The comet assay: what can it really tell us?

https://doi.org/10.1016/S0027-5107(97)00013-4Get rights and content

Abstract

A range of applications of the alkaline comet assay is covered, from investigations of the physicochemical behaviour of DNA, through studies of cellular responses to DNA damage, to biomonitoring of human populations. The underlying principles of this assay are discussed, and new evidence presented which supports the concept of relaxation of supercoiled loops, rather than alkaline unwinding, as the primary reason for comet tail formation. DNA-damaging agents that do not induce strand breaks directly can be detected when cellular repair processes convert lesions to transient strand breaks; an approach is outlined here which maximises this effect and thus widens the scope of the assay. Purified repair enzymes, applied to DNA during the course of the comet assay procedure, greatly increase the sensitivity and specificity of the assay; recent developments with formamidopyrimidine glycosylase (recognising 8-OH-gua and other damaged purines) and uvrABC (for bulky lesions) are presented. The kinetics of cellular repair after low doses of oxidative damage have been followed with this modified comet assay. Finally, the successful measurement of biomarkers of oxidative damage in human populations establishes the comet assay as a valuable tool in molecular epidemiology.

Introduction

The comet assay (single cell gel electrophoresis or SCGE) is attractive for many reasons. Apart from the appeal of the images it produces, it is a quick, simple, sensitive, reliable and fairly inexpensive way of measuring DNA damage. But the most pleasing feature is the scope of its applications. It has made its name as a test for genotoxicity. However, it is also an invaluable tool for investigating fundamental aspects of DNA damage and cellular responses to this damage. At the other end of the scale, it is being used successfully to monitor DNA damage in human populations. In this article, we will provide, mostly from our own work, examples of investigations that span this range from molecular to epidemiological studies.

First, some thoughts about the behaviour of DNA in the comet assay. It should be obvious that DNA is not migrating as fragments, as in conventional electrophoresis, where the distance travelled is inversely related to fragment size. The alkaline comet assay resolves break frequencies up to a few thousand per cell, so the distances between breaks are of the order of 109 Da, definitely well beyond the range of fragment size for which conventional electrophoresis is suitable. The length of such a `fragment' is about 1 mm; the length of the tail of a comet is a few hundredths of this. So, are we perhaps seeing just the free, broken ends of DNA being pulled out into the tail? An analogy would be a tangled ball of string where a few judicious cuts can make pulling the ball apart much easier.

But nuclear DNA is not a tangle of string. Even after treatment with detergent and strong salt solution, as in the standard SCGE procedure, the nucleus (or nucleoid) has a structure; the DNA is organised as loops, which retain the supercoils that were formerly contained in the nucleosomes. The presence of supercoiled loops was deduced by Cook et al. [1], and they observed that, when DNA was broken by irradiation, supercoiling was relaxed and loops spilled out into a `halo' around the nucleoid core. By analogy, we can assume that the comet tail is made up of relaxed loops, and that the number of loops in the tail (or, simply, the relative tail intensity) indicates the number of DNA breaks. This model certainly fits with the observation that, with increasing amount of damage, the tail intensity rather than length increases, and tail length is presumably determined primarily by the length of the loops.

Whether the tail consists of relaxed DNA loops or of free ends could perhaps be decided by looking at the distribution of ends using an end-labelling technique to incorporate tagged nucleotides with subsequent immunofluorescent detection, since differing patterns would be predicted by the two models. Meanwhile, some evidence is presented in this paper which supports the loop model.

The alkaline comet assay detects single (and double) DNA strand breaks. But single-strand breaks (SSBs) are not the most interesting of lesions. They are quickly repaired, and are not regarded as a significant lethal or mutagenic lesion. Many genotoxic agents do not induce strand breaks directly. They may create AP sites, which are alkali-labile and are probably converted to breaks while DNA is in the electrophoresis solution at high pH. Furthermore, breaks will be transiently present when cells repair lesions via base excision or nucleotide excision, and so a high level of breaks in the comet assay may indicate either high damage or efficient repair. In fact, much useful information can be obtained by exploiting cellular repair to produce DNA breaks and thus to reveal or amplify the effects of genotoxins that otherwise would not show positive effects in the comet assay, as we will describe.

We have pioneered a modification of the comet assay which enormously increases its range, sensitivity and specificity. The DNA in the gel, following lysis, is digested with a lesion-specific repair endonuclease, which introduces breaks at sites of damage. In principle, any lesion for which a repair endonuclease exists can be detected in this way. We first showed this effect with endonuclease III, whose substrate is oxidised pyrimidines. Analogous trials with formamidopyrimidine glycosylase (FPG, active on 8-OH-gua) and uvrABC (recognising bulky lesions) have recently been carried out.

The ability to detect specific lesions opens up a new area of investigation – cellular DNA repair. It is generally true that the only unambiguous and quantitative indicator of cellular repair is the removal of damage from DNA. We have compared the kinetics of repair of oxidative damage in normal human lymphocytes and transformed epithelial (HeLa) cells.

Repair processes can be studied in subcellular systems, particularly useful in fractionation and reconstitution studies, with the aim of purifying repair proteins, and in characterising repair-defective mutant cells. Here again, the comet assay provides a simple assay system with the advantage of allowing examination of the sequential steps of incision/excision, and resynthesis/ligation.

Finally, at the other end of the scale, we have applied the modified comet assay, making use of lesion-specific enzymes, to the monitoring of human populations for oxidative DNA damage. The comet assay has proved to be a valuable tool in molecular epidemiology.

Section snippets

Cells: culture and treatment with DNA-damaging agents

HeLa (transformed human epithelial) cells were routinely cultured as monolayers in Glasgow-modified Eagle's Minimal Essential Medium (ICN Flow) with 5% foetal calf serum, 5% newborn calf serum, penicillin and streptomycin, at 37°C in a 5% CO2 atmosphere. Oxidative damage was introduced by replacing the medium with phosphate-buffered saline (PBS) containing H2O2 at the required concentration; treatment was for 5 min on ice. Cells were treated with methylmethane sulphonate (MMS) in medium for 30

DNA loops and alkaline unwinding

The comet assay, as now generally practised, includes incubation of DNA at high pH before and during electrophoresis, although the original work of Ostling and Johanson [8]employed near-neutral pH. The idea has developed that SCGE is related to methods for DNA break analysis, such as alkaline elution and alkaline unwinding. In these procedures, the separation of DNA strands at high pH is facilitated by the presence of strand breaks, and the extent or rate of separation is the index of DNA

Acknowledgements

We thank Dr. Serge Boiteux and Dr. Geoff Margison for gifts of FPG, and Dr. Richard Cunningham for the endonuclease III over-producing cell strain. The UvrABC proteins were obtained from Dr. P. van de Putte; purification of these proteins was done with the support of the EC Concerted Action on DNA Repair and Cancer. We are grateful to Dr. Susan Duthie, Catherine Gedik, Iona Fleming and Ma Ai-guo for their substantial contributions to the work described here. M.D. was in receipt of an ICRETT

References (20)

  • Cook, P.R., I.A. Brazell and E. Jost (1976) Characterization of nuclear structures containing superhelical DNA, J. Cell...
  • Singh, N.P., M.T. McCoy, R.R. Tice and E.L. Schneider (1988) A simple technique for quantitation of low levels of DNA...
  • Gedik, C.M., S.W.B. Ewen and A.R. Collins (1992) Single-cell gel electrophoresis applied to the analysis of UV-C damage...
  • Collins, A.R., S.J. Duthie and V.L. Dobson (1993) Direct enzymic detection of endogenous oxidative base damage in human...
  • Collins, A.R., A.-G. Ma and S.J. Duthie (1995) The kinetics of repair of oxidative DNA damage (strand breaks and...
  • Olive, P.L., J.P. Banáth and R.E. Durand (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and...
  • Collins, A.R., M. Dušinská, C.M. Gedik and R. Štětina (1996) Oxidative damage to DNA: do we have a reliable biomarker?...
  • Ostling, O. and K.J. Johanson (1984) Microelectrophoretic study of radiation-induced DNA damages in individual...
  • Green, M.H.L., A.P.W. Waugh, J.E. Lowe, S.A. Harcourt, J. Cole and C.F. Arlett (1994) Effect of deoxyribonucleosides on...
  • Yew, F.F.-H. and R.T. Johnson (1979) Ultraviolet-induced DNA excision repair in human B and T lymphocytes. II. Effect...
There are more references available in the full text version of this article.

Cited by (620)

  • DNA integrity under alkaline conditions: An investigation of factors affecting the comet assay

    2023, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
  • Measurement of oxidatively damaged DNA in mammalian cells using the comet assay: Reflections on validity, reliability and variability

    2022, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
    Citation Excerpt :

    This article is part of the Mutation Research special issue that pays tribute to Andrew Collins and his contribution to use of the comet assay. It circles around two questions that Andrew Collins asked about the comet assay in the 1990s, namely “What can it really tell us?” [10] and “Do we have a reliable biomarker?” [6].

View all citing articles on Scopus
View full text