Sergey Shaposhnikov

DNA oxidation : Investigating its key role in environmental mutagenesis with the comet assay

DNA oxidation, which can have potentially serious mutagenic consequences, commonly accompanies exposure to environmental mutagens. Oxidised bases can be measured chromatographically, but spurious oxidation during sample preparation leads to serious over-estimation of low levels of damage. A more reliable approach is to employ endonucleases specific for oxidised bases, to introduce breaks in cellular DNA that are then most commonly measured using the comet assay (alkaline single cell gel electrophoresis). The two enzymes in general use are formamidopyrimidine DNA glycosylase, which detects primarily 8-oxo-7,8-dihydroguanine (8-oxoGua), and endonuclease III which recognises oxidised pyrimidines. We give a brief account of the recommended experimental procedures, and then describe applications in various areas of environmental research. Cultured cell lines or white blood cells have been exposed to a range of environmental mutagens, including natural products, industrial chemicals, radiation and nanoparticles. In vivo exposure of animals and humans to pollutants is more challenging but can give particularly valuable information in relation to real life exposure. Possibly the most useful application is in biomonitoring of human population groups suffering actual exposure to environmental or occupational mutagens. Finally, the potential use of this technique to monitor effects of contaminants in the natural environment has yet to be fully exploited.

Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation

DNA repair is one of the important determinants of susceptibility to cancer. It is therefore useful to be able to measure DNA repair capacity in samples from population studies. Our aim was, first, to develop a simple comet-based in vitro assay for nucleotide excision repair (NER), similar to that already in use for base excision repair (BER), and then to apply these in vitro assays to lymphocyte samples collected on several occasions from healthy subjects, to gain an impression of the degree of intra- and inter-individual variability. The in vitro assay consists of an incubation of lymphocyte extract with substrate nucleoid DNA from cells pretreated with specific damaging agent; either photosensitiser plus light to induce 8-oxoguanine, for BER, or short wavelength ultraviolet light irradiation for NER. In the new NER assay, which requires magnesium but not adenosine triphosphate, there was significant accumulation of UV-dependent incisions during a 30-min incubation of extract with DNA. We found significant correlations between individual repair rates from samples taken on different occasions; i.e. individuals have a characteristic repair capacity. There was also significant variation between individuals, to the extent of about fourfold for BER and tenfold for NER. There was no correlation between BER and NER rates. The BER and NER assays are simple to perform and can provide valuable information in molecular epidemiological studies in which DNA instability is an endpoint.

Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation

The comet assay is widely used to measure DNA damage and repair in basic research, genotoxicity testing and human biomonitoring. The conventional format has 1 or 2 gels on a microscope slide, 1 sample per slide. To increase throughput, we have designed and tested a system with 12 smaller gels on one slide, allowing incubation of individual gels with different reagents or enzymes. Thus several times more samples can be analysed with one electrophoresis run, and fewer cells and smaller volumes of test solutions are required. Applications of the modified method include treatment with genotoxic agents at different concentrations; simultaneous analysis of different lesions using a range of enzymes; analysis of cell extracts for DNA repair activity; and fluorescent in situ hybridisation (FISH) to comet DNA with specific labelled probes.

High throughput toxicity screening and intracellular detection of nanomaterials

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

Single-cell gel electrophoresis (the comet assay): loops or fragments?

Single‐cell gel electrophoresis, or the comet assay, is widely used to measure DNA damage and repair. Upon electrophoresis, the DNA of lysed, agarose‐embedded cells known as nucleoids, extends towards the anode in a structure resembling a comet, the relative intensity of the tail reflecting the frequency of DNA breaks. The structural organization of the DNA within comet preparations is not fully understood. We have used fluorescent in situ hybridization with large‐insert genomic probes and human Cot‐I DNA to investigate whether the production of the comet tail is simply explained by the relaxation of supercoiled DNA loops. We find that, under neutral electrophoresis conditions, when the tail and head DNA are double‐stranded, the probed sequence of DNA is seen as a linear array, consistent with extension from a fixed point on the nuclear core or matrix. After alkaline electrophoresis, the appearance of the fluorescent probes suggests that linear DNA has coalesced into a granular form.

Increasing the resolution of the comet assay using fluorescent in situ hybridization—a review

The comet assay (single-cell gel electrophoresis) is now the most popular method for measuring low levels of damage in cellular DNA. Cells are embedded in agarose on a microscope slide and lysed to produce nucleoids of supercoiled DNA attached to the nuclear matrix. Breaks in the DNA relax the supercoiling and allow DNA loops to expand, and on electrophoresis to move towards the anode, giving the appearance of a comet tail. The % of DNA in the tail reflects the break frequency. Digestion of nucleoid DNA with lesion-specific endonucleases extends the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with a genotoxic agent, incubating them and using the comet assay to follow the removal of the damage. An important feature of the assay is that damage is detected at the level of individual cells. The comet assay can be combined with fluorescent in situ hybridization, using labelled probes to particular DNA sequences, and DNA damage and repair can be examined at an even finer level of resolution. Here, we provide a general review of the technique, answer some technical and theoretical questions and give examples of applications of the method.

Controlling variation in the comet assay

Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period—for example, analysing samples of white blood cells from a large human biomonitoring trial—to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

In vitro genotoxicity testing of four reference metal nanomaterials, titanium dioxide, zinc oxide, cerium oxide and silver: towards reliable hazard assessment

There is serious concern about the potential harmful effects of certain nanomaterials (NMs), on account of their ability to penetrate cell membranes and the increased reactivity that results from their increased surface area compared with bulk chemicals. To assess the safety of NMs, reliable tests are needed. We have investigated the possible genotoxicity of four representative NMs, derived from titanium dioxide, zinc oxide, cerium oxide and silver, in two human cell lines, A549 alveolar epithelial cells and lymphoblastoid TK6 cells. A high-throughput version of the comet assay was used to measure DNA strand beaks (SBs) as well as oxidised purines (converted to breaks with the enzyme formamidopyrimidine DNA glycosylase). In parallel, cytotoxicity was measured with the alamarBlue® assay, and the ability of NM-treated cells to survive was assessed by their colony-forming efficiency. TiO2 and CeO2 NMs were only slightly cytotoxic by the alamarBlue® test, and had no long-term effect on colony-forming efficiency. However, both induced DNA damage at non-cytotoxic concentrations; the damage decreased from 3 to 24-h exposure, except in the case of CeO2-treated A549 cells. ZnO and Ag NMs affected cell survival, and induced high levels of DNA damage at cytotoxic concentrations. At lower concentrations, there was significant damage, which tended to persist over 24 h. The implication is that all four reference metal NMs tested—whether cytotoxic or not—are genotoxic. A full assessment of NM toxicity should include tests on different cell types, different times of incubation and a wide range of (especially non-cytotoxic) concentrations; a test for cell viability should be performed in parallel. Inclusion of Fpg in the comet assay allows detection of indirect genotoxic effects via oxidative stress.

Combining fluorescent in situ hybridization with the comet assay for targeted examination of DNA damage and repair.

The comet assay is a simple and sensitive method for measuring DNA damage. Cells are embedded in agarose on a microscope slide, lysed, and electrophoresed; the presence of strand breaks allows the DNA to migrate, giving the appearance of a comet tail, the percentage of DNA in the tail reflecting the break frequency. Lesion-specific endonucleases extend the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with damaging agent and monitoring the damage remaining at intervals during incubation. An important feature of the assay is that damage is detected at the level of individual cells. By combining the comet assay with fluorescent in situ hybridization (FISH), using labeled probes to particular DNA sequences, we can examine DNA damage and repair at the level of single genes or DNA sequences. Here we provide protocols for the comet assay and the FISH modification, answer some technical questions, and give examples of applications of the technique.

High throughput sample processing and automated scoring.

The comet assay is a sensitive and versatile method for assessing DNA damage in cells. In the traditional version of the assay, there are many manual steps involved and few samples can be treated in one experiment. High throughput (HT) modifications have been developed during recent years, and they are reviewed and discussed. These modifications include accelerated scoring of comets; other important elements that have been studied and adapted to HT are cultivation and manipulation of cells or tissues before and after exposure, and freezing of treated samples until comet analysis and scoring. HT methods save time and money but they are useful also for other reasons: large-scale experiments may be performed which are otherwise not practicable (e.g., analysis of many organs from exposed animals, and human biomonitoring studies), and automation gives more uniform sample treatment and less dependence on operator performance. The HT modifications now available vary largely in their versatility, capacity, complexity, and costs. The bottleneck for further increase of throughput appears to be the scoring.