Annibal P. Campello

Methotrexate: pentose cycle and oxidative stress

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 γ‐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. Copyright

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.

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.

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 to 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 μM) 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.

Effects of propranolol on heart muscle mitochondria

Abstract 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.

Occurrence of the Crabtree effect in HeLa cells

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.

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+.

Studies of schistosomicides antimonials on isolated mitochondria—I: Sodium antimony gluconate (triostib)

Abstract 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.

Possible mechanism of action of perhexiline maleate on heart mitochondria.

Abstract 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.

The effect of chlorobutanol on the respiratory metabolism and on the normal properties of isolated mitochondria

Abstract 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.