Oxidative stress in ARPE-19 cultures: Do melanosomes confer cytoprotection?

Abstract

The pigment melanin has antioxidant properties that could theoretically reduce oxidative damage to the retinal pigment epithelium (RPE), perhaps protecting against retinal diseases with an oxidative stress component like age-related macular degeneration. To determine whether melanin confers cytoprotection on RPE cells, melanosomes or control particles were introduced by phagocytosis into the human cell line ARPE-19 and oxidative stress was induced chemically (H 2O 2 or tert-butyl hydroperoxide) or with visible light. Since the iron-binding capacity of melanin is important for its antioxidant function, experiments were performed to confirm that the melanosomes were not iron saturated. Cytotoxicity was assessed by measures of plasma or lysosomal membrane integrity, mitochondrial function, and cell-substrate reattachment. Oxidative stress protocols were critically evaluated to produce modest cytotoxicity, which might allow detection of a small cytoprotective effect as expected for melanosomes. Particle internalization alone had no effect on baseline metabolic activity or on major RPE antioxidants. Particles were tested in multiple oxidative stress experiments in which culture conditions known to affect stress-induced cytotoxicity, notably culture density, were varied. No testing condition or outcome measure revealed a consistent protective (or cytotoxic) effect of melanosomes, indicating that measures of lysosome stability or whole cell viability do not demonstrate an antioxidant role for RPE melanosomes. If the melanosome, an insoluble particle, performs a cytoprotective function within cells, its effects may be limited to the local environment of the organelle and undetectable by conventional methods.