RESUMO
A nutritional characteristic of trypanosomatid protozoa is that in vitro they need a haem-compound as a growth factor, which is supplied as haemoglobin, haematin or haemin. Because haemin and related porphyrins are an important source of oxidative stress in biological systems, the effect of haemin on growth, protein content and the antioxidant defence system in Trypanosoma cruzi was evaluated. We have observed that, in epimastigotes grown under different haemin concentrations in the culture medium (0-30 mg/l), 5 mg/l was the haemin concentration yielding optimum growth. Above 15 mg/l there was a clear decrease in growth rate, producing the epimastigote to amastigote transformation. Such morphological change was observed together with a marked injury of the enzymatic machinery of the parasite, leading to diminished protein synthesis as well as lower activity of the antioxidant enzymes (superoxide dismutase, ascorbate peroxidase and trypanothione reductase), reduced total thiol content and a marked increase in the HaemOx-1 activity and expression. The current work demonstrates that there is a correlation between higher haemin concentrations in the culture medium and oxidative damage in the cells. Under these conditions induction of HaemOx-1 would indicate the important role of this enzyme as an antioxidant defence response in Trypanosoma cruzi.
Assuntos
Hemina/farmacologia , Estresse Oxidativo/fisiologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/fisiologia , Animais , Ascorbato Peroxidases , Meios de Cultura , Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/análise , Heme Oxigenase-1/biossíntese , NADH NADPH Oxirredutases/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peroxidases/efeitos dos fármacos , Peroxidases/metabolismo , Proteínas/análise , Proteínas/efeitos dos fármacos , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Fatores de Tempo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.
Assuntos
Acanthamoeba/efeitos dos fármacos , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antipsicóticos/farmacologia , NADH NADPH Oxirredutases/efeitos dos fármacos , Naegleria fowleri/efeitos dos fármacos , Acanthamoeba/enzimologia , Animais , Cromatografia Líquida de Alta Pressão , Glutationa Redutase/efeitos dos fármacos , Glutationa Redutase/metabolismo , Humanos , NADH NADPH Oxirredutases/metabolismo , Naegleria fowleri/enzimologiaRESUMO
Trypanosoma cruzi trypanothione reductase is an enzyme that has been identified as a potential target for chemotherapy. Thioridazine inhibits it and prevented cardiopathy in mice infected with T. cruzi Tulahuen strain. As not all T. cruzi strains respond to treatment in the same way, an isolate from a chronic patient (SGO Z12) was used; parasitaemias were studied along with, survival, serology, electrocardiography, histology and cardiac beta receptor function. Parasitaemia in thioridazine (80 mg/(kg day) for 3 days) treated mice was less and lasted for a shorter period (P < 0.01), there were reduced electrocardiographic and histological alterations and significantly improved survival (80% of non-treated died). Treated mice had lower receptor affinity and higher density as a compensatory mechanism, modifying the course of T. cruzi infection (SGO Z12 isolate) and preventing the consequent cardiopathy.
Assuntos
Cardiomiopatia Chagásica/prevenção & controle , Doença de Chagas/complicações , Doença de Chagas/tratamento farmacológico , Tioridazina/farmacologia , Tioridazina/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Animais , Anticorpos Antiprotozoários/sangue , Modelos Animais de Doenças , Eletrocardiografia , Masculino , Camundongos , Miocárdio/patologia , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/efeitos dos fármacos , Parasitemia , Análise de Sobrevida , Tripanossomicidas/farmacologiaRESUMO
The aim of the present study was to evaluate the changes caused by adjuvant-induced arthritis in liver mitochondria and to investigate the effects of the nonsteroidal anti-inflammatory drug nimesulide. The main alterations observed in liver mitochondria from arthritic rats were: higher rates of state IV and state III respiration with beta-hydroxybutyrate as substrate; reduced respiratory control ratio and impaired capacity for swelling dependent on beta-hydroxybutyrate oxidation. No alterations were found in the activities of NADH oxidase and ATPase. Nimesulide produced: (1) stimulation of state IV respiration; (2) decrease in the ADP/O ratio and in the respiratory control ratio; (3) stimulation of ATPase activity of intact mitochondria; (4) inhibition of swelling driven by the oxidation of beta-hydroxybutyrate; (5) induction of passive swelling due to NH(3)/NH(4)+ redistribution. The activity of NADH oxidase was insensitive to nimesulide. Mitochondria from arthritic rats showed higher sensitivity to nimesulide regarding respiratory activity. The results of this work allow us to conclude that adjuvant-induced arthritis leads to quantitative changes in some mitochondrial functions and in the sensitivity to nimesulide. Direct evidence that nimesulide acts as an uncoupler was also presented. Since nimesulide was active in liver mitochondria at therapeutic levels, the impairment of energy metabolism could lead to disturbances in the liver responses to inflammation, a fact that should be considered in therapeutic intervention.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/metabolismo , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Sulfonamidas/farmacologia , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Artrite Experimental/induzido quimicamente , Técnicas In Vitro , Masculino , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mitocôndrias Hepáticas/metabolismo , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Polarografia , Ratos , Ratos Wistar , Desacopladores/metabolismoRESUMO
Trypanosoma cruzi trypanothione reductase (TR) was irreversibly inhibited by peroxidase/H2O2 /phenothiazine (PTZ) systems. TR inactivation depended on (a) time of incubation with the phenothiazine system; (b) the peroxidase nature and (c) the PTZ structure and concentration. With the most effective systems, TR inactivation kinetics were biphasic, with a relatively fast initial phase during which about 75% of the enzyme activity was lost, followed by a slower phase leading to total enzyme inactivation. GSH prevented TR inactivation by the peroxidase/H2O2/PTZ+* systems. Production of PTZ+* cation radicals by PTZ peroxidation was essential for TR inactivation. Horseradish peroxidase, leukocyte myeloperoxidase (MPO) and the pseudo-peroxidase myoglobin (Mb) were effective catalysts of PTZ+* production. Promazine, thioridazine, chlorpromazine, propionylpromazine prochlorperazine, perphenazine and trimeprazine were effective constituents of the HRP/H2O2 /PTZ system. The presence of substituents at the PTZ nucleus position 2 exerted significant influence on PTZ activity, as shown by the different effects of 2-trifluoromethyl and 2-H or 2-chlorophenothiazines. The PTZ+* cation radicals disproportionation regenerated the non-radical PTZ molecule and produced the PTZ sulfoxide that was inactive on TR. Thiol compounds including GSH interacted with PTZ+* cation radicals transferring an electron from the sulfide anion to the PTZ+*, thus nullifying the PTZ+* biological and chemical activities.
Assuntos
Inibidores Enzimáticos/farmacologia , NADH NADPH Oxirredutases/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Cátions Monovalentes , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Peroxidase do Rábano Silvestre/metabolismo , Peroxidase do Rábano Silvestre/farmacologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Cinética , Mioglobina/metabolismo , Mioglobina/farmacologia , Peroxidase/metabolismo , Peroxidase/farmacologia , Fenotiazinas/metabolismo , Fenotiazinas/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-Atividade , Compostos de Sulfidrila/farmacologia , Fatores de Tempo , Trypanosoma cruziRESUMO
Over the last two decades, progress towards new drugs for the treatment of Chagas' disease has been disappointing. However, as a result of the parasite genome sequencing projects, the possibility of identifying novel drug targets through genomics, proteomics and bioinformatics has never been better. Progress towards the development of novel therapeutics, from target identification and validation by chemical and genetic means through to rational drug design, is illustrated with reference to the metabolism and functions of trypanothione, with particular emphasis on trypanothione reductase, one current drug target of choice.
Assuntos
Doença de Chagas/tratamento farmacológico , Desenho de Fármacos , NADH NADPH Oxirredutases/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Animais , Previsões , Humanos , NADH NADPH Oxirredutases/metabolismo , Trypanosoma cruzi/efeitos dos fármacosRESUMO
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.
Assuntos
Mitocôndrias/efeitos dos fármacos , Zidovudina/farmacologia , Animais , Relação Dose-Resposta a Droga , Transporte de Elétrons/efeitos dos fármacos , Rim/enzimologia , Masculino , Mitocôndrias/enzimologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/enzimologia , Complexos Multienzimáticos/efeitos dos fármacos , Músculo Esquelético/enzimologia , NADH NADPH Oxirredutases/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , ATPases Translocadoras de Prótons/efeitos dos fármacos , Ratos , Ratos Wistar , Ácido Succínico/metabolismo , Zidovudina/efeitos adversosRESUMO
Liver microsomal functions related to xenobiotic biotransformation and free radical production were studied in control rats and in animals subjected to L-3,3',5-triiodothyronine (T3) and/or lindane administration as possible mechanisms contributing to oxidative stress, in relation to the activity of enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glucose-6-phosphate dehydrogenase (G-6PDH)) and content of lipid-soluble vitamins (alpha-tocopherol, beta-carotene, and lycopene) affording antioxidant protection. Lindane treatment in euthyroid rats at a dosage of 20mg/kg did not modify the content of liver microsomal cytochromes P450 and b5, the activity of NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase, and the production of superoxide radical (O2.-), as well as antioxidant systems, except for the reduction in lycopene levels. Hyperthyroidism elicited a calorigenic response and increased specific and molecular activities of NADPH-cytochrome P450 reductase, O2.- generation, and G-6PDH activity, concomitantly with diminution in liver SOD and catalase activities and in alpha-tocopherol, beta-carotene, and lycopene levels. The administration of lindane to hyperthyroid animals led to a further increase in the molecular activity of NADPH-cytochrome P450 reductase and in the O2.- production/SOD activity ratio, and decrease of hepatic alpha-tocopherol content, in a magnitude exceeding the sum of effects elicited by the separate treatments, as previously reported for reduced glutathione depletion. Collectively, these data support the contention that the increased susceptibility of the liver to the toxic effects of acute lindane treatment in hyperthyroid state is conditioned by potentiation of the hepatic oxidative stress status.
Assuntos
Antioxidantes/metabolismo , Hexaclorocicloexano/farmacologia , Hipertireoidismo/metabolismo , Microssomos Hepáticos/metabolismo , Estresse Oxidativo , Animais , Biotransformação , Carotenoides/metabolismo , Catalase/efeitos dos fármacos , Catalase/metabolismo , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Glucosefosfato Desidrogenase/efeitos dos fármacos , Glucosefosfato Desidrogenase/metabolismo , Glutationa Peroxidase/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Hipertireoidismo/tratamento farmacológico , Licopeno , Masculino , Microssomos Hepáticos/efeitos dos fármacos , NADH NADPH Oxirredutases/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , NADPH-Ferri-Hemoproteína Redutase , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Tri-Iodotironina/farmacologia , Vitamina E/metabolismo , beta Caroteno/metabolismoRESUMO
In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function.
Assuntos
Bradicinina/farmacologia , NADH NADPH Oxirredutases/sangue , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fosfatidilinositóis/sangue , Fosfolipase D/sangue , Fosfolipases Tipo C/sangue , Sequência de Aminoácidos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Dados de Sequência Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADH NADPH Oxirredutases/efeitos dos fármacos , NADPH Oxidases , Neutrófilos/enzimologia , Óxido Nítrico/biossíntese , Óxido Nítrico/sangue , Fosfolipase D/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Estimulação Química , Superóxidos/sangue , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/efeitos dos fármacosRESUMO
We are studying the action of tert-butylhydroperoxide (t-BOOH) on Escherichia coli as a model system for peroxide toxicity. In our previous report (De la Cruz-Rodriguez, L.C., Farías, R.N. and Massa, E.M. (1990) Biochim. Biophys. Acta 1015, 510-516), the respiratory chain was identified as a major target of t-BOOH. In the present paper, we study further the effect of t-BOOH on the NADH oxidase of the E. coli respiratory chain to clarify the mechanism of damage, especially regarding the identity and role of the metal ion involved. The results are: (a) t-BOOH toxicity is mediated by membrane-bound copper ions; (b) a small pool of the membrane-bound copper is reduced from Cu(II) to Cu(I) in the presence of NADH and other respiratory substrates (succinate, D-lactate); (c) this reduction of copper occurs at 37 degrees C but not at 0 degrees C or when the membranes are inactivated by previous heating; (d) the Cu(I) generated by reduction of Cu(II) during membrane preincubation with NADH, is oxidized by t-BOOH with simultaneous inactivation of the NADH oxidase, whereas treatment with only t-BOOH (without NADH) has no effect on the oxidase. It is concluded that the effect of t-BOOH on the respiratory chain is mediated by redox cycling of copper. It is proposed that the damage results from activation of the hydroperoxide through its interaction with Cu(I) in a site-specific Fenton-type reaction.