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, γ-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.

Figure 1. Distribution of total metabolites identified in Trypanosoma cruzi epimastigote samples.

The pie chart depicts the percentages of putatively identified metabolites from each of the metabolite classes. A total of 1,069 metabolites were analysed.

Figure 2. Putatively identified metabolites mapped onto the KEGG metabolic pathways.

Coloured nodes (putatively identified metabolites) demonstrate coverage of diverse metabolic pathways (grey lines indicate annotated T. cruzi pathways). Node colour represents metabolite abundance in Bzn-treated T. cruzi relative to untreated controls on a continuous colour scale: blue: 0.5, yellow: 1 (no change), red: 2-fold increase. Node size indicates P-value from unpaired Welch's t-test: large: p<0.01, medium: p<0.05, small: P≥0.05. The large blue spot in the lower-right corner represents trypanothione disulphide (relative abundance = 0.7, P-value<0.01). Image generated with iPath2.0 .

Figure 3. Metabolites displaying significant differences between control and 20 µM Bzn treated T. cruzi samples.

Metabolites displaying significant differences between control samples (cBc) and 20 µM Bzn treated samples (cBt) with p<0.05 (unpaired T-test) and fold change >1.4. Bzn derived metabolites were not included.

Figure 4. Bzn-derived metabolites.

A number of Bzn derived molecules detected after treatment of T. cruzi epimastigotes with 20 µM and/or 50 µM Bzn are represented. A putative pathway for their in vivo formation is shown; double arrows indicate the existence of possible intermediates such as nitroso or nitrenium derivatives. The observed m/z values corrected for proton gain or loss (observed m/z ± 1.007276) are included. Cys, G, Ov, γGC, T and Gsp refer to cysteine, glutathione, ovothiol A, gamma-glutamylcysteine, trypanothione and glutathionylspermidine respectively. All thiols are represented with their functional -S- groups separately. Bold numbers in parenthesis are the metabolite reference numbers (Tables 1 and 2).

Figure 5. Identification of Bzn-glutathione adducts.

A. Upper plot is a chromatogram obtained from a sample of Bzn treated parasites corresponding to m/z 536.1922 within a 3 ppm mass range (single-charged ion). Middle and bottom plots are magnified mass spectra in the regions of interest within RT 18.5–19. B. Chromatograms and m/z plots corresponding to m/z 268.5997 (di-charged ion) in a sample of Bzn treated parasites. In both A and B each ion is detected in two different but closely eluting chromatographic peaks with retention times centred at 17.8 and 18.6 minutes. Ionization charge states of the molecules are confirmed by the m/z difference for the isotopic ¹³C peaks, which are expected to be 1.0034 for mono charged ions and 0.5017 for di-charged ions (bottom plots). ³⁴S isotopic peaks are also observed for both ions which are expected at 1.9959 m/z difference for mono-charged ions and 0.9979 for di-charged ions. C. MSMS fragmentation spectrum for precursor ion m/z 536.19. The m/z_c = m/z−1.007276 (proton mass). The proposed structures for the precursor ion (Bzn-glutathione adduct) and for some of the most intense fragments are shown. Exact theoretical mass and formula are included together with each fragment structure. A 4-C adduct is depicted though a 5-C adduct could be represented.