The Effect of Chemical Therapy with Bleomycin

Carlos Alberto da Silva
1
, Karina M Cancelliero
2
, Dirceu Costa
1,2
1Department of Physiotherapy, Methodist University of Piracicaba (UNIMEP). Piracicaba, Brazil.
2Department of Physiotherapy, Federal University of São Carlos (UFSCar). São Carlos, Brazil
ABSTRACT
Objective The objective of the present study was to evaluate the effect of bleomycin sulfate on parameters related to the
functionality of pancreatic tissue, with emphasis on the glucose tolerance test, insulin tolerance test, insulinemia and static secretion
of insulin as well as the insulin receptor, and PKA, PKC and GLUT2 concentrations in the pancreatic islets. Design Twenty-four
male rats were divided into 2 groups: control and treated with bleomycin (2.5 mg/kg, intratracheal mode). After 7 days, the animals
were euthanized and the analyses were carried out. Statistics The normality and the homoscedasticity of the data distribution were
tested and ANOVA was applied. The Tukey post hoc test followed ANOVA for the comparison of the static insulin secretion test at
different glucose concentrations. Results In the glucose tolerance test, the bleomycin group showed a larger area (17,306±539
mg/dLx60min) than that of the control group (9,151±517 mg/dLx60min) and in the insulin tolerance test, there was a greater
percentage fall in glycemia (8.08±0.56%) in the bleomycin than in the control group (3.87±1.14%). The bleomycin group also
presented a reduction in insulin secretion and an increase in plasmatic insulin concentration in the static insulin secretion test. With
respect to the concentrations of the insulin receptor, GLUT2, PKC and PKA in the pancreatic islets of the bleomycin group, there
was an increase in GLUT2 (48.4%) and PKC (70.8%) and a reduction in PKA (38.5%). Conclusion During treatment with
bleomycin, innumerable chemical-metabolic alterations were unleashed in the tissues which were not primary targets of the chemical
therapy and which could compromise the homeostasis of the systems taking part in the glycemic adjustment, predisposing the
organism to the development of a pre-diabetic pattern whose degree of incidence or reversibility is still unknown to the scientific
community.
INTRODUCTION
Pulmonary fibrosis is a respiratory disease which can
be caused by the administration of bleomycin sulfate,
an antineoplastic agent which can cause this disease as
a side effect in the treatment of humans [1]. This toxic
effect has been used in experimental models in animals
[2], but the mechanism involved in the induction of the
disease is still not completely understood [3].
It is important to point out that the time of
administration of the bleomycin can influence the
respiratory disease induced. According to Borzone et
al. [4], the earlier stages of the pulmonary injury
induced by bleomycin are associated with biochemical
and functional changes which are similar to those of
human pulmonary fibrosis whereas, in studies of more
chronic stages, the pulmonary function changes are not
compatible with a restrictive disease, but are more
similar to those described in studies on humans with
chronic obstructive pulmonary disease in which the
mural inflammation and fibrosis of the bronchioles are
associated with emphysematous changes.
There are various studies in the literature related to
treatment with bleomycin, dealing with analyses
related to the pulmonary tissue and the local and
systemic effects resulting from the pathological
condition of this tissue. However, presently, no study
has been found concerning the effect of bleomycin on
the pancreas, an organ of extreme importance to the
organism, which could be influenced by treatment with
this chemical therapeutic agent since it can cause
alterations in tissue DNA due to its antineoplastic
effect, and without tissue specificity, could cause
pancreatic injury.
Thus, in the knowledge that treatment with bleomycin
is widely used, and has already been described in
experimental models with animals focused on the
pulmonary fibrosis condition, the objective of the
present study was to evaluate the effect of bleomycin
Received November 19th, 2008 - Accepted March 17th, 2009
Key words Bleomycin; Blood Glucose; Insulin; Pancreas;
Pulmonary Fibrosis
Abbreviations SLC2A2: solute carrier family 2 (facilitated
glucose transporter), member 2 (also known as GLUT2)
Correspondence Dirceu Costa
Universidade Federal de São Carlos (UFSCar), Departamento de
Fisioterapia, Rodovia Washington Luís (SP-310), km 235, CEP
13565-905, São Carlos, São Paulo, Brazil
Phone: +55-16.3351.8343; Fax: +55-16.3361.2081
E-mail: dirceu@power.ufscar.br
Document URL http://www.joplink.net/prev/200905/15.html

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sulfate on parameters related to the functionality of
pancreatic tissue, with emphasis on the glucose
tolerance test, insulin tolerance test, insulinemia and
static secretion of insulin as well as the insulin
concentration and the concentrations of protein kinase
A (PKA), protein kinase C (PKC) and SLC2A2 [solute
carrier family 2 (facilitated glucose transporter),
member 2; also known as GLUT2] in the pancreatic
islets.
The importance of this work in the evaluation of
animal models should be emphasized, due to invasive
analyses and the need for tissue biopsies, respecting the
norms of the ethics committee on animal
experimentation.
MATERIALS AND METHODS
Animals
Twenty-four male Wistar rats (3 to 4 months old; 250-
300 g) were maintained under controlled vivarium
conditions, with free access to food and water. The
animals were divided into 2 groups (12 in each group):
control and treated with bleomycin.
Bleomycin Treatment
The treatment was carried out using a single dose of
bleomycin (Oncoprod, São Paulo, Brazil; 2.5 mg/kg
weight, intratracheal mode) [5] and, after a seven-day
period, the animals were euthanized and the analyses
were carried out.
Sampling
After the experimental period, the animals were
sacrificed. The blood was collected and centrifuged,
and the plasma was separated and dispatched for
analysis. The pancreatic tissue was isolated and
dispatched for evaluation. The animals of each group
were divided into two subgroups (6 eah): one subgroup
for the analysis of the static secretion of insulin and the
other subgroups for Western Blot analysis (Sigma
Chemical Co., St. Louis, MO, U.S.A.).
Glucose Tolerance Test
For the glucose tolerance test, the animals were
anaesthetized with sodium pentobarbital (40 mg/kg
weight, i.p.) and, after 40 minutes, an incision was
made close to the femoral vein from where the blood
sample was collected. After the first collection, glucose
was injected (1 g/kg weight), new samples were
collected after 5, 10, 15, 20, 30 and 60 minutes, and
glycemia evaluated using a glucosimeter (Accu
Check®, Roche, São Paulo, Brazil). The area under the
blood glucose curve was evaluated.
Insulin Tolerance Test
Since the beta cell response to an overload of glucose
was shown to be altered, an insulin tolerance test was
carried out to evaluate tissue sensitivity. For the insulin
tolerance test, the animals were anaesthetized with
sodium pentobarbital (40 mg/kg weight, i.p.) and, after
40 minutes, an incision was made close to the femoral
vein from where the blood sample was collected. After
the first collection, 1 U/kg weight (1 U/mL) of regular
insulin (Biobrás®, Montes Claros, Brazil) was injected;
new samples were collected after 0, 2.5, 5, 10 and 20
minutes, and the glycemia was evaluated using a
glucosimeter (Accu Check®, Roche, São Paulo, Brazil).
Insulin tolerance test was represented by a percentage
fall in glycemia (KiTT) in the presence of insulin.
Plasmatic Insulin
Plasma samples obtained by centrifuging the blood
were sent for an analysis of insulinemia (ng/mL),
carried out using a radioimmune assay (Du Pont New
Research Products, Boston, MA, U.S.A.).
Static Secretion of Insulin
Islets of Langerhans were isolated using the technique
described by Moskalewski [6] as applied to the murine
pancreas by Lacy and Kostianovsky [7], with the
modifications of Boschero et al. [8] and Sutton et al.
[9]. After laparotomy and localization of the common
bile duct, this was occluded at the extreme distal end
close to the duodenum, and dissected close to the
hepatic pedicle, where a polyethylene cannula was
introduced in the discharge direction. About 8 mL of
Hanks solution containing 8 mg of collagenase was
injected via the cannula, causing rupture of the acinous
tissue by retrograde flow. The pancreas was then
ablated and transferred to a glass test tube (12x12 cm)
and incubated for 18 minutes at 37ºC.
The contents, still at 37ºC, were then shaken vigorously
for one minute and poured into a beaker. After mixing
with Hanks solution, the contents were stirred slowly,
ejected with a syringe and decanted for 3 minutes. The
supernatant was discarded and the sediment re-
suspended in Hanks solution. After repeating this
procedure 4 times, the final product was transferred to
a Petri dish, from which, under a magnifying glass, the
islets were collected by aspiration using a glass pipette
with a long tapered tip. The isolated rat islets were
collected in a polyethylene plate containing 24 wells,
each containing 0.5 mL of Krebs-Ringer buffer-
solution supplemented with bovine albumin, to which
glucose (5.6 mM) was added. All isolated pancreatic
islets were collected and formed one pull of islets and
each well contained 5 pancreatic islets. After
incubating for 45 minutes (pre-incubation) at 37ºC in a
carbogenic atmosphere (pH 7.4), the Krebs solution
was substituted by 1.0 mL of the same buffer
containing different glucose concentrations: 2.8, 5.6,
8.3, and 16.7 mmol/L. The plate was then incubated for
an additional 90 minutes under the same conditions as
above. After this period, the plates were placed in a
freezer (-20ºC) for 10 minutes, and the supernatant in
each well, separated from the cell, was transferred to
polyethylene tubes and maintained at -20ºC until the
secreted insulin was dosed one hour after the islets
were collected. The normalization of the method was
related to the use of the same number of pancreatic
islets/well. During freezing, no evidence of cell

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breakage was found. The insulin secreted by the islets
after incubation or plasmatic insulin was evaluated
using the method described by Herbet [10], as modified
by Scott et al. [11].
Dosage of the Insulin Receptor, PKA, PKC and
GLUT2 in the Pancreatic Islets
Western Blot methodology was used to detect the cell
expression levels of the insulin receptor, the extruded
proteins, and the movement of the PKC and PKA
insulin granules and of the GLUT2. Groups of 500
recently-isolated islets, incubated for 2.5 hours in
Krebs solution containing 8.3 mM glucose, were
centrifuged quickly and the supernatant discarded.
Two-hundred μL of buffer specific for immuno-
precipitation was then added and the islets were then
polytenized in this solution for approximately 10
seconds, after which thhe homogenate was centrifuged
at 3000 g for 10 min.
The precipitate was discarded and the protein dosed in
the supernatant obtained, using the BioRad Protein
Assay-Dye Reagent Concentrate (Melville, NY,
U.S.A.). An albumin standard curve was used as the
reference.
The samples were then incubated at 37ºC for 1 h in
20% by volume of Laemmli 5X buffer (0.1%
bromphenol blue, 1 M sodium phosphate, 50% glycerol
and 10% sodium dodecyl sulfate (SDS)).
The following biphasic gel was used in the
electrophoretic run: stacking gel (4 mM EDTA, 2%
SDS, 750 mM trizma base, pH 6.7) and running gel (94
mM EDTA, 2% SDS, 50 mM trizma base, pH 6.7).
The run was carried out at 200 V for approximately 30
min with running buffer (200 mM trizma base, 1.52 M
glycine, 7.18 mM EDTA and 0.4% SDS), diluted 1:4.
The samples were transferred to a nitrocellulose
membrane (BioRad, Melville, NY, U.S.A.). The
transfer was carried out for 60 min at 30 V on ice,
bathed by transfer buffer (25 mM trizma base, 192 M
glycine).
After transfer, the membrane was blocked with 5%
skimmed milk in tris saline solution (TBS) (1 M trizma
base, 5 M NaCl, 0.5% tween 20) overnight at 4ºC. The
proteins related to the study were detected on the
nitrocellulose membrane by incubating at room
temperature for 2 h, with the following specific
monoclonal antibodies: anti-insulin receptor, anti-PKA,
anti-PKC, and anti-GLUT2 (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA, U.S.A.; 1:500
dilution in TBS with 3% skimmed milk). The
membrane was then incubated with the antibody
conjugated with peroxidase HPB (1:5,000 dilution or 2
μg/mL in TBS buffer). The reaction with peroxidase
was detected by autoradiography, soon after the
reaction with Pierce’s Super Signal kit (Thermo Fisher
Scientific, Rockford, IL, U.S.A.).
RT-PCR
PCR was carried out on 12.5 μL of a mixture
containing the following components: Taq polymerase
buffer, 50 mM MgCl2, 10 mM of each
deoxynucleoside triphosphate, 2.5 U/μL Taq DNA
polymerase, 10 pmol of the primer sense, 10 pmol of
the primer anti-sense and cDNA. Five μL aliquots of
the PCR product were analyzed by gel electrophoresis
in 1.0% agarose prepared in TBE buffer. After staining
with ethidium bromide (0.5 μg/mL), the gel was
photographed under UV light and quantified in an
Eagle Eye II apparatus.
ETHICS
The animals were treated in accordance with the
recommendations of the animal ethics committee
which approved the project (protocol nº 754-2).
STATISTICS
Data are reported as mean±SD. The data were initially
tested for normality (Kolmogorov-Smirnov test) and
homoscedasticity (Barlett criterion). Since the data
presented normal distribution and homoscedasticity,
analysis of variance (ANOVA) was used. The Tukey
post hoc test followed ANOVA for comparison of the
static insulin secretion test at glucose concentrations of
2.8 mM, 8.3 mM, and 22.2 mM, respectivelyA two-
tailed significant level of 5% (P<0.05) was fixed. The
software used was Origin 6.0® (Microcal Software,
Inc., Northampton, MA, U.S.A.) and Prism®
3.0
(GraphPad Software, Inc., San Diego, CA, U.S.A.).
RESULTS
The bleomycin-treated group showed a statistically
larger glucose tolerance test area (17,306±1,205
mg/dLx60min, P<0.001) than that of the control group
(9,151±1,463 mg/dLx60min), as shown in Figure 1.
The insulin tolerance test is shown in Figure 2. The
treated group presented a greater percentage fall in
glycemia (-8.08±1.36% P<0.001) as compared to the
control group (-3.87±0.92%). This shows that
bleomycin altered the insulin sensitivity since the speed
of reduction of the glycemia was greater.
Figure 1. Glucose tolerance test (GTT) applied to the control and
bleomycin-treated groups. The mean blood glucose analyzed 0, 5,
10, 15, 20, 30 and 60 minutes after a glucose overload is shown. The
treated group (n=6) presented a significant difference (P<0.001)
from the control group (n=6).

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Since there was an alteration in pancreatic functionality
as shown by the glucose tolerance test, the study
moved in the direction of a specific evaluation of the
pancreatic islets, carrying out a static insulin secretion
test where a significant reduction in insulin secretion at
the three glucose concentrations (2.8 mM, 8.3 mM, and
22.2 mM) was observed (representing -40.0%, -35.5%
and -55.5%, respectively) when compared with the
control group (P=0.036, P=0.017, P<0.001,
respectively) (Figure 3).
With the focus still on the pancreatic islets of the group
treated with bleomycin, the concentrations of the
insulin receptor, GLUT2, PKC and PKA were
determined, observing significant increases in GLUT2
(48.4%) and PKC (70.8%) and a significant reduction
in PKA (38.5%), as shown in Table 1.
The plasmatic insulin concentration was also evaluated,
showing a significant increase (P<0.001) in the treated
group (7.81±1.71 ng/mL) as compared to the control
group (0.83±0.19 ng/mL).
DISCUSSION
Insulin is undoubtedly an extremely important
hormone in the regulation of glycemia homeostasis,
and, thus, any physiological alteration of the endocrine
pancreas reflects directly on the equilibrium of the
synthesis/degradation ratio in the principal energy
reserves [12].
Innumerable evaluation methods have been developed
with the aim of determining the responsiveness of beta
pancreatic cells, amongst which the glucose tolerance
test stands out [13]. The action mechanisms of
antineoplastic agents have long been a target of study
and a guiding pivot of research studies [14]. In this
context, the antineoplastic agent bleomycin deserves
special attention since, even at low concentrations, this
substance can induce oxidative stress and DNA
alterations, particularly those related to the
nitrogenized base thymine, expressively altering the
cell cycles, a condition which stabilizes and/or inhibits
the growth of neoplastic cells [15, 16].
In the present study, the glucose tolerance test showed
that treatment with bleomycin induced supra-
unevenness of the area, indicating that the sensitivity of
the beta pancreatic cells had been compromised. In an
attempt to explain this, it is important to reflect on the
changes in the secreting behavior of the insulin. In this
context, the study was initially focused on the fact that
the bleomycin expressed its action on the DNA,
indicating that it was a highly fat soluble substance,
Figure 2. Insulin tolerance test (ITT) applied to the control and
bleomycin-treated groups. The mean blood glucose analyzed 0, 2.5,
5, 10, 15 and 20 minutes after applying the insulin is shown. The
treated group (n=6) presented a significant difference (P<0.001)
from the control group (n=6).
Table 1. Insulin receptor, GLUT2, PKC and PKA concentration and immunoblotting in islets isolated from the control and bleomycin-treated groups.
Control
Bleomycin
P value
Immunoblotting
(Left: control; right: bleomycin)
Insulin receptor
2.22±0.16
2.26±0.16
0.618
GLUT2
2.27±0.19
3.37±0.48
<0.001
PKC
1.27±0.30
2.17±0.61
0.009
PKA
4.24±0.47
2.59±0.67
<0.001
Data are reported as mean±SD values of arbitrary units (n=6).
Figure 3. Insulin concentrations (ng.ilh.h) found in the control and
bleomycin-treated groups when undergoing the static insulin
secretion test at glucose concentrations of 2.8 mM, 8.3 mM, and 22.2
mM. The values correspond to the mean±SD (n=6).

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with access to the nuclei of different cells and which
possibly presented a greater intensity of action in cells
with high metabolic activity, such as tumour cells.
However, one should not ignore the possibility of its
action in non-cancer cells whose homeostasis could
also be affected by bleomycin.
In the context of this discussion, it is well known that
amongst the mechanisms related to the action of the
antineoplastic substance bleomycin, great relevance
has been attributed to its capacity of promoting the
formation of free radicals [17]
It is known that beta pancreatic cells possess high
metabolic activity, regulated in a multi-factorial way
especially when faced with an increase in extra-cellular
glucose concentration, adenyl cyclase activators or
phospholipase C activators (acetyl choline and
cholecystokinin) [18, 19].
With respect to the action of bleomycin on the insulin
secretion process, it was observed that islets isolated
from rats treated with bleomycin presented an increase
in the concentration of GLUT2, a transporter which
expresses itself with high intensity in beta cells,
indicating that, when faced with an overload of
glucose, it would rapidly reach the Kof GLUT2,
making the capture of large amounts of glucose
possible, a fact which predisposes the organism to an
increase in glucolytic flow, change in the ATP/ADP
ratio, change in the cytosolic calcium concentration
and consequent increase in insulin secretion, associated
events which could induce an increase in the
generation of reactive oxygen species [20]. In this way,
concomitant with the increase in the cytosolic glucose
content in the beta cells, there is also an increase in free
radical formation and changes in the responsiveness of
the insulin secretion process represented by the larger
area under the curve after a glucose overload in
agreement with various studies which suggest these
changes [21, 22]. In this way, bleomycin can reduce
the efficiency of the secretion process by the beta cells
and possibly unleash a pre-diabetic status.
The oxidative stress generated by treatment with
bleomycin is also one of the factors contributing to
injury of the beta cells of the pancreatic islets which,
on presenting increased activity of the AMP-activated
protein kinase (AMPK) system, start producing a large
amount of reactive oxygen species which, on activating
the caspase enzyme and the B-cell CLL/lymphoma 2
(BCL2) gene segment, become predisposed to cell
apoptosis, compromising the responsiveness of the
insulin secretion cells, reinforcing the supra-
unevenness of the glucose tolerance test curve
described above even more [23, 24, 25, 26].
Of the insulin secretion-inducing mechanisms, the
importance of the mitochondria as integrating agents in
generating energy should be emphasized since this
determines the efficiency of the secretion process, and
the mutations and alterations in mitochondrial function
inducing a considerable production of reactive oxygen
species which could take part in injuring the beta cells
as has already been identified in diabetes [27, 28].
Due to the alteration of the responsiveness of the beta
cells as indicated by the glucose tolerance test, the next
objective was to evaluate insulin secretion by islets
isolated from control animals, and compare them with
islets isolated from bleomycin-treated rats. A reduction
in the secretory response was found even in the
presence of secretagogue concentrations of glucose,
such as 8.3 mM and 22.2 mM, and, thus, the results
presented here are of extreme importance since they
show that treatment with bleomycin compromises the
insulin secretory process induced by glucose and could
be a reflex of possible oxidative stress resulting from
the toxicity of the substance in the beta cells or could
even strengthen the glucotoxicity produced by an
elevated hexose capture [29, 30].
Thus, a more judicious evaluation of the insulin
secretory process was carried out, evaluating the
concentrations of the C (PKC) and A (PKA) protein
kinases. The data showed a lack of functional
equilibrium due to a significant increase in the PKC
concentration accompanied by a reduction in PKA
enzymes which, in an integrated and synergic way,
regulate the steps of the insulin secretory process [31].
It is worth pointing out that an increase in the GLUT2
population was observed in the pancreatic beta cells
isolated from the bleomycin-treated rats, offering ideal
conditions for an increase in the rate of glucose capture
in this micro-environment, concomitant with an
increase in the cytosol glucose concentration. In this
context, it has been described that an increase in
glucose concentration is a strong PKC activating
stimulus and, thus, the present study accompanies and
corroborates the suggestion of Ha et al. [32].
One important point to be made is that the action of
bleomycin is possibly related to alterations in the
efficiency of metabolic pathways since PKA acts on
the speed of movement of the vesicles containing
insulin granules, and PKC acts in the co-localization of
the enzymes involved in the secretory process and of
the insulin granules, aiding in the movement of the
vesicles to the cell periphery [33]. Thus, transporting
these observations to the target cells, that is, to the
tumour cells, this suggests that bleomycin causes
disorder in the synergism between the kinases and, due
to this, causes a lack of equilibrium in the efficiency of
the energy-generating processes which is essential in
keeping the cell alive. It is interesting to point out that
Lee et al. [34] showed that reactive oxygen species
amplified the expression and action of PKC when one
considers the behavior of PKC in the functionality of
the pancreatic beta cell. The action of bleomycin as an
agent which increases the formation of free radicals
can indirectly compromise the secretory process and
implant a pre-diabetic status, a result which was
recently confirmed in the study of Pi et al. [35].
On the other hand, on examining plasmatic
insulinemia, an increase was shown in the bleomycin
treated group, a fact which led to the hypothesis that
bleomycin could induce apoptosis in the pancreatic
beta cells promoting hyperinsulinemia since, when an

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islet dies, it transfers its insulin content to the plasma,
as has already beenreported in the initial phases of the
development of diabetes mellitus. In this sense, one can
expect to find the implantation of a diabetogenic status
connected to the treatment.
Another evaluation carried out was the insulin
tolerance test with the objective of evaluating the
behavior of the peripheral tissues, using the proposal of
Cobelli et al. [13]. The analysis of the index of the fall
in glycemia showed that there was a significant
increase in the rate of glucose capture by the peripheral
tissues, indicating changes in tissue responsiveness.
This data suggest that insulin resistance could develop
with a constant stimulus and, in this sense, some
considerations are important. Two hypotheses were
initially raised, that is, faced with a reduction in insulin
secretion induced by the bleomycin, there is an up-
regulation of the insulin receptor population in the
peripheral tissues, causing hypersensitivity of the
glucoregulatory system so as to compensate and
provoke an increase in the hexose capture speed,
justifying the results found in the insulin tolerance test.
On the other hand, one cannot discard the hypothesis
that the free radicals generated by treatment with
bleomycin injured the energy-generating systems, and
could, in a second phase, compromise the insulin
sensitivity of the peripheral tissues. In this way, recent
studies have demonstrated that, in the insulin resistance
condition, a reduction in mitochondrial activity and
deficiency in oxidative phosphorylation were observed
as well as the induction of morphological modifications
of the mitochondria [36, 37, 38, 39].
Thus, during treatment with bleomycin, innumerable
chemical-metabolic alterations are unleashed in the
tissues, which are not the primary targets of the
chemical therapy, and which could compromise the
homeostasis of the systems taking part in the glycemia
adjustment, and predispose the organism to the
development of a pre-diabetic pattern whose degree of
incidence or reversibility are still unknown to the
scientific community.
It is important to point out the limitations of this study,
one of which being the dosage used, which, although
according to the literature, is the sole limitation. On the
other hand, the period of treatment characterized the
induction phase of the restrictive pattern, and other
periods preceding the chronic phase, should also be
analyzed.
Acknowledgement Research supported by FAPESP
(Fundação de Amparo à Pesquisa do Estado de São
Paulo) (protocol 04/14798-5).
Conflict of interest The authors have no potential
conflicts of interest
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