Dynamic analyses reveal cytoprotection by RPE melanosomes against non-photic stress.

PURPOSE: Isolated melanosomes are known to have antioxidant properties but whether the granules perform an antioxidant function within cells is unclear. The aim of this study was to determine whether retinal pigment epithelium (RPE) melanosomes are competent to protect cultured cells against non-photic oxidative stress induced by treatment with H(2)O(2). METHODS: Porcine melanosomes, either untreated or irradiated with visible light to simulate age-related melanin photobleaching, were introduced by phagocytosis into ARPE-19 cells. Cells were treated with H(2)O(2) using two delivery methods: as a pulse, or by continuous generation following addition of glucose oxidase to the medium. Cell survival in melanosome-containing cells was compared to survival in cells containing phagocytosed control latex beads using two real-time cell death assays. RESULTS: Following H(2)O(2) delivery by either method, greater resistance to critical concentrations of H(2)O(2) was seen for cells containing melanosomes than for cells containing beads. Melanosome-mediated protection manifested as a delay in the time of onset of cell death and a slower rate of cell death over time. Photobleaching diminished the stress resistance conferred by the pigment granules. Individual cells in co-cultures were differentially sensitive to oxidative stress depending upon their particle content. Additional features of the time course of the cell death response were revealed by the dynamic analyses conducted over hours post oxidant treatment. CONCLUSIONS: The results show, for the first time, that melanosomes perform a cytoprotective function within cultured cells by acting as an antioxidant. The outcomes imply that melanosomes perform functions within RPE cells aside from those related to light irradiation, and also suggest that susceptibility to ubiquitous pro-oxidizing agents like H(2)O(2) is partly determined by discrete features of individual RPE cells such as their granule content.

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

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 (H2O2 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.