Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of 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 a human conjunctival cell line. 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 and free radi cals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. The comparison was done with an oxidative stress model of cells treated with 0 .001-0.000001% hydrogen peroxide (H2O2). In addition, cell size and the exp ression of an apoptotic marker Apo2.7 were evaluated 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 initial decr ease in cell viability was also observed with all tested concentrations of timolol-BAC(-) but, 24h later, cell viability either tended to remain const ant or cells completely recovered. Cell viability fell down after 24h expos ure to 0.001% H2O2 whereas it was not modified at lower concentrations. 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was re duced by 55% whereas it did not vary after 0.25% timolol -BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation and cell size reduction . Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic m arker Apo2.7. Both timolol-BAC(+) and BAC(-) induced reactive oxygen specie s (ROS) production which was significantly more important when 0.25% or 0.4 % timolol-BAC(+) were applied. Only 0.001% and 0.0001% H2O2 generated a sig nificant free radicals production. Conclusion. In our model of conjunctival cells in vitro timolol-BAC(+) indu ced irreversible cytotoxic damage with some characteristics of apoptosis. T he active compound of timolol-BAC(-) could be responsible for reactive oxyg en species production and for cell viability variations. The role of oxidat ive stress in timolol-BAC(+)- induced toxicity seems not to be predominant. In vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in longterm treated patients.