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1 1
Supplementation with vitamin A enhances oxidative stress in the lungs of rats submitted
2
to aerobic exercise
3 a*
a
a
a
4
Juciano Gasparotto , Lyvia Lintzmaier Petiz , Carolina Saibro Girardi , Rafael Calixto Bortolin ,
5
Amanda Rodrigues de Vargasa, Bernardo Saldanha Henkina, Paloma Rodrigues Chavesa,
6
Sabrina Roncato , Cristiane Matté , Alfeu Zanotto-Filho , José Cláudio Fonseca Moreira ,
7
Daniel Pens Gelaina
a
a
a
a
8 9 10
*Correspondence adress: Rua Ramiro Barcelos, 2600 – anexo, CEP 90035-003, Porto Alegre,
11
RS, Brazil. Phone: +55 51 3308-5577, Fax: +55 51 3308-5535.
12
E-mail: [email protected]
13 14
a
15
Básicas da Saúde, Universidade Federal do Rio Grande do Sul – Porto Alegre, RS – Brazil;
16
E-mail address (each author):
17
[email protected] (Correspondence author)
18
[email protected]
19
[email protected]
20
[email protected]
21
[email protected]
22
[email protected]
23
[email protected]
24
[email protected]
25
[email protected]
26
[email protected]
27
[email protected]
28
[email protected]
29 30
Centro de Estudos em Estresse Oxidativo, Departamento de Bioquímica, Instituto de Ciências
Page 2 of 24
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2 31
Abstract
32 33
Exercise training induces ROS production and low levels of oxidative damage, which are
34
required for induction of antioxidant defenses and tissue adaptation. This process is
35
physiological and essential to improve physical conditioning and performance. During exercise,
36
endogenous antioxidants are recruited to prevent excessive oxidative stress, demanding
37
appropriate intake of antioxidants from diet or supplements; in this context, the search for
38
vitamin supplements that enhance the antioxidant defenses and improve exercise performance
39
has been continuously increasing. On the other hand, excess of antioxidants may hinder the
40
pro-oxidant signals necessary for this process of adaptation. The aim of this study was to
41
investigate the effects of vitamin A supplementation (2000 IU/kg, oral) upon oxidative stress and
42
parameters of pro-inflammatory signalling in lungs of rats submitted to aerobic exercise
43
(swimming protocol). When combined to exercise, vitamin A inhibited biochemical parameters of
44
adaptation/conditioning by attenuating exercise-induced antioxidant enzymes (superoxide
45
dismutase and glutathione peroxidase) and decreasing the content of the receptor for advanced
46
glycation endproducts (RAGE). Increased oxidative damage to proteins (carbonylation) and
47
lipids (lipoperoxidation) was also observed in these animals. In sedentary animals, vitamin A
48
decreased superoxide dismutase and increased lipoperoxidation. Vitamin A also enhanced the
49
levels of TNF-α and decreased IL-10, effects partially reversed by aerobic training. Taken
50
together, the herein presented results point to negative effects associated with vitamin A
51
supplementation at the specific dose here used upon oxidative stress and pro-inflammatory
52
cytokines in lung tissues of rats submitted to aerobic exercise.
53 54 55 56 57 58 59 60 61
Key words: Aerobic exercise; Vitamin A; Oxidative stress; Cytokines; Lung
Page 3 of 24
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3 62
Introduction
63 64
Vitamin A (retinol) and its metabolites (retinoids) have long been considered as
65
important redox modulators in lung, heart and liver tissues, among others (Palace et al. 1999,
66
Pasquali et al. 2009a). At physiological concentrations, vitamin A regulates several primordial
67
functions, such as visual acuity, cellular proliferation and differentiation (Ribeiro Nogueira et al.
68
2009). For instance, retinoids inhibit cell growth and induce differentiation and apoptosis in
69
normal or tumoral cells (Niles 2000, Massaro and Massaro 2010). Particularly in lungs, retinoids
70
participate in the molecular mechanisms governing embryogenesis and organogenesis during
71
the fetal period, and are required for appropriate maturation and remodeling during perinatal
72
and postnatal periods and for maintenance of the fully matured lungs (Massaro and Massaro
73
2010, Chytil 1996). The importance of vitamin A is evident in geographical regions where the
74
local diet is deficient in this compound (or the so-called pro-vitamin A compounds, such as β-
75
and α-carotenes). In such areas, increased infant death and vision loss was attributed to poor
76
levels of vitamin A, and programs to promote supplementation with high doses demonstrated to
77
be effective in reducing these problems, thus leading the World Health Organization to
78
recommend vitamin A supplementation to populations considered “at risk” (Mason et al. 2014,
79
Sommer and Davidson 2002, Schooling and Jones 2015, WHO 2009).
80 81
While much of the vitamin A effects were initially attributed to retinoids (retinoic acid
82
isomers) binding to nuclear receptors, subsequent studies showed that pro-vitamin A
83
compounds and retinol may also act as free radicals/reactive oxygen species (ROS)
84
scavengers (Hix et al. 2004), thus preventing oxidative stress-induced damage and tissue
85
function impairments (Gray et al. 2005). Indeed, vitamin A administration demonstrated to be
86
effective to attenuate oxidative stress and inflammatory parameters related to some diseases,
87
such as bronchopulmonary dysplasia (Strueby and Thebaud 2014) and allergic asthma (Guo et
88
al. 2012a). This antioxidant effect of vitamin A stimulated the recommendation of vitamin A
89
intake to prevent diseases associated to free increased free radicals production and oxidative
90
stress, such as cancer, cardiovascular diseases and neurodegenerative conditions. In a
91
particularly famous clinical study, the carotene and retinol efficacy trial (CARET), designed to
Page 4 of 24
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4 92
evaluate the effect of vitamin A and oral carotene intake to prevent lung cancer in smokers and
93
workers exposed to asbestos, the incidence of lung cancer and risk of death from
94
cardiovascular diseases were actually increased, and the study had to be discontinued (Omenn
95
et al. 1996, Cui et al. 2008). Since then, several studies have reported that the excess of
96
vitamin A intake may also induce deleterious effects at specific situations, probably due to an
97
imbalance in reactive species production. Studies using in vivo and in vitro approaches showed
98
that high concentrations of vitamin A enhance mitochondrial superoxide production (Gelain et
99
al. 2006) and cause oxidative damage to biomolecules in the brain, lungs and cardiovascular
100
system (Schnorr et al. 2015, Da Rocha et al. 2010). These data, along with evidence that high
101
intake of vitamin A may cause different levels of toxic effects (Castano et al. 2006, Allen and
102
Haskell 2002, Block et al. 2008, Penniston and Tanumihardjo 2006), indicate that the effects of
103
supplementation with vitamin A and other retinoids must be better evaluated and understood.
104 105
Exercise is a physiological source of ROS in many tissues, and the accumulation of
106
such reactive species plays an important role in cell metabolism, adaptation and acquired
107
resistance to subsequent oxidative challenges (Niess and Simon 2007, Peternelj and Coombes,
108
2011 Paulsen et al. 2014). Noteworthy, while moderated levels of reactive species appear to be
109
necessary for various physiological processes (pre-conditioning), excessive ROS production
110
may cause oxidative damage if antioxidant systems do not appropriately adapt to such as
111
insults (Gomez-Cabrera et al. 2008b, Paulsen et al. 2014). Exercise poses cells to extensive
112
metabolic activity which, consequently, increases the need for some metabolic substrates as
113
well as vitamins. In this context, even though basically all vitamins can be obtained from a
114
balanced diet - thus minimizing the real need of vitamin and mineral supplements (Maughan et
115
al. 2007) - many athletes unknowingly consume higher-than-necessary amounts of
116
supplements in order to prevent the deleterious effects of exercise-induced oxidative stress as
117
well as to improve recovery of muscle function and, consequently, optimize performance.
118
However there are neither official guidelines nor significant scientific evidence regarding the
119
biological requirement, efficacy and safety of vitamin intake in athletes (Paulsen et al. 2014,
120
Maughan et al. 2007).
121
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5 122
Our group has extensively shown the impact of vitamin A supplementation in different
123
biological models (Gelain et al. 2011, Schnorr et al. 2011), with the lungs being one of the most
124
susceptible organs to vitamin A (Pasquali et al. 2009b, Pasquali et al. 2010). Given that
125
exercise increases oxygen pressure with lung tissues, that vitamin A intake was previously
126
associated with lung cancer incidence and considering the increasing number of people
127
adopting the use of vitamin supplements to enhance exercise performance, we decided here to
128
investigate the effects of vitamin A supplementation at therapeutically recommended doses
129
(2000 IU/kg) upon markers of oxidative stress and pro-inflammatory parameters in lungs of rats
130
under exercise training protocol.
131 132
Materials and Methods
133
Chemicals
134 135
Glycine, H2O2, thiobarbituric acid (TBARS), AAPH (2,2’-azobis[2-methylpropionamidine]
136
dihydrochloride), 5,5′-dithionitrobis 2-nitrobenzoic acid (DTNB), Bile salts, sodium dodecyl
137
sulfate. Electrophoresis and immunoblot reagents were from Bio-Rad (California, USA), GE
138
Healthcare Brazilian Headquarter (São Paulo, Brazil) and Sigma-Aldrich® (St. Louis, USA).
139
Polyclonal and monoclonal antibodies from Cell Signalling technology (Beverly, USA):
140
Receptor for advanced glycation endproducts (RAGE) (#4679), HSP70 (#4872), glutathione S-
141
transferase (GST) (#2622) and glycogen synthase kinase-3 beta (GSK-3β) (#9336). β-actin
142
(A1978) monoclonal antibody was from Sigma-Aldrich® (St. Louis, USA). Superoxide dismutase
143
2 (SOD2) (ab13533), superoxide dismutase 1 (SOD1) (ab16831), interleukin-10 (IL-10)
144
(ab9969), tumor necrosis factor alpha (TNF-α) (ab6671), interleukin-1 beta (IL-1β) (ab9722) are
145
polyclonal antibodies from Abcam® (Cambridge, UK). Catalase (CAT) (219010) polyclonal
146
antibody was from Merck Millipore (Massachusetts, EUA). Glutathione peroxidase (GPx-1) (sc-
147
22145), Santa Cruz Biotechnology®, (Texas, USA). Biotinylated protein ladder from Cell
148
Signaling technology (Beverly, USA). Anti-Rabbit IgG, peroxidase conjugated (#AP132P) and
149
Anti-Mouse IgG, peroxidase conjugated, H+L (#AP124P) from Merck Millipore (Massachusetts,
150
EUA). Immunoblot chemiluminescence detection was carried out with the West Pico detection
®
®
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6 151
kit from Thermo Scientific Pierce Protein Biology Products (Rockford, USA). All other reagents
152
used in this study were of analytical or HPLC grade.
153 154
Animals
155
Male Wistar rats (60 days-old) were obtained from our breeding colony. They were caged in
156
groups of four animals with free access to standard commercial food (Chow Nuvilab® CR-1
157
type; Curitiba, PR, Brazil). Chow nutritional composition consisted in: total protein (22 %),
158
vegetal fiber (8 %), minerals (10 %), calcium (1.4 %), and phosphorous (0.8 %). Enrichment by
159
kilograms: vitamin A (12,000 IU), vitamin D3 (1,800 IU), vitamin E (30 IU), vitamin K3 (3 mg),
160
vitamin B1 (5 mg), vitamin B2 (6 mg),vitamin B6 (7mg), vitamin B12 (20 µg), niacin (60 mg),
161
folic acid (1 mg), biotin (0.05 mg), choline (600 mg), iron (50 mg), copper (10 mg), zinc (60 mg),
162
manganese (60 mg), cobalt (1.5 mg), iodine (2 mg), selenium (0.05 mg), lysine (100 mg), and
163
methionine (300 mg). Rats were maintained in a 12-hour light–dark cycle in a temperature-
164
controlled room (21° C). All experimental procedures were performed in accordance with the
165
guidelines of the National Research Council (NCR 2011) and Canadian Council on Animal
166
Care (CCAC) guidelines (CCAC 1993). Our animal research protocol (n° 25837) was approved
167
by the Ethical Committee for Animal Experimentation of the Universidade Federal do Rio
168
Grande do Sul (UFRGS). A total of twenty eight healthy animals were utilized for this study.
169 170
Exercise training (swimming protocol)
171
Briefly, sixty days-old male Wistar rats were individually submitted to water adaptation during 8
172
days (with increasing time exposure to water (10, 20, 40, 60 minutes) every 2 days). After this
173
period, the animals were introduced to swimming adaptation. This protocol took 12 days, and an
174
overload time was increased each 3 days (10, 20, 40 and 60 minutes) to improve performance.
175
After adaptation period, the rats started the swimming training protocol, which consisted of
176
continuous swimming for 60 minutes per day in an individual swimming pool during 30 days.
177
Overload was calculated based on body weight (% B.W) as follows: 5 days no weight just
178
wearing mini sandbags attached to their thorax; 5 days - 1% B.W; 5 days - 2% B.W; 5 days -
179
4% B.W; 10 days – 6% B.W. Pool characteristics: glass swimming pool with warm water 28-29
180
ºC, 32-cm deep, 20-cm long and 15-cm wide. Animals were randomly divided in 4 groups (n=7
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7 181
per group), sedentary (SED), sedentary + vitamin A 2000 IU/Kg (SED+VIT.A), exercise (EXE),
182
exercise + vitamin A 2000 IU/Kg (EXE+VIT.A). Timeline and sub-groups are detailed in Fig.1.
183 184
Vitamin A treatment
185
The animals were treated once a day for a 42 days period, which was administered after the
186
exercise training. The control group received vehicle (0.15 M saline) and the others received
187
2000 IU/kg of oral Arovit - retinol palmitate (vitamin A) administrated by gavage in a maximum
188
volume of 0.8 mL. Standard procedures were taken to minimize animal pain or discomfort.
189 190
Protein assay
191
Total protein was quantified by Bradford assay and used to normalize the data (Bradford, 1976).
192 193
Antioxidant Enzymes (CAT, SOD and GPx)
194
CAT (EC 1.11.1.6), SOD (EC 1.15.1.1) and GPx (EC 1.11.1.9) activities were quantified in lung
195
homogenates. Lungs were homogenized with phosphate buffer (PB) 50 mM (KH2PO4 and
196
K2HPO4, pH 7.4). CAT activity was evaluated by assessing the rate of hydrogen peroxide
197
(H2O2) absorbance decrease at 240 nm (Aebi 1984). Results are expressed as units of CAT/mg
198
of protein. The SOD activity was measured by monitoring the inhibition of superoxide-
199
dependent adrenaline auto-oxidation to adrenochrome at 480 nm for 10 min (32 °C) in a
200
spectrophotometer (Misra and Fridovich 1972). Results are expressed as units of SOD/mg of
201
protein. GPx activity was determined by measuring the rate of NADPH oxidation in a
202
spectrophotometer at 340 nm as previously described (Wendel 1981). GPx activity was
203
expressed as Units (µmol NADPH oxidized/min)/mg protein.
204 205
Oxidative damage markers (carbonyl, TBARS, sulfhydril)
206
As an index of protein oxidative damage, the carbonyl groups were determined based on its
207
reaction with 2,4-dinitrophenylhydrazine (DNPH) as previously described (Levine et al. 1990).
208
Lipoperoxidation was determined from the quantification of TBARS originated from reaction of
209
thiobarbituric acid with lipoperoxides in an acid-heating medium (Draper and Hadley 1990).
210
After precipitation with trichloroacetic acid 10% (TCA), supernatant was mixed with 0.67%
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8 211
thiobarbituric acid and heated in a boiling water bath for 25 min. TBARS were determined in a
212
spectrophotometer set at 532 nm. The oxidative status of thiol groups (SH) was assessed by
213
quantification of total reduced SH groups in the samples (Ellman 1959). Briefly, a 60 µg sample
214
aliquot was diluted in PB 10 mM 5,5′-dithionitrobis 2-nitrobenzoic acid for 60 min at room
215
temperature, and read in a spectrophotometer at 412 nm.
216 217
Total reactive antioxidant potential (TRAP assay)
218
Complimentary to enzymatic assays, the total reactive antioxidant potential (TRAP) was used as
219
an index of non-enzymatic antioxidant capacity. This assay is based on the quenching of
220
peroxyl radicals generated by AAPH (2,2 azobis[2- amidinopropane]) by antioxidants present in
221
the sample (Lissi et al. 1992). Briefly, a chemical system that generates peroxyl radicals at a
222
constant rate (AAPH dissolved in glycine buffer) is coupled to the luminescent reactant luminol
223
which emits chemiluminescence proportionally to its oxidation at a constant rate. Equal amounts
224
of samples are then added to this reaction system, and the luminescence emission at the
225
moment following sample injection (t=0) was recorded. This initial emission reflects the
226
production of free radicals by AAPH at the first moment right after sample addition and is related
227
to the endogenous oxidant state of the sample. For each sample, we monitored TRAP
228
luminescence emission for 100 minutes in a MicroBeta luminescence counter (Perkin Elmer,
229
USA), and calculated the area under the curve (AUC) relative to the system without samples
230
(which was considered as 100 % of luminescence emission) recorded.
®
231 232
Indirect ELISA
233
Cytokines IL-1β, TNF-α and IL-10 levels were quantified by indirect ELISA. Lung homogenates
234
(50 µg) were incubated in an ELISA plate for 24 h. Afterwards, the plates were washed three
235
times with Tween-Tris buffered saline (TTBS, 100 mM Tris – HCl, pH 7.5, containing 0.9 %
236
NaCl, and 0.1 % Tween-20), 200 µL of primary antibody (1:1,000) was added, and incubation
237
was carried out for 24 hours at 4 °C. The plates were washed three times with TTBS and
238
incubated with rabbit or mouse IgG peroxidase-linked secondary antibody (1:1,000) for 2 h
239
according with manufacturer instructions. After washing the plate three times with TTBS, 200 µL
240
of substrate solution (TMB spectrophotometric ELISA detection kit) was added to each well and
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9 241
incubated for 15 min. The reaction was stopped with 50 µL of 12 M sulfuric acid, and the plate
242
read at 450 nm in a microplate reader.
243 244
Western blotting
245
To perform immunoblot experiments, the tissues were homogenized with 1X RIPA buffer (50
246
mM Tris–HCl at pH 8, 150 mM NaCl, 1% IGEPAL, 0,5% biliary salts, 0,1% SDS, 1µg/mL
247
leupeptin), centrifuged at 14,000 g for 10 minutes at 4oC, and supernatant proteins were
248
measured by the Bradford method (Bradford, 1976). Laemmli-sample buffer (62.5 mM Tris–HCl,
249
pH 6.8, 1% (w/v) SDS, 10% (v/v) glycerol) was added to complete volume according the protein
250
content of each sample and equal amounts of cell protein (30 µg/well) were fractionated by
251
SDS-PAGE and electro-blotted onto nitrocellulose membranes with Trans-Blot® SD Semi-Dry
252
Electrophoretic Transfer Cell, Bio-Rad (California, USA). Protein loading and electro-blotting
253
efficiency were verified through Ponceau S staining, and the membrane was washed with
254
Tween–Tris buffered saline (Tris 100 mM, pH 7.5, 0.9% NaCl and 0.1% Tween-20). Membranes
255
were incubated for 20 minutes at room temperature in SNAP i.d.® 2.0 Protein Detection System
256
Merck Millipore (Massachusetts, EUA) with primary antibodies (RAGE, HSP70, β-Actin, SOD1,
257
SOD2, GST, GPX1, CAT and GSK-3β - 1:2,000 dilution), washed with TTBS, following
258
incubation with IgG peroxidase-linked secondary antibodies for additional 20 minutes in the
259
SNAP system (1:5,000 dilution range). The immunoreactivity was detected by enhanced
260
chemiluminescence using Supersignal West Pico Chemiluminescent kit from Thermo Scientific
261
(Luminol/Enhancer and Stable Peroxide Buffer). Molecular weight was monitored through the
262
biotinylated protein ladder kit (Cell Signaling, #7727). Densitometric analysis was performed by
263
Image J. software. All results were expressed as a relative ratio to the β-actin.
264 265
Statistical analysis
266
Statistical analysis was performed with GraphPad 5.0 software. One-way analysis of variance
267
(ANOVA) followed by Tukey's post hoc test was applied. The results were expressed as mean ±
268
standard error (SEM). Differences were considered significant at p < 0.05.
269 270
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10 271
Results
272 273
Antioxidant enzymes (SOD, CAT and GPx) activities in the lung
274
Sedentary animal that received vitamin A supplementation showed a significant decrease in the
275
activity of SOD in lungs when compared to the SED group (p
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 08/19/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.
1 1
Supplementation with vitamin A enhances oxidative stress in the lungs of rats submitted
2
to aerobic exercise
3 a*
a
a
a
4
Juciano Gasparotto , Lyvia Lintzmaier Petiz , Carolina Saibro Girardi , Rafael Calixto Bortolin ,
5
Amanda Rodrigues de Vargasa, Bernardo Saldanha Henkina, Paloma Rodrigues Chavesa,
6
Sabrina Roncato , Cristiane Matté , Alfeu Zanotto-Filho , José Cláudio Fonseca Moreira ,
7
Daniel Pens Gelaina
a
a
a
a
8 9 10
*Correspondence adress: Rua Ramiro Barcelos, 2600 – anexo, CEP 90035-003, Porto Alegre,
11
RS, Brazil. Phone: +55 51 3308-5577, Fax: +55 51 3308-5535.
12
E-mail: [email protected]
13 14
a
15
Básicas da Saúde, Universidade Federal do Rio Grande do Sul – Porto Alegre, RS – Brazil;
16
E-mail address (each author):
17
[email protected] (Correspondence author)
18
[email protected]
19
[email protected]
20
[email protected]
21
[email protected]
22
[email protected]
23
[email protected]
24
[email protected]
25
[email protected]
26
[email protected]
27
[email protected]
28
[email protected]
29 30
Centro de Estudos em Estresse Oxidativo, Departamento de Bioquímica, Instituto de Ciências
Page 2 of 24
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2 31
Abstract
32 33
Exercise training induces ROS production and low levels of oxidative damage, which are
34
required for induction of antioxidant defenses and tissue adaptation. This process is
35
physiological and essential to improve physical conditioning and performance. During exercise,
36
endogenous antioxidants are recruited to prevent excessive oxidative stress, demanding
37
appropriate intake of antioxidants from diet or supplements; in this context, the search for
38
vitamin supplements that enhance the antioxidant defenses and improve exercise performance
39
has been continuously increasing. On the other hand, excess of antioxidants may hinder the
40
pro-oxidant signals necessary for this process of adaptation. The aim of this study was to
41
investigate the effects of vitamin A supplementation (2000 IU/kg, oral) upon oxidative stress and
42
parameters of pro-inflammatory signalling in lungs of rats submitted to aerobic exercise
43
(swimming protocol). When combined to exercise, vitamin A inhibited biochemical parameters of
44
adaptation/conditioning by attenuating exercise-induced antioxidant enzymes (superoxide
45
dismutase and glutathione peroxidase) and decreasing the content of the receptor for advanced
46
glycation endproducts (RAGE). Increased oxidative damage to proteins (carbonylation) and
47
lipids (lipoperoxidation) was also observed in these animals. In sedentary animals, vitamin A
48
decreased superoxide dismutase and increased lipoperoxidation. Vitamin A also enhanced the
49
levels of TNF-α and decreased IL-10, effects partially reversed by aerobic training. Taken
50
together, the herein presented results point to negative effects associated with vitamin A
51
supplementation at the specific dose here used upon oxidative stress and pro-inflammatory
52
cytokines in lung tissues of rats submitted to aerobic exercise.
53 54 55 56 57 58 59 60 61
Key words: Aerobic exercise; Vitamin A; Oxidative stress; Cytokines; Lung
Page 3 of 24
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3 62
Introduction
63 64
Vitamin A (retinol) and its metabolites (retinoids) have long been considered as
65
important redox modulators in lung, heart and liver tissues, among others (Palace et al. 1999,
66
Pasquali et al. 2009a). At physiological concentrations, vitamin A regulates several primordial
67
functions, such as visual acuity, cellular proliferation and differentiation (Ribeiro Nogueira et al.
68
2009). For instance, retinoids inhibit cell growth and induce differentiation and apoptosis in
69
normal or tumoral cells (Niles 2000, Massaro and Massaro 2010). Particularly in lungs, retinoids
70
participate in the molecular mechanisms governing embryogenesis and organogenesis during
71
the fetal period, and are required for appropriate maturation and remodeling during perinatal
72
and postnatal periods and for maintenance of the fully matured lungs (Massaro and Massaro
73
2010, Chytil 1996). The importance of vitamin A is evident in geographical regions where the
74
local diet is deficient in this compound (or the so-called pro-vitamin A compounds, such as β-
75
and α-carotenes). In such areas, increased infant death and vision loss was attributed to poor
76
levels of vitamin A, and programs to promote supplementation with high doses demonstrated to
77
be effective in reducing these problems, thus leading the World Health Organization to
78
recommend vitamin A supplementation to populations considered “at risk” (Mason et al. 2014,
79
Sommer and Davidson 2002, Schooling and Jones 2015, WHO 2009).
80 81
While much of the vitamin A effects were initially attributed to retinoids (retinoic acid
82
isomers) binding to nuclear receptors, subsequent studies showed that pro-vitamin A
83
compounds and retinol may also act as free radicals/reactive oxygen species (ROS)
84
scavengers (Hix et al. 2004), thus preventing oxidative stress-induced damage and tissue
85
function impairments (Gray et al. 2005). Indeed, vitamin A administration demonstrated to be
86
effective to attenuate oxidative stress and inflammatory parameters related to some diseases,
87
such as bronchopulmonary dysplasia (Strueby and Thebaud 2014) and allergic asthma (Guo et
88
al. 2012a). This antioxidant effect of vitamin A stimulated the recommendation of vitamin A
89
intake to prevent diseases associated to free increased free radicals production and oxidative
90
stress, such as cancer, cardiovascular diseases and neurodegenerative conditions. In a
91
particularly famous clinical study, the carotene and retinol efficacy trial (CARET), designed to
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4 92
evaluate the effect of vitamin A and oral carotene intake to prevent lung cancer in smokers and
93
workers exposed to asbestos, the incidence of lung cancer and risk of death from
94
cardiovascular diseases were actually increased, and the study had to be discontinued (Omenn
95
et al. 1996, Cui et al. 2008). Since then, several studies have reported that the excess of
96
vitamin A intake may also induce deleterious effects at specific situations, probably due to an
97
imbalance in reactive species production. Studies using in vivo and in vitro approaches showed
98
that high concentrations of vitamin A enhance mitochondrial superoxide production (Gelain et
99
al. 2006) and cause oxidative damage to biomolecules in the brain, lungs and cardiovascular
100
system (Schnorr et al. 2015, Da Rocha et al. 2010). These data, along with evidence that high
101
intake of vitamin A may cause different levels of toxic effects (Castano et al. 2006, Allen and
102
Haskell 2002, Block et al. 2008, Penniston and Tanumihardjo 2006), indicate that the effects of
103
supplementation with vitamin A and other retinoids must be better evaluated and understood.
104 105
Exercise is a physiological source of ROS in many tissues, and the accumulation of
106
such reactive species plays an important role in cell metabolism, adaptation and acquired
107
resistance to subsequent oxidative challenges (Niess and Simon 2007, Peternelj and Coombes,
108
2011 Paulsen et al. 2014). Noteworthy, while moderated levels of reactive species appear to be
109
necessary for various physiological processes (pre-conditioning), excessive ROS production
110
may cause oxidative damage if antioxidant systems do not appropriately adapt to such as
111
insults (Gomez-Cabrera et al. 2008b, Paulsen et al. 2014). Exercise poses cells to extensive
112
metabolic activity which, consequently, increases the need for some metabolic substrates as
113
well as vitamins. In this context, even though basically all vitamins can be obtained from a
114
balanced diet - thus minimizing the real need of vitamin and mineral supplements (Maughan et
115
al. 2007) - many athletes unknowingly consume higher-than-necessary amounts of
116
supplements in order to prevent the deleterious effects of exercise-induced oxidative stress as
117
well as to improve recovery of muscle function and, consequently, optimize performance.
118
However there are neither official guidelines nor significant scientific evidence regarding the
119
biological requirement, efficacy and safety of vitamin intake in athletes (Paulsen et al. 2014,
120
Maughan et al. 2007).
121
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5 122
Our group has extensively shown the impact of vitamin A supplementation in different
123
biological models (Gelain et al. 2011, Schnorr et al. 2011), with the lungs being one of the most
124
susceptible organs to vitamin A (Pasquali et al. 2009b, Pasquali et al. 2010). Given that
125
exercise increases oxygen pressure with lung tissues, that vitamin A intake was previously
126
associated with lung cancer incidence and considering the increasing number of people
127
adopting the use of vitamin supplements to enhance exercise performance, we decided here to
128
investigate the effects of vitamin A supplementation at therapeutically recommended doses
129
(2000 IU/kg) upon markers of oxidative stress and pro-inflammatory parameters in lungs of rats
130
under exercise training protocol.
131 132
Materials and Methods
133
Chemicals
134 135
Glycine, H2O2, thiobarbituric acid (TBARS), AAPH (2,2’-azobis[2-methylpropionamidine]
136
dihydrochloride), 5,5′-dithionitrobis 2-nitrobenzoic acid (DTNB), Bile salts, sodium dodecyl
137
sulfate. Electrophoresis and immunoblot reagents were from Bio-Rad (California, USA), GE
138
Healthcare Brazilian Headquarter (São Paulo, Brazil) and Sigma-Aldrich® (St. Louis, USA).
139
Polyclonal and monoclonal antibodies from Cell Signalling technology (Beverly, USA):
140
Receptor for advanced glycation endproducts (RAGE) (#4679), HSP70 (#4872), glutathione S-
141
transferase (GST) (#2622) and glycogen synthase kinase-3 beta (GSK-3β) (#9336). β-actin
142
(A1978) monoclonal antibody was from Sigma-Aldrich® (St. Louis, USA). Superoxide dismutase
143
2 (SOD2) (ab13533), superoxide dismutase 1 (SOD1) (ab16831), interleukin-10 (IL-10)
144
(ab9969), tumor necrosis factor alpha (TNF-α) (ab6671), interleukin-1 beta (IL-1β) (ab9722) are
145
polyclonal antibodies from Abcam® (Cambridge, UK). Catalase (CAT) (219010) polyclonal
146
antibody was from Merck Millipore (Massachusetts, EUA). Glutathione peroxidase (GPx-1) (sc-
147
22145), Santa Cruz Biotechnology®, (Texas, USA). Biotinylated protein ladder from Cell
148
Signaling technology (Beverly, USA). Anti-Rabbit IgG, peroxidase conjugated (#AP132P) and
149
Anti-Mouse IgG, peroxidase conjugated, H+L (#AP124P) from Merck Millipore (Massachusetts,
150
EUA). Immunoblot chemiluminescence detection was carried out with the West Pico detection
®
®
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6 151
kit from Thermo Scientific Pierce Protein Biology Products (Rockford, USA). All other reagents
152
used in this study were of analytical or HPLC grade.
153 154
Animals
155
Male Wistar rats (60 days-old) were obtained from our breeding colony. They were caged in
156
groups of four animals with free access to standard commercial food (Chow Nuvilab® CR-1
157
type; Curitiba, PR, Brazil). Chow nutritional composition consisted in: total protein (22 %),
158
vegetal fiber (8 %), minerals (10 %), calcium (1.4 %), and phosphorous (0.8 %). Enrichment by
159
kilograms: vitamin A (12,000 IU), vitamin D3 (1,800 IU), vitamin E (30 IU), vitamin K3 (3 mg),
160
vitamin B1 (5 mg), vitamin B2 (6 mg),vitamin B6 (7mg), vitamin B12 (20 µg), niacin (60 mg),
161
folic acid (1 mg), biotin (0.05 mg), choline (600 mg), iron (50 mg), copper (10 mg), zinc (60 mg),
162
manganese (60 mg), cobalt (1.5 mg), iodine (2 mg), selenium (0.05 mg), lysine (100 mg), and
163
methionine (300 mg). Rats were maintained in a 12-hour light–dark cycle in a temperature-
164
controlled room (21° C). All experimental procedures were performed in accordance with the
165
guidelines of the National Research Council (NCR 2011) and Canadian Council on Animal
166
Care (CCAC) guidelines (CCAC 1993). Our animal research protocol (n° 25837) was approved
167
by the Ethical Committee for Animal Experimentation of the Universidade Federal do Rio
168
Grande do Sul (UFRGS). A total of twenty eight healthy animals were utilized for this study.
169 170
Exercise training (swimming protocol)
171
Briefly, sixty days-old male Wistar rats were individually submitted to water adaptation during 8
172
days (with increasing time exposure to water (10, 20, 40, 60 minutes) every 2 days). After this
173
period, the animals were introduced to swimming adaptation. This protocol took 12 days, and an
174
overload time was increased each 3 days (10, 20, 40 and 60 minutes) to improve performance.
175
After adaptation period, the rats started the swimming training protocol, which consisted of
176
continuous swimming for 60 minutes per day in an individual swimming pool during 30 days.
177
Overload was calculated based on body weight (% B.W) as follows: 5 days no weight just
178
wearing mini sandbags attached to their thorax; 5 days - 1% B.W; 5 days - 2% B.W; 5 days -
179
4% B.W; 10 days – 6% B.W. Pool characteristics: glass swimming pool with warm water 28-29
180
ºC, 32-cm deep, 20-cm long and 15-cm wide. Animals were randomly divided in 4 groups (n=7
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7 181
per group), sedentary (SED), sedentary + vitamin A 2000 IU/Kg (SED+VIT.A), exercise (EXE),
182
exercise + vitamin A 2000 IU/Kg (EXE+VIT.A). Timeline and sub-groups are detailed in Fig.1.
183 184
Vitamin A treatment
185
The animals were treated once a day for a 42 days period, which was administered after the
186
exercise training. The control group received vehicle (0.15 M saline) and the others received
187
2000 IU/kg of oral Arovit - retinol palmitate (vitamin A) administrated by gavage in a maximum
188
volume of 0.8 mL. Standard procedures were taken to minimize animal pain or discomfort.
189 190
Protein assay
191
Total protein was quantified by Bradford assay and used to normalize the data (Bradford, 1976).
192 193
Antioxidant Enzymes (CAT, SOD and GPx)
194
CAT (EC 1.11.1.6), SOD (EC 1.15.1.1) and GPx (EC 1.11.1.9) activities were quantified in lung
195
homogenates. Lungs were homogenized with phosphate buffer (PB) 50 mM (KH2PO4 and
196
K2HPO4, pH 7.4). CAT activity was evaluated by assessing the rate of hydrogen peroxide
197
(H2O2) absorbance decrease at 240 nm (Aebi 1984). Results are expressed as units of CAT/mg
198
of protein. The SOD activity was measured by monitoring the inhibition of superoxide-
199
dependent adrenaline auto-oxidation to adrenochrome at 480 nm for 10 min (32 °C) in a
200
spectrophotometer (Misra and Fridovich 1972). Results are expressed as units of SOD/mg of
201
protein. GPx activity was determined by measuring the rate of NADPH oxidation in a
202
spectrophotometer at 340 nm as previously described (Wendel 1981). GPx activity was
203
expressed as Units (µmol NADPH oxidized/min)/mg protein.
204 205
Oxidative damage markers (carbonyl, TBARS, sulfhydril)
206
As an index of protein oxidative damage, the carbonyl groups were determined based on its
207
reaction with 2,4-dinitrophenylhydrazine (DNPH) as previously described (Levine et al. 1990).
208
Lipoperoxidation was determined from the quantification of TBARS originated from reaction of
209
thiobarbituric acid with lipoperoxides in an acid-heating medium (Draper and Hadley 1990).
210
After precipitation with trichloroacetic acid 10% (TCA), supernatant was mixed with 0.67%
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8 211
thiobarbituric acid and heated in a boiling water bath for 25 min. TBARS were determined in a
212
spectrophotometer set at 532 nm. The oxidative status of thiol groups (SH) was assessed by
213
quantification of total reduced SH groups in the samples (Ellman 1959). Briefly, a 60 µg sample
214
aliquot was diluted in PB 10 mM 5,5′-dithionitrobis 2-nitrobenzoic acid for 60 min at room
215
temperature, and read in a spectrophotometer at 412 nm.
216 217
Total reactive antioxidant potential (TRAP assay)
218
Complimentary to enzymatic assays, the total reactive antioxidant potential (TRAP) was used as
219
an index of non-enzymatic antioxidant capacity. This assay is based on the quenching of
220
peroxyl radicals generated by AAPH (2,2 azobis[2- amidinopropane]) by antioxidants present in
221
the sample (Lissi et al. 1992). Briefly, a chemical system that generates peroxyl radicals at a
222
constant rate (AAPH dissolved in glycine buffer) is coupled to the luminescent reactant luminol
223
which emits chemiluminescence proportionally to its oxidation at a constant rate. Equal amounts
224
of samples are then added to this reaction system, and the luminescence emission at the
225
moment following sample injection (t=0) was recorded. This initial emission reflects the
226
production of free radicals by AAPH at the first moment right after sample addition and is related
227
to the endogenous oxidant state of the sample. For each sample, we monitored TRAP
228
luminescence emission for 100 minutes in a MicroBeta luminescence counter (Perkin Elmer,
229
USA), and calculated the area under the curve (AUC) relative to the system without samples
230
(which was considered as 100 % of luminescence emission) recorded.
®
231 232
Indirect ELISA
233
Cytokines IL-1β, TNF-α and IL-10 levels were quantified by indirect ELISA. Lung homogenates
234
(50 µg) were incubated in an ELISA plate for 24 h. Afterwards, the plates were washed three
235
times with Tween-Tris buffered saline (TTBS, 100 mM Tris – HCl, pH 7.5, containing 0.9 %
236
NaCl, and 0.1 % Tween-20), 200 µL of primary antibody (1:1,000) was added, and incubation
237
was carried out for 24 hours at 4 °C. The plates were washed three times with TTBS and
238
incubated with rabbit or mouse IgG peroxidase-linked secondary antibody (1:1,000) for 2 h
239
according with manufacturer instructions. After washing the plate three times with TTBS, 200 µL
240
of substrate solution (TMB spectrophotometric ELISA detection kit) was added to each well and
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9 241
incubated for 15 min. The reaction was stopped with 50 µL of 12 M sulfuric acid, and the plate
242
read at 450 nm in a microplate reader.
243 244
Western blotting
245
To perform immunoblot experiments, the tissues were homogenized with 1X RIPA buffer (50
246
mM Tris–HCl at pH 8, 150 mM NaCl, 1% IGEPAL, 0,5% biliary salts, 0,1% SDS, 1µg/mL
247
leupeptin), centrifuged at 14,000 g for 10 minutes at 4oC, and supernatant proteins were
248
measured by the Bradford method (Bradford, 1976). Laemmli-sample buffer (62.5 mM Tris–HCl,
249
pH 6.8, 1% (w/v) SDS, 10% (v/v) glycerol) was added to complete volume according the protein
250
content of each sample and equal amounts of cell protein (30 µg/well) were fractionated by
251
SDS-PAGE and electro-blotted onto nitrocellulose membranes with Trans-Blot® SD Semi-Dry
252
Electrophoretic Transfer Cell, Bio-Rad (California, USA). Protein loading and electro-blotting
253
efficiency were verified through Ponceau S staining, and the membrane was washed with
254
Tween–Tris buffered saline (Tris 100 mM, pH 7.5, 0.9% NaCl and 0.1% Tween-20). Membranes
255
were incubated for 20 minutes at room temperature in SNAP i.d.® 2.0 Protein Detection System
256
Merck Millipore (Massachusetts, EUA) with primary antibodies (RAGE, HSP70, β-Actin, SOD1,
257
SOD2, GST, GPX1, CAT and GSK-3β - 1:2,000 dilution), washed with TTBS, following
258
incubation with IgG peroxidase-linked secondary antibodies for additional 20 minutes in the
259
SNAP system (1:5,000 dilution range). The immunoreactivity was detected by enhanced
260
chemiluminescence using Supersignal West Pico Chemiluminescent kit from Thermo Scientific
261
(Luminol/Enhancer and Stable Peroxide Buffer). Molecular weight was monitored through the
262
biotinylated protein ladder kit (Cell Signaling, #7727). Densitometric analysis was performed by
263
Image J. software. All results were expressed as a relative ratio to the β-actin.
264 265
Statistical analysis
266
Statistical analysis was performed with GraphPad 5.0 software. One-way analysis of variance
267
(ANOVA) followed by Tukey's post hoc test was applied. The results were expressed as mean ±
268
standard error (SEM). Differences were considered significant at p < 0.05.
269 270
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10 271
Results
272 273
Antioxidant enzymes (SOD, CAT and GPx) activities in the lung
274
Sedentary animal that received vitamin A supplementation showed a significant decrease in the
275
activity of SOD in lungs when compared to the SED group (p
