Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

Abstract

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2- generation in mitochondria respiring on the complex I substrates pyruvate+malate, an effect fully inhibited by rotenone. Antimycin A increased O2- production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2- production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2- formation driven with the complex II substrate succinate. At 0.6 microM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2- formation; however, at 40 microM myxothiazol (which completely inhibits both complexes I and III) eliminated O2- production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2- from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.

Effects of mitochondrial poisons on glutathione redox potential and carotid body chemoreceptor activity

“…The intimate mechanism mediating the mitochondrial production of superoxide by 3-NP is controversial (Bacsi et al, 2006). Yet, it is well established that 3-NP is an irreversible inhibitor of succinate dehydrogenase capable of producing marked amounts of superoxide and oxidative damage when applied to isolated cell systems or injected in intact animals (Bacsi et al, 2006;Herrera-Mundo et al, 2006).…”

“…Yet, it is well established that 3-NP is an irreversible inhibitor of succinate dehydrogenase capable of producing marked amounts of superoxide and oxidative damage when applied to isolated cell systems or injected in intact animals (Bacsi et al, 2006;Herrera-Mundo et al, 2006). There are many studies indicating that the neurotoxic manifestations of 3-NP injections are prevented or ameliorated by concomitant antioxidant treatment (Nam et al, 2005) and attenuated in transgenic mice overexpressing superoxide dismutase (Beal et al, 1995).…”

Oxidative modification enhances the immunostimulatory effects of extracellular mitochondrial DNA on plasmacytoid dendritic cells

“…Antimycin A, a well-known specific inhibitor of mitochondrial electron transport chain at complex III [24], was applied to induce oxidative modification of mtDNA in both human HL-60 and murine SP2/0-Ag14 cells. To determine the optimal amount for generation of mtDNA damage, cells were treated with increasing concentrations of antimycin A for 2 h.…”

Increased ROS generation in subsets of OGG1 knockout fibroblast cells

“…To define the intracellular site(s) of ROS generation the cells were loaded with 2 μM dihydroethidium (H 2 Et; Invitrogen Co., Carlsbad, CA) for 10 min and placed in a thermocontrolled microscopic chamber (Bacsi et al, 2006). MitoTracker Red (Invitrogen Co., Carlsbad, CA), a cell-permeable fluorescent probe that accumulates in active mitochondria was used to stain mitochondria at a final concentration of 25 nM.…”

“…Amplex Red assays were used to measure H 2 O 2 production of isolated mitochondria (Bacsi et al, 2006). Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine; Molecular Probes) reacts with H 2 O 2 in the presence of horseradish peroxidase to generate a stable fluorescent product, resorufin (Zhou et al, 1997).…”