Abstract
Purpose: :
Exposure to ultraviolet light is known to cause oxidative damage through the production of reactive oxygen species (ROS), which cause damage to retinal cells. The aim of this study is to evaluate UV light induced phototoxicity in both retinal ganglion (RGC-5) and retinal pigment epithelial (ARPE-19) cells by quantifying ROS and pro-apoptotic markers.
Methods: :
Both RGC-5 and ARPE-19, 100,000 cells/well were maintained in 24 well plates. Cells were exposed to UV light with a midrange wavelength of 300nm for two separate time intervals of 15 and 30 minutes. Cells not exposed to UV light served as a control. Cell viability and structural morphology was assessed by neutral red (NR) uptake assay and by phase contrast imaging after 48hrs. Oxidative stress was measured immediately after UV light exposure and re-measured 24hrs later, by quantifying the fluorescence of dihydrorhodamine-123. Statistical analysis was performed using the student's t-test. RT-PCR was performed to measure both pro-apoptotic (BAX) and anti-apoptotic (Bcl-2) markers.
Results: :
Cell viability, assessed by NR uptake, was reduced to 41% and 35% in RGC-5 and 55% and 15% in ARPE-19 following 15 and 30 minute UV exposure, respectively, as compared with control (p<0.05).ROS increased by 83% and 114% in RGC-5 and 109% and 157% in ARPE-19 at 24hrs following 15 and 30 minute exposure respectively (p<0.0001). Phase contrast imaging revealed cell blebbing and the presence of apoptotic bodies following 30 minute exposure to UV light in both RGC-5 and ARPE-19 cells.RT-PCR demonstrated increased expression of BAX following 15 and 30 minute exposure and a decrease level of Bcl-2 after 15 minutes of UV light exposure.
Conclusions: :
UV light was shown to reduce cell viability in retinal ganglion and pigment epithelial cells through an increase in ROS and pro-apoptotic markers.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • apoptosis/cell death