © Springer Science + Business Media New York 2014