The Comet Assay in Toxicology
Chapter 2: High-throughput Measurement of DNA Breaks and Oxidised Bases with the Comet Assay
Published:07 Oct 2016
Special Collection: 2016 ebook collectionSeries: Issues in Toxicology
A. Azqueta, I. C. Costa-Amaral, and A. R. Collins, in The Comet Assay in Toxicology, ed. D. Anderson and A. Dhawan, The Royal Society of Chemistry, 2nd edn, 2016, ch. 2, pp. 65-92.
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DNA is continuously under attack, from environmental chemicals and radiation and also from intrinsic damaging agents, notably reactive oxygen species. In the case of the latter, potential damage is kept under control by antioxidant defences. The damage that does occur is mostly removed by efficient repair pathways, but damage that remains when cells replicate DNA can lead to mutations and possible cell transformation. Oxidative damage to DNA is elevated in various human diseases, including cancers, though this does not necessarily indicate a causal relationship; oxidative stress resulting from the disease could cause DNA damage as a secondary effect. The comet assay is a sensitive and popular method for measuring DNA damage. The underlying principle is that, after cell lysis and removal of nuclear membranes and histones, DNA remains attached to a matrix in the form of supercoiled loops; a strand break in one loop relaxes supercoiling in that loop; on subsequent electrophoresis (normally at high pH), relaxed loops are pulled towards the anode, forming the tail of a comet-like image viewed by fluorescence microscopy. The percentage of DNA in the tail is proportional to break frequency. Oxidised bases are measured by including, after lysis, a digestion with an enzyme with endonuclease activity specific for oxidised pyrimidines (EndoIII or Nth) or for 8-oxoguanine and other products of purine oxidation (formamidopyrimidine DNA glycosylase, Fpg). The comet assay with Fpg has been shown to be more accurate than chromatographic techniques at measuring low levels of 8-oxoguanine, and it is the method of choice in human biomonitoring studies which aim to detect effects of occupational or environmental exposure, or of nutritional supplementation, e.g. with antioxidant-rich foods. A major application of the comet assay is in testing chemicals for genotoxic effects. OECD guidelines exist for use of the comet assay in in vivo experiments, and it is also widely used in in vitro testing. The inclusion of Fpg in the in vitro assay greatly increases the sensitivity of detection of various types of DNA-damaging agent (not only those that induce oxidation damage). In this chapter we provide a detailed protocol, covering all steps from embedding cells in agarose to scoring comets, and including the use of mini-gels (12 per slide) to increase throughput.