1. Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor activation and apoptosis
- Author
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Patrice Rat, Isabelle Larosche, Jean-Michel Warnet, Franc¸ois Pouzaud, Mélody Dutot, and Franc¸oise Brignole-Baudouin
- Subjects
Programmed cell death ,Ofloxacin ,Apoptosis ,Biology ,medicine.disease_cause ,Cell Line ,Cornea ,chemistry.chemical_compound ,Benzalkonium chloride ,Superoxides ,medicine ,Animals ,Humans ,chemistry.chemical_classification ,Reactive oxygen species ,Superoxide ,Receptors, Purinergic P2 ,Preservatives, Pharmaceutical ,Epithelial Cells ,Phosphatidylserine ,Flow Cytometry ,Molecular biology ,Anti-Bacterial Agents ,Oxidative Stress ,Biochemistry ,chemistry ,Microscopy, Fluorescence ,Rabbits ,Receptors, Purinergic P2X7 ,Benzalkonium Compounds ,Reactive Oxygen Species ,Conjunctiva ,Oxidative stress ,medicine.drug - Abstract
PURPOSE. To investigate in vitro whether eye toxicity is attributable to the preservative or the fluoroquinolone used in ophthalmic formulations. METHODS. 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. RESULTS. 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. CONCLUSIONS. 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. (Invest Ophthalmol Vis Sci. 2006;47:2812‐2819) DOI:10.1167/iovs.06-0224
- Published
- 2006