Acute hyperoxia increases lipid peroxidation and induces plasma membrane blebbing in human U87 glioblastoma cells

Atomic force microscopy (AFM), malondialdehyde (MDA) assays, and amperometric measurements of extracellular hydrogen peroxide (H(2)O(2)) 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) O(2), normobaric hyperoxia (0.95 ATA O(2)) or hyperbaric hyperoxia (HBO(2), 3.25 ATA O(2)) for 60 min. H(2)O(2) (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 H(2)O(2). Average membrane roughness (R(a)) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The R(a) of the plasma membrane was 34+/-3, 57+/-3 and 63+/-5 nm in 0.20 ATA O(2), 0.95 ATA O(2) and HBO(2), respectively. R(a) was 56+/-7 and 138+/-14 nm in 0.2 and 2 mM H(2)O(2). Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H(2)O(2) and correlated with O(2)-induced membrane blebbing (r(2)=0.93). Coapplication of antioxidant, Trolox-C (150 microM), significantly reduced membrane R(a) and MDA levels during hyperoxia. Hyperoxia-induced H(2)O(2) production increased 189%+/-5% (0.95 ATA O(2)) and 236%+/-5% (4 ATA O(2)) above control (0.20 ATA O(2)). We conclude that MLP and membrane blebbing increase with increasing O(2) 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.