Dutot Mélody, Pouzaud François, Larosche Isabelle, Brignole-Baudouin Françoise, Warnet Jean-Michel, Rat Patrice
Laboratoire de Toxicologie, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes-Paris, and Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.
Invest Ophthalmol Vis Sci. 2006 Jul;47(7):2812-9. doi: 10.1167/iovs.06-0224.
To investigate in vitro whether eye toxicity is attributable to the preservative or the fluoroquinolone used in ophthalmic formulations.
Corneal and conjunctival cell lines were incubated with preserved (benzalkonium chloride [BAC]) or unpreserved ofloxacin solutions for 15 minutes. Several concentrations of BAC were also tested (0.0025%-0.01%). Membrane integrity, reactive oxygen species, and superoxide anion production were assessed with the neutral red test, the 2',7'-dichlorofluorescein diacetate test, and the dihydroethidium test, respectively. P2X7 cell death receptor activation was evaluated using the YO-PRO-1 assay and apoptosis (chromatin condensation and translocation of phosphatidylserine) using the Hoechst 33342 and annexin V-FITC dyes. Tests were performed with microplate cytofluorometry, inverted fluorescence microscopy, and flow cytometry.
The preserved solution and all tested BAC concentrations induced a significant decrease in membrane integrity, unlike the unpreserved ofloxacin. Reactive oxygen species and superoxide anion productions observed for all solutions were significantly higher for the preserved ofloxacin and BAC solutions, which also induced apoptosis (chromatin condensation and translocation of phosphatidylserine) through P2X7 pore opening, whereas unpreserved ofloxacin did not.
The cytotoxicity observed with fluoroquinolone eye drops seems to be caused mainly by the preservative, which induced P2X7 cell death receptor activation associated with oxidative stress and apoptosis. Therefore, it is recommended that fluoroquinolone be used in preservative-free formulations.
在体外研究眼部毒性是归因于眼科制剂中使用的防腐剂还是氟喹诺酮类药物。
将角膜和结膜细胞系与含有防腐剂（苯扎氯铵[BAC]）或不含防腐剂的氧氟沙星溶液孵育15分钟。还测试了几种浓度的BAC（0.0025%-0.01%）。分别用中性红试验、2',7'-二氯荧光素二乙酸酯试验和二氢乙锭试验评估膜完整性、活性氧和超氧阴离子的产生。使用YO-PRO-1检测评估P2X7细胞死亡受体激活情况，使用Hoechst 33342和膜联蛋白V-FITC染料评估凋亡（染色质浓缩和磷脂酰丝氨酸易位）。通过微孔板细胞荧光测定法、倒置荧光显微镜和流式细胞术进行检测。
与不含防腐剂的氧氟沙星不同，含有防腐剂的溶液和所有测试浓度的BAC均导致膜完整性显著降低。所有溶液中观察到的活性氧和超氧阴离子产生在含有防腐剂的氧氟沙星和BAC溶液中显著更高，这也通过P2X7孔开放诱导了凋亡（染色质浓缩和磷脂酰丝氨酸易位），而不含防腐剂的氧氟沙星则没有。
氟喹诺酮眼药水观察到的细胞毒性似乎主要由防腐剂引起，防腐剂诱导了与氧化应激和凋亡相关的P2X7细胞死亡受体激活。因此，建议使用不含防腐剂的氟喹诺酮制剂。
