Mitochondrial activity and glutathione injury in apoptosis induced by unpreserved and preserved beta-blockers on Chang conjunctival cells.
OBJECTIVE
Quaternary ammonium ions have been demonstrated to induce apoptosis correlated with superoxide anion production in vitro. The purpose of this study was to further investigate the mechanisms of benzalkonium chloride (BAC), unpreserved and preserved beta-blocker eye-drops-induced programmed cell death, with special attention to the roles of mitochondrial transmembrane potential and intracellular reduced glutathione.


METHODS
Chang conjunctival cells were incubated with different concentrations of unpreserved or preserved timolol (0.1%, 0.25%, and 0.4%), or carteolol (1% and 2%), or BAC (0.0001% to 0.01%) for 15 minutes, or for 15 minutes with a 24-hour recovery period in normal medium. Cellular viability (neutral red test), mitochondrial activity (rhodamine 123 test), intracellular reduced glutathione (monochlorobimane test), DNA condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests) were evaluated using microplate cold-light cytofluorometry.


RESULTS
A significant, concentration-dependent decrease in cellular viability was found with preserved beta-blockers and with BAC alone, whereas unpreserved preparations did not show any toxicity. Only preserved beta-blockers induced chromatin condensation associated with an alteration of mitochondrial activity and a decrease of glutathione, suggesting an apoptotic phenomenon. BAC increased glutathione after 15 minutes, whereas a decrease was observed after a recovery period. ROS production was found with preserved formulations at significantly higher levels than those observed with unpreserved drugs.


CONCLUSIONS
This in vitro study demonstrates that oxidative stress, evidenced by enhanced ROS production and mitochondrial injury rather than by cellular glutathione depletion, is a mechanism involved in apoptosis induced by preservative-containing eye-drops.
Debbasch
C
C
Unité de Pharmaco-Toxicologie Cellulaire, Centre Hospitalier National d'Ophtalmologie (CHNO) des Quinze-Vingts, Paris, France. debbasch@caramail.com
Pisella
P J
PJ
De Saint Jean
M
M
Rat
P
P
Warnet
J M
JM
Baudouin
C
C
D000319:Adrenergic beta-Antagonists / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001548:Benzalkonium Compounds / Q000494:pharmacology*; D002354:Carteolol / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003228:Conjunctiva / Q000187:drug effects* / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000502:physiology*; D018384:Oxidative Stress; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013999:Timolol / Q000494:pharmacology
D000319:Adrenergic beta-Antagonists; D001548:Benzalkonium Compounds; D011310:Preservatives, Pharmaceutical; D017382:Reactive Oxygen Species; D013999:Timolol; D002354:Carteolol; D004247:DNA; D005978:Glutathione