Comparative Biochemistry and Physiology, Part C 151 (2010) 204–208

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part C

j ourna l homepage: www.e lsev ie r.com/ locate /cbpc

Effects of acute heat stress and subsequent stress removal on function of hepaticmitochondrial respiration, ROS production and lipid peroxidation in broiler chickens

Lin Yang b,1, Gao-Yi Tan a,b,1, Yu-Qiang Fu a,b, Jin-Hai Feng a, Min-Hong Zhang a,⁎a State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, PR Chinab Department of Animal Nutrition and Feed Science, College of Animal Science, South China Agriculture University, Guangzhou 510642, PR China

⁎ Corresponding author. Tel.: +86 10 6289 5517; faxE-mail address: minhong_zhang@yahoo.com (M.-H.

1 Contributed equally to this publication.

1532-0456/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.cbpc.2009.10.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 June 2009Received in revised form 21 October 2009Accepted 22 October 2009Available online 31 October 2009

Keywords:Acute heat stressMitochondrial respiratory complexReactive oxygen speciesLipid peroxidationBroiler chicken

In order to investigate the effects of acute heat stress and subsequent stress removal on function of hepaticmitochondrial respiration, production of reactive oxygen species (ROS) and lipid peroxidation in broilerchickens, 128 six-week-old broiler chickens were kept in a controlled-environment chamber. The broilerchickens were initially kept at 25 °C (relative humidity, RH, 70±5%) for 6 d and subsequently exposedto 35 °C (RH, 70±5%) for 3 h, then the heat stress was removed and the temperature returned to 25 °C (RH,70±5%). Blood and liver samples were obtained before heat exposure and at 0 (at the end of the three-hourheating episode, this group is also abbreviated as the HT group), 1, 2, 4, 8, 12 h after the stress was removed.The results showed that acute heat stress induced a significant production of ROS, function of themitochondrial respiratory chain, antioxidative enzymes [superoxide dismutase (SOD), catalase (CAT) andglutathione peroxidase (GSH-Px)] activity, and formation of malondialdehybe (MDA). Within the first 12 hafter removal of the heat stress, the acute modification of the above parameters induced by heat stressgradually approached to pre-heat levels. The results of the present study suggest that acute exposure to hightemperatures may depress the activity of the mitochondrial respiratory chain. This leads to over-productionof ROS, which ultimately results in lipid peroxidation and oxidative stress. When the high temperature wasremoved, the production of ROS, mitochondrial respiratory function and oxidative injury that were inducedby acute heat exposure gradually approached the levels observed before heating, in a time-dependentmanner.

: +86 10 6281 5990.Zhang).

l rights reserved.

© 2009 Elsevier Inc. All rights reserved.

1. Introduction

Previous in vivo and in vitro studies have demonstrated that heatstress can disturb the balance between the production of reactiveoxygen species (ROS) and the antioxidant systems, and may furtherstimulate the formation of ROS (Mahmoud and Edens, 2003; Lin et al.,2006; Feng et al., 2008). Superfluous ROS induced by heat stress cancause oxidative injury, such as lipid peroxidation and oxidative damageto proteins and DNA (Halliwell and Aruoma, 1991; Mujahid et al.,2007b). In broiler chickens, oxidative injury induced by high ambienttemperatures has been demonstrated in a number of studies (Altan etal., 2003;Mujahid et al., 2005, 2006, 2007b; Lin et al., 2004, 2006, 2008).

The electron transport chain of the mitochondria is the majorsource of cellular ROS (Boveris et al., 1972). The inhibition of therespiratory chain by damage, mutation, ischaemia or loss of cyto-chrome c will lead to the formation of ROS (Kudin et al., 2005;

Kussmaul and Hirst, 2006; Murphy, 2009). However, the mechanismof ROS production in chickens subjected to heat stress requireselucidation. Mujahid et al. (2005) speculated that the mechanismsthroughwhich increased ROS production occurs in heat-treatedmeat-type chickens may be associated with mitochondrial damage, suchas a reduction in the activity of the mitochondrial respiratory chaincomplex, or a down-regulation of the synthesis of avian uncouplingprotein (avUCP) (Mujahid et al., 2005, 2006). Subsequent studies haveshown that over-production of mitochondrial ROS in chicken skeletalmuscle under conditions of heat stress may result from enhancedsubstrate oxidation and down-regulation of avUCP in a time-dependent manner (Mujahid et al., 2007a). However, to date therehave been limited information about the function of themitochondrialrespiratory chain in broiler chickens exposed to heat stress.

In view of these considerations, the present study investigated theeffect of acute heat stress and subsequent removal on function ofhepatic mitochondrial respiration, ROS production, antioxidativeenzymes activity and lipid peroxidation in broiler chickens. Inaddition, the recovery from the effects of oxidative injury inducedby acute heat stress was evaluated during the subsequent period,when the heat stress had been removed.

205L. Yang et al. / Comparative Biochemistry and Physiology, Part C 151 (2010) 204–208

2. Materials and methods

2.1. Animals and experimental design

128 6-week-old male broiler chickens (Arbor Acres) were used inthe current study. All the chickens were randomly divided into eightreplicates; each replicate consisted of a cage containing 16 birds. Thebirds were housed in a controlled-environment chamber at 25 °C and70±5% relative humidity (RH) for 6 days. After the adaptation period,the chickens were exposed to 35 °C (RH, 70±5%) for 3 h, then theheat stress was removed immediately and the temperature returnedto 25 °C (RH, 70±5%). Feed and water were offered ad libitum duringthe whole experimental procedure.

Blood and liver sampleswere obtained before heat exposure and at0 (at the end of the three-hour heating episode, this group is alsoabbreviated as the HT group), 1, 2, 4, 8, 12 h after the stress wasremoved. Eight birds were sampled at each of the sampling times. Theserum was isolated by centrifugation for 10 min at 2500×g and thenstored at −80 °C for analyses. The liver samples used for enzymesassay and lipid peroxidation assay were collected and frozenimmediately in liquid nitrogen, then stored at −80 °C.

2.2. Isolation of hepatic mitochondria

Hepatic mitochondria were isolated by differential centrifugationas described by previous studies (Rustin et al., 1994; Cawthon et al.,1999), with modifications. Briefly, 1 g of liver was placed in 10 mL ofisolation medium [210 mM mannitol, 70 mM sucrose, 5 mM N-2-hydroxyethyl piperazine-N′-ethanesulfonic acid (Hepes), 0.2 mg/mLof bovine serum albumin (BSA), and 1 mM ethylene glycol tetraaceticacid (EGTA), pH 7.4], minced, and homogenized with a tissue grinder(10,000 cycles/min, twice for 15 s). The homogenate was thencentrifuged twice for 10 min at 1300×g, and the supernatant wascentrifuged again at 8700 ×g for 10 min. The final mitochondrialpellet was resuspended in medium containing 210 mM mannitol,70 mM sucrose, and 5 mM Hepes, pH 7.4. Mitochondrial proteinconcentrations were determined using the Coomassie Brilliant Blue G-250 reagent (27816, Fluka, Buchs, Switzerland) with BSA as astandard.

2.3. Assays for activity of mitochondrial respiratory enzymes

The mitochondrial respiratory enzymes assay was carried outaccording to previous studies (Chuang et al., 2002; Chan et al., 2005).All enzyme assays were performed using a UV/visible spectropho-tometer (TU-1810, Pgeneral, China). At least duplicate determinationwas carried out for each tissue sample in all enzyme assays. Allreagents used in enzyme assays were purchased from Sigma.

Nicotinamide adenine dinucleotide (NADH) cytochrome c reduc-tase (NCCR; Complexes I+III) activity was determined by thereduction of oxidized cytochrome c measured at 550 nm, and wascalculated as the difference in the presence or absence of rotenone.The activity was assayed in 50 mM K2HPO4 buffer (pH 7.4) containing1.5 mM KCN, 1.0 mM NADH and 5 μL of mitochondrial suspension inthe presence or absence of rotenone (20 μM). The reaction wasinitiated after 2 min of stabilization by adding 0.1 mM cytochrome c,and absorbance at 550 nm was measured at 5-second intervals overthe first 3 min at 37 °C. Themolar extinction coefficient of cytochromec at 550 nm is 28 mM−1cm−1.

Determination of succinate cytochrome c reductase (SCCR;Complexes II+III) activity was performed in 40 mM K2HPO4 buffer(pH 7.4) containing 20 mM succinate, 1.5 mM KCN and 5 μL ofmitochondrial suspension. After 5 min of incubation at 37 °C, thereaction was initiated by adding 50 μM cytochrome c, and absorbanceat 550 nm was measured at 5-second intervals over the first 2 min at37 °C.

Cytochrome c oxidase (CCO; Complex IV) activity was measuredby recording the oxidation of reduced cytochrome c at 550 nm. Theactivity of CCO is defined as the first order rate constant and iscalculated from the known concentration of ferrocytochrome c andthe amount of enzyme in the assay mixture. The activity was assayedin 10 mM K2HPO4 buffer (pH 7.4) containing 1.5 mM KCN, 20 mMsuccinate, and 5 μL of mitochondrial suspension. After 5 min ofincubation at 30 °C, the reaction was initiated by adding 45 μMferrocytochrome c, and absorbance at 550 nm was measured at 5-second intervals over the first 3 min.

2.4. Hepatic reactive oxygen species (ROS) determination

ROS formation in liver was evaluated by ESR spectroscopy(Mujahid et al., 2005; Lin et al., 2008). After chickens wereslaughtered, the fresh liver sample was prepared immediately in airor nitrogen. Liver tissue (1.5 g) was homogenized for 6 min afteraddition of 1.5 mL α-phenyl-N-tert-butylnitrone (PBN, Sigma-Aldrich, USA) in 3 mL toluene. Then the homogenate was centrifugedat 4000×g for 5 min at 4 °C and the supernatant of the homogenizedtissue was taken into low temperature vials and stocked in liquidnitrogen for electron spin resonance (ESR) measurement. The ESRspectra were recorded with Bruker ESP300 X-band spectrometer(Bruker, Germany) at room temperature. Typical instrument settingswere: modulation frequency 100 kHz; modulation amplitude, 1.14 G;microwave power, 12.9 mW; receiver gain, 8.00 e; time constant,163.84 ms; sweep time, 83.89 s; center field, 3486.95 G; field sweep,60 G. The ESR signal obtained with these parameters was used tocalculate the height of the central peak as an indication of ROS level.

2.5. Antioxidative enzymes activity and lipid peroxidation

The activities of superoxide dismutase (SOD), catalase (CAT),glutathione peroxidase (GSH-Px) and malondialdehybe (MDA) inliver, and serum were determined (TU-1810, Pgeneral, China) by thecorresponding assay kits (Nanjing Jiancheng Bioengineering Institute,Nanjing, China) according to the manufacturer's instructions. Nitritecolouration method was used to determine SOD activity with awavelength of 550 nm to determine absorbance. CAT activity wasmeasured at 405 nm according to Góth (1991) with the assay kit.GSH-Px activity was measured at 412 nm by quantifying the rate ofoxidation of reduced GSH to oxidized glutathione. Thiobarbitalmethod was used to determine MDA concentration with wavelength532 nm to determine absorbance. The liver tissue protein concentra-tions were determined using the Coomassie Brilliant Blue G-250reagent with bovine serum albumin as a standard.

2.6. Statistical analysis

Statistical analyses were carried out using the SPSS version 17.0software. Data for all the groups of birds were compared using one-way analysis of variance (ANOVA) followed by Duncan's multiplecomparison tests. The data were expressed as means±standard errorof mean (SEM) setting P<0.05 as criterion of statistical significance.

3. Results

The production of hepatic ROS at different time points is shown inFig. 1. Acute heat stress (temperature, 35 °C; RH, 70%; treatment time,3 h) induced significant hepatic production of ROS in comparison tocontrol (25 °C, 70%) values at the P<0.05 level. When the heat stresswas removed (recovery time) and the temperature returned to 25 °C(RH, 70±5%), the production of ROS gradually approaching pre-heatlevels with the progression of recovery time. These results demon-strated that acute heat stress caused a significant increase in thehepatic generation of ROS.

Fig. 1. Production of hepatic ROS at different time points. Values are mean±SEM ofeight replications. Data points with different superscripts are significantly different(one-way ANOVA) at the level of P<0.05 by Duncan's multiple comparison test.

Fig. 3. SOD activity in serum and liver tissue at different time points. Values are mean±SEM of eight replications. Data points with different superscripts are significantlydifferent (one-way ANOVA) at the level of P<0.05 by Duncan's multiple comparisontest.

206 L. Yang et al. / Comparative Biochemistry and Physiology, Part C 151 (2010) 204–208

The changes in mitochondrial function in liver tissue during theacute heat stress and subsequent stress removal phases wereevaluated by examining the activity of key enzymes in respiratorycomplexes I+III (NCCR), II+III (SCCR) and IV (CCO) at the differenttime points. The activities of NCCR, SCCR and CCO in hepaticmitochondria are shown in Fig. 2. In comparison to control, it can beseen that while the activity of NCCR and CCO in hepatic mitochondriaunderwent a significant decrease during the heat stress period(P<0.05), SCCR remained essentially unchanged (P>0.05). Whenthe heat stress was removed, the reduction in the activity of NCCR andCCO that was induced by heat stress also recovered gradually with theprogression of recovery time. Thus, acute heat stress affected theactivity of NCCR and CCO significantly.

The activity of SOD, CAT, GSH-Px and level of MDA are shown inFigs. 3–6, respectively. In comparison to controls, acute heat stressinduced a significant up-regulation of all the antioxidative enzymesactivity (P<0.05) and MDA production (P<0.05). When the heatstress was removed, both the SOD activity and the MDA level alsogradually approaching pre-heat levels with the progression ofrecovery time. These results demonstrate that acute heat stress cancause a compensatory increase in SOD activity and lipid peroxidation.

Fig. 2. Activities of NCCR, SCCR and CCO in hepatic mitochondria at different timepoints. Values are mean±SEM of eight replications. Data points with differentsuperscripts are significantly different (one-way ANOVA) at the level of P<0.05 byDuncan's multiple comparison test.

4. Discussion

The relative long lifespan of birds, together with their metaboliccharacteristics of high body temperature, high plasma level of glucoseand rapid metabolic rate, makes birds an interesting model for studiesof oxidative stress (Barja, 2002; Simoyi et al., 2002). Moreover, thecontinuous selection of broiler chickens for fast growth has beenassociated with increased susceptibility to heat stress (Washburnet al., 1980; Cahaner et al., 1995; Berrong and Washburn, 1998). TheArbor Acres broiler chicken, an example of a meat-type broiler breedthat shows rapid growth, was used as the subject of this study.

The liver is known to be the hub of themetabolism; it plays amajorrole in controlling glucose storage and flux. It is also known that,during heat stress, both lipids and carbohydrate stores can bemobilized for energy generation to attenuate the stress response(Manoli et al., 2007). In addition, many biochemical studies have beenperformed using mitochondria from liver cells; each hepatocytecontains 1000–2000 mitochondria, which in total occupy about one-fifth of the cell volume (Alberts et al., 2002). In this study, parametersrepresenting the function of hepatic mitochondria in response toacute heat exposure were measured, and corresponding indicators ofthe serum response were also determined.

Fig. 4. CAT activity in serum and liver tissue at different time points. Values are mean±SEM of eight replications. Data points with different superscripts are significantlydifferent (one-way ANOVA) at the level of P<0.05 by Duncan's multiple comparisontest.

Fig. 5. GSH-Px activity in serum and liver tissue at different time points. Values aremean±SEM of eight replications. Data points with different superscripts aresignificantly different (one-way ANOVA) at the level of P<0.05 by Duncan's multiplecomparison test.

207L. Yang et al. / Comparative Biochemistry and Physiology, Part C 151 (2010) 204–208

During the initial phase of the acute stress response, energyexpenditure is initially enhanced by as much as 200%. Manoli et al.(2007) have documented that acute and limited exposure to stressmediators is associated with increases in mitochondrial biogenesisand the enzymatic activity of selected subunits of the respiratorychain complexes, to meet the increased energy demands of the cell.However, excessive acute or prolonged challenges to mitochondrialhomeostasis can lead to respiratory chain dysfunction and decreasedproduction of ATP (Duclos et al., 2004). According to the results of thepresent study, through the activity of the mitochondrial respiratorychain was depressed significantly by acute heat exposure, this result isinsufficient to infer compromise of ATP generation, because severalauthors have shown that the activity of the respiratory chain complexhas a lower limit; stress may occur up to a critical value withoutaffecting the rate of mitochondrial respiration or ATP synthesis(Letellier et al., 1994; Davey and Clark, 1996; Rossignol et al., 1999,2003). Once the inhibition exceeds the critical value, ATP synthesiswill be compromised; this will lead to the formation of superoxideanion radicals (Boveris et al., 1972; Liu et al., 2002; Kudin et al., 2005;

Fig. 6. The formation of MDA in serum and liver tissue at different time points. Valuesare mean±SEM of eight replications. Data points with different superscripts aresignificantly different (one-way ANOVA) at the level of P<0.05 by Duncan's multiplecomparison test.

Szabo et al., 2007; Murphy, 2009). Although our present study did notmeasure the production of ATP under conditions of acute heatexposure, the over-production of ROS in the liver tissue of broilerchickens under conditions of heat stress provides a clue that acuteheat exposure may induce the production of ROS by inhibiting theactivity of mitochondrial respiration and subsequently compromisingATP generation. Furthermore, in the current study the modification ofmitochondrial respiratory activity and ROS production showed aconsistent response, both under conditions of heat exposure andwithout heat exposure. It should also be noted here that the activity ofsuccinate cytochrome c reductase (complexes II+III) remainedunaltered. Previous studies have reported similar results in the rostralventrolateral medulla of rats during endotoxaemia (Chuang et al.,2002; Chan et al., 2005). Nevertheless, the reason why acute heatstress does not alter the function of complexes II+III deserves furtherinvestigation.

It is known that ROS are highly toxic, and they can produce avariety of pathological changes through lipid peroxidation and DNAdamage. Exposure to heat stress increased lipid peroxidation, as aconsequence of the increased generation of ROS, as indicated by theconcentration of MDA. Therefore, the content of MDA in serum andtissue can reflect indirectly the extent of lipid peroxidation and over-production of ROS in the body. In contrast, SOD can protect cells fromoxidative injury by clearing superoxide anions. McArdle and Jackson(2000) have also documented that a small increase in ROS can inducethe expression of antioxidants. Therefore, the activity of SOD and theconcentration of MDA are commonly combined in the investigation ofthe extent of damage to the whole body. In the present study, theactivity of SOD increased during acute heat exposure and thendecreased again during recovery at 25 °C. It is known that there arenumerous antioxidant enzymes in liver cells that are also affected byhigh temperature stressors. Therefore, the activity of CAT and GSH-Pxwas tested in serum and tissue, and similar responses to heat werefound. The results also showed that the concentrations of MDA inserum and liver increased concomitantly with an increase inantioxidant enzyme activities. A similar result has been reportedpreviously in heat-stressed chickens (Altan et al., 2003). As a result, allthe antioxidative enzymes and MDA in serum and liver tissue showeda consistent trend that was dependent directly on the production ofROS. This result also confirmed that acute heat exposure induced theover-production of ROS, and over-production of ROS induced lipidperoxidation, which was consistent with previous reports (Mujahidet al., 2007b).

In the present study, results showed that the activity of mito-chondrial respiratory enzymes, production of ROS and lipid peroxida-tion were affected significantly by acute heat exposure; with theprogression of the recovery period, all the related parametersgradually recovered. The results show that, in the broiler chickenmodel used in the present study, acute exposure to high temperaturesmay depress the activity of the mitochondrial respiratory chain. Thisleads to the over-production of ROS, which ultimately results in lipidperoxidation and oxidative stress. However, this hypothesis needs tobe evaluated rigorously in future studies. It has also been shown that,when the high temperature was removed, the ROS production,mitochondrial respiratory dysfunction and oxidative injury thatwere induced by acute heat exposure recovered in a time-dependentmanner.

Acknowledgements

This study was supported by the National Basic Research Programof China (Grant No. 2004CB117507). The authors also gratefullyacknowledge the financial support provided by the Program Spon-sored by State Key Laboratory of Animal Nutrition [2004DA125184(Group) 0807]. In addition, we sincerely thank the editors and ano-nymous reviewers.

208 L. Yang et al. / Comparative Biochemistry and Physiology, Part C 151 (2010) 204–208

References

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Roberts, P., 2002. Molecular Biologyof the Cell, 4th ed. Garland Science Publishing, New York, USA.

Altan, O., Pabuçcuoğlu, A., Altan, A., Konyalioğlu, S., Bayraktar, H., 2003. Effect ofheat stress on oxidative stress, lipid peroxidation and some stress parameters inbroilers. Br. Poult. Sci. 44, 545–550.

Barja, G., 2002. Rate of generation of oxidative stress-related damage and animallongevity. Free Radic. Biol. Med. 33, 1167–1172.

Berrong, S.L., Washburn, K.W., 1998. Effects of genetic variation on total plasma protein,body weight gains, and body temperature responses to heat stress. Poult. Sci. 77,379–385.

Boveris, A., Oshino, N., Chance, B., 1972. The cellular production of hydrogen peroxide.Biochem. J. 128, 617–630.

Cahaner, A., Pinchasov, Y., Nir, I., Nitsan, Z., 1995. Effects of dietary protein under highambient temperature on body weight, breast meat yield, and abdominal fatdeposition of broiler stocks differing in growth rate and fatness. Poult. Sci. 74,968–975.

Cawthon, D., McNew, R., Beers, K.W., Bottje, W.G., 1999. Evidence of mitochondrialdysfunction in broilers with pulmonary hypertension syndrome (Ascites): effect oft-butyl hydroperoxide on hepatic mitochondrial function, glutathione, and relatedthiols. Poult. Sci. 78, 114–124.

Chan, S.H., Wu, K.L., Wang, L.L., Chan, J.Y., 2005. Nitric oxide- and superoxide-dependent mitochondrial signaling in endotoxin-induced apoptosis in the rostralventrolateral medulla of rats. Free Radic. Biol. Med. 39, 603–618.

Chuang, Y.C., Tsai, J.L., Chang, A.Y., Chan, J.Y., Liou, C.W., Chan, S.H., 2002. Dysfunction ofthe mitochondrial respiratory chain in the rostral ventrolateral medulla duringexperimental endotoxemia in the rat. J. Biomed. Sci. 9, 542–548.

Davey, G.P., Clark, J.B., 1996. Threshold effects and control of oxidative phosphorylationin nonsynaptic rat brain mitochondria. J. Neurochem. 66, 1617–1624.

Duclos, M., Gouarne, C., Martin, C., Rocher, C., Mormede, P., Letellier, T., 2004. Effects ofcorticosterone on muscle mitochondria identifying different sensitivity toglucocorticoids in Lewis and Fischer rats. Am. J. Physiol. Endocrinol. Metab. 286,E159–E167.

Feng, J.H., Zhang, M.H., Zheng, S.S., Xie, P., Ma, A.P., 2008. Effects of high temperature onmultiple parameters of broilers in vitro and in vivo. Poult. Sci. 87, 2133–2139.

Góth, L., 1991. A simple method for determination of serum catalase and revision ofreference range. Clin. Chim. Acta. 196, 143–152.

Halliwell, B., Aruoma, O.I., 1991. DNA damage by oxygen-derived species. Itsmechanism and measurement in mammalian systems. FEBS Lett. 281, 9–19.

Kudin, A.P., Debska-Vielhaber, G., Kunz, W.S., 2005. Characterization of superoxideproduction sites in isolated rat brain and skeletal muscle mitochondria. Biomed.Pharmacother. 59, 163–168.

Kussmaul, L., Hirst, J., 2006. The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc.Natl. Acad. Sci. 103, 7607–7612.

Letellier, T., Heinrich, R., Malgat, M., Mazat, J.P., 1994. The kinetic basis of thresholdeffects observed in mitochondrial diseases: a systemic approach. Biochem. J. 302,171–174.

Lin, H., Decuypere, E., Buyse, J., 2004. Oxidative stress induced by corticosteroneadministration in broiler chickens (Gallus gallus domesticus): 2. Short-term effect.Comp. Biochem. Physiol. B Biochem. Mol. Biol. 139, 745–751.

Lin, H., Decuypere, E., Buyse, J., 2006. Acute heat stress induces oxidative stress inbroiler chickens. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 144, 11–17.

Lin, H., De Vos, D., Decuypere, E., Buyse, J., 2008. Dynamic changes in parameters ofredox balance after mild heat stress in aged laying hens (Gallus gallus domesticus).Comp. Biochem. Physiol. C Toxicol. Pharmacol. 147, 30–35.

Liu, Y., Fiskum, G., Schubert, D., 2002. Generation of reactive oxygen species by themitochondrial electron transport chain. J. Neurochem. 80, 780–787.

Mahmoud, K.Z., Edens, F.W., 2003. Influence of selenium sources on age-related andmild heat stress-related changes of blood and liver glutathione redox cycle inbroiler chickens (Gallus domesticus). Comp. Biochem. Physiol. B Biochem. Mol. Biol.136, 921–934.

Manoli, I., Alesci, S., Blackman, M.R., Su, Y.A., Rennert, O.M., Chrousos, G.P., 2007.Mitochondria as key components of the stress response. Trends. Endocrinol. Metabol.18, 190–198.

McArdle, A., Jackson, M.J., 2000. Exercise, oxidative stress and ageing. J. Anat. 197,539–541.

Mujahid, A., Yoshiki, Y., Akiba, Y., Toyomizu, M., 2005. Superoxide radical production inchicken skeletal muscle induced by acute heat stress. Poult. Sci. 84, 307–314.

Mujahid, A., Sato, K., Akiba, Y., Toyomizu, M., 2006. Acute heat stress stimulatesmitochondrial superoxide production in broiler skeletal muscle, possibly via downregulation of uncoupling protein content. Poult. Sci. 85, 1259–1265.

Mujahid, A., Akiba, Y., Warden, C.H., Toyomizu, M., 2007a. Sequential changes in super-oxide production, anion carriers and substrate oxidation in skeletal muscle mito-chondria of heat-stressed chickens. FEBS Lett. 581, 3461–3467.

Mujahid, A., Pumford, N.R., Bottje, W., Nakagawa, K., Miyazawa, T., Akiba, Y., Toyomizu,M., 2007b. Mitochondrial oxidative damage in chicken skeletal muscle induced byacute heat stress. J. Poult. Sci. 44, 439–445.

Murphy, M.P., 2009. How mitochondria produce reactive oxygen species. Biochem. J.417, 1–13.

Rossignol, R., Malgat, M., Mazat, J.P., Letellier, T., 1999. Threshold effect and tissuespecificity. Implication formitochondrial cytopathies. J. Biol. Chem. 274, 33426–33432.

Rossignol, R., Faustin, B., Rocher, C., Malgat, M.,Mazat, J.P., Letellier, T., 2003.Mitochondrialthreshold effects. Biochem. J. 370, 751–762.

Rustin, P., Chretien, D., Bourgeron, T., Gerard, B., Rotig, A., Saudubray, J.M., Munnich, A.,1994. Biochemical and molecular investigations in respiratory chain deficiencies.Clin. Chim. Acta. 228, 35–51.

Simoyi, M.F., Van Dyke, K., Klandorf, H., 2002. Manipulation of plasma uric acid inbroiler chicks and its effect on leukocyte oxidative activity. Am. J. Physiol. Regul.Integr. Comp. Physiol. 282, R791–R796.

Szabo, C., Ischiropoulos, H., Radi, R., 2007. Peroxynitrite: biochemistry, pathophysiologyand development of therapeutics. Nat. Rev. Drug. Discov. 6, 662–680.

Washburn, K.W., Peavey, R., Renwick, G.M., 1980. Relationship of strain variation andfeed restriction to variation in blood pressure and response to heat stress. Poult. Sci.59, 2586–2588.