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Caroline Debbasch, Françoise Brignole, Pierre-Jean Pisella, Jean-Michel Warnet, Patrice Rat, Christophe Baudouin; Quaternary Ammoniums and Other Preservatives’ Contribution in Oxidative Stress and Apoptosis on Chang Conjunctival Cells. Invest. Ophthalmol. Vis. Sci. 2001;42(3):642-652. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
purpose. To investigate some of the toxicity mechanisms of 10 preservatives
currently used in ophthalmic solutions in vitro.
methods. A continuous human conjunctival cell line was treated with different
concentrations of various preservatives for 15 minutes and for 15
minutes followed by 24 hours of cell recovery: three benzalkonium
chlorides (BACs) with different hydrocarbon chain length,
benzododecinium bromide (BOB), cetrimide (Cet), phenylmercuric nitrate
(PM), thimerosal (thi), methyl parahydroxybenzoate (MPHB),
chlorobutanol (clb), and EDTA. An inhibition study was then conducted
using a 1-hour vitamin E pretreatment followed by a 15-minute BAC
treatment. Membrane integrity was assessed using a neutral red test and
chromatin condensation with a Hoechst 33342 test. Reactive oxygen
species were measured using dichlorofluorescein diacetate test for
H2O2 production and hydroethidine test for
O2 .− production. These tests were
performed using microplate cold light cytofluorometry. Cell size and
DNA content were also analyzed using flow cytometry. Confocal
microscopy was used to explore morphologic changes.
results. A significant decrease of membrane integrity with chromatin
condensation was observed with all the quaternary ammoniums tested at
concentrations of 0.005% and higher. The effect was amplified after 24
hours of cell recovery. The other preservatives tested did not decrease
membrane integrity. H2O2 production was
observed with all the preservatives, whereas
O2 .− production was significantly
higher with the quaternary ammoniums at 0.005% and 0.01%, compared
with the other preservatives. Flow cytometry results confirmed the
cytotoxicity observed with cold light cytofluorometry.
conclusions. The quaternary ammoniums tested (BAC, BOB, and Cet) were the most
cytotoxic preservatives in the current model. An apoptotic mechanism
appeared to be present at low concentrations of quaternary ammoniums,
whereas a necrotic process appeared at higher concentrations.
Superoxide anions may play an important role in tissue damage induced
by preservatives in ocular surface disorders.
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