April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Hyperosmolarity Potentiates The Toxic Effect Of Benzalkonium Chloride On Conjunctival Epithelial Cells
Author Affiliations & Notes
  • Chloe Clouzeau
    THERAPEUTIC, INSTITUT DE LA VISION, PARIS, France
  • David Godefroy
    THERAPEUTIC, INSTITUT DE LA VISION, PARIS, France
  • Luisa Riancho
    THERAPEUTIC, INSTITUT DE LA VISION, PARIS, France
  • William Rostene
    THERAPEUTIC, INSTITUT DE LA VISION, PARIS, France
  • Christophe Baudouin
    THERAPEUTIC, INSTITUT DE LA VISION, PARIS, France
  • Françoise Brignole-Baudouin
    THERAPEUTIC, INSTITUT DE LA VISION, PARIS, France
  • Footnotes
    Commercial Relationships  Chloe Clouzeau, grant ANRT & Horus Pharma (F); David Godefroy, None; Luisa Riancho, None; William Rostene, None; Christophe Baudouin, None; Françoise Brignole-Baudouin, None
  • Footnotes
    Support  ANRT Grant Horus Pharma
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1130. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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).

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: conjunctiva • apoptosis/cell death • inflammation 
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