Guido Scutari

Induction of mitochondrial permeability transition by auranofin, a Gold(I)-phosphine derivative

Gold(I)‐thiolate drugs are compounds that specifically interact with thiol and/or selenol groups and are essentially utilized in the treatment of rheumatoid arthritis. Considering the importance of thiol groups in regulating mitochondrial membrane permeability, the effects of auranofin (S‐triethylphosphinegold(I)‐2,3,4,6‐tetra‐O‐acetyl‐1‐thio‐β‐D‐glucopyranoside), a second‐generation gold drug, were studied on mitochondria isolated from rat liver. Auranofin, at submicromolar concentrations, was able to induce the mitochondrial membrane permeability transition observed as swelling and loss of membrane potential. Both events are completely inhibited by cyclosporin A, the specific inhibitor of mitochondrial permeability transition. Calcium ions and energization by succinate are required for the occurrence of permeability transition. By interacting with the active site selenol group, auranofin results as an extremely potent inhibitor of mitochondrial thioredoxin reductase, both isolated and in its mitochondrial environment. It is concluded that auranofin, in the presence of calcium ions, is a highly efficient inducer of mitochondrial membrane permeability transition, potentially referable to its inhibition of mitochondrial thioredoxin reductase.

Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase.

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.

Mitochondrial permeability transition and release of cytochrome c induced by retinoic acids

Retinoic acids, structurally related to vitamin A, inhibit the in vitro proliferation of different types of normal and neoplastic cells. The effects of all-trans, 9-cis, and 13-cis retinoic acids were tested on mitochondria isolated from rat liver. All the compounds were able to induce the membrane permeability transition observed as swelling and decrease in membrane potential, but 13-cis retinoic acid appeared to be the most effective. The latter was also shown to stimulate the release of cytochrome c from mitochondria, suggesting a potential target of retinoids in the induction of cell apoptosis. Interestingly, EGTA and cyclosporin A, which strongly inhibit the permeability transition induced by 13-cis retinoic acid, were without effect on the release of cytochrome c from the mitochondrial intermembrane space.

Influence of the anesthetic 2,6-diisopropylphenol on the oxidative phosphorylation of isolated rat liver mitochondria.

Isolated rat liver mitochondria have been incubated in the presence of the general anesthetic 2,6-diisopropylphenol (0-100 microM) and the efficiency of oxidative phosphorylation has been evaluated by measuring the respiratory rates, the rates of ATP synthesis or hydrolysis and the magnitude of the transmembrane electrical potential. The results obtained indicate that: (a) in mitochondria energized either by succinate or by ATP, 2,6-diisopropylphenol decreased the transmembrane electrical potential and increased the rates of either electron transfer or ATP hydrolysis; (b) in succinate-energized mitochondria 2,6-diisopropylphenol, at concentrations causing substantial depression of the transmembrane electrical potential, did not modify either the rate of phosphorylation of added ADP or the rate of ADP-stimulated respiration: (c) in succinate-energized mitochondria 2,6-diisopropylphenol caused a concentration-dependent inhibition of the uncoupler-stimulated rate of succinate oxidation. These findings suggest that under the experimental conditions reported 2,6-diisopropylphenol affected the generation and/or maintenance of the transmembrane electrical potential while leaving unchanged the coupling between the electron flow in the respiratory chain and the synthesis of ATP.

ANTIOXIDANT PROPERTIES OF CLOZAPINE AND RELATED NEUROLEPTICS

The antioxidant properties of clozapine and other related molecules were evaluated with the crocin bleaching test both in aqueous and non-aqueous environment. The tests of microsomal lipid peroxidation and carbonyl formation were also used. In aqueous solution, chlorpromazine and trifluoperazine appear particularly effective in the bleaching of crocin, while serotonin has an efficacy intermediate between those of phenothiazines and clozapine. The latter drug, on the other hand, in a non-aqueous medium shows an antioxidant power comparable to that of butylated hydroxytoluene, indicating that its antioxidant properties are better expressed in a hydrophobic environment of the type present in a biological membrane. In fact, in lipid peroxidation induced in microsomal membranes, clozapine, chlorpromazine, trifluoperazine and serotonin act as very good antioxidants; at low concentrations, clozapine appears to be the most efficient after butylated hydroxytoluene. Similarly, all these compounds markedly inhibit protein carbonyl formation, clozapine being one of the most efficient. Thus, under different in vitro experimental conditions, the neuroleptic drugs chlorpromazine and trifluoperazine and the antipsychotic substance clozapine act as very effective antioxidants; this property might, at least in part, be responsible for the physiological and clinical effects observed in vivo.

Gold(I) complexes determine apoptosis with limited oxidative stress in Jurkat T cells.

In Jurkat T cells, S-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-beta-d-glucopyranoside (auranofin) and triethylphosphine gold(I) chloride (TepAu) induced apoptosis, as estimated by DNA fragmentation and visualised by fluorescence microscopy. Apoptosis was characterised by mitochondrial cytochrome c release which was not prevented by cyclosporin A. Apoptosis appeared to be triggered by inhibition exerted by gold(I) compounds on the cytosolic and mitochondrial isoforms of thioredoxin reductase, which determined a definite increase in hydrogen peroxide, whereas glutathione and its redox state were not modified. Total thiols showed a slight decrease, particularly in the presence of auranofin. However, no significant lipid peroxidation or nitric oxide formation were observed after incubation with gold(I) complexes, indicating that the cells had not been subjected to extensive oxidative stress. Interestingly, the gold(I) compound aurothiomalate was poorly effective, both in inhibiting thioredoxin reductase and in inducing apoptosis. These results demonstrate that the increased production of hydrogen peroxide determines an oxidative shift responsible for the occurrence of apoptosis and not involving lipid peroxidation.

Uncoupling effect of the general anesthetic 2,6-diisopropylphenol in isolated rat liver mitochondria

2,6-Diisopropylphenol, a general anesthetic, was previously reported to reduce the transmembrane electrical potential in isolated rat liver mitochondria without affecting the rate of ATP production. This effect appeared to contrast with the generally accepted chemiosmotic mechanism for oxidative phosphorylation. In this study we further examined the influence of 2,6-diisopropylphenol on the production of ATP by isolated mitochondria and we studied its effect on the permeability of the inner mitochondrial membrane to protons. In order to clarify the effects of 2,6-diisopropylphenol on mitochondrial ATP production the activities of the adenine nucleotide translocator and the ATP synthetase were evaluated. The results obtained indicate that the depression of the transmembrane electrical potential elicited by 2,6-diisopropylphenol decreased the activity of the ATP synthetase (as expected in the chemiosmotic model for energy coupling), but not that of the adenine nucleotide translocator. The decrease of the ATP synthetase activity, however, did not result in an apparent inhibition of the overall rate of ATP production in isolated mitochondria due to the rate-limiting effect of the adenine nucleotide translocator in this process. Moreover 2,6-diisopropylphenol was found to increase the permeability to protons of the inner mitochondrial membrane; this effect became more marked as the pH of the incubation medium was increased, demonstrating that it involved the dissociated form of 2,6-diisopropylphenol. These observations suggested that 2,6-diisopropylphenol affected oxidative phosphorylation by acting as a mild protonophore and that its effectiveness was limited by the low fraction of phenol dissociated at near-physiological pH.

Evaluation of the antioxidant properties of propofol and its nitrosoderivative. comparison with homologue substituted phenols.

Propofol (2,6-diisopropylphenol), some substituted phenols (2,6-dimethylphenol and 2,6-ditertbutylphenol) and their 4-nitrosoderivatives have been compared for their scavenging ability towards 1,1-diphenyl-2-picrylhydrazyl and for their inhibitory action on lipid peroxidation. These products were also compared to the classical antioxidants butylated hydroxytoluene and butylated hydroxyanisole. When measuring the reactivity of the various phenolic derivatives with 1,1-diphenyl-2-picrylhydrazyl the following order of effectiveness was observed: butylated hydroxyanisole>propofol>2,6-dimethylphenol>2,6-di-tertbutylphenol > butylated hydroxytoluene. In cumene hydroperoxide-dependent microsomal lipid peroxidation, propofol acts as the most effective antioxidant, while butylated hydroxyanisole, 2,6-di-tertbutylphenol and butylated hydroxytoluene exhibit a rather similar effect, although lower than propofol. In the iron/ascorbate-dependent lipid peroxidation propofol, at concentrations higher than 10 μM, exhibits antioxidant properties comparable to those of butylated hydroxytoluene and butylated hydroxyanisole. 2,6-Dimethylphenol is scarcely effective in both lipoperoxidative systems. The antioxidant properties of the various molecules depend on their hydrophobic characteristics and on the steric and electronic effects of their substituents. However, the introduction of the nitroso group in the 4-position almost completely removes the antioxidant properties of the examined compounds. The nitrosation of the aromatic ring of antioxidant molecules and the consequent loss of antioxidant capacity can be considered a condition potentially occurring in vivo since nitric oxide and its derivatives are continuously formed in biological systems.

Effect of Metal Complexes on Thioredoxin Reductase and the Regulation of Mitochondrial Permeability Conditions

Abstract: Gold(I) compounds such as auranofin, chloro(triethylphosphine) gold(I), and aurothiomalate act on mitochondrial functional parameters by determining an extensive permeability transition and a decrease of membrane potential. On the contrary, pyridine nucleotides and glutathione are not modified, whereas a slight but significant decrease of total thiols is apparent. The effect of gold(I) compounds is essentially referable to the inhibition, in the nanomolar range, of thioredoxin reductase activity and to an increase of hydrogen peroxide production. Metal ions and metal complexes (zinc and cadmium acetate, cisplatin, tributyltin) are also good inhibitors of thioredoxin reductase, although in the micromolar range, and in addition, they act as inducers of permeability transition and of membrane potential decrease. At variance with gold(I) compounds, which appear to work almost exclusively on thioredoxin reductase, metal ions and complexes are less specific, since they are active on different mitochondrial targets, including the respiratory chain.

AN IN VITRO STUDY ON THE TOXIC EFFECTS OF NONYLPHENOLS (NP) IN MITOCHONDRIA

This paper is focused on alkylphenols, compounds which are formed by the biodegradation of polyethoxilatedalkylphenols detergents. Our experiments show that alkylphenols act not only as detergents, but also as uncouplers of the oxidative phosphorylation. This effect, can be observed at very low doses, thus suggesting that the preferential target of nonylphenols in living organisms are mitochondria.