Mutagenesis - current issue

The comet assay: topical issues

2008-05-02

The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo—or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.

The use of the comet assay in the study of human nutrition and cancer

Wasson, G. R., McKelvey-Martin, V. J., Downes, C. S. — 2008-05-02

The influence of diet on carcinogenesis is a hugely complex area; not only is the consumption of major dietary factors such as meat, fat and fruits and vegetables associated with increased or decreased risk of a range of cancers but also an increasing number of specific nutrients such as vitamins, minerals and phytochemicals are being proposed as the next ‘superfoods’ to combat the development of cancer. As well as epidemiological studies to determine the association of these dietary factors with cancer risk, it is also essential to investigate the underlying mechanisms through which these factors may causally influence carcinogenesis. The comet assay provides a relatively simple, cheap and rapid method to examine DNA damage and repair and is, therefore, an ideal biomarker for the study of the effects of nutrition on cancer. This review focuses on the use of the comet assay in studies involving human subjects or human cell lines, which investigate the effects of various nutrients on biomarkers relevant to carcinogenesis, and discusses the potential of the comet assay and its various modifications for use as cancer-related biomarkers suitable for use in nutritional studies.

Using the alkaline comet assay in prognostic tests for male infertility and assisted reproductive technology outcomes

Lewis, S. E. M., Agbaje, I. M. — 2008-05-02

Infertility affects one in six couples in Europe during their reproductive years with dysfunctional sperm being one of the most common causes. Conventional semen analysis has proven variable and lacking in prognostic value so, over the past decade, more useful molecular fertility biomarkers have been explored. Among the tests showing most promise are those measuring sperm DNA quality. Sperm DNA damage has been closely associated with numerous indicators of reproductive health, including, fertilization, embryo quality, implantation, spontaneous abortion and childhood diseases. It therefore has great potential as a prognostic test for assisted reproductive treatment (ART), when couples are presenting with male infertility. Unlike somatic cells, sperm have a unique tightly compacted chromatin structure. Our group has modified the alkaline comet assay for use with sperm. Sperm DNA also differs from somatic cells in its high susceptibility to oxidative damage; this is largely due to the presence of abundant polyunsaturated fatty acids acting as substrates for reactive oxygen species (ROS) and its lack of repair mechanisms. Consequently, the effects of ROS and antioxidant protection on sperm DNA fragmentation have been widely investigated. In this review, the relationship between actual sperm DNA damage as determined by the alkaline comet assay and potential DNA damage as measured by DNA adduct testing will also be examined and the potential of routine clinical practices such as cryopreservation and prolonged incubation to induce further DNA damage was investigated. Finally, the usefulness of sperm DNA tests as prognostic markers and in particular, the opportunities and challenges provided by DNA testing in male fertility determination will be discussed.

Statistical issues in the use of the comet assay

Lovell, D. P., Omori, T. — 2008-05-02

The comet or single-cell gel electrophoresis assay is now widely used in regulatory, mechanistic and biomonitoring studies using a range of in vitro and in vivo systems. Each of these has issues associated with the experimental design which determine to a large extent the statistical analyses than can be used. A key concept is that the experimental unit is the smallest ‘amount’ of experimental material that can be randomly assigned to a treatment: the animal for in vivo studies and the culture for in vitro studies. Biomonitoring studies, being observational rather than experimental, are vulnerable to confounding and biases. Critical factors in any statistical analysis include the identification of suitable end points, the choice of measure to represent the distribution of the comet end point in a sample of cells, estimates of variability between experimental units and the identification of the size of effects that could be considered biologically important. Power and sample size calculations can be used in conjunction with this information to identify optimum experimental sizes and provide help in combining the results of statistical analyses with other information to aid interpretation. Interpretation based upon the size of effects and their confidence intervals is preferred to that based solely upon statistical significance tests. Statistical issues associated with the design and subsequent analyses of current validation studies for the comet assay include the identification of acceptable levels of intra- and inter-laboratory repeatability and reproducibility and criteria for dichotomizing results into positive or negative.

Potential use of the comet assay in the clinical management of cancer

McKenna, D. J., McKeown, S. R., McKelvey-Martin, V. J. — 2008-05-02

The comet assay has been widely used to measure a range of cellular responses to DNA damage and has found applications in genotoxicity studies, bio-monitoring, ecological testing and in the study of human disease. This review discusses how the comet assay has been applied to the study of DNA damage and repair associated with cancer. The potential of the assay as a tool for predicting an individual's tumour sensitivity to radiation and to various chemotherapeutic drugs is examined, as well as outlining the usefulness of the assay in assessing oxidative stress within tumours. In addition, we review the use of the comet assay in investigations of the DNA-damaging effect of anti-neoplastic drugs and radiation used during cancer therapy. The advantages and limitations of the comet assay in carrying out all these studies are outlined, and the suitability of the comet assay for use in the clinical management of cancer is discussed.

The comet assay in human biomonitoring: gene-environment interactions

Dusinska, M., Collins, A. R. — 2008-05-02

The comet assay is the method of choice for measuring DNA damage, of various sorts, in human cells such as lymphocytes obtained in the course of population-based studies of environmental and occupational exposure to different genotoxic agents, including radiation, chemicals and oxidative stress. It is noted for its versatility and the breadth of its possible applications. In terms of simplicity, cost, small amount of material required, sensitivity and reliability, the comet assay in its various modifications has few serious competitors. When standardized and validated, the comet assay can provide invaluable information in the areas of hazard identification and risk assessment of environmental and occupational exposure, diseases linked with oxidative stress (e.g. diabetes and cardiovascular disease), nutrition, monitoring the effectiveness of medical treatment and investigating individual variation in response to DNA damage that may reflect genetic or environmental influences. The information obtained could lead to individual advice on lifestyle changes to promote health and especially on relative risks of genotoxic exposure to environmental pollution.

Ecotoxicological applications and significance of the comet assay

Jha, A. N. — 2008-05-02

Application of the single-cell gel electrophoresis or comet assay has revolutionized the field of genetic ecotoxicology or eco-genotoxicology. It is a rapid, sensitive and relatively inexpensive method providing the opportunity to study DNA damage (including oxidative damage), repair and cell death (apoptosis) in different cell types without prior knowledge of karyotype and cell turnover rate. The assay has, however, often attracted criticism for its lack of ecotoxicological relevance. In addition, in contrast to genetic toxicology where rapid technical progress has been made to improve cell- and tissue-specific adoption of the assay, only limited advancement has been made to transfer the methodologies to ecotoxicological studies. While reviewing the recent information available in the literature and underscoring the importance of induced genetic damage in natural species, the aims of this article are to (i) highlight and judiciously analyse the ecotoxicological relevance of the assay; (ii) attempt to correlate the comet response with other relevant biological responses or biomarkers; (iii) identify the technical challenges and various factors affecting its application in order to make it reliable, reproducible and robust; (iv) critically compare the technical developments in genetic toxicology and genetic ecotoxicology and (v) evaluate the future developments with respect to applications of the assay. It is suggested that while complementing other ecotoxicological parameters and further improving the methodologies, the comet assay will continue to play an important role in genetic ecotoxicology to determine induced genetic damage, which has significant consequences for short- and long-term survival of the natural or wild species. Information obtained through integrated studies using simultaneous applications of multiple biomarkers on different wild organisms could also provide an holistic dimension of toxicological impact of environmental contaminants for the protection of human health.

Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

2008-05-02

The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose–response relationships of cells exposed to -radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10⁶ bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14–0.33] and 0.31 (95% CI: 0.20–0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.

Recommendations for design of the rat comet assay

Smith, C. C., Adkins, D. J., Martin, E. A., O'Donovan, M. R. — 2008-05-02

Although the rodent comet assay is gaining acceptance as a standard technique for evaluating DNA damage in vivo, there is no internationally accepted guideline for its conduct and several aspects of its experimental design have not been optimized. For example, no standard positive control is used, there is no agreement on how tissue toxicity should be measured and sources of experimental variability have not been considered in relation to experimental design. This study showed that methylnitrosourea is a good alternative positive control inducing DNA damage in all tissues examined (stomach, liver, blood and bone marrow) over a dose range of 25–100 mg/kg at both 3 and 24 h after treatment. At the highest dose, significant toxicity was seen in all tissues using the neutral diffusion assay and also by histopathological/haematological analysis, except in the liver where no change was seen even 7 days after dosing. Analyses using control data pooled from several studies showed that, as expected, the greatest variability was seen between tissue preparations from different animals and that different numbers of animals were required to detect the same fold increases in different tissues. Power analyses showed that, preparing three gels for each tissue and scoring 50 nuclei per gel, a group of six animals allows 2-fold increases over control in the liver, bone marrow and stomach and a 3-fold increase in blood to be detected with 80% probability. It is recommended that similar investigations of experimental variability should be performed to determine optimal experimental design in any laboratory using the rodent comet assay.