Abstract
Background
The mechanisms of trabecular cell loss in glaucoma patients are poorly understood. In order to determine whether drug-induced apoptosis could be one of the mechanisms by which trabecular cells die in glaucoma, we evaluated the effect of benzalkonium-preserved (BAC+) or preservative-free (BAC−) antiglaucoma medications on apoptotic marker expression by cultured human trabecular meshwork (HTM) cells.
Methods
Normal and glaucomatous trabecular cell lines were treated for 15 min with antiglaucoma drugs (1/100 and 1/10 dilutions): timolol BAC+ or BAC−, betaxolol BAC+ or BAC−, latanoprost BAC+ or pure BAC. Apo2.7 expression, annexin V binding and DNA content were evaluated by flow cytometry and confocal microscopy.
Results
Results obtained in the two cell lines were similar for all tested drugs and criteria. In a 1/100 dilution, unpreserved beta-blockers had no apoptotic effect, preserved beta-blockers and latanoprost significantly increased Apo2.7 expression only, while BAC significantly increased all three apoptotic markers. When tested in a 1/10 dilution, all drugs except unpreserved timolol triggered a 2- to 3.5-fold increase in apoptotic features, whereas up to 95% of the cells underwent apoptosis upon treatment with BAC (representing a 9-fold increase over the background level).
Conclusion
At concentrations higher than those supposed to be found in the aqueous humor after instillation (1/100 dilution), unpreserved beta-blocker exhibited no proapoptotic activity on HTM cells in vitro. Benzalkonium-containing beta-blockers and prostaglandin analogue triggered mild expression of one out of three apoptotic markers, while the pro-apoptotic effect observed with BAC appeared to be largely hindered by active compounds in the preserved eyedrops.
Similar content being viewed by others
References
Alvarado J, Murphy C, Polansky J, Juster R (1981) Age-related changes in trabecular meshwork cellularity. Invest Ophthalmol Vis Sci 21:714–727
Alvarado J, Murphy C, Juster R (1984) Trabecular meshwork cellularity in primary open-angle glaucoma and non-glaucomatous normals. Ophthalmology 91:564–579
Baudouin C, Pisella PJ, Fillacier K, Goldschild M, Becquet F, De Saint Jean M, Béchetoille A (1999) Ocular surface inflammatory changes induced by topical antiglaucoma drugs: human and animal studies. Ophthalmology 106:556–563
Brignole F, De Saint Jean M, Goldschild M, Becquet F, Baudouin C (1998) Expression of Fas-ligand antigens and apoptotic marker APO2.7 by the human conjunctival epithelium: positive correlation with class II HLA DR expression in inflammatory ocular surface disorders. Exp Eye Res 67:687–697
Champeau EJ, Edelhauser HF (1986) Effect of ophthalmic preservatives on the ocular surface: conjunctival and corneal uptake of benzalkonium chloride and chlorhexidine disconjugate. In: Holly FJ (ed). The preocular tear film in health, disease and contact lens wearers. The Dry Eye Institute, Lubbock, Texas, pp 292–302
De Saint Jean M, Debbasch C, Brignole F, Rat P, Warnet JM, Baudouin C (2000) Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of conjunctival cells. Curr Eye Res 20:85–94
Debbasch C, Brignole F, Pisella PJ, Warnet JM, Rat P, Baudouin C (2001a) Quaternary ammoniums and other preservatives’ contribution in oxidative stress and apoptosis on Chang conjunctival cells. Invest Ophthalmol Vis Sci 42:642–652
Debbasch C, Pisella PJ, De Saint Jean M, Rat P, Warnet JM, Baudouin C (2001b) Mitochondrial activity and glutathione injury in apoptosis induced by unpreserved and preserved β-blockers on Chang conjunctival cells. Invest Ophthalmol Vis Sci 42:2525–2533
Ellis PP, Wu PY, Riegel M (1991) Aqueous humor pilocarpine and timolol levels after instillation of the single drug or in combination. Invest Ophthalmol Vis Sci 32:520–522
Grierson I (1987) What is open angle glaucoma? Eye 1:15–28
Grierson I, Howes RC (1987) Age-related depletion of the cell population in the human trabecular meshwork. Eye 1:204–210
Hamard P, Sourdille P, Valtot F, Baudouin C (2002) Confocal microscopic examination of trabecular meshwork removed during ab externo trabeculectomy. Br J Ophthalmol 86:1046–1052
Hogg P, Calthorpe M, Batterbury M, Grierson Y (2000) Aqueous humor stimulates the migration of human trabecular meshwork cells in vitro. Invest Ophthalmol Vis Sci 41:1091–1098
Johnson DH, Richardson TM, Epstein DL (1989) Trabecular meshwork recovery after phagocytic challenge. Curr Eye Res 8:1121–1130
Johnson MC, Kamm RD (1983) The role of Schlemm’s canal in aqueous outflow from the human eye. Invest. Ophthalmol Vis Sci 24:320–325
Kawa JE, Higginbotham EJ, Chang IL, Yue BY (1993) Effects of antiglaucoma medications on bovine trabecular meshwork cells in vitro. Exp Eye Res 57:557–565
Mäepea O, Bill A (1989) The pressures in the episcleral veins, Schlemm’s canal, and the trabecular meshwork in monkeys: effects of changes in intraocular pressure. Exp Eye Res 49:645–663
Mäepea O, Bill A (1992) Pressure in the juxtacanalicular tissue and Schlemm’s canal in monkeys. Exp Eye Res 54:879–883
Miyake K, Ota I, Ibaraki N, Ibaraki N, Akura J, Ichihashi S, Shibuya Y, Maekubo K, Miyake S (2002) Enhanced disruption of the blood-aqueous barrier and the incidence of angiographic cystoid macular edema by topical timolol and its preservative in early postoperative pseudophakia. Arch Ophthalmol 119:397–394
Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279
Ormerod MG (1998) The study of apoptotic cells by flow cytometry. Leukemia 12:1013–1025
Pang IH, Shade DI, Clark AL, Steely HT, DeSantis L (1994) Preliminary characterization of a transformed cell strain derived from trabecular meshwork. Curr Eye Res 13:51–63
Phan TM, Nguyen KP, Giacomini JC, Lee DA (1991) Ophthalmic beta-blockers: determination of plasma and aqueous humor levels by radioreceptor assay following multiple doses. J Ocul Pharmacol 7:243–252
Rohen JW, Lutjen-Drecoll E, Flugel C, Meyer M, Grierson I (1993) Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma. Exp Eye Res 56:683–692
Samples JR, Binder PS, Nayak S (1989) The effect of epinephrine and benzalkonium chloride on cultured corneal endothelial and trabecular meshwork cells. Exp Eye Res 49:1-12
Sibayan SA, Latina MA, Sherwood ME, Flotte TJ, White K (1998) Apoptosis and morphologic changes in drug-treated trabecular meshwork cells in vitro. Exp Eye Res 66:521–529
Sjoquist B, Sternschantz J (2002) Ocular and systemic pharmacokinetics of latanoprost in human. Surv Ophthalmol 47 [Suppl] 1:S6–12
Tripathi BJ, Tripathi R, Millard CB (1989) Epinephrine-induced toxicity of human trabecular cells in vitro. Lens Eye Toxic Res 6:141–156
Verhoven AJ, Schlegel RA, Williamson P (1995) Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182:1597–1601
Wood JP, Desantis L, Chao H, Osborne NN (2001) Topically applied betaxolol attenuates ischaemia-induced effects to the rat retina and stimulates BDNF mRNA. Exp Eye Res 72:79–86
Zhang C, Ao Z, Seth A, Schlossmann SF (1996) A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells. J Immunol 157:3980–3987
Zhou L, Fukuchi T, Kawa JE, Higginbotham EJ, Yue BY(1995) Loss of cell-matrix cohesiveness after phagocytosis by trabecular meshwork cells. Invest Ophthalmol Vis Sci 36:787–795
Zhou L, Li Y, Yue BY (1999) Alteration of cytoskeletal structure, integrin distribution, and migratory activity by phagocytic challenge in cells from an ocular tissue—the trabecular meshwork. In Vitro Cell Dev Biol Anim 35:144–149
Acknowledgements
The authors thank Alcon Laboratories, Fort Worth, Texas, USA for kindly providing the cell lines HTM-5 and HTM-3.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hamard, P., Blondin, C., Debbasch, C. et al. In vitro effects of preserved and unpreserved antiglaucoma drugs on apoptotic marker expression by human trabecular cells. Graefe's Arch Clin Exp Ophthalmol 241, 1037–1043 (2003). https://doi.org/10.1007/s00417-003-0777-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00417-003-0777-7