Eva Gunilla Skare Carnieri

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

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-induced mitochondrial permeability transition

The effects of mycotoxin citrinin on Ca2+ efflux and membrane permeabilization were studied in isolated rat liver mitochondria. The efflux rate observed when in presence of ruthenium red was higher when citrinin was added. Swelling experiments demonstrated Ca2+‐dependent membrane permeabilization by citrinin. Catalase, butylhydroxitoluene (BHT), and dithiothreitol (DTT) did not protect swelling caused by Ca2+ plus citrinin. The protection conferred by ATP–Mg2+ and cyclosporin A in the latter experiments are strong indications of pore formation. These results suggest that citrinin can induce permeability transition by a mechanism that does not involve oxidative damage.

Mitochondrial sensitivity to AZT.

The possibility of tissue‐specific effects regarding mitochondrial sensitivity to AZT was evaluated in this study. When mitochondria isolated from liver, kidney, skeletal and cardiac muscle were oxidizing glutamate, a dose‐dependent inhibition by AZT of state 3 respiration was observed; using succinate as substrate the inhibition occurred only in skeletal and cardiac muscle mitochondria. The same results were obtained with FCCP‐uncoupled mitochondria. NADH oxidase of intact and disrupted mitochondria, isolated from all four tissues was strongly inhibited. Succinate oxidase activity was inhibited by AZT only in intact mitochondria from skeletal and cardiac muscles, suggesting the involvement of succinate transport systems. Similarly, inhibition by the drug of the hydrolytic activity of H+‐ATPase was observed only in mitochondria of these tissues. These effects taken together, indicate a tissue/carrier‐specific inhibition in vitro, although its precise mechanism requires further research.

Hispidulin: Antioxidant properties and effect on mitochondrial energy metabolism†

Hispidulin (6-methoxy-5,7,4′-trihydroxyflavone) and eupafolin (6-methoxy-5,7,3′,4′-tetrahydroxyflavone), are flavonoids found in the leaves of Eupatorium litoralle. They have recognized antioxidant and antineoplastic properties, although their action mechanisms have not been previously described. We now report the effects of hispidulin on the oxidative metabolism of isolated rat liver mitochondria (Mit) and have also investigated the prooxidant and antioxidant capacity of both flavonoids. Hispidulin (0.05–0.2 mM) decreased the respiratory rate in state III and stimulated it in state IV, when glutamate or succinate was used as oxidizable substrate. Hispidulin inhibited enzymatic activities between complexes I and III of the respiratory chain. In broken Mit hispidulin (0.2 mM) slightly inhibited ATPase activity (25%). However, when intact Mit were used, the flavonoid stimulated this activity by 100%. Substrate energized mitochondrial swelling was markedly inhibited by hispidulin. Both hispidulin and eupafolin were able to promote iron release from ferritin, this effect being more accentuated with eupafolin with the suggestion of a possible involvement of H2O2 in the process. Hispidulin was incapable of donating electrons to the stable free radical DPPH, while eupafolin reacted with it in a similar way to ascorbic acid. The results indicate that hispidulin as an uncoupler of oxidative phosphorylation, is able to release iron from ferritin, but has distinct prooxidant and antioxidant properties when compared to eupafolin.

Effect of MI-D, a new mesoionic compound, on energy-linked functions of rat liver mitochondria.

MI‐D (4‐phenyl‐5‐(4‐nitro‐cinnamoyl)‐1,3,4‐thiadiazolium‐2‐phenylamine chloride), a new mesoionic compound, depressed the phosphorylation efficiency of liver mitochondria as deduced from an accentuated decrease of the respiratory control coefficient and ADP/O ratio. Analysis of segments of the respiratory chain suggested that the MI‐D inhibition site is further on than complex I and between complexes II and III. The transmembrane electrical potential (Δψ) was collapsed dependent on MI‐D concentration. ATPase activity was dramatically increased by MI‐D in intact mitochondria, but inhibited in carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP)‐uncoupled mitochondria. These results suggest that MI‐D acts as an uncoupler agent, a property closely related to its structural characteristics.

Production of cachexia mediators by Walker 256 cells from ascitic tumors

In neoplasic cachexia, chemical mediators seem to act as initiators or perpetuators of this process. Walker 256 cells, whose metabolic properties have so far been little studied with respect to cancer cachexia, are used as a model for the study of this syndrome. The main objective of this research was to pinpoint the substances secreted by these cells that may contribute to the progression of the cachectic state. Since inflammatory mediators seem to be involved in the manifestation of this syndrome, the in vitro production of nitric oxide (NO), cytokines (tumor necrosis factor alpha (TNF‐α) and interleukin‐6 (IL‐6)), and prostaglandin E2 (PGE2) was evaluated in Walker 256 cells isolated from ascitic tumors. After 4 or 5 h, a significant increase in NO production was observed (2.55 ± 1.56 and 4.05 ± 1.99 nmol NO per 107 cells, respectively). When isolated from a 6‐day‐old tumor, a significantly lower production of IL‐6 and higher production of TNF‐α than in cells from a 4‐day‐old tumor were observed, indicating a relationship between the production of cytokines and the time of tumor development after implantation. Considerable production of PGE2 by Walker 256 cells isolated from the 6‐day‐old tumor was also observed. Polyamines were also determined in Walker 256 cells. Levels of putrescine, spermidine, and spermine did not show significant differences in tumors developed during 4 or 6 days. Direct evidence of the release of proinflammatory cytokines and PGE2 by Walker 256 cells suggests that these mediators can drive the cachectic syndrome in the host, the effect being dependent on tumor development time. Copyright

Activity of isosteviol lactone on mitochondrial metabolism.

Isosteviol lactone (LAC), a lactone derivative of the diterpenic acid isosteviol (ISO) was evaluated for its effect on the oxidative metabolism of mitochondria isolated from rat liver. In this model, LAC (1 mM) depressed the phosphorylation efficiency, as shown by the decreased respiratory control coefficient (RCC) and ADP/O ratio. LAC (1 mM) inhibited NADH oxidase (45%), succinate oxidase (34%) and promoted low-level inhibitions on succinate dehydrogenase (13%), succinate-cytochrome c oxide-reductase (23%), cytochrome c oxidase (10%), and NADH dehydrogenase (13%). Glutamate dehydrogenase was also a target for LAC, as it was 85% inhibited by 1 mM LAC. Cyclic voltammetry data showed that LAC, as well as ISO, does not undergo redox reactions under current experimental conditions. LAC (0.05-0.75 mM) inhibited the swelling dependent on the glutamate oxidation, 50% of the effect occurring at 0.5 mM LAC. Swelling supported by KNO(3) and valinomycin was also inhibited over all concentrations used of LAC and ISO, the effect being of a lower intensity for LAC, suggesting that the modification of the structure of ISO by lactonization diminished its interaction with the membrane. This could contribute to attenuation of the toxic effects described for ISO on mitochondrial function, such as those on respiratory chain enzymatic complexes and phosphorylating activity.

Effects of natural flavones on membrane properties and citotoxicity of HeLa cells

The aim of this study was to determine whether eupafolin and hispidulin, flavones extracted from Eupatorium littorale Cabrera, Asteraceae, have the ability to change properties of biological membranes and promote cytotoxic effects. Eupafolin (50-200 µM) decreased approximately 30% the rate and total amplitude of valinomycin induced swelling and 60-100% the energy-dependent mitochondrial swelling. Moreover, eupafolin (200 µM) reduced 35% the mitochondrial permeability transition, and hispidulin did not change this parameter in any of the doses tested. The evaluation of phase transition of DMPC liposomes with the probe DPH demonstrated that hispidulin and eupafolin affect gel and fluid phase. With mitochondrial membrane as model, hispidulin increased the polarization of fluorescence when used DPH-PA probe. Eupafolin and hispidulin (100 µM) promoted a reduction of 40% in cellular viability of HeLa cells in 24 h. Our results suggest that eupafolin and hispidulin have cytotoxic effects that can be explained, in part, by alterations promoted on biological membranes properties and mitochondrial bioenergetics.

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.