Agent-specific DNA repair kinetic in 3T3 cells: a comparative study using the in vitro comet assay

Alkaline comet assay is a widely used technique for assessing DNA damage and repair at the single-cell level. However, the lack of standardized protocols, particularly for in vitro applications, makes it difficult to reproduce and compare the results from different laboratories. This study aimed to evaluate time-dependent DNA repair kinetics in 3T3 cells following exposure to six commonly used genotoxic agents, hydrogen peroxide (H₂O₂), potassium bromate (KBrO₃), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), and etoposide (EP). The cells were subjected to concentrations and exposure durations previously determined to induce maximum DNA damage. DNA repair was subsequently assessed at various intervals over a 24-h duration utilizing the comet assay. Each agent displayed a unique repair profile based on the type of DNA damage generated and cellular repair mechanisms involved. H₂O₂ and EMS induced rapid repair, whereas ENU- and EP-induced lesions were more persistent. These findings underscore the critical role of time as a variable in comet assay-based DNA repair assessments, contributing valuable reference data to support the standardization and validation of this assay for genotoxicity testing.