Selenium Derivatives as Promising Therapy for Chagas Disease: In Vitro and In Vivo Studies

Chagas disease is a tropical infection caused by the protozoan parasite Trypanosoma cruzi and a global public health concern. It is a paradigmatic example of a chronic disease without an effective treatment. Current treatments targeting T. cruzi are limited to two obsolete nitroheterocyclic drugs, benznidazole and nifurtimox, which lead to serious drawbacks. Hence, new, more effective, safer, and affordable drugs are urgently needed. Selenium and their derivatives have emerged as an interesting strategy for the treatment of different prozotoan diseases, such as African trypanosomiasis, leishmaniasis, and malaria. In the case of Chagas disease, diverse selenium scaffolds have been reported with antichagasic activity in vitro and in vivo. On the basis of these premises, we describe the in vitro and in vivo trypanocidal activity of 41 selenocompounds against the three morphological forms of different T. cruzi strains. For the most active selenocompounds, their effect on the metabolic and mitochondrial levels and superoxide dismutase enzyme inhibition capacity were measured in order to determine the possible mechanism of action. Derivative 26, with a selenocyanate motif, fulfills the most stringent in vitro requirements for potential antichagasic agents and exhibits a better profile than benznidazole in vivo. This finding provides a step forward for the development of a new antichagasic agent.

C hagas disease (CD) or American trypanosomiasis is a lifethreatening tropical infection caused by the insecttransmitted protozoan parasite Trypanosoma cruzi. CD is an important public health problem in Latin America, being the major cause of morbimortality in many endemic regions: It affects 6−7 million people, causing about 14 thousand deaths annually, and it is hypothesized that about 100 million people are living at risk of infection worldwide. 1,2 Blood-sucking triatomine bugs (vectors) are the main transmission route, although the oral route involving parasite-contaminated food and drink, the congenital route, as well as blood transfusion, transplantation, and laboratory accidents are also important. 3 CD has recently spread to nonendemic areas as a result of migratory flows, particularly in the United States and Europe. 4,5 In mammalian hosts, T. cruzi is an obligate intracellular parasite which can infect most nucleated cells. 6 The parasites become widely disseminated in tissues and organs and can be detected in the bloodstream during the initial acute phase of CD. Following suppression by the adaptive immune response, 7 CD progresses to a long-lasting asymptomatic chronic stage with an extremely low parasite burden. However, about 30% of patients will progress to a symptomatic chronic CD, developing cardiomyopathy and digestive tract megasyndromes, among others, for which there are few therapeutic options. 8 Because of the gaps in knowledge about T. cruzi, the longterm nature of CD, and its complex pathology, no vaccines are available. Currently, the front-line chemotherapy used to treat CD is limited to two obsolete drugs for more than 50 years: benznidazole (BZN) and nifurtimox (NFX). These drugs lead to serious drawbacks, such as a range of toxic side-effects, extended treatment length and frequent treatment failures. 9,10 Furthermore, the well-known cross-resistance−both drugs require metabolic activation within the parasite by the same mitochondrial nitroreductase 11 − and the natural variation in susceptibility to drugs due to the extreme diversity of species 12 make crucial the international effort aimed at developing new drugs against CD.
In this context, selenium (Se) and their derivatives have emerged as an interesting strategy for the treatment of different trypanosomiasis. Previous studies of our research group have shown that different selenocompounds exhibited promising activity against visceral leishmaniasis. 13−17 Moreover, selenocompounds have displayed activity against malaria, 18 African trypanosomiasis, 19 or intestinal schistosomiasis. 20 In the case of CD, several studies confirmed an association between the chronification of the disease and the decrease of Se plasma levels. 21 In addition, Se supplementation therapies modulated the antioxidant, immune, and inflammatory responses, thus improving the intestinal megasyndrome, 22 the placental immune response in pregnancy cases, 22 and especially Chagasic cardiomyopathy problems. 23,24 During the last years, diverse selenium scaffolds such as selenosemicarbazones, 25 Se lapachones, 26 Se quinones, 27 Se naphtoquinones, 28 and selenocyanates 29 have been reported with antichagasic activity in vitro and in vivo. On the basis of these premises, and in connection with the interesting properties of selenium derivatives for the treatment of CD 30 in the present contribution, we describe the in vitro and in vivo trypanocidal activity of 41 selenocompounds against the three morphological forms of three different T. cruzi strains. In order to expand the chemical space and the molecular diversity several selenoamides and selenophosphoramidates containing seleno-cyanate and diselenide motifs have been selected (Figure 1). For the most active and selective selenocompounds, its effect on the metabolite excretion, mitochondrial membrane potential, DNA or RNA alteration, and Fe-superoxide dismutase (SOD) enzyme inhibition capacity were measured in order to determine the possible mechanism of action.

■ RESULTS AND DISCUSSION
In Vitro Evaluation. Taking into account the parasite's genetic diversity, drug resistance, the different sensitivities against moieties, and the CD target product profile established by the Drugs for Neglected Diseases initiative (DNDi), 10,31 compounds and BZN were evaluated against three different T. cruzi strains belonging to different discrete typing units (DTUs) associated with the human parasitosis. Moreover, the compounds' effects on the viability and growth of the host cells was evaluated against Vero cells.
First, in vitro evaluation screening was performed using the extracellular epimastigotes because of their simple culture. The results, summarized in Table S1, are expressed as the inhibitory concentration 50 (IC 50 ). Otherwise, selectivity index (SI) was calculated as the ratio between IC 50 values of compounds against Vero cells relative to those against T. cruzi epimastigotes.
Compounds with SI values higher than 10 in at least one T. cruzi strain advanced to the in vitro activity assessment against the developed forms in vertebrate hosts (amastigotes and trypomastigotes); these are the relevant forms from a clinical   50 and SI values against the amastigotes and trypomastigotes over the three strains (Tables  1 and 2). Interestingly, diselenide derivatives did not show activity at all against T. cruzi epimastigotes as it happens against Leishmania infantum. 33 In contrast, 14 out of the 20 selenocyanate derivatives presented IC 50 values lower than 20 μM at least in one of the strains, and 12 of them presented IC 50 values lower than 10 μM in at least in one of the strains. The criteria established for this in vitro screening as the cutoff of this step were the same as those according to the literature: 34,35 IC 50 < 10 μM and SI > 50 (in at least two strains and one parasitic form). Therefore, compounds 26 and 28 were prioritized as potential compounds, and they were chosen for the subsequent in vivo evaluation. Moreover, these compounds were active against the three T. cruzi strains (including the BZN-resistant SN3 strain), devoid of drug resistance.
Before going ahead with in vivo studies, the infection rates in Vero cells were evaluated by counting the infected cells after 72 h of treatment with compounds 26 and 28. Results are summarized in Figure 2, together with the previously determined IC 50 values against amastigotes and trypomastigotes of the Arequipa strains. It was found that the rates of infection (and the total number of amastigotes and trypomastigotes) decreased as the concentrations of compounds and BZN increased.
The average number of amastigotes per cell was also measured ( Figure S1), giving an idea of the killing rate. This value was measured after 72 h of exposure at different concentrations of compounds and BZN, and the data show that compounds exhibit behavior similar to BZN, which is considered as a fast-acting compound. 36 The number of amastigotes per cell gradually decreased in all cases after 72 h of exposure, highlighting that the effect of tested compounds at 50 μM reduced the number of amastigotes to practically zero. These data show that the compounds not only inhibit parasite multiplication but also cause its death. All these features are additional advantages since fast-acting and trypanocidal compounds can eliminate the parasite in a few doses, 37 as well as act against the newfound quiescent or dormant amastigote form (responsible form of relapses after chemotherapy). 38 In Vivo Anti-T. cruzi Activity in BALB/c Mice. Considering the results obtained from in vitro assays, compounds 26 and 28 were selected as promising drugs for in vivo evaluation in BALB/c mice. These trials were performed using only the T. cruzi Arequipa strain in order to reduce the number of animals tests, as there were no significant differences for the other evaluated strains.
As currently used clinical drugs presented variable activity in the acute and the chronic phase of the disease, 39 compounds were evaluated in both phases according to the experiments established in Scheme 1. It should be noted that most in vivo testing has focused on acute CD, partially because it is simpler to monitor parasite burden. 40 However, the ability to cure chronic CD is the main need from a clinical point of view. 31 Therefore the different phase-specific drugs responses highlight that chronic CD should be the main research focus in animal models.
BALB/c mice were intraperitoneally infected according to a literature procedure. 41 Compounds 26 and 28 or BZN were administered intraperitoneally from 9 days post-infection  (dpi), once daily for 5 consecutive days at a dose of 20 mg· kg 1− . This dosage represents the subcurative dose of BZN, so the experiment demonstrated whether the studies compound are more effective or not than the reference drug during the disease progress. In addition, it is shown that a compound showing a reasonable reduction in parasitaemia following 5 consecutive days of treatment can be considered a lead compound. 36 First, parasitaemia levels of the four studies groups during the acute phase were determined by counting bloodstream trypomastigotes (BTs) as described in the "Methods" section ( Figure 3A). Compound 28 showed a slight antiparasitic activity over the first month becoming similar to the BZN activity after 40 days. Compound 26 demonstrated a better activity profile compared to that of BZN, with low parasitaemia levels over the completely acute phase for 48 days. It is important to note that compound 26 caused an evident reduction of parasitaemia from the beginning of the treatment, even disappearing on 12 dpi. In addition, the parasitaemia of mice treated with compound 26 was resolved at 48 dpi, that is, 7 days before the parasitaemia of the untreated mice and those treated with BZN.
Second, the experimental cure was evaluated using a double checking widely used in animal models to evaluate the treatment effectiveness: immunosuppression (IS) and PCR of the target organs/tissues. Animals whose parasitaemia reactivation does not reappear and show negative PCR data after IS are considered cured. 42 At 115 dpi, mice were immunosuppressed with cyclophosphamide monohydrate (CP) in order to assess the effectiveness of the treatments in acute and chronic CD. This procedure expands the residual infection to detectable levels in there is still presence of parasite after treatment. 43 Figure 3B shows the reactivation percentages of infection for each group of mice compared to those of the untreated (control) group after the immunosuppression. As observed, mice treated with BZN and compound 26 showed a similar parasitaemia reactivation. Compound 26 showed a reactivation of approximately 65 and 60% in acute and chronic phases respectively, being slightly more effective than BZN. However, mice treated with compound 28 showed a high parasitaemia ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article reactivation (approximately 95 and 85% in acute and chronic phase, respectively). With this result, we hypothesized that compound 26 could be an effective antichagasic treatment. These results will be lately confirmed with the results obtained from tissue PCR. In order to further evaluate the effectiveness of the compounds in acute and chronic CD, the presence of nested T. cruzi parasites in target organs/tissues 42 was measured by PCR after necropsy (145 dpi). Figure 3C resumes the PCR results for the different target organs/tissues in the 4 mice groups in both phases. As expected, untreated mice ( Figure  3C(a)) showed the presence of parasites in every analyzed organs/tissues for both phases. In mice treated with BZN, brain and lung appeared free of parasites for the acute phase, and brain, esophagus and lung appeared free of parasites for the chronic phase (28.6 and 42.9% of parasite-free organs/ tissues, respectively). In accordance with previously obtained results, mice treated with compound 26 showed better results than those treated with BZN. In this case, adipose tissue, brain, and lung appeared free of parasites for the acute phase, and bone marrow, brain, esophagus for the chronic phase (42.9% of parasite-free organs/tissues in both phases). In contrast, treatment with compound 28 did not decrease enough parasite levels in the analyzed tissues. As observed, compound 26 showed the best trypanocidal activity after the double checking of the cure, even better than that shown by BZN, confirming the partial curative effect in both phases of CD at the tested dosage.
Otherwise, the immune response to T. cruzi infection was assessed by counting the immunoglobulin G (IgG) levels by indirect enzyme-linked immunosorbent assay (ELISA). 44 The amount of IgG is directly associated with the parasitic load, and these experiments allowed the effectiveness of compounds evaluation in combination with the innate protection of the mice. 42 The titer of anti-T. cruzi IgG in the different groups and the respective controls are shown in Figure S2. As observed, in the acute phase mice treated with compounds 26 or 28 showed lower IgG values than the treated with BZN at the beginning of the treatment becoming similar at the end, before and after IS. In the chronic phase, IgG values were similar between mice treated with compounds 26 and 28, treated with BZN and the control mice. These results confirm  1) adipose, (2) bone marrow, (3) brain, (4) esophagus, (5) heart, (6) lung, (7) muscle. ■ , 1 / 3 of the corresponding organ/tissue PCR products showed banding on electrophoresis; *, 2 / 3 of the corresponding organ/tissue PCR products showed banding on electrophoresis.
the antichagasic activity of both compounds 26 and 28, with compound 26 being more effective, especially in the acute phase. It has to be mentioned that the samples obtained after IS do not reflect data indicating infection rates, but rather confirm the IS suffered by the mice.
Moreover, splenomegaly is manifested in both acute and chronic phases on T. cruzi infected mice. This indicated the direct link between the enlargement of the spleen and the parasitic load. Figure S3 shows the weight percentage of the spleens for each group of mice. As observed, treated groups of mice demonstrated a smaller increase in the spleen weight when compared with the untreated (control) ones. Mice treated with compound 26 showed splenomegaly reduced by 49 and 38% in acute and chronic phases, respectively, and mice treated with compound 28 showed splenomegaly reduced by 37 and 48%, in comparison with the untreated (control) mice. Both compounds showed spleen weight percentage similar to the BZN-treated mice, with mice treated with compound 26 being more active in the acute phase, as previous results demonstrated.
Finally, the metabolic abnormalities associated with the treatment were determined by measuring kidney, heart, and liver biochemical markers (Table S2), including values for uninfected mice. Although most of the clinical parameters showed alteration at 2 days post-treatment, they returned to normal levels in the measurements obtained on the necropsy day. Moreover, it is noteworthy that none of the mice died or lost more than 10% body mass during and/or after treatment. The low toxicity allows these compounds to be studied at higher doses, establishing an improved treatment schedule based on pharmacokinetic studies in order to reach a sterile cure.
Mode of Action (MoA) Studies. Metabolite Excretion. T. cruzi parasites are known to significantly reduce glucose metabolism catabolites to pyruvate, acetate, and succinate, instead of degrading glucose to CO 2 and water, 45 so epimastigotes of T. cruzi Arequipa strain (untreated and treated with 26 and 28 at IC 25 concentrations) were analyzed by 1 H nuclear magnetic resonance ( 1 H NMR) in order to measure different glucose metabolism catabolites. Figure 4 shows the percentage variation of excreted catabolites in treated parasites in comparison with control (untreated) parasites. As shown, excretion of all analyzed catabolites was altered, with succinate being the most altered for both treatments with values > 250%. According to the literature, an increase in excreted succinate may be closely related to a mitochondrial dysfunction. 45,46 Therefore, mitochondrial stability assays were performed.
Mitochondrial Membrane Potential Measurement. Mitochondria play an essential role in the maintenance of the electrochemical gradient and disturbances in the membrane potential could lead to less DNA replication, RNA transcription, and therefore cell apoptosis and/or necrosis. In view of the possible mitochondrial dysfunction, acridine orange (AO) and rhodamine 123 (Rho) staining were performed to evaluate the integrity of this organelle by flow cytometry. Figure 5 shows the measured percentage variation after treatment at IC 25 concentrations, as stated before. BZN reduces the mitochondrial membrane potential (35.4%) due to its MoA. 47 Parasites treated with compound 26 suffered a mitochondrial membrane depolarization of 40.6%, while parasites treated with compound 28 reached a higher depolarization of 67%. These results lead us to hypothesize that the antichagasic activity of the presented compounds may be related to an effect at the mitochondrial level: They could produce bioenergetic collapses, which precede T. cruzi death via necrosis in a mitochondrion-dependent manner, being the cause of the fast-acting trypanocidal activities of these compounds.
DNA and RNA Levels Measurement. As mentioned before, mitochondrial membrane potential alteration affects DNA replication and RNA transcription because of a decrease in ATP levels and a NADH/NAD + imbalance. 45 Thus, DNA and RNA levels were quantified by flow cytometry, with their percentages summarized in Figure S4. Significant alteration was observed in every treatment, with BZN, 26, and 28 compared with the nontreated parasites. BZN inhibited nucleic acid levels up to 22%. Meanwhile, treatments with compounds 26 and 28 produced an inhibition of nucleic acid levels of 47 and 62%, respectively. These results bear out the possible MoA previously described. It must be noted that these inhibitions are due to not only an ATP deficit but also random nucleic acids degradation as a feature usually attributed to necrosis. 48 T. cruzi Fe-SOD Enzyme Inhibition. Previous studies of our research group demonstrated the selective inhibition against Fe-SOD of selenium derivatives, 13 one of the most relevant targets for CD treatment. Therefore, in view of the previous results and the well-known antioxidant system modulator activity of these compounds, 33 compounds 26 and 28 ability to inhibit Fe-SOD and/or Cu/SOD enzyme was assessed. Figure  6 reveals the inhibition curve of both compounds against Fe-SOD and human erythrocytes CuZn-SOD. Both compounds inhibit Fe-SOD enzyme activity, reaching IC 50 values of 51.9 μM for compound 26 and 9.4 μM for compound 28. Moreover, compounds did not reach 50% inhibition of Cu/ SOD even at 100 μM. These results indicated the high selectivity of compound 28 against the parasitic enzyme, allowing us to propose it as one of the possible targets or MoA. Even when its trypanocidal effect can be ultimately ascribed to the inhibition of this enzyme, the possibility of multitarget activity should however not be rejected.

■ CONCLUSION
In response to a dire need for new medications to treat CD, one chemical library of 41 leishmanicidal selenium amides and phophoramidate derivatives were screened against T. cruzi. The most active and selective compounds against the epimastigote form (compounds 25, 26, 28, 29, and 33) were tested in the developed forms in vertebrate hosts (amastigotes and trypomastigotes). Taking into account the activity and selectivity of the derivatives, 26 and 28 were selected for the in vivo studies. Compound 26 exhibits a better profile than 28 and BZN, and it fulfils the most stringent in vitro requirements for potential antichagasic agents. It showed higher activity and lower toxicity than BZN after in vivo treatment, as indicated by different trials such as parasitemia monitoring, PCR, IS, or biochemical analysis. MoA analysis suggests a trypanocidal activity via necrosis in a mitochondrion-dependent manner through a bioenergetic collapse caused by a mitochondrial membrane depolarization. These findings suggest that these derivatives could be exploited and provide a step forward in the development of new antichagasic agents. It is worth considering higher doses and/or different treatment schedules, even combined therapies, to obtain a sterile cure.

■ METHODS
Chemistry. The compound library consists of 41 compounds containing bis(4-aminophenyl)diselenide 33 or 4aminophenylselenocyanate 33 entities as nucleus that have been decorated with aliphatic, cycloaliphatic, aromatic, and heteroaromatic substituents linked by an amide or phosphoramidate group. Structures are summarized in Figure 6. The synthesis and characterization of the molecules are reported in the literature. 33 Briefly, amides derived from diselenide (compounds 4, 11 and 13) and selenocyanate (compounds 21−34) were synthesized by an amide bond coupling with the corresponding amino groups of these scaffolds and the corresponding carbonyl of the acid chlorides. 33 Compounds 1−3, 5−10, 12, and 14 were prepared or by the reduction with sodium borohydride of the corresponding selenocyanate analogues. 33 Finally, phosphoramidate derivatives were obtained by reaction between the phosphoryl chlorides and bis(4aminophenyl)diselenide (compounds 15−20) or 4aminophenylselenocyanate (compounds 35−41), respectively  (unpublished). Briefly, derivatives 15−20 were obtained by reaction of a solution of bis(4-aminophenyl)diselenidein acetonitrile with the corresponding commercially available phosphoryl chlorides in a molar ratio (1:1) and triethylamine at 80°C for 1 h under an inert atmosphere of nitrogen. Compounds 35−41 were obtained reacting 4-aminophenylselenocyanate, the corresponding phosphoryl chloride, in a molar ratio of 1:2, and trimethylamine in chloroform under nitrogen at 60°C for 5 h. Phosphoramidate derivatives (15− 20 and 35−41) were purified by flash chromatography using different gradients of hexane/ethyl acetate as eluents.
Cytotoxicity Test. Mammalian Vero cells (EACC No. 84113001), cultured as previously reported, 41 were used to determine the cytotoxicity of the selenocompounds. 42 In short, 1.25 × 10 4 Vero cells·mL −1 were treated by adding the tested selenocompounds and BZN at a concentration range of 2000− 50 μM in 96-well plates (200 μL·well −1 ) at 37°C for 48 h. Untreated controls were also included. Subsequently, resazurin sodium salt (Sigma-Aldrich) was added, and the cells were incubated for a further 24 h. Finally, cell viability was determined following the same procedure as described to assess the trypanocidal activity in the epimastigotes. Each compound concentration was tested in triplicate in three independent experiments.
Screening against Intracellular Amastigotes and Infected Cells. Trypanocidal activity against amastigotes was determined according to the literature reported previously. 41 In short, 1 × 10 4 Vero cells·well −1 were seeded in 24-well plates and then infected with culture-derived trypomastigotes (obtained as previously described) 42 at a multiplicity of infection (MOI) ratio of 1:10. After 24 h of infection, nonphagocyted trypomastigotes were washed, and the plates were treated by adding the tested selenocompounds and BZN to the corresponding well at a concentration range of 50−0.1 μM in 500 μL·well −1 . Untreated controls were also included. After 72 h of incubation, the trypanocidal effect was determined based on the counting of amastigotes and infected cells in methanol-fixed and Giemsa-stained preparations, and the activity was expressed as the IC 50 using GraphPad Prism 6 software. Each compound concentration was tested in triplicate in three independent experiments.
Screening against Bloodstream Trypomastigotes. BTs (2 ×10 6 BTs·mL −1 , obtained as previously described from infected BALB/c mice) 51 were treated by adding the tested selenocompounds and BZN at a concentration range of 50− 0.1 μM in 96-well plates (200 μL·well −1 ) at 37°C. Untreated controls were also included. After 24 h of treatment, resazurin sodium salt (Sigma-Aldrich) was added, and the cells were incubated for a further 4 h. Finally, trypanocidal activity was determined following the same procedure as described to assess the trypanocidal activity in the epimastigotes. Each compound concentration was tested in triplicate in three independent experiments.
In Vivo Trypanocidal Activity on BALB/c Mice. Ethics Statement. All animal work and maintenance was performed under RD53/2013 and approved by the Ethics Committee on Animal Experimentation (CEEA) of the University of Granada, Spain.
Infection and Treatment. Female BALB/c mice aged 10− 12 weeks and with a size of 20−24 g were divided into five groups (n = 3 per group): 0, negative control group (uninfected and untreated mice); I, positive control group (infected and untreated mice); II, BZN group (infected mice treated with BZN); III, 26 group (infected mice treated with 26); and IV, 28 group (infected mice treated with 28).
Mice were infected by intraperitoneal injection of 5 × 10 5 BTs of T. cruzi Arequipa strain per mouse in 200 μL PBS. 42 Subsequently, the treatment was intraperitoneally administered (∼200 μL) once daily for 5 consecutive days.
The tested selenocompounds and BZN were prepared at 2 mg·mL −1 in an aqueous suspension vehicle containing 5% (v/ v) DMSO and 0.5% (w/v) hydroxypropyl methylcellulose, as previously reported. 52 Compounds (20 mg·kg −1 per day) were administered for 5 consecutive days (total dosages of 100 mg· kg −1 ), and vehicle only was administered in the negative and positive control groups. Treatments began when the infection was confirmed (9 dpi) for acute-phase-treated mice, and when it was established that the animals moved into the chronic phase (100 dpi, for chronic-phase-treated mice; Scheme 1).
Monitoring of Parasitaemia during the Acute Phase Treatment. Parasitemia levels were measured by counting BTs from peripheral blood drawn from the mandibular vein and diluted at a ratio of 1:100, as previously described. 42 Fresh blood microscopic examination was performed until the day the parasitemia was undetected (Scheme 1). Parasitemia was expressed as parasites·mL −1 .
Immunosuppression. Immunosuppression was performed by intraperitoneal injection of three doses of 200 mg·kg −1 of ISOPAC CP at 3−4 day intervals (Scheme 1), as previously reported. 53 Mice were closely monitored for side effects or for secondary infections due to immunosuppression. Within 7 days after the last CP injection, parasitemia reactivation was determined by counting BTs according the procedure described for parasitemia in the acute phase. 42 Mice Sacrifice, Blood Collection, and Organs/Tissues Extraction. On 145 dpi, the mice were euthanized using CO 2 , followed by exsanguination via cardiac puncture, and blood was collected. Nine target organs/tissues (adipose, bone marrow, brain, esophagus, heart, lung, muscle, spleen, and stomach) were then harvested 42 and perfused with prewarmed PBS to avoid contamination with BTs. 54 Finally, they were stored at −80°C until DNA extraction. In addition, spleens were weighed to assess splenomegaly. 42 Tissue DNA Extraction, PCR, and Electrophoresis. DNA extraction of the post-mortem organs/tissues was performed using Wizard Genomic DNA Purification Kit, 41 and the extracted DNA was subjected to amplification by PCR based on the spliced leader (SL) intergenic region sequence of T. cruzi (for detailed description, see literature reported previously). 55 Finally, the PCR products were resolved by electrophoresis on a 2% agarose gel (containing GelRed nucleic gel stain) for 90 min at 90 V.
ELISA Test. Serum samples were obtained from blood collected on several days post-infection (Scheme 1), processed according the method previously described, 42 and aliquoted to the ELISA test and biochemical analysis, as mentioned below.
Circulating antibodies in serum against both antigens were quantitatively evaluated in triplicate by the ELISA test in 96well plates using diluted serum samples (1:80 in PBS), as previously described. 41 Toxicity Test by Clinical Analysis. Serum samples obtained from blood in several days post-infection (Scheme 1) were sent to the Biochemical Service (University of Granada) to measure a series of biochemical parameters with the commercial Cromakit using a clinical chemistry analyzer (BS-200, Shenzhen Mindray Biomedical Electronics Co., LTD), as previously described. 41 Mode of Action Studies. 1 H NMR Analysis of Excreted Metabolites. T. cruzi Arequipa (5 ×10 5 epimastigotes·mL −1 ) were treated by adding the tested compounds at IC 25 concentrations in 25 cm 2 cell culture flasks at 28°C for 72 h. Untreated controls were also included. Cultures were then centrifuged and filtered, and the metabolites of the supernatants were analyzed using a 1 H NMR spectrometer (Varian Direct Drive 500 MHz Bruker) with AutoX probe, D 2 O as solvent, and 2,2-dimethyl-2-silapentane-5-sulfonate as the reference signal. 56 Chemical shifts were expressed in parts per million (ppm), and analyses were conducted as previously reported. 57 Flow Cytometry Analysis of Mitochondrial Membrane Potential and Nucleic Acid Levels. The untreated and treated epimastigotes of T. cruzi Arequipa described in the 1 H NMR analysis were collected by centrifugation, washed three times in PBS, and stained with 10 mg·mL −1 Rho (Sigma-Aldrich) or AO (Sigma-Aldrich) dyes in 0.5 mL of PBS for 20 min. 58 Control epimastigotes with a fully depolarized mitochondrion were obtained by incubation for 40 min with 10 Mm KCN prior to Rho loading. 59 Nonstained parasites were also included. After the elapsed time, epimastigotes were processed and analyzed by flow cytometry as previously reported. 57 SOD Enzymatic Inhibition Analysis. The in vitro activities of either excreted Fe-SOD from T. cruzi (obtained as previously described) 57 and commercial copper/zinc superoxide dismutase (Cu/Zn-SOD) from human erythrocytes (Sigma-Aldrich) were evaluated using the method previously described 60 after incubation with a concentration range of compound from 100 to 0.1 μM.
Statistical Analyses. Statistical analyses were performed with the SPSS v21.0 software (IBM Corp; Armonk, NY). The t-test for paired samples was used to verify whether there were differences between the assays used. Differences were considered significant when the p-value was less than 0.05. Also statistical studies based on contingency tables (prevalence) were conducted.