Abstract
Purpose: :
Benzalkonium Chloride (BAK), a common preservative in many ophthalmic formulations, is known to cause desquamation of corneal epithelial cells. This study examined if this loss of cellular adhesion is secondary to reduced actin contractility.
Methods: :
Experiments were conducted in primary cultures of bovine corneal epithelial cells at second to third passage. Cell adhesion was assayed in ninety–six–well tissue culture plates using a fluorescence technique. MLC phosphorylation was assayed as a biochemical measure of actin contractility by urea–glycerol gel electrophoresis followed by Western blotting. RhoA activity was assayed by Western blotting. The ratios of apoptotic and necrotic cells were measured using flow cytometry with staining of annexin–V and propidium iodide.
Results: :
The % of adherent cells increased by 28% upon coating of the culture plates with fibronectin and collagen I (n=10; p<0.001). Treatment with BAK (0.001%) caused a significant reduction in cell adhesion (n=10; p<0.001). There was no effect of BAK on cell adhesion at a concentration of 0.0001% (n=10; p>0.05). Treatment of BAK at concentrations of 0.0005% (n=6), 0.001% (n=8) and 0.003% (n=8) did not affect the ratio of apoptotic or necrotic cells significantly (p>0.05) at 20 minutes. Treatment of cells with BAK at 0.001% for 20 min led to significant MLC dephosphorylation and inactivation of RhoA.
Conclusions: :
BAK induces loss of cell adhesion at a concentration of 0.001% possibly due to reduced actin contractility as indicated by MLC dephosphorylation. At this sub–clinical concentration, cell death was not apparent suggesting that inactivation of RhoA and MLC dephosphorylation may be caused by depletion of GTP as suggested previously (Ying et al, ARVO, 2001).
Keywords: cytoskeleton • cell adhesions/cell junctions • drug toxicity/drug effects