title: Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of 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 a human conjunctival cell line.
  
  
  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 and free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. The comparison was done with an oxidative stress model of cells treated with 0.001-0.000001% hydrogen peroxide (H(2)O(2)). In addition, cell size and the expression of an apoptotic marker Apo2.7 were evaluated 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 initial decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-) but, 24h later, cell viability either tended to remain constant or cells completely recovered. Cell viability fell down after 24h exposure to 0.001% H(2)O( 2) whereas it was not modified at lower concentrations. 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 and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Both timolol-BAC(+) and BAC(-) induced reactive oxygen species (ROS) production which was significantly more important when 0.25% or 0.4% timolol-BAC(+) were applied. Only 0.001% and 0.0001% H(2)O(2) generated a significant free radicals production.
  
  
  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 reactive oxygen species production and for cell viability variations. The role of oxidative stress in timolol-BAC(+)-induced toxicity seems not to be predominant. in vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.
authors.affiliation: Department of Ophthalmology, University Paris 5, Ambroise Paré Hospital, Boulogne, France. 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 / Q000494:pharmacology*; D001548:Benzalkonium Compounds / Q000633:toxicity*; D002452:Cell Count / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005609:Free Radicals / Q000378:metabolism; D005901:Glaucoma / Q000517:prevention & control; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D011310:Preservatives, Pharmaceutical / Q000633:toxicity*; D013999:Timolol / Q000494:pharmacology*; D018675:Toxicity Tests
chemical_list: D000319:Adrenergic beta-Antagonists; D001548:Benzalkonium Compounds; D002843:Chromatin; D005609:Free Radicals; D011310:Preservatives, Pharmaceutical; D013999:Timolol; D006861:Hydrogen Peroxide