Alizadeh et al.
40 demonstrated that the oxidants
tert-butyl hydroperoxide (tBH) and H
2O
2 upregulates HO-1 and fibroblast growth factor receptor (FGFR1) mRNA in differentiated ARPE-19 cells with concurrent downregulation of specific genes, including cellular retinaldehyde-binding protein (CRALBP) and the RPE-specific 65-kDa protein (RPE65). Similarly, in our study, low doses of H
2O
2 induced a proliferative response in ARPE-19 cultures where nonquiescent cells may remain (9 days), whereas higher doses induced cell death. It is well established that markedly different responses to oxidative stress by proliferating and nonproliferating mammalian cells are dependent on the degree of cellular differentiation and on the magnitude of the stress stimulus.
41 42 In this regard, although the threshold of viability to the H
2O
2 challenge in fully differentiated RPE cells in our study was relatively high, the slope of the viability curves were quite steep. These experiments show that bilberry anthocyanins can be preloaded into cells and quench intracellular radicals, albeit at supraphysiologic levels; however, this radical quenching activity was insufficient to prevent cell death caused by a 500 μM H
2O
2 challenge. High levels of H
2O
2 may render catastrophic oxidant damage to surface proteins and receptors representing an insult disproportionate to in vivo conditions. Lethal cellular injury mediated by acute H
2O
2-induced oxidative stress involves an elevation of intracellular cytosolic Ca
2+ associated with collapse of the mitochondrial membrane potential. The viability assay chosen for these experiments is an indicator of mitochondrial dysfunction leading to failure of reductive potential in the cell. Sequestration of Ca
2+ in the mitochondrion depends on NADPH and GSH, both of which are consumed in the metabolism of H
2O
2. GSH was depleted rapidly by 500 μM H
2O
2 in our 10-day RPE cultures (data not shown). Catastrophic oxidative defense failure, observed in in vitro experiments where it is necessary to use high levels of H
2O
2, may not adequately mimic in vivo oxidative stress associated with aging. These experiments should be repeated in an optimal in vivo model over a longer time period with lower levels of oxidative stress to test the impact of long-term nutrient intakes on upregulation of protective enzymes and potential reduction in risk of chronic diseases like AMD.