Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor activation and apoptosis.
OBJECTIVE
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.
Dutot
Mélody
M
Laboratoire de Toxicologie, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes-Paris, and Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.
Pouzaud
François
F
Larosche
Isabelle
I
Brignole-Baudouin
Françoise
F
Warnet
Jean-Michel
JM
Rat
Patrice
P
D000818:Animals; D000900:Anti-Bacterial Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D001548:Benzalkonium Compounds / Q000633:toxicity*; D002460:Cell Line; D003228:Conjunctiva / Q000187:drug effects* / Q000378:metabolism; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D015242:Ofloxacin / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects; D011310:Preservatives, Pharmaceutical / Q000633:toxicity*; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D013481:Superoxides / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001548:Benzalkonium Compounds; C546468:P2RX7 protein, human; C546470:P2rx7 protein, rat; D011310:Preservatives, Pharmaceutical; D017382:Reactive Oxygen Species; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D013481:Superoxides; D015242:Ofloxacin