Cetalkonium Chloride Induced a Dose-Dependent Toxicity on Corneal Endothelial Cells

<b><i>Purpose:</i></b> Cetalkonium chloride (CKC) is a cationic agent used in ophthalmical emulsions. Despite its growing application in ocular drug delivery, its safety profile on corneal endothelial cells remains unclear. This study evaluated the <i>in vitro</i> cytotoxic effects of CKC on human corneal endothelial cells (HCEnCs). <b><i>Methods:</i></b> HCEnCs were exposed to CKC at concentrations ranging from 0.03125 to 4.0 × 10^-4% (w/v) for 24-72 h. Cell viability was assessed using Cell Counting Kit-8 and lactate dehydrogenase (LDH) assays. Live/dead cell staining was performed for morphological confirmation. Reactive oxygen species (ROS) production and mitochondrial function were evaluated using DCFDA and MitoTracker assays. Western blot analysis was conducted to examine CKC-induced changes in cell survival pathways, including mammalian target of rapamycin (mTOR), protein kinase B (Akt), extracellular signal-regulated kinase (ERK), Bcl-2-associated X protein (BAX), and B-cell lymphoma-extra-large (Bcl-xL). <b><i>Results:</i></b> CKC induced dose- and time-dependent cytotoxicity in HCEnCs. Exposure to CKC at concentrations ≥0.25 × 10^-4 % for over 48 h significantly reduced cell viability and increased LDH release and ROS production. At concentrations ≥1.0 × 10^-4 %, cell viability was reduced by more than 50% at both 48 and 72 h. In surviving cells, mitochondria showed minimal structural alterations. CKC exposure inhibited cell survival pathways such as mTOR, Akt, Bcl-xL, and ERK, while the proapoptotic pathway marker BAX was upregulated. <b><i>Conclusion:</i></b> CKC exhibits dose- and time-dependent toxicity in HCEnCs, mediated by oxidative stress and the modulation of survival and apoptotic signaling pathways. However, it is challenging to directly extrapolate laboratory conditions to the clinical setting. Therefore, these findings should be interpreted with caution, particularly in scenarios where direct exposure of the corneal endothelium to CKC-containing formulations is anticipated.