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Rafael Migon, Saffar Mansoor, Baruch D. Kuppermann, Maria C. Kenney; Effects Of Chrysene On Human Arpe-19 Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4429.
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To evaluate the in vitro toxicity of chrysene on human retinal pigment epithelial cells (ARPE-19).
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).
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.
Chrysene in all concentrations used in this study significantly decreases retinal cell viability in vitro, induces JC1, ROS, and caspase 3/7 activity.
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