RESUMO
Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.
Assuntos
Proliferação de Células , Hidroxiureia , Taenia , Hidroxiureia/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Taenia/efeitos dos fármacos , Taenia/genética , Taenia/crescimento & desenvolvimento , Taenia/metabolismo , Proteômica/métodos , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Proteoma/metabolismo , Cysticercus/efeitos dos fármacos , Cysticercus/metabolismoRESUMO
The benzimidazole derivative, 6-chloro-5-(2,3-dichlorophenoxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB15), has a similar mode of action and efficacy as albendazole, a commonly used anthelminthic drugs. The aim of this study was to evaluate its influence on the tricarboxylic acid cycle in Taenia crassiceps cysticerci. The parasites were cultured in supplemented RPMI medium containing albendazole sulfoxide (ABZSO) or RCB15, for 24 h. Then, frozen in liquid nitrogen for organic metabolites extraction. Samples were analyzed by high performance liquid chromatography and organic acids of the tricarboxylic acid cycle were detected. It was possible to observe changes in the concentrations of all acids involved in this metabolic pathway, with the exception of α-ketoglutarate, which was not detected in the control group neither in most of the treated groups. It indicates that the parasite presented a partial inhibition of the tricarboxylic acid cycle. The significant increase in the concentration of citrate, oxaloacetate and succinate in the RCB15 treated groups may indicate an activation of the fumarate reductase pathway, leading to metabolic distress. Therefore RCB15 may be considered an alternative for the treatment of tissue parasitic diseases, since it induced changes in the main metabolic pathway of the parasite.
Assuntos
Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Cysticercus/efeitos dos fármacos , Taenia/efeitos dos fármacos , Animais , Cysticercus/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Taenia/metabolismoRESUMO
Nitazoxanide (NTZ) is a broad-spectrum drug used in intestinal infections, but still poorly explored in the treatment of parasitic tissular infections. This study aimed to evaluate the in vitro responses of the energetic metabolism of T. crassiceps cysticerci induced by NTZ. The organic acids of the tricarboxylic acid cycle, products derived from fatty acids oxidation and protein catabolism were analyzed. These acids were quantified after 24â¯h of in vitro exposure to different NTZ concentrations. A positive control group was performed with albendazole sulfoxide (ABZSO). The significant alterations in citrate, fumarate and malate concentrations showed the NTZ influence in the tricarboxylic acid (TCA) cycle. The non-detection of acetate confirmed that the main mode of action of NTZ is effective against T. crassiceps cysticerci. The statistical differences in fumarate, urea and beta-hydroxybutyrate concentrations showed the NTZ effect on protein catabolism and fatty acid oxidation. Therefore, the main energetic pathways such as the TCA cycle, protein catabolism and fatty acids oxidation were altered after in vitro NTZ exposure. In conclusion, NTZ induced a significant metabolic stress in the parasite indicating that it may be used as an alternative therapeutic choice for cysticercosis treatment. The use of metabolic approaches to establish comparisons between anti parasitic drugs mode of actions is proposed.
Assuntos
Antiparasitários/farmacologia , Taenia/efeitos dos fármacos , Tiazóis/farmacologia , Albendazol/análogos & derivados , Albendazol/farmacologia , Análise de Variância , Animais , Anti-Helmínticos/farmacologia , Citratos/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Meios de Cultura/química , Cysticercus/efeitos dos fármacos , Cysticercus/metabolismo , Metabolismo Energético/efeitos dos fármacos , Fumaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Neurocisticercose/tratamento farmacológico , Nitrocompostos , Ácido Oxaloacético/metabolismo , Ácido Succínico/metabolismo , Taenia/metabolismoRESUMO
Benzimidazole derivatives such as albendazole (ABZ) and mebendazole are important molecules used in helminthic treatment. Neurocysticercosis is the main cause of acquired epilepsy throughout the world and is currently treated with ABZ. New molecules have been studied in order to aid in the treatment of this neglected tropical disease, among them RCB15 and RCB20. The aim of this study was to evaluate the metabolic impact of RCB15 and RCB20 on Taenia crassiceps cysticerci intracranially inoculated in Balb/c mice. Thirty days after the inoculation the mice were treated with 50 mg kg-1 of RCB15, RCB20, ABZ or NaCl 0.9%. The euthanasia and cysticerci removal were performed 24 h after the treatment. The cysticerci were analysed through high performance liquid chromatography. After the treatments, there was an impairment in the main energetic pathways such as glycolytic pathway, homolactic fermentation or in mitochondrion energy production detected through the decrease in pyruvate, lactate, oxaloacetate, malate and fumarate concentrations. This induced the parasite to resort to alternative energetic pathways such as proteins catabolism, propionate fermentation and fatty acids oxidation. Therefore, benzimidazole derivatives are a promising alternative to ABZ use as they also reach the brain tissue and induce a metabolic stress in the cysticerci.
Assuntos
Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Cysticercus/efeitos dos fármacos , Neurocisticercose/tratamento farmacológico , Animais , Cysticercus/fisiologia , Metabolismo Energético/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Androgens have been shown to exert a cysticidal effect upon Taenia crassiceps, an experimental model of cysticercosis. To further inquire into this matter, the Taenia crassiceps model was used to evaluate the expression of several proteins after testosterone (T4) and dihydrotestosterone (DHT) in vitro treatment. Under 2-D proteomic maps, parasite extracts were resolved into approximately 130 proteins distributed in a molecular weight range of 10-250 kDa and isoelectrical point range of 3-10. The resultant proteomic pattern was analysed, and significant changes were observed in response to T4 and DHT. Based on our experience with electrophoretic patterns and proteomic maps of cytoskeletal proteins, alteration in the expression of isoforms of actin, tubulin and paramyosin and of other proteins was assessed. Considering that androgens may exert their biological activity in taeniids through the non-specific progesterone receptor membrane component (PGRMC), we harnessed bioinformatics to propose the identity of androgen-regulated proteins and establish their hypothetical physiological role in the parasites. These analyses yield a possible explanation of how androgens exert their cysticidal effects through changes in the expression of proteins involved in cytoskeletal rearrangement, dynamic vesicular traffic and transduction of intracellular signals.
Assuntos
Androgênios/farmacologia , Morte Celular , Proteoma , Taenia/efeitos dos fármacos , Taenia/fisiologia , Actinas/genética , Animais , Biologia Computacional , Cisticercose/patologia , Cysticercus/efeitos dos fármacos , Cysticercus/fisiologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/genética , Di-Hidrotestosterona/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Progesterona/genética , Testosterona/farmacologia , Tropomiosina/genética , Tubulina (Proteína)/genéticaRESUMO
Neurocysticercosis is the most common parasitic infection of the nervous system and currently represents a serious public health issue in many regions of Latin America, Asia, and Africa. To date, praziquantel is one of the chosen drugs for the treatment of neurocysticercosis. Its mechanism of action is based on the inhibition of different biochemical pathways within the parasite which contribute to its death. Thus, the aim of this work was to analyze, for the first time, whether the nanoformulations of praziquantel would modify the energetic pathway of Taenia crassiceps cysticerci, after an intracranial inoculation in BALB/c mice. Praziquantel nanosuspensions were formulated with polyvinyl alcohol, poloxamer 188, and poloxamer 407, as stabilizers. These formulations exhibited particle size in a range of 74-285 nm and zeta potential values in a range of - 8.1/- 13.2 depending on the type of stabilizer. Physical stability study at both 4 ± 2 and 25 ± 2 °C indicated that praziquantel (PZQ) nanoparticles were stable in terms of solubility and particle size after 120-day storage. In vivo studies demonstrated that those nanosystems were able to produce significant modifications on the concentrations of oxaloacetate, citrate, pyruvate, alpha-ketoglutarate, malate, succinate, lactate, beta-hydroxybutyrate, fumarate, and propionate involved in the metabolism of Taenia crassiceps cysticerci. Therefore, these nanoformulations may be considered as a promising tool to deliver praziquantel to the brain for the effective management of neurocysticercosis.
Assuntos
Anti-Helmínticos/administração & dosagem , Metaboloma/efeitos dos fármacos , Neurocisticercose/tratamento farmacológico , Praziquantel/administração & dosagem , Animais , Anti-Helmínticos/química , Anti-Helmínticos/uso terapêutico , Cysticercus/efeitos dos fármacos , Cysticercus/fisiologia , Modelos Animais de Doenças , Estabilidade de Medicamentos , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/química , Neurocisticercose/metabolismo , Tamanho da Partícula , Praziquantel/química , Praziquantel/uso terapêuticoRESUMO
The aim of this work was to develop nanosuspensions of praziquantel (PZQ) and to evaluate their influence on the energetic metabolism of cysticerci inoculated in BALB/c mice. We analyzed metabolic alterations of glycolytic pathways and the tricarboxylic acid cycle in the parasite. The nanosuspensions were prepared by precipitation and polyvinyl alcohol (PVA), poloxamer 188 (P188) and poloxamer 407 (P407) were used as stabilizers. Nanosuspension prepared with PVA had a particle size of 100nm, while P188- and P407-based nanosuspensions had particle sizes of 74nm and 285nm, respectively. The zeta potential was -8.1, -8.6, and -13.2 for the formulations stabilized with PVA, P188 and P407, respectively. Treatments of T. crassiceps cysticerci-infected mice resulted in an increase in glycolysis organic acids, and enhanced the partial reversion of the tricarboxylic acid cycle, the urea cycle and the production of ketonic bodies in the parasites when compared to the groups treated with conventional PZQ. These data suggest that PZQ nanosuspensions greatly modified the energetic metabolism of cysticerci in vivo. Moreover, the remarkable metabolic alterations produced by the stabilizers indicate that further studies on nanoformulations are required to find potentially suitable nanomedicines.
Assuntos
Cisticercose/tratamento farmacológico , Cisticercose/fisiopatologia , Cysticercus/efeitos dos fármacos , Cysticercus/metabolismo , Praziquantel/uso terapêutico , Taenia/efeitos dos fármacos , Taenia/metabolismo , Animais , Camundongos , Camundongos Endogâmicos BALB C , NanopartículasRESUMO
In the search of new alternatives for neurocysticercosis treatment, the cysticidal activity of organic extracts of Teloxys graveolens was evaluated. The in vitro activity of hexane, ethyl acetate and methanol extracts against Taenia crassiceps cysts was tested and the selectivity index relative to human fibroblasts was determined. Subsequently, the in vivo efficacy of the methanolic extract at doses of 200 and 500 mg/kg in the murine cysticercosis model was evaluated. The ultrastructural effects in vitro and in vivo of the methanolic extract were also investigated using scanning electron microscopy. Additionally, a bioassay-guided fractionation for the isolation of the cysticidal components was performed. Our in vitro findings revealed that all extracts exhibited good cysticidal activity with EC50 values from 44.8 to 67.1 µg/mL. Although the ethyl acetate and methanolic extracts displayed low cytotoxicity, the methanolic extract was the most selective. The methanolic extract also showed in vivo efficacy which was similar to that obtained with ABZ. Significant alterations were found on the germinal layer of the cysts, with a high accumulation of granules of glycogen and vacuoles. The bioguided fractionation of methanolic extract led to the isolation of three flavonoids: chrysin, pinocembrin and pinostrobin; among them, pinocembrin was the compound that displayed cysticidal activity. This is the first study which reveals that T. graveolens could be a potential source for cysticidal and non-toxic compounds.
Assuntos
Amaranthaceae/química , Cisticercose/tratamento farmacológico , Cysticercus/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Bioensaio , Cysticercus/ultraestrutura , Feminino , Fibroblastos/efeitos dos fármacos , Flavanonas/química , Flavanonas/isolamento & purificação , Flavanonas/farmacologia , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Gengiva/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Extratos Vegetais/toxicidadeRESUMO
Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites.
Assuntos
Auranofina/toxicidade , Glutationa/análise , Oxidantes/toxicidade , Estresse Oxidativo , Taenia/química , Taenia/efeitos dos fármacos , Acetilcisteína/metabolismo , Animais , Antioxidantes/metabolismo , Butionina Sulfoximina/metabolismo , Cysticercus/química , Cysticercus/efeitos dos fármacos , Cysticercus/fisiologia , Espécies Reativas de Oxigênio/análise , Análise de Sobrevida , Taenia/fisiologiaRESUMO
Helminth ß-tubulins are the targets of benzimidazole (BZM) carbamate compounds. The specificity of the interactions between such compounds and their in vivo targets depends on the presence of specific amino acid residues in the target molecules. To discover new and effective anthelmintic drugs, we used a medicinal chemistry approach to synthesize a series of BZM derivatives that exploited the BZM moiety as a template. We have previously found that one compound, 2-(trifluoromethyl)-1H-benzimidazole (RCB20), has better in vitro and in vivo activity than albendazole sulfoxide (ABZSO). In the present study, the effect of RCB20 and ABZSO treatment on expression of Taenia crassiceps cysticerci cytoskeletal proteins such as actin, myosin II, and tubulin isoforms was examined. The effects of RCB20 and ABZSO after 11 days treatment of the parasites was evaluated by light, confocal, and electron microscopy, and by immunochemistry and immunohistochemistry. The RCB20-induced effects were more rapid than the ABZSO-induced effects on the parasites. In the RCB20-treated parasites, we observed gross-structural damage at the whole parasite level, particularly in the inner tissues and flame cells. Changes in the expression patterns of the cytoskeletal proteins, as assessed by immunohistochemistry and immunoblotting, revealed that the most important drug-induced effect on the parasites was a reduction in the expression level of tyrosinated α-tubulins. Our research findings suggest that RCB20 treatment affected posttranslational modification of parasite α-tubulin molecules, which involved removal of the α-tubulin carboxy-terminal tyrosine.