Peroxide-induced cell death and lipid peroxidation in C6 glioma cells

Abstract

Peroxides are often used as models to induce oxidative damage in cells in vitro. The aim of the present study was to elucidate the role of lipid peroxidation in peroxide-induced cell death. To this end (i) the ability to induce lipid peroxidation in C6 rat astroglioma cells of hydrogen peroxide (H2O2), cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BuOOH) (ii) the relation between peroxide-induced lipid peroxidation and cell death in terms of time and concentration dependency and (iii) the capability of the lipid peroxidation chain breaking alpha-tocopherol to prevent peroxide-induced lipid peroxidation and/or cell death were investigated. Lipid peroxidation was characterised by measuring thiobarbituric acid reactive substances (TBARS) and, by HPLC, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and hexanal. Within 2 h CHP, t-BuOOH and H2O2 induced cell death with EC50 values of 59+/-9 microM, 290+/-30 microM and 12+/-1.1 mM, respectively. CHP and t-BuOOH, but not H2O2 induced lipid peroxidation in C6 cells with EC50 values of 15+/-14 microM and 130+/-33 microM, respectively. The TBARS measured almost exclusively consisted of MDA. 4-HNE was mostly not detectable. The concentration of hexanal slightly increased with increasing concentrations of organic peroxides. Regarding time and concentration dependency lipid peroxidation preceded cell death. Pretreatment with alpha-tocopherol (10 microM, 24 h) prevented both, peroxide-induced lipid peroxidation and cell death. The results strongly indicate a major role of lipid peroxidation in the killing of C6 cells by organic peroxides but also that lipid peroxidation is not involved in H2O2 induced cell death.