Maria Benigna M. Oliveira

Effect of an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colubrina (Angico branco) on peritoneal macrophage functions

Brazilian flora are a source of interesting polysaccharides which, either in their native state or when submitted to structural modifications, might have potential applications as biological response modifiers (BRM). A complex acidic heteropolysaccharide, containing mainly galactose and arabinose (ARAGAL), isolated from the gum of the native leguminous tree Anadenanthera colubrina (Angico branco), was studied for its immunological properties on peritoneal exudate cells, namely their superoxide anion production, phagocytic activity, morphological alterations and percentage content of activated macrophages. Activation of macrophages showing increased cytoplasm, bright and large nuclei, various cytoplasmatic projections and spreading ability, was detected following in vitro cell exposure to ARAGAL or in cells obtained from treated animals. In vitro exposure to ARAGAL increased the occurrence of activated macrophages in a time- and a dose-dependent pattern, since approximately 82% of the cells were activated in the presence of 300 microg/ml of ARAGAL after 24 h of incubation and approximately 91% after 48 h. The occurrence of activated macrophages was also evident in cell preparations from ARAGAL-treated mice, their percentage showing a dose-dependent pattern. There were approximately 60, 75 and 75% following treatment with 100, 250 and 500 mg/kg of ARAGAL, respectively. A phagocytic assay showed that 25 microg/ml ARAGAL was sufficient to impose a maximum phagocytic ability, although this effect was dose-dependent. O(2)(-) production by macrophages from ARAGAL-treated mice was 70% higher than that of cells from untreated mice. Moreover, cells from treated mice responded to PMA, the effect being 25% higher than that of the control using untreated mice. These results thus suggest a possible role of ARAGAL from A. colubrina as a BRM.

Effects of a lichen galactomannan and its vanadyl (IV) complex on peritoneal macrophages and leishmanicidal activity

A galactomannan (GMPOLY) isolated from lichen Ramalina celastri was complexed with vanadyl ion (IV;VO) forming the complex GMPOLY-VO. Both GMPOLY and GMPOLY-VO diminished the superoxide anion production by macrophages triggered with PMA, the complex giving rise to this effect at concentrations 100 times lower than GMPOLY. Macrophages treated with GMPOLY enhanced the nitric oxide production (40%), this effect not being observed when interferon-γ ((IFN-γ) or IFN-γ plus lipopolysaccharide (LPS) were present. No effect on nitric oxide production was observed by treatment of macrophage with GMPOLY-VO. Both GMPOLY and GMPOLY-VO exhibited leishmanicidal effects on the amastigote form of Leishmania amazonesis, but only GMPOLY-VO inhibited the growth of promastigote form.

Effect of a soluble α-d-glucan from the lichenized fungus Ramalina celastri on macrophage activity

An alpha-glucan from the lichen Ramalina celastri has previously been demonstrated to have cytotoxic effects against HeLa cells. This polysaccharide was studied using Sarcoma-180 cells as tumoral model, and its effects on peritoneal exudate cells, namely, hydrogen peroxide production, phagocytic activity and cell eliciting activity are evaluated. Tumors developing in animals treated with the glucan at a dose of 200 mg kg(-1), had a tumor size approximately 80% smaller than that of the control group, showing an impairment of tumor establishment. The polysaccharide was injected into mice not bearing a tumor and after 7, 15 and 30 days the cells were collected from the peritonea. The number of peritoneal cells increased approximately 130% 7 days after inoculation, and then gradually decreased. Hydrogen peroxide production was 75% greater 7 and 15 days after inoculation, on in vitro phorbol myristate acetate (PMA) triggering. Without PMA, the difference in hydrogen peroxide production was not significant. Phagocytic assays using fluorescent beads showed that the uptake increased 7 and 15 days after inoculation, when compared with the control. These results thus suggest a possible role of the R. celastri glucan as a biological response modifier (BRM).

Antimelanoma activity of 1,3,4-thiadiazolium mesoionics: a structure-activity relationship study.

The effect of a series of 4-phenyl-5-(2′-Y, 4′-X or 4′-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chlorides was evaluated against B16-F10 murine melanoma cells in vitro and against tumors resulting from implanted B16-F10 cells in C57BL/6 mice. These compounds differ from each other only at the cinnamoyl ring substituent (MI-J, X=OH; MI-2,4diF, X=Y=F; MI-4F, X=F and MI-D, X=NO2). The results were compared with those obtained for MI-D, which has already been shown to be a potent and promising drug against melanoma. On exposure of B16-F10 cells to MI-D, MI-2,4diF and MI-4F, all of them at the same micromolar concentration (50 μM) decreased the cell viability to 8, 50 and 22%, respectively, while MI-J did not show any significant effect under the same conditions. However, low doses such as 10 μM MI-D were sufficient to impair cell growth over 72 h, but for MI-2,4diF and MI-4F the effect on B16-F10 proliferation was only observed at a concentration of 25 μM. Furthermore, MI-4F had a slightly better effect than MI-2,4diF in vitro; its effect on tumor growth in vivo was not significant. MI-D inhibited tumor growth by 77%. The greater effectiveness of MI-D compared with MI-2,4diF, MI-4F and MI-J against B16-F10 melanoma cells is probably due to its stronger electron-withdrawing group (NO2), which increases the positive charge on the mesoionic ring and allows extensive conjugation of the side-chain with the exocyclic moiety. This seems to be important for degree of anti-tumor activity of these compounds.

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.

Cytotoxic effect of a new 1,3,4-thiadiazolium mesoionic compound (MI-D) on cell lines of human melanoma.

The structural characteristics of mesoionic compounds, which contain distinct regions of positive and negative charges associated with a poly-heteroatomic system, enable them to cross cellular membranes and interact strongly with biomolecules. Potential biological applications have been described for mesoionic compounds. 1,3,4-Thiadiazolium mesoionic compound (MI-D), a new mesoionic compound, has been demonstrated to be extremely cytotoxic to B16-F10 murine melanoma cells when compared to fotemustine and dacarbazine, drugs of reference in melanoma treatment protocols, describing inhibition of tumours grown in vitro and in vivo. We now evaluate the effects of mesoionic compound MI-D on different human melanoma cell lines. The drug decreased the viability and proliferation of MEL-85, SK-MEL, A2058 and MEWO cell lines in vitro, showing a considerable cytotoxic activity on these human cells. Adhesion of MEL-85 cells was evaluated in the presence of the drug using different extracellular matrix (ECM) constituents. MI-D decreased MEL-85 adhesion to laminin, fibronectin and matrigel. The morphology and actin cytoskeleton organisation of MEL-85 cells were also modified on MI-D treatment. These results on human melanoma cell lines indicate that MI-D is a very encouraging drug against melanoma, a tumour that is extremely resistant to chemotherapy.

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.

Chemical and immunological modifications of an arabinogalactan present in tea preparations of Phyllanthus niruri after treatment with gastric fluid

An arabinogalactan (AG) obtained from tea preparations of Phyllanthus niruri was previously investigated and presented immunological properties when tested with peritoneal mice macrophages. AG was now submitted to acidic and neutral gastric conditions using human gastric fluids and aq. HCl solution. Since the acidic procedures gave rise to the same free monosaccharidic composition, the acid hydrolyzate of AG at pH 2.00 was treated with ethanol to form insoluble (AG-P) and soluble fractions (AG-S). These were analyzed using (13)C NMR, HPSEC, and GC-MS for monosaccharide composition and methylation analyses. The results showed an intense partial degradation, including cleavages of the main chain. AG-S presented the monosaccharides released from the native polymer and some oligosaccharides as shown by methylation data. AG-P contained larger molecular fragments comprising the internal units from AG, which were not attacked by the hydrolysis condition. Both fractions were tested in peritoneal mice macrophages and remained active, promoting an increase of superoxide anion production of 2.0 and 2.3-fold, at 250 microg/mL, for AG-S and AG-P, respectively. When compared to AG, a slight diminished response was observed, revealing a structure-activity relation. The significance of the results is that most plant extracts are orally ingested and will reach the gastrointestinal tract before performing a biological function, so checking these changes is crucial to propose future clinical therapies based on the rational use of phytomedicine.

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.