RESUMO
The main form of control of leishmaniasis is the treatment, however various side effects and poor efficacy are associated with presently available drugs. The investigation of bioactive natural products for new antileishmanial drugs is a valid approach. The present study reports the in vitro efficacy of natural isoflavonoids and terpenes against Leishmania infantum and L. amazonensis and their cytotoxicity against HepG2 cells. L. infantum and L. amazonensis promastigotes were exposed to the terpenes kaurenoic acid, xylopic acid, and (-)-α-bisabolol and to the isoflavonoids (-)-duartin and (3R)-claussequinone for antileishmanial activity and to cytotoxicity to HepG2 cells. The most effective substance against both L. infantum and L. amazonensis species was (3R)-claussequinone (IC50 = 3.21 µg/mL and 2.47 µg/mL, respectively) that disclosed low cytotoxicity against HepG2 cells (CC50 = 387.79 µg/mL). The efficacy of (3R)-claussequinone against intracellular amastigotes of L. infantum and the externalization of phosphatidylserine in promastigotes of this isoflavanoid were investigated by infection of Raw 264.7 macrophages and marking with Annexin V-FITC and propidium Iodide for flow cytometry analysis. The results for amastigotes showed that (3R)-claussequinone was able to reduce the rate of infection with IC50 = 4.61 µg/mL and did not alter the externalization of phosphatidylserine. In conclusion it is presently reported, for the first time, the striking antileishmanial activity of (3R)-claussequinone against L. infantum and L. amazonensis associated to low cytotoxicity. Furthermore, these results suggest that (3R)-claussequinone is a new hit aiming to develop new therapeutic alternatives.
Assuntos
Antiprotozoários , Produtos Biológicos , Leishmania infantum , Camundongos , Animais , Terpenos/farmacologia , Fosfatidilserinas , Propídio , Camundongos Endogâmicos BALB C , Antiprotozoários/toxicidade , Antiprotozoários/uso terapêutico , Produtos Biológicos/farmacologiaRESUMO
BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.
Assuntos
Antimônio/toxicidade , Proteínas de Escherichia coli/genética , Escherichia coli , Guanina/análogos & derivados , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Pirofosfatases , Superóxido Dismutase/metabolismo , Animais , Antiprotozoários/farmacologia , Proteínas de Escherichia coli/metabolismo , Guanina/farmacologia , Humanos , Peróxido de Hidrogênio/toxicidade , Leishmania braziliensis/enzimologia , Leishmania infantum/enzimologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pirofosfatases/genética , Pirofosfatases/metabolismo , Coelhos , Ratos , Superóxido Dismutase/genéticaRESUMO
Leishmaniasis is one of the most important neglected diseases worldwide. It is a life-threatening disease and causes significant morbidity, long-term disability, and early death. Treatment involves disease control or use of intervention measures, although the currently used drugs require long-lasting therapy, and display toxicity and reduced efficacy. The use of natural products isolated from plants, such as lapachol, an abundant naphthoquinone naturally occurring in South American Handroanthus species (Tabebuia, Bignoniaceae), is a promising option for the treatment of leishmaniasis. In this study, we investigated the leishmanicidal activity of lapachol in vitro and in vivo against Leishmania infantum and L. amazonensis, causative agents of visceral and cutaneous leishmaniasis, respectively. Low cytotoxicity in HepG2 cells (3405.8⯱â¯261.33⯵M), good anti-Leishmania activity, and favorable selectivity indexes (SI) against promastigotes of both L. amazonensis (IC50â¯=â¯79.84⯱â¯9.10⯵M, SIâ¯=â¯42.65) and L. infantum (IC50â¯=â¯135.79⯱â¯33.04⯵M, SIâ¯=â¯25.08) were observed. Furthermore, anti-Leishmania activity assays performed on intracellular amastigotes showed good activity for lapachol (IC50â¯=â¯191.95⯵M for L. amazonensis and 171.26⯵M for L. infantum). Flow cytometric analysis demonstrated that the cytotoxic effect of lapachol in Leishmania promastigotes was caused by apoptosis-like death. Interestingly, the in vitro leishmanicidal effect of lapachol was confirmed in vivo in murine models of visceral and cutaneous leishmaniasis, as lapachol (25â¯mg/kg oral route for 24â¯h over 10 days) was able to significantly reduce the parasitic load in skin lesions, liver, and spleen, similar to amphotericin B, the reference drug. These results reinforce the therapeutic potential of lapachol, which warrants further investigations as an anti-leishmaniasis therapeutic.
Assuntos
Antiprotozoários/uso terapêutico , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Visceral/tratamento farmacológico , Naftoquinonas/uso terapêutico , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Anfotericina B/toxicidade , Animais , Antiprotozoários/farmacologia , Antiprotozoários/toxicidade , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Células Hep G2/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Leishmania infantum/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Fígado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Naftoquinonas/farmacologia , Naftoquinonas/toxicidade , Carga Parasitária , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Extratos Vegetais/toxicidade , Células RAW 264.7/efeitos dos fármacos , Células RAW 264.7/parasitologia , Distribuição Aleatória , Pele/parasitologia , Baço/parasitologia , Tabebuia/químicaRESUMO
Trypanosoma cruzi is exposed to oxidative stresses during its life cycle, and amongst the strategies employed by this parasite to deal with these situations sits a peculiar trypanothione-dependent antioxidant system. Remarkably, T. cruzi's antioxidant repertoire does not include catalase. In an attempt to shed light on what are the reasons by which this parasite lacks this enzyme, a T. cruzi cell line stably expressing catalase showed an increased resistance to hydrogen peroxide (H2O2) when compared with wild-type cells. Interestingly, preconditioning carried out with low concentrations of H2O2 led untransfected parasites to be as much resistant to this oxidant as cells expressing catalase, but did not induce the same level of increased resistance in the latter ones. Also, presence of catalase decreased trypanothione reductase and increased superoxide dismutase levels in T. cruzi, resulting in higher levels of residual H2O2 after challenge with this oxidant. Although expression of catalase contributed to elevated proliferation rates of T. cruzi in Rhodnius prolixus, it failed to induce a significant increase of parasite virulence in mice. Altogether, these results indicate that the absence of a gene encoding catalase in T. cruzi has played an important role in allowing this parasite to develop a shrill capacity to sense and overcome oxidative stress.
Assuntos
Catalase/metabolismo , Estresse Oxidativo , Transdução de Sinais , Trypanosoma cruzi/metabolismo , Animais , Catalase/genética , Linhagem Celular , Doença de Chagas/parasitologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , NADH NADPH Oxirredutases/metabolismo , Rhodnius/parasitologia , Superóxido Dismutase/metabolismo , Transfecção , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/patogenicidadeRESUMO
The objective of this study was to evaluate the feeding behavior of Triatoma vitticeps through the identification of its food sources and the characterization of the blood ingestion process. In addition, we aimed to verify if the saliva of this vector interferes with the perception of the host during the feedings by creating a nervous impulse. Here, we demonstrated that the T. vitticeps saliva reduces, gradually and irreversibly, the amplitude of the compound action potential of the nervous fibre, which helps decrease the perception of the insect by the host. The precipitin reaction demonstrated the feeding eclecticism of this vector, with the identification of eight food sources - most of them found simultaneously in the same insect. The analysis of the electrical signals produced by the cibarial pump during meals demonstrated that the best feeding performance of T. vitticeps nymphs that fed on pigeons is mainly due to the higher contraction frequency of the pump. The longer contact period with the host to obtain a complete meal compared with other triatominae species of the same instar could favor the occurrence of multiple blood sources in T. vitticeps under natural conditions, as it was evidenced by the precipitin test.
Assuntos
Conteúdo Gastrointestinal , Insetos Vetores/fisiologia , Saliva/fisiologia , Triatoma/fisiologia , Animais , Tatus , Aves , Brasil , Bovinos , Cães , Comportamento Alimentar/fisiologia , Humanos , Lagartos , Gambás , Roedores , Saliva/químicaRESUMO
Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 - a recombinase involved in homologous recombination - in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism.
Assuntos
Recombinação Homóloga/fisiologia , Proteínas de Protozoários/metabolismo , Rad51 Recombinase/metabolismo , Trypanosoma cruzi/enzimologia , Proteínas de Protozoários/genética , Rad51 Recombinase/genética , Trypanosoma cruzi/genéticaRESUMO
Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. Like most living organisms, it is susceptible to oxidative stress, and must adapt to distinct environments. Hence, DNA repair is essential for its survival and the persistence of infection. Therefore, we studied whether T. cruzi has a homolog counterpart of the MutY enzyme (TcMYH), important in the DNA Base Excision Repair (BER) mechanism. Analysis of T. cruzi genome database showed that this parasite has a putative MutY DNA glycosylase sequence. We performed heterologous complementation assays using this genomic sequence. TcMYH complemented the Escherichia coli MutY- strain, reducing the mutation rate to a level similar to wild type. In in vitro assays, TcMYH was able to remove an adenine that was opposite to 8-oxoguanine. We have also constructed a T. cruzi lineage that overexpresses MYH. Although in standard conditions this lineage has similar growth to control cells, the overexpressor is more sensitive to hydrogen peroxide and glucose oxidase than the control, probably due to accumulation of AP sites in its DNA. Localization experiments with GFP-fused TcMYH showed this enzyme is present in both nucleus and mitochondrion. QPCR and MtOX results reinforce the presence and function of TcMYH in these two organelles. Our data suggest T. cruzi has a functional MYH DNA glycosylase, which participates in nuclear and mitochondrial DNA Base Excision Repair.
Assuntos
DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Estresse Oxidativo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Dano ao DNA , DNA Glicosilases/química , Reparo do DNA , DNA Mitocondrial , Ativação Enzimática , Glucose Oxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Mutação , Transporte Proteico , Análise de Sequência de DNARESUMO
Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination-mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double-stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double-stranded DNA breaks in T. cruzi DNA.
Assuntos
Enzimas Reparadoras do DNA/genética , Resistência a Medicamentos/genética , Nitroimidazóis/farmacologia , Proteínas de Protozoários/genética , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Sobrevivência Celular , Doença de Chagas/tratamento farmacológico , Doença de Chagas/genética , Doença de Chagas/parasitologia , DNA Glicosilases/genética , Reparo do DNA/efeitos dos fármacos , DNA de Protozoário/efeitos dos fármacos , Guanina/análogos & derivados , Guanina/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/genéticaRESUMO
The objective of this study was to evaluate the feeding behavior of Triatoma vitticeps through the identification of its food sources and the characterization of the blood ingestion process. In addition, we aimed to verify if the saliva of this vector interferes with the perception of the host during the feedings by creating a nervous impulse. Here, we demonstrated that the T. vitticeps saliva reduces, gradually and irreversibly, the amplitude of the compound action potential of the nervous fibre, which helps decrease the perception of the insect by the host. The precipitin reaction demonstrated the feeding eclecticism of this vector, with the identification of eight food sources - most of them found simultaneously in the same insect. The analysis of the electrical signals produced by the cibarial pump during meals demonstrated that the best feeding performance of T. vitticeps nymphs that fed on pigeons is mainly due to the higher contraction frequency of the pump. The longer contact period with the host to obtain a complete meal compared with other triatominae species of the same instar could favor the occurrence of multiple blood sources in T. vitticeps under natural conditions, as it was evidenced by the precipitin test.