Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of conjunctival cells
Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of conjunctival cells
复制标题
DOI：
10.1076/0271-3683(200002)20:2;1-d;ft085
复制
发表时间：
2000-02-01
影响因子：
2
通讯作者：
Baudouin, C
中科院分区：
文献类型：
--
作者：
De Saint Jean, M;Debbasch, C;Baudouin, C
目的。比较噻洛尔与苯扎氯铵（噻洛尔- bac +）和未保存的噻洛尔（噻洛尔- bac -）短时应用对人结膜细胞系的毒性。用0.1%、0.25%或0.4%噻莫罗尔-BAC（+）或BAC（-）处理Chang’s结膜细胞系（ATCC CCL 20.2） 15分钟，然后立即或24小时后检查。用微孔板冷光细胞术研究细胞活力、染色质凝聚和自由基生成。用结晶紫比色法测定相对细胞数。与0.001-0.000001%过氧化氢（H2O2）处理的细胞氧化应激模型进行比较。流式细胞术检测细胞大小和凋亡标志物Apo2.7的表达。Timolol-BAC（+）诱导细胞活力迅速下降，从治疗后立即下降40%到24小时后下降85%。在所有测试浓度的timolol-BAC（-）中也观察到细胞活力的微小初始下降，但24小时后，细胞活力趋于保持不变或细胞完全恢复。0.001% H2O2处理24h后，细胞活力下降，而在较低浓度下不进行修饰。0.25%噻莫洛尔- bac（+）处理24h后，相对细胞数量减少55%，而0.25%噻莫洛尔- bac（-）处理24h后，相对细胞数量没有变化。只有噻莫罗尔- bac（+）诱导染色质凝聚和细胞大小减小。此外，替莫洛尔- bac（+）处理的细胞过表达凋亡标志物Apo2.7。timolol-BAC（+）和BAC（-）均能诱导活性氧（ROS）的产生，且在0.25%和0.4% timolol-BAC（+）浓度下ROS的产生更为显著。只有0.001%和0.0001%的H2O2产生了显著的自由基。在体外结膜细胞模型中，噻莫洛尔- bac（+）可诱导不可逆的细胞毒性损伤，并伴有一定的细胞凋亡特征。timolol-BAC（-）的活性化合物可能负责活性氧的产生和细胞活力的变化。氧化应激在噻莫洛尔- bac(+)-诱导的毒性中的作用似乎不是主要的。抗青光眼药物的体外毒性作用可以部分解释长期治疗患者的一些眼表疾病。
Purpose. To compare the toxicity of a short-time application of timolol with 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 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation and free radicals 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 expression 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 decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-) but, 24h later, cell viability either tended to remain constant or cells completely recovered. Cell viability fell down after 24h exposure 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 reduced 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 marker Apo2.7. Both timolol-BAC(+) and BAC(-) induced reactive oxygen species (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 significant free radicals production.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 reactive oxygen species production and for cell viability variations. The role of oxidative 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.