You are on page 1of 11

See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/276108599

Heat stress impairs mitochondria functions


and induces oxidative injury in broiler chickens
ARTICLE in JOURNAL OF ANIMAL SCIENCE MAY 2015
Impact Factor: 2.11 DOI: 10.2527/jas.2014-8739

CITATION

READS

45

6 AUTHORS, INCLUDING:
Zhigang Song

Huameng Lin

Shandong Agricultural University

Shandong Agricultural University

40 PUBLICATIONS 367 CITATIONS

37 PUBLICATIONS 803 CITATIONS

SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate,


letting you access and read them immediately.

SEE PROFILE

Available from: Zhigang Song


Retrieved on: 11 February 2016

Published May 15, 2015

Heat stress impairs mitochondria functions


and induces oxidative injury in broiler chickens1
C. Huang, H. Jiao, Z. Song, J. Zhao, X. Wang, and H. Lin2
Department of Animal Science, Shandong Agricultural University, Taian, Shandong 271018, P.R. China

ABSTRACT: The objective of this study was to


explore the linkage of oxidative stress occurring in
mitochondria, skeletal muscles, and plasma in heat
stresschallenged broilers. At d 35, 24 broilers were
randomly assigned to 2 treatments: rearing at high
temperature (32 1C; heat stress group) or normal
temperature (21 1.2C; control) for 7 d. The oxidative
damage of lipid, DNA, and protein and the activities of
antioxidative enzymes were measured, respectively, in
plasma, skeletal muscles (breast and thigh muscles),
and skeletal muscle mitochondria. The result showed
that heat exposure increased (P < 0.01) plasma concentrations of thiobarbituric acid reacting substances
(TBARS) and 8-hydroxydeoxyguanosine (8-OHdG)
whereas it deceased total antioxidant capacity (P <
0.05) and ability to inhibit hydroxyl radicals (AIHR;
P < 0.001). Protein carbonyl and TBARS levels were
increased (P < 0.001) by heat stress in breast and thigh
muscles. In skeletal muscle mitochondria, heat stress
increased (P < 0.05) 8-OHdG and suppressed AIHR.
Plasma activity of superoxide dismutase (SOD) was
increased (P < 0.001) whereas glutathione peroxidase
(GSH-Px) was suppressed by heat stress (P< 0.001).

Heat exposure increased SOD and catalase activities


in breast muscle (P < 0.01) but the reverse was true
in thigh muscle (P < 0.05). Glutathione peroxidase
was increased in thigh muscle (P < 0.001) but was
not changed in breast muscle (P > 0.05). Heat stress
increased SOD (P < 0.05) and decreased GSH-Px
activities (P < 0.05) of mitochondria regardless of muscle types. Plasma allantoin level increased (P < 0.01)
correspondingly with urate (P < 0.001) in heat-stressed
broilers, indicating that urate could serve as an antioxidant to enhance the antioxidative capacity during stress
in a concentration-dependent manner. The activities of
respiratory chain complexes I and III were estimated
in skeletal muscle mitochondria. Mitochondrial complex I activity was suppressed (P < 0.01) by heat
exposure in breast and thigh muscles but complex III
activity was elevated only in breast muscle (P < 0.01)
of heat-stressed broiler. The fatty acid composition in
skeletal muscle was not influenced by heat stress. In
conclusion, suppressed mitochondrial complex I activity is associated with oxidative stress induced by heat
exposure, which, in turn, is linked with the oxidative
damages in muscle tissues and plasma.

Key words: antioxidant enzyme, heat stress,


lipid peroxidation, respiratory chain complex, skeletal muscle, urate
2015 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2015.93:21442153
doi:10.2527/jas2014-8739
INTRODUCTION
Heat stress causes production loss and a series
of metabolic changes in farm animals (Rhoads et al.,
1This work was supported by grants from the Natural Science

Foundation of China (31272467 and 31472114) and the Specific Fund


for Agro-scientific Research in the Public Interests (201003011).
The authors report no conflicts of interest. The authors alone are
in charge of the content and writing of the paper.
2Corresponding author: hailin@sdau.edu.cn
Received November 20, 2014.
Accepted February 22, 2015.

2013). Heat stress induces oxidative injury (Lin et al.,


2000; Mahmoud and Edens, 2003) that may result
from increased production of reactive oxygen species (ROS; Lin et al., 2008). Increased body temperature (Mujahid et al., 2005) and circulating glucocorticoids (Lin et al., 2004a,b) are associated with heat
stressinduced oxidative damage. Oxidative stress
occurs when the subtle balance between the oxidation and antioxidant defense system is disturbed
within cells, resulting in lipid peroxidation and
oxidative damage to proteins and DNA (Halliwell
and Gutteridge, 1989; Yu, 1994; Drge, 2002). The
maintenance of redox balance is important for health

2144

Heat stress induces oxidative injury

and nutrient metabolism of farm animals (Bottje et al.,


1998; Chauhan et al., 2014).
The mitochondrial respiratory chain is the major
site of ROS formation (Chance et al., 1979). Acute heat
stress results in increased levels of ROS in mitochondria of skeletal muscle (Mujahid et al., 2006). The enhanced substrate oxidation, downregulation of uncoupling protein (Mujahid et al., 2007a,b), and depressed
activity of the mitochondrial respiratory chain (Yang et
al., 2010) are involved in the augmented production of
ROS. However, the relationship between mitochondria
function and muscle tissue and whole body oxidative
stress induced by heat stress remains unclear.
Urate is a product of purine metabolism in mammals or of nitrogen metabolism in birds. In the redox
system, urate has a paradox role. Urate serves as a major antioxidant in blood that protects against oxidative
stress and can be a prooxidant within cells under certain circumstances (Corte and Stirpe, 1972; Hellsten et
al., 1997; Sautin and Johnson, 2008). In poultry, urate
can serve as a hydroxyl radical scavenger (Simoyi et
al., 2002; Carro et al., 2010). Urate oxidase, an enzyme
that catalyzes urate to allantoin, is not present in birds
(De Boeck and Stockx, 1978). Therefore, the plasma
concentration of allantoin may reflect a result of the reactions between oxidants and uric acid (Simoyi et al.,
2003). During stress, the role of increased plasma concentration of urate as the result of protein catabolism
remains unclear. Therefore, the major objective of the
present study was to explore the linkage of oxidative
stress in mitochondria, skeletal muscle, and plasma in
broilers exposed to high temperature. The antioxidant
enzyme activities, antioxidative scavenge capacity, and
oxidative damage parameters were determined.
MATERIALS AND METHODS
Animals and Experimental Design
Day-old male chickens (Arbor Acres) were obtained from a local hatchery (Dabao Breeding Co.,
Xintai, Shandong, P.R. China). Chickens were reared
in an environmentally controlled room containing 18
floor pens (2 by 2 m). The brooding temperature was
maintained at 35C (65% relative humidity) for the
first 2 d, then decreased gradually to 21C (45% relative humidity) at Day 28, and maintained as such until
the end of the experiment (Day 42). The light regime
was 23:1 light:dark. All chickens received a starter diet
with 21.5% CP and 12.37 MJ/kg of ME from Day 1
to 21. Thereafter, the chickens received a grower diet
with 19.5% CP and 12.90 MJ/kg of ME. The study was
approved by the Shandong Agricultural University,
Taian, Shandong, and performed in accordance with the

2145

Guidelines for Experimental Animals of the Ministry of


Science and Technology (Beijing, P.R. China).
At 32 d of age, 24 broilers with similar BW were
divided into 2 groups. At 35 d of age, all the chickens
were randomly subjected to 1 of the 2 treatments: rearing at high temperature (32 1.0C; heat stress group)
or in the normal environment (21 1.2C; control) for
7 d. All the birds had free access to feed and water during the entire experimental period.
At d 42, 8 chickens around the mean BW were randomly selected from each treatment. A blood sample
was drawn from a wing vein within 30 s using a heparinized syringe. Plasma was obtained by centrifugation of
blood sample at 400 g for 10 min at 4C. Aliquots of
1.0 mL were pipetted into Eppendorf tubes and stored at
20C for further analysis. Thereafter, all the chickens
were killed by exsanguination after cervical dislocation.
Breast and thigh muscles were harvested and weighed.
Muscle samples were obtained from the left pectoralis major (fast-twitch glycolytic fiber type muscle) and
the left biceps femoris (slow-twitch oxidative fiber type
muscle). The muscle samples were divided into 2 parts,
and one part was used for mitochondria isolation and
another part was snap-frozen in liquid nitrogen and
stored at 80C for further analysis.
Oxidative Injury and Enzyme Activity in Plasma
Plasma concentrations of lipid peroxidation were
estimated by spectrophotometric determination of
thiobarbituric acid reacting substances (TBARS)
with the method of Lin et al. (2004a). Thiobarbituric
acid reacting substances were expressed as nanomoles of malonaldehyde (MDA) per milliliter plasma.
8-Hydroxydeoxyguanosine (8-OHdG), an oxidized
nucleoside of DNA, is the most frequently detected and
studied DNA lesion (Wu et al., 2004). The concentration
of urate (kit number C012); total antioxidant capacity
(TAC; kit number A015); 8-OHdG (kit number H165);
the ability to inhibit hydroxyl radicals (AIHR; kit number A018); and activities of catalase (CAT; kit number
A007), superoxide dismutase (SOD; kit number A001),
and glutathione peroxidase (GSH-Px; kit number A005)
were measured by commercial diagnostic kits (Jiancheng
Bioengineering Institute, Nanjing, P.R. China). All the
kits have been successfully used in poultry study (Lin et
al., 2009; Gao et al., 2010).
Allantoin was determined by HPLC (SPD-10AVP;
Shimadzu Corp., Kyoto, Japan) using a modified method of Grootveld and Halliwell (1986). Briefly, plasma
was deproteinized with ice-cold 0.6 mol/L of HClO4
and the supernatant was neutralized with 2.0 mol/L of
K3PO4. An aliquot (20 L) of supernatant was injected on to an Anachem S5 ODS-2 column (Anachem

2146

Chunxu et al.

Ltd., Anachem House, Luton, Bedfordshire, LU4 8EF,


U.K.) with a mobile phase of 96.7% (vol/vol) 30 mM
sodium citrate/27.7 mM sodium acetate buffer, pH
4.75, and 3.3% (vol/vol) methanol at a flow rate of
0.9 mL/min. Allantoin was detected at 224 nm and
the typical retention times for allantoin were 3.80 min.
Standard solutions of allantoin (0100 mol/L) were
prepared by dissolving pure allantoin (Sigma-Aldrich,
St. Louis, MO) in deionized water.
Oxidative Injury and Enzyme
Activity in Breast and Thigh Muscles
Tissue samples were homogenized in 9 volumes of
10 mM sodium phosphate buffer (pH 7.4) containing
1.15% potassium chloride. After the homogenates were
centrifuged (400 g for 10 min at 4C), the supernatants were used for further measurements. The concentration of TBARS in skeletal muscle tissues was measured according to the description by Lin et al. (2004b)
and expressed as nanomoles of MDA/milligram protein.
The content of protein carbonyl and cellular activities
of CAT, SOD, and GSH-Px were measured with commercial kits as described above. The concentration of
8-OHdG was determined with a commercial kit (number 201005; R&D Systems, 614 McKinley Place NE,
Minneapolis, MN 55413).
Muscle fatty acid composition was measured according to Gao et al. (2010). Briefly, total lipid extracts
of muscle samples were transmethylated into fatty acid
methyl esters and separated by using a gas chromatograph
(GC2014B; Shimadzu Corp.). Aliquots of 1 L were injected into a DB-23 capillary column (30 m by 0.25 mm
i.d. and 0.25-m thickness; Agilent J&W Technologies,
Hangzhou, Zhejiang 310030, P. R. China). Nitrogen was
used as the carrier gas at a constant flow rate of 24 mL/
min. The following oven temperature program was used:
150C held for 2 min, increased to 180C at 6C/min and
held for 2 min, then increased to 210C at 5C/min, and
210C held for 2 min. Peaks were separated by using a
flame-ionization detector and quantified and identified
with an electric integrator (CR-8A; Shimadzu Corp.) by
using pure standard mixtures (Sigma-Aldrich). The contents of SFA, MUFA, and PUFA were measured and the
ratios of MUFA to SFA and PUFA to SFA were calculated.
Oxidative Injury and Enzyme
Activity in Mitochondria
Breast and leg muscle mitochondria were isolated
according to Bhattacharya et al. (1991) and Bottje et al.
(2002), with modifications. All media were ice-cold, and
procedures were done on ice or at 4C. Muscles were
cleaned of connective tissue and fat and finely minced in

an empty dish. Minced tissues were incubated in 10 mL


of medium A (100 mM sucrose, 10 mM EDTA, 100 mM
Tris-HCl, and 46 mM KCl, pH 7.4) containing 0.02%
nagarase for 5 min on ice. The tissues were washed and
resuspended with 10 mL medium A plus 0.5% BSA.
The minced tissue was homogenized. The homogenate
was centrifuged (500 g for 10 min at 4C) for suspension and the resulting supernatant was centrifuged
(12,000 g for 10 min at 4C) to obtain the mitochondrial pellet that was resuspended and washed in 10 mL
of isolation medium A plus 0.5% BSA. Mitochondria
were pelleted by centrifugation (12,000 g for 10 min at
4C) in incubation medium (230 mM mannitol, 70 mM
sucrose, 20mM Tris-HCl, and 5 mM KH2PO4, pH 7.4).
The resulting pellet was resuspended in 2 mL of incubation medium and placed on ice for the functional and
respiratory inhibitor studies described below.
Protein carbonyl contents; activities of CAT, SOD,
and GSH-Px; and 8-OHdG were determined with a commercial kit as described above. The activities of the respiratory chain complexes (I and III) were assessed with
an UV spectrophotometer using the method described by
Bottje et al. (2002) and Iqbal et al. (2004). The respiratory
chain complex activities are expressed in units of activity
per minute per milligram of mitochondrial protein.
Statistical Analyses
The data are presented as means SEM (n = 8).
All data were analyzed with 1-way ANOVA to test the
main effect of heat stress. The effect was considered
statistically significant when P < 0.05
RESULTS
Blood Variables
Plasma concentrations of TBARS (P < 0.01) and
8-OHdG (P < 0.001) were increased by heat exposure
treatment (Fig. 1a and 1b). Heat exposure decreased
plasma concentrations of TAC (P < 0.01) and AIHR (P<
0.001; Fig. 1c and 1d). In contrast, urate (P < 0.001) and
allantoin (P < 0.01) levels were increased by heat exposure treatment (Fig. 1e). The activities of antioxidant enzymes were differently affected. Superoxide dismutase
activity increased (P < 0.001) and GSH-Px activity decreased (P < 0.001) in heat-challenged broilers, whereas
CAT activity was not significantly altered (P > 0.05;
Fig.1f) by heat exposure treatment.
Muscle
In breast muscle, activities of SOD (P < 0.001) and
CAT (P < 0.01) were higher in heat-stressed chickens

Heat stress induces oxidative injury

2147

Figure 1. Effect of heat exposure treatment (32 1C for 7 d) on plasma activities of antioxidative enzymes and oxidative damage biomarkers of
broilers. 8-OHdG = 8-hydroxydeoxyguanosine; AIHR = ability to inhibit hydroxyl radicals; CAT = catalase; GSH-Px = glutathione peroxidase; SOD =
superoxide dismutase; TAC = total antioxidant capacity; TBARS = thiobarbituric acid reacting substances.

compared to controls, whereas the activity of GSHPx was not affected (P > 0.05; Fig. 2a). In thigh muscle, however, SOD (P = 0.001) and CAT (P < 0.05)
activities were decreased whereas GSH-Px activity
was increased (P < 0.001) by heat exposure (Fig. 2b).
Compared to control birds, heat-stressed broilers had
higher concentrations of protein carbonyl (P < 0.001)
and TBARS (P < 0.001) in breast and thigh muscles

(Fig. 2c and 2d). Heat exposure treatment increased


the contents of 8-OHdG in breast muscle (P < 0.05)
but not in thigh muscle (P = 0.104; Fig. 2c and 2d).
Fatty acid composition of breast and thigh muscle
tissues was not significantly (P > 0.05) affected by
heat exposure treatment (Supplemental Table S1 and
S2). The contents of PUFA and ratios of MUFA to SFA
and PUFA to SFA in breast and thigh muscles of heat-

2148

Chunxu et al.

Figure 2. Effect of heat exposure treatment (32 1C for 7 d) on activities of antioxidant enzymes and contents of oxidative damage biomarkers in
breast and thigh muscles of broilers. 8-OHdG = 8-hydroxydeoxyguanosine; CAT = catalase; GSH-Px = glutathione peroxidase; SOD = superoxide dismutase; TBARS = thiobarbituric acid reacting substances.

stressed broilers were not significantly different (P >


0.05) compared with control ones.
Mitochondria
Heat exposure increased SOD activity but decreased GSH-Px activity (P < 0.05) in both breast and
thigh muscles (Fig. 3a and 3b). In contrast, CAT activity was not influenced (P > 0.05; Fig. 3a and 3b).
Heat exposure reduced AIHR level (P < 0.05) and increased 8-OHdG (P< 0.05) in either breast or thigh
muscle (Fig. 3c and 3d).
The respiratory chain complex activities were differently affected by heat stress. The activity of complex I was suppressed (P < 0.01) by heat exposure in
both breast and thigh muscles (Fig. 4a). Complex III
activity, however, was elevated in breast (P < 0.01)
but not in thigh muscle (P > 0.05) of heat-stressed
broilers (Fig. 4b).

DISCUSSION
In the present study, the effect of heat exposure
on the oxidative stress in mitochondria, skeletal muscle, and plasma was investigated. The result indicated
that heat stressinduced oxidative injuries occurred in
mitochondria, skeletal muscles, and the whole body.
Antioxidant enzyme activity responded to heat stress
in a tissue-specific manner. Increased circulating allantoin level demonstrates that urate functions as an
antioxidant during heat stress. The suppressed mitochondrial complex I activity is suggested to be associated with induced oxidative stress by heat exposure.
Heat ExposureInduced Whole Body Oxidative Stress
The antioxidant enzyme system, including SOD,
CAT, and GSH-Px, works in concert with free radical
scavengers to quench ROS and to protect cells from

Heat stress induces oxidative injury

2149

Figure 3. Effect of heat exposure treatment (32 1C for 7 d) on antioxidant enzymatic activities, the contents of 8-hydroxydeoxyguanosine (8OHdG), and ability to inhibit hydroxyl radicals (AIHR) in mitochondria of breast and thigh muscles of broilers. CAT = catalase; GSH-Px = glutathione
peroxidase; SOD = superoxide dismutase.

oxidative damage (Weiss, 1986). The balance between


the production of free radicals and the antioxidant systems could be disturbed by heat stress in sheep (Chauhan
et al., 2014), chickens (Lin et al., 2000, 2006b, 2008),
and Japanese quails (Sahin et al., 2003; Del Vesco et
al., 2014). Our previous work demonstrated that heat
exposure augmented the production of ROS in laying
hens (Lin et al., 2008). Acute heat exposure (32C for
6 h) resulted in oxidative damage in the liver of 5-wkold broiler chickens (Lin et al., 2006a,b). In the present
study, broilers were exposed to high temperature (32
1.0C) for a relatively long period (7 d). The oxidative
damage in lipid, protein, and nucleoside was estimated,
respectively, by TBARS, protein carbonyl contents, and
8-OHdG. The elevated plasma TBARS and 8-OHdG in
heat-stressed broilers indicated that heat exposureinduced oxidative injuries, in line with the aforementioned
previous work.
In this study, we evaluated the nonenzymatic and
enzymatic systems, respectively. The decreased TAC

in heat-stressed broilers indicated that the nonenzymatic scavenge capacity was lowered by heat exposure. This speculation was supported by the observation that heat-stressed chickens had a lower AIHR,
which was determined to reflect the ability to inhibit
hydroxyl radicals (Liu et al., 2013).
For enzymatic system, the increased SOD, decreased GSH-Px, and unaltered CAT activities imply
that the balance between the production and scavenging of hydroxyl radicals is disrupted, resulting in the
accumulation of hydroxyl radicals. The responses of
antioxidant enzyme activity during heat exposure were
different in previous studies. For example, in laying
ducks, heat exposure decreased plasma activities of
SOD and GSH-Px (Ma et al., 2014). In contrast, plasma SOD activity was increased in heat-stressed laying hens (Lin et al., 2008). Acute heat stress increased
serum activities of SOD, CAT, and GSH-Px (Yang et
al., 2010). In the present study, the decreased GSH-Px
and unaltered CAT activities may be related the high

2150

Chunxu et al.

Figure 4. Effect of heat exposure treatment (32 1C for 7 d) on respiratory chain complex activities in breast and thigh muscle of broilers.

Michaelis constant (Km) of CAT rather than that of


GSH-Px (Little et al., 1970; Austin et al., 1988). Hence,
the result suggests that the suppressed ROS scavenging capacity should be responsible, at least partially, for
the oxidative injury induced by heat stress.
Heat ExposureInduced Oxidative
Injuries in Skeletal Muscles
Heat stress induces oxidative stress in skeletal muscle (Azad et al., 2010). In humans, exercise-associated
rises in core and muscle temperatures causes an increased production of ROS and oxidative stress (Salo et
al., 1991). Heat stress stimulates intracellular and extracellular superoxide production in the diaphragm (Zuo
et al., 2000). Induction of oxidative stress was associated with a heat stressinduced increase in rectal and
muscle temperatures in poultry as well (Mujahid et al.,
2005; Lin et al., 2008). In line with previous works, the
increased levels of protein carbonyls and TBARS and

8-OHdG concentrations in breast and thigh muscles indicated that heat stress resulted in oxidative injury in
skeletal muscles of broilers, regardless of muscle type.
The altered antioxidative enzyme activities were
involved in the development of oxidative injury. The elevated SOD and CAT activities and unaffected GSH-Px
activity in breast muscle was in line with previous work
of Ghazi Harsini et al. (2012), who reported that GSHPx activity remained relatively constant whereas SOD
activity levels in breast muscle (pectoralis superficialis) were enhanced on exposure to heat stress. Similarly,
there was a temperature-dependent elevation in Cu/ZnSOD activity whereas GSH-Px activity remained relatively constant during heat stress (Azad et al., 2010).
In contrast, the increased GSH-Px but decreased SOD
and CAT activities in thigh muscle indicated there is a
tissue-specific response in the antioxidative enzymatic
system. As oxidative injuries were detected in breast
and thigh muscle tissues, the result imply that the activation of antioxidative enzymes cannot prevent the oxidative injury induced by heat exposure in breast, and on
the other hand, the suppressed enzyme activities suggests the suppressed radical elimination.
We further evaluated the redox balance in mitochondria of muscle tissues. The higher concentration
of 8-OHdG in breast and thigh muscles of heat stress
chickens compared with control ones indicated that
oxidative stress occurred in mitochondria, in line with
the work of Mujahid et al. (2007c), who reported that
oxidative damage in mitochondrial lipids and proteins
occurred in muscles of heat-stressed chickens (Mujahid
et al., 2007c). Moreover, the decreased AIHR and elevated SOD and decreased GSH-Px and CAT activities
may further imply the arrested reduction capacity for
hydrogen peroxide in mitochondria of skeletal muscles.
During the movement of electrons in the transport
chain, electrons may leak from the respiratory chain before they reach the terminal electron acceptor (Chance
et al., 1979). The overproduction of mitochondrial ROS
in chicken skeletal muscle under heat stress might result
from enhanced substrate oxidation and downregulation
of avUCP (Mujahid et al., 2006, 2007a,b). The oxidative
phosphorylation system consists of 4 multiprotein complexes (I to IV) and ATP synthase (complex V), through
which electrons from reduced substrates pass to oxygen.
Complex I and III are the 2 principal sites of superoxide
generation in mitochondria (St-Pierre et al., 2002; Brand
et al., 2004). The lowered activity of complex I in breast
and thigh muscle tissues of heat-stressed broilers compared to control birds and the differently influenced activity of complex III suggest that complex I is involved
in the enhanced leakage of superoxide during heat stress.
In mammals, complex I inhibition resulted in greater
ROS production and oxidative stress (Pitknen and

2151

Heat stress induces oxidative injury

Robinson, 1996; Barrientos and Moraes, 1999). More


recently, it was shown that the quinol site in complex I
and the flavin site in complex II generated about half of
the hydrogen peroxides in rat muscle mitochondria in an
ex vivo system (Goncalves et al., 2015). In accordance
with the previous reports in mammals, the result suggests that heat stress could stimulate the production of
ROS by suppressing the activity of complex I. Oxidative
stressinduced mitochondrial inefficiency in muscle is
linked with low feed efficiency of broilers (Bottje et al.,
2002, 2006). The present result suggests that mitochondrial oxidative damage induced by heat stress should be
responsible, at least partially, for the retarded growth performance of heat-stressed broilers.
The degree of fatty acid unsaturation in biomembranes is associated with their sensitivity to lipid peroxidation (Rebol et al., 2006). Our previous work
indicated that exogenous glucocorticoid administration simultaneously induced oxidative damage and increased the saturation level of skeletal muscle fatty acids (Gao et al., 2010). In the present study, unchanged
SFA, MUFA, and PUFA concentrations and their ratios indicate that the fatty acid composition is not influenced by oxidative stress induced by heat exposure.
Role of Urate in the Nonenzymatic Antioxidative System
Birds have a more potent antioxidant system than
mammals as the high circulating levels of urate (Klandorf
et al., 1999; Simoyi et al., 2002, 2003; Machn et al.,
2004). Urate may serve as an antioxidant in mammals
(Hellsten et al., 1997), although it is not a major factor controlling oxidative stress in vivo (Hershfield et al.,
2010). During stress, increased plasma urate as a result
of protein catabolism could enhance the nonenzymatic
antioxidant capacity in chickens (Lin et al., 2004a,b).
High uric acid concentrations are associated with increased serum antioxidant capacity and reduced oxidative stress during acute physical exercise in healthy
subjects, indicating that the antioxidant properties of
uric acid are of biological importance in vivo (Waring
et al., 2003). Therefore, in the present study, we further
investigated that if the increased plasma concentration
of urate could play a protective role during oxidative
stress. Compared with control chickens, a higher allantoin concentration (130.8%) was matched to a higher
urate concentration (132.4%) in heat-stressed broilers,
suggesting that the augmented oxidative stress broilers
during heat exposure. However, the similar ratio of allantoin to urate in heat stress broilers (8.09%) and control chickens (8.18%) implies that the oxidation of urate
is concentration dependent during oxidative stress. To
our best knowledge, this is the first report that showed
urate could serve as antioxidant during stress.

Under certain circumstances, however, urate can


be a prooxidant within cells (Corte and Stirpe, 1972;
Hellsten et al., 1997; Sautin and Johnson, 2008). During
inflammation, myeloperoxidase oxidized urate to a reactive hydroperoxide in combination with superoxide and
hydrogen peroxide (Meotti et al., 2011). These results indicated that urate is a physiological peroxidase substrate
and could be oxidized to the urate radical. Urate should
not necessarily be considered the end product of purine
metabolism, and elevated allantoin levels may reflect
peroxidase activity rather than nonspecific scavenging
of oxidants by urate (Meotti et al., 2011). In contrast, it
is also possible that hyperuricemia represents a compensatory or protective mechanism to try to prevent or
correct oxidative damage (lvarez-Lario and MacarrnVicente, 2011). Therefore, whether the antioxidant effects of urate are predominant over the prooxidant effect
during heat stress needs to be investigated further.
In conclusion, heat stress induced oxidative damages in mitochondria, skeletal muscles, and the whole
body. The suppressed mitochondrial complex I activity is associated with oxidative stress induced by heat
exposure, which, in turn, is linked with the oxidative
damages in muscle tissues and plasma. Urate could
serve as an antioxidant during heat stress in a concentration dependent manner.
LITERATURE CITED
lvarez-Lario, B., and J. Macarrn-Vicente. 2011. Is there anything
good in uric acid? Q. J. Med. 104:10151024. doi:10.1093/
qjmed/hcr159.
Austin, L., H. Arthur, M. de Niese, A. Gurusinghe, and M. S. Baker.
1988. Micromethods in single muscle fibers. 1. Determination
of catalase and superoxide dismutase. Anal. Biochem.
174:568574. doi:10.1016/0003-2697(88)90057-7.
Azad, M. A. K., M. Kikusato, T. Maekawa, H. Shirakawa, and
M. Toyomizu. 2010. Metabolic characteristics and oxidative
damage to skeletal muscle in broiler chickens exposed to
chronic heat stress. Comp. Biochem. Physiol. A Mol. Integr.
Physiol. 155:401406. doi:10.1016/j.cbpa.2009.12.011.
Barrientos, A., and C. T. Moraes. 1999. Titrating the effects of mitochondrial complex I impairment in the cell physiology. J. Biol.
Chem. 274:1618816197. doi:10.1074/jbc.274.23.16188.
Bhattacharya, S. K., J. H. Thakar, P. L. Johnson, and D. R. Shanklin.
1991. Isolation of skeletal muscle mitochondria from hamsters using an ionic medium containing ethylenediaminetetraacetic acid and nagarse. Anal Biochem. 192: 344-349.
Bottje, W., M. Iqbal, Z. X. Tang, D. Cawthon, R. Okimoto, T. Wing,
and M. Cooper. 2002. Association of mitochondrial function
with feed efficiency within a single genetic line of male broilers. Poult. Sci. 81:546555. doi:10.1093/ps/81.4.546.
Bottje, W., N. R. Pumford, C. Ojano-Dirain, M. Iqbal, and K.
Lassiter. 2006. Feed efficiency and mitochondrial function.
Poult. Sci. 85:814. doi:10.1093/ps/85.1.8.
Bottje, W. G., S. Wang, F. J. Kelly, C. Dunster, A. Williams, and
I. Mudway. 1998. Antioxidant defenses in lung lining fluid
of broilers: Impact of poor ventilation conditions. Poult. Sci.
77:516522. doi:10.1093/ps/77.4.516.

2152

Chunxu et al.

Brand, M. D., J. A. Buckingham, T. C. Esteves, K. Green, A. J.


Lambert, S. Miwa, M. P. Murphy, J. L. Pakay, D. A. Talbot,
and K. S. Echtay. 2004. Mitochondrial superoxide and aging: Uncoupling-protein activity and superoxide production.
Biochem. Soc. Symp. 71:203213.
Carro, M. D., E. Falkenstein, W. J. Radke, and H. Klandorf. 2010.
Effects of allopurinol on uric acid concentrations, xanthine
oxidoreductase activity and oxidative stress in broiler chickens.
Comp. Biochem. Physiol. C Toxicol. Pharmacol. 151:1217.
Chance, B., H. Sies, and A. Boveris. 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527605.
Chauhan, S. S., P. Celi, B. J. Leury, I. J. Clarke, and F. R. Dunshea.
2014. Dietary antioxidants at supranutritional doses improve
oxidative status and reduce the negative effects of heat stress in
sheep. J. Anim. Sci. 92:33643374. doi:10.2527/jas.2014-7714.
Corte, E. D., and F. Stirpe. 1972. The regulation of rat liver xanthine
oxidase. Involvement of thiol groups in the conversion of the
enzyme activity from dehydrogenase (type D) into oxidase (type
O) and purification of the enzyme. Biochem. J. 126:739745.
De Boeck, S., and J. Stockx. 1978. A purine N1-C6 hydrolase activity in the chicken egg yolk: a vestigial enzyme? Enzyme
23: 56-63. doi=ijps.2009.545.552.
Del Vesco, A. P., E. Gasparino, D. O. Grieser, V. Zancanela, F.
R. S. Gasparin, J. Constantin, and A. R. Oliveira Neto. 2014.
Effects of methionine supplementation on the redox state of
acute heat stressexposed quails. J. Anim. Sci. 92:806815.
doi:10.2527/jas.2013-6829.
Drge, W. 2002. Free radicals in the physiological control of cell
function. Physiol. Rev. 82:4795.
Gao, J., H. Lin, X. J. Wang, Z. G. Song, and H. C. Jiao. 2010. Vitamin
E supplementation alleviates the oxidative stress induced by
dexamethasone treatment and improves meat quality in broiler
chickens. Poult. Sci. 89:318327. doi:10.3382/ps.2009-00216.
Ghazi Harsini, S., M. Habibiyan, M. M. Moeini, and A. R.
Abdolmohammadi. 2012. Effects of dietary selenium, vitamin E, and their combination on growth, serum metabolites,
and antioxidant defense system in skeletal muscle of broilers under heat stress. Biol. Trace Elem. Res. 148:322330.
doi:10.1007/s12011-012-9374-0.
Goncalves, R. L. S., C. L. Quinlan, I. V. Perevoshchikova, M. HeyMogensen, and M. D. Brand. 2015. Sites of superoxide and
hydrogen peroxide production by muscle mitochondria assessed ex vivo under conditions mimicking rest and exercise.
J. Biol. Chem. 290:209227. doi:10.1074/jbc.M114.619072.
Grootveld, M., and B. Halliwell. 1986. Aromatic hydroxylation
as a potential measure of hydroxyl- radical formation in vivo.
Identification of hydroxylated derivatives of salicylate in human body fluids. Biochem. J. 237:499504.
Halliwell, B., and J. M. C. Gutteridge. 1989. Free radicals in biology and medicine. 2nd ed. Clarendon, Oxford, UK.
Hellsten, Y., P. C. Tullson, E. A. Richter, and J. Bangsbo. 1997. Oxidation
of urate in human skeletal muscle during exercise. Free Radic.
Biol. Med. 22:169174. doi:10.1016/S0891-5849(96)00286-9.
Hershfield, M. S., L. J. Roberts II, N. J. Ganson, S. J. Kelly, I. Santisteban,
E. Scarlett, D. Jaggers, and J. S. Sundy. 2010. Treating gout with
pegloticase, a PEGylated urate oxidase, provides insight into the
importance of uric acid as an antioxidant in vivo. Proc. Natl. Acad.
Sci. USA 107:1435114356. doi:10.1073/pnas.1001072107.
Iqbal, M., N. R. Pumford, Z. X. Tang, K. Lassiter, T. Wing, M.
Cooper, and W. Bottje. 2004. Low feed efficient broilers within
a single genetic line exhibit higher oxidative stress and protein
expression in breast muscle with lower mitochondrial complex
activity. Poult. Sci. 83:474484. doi:10.1093/ps/83.3.474.
Klandorf, H., I. L. Probert, and M. Iqbal. 1999. In the defence
against hyperglycaemia: An avian strategy. Worlds Poult. Sci.
J. 55:251268. doi:10.1079/WPS19990019.

Lin, H., E. Decuypere, and J. Buyse. 2004a. Oxidative stress


induced by corticosterone administration in broiler chickens (Gallus gallus domesticus) 1. Chronic exposure. Comp.
Biochem. Physiol. B Biochem. Mol. Biol. 139:737744.
doi:10.1016/j.cbpc.2004.09.013.
Lin, H., E. Decuypere, and J. Buyse. 2004b. Oxidative stress
induced by corticosterone administration in broiler chickens (Gallus gallus domesticus) 2. Short-term effect. Comp.
Biochem. Physiol. B Biochem. Mol. Biol. 139:745751.
doi:10.1016/j.cbpc.2004.09.014.
Lin, H., E. Decuypere, and J. Buyse. 2006a. Acute heat stress induces oxidative stress in broiler chickens. Comp. Biochem.
Physiol. A Mol. Integr. Physiol. 144:1117. doi:10.1016/j.
cbpa.2006.01.032.
Lin, H., D. De Vos, E. Decuypere, and J. Buyse. 2008. Dynamic
changes in parameters of redox balance after mild heat
stress in aged laying hens (Gallus gallus domesticus). Comp.
Biochem. Physiol. C Toxicol. Pharmacol. 147:3035.
Lin, H., R. Du, X. H. Gu, F. C. Li, and Z. Y. Zhang. 2000. A study
on the plasma biochemical indices of heat-stressed broilers.
Asian-Australas. J. Anim. Sci. 13:12101218. doi:10.5713/
ajas.2000.1210.
Lin, H., J. Gao, Z. G. Song, and H. C. Jiao. 2009. Corticosterone administration induces oxidative injury in skeletal muscle of broiler
chickens. Poult. Sci. 88:10441051. doi:10.3382/ps.2008-00312.
Lin, H., H. C. Jiao, J. Buyse, and E. Decuypere. 2006b. Strategies
for prevention of heat stress in poultry. Worlds Poult. Sci. J.
62:7185. doi:10.1079/WPS200585.
Little, C., R. Olinescu, K. G. Reid, and P. J. OBrien. 1970.
Properties and regulation of glutathione peroxidase. J. Biol.
Chem. 245:36323636.
Liu, X. F., L. M. Zhang, H. N. Guan, Z. W. Zhang, and S. W. Xu.
2013. Effects of oxidative stress on apoptosis in manganeseinduced testicular toxicity in cocks. Food Chem. Toxicol.
60:168176. doi:10.1016/j.fct.2013.07.058.
Ma, X., Y. Lin, H. Zhang, W. Chen, S. Wang, D. Ruan, and Z. Jiang.
2014. Heat stress impairs the nutritional metabolism and reduces the productivity of egg-laying ducks. Anim. Reprod.
Sci. 145:182190. doi:10.1016/j.anireprosci.2014.01.002.
Machn, M., M. F. Simoyi, K. P. Blemings, and H. Klandorf. 2004.
Increased dietary protein elevates plasma uric acid and is associated with decreased oxidative stress in rapidly-growing
broilers. Comp. Biochem. Physiol. B Biochem. Mol. Biol.
137:383390. doi:10.1016/j.cbpc.2004.01.002.
Mahmoud, K. Z., and F. W. Edens. 2003. Influence of selenium sources on age-related and mild heat stress-related changes of blood
and liver glutathione redox cycle in broiler chickens (Gallus
domesticus). Comp. Biochem. Physiol. B Biochem. Mol. Biol.
136:921934. doi:10.1016/S1096-4959(03)00288-4.
Meotti, F. C., G. N. L. Jameson, R. Turner, D. T. Harwood, S. Stockwell,
M. D. Rees, S. R. Thomas, and A. J. Kettle. 2011. Urate as a physiological substrate for myeloperoxidase: implications for hyperuricemia and inflammation. J. Biol. Chem. 286: 12901-12911.
Mujahid, A., Y. Akiba, and M. Toyomizu. 2007a. Acute heat stress
induces oxidative stress and decreases adaptation in young
white leghorn cockerels by downregulation of avian uncoupling protein. Poult. Sci. 86:364371. doi:10.1093/ps/86.2.364.
Mujahid, A., Y. Akiba, C. H. Warden, and M. Toyomizu. 2007b.
Sequential changes in superoxide production, anion carriers and substrate oxidation in skeletal muscle mitochondria of heat-stressed chickens. FEBS Lett. 581:34613467.
doi:10.1016/j.febslet.2007.06.051.
Mujahid, A., N. R. Pumford, W. Bottje, K. Nakagawa, T. Miyazawa,
Y. Akiba, and M. Toyomizu. 2007c. Mitochondrial oxidative
damage in chicken skeletal muscle induced by heat stress.
Jpn. Poult. Sci. 44:439445. doi:10.2141/jpsa.44.439.

Heat stress induces oxidative injury


Mujahid, A., K. Sato, Y. Akiba, and M. Toyomizu. 2006. Acute
heat stress stimulates mitochondrial superoxide production in broiler skeletal muscle, possibly via downregulation
of uncoupling protein content. Poult. Sci. 85:12591265.
doi:10.1093/ps/85.7.1259.
Mujahid, A., Y. Yoshiki, Y. Akiba, and M. Toyomizu. 2005.
Superoxide radical production in chicken skeletal muscle induced by acute heat stress. Poult. Sci. 84:307314.
doi:10.1093/ps/84.2.307.
Pitknen, S., and B. H. Robinson. 1996. Mitochondrial complex I
deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase. J. Clin. Invest.
98:345351. doi:10.1172/JCI118798.
Rebol, A., M. L. Rodrguez, L. T. Ortiz, C. Alzueta, C. Centeno,
A. Viveros, A. Brenes, and I. Arija. 2006. Effect of dietary
high-oleic acid sunflower seed, palm oil and vitamin E supplementation on broiler performance, fatty acid composition
and oxidation susceptibility of meat. Br. Poult. Sci. 47:581
591. doi:10.1080/00071660600939727.
Rhoads, R. P., L. H. Baumgard, and J. K. Suagee. 2013. Metabolic
priorities during heat stress with an emphasis on skeletal muscle. J. Anim. Sci. 91:24922503. doi:10.2527/jas.2012-6120.
Sahin, K., M. Onderci, N. Sahin, M. F. Gursu, and O. Kucuk. 2003.
Dietary vitamin C and folic acid supplementation ameliorates
the detrimental effects of heat stress in Japanese quail. J. Nutr.
133:18821886.
Salo, D. C., C. M. Donovan, and K. J. A. Davies. 1991. HSP70
and other possible heat shock or oxidative stress proteins
are induced in skeletal muscle, heart, and liver during exercise. Free Radic. Biol. Med. 11:239246. doi:10.1016/08915849(91)90119-N.
Sautin, Y. Y., and R. J. Johnson. 2008. Uric acid: The oxidantantioxidant paradox. Nucleosides Nucleotides Nucleic Acids
27:608619. doi:10.1080/15257770802138558.

2153

Simoyi, M. F., K. V. Dyke, and H. Klandorf. 2002. Manipulation


of plasma uric acid in broiler chicks and its effect on leukocyte oxidative activity. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 282:R791R796.
Simoyi, M. F., E. Falkenstein, K. V. Dyke, K. P. Blemings, and H.
Klandorf. 2003. Allantoin, the oxidation product of uric acid
is present in chicken and turkey plasma. Comp. Biochem.
Physiol. B Biochem. Mol. Biol. 135:325335. doi:10.1016/
S1096-4959(03)00086-1.
St-Pierre, J., J. A. Buckingham, S. J. Roebuck, and M. D. Brand.
2002. Topology of superoxide production from different sites
in the mitochondrial electron transport chain. J. Biol. Chem.
277:4478444790. doi:10.1074/jbc.M207217200.
Waring, W. S., A. Convery, V. Mishra, A. Shenkin, D. J. Webb, and
S. R. J. Maxwell. 2003. Uric acid reduces exercise-induced
oxidative stress in healthy adults. Clin. Sci. 105:425430.
doi:10.1042/CS20030149.
Weiss, J. S. 1986. Oxygen, ischemia and inflammation. Acta
Physiol. Scand (Suppl.) 548: 9-37.
Wu, L. L., C. C. Chiou, P. Y. Chang, and J. T. Wu. 2004. Urinary
8-OHdG: A marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clin. Chim. Acta
339:19. doi:10.1016/j.cccn.2003.09.010.
Yang, L., G. Y. Tan, Y. Q. Fu, J. H. Feng, and M. H. Zhang. 2010.
Effects of acute heat stress and subsequent stress removal
on function of hepatic mitochondrial respiration, ROS production and lipid peroxidation in broiler chickens. Comp.
Biochem. Physiol. C Toxicol. Pharmacol. 151:204208.
Yu, Byung P. 1994. Cellular defenses against damage from reactive oxygen species, Physiol.Rev. 74: 139-162.
Zuo, L., F. L. Christofi, V. P. Wright, C. Y. Liu, A. J. Merola, L.
J. Berliner, and T. L. Clanton. 2000. Intra-and extracellular
measurement of reactive oxygen species produced during
heat stress in diaphragm muscle. Am. J. Physiol. Cell Physiol.
279:10581066.

You might also like