Blue light decreases oxidative stress defenses in an in vitro model of AMD.

Abstract

Purpose : Blue light is an identified risk factor for age-related macular degeneration (AMD). Using a custom-made illumination system delivering narrow illumination bands within the blue-green range, we recently showed that the narrow range 415-455 nm was the most toxic for A2E-loaded RPE cells (Arnault et al., 2013). Then we demonstrated that under blue light exposure, these cells displayed high levels of both hydrogen peroxide and superoxide anion. To further understand the involved mechanisms in light-induced retinal damage, we investigated the expression of several antioxidant defenses in the blue-green spectral range.

Methods : Primary cultures of porcine retinal pigment epithelium cells (RPE) were incubated for 6 hours with A2E and then exposed for 15 hours to 10 nm-wide illumination bands centered from 390 to 520 nm in 10 nm increments; one additional band centered at 630 nm was used as a light control. Light irradiances were normalized with respect to the natural sunlight reaching the retina after being filtered by the ocular structures. Antioxidant defenses were quantified at the end of light exposure or after a rest period in darkness by qPCR or by western blot. Results were normalized to the ribosomal gene 18S for qPCR and to actin for western blot.

Results : In A2E-loaded RPE cells exposed to blue light, mRNA expression of various proteins involved in oxidative stress defense such as catalase, glutathione peroxidase1 and SOD2 significantly decreased. However, the cytoplasmic SOD1 mRNA level did not seem to be affected. Preliminary results showed that blue light also induced a downregulation at protein level at the end of exposure and after 24-hour rest in darkness. Furthermore, we observed that mitochondria which contain antioxidant proteins exhibited a globular shape and a perinuclear clustering after blue light exposure as compared to red light exposure.

Conclusions : Using our in vitro model of AMD, we showed here that antioxidant defenses are downregulated after blue light exposure, which could contribute to the concomitant increase in reactive oxygen species (ROS). All our photobiology results support blue light as an inducer of ROS and as an inhibitor of ROS elimination process, resulting in an increase of oxidative stress and thus cell death. These findings provide us with more precise tools to investigate the prevention of oxidative stress generated by blue light in retinal photoaging and AMD.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.