Lung Mitochondrial Dysfunction in Pulmonary Hypertension Syndrome ...

Lung Mitochondrial Dysfunction in Pulmonary Hypertension Syndrome. II. Oxidative Stress and Inability to Improve Function with Repeated Additions of Adenosine Diphosphate M. Iqbal, D. Cawthon, R. F. Wideman, Jr., and W. G. Bottje1 Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 tive stress [lower α-tocopherol and reduced glutathione (GSH) and higher oxidized glutathione levels (GSSG)]. High dietary α-tocopherol had no effect on lung mitochondrial function in healthy broilers (VE vs. controls) but attenuated dysfunction in VE-PHS mitochondria. In Experiment 2, there were no differences in selected and nonselected mitochondrial function following a single addition of ADP, but nonselected mitochondria exhibited lower RCR and ADP:O values with repeated energy demand. Higher GSSG levels were also observed in nonselected lung. The results indicate that lung mitochondrial dysfunction present in broilers with PHS was associated with oxidative stress and may be attenuated by high dietary vitamin E. Furthermore, genetic resistance to PHS was associated with more efficient oxidative phosphorylation in lung mitochondria and an inherently lower degree of oxidative stress.

ABSTRACT The major objective of this study was to examine lung mitochondrial dysfunction and antioxidants in pulmonary hypertension syndrome (PHS) in broilers. Lung mitochondria were obtained from broilers fed diets containing 15 IU (control) and 100 IU dl-α-tocopherol acetate, i.e., vitamin E (VE)/kg with and without PHS; the four treatment groups were control, VE, PHS, and VE-PHS, respectively (Experiment 1), or from healthy broilers genetically selected or not selected for resistance to PHS (Experiment 2). Mitochondrial function was assessed with sequential additions of adenosine diphosphate (ADP) to mimic a repeated demand for energy. Compared to controls, PHS mitochondria in Experiment 1 exhibited mitochondrial dysfunction [lower respiratory control (RCR) and ADP:O ratios and an inability to improve function with repeated energy demand] and oxida-

(Key words: pulmonary hypertension syndrome, mitochondrial function, glutathione, tocopherol, broiler) 2001 Poultry Science 80:656–665

Pulmonary hypertension syndrome (PHS), a costly metabolic disease in broilers, is a basic problem of oxygen supply and demand that develops in response to cardiopulmonary insufficiency (Wideman and Bottje, 1993; Wideman and Kirby, 1995a,b; Wideman et al., 1997). Systemic hypoxia triggers a series of events including peripheral vasodilation, increased cardiac output and pulmonary arterial pressure, and right ventricular hypertrophy (Wideman and Bottje, 1993; Bottje and Wideman, 1995). The right ventricular hypertrophy that occurs in response to pulmonary hypertension (Burton et al., 1968; Peacock et al., 1990) leads to incompetency of the right monocuspid valve and congestive heart failure. Although PHS may be triggered by environmental factors such as cold tem-

perature, lung damage, or high altitude hypoxia, it also occurs in optimal conditions simply in response to rapid growth (e.g., Huchzermeyer and DeRuyck, 1986; Hernandez, 1987; Huchzermeyer et al., 1988; Enkvetchakul et al., 1993). We have hypothesized that mitochondrial dysfunction contributes to systemic hypoxia that develops in PHS (Bottje and Wideman, 1995). At the cellular level, 85 to 90% of oxygen is consumed by mitochondria during aerobic respiration (Shigenaga et al., 1994). The mitochondrial respiratory chain uses electron flow from various energy substrates to create a proton gradient (proton motive force) that is used to drive adenosine triphosphate (ATP) synthesis (Lehninger et al., 1992). Oxygen is the terminal electron acceptor in the chain of oxidation-reduction reactions that occur in the inner mitochondrial membrane

2001 Poultry Science Association, Inc. Received for publication July 14, 2000. Accepted for publication December 6, 2000. 1 To whom correspondence should be addressed: wbottje@comp. uark. edu.

Abbreviation Key: ADP = adenosine diphosphate; EGTA = ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′ tetraacetic acid; GSH = reduced glutathione; GSSG = oxidized glutathione; MOPS = morpholinopropane sulfate; PHS = pulmonary hypertension syndrome; ROS = reactive oxygen species; RCR = respiratory control ratio; RV = right ventricle; TV = total ventricle; VE = vitamin E.

INTRODUCTION

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(Lehninger et al., 1992). Proton movement through the f1f0 adenosine ATP synthase effectively couples the energyreleasing reactions of oxidation to the energy-storing reaction of phosphorylation. Consequently, the addition of adenosine diphosphate (ADP) to isolated mitochondria increases electron flow and oxygen consumption until ADP has been phosphorylated to ATP. Lower respiratory control (RCR) and ADP:O ratios, indices of electron transport chain coupling and of oxidative phosphorylation, respectively (Estabrook, 1967), were observed in PHS liver mitochondria (Cawthon et al., 1999). Furthermore, repeated additions of ADP improved mitochondrial function in Control, but not in PHS mitochondria (Cawthon et al., 2001), representing further evidence of inefficient use of oxygen at the cellular level. Besides being a major site of cellular oxygen consumption, mitochondria also are a major site of cellular oxidative stress due to the generation of reactive oxygen species (ROS) and therefore could contribute to oxidative stress observed in broilers with PHS (Enkvetchakul et al., 1993; Bottje and Wideman, 1995; Bottje et al., 1995; Diaz-Cruz et al., 1996). Rather than being completely reduced to water, it has been estimated that 1 to 4% of oxygen consumed by mitochondria is incompletely reduced to ROS (e.g., O2ⴢ− and H2O2) due to leakage of electrons from the respiratory chain (Boveris and Chance, 1973; Chance et al., 1979). With regard to PHS, electron leakage and ROS production are accentuated during relative hypoxia (Dawson et al., 1993). Thus, due to the great demand that is placed on mitochondria to support the rapid rates of growth in commercial broilers, combined with the propensity of mitochondria to produce ROS, broiler mitochondria may be extremely important in contributing to oxidative stress associated with PHS. Evidence of H2O2 accumulation in heart mitochondria in broilers of PHS (Maxwell et al., 1996) and of oxygen radical production in PHS liver and lung mitochondria (Cawthon et al., 2001; Iqbal et al., 2001) lends support to this hypothesis. Therefore, mitochondrial dysfunction in PHS (Cawthon et al., 1999, 2001) could be attributed to increased electron leakage from the respiratory chain. Oxidative stress in PHS has been observed as a decrease in tissue antioxidants glutathione and vitamin E (VE; αtocopherol) (Enkvetchakul et al., 1993; Bottje et al., 1995; Maynard et al., 1995) and as an increase in lipid peroxidation in broilers (Bottje et al., 1995; Diaz-Cruz et al., 1996). Vitamin E was also depressed in heart tissue of a monocrotaline-induced model of pulmonary hypertension in rats (Pichardo et al., 1999). Vitamin E has been reported to be effective in lowering PHS mortality in some studies (Bottje et al., 1995; Roch and Boulianne, 2000) but not in all cases (Dale and Villacres, 1986; Bottje et al., 1997). αTocopherol is found in particularly high levels in mito-

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chondrial membranes (Bjorneboe et al., 1991) and is depressed in liver mitochondria obtained from broilers with PHS (Cawthon et al., 2001). Furthermore, VE was effective in attenuating H2O2 production in lung mitochondria of broilers with PHS in a companion study (Iqbal et al., 2001). Thus, it would be of considerable interest to determine the effect of high levels of VE on lung mitochondrial function and antioxidant capacity in broilers with and without PHS. In light of the evidence outlined above, major objectives of the present study were to determine if there is a dysfunction associated with PHS in lung mitochondria, and if high dietary VE could attenuate mitochondrial dysfunction in PHS. Because genetic resistance to PHS was recently reported to be associated with a decrease in H2O2 production in lung mitochondria (Iqbal et al., 2001), a third objective was to determine if lung mitochondrial function is improved in broilers selected for genetic resistance to PHS. A final objective was to determine the effect of repeated additions of ADP on lung mitochondrial function in this study, as repeated energy demand accentuated liver mitochondrial dysfunction associated with PHS (Cawthon et al., 2001).

MATERIALS AND METHODS Birds and Management In Experiment 1, male broiler chicks (Cobb 500)2 obtained from a local hatchery3 at 1 d of age were placed in an environmental chamber (8 m2 floor space) on wood shaving litter. Birds were provided access ad libitum to water and to a diet (23.7% protein, 3,200 kcal ME) supplemented with 15 (control) or 100 IU dl-α-tocopherol acetate4 (VE) per kg. Temperature in the chamber was 32 and 30 C during Weeks 1 and 2, lowered to 15 C during Week 3, and maintained between 10 and 15 C for the rest of the study. Initially, 120 chicks were placed in the chamber, but this number was reduced to 100 by 21 d due to removal of those with deformed legs or that had failed to thrive or from early chick mortality. The cool temperatures combined with feed to support rapid growth rate have been shown to induce a high incidence of PHS (Wideman et al., 1995). The study was replicated using identical conditions. In Experiment 2, lung mitochondria were obtained from male broilers5 that had been genetically selected for resistance to PHS (Wideman and French, 1999, 2000). These birds were third generation progeny of breeding stock that did not develop PHS following unilateral pulmonary arterial occlusion. These birds were provided the control diet and water ad libitum and were maintained under the environmental conditions as described above.

Sampling Procedure 2

Cobb Vantress, Siloam Springs, AR 74364. Randall Road, Tyson, Springdale, AR 72762. 4 Roche Vitamins, Inc., Nutley, NJ07110. 5 Hubbard Farms, Walpole, NH 03608. 3

In Experiment 1, birds were randomly selected that exhibited overt PHS symptoms (e.g., systemic cyanosis of the comb, wattle and skin, or abdominal fluid accumu-

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lation) or appeared clinically healthy (i.e., no cyanosis) as previously described (Cawthon et al., 1999). Only one bird was sampled per day due to the length of time required for mitochondrial isolation and functional studies described below. After weighing the birds, each bird was killed with an overdose of sodium pentobarbital by i.v. injection into the wing vein. The lungs were prepared for mitochondrial isolation as described below. The heart was also obtained and after careful removal of the atria, the right ventricle (RV) and total ventricle (TV) weights were determined to calculate RV:TV weight ratio, a sensitive indicator of prior exposure of the heart to elevated pulmonary arterial pressure (Burton et al., 1968). Birds with an RV:TV > 0.30 were classified as having PHS, whereas those with an RV:TV ratio ≤ 0.27 that did not have abdominal or pericardial fluid were classified as non-PHS birds. In the 2 × 2 factorial design, the four treatment groups consisted of the two dietary treatments, control (n =10) and high dietary VE (n = 8) for healthy birds without PHS and PHS (n = 7) or VE-PHS (n = 7) for birds with PHS. The higher RV:TV in PHS and VE-PHS groups confirmed the presence of prolonged pulmonary arterial hypertension in these groups (Burton et al., 1968). Mortality from PHS was not monitored in Experiment 1. In Experiment 2, birds without PHS were randomly chosen from nonselected (n = 4) and those selected (n = 4) for resistance to PHS. These birds were raised under cold conditions similar to Experiment 1. Sampling of birds occurred between 49 and 57 d of age (as opposed to continuous sampling between 21 and 50 d of age in Experiment I) after 4 wk of exposure to cold conditions. The mortality from PHS in selected and nonselected broilers after 4 wk of cold exposure was 5 and 38%, respectively (Wideman and French, 2000). However, because birds used in Experiment 2 did not exhibit symptoms of PHS, they would likely be the most resistant to PHS within their respective populations.

Isolation of Lung Mitochondria Lung mitochondria were isolated as described by Fisher et al. (1975) with modifications. The pulmonary vasculature was perfused immediately with 10 mL of isotonic heparinized saline (0.85% NaCl, 200 U heparin/ mL; 40 C). All chemicals used for mitochondrial isolation and incubation studies were purchased from Sigma Chemical Co.6 The lungs were removed, trimmed of extraparenchymal tissue and washed several times with an ice-cold primary isolation medium (225 mM D-mannitol, 75 mM sucrose, and 2 mM EDTA), pH 7.3. The lungs were then weighed, minced, and transferred to an icecold secondary isolation medium (tissue to medium ratio

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Sigma Chemical Co., St. Louis, MO 63178. Thomas Scientific, Swedesboro, NJ 08085-0099. 8 Tekmar Co., Cincinnati, OH 45222-1856. 9 Sigma kit # 610-A, Sigma Diagnostics, St. Louis, MO 63178-9916. 10 Instech Labs, Plymouth Meetings, PA 19462-1216. 7

of 1:20), containing 225 mM D-mannitol, 75 mM sucrose, 0.8% (wt/vol) lipid-free BSA, 20 mM ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′ tetraacetic acid (EGTA) and 5 mM morpholinopropane sulfate (MOPS), pH 7.3. The minced tissue was homogenized in a Potter-Elvehjem vessel with a Teflon pestle of 0.16 mm clearance,7 followed by a second homogenization with a Tekmar homogenizer8 for 10 s. The homogenized tissue was centrifuged for 5 min (1,500 × g), and pellets containing nuclei and cell debris were discarded. The supernatant was strained through double-layer cheesecloth and centrifuged (for 10 min at 10,000 × g). The resulting pellet was resuspended in secondary isolation medium, centrifuged for 3 min × 1,500 × g to remove blood remnants, and centrifuged again (10 min × 10,000 × g). The final mitochondrial pellet was resuspended in incubation medium (300 to 500 µL, depending upon the size of the mitochondrial pellet) containing 225 mM D-mannitol, 75 mM sucrose, 20 mM EGTA, and 5 mM MOPS, pH 7.3 (Fisher et al., 1975). Mitochondrial protein concentration was measured spectrophotometrically9 to enable all mitochondrial variables (function, respiration, and antioxidant levels, see below) to be based on mitochondrial protein (Fisher et al., 1975).

Mitochondrial Function Mitochondrial function was assessed by monitoring oxygen consumption in isolated mitochondrial preparations using succinate as a substrate (Estabrook, 1967). Oxygen consumption was measured polarographically with an oxygen electrode in a heated water-jacketed chamber (Model 600) that was maintained at 40 C and equipped with magnetic stirring.10 An aliquot of mitochondrial suspension (60 µL) was added to the chamber containing 440 µL of RCR reaction buffer comprising 145 mM KCl, 5 mM KH2PO4, 20 mM Tris-Cl, 3.4 µM rotenone, and 23 mM succinate, pH 7.2. In pilot studies, it was determined that the yield of mitochondria (based on protein concentration) isolated from lungs was much less than that obtained from liver (Cawthon et al., 1999). To conserve lung mitochondria used for functional studies, we switched from larger incubation chambers used previously (Cawthon et al., 1999) to smaller incubation chambers (Model 600, described above). Due to technical difficulties encountered with these smaller chambers, functional studies on lung mitochondria were carried out successfully in four to six mitochondrial preparations for each group in Experiment 1. Oxygen consumption was monitored during State III (following the addition of 0.2 mM ADP) and State IV respiration (ADP limiting), and the RCR (an index of electron chain coupling) was calculated by dividing State III by State IV respiration rates (Estabrook, 1967). State III and IV respiration rates are expressed as nanomoles monomeric oxygen per minute per milligram mitochondrial protein. The ADP:O ratio, the efficiency of ATP synthesis coupled to cell respiration, was calculated by dividing the amount of monomeric oxygen consumed per minute during State III respiration by the amount of


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ADP added to the reaction vessel (nmol ADP:nmol O) (Estabrook, 1967). To determine the effect of repeated energy demand on mitochondrial function, a second set of functional parameters were determined following a second sequential addition of 0.4 mM ADP in the same chamber (Cawthon et al., 2001).

were accomplished using the general linear models procedure of SAS威 software (SAS Institute, 1996). Data are presented as means ± SEM. A probability level of P ≤ 0.05 was considered statistically significant.

Biochemical Analyses

Experiment 1: VE and Mitochondrial Function

Glutathione Analysis. Acid-soluble reduced glutathione (GSH) and oxidized glutathione (GSSG) were analyzed by HPLC using a Waters system11 following thiol derivatization as described by Fariss and Reed (1987). Briefly, the mitochondrial pellet and supernatant were treated with 10% perchloric acid to precipitate proteins. This step was followed by reaction of iodoacetic acid with thiols to form S-carboxy-methyl derivatives and derivatization of amino groups in the supernatant with 1-fluoro2,4-dinitrobenzene. Derivatized thiols were separated by ion-exchange column chromatography. Thiols were identified by retention times of authentic standards and concentrations calculated from peak integrated areas. Tocopherol Analysis. Tocopherols (α- and γ-) were detected using a modified method of Warren and Reed (1991). Protein was precipitated in mitochondrial suspensions using ice-cold ethanol containing ascorbic acid (1 g/L). After extracting the sample homogenate twice with hexane,12 the combined organic layer was evaporated under nitrogen. The tocopherols were redissolved in methanol/acetonitrile12 (1:3) and then centrifuged (5 min × 12,000 × g); the supernatant used for reverse-phase HPLC using a Waters system12 with a C18 Nova-Pak13 column (3.9 × 150 cm). Tocopherols were separated using isocratic conditions with a mobile phase of 25% methanol and 75% acetonitrile at a flow rate of 1 mL/min and were monitored with a fluorescence detector set at excitation/ emission wavelengths of 298/328 nm, respectively. Tocopherols were identified and quantified by comparison to the retention times and peak areas of authentic standards. Extraction efficiency of tocopherol was based on the theoretical response of the internal standard.

Statistical Analyses The data of the first experiment were analyzed using 2 × 2 factorial design. Selection of birds from each treatment group during each week of the study was randomized, and one bird was selected from each group each week. In Experiment 1, there were no main effects due to replicate; therefore, these data were pooled. The data of the second experiment were analyzed by t-tests. The effects of sequential additions of ADP on within group mitochondrial respiration measurements in both experiments were also determined by t-test. All of the above statistical analyses

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Waters Corp., Milford, MA 01757. Fisher Scientific, Fairlawn, NJ 07410. Hoffman-La Roche Inc., Nutley, NJ 07110.

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RESULTS

There were no differences in BW between the four treatment groups of birds selected in Experiment 1 (Table 1). Lung weight was lower in the PHS group than in the controls but was not different from the VE or VE-PHS group. However, there were no differences between groups when lung weight was expressed as a percentage of BW (data not shown). The elevation in the RV:TV in the PHS and VE-PHS groups clearly indicated the presence of pulmonary hypertension in these groups. There was no main effect due to weeks (age) of any mitochondrial variable in Experiment 1. There were no differences in lung mitochondrial protein content between groups (Table 1). Function was clearly compromised in PHS lung mitochondria as indicated by the lower RCR and ADP:O ratios compared to controls (Table 1). High dietary VE had no effect on mitochondrial function in healthy broilers (VE vs. control), but supplemental VE was effective in attenuating depressions in mitochondrial function, because there were no differences in RCR or ADP:O among VE-PHS, control, and VE mitochondria. Uncoupling of the electron transport chain (RCR < 2.0) was only observed in PHS mitochondria. There were no differences in State III oxygen consumption among groups, with the exception that State III was higher in VE-PHS than in VE mitochondria. The lower RCR in PHS mitochondria was due to a higher State IV (resting) oxygen consumption rate compared to control and VE values. Higher State IV respiration observed in PHS mitochondria was attenuated in VE-PHS mitochondria, i.e., State IV respiration was higher in PHS but not in VE-PHS mitochondria compared to values in control and VE mitochondria. Sequential additions of ADP (repeated energy demand) improved mitochondrial electron transport chain coupling (RCR, Figure 1A) and efficiency of oxidative phosphorylation (ADP:O, Figure 1B) in control and VE mitochondria (P < 0.05). The VE-PHS mitochondria also exhibited increased RCR and ADP:O (P < 0.10) in response to the sequential addition of ADP (0.2 vs. 0.4 mM), but similar improvements were not observed in PHS lung mitochondria. Changes in State III and State IV respiration following sequential additions of ADP for Experiment 1 are shown in Figure 2. In general, both State III and IV respiration decreased following the addition of 0.4 mM ADP (III2 and IV,2 respectively) with the exception of State III2 in VE mitochondria. Although State IV2 respiration declined in PHS lung mitochondria, a very large decrease in State III2 respiration in PHS mitochondria accounted

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IQBAL ET AL. TABLE 1. Body and lung weights (LW), right ventricular weight ratio (RV:TV), and lung mitochondrial protein, function, and respiration obtained from broilers in Experiment 1 and 21 Treatment groups2 Experiment 1 3

Variables

Control (n)

BW (kg) LW (g) RV:TV Protein (mg/mL) RCR ADP:O State III State IV

2.49 15.1 0.22 12.4 3.45 1.79 107 31

± ± ± ± ± ± ± ±

0.33 (10) 1.5a (10) 0.01c (10) 0.9 (10) 0.55a (6) 0.05a (6) 6ab (6) 6b (6)

PHS (n) 1.95 12.0 0.32 9.4 1.83 1.55 110 60

± ± ± ± ± ± ± ±

0.23 (8) 1.4b (8) 0.02b (8) 3.3 (10) 0.45b (4) 0.09b (4) 7ab (4) 14a (4)

Experiment 2

VE (n) 2.16 13.5 0.21 13.2 3.39 1.84 95 28

± ± ± ± ± ± ± ±

VE-PHS (n) 0.24 (7) 1.5ab (7) 0.02c (7) 1.6 (7) 0.11a (4) 0.07a (4) 4b (4) 4b (4)

2.30 13.5 0.41 13.0 2.57 1.71 126 49

± ± ± ± ± ± ± ±

0.42 (7) 1.5ab (7) 0.02a (7) 2.1 (7) 0.08ab (4) 0.08ab (4) 12a (4) 13ab (4)

S (n = 4) 3.95 25.6 0.22 16.9 2.80 1.67 175 63

± ± ± ± ± ± ± ±

0.17 1.1a 0.02 1.8 0.35 0.13 32 8

NS (n = 4) 3.39 19.3 0.23 15.1 3.19 1.43 143 63

± ± ± ± ± ± ± ±

0.26 1.2b 0.02 1.5 0.64 0.08 37 20

Means in the same row and experiment with no common superscripts differ significantly (P < 0.05). All values are the means (number given in parentheses) ± SEM. 2 PHS = pulmonary hypertension syndrome; VE = high dietary vitamin E (no PHS); VE-PHS = high dietary vitamin E with PHS; S = selected; NS = nonselected for resistance to PHS birds fed only with control feed. 3 RCR = respiratory control ratio; ADP:O = adenosine diphosphate to oxygen ratio; State III = mitochondrial respiration in the presence of excess substrate (succinate) and ADP; State IV = mitochondrial respiration in the presence of excess substrate but in the absence of ADP. State III and IV respiration rates are expressed in nanomoles of oxygen per minute per milligram protein. a–c 1

for the low RCR observed in this group following 0.4 mM ADP (Figure 1A). Values of tocopherol and GSH, representing lipid and water-soluble antioxidants respectively, for lung mitochondria in Experiment 1 are presented in Figure 3. Oxidative stress was apparent in PHS lung mitochondria as indicated by lower α- and γ-tocopherol levels (Figure 3A), by depletion of mitochondrial GSH (Figure 3B), and an increase in the GSSG/GSH ratio (Figure 3B, inset) compared to controls. As expected, mitochondria from birds fed high VE exhibited higher α-tocopherol, but not γtocopherol, compared to controls (Figure 3A). Despite higher α-tocopherol levels in VE-PHS than in control and PHS mitochondria, oxidative stress was still apparent in VE-PHS mitochondria as indicated by depletion of GSH to levels nearly identical to that in the PHS mitochondria (Figure 3B) and lower α-tocopherol levels than in VE mitochondria (Figure 3A). There was no difference, however, in the GSSG/GSH ratio between VE and VE-PHS mitochondria (Figure 3B inset). The reason for the higher GSSG/GSH ratio in VE mitochondria compared to controls is not apparent at this time. Analysis of mitochondrial fatty acid content indicates that feeding high dietary VE may cause shifts in saturated and unsaturated fatty acids (unpublished observations) that could affect susceptibility of mitochondria to oxidative stress. This shift in fatty acid content, however, would affect the lipid component of cells and not the aqueous compartment of mitochondria where GSH would exert its antioxidant protection.

Experiment 2: PHS Resistance and Mitochondrial Function There were no differences in BW, lung weight, and the RV:TV of broilers selected or not selected for PHS resistance in Experiment 2 (Table 1). The higher BW and lung weights in birds in Experiment 2 reflect the older

age of these birds (49 to 57 d) compared to 21 to 50 d in Experiment 1. There were no differences in the protein content, respiration, or function between selected and not selected lung mitochondria (Table 1). Respiration rates of lung mitochondria in Experiment 2 were somewhat higher than in Experiment 1. The reason for this difference is not apparent in this study but could be related to the higher mitochondrial yield (protein content) in Experiment 2 or might be due to differences in breed of birds (Cobb vs. Hubbard) between the two experiments. The sequential addition of ADP improved the RCR and ADP:O in both groups, but values were significantly higher in the selected mitochondria (Figure 4). Repeated energy demand lowered State IV respiration (IV2) in both groups but had no effect on State III (III2) respiration (Figure 5). There were no differences in α- and γ-tocopherols (Figure 6A) or in GSH (Figure 6B) between selected and not selected mitochondria. However, selected lung mitochondria exhibited lower GSSG (Figure 6B) and GSSG/GSH ratio (inset), indicating that selected lung mitochondria may have an inherently lower degree of oxidative stress compared to lung mitochondria obtained from broilers that were not selected for PHS resistance.

DISCUSSION Mitochondrial Function The major indices to measure the functionality of mitochondria are the RCR and ADP:O, which represent efficiencies in electron transport chain coupling and oxidative phosphorylation, respectively (Estabrook, 1967). The lower RCR and ADP:O in PHS lung mitochondria (Table 1) concur with observations in liver mitochondria (Cawthon et al., 1999, 2001) and clearly indicates that lung mitochondrial function is compromised in broilers with PHS. The apparent uncoupling of PHS lung mitochondria


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(RCR < 2.0) and its association with oxidative stress (Figure 3) also concurs with observations that PHS liver mitochondria were more prone to become uncoupled in response to treatment with t-butyl hydroperoxide to induce oxidative stress (Cawthon et al., 1999). As no attempt was made to separate cell types in this study, we do not know if PHS adversely impacted mitochondria in all cells, or if differences may exist in mitochondrial function of the various types of cells in the broiler lung. The lower ADP:O in PHS lung mitochondria could represent functional damage to oxidative phosphorylation (Nakahara et al., 1998) and indicates that more oxygen would have to be consumed to synthesize ATP equivFIGURE 2. Oxygen consumption in lung mitochondria during State III and IV respiration: before (III1 and IV1) and after (III2 and IV2) sequential additions of adenosine diphosphate (ADP) in broilers with and without pulmonary hypertension syndrome (PHS) fed diets with standard or high vitamin E (VE) supplementation (Experiment 1). Each bar represents the mean ± SE for broilers without PHS fed the normal diet (C; n = 6), broilers with PHS (PHS; n = 4), for broilers without PHS fed the diet with high levels of vitamin E (VE; n = 4), and for broilers fed high dietary vitamin E that developed PHS (VE-PHS, n = 4). a– c Means with no common letters differ significantly (P < 0.05) in all treatment groups for each state of respiration. xWithin a treatment group, State III2 and IV2 respiration were lower (P < 0.05) than State III1 and IV1 respiration.

FIGURE 1. Respiratory control ratio (RCR) (A) and the adenosine diphosphate (ADP) to oxygen ratio (ADP:O) (B) in lung mitochondria after sequential additions of ADP (0.2 and 0.4 mM) obtained from broilers with and without pulmonary hypertension syndrome (PHS) fed diets with standard or high vitamin E (VE) supplementation (Experiment 1). Each bar represents the mean ± SE for broilers without PHS fed the standard VE supplementation (control: C; n = 6), broilers with PHS (PHS; n = 4), for broilers without PHS fed the diet with high vitamin E supplementation (VE; n = 4), and for broilers fed vitamin E that developed PHS (VE-PHS; n = 4). a–c Means with no common letters differ significantly (P < 0.05) in 0.2 and 0.4 mM additions of ADP. xMitochondrial function (RCR and ADP:O) was different (P < 0.05) after sequential additions of ADP (0.2 vs. 0.4 mM). †Mitochondrial function (RCR and ADP:O) was different (P < 0.10) after sequential additions of ADP (0.2 vs. 0.4 mM).

alent to that in controls. Thus, we have now observed oxygen misuse by mitochondria in the liver (Cawthon et al., 1999, 2001), a metabolically active organ that accounts for 15% of total oxygen consumption in the body (Field et al., 1937) and in the lung (present study). Although not contributing significantly to total body oxygen consumption, the lung remains vitally important in PHS pathophysiology due to its role in blood oxygenation. We have also observed dysfunction in skeletal and cardiac muscle (unpublished observations), which indicates that there may be a pervasive mitochondrial dysfunction in many organs in broilers with PHS. An explanation for the lower ADP:O ratio in PHS lung mitochondria could be due to electron leakage from the respiratory chain. In fact, electron leakage can adversely effect mitochondria by a) reducing electron flux through the respiratory chain that in turn lowers the proton motive force and ATP production (Esposito et al., 2000), and b) increasing the production of ROS, e.g., superoxide, H2O2 (Chance et al., 1979; Kristal et al., 1997). Increased mitochondrial production of ROS has been linked to metabolic diseases (e.g., cystic fibrosis, diabetes) and aging (Fiegal and Shapiro, 1979; Hagen et al., 1997; Herrero and Barja, 1997, 1998; Kristal et al., 1997; Lass et al., 1998) and more recently to PHS in broilers (Cawthon et al., 2001; Iqbal et al., 2001). As electrons that leak from the electron transport chain can not be used to support ATP synthesis, the lower ADP:O observed in PHS lung mitochondria could be due to site-specific leakage of electrons from the respiratory chain (Iqbal et al., 2001). The release of ROS could in turn result in functional damage to critical components

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of the respiratory chain and impair mitochondrial oxidative phosphorylation (Nakahara et al., 1998). As the coupling between ADP phosphorylation and oxygen use is represented by the ADP:O, the ADP:O can be used to calculate the approximate amount of oxygen consumed by electron transport activity that is not used to support ATP synthesis (Kristal et al., 1997). Thus, assuming there is no ROS generation in perfectly coupled mitochondria provided succinate (theoretical ADP:O = 2.0), and that 1 to 4% of the oxygen consumed by control mitochondria results in ROS production (Chance et al., 1979), radical production in PHS mitochondria would account for 2.5 to 9% of total oxygen consumption in PHS mitochondria (Figure 7). The calculated amount of ROS generation in VE mitochondria would range from 0.8 to 3.2% representing a 20% reduction compared to controls. In VE-PHS mitochondria, ROS production would repre-

FIGURE 4. The respiratory control ratio (RCR) (A) and adenosine diphosphate to oxygen ratio (ADP:O) (B) following sequential additions of ADP (0.2 and 0.4 mM) obtained from broilers genetically selected (S) or not selected (NS) for resistance to PHS (Experiment 2). Each bar represents the mean (n = 4) ± SE. a,bMeans with no common letters differ significantly (P < 0.05). xMitochondrial function (RCR and ADP:O) differed (P < 0.05) after sequential additions of ADP (0.2 vs. 0.4 mM).

FIGURE 3. Lung mitochondrial concentrations of A) α- and γ-tocopherol, B) reduced (GSH) and oxidized (GSSG) glutathione and (inset) GSSG/GSH ratio (Experiment 1) obtained from broilers with and without pulmonary hypertension syndrome (PHS) fed diets with standard or high vitamin E (VE) supplementation. Each bar represents the mean ± SE for broilers without PHS fed the standard diet (C; n = 7), broilers with PHS (PHS; n = 8), for broilers without PHS fed the diet with high levels of VE (VE; n = 8), and for broilers fed high dietary vitamin E that developed PHS (VE-PHS; n = 7). a–dMeans with no common letters are different (P < 0.05).

FIGURE 5. Oxygen consumption in lung mitochondria during State III and IV respiration: before (III1 and IV1) and after (III2 and IV2) sequential additions of adenosine diphosphate (ADP) in broilers selected (S; n = 4) and not selected (NS; n = 4) for resistance to PHS (Experiment 2). xMitochondrial respiration was different (P < 0.05) after sequential additions of ADP (III1 vs. III2, and IV1 vs. IV2).


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sent 1.5 to 6% of total oxygen consumption in VE-PHS mitochondria or would be 30% lower when compared to PHS mitochondria but 50% higher when compared with controls.

Sequential Addition of ADP and Mitochondrial Function

FIGURE 6. Lung mitochondrial concentrations of A) α- and γ-tocopherol, B) reduced (GSH) and oxidized (GSSG) glutathione and the GSSG/ GSH ratio (inset) obtained from broilers that were selected (S) or not selected (NS) for resistance to pulmonary hypertension syndrome (PHS) in Experiment 2. Each bar represents the mean (n = 4) ± SE. a,bMeans with no common letters are different (P < 0.05).

To our knowledge, improvements in mitochondrial function in healthy broilers observed in the present study and a recent report (Cawthon et al., 2001) have not been previously reported. In Experiment 1, the inability of PHS lung mitochondria to improve the RCR and ADP:O in response to sequential additions of ADP (repeated energy demand) (Figure 1) concurs with that observed previously in liver mitochondria (Cawthon et al., 2001). Interestingly, in addition to attenuating the decline in RCR and ADP:O following the initial addition of ADP, the VEPHS lung mitochondria RCR and ADP:O after the second addition of ADP were not different from those in the control and VE treatment groups. Thus, although the effects of VE on PHS mortality is equivocal (Bottje et al., 1995, 1997), results from this and a recent study (Iqbal et al., 2001) indicate that high levels of dietary VE are beneficial with regard to lung mitochondrial function in broilers that develop PHS. Sequential additions of ADP also resulted in increasing RCR and ADP:O values in selected and not selected mitochondria in Experiment 2, but these functional indices were higher in selected than in not selected lung mitochondria (Figure 4). Thus, genetic resistance to PHS may be associated with more efficient oxygen utilization and energy production in response to continued need for energy production. It is important to emphasize that differences in mitochondrial function due to PHS were magnified with the paradigm of repeated additions of ADP used in this and a previous study (Cawthon et al., 2001). Thus, it is not unreasonable to hypothesize that inefficient oxygen utilization in PHS mitochondria may be even greater in vivo. It should also be pointed out that the measurements presented here are essentially a snapshot of mitochondrial function obtained at a single point in time. Thus, even relatively small differences between groups would be greatly magnified if measurements could be made over a 24-h period. Over longer periods of time, this condition would result in more efficient oxygen usage and in turn lower the systemic drive for development of systemic hypoxia that is characteristic of PHS.

VE: Antioxidant Status and Mitochondrial Function

FIGURE 7. Calculated reactive oxygen species (ROS) generation [as a percentage of total oxygen (O2 consumed)] for each of the treatment groups using the assumptions that a) 1 to 4% of oxygen consumed in control mitochondria resulted in ROS generation (Chance et al., 1979), and b) that all uncoupled oxygen consumption resulted in the generation of ROS. Mean values shown were calculated from the adenosine diphosphate to oxygen (ADP:O) ratios presented in Table 1.

Vitamin E has long been recognized as the major lipidsoluble chain-breaking antioxidant that prevents free radical-initiated peroxidative tissue damage (Tappel, 1972; Burton and Traber, 1990). As mitochondrial membranes have high levels of α-tocopherol (Bjorneboe et al., 1991), and VE supplementation may lower PHS mortality under certain conditions (Bottje et al., 1995; Roch and Boulianne,

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2000), it is of interest to determine the effect of high dietary VE on mitochondrial function in broilers. Vitamin E improved mitochondrial function in birds that developed PHS (VE-PHS) but had no affect on mitochondrial function in broilers without PHS (Figure 1). High dietary VE did attenuate H2O2 production in lung mitochondria (Iqbal et al., 2001), as well as the calculated amount of ROS production, based on differences in the ADP:O ratio between PHS and VE-PHS mitochondria (Figure 7) as indicated above. These findings indicate a beneficial effect of VE in lowering mitochondrial and therefore cellular oxidative stress in broilers with PHS. Additionally, supplementation of VE to VE-deficient mice has also been reported to decrease oxidative stress in dose-dependent manner in muscle and liver mitochondria. (Thomas et al., 1993; Chow et al., 1999). However, high dietary VE did not prevent GSH depletion and oxidation despite elevations in α-tocopherol in VE-PHS lung mitochondria (Figure 3). Thus, elevating lipid antioxidant levels did not prevent mitochondrial aqueous oxidation in lung mitochondria of broilers with PHS in the present study, which in turn could be a partial explanation as to why feeding high levels of dietary VE is not always effective in attenuating PHS mortality (Dale and Villacres, 1986; Bottje et al., 1997). In Experiment 2, mitochondrial tocopherol (α- and γ-) levels were not different between selected and not selected mitochondria, suggesting that protection against lipid oxidation was equivalent in these groups. However, not selected mitochondria appeared to have an inherently higher degree of oxidative stress in the mitochondrial matrix due to higher GSSG and the GSSG/GSH ratio compared to selected mitochondria. Two enzymes critical to protection of the mitochondria from oxidative stress are GSH peroxidase and GSH reductase. Lipid and H2O2 are catabolized by GSH peroxidase using the reducing equivalents from GSH with subsequent formation of GSSG. Elevations in GSSG in mitochondria can be toxic due its ability to initiate protein thiol formation and oxidize proteins, rendering them nonfunctional (Olafsdottir and Reed, 1988). Furthermore, elevations in GSSG are especially detrimental in mitochondria. Unlike cells (e.g., hepatocytes and erythrocytes) that have the capability of exporting GSSG out of the cell, mitochondria are unable to release GSSG into the cytosol, and therefore are particularly vulnerable to GSSG-induced oxidation (Olafisdottir and Reed, 1988). Thus, GSH reductase is especially important for maintaining mitochondrial function due to its action in reducing GSSG to GSH. In light of the findings of the present study, it is possible that susceptibility or resistance to PHS may be due in part to the activity or expression of GSH peroxidase and reductase, especially within mitochondria. In summary, lung mitochondria obtained from birds with PHS exhibited mitochondrial dysfunction as evidenced by lower RCR and ADP:O values. This mitochondrial dysfunction was associated with oxidative stress indicated by lower GSH and α- and γ-tocopherol and an increase in the GSSG/GSH ratio compared to controls.

High dietary VE increased mitochondrial tocopherol levels but had no effect on function of mitochondria obtained from lungs of healthy broilers. High dietary VE did attenuate lung mitochondrial dysfunction in broilers that developed PHS. Lower oxidative stress and more efficient oxidative phosphorylation were observed in lung mitochondria obtained from broilers selected for genetic resistance to PHS. In conclusion, the results of this study provide further insight into the mechanisms associated with mitochondrial dysfunction in PHS as well as genetic resistance to PHS.

ACKNOWLEDGMENTS This research was published with support by the Director of the Agriculture Research Experiment Station, University of Arkansas, Fayetteville, AR, and was funded by a USDA-NRI grant (#99-2123 to W. Bottje). Appreciation is extended to R. McNew (Agriculture Statistics Lab, University of Arkansas) for statistical consultation, Hubbard ISA (Walpole, NH) for providing broilers (Experiment 2), and to H. Brandenburger for technical editing of the manuscript. Parts of this study were reported at the 6th Annual Oxygen Society meeting in New Orleans, LA (November 18–22, 1999).

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