Evaluation of DNA damage after tourniquet-induced ischaemia/reperfusion injury during lower extremity surgery

Ischemia/reperfusion (I/R) injury represents a source of substantial morbidity and mortality in various statuses that is, coronary bypass, myocardial infarction, and so on. Oxygen free radicals, formed during I/R, have been proposed as one of the main causes of tissue injury and play important role in I/R injury. Leucocytes have been shown to play an important role in the development of tissue injury after I/R. Accordingly, numerous studies have shown that even short-time I/R-induced DNA damage can be investigated in human peripheral leucocytes using the alkaline single-cell gel-electrophoresis assay (comet assay). After ischaemia, the genotoxic damage detected in human peripheral leucocytes locally in the reperfused tissue results in numerical, morphologic and biochemical alterations of all circulating white blood cells in the human organism. It leads particularly to the release of substantial amounts of oxygen radicals and other reactive agents. Simultaneously, local ischaemia in the reperfused tissue is extended to the whole body systemically through these activated inflammatory cells and, possibly, results in secondary detectable tissue damage in endothelial cells of the systemic circulation inducing prolonged DNA damage even in the early reperfusion period. Thus, we aimed to investigate whether the I/R during the routinely practised operation causes DNA damage, since other published studies were on animal and in vitro models and did not exactly reflect the operation procedure in the clinic. We measured DNA damage (single-strand breaks, oxidised purines and pyrimidines) by modified alkaline comet assay using two bacterial enzymes (formamidopyrimidine glycosylase (Fpg), endonuclease III (EndoIII)), which recognise oxidised purines and pyrimidine bases, in patients who had lower extremity surgery. There was no statistically significant difference in DNA damage between time periods (before surgery, after I/R; T1, T2 and T3). According to our findings, we indicate that at the molecular level DNA is damaged due to the I/R during the routinely practised operation; however, this effect could not be determined phenotypically. Thus, we may suggest that the surgeons and anaesthetist/operation room personnel could be informed about that fact and encouraged to use antioxidants and/or apply prophylaxis. Our findings showed that I/R injury did not induce DNA damage. Further studies of this approach are needed.