Oxidative damage of mitochondria induced by 5-aminolevulinic acid: role of Ca2+ and membrane protein thiols.

Reactive oxygen species (ROS) generated by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation have been shown to damage the inner membrane of isolated rat liver mitochondria by a Ca(2+)-dependent mechanism. The present work describes experiments indicating that this damage can be prevented, but not completely reversed by the additions of catalase, ADP, cyclosporin A and dithiothreitol, as judged by the extent of delta psi regeneration by the injured mitochondria. In contrast, the addition of EGTA, which removes free Ca2+ and, possibly, Fe2+ present both in the intra- and extramitochondrial compartments, causes a prompt and complete regeneration of delta psi, even after long periods of mitochondrial incubations in the presence of ALA. This reversibility suggests that protein alterations such as protein thiol cross-linkings, evidenced by SDS-polyacrylamide gel electrophoresis, are the main cause of increased membrane permeability promoted by ALA oxidation. The inhibition of protein aggregation and fast regeneration of delta psi promoted by EGTA suggest that the binding of Ca2+ to some membrane proteins plays a crucial role in the mechanism of both protein polymerization (pore assembly) and pore opening. The implication of these results with the molecular pathology of acute intermittent porphyria is also discussed.

Permeabilization of the inner mitochondrial membrane by Ca2+ ions is stimulated by t-butyl hydroperoxide and mediated by reactive oxygen species generated by mitochondria.

The extent of swelling undergone by deenergized mitochondria incubated in KCl/sucrose medium in the presence of Ca2+ alone or Ca2+ and t-butyl hydroperoxide decreases by decreasing molecular oxygen concentration in the reaction medium; under anaerobiosis no swelling occurs. This swelling is also inhibited by the presence of exogenous catalase or by the Fe2+ chelator o-phenanthroline in a time-dependent manner. The production of protein thiol cross-linking that leads to the formation of protein aggregates induced by Ca2+ and t-butyl hydroperoxide does not occur when mitochondria are incubated in anaerobic medium or in the presence of o-phenanthroline. In addition, it is also shown that the yield of stable methyl radical adducts, obtained from rat liver mitochondria treated with t-butyl hydroperoxide and the spin trap DMPO, is reduced by addition of EGTA and increases by addition of Ca2+ ions. These data support the hypothesis that Ca2+ ions stimulate electron leakage from the respiratory chain, increasing the mitochondrial production of reactive oxygen species.

Oxidative damage of mitochondria induced by Fe(II)citrate or t-butyl hydroperoxide in the presence of Ca2+: effect of coenzyme Q redox state.

The role of coenzyme Q on the process of mitochondrial membrane damage associated with oxidative stress was studied in a suspension of uncoupled mitochondria exposed to Ca2+ in the presence of Fe(II)citrate or t-butyl hydroperoxide. Reduction of coenzyme Q by succinate was shown to inhibit both inner membrane lipid peroxidation and permeabilization induced by Fe(II)citrate. In contrast, the inner membrane permeabilization induced by Ca2+ alone or Ca2+ plus t-butyl hydroperoxide was potentiated by the presence of succinate. These results support our previous proposition that the mitochondrial damage associated with oxidative stress generated by these pro-oxidants in the presence of Ca2+ is mediated by different mechanisms.

The iron chelator pyridoxal isonicotinoyl hydrazone inhibits mitochondrial lipid peroxidation induced by Fe(II)-citrate.

Pyridoxal isonicotinoyl hydrazone (PIH) is able to prevent iron-mediated hydroxyl radical formation by means of iron chelation and inhibition of redox cycling of the metal. In this study, we investigated the effect of PIH on Fe(II)-citrate-mediated lipid peroxidation and damage to isolated rat liver mitochondria. Lipid peroxidation was quantified by the production of thiobarbituric acid-reactive substances (TBARS) and by antimycin A-insensitive oxygen consumption. PIH at 300 microM induced full protection against 50 microM Fe(II)-citrate-induced loss of mitochondrial transmembrane potential (deltapsi) and mitochondrial swelling. In addition, PIH prevented the Fe(II)-citrate-dependent formation of TBARS and antimycin A-insensitive oxygen consumption. The antioxidant effectiveness of 100 microM PIH (on TBARS formation and mitochondrial swelling) was greater in the presence of 20 or 50 microM Fe(II)-citrate than in the presence of 100 microM Fe(II)-citrate, suggesting that the mechanism of PIH antioxidant action is linked with its Fe(II) chelating property. Finally, PIH increased the rate of Fe(II) autoxidation by sequestering iron from the Fe(II)-citrate complex, forming a Fe(III)-PIH, complex that does not participate in Fenton-type reactions and lipid peroxidation. These results are of pharmacological relevance since PIH is a potential candidate for chelation therapy in diseases related to abnormal intracellular iron distribution and/or iron overload.

Inhibition of membrane lipid peroxidation by a radical scavenging mechanism: a novel function for hydroxyl-containing ionophores.

In the present study we show that K+/H+ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe2+-citrate and 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidylcholine (PC) liposomes containing negatively charged lipids--dicetyl phosphate (DCP) or cardiolipin (CL)--and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na+ than for K+, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O2 consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by butylated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, valinomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (Seff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe2+-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe2+-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidylcholine (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.

The role of reactive oxygen species in mitochondrial permeability transition.

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca(2+)-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca2+ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca2+ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.

Ruthenium red-catalyzed degradation of peroxides can prevent mitochondrial oxidative damage induced by either tert-butyl hydroperoxide or inorganic phosphate.

We have recently shown that ruthenium red, a non-competitive inhibitor of the mitochondrial Ca2+ uniporter, can reduce tert-butyl hydroperoxide via a Fenton-type reaction. In respiring mitochondrial preparations containing tert-butyl hydroperoxide, redox cycling of ruthenium red occurs and causes the amplification of methyl radical generation (Meinicke, A. R., Zavan, S. S., Ferreira, A. M. C., Vercesi, A. E., and Bechara, E. J. H. (1996) Arch. Biochem. Biophys. 328, 239-244). In this study we show that ruthenium red can act as an antioxidant preventing mitochondrial damage when the respiratory chain is reduced or when ascorbate is present. Ruthenium red can catalyze the degradation of hydrogen peroxide into H2O and O2. We show here that ruthenium red prevents both accumulation of mitochondrial generated H2O2 and swelling in the presence of the Ca2+ ionophore A23187. Under these conditions the damage induced by Ca2+ ions and either tert-butyl hydroperoxide or inorganic phosphate is promoted by mitochondrial-generated reactive oxygen species. Swelling induced by phenylarsine oxide, a thiol cross-linker, by a mechanism independent of free radicals is not inhibited by ruthenium red. These data provide evidence that the antioxidant behavior of ruthenium red under our conditions is due to its ability to destroy peroxides, which is related to its redox cycling and is prevalent over the Fenton-type reaction.

Characteristics of Fe(II)ATP complex-induced damage to the rat liver mitochondrial membrane.

It is well established that several iron complexes can induce oxidative damage in hepatic mitochondrial membranes by catalyzing the formation of OH radicals and/or by promoting lipid peroxidation. This is a relevant process for the molecular basis of iron overload diseases. The present work demonstrates that Fe(II)ATP complexes (5-50 microM) promote an oxygen consumption burst in a suspension of isolated rat liver mitochondria (either in the absence or presence of Antimycin A), caused mainly by lipid peroxidation. Fe(II)ATP alone induced small levels of oxygen uptake but no burst. The time course of Fe(II)ATP oxidation to Fe(II)ATP in the extramitochondrial media also reveals a simultaneous 'burst phase'. The iron chelator Desferal (DFO) or the chain-break antioxidant butylated hydroxytoluene (BHT) fully prevented both lipid peroxidation (quantified as oxygen uptake burst) and mitochondrial swelling. DFO and BHT were capable of stopping the ongoing process of peroxidation at any point of their addition to the mitochondrial suspension. Conversely, DFO and BHT only halted the Fe(II)ATP-induced mitochondrial swelling at the onset of the process. Fe(II)ATP could also cause the collapse of mitochondrial potential, which was protected by BHT if added at the onset of the damaging process. These results, as well as correlation studies between peroxidation and mitochondrial swelling, suggest that a two phase process is occurring during Fe(II)ATP-induced mitochondrial damage: one dependent and another independent of lipid peroxidation. The involvement of lipid peroxidation in the overall process of mitochondrial membrane injury is discussed.

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids.

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used (< or = 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.

The participation of reactive oxygen species and protein thiols in the mechanism of mitochondrial inner membrane permeabilization by calcium plus prooxidants.

We have recently shown that permeabilization of the inner mitochondrial membrane by calcium plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates [Fagian, M. M., Pereira da Silva, L., Martins, I. S. and Vercesi, A. E. (1990) J. Biol. Chem. 265, 19955-19960]. In this study we show that mitochondria could regenerate and sustain a membrane potential (delta psi) comparable to the control experiment after the protein aggregates were cleaved by dithiothreitol. The addition of ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, which removes Ca2+ but does not eliminate the protein aggregates, caused an incomplete and nonsustainable recovery of delta psi. Exogenous catalase prevented the disruption of membrane potential and decreased the production of membrane protein aggregates when mitochondria were incubated in the presence of Ca2+ alone or Ca2+ plus a prooxidant. This strongly indicates that H2O2 and possibly other H2O2-derived reactive oxygen species are involved in the mechanism of membrane protein aggregates production that may result in the process of membrane pore formation.

The calcium sensor ruthenium red can act as a Fenton-type reagent.

Ruthenium red (RR), an ammoniated form of tris-ruthenium(III,IV,III) oxychloride, has been widely used in the micromolar range as a strong and specific inhibitor of in vitro and in vivo Ca(2+)-mediated biochemical processes without regard for its redox properties. We show here that in the presence of tert-butyl hydroperoxide (TBHP) and an electron source, either succinate-energized rat liver mitochondria or ascorbate, RR amplifies the generation of methyl radicals. The EPR spin trapping signal of the 5,5-dimethyl-1-pyrroline-N-oxide/methyl radical (DMPO/.CH3) adduct obtained from incubations of TBHP (1.5 mM) and mitochondria (5 mg protein/ml) in an adequate medium increases upon addition of RR in a concentration-dependent fashion: sixfold at 10 microM RR. Respiring mitochondria can be replaced by ascorbate (1 mM), the characteristic EPR signal of the ascorbyl radical also being observed (aH = 0.18 mT). Spectrophotometric, cyclic voltammetric and spectroelectrochemical studies unequivocally show oxidation of RR(III,IV,III) (lambda max = 538 nm) to the ruthenium(IV,III,IV) species ("ruthenium brown," RB; lambda max = 464 nm) by TBHP, followed by its one-electron back reduction to RR by the respiratory chain or ascorbate. The calcium chelator EGTA (1 mM) strongly binds and stabilizes the RR form, slowing down its recycling by TBHP and either ascorbate or the mitochondrial electron chain. These data clearly show that Ru(III) in the RR complex can reduce TBHP via a Fenton-type reaction and thus must be considered when RR is used as a tool to study biological processes simultaneously involving Ca2+ ions and peroxides.

Oxidative damage of mitochondria induced by Fe(II)citrate is potentiated by Ca2+ and includes lipid peroxidation and alterations in membrane proteins.

Isolated rat liver mitochondria exposed to Fe(II)citrate undergo lipid peroxidation and alterations in membrane proteins. These processes were associated with irreversible decrease in membrane potential and mitochondrial swelling. Lipid peroxidation was evidenced by the production of thiobarbituric acid-reactive substances and also by the reaction of these products with membrane proteins, through the formation of Schiff bases. Alterations in membrane proteins were also characterized by the loss of specific proteins that could be recovered from the mitochondrial supernatant as shown by SDS-polyacrylamide gel electrophoresis. The degree of both lipid peroxidation and alterations in membrane proteins were diminished by EGTA, ruthenium red, or dibucaine. This strongly indicates that Ca2+ potentiates the oxidative damage of mitochondria exposed to Fe(II)citrate.

Thapsigargin causes Ca2+ release and collapse of the membrane potential of Trypanosoma brucei mitochondria in situ and of isolated rat liver mitochondria.

Thapsigargin, previously reported to release Ca2+ from non-mitochondrial stores of different cell types, as well as nigericin, were found, when used at high concentrations, to release Ca2+ and collapse the membrane potential of Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria in situ. At similarly high concentrations (> 10 microM), thapsigargin was also found to release Ca2+ and collapse the membrane potential of isolated rat liver mitochondria. These results indicate that care should be taken when attributing the effects of thapsigargin in intact cells to the specific inhibition of the sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPase family of calcium pumps. In addition, we have found no evidence for an increase in intracellular Ca2+ by release of the ion from intracellular stores by nigericin, measuring changes in cytosolic Ca2+ by dual wavelength spectrofluorometry in fura-2-loaded T. brucei bloodstream trypomastigotes or measuring Ca2+ transport in digitonin-permeabilized cells.