The comet assay is robust, sensitive, and easy to set up in a lab, requiring simple equipment and reagents. As a result, it has been widely adopted as a method for measuring DNA damage in human trials, including studies of occupational and environmental exposure, case-control studies, nutritional intervention trials….
There are now enormous amounts of human comet assay data. In addition to DNA strand breaks, many studies have included measurement of DNA base damage (specifically oxidation), using lesion-specific enzymes such as formamidopyrimidine DNA glycosylase (Fpg).
In a smaller number of studies, DNA repair capacity has also been measured.
Several years ago, we launched a project, ComNet, to collect comet assay data from human trials into a common database and to carry out a pooled analysis.
The over-riding aim of ComNet was to find definitive answers to questions such as:
Does DNA damage increase with age?
Are there differences in DNA damage between men and women? smokers and non-smokers?
What are the effects of exposure to various environmental and occupational mutagens?
There is a further, challenging, question: Is DNA damage a biomarker of cancer risk?
• While DNA damage might be elevated in cancer patients, this could be an effect rather than a cause.
• DNA damage is, of course, an initiating event in carcinogenesis – but almost all DNA damage is repaired, and so the link with cancer is tenuous.
• Prospective (or retrospective) cohort studies are required.
Our COST Action is carrying out the aims of ComNet – and also attempting to standardise the comet assay so that inter-laboratory comparisons are easier and more reliable.
hCOMET has members from 25 countries.
The 4-year Action ended in October 2020, but collaborations are continuing.
See the hCOMET website for more details: www.hcomet.eu
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What next? One possibility - a retrospective human trial to determine whether DNA damage measured on blood cells can predict the risk of cancer.
The COST Action hCOMET succeeded in collecting comet assay data from almost 20,000 individuals taking part in various human biomonitoring trials. Our statistician members managed to analyse the data, taking account of the technical differences in the way the assay was performed in different labs, and concentrating on demographic factors. DNA damage (strand breaks) increases with age, and there is no significant difference in damage between males and females. The most exciting finding comes from a subset of data, from trials where there was follow-up of the participants, so that we have data on subsequent disease and death. It turns out that those individuals with higher levels of damage show increased mortality in the following decades. With a limited number of subjects, it was not possible to link DNA damage with risk of specific diseases - and there were not enough data on DNA damage other than strand breaks. So further studies are necessary. These results have been submitted for publication.
One option to investigate this further would be to set up a dedicated prospective study, measuring DNA damage and monitoring the subjects for disease and death. This would be a long-term project, involving thousands of subjects, and very expensive. An alternative is to work with existing prospective cohort studies in which biobanks have been established. A so-called 'nested case-control study' would be performed; a group of patients with a specific disease would be selected, along with a control group matched for age, sex, etc., and samples taken early in the trial would be retrieved from the biobank and analysed for DNA damage. Several hCOMET researchers have been looking at the feasibility of performing the comet assay on blood samples frozen for long periods; leukocytes from whole blood or buffy coat (the upper layer of the red cell pellet when blood is centrifuged, enriched in white cells) give good results, without any indication of additional damage caused by storage. We are hoping to link up with one or more suitable long-term human trials to investigate the important question, whether DNA damage measured with the comet assay can serve as a predictive maker of risk of cancer (or other diseases).
