Optimization of Alkaline Single Cell Gel Electropohoresis (Comet Assay) Protocol for Cells from Oral Cavity

Abstract

Comet assays or single cell gel electrophoresis for detection of DNA damage is a test that has been widely utilized to assess the effects of expositions to environmental genotoxicants. The test is also used to evaluate DNA damage related to chronic inflammation or preneoplastic and neoplastic conditions. The cells more frequently assessed in comet assay in humans are the peripheral blood lymphocytes, but there are other cell types that have been considered for that purpose. Among those, buccal cells have received attention for its suitability for comet assay, but there have been relatively few studies on comet assay in buccal epithelial cells. However, there are technical difficulties related to comet assay in buccal epithelial cells that justifies the attempts to develop or optimize protocols that could contribute to standardize the test allowing more widespread use of buccal cells in biomonitoring or clinical trials. In the present work, we compared three protocols: the standard technique of alkaline comet from Tice et al. 1999 and the protocols developed specifically for oral cavity cells from Valverde et al. 1997 and Szeto et al. 2005. We introduced modifications in the protocols related to, a device utilized to scrape the cells from the mucosa, the place and volume of sample enzymatic digestion, trypsin concentration, and also, the times for lysis incubation and unwinding. This modified protocol is a contribution to the optimization of comet assay for buccal cells and contributes to its utilization in biomonitoring human DNA damage.