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The effect of methotrexate (MTX) and leucovorin (LCV) on pentose cycle enzymes and the activity of enzymes involved in enzyme defence mechanisms against ROS in HeLa cells, were studied. The effect of MTX was also investigated on the cellular levels of glutathione. MTX inhibited the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. The activities of glutathione reductase and gamma-glutamylcysteine synthetase were also inhibited by the drug. No effect was observed on the activities of catalase, superoxide dismutase or transketolase. LCV had no effect on any of the enzymes studied. MTX decreased the cellular levels of glutathione (70 per cent), while the presence of LCV and glutamine did not interfere with the effect of MTX. The net results appear to show that the biological situation resulting from treatment with MTX leads to a reduction of effectiveness of the antioxidant enzyme defence system.

The occurrence of a Crabtree effect in HeLa cells was detected. Some properties of pyruvate kinase (PK) were also evaluated. Hexose phosphate, triose-phosphate and phosphoenolpyruvate (PEP) significantly decreased the oxygen consumption of digitonin-permeabilized HeLa cells, which were oxidizing succinate. The Crabtree effect promoted by PEP was concentration-dependent and was lowered by an increase of ADP concentration, suggesting a participation of PK. The dependence of fructose-1,6-bisphosphate (FDP) by HeLa cell PK was observed. The PK of HeLa cells was inhibited by L-alanine only in the absence of FDP, while in the presence of the metabolite, an increase in the activity was observed. PK was also inhibited in the presence of L-histidine and L-leucine, while L-serine promoted activation. L-Cysteine and L-phenylalanine also inhibited the PK of HeLa cells. This, together with the sigmoidal character in relation to substrate concentration, suggests the presence of the K-type of PK in HeLa cells. © 1998 John Wiley & Sons, Ltd.

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+. © 1998 John Wiley & Sons, Ltd.

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.

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.

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.

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.

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.

Tordon herbicide, which is a mixture of 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D), depresses the phosphorylation efficiency of the rat liver mitochondria, as inferred from the decrease of the respiratory control coefficient and the ADP/O ratios when NAD(+)-dependent substrates were used; NADH oxidase and NADH cytochrome c reductase were also inhibited, without any effect on the other enzymatic complexes of the respiratory chain. Tordon (66.2 nmol picloram + 270 nmol 2,4-D mg-1 protein) affected the amplitude of swelling induced by glutamate, succinate, (N,N,N',N'-tetramethyl-p-phenyldiamine + sodium ascorbate and ATP. These results characterize an interaction of Tordon with complex I of the respiratory chain and also a partial collapse of the proton motive force of the mitochondrial inner membrane without affecting its elasticity.

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.

Foi determinada a atividade das enzimas NADH desidiogenase, NADH citocromo e redutase, succinato desidiogenase, succinato citocromo e redutase, citocromo e oxidase e citrato sintase em mitocôndrias de músculo esquelético humano normal e doente (suspeito de miopatia mitocondrial). O grupo controle foi constituído de 13 indivíduos normais e que não faziam uso contínuo de fárrnacos. O grupo doente era constituído de 10 pacientes cujo diagnóstico anatomopatológico indicava suspeita de miopatia mitocondrial. Observou-se redução na atividade das enzimas em todos os pacientes: 7 com anormalidades em todas as enzimas ensaiadas; 2 com deficiências em todas as enzimas exceto na citocromo e oxidase; e 1 paciente com disfunção apenas na atividade da succinato desidiogenase e succinato citocromo e redutase. Este perfil possibilitou caracterizar múltiplas deficiências ou deficiência combinada da cadeia respiratória, além da disfunção na citrato sintase em 9 pacientes. Um dos casos constituiu exceção, sendo a deficiência enzimática restrita ao complexo II. Foi possível concluir que a metodologia usada é adequada e facilmente aplicável aos objetivos clínicos. Os resultados obtidos possibilitam a caracterização dos complexos enzimáticos mitocondriais deficientes, mostrando que tais enfermidades são originadas de disfunção no metabolismo energético. The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome e 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.

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.

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 (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.

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.

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.

Rat liver mitochondria, stored with the energy-linked functions preserved or in aging conditions, were used to assay the activity of various enzymes during five days. The preservation of energy-linked functions was monitored by the respiratory control coefficient. ATPase, cytochrome oxidase and NADH dehydrogenase showed increased activity when the energy-linked functions were preserved. In aging conditions, cytochrome oxidase, NADH dehydrogenase and ATPase showed decreased activity. The ATPase activity increased only when mitochondria were stored in the presence of inhibitors of the electron transport chain. The activity of NADH oxidase did not change, and succinate oxidase and succinate dehydrogenase showed a small decrease in their activity. The enzymes of the matrix, alpha-ketoglutarate dehydrogenase, malate dehydrogenase and aspartate aminotransferase showed little decrease in activity under either of the conditions of storage. The total protein content decreased slightly under both conditions of storage. These results show that the activity of the enzymes analysed was maintained at reasonable levels, when the energy-linked functions of isolated mitochondria were preserved.

The effects of sodium antimony gluconate (Triostib) on oxidative phosphorylation and oxidation of succinate, glutamate and α-ketoglutarate by rat liver mitochondria were studied. Oxidative phosphorylation and oxidation of NAD + -linked substrates by liver mitochondria were depressed by 4.14 × 10 −3 M Triostib. Oxidative phosphorylation accompanying succinate oxidation was not significantly affected. Submitochondrial particles with NADH-oxidase activity were prepared and the NADH oxidation inhibited by Triostib was restored in the presence of methylene blue. It is hypothesized that Triostib acts at the NAD + -oxidase segment of the chain, its site of action being localized between NAD + and the flavoprotein.

The effect of methotrexate (MTX) on transplasma-membrane electron transport and ferricyanide-induced proton extrusion by HeLa cells was studied. Both systems were inhibited by MTX. It is suggested that inhibition of electron transport and proton extrusion caused by MTX could be associated with other metabolic alterations such as response to the increase in NADH levels and decrease in intracellular pH.

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.

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.

Rat liver mitochondria were stored at 0-4 degrees C for several days using an appropriate medium and energy source. The elimination of the majority of microsomes and lysosomes, that normally contaminate isolated mitochondria, had a positive effect in preservation of respiratory control, P:O ratio, and monoamine oxidase activity during long term storage.

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).

Oxidative phosphorylation and oxidation of NAD+-linked substrates by rat heart mitochondria were depressed by 6.75 × 10−5 M perhexiline maleate (PM), while the succinate oxidation was increased to 320 per cent activity. The drug had no effect on mitochondrial succinic and malate dehydrogenase, NADH-ferricyanide reductase and NADH-CoQ reductase (340 nm); therefore, NADH-oxidase, mitochondrial electron-transporting particles (EP1), NADH-CoQ reductase (550 nm) and aspartate aminotransferase were inhibited. It is suggested that PM would act. preventing the reoxidation of NADH+ + H+ through the respiratory chain.

A study of respiratory chain of molluscan ( Strophoceilus oblongus ) heart mitochondria at room temperature was carried out. The concentration of cytochromes was determined. Oxidative phosphorylation was assayed by polarographic technique. It was found that ADP/0 ratios were the same in the presence of either glutamate or α-ketoglutarate (about 2.9), whereas for malate it was about 2.5. By Warburg respirometry the oxidation of malate was higher than that of succinate. The oxidation of α-glycerophosphate was similar to that of succinate.

The possible site of action of chlorobutanol upon the respiratory metabolism was investigated in rat heart, cerebrum, and cerebellum preparations. The behavior of this compound on respiration of slices, homogenates, and mitochondria showed that its inhibitory effect was more evident when NAD-dependent dehydrogenases were involved in the primary oxidation of substrates, although a more drastic effect of chlorobutanol on the mitochondria could be obtained, since chlorobutanol is also an antiseptic. Nevertheless, chlorobutanol in suitable amounts was used to determine its effect on the normal properties of mitochondria-respiration, oxidative phosphorylation, respiratory control coefficient—and on the steady-state levels of cytochrome b from heart sarcosomes.

The effects of propranolol on oxidative phosphorylation and oxidation of succinate, α-ketoglutarate, glutamate and l -malate coupled with pyruvate by rat heart mitochondria were studied. Oxidative phosphorylation and oxidation of NAD + -linked substrates by heart mitochondria were depressed by 1.44 × 10 −3 M propranolol. The activity of NADH-oxidase, NADH-cytochrome c reductase and heart mitochondrial transporting particles (inner membrane) were depressed by propranolol. Dibutyryl cyclic AMP (DBc AMP) does not overcome the inhibition caused by propranolol. It is hypothesized that propranol acts at the NAD + -oxidase segment of the chain, its site of action being localized between NAD + and flavoprotein.