A.C.S. Bombaça et al.
Biomedicine & Pharmacotherapy 135 (2021) 111186
ˆ
non-infected male Swiss mice (5–6 weeks) were obtained as previously
Eletronica Rudolf Barth (Instituto Oswaldo Cruz, Fiocruz).
described [32]. This work is in accordance with the guidelines of the
´
˜
Colegio Brasileiro de Experimentaçao Animal (COBEA) and was per-
formed in biosafety conditions. All animal procedures were reviewed
and approved by the Fiocruz Committee of Ethics in Animal Research
(L-005/2017) according to resolution 196/96 of the National Health
Council of Brazilian Ministry of Health.
2.6. Flow cytometry analysis
Epimastigotes (5 × 106 parasites/mL) were treated with 1.2 and 2.4
μ
M N4 for 24 h, washed with PBS, and labeled with 32 ng/mL rhoda-
mine 123 (Rh123) and 10 μg/mL propidium iodide (PI) (Sigma-Aldrich)
for 30 min to assess mitochondrial membrane potential (ΔΨm) and
2.3. Direct effect analysis
plasma membrane integrity, respectively. As positive controls, parasites
were preincubated with 10 μM carbonyl cyanide 4-(trifluoromethoxy)
A solution (100
μ
L) of bloodstream trypomastigotes (1 × 107 para-
phenylhydrazone (FCCP, Sigma-Aldrich) or 0.1 % saponin (Sigma-
Aldrich) for 30 min at 28 ◦C. All Rh123 data were normalized by the
addition of FCCP at each experimental condition. Rh123 fluorescence
was monitored by variation index (VI), which corresponds to the
equation (MT - MC)/MC, with MT being the fluorescence median of
treated parasites and MC the fluorescence median of untreated parasites.
Negative VI values represent the mitochondrial membrane depolariza-
tion. All data were acquired with a FACSCalibur flow cytometer (Becton
Dickinson, Franklin Lakes, USA) equipped with Cell Quest software
(Joseph Trotter, Scripps Research Institute, La Jolla, USA), totaling
10,000 events in the region previously established to correspond to
epimastigotes. The analyses were performed using the Summit 6.1
software (Beckman Coulter, Brea, USA).
sites/mL) resuspended in RPMI medium (Sigma-Aldrich, St. Louis, USA)
containing 10 % FBS and 10 % mouse blood was added to the same
volume of N4 at twice the desired final dose and incubated in 96-well
microplates (Nunc Inc., Rochester, USA) at 4 ◦C for 24 h. Alternatively,
the assays were also performed in the absence of blood at 37 ◦C. The
experiments with epimastigotes were performed in LIT at 28 ◦C for 24 h.
After the incubation time, the quantification was performed using the
Neubauer chamber, and the activity of the compounds was expressed as
IC50/24 h, corresponding to the concentration that leads to 50 % para-
site lysis. An N4 stock solution was prepared in dimethylsulfoxide
(DMSO, Merck, Darmstadt, Germany), and the solvent final concentra-
tion never exceeded 0.1 %, with no deleterious effect on T. cruzi [33].
2.4. Effect on amastigotes and mammalian toxicity analysis
2.7. High resolution oxygraphy and ATP production analysis
Peritoneal macrophages (3 × 105 cells/well) plated in 24-well plates
(Nunc Inc.) were infected with bloodstream trypomastigotes (10 para-
sites/host cell) for 24 h. Then, the cultures were washed in phosphate
buffered saline (PBS, pH 7.4, Sigma-Aldrich) to remove non-internalized
Epimastigotes (1 × 107 parasites/mL) were treated with 1.2 and 2.4
μ
M N4 for 24 h and washed with PBS. Then, 5 × 107 parasites were
resuspended in 2 mL of respiration buffer (125 mM sucrose, 5 mM suc-
cinate, 65 mM potassium chloride, 10 mM Tris-HCl pH 7.2, 1 mM mag-
nesium chloride, and 2.5 mM monobasic potassium phosphate) to
evaluate the O2 flux and concentration. The analysis was made in a high
resolution respirometry 2 K (Oroboros Instruments, Innsbruck, Austria)
at 28 ◦C and under continuous stirring. The data were acquired by
parasites, and N4 (0.5–2.0 μ
M) was added for 24 h at 37 ◦C. After this
period, the cells were fixed and stained with Panotic dye kit (Laborclin,
Rio de Janeiro, Brazil), and the parameters of percentage of infection,
parasites per cell and infection index (percentage of infected host cells
multiplied by the number of parasites per cell) were calculated after the
quantification of 300 cells per coverslip using Zeiss Axioplan microscope
(Oberkochen, Germany) [34].
DatLab 5.1 software, and the addition of 2 μM antimycin A (AA) was
used to assess residual oxygen consumption (ROX), as previously re-
ported [30]. For ATP analysis, 1 × 108 parasites were resuspended in
In parallel, uninfected macrophages (5 × 104 cells/well) were
employed to evaluate the toxicity of N4 to the host cells. After 24 h
100 μL PBS, added to the same volume of luminescent reagent
CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison,
USA) in 96-well microplates (Nunc Inc.) and incubated for 15 min at
room temperature. The bioluminescence was measured according to
manufacturer’s instructions in a SpectraMax M3 Microplate Reader
(Molecular Devices) and a standard curve was made with ATP.
treatment at 37 ◦C, 10
μL PrestoBlue (Invitrogen, Carlsbad, USA) was
added to the cells (final concentration of 10 %) for 2 h, and the data
acquired at 560 and 590 nm in SpectraMax M3 Microplate Reader
(Molecular Devices, San Jose, USA). These results were expressed as
LC50/24 h, which corresponds to the concentration that leads to a
damage of 50 % in macrophages, and SI was calculated by the ratio of
LC50/24 h (uninfected macrophages) and IC50/24 h (infection index
parameter).
2.8. Biochemical analysis
Epimastigotes (1 × 107 parasites/mL) were treated with 1.2 and 2.4
μ
M N4 for 2, 4 and 24 h, washed with PBS and thawed. After that, the
2.5. Ultrastructural analysis
protozoa were disrupted by sonication, as previously described [35].
Protein concentration was determined using the Pierce™ BCA Protein
Assay Kit (Thermo Fisher, Waltham, EUA) in the soluble fraction (16,
000 g for 10 min). Alternatively, epimastigotes were pre-treated with
10 nM MitoTEMPO (Santa Cruz Biotechnology, Dallas, Texas, USA) for
30 min in the same conditions previously described and treated for
Epimastigotes (5 × 107 parasites/mL) were treated with 1.2 and 2.4
μ
M N4 in LIT medium at 28 ◦C for 24 h. After this period, control and
treated parasites were fixed with 2.5 % glutaraldehyde (Sigma-Aldrich)
diluted in 0.1 M Na-cacodylate buffer (pH 7.2) for 40 min and post fixed
with 1 % osmium tetroxide (Sigma-Aldrich) diluted in the same buffer,
adding 2.5 mM calcium chloride and 0.8 % potassium ferricyanide for
20 min, both at 25 ◦C. After the washings, dehydration was performed in
acetone (50, 70, 90 and 100 %) before embedding in PolyBed 812
(Polysciences Inc., Warrington, USA) resin for 72 h. Ultrathin sections
were obtained and stained with uranyl acetate and lead citrate for the
examination with a JEM1011 transmission electron microscope (Jeol,
Tokyo, Japan). Alternatively, fixed epimastigotes were dehydrated in
ethanol (50, 70, 90 and 100 %), dried by the critical point method with
CO2, mounted on aluminum stubs, coated with a 20-nm-thick gold layer,
and analyzed with a Jeol JSM6390LV scanning electron microscope.
Both electron microscopes are located in Plataforma de Microscopia
another 2 or 4 h with 1.2 and 2.4 μM N4. The AA-sensitive succinate:
cytochrome c oxidoreductase activity (complexes II–III) is based in fer-
rocytochrome c reduction at 550 nm (
ε
=19 mMꢀ 1 cmꢀ 1) and was
measured in a reaction mixture consisted of 100 mM potassium phos-
phate buffer (pH 7.4), 150 μM bovine heart cytochrome c, 3 mM succinic
acid and 1 mM KCN. The measurement of KCN-sensitive cytochrome c
oxidase (complex IV) activity is based on the ferrocytochrome c oxida-
tion at 550 nm (
ε
=19 mMꢀ 1 cmꢀ 1) and the reaction mixture consisted of
100 mM potassium phosphate buffer pH 7.4 and 50 μM sodium
dithionite reduced equine heart cytochrome c. In both assays, the re-
actions started with the addition of 0.5 mg/mL, and specific controls
were made with 2 μM AA or 1 mM KCN to complexes II-III and IV,
3