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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2018 Oct 15;99(5):236–248. doi: 10.1111/iep.12289

Hydroxymethylnitrofurazone treatment in indeterminate form of chronic Chagas disease: Reduced intensity of tissue parasitism and inflammation—A histopathological study

Cauê B Scarim 1,✉, Cleverton R de Andrade 2, João A da Rosa 3, Jean L dos Santos 1, Chung M Chin 1,✉
PMCID: PMC6302791  PMID: 30320480

Summary

Hydroxymethylnitrofurazone (NFOH) is a nitrofurazone prodrug effective in vivo during acute infections, and it has less hepatotoxicity effect than the standard drug benznidazole (BZN) which has been used during short‐ and long‐term treatment. In the present study, we induced the indeterminate form of Chagas disease in mice with a Y strain of Trypanosoma cruzi and analysed the histopathological data about the effects of NFOH and BZN on different tissues, including the heart, skeletal muscle, liver, kidney, colon, spleen and brain. After infection, BALB/c mice were treated with NFOH (150 mg/kg) and BZN (60 mg/kg) for 60 days and then submitted to immunosuppression using dexamethasone (5 mg/kg) for 14 days. Two trained analysts, as part of a blind evaluation, examined the results using serial sections of 3 mm diameter in two different moments. The results showed reactivation of the disease only in the infected nontreated group (POS). After treatment, amastigote nests were found in the heart, colon, liver and skeletal muscle in the POS group and in the heart and liver of the BZN group. Interestingly, amastigote nests were not found in the NFOH and NEG groups. The histopathological analysis showed fewer tissue lesions and parasite infiltrates in the NFOH group when compared with the BZN and POS groups. We have not observed any increase in the levels of hepatocellular injury biomarkers (AST/ALT) in the NFOH group. These in vivo studies show the potential for NFOH as an effective and safe compound useful as an anti‐T. cruzi agent.

Keywords: benznidazole, Chagas disease, chronic phase, hydroxymethylnitrofurazone, indeterminate form, Trypanosoma cruzi

1. INTRODUCTION

American trypanosomiasis or Chagas disease is a tropical disease caused by the Trypanosoma cruzi (T. cruzi) that affects approximately 5‐6 million people in the world.1 The globalized transport system has led to the disease being seen in developed countries such as the United States, Japan, Germany, Spain, the Netherlands, France, Portugal, Sweden, Switzerland, the United Kingdom, Italy, Belgium, and Australia. Despite this, only two drugs remain available for treatment during the acute phase of Chagas disease: nifurtimox and benznidazole (BZN).2, 3 For both adults and children, these drugs have demonstrated limited action in the indeterminate form.4, 5, 6, 7, 8, 9, 10, 11, 12

Chung et al13 obtained the hydroxymethylnitrofurazone (NFOH), as a synthetic intermediate aiming to obtain nitrofurazone (NF)‐primaquine prodrugs. The NFOH exhibited trypanocidal activity against trypomastigotes and amastigote forms of T. cruzi using infected LLC‐MK2 cells.13 The NFOH was four times less mutagenic than NF through the AMES test,14 and it did not show in vivo genotoxicity.15 In addition, the pharmacokinetic study has shown an increased bioavailability that is around 20 times more in rats and 10% in rabbits.16, 17

The improved activity of NFOH in comparison with NF can be explained by the nucleophilic attack of Cys25 residue present in the enzyme cruzain of T. cruzi to the carbonyl group present in the semicarbazone subunit of NFOH. At the same concentration, NF inhibited 30% of cruzain, while for NFOH, a value of 60% was recorded.18, 19, 20

Two studies described the in vivo efficacy of NFOH in the acute phase of Chagas disease. Ekins et al,21 using infected transgenic T. cruzi Brazil (Luc strains) with firefly luciferase, showed 78.5% of efficacy after four days of treatment with NFOH (50 mg/kg).21 In another study, Davies et al,22 which used the Tulahuen strain of T. cruzi, demonstrated negative PCR after six months of the last dose of NFOH treatment (150 mg/kg, 60 days). In addition, NFOH caused low animal mortality after experiments (NFOH, 16%; BZN, 33%; NF, 75%; positive control, 66%).22

Melo23 reported an increase in the rat lethal dose (LD50) for NFOH (2000 mg/kg) in comparison with NF (556.3 mg/kg).23 A cytotoxicity assay using HepG2 cells revealed that BZN was able to reduce 30% of the cells, while NFOH did not cause cell death. Moreover, in vivo analysis for short‐term (ST, 21 days) and long‐term (LT, 60 days) treatment demonstrated that NFOH (150 mg/kg/d) did not show hepatotoxicity, while BZN (150 mg/kg/d) showed extensive liver inflammation and fibrosis.24

In summary, the previous studies showed the in vitro and in vivo efficacy of NFOH against trypomastigotes forms of T. cruzi in the early acute phase of Chagas disease with decreased toxicity compared to BZN. The current available drugs are effective in the acute phase of the disease and act against amastigotes of some strains of T. cruzi in late acute, indeterminate, and chronic phases of Chagas disease. But they do not exhibit activity against the Y strain of T. cruzi during the later phases.25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 So, the purpose of this work was to evaluate the effect of NFOH in several tissues (heart, colon, liver, kidney, spleen, brain and skeletal muscle) during the indeterminate form of the disease, induced by the Y strain of T. cruzi in BALB/c male mice.

2. MATERIAL AND METHODS

2.1. Ethical approval

The study and all the procedures were approved by the Research Ethics Committee of Animal Experimentation of the School of Pharmaceutical Sciences of Araraquara, São Paulo State, Brazil—UNESP (39/2014). Male mice were used, BALB/c isogenic, with weights between 18 and 21 g. All animals were kept under controlled temperatures (23 ± 1°C), automatic lighting (12/12 hours cycle), humidity (55% ± 5%), ration and water ad libitum.

2.2. Parasites and animal infection

The induction of the Chagas disease was performed with an inoculum of 102 trypomastigotes forms (blood transfusions),28, 31, 36 Y strain of T. cruzi, by intraperitoneal injection. This inoculum was obtained, standardized and maintained by the Parasitology Laboratory of the School of Pharmaceutical Sciences, São Paulo State University—UNESP, Araraquara/SP—Brazil.

2.3. Experimental design and treatment

Figure 1 shows the experimental design. After three days of inoculation, 5 μL of fresh blood samples from the tails of the mice was collected weekly until no parasites were observed in the blood, which was confirmed by three negative parasitaemia on alternate days, showing subpatent parasitaemia and a simulation of the indeterminate form of chronic Chagas disease. Using a parasitaemia curve, in order to establish the infection pattern, 5 μL of blood obtained from the Balb/c mice's tails was examined microscopically (100 fields), and the number of parasites was established according to Brener's technique (1962).

Figure 1.

Figure 1

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Experimental protocol. A, Infection and induction of chronic phase with a Y strain of T. cruzi; B, treatment period with NFOH and BZN (indeterminate form of chronic phase); C, immunosuppression [Colour figure can be viewed at wileyonlinelibrary.com]

Four groups were separated into positive (POS, infected), negative (NEG, not infected) control, treatment with BZN (infected, 60 mg/kg/d) and treatment with NFOH (infected, 150 mg/kg/d)22 (N = 42; each infected groups, n = 12; no infected group, n = 6). The drug was administrated by gavage in a suspension of arabic gum (4%)37, 38, 39 once a day for 60 consecutive days.22 The experiment was performed with all groups at one time.

2.4. Recrudescence induced

Twenty‐four hours after treatment, the immunosuppression was induced by dexamethasone (5 mg/kg/d) through i.p. route for 14 consecutive days. The parasitaemia analyses were observed in three distinct periods, analysed at the first, seventh and fourteenth day.40

2.5. Anatomopathological evaluation—Relative weight

The weight of animals was measured weekly to identify gain or loss of weight during the experimental protocol, as well as any change at any moment of research. The weight of animals was measured weekly to identify any change at any moment of research. After euthanizing, the organs, heart, liver, kidney, spleen and brain, were weighed to establish the relative weight ([organ/animal weight] × 100), and the results were expressed as percentage.41, 42, 43, 44, 45 The colon and skeletal muscle were excluded from this analysis due to the importance of weight of bone and faeces (bias).

2.6. Macroscopic analysis

After euthanizing, the organs were immersed in tamponed formaldehyde (10%). All organs and tissues were codified to blind all groups on the subsequently analysis. The macroscopic analyses were carried out to identify any alterations on surface of organs and tissues. Specifically, relevant to the liver, the gallbladder was measured and classified into normal (+, <1 mm), enhanced (++, 1‐3 mm) and mega‐gallbladder (+++, >3 mm).

2.7. Histopathological analysis

The organs were analysed as follows: heart (all cardiac tissue—portions transversal); brain (the internal half of the both hemispheres—portions transversal); large intestine (colon segment—fragment collected just after the ileocecal valve—portions transversal); kidney (both ventral half—portions transversal); spleen (all distal part—portions transversal); liver (biggest lobule—portions transversal); gallbladder (entire organ—macroscopic analysis); skeletal muscle (quadriceps muscles—portions transversal). All organs/tissues were sectioned each 3 mm for analysis of whole organ/tissue (Figure 2).

Figure 2.

Figure 2

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Representation of serial histopathological sections (3 mm). (I) Example of an anatomical organ; (II) geometric representation of the organ/tissue; (III) serial sections of 3 mm throughout the organ/tissue; and (IV) representation of organs/tissue in paraffin for histopathological sections. In this study, the following organs/tissues were serially analysed: heart, colon, spleen, liver, kidney, brain and skeletal muscle [Colour figure can be viewed at wileyonlinelibrary.com]

All codified organs and tissues were placed in paraffin (in a 4‐μmol/L section), and haematoxylin and eosin staining (H and E) was performed. The histopathological analyses were performed by optical microscopy (Primo Star; Carl Zeiss Mycroscopy® GmbH, Gottingen, Germany) at 10×, 20×, 40× and 100× magnifications. A pathologist carried out the analysis, and 15% of samples were used to calculate the intra‐examined index (Kappa index, 0.83). Photographs were taken at 400 × with an Olympus X‐785 digital camera coupled to the microscope. The histopathology analysis was based in the following pathological alterations:

In the heart, the following aspects were identified: cellular hypertrophy, inflammatory infiltrate (Table 1), dystrophic calcification and amastigote nests.

Table 1.

Numerical criteria for the classification of inflammatory parameters according to the type of cell found during histopathological analysis

Infiltrates parameters Intensity Type
Absent (0%) Mild (1%‐33%) Moderate (33%‐66%) Intense (>66%) Acute Mixed Chronic
Inflammatory Infiltrates −1 0 1 2 −1 to 0 0.1‐1 1.1‐2
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In the colon, epithelial mucosa, gut‐associated lymphoid tissue (GALT), dystrophic calcification and amastigote nests were analysed. The ascending, transverse, descending and sigmoid colon was all examined.

In the spleen, the presence of capsule, white/red pulp, marginal zone and follicles in the organ (Table 2); dystrophic calcification; and amastigote nests were analysed.

Table 2.

Numerical criteria for the classification of splenic parameters according to the type found during histopathological analysis

Lymphoid parameters (spleen) Decreased Increased
White pulp 1‐2 2.1‐3
Red pulp 1‐2 2.1‐3
−1 to 0 0.1‐1
Lymphoid follicles Disorganized Organized
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The liver analyses involved the general structure, hepatocyte morphology, necrosis, lipidosis (Table 3), inflammatory infiltrate (Table 1), dystrophic calcification and presence of amastigote nests.

Table 3.

Numerical criteria for the classification of hepatic parameters according to the histopathological measures

Hepatic parameters (Liver) Intensity
Absent (0%) Mild (1%‐33%) Moderate (33%‐66%) Intense (>66%)
Lipidosis −1 0 1 2
Necrosis −1 0 1 2
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In the kidneys, the presence of Bowman's capsule space, the proximal and distal ducts, circulatory alteration (haemorrhage), dystrophic calcification and amastigote nests were analysed.

In the brain, Purkinje cells, circulatory alteration (haemorrhage), dystrophic calcification and amastigote nests were analysed.

The skeletal muscle analyses involved the general structure of the muscle cells, inflammatory infiltrate (Table 1), dystrophic calcification and amastigote nests.

2.8. Cellular damage markers

The activities of alanine (ALT) and aspartate (AST) aminotransferases at the end of the treatment followed by immunosuppression were determined in BALB/c mice by using commercially available assay kits (Labtest®, Lagoa Santa, MG, Brazil). The data are expressed as average ± SD.

2.9. Statistical analysis

For statistical analysis, the GraphPad Prism Software® (La Jolla, CA, USA) statistical program 6.0 was used by applying analysis of variance (P < 0.05, P < 0.001) by Tukey multiple comparisons and by ANOVA parametric test and performed using analysis of the Kruskal‐wallis nonparametric test (P < 0.05).

3. RESULTS

3.1. NFOH does not cause mortality during the treatment

The acute phase showed 25% of mortality. By the end of the immunosuppressive phase, one animal from each treated group died (NFOH and BZN), due to alpha male attacks.46, 47, 48, 49, 50 No animal died during the treatment phase.

3.2. NFOH is able to prevent parasitaemia recrudescence

The parasitaemia was accomplished, as shown in Figure 3. After immunosuppression, the highlighted area details the reactivation of parasitaemia. Parasitaemia reactivation was found in the nontreated group (POS), which demonstrated the efficacy of immunosuppression to induce the parasitaemia during the indeterminate form of chronic Chagas disease in mice. Both NFOH and BZN groups did not exhibit parasitaemic reactivation (Figure 3).

Figure 3.

Figure 3

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Trypanosoma cruzi (Y strain) parasitaemia curves from infected isogenic BALB/c mice (POS and NFOH and BZN treatments), expressed as logarithmic mean (Mlog). *Statistically different of NFOH, BZN and NEG groups (P < 0.0001) [Colour figure can be viewed at wileyonlinelibrary.com]

3.3. NFOH is able to prevent the formation of splenomegaly, mega‐kidney and mega‐gallbladder (without mega‐stone) compared to POS

Organ relative weight was another parameter evaluated in order to verify the health of the animals. The mean of relative weights of organs, including the heart, brain, liver, kidney and spleen, is shown in Figure 4. The POS group had increased weight of the brain, liver, kidney and spleen compared with the NEG group. The NFOH group exhibited low weights of organs, such as the kidney and spleen, when compared to the POS and BZN groups. The BZN group showed an increased weight of the brain, kidney and spleen compared with the NEG group. In addition, this group exhibited a decreased liver weight, compared with the NFOH group, and a reduced spleen weight when compared to the POS group (Tables S1‐S8).

Figure 4.

Figure 4

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Relative weight of the organs/tissue (spleen, kidneys, heart, brain and liver). (A) POS, (B) NFOH, (C) BZN and (D) NEG. *Statistically significant in the NEG; **Statistically significant in the NFOH; ***Statistically significant in the BZN (P < 0.05); (a) spleen photograph (Tables S1‐S8) [Colour figure can be viewed at wileyonlinelibrary.com]

Macroscopic analyses have shown a statistical difference in the gallbladder size. The NEG group exhibited smaller gallbladders compared with the POS and BZN groups. The POS group showed enhanced gallbladders in 22.2% and mega‐gallbladder in 44.4% of animals. For the BZN group, enhanced gallbladders and mega‐gallbladder were observed in 12.5% of animals. In the NFOH group, no alterations of this organ were observed, being 100% normal gallbladder; therefore, it is statistically different from the group POS (Table 4).

Table 4.

Macroscopic analysis of the gallbladder (size) expressed in percentage (%) of animals per group

Groups Gallbladder size (macroscopic analysis)
Normal (+, <1 mm) Enhanced (++, 1‐3 mm) Mega‐gallbladdera (+++, >3 mm)
POS 33.3% 22.2% 44.4%
NFOH 100% 0% 0%
BZN 75% 12.5% 12.5%
NEG 100% 0% 0%
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a

Presence of mega‐stones in gallbladder.

3.4. NFOH reduced intensity of tissue parasitism and inflammation compared to POS

The histopathological analyses were performed, and the results are shown in Figures 5 and 6. All infected animals (POS, BZN and NFOH) showed cardiac hypertrophy and mild and chronic inflammatory infiltrates that were statistically different from the NEG group. There was no statistically significant difference between the groups infected in relation to these cardiac parameters. Dystrophic calcification was found in the cardiac tissue of 44.4% of animals in the POS group, 25% in the NFOH group and 12.5% in the BZN group, but it was absent in the animals of the NEG group. In addition, the presence of amastigote nests was found in 55.5% of the animals in the POS group and 50% in the BZN group, but none were recorded in the NFOH group (Figures 5 and 7).

Figure 5.

Figure 5

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Histopathological analysis of the organs (H&E). A, Percentage of animals/organs with amastigote nests; B, percentage of animals/organs with dystrophic calcification. *Statistically different of NEG; **Statistically different of NFOH; ***Statistically different of BZN (P < 0.05)

Figure 6.

Figure 6

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Histopathological analysis of the organs (H&E). A, Percentage of animals with disorganization of lymphoid tissues; B, quantification of inflammatory infiltrates in hepatic tissue (according to Tables 1, 2, 3). *Statistically different of POS (P < 0.05); **Statistically significant in the NEG; ***Statistically significant in the NFOH (P < 0.05)

Figure 7.

Figure 7

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Photograph of the heart (I‐II), skeletal muscle (H&E, 40 X) and colon tissues (H&E, 10X). Heart: I—POS and BZN presence of amastigote nest (small arrow), NFOH and NEG: healthy cardiac muscle cells; II—dystrophic calcification (large arrow) presence in the POS. The BZN, NFOH and NEG: healthy cardiac muscle cells. Skeletal muscle: POS presence of amastigote nest (small arrow) and dystrophic calcification region (large arrow), NFOH, BZN and NEG: normal tissue. Colon: POS presence of amastigote nest (small arrow) and hypertrophy of the muscle layer (large arrow), NFOH, BZN and NEG normal colon [Colour figure can be viewed at wileyonlinelibrary.com]

In the colon, amastigotes were found only in the POS group. The presence of amastigote nests was found around the myenteric plexus, and focal inflammatory infiltrates were recorded in the POS group in 22.2% of the animals. Amastigotes and inflammatory infiltrates were absent in the NFOH, BZN and NEG groups (Figures 5 and 7).

The analysis of skeletal muscle identified amastigote nests and dystrophic calcification in all animals of the POS group. The treatment with NFOH and BZN groups showed 87.5% and 100% of animals with dystrophic calcification respectively. However, amastigote nests were not found in these groups (Figures 5 and 7).

For the liver, all groups showed normal hepatocytes and normal sinusoid spaces. Focal necrotic regions were identified, mainly in the POS and BZN groups, but there was no statistical difference between the infected groups. The presence of acute, chronic and mixed inflammatory infiltrates was observed in the POS group, and this was statistically increased, compared with the BZN and NFOH groups. The presence of amastigote nests was verified in 33.3% of animals in the POS group and 12.5% in the BZN group. There were no amastigote nests found in the NFOH group (Figures 6 and 8).

Figure 8.

Figure 8

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Photograph of the spleen and liver tissues (H&E, 10×). Spleen: POS and BZN lymphoid follicles unorganized, NFOH and NEG lymphoid follicles organized (small arrow); liver: POS and BZN presence of inflammatory infiltrates (larger arrow) and necrosis areas (small arrow), NFOH and NEG healthy hepatocytes [Colour figure can be viewed at wileyonlinelibrary.com]

A disorganized lymphoid follicle in the POS group was found in all animals during splenic tissue analysis. The BZN group showed that 50% of the animals had disorganized lymphoid tissue. In addition, organized lymphoid follicles were found for both the NEG and NFOH groups (Figures 6 and 8). Kidney analysis showed normal proximal, distal ducts and glomerular structure, and a focal region was identified containing inflammatory infiltrates (acute and mild) in all infected groups. The brain analysis identified one animal in the POS group exhibiting dystrophic calcification. The BZN and NFOH groups did not show dystrophic calcification. Atrophy of the granular layer was observed in all infected groups. However, the data analysis did not show statistical differences among the groups.

3.5. NFOH during long period of treatment does not cause increased serum alanine to aspartate aminotransferase (ALT/AST) levels

The BALB/c mice received an oral dose of NFOH (150 mg/kg) and BZN (60 mg/kg) during 60 days of treatment to determine the liver toxicity of antiparasite drugs. All groups showed normal AST levels. NFOH group, as well as BZN group, does not cause increase in AST levels when compared to POS respectively (Figure 9).

Figure 9.

Figure 9

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Toxicity evaluation in BALB/c mice after treatment an oral dose of 150 mg/kg/d of NFOH, 60 mg/kg/d of BZN. Serum levels of aspartate aminotransferase (AST) (A) and alanine aminotransferase (ALT) (B). The data are expressed as average ± SD. *Statistically significant difference with the infected animals (POS; P < 0.05)

4. DISCUSSION

The current drugs in the global market for the treatment of Chagas diseases have been used for more than 40 years. Their common problem is inefficacy during indeterminate and chronic phases of the disease, which culminates with the appearance of the latent form of the parasite.51, 52, 53, 54, 55 Silva and Nussenzweig56 isolated the Y strain (T. cruzi) from an acute human case reported in the city of Marília, São Paulo State, Brazil. Because of its high virulence (type II) and resistance to the current drugs, most of the animals died in the experiments during the acute phase.56, 57, 58, 59, 60, 61

According to the literature, the pre‐manifestation period occurs before the acute phase, and it lasts until the 7th day postinfection (dpi). The parasitaemia peaks after inoculation of the Y strain (T. cruzi) and occurs in 21‐28 dpi. The acute phase is considered to be under 30 dpi, indeterminate form (latent period) between 30 and 90 dpi and chronic phase at more than 120 dpi.62, 63, 64, 65

The immunosuppression can be reactivated and induced by drugs for animals and humans with a T. cruzi infection, after transplantation or even in association with autoimmune diseases, such as AIDS.10, 66, 67, 68 According to Pereira et al,40 dexamethasone can be used to induce reactivation of disease in animals, and Andrade et al69 reported 6%‐25% of deaths after immunosuppression of animals.

In this study, we inoculated 102 cells of trypomastigotes forms. We noticed that 25% of infected animals died during the acute phase, as expected. We used a dose of 60 mg/kg/d for the BZN group and 150 mg/kg/d for the NFOH group, following a previous report from Davies et al,22 to guarantee the maximal number of surviving animals up until the end of the procedure, after the immunosuppression stage.22, 24 In animal models, the BZN concentration in the chronic phase of Chagas disease, is directly related to side effects; this is favourable to use lower concentration in order to increase the period of treatment.70

The treatment started after 7 weeks of infection during the period of subacute parasitaemia. The period of drug treatment was performed according to previous procedures described in the literature.22, 24, 35 For the reactivation of the parasitaemia, 5 mg/kg of dexamethasone was administered for 14 days.40 The results showed that duration of 60 days of treatment using both drugs (BZN and NFOH) could inhibit the parasite growth even after immunosuppression.

Histopathological analysis of hearts from patients with chronic Chagas cardiomyopathy reveals a diffuse infiltrating inflammatory, hypertrophy, fibrosis and myocarditis with scarce tissue parasitism.71, 72, 73 In the previous literature, the immunosuppression during the chronic phase of Chagas disease (Y strain) caused development of myocardial lesions in mice.69 In our study, 100% of the infected animals were found to have cardiac hypertrophy and inflammatory infiltrates that were statistically different from the NEG group. Amastigote nests were found in 55.5% of cardiac tissue for the POS group and 50% for the BZN group. Interestingly, amastigote nests were not observed in NFOH and NEG groups.

The dystrophic calcification is a physiological condition that appears after necrosis, and occurs in either the acute or the chronic phase of infection.74, 75, 76, 77 However, heart sizes were not significantly different across all groups. Herein, we have observed the appearance of this dystrophic calcification in cardiac muscle tissues in 25% of animals treated with NFOH and 12.5% of animals from the BZN group, while for the POS group, it was identified in 44.4% of animals. These data suggest that all infected animals had heart necrosis that was most likely promoted by the amastigote infection and that the treatment with the NFOH was more effective than with the BZN (even though this group had less dystrophic calcification, it was found in 50% of animals with amastigote nests).

In histological analysis of skeletal tissues, amastigote nests and dystrophic calcifications were found in 100% of the POS animals. In the NFOH and BZN groups, we observed the appearance of this dystrophic calcification in 87.5% and 100% respectively. No amastigote nests were found after treatments.

In the liver, inflammatory infiltrate was observed with the presence of amastigote nests, focal regions of inflammatory infiltrate and necrosis in 33.3% of animals of the POS group and 12.5% of the BZN group. However, through observation, we saw that the area of necrosis was greater in the POS group than in the BZN group. The treatment with NFOH showed similar tissue profiles as the negative group. These results are in accordance with the results of Davies et al,24 who studied the in vivo hepatotoxicity using the NFOH and BZN at identical concentrations (150 mg/kg/d) during short‐term (ST: 21 days) and long‐term treatments (LT: 60 days). These authors have reported that the NFOH effect on the liver causes lower quantities of inflammatory infiltrates than in the BZN group in both periods (ST and LT). The same is observed for liver fibrotic remodelling during ST.24

Moreover, the NFOH treatment (150 mg/kg/d) did not cause liver injury in T. cruzi‐infected BALB/c mice, as demonstrated by levels of the ALT/AST assay when compared to reported references values.24 On the other hand, the NFOH group showed lower levels of AST compared to POS. It is interesting to note that biomarkers of hepatic levels (ALT/AST) are proportional to the increased liver damage (histopathological analysis) and can be attributed to T. cruzi tropism in this organ.

In the colonic tissue, amastigote nests were found in 22.2% of animals, only in the POS group. Ganglioneuritis in organs such as the colon, oesophagus and stomach has been reported,78 but it was not observed in this work. However, a significant increase in relative weight was observed in the spleen, liver and kidney. In fact, the infection enlarges secondary organs such as the spleen, liver, kidneys, gallbladder, pancreas, stomach, duodenum and jejunum,44, 79, 80 by destruction of the enteric nervous system (ESN) neurons which are present throughout the gastrointestinal tract. Once the ESN is dependent on the parasympathetic functions of the autonomic nervous system (ASN), the destruction of ESN alters the secretion regulations, motility, blood flow and immunological control of gastrointestinal organs.79, 81, 82 The parasympathetic denervation of the digestive tract and the consequent morphological and functional alterations have been extensively described.83, 84, 85, 86 The involvement of the glands and secondary organs of the digestive tract in Chagas disease has not been studied much.81, 82, 87 When ESN is destroyed, the bile secretion is blocked, increasing the gallbladder size.79, 81, 82 In our experiments, macroscopic analysis of the gallbladder revealed that no treated animal belonging to the NFOH group had an increase to this specific organ. On the other hand, an increase was observed in 66.6% of the POS and 25% of the BZN animals, suggesting the possible action of NFOH in intracellular parasites of T. cruzi and its ability to prevent the destruction of the myenteric plexus of this organ.

The congestive splenomegaly is a haemodynamic change caused from congestive heart failure in Chagas disease and is characterized by hyperplasic cells and disorganization of the spleen lymphoid follicles. The alteration of the lymphoid follicles also indicates an ongoing inflammatory process.45, 88, 89 Our results showed splenomegaly with a disorganization of lymphoid follicles in all the POS animals and 50% of the BZN animals. For the NFOH group, a reversible effect, a normal morphologic condition, was observed, suggesting a haemodynamic heart condition recovery and decrease in the inflammation process in 100% of the animals.

The BZN showed limited effectiveness in the disease. Several drawbacks, including its high toxicity, inability to eliminate amastigote forms present in tissues and absence of a protective effect in the tissues caused by the chronic stage of the disease, are still serious limitations that justify the discovery of new drugs.4, 7, 8, 9, 10, 12, 35, 90, 91 Even after more than 40 years since its discovery, there is not an efficient and safe treatment for chronic phase lesions. In some countries such as Brazil, BZN is the only drug available.

In this work, we describe a new alternative drug candidate for the indeterminate form of Chagas disease which is capable of preventing the reactivation of parasitaemia after immunosuppression, reducing lesions, and decreasing parasite infiltrates on organs/tissues. Moreover, NFOH has presented easy synthetic preparation, low cost and high yield.

5. CONCLUSION

We have succeeded in inducing the experimental indeterminate form of chronic Chagas disease, with reactivation of parasitaemia only in the POS group after immunosuppression. The histopathological studies demonstrate that animals without treatment (POS) showed the most severe parasite infiltrates in organs such as the heart, skeletal muscle, colon and liver tissue. The BZN treatment showed the presence of heart and liver amastigote infiltrates. The NFOH treatment removed amastigotes from all tissues studied; being intact fixed organism, these parasites were carefully analysed, as well as seen directly in situ. Furthermore, the gallbladder of animals treated with NFOH exhibited similar profiles to noninfected animals. Concerning inflammatory infiltrates of the liver, we identified the following order: POS > BZN ≅ NFOH > NEG. In this model, NFOH was not able to increase the levels of liver biomarkers. These results suggest that the NFOH is a promising drug candidate and alternative to treat Chagas disease.

AUTHORS’ CONTRIBUTIONS

All authors contributed equally to the manuscript.

Supporting information

 

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ACKNOWLEDGEMENTS

The authors would like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Proc. 2014/14980‐0, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Proc. 312404/2013‐1, Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP (PADC/FCF‐UNESP) and Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES) for research fellowships and financial assistance. Also, the authors are grateful for the technical assistance of Isabel Martinez for the T. cruzi inoculation and to Rafael Consolin Chelucci and Diego Eidy Chiba who kindly helped in the euthanasia of the animals.

Scarim CB, de Andrade CR, da Rosa JA, dos Santos JL, Chin CM. Hydroxymethylnitrofurazone treatment in indeterminate form of chronic Chagas disease: Reduced intensity of tissue parasitism and inflammation—A histopathological study. Int J Exp Path. 2018;99:236–248. 10.1111/iep.12289

Contributor Information

Cauê B. Scarim, Email: cauebenitos@gmail.com.

Chung M. Chin, Email: chung@fcfar.unesp.br

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