Abstract
Purpose: :
To evaluate the in vitro safety of bromfenac ophthalmic solution (BOS) on human retinal pigment epithelial cells (ARPE-19).
Methods: :
ARPE-19 cells were treated with 0.09% BOS at 22.5mcg/ml (1X clinical dose), 45.0mcg/ml (2X), 90.0mcg/ml (4X), 11.25mcg/ml (X/2) or 5.625mcg/ml (X/4). Clinical dose was defined as the concentration of drug in 0.1ml of 0.09% BOS injected into the 4ml human vitreous assuming equal drug distribution throughout the vitreous. Cells were incubated at 37º C until confluent. Before drug exposure, the cells were incubated for 24 hours in fetal bovine serum free medium to make them relatively non-proliferating. After 24 hours of drug exposure, the following assays were performed: trypan blue dye exclusion to measure cell viability (CV), JC-1 to measure changes in mitochondrial membrane potential (ΔΨM), caspase-3/7 as an indicator of apoptotic activity and the 2’, 7’-dichlorodihydrofluorescin diacetate (DCFH-DA) assay to measure accumulation of reactive oxygen species (ROS).
Results: :
ARPE-19 cells treated with 4X and 2X BOS showed mean CV of 16.95±3.04% (p<0.001), and 28.45±2.33% (p0.05). The ΔΨM was decreased and the caspase 3 /7 activity increased in ARPE-19 cells treated with all BOS concentrations when compared to untreated ARPE 19 controls (15.15±1.05). The ΔΨM of ARPE-19 cells were 2.74±0.36 (p<0.001), 2.35±0.33 (p<0.001), 2.63±0.20 (p<0.001), 5.30±0.18 (p<0.001) and 5.22±0.15 (p<0.001) for 4X, 2X, X, X/2 and X/4 concentrations respectively. The ROS activity was significantly increased for all BOS concentrations when compared to untreated control. Caspase 3/7 activity was significantly increased for all BOS concentrations.
Conclusions: :
Bromfenac ophthalmic solution at concentrations 2X clinical dose or higher decreases retinal cell viability in vitro and all BOS concentrations significantly induce JC1, ROS, and caspase 3/7 activity.
Keywords: retinal culture • drug toxicity/drug effects • cell survival