Changes of Vital Parameters of Human RPE Cells During Oxidative Stress as Early Indicators of Acute Cell Injury

Abstract

Abstract: : Purpose: The RPE cell layer in vivo is subjected to continuous oxidative stress throughout life. Aim of the study was to demonstrate and quantify early functional and morphological changes in different cellular compartments of RPE cells in relation to intensity and duration of oxidative stress exposure in vitro. Methods: SV40–transfected human RPE cells were exposed to oxidative stress by incubation in H₂O₂ at increasing concentrations from 100 µM to 800 µM, staurosporin (apoptosis model) or ethanol (necrosis model). The induced cellular changes were observed by fluorescence microscopy and quantified by FACS analysis. Depolarization of mitochondrial membranes was assessed by vital staining with JC–1. Membrane blebbing was evidenced using FITC–conjugated Annexin V. Apoptotic cells were detected using YO–PRO–1 and propidium iodide, and by determination of the Sub–G1–peak. Production of reactive oxygen species (ROS) was traced by vital staining with DCFDA–AM. Results: Vital staining with JC–1 revealed mitochondrial membrane depolarization at H₂O₂ concentrations of 400µM and above over a period of 5h. Prolonged incubation induced membrane depolarization already at lower concentrations (200µM), while at 400µM a perinuclear rearrangement of mitochondria was observed. The typical fragmented nuclei of apoptotic cells appeared at concentrations of 400µM and above after 5h of H₂O₂ exposure, as determined with YO–PRO vital staining. At this concentration initial cell clumping and necrotic cells (visualized with propidium iodide) could also be observed, with sharp rise of their number at increasing H₂O₂ concentrations. After 5h exposure to H₂O₂ FACS analysis of Annexin V staining revealed increasing fluorescence beginning at concentrations as low as 200µM, which was intensified after exposures of 24h. After 5h of exposure at 200µM a distinct ROS–producing cell subpopulation was observed. At a concentration of 800µM the cell population divided into two subgroups, one generating less and the other generating more ROS. After prolonged exposure up to 24h the ROS production at 200µM H₂O₂ concentration was comparatively increased, but at higher concentrations ROS production was clearly reduced. Conclusions: Detection of mitochondrial membrane depolarization, membrane blebbing, ROS–production and nuclear fragmentation by vital staining represent early and sensitive markers of cellular injury caused by oxidative stress in a RPE–cell line.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • stress response