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Chloe Clouzeau, David Godefroy, Luisa Riancho, William Rostene, Christophe Baudouin, Françoise Brignole-Baudouin; Hyperosmolarity Potentiates The Toxic Effect Of Benzalkonium Chloride On Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1130.
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To investigate the proapoptotic and proinflammatory effects of NaCl-induced hyperosmolar conditions on conjunctival epithelial cells in association or not with the preservative benzalkonium chloride (BAK).
Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured in hyperosmolar conditions (400-425-500 mOsM) or in BAK (5.10-4%) or in combination of both. Cell viability (Neutral red assay), apoptosis (P2X7 receptor activation-induced membrane permeability, YO-PRO-1), chromatin condensation (Hoechst33342/propidium iodide), oxidative stress (H2DCFDA, Hydroethidine) and chemokine expression (CXCL8/CCL2) were assessed by spectrofluorimetry and/or flow cytometry. Immunohistochemistry was performed for active caspase-3, PARP-1 and cytochrome c. CXCL8 and CCL2 secretion was quantified using ELISA.
Hyperosmolarity induced a decrease in cell proliferation and viability, illustrated by an increase in membrane permeability, cell shrinkage, cell blebbing and chromatin condensation. A caspase-dependent apoptosis was observed with cytochrome c release from mitochondria to cytoplasm, leakage of PARP-1 from nucleus and caspase-3 activation. IL-8 was not induced by hyperosmolarity alone and slightly by BAK while MCP1 was induced by BAK; the combination of both stresses potentiated the secretion of both chemokines.
This study shows that the cytotoxic and proinflammatory effects of BAK were increased in hyperosmotic conditions, with cell death and chemokine release in a concentration-dependent manner. As BAK is known to induce evaporative dry eye syndrome and to promote tear hyperosmolarity, this in vitro hyperosmolarity model highlights the risk of inducing a vicious circle and the importance of avoiding BAK in clinical dry eye conditions.
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