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 <i>in vitro</i> 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.