[Quaternary ammonium cytotoxicity in a human conjunctival cell line].
OBJECTIVE
Ophthalmic preparations can induce conjunctival toxicity, often caused by preservatives. The aim of this study was to evaluate in vitro cytotoxicity of quaternary ammonium.


METHODS
Cytotoxicity tests were done on a continuous human conjunctival cell line using microplate cold light cytofluorimetry. Membrane integrity (neutral red test), DNA condensation (Hoechst 33342 test) and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests) were evaluated on living cells treated with different concentrations of benzalkonium chloride, benzododecinium bromide and cetrimide (0.00001 to 0.01%) after 15 minutes of treatment or 15 minutes and 24 hours of cell recovery.


RESULTS
All the compounds tested showed similar in vitro effects. Using the neutral red test, we observed a decrease in membrane integrity even at 0.005% and 0.01% (p < 0.001) and after a short time (15 minutes). A stimulation of ROS production (H2O2 and O2) was observed at 0.00001% and above (p < 0.001), associated with a chromatine condensation due to an apoptotic phenomenon.


CONCLUSIONS
A necrotic phenomenon is suggested at high concentrations of quaternary ammonium preservatives whereas an apoptotic mechanism exists for lower concentrations. This toxicity observed in vitro can explain some of the ocular surface damage caused by long-term use of preserved eye-drops.
Debbasch
C
C
Unité de Pharmaco-Toxicologie Cellulaire, CHNO des Quinze-Vingts, Paris.
de Saint Jean
M
M
Pisella
P J
PJ
Rat
P
P
Warnet
J M
JM
Baudouin
C
C
D000891:Anti-Infective Agents, Local / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D001548:Benzalkonium Compounds / Q000633:toxicity; D002460:Cell Line / Q000187:drug effects; D002470:Cell Survival; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000633:toxicity; D002843:Chromatin / Q000187:drug effects; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects*; D003902:Detergents / Q000633:toxicity; D004202:Disinfectants / Q000633:toxicity; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D009499:Neutral Red; D009883:Ophthalmic Solutions / Q000494:pharmacology / Q000633:toxicity*; D011310:Preservatives, Pharmaceutical / Q000633:toxicity*; D000644:Quaternary Ammonium Compounds / Q000633:toxicity*; D017382:Reactive Oxygen Species; D013997:Time Factors
D000891:Anti-Infective Agents, Local; D001548:Benzalkonium Compounds; D002593:Cetrimonium Compounds; D002843:Chromatin; D003902:Detergents; D004202:Disinfectants; D009883:Ophthalmic Solutions; D011310:Preservatives, Pharmaceutical; D000644:Quaternary Ammonium Compounds; D017382:Reactive Oxygen Species; D009499:Neutral Red; C015163:dimethyldodecylbenzylammonium; D000077286:Cetrimonium