Increase of reactive oxygen species by desferrioxamine during experimental Chagas' disease.

Abstract

Oxidative stress is common in inflammatory processes associated with many diseases including Chagas' disease. The aim of the present study was to evaluate, in a murine model, biomarkers of oxidative stress together with components of the antioxidant system in order to provide an overview of the mechanism of action of the iron chelator desferrioxamine (DFO). The study population comprised 48 male Swiss mice, half of which were treated daily by intraperitoneal injection of DFO over a 35-day period, while half were administered sterile water in a similar manner. On the 14th day of the experiment, 12 DFO-treated mice and an equal number of untreated mice were experimentally infected with Trypanosoma cruzi. Serum concentrations of nitric oxide and superoxide dismutase and hepatic levels of total glutathione, thiobarbituric acid reactive species and protein carbonyl, were determined on days 0, 7, 14 and 21 post-infection. The results obtained revealed that DFO enhances antioxidant activity in the host but also increases oxidative stress, indicating that the mode of action of the drug involves a positive contribution to the host together with an effect that is not beneficial to the parasite.

Full text

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Increase of reactive oxygen species by
desferrioxamine during experimental Chagas’
disease
Amanda Fortes Francisco1, Paula Melo de Abreu Vieira1, Jerusa Marilda
Arantes3,4, Maisa Silva2, Maria Lúcia Pedrosa2, Silvana Maria Elói-Santos5,
Olindo Assis Martins-Filho3, Andréa Teixeira-Carvalho3, Márcio Sobreira Silva
Araújo3, Washington Luiz Tafuri1, Cláudia Martins Carneiro1,6
1Laboratório de Imunopatologia and2Laboratório de Bioquímica e Biologia Molecular, Núcleo de
Pesquisas em Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de
Ouro Preto, Ouro Preto, Brazil
3Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas René Rachou,
Fundação Osvaldo Cruz, Belo Horizonte, Brazil
4Pós-Graduação em Patologia, and5Departamento de Propedêutica Complementar, Faculdade de
Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
6DepartamentodeAnálisesClinicas,EscoladeFarmácia,UniversidadeFederaldeOuroPreto,OuroPreto,Brazil
Oxidative stress is common in inflammatory processes associated with many diseases including
Chagas’ disease. The aim of the present study was to evaluate, in a murine model, biomarkers of
oxidative stress together with components of the antioxidant system in order to provide an overview
of the mechanism of action of the iron chelator desferrioxamine (DFO). The study population
comprised 48 male Swiss mice, half of which were treated daily by intraperitoneal injection of DFO
over a 35-day period, while half were administered sterile water in a similar manner. On the 14th
day of the experiment, 12 DFO-treated mice and an equal number of untreated mice were
experimentally infected with Trypanosoma cruzi. Serum concentrations of nitric oxide and
superoxide dismutase and hepatic levels of total glutathione, thiobarbituric acid reactive species
and protein carbonyl, were determined on days 0, 7, 14 and 21 post-infection.The results obtained
revealed that DFO enhances antioxidant activity in the host but also increases oxidative stress,
indicating that the mode of action of the drug involves a positive contribution to the host together
with an effect that is not beneficial to the parasite.
Keywords: desferrioxamine, oxidative stress, Chagas’ disease, Trypanosoma cruzi
Research article
Correspondence to: Dr C.M. Carneiro, Laboratório de Imunopatologia (sala 40, ICEB II), Nucleo de Pesquisas em Ciências Biológicas (NUPEB), Instituto
de Ciências Exatas e Biológicas (ICEB), Universidade Federal de Ouro Preto (UFOP), Campus Universitário Morro do Cruzeiro, 35400-000 Ouro Preto,
MG, Brazil. Tel: +55 31 35591694; Fax: +55 31 35591680; E-mail: carneirocm@gmail.com or carneiro@ef.ufop.br
Received 13 February 2010, revised manuscript accepted 18 May 2010
© W. S. Maney and Son Ltd 2010
DOI 10.1179/174329210X12650506623528
MORE OpenChoice articles are open access and distributed under the terms of the
Creative Commons Attribution License 3.0
Redox Report
2010 Vol 15No 4
185
Abbreviations: DFO, desferrioxamine; dpi, days post-infection; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, glutathione disulfide; GT,
total glutathione; INT, infected with T. cruzi but not treated with DFO; IP, intraperitoneal; IT, infected with T. cruzi and treated with DFO; NIT, not infected
with T. cruzi but treated with DFO; NINT, not infected with T. cruzi and not treated with DFO; NO, nitric oxide; NOS, nitric oxide synthase; PC, carbonyl
protein; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TNB, 5-thio-2-nitrobenzoic acid.

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Introduction
Chagas’ Disease continues to pose a serious threat to
human health in much of Latin America, and has
become the most important emerging parasitic disease
in developed countries. There is growing evidence to
suggest that during the course of infection by
Trypanosoma cruzi, the myocardium is exposed to
injuries induced by sustained oxidative stress, and that
these may contribute to disease progression. The
principal causes of oxidative stress are reactive oxygen
species (ROS), which may be broadly defined as
derivatives of molecular oxygen, e.g. O2
hydrogen peroxide. ROS are unstable and react rapidly
with other free radicals and macromolecules in chain
reactions to generate increasingly harmful oxidants.1
Acute T. cruzi infection induces H2O2release by
peripheral blood monocytes and spleen macrophages
in rats infected with the Y strain.2During the course
of development of Chagas’disease, high levels of ROS
canbeproduced asa
destruction caused by toxic secretions from the
parasite, immune-mediated cytotoxic reactions and
secondary damage to mitochondria.1
All aerobic organisms are continuously subjected to
ROSgeneratedby oxidative
detoxificationof xenobiotics
ultraviolet radiation and several other mechanisms,
because anaerobic organisms do not need such
antioxidant adaptations, and there are many other
mechanisms that generate ROS in animals, fungi,
algae, protists and plants. In response, organisms have
developed a variety of antioxidant defense systems to
cope with ROS, although it is important to note that
the defense strategies of parasitic protozoa exhibit major
differences one from another and also in comparison
with their mammalian hosts. Many antioxidant defense
strategies depend on the action of antioxidant enzymes,
including superoxide dismutase (SOD), glutathione
peroxidase (GPx) and catalase, or low molecular weight
antioxidants due to their importance in ROS (hydrogen
peroxide) detoxification3. SOD catalyses the conversion
of the superoxide anion into hydrogen peroxide and
oxygen, and exists in a number of different isoforms.
Unlike other eukaryotic parasites, the trypanosomatids
(Leishmania spp. and Trypanosoma spp.) together with
Plasmodium spp. have an iron-containing SOD isoform
that is normally found only in bacteria.4,5GPx exists in
five isoforms and catalyses reduction by glutathione of
hydrogen peroxide or alkyl peroxide to water or alcohol,
respectively.1
A number of assays are available that enable
assessment of the level of oxidative stress induced by
•–, HO•and
consequenceoftissue
metabolism,
orthe
the
ofaction
different biological agents within an organism. The
determination of lipid peroxidation in terms of
thiobarbituric acid reactive substances (TBARS) is
considered to be a very useful, cheap and easy assay
for evaluating oxidative stress. However, this assay,
despite common use in evaluating oxidative stress, has
somelimitations,especially
spectrophotometry and not by fluorescence or HPLC.
Additionally, the direct attack of protein by ROS leads
to the formation of carbonyl groups, such that the
estimation of protein carbonyl (PC) provides an
additional (or complementary) measure of the effect
of oxidative stress.
Desferrioxamine (DFO; Desferal®, Novartis, Basel,
Switzerland) is a hexadentate iron chelator that
complexes with iron in a 1:1 molar ratio to yield the
stable complex ferrioxamine (stability constant 1031).6
DFO is believed to access the pools of iron in the liver
and in the reticulo-endothelial system that are together
responsible for the major proportion of the body iron
burden. However, since DFO permeates into cells
relatively slowly, because of its large molecular size and
hydrophilic nature, its efficiency as an intracellular
chelatorislimited.5,7Variousstudies2,8–11haveestablished
that administration of DFO in T. cruzi infection
modifies the course of Chagas’ disease, reducing
parasitemia and mortality. Results obtained using a
murine model have shown, however, that whilst there
were reductions in the number of circulating parasites
and in mortality, the animals did not suffer from
anemia.8,9It was concluded, therefore, that DFO had a
direct effect on the parasite and not on the reduction of
iron supply in the host.
The aim of the present study was to evaluate the
relationship between oxidative stress (assessed by
TBARS, PC and nitrate/nitrite assays) and antioxidant
defenses (evaluated in terms of SOD and GPx levels) in
T.cruzi-infectedmicethathadbeentreatedwithDFOin
order to clarify the mode of action of the drug.
whenmeasuredby
Materials and methods
Details of the project were submitted to, and approved
by, the Ethical Committee on Animal Research of the
Universidade Federal de Ouro Preto and by the
institutional Animal Care and Use Committee
(CETEA 153/07) of the Universidade Federal de
Minas Gerais. All procedures were carried out in
compliance with current Brazilian regulations relating
to Experimental Biology and Medicine as described in
the guidelines issued by the Colégio Brasileiro de
Experimentaçao Animal (COBEA) in 2006.
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Increase of reactive oxygen species by desferrioxamine during experimental Chagas’disease

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Experimental animals comprised 48 male Swiss mice,
each approximately 30 days old. Throughout the 35-day
period of the study, animals received a commercial diet in
pellet form (Nuvilab CR1) together with sterilized water
ad libitum. Animals were distributed randomly into four
equal groups (n = 12) as follows: control animals (NINT
group)werenotinfectedwithT.cruziandwerenottreated
withDFO;groupINTanimalswereinfectedwithT.cruzi
but were not treated with DFO; group NIT animals were
not infected with T. cruzi but were treated with DFO; and
group IT animals were infected with T. cruzi and treated
with DFO. Animals belonging to the treatment groups
received daily doses of DFO (5 mg; 0.05 ml) by
intraperitoneal (IP) injection for 14 days prior to infection
andforafurther21dayspost-infection(dpi).
Experimental animals in the NINT and INT groups
were submitted to the same conditions of stress as the
treated animals by daily administration of sterile water
(0.05 ml) by IP injection. On day 14 of the study period,
the 24 animals comprising IT and INT groups were
infected by IP injection of 500 blood-stream forms of T.
cruzi Y strain,12which is considered to be partially
resistant to chemotherapeutic treatment.5,13
Peripheral blood samples were collected from the
orbital plexus of animals at 0, 7, 14 and 21 dpi. In each
case, the serum was separated and stored frozen (–70ºC)
untilrequiredfornitrate/nitriteandSODanalysis.Inorder
to determine the levels of GT, TBARS and PC in
hepatocytes, 12 animals in each group were sacrificed by
cervical dislocation at 0, 7, 14 and 21 dpi, their livers were
removed and immediately frozen to –70ºC and stored at
thistemperatureuntilrequiredforanalysis.
Assay of antioxidant enzymes
SOD activity was quantified using Fluka (St Louis,
MO,USA) SOD determination
#19160) based on the capacity of the antioxidant
enzyme to inhibit the reduction of 2-(4-iodophenyl)-3-
(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium salt by superoxide anions (generated in
situ by a xanthine/xanthine oxidase system) to yield
the corresponding formazan dye (λmax450 nm).
Total glutathione (GT), i.e. reduced glutathione
(GSH) and glutathione disulfide (GSSG), was assayed
using a kinetic method (Sigma, St Louis, MO, USA;
product code CS0260) in which GSH reduced 5,5’-
dithiobis(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic
acid (TNB) and GSSG, whilst the latter was recycled
in situ by glutathione reductase and NADPH. TNB
was determined spectrophotometrically at 412 nm.
kit (catalogue
Assay of protein carbonyls
The carbonyl moieties formed by the oxidation of
protein by ROS were assayed using a conventional
spectrophotometric procedure based on reaction with
2,4-dinitrophenylhydrazine
corresponding hydrazone derivatives. Determination
of serum PC concentration was carried out according
to a published procedure,14whilst total proteins were
assayed according to the method of Lowry et al.15PC
was determined spectrophotometrically at 370 nm.
toform the
Determination of lipid peroxidation
Determination of TBARS concentration was based
on the ability of the thiobarbituric acid to bind to
oxidizedlipids, thisdosage
described by Buege and Aust.16
determined spectrophotometrically at 535 nm.
wasperformed
TBARS were
as
Determination of serum nitrate/nitrite
Levels of endogenously indirect production of NO
weremeasured bynitrate/nitrite
assessed by reducing serum nitrate to nitrite with
nitrate reductase (1 U/ml) followed by the Griess
reaction.17Nitrite concentrations were determined by
extrapolation from standard curves, constructed using
various concentrations of sodium nitrite, and the
results were expressed as micromolar. Nitrate/nitrite
concentrations was measured by an ELISA plaque
(Molecular Devices, E Max, USA) at 570 nm.
concentrations
Statistical analysis
Between-group differences (NINT × NIT, INT × IT,
NINT × INT and NIT × IT) in the levels of SOD, GT,
nitrate/nitrite, TBARS and PC and between days after
infection were analyzed by one-way analysis of
variance and Tukey post-tests. In all cases, differences
were considered statistically significant when P values
were < 0.05.
Results
No differences between the experimental and control
groups were observed in the levels of nitrate/nitrite at
0 and 7 dpi, PC at 0, 14 and 21 dpi and TBARS at 0
and 14 dpi. A significant increase in the level of
TBARS in the INT group compared with the control
(NINT group) was observed at 7 dpi (Fig. 1A), whilst
at 21 dpi TBARS production in IT group animals was
significantly increased compared with the NIT and
INT groups (Fig. 1C).
Figure 1B reveals that at 7 dpi, treatment with DFO
had induced a significant increase in the levels of PC
in the IT group compared with the NIT group.
Significant increases in serum nitrate/nitrite levels
were also observed at 21 dpi in animals of the IT
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Francisco et al.Increase of reactive oxygen species by desferrioxamine during experimental Chagas’disease

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group when compared with the NIT and INT groups
(Fig. 1D). Figure 2A reveals that at 21 dpi, treatment
with DFO in animals of IT group had induced a
significant increase in serum nitrate/nitrite levels
compared with 0, 7 and 14 dpi. Additionally, serum
nitrate/nitrite levels in infected but untreated mice
(INT group) were significantly higher at 14 dpi than
those of the control animals (NINT group; data not
shown). Figure 2B reveals that at 21 dpi, treatment
with DFO in animals of IT group had induced a
significant increase in SOD activity compared with 0,
7 and 14 dpi.
No differences between the experimental and
control groups were observed in the levels of GT at 0,
7 and 21 dpi or in SOD activity at 0, 7 and 14 dpi (data
not shown). At 14 dpi, GT production in the livers of
T. cruzi-infected mice that had been treated with DFO
(IT group) was reduced significantly in comparison
with non-infected but treated animals (NIT group) as
shown in Figure 3A.
At 21 dpi, SOD activity in serum of mice belonging
to the IT group was increased significantly compared
with those of NIT and INT group animals, indicating
an antioxidant effect of DFO when associated with
infection (Fig. 3B).
No differences at 0, 7, 14 and 21 dpi were observed
in animals of the NINT, NIT and INT groups (data
not shown).
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Increase of reactive oxygen species by desferrioxamine during experimental Chagas’disease
Figure 1 TBARS and PC levels in hepatocytes and serum levels of nitrate/nitrite in mice not infected or infected with T. cruzi
and not treated or treated with desferrioxamine at days 7 and 21. Significant differences (P < 0.05) between groups
are indicated by connecting lines
Figure 2 Serum levels of nitrate/nitrite and serum activities
of SOD in mice infected with T. cruzi and treated
with desferrioxamine at days 0, 7, 14 and 21.
Significant differences (P < 0.05) between days are
indicated by connecting lines

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Discussion
In the present study, levels of PC were elevated in the
IT group (Fig. 1B) suggesting that infection and DFO
together increased oxidative stress in the host in order
to exert some damage on the parasite. Additionally,
the increases in production of TBARS in animals of
the INT group that were observed at 7 dpi (Fig. 1A)
indicate that infection initially augmented these
reactive species. It must be pointed out that different
organs respond in different ways in relation to ROS
production, and that a high degree of oxidative stress
has been detected in the peripheral blood of chagasic
mice.5However, treatment with DFO promoted an
increase in oxidative stress over a longer period of time
(i.e. up to 21 dpi), suggesting that this may represent a
possible mechanism in the reduction of parasitemia.
Nitric oxide (NO) is known to exert microbicidal or
microbiostatic effects against many micro-organisms.3
Gradual increases in the levels of nitrate/nitrite in
animals of the IT group occurred throughout the post-
infection treatment period (Fig. 1D) indicating that the
action of DFO must be associated with infection. The
increase of nitrate/nitrite in the INT group of animals
(data not shown) was expected since T. cruzi infection
stimulates the production of tumor necrosis factor-α
and interferon-γ, and this culminates in the increased
expression of inducible nitric oxide synthase (iNOS) and
the subsequent production of NO by macrophages.18
Although details of the mechanism by which iNOS is
activated are unclear, infection typically increases NO
production by 30%.19
The decrease in hepatic GT concentration in infected
animals that had been treated with DFO (Fig. 3A)
indicates the ability of the drug to increase the defense of
the host against oxidative stress associated infection.
Since GT levels in the NIT group were unchanged in
comparison with both untreated groups, it is likely that
DFO up-regulates GPx only in the presence of T. cruzi
infection. Experimental studies have shown that host
response to acute infection by T. cruzi involves the up-
regulation of the glutathione antioxidant defense system
comprisingGPx,GSH
glutathione.1However,duringtheprogressionof Chagas’
disease in mice, glutathione defense becomes non-
responsive to chronic oxidative stress and cardiac levels
of GPx are reduced; a systemic GPx and SOD down-
regulation is also observed in mice and humans under
chronic conditions.20,21It is important to note that
trypanosomatids do not possess the selenium-dependent
glutathione peroxidase/glutathione reductase system
found in other cells.22
In the present study, the levels of SOD in animals of
the IT group occurred throughout the post-infection
treatment period (Fig. 3B) indicating that the action of
DFO must be associated with infection. In this
respect, the infection-related effect of
important in increasing the level of the antioxidant
enzyme since SOD represents the first line of defense
against ROS. It has previously been established that
SOD activity in the erythrocytes of patients with mild
cardiac impairment was higher than that found in
controls, but no differences were detected in SOD
activity in different chagasic groups.21
In terms of the glutathione and SOD defense
systems, it is clear that DFO exerts an antioxidant
action on the hepatocytes of the host that contributes
to the protection against oxidative damage caused by
Chagas’ disease. Moreover, it would appear that the
effects of DFO are apparent at the start of the
parasitemia peak, leading to a reduction in the
parasitemia curve of T. cruzi in DFO-treated animals
as has been shown previously.8,9It is also possible that
DFO causes an increase in the generation of ROS
and oxidizedformsof
DFO is
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2010 Vol 15No 4
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Francisco et al.Increase of reactive oxygen species by desferrioxamine during experimental Chagas’disease
Figure 3 GT levels in hepatocytes and serum activities of
SOD in mice not infected or infected with T. cruzi
and not treated or treated with desferrioxamine at
days 14 and 21. Significant differences (P < 0.05)
between groups are indicated by connecting lines

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through Fenton-type mechanisms since iron is an
essential catalytic agent in such reactions. The use of
this chelator may provoke a release of iron from
ferritin as ‘free iron’ that was used in this reaction. It
must be concluded, therefore, that the action of DFO
increases the protection of the host against infection
and also acts directly on the parasite itself, thus
explaining the reductions in parasitemia and the rate
of mortality in DFO-treated animals that have been
reported earlier.8,9
However, DFO also delayed
increased production of nitrate/nitrite until 21 dpi,
and this could be the reason why mortalities of
infected animals treated with DFO have been
observed by our group.9
Conclusions
On the basis of the results presented herein, it is
concluded that DFO possesses the capacity to increase
antioxidant defense systems in the host, particularly
by up-regulating SOD, as well as promoting a
measured increase in oxidative stress that exerts
pressure on the parasite. This suggests that DFO
offers a balance of effects with respect to its capacity
to promote protection in the host. The differences
between the parasite and host in terms of the
biochemical systems that afford the antioxidant
defensesofthetwoorganisms
exploitable targets for the development of an effective
chemotherapy for Chagas’ disease.
couldprovide
Acknowledgements
This work was supported by the Coordenaçao de
Aperfeiçoamento de Pessoal de Nivel Superior
(CAPES),Centrode Pesquisas
Fundação Osvaldo Cruz – FIOCRUZ (CPqRR),
Fundação de Amparo a Pesquisa do Estado de Minas
Gerais (FAPEMIG), Rede Mineira de Biotérios
(RMB) and by the Universidade Federal de Ouro
Preto (UFOP).
René Rachou,
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