DNA damage is an important biomarker, in relation to human health and disease. It is primarily associated with cancer, as DNA damage is the precursor for mutations and chromosomal alterations, and they in turn can lead to cell transformation and tumorigenesis. However, the link is not straightforward, since almost all incident damage is repaired, and mutations and chromosome changes are effective only if they influence those genes - oncogenes, tumour suppressor genes and susceptibility genes - that are involved in the pathway to carcinogenesis. DNA damage is elevated in various diseases apart from cancer, whether as cause or effect.A common factor in disease aetiology and progression is oxidative stress, caused by overproduction of ROS or deficient antioxidant defences. The level of oxidation of bases in DNA - typically measured in PBMN cells, but also sometimes in tissues, or buccal epithelial cells, or cells retrieved from alveolar lavage - serves as a convenient marker for general oxidative damage, and thus for oxidative stress. The method of choice for measuring DNA damage in human biomonitoring is the comet assay, or single cell gel electrophoresis (Figure 1).
Figure 1. The comet assay, as used to measure DNA base damage (such as oxidised guanine)
Briefly, cells are embedded in agarose on a microscope slide and lysed in a buffer containing detergents and high salt which together dissolve cell and nuclear membranes, release soluble constituents, and strip histones from the DNA, leaving the DNA attached at intervals to a nuclear matrix, effectively as a series of supercoiled loops. Alkaline incubation follows, converting alkali-labile sites to strand breaks (SBs). During electrophoresis at high pH, DNA loops that have lost their supercoiling because of a SB are drawn towards the anode, forming a comet-like image when viewed by fluorescence microscopy. The % of DNA in the comet tail reflects the frequency of DNA breaks (calibrated using ionising radiation which introduces a known break frequency per Gray). The lysis step can be followed by digestion of DNA with an enzyme that recognises specific lesions and converts them to DNA breaks. The most commonly used enzyme is formamidopyrimidine DNA glycosylase (FPG), which recognises 8- oxoguanine (and some other altered guanines). Thus the comet assay can be used to measure not just SBs but oxidised bases - a far more specific indicator of ROS attack and oxidative stress.
Recently developed high throughput versions of the comet assay, with automated scoring, make it far more amenable for use as a biomonitoring tool, since epidemiological studies demand large numbers of samples. The comet assay has been further modified to measure the capacity of cells to repair DNA damage - an essential element in our defences against cancer. In an in vitro assay, a cell extract is incubated with substrate DNA containing specific lesions; oxidised bases (to measure base excision repair, BER), or UV induced pyrimidine dimers (to measure nucleotide excision repair, NER). The DNA breaks induced by repair enzymes in the extract are measured with the comet assay.
hCOMET [human Comet], is centred on the use of the comet assay in human studies, and most of what follows will be exclusively about this application.