Oxidative damage to mitochondria and protection by ebselen and other antioxidants

Iron/ascorbate induced lipid peroxidation in liver mitochondria isolated from normal and glutathione-depleted rats was monitored by low-level chemiluminescence and by accumulation of thiobarbituric acid-reactive substances (TBARS). Antioxidant capacity was assessed by the duration of the lag phase preceding the onset of active peroxidation. The lag phases in state 4 and in the presence of uncouplers were similar, but shorter in the presence of ADP (state 3). In glutathione-depleted rats the lag periods were less than those in normal mitochondria. A biphasic pattern of loss of membrane alpha-tocopherol was typical in state 4 with about 55% remaining after 40 min, while in presence of ADP there was a steady and rapid loss to about 30% of the initial level. Synthetic antioxidants such as ebselen or its glutathione adduct protected mitochondrial membranes against peroxidative reactions. There was a 5-fold increase in the lag phase with 1 microM ebselen in state 4 (lag doubling concentration, 0.4 microM) and a significantly lower rate of loss of alpha-tocopherol with about 90% of the initial level still remaining after 40 min. Likewise, the lag doubling concentrations were 0.04 microM for diethyldithiocarbamate, 0.3 microM for 5-hydroxyindole, 10 microM for dihydroxyphenylalanine and serotonin, and about 40 microM for epinephrine and norepinephrine.