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Arch Toxicol
DOI 10.1007/s00204-011-0652-y
123
GENOTOXICITY AND CARCINOGENICITY
Protective properties of quercetin against DNA damage
and oxidative stress induced by methylmercury in rats
Gustavo Rafael Mazzaron Barcelos · Denise Grotto · Juliana Mara Serpeloni · José Pedro Friedmann Angeli ·
Bruno Alves Rocha · Vanessa Cristina de Oliveira Souza · Juliana Tanara Vicentini · Tatiana Emanuelli ·
Jairo Kenupp Bastos · Lusânia Maria Greggi Antunes · Siegfried Knasmüller · Fernando Barbosa Jr
Received: 1 September 2010 / Accepted: 13 January 2011
© Springer-Verlag 2011
Abstract Aim of the study was to Wnd out whether con-
sumption of quercetin (QC), an abundant Xavonoid in the
human diet, protects against DNA damage caused by expo-
sure to organic mercury. Therefore, rats were treated orally
with methylmercury (MeHg) and the Xavonoid with doses
that reXect the human exposure. The animals received
MeHg (30 g/kg/bw/day), QC (0.5–50 mg/kg/bw/day), or
combinations of both over 45 days. Subsequently, the glu-
tathione levels (GSH) and the activities of glutathione per-
oxidase (GPx) and catalase (CAT) were determined, and
DNA damage was measured in hepatocytes and peripheral
leukocytes in single cell gel electrophoresis assays. MeHg
decreased the concentration of GSH and the activity of GPx
by 17 and 12%, respectively and caused DNA damage to
liver and blood cells, while with QC no such eVects were
seen. When the Xavonoid was given in combination with
MeHg, the intermediate and the highest concentrations (5.0
and 50.0 mg/kg/bw/day) were found to cause DNA protection;
DNA migration was reduced by 54 and 65% in the hepato-
cytes and by 27 and 36% in the leukocytes; furthermore, the
reduction in GSH and GPx levels caused by MeHg treat-
ment was restored. In summary, our results indicate that
consumption of QC-rich foods may protect Hg-exposed
humans against the adverse health eVects of the metal.
Keywords Quercetin · Methylmercury · DNA damage ·
Antioxidant · Redox status · Comet assay
Introduction
Mercury (Hg) is one of the most hazardous metals in the
environment, and it is well documented that its toxicity
involves oxidative damage to macromolecules (Beyersmann
and Hartwig 2008; Chuu et al. 2008; Mori et al. 2007). Several
experimental and epidemiological studies demonstrated
G. R. M. Barcelos · D. Grotto · J. M. Serpeloni ·
V. C. de Oliveira Souza · L. M. G. Antunes · F. Barbosa Jr
Departamento de Análises Clínicas,
Toxicológicas e Bromatológicas,
Faculdade de Ciências Farmacêuticas de Ribeirão Preto,
Universidade de São Paulo, Av. do Café, s/no,
Ribeirão Preto, São Paulo CEP 14040-903, Brazil
J. P. F. Angeli
Departamento de Bioquímica, Instituto de Química,
Universidade de São Paulo, Av. Professor Lineu Prestes 748,
São Paulo CEP 05508-900, Brazil
B. A. Rocha · J. K. Bastos
Departamento de Ciências Farmacêuticas,
Faculdade de Ciências Farmacêuticas de Ribeirão Preto,
Universidade de São Paulo, Av. do Café, s/no,
Ribeirão Preto, São Paulo CEP14040-903, Brazil
J. T. Vicentini
Departamento de Analises Clínicas e Toxicológicas,
Centro de Ciencias da Saúde, Universidade Federal de Santa
Maria, Av. Roraima 1000, Santa Maria, Rio Grande
do Sul CEP 97105-900, Brazil
T. Emanuelli
Departamento de Tecnologia e Ciência dos Alimentos,
Centro de Ciências Rurais, Universidade Federal de Santa Maria,
Av. Roraima 1000, Santa Maria,
Rio Grande do Sul CEP 97105-900, Brazil
S. Knasmüller (&)
Department of Inner Medicine I,
Institute of Cancer Research, Medical University of Vienna,
Borschkegasse 8A, 1090 Vienna, Austria
e-mail: siegfried.knasmueller@meduniwien.ac.at
Arch Toxicol
123
that exposure to its organic form, methylmercury (MeHg),
which is found in foods causes neurotoxic eVects (Dolbec
et al. 2000), damage to the immune and renal system
(Moszczynski et al. 1998; Rutowski et al. 1998), infertility
(Boujbiha et al. 2009), cardiovascular diseases (Virtanen
et al. 2007), and cancer (IARC 1993). Furthermore, it is
notable that several studies indicated associations between
genetic damage induced by MeHg and alterations of the
redox status (Ben-Ozer et al. 2000; Grotto et al. 2009;
Grotto et al. 2010), and it is conceivable that DNA damage
may play a role in its adverse health eVects.
It has been demonstrated that consumption of foods con-
taining antioxidant nutrients such as Xavonoids protects
against the toxicity of heavy metals (Fang et al. 2002;
Giuliano 2000; Kahkonen et al. 1999; Passos et al. 2007). A
highly promising chemopreventive dietary constituent is
quercetin (QC), which is one of the most abundant Xavo-
noids in human foods and found in fruits and vegetables,
including blueberries, onions, curly kale, broccoli, and leek
(Manach et al. 2004). Comparisons of the antioxidant
activities of QC with that of other Xavonoids indicated that
QC is a more potent radical scavenger than other structurally
related compounds (Boots et al. 2008; Haenen et al. 1997);
furthermore, it was shown that it activates also antioxidant
as well as drug-metabolizing enzymes (Noroozi et al. 1998).
Humans are exposed to mercury compounds at the work-
places, i.e., in chlorine-producing (Cebulska-Wasilewska
et al. 2005) and light bulb factories (Zavariz and Glina
1993) or via consumption of contaminated foods. An exam-
ple for the latter form of exposure is the intake of contami-
nated Wsh by populations in the Amazon basin (Passos et al.
2007). In this context, it is notable that Fillion et al. (2006)
reported a signiWcant dose-dependent relation between Hg
exposure and elevated blood pressure in populations living
in the Brazilian Amazon, and Amorim et al. (2000) found
cytogenetic damage to blood cells of the same population.
Aim of the present study was the investigation into
potential protective eVects of QC against the toxic eVects
caused by methylmercury. Therefore, experiments were
carried out under controlled laboratory conditions to study
the impact of the consumption of the Xavonoid on DNA
damage to single cell gel electrophoresis (SCGE) assays in
liver and blood cells of rats. These experiments are based
on the determination of DNA migration in an electric Weld
and reXect the formation of single- and double-strand
breaks and apurinic sites (Tice et al. 2000). As they are
time- and cost-eVective, they are increasingly used to assess
genotoxic eVects in inner organs and blood cells in animal
and human studies (Cemeli et al. 2009; Hoelzl et al. 2009).
Furthermore, we studied in the same experiments also
the eVects of QC on alterations of several parameters of the
redox status caused by the metal, namely on the activities
of the antioxidant enzymes catalase (CAT) and glutathione
peroxidase (GPx) and also on the concentrations of reduced
glutathione (GSH), which is one of the most potent endoge-
nous antioxidants (Singh 2002).
The experiments were conducted in such way that the
treatment of the animals with the metal compound and with
the Xavonoid reXects the exposure situation of human
population.
Materials and methods
Chemicals
Methylmercury chloride (CAS 115-09-3), hydrogen perox-
ide (CAS 772-84-1), nicotinamide adenine dinucleotide
phosphate-oxidase (NADPH, CAS 100929-71-3), reduced
glutathione (GSH, CAS 70-18-8), reductase glutathione
(GSHR, CAS 9001-48-3), sodium azide (CAS 26628-22-
8), trypan blue (CAS 72-57-1), ethidium bromide (CAS
1239-45-8), tetramethylammonium hydroxide (CAS 75-59-
2), and dimethyl sulfoxide (DMSO) (CAS 67-68-5) came
from Sigma–Aldrich (St. Louis, MO, USA). Quercetin
(3,3⬘,4⬘,5,7-pentahydroxyXavone, CAS 6151-25-3) was
obtained from Merck (Darmstadt, Germany). Ketamine and
xilazine were purchased from Bayer (São Paulo, Brazil).
Low melting point agarose (LMP) and normal melting
point (NMP) agarose were obtained from Invitrogen (Cali-
fornia, CA, USA). All other chemicals, reagents and buVers
were analytical grade products from Sigma (St Louis, MO,
USA).
Animals
The experiments were carried out with male Wistar rats
weighting on average 200 §20 g, which were obtained
from the Central Bioterium (University of São Paulo,
Ribeirão Preto, Brazil). The animals were kept under a 12-h
light/dark cycle in an acclimatized room at 22–25°C and
had free access to food (standard ration from Guabi, Cam-
pinas, Brazil) and water. The animals were used according
to the guidelines of the Committee on Care and Use of
Experimental Animal Resources, University of São Paulo,
Brazil (Approved protocol number: 09.1.457.53.1).
Dose selection and experimental groups
The exposure dose of the animals to MeHg was chosen on
the basis of a recent study of Passos et al. (2007), in which
the daily exposure to MeHg of riparian Amazon popula-
tions that are environmentally exposed to the metal via Wsh
consumption was estimated. The QC doses were chosen on
the basis of daily intake estimates from human studies
(Formica and Regelson 1995; Lamson and Brignall 2000).
Arch Toxicol
123
The animals were divided into eight groups (I: control
(H2O); II: MeHg (30 g/kg/bw); III: QC 1 (0.5 mg/kg/bw);
IV: QC 2 (5.0 mg/kg/bw); V: QC 3 (50.0 mg/kg/bw); VI:
QC 1 + MeHg; VII: QC 2 + MeHg; and VIII: QC
3 + MeHg) and were treated by gavage for 45 days. After
the treatment, the rats were killed by an overdose with keta-
mine and xylazine (300 and 30 mg/kg/bw, respectively).
Subsequently, blood was collected by decapitation, and the
livers washed with phosphate-buVered saline (PBS, pH 7.4)
before removal. Part of the total blood was used for comet
assays and to measure the hemoglobin concentrations; it
was aliquoted in heparinized microcentrifuge tubes after
collection and stored at ¡80°C until analysis.
Comet assay in peripheral leukocytes and hepatocytes
Whole blood was used for the evaluation of DNA damage
to leukocytes (da Silva et al. 2000). The nuclei were iso-
lated from the liver cells by homogenization with a Potter–
Elvehjem (Krackeler ScientiWc Inc., Albany, USA) and
subsequent centrifugation (for details, see Sekihashi et al.
2002).
The SCGE assays were carried out according to the pro-
tocol of Singh et al. (1988). After exposure to the com-
pounds, 20 l of blood or nuclei from the liver suspensions
was mixed with agarose and transferred to agarose-coated
slides which were coverslipped and cooled at 4°C for
20 min. After removal of the coverslips, the slides were
immersed in fresh lysis solution for 1 h at 4°C. Thereafter,
they were transferred to an electrophoresis chamber with
buVer (300 mM NaOH and 1.0 mM EDTA pH > 13), and
electrophoresis was conduced under standard conditions
(25 V; 300 mA; 1.25 Vcm¡1) for 20 min. Subsequently, the
slides were neutralized, air-dried, and Wxed in absolute eth-
anol for 10 min.
The slides were stained with ethidium bromide and eval-
uated under a Xuorescence microscope (Nikon, Japan)
under 40£ magniWcation. From each sample, two slides
were made, and from each, 50 cells were evaluated per ani-
mal. Comets were scored using the Comet Assay IV soft-
ware (Perceptive Instruments, Haverhill, England); the
percentage of DNA in tail was determined as a parameter of
DNA damage. All experiments were carried out according
to the guidelines for SCGE assays (Hartmann et al. 2003).
Catalase (CAT) activity
The activity of the antioxidant enzyme catalase (CAT) was
measured in red blood cells as described previously by
Aebi (1984). The method is based on changes in the absor-
bance at 240 nm due to the catalase-dependent decomposi-
tion of H2O2. The activity of the enzyme was related to the
hemoglobin content (/g of Hg). Hemoglobin was deter-
mined by the use of a commercial kit (Hemoglobina Mono-
test, Inlab Diagnostica, São Paulo, Brazil) according to the
manufacture’s description.
Glutathione peroxidase (GPx) activity
The activity of the glutathione peroxidase (GPx) was deter-
mined spectrophotometrically. This method is based on the
oxidation of NADPH, which can be measured as the
decrease in absorbance at 340 nm (Paglia and Valentine
1967). Results are expressed in nmol NADPH/min/ml
erythrocytes.
Reduced-glutathione (GSH) levels
Reduced-glutathione (GSH) levels were determined in
erythrocytes by addition of 5-5⬘-dithio-bis(2-nitrobenzoic
acid) (DTNB), as described by Ellman (1959). DTNB, a
symmetric aryl disulWde, reacts with free thiols to form
disulWde plus 2-nitro-5-thiobenzoic acid, the latter reaction
product can be quantiWed by its absorbance at 412 nm.
Results are expressed as micromoles per milliliter (mol/
ml) erythrocytes.
Measurements of mercury in liver and blood
Determination of Hg concentrations in blood and liver was
performed as described by Palmer et al. (2006) and Batista
et al. (2009), respectively, using inductively coupled
plasma mass spectrometry (ICP-MS) (ELAN DRCII, Perkin
Elmer, USA). Results are expressed in mg/l and g/g.
Statistical analysis
All data analyses were performed with GraphPad Prism 5
Project software system (La Jolla, CA, USA). Results are
reported as means §standard deviations (SD). The results
of diVerent experiments were analysed using one-way
ANOVA and Dunnett’s test. Pvalues ·0.05 were con-
sidered as statistically signiWcant.
Results
Treatment of the animals with the mercury compound
caused a clear increase in the Hg levels in liver and blood.
It can be seen in Table 1 that the metal concentrations in the
blood stream were higher than those found in the liver; fur-
thermore, it is notable that co-treatment of the animals with
the Xavonoid had no signiWcant impact on the metal levels.
Experiments concerning the evaluation of the eVects of
QC and on the genotoxic eVects induced by the mercury
compound are summarized in Fig. 1. Treatment of the animals
Arch Toxicol
123
with MeHg increased the extent of DNA migration in liver
cells sixfold over the background values found in the con-
trol animals, a similar eVect was observed in the leuko-
cytes. QC itself did not cause DNA damage in the indicator
cells in the range between 0.5 and 50.0 mg/kg/bw.
When the Xavonoid was given in combination with the
metal, comet formation was reduced. In hepatocytes, the
reduction was 65% at the highest dose tested (50 mg/kg/
bw), at the medium dose (5.0 mg/kg/bw) the decrease was
54%, respectively; at the lowest dose (0.5 mg/kg/bw) no
signiWcant protective eVect was seen. Also, in the blood
cells a clear reduction in comet formation was seen, which
was signiWcant at the higher concentrations.
The results of the measurements of the antioxidant
enzymes and of the GSH levels are summarized in Table 2.
It can be seen that QC treatment had no impact at the
CAT levels, while signiWcant induction of GPx after the
treatment of the animal with the Xavonoid was observed;
likewise also the GSH concentrations were increased. At
the highest Xavonoid dose tested (50.0 mg/kg/bw), the
induction of GPx was 34% and that of the tripeptide 16%.
The metal compound caused clear adverse eVects, i.e., it
reduced the levels of GPx (12%) and of GSH (17%); fur-
thermore, also a reduction in the activity of CAT was
found, but this latter result did not reach signiWcance.
Table 2 summarizes also the results that were obtained
after combined treatment of the animals with the metal and
with diVerent doses of the Xavonoid. In all groups, the pat-
tern of changes caused by the metal was reversed, i.e., the
GSH levels were restored and also the decline of the activi-
ties of the antioxidant enzymes CAT and GPx was reverted.
In the latter case, a signiWcantly higher level compared with
the group that had been treated with the metal compound
alone was seen with all three doses of Xavonoid.
Discussion
The results of the present study indicate that QC, which is
one of the most abundant Xavonoids in plant derived foods,
protects against DNA damage and alterations of the redox
status induced by organic mercury under experimental con-
ditions that are relevant for humans (see below).
It is known from earlier studies that MeHg induces DNA
damage to diVerent experimental systems (De Flora et al.
1994), and the present Wndings are in agreement with the
results obtained in earlier SCGE experiments which
showed that the metal induces DNA migration in inner
organs of rats (Ariza et al. 1998). Also formation of 8-OHdG
was induced by mercury in inner organs such as liver and
kidneys (Jin et al. 2008). This latter Wndings support that
assumption that oxidative damage accounts (at least partly)
for the comet formation which was seen in the present
study.
It is notable that no DNA-damaging eVects of QC were
seen in the same experimental system (Fig. 1a, b). This is
an interesting observation as the Xavonoid induced DNA
damage to various in vitro models (Caria et al. 1995; RueV
Table 1 Mercury (Hg) concentrations in blood and liver of male
Wistar rats. Six animals per group were treated daily with the diVerent
compounds by gavage over a period of 45 days
aAmount in parenthesis indicates the daily dose. QC was dissolved in
a 1.0% aqueous solution of DMSO; MeHg was dissolved in distilled
water
GroupsaHg in blood (mg/l)
(Mean §SD)
Hg in liver (g/g)
(Mean §SD)
Control (water) 0.004 §0.003 0.03 §0.01
MeHg (30 g/kg/bw) 11.21 §2.67 0.48 §0.08
QC 1 (0.5 mg/kg/bw) 0.00 §0.00 0.02 §0.01
QC 2 (5.0 mg/kg/bw) 0.00 §0.00 0.01 §0.01
QC 3 (50.0 mg/kg/bw) 0.00 §0.00 0.02 §0.01
MeHg + QC 1 12.01 §3.76 0.51 §0.11
MeHg + QC 2 12.57 §3.21 0.50 §0.15
MeHg + QC 3 10.51 §2.67 0.49 §0.13
Fig. 1 Impact of quercetin (QC) on induction of DNA damage by
methylmercury (MeHg) in hepatocytes (a) and lymphocytes (b)of
rats. The animals (6 per group) were treated by gavage with diVerent
doses of the Xavonoid (QC 0.5; 5.0, and 50.0 mg/kg/bw) in combina-
tion with the metal compound (30 g/kg/bw) over a period of 45 days.
Bars indicate means §SD, stars indicate statistical diVerence in the
metal group (P·0.05; one-way ANOVA and Dunnett’s test)
Arch Toxicol
123
et al. 1992), but it was in general, not active in rodents
(Caria et al. 1995; Utesch et al. 2008). This diVerence may
be due to release of ROS under in vitro conditions, which
does not take place under in vivo conditions (Gaspar et al.
1994; RueV et al. 1992).
As described above (Table 1), the results of the Hg mea-
surements in blood and liver show that treatment of the ani-
mals with QC has no impact on the metal levels. This
indicates that the Xavonoid does not alter the absorption
and/or excretion of organic mercury. In this context, it is
notable that Passos et al. (2007) reported that increased
uptake of fruits and vegetables, which are sources of dietary
uptake of QC in Hg-exposed humans, leads to a decrease in
the levels of the metal in the blood. The authors hypothe-
size that this observation may be due to decreased uptake of
the metal in the gastrointestinal tract.
It is well documented that MeHg exposure leads to oxi-
dative damage to macromolecules due to formation of ROS
(for review, see Clarkson and Magos 2006). Furthermore, it
was also shown that the metal binds to endogenous biomol-
ecules with –SH groups which explains the decrease in the
GSH levels and also the decline of GPx activity which was
seen in earlier trials (Chen et al. 2005; Grotto et al. 2010)
and also in the present study (Table 2). In this context, it is
notable that also in a human study increased levels of the
tripeptide and of the enzyme were seen in individuals that
were occupationally exposed to the metal (Bulat et al.
1998).
It is well documented that QC is a potent antioxidant and
that its action involves direct scavenging of ROS as well as
indirect eVects such as induction of antioxidant enzymes
(Kang et al. 2009; Lavoie et al. 2009). Our Wndings con-
cerning the improvement in the GSH status and the induc-
tion of GPx are in agreement with earlier Wndings obtained
with rodents. For example, Meyers et al. (2008) showed
such eVects in mice and demonstrated that the Xavonoid
counteracts the eVects caused by mercury; on the contrary,
Fiorani et al. (2001) reported that the Xavonoid is not able
to prevent the reduction in the concentrations of the tripep-
tide in rabbit erythrocytes after treatment with dehydroa-
scorbic acid.
As described above, no alterations of the CAT activity
were observed in the present experiments. This observation
is in agreement with the Wndings of a chronic treatment
study with rats in which a low dose of the Xavonoid
(50 mg/kg/bw) was given to the animals (Vidhya and Indira
2009), while in another trial with a shorter treatment time
and higher levels of QC a signiWcant decrease in the activ-
ity of this enzyme was observed (Breinholt et al. 1999).
The results of the biochemical measurements show very
clearly that QC improves the disturbance of the redox status
caused by exposure of the animals to the metal. This obser-
vation provides a possible explanation for the prevention of
comet formation in liver and blood cells.
As mentioned in the introduction section, it is conceivable
that the adverse eVects of mercury in humans are associated
with oxidative damage, and it is also likely that damage to
the genetic material is responsible for the long-term eVects
of the metal such as infertility (Boujbiha et al. 2009) and
cancer (IARC 1993). Extrapolation of the results of the ani-
mal experiments indicates that the daily uptake of QR
required to cause protective eVects is ¸350 mg/P/day.
According to an estimate of Lamson and Brignall (2000),
the daily ingestion in Western countries lies in the range
between 25 and 50 mg per person. For Brazil, no uptake
assessments were found in the literature, but comparisons
between diVerent studies indicated that the overall con-
sumption of Xavonoids is in this country two- to fourfold
higher as that in Europe (Arabbi et al. 2004; Hertog et al.
1992, 1993); since QR is the most abundant compound in
this group, this can be taken as an indication that its overall
daily uptake is higher in Brazil. In this context, it is notable
Table 2 Impact of quercetin (QC) and methylmercury (MeHg) treatment on the activities of antioxidant enzymes (CAT and GPx) and GSH. The
animals (male Wistar rats; 6/group) were treated daily with the diVerent compounds by gavage over a period of 45 days
aAmount in parenthesis indicates the daily dose; b/g of Hb; cmmol NADPH//min/ml of erythrocytes; dmol/ml erythrocytes
* Statistically diVerent from the control group; #statistically diVerent from the MeHg-treated group (P·0.05; ANOVA and Dunnett’s test)
GroupsaCAT activityb
(mean §SD) (%) GPx acitivityc
(mean §SD) (%) Reduced-GSH levelsd
(mean §SD) (%)
Control (water) 274.7 §98.1 100.0 33.2 §2.4 100.0 0.81 §0.05 100.0
MeHg (30 g/kg/bw) 201.9 §36.9 73.5 29.2 §5.0* 87.9 0.67 §0.07* 82.7
QC 1 (0.5 mg/kg/bw) 290.1 §91.7 105.6 39.7 §3.2#119.5 0.74 §0.04 91.3
QC 2 (5.0 mg/kg/bw) 277.1 §46.3 100.8 45.0 §3.4*#135.5 0.98 §0.10*#120.9
QC 3 (50.0 mg/kg/bw) 283.9 §47.8 103.3 44.8 §5.4*#134.9 0.94 §0.08*#116.0
MeHg + QC 1 224.2 §52.6 81.6 35.3 §5.5#106.3 0.63 §0.09* 77.8
MeHg + QC 2 260.2 §37.3 94.7 37.4 §5.0#112.6 0.73 §0.05 90.1
MeHg + QC 3 248.2 §38.2 90.3 36.8 §6.9#110.8 0.76 §0.10#93.8
Arch Toxicol
123
that chemical analyses indicate that the QR concentrations in
certain vegetables such as kale and red onions that contain
high levels are higher in Brazil as those detected in these veg-
etables in other countries (Huber et al. 2009). It was found
that onion cultivars grown in Brazil contain more than
900 mg/100 g (Arabbi et al. 2004) and similar levels were
found in speciWc types of lettuce (Crozier et al. 1997). Con-
sumption of such cultivars and of QR-rich foods in general
will lead to uptake levels that correspond to those used in the
present study. Another possible option is the consumption of
QR or its glucoside as a dietary supplement; in a number of
intervention studies, the Xavonoid was given at a daily dose
of 1,000 mg/P without any adverse side eVects (Davis et al.
2010; Cureton et al. 2009; Ganio et al. 2010). The present
Wndings can be taken as an indication that QR may protect
against the adverse eVects caused by mercury in man pro-
vided that the uptake levels are suYciently high.
Acknowledgments We would like to thank Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional para
o Desenvolvimento CientíWco e Tecnológico (CNPq) and Coorde-
nação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES/
DS) for Wnancial support.
ConXict of interest The authors declare that they have no conXict of
interest.
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