Debbasch C, Brignole F, Pisella P J, Warnet J M, Rat P, Baudouin C
Unit of Cellular Pharmacotoxicology, Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, the Toxicology Laboratory, University of Paris-V., France.
Invest Ophthalmol Vis Sci. 2001 Mar;42(3):642-52.
To investigate some of the toxicity mechanisms of 10 preservatives currently used in ophthalmic solutions in vitro.
A continuous human conjunctival cell line was treated with different concentrations of various preservatives for 15 minutes and for 15 minutes followed by 24 hours of cell recovery: three benzalkonium chlorides (BACs) with different hydrocarbon chain length, benzododecinium bromide (BOB), cetrimide (Cet), phenylmercuric nitrate (PM), thimerosal (thi), methyl parahydroxybenzoate (MPHB), chlorobutanol (clb), and EDTA. An inhibition study was then conducted using a 1-hour vitamin E pretreatment followed by a 15-minute BAC treatment. Membrane integrity was assessed using a neutral red test and chromatin condensation with a Hoechst 33342 test. Reactive oxygen species were measured using dichlorofluorescein diacetate test for H2O2 production and hydroethidine test for O2.- production. These tests were performed using microplate cold light cytofluorometry. Cell size and DNA content were also analyzed using flow cytometry. Confocal microscopy was used to explore morphologic changes.
A significant decrease of membrane integrity with chromatin condensation was observed with all the quaternary ammoniums tested at concentrations of 0.005% and higher. The effect was amplified after 24 hours of cell recovery. The other preservatives tested did not decrease membrane integrity. H2O2 production was observed with all the preservatives, whereas O2.- production was significantly higher with the quaternary ammoniums at 0.005% and 0.01%, compared with the other preservatives. Flow cytometry results confirmed the cytotoxicity observed with cold light cytofluorometry.
The quaternary ammoniums tested (BAC, BOB, and Cet) were the most cytotoxic preservatives in the current model. An apoptotic mechanism appeared to be present at low concentrations of quaternary ammoniums, whereas a necrotic process appeared at higher concentrations. Superoxide anions may play an important role in tissue damage induced by preservatives in ocular surface disorders.
体外研究目前眼科溶液中使用的10种防腐剂的一些毒性机制。
用不同浓度的各种防腐剂处理连续的人结膜细胞系15分钟，然后恢复24小时：三种具有不同烃链长度的苯扎氯铵（BAC）、苯度氯铵（BOB）、西曲溴铵（Cet）、硝酸苯汞（PM）、硫柳汞（thi）、对羟基苯甲酸甲酯（MPHB）、三氯叔丁醇（clb）和乙二胺四乙酸（EDTA）。然后进行一项抑制研究，先用维生素E预处理1小时，随后用BAC处理15分钟。使用中性红试验评估膜完整性，用Hoechst 33342试验评估染色质凝聚。使用二氯荧光素二乙酸酯试验检测过氧化氢（H2O2）的产生来测量活性氧，用氢乙锭试验检测超氧阴离子（O2.-）的产生。这些试验使用微孔板冷光细胞荧光测定法进行。还使用流式细胞术分析细胞大小和DNA含量。使用共聚焦显微镜探索形态学变化。
在所测试的所有季铵盐浓度为0.005%及更高时，观察到膜完整性显著下降并伴有染色质凝聚。在细胞恢复24小时后，这种效应增强。所测试的其他防腐剂未降低膜完整性。所有防腐剂均观察到H2O2的产生，而在0.005%和0.01%浓度下，季铵盐产生的O2.-明显高于其他防腐剂。流式细胞术结果证实了冷光细胞荧光测定法所观察到的细胞毒性。
在所测试的季铵盐（BAC、BOB和Cet）是当前模型中细胞毒性最大的防腐剂。在低浓度季铵盐时似乎存在凋亡机制，而在高浓度时出现坏死过程。超氧阴离子可能在眼表疾病中防腐剂诱导的组织损伤中起重要作用。
