Purpose: To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in an in vitro model of human conjunctival cells.
Methods: Chang's conjunctival cell line (ATCC CCL 20.2) was treated for 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation, mitochondrial mass and activity, free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. In addition, cell size and the expression of an apoptotic marker Apo2.7 were studied by flow cytometry.
Results: Timolol-BAC(+) induced a rapid decrease in cell viability ranging from 40% immediately after treatment to 85% 24h later. A small, significantly less important decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-). 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was reduced by 55% whereas it did not vary after 0.25% timolol -BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation, decrease in mitochondrial membrane potential and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Also reactive oxygen species (ROS) production was significantly more important after cell exposure to timolol-BAC(+).
Conclusion: In our model of conjunctival cells in vitro, timolol-BAC(+) induced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for ROS production and for cell viability variations. Oxidative stress could also play a role in timolol-BAC(+)-induced toxicity. In vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.