5-Fluorouracil (5-FU) is widely used in treating colorectal cancers, yet its therapeutic efficacy is often limited by its tendency to trigger excess reactive oxygen species (ROS), resulting in damage to healthy tissues. Riboflavin (RF), a natural and safe photosensitizer, has previously shown protective effects in our studies on cisplatin-induced toxicity under photoillumination, both in cell-based and animal models. Building on these findings, the present work examines whether RF can modulate 5-FU–associated oxidative stress.

Protein carbonyl analysis revealed that carbonyl levels increased only up to a certain 5-FU concentration, after which additional oxidative protein modification did not occur. When RF was introduced into the reaction system, the photoactivation of 5-FU was suppressed at lower drug concentrations. Plasmid DNA nicking experiments further supported this observation: high concentrations of 5-FU showed minimal impact on DNA integrity, whereas the presence of RF at lower 5-FU levels effectively masked plasmid nicking.

Moreover, lymphocytes exposed to low concentrations of 5-FU along with RF exhibited a marked reduction in DNA tail length in the comet assay, indicating reduced genotoxicity compared to cells treated with 5-FU alone. The overall pattern suggests that 5-FU displays biphasic behavior—its photoinduced ROS generation is quenched by RF at low drug levels, while at higher concentrations, dimer formation may explain the reduced toxicity.   Collectively, these findings indicate that RF, under white-light illumination, can significantly lessen 5-FU-induced oxidative and genetic damage, highlighting its potential as a supportive agent during chemotherapy.