Mechanism of citrinin-induced dysfunction of mitochondria. VI. Effect on iron-induced lipid peroxidation of rat liver mitochondria and microsomes.

The inhibition by citrinin (CTN) of lipid peroxidation of mitochondria, sub-mitochondrial particles (SMP) and microsomes was studied. This effect was reversed by the presence of high concentrations of Fe3+ (0.4 and 0.5 mM), suggesting chelation of the mycotoxin with iron or interference in the reduction of Fe3+.

Effect of amiodarone (AMD) on the antioxidant enzymes, lipid peroxidation and mitochondrial metabolism.

The effects of amiodarone (AMD) on lipid peroxidation of rat liver mitochondria, the formation of superoxide anions at the respiratory chain level, and the cytosolic and mitochondrial enzymatic protective mechanisms of oxidative stress were studied. An attempt of classify AMD according to its toxic ability to interfere with the integrated function of electron transport enzymes was also investigated. The results confirm the effects of AMD on complex I and permit the placing of this drug in class A of the classification of Knobeloch, together with rotenone, amytal and chaotropic agents. AMD has no effect on the activity of the enzymes superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase, nor on glucose 6-phosphate dehydrogenase. AMD did not promote an increase in the formation of anion superoxide at the respiratory chain level. Pre-incubation with AMD (16.6 microM) inhibited about 70 per cent of lipid peroxidation. The results suggest a protective effect of AMD against lipid peroxidation in mitochondrial membranes by iron-dependent systems.

Mechanism of citrinin-induced dysfunction of mitochondria. V. Effect on the homeostasis of the reactive oxygen species.

The effects of citrinin in the maintenance of the homeostasis of the reactive oxygen species in rat liver cells were evaluated. Citrinin (CTN) modifies the antioxidant enzymatic defences of cells through the inhibition of GSSG-reductase and transhydrogenase. No effect was observed on GSH-peroxidase, catalase, glucose 6-phosphate and 6 phosphogluconate dehydrogenases, and superoxide dismutase. The mycotoxin increased the generation of reactive oxygen species, stimulating the production of the superoxide anion in the respiratory chain. The results suggest that oxidative stress is an important mechanism, side by side with other effects previously shown, in the establishment of the cytotoxicity and cellular death provoked by CTN in several tissues.

[Characterization of mitochondrial myopathies through the evaluation of the enzymatic activities involved in energy metabolism]. / Caracterização de miopatias mitocondriais através da avaliação das atividades enzimáticas envolvidas no metabolismo energético.

The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome c oxidase and citrate synthase in normal and sick human skeletal muscle mitochondria were determined. A control group was formed by 13 normal people and without using continuous medication. The patient group was formed by 10 people whose pathological diagnosis indicated suspicion of mitochondrial myopathy. A decrease in the activity of the enzymes in all patient was observed: 7 with abnormality in all the tested enzymes; 2 with deficiencies in all the enzymes except cytochrome e oxidase; and 1 with dysfunction only in the activities of succinate dehydrogenase and succinate cytochrome e reductase. The results indicate multiple or combined deficiencies in the respiratory chain, besides dysfunction of citrate synthase in 9 patients. In one exceptional case, the enzymatic deficiency was restricted to complex II. It is possible to conclude that the methodology used herein is adequate and easily applicable to clinical objectives, and that the results obtained allow characterization of the deficient mitochondrial enzymatic complexes, thus showing that the origin of the diseases is an energetic metabolic dysfunction.

Effect of methotrexate (MTX) on NAD(P)+ dehydrogenases of HeLa cells: malic enzyme, 2-oxoglutarate and isocitrate dehydrogenases.

The effects of methotrexate (MTX) on oxygen uptake by permeabilized HeLa cells were evaluated. MTX did not inhibit state III respiration when the oxidizable substrate was succinate, but when the substrates were 2-oxoglutarate or isocitrate the respiration decreased about 50 per cent at 1.0 mM concentration of the drug. This effect was explained by inhibition of 2-oxoglutarate and isocitrate dehydrogenases by MTX. No effect was observed on succinate dehydrogenase. An evaluation of the effects of MTX on malic enzyme activity as measured by pyruvate plus lactate production in intact cells supplied with malate showed a decrease of about 40 per cent in metabolite production using 0.4 mM MTX. HeLa cell malic enzyme, as observed for other tumour cells, is compartmentalized in mitochondria and cytosol, and is another example of a dehydrogenase inhibited by MTX.

Citrinin affects the oxidative metabolism of BHK-21 cells.

The effects of citrinin on energy production along the respiratory chain and on glycolytic lactate production were examined in BHK-21 cultured cells. Citrinin inhibited the oxygen consumption rate by about 45 per cent. The respiratory rate of digitonin-treated cells energized with succinate, in the presence of ADP, was reduced by about 39 per cent. The mycotoxin inhibited the glucose utilization of BHK-21 cells by about 86 per cent. Cells treated with citrinin produced a small quantity of pyruvate, but were unable to produce lactate. It is concluded that BHK-21 cells cannot generate lactate when oxidative metabolism is inhibited by citrinin. The perturbations in BHK-21 cells caused by citrinin are due to alterations in mitochondrial function and in the glycolytic anaerobic pathway.

Amiodarone effects on membrane organization evaluated by fluorescence polarization.

The effects of amiodarone (0-100 microM) on the physical state of synthetic and native membranes were investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and of its anionic propionic acid derivative (DPH-PA), probing the outer regions of the bilayer. In the gel phase of dimyristoylphosphatidylcholine (DMPC) bilayers, amiodarone broadens the transition profile and shifts the phase transition midpoint to lower temperature values, as evaluated by both probes. On the other hand, the drug orders the fluid phase of the lipid either in hydrophobic core or in the outer regions of the bilayer, as detected by DPH and DPH-PA, respectively. The effects of amiodarone on the thermotropic behaviour of DPPC confirm and extend data in DMPC. Cholesterol concentration modulates to a great extent the effects of amiodarone in the fluid phase of DMPC. Thus, both probes, DPH and DPH-PA, detect either ordering effects of amiodarone for low cholesterol concentrations (< or = 20 mol%) or disordering amiodarone effects at higher cholesterol levels (> 20 mol%). In agreement with the results in models of synthetic lipids, the ordering effects of amiodarone in fluid native membranes of mitochondria and brain microsomes are depressed with the increase in intrinsic cholesterol. The ordering effects in mitochondria may induce bioenergetic dysfunctions and consequently disturbances in the electromechanic functioning of myocardium.

Mechanism of citrinin-induced dysfunction of mitochondria. III. Effects on renal cortical and liver mitochondrial swelling.

The effects of the mycotoxin citrinin on renal cortical and liver mitochondrial swelling were studied. Citrinin decreases the rate of swelling induced by the valinomycin-K+ complex, suggesting that the mycotoxin interferes with the mitochondrial membrane fluidity. Citrinin promotes reduction of the amplitude of swelling in the presence of Na+ ions. This alteration reflects interference with complex I of the respiratory chain and ATP synthase complex activity without disarranging the inner mitochondrial membrane, in view of the fact that the shrinkage was not affected. The effect increases with citrinin concentration. Renal tissue is more susceptible than hepatic tissue.

Mechanism of citrinin-induced dysfunction of mitochondria. IV--Effect on Ca2+ transport.

The effect of citrinin on Ca2+ transport was studied in isolated kidney cortex and liver mitochondria, and baby hamster kidney cultured cells. The mycotoxin significantly inhibited the activity of 2-oxoglutarate and pyruvate dehydrogenases in both kidney cortex and liver mitochondria. Citrinin promoted a decrease in the velocity and in the total capacity of Ca2+ uptake, in both mitochondria. Apparently, citrinin acts by a mechanism similar to ruthenium red. In intact cultured cells, citrinin also had a preferential effect on mitochondrial Ca2+ fluxes. Citrinin promoted a marked decrease in the Ca2+ level in the mitochondrial matrix, whereas that of the extramitochondiral fraction became less affected. All the observed effects were dependent on the citrinin concentration.

Alterations induced by citrinin in cultured kidney cells.

The cytotoxicity of citrinin was evaluated in an established cell line of baby hamster kidney cells. The primary effect of the mycotoxin was on the adherence of the cells to the culture bottles. Microscopic evaluation of morphological alterations indicated that the cells which were originally elongated and flattened, became swollen and round. Electron microscopic examination showed that citrinin (0.1, 0.5 and 1.0 mM) incubated for 10 hours with cultured cells, promoted drastic alterations of normal mitochondria, with swelling and cell death. Transplasma membrane redox system is inhibited by citrinin (81%). This effect is dependent not only on the toxin concentration, but also on the time of exposure to the cells.

Enalapril maleate affects 2-oxoglutarate metabolism in mitochondria from the rat kidney cortex.

Enalapril maleate (EM) is the salt of N-[(S)-1-ethoxycarbonyl)-3-phenylpropyl]-L-alanyl-L-proline, used therapeutically as an anti-hypertensive agent. The effects of EM on some aspects of the energy metabolism and membrane properties of mitochondria from rat liver and kidney cortex were studied, but only the latter were significantly affected. With 0.8 mM of EM and 2-oxoglutarate as oxidizable substrate for isolated mitochondria from rat kidney cortex, the findings were: (a) inhibition of the respiratory rate in state III (37 per cent) and decrease (45 per cent) in respiratory control ratio (RCR), with only one addition of ADP; (b) reinforcement of the inhibition when a second addition of ADP was made; (c) no significant effect either on the rate of respiration in state IV or on the ADP/O ratio; (d) no effect on the ATPase activity of mitochondria from liver or kidney cortex; (e) inhibition of the transmembrane potential (delta psi) after a second addition of ADP; (f) inhibition of the 2-oxoglutarate dehydrogenase complex. It is suggested that in kidney mitochondria, EM interferes in the gluconeogenesis dependence of at least five substrates: 2-oxoglutarate, glutamine, glutamate, lactate, and pyruvate. Also, EM may inhibit Na+/H+ exchange causing natriuresis.

Mechanism of citrinin-induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria.

The mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficient and of the ADP/O ratio. Citrinin (1.0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2-oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (delta psi), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4-dinitrophenol.

Mechanism of citrinin-induced dysfunction of mitochondria. I. Effects on respiration, enzyme activities and membrane potential of renal cortical mitochondria.

Citrinin depresses the phosphorylation efficiency of rat renal cortical mitochondria, as inferred from the decrease of the respiratory control coefficient (RC) and ADP/O ratios. The transmembrane potential (delta psi) developed by energized mitochondria and the depolarization upon ADP addition are also decreased. Citrinin (1.0 mM) inhibits almost all enzymes linked to the respiratory chain and increases the activity of succinate cytochrome c reductase and succinate oxidase (coupled). Malate and glutamate dehydrogenases are also inhibited. The inhibitory action of citrinin on phosphorylation efficiency could be related to the following findings: the effect on complex I; the action on the ATP synthetase complex; the partial inhibition of the transmembrane potential.

Calcium transport by HeLa cell mitochondria.

1. We studied mitochondria of digitonin-permeabilized HeLa cells, since digitonin (60 micrograms/10(6) cells) increases plasma membrane Ca2+ permeability, in order to avoid problems such as low mitochondrial yield and the possibility of obtaining damaged or uncoupled mitochondria from tumor cells. 2. Addition of Ca2+ to digitonin-permeabilized HeLa cells gave rise to a cycle of respiratory stimulation. Ca2+ uptake was almost totally inhibited by antimycin A (6.0 micrograms/ml). 3. Ca2+ release occurred upon addition of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) to digitonin-permeabilized HeLa cells, under steady-state conditions. An antimycin A- and FCCP-insensitive Ca2+ uptake was also detected in this preparation when ATP was added, which reflects Ca2+ capture by other organelles. 4. The characteristics of the mitochondrial Ca2+ transport system in HeLa cells are similar to those of other previously studied tumor cells. Mitochondria from HeLa cells are resistant to the deleterious effects of massive Ca2+ loads.

Calcium transport by Hela cell mitochondria

We studied mitochondria of digitonin-permeabilized Hela cells, since digitonin (60 ug/10 6 cells) increases plasma membrane Ca2+ permeability, in order to avoid problems such as low mitochondrial yield and the possibility of obtaining damaged or uncoupled mitochondria from tumor cells. Addition of Ca2+ to digitonin-permeabilized Hela cells gave rise to a cycle of respiratory stimulation. Ca2+ uptake was almost totally inhibited by antimycin A (6.0 ug/ml). Ca2+ release occurred upon addition of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) to digitonin-permeabilized Hela cells, under state conditions. An antimycin A-and FCCP-insensitive Ca2+ uptake was also detected in this preparation when ATP was added, which reflects Ca2+ capture by other organelles. The characteristics of the mitochondrial Ca2+ transport system in Hela cells are similar to those of other previously studied tumor cells. Mitochondria from Hela cells are resistant to the deleterious effects of massive Ca2+ loads

Methotrexate: studies on cellular metabolism. IV. Effect on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells.

The effect of methotrexate (MTX) on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells was studied. MTX inhibited (100 per cent) malate dehydrogenase activity, but no effect was observed on that of GOT. MTX (0.5 mM) inhibited (100 per cent) the activity of reconstituted enzymatic system MDH-GOT, probably as a consequence of inhibition of malate dehydrogenase activity. MTX decreased pyruvate production (54 per cent), demonstrating its inhibitory action on the malate-aspartate shuttle. Blockage of the malate-aspartate shuttle by MTX accounts for the decrease in cellular energetic gain. The results obtained are consistent with the view that in HeLa cells, as well as in other tumour cells, the transport of reducing equivalents from cytoplasmic NADH into the respiratory chain of mitochondria is via the malate-aspartate shuttle.

Methotrexate: studies on cellular metabolism. II--Effects on mitochondrial oxidative metabolism and ion transport.

Effects of methotrexate (MTX) on mitochondrial oxidative metabolism and ion transport were studied. MTX decreases the membrane potential (delta psi) upon energization of the mitochondrial membrane by NAD+-linked substrates and decreases the amplitude and velocity of swelling induced by glutamate and alpha-ketoglutarate. MTX also has an inhibitory effect on the activities of the oxidation enzymes of NAD+-linked substrates without interfering with the oxidation systems of FAD-linked substrates. The effects of MTX could be interpreted as a consequence of a decrease in the ionic conductivity of the mitochondrial inner membrane.

Methotrexate: studies on the cellular metabolism. I. Effect on mitochondrial oxygen uptake and oxidative phosphorylation.

Effect of methotrexate (MTX) on mitochondrial oxygen uptake, oxidative phosphorylation and on the activity of several enzymes linked to respiratory chain was studied. MTX was able to inhibit state III respiration activated by ADP and to decrease the respiratory coefficient with the substrates alpha-ketoglutarate and glutamate; these effects became pronounced when mitochondria were pre-incubated with MTX for 10 min. No effect was observed on ATPase activity of undamaged or broken mitochondria; the same was true for NADH-oxidase, NADH-dehydrogenase, NADH-cytochrome c reductase, succinate oxidase, and cytochrome c oxidase activity. The effect on the steady-state of cytochrome b, as well as, the inhibitory effect on state III of respiration with NAD+-linked substrates, offers a reasonable possibility to suggesting that the inhibition site of MTX could be in a place anterior to cytochrome b region, and not linked to respiratory chain.

Biochemical aspects of the mechanism of action of antiarrhythmic drugs on mitochondria. VIII. Effect on Ca2+ transport.

The effect of antiarrhythmic drugs (propranolol, lidoflazine, perhexiline maleate and iproveratril) on Ca2+ uptake and Na+-induced Ca2+ release by isolated rat heart mitochondria has been studied using arsenazo III as Ca2+ indicator. It was concluded that those drugs are not selective inhibitors either for Na+-induced Ca2+ release or for Ca2+ uptake. It seems possible that those drugs act by complexation with some components of the membrane, presumably with phospholipids.

Biochemical aspects of the mechanism of action of antiarrhythmic drugs on mitochondria. VII. Effect on energy-linked reactions and on membrane potential.

Effects of the antiarrhythmic drugs (propranolol, perhexiline maleate, lidoflazine and iproveratril) on energy-linked reactions and on membrane potential were studied. Propranolol, perhexiline maleate and lidoflazine inhibit the ATPase activity of undamaged and broken mitochondria, and of submitochondrial particles. All drugs are inhibitors of either ATP-driven or of succinate-driven reduction of NADP+. The antiarrhythmics promote a decrease in the membrane potential upon energization of the mitochondrial membrane by alpha-ketoglutarate, succinate, or ATP. It was suggested that these drugs have a primary action on the mitochondrial membrane, thus altering the activities of membrane proteins (channels and enzymes).