Atomic Force Microscopy (AFM) Analysis of Lipid Peroxidation Following Hyperoxia and Hydrogen Peroxide Treatment in Human U87 Glioblastoma Cells

The atomic force microscope (AFM) is capable of resolving the plasma membrane with nanometer resolution. In this study we used the AFM to characterize hyperoxia-induced oxidative damage to changes in the plasma membranes of cultured human glioma cells (U87). U87 cells were exposed to 0.20 ATA O ₂ , normobaric hyperoxia (0.95 ATA O ₂ ) or hyperbaric hyperoxia (HBO ₂ , 3.25 ATA O ₂ ) for 30 min. In separate experiments H ₂ O ₂ (200 µM and 2 mM) was used as a positive control. Malondialdehyde (MDA) was measured to confirm lipid peroxidation. Following treatment, the cells were fixed with 2% glutaraldehyde and scanned in air or fluid. Individual cells from each group (n = 35 to 45 cells/group) were scanned and analyzed to assess average membrane roughness (R _a ). The R _a of the plasma membrane was 34 ± 3 nm, 57 ± 3 nm and 63 ± 5 nm in 0.20 ATA O ₂ , 0.95 ATA O ₂ and HBO ₂ , respectively. In H ₂ O ₂ treated cells R _a was 28 ± 4 nm, 56 ± 7 nm and 138 ± 14 nm in air (air in 5% CO ₂ ), 200 µM and 2 mM H ₂ O ₂ . Co-treatment with antioxidant Trolox C (150 µM) significantly reduced R _a during exposure to hyperoxia and H ₂ O ₂ , suggesting that the amount of membrane blebbing was proportional to the level of oxidative stress. Furthermore, measurement of MDA confirmed that H ₂ O ₂ and hyperoxia increased lipid peroxidation, suggesting that membrane blebbing is related to oxidative stress. In conclusion, these data demonstrate oxidative damage from lipid peroxidation increases with oxygen concentration.