This study aimed to provide integrative mechanistic insights into benzalkonium chloride (BAK)-induced cytotoxicity in human corneal epithelial (HCE-T) cells, with a particular focus on the crosstalk between oxidative stress, DNA damage, ferroptosis-related events, apoptosis, and inflammatory signaling. HCE-T cells were exposed to increasing concentrations of BAK. Cell viability and membrane integrity were evaluated using MTT and lactate dehydrogenase assays, respectively. Oxidative stress was assessed by measuring intracellular reactive oxygen species (ROS) and antioxidant defense markers, including superoxide dismutase, catalase, glutathione peroxidase, and total antioxidant capacity. Genotoxic effects were analyzed using the comet assay, while lipid peroxidation was quantified by malondialdehyde levels. To distinguish cell death mechanisms, ferroptosis-related events (iron accumulation) and apoptotic markers (caspase-3/7 activity and DNA fragmentation via TUNEL assay) were examined. Additionally, inflammatory responses were evaluated by measuring the expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α. The results demonstrate that BAK induces dose-dependent cytotoxicity in HCE-T cells, characterized by excessive ROS generation, impaired antioxidant defenses, significant DNA damage, and enhanced lipid peroxidation. Iron accumulation and oxidative lipid damage suggested a prominent role for ferroptosis-related events, occurring alongside apoptosis. Moreover, BAK exposure markedly activated inflammatory signaling pathways. Collectively, these findings highlight a complex, interconnected network of oxidative stress, DNA damage, ferroptosis-related events, and inflammation underlying BAK-induced corneal epithelial toxicity. This study provides mechanistic evidence relevant to ocular safety and preservative formulation strategies.
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