Acute Hyperoxia Increases Lipid Peroxidation and Induces Plasma Membrane Blebbing in Human U87 Glioblastoma Cells

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Atomic force microscopy (AFM), malondialdehyde (MDA) assays, and amperometric measurements of extracellular hydrogen peroxide (H2O2) were used to test the hypothesis that graded hyperoxia induces measurable nanoscopic changes in membrane ultrastructure and membrane lipid peroxidation (MLP) in cultured U87 human glioma cells. U87 cells were exposed to 0.20 atmospheres absolute (ATA) O2, normobaric hyperoxia (0.95 ATA O2) or hyperbaric hyperoxia (HBO2, 3.25 ATA O2) for 60 min. H2O2 (0.2 or 2 mM; 60 min) was used as a positive control for MLP. Cells were fixed with 2% glutaraldehyde immediately after treatment and scanned with AFM in air or fluid. Surface topography revealed ultrastructural changes such as membrane blebbing in cells treated with hyperoxia and H2O2. Average membrane roughness (Ra) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The Ra of the plasma membrane was 34±3, 57±3 and 63±5 nm in 0.20 ATA O2, 0.95 ATA O2 and HBO2, respectively. Ra was 56±7 and 138±14 nm in 0.2 and 2 mM H2O2. Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H2O2 and correlated with O2-induced membrane blebbing (r2=0.93). Coapplication of antioxidant, Trolox-C (150 μM), significantly reduced membrane Ra and MDA levels during hyperoxia. Hyperoxia-induced H2O2 production increased 189%±5% (0.95 ATA O2) and 236%±5% (4 ATA O2) above control (0.20 ATA O2). We conclude that MLP and membrane blebbing increase with increasing O2 concentration. We hypothesize that membrane blebbing is an ultrastructural correlate of MLP resulting from hyperoxia. Furthermore, AFM is a powerful technique for resolving nanoscopic changes in the plasma membrane that result from oxidative damage.



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