Blue light negatively affects the survival of ARPE19 cells through an action on their mitochondria and blunted by red light

Abstract

Purpose: To ascertain whether red light, known to enhance mitochondrial function, can blunt a blue light insult to ARPE19 cells in culture.

Methods: Semi-confluent ARPE19 cells cultured in 10% FBS were subjected to various regimes of treatment with blue (465-475 nm, 800 lux, 26 W/m² ) and red (625-635 nm, 950 lux, 6.5 W/m² ) light, as well as with toxins that inactivate specific enzymes associated with mitochondrial oxidative phosphorylation. Cultures were then analysed for cell viability (MTT assay), mitochondrial status (JC-1), ROS formation, immunocytochemistry and the activation of specific proteins by electrophoresis/Western blotting. In addition, ARPE19 cells were cultured in polycarbonate membrane inserts in culture medium containing 1% FBS. Such cultures were exposed to cycles of red, blue or a combination of red and blue light for up to 6 weeks. Culture medium was changed and the trans-epithelium membrane resistance (TER) of the inserts-containing cells was measured twice weekly.

Results: ARPE19 cells in culture are affected negatively when exposed to blue light. This is indicated by a loss of viability, a depolarization of their mitochondria and a stimulation of ROS. Moreover, blue light causes an up-regulation of HO-1 and phospho-p-38-MAPK and a cleavage of apoptosis inhibitory factor, proteins which are all known to be activated during cell death. All of these negative effects of blue light are significantly blunted by the red light administered after the blue light insult in each case. ARPE19 cell loss of viability and mitochondrial potential caused by toxins that inhibit specific mitochondrial enzyme complexes was additive to an insult delivered by blue light in each case. After a time, ARPE19 cells in culture express the tight junction protein ZO-1, which is affected by blue light. The development of tight junctions between ARPE19 cells grown in inserts reached a steady peak of resistance after about 40 days and then increased very slightly over the next 40 days when still in darkness. However, maximum resistance was significantly attenuated, when cultures were treated with cycles of blue light after the initial 40 days in the dark and counteracted significantly when the blue light cycle insult was combined with red light.

Conclusion: Blue light affects mitochondrial function and also the development tight junctions between ARPE19 cells, which results in a loss of cell viability. Importantly, red light delivered after a blue light insult is significantly blunted. These findings argue for the therapeutic use of red light as a noninvasive procedure to attenuate insults caused by blue light and other insults to retinal pigment epithelial cell mitochondria that are likely to occur in age-related macular degeneration.

Keywords: ARPE19 cells; blue light toxicity; mitochondria; neuroprotection; red light; trans-epithelial membrane resistance.