Experimental Parasitology 124 (2010) 167–171

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole

Rodrigo López-Muñoz a, Mario Faúndez d, Sebastián Klein d, Sebastián Escanilla d, Gloria Torres a,
Dasfne Lee-Liu a, Jorge Ferreira a, Ulrike Kemmerling b, Myriam Orellana a, Antonio Morello a,
Arturo Ferreira c, Juan D. Maya a,*
a
Program of Molecular and Clinical Pharmacology, ICBM, School of Medicine, University of Chile, Chile
b
Program of Anatomy and Developmental Biology, ICBM, School of Medicine, University of Chile/School of Health Sciences, Talca University, Chile
c
Program of Immunology, ICBM, School of Medicine, University of Chile, Chile
d
Facultad de Química, Pontificia Universidad Católica de Chile, Chile

a r t i c l e i n f o a b s t r a c t

Article history: Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have
Received 9 March 2009 limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an
Received in revised form 1 September 2009 antiparasitic response, which the parasite evades through an increase of tumor growth factor b and
Accepted 2 September 2009
PGE2 activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin
Available online 6 September 2009
might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect.
Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolo-
Keywords:
graphic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic
Trypanosoma cruzi
Aspirin
effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively.
Nifurtimox The observed effect of aspirin upon T. cruzi infection was not related with the PGE2 synthesis inhibition.
Benznidazole Nevertheless, NO levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macro-
Synergism phage antiparasitic activity improvement.
Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to
increase antiparasitic activity of macrophages.
Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction 1998; Peluffo et al., 2004). Simultaneously, T. cruzi induces an anti-

In Latin America, Chagas’ disease, caused by the protozoan Try-
panosoma cruzi, represents a health threat for an estimated 10–20
million people and is the second highest burden of disease among
Tropical Diseases in the Americas (Reithinger et al., 2009).
Current Chagas’ disease treatment is limited to nifurtimox and
benznidazole. However, these drugs have limited efficacy and fre-
quent and significant side effects. Hundreds of natural and syn-
thetic compounds have been tested against T. cruzi. However,
very few are devoid of host’s cytotoxic activity and are more effec-
tive than nifurtimox and benznidazole, especially against the intra-
cellular amastigotes (Maya et al., 2007).
During primoinfection, T. cruzi trypomastigotes invade macro-
phages, and transform in the intracellular amastigote forms. In this
setting, macrophages mount an antiparasitic response through an
inflammatory activity including increasing nitric oxide production
and oxidative stress through peroxynitrite formation (Borges et al.,
* Corresponding author. Address: University of Chile, Faculty of Medicine,
Molecular and Clinical Pharmacology Program, P.O. Box 70000, Santiago 7, Chile.
Fax: +562 7355580.
E-mail address: jmaya@med.uchile.cl (J.D. Maya).
inflammatory response through activation of cellular mediators
such as tumor growth factor b (TGF-b) (Li et al., 2006) and prosta-
glandins, specifically, PGE2 (Abdalla et al., 2008). TGF-b and PGE2
decrease iNOS activity and, consequently, increase polyamine pro-
duction, by increasing L-arginine availability (Freire-de-Lima et al.,
2006; Peluffo et al., 2004). Polyamines are essential for T. cruzi
amastigote proliferation and trypanothione (T(SH)2) synthesis.
T(SH)2 is the most important oxidative aggression scavenger in try-
panosomatids (Maya et al., 2007). Thus, the parasite evades the in-
nate immune response attenuating the antiparasitic response of
macrophages by decreasing nitric oxide production, improving
polyamine availability and providing a favorable intracellular re-
dox environment.
Freire-de-Lima et al. (2000) showed that prostaglandin synthe-
sis inhibition in T. cruzi infected macrophages, which have ingested
apoptotic bodies, produced a significant decrease in infection. Fur-
ther evidence indicates that prostaglandin synthesis inhibition
could be important in acute infection therapy (Freire-de-Lima
et al., 2000; Hideko Tatakihara et al., 2008; Michelin et al., 2005;
Pinge-Filho et al., 1999).
In this work, we show that a COX inhibitor like aspirin (ASA) in-
creases the trypanocidal activity of nifurtimox and benznidazole in

0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2009.09.005
168 R. López-Muñoz et al. / Experimental Parasitology 124 (2010) 167–171
an in vitro model of T. cruzi infection. Thus, the experimental basis
for a potentially new therapeutic strategy for the control of this
disease is established.
2. Materials and methods
2.1. Cell culture and in vitro infection with T. cruzi
RAW (1  105) 264.7 cells/cm2 were cultured in 0.22% sodium
bicarbonate, 5% fetal bovine serum supplemented RMPI 1640 med-
ium in humidified air with 5% CO2, at 37 °C. RAW 264.7 cells were
infected with Dm28c trypomastigotes, at a 1:3 (cell:parasite) ratio.
T. cruzi trypomastigotes were initially obtained from primary cul-
tures of peritoneal macrophage from chagasic mice.
2.2. Cell viability assay
The effect of drug treatments on RAW 264.7 cells was evalu-
ated through the MTT assay as viability test (Mosmann, 1983).
Briefly, 10 ll of 5 mg/ml tetrazolium dye (MTT; 3[4,5-dimethylthi-
azol-2-yl]-2,5-diphenyltetrazolium bromide) plus 0.22 mg/ml
Phenazine metosulfate (electron carrier), were added to each well
containing RAW 264.7 cell culture in 100 ll RPMI 1640 without
phenol red. Drugs, dissolved in DMSO, were added to culture media
at the concentrations shown in figures and tables. DMSO final con-
centration was less than 0.25% v/v. After incubation for 4 h at 37 °C,
the generated water insoluble formazan dye was dissolved by
addition of 100 ll of 10% w/v SDS in 0.01 M HCl. The plates were
further incubated overnight at 37 °C, and optical density (OD) of
the wells was determined using a microplate reader (Labsystems
Multiskan MS, Finlandia) at 570 nm. Under these conditions, the
OD is directly proportional to the viable cell number in each well.
All experiments were performed at least three times and data are
shown as the means and their standard deviations from triplicate
cultures.
2.3. Prostaglandin E2 measurement
The PGE2 levels were measured in the supernatant of an in-
fected RAW cells culture. The ‘‘Prostagladin E2 EIA Kit – Monoclo-
nal” (CaymanÒ Chemical Co., USA) kit was used, according to the
manufacturer’s instructions. RAW 264.7 cells were infected with
T. cruzi Dm28 clone trypomastigotes. After 72 h post-infection,
50 ll supernatant were tested using this immunoassay.
2.4. Nitric oxide measurement
NO synthesis was evaluated indirectly, through nitrite content
in supernatants from T. cruzi-infected RAW 264.7 cultures. Griess’
reactive was used to measure colorimetrically the nitrite content
at 570 nm. In each experiment, nitrite concentration was calcu-
lated from a NaNO2 standard curve (Tsikas, 2007).
2.5. Effect of drugs on infected RAW 264.7 cells
The effect of aspirin, nifurtimox and benznidazole on T. cruzi-in-
fected RAW 264.7 cells was assessed by the number of trypom-
astigotes released to the culture supernatants. Twenty-four hours
after infection, aspirin, nifurtimox, benznidazole treatments and
their combinations were started at concentrations described in re-
sults. Every 24 h culture media was removed and fresh medium
was added together with the drugs at the same concentrations.
To show the influence upon NOS and COX, aminoguanidine
(5 mM) and PGE2 (1 mM), respectively, were added in some exper-
iments. Cell culture medium was harvested at the third day post
treatment and centrifuged at 500 g during 5 min. Supernatants
were discarded, pellets resuspended in 1 ml of fresh RPMI 1640
and trypomastigotes were counted using direct microscopy
(Freire-de-Lima et al., 2000; Nunes et al., 1998).
2.6. Statistical analysis
IC50 and growth rate calculations were analyzed by non-linear
regression. When necessary, data were also analyzed through
two-way ANOVA. The drug combination treatments were analyzed
by isobologram constructions through sum-of-squares plus Fisher
test. Values of p equal or less than 0.05 were considered significant.
All statistical analyses were performed using GraphPad Prism 5.0.
All experiments were done in triplicate and results correspond to
means ± SD from at least three independent experiments.
3. Results
3.1. Aspirin decreases trypomastigote release to culture supernatants
from T. cruzi-infected RAW 264.7 cells
At fourth day of RAW cells infection, trypomastigotes began to
be released to the supernatants. Aspirin showed a concentration-
dependent effect, with an IC50 of 313.1 ± 36.0 lM (Table 1). This
concentration is below the steady-state concentration (Css, 1.1–
2.2 mM after oral administration of 3–4 g/day) found in humans
treated with aspirin at high concentrations, as is the highest aspirin
concentration used in other experiments (1 mM) (Anderson et al.,
2002; Keystone et al., 1982). This result is in agreement with pre-
vious reports, showing that aspirin and other COX inhibitors de-
creased T. cruzi infection (Abdalla et al., 2008; Freire-de-Lima
et al., 2000; Kubata et al., 2002; Michelin et al., 2005; Paiva et al.,
2007).
3.2. Aspirin improves the activity of nifurtimox and benznidazole upon
infected RAW 264.7 cells in a synergistic way
As expected, nifurtimox and benznidazole had a concentration-
dependent effect on trypomastigote release with an IC50 of
0.083 lM and 0.336 lM, respectively (Table 1). The addition of a
low concentration of aspirin (IC12.5 of aspirin) reduces the IC50 of nif-
urtimox from 0.083 to 0.023 lM (reduction by 72.3%) while the IC50
of benznidazole was reduced by 61.7%. In addition, when aspirin was
combined with nifurtimox or benznidazole (at its IC12.5 concentra-
tions), the IC50 value for aspirin was decreased by 57.2% and 76.7%,
respectively. The observed IC50 value is significantly lower than that
expected for the combinations if the interaction was additive
(p < 0.002) (Table 1). The graphical representation of this interaction
is shown in the isobolograms of Fig. 1. These isobolograms allow the
comparison of theoretical additive and observed experimental ef-
fects. Combinatory indexes (Chou, 2006) equal, lower or larger than
1, correspond to additive, synergistic or antagonic behavior, respec-
tively. Combinatory indexes for aspirin and nifurtimox or benzni-
dazole combinations are described in Table 1.
Neither aspirin, nifurtimox, benznidazole nor their combina-
tions, at the highest trypanocidal concentrations, showed cytotox-
icity against uninfected RAW 264.7 cells (data not shown).
3.3. Aspirin effect on infected RAW cells is not dependent of PGE2
synthesis inhibition
Trypanosoma cruzi-infected RAW 264.7 cells presented a signifi-
cantly higher prostaglandin (PGE2) production compared to unin-
fected RAW cells. ASA reverted prostaglandin levels to baseline in
a dose–response way (Fig. 2A). Note that the maximal effect of ASA
 R. López-Muñoz et al. / Experimental Parasitology 124 (2010) 167–171 169

Table 1
Combinatory index of the aspirin effect together with nifurtimox and benznidazole upon RAW 264.7 cells infected with T. cruzi. The effect of drugs (IC50 values) was determined
by trypomastigote release from T. cruzi-infected RAW 264.7 cells.

IC50 (lM) IC50 of the combination (lM) Combinatory index

Theoreticala Observed p-Value
Aspirin 313.1 ± 36.0 250 134.0 ± 13.4 <0.001 0.71
Nifurtimox 0.083 ± 0.005 0.066 0.023 ± 0.008 0.002
Aspirin 313.1 ± 36.0 250 73.1 ± 11.4 <0.0001 0.61
Benznidazole 0.34 ± 0.002 0.27 0.13 ± 0.02 <0.0001
a
Theoretical IC50 corresponds to the value predicted from isobolograms depicted in Fig. 1. Nifurtimox, aspirin and benznidazole were added at their estimated IC12.5
concentrations. p values correspond to the comparisons between theoretical and observed IC50 values through sum-of-squares plus F test. Combinatory index was calculated
according to Chou (2006). Results are expressed as means ± SD from three independent experiments.

Fig. 2. Relationship between prostaglandin synthesis inhibition and anti-T. cruzi

Fig. 1. Isobolographic analysis of the effect of aspirin, nifurtimox, benznidazole
and their combinations on trypomastigote release. RAW 264.37 cells were
infected with T. cruzi Dm28c trypomastigotes and incubated with (A) aspirin
alone, aspirin plus nifurtimox at their IC12.5 and, nifurtimox plus aspirin at their
IC12.5; (B) aspirin alone, aspirin plus benznidazole at their IC12.5 and, benznidaz-
ole plus aspirin at their IC12.5. After 3 days of treatment, trypomastigotes released
to the supernatant were counted by direct microscopy. Continuous line corre-
sponds to the predicted positions of the experimental points for additive effects.
Dots in the discontinuous line correspond to the experimental findings depicted
in Table 1.
aspirin effect on infected RAW 264.7 cells. (A) Effect of aspirin upon prostaglandin
production in RAW 264.7 cells. Macrophages were infected with T. cruzi trypom-
astigotes and PGE2 was measured in the supernatant, after 72 h treatment with
aspirin (ASA, at 0.065, 0.125, 0.25, 0.5 mM). Results are expressed as means ± SD
from three independent experiments, ***p < 0.001 from Tukey post-test. (B) Effect of
PGE2 upon trypomastigote release in aspirin treated RAW 264.7 cells infected with
T. cruzi. Macrophages were infected with T. cruzi trypomastigotes and, after 72 h
treatment with 0.25 mM ASA alone or combined with 1 mM PGE2, trypomastigote
release to supernatants were measured. Results are expressed as means ± SD from
three independent experiments. ***p < 0.001 from Tukey post-test. ns, no significant
differences.

over the PGE2 synthesis is achieved at 0.25 mM, a dose–response fit
of this data showed an IC50 of 7.7 ± 2.54 lM. PGE2 was added to re-
verse the effect of ASA 0.25 mM. The lack of effect of PGE2 over
ASA treatment might indicate that PGE2 is not involved in the ASA ef-
fect on infected cells (Fig. 2B).
3.4. Aspirin restores NO levels in T. cruzi-infected RAW 264.7 cells
The effect of aspirin upon NO production was evaluated by ni-
trite detection in supernatants from T. cruzi-infected RAW 264.7
cells by the Griess reaction. While T. cruzi infection decreases NO
170 R. López-Muñoz et al. / Experimental Parasitology 124 (2010) 167–171
Fig. 3. Effect of aspirin upon nitrite production and trypomastigote release in T.
cruzi-infected RAW 264.7 cells. (A) Nitrite levels (white bars) and trypomastigote
release (black bars) in aspirin treated Raw 264.7 cells infected with T. cruzi
trypomastigotes. After 72 h nitrite levels in supernatant were determined by the
Griess reaction. §p < 0.001 compared with uninfected control (RAW). àp < 0001
compared with infected control (ASA 0 mM). (B) Effect of aminoguanidine (AMG)
upon trypomastigote release in aspirin-treated T. cruzi-infected RAW 264.7 cells.
Macrophages were infected with T. cruzi trypomastigotes, and after 72 h treatment
with 1 mM aspirin (ASA), 5 mM AMG or ASA plus AMG trypomastigote release to
supernatants was measured. Results are expressed as means ± SD from three
independent experiments. *p < 0.05; ***p < 0.001 from Tukey post-test.
production in RAW 264.7 cells, aspirin restores it (Fig. 3A). The in-
crease in NO production, induced by aspirin has a dose–response
behavior, similar to trypomastigote release inhibition (Fig. 3A). At
1 mM aspirin, the highest effective concentration evaluated, NO
production was similar to non-infected cells (p < 0.001) (Fig. 3A).
To corroborate that the effect of aspirin on macrophage activity de-
pends upon restoring NO levels, we assessed the trypomastigote
release to culture medium when RAW 264.7 cells were incubated
with aminoguanidine (AMG), an iNOS inhibitor (Fig. 3C). Thus,
5 mM AMG reverts aspirin effect by 50%.
4. Discussion
Different studies have shown the effect of COX inhibitors, such
as aspirin, on several T. cruzi infection models (Freire-de-Lima
et al., 2000; Hideko Tatakihara et al., 2008; Michelin et al., 2005;
Pinge-Filho et al., 1999). However, COX inhibitors concentration-
dependent responses and mechanisms affecting T. cruzi prolifera-
tion inside host cells have not been studied. Herein, we studied
the improvement of the macrophage antiparasitic activity induced
by aspirin, the synergy of this effect with the activity of the antich-
agasic drugs nifurtimox and benznidazole and, possible mecha-
nisms involved in those effects.
Attention can be drawn to the low reported IC50 values of nifur-
timox (0.0830 lM) and benznidazole (0.336 lM). However, this
might be due to the low parasite density used in the experiments.
In previous experiments carried out on VERO cells (Faundez et al.,
2005), parasite density was an order of magnitude higher and the
highest concentration used for nifurtimox and benznidazole was
1 lM. Nevertheless, this situation is not an obstacle to perform
synergism studies with other drugs.
Herein, the relationship of aspirin with classic antichagasic
therapy is further substantiated. Thus, the IC50 value of aspirin in
infected macrophages is several folds higher than those deter-
mined for nifurtimox and benznidazole (Table 1). However, from
the isobolographic analysis, we can conclude that aspirin acts syn-
ergistically with these classical drugs. In fact, the IC50 of both nif-
urtimox and benznidazole, decreased in average by 67% when
combined with aspirin (Table 1). However, the observed synergy
is not related to a direct effect of both drugs upon the same target
or pathway, i.e. parasite viability. Instead, the combined effects of
aspirin on the macrophage and the toxic effects of antichagasic
drugs on the parasite are complementary mechanisms that explain
the isobolographic result. This concept is in agreement with syner-
gistic mechanisms reported for other drug combinations (Jia et al.,
2009).
Fig. 2A shows that PGE2 production increases significantly in T.
cruzi infected macrophages as compared with healthy macro-
phages. Thus, PGE2 synthesis inhibition using aspirin could im-
prove macrophage response against T. cruzi infection. In fact, ASA
reduces PGE2 levels in a dose–response way, with an IC50 = 7.7 lM.
This value is 40-fold lower than the ASA IC50 observed on trypo-
mastigote release experiments (IC50 = 313 lM). Therefore, com-
plete PGE2 synthesis inhibition is achieved at concentration
levels that have no significant effect on trypomastigote release.
Moreover, there is no evidence supporting that PGE2 itself could in-
crease T. cruzi infection. We attempted to determine whether PGE2
is involved in the effect of ASA upon T. cruzi infected cells. We did
not find reversion of ASA effect, even at PGE2 concentrations as
high as 1 mM (Fig. 2B), indicating that PGE2 does not have a main
role on ASA effect. Other COX inhibitors, such as indomethacin and
meloxicam were tested, both inhibiting trypomastigote release
(IC50 = 97.6 ± 11.1 and 42.7 ± 0.36, respectively). These data sug-
gest a pivotal function of COX in intracellular amastigote prolifer-
ation. However, there might be other prostaglandins playing key
roles in parasite proliferation. Cardoni and Antunez (2004) showed
that T. cruzi infected mice have increased levels of other COX
metabolites, such as PGI2 and TXA2, yet their role on parasite infec-
tion is still unknown (Cardoni and Antunez, 2004).
Trypanosoma cruzi induces TGF-b and PGE2 production by mac-
rophages, especially in the presence of apoptotic bodies (Freire-de-
Lima et al., 2000). Thus, the parasite induces an anti-inflammatory
response, attenuating macrophage activation and evading its anti-
parasitic activity. In macrophages exposed to apoptotic bodies,
TGF-b increases PGE2 synthesis and decreases NO (Freire-de-Lima
et al., 2006). Inhibition of COX activity may increase NO levels,
thus restoring the antiparasitic activity of macrophages. Our re-
sults are in agreement with this proposal (Fig. 3A and B). It is
important to note that all these experiments were performed with-
out exposure to apoptotic bodies. Consequently, T. cruzi itself can
suppress macrophage antiparasitic activity in an apoptosis mim-
icry fashion, through, perhaps, T. cruzi calreticulin–C1q interactions
(Ferreira et al., 2004). Thus, macrophage antiparasitic activity res-
toration can be pharmacologically modulated.
Finally, in T. cruzi infected macrophages, COX is related to the
increase of ornithine decarboxylase (ODC) activity (Freire-de-Lima
et al., 2000), which might increase the polyamine content in mac-
rophages. Since T. cruzi uses these polyamines to synthesize trypa-
nothione, the inhibition of COX by ASA, indirectly contribute to
 R. López-Muñoz et al. / Experimental Parasitology 124 (2010) 167–171
decrease trypanothione synthesis in T. cruzi. This mechanism, to-
gether with the restoring of NO levels in macrophages through
COX inhibition, could be involved in the activity of ASA on the mac-
rophage response to T. cruzi infection.
In conclusion, the observed synergic effect of aspirin with nifur-
timox and benznidazole is explained by the capability of aspirin to
improve the antiparasitic activity of macrophages.
Acknowledgments
We thank Dr. Norbel Galanti (Cellular and Molecular Biology
Program, ICBM, Faculty of Medicine, University of Chile) for critical
review of this paper. FONDECYT 1090078, PBCT ANILLO ACT 29 and
REDES-07, Chilean grants supported this research.
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