Received: 30 December 2023 | Revised: 26 April 2024 | Accepted: 30 April 2024

DOI: 10.1111/mmi.15279

RESEARCH ARTICLE

The effect of nutritional and oxidative stress on the

metabolome of Trypanosoma cruzi
1 2 1 3
Michel Augusto Silva | Mario Augusto Izidoro | Mirella Aricó | Luiz Juliano |
Sergio Schenkman 1

1
Department of Microbiology,
Immunology and Parasitology, Escola Abstract
Paulista de Medicina, Universidade
Trypanosoma cruzi, a flagellated protozoan, is the causative agent of Chagas disease.
Federal de São Paulo, São Paulo, Brazil
2
Hospital São Paulo, Brazil
The parasite has developed various mechanisms to get through its intricate life cycle
3
Department of Biophysics, Escola and adapt to different evolutionary phases. T. cruzi proliferates in the insect vector's
Paulista de Medicina, Universidade digestive tract as an epimastigote form, encountering fluctuating nutrient availability
Federal de São Paulo, São Paulo, Brazil
and oxidative stress caused by the digestion of red blood cells from the mammalian
Correspondence host blood meal. To unravel how the parasite's metabolism adapts to these changing
Sergio Schenkman, Department
of Microbiology, Immunology and conditions, we conducted an analysis of the chemical species present in epimastigote
Parasitology, Universidade Federal de forms. This involved comparing cultured parasites with those subjected to nutritional
São Paulo, R. Pedro de Toledo 669 L6A,
04039-­032, São Paulo, SP, Brazil. deficiency or oxidative stress using untargeted metabolomics. We looked at 21 sam-
Email: [email protected] ples: seven biological copies of parasites that were actively growing, seven samples
Funding information that were put in a medium without nutrients for 3 h, and seven samples that were
Coordenação de Aperfeiçoamento treated with glucose oxidase for 30 min to make H2O2 continuously. Importantly, in all
de Pessoal de Nível Superior, Grant/
Award Number: 88887.900282/2023-­ conditions, parasite viability was maintained when the samples were collected. Upon
00; Fundação de Amparo à Pesquisa nutrient removal, we observed a substantial decrease in amino acids and carbohy-
do Estado de São Paulo, Grant/Award
Number: 2020/07870-­4, 2021/12515-­ drate metabolites, accompanied by the accumulation of fatty acids and steroids, with
1 and 2021/12515-­1 CEPID-­ARIES; the predominance of inositol and sphingolipid metabolism, along with a simultaneous
Conselho Nacional de Desenvolvimento
Científico e Tecnológico, Grant/Award decrease in the levels of H2O2. In the presence of H2O2, a significant rise in compo-
Number: 303788/2020-­8 and INCTV-­ nents of the pentose pathway and specific amino acids such as methionine and serine
CNPq
occurred, along with pathways related to an increase in antioxidant species metabo-
lism such as ribulose 5-­phosphate and glyceric acid. Conversely, fatty acid and steroid
levels decrease. We found no common increase in metabolites or lipids. In contrast,
eight species (succinic acid, glutamic acid, valine, 2-­hydroxyisocaproic acid, alanine,
indolelactic acid, proline, and lanosterol) were consumed under both stresses. These
findings underscore the rapid and distinct enrichment responses in amino acids, lipids,
and carbohydrates required to cope with each different environmental condition. We
concluded that T. cruzi presents a flexible metabolism that rapidly adapts to variable
changes in the environment.

KEYWORDS
growth, metabolomic, nutritional stress, oxidative stress, pentose phosphate pathway,
Trypanosoma cruzi

704 | © 2024 John Wiley & Sons Ltd. wileyonlinelibrary.com/journal/mmi Molecular Microbiology. 2024;122:704–719.
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SILVA et al. 705

1 | I NTRO D U C TI O N and succinate. Pyruvate is then used in the mitochondria that, de-
pending on the parasite stage, leads to alanine and acetate (Michels
Trypanosoma cruzi is a flagellar-­bearing protozoan that causes Chagas' et al., 2021). Within these structures, in addition to glycolysis,
disease, a tropical and neglected tropical disease that distresses other metabolic processes occur, including the pentose phosphate
about 7 million people around the world, most of them in South pathways (Maugeri et al., 2011; Michels et al., 2021). Furthermore,
and Central America, though hundreds of cases have been found for efficient proliferation, the parasite requires polyamines (Saye
in North America, Asia, and Europe. The parasite has a complex life et al., 2019), purines (Berens et al., 1981), and heme (Nogueira
cycle that uses insect vectors to infect several mammalian species et al., 2017; Salzman et al., 1982).
including humans through its feces and urine through exposed mu- Oxidative molecules may trigger dynamic changes and reactions
cosae or by oral transmission (Yoshida, 2008). However, parasites in the metabolic homeostasis of T. cruzi. For example, the absence
can also be transmitted by congenital contact, blood transfusion, or of heme results in the accumulation of hydrogen peroxide and in-
organ transplants (Echeverria & Morillo, 2019). In the insect, vector, creased cytosolic and mitochondrial ROS, along with compro-
the parasite develops in the gut, whereas, in mammals, it invades mised ATP synthesis, essential for the proliferation of the parasite
host cells, forming a parasitophorous vacuole that is subsequently (Nogueira et al., 2017). Conversely, the parasite possesses robust
ruptured to then proliferate in the host cell's cytosol. After that, it detoxification machinery crucial for maintenance and adaptation,
differentiates in non-­proliferative but infective stages that circulate involving responses to ROS. This is based on the reducing capability
through the bloodstream. Therefore, T. cruzi encounters different of trypanothione, a unique molecule of the Kinetoplastids, formed
conditions that require fast metabolic and physiological adaptations. by the conjugation of two glutathione molecules, serving as a sub-
Among them are changes in nutrient availability and the presence of strate for the reductive activity of the trypanothione reductase
reactive oxygen species (ROS; Lander et al., 2021; Liu et al., 2021). (Fairlamb et al., 1985), a key enzyme in the antioxidant metabolism
The epimastigote form, which is the stage of T. cruzi that repli- (Jockers-­Scherubl et al., 1989). In addition, the redox potential re-
cates the insect vector midgut, undergoes differentiation into infec- lies on the combined activity of Fe2+ superoxide dismutases (Ismail
tive and non-­dividing trypomastigotes known as metacyclic forms. et al., 1997; Temperton et al., 1996), tryparedoxin peroxidases
This transformation occurs when the nutrients are removed either (Pineyro et al., 2011), ascorbate peroxidase (Wilkinson et al., 2002),
when growing in culture or when parasites reach the stationary and glutathione reductase (Wilkinson et al., 2000), as reviewed in
growth phase, mimicking what occurs in the final portions of the in- Piacenza et al. (Piacenza et al., 2013). These antioxidant enzymes
sect gut (Goncalves et al., 2018). It has been shown that in T. cruzi, play crucial roles in reducing protein disulfide bonds, DNA synthesis
nutrient availability at the stationary phase leads to large changes in and repair pathways, detoxifying ROS, regulating the cell cycle and
the metabolic profile (Barison et al., 2017; De-­Simone et al., 2022). differentiation, and responding to stress induced by the extracellular
The parasite ceases the utilization of carbohydrates and shifts to environment, indicating robust antioxidant activity.
using lipids and amino acids for ATP production. These adaptations In this study, we employed untargeted metabolomic analysis em-
correlate with changes in the parasite morphology and expression of ploying a gas chromatography coupled to mass spectrometry (GC–
different set of proteins facilitating adhesion and invasion of mam- MS) system. This allowed us to gather qualitative and comparative
malian cells (Amorim et al., 2017; Araya et al., 2008; Parodi-­Talice data regarding the existence of small molecules and metabolites in
et al., 2007). Additionally, as the insect acquires the parasite within the parasites. We conducted the analysis after subjecting the para-
the host blood, after the digestive process, it accumulates oxidized sites to short periods of incubation under nutrient depletion and in
products derived from red blood cells, such as hemin, creating a the presence of oxidative stress. Our results demonstrated that T.
harsh environment for the parasite (Nogueira et al., 2017). This re- cruzi quickly and distinctly responds to these two dissimilar situa-
sults in an oxidative environment that also contribute to changes tions, implying that it rapidly adjusts its metabolism to each environ-
in the metabolic profile, and several works have demonstrated mental circumstance.
the possible requirement of ROS for parasite differentiation (Dick
et al., 2021; Nogueira et al., 2015). However, there is limited knowl-
edge regarding nutrition depletion and how the increase in ROS lev- 2 | R E S U LT S A N D D I S CU S S I O N
els affects the parasite's metabolic profile.
Trypanosoma cruzi metabolism can rely on the consumption of 2.1 | Nutritional and oxidative stresses
various available energy sources. In insect growing forms, it primar- differentially inhibited parasite growth and viability
ily utilizes amino acids derived from the digestion of the blood meal
(Barison et al., 2016; Bringaud et al., 2006; Marchese et al., 2018). To obtain significant comparisons of the effect of nutrient depletion
Proline, an essential amino acid, plays a critical role in cell growth, and oxidative stress, it was necessary to carefully obtain parasite
survival, and differentiation processes (Mantilla et al., 2015). samples during the exponential growth phase under very similar
Additionally, the parasite has the capacity to use glucose, when conditions. We harvested parasites at a concentration of 1 × 107/
available, as an energy source. Glycolysis is compartmentalized in mL. In one set of experiments, cells were incubated for 3 h at the
organelles known as glycosomes, generating glycerol, pyruvate, same concentration in TAU media, which is devoid of carbon, lipids,
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706 SILVA et al.

and amino acids. In the other case, the parasites were incubated for 2.2 | Nutritional and oxidative stress did not affect
30 min with 6 U/mL of glucose oxidase (GOX) in regular LIT to contin- major protein levels at short incubation periods
uously generate H2O2. Respective controls were maintained for the
same periods in regular LIT medium. After these treatments, we col- We assessed initial variations in H2O2 compared to control para-
lected the samples for analysis as the parasite retained full viability sites in each case to confirm that the parasites responded to diverse
(Figure 1a,b, respectively). Following these incubations, TAU-­treated stressors. As anticipated, H2O2 significantly dropped in TAU, indi-
parasites remained fully motile and presented a typical epimastig- cating a lower metabolic rate. However, the parasites treated with
ote morphology (Figure S1). Subsequently, the cultures were cen- GOX generated an eightfold rise (Figure 2a). This could result from
trifuged, the parasites were washed, and 2 × 10 8 cells were used for oxidation of numerous cellular components to serve as substrates
metabolomic analysis. The rest of the sample was resuspended in for peroxidase during the H2O2 assay, or, eventually, to the increased
the regular media at 1 × 107/mL. Growth was fully inhibited in the metabolism to detoxify free H2O2 by endogenous reductase.
parasites subjected to nutritional stress (Figure 1c), resulting in a Next, we evaluated the protein levels in each case. In nutrient-­
25% decrease in cell viability after 24 h (Figure 1a). This condition poor conditions and in the presence of GOX, minimal changes in
was previously found to induce the transformation of epimastig- the total protein levels were observed, as indicated by Ponceau S
otes into infective metacyclic trypomastigotes (Contreras, Morel, (Figure 2b) and Coomassie blue staining (Figure S1b). The levels of
et al., 1985; Contreras, Salles, et al., 1985), as observed in the feces HSP70, highly expressed in proliferating epimastigotes and reduced
and urine of the insect vector. upon differentiation (Olson et al., 1994), remained constant, as de-
In the samples incubated with GOX for 30 min, the parasites were tected by immunoblot (Figure 2b). Additionally, the endoplasmic
centrifuged, washed in PBS, and resuspended in regular LIT. One reticulum chaperone (BIP or GRP78), which is normally unaffected
hour later, the parasites continued to proliferate, albeit slower than during growth and heat shock of T. cruzi cells (Tibbetts et al., 1994),
control cells, and started to show a decreased viability (Figure 1d). remained constant. Fructose 1,6-­biphosphate aldolase also ex-
Four h later, viability decreased, with most of the cells becoming hibited similar levels, further suggesting that both types of stress
round and disintegrating. As GOX induces continuous production of resulted in minimal changes in the majority of proteins, as previ-
H2O2 and may be present after centrifugation and washes (Khatami ously demonstrated by proteomic analysis (de Godoy et al., 2012).
et al., 2022), potential death occurs as H2O2 levels become toxic for Nonetheless, changes in various sets of protein kinases were fre-
the parasites upon extended incubation, as demonstrated previously quently seen following nutritional stress (Amorim et al., 2017),
(Finzi et al., 2004). Interestingly, we observed that both stressed par- implying that post-­translational protein modifications, such as phos-
asites exhibited more visible structures in their bodies, as shown by phorylation, acetylation, and methylation, among others, might have
differential interference contrast (Figure S1a). Stress has been asso- occurred, as shown before (de Godoy et al., 2012). A different set
ciated with parasite damage and the release of extracellular vesicles of modifications also occur during oxidative and ionization radiation
(Vasconcelos et al., 2021). stress damage (Finzi et al., 2004; Vieira et al., 2014).

F I G U R E 1 Effect of nutritional
and oxidative stress on the viability
and growth of Trypanosoma cruzi
epimastigotes. Exponentially growing
epimastigotes were collected and
resuspended to 1 × 107/mL either in
LIT with 10% FBS (complete LIT) or in
TAU (a) or just in complete LIT with or
without 0.6 U/mL of GOX (b). At the
indicated times at 28°C, the viability
was determined and expressed as the
percentage of live relative to death as
described in the “Methods” section.
Panels (c) and (d) show the respective
parasite density (n = 3). Asterisks indicate
statistically significant differences
(p < 0.05) using the Student t-­test.
Arrows indicate the time of collection for
metabolomic analysis.
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SILVA et al. 707

F I G U R E 2 The levels of H2O2 decrease

in parasites subjected to nutritional
stress and increase after incubation
with GOX without major changes in
the protein levels. (a) Trypanosoma cruzi
epimastigotes were incubated either
in complete LIT, TAU for 2 h, or 0.6 U/
mL of GOX. The levels of H2O2 in the
parasites were subsequently determined
using the Amplex reagent as described
in the “Methods” section. The values are
means of triplicates ± standard deviation.
Asterisks indicate significant differences
using a 2-­way ANOVA test (p < 0.05). (b)
The equivalent of 2 × 106 of the samples
above were loaded in each gel lane. After
electrophoresis, the membranes were
transferred to nitrocellulose membranes
and stained with Ponceau S, or incubated
with the indicated antibodies, further
revealed by using anti-­Rabbit IgG coupled
to IrDye 800.

2.3 | Assessment of metabolomic T. cruzi data (Figure S5c), to allow the analysis. Similar separation of the metab-
olites was obtained by the seven samples incubated for 30 min with
The analyses were made using parasite samples maintained in TAU GOX added to LIT in comparison with seven samples maintained for
and LIT in one experiment and in a second experiment in GOX and the same period in LIT (Figure S5d–f).
LIT. The fingerprint profiles of these samples generated a matrix
of 65 and 78 molecules, respectively, which were identified using
NIST 17 MASS (v.1.00.1) and GCMS Smart Metabolite (v.3.01), all 2.4 | Metabolic changes in epimastigotes
developed by Shimadzu Co. The identifications were confirmed by submitted to nutrient deprivation
the Human Metabolome Database (https://​hmdb.​ca/​). The multi-
variate analyses (MVA) were performed with quality control (QC) The multivariate analyses (MVA) allowed us to generate a compara-
samples, which were a mixture of paired samples processed and tive assessment of the molecules identified in each experiment. Two
injected in the GC–MS equipment. Both analyses showed a unique major clusters were observed in the heatmaps of the TAU/LIT ex-
cluster of these QC assembled in a group separated from the LIT and periment. One group includes 19 molecules that decreased in the
samples submitted to nutritional (Figure S2a) and oxidative stress parasites incubated in TAU compared to LIT (green in Figure 3a). The
(Figure S2b). The same results were obtained by a supervised mul- other cluster consisted of at least two separate groups. One group
tivariate analysis (PLS) for the TAU/LIT (Figure S3a,b) and GOX/LIT included molecules that increased in TAU (blue), while the other had
(Figure S4a,b), both showing a predictive Q2 value of 4 and 5 com- molecules with variable and indistinguishable levels between the
ponents with a performance close to 1.0, respectively. two conditions (red). Major differences could be seen by the Volcano
Our analysis also showed a clear separation of the seven LIT plot considering the medians using the t-­test analysis, revealing in-
samples from the six samples of parasites subjected to nutritional creased levels of inositol, glycolic acid, glycine, fatty acids, and cera-
stress (Figure S5a). One of the samples incubated in TAU showed a mide (Figure 3b and Table 1). This suggests that lipid metabolism
distinct pattern and was discarded in the analysis. The populations occurs preferentially in nutrient-­deprived cells. In contrast, succinic
were grouped separately (Component 1 = 66.2% and Component acid and alanine, major products of the carbohydrate metabolism
2 = 8.8%). The principal component analysis (PCA) was also clearly (Marchese et al., 2018), decreased. Additionally, the levels of sev-
distinguishable (Figure S5b) with sufficient T scores (56.6%) eral amino acids, also a source of energy, were reduced, indicating
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708 SILVA et al.

F I G U R E 3 (a) Heat map plot for LIT/

TAU built from the parametric Students
t-­test significant variables (p-­value < 0.05).
The plot used column raw data from
Table S1, without the QC samples, after
median normalization, log transformation
(base 10), and Pareto scaling with the
Metaboanalyst 6.0. Fifty-­eight significant
molecules (p < 0.05) were detected. The
heat map was generated by the Ward
clusterization and distance measurements
of Minkowsky. Green, red, and blue
colors were used to indicate the major
metabolic clusters. (b) Volcano Plot of
metabolites from the sample groups LIT/
TAU. The y-­axis shows the -­log10 (p-­value),
while the x-­axis presents log2 (fold change
(FC) of TAU minus LIT). Red, purple, and
gray circles represent the upregulated
metabolites, downregulated metabolites,
and non-­significant changes, respectively.
The vertical dashed lines represent FC
equals 1.5, and the horizontal dashed lines
represent the fold discovery rate (FDR) for
p < 0.05). The most significant molecules
are named.

that the cells may rely on lipid oxidation to generate energy. The (Figure 4a–c). By looking at the corresponding KEGG in the T. cruzi
increased role of lipids is also illustrated by performing a feature im- database, the data indicate the predominance of sphingolipid and
portance analysis (Figure S5), which included several fatty acids, as inositol, followed by glycerophospholipid metabolism (Figure 4c).
previously found in stationary phase parasites (Souza et al., 2021). These findings additionally support the notion that under nutritional
To further assess the predominant metabolic pathways after stress, lipid metabolism predominates in the parasites in a condition
incubation with TAU, we performed enriched analysis using both that causes growth arrest and differentiation. Indeed, a previous
the HMDB database of metabolites or lipids separately, employ- report demonstrated changes in the lipid anchors of major surface
ing the Metaboanalyst 6.0. Both showed a predominant linoleic glycoproteins from 1-­O-­hexadecyl-­2-­O-­hexadecanoylphosphatidyli
acid metabolism, followed by inositol and glycerolipid metabolism nositol in epimastigotes, to inositol phosphoceramides in metacyclic
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SILVA et al. 709

TA B L E 1 Statistical analysis of compound variation in LIT × TAUa.

Compound t stat p-­value LOG10(p) FDR

Inositol −13.854 2.62E-­0 8 7.58E+00 2.31E-­07

Linoleic acid −9.5248 1.20E-­06 5.92E+00 9.60E-­06
Glycolic acid −8.5748 3.36E-­06 5.47E+00 2.20E-­05
3-­Hydroxyisovaleric acid −8.5376 3.50E-­06 5.46E+00 2.20E-­05
Inositol phosphate −8.3668 4.25E-­06 5.37E+00 2.34E-­05
Phosphoric acid −8.3033 4.58E-­06 5.34E+00 2.37E-­05
Ergosterol −8.1985 5.17E-­06 5.29E+00 2.38E-­05
Tris(2,4-­di-­tert-­butylphenyl) phosphate −8.1798 5.28E-­06 5.28E+00 2.38E-­05
1,5-­Heptadiene-­3,4-­diol −7.9302 7.10E-­06 5.15E+00 2.84E-­05
Cholesterol −7.7786 8.52E-­06 5.07E+00 3.26E-­05
N-­Tetracosane −7.3548 1.44E-­05 4.84E+00 5.28E-­05
Tetracosanoic acid −7.2969 1.55E-­05 4.81E+00 5.45E-­05
Octadecanamide −7.0364 2.16E-­05 4.66E+00 7.33E-­05
cis-­9-­Hexadecenoic acid −6.4344 4.85E-­05 4.31E+00 1.48E-­0 4
Glycine −6.4308 4.87E-­05 4.31E+00 1.48E-­0 4
Docosanoic acid −6.3155 5.71E-­05 4.24E+00 1.68E-­0 4
(Z)6,(Z)9-­Pentadecadien-­1-­ol −6.108 7.65E-­05 4.12E+00 2.17E-­0 4
15-­Tetracosenoic acid −5.9222 9.99E-­05 4.00E+00 2.75E-­0 4
Pipecolic acid −5.7703 1.25E-­0 4 3.90E+00 3.32E-­0 4
Glycerol 3-­phosphate −5.5413 1.75E-­0 4 3.76E+00 4.53E-­0 4
Arachidic acid −5.4875 1.90E-­0 4 3.72E+00 4.77E-­0 4
L-­Lyxulose −5.3121 2.48E-­0 4 3.61E+00 6.06E-­0 4
O-­Phosphoethanolamine −5.0713 3.60E-­0 4 3.44E+00 8.33E-­0 4
Stearic acid −4.7188 6.31E-­0 4 3.20E+00 1.39E-­03
N-­Heneicosane −4.6694 6.83E-­0 4 3.17E+00 1.47E-­03
Docosahexaenoic acid −4.452 9.76E-­0 4 3.01E+00 2.04E-­03
Cer(D18:1/16:0) −4.3512 1.15E-­03 2.94E+00 2.36E-­03
MG(18:0/0:0) −4.1465 1.63E-­03 2.79E+00 3.25E-­03
MG(0:0/18:0) −4.0086 2.06E-­03 2.69E+00 4.02E-­03
Lactose −3.8403 2.74E-­03 2.56E+00 5.25E-­03
Uracil −3.8255 2.82E-­03 2.55E+00 5.27E-­03
Heptadecanenitrile −3.8004 2.94E-­03 2.53E+00 5.39E-­03
8,11,14-­Eicosatrienoic acid −3.6295 3.96E-­03 2.40E+00 7.07E-­03
MG(18:2/0:0) −3.6211 4.02E-­03 2.40E+00 7.07E-­03
Palmitaldehyde −3.5161 4.83E-­03 2.32E+00 8.03E-­03
Phosphonic acid −3.4657 5.28E-­03 2.28E+00 8.60E-­03
Palmitoleic acid −3.306 7.00E-­03 2.15E+00 1.12E-­02
Ribose 5-­phosphate −3.2607 7.59E-­03 2.12E+00 1.19E-­02
1-­Tricosene −2.8 1.73E-­02 1.76E+00 2.62E-­02
Palmitic acid −2.6489 2.26E-­02 1.65E+00 3.38E-­02
Niacinamide −2.6229 2.37E-­02 1.63E+00 3.43E-­02
1-­Monopalmitin −2.6209 2.38E-­02 1.62E+00 3.43E-­02
Oleic acid −2.532 2.79E-­02 1.55E+00 3.96E-­02
Tetradecanoic acid −2.4866 3.02E-­02 1.52E+00 4.22E-­02
5,8,11,14,17-­Eicosapentaenoic acid −2.4198 3.40E-­02 1.47E+00 4.68E-­02
LPA(18:1/0:0) −2.3937 3.56E-­02 1.45E+00 4.82E-­02
Lanosterol 3.0196 1.17E-­02 1.93E+00 1.80E-­02
(Continues)
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710 SILVA et al.

TA B L E 1 (Continued)

Compound t stat p-­value LOG10(p) FDR

Arabinofuranose 3.5154 4.84E-­03 2.32E+00 8.03E-­03

Ribitol 3.5657 4.43E-­03 2.35E+00 7.64E-­03
D-­Ribulose 5-­phosphate 4.838 5.21E-­0 4 3.28E+00 1.17E-­03
5-­Oxoproline 5.2724 2.63E-­0 4 3.58E+00 6.26E-­0 4
Fructose 6-­phosphate 6.6177 3.77E-­05 4.42E+00 1.23E-­0 4
L-­Methionine 8.0481 6.17E-­06 5.21E+00 2.59E-­05
Indolelactic acid 8.1582 5.42E-­06 5.27E+00 2.38E-­05
Citric acid 8.3986 4.10E-­06 5.39E+00 2.34E-­05
L-­Serine 9.2308 1.64E-­06 5.79E+00 1.20E-­05
L-­Alanine 15.032 1.11E-­0 8 7.95E+00 1.09E-­07
L-­Phenylalanine 17.282 2.55E-­09 8.59E+00 2.80E-­0 8
L-­Threonine 17.894 1.76E-­09 8.76E+00 2.21E-­0 8
Succinic acid 19.911 5.61E-­10 9.25E+00 8.22E-­09
L-­Valine 20.746 3.61E-­10 9.44E+00 6.35E-­09
L-­Glutamic acid 21.611 2.32E-­10 9.63E+00 5.11E-­09
L-­Leucine 21.667 2.26E-­10 9.65E+00 5.11E-­09
L-­Proline 29.198 8.95E-­12 1.10E+01 3.94E-­10
2-­Hydroxyisocaproic acid 32.082 3.21E-­12 1.15E+01 2.83E-­10
a
Two-­sample t-­tests & Wilcoxon rank-­sum test using a fold discovery rate (FDR) of unpaired samples for p < 0.05.

trypomastigotes (Acosta-­Serrano et al., 1995). Moreover, the use substantial reduction in succinate, which is produced by carbohy-
of lipids for energy production and changes in the membrane com- drate metabolism in the glycosome of T. cruzi (Michels et al., 2021).
position among different stages have been described in T. cruzi and A moderate decrease was also detected for amino acids such as
other Trypanosomatids, as recently reviewed (Booth & Smith, 2020; valine, glutamic acid, aspartic acid, and glycine, supporting the no-
Parreira de Aquino et al., 2021). These results suggest that inositol-­ tion of an attenuation of the energy production by amino acids.
phosphates contribute to the metabolic control in T. cruzi (Mantilla Similarly, some lipids and steroids are moderately decreased in
et al., 2021). GOX-­t reated cells, collectively indicating a significant variation as
evaluated by the feature importance analysis (Figure S7). Pathway
analysis using the KEGG of T. cruzi further confirmed an increased
2.5 | Metabolic changes in epimastigotes glyoxylate/dicarboxylate metabolism, in addition to the increased
submitted to oxidative stress levels of pentose phosphate pathway and sulfur metabolism in this
parasite (Figure 6a,b).
The comparative analysis of the molecules identified in parasites
incubated with GOX clearly showed three main clusters (Figure 5a).
The first cluster had two subgroups formed by metabolites con- 2.6 | Final remarks and conclusions
sistently increased in GOX-­t reated parasites, including glyceric
acid, ribulose, ribulose 5-­p hosphate, sedoheptulsose 7-­p hosphate, Our results disclosed rapid and distinct changes in the metabolic
and ribitol, as also demonstrated in the Volcano plot of t-­test me- profile of T. cruzi epimastigotes from the DM28c strain incubated
dians (Figure 5b and Table 2). The increased role of lipids is also il- in the absence of nutrients compared to parasites subjected to
lustrated by performing a feature importance analysis (Figure S6), oxidative stress. We found only 33 common metabolites in both
which included several fatty acids, as previously found in station- experimental settings (Figure 7a), without common increased
ary phase parasites (Souza et al., 2021). These results indicated molecules or similar pathways activated for these two types of
a clear increase in the pentose and glucoronate intermediates stress (Figure 7b). Therefore, distinct responses occur in these
from the pentose pathway, which is required for the production of two different conditions, suggesting a multifaceted capacity of
NADPH to regenerate oxidized molecules in general (Kruger & von T. cruzi epimastigotes to rapidly adapt their metabolism. While a
Schaewen, 2003). Increased levels of methionine and serine were lipid metabolism predominates upon nutrient depletion to gener-
also detected, serving as antioxidant defense through the pentose ate parasite energy, the pentose pathway is boosted, possibly pro-
pathway, a mechanism observed in several organisms (Campbell moting the generation of NADPH to cope with oxidative stress.
et al., 2016) as in T. cruzi (Igoillo-­E steve et al., 2007). In contrast, a In contrast, we detected eight commonly decreased metabolites,
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SILVA et al. 711

F I G U R E 4 Analysis of metabolites enriched in TAU groups in the comparative experiments of LIT versus TAU. The analysis was
performed against the 99 metabolite sets based on normal human metabolic pathways with metabolites (a) or lipids (b). Panel (c) shows the
scatter plot testing the pathway impact using the Trypanosoma cruzi KEGG database in the Metaboanalyst.

particularly molecules of the alanine, aspartate, and glutamate 3 | E X PE R I M E NTA L PRO C E D U R E S

metabolism, in the two different experiments (Figure 7c). This
suggests that the shutdown of these pathways is associated with 3.1 | Parasites and stress induction
a general stress response in T. cruzi, possibly signaling changes
in gene expression that will occur later if the parasite survives T. cruzi epimastigotes of the DM28c strain (Sturm et al., 2003) were
(Machado-­Silva et al., 2016; Parab & McCall, 2021). maintained in liver infusion tryptose medium (LIT; Camargo, 1964)
The rapid metabolic changes in insect proliferating forms containing 10% fetal bovine serum, 0.2% glucose, 10 μg/mL hemin,
are important to survive for long periods in the gut of the insect 0.5 and 0.1 μg/mL penicillin and streptomycin, respectively, at 28°C.
vector, which does not feed continuously. Moreover, it gives this Parasite numbers were determined by using a Neubauer chamber.
parasite the capability to colonize different blood-­sucking in- For nutritional stress, exponentially growing cultures (1 × 107 para-
sects. Although our experiments were observed with epimastig- sites/mL) were collected by centrifugation at 2000× g for 5 min,
otes maintained in culture, the rapid metabolic adaptation of the washed once with TAU solution (190 mM NaCl, KCl 17 mM, 2 mM
parasite might also occur in the mammalian forms of the parasite, CaCl2, 2 mM MgCl2, 4,1 mM NaHCO3, 47 μM NaH2PO 4, 15 mM
able to infect and proliferate in different types of cells, which can NaHPO 4, pH 6,2), resuspended in the same solution to the origi-
be taken into consideration during drug design to treat Chagas' nal parasite concentration and (TAU), and maintained at 28°C for
disease. Whether the same changes occur in all types of T. cruzi, 3 h as indicated. To induce oxidative stress, parasites in the expo-
defined as discrete typing units (Breniere et al., 2016), should be nential growth phase were centrifuged and resuspended in a com-
further examined in this respect. plete LIT medium containing 0.6 U/mL of glucose oxidase (GOX,
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712 SILVA et al.

F I G U R E 5 (a) Heat map plot for LIT/GOX built from the parametric Students t-­test significant variables (p-­value < 0.05). The plot used raw data
generated without the QC samples (Table S2), after median normalization, log transformation (base 10), and Pareto scaling with the Metaboanalyst
6.0. Fifty-­eight significant molecules (p < 0.05) were detected. The heat map was generated by the Ward clusterization and distance measurements
of Minkowsky. Green, red, and blue colors were used to indicate the major metabolic clusters. (b) Volcano Plot of metabolites from the sample
groups LIT/GOX. The y-­axis shows the -­log10(p-­value), while the x-­axis presents log2 (fold change (FC) of GOX minus LIT). Red, purple, and gray circles
represent the upregulated metabolites, downregulated metabolites, and non-­significant changes, respectively. The vertical dashed lines represent FC
equals 1.5, and the horizontal dashed lines represent the fold discovery rate (FDR) for p < 0.05). The most significant molecules are named.
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SILVA et al. 713

TA B L E 2 Statistical analysis of compound variation in GOX × LITa.

Compound t stat p-­value LOG10(p) FDR

Succinic acid −22.199 4.11E-­11 1.04E+01 1.60E-­09

L-­Glutamic acid −18.580 3.29E-­10 9.48E+00 6.42E-­09
Malic acid −16.876 1.00E-­09 9.00E+00 1.48E-­0 8
L-­Valine −16.417 1.38E-­09 8.86E+00 1.48E-­0 8
2-­Hydroxyisocaproic acid −14.316 6.62E-­09 8.18E+00 5.73E-­0 8
Phosphoethanolamine −11.235 1.00E-­07 7.00E+00 7.83E-­07
L-­5-­Oxoproline −11.023 1.24E-­07 6.91E+00 8.05E-­07
9-­Hexadecenoic acid −10.480 2.15E-­07 6.67E+00 1.17E-­06
2,3-­Butanediol −9.910 3.95E-­07 6.40E+00 1.92E-­06
Ergosterol −9.583 5.67E-­07 6.25E+00 2.46E-­06
Pyroglutamic acid −9.389 7.04E-­07 6.15E+00 2.89E-­06
Lactose −9.015 1.08E-­06 5.96E+00 3.85E-­06
L-­A spartic acid −9.014 1.09E-­06 5.96E+00 3.85E-­06
Inositol phosphate −8.326 2.49E-­06 5.60E+00 8.10E-­06
Docosahexaenoic acid −8.082 3.39E-­06 5.47E+00 1.06E-­05
Glycine −7.653 5.90E-­06 5.23E+00 1.77E-­05
4-­Hydroxyphenyllactic acid −7.476 7.47E-­06 5.13E+00 2.16E-­05
Phosphoric acid −7.397 8.31E-­06 5.08E+00 2.31E-­05
Lignoceric acid −6.659 2.33E-­05 4.63E+00 6.27E-­05
Sorbitol −6.477 3.04E-­05 4.52E+00 7.89E-­05
Adenosine monophosphate −6.335 3.75E-­05 4.43E+00 9.43E-­05
L-­Alanine −6.134 5.07E-­05 4.30E+00 1.23E-­0 4
2-­Monostearin −5.982 6.39E-­05 4.19E+00 1.51E-­0 4
Cholesterol −5.899 7.27E-­05 4.14E+00 1.67E-­0 4
Indolelactic acid −5.826 8.14E-­05 4.09E+00 1.81E-­0 4
Palmitic acid −5.496 1.37E-­0 4 3.86E+00 2.97E-­0 4
Phosphorylethanolamine −5.421 1.55E-­0 4 3.81E+00 3.26E-­0 4
Ethylphosphonic acid −5.377 1.66E-­0 4 3.78E+00 3.37E-­0 4
myo-­inositol −5.368 1.69E-­0 4 3.77E+00 3.37E-­0 4
Pipecolic acid −4.705 5.10E-­0 4 3.29E+00 9.70E-­0 4
alpha.-­D-­Mannopyranose −4.639 5.71E-­0 4 3.24E+00 1.06E-­03
1-­Monopalmitylglycerol −4.290 1.05E-­03 2.98E+00 1.78E-­03
L-­Proline −4.161 1.32E-­03 2.88E+00 2.19E-­03
Behenic acid −4.125 1.41E-­03 2.85E+00 2.26E-­03
Cer(d18:1/16:0) −3.814 2.46E-­03 2.61E+00 3.84E-­03
Sucrose −3.784 2.61E-­03 2.58E+00 3.98E-­03
Succinimide −3.688 3.10E-­03 2.51E+00 4.65E-­03
Stearic acid −2.841 1.49E-­02 1.83E+00 2.11E-­02
Arachidic acid −2.769 1.70E-­02 1.77E+00 2.37E-­02
Lanosterol −2.751 1.76E-­02 1.76E+00 2.41E-­02
Monostearin −2.721 1.86E-­02 1.73E+00 2.46E-­02
Sebatic acid −2.511 2.73E-­02 1.56E+00 3.56E-­02
Octadecanol −2.498 2.80E-­02 1.55E+00 3.58E-­02
1-­Monopalmitin −2.412 3.28E-­02 1.48E+00 4.13E-­02
Arabinofuranose −1.964 7.31E-­02 1.14E+00 8.91E-­02
Palmitoleic acid −1.829 9.23E-­02 1.03E+00 1.11E-­01
Myristic acid −1.590 1.38E-­01 8.60E-­01 1.63E-­01

(Continues)
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714 SILVA et al.

TA B L E 2 (Continued)

Compound t stat p-­value LOG10(p) FDR

Octadecanamide −1.416 1.82E-­01 7.40E-­01 2.12E-­01

Monoethylhexyl phthalic acid −1.236 2.40E-­01 6.19E-­01 2.72E-­01
Batyl alcohol −0.963 3.55E-­01 4.50E-­01 3.85E-­01
L-­Hydroxyproline −0.736 4.76E-­01 3.23E-­01 5.08E-­01
Ureidosuccinic acid −0.687 5.05E-­01 2.97E-­01 5.25E-­01
Glucose −0.676 5.12E-­01 2.91E-­01 5.25E-­01
Uracil −0.062 9.52E-­01 2.15E-­02 9.64E-­01
D-­Allose 0.020 9.84E-­01 6.86E-­03 9.84E-­01
9,12-­Octadecadienoic acid 0.716 4.88E-­01 3.12E-­01 5.14E-­01
L-­Leucine 0.962 3.55E-­01 4.50E-­01 3.85E-­01
Glycerol 3-­phosphate 1.132 2.80E-­01 5.53E-­01 3.12E-­01
L-­Tyrosine 1.393 1.89E-­01 7.24E-­01 2.17E-­01
L-­Threonine 2.270 4.25E-­02 1.37E+00 5.26E-­02
Fructose 6-­phosphate 2.740 1.79E-­02 1.75E+00 2.41E-­02
O-­Acetylserine 3.313 6.19E-­03 2.21E+00 8.95E-­03
L-­Serine 3.373 5.54E-­03 2.26E+00 8.15E-­03
Tagatose 4.121 1.42E-­03 2.85E+00 2.26E-­03
3-­Phosphoglyceric acid 4.435 8.14E-­0 4 3.09E+00 1.41E-­03
2-­Phosphoglyceric acid 4.441 8.06E-­0 4 3.09E+00 1.41E-­03
Arabitol 4.456 7.85E-­0 4 3.11E+00 1.41E-­03
Mannitol 4.753 4.70E-­0 4 3.33E+00 9.16E-­0 4
D-­fructose 8.962 1.16E-­06 5.94E+00 3.92E-­06
L-­methionine 9.214 8.61E-­07 6.06E+00 3.36E-­06
D-­gluconic acid 9.823 4.34E-­07 6.36E+00 1.99E-­06
Ribitol 10.441 2.24E-­07 6.65E+00 1.17E-­06
Citric acid 10.539 2.02E-­07 6.69E+00 1.17E-­06
3-­Methylvalerate 11.055 1.20E-­07 6.92E+00 8.05E-­07
Sedoheptulose 7-­phosphate 16.280 1.52E-­09 8.82E+00 1.48E-­0 8
Ribulose 5-­phosphate 16.454 1.34E-­09 8.87E+00 1.48E-­0 8
Ribulose 19.961 1.43E-­10 9.85E+00 3.71E-­09
Glyceric acid 32.454 4.63E-­13 1.23E+01 3.61E-­11
a
Two-­sample t-­tests & Wilcoxon rank-­sum test using a fold discovery rate (FDR) of unpaired samples for p < 0.05.

Sigma-­Aldrich) for 30 min at 28°C. GOX was added from 6 U/mL 3.2 | Immunoblotting
stock aliquots in 80 mM PIPES (Sigma-­Aldrich) pH 6.9, 1 mM EGTA,
1 mM MgCl2, kept at –80°C. The equivalent of 5 × 10 8 parasites were collected by centrifu-
Microscope images were obtained by attaching PBS washed gation at 2000 g for 5 min, supernatants removed, and precipi-
and 4% paraformaldehyde in PBS fixed parasites to glass round tates washed once with 1 mL PBS, before resuspension in 1 mL
coverslips pre-­coated with 0.1% poly-­L-­lysine for 5 min. Following of TDB (80 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 10 mM Na 2 HPO 4 ,
attachment, the coverslips were washed with PBS and incubated 2 mM NaH 2 PO 4 , 20 mM glucose, 2% Nonidet P-­4 0, 5 mM NaF,
30 min in PBS containing 10 μg/mL 4′,6-­D iamidino-­2-­fenilindol, 2-­ 1 mM Na 3 VO 4 , 1 mM Na 4 P 2O7, 1 mM, 1X Complete, EDTA-­f ree
(4-­A midinofenil)-­6 -­indolecarbamidine (DAPI). The coverslips were protease inhibitors (Sigma-­A ldrich), 1X Phostop (Sigma-­A ldrich),
mounted in Prolong Gold (ThermoFisher Scientific), and images pH 7.4). The protein content was determined by Bradford assay
were acquired using an Orca R2 CCD camera (Hamamatsu) cou- (Bradford, 1976). Forty micrograms of proteins in the lysates
pled to an Olympus BX-­61 microscope and a ×100 plan Apo-­oil were adjusted to contain 2% SDS (30 mM Tris–HCl pH 6.8, 10%
objective (NA 1.4). glycerol, 0.01% bromophenol blue, and 5 mM dithiothreitol,
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SILVA et al. 715

F I G U R E 6 Analysis of metabolites
enriched in GOX groups in the
comparative experiments of LIT versus
GOX. (a) The analysis was performed
against the 99 metabolite sets based
on normal human metabolic pathways
with significant features (p values <0.05)
reflecting the enrichment ratio in each
case. Panel (b) shows the scatter plot
testing the pathway impact using the
Trypanosoma cruzi KEGG database in the
Metaboanalyst.

DTT), boiled for 5 min and loaded in SDS-­p olyacrylamide gels 3.3 | Cell viability
(Laemmli, 1970). After electrophoresis, the proteins were trans-
ferred to 0.45 μm nitrocellulose in a semidry Biorad apparatus at The equivalent of 1 × 107 parasites obtained from all treatments was
25 V for 40 min. Membranes were stained with 0.3% Ponceau S collected by centrifugation and resuspended in 1 mL of complete
in 3% trichloroacetic acid and washed in 10 mM Tris–HCl, 0.15 M LIT medium. Then, 20 μL of each sample was mixed with 380 μL of
NaCl, pH 7.4 (TBS) containing 0.05% Tween 20 (TBS-­T ) before Muse® Count & Viability Reagent (Merck-­Millipore) in each tube
incubation with 5% BSA in TBS-­T for 1 h and primary antibodies and incubated on ice for 10 min. After the incubation, the samples
also diluted in 5% BSA in TBS-­T. The following antibodies were were analyzed on the Muse® Cell Analyzer (Merck-­Millipore) fol-
used: rabbit anti-­β -­t ubulin, at 1:3000 dilution (Cell signaling), lowing the manufacturer's recommendations. In this kit, non-­viable
rabbit anti-­B IP at 1:40.000 dilution (Bangs et al., 1993), rab- parasites are detected by dye incorporation.
bit anti-­h eat shock 70 (HSP70) at 1:10,000 (Bangs et al., 1996),
and rabbit anti-­a ldolase at 1:3000 dilution (Barbosa Leite
et al., 2020). After antibody incubation, membranes were 3.4 | Hydrogen peroxide detection
washed 3X, 10 min each, with TBS-­T followed by 1 h incubation
with 1:10.000 dilution of goat anti-­R abbit IgG coupled to Alexa Cell hydrogen peroxide was detected by using the Amplex Red Kit
Fluor® 680 (ThermoFisher Scientific) in TBS-­T for 40 min. After (ThermoFisher Scientific). Briefly, equivalents of 1 × 107 parasites
new washes as above, bound antibodies were detected by im- were collected by centrifugation, resuspended in 50 μL of reaction
aging in an Odyssey Imaging Systems—LI-­COR (Biosciences) at buffer (0.05 M NaHPO4, pH 7.4), and lysed by three rounds of freezing
700 nm. Quantifications were made using the Image Studio Lite and thawing. Each sample was then added to one well of a black 96-­
(LICOR) software in triplicate experiments. well plate, which then received 50 μL of the Amplex Red mix and the
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716 SILVA et al.

F I G U R E 7 Venn diagrams depicting the presence and changes in several compounds under nutritional and oxidative stress conditions.
(a) The graphic depicts the total number of metabolites discovered in each test. (b) A diagram comparing increased and decreased molecules
under each form of stress. (c) Molecules frequently decreased in both situations and statistical values.

peroxidase according to the manufacturer's instructions. After 30 min, vials containing glass inserts and dried in SpeedVac at 30°C. After
the emitted relative fluorescence at 590 nm with excitation at 544 nm complete drying, the samples were submitted to methoxification by
was measured by using a M3 SpectraMax (Molecular Devices) plate the addition of 15 μL of 15 mg/mL of O-­metoxiamine hydrochloride
reader. The amount of H2O2 produced was determined by comparing in pyridine and homogenized by vortex and ultrasound before incu-
the relative fluoresce with a H2O2 standard curve. bation in the dark at room temperature for 16 h. After incubation,
15 μL N,O-­bis(trimetilsilil)trifluoroacetamida (BSTFA) with 1% chlo-
drotrimethylsilane (TMCS, v/v) were added, the samples mixed by
3.5 | Gas chromatography-­mass spectrometry vortex, and incubated for 1 h at 70°C to achieve silylation. At the end
(GC–MS) analysis in scan mode of the procedure, 20 ppm of pentadecanoic acid (diluted in heptane)
was added to each sample. Three blank samples and two for quality
For metabolomic analysis, pellets containing 2 × 108 unwashed controls made from a pool of all samples were prepared in the same
parasites were resuspended in 500 μL of cold 100% methanol (hy- way (Ballari et al., 2021; Fall et al., 2022).
pergrade LC–MS/MS) to allow complete deproteinization of the The samples were analyzed in a system containing a gas chromato-
samples. The samples were agitated on a vortex for 1 min and then graph coupled to a quadrupole mass spectrometer (GCMS-­QP2020
centrifuged for 15 min at 17000× g at 4°C. Two hundred microlit- NX, Shimadzu Co., Kyoto, Japan). One microliter of each sample was
ers of the resulting supernatants were then transferred to GV-­MS loaded into a DB5-­MS column (30 m × 0.25 mm, 0.25 μm, Restek)
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SILVA et al. 717

in spitless mode at 20 mL/min of helium gas and the carrier gas at editing; writing – original draft; formal analysis; supervision; re-
1.36 mL/min. The initial temperature of the column was maintained sources; data curation; project administration.
at 80°C, and progressively increased by 15°C to reach 300°C, then
kept at this temperature for 8 min before cooling it. The injector, AC K N OW L E D G M E N T S
transfer line, power filament, and quadrupole were adjusted to 280, We thank Claudio Rogerio Oliveira and Claudeci Costa Medeiro da
200, 150, and 150°C, respectively. The system operated in full scan Costa for the general technical assistance in the sample prepara-
(m/z 40–650), generating three spectra per second, configured at tions. This work was supported by Grants of Fundação de Amparo
70 eV. Therefore, a close retention method (CRT) was applied to de- à Pesquisa do Estado de São Paulo, Grants 2020/07870-­4 to
crease the time of retention (TR) in all analyses as described (Barison SS, 2021/12515-­1 to MA, 2021/12515-­1 CEPID-­ARIES, to ALC;
et al., 2022). We used LabSolutions (GCMS version 4.5, Shimadzu Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Co., Japan) for instrument control, data acquisition, and processing, (CAPES) grant 88887.900282/2023-­0 0 to MAS; Conselho Nacional
which allowed us a real-­time control for each identified analyte and de Desenvolvimento Científico e Tecnológico (CNPq) grants
metabolite in SIM and Scan mode. 303788/2020-­8 and INCTV-­CNPq to SS.

C O N FL I C T O F I N T E R E S T S TAT E M E N T
3.6 | Molecule identification The authors declare that they have no conflicts of interests.

For scan mode analysis, the detected metabolites were processed DATA AVA I L A B I L I T Y S TAT E M E N T
by creating a unified matrix of variables from different charge states All data obtained in this study is included in the main figures and
and groups of the same analytes through all samples using the GCMS Supplemental Information. Primary GC–MS data are available upon
Solution (version 3.30), NIST17 MASS (version 1.00.1), and GCMS request.
Smart Metabolite (version 3.01) (Shimadzu Co.). The software was
set to process all detected peaks, considering the equipment noise. E T H I C S S TAT E M E N T
The molecules were identified based on NIST (Kind et al., 2009) and This work did not include animal or experimental subjects requir-
Smart Metabolite and exported to Excel (Microsoft Office) for statis- ing formal approval. Antibodies were available commercially or were
tical treatment. We also used a public database (https://​w ww.​ge- published previously. The work was approved by the ethical review
nome.​jp/​kegg/​ and http://​w ww.​hmdb.​ca) to identify and check the board of the Federal University of São Paulo from Brazil, under the
GC–MS spectra conformations (Mastrangelo et al., 2015). number CEUA 266629105.

ORCID
3.7 | Statistical analysis Michel Augusto Silva https://orcid.org/0000-0002-5349-7810
Mario Augusto Izidoro https://orcid.org/0000-0002-4375-6719
Uni-­ and multivariate analyses were made using Metaboanalyst 6.0 Mirella Aricó https://orcid.org/0000-0003-4905-3982
(http://​w ww.​metab​oanal​yst.​ca/​), following parametric and non-­ Luiz Juliano https://orcid.org/0000-0002-5589-2822
parametric algorithms as Student's t-­test, parametric ANOVA and Sergio Schenkman https://orcid.org/0000-0001-9353-8480
its non-­parametric version (Kruskal-­Wallis), creating multivariate
models with logarithmic transformation for data normalization, and
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