Abstract
Purpose: :
To evaluate the in vitro toxicity of chrysene on human retinal pigment epithelial cells (ARPE-19).
Methods: :
ARPE-19 cells were exposed to different concentrations of Chrysene diluted in DMSO (250µM, 500µM, 750µM and 1000µM). 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 chrysene 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’ dichlorodihydrofluorescein diacetate (DCFH-DA) assay to measure accumulation of reactive oxygen species (ROS).
Results: :
ARPE-19 cells treated with chrysene mean % CV was 93.65%, 81.10%, 77.55% and 60.05% for 250, 500, 750 and 1000 µM respectively, the later showed significantly reduction in CV% compared to untreated ARPE19 controls. The ΔΨM was decreased in all chrysene concentrations when compared to untreated ARPE 19 controls (14.56±0.167). The mean ΔΨm values were 4.5±0.19, 3.4±0.14, 4.3±0.22 and 3.5±0.5 for Chrysene 250, 500, 750 and 1000µM respectively. ROS activity was significantly increased for all chrysene concentrations when compared to untreated control. The mean fluorescence values were 36717±627 msi, 40097±620 msi, 37044±511 msi and 38040±1743, for Chrysene 250, 500, 750 and 1000µM respectively as compared to untreated controls (18064±1484 msi). Caspase 3/7 activity was significantly increased for all chrysene concentrations. The mean fluorescence values were 6179±316 msi, 6424±334 msi, 8345±551 msi and 11235±539 for Chrysene 250, 500, 750 and 1000 µM respectively as compared to untreated cells (2564±425 msi). Toxicity for the chrysene solvent DMSO was tested for all treatment groups and no statistically significant change was seen.
Conclusions: :
Chrysene in all concentrations used in this study significantly decreases retinal cell viability in vitro, induces JC1, ROS, and caspase 3/7 activity.
Keywords: retinal culture • oxidation/oxidative or free radical damage • apoptosis/cell death