De Saint Jean M, Brignole F, Bringuier A F, Bauchet A, Feldmann G, Baudouin C
Laboratoire de Biologie Cellulaire, INSERM U327, Faculté de Médecine Xavier Bichat, Université Paris VII, France.
Invest Ophthalmol Vis Sci. 1999 Mar;40(3):619-30.
The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro.
A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel.
Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative.
These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs.
本研究旨在调查苯扎氯铵（BAC），这种在大多数眼科局部用药溶液中用作防腐剂的物质，对体外结膜上皮细胞的作用。
将连续传代的人结膜细胞系（张氏结膜的黄 - 基尔伯恩衍生物）暴露于不同浓度（0.1% - 0.0001%）的BAC溶液中10分钟。在重新暴露于正常细胞培养条件下之前、处理后3小时、24小时、48小时和72小时对细胞进行检查。用结晶紫和3 - （4,5 - 二甲基噻唑 - 2 - 基） - 2,5 - 二苯基四氮唑溴盐比色法评估细胞数量和活力。通过流式细胞术研究凋亡标志物Apo 2.7、核抗原p53、膜蛋白Fas和Fas配体的表达以及DNA含量。用核酸特异性染料4',6'-二脒基 - 2 - 苯基吲哚二盐酸盐在载玻片上分析细胞核的形态学特征。用单克隆抗全细胞角蛋白抗体标记细胞骨架。此外，通过琼脂糖凝胶中的DNA电泳分析来检测凋亡。
细胞暴露于0.1%和0.05%的BAC后，处理后立即诱导细胞裂解。所有用0.01% BAC处理的细胞（100%）在24小时内延迟死亡，具有大多数凋亡特征（染色质浓缩和DNA片段化、细胞体积减小、凋亡标志物Apo 2.7的表达以及DNA含量的凋亡变化）。0.005%、0.001%、0.0005%和0.0001%的BAC等分试样在处理后24至72小时以剂量依赖方式诱导生长停滞和凋亡性细胞死亡。BAC处理后Fas和p53的表达没有变化。Fas配体始终为阴性。
这些结果表明，BAC在低至0.0001%的浓度下即可诱导细胞生长停滞和死亡。BAC诱导细胞死亡的方式是剂量依赖性的。高浓度BAC处理后细胞通过坏死死亡，而低浓度BAC处理时细胞通过凋亡死亡。BAC体外毒性的这一新方面可以部分解释长期使用含防腐剂药物进行局部治疗的患者中观察到的一些眼表疾病。
