title: [Toxicity of preserved and unpreserved beta-blocker eyedrops in an in vitro model of human conjunctival cells].
abstract: OBJECTIVE
  To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in an in vitro model of human conjunctival cells.
  
  
  METHODS
  Chang's conjunctival cell line (ATCC CCL 20.2) was treated for 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation, mitochondrial mass and activity, free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. In addition, cell size and the expression of an apoptotic marker Apo2.7 were studied by flow cytometry.
  
  
  RESULTS
  Timolol-BAC(+) induced a rapid decrease in cell viability ranging from 40% immediately after treatment to 85% 24h later. A small, significantly less important decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-). 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was reduced by 55% whereas it did not vary after 0.25% timolol -BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation, decrease in mitochondrial membrane potential and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Also reactive oxygen species (ROS) production was significantly more important after cell exposure to timolol-BAC(+).
  
  
  CONCLUSIONS
  In our model of conjunctival cells in vitro, timolol-BAC(+) induced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for ROS production and for cell viability variations. Oxidative stress could also play a role in timolol-BAC(+)-induced toxicity. In vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.
authors.affiliation: Service d'Ophtalmologie, Hôpital Ambroise Paré, AP-HP, Université Paris V, 9, avenue Charles de Gaulle, 92120 Boulogne. destjean@club-internet.fr
authors.forename: M
authors.initials: M
authors.lastname: de Saint Jean
authors.forename: C
authors.initials: C
authors.lastname: Debbasch
authors.forename: F
authors.initials: F
authors.lastname: Brignole
authors.forename: P
authors.initials: P
authors.lastname: Rat
authors.forename: J M
authors.initials: JM
authors.lastname: Warnet
authors.forename: C
authors.initials: C
authors.lastname: Baudouin
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D001548:Benzalkonium Compounds / Q000633:toxicity*; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D009883:Ophthalmic Solutions; D011310:Preservatives, Pharmaceutical / Q000633:toxicity*; D013999:Timolol / Q000633:toxicity*
chemical_list: D000319:Adrenergic beta-Antagonists; D001548:Benzalkonium Compounds; D009883:Ophthalmic Solutions; D011310:Preservatives, Pharmaceutical; D013999:Timolol