Abstract
Abstract: :
Purpose: Melanosomes could theoretically protect RPE cells from oxidative stress since melanin has antioxidant properties, but any protective effect is expected to be very small (though significant over a lifetime) and difficult to detect experimentally. Here the effect of melanosomes in oxidatively stressed RPE cells was tested after extensive refinement of a culture model. Methods: ARPE–19 cells were tested in multiple protocols for effects of oxidants (H2O2 and t–butylhydroperoxide [TBH]) added to the medium, followed by several measures of cytotoxicity. The effect of bovine melanosomes or control particles (silica or charcoal), introduced into cells by phagocytosis, was then tested using refined assay conditions. Results: Protocol variations with the greatest effects on sensitivity of cells to oxidants were time post plating (endogenous antioxidants catalase and glutathione showed large variations over time) and culture density. Denser cultures were more resistant, due largely to humoral factor(s) (conditioned medium of dense cultures protected sparser ones) rather than to cell–cell contact mediated mechanisms (cell separation by calcium reduction had no effect). Outcome measures of oxidant effects showed the following sensitivity hierarchy: mitochondrial MTT> cell attachment>total cellular ATP>propidium iodide fluorescence. Cells without particles or with high numbers of internalized melanosomes or control particles were exposed in multiple experiments to 100–400 µM H2O2 or TBH in complete medium (with serum) at densities of 110,000 or 170,000 cells/cm2 followed by assay at 24 hr. Reproducible dose dependent cytotoxic effects of the oxidants were seen with the sensitive measures, but no particle type, including melanosomes, had a consistent effect. Conclusions: Melanosomes did not display an anti– (or pro–) oxidant effect in RPE cells exposed to humoral oxidants in vitro when measures of total cell toxicity were used. It cannot be concluded yet, however, that they are without effect in the microenvironment of the organelle.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • aging