RESUMO
Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.
Assuntos
Crithidia/efeitos dos fármacos , Crithidia/microbiologia , Fosforilcolina/análogos & derivados , Simbiose , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Crithidia/metabolismo , Crithidia/ultraestrutura , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Fosfatidilcolinas/biossíntese , Isótopos de Fósforo/metabolismo , Fosforilcolina/farmacologiaRESUMO
Surface charge of wild-type Crithidia fasciculata and three drug-resistant mutants (TR3, TFRR1, and FUR11) was studied by direct zeta-potential determination and ultrastructural cytochemistry. Surface tension was also investigated by measurements of the advancing contact angle formed by the protozoa monolayers with drops of liquids of different polarities. The individual zeta potential varies markedly among the C. fasciculata cells. The wild and FUR11 mutant strains displayed lower negative surface charge (-12.5 and -9.5 mV, respectively) as compared with the TR3 (-14.8 mV) and TFRR1 (-14.7 mV) mutant strains. Binding of cationized ferritin (CF) was observed at the cell surface of wild and mutant strains of C. fasciculata. Neuraminidase treatment reduced the negative surface charge in the TFRR1 and TR3 mutants in about 37 and 29%, respectively, whereas no significant change was observed with the wild and FUR11 mutant strains. These findings suggest that sialic acid residues are the major anionogenic groups on the surface of C. fasciculata. The density of sialic acid residues per cell in wild and mutant strains of C. fasciculata falls in a range of 1.4 x 10(4) to 3.6 x 10(4). Marked differences of hydrophobicity were also observed. For example, the TFRR1, FUR11, and TR3 drug-resistant mutant strains showed higher contact angle values (55.4, 54.2, and 49.3, respectively) than the wild-type (35.6), as assessed by alpha-bromonaphtalene.
Assuntos
Ânions , Crithidia/química , Água/química , Animais , Crithidia/efeitos dos fármacos , Crithidia/genética , Histocitoquímica , Potenciais da Membrana/fisiologia , Metilnitronitrosoguanidina , Mutação , Valores de Referência , Solubilidade , Propriedades de SuperfícieRESUMO
The use of digitonin to permeabilize Leishmania mexicana mexicana, Leishmania agamae, and Crithidia fasciculata plasma membranes enabled us to study Ca2+ transport in situ. The present results show that the mitochondria of these trypanosomatids are able to build up and retain a membrane potential as indicated by a tetraphenylphosphonium-sensitive electrode. Ca2+ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2+ transport mechanism in these mitochondria. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by inorganic phosphate. Large amounts of Ca2+ were retained by C. fasciculata mitochondria even after addition of thiols and NAD(P)H oxidants such as t-butylhydroperoxide and diamide. In contrast, Ca2+ was not retained in the matrix of Leishmania sp. mitochondria for long periods of time. In addition to the mitochondrial Ca2+ uptake, a vanadate-sensitive Ca2(+)-transporting system was also detectable in these trypanosomatids.
Assuntos
Cálcio/metabolismo , Digitonina/farmacologia , Trypanosoma/metabolismo , Animais , Transporte Biológico , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/metabolismo , Potenciais da Membrana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Trypanosoma/efeitos dos fármacosRESUMO
Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis in Trypanosoma cruzi and stimulated O2 uptake and O2-. generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and O2-. generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H2O2 production were obtained with Crithidia fasciculata despite the presence of catalase in this organism.
Assuntos
Antiprotozoários , Crithidia/efeitos dos fármacos , Iminas/farmacologia , Isoxazóis/farmacologia , Naftoquinonas/farmacologia , Oxazóis/farmacologia , Oxigênio/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Animais , Crithidia/crescimento & desenvolvimento , Crithidia/metabolismo , DNA/biossíntese , Radicais Livres , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
Se ensayaron varias aminoisoxazolil-1,2-naftoquinomas sobre la formación de anión superóxido (O2) y peróxido de hidrógeno (H2O2) por Crithidia fasciculata y Leptomonas seymouri. Los compuestos IVD (N-(5-metil-3-isoxazolil)-4 amino-1,2-naftoquinona; IIID (2-hidroxi-N-(5-metil-3-isoxazoli)-1,4-naftoquinona-4-imina) y IIIE (2-hidroxi-N-(3-metil-5-isoxazolil)-1,4-naftoquinona-4-imina) estimularon la produción de H2O2 mientras que IIIE, IIIC (2-hidroxi-N-(3,5-dimetil-4-isoxazoli)-1,4-naftoquinona-4-imina) y IIIA (2-hidroxi-N-3,4-dimetil-5-isoxazolil)-1,4-naftoquinona-4-imina), pero no IVD, estimularon la formación de O2 en los organismos estudiados. El ciclo redox de las quinona-iminas se verificó por a) la variación de su absorbancia en función de la concentración de oxígeno en el medio de suspensión de las células; b) su reducción anaeróbica, relativamente rápida, con velocidad máxima para IVD; c) la oxidación de los quinoles correspondientes, obtenidos por reducción con borohidruro. C. fasciculata y L. seymouri contenían superóxido dismutasa, enzima esencial para la formación de peróxidos como consecuencia del ciclo redox de las quinonas, y también catalasa, cuya actividad fue seis veces mayor en C. fasciculata. La presencia de catalasa no impidió la formación de H2O2, como producto metabólico de las quinona-iminas. No se pudo demostrar actividad de ascorbato peroxidasa, benzidina peroxidasa o guayacol peroxidasa en los organismos estudiados
Assuntos
Animais , Peróxido de Hidrogênio/metabolismo , Naftoquinonas/farmacologia , Trypanosomatina/efeitos dos fármacos , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Iminas/farmacologia , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismo , Trypanosomatina/metabolismoRESUMO
Se ensayaron varias aminoisoxazolil-1,2-naftoquinomas sobre la formación de anión superóxido (O2) y peróxido de hidrógeno (H2O2) por Crithidia fasciculata y Leptomonas seymouri. Los compuestos IVD (N-(5-metil-3-isoxazolil)-4 amino-1,2-naftoquinona; IIID (2-hidroxi-N-(5-metil-3-isoxazoli)-1,4-naftoquinona-4-imina) y IIIE (2-hidroxi-N-(3-metil-5-isoxazolil)-1,4-naftoquinona-4-imina) estimularon la produción de H2O2 mientras que IIIE, IIIC (2-hidroxi-N-(3,5-dimetil-4-isoxazoli)-1,4-naftoquinona-4-imina) y IIIA (2-hidroxi-N-3,4-dimetil-5-isoxazolil)-1,4-naftoquinona-4-imina), pero no IVD, estimularon la formación de O2 en los organismos estudiados. El ciclo redox de las quinona-iminas se verificó por a) la variación de su absorbancia en función de la concentración de oxígeno en el medio de suspensión de las células; b) su reducción anaeróbica, relativamente rápida, con velocidad máxima para IVD; c) la oxidación de los quinoles correspondientes, obtenidos por reducción con borohidruro. C. fasciculata y L. seymouri contenían superóxido dismutasa, enzima esencial para la formación de peróxidos como consecuencia del ciclo redox de las quinonas, y también catalasa, cuya actividad fue seis veces mayor en C. fasciculata. La presencia de catalasa no impidió la formación de H2O2, como producto metabólico de las quinona-iminas. No se pudo demostrar actividad de ascorbato peroxidasa, benzidina peroxidasa o guayacol peroxidasa en los organismos estudiados (AU)
Assuntos
Animais , Naftoquinonas/farmacologia , Trypanosomatina/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Iminas/farmacologia , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismo , Trypanosomatina/metabolismoRESUMO
Nifurtimox (NF) and benznidazole (BZ), drugs used in the treatment of Chagas' disease, did not inhibit protein biosynthesis in in vitro homologous cell-free systems isolated from Trypanosoma cruzi and Crithidia fasciculata; nevertheless, their addition to growing cultures caused polyribosomal depolymerization. On the other hand, Berenil, Antrycide and suramin, used against African trypanosomiasis, inhibited protein biosynthesis in vitro but did not affect ribosomal distribution, probably due to low permeability to the drugs. The results suggest that the inhibition by NF and BZ of protein synthesis, measured as [14C]leucine incorporation by other authors, is indirect, probably through inhibition of nucleic acid synthesis and energy metabolism.
Assuntos
Nifurtimox/farmacologia , Nitrofuranos/farmacologia , Nitroimidazóis/farmacologia , Biossíntese de Proteínas , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Crithidia/efeitos dos fármacos , Técnicas In Vitro , Trypanosoma cruzi/metabolismoRESUMO
It has been synthesized a series of 4-aminoisoxazol-1,2-naftoquinones, which inhibit growth and DNA synthesis in Trypanosoma cruzi. This effect was related to quinone induction of oxyradical generation. N-(5-methyl-3-isoxazolyl)-4-amino-1,2-naftoquinone and 2-hydroxy-N-(3,5-dimethyl-1-4-isoxazolyl)-1,4-naftoquinone-4 -imine inhibited also growth of Crithidia fasciculata and Leptomonas symouri, two organisms that may prove useful for the assay of trypanocidal drugs. In order to establish the role of oxyradicals for quinone-imine toxicity, the latter redox-cycling was demonstrated by a) reversible change in their spectra; b) reduction under anaerobic conditions, and c) quinol oxidation by oxygen. Production of H2O2, measured by the microperoxidase method, was maximal with N-(5-methyl-3-isoxazolyl)-4-amine-1,2-naftoquinone (IVD) (0.29-0.26 nmol/min/mg of cell protein), either with C. fasciculata or L. seymouri. Lesser, though significant activities were obtained with 2-hydroxy-N-(5-methyl-3-isoxazolyl)-1,4-naftoquinone-4-imine (IIID) and 2-hydroxy-N-(3-methyl-5-isoxazolyl)-1,4-naftoquinone-4-imine (IIIE) whereas IIIA and IIIC were inactive. O2-. generation, measured by the adrenochrome method, was induced by naftoquinone-imines with a hydroxyl group at C-2 (IIIE, 2-hydroxy-N-(3,5-dimethyl-4-isoxazolyl)-1,4-naftoquinone-4-i mine (IIIC) and 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naftoquinone-4-i mine (IIIA), but no by IVD, with a carbonyl group at C-2. Measurement of catalase and superoxide dismutase in both organisms yielded significant activities, but ascorbate peroxidase, guaiacol peroxidase and bencidine peroxidase were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Peróxido de Hidrogênio/metabolismo , Naftoquinonas/farmacologia , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/metabolismo , Animais , Crithidia/efeitos dos fármacos , Crithidia/metabolismo , Iminas/farmacologia , Oxirredução/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
Demonstration of trypanocidal effects in vitro is a first step for the development of new antichagasic drugs. In order to obtain an experimental model allowing the pre-screening of potential trypanocides for Trypanosoma cruzi in a short time and under safe conditions, the trypanosomatids T. mega and C. fasciculata were assayed for their response to a) compounds known for their action on T. cruzi, and b) compounds not tested before on the latter. The drugs were assayed on the organisms growth in a liquid culture medium, cell multiplication being measured by the medium turbidity increase, using a photoelectric colorimeter previously calibrated with cell suspensions of known concentration. A series of quinones (Lapachones and related compounds), naftoquinone-imines, benzoquinones (perezone and dihydroperezone), a quinol (miconidine) and several nitrofurans, including nifurtimox and (5-nitro-2-furfurylidene)-amino (NF-group) derivatives, inhibited the flagellates growth, specially T. mega, with half-maximal inhibitory concentrations lesser than 5.0 microM, for the most active compounds. T. mega response to nifurtimox, NF-derivatives and beta-lapachone was in close agreement with that of T. cruzi. Cultures of T. mega in the presence of NF-pyrazole, NF-indazoles and NF-imidazole but not nifurtimox, showed irreversible damage since, after re-incubation in fresh medium without inhibitor, these cells grew significantly less than their corresponding controls. Similar effects were observed in C. fasciculata, with beta-lapachone and one naftoquinone-imine. Our results qualify T. mega as an adequate experimental model for the assay of antichagasic agents, as C. fasciculata and T. brucei brucei do for the african trypanosomes.
Assuntos
Crithidia/efeitos dos fármacos , Nitrofuranos/farmacologia , Quinonas/farmacologia , Trypanosoma/efeitos dos fármacos , Animais , Crithidia/crescimento & desenvolvimento , Trypanosoma/crescimento & desenvolvimentoRESUMO
La valoración in vitro de tripanocidas activos sobre Trypanosoma cruzi es el primer paso para el desarrollo de nuevos agentes terapéuticos contra la enfermedad de Chagas. Para verificar si una información equivalente puede lograrse con organismos no patógenos, se estudió la acción de varios tripanocidas de t. cruzi sobre T. mega y Crithidia fasciculada. Las drogas se ensayaron sobre el crecimiento de los protozoarios que se midió por la turbiedad de la suspensión celular en medio de cultivo líquido. Una serie de quinonas, incluyendo quinonas lipofílicas, lapachonas, quinina-iminas, benzoquinonas, un quinol (miconidina) y nitrofuranos (nifurtimox y análogos derivados del grupo (5-nitro-2-furfurilideno)-amino (grupo NF) inhibieron el crecimiento de los organismos, especialmente el de T. mega, con I50 menores de 5 micronM, para los compuestos mas activos. La sensibilidad de T. mega fue similar a la de T. cruzi y significativamente mayor que la de C. fasciculata. El cultivo de muestras de T. mega, preincubados con las mismas drogas,d emostró efectos irreversibles con los NF-derivados del pirazol, imidazol, indazol y benzimidazol pero no con el nifurtimox. En iguales condiciones, C. fasciculata fue afectada solamente por la ß-lapachona y una naftoquinona-imina. Estos resultados califican a T. mega como un modelo adecuado para el ensayo inicial de quimioterápicos anti-chagásicos, como lo son C. fasciculata y T. brucei para los tripanosomas africanos
Assuntos
Animais , Crithidia/efeitos dos fármacos , Nitrofuranos/farmacologia , Quinonas/farmacologia , Trypanosoma/efeitos dos fármacos , Crithidia/crescimento & desenvolvimento , Trypanosoma/crescimento & desenvolvimentoRESUMO
La valoración in vitro de tripanocidas activos sobre Trypanosoma cruzi es el primer paso para el desarrollo de nuevos agentes terapéuticos contra la enfermedad de Chagas. Para verificar si una información equivalente puede lograrse con organismos no patógenos, se estudió la acción de varios tripanocidas de t. cruzi sobre T. mega y Crithidia fasciculada. Las drogas se ensayaron sobre el crecimiento de los protozoarios que se midió por la turbiedad de la suspensión celular en medio de cultivo líquido. Una serie de quinonas, incluyendo quinonas lipofílicas, lapachonas, quinina-iminas, benzoquinonas, un quinol (miconidina) y nitrofuranos (nifurtimox y análogos derivados del grupo (5-nitro-2-furfurilideno)-amino (grupo NF) inhibieron el crecimiento de los organismos, especialmente el de T. mega, con I50 menores de 5 micronM, para los compuestos mas activos. La sensibilidad de T. mega fue similar a la de T. cruzi y significativamente mayor que la de C. fasciculata. El cultivo de muestras de T. mega, preincubados con las mismas drogas,d emostró efectos irreversibles con los NF-derivados del pirazol, imidazol, indazol y benzimidazol pero no con el nifurtimox. En iguales condiciones, C. fasciculata fue afectada solamente por la ß-lapachona y una naftoquinona-imina. Estos resultados califican a T. mega como un modelo adecuado para el ensayo inicial de quimioterápicos anti-chagásicos, como lo son C. fasciculata y T. brucei para los tripanosomas africanos (AU)