Metabolism of acetaldehyde to methyl and acetyl radicals: in vitro and in vivo electron paramagnetic resonance spin-trapping studies.

Acetaldehyde oxidation by enzymes and cellular fractions has been previously shown to produce radicals that have been characterized as superoxide anion, hydroxyl, and acetyl radicals. Here, we report that acetaldehyde metabolism by xanthine oxidase, submitochondrial particles and whole rats produces both the acetyl and the methyl radical, although only the latter was unambiguously identified in vivo. Electron paramagnetic resonance (EPR) characterization of both radicals was possible by the use of two spin traps, 5,5-dimethyl 1-pyrroline N-oxide (DMPO) and alpha-(4-pyridyl 1-oxide)-N-t-butylnitrone (POBN), and of acetaldehyde labeled with (13)C. The POBN-acetyl radical adduct proved to be unstable, but POBN was employed to monitor acetaldehyde metabolism by Sprague-Dawley rats because previous studies have shown its usefulness for in vivo spin trapping. EPR analysis of the bile collected from treated and control rats showed the presence of the POBN-methyl and of an unidentified, biomolecule-derived, POBN adduct. Because decarbonylation of the acetyl radical is one of the routes for methyl radical formation from acetaldehyde, detection of the latter in bile provides strong evidence for the production of both radicals in vivo. The results may be relevant to understanding the toxic effects of acetaldehyde itself and of its more relevant biological precursor, ethanol.

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.

Ca2+-induced increased lipid packing and domain formation in submitochondrial particles. A possible early step in the mechanism of Ca2+-stimulated generation of reactive oxygen species by the respiratory chain.

Ca2+ and P(i) accumulation by mitochondria triggers a number of alterations leading to nonspecific increase in inner membrane permeability [Kowaltowski, A. J., et al. (1996) J. Biol. Chem. 271, 2929-2934]. The molecular nature of the membrane perturbation that precedes oxidative damage is still unknown. EPR spectra of spin probes incorporated in submitochondrial particles (SMP) and in model membranes suggest that Ca(2+)-cardiolipin (CL) complexation plays an important role. Ca(2+)-induced lipid domain formation was detected in SMP but not in mitoplasts, in SMP extracted lipids, or in CL-containing liposomes. The results were interpreted in terms of Ca2+ sequestration of CL tightly bound to membrane proteins, in particular the ADP-ATP carrier, and formation of CL-enriched strongly immobilized clusters in lipid shells next to boundary lipid. The in-plane lipid and protein rearrangement is suggested to cause increased reactive oxygen species production in succinate-supplemented, antimycin A-poisoned SMP, favoring the formation of carbon-centered radicals, detected by EPR spin trapping. Removal of tightly bound CL is also proposed to cause protein aggregation, facilitating intermolecular thiol oxidation. Lipid peroxidation was also monitored by the disappearance of the nitroxide EPR spectrum. The decay was faster for nitroxides in a more hydrophobic environment, and was inhibited by butylated hydroxytoluene, by EGTA, or by substituting Mg2+ for Ca2+. In addition, Ca2+ caused an increase in permeability, evidenced by the release of carboxyfluorescein from respiring SMP. The results strongly support Ca2+ binding to CL as one of the early steps in the molecular mechanism of Ca(2+)-induced nonspecific inner mitochondrial membrane permeabilization.

Inhibition of Ca2+ release from Trypanosoma brucei acidocalcisomes by 3,5-dibutyl-4-hydroxytoluene: role of the Na+/H+ exchanger.

Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain a considerable fraction of intracellular Ca2+. They possess a vacuolar-type H+-ATPase for H+ uptake, a Ca2+/H+ countertransporting ATPase for Ca2+ uptake and a Ca2+/nH+ antiporter for Ca2+ release. Trypanosoma brucei procyclic trypomastigotes acidocalcisomes possess, in addition, an Na+/H+ antiporter that may participate in Ca2+ release from these organelles. In this work we show that the hydrophobic antioxidant 3,5-dibutyl-4-hydroxy toluene (BHT), at concentrations in the range 1-20 microM, inhibits Na+-induced Ca2+ release from the acidocalcisomes of digitonin-permeabilized procyclic trypomastigotes in a concentration-dependent manner. This effect supports the notion that Ca2+ release from this compartment is regulated by the activity of the Na+/H+ antiporter. In the presence of BHT, Ca2+ release could still be obtained by nigericin-mediated alkalinization of the acidocalcisomes, clearly demonstrating that the action of BHT is not at the level of the Ca2+/nH+ antiporter but on that of the Na+/H+ antiporter. In the same range of concentrations and depending on the preincubation time, BHT had an stimulatory or an inhibitory effect on the vacuolar H+-ATPase present in T. brucei acidocalcisomes. Since these effects of BHT were obtained at concentrations which are commonly used for its antioxidant properties, these results indicate that care should be exercised when attributing effects of BHT to only these properties.

Effect of inorganic phosphate concentration on the nature of inner mitochondrial membrane alterations mediated by Ca2+ ions. A proposed model for phosphate-stimulated lipid peroxidation.

Addition of high concentrations (>1 mm) of inorganic phosphate (Pi) or arsenate to Ca2+-loaded mitochondria was followed by increased rates of H2O2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This swelling was totally prevented by the simultaneous presence of these compounds. At lower Pi concentrations (1 mm), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, at low Pi concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition pore, whereas at high Pi concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions, lipid peroxidation is strongly inhibited by sorbate, a putative quencher of triplet carbonyl species. This suggests that high Pi or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because these anions are able to catalyze Cn-aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower Cn - 1-aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate liposomes and the triplet acetone-generating system isobutanal/horseradish peroxidase, where phosphate and Ca2+ cooperate to increase the yield of thiobarbituric acid-reactive substances.