Toxicity of preserved and unpreserved beta-blocker eyedrops in an in vitromodel of human conjunctival cells

Purpose: To compare the toxicity of a short-time application of timolol wit h 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 15m in. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined imm ediately or 24h later. Cell viability, chromatin condensation, mitochondria l mass and activity, free radicals production were studied by microplate co ld 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, significant ly less important decrease in cell viability was also observed with all tes ted concentrations of timolol-BAC(-). 24h after treatment with 0.25% timolo l-BAC(+), the relative cell number was reduced by 55% whereas it did not va ry after 0.25% timdol -BAC(-) treatment. Only timolol-BAC(+) induced chroma tin condensation, decrease in mitochondrial membrane potential and cell siz e 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(+) ind uced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for ROS producti on and for cell viability variations. Oxidative stress could also play a ro le in timolol-BAC(+)-induced toxicity. In vitro toxic effects of antiglauco ma drugs could, in part, explain some ocular surface disorders in long-term treated patients.