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Abstract
Purpose
To determine the possible effects of chronic exposure of low dose benzalkonium chloride (BAK) on trabecular meshwork cells, and to characterize the pathways involved in the effects.
Methods
Trabecular meshwork cells were treated with 0.0005%, 0.00075%, 0.001%, and 0.0025% BAK for 10 minutes; then, the cells were transferred to a new medium for 24 hours. This process was repeated three times. Cell survival was assessed using the MTT assay to determine the non-apoptotic BAK concentration. Senescence-associated (SA)-β-gal staining was performed to compare quantitatively the cellular senescence of BAK-treated cells with the control group. Cells treated with BAK were analyzed by western blot to determine whether the expressions of cell cycle regulators were affected.
Results
Two concentrations (0.0005% and 0.00075%) showed persistent cell viability and were chosen for further experiments. After SA-β-gal staining, cells treated with 0.0005% and 0.00075% BAK showed 28% (± 2.08), 37% (± 2.08) increases in cellular senescence expression, respectively, when compared with control cells (p < 0.05). To identify the molecular pathways involved in cell cycle arrest via BAK, western blot analysis was performed on trabecular meshwork cells, resulting in decreased expressions of cyclin E/CDK2, and increased expressions of the upper stream control molecules, p53 and p21.
Conclusions
Chronic exposure to low dose BAK accelerated cell senescence through cell cycle arrest. Because senescent cells of the trabecular meshwork can inhibit its outflow pathway function and ultimately worsen the glaucomatous process, long-term usage of topical glaucoma medications containing BAK should be conducted with caution.
Figures and Tables
Figure 1
Cell viability was measuresd by MTT assay. BAK decreased the survival of trabecular meshwork cells significantly from concentrations 0.001% to 0.0025% (*p < 0.05, **p < 0.001). Veh = vehicle; MTT assay = 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay; BAK = benzalkonium chloride.
Figure 2
Senescence detection by senescence-associated (SA) β-galactosidase staining. Photograph of SA-β-gal positive senescent trabecular meshwork cells (blue color) after being exposed to 0.0005%, 0.00075% of BAK. Both 0.0005% and 0.00075% BAK showed concentration depedent increase of blue stained senescent cells compared to non-exposed control (×200). Veh = vehicle; BAK = benzalkonium chloride.
Figure 3
Effect of BAK on senescence of trabecular meshwork cells. BAK concentrations more than 0.0005% increased cell senescence significantly (*p < 0.05). Veh = vehicle; BAK = benzalkonium chloride.
Figure 4
Effect of BAK on cell cycle regulator molecules of trabecular meshwork cells. β-actin, also known as a “housekeeping” protein, acts as a loading control. (A) Expressions of cyclin E and CDK2 are decreased with 0.00075% BAK while expressions of cyclin D1 and CDK4 seem to be unaffected. (B) The expressions of phosphorylated p53 and p21 show concentration dependent increase with 0.0005% and 0.00075% BAK. BAK = benzalkonium chloride; CDK = cyclin-dependent kinase.
Notes
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