The use of Comet assay for detection of genotoxic effect of benzo[a]pyrene

Introduction: The Comet assay, or single-cell gel electrophoresis, is a highly sensitive technique for detecting DNA damage at the single-cell level. It enables the identification of strand breaks, alkali-labile sites, and cross-linking in individual cells. This assay is extensively used in genotoxicity testing, cancer research, and studies on DNA repair mechanisms. Benzo[a]pyrene, a known genotoxic compound detected in cigarette smoke, diesel exhaust, and industrial waste, poses significant health risks.
Objective: This study aims to evaluate the applicability of the Comet assay in detecting DNA damage induced by exposure to this cytotoxic agent.
Materials and Methods: Peripheral blood mononuclear cells were treated with varying concentrations of benzo[a]pyrene, followed by Comet assay under alkaline conditions. Cells were embedded in agarose, lysed, and subjected to electrophoresis. DNA migration was visualized using silver staining, and damage was quantified by visually scoring 50 cells per slide.
Results: The results revealed a dose-dependent increase in DNA damage, as reflected by a significant increase in comet tail arbitrary units. Higher concentrations of benzo[a]pyrene resulted in pronounced DNA fragmentation, indicating increased genotoxicity.
Conclusion: These findings affirm the effectiveness of the Comet assay in detecting DNA damage and monitoring cellular responses to genotoxic agents. The study highlights benzo[a]pyrene as a potent genotoxic agent, reinforcing its classification as a Group I carcinogen. This research underscores the importance of genotoxic assessments in evaluating and understanding the cellular impact of environmental and industrial toxins.