Dataset Information

Benznidazole biotransformation and multiple targets in Trypanosoma cruzi revealed by metabolomics.

ABSTRACT:

Background

The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.

Methodology/principal findings

Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, ?-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.

Conclusions/significance

Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi.

SUBMITTER: Trochine A

PROVIDER: S-EPMC4031082 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Publications

Benznidazole biotransformation and multiple targets in Trypanosoma cruzi revealed by metabolomics.

Trochine Andrea A Creek Darren J DJ Faral-Tello Paula P Barrett Michael P MP Robello Carlos C

PLoS neglected tropical diseases 20140522 5

<h4>Background</h4>The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.<h4>Methodology/principal findings</h4>Parasite ...[more]

PMID: 24853684

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Project description:BackgroundDrugs currently used for the treatment of Chagas' disease, nifurtimox and benznidazole, have a limited effectiveness and toxic side effects. With the aim of finding new therapeutic approaches, in vitro and in vivo anti-Trypanosoma cruzi activity of vitamin C alone and combined with benznidazole were investigated.Methodology/principal findingsThe trypanocidal activity on epimastigote and trypomastigote forms was evaluated by counting parasites in a Neubauer chamber after treatment with the compounds. For the amastigote stage, transgenic parasites expressing β-galactosidase were used and quantified by measuring the β-galactosidase activity. The cytotoxicity of compounds was tested on Vero cells. The redox state of the parasite was evaluated by determining the reduced thiol levels (spectrophotometric assay) and the intracellular oxidative state (by flow cytometry). The in vivo trypanocidal activity was evaluated on a murine model of Chagas' disease. The trypanocidal activity of vitamin C and benznidazole was similar for the three parasite forms. When combining both drugs, vitamin C did not induce any change in the antiparasitic activity of benznidazole on trypomastigotes; however, on mammal cells, vitamin C diminished the cytotoxicity degree of benznidazole. Two mechanisms of action may be postulated for vitamin C: a lethal pro-oxidant effect on the parasite when used alone, and an antioxidant effect, when combined with benznidazole. A similar behavior was observed on infected mice; i.e., parasite counts in infected mice treated with vitamin C were lower than that of the control group. Animals treated with benznidazole presented lower parasitemia levels, as compared with those treated with vitamin C alone. Again, vitamin C did not cause any effect on the antiparasitic profile of benznidazole. Even though a combined treatment was employed, the antioxidant effect of vitamin C on the host was evidenced; a 100% survival was observed and the weight loss occurring during the acute phase of the infection was reduced.Conclusions/significanceBased on these results, the combination of vitamin C with benznidazole could be considered as an alternative treatment for Chagas' disease. These preliminary results encourage further research to improve the treatment of Chagas' disease.

Project description:Hexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. Previous studies employing the Differential Display technique have shown that the transcription level of the HT gene from T. cruzi (TcrHT) is higher in an in vitro-induced benznidazole (BZ)-resistant population of the parasite (17 LER) than in its susceptible counterpart (17 WTS).In the present study, TcrHT has been characterized in populations and strains of T. cruzi that are resistant or susceptible to BZ. We investigated the copy number and chromosomal location of the gene, the levels of TcrHT mRNA and of TcrHT activity, and the phylogenetic relationship between TcrHT and HTs from other organisms.In silico analyses revealed that 15 sequences of the TcrHT gene are present in the T. cruzi genome, considering both CL Brener haplotypes. Southern blot analyses confirmed that the gene is present as a multicopy tandem array and indicated a nucleotide sequence polymorphism associated to T. cruzi group I or II. Karyotype analyses revealed that TcrHT is located in two chromosomal bands varying in size from 1.85 to 2.6?Mb depending on the strain of T. cruzi. The sequence of amino acids in the HT from T. cruzi is closely related to the HT sequences of Leishmania species according to phylogenetic analysis. Northern blot and quantitative real-time reverse transcriptase polymerase chain reaction analyses revealed that TcrHT transcripts are 2.6-fold higher in the resistant 17 LER population than in the susceptible 17 WTS. Interestingly, the hexose transporter activity was 40% lower in the 17 LER population than in all other T. cruzi samples analyzed. This phenotype was detected only in the in vitro-induced BZ resistant population, but not in the in vivo-selected or naturally BZ resistant T. cruzi samples. Sequencing analysis revealed that the amino acid sequences of the TcrHT from 17WTS and 17LER populations are identical. This result suggests that the difference in glucose transport between 17WTS and 17LER populations is not due to point mutations, but probably due to lower protein expression level.The BZ resistant population 17 LER presents a decrease in glucose uptake in response to drug pressure.

Dataset Information

Benznidazole biotransformation and multiple targets in Trypanosoma cruzi revealed by metabolomics.

Background

Methodology/principal findings

Conclusions/significance

Publications

Benznidazole biotransformation and multiple targets in Trypanosoma cruzi revealed by metabolomics.

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