Application of the comet assay for assessment of oxidative DNA damage in circulating lymphocytes of Tetralogy of Fallot patients

Abstract

Tetralogy of Fallot (TOF) is a common and severe cyanotic congenital heart defect characterized by frequent episodes of hypoxia due to cyanosis. The hypoxia of cyanotic heart disease results in a down-regulation of antioxidant defenses, making cells vulnerable to oxidant damage, which subsequently leads to the single strand breaks and oxidative DNA damage. Quantification of DNA damage was performed in circulating lymphocytes of Tetralogy of Fallot patients ( n = 63) and healthy controls ( n = 65). The damage of DNA was assessed by alkaline comet assay in lymphocytes isolated from all children followed by silver staining. The DNA migrates out of the nucleus forming a tail, which represents the extent of DNA damage in individual cells. TOF patients exerted a higher percent of comet tails, which are indicative of DNA damage, when compared to control children ( p < 0.001). The mean comet tail length was significantly higher in TOF patients (2.57 ± 0.29) when compared with healthy controls (1.28 ± 0.32). The results showed that hypoxia is associated with an increase in the level of oxidants and a simultaneous decrease in the level of antioxidants in patients. Hence, the present study concludes unequivocally that hypoxia causes oxidative DNA damage in TOF patients.