title: Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro.
abstract: OBJECTIVE
  Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays.
  
  
  METHODS
  The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400-425-500 mOsM), in low concentrations of BAK (10(-4)%, 3.10(-4)%, and 5.10(-4)%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells.
  
  
  RESULTS
  As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from mitochondria to the nucleus in both conditions.
  
  
  CONCLUSIONS
  This study showed increased cytotoxic effects of BAK in hyperosmotic conditions, with characteristic cell death processes, namely caspase-dependent and independent apoptosis and oxidative stress. As BAK is known to disrupt tear film, which could promote evaporative dry eye and tear hyperosmolarity, BAK could promote the conditions enhancing its own cytotoxicity. This in vitro hyperosmolarity model thus highlights the risk of inducing a vicious cycle and the importance of avoiding BAK in patients with dry eye conditions.
authors.affiliation: INSERM, U968, Paris, France. chloe.clouzeau@gmail.com
authors.forename: Chloé
authors.initials: C
authors.lastname: Clouzeau
authors.forename: David
authors.initials: D
authors.lastname: Godefroy
authors.forename: Luisa
authors.initials: L
authors.lastname: Riancho
authors.forename: William
authors.initials: W
authors.lastname: Rostène
authors.forename: Christophe
authors.initials: C
authors.lastname: Baudouin
authors.forename: Françoise
authors.initials: F
authors.lastname: Brignole-Baudouin
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D051033:Apoptosis Inducing Factor / Q000032:analysis; D001548:Benzalkonium Compounds / Q000009:adverse effects*; D053148:Caspase 3 / Q000032:analysis; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002843:Chromatin / Q000378:metabolism; D003228:Conjunctiva / Q000187:drug effects* / Q000473:pathology; D045304:Cytochromes c / Q000032:analysis; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D008858:Microscopy, Phase-Contrast; D008928:Mitochondria / Q000187:drug effects; D009883:Ophthalmic Solutions / Q000009:adverse effects*; D009994:Osmolar Concentration; D018384:Oxidative Stress; D011310:Preservatives, Pharmaceutical / Q000009:adverse effects*; D012965:Sodium Chloride / Q000737:chemistry; D014985:Xerophthalmia / Q000188:drug therapy / Q000473:pathology
chemical_list: D051033:Apoptosis Inducing Factor; D001548:Benzalkonium Compounds; D002843:Chromatin; D009883:Ophthalmic Solutions; D011310:Preservatives, Pharmaceutical; D012965:Sodium Chloride; D045304:Cytochromes c; D053148:Caspase 3