Frontiers in Genetics (Jun 2015)
The comet assay in oral leucocytes: a non-invasive alternative in human studies?
Abstract
The comet assay is the method of choice for measuring DNA damage. In human studies mostly peripheral blood mononuclear cells (PBMC) are used to conduct this single cell gel electrophoresis (Azqueta and Collins, 2013) and also a method for whole blood was described recently (Al-Salmani et al., 2013). However, oral viable leucocytes, obtained via mouth washes, might serve as a non-invasive alternative for the comet assay (Osswald et al., 2003). During a cross-sectional human study, comet assay of oral leucocytes in addition to PBMC and whole blood was performed. In total 146 female patients with diabetes mellitus type 2 donated oral leucocytes. Therefore, they rinsed their mouth with tap water which than was added to a sodium chloride solution. After washing the cells, the separation of oral leucocytes and buccal cells was performed with density gradient centrifugation. Oral leucocytes were counted and comet assay (strand breaks, resistance to H2O2, FPG-sensitive sites) was performed. Cell counts of oral leucocytes differed substantially and therefore are the limiting step. Only 110 patients (75 %) donated enough cells to perform the Comet assay. Resulting % Tail DNA (strand breaks, resistance to H2O2, FPG-sensitive sites) differed between PBMC vs. oral leucocytes and whole blood vs. oral leucocytes, but also between PBMC vs. whole blood. Nevertheless, values of % Tail DNA of all three matrices were in a biological comparable range. If sampling of oral leucocytes gets more reliable, it could be a fast alternative method to measure DNA damage in larger cohorts without any invasive needs.
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