Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.

Disulfiram (Ds), a clinically employed alcohol deterrent of the thiuram disulfide (TD) class of compounds, is known to cause hepatitis and neuropathies. Although this drug has been shown to inhibit different thiol-containing enzymes, the actual mechanism of Ds toxicity is not clear. We have previously demonstrated that Ds impairs the permeability of inner mitochondrial membrane (IMM) [Arch. Biochem. Biophys. 356 (1998) 46]. In this report, the effect of Ds and its structural analogue thiram (Th) on mitochondrial functions was studied in detail. We found that mitochondria metabolize TDs in a NAD(P)H- and GSH-dependent manner. At the concentration above characteristic threshold, TDs induced irreversible oxidation of NAD(P)H and glutathione (GSH) pools, collapse of transmembrane potential, and inhibition of oxidative phosphorylation. The presence of Ca(2+) and exhaustion of mitochondrial glutathione (GSH+GSSG) decreased the threshold concentration of TDs. Swelling of the mitochondria and leakage of non-transported fluorescent dye BCECF from the matrix indicated that TDs induced the mitochondrial permeability transition (MPT). Mitochondrial permeabilization by TDs involves two, apparently distinct mechanisms. In the presence of Ca(2+), TDs produced cylosporin A-sensitive swelling of mitochondria, which was inhibited by ADP and accelerated by carboxyatractyloside (CATR) and phosphate. In contrast, the swelling produced by TDs in the absence of Ca(2+) was not sensitive to cyclosporin A (CsA), ADP and CATR but was inhibited by phosphate. Titration with N-ethylmaleimide revealed that these two mechanisms involve different SH-groups and probably different transport proteins on the IMM. Our findings indicate that at pharmacologically relevant concentrations TDs may cause an irreversible mitochondrial injury as a result of induction of the MPT.

Lipoic acid-derived amphiphiles for redox-controlled DNA delivery.

BACKGROUND: Intracellular release of free DNA from the vector complex is one of the critical steps limiting the efficiency of non-viral gene delivery. The complex should be stable enough to prevent DNA degradation but it should be destabilized inside the cell to allow DNA release and transcription. Destabilization and degradation of synthetic vectors is also required to reduce their cytotoxicity and augment the life-time of transfected cells. RESULTS: Here we describe new cationic amphiphiles made from the natural pro-vitamin, lipoic acid, that reversibly binds and releases DNA, depending on the redox state of the lipoate moieties. In the oxidized state these amphiphiles condense DNA into homogeneous spherical particles, which, upon reduction, swell into DNA toroids with subsequent release of free DNA. Complex reduction and DNA release can be induced by various thiols as well as enzymatically, by thioredoxin reductase. Transfection with amphiphile-DNA complexes in vitro shows a several fold increase of transgene expression compared with DOTAP, and can be further augmented by attachment of the nucleus-targeting peptide to the amphiphile. The increase of transfection efficiency results from GSH- and NAD(P)H-dependent complex reduction and release of free DNA inside the cells. CONCLUSIONS: The present work demonstrates the principle of a redox-controlled gene delivery system that uses the reversibility of thiol-disulfide exchange reaction. Our data suggest that the efficiency of synthetic vectors can be augmented by their controlled destabilization inside the cells. Being formed from the natural non-toxic compound lipoic acid, these cationic amphiphiles provide a new promising class of synthetic vectors for gene delivery.

Gradual changes in permeability of inner mitochondrial membrane precede the mitochondrial permeability transition.

Some compounds are known to induce solute-nonselective permeability of the inner mitochondrial membrane (IMM) in Ca2+-loaded mitochondria. Existing data suggest that this process, following the opening of a mitochondrial permeability transition pore, is preceded by different solute-selective permeable states of IMM. At pH 7, for instance, the K0.5 for Ca2+-induced pore opening is 16 microM, a value 80-fold above a therapeutically relevant shift of intracellular Ca2+ during ischemia in vivo. The present work shows that in the absence of Ca2+, phenylarsine oxide and tetraalkyl thiuram disulfides (TDs) are able to induce a complex sequence of IMM permeability changes. At first, these agents activated an electrogenic K+ influx into the mitochondria. This K+-specific pathway had K0.5 = 35 mM for K+ and was inhibited by bromsulfalein with Ki = 2.5 microM. The inhibitors of mitochondrial KATP channel, ATP and glibenclamide, did not inhibit K+ transport via this pathway. Moreover, 50 microM glibenclamide induced by itself K+ influx into the mitochondria. After the increase in K+ permeability of IMM, mitochondria become increasingly permeable to protons. Mechanisms of H+ leak and nonselective permeability increase could also be different depending on the type of mitochondrial permeability transition (MPT) inducer. Thus, permeabilization of mitochondria induced by phenylarsine oxide was fully prevented by ADP and/or cyclosporin A, whereas TD-induced membrane alterations were insensitive toward these inhibitors. It is suggested that MPT in vivo leading to irreversible apoptosis is irrelevant in reversible ischemia/reperfusion injury.

Effect of the triaminopyridine flupirtine on calcium uptake, membrane potential and ATP synthesis in rat heart mitochondria.

1. Flupirtine is an analgesic agent which exhibits neuronal cytoprotective activity and may have value in the treatment of conditions involving cell injury and apoptosis. Since flupirtine has no action on known receptor sites we have investigated the effect of this drug on mitochondrial membrane potential, and the changes in intramitochondrial calcium concentration in particular. 2. The findings show that flupirtine increases Ca2+ uptake in mitochondria in vitro. At clinically relevant flupirtine concentrations, corresponding to flupirtine levels in vitro of 0.2 to 10 nmol mg(-1) mitochondrial protein, there was a 2 to 3 fold increase in mitochondrial calcium levels (P<0.01). At supra-physiological flupirtine concentrations of 20 nmol mg(-1) mitochondrial protein and above, the mitochondrial calcium concentrations were indistinguishable from those in untreated mitochondria. 3. Mitochondrial membrane potential closely paralleled the changes in mitochondrial calcium levels showing a 20% (P<0.01) increase when the flupirtine concentration was raised from 0.2 nmol to 10 nmol mg(-1) mitochondrial protein and a return to control values at 20 nmol mg(-1) protein. 4. The increase in mitochondrial calcium uptake and membrane potential were accompanied by an increase in mitochondrial ATP synthesis (30%; P<0.05) and a similar percentage reduction in mitochondrial volume. 5. Calcium at 80 and 160 nmol mg(-1) mitochondrial protein decreased ATP synthesis by 20-25% (P<0.001). This decrease was prevented or diminished if flupirtine at 10 nmol mg(-1) protein was added before the addition of calcium. 6. Since intracellular levels of flupirtine in intact cells never exceeded 10 nmol mg(-1) mitochondrial protein, these findings are supportive evidence for an in vivo cytoprotective action of flupirtine at the mitochondrial level.