Calcium overload is a critical step in programmed necrosis of ARPE-19 cells induced by high-concentration H₂O₂.

OBJECTIVE: Oxidative stress plays an important role in retinal pigmental epithelium (RPE) death during aging and the development of age-related macular degeneration. Although early reports indicate that reactive oxygen species (ROS) including H₂O₂ can trigger apoptosis at lower concentrations and necrosis at higher concentrations, the exact molecular mechanism of RPE death is still unclear. The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS, especially at higher concentrations.

METHODS: Cultured ARPE-19 cells were treated with H₂O₂ at different concentrations and cell viability was measured with the MTT assay. Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining. Furthermore, the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG. Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects.

RESULTS: H₂O₂ reduced the viability of ARPE-19 cells in a concentration-dependent manner, which was presented as a typical s-shaped curve. Cell death caused by high concentrations of H₂O₂ was confirmed to be programmed necrosis. Morphologically, dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane, positively detected with APOPercentage assay and PI staining. 24-hour treatment with 500 μmol/L H₂O₂ induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells. Moreover, antioxidant treatment using EGCG effectively protected cells from H₂O₂-induced injury, increasing cell viability from 14.17%±2.31% to 85.77%±4.58%. After H₂O₂ treatment, intracellular calcium levels were highly elevated with a maximum concentration of 1200 nM. Significantly, the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway, rescuing the ARPE-19 cell from H₂O₂-induced death.

CONCLUSIONS: At high concentrations, H₂O₂ induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.