Lung Mitochondrial Dysfunction in Pulmonary Hypertension Syndrome ...

Lung Mitochondrial Dysfunction in Pulmonary Hypertension Syndrome. I. Site-Specific Defects in the Electron Transport Chain1 M. Iqbal, D. Cawthon, R. F. Wideman, Jr., and W. G. Bottje2 Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 production was higher in PHS than in control mitochondria. Differences in H2O2 production between control and PHS were magnified by inhibition of Complexes I and III (Coenzyme Q) of the respiratory chain in mitochondria. Functional defects in PHS mitochondria were attenuated by high dietary VE. In Experiment 2, basal H2O2 production and that following inhibition of Complexes I and III were lower in lung mitochondria isolated from broilers selected for genetic resistance to PHS than in nonselected birds in the base population. The results of this study indicate that site-specific defects in Complexes I and III may underlie lung mitochondrial dysfunction in broilers with PHS, that these defects are attenuated by high dietary vitamin E, and that these defects may be related to genetic predisposition to PHS.

ABSTRACT The main objectives of this study were to determine a) site-specific defects in the electron transport chain of lung mitochondria of broilers with pulmonary hypertension syndrome (PHS), b) if these defects are attenuated by high dietary vitamin E, and c) if these defects have a genetic basis. In Experiment 1, lung mitochondria were isolated from broilers with and without PHS fed diets containing 15 IU and 100 IU dl-α-tocopherol acetate/kg (VE); the four treatments were control, VE, PHS, and VE-PHS, respectively. Hydrogen peroxide (H2O2) generation in isolated lung mitochondria was monitored by dichlorofluorosein (DCF) fluorescence in response to chemicals that inhibit electron flow at specific sites on the electron transport chain using a 96-well microplate with Cytoflour (excitation/emission 480/530 nm). Basal H2O2

(Key words: mitochondria, H2O2, vitamin E, pulmonary hypertension syndrome, broiler chicken) 2001 Poultry Science 80:485–495

Mitochondrial dysfunction may contribute to oxidative stress and development of systemic hypoxia that occurs in pulmonary hypertension syndrome (PHS), a devastating metabolic disease in commercial meat chickens (broilers) (Bottje and Wideman, 1995; Cawthon and Bottje, 1999; Cawthon et al., 1999, Iqbal et al., 1999a). PHS is a basic problem of oxygen supply and demand that develops in response to cardiopulmonary insufficiency (Wideman and Kirby, 1995). Systemic hypoxia triggers a series of events that includes peripheral vasodilation, increased cardiac output and pulmonary arterial pressure, and right ventricular hypertrophy (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 temperature, lung damage, or high altitude hypoxia, it also can occur in response to rapid growth (Huchzermeyer and DeRuyck, 1986; Hernandez, 1987; Huchzermeyer et al., 1988, Enkvetchakul et al., 1993). Typical PHS mortality can range between 2 to 5% under optimal growing conditions and as high as 30% under suboptimal conditions. Lower respiratory control ratios and adenosine diphosphate (ADP):O ratios, indices of electron transport chain coupling and of oxidative phosphorylation, respectively (Estabrook, 1967), were observed in PHS liver lung mitochondria (Cawthon et al., 1999; Iqbal et al., 1999a). Thus, greater demand for oxygen in broilers with PHS may stem from inefficient use of oxygen at the cellular level. Mitochondria are a major site of oxygen consumption and a major site of oxidative stress due to generation of reactive oxygen species (ROS). Rather than being completely reduced to water, it has been estimated that 2 to

Received for publication May 26, 2000. Accepted for publication December 6, 2000. 1 This research is published with support by the Director of the Agriculture Research Experiment Station, University of Arkansas, Fayetteville, AR and funded by USDA-NRI grant (#99-2123to W. Bottje). 2 To whom correspondence should be addressed: wbottje@comp. uark.edu.

Abbreviation Key: ADP = adenosine diphosphate; CoQ = Coenzyme Q; DCF = 2′,7′-dichlorofluorescein; DCFH = 2′, 7′-dichlorofluorescin diacetate; EGTA = ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′ tetraacetic acid; NS = nonselected; O2• − = superoxide anion; PHS = pulmonary hypertension syndrome; ROS = reactive oxygen species; RV:TV = right to total ventricular weight ratio; SOD = superoxide dismutase; TTFA = thenoyltrifluroacetone; VE = vitamin E.

INTRODUCTION

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4% of oxygen consumed by mitochondria is incompletely reduced to ROS (e.g., O2• − 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, but not absolute, hypoxia (Dawson et al., 1993). Thus, due to the demand that is placed on mitochondria to support broiler growth in commercial broilers, combined with the propensity of mitochondria to produce ROS, broiler mitochondria may contribute to the oxidative stress associated with PHS. Indeed, histological evidence of H2O2 accumulation in PHS heart mitochondria (Maxwell et al., 1996) lends support to this hypothesis. Within the broiler lung, mitochondrial ROS generation could be particularly detrimental in PHS by stimulating the release of pulmonary vaso- and bronchoconstrictors such as thromboxanes or leukotrienes (Gurtner et al., 1987; Atzori et al., 1991) that would exacerbate the existing cardiopulmonary insufficiency. Increased mitochondrial ROS production has been linked to metabolic diseases such as cystic fibrosis and diabetes and aging (Fiegal and Shapiro, 1979; Hagen et al., 1997; Kristal et al., 1997; Herrero and Barja, 1998, Lass et al., 1998). Free radicals are produced mainly within Complex I or III of the electron transport chain (Turrens and Boveris, 1980; Nohl et al., 1996; Hansford et al., 1997; Herrero and Barja, 1998). Kristal et al. (1997) identified an electron leak in Complex III of diabetic rat mitochondria that would increase O2• − and H2O2 generation, whereas Kwong and Sohal (1998) demonstrated that sites of H2O2 production in the electron transport chain are tissue dependent. Because electrons that leak from the electron transport chain cannot be used to support ATP synthesis, the lower ADP:O observed in PHS liver mitochondria (Cawthon and Bottje, 1999; Cawthon et al., 1999) could also be due to site-specific leakage of electrons from the respiratory chain as well as functional damage to mitochondrial oxidative phosphorylation (Nakahara et al., 1998). Additionally, it is now clear that there is a genetic basis for PHS. A high incidence of PHS mortality can be induced by placing a clamp on one pulmonary artery, which accentuates the cardiopulmonary insufficiency by forcing the entire cardiac output through one lung (Wideman and Kirby, 1995). Within 4 wk, up to 90% of the clamped birds died from PHS. Interestingly, progeny raised from the remaining 10% that did not die following pulmonary arterial clamp procedures are highly resistant to developing PHS. Mortality from PHS was attenuated in first generation progeny (Wideman and French, 1999), whereas third generation progeny selected for PHS resistance to pulmonary artery clamp exhibited very low PHS mortality (4%) compared to 38% in the unselected base

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Cobb Vantress, Siloam Springs, AR 74364. Randall Road, Tyson, Springdale, AR 72762. Roche Vitamins, Inc., Nutley, NJ 07110.

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population upon exposure to cold temperatures (Wideman and French, 2000). Mitochondrial dysfunction has been observed in mitochondria obtained from the liver and lung of broilers with PHS (Cawthon and Bottje, 1999; Cawthon et al., 1999; Iqbal et al., 1999b). As described above, this dysfunction could be due in part to leakage of electrons from the respiratory chain of PHS mitochondria. Thus, the objectives of the present study were to determine if site-specific defects exist in electron transport of lung mitochondria obtained from broilers with PHS, if such a defect could be attenuated by feeding of high levels of dietary vitamin E, and if such a defect might have a genetic basis.

MATERIALS AND METHODS Birds and Management In Experiment 1, male broiler chicks (Cobb 500)3 were obtained from a local hatchery4 at 1 d of age and were placed in an environmental chamber (8 m2 floor space per 100 chicks) on wood shaving litter. Birds were provided access ad libitum to water and a diet (23.7% protein, 3,200 kcal ME) supplemented with 15 (control) or 100 IU dl-αtocopherol acetate5 (vitamin E, VE) per kilogram. Temperatures in the chamber were 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. 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). Upon completion, the study was replicated using identical conditions. In Experiment 2, lung mitochondria were obtained from male broilers (Hubbard Farms, Walpole, NH 03608) 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 fed the control diet, provided with water ad libitum, and maintained under the same environmental conditions as described above.

Sampling Procedure Birds were randomly selected that exhibited overt symptoms of PHS (including systemic cyanosis of the comb, wattle and skin, or abdominal fluid accumulation) or appeared clinically healthy (no cyanosis) as previously described (Cawthon et al., 1999). After being weighed, 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. After removal of the atria, the right ventricle (RV) and total ventricle (TV) weights were determined to calculate the RV:TV weight ratio, a sensitive indicator of prior exposure of the heart to increased pulmonary arterial pressures (Burton et al., 1968). Birds with an RV:TV > 0.30 were classified as having PHS, whereas those with an RV:TV ≤ 0.27 that did

MITOCHONDRIAL DYSFUNCTION IN PULMONARY HYPERTENSION SYNDROME

not have abdominal or pericardial fluid were classified as non-PHS birds. In the 2 × 2 factorial design in Experiment 1, 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 = 8) or VE-PHS (n = 7), for birds with PHS. 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 (S, n = 4) for resistance to PHS. These birds were raised under cold conditions similar to those of Experiment 1. Sampling of birds occurred after 4 wk of exposure to cold between 49 and 57 d of age. Mortalities from PHS in selected and nonselected were 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 of the killed bird was perfused immediately with 10 mL of isotonic heparinized saline (0.85% NaCl, 200 U heparin/mL) (40 C). The lungs were removed, trimmed of extraparenchymal tissue, and washed several times with an ice-cold initial isolation medium containing 225 mM D-mannitol, 75 mM sucrose, and 2 mM EDTA,6 pH 7.3. The lungs were then weighed, minced, and transferred to an ice-cold secondary isolation medium (tissue to medium ratio 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),5 pH 7.3. The minced tissue was homogenized in a Potter-Elvehjem vessel with a Teflon pestle of 0.16 mm clearance7 followed by a second homogenization with a Tekmar homogenizer8 for 10 s. The homogenate was centrifuged for 5 min at 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 pellet was resuspended in secondary isolation medium, centrifuged for 3 min at 1,500 × g to remove blood remnants, and centrifuged again for 10 min at 10,000 × g. The final mitochondrial pellet was resuspended in 250 to 500 µL incubation medium containing 225 mM D-mannitol, 75 mM sucrose, 20 mM EGTA, and 5 mM morpholinopropane sulfate, pH 7.3.

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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 Molecular Probes Inc., Eugene, OR 97402. 10 Cytofluor 2350, Millipore Corporation, Bedford, MA 01730. 7

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Determination of Mitochondrial H2O2 Production Generation of H2O2 was determined using the 2′, 7′dichlorofluorescin diacetate (DCFH-DA)9 chemical probe. Amounts of H2O2 were measured in 96-well microplates, by a photo-fluorometric detector10 at a sensitivity of 3 and an excitation/emission wavelength at 480/ 530 nm. Reaction conditions for H2O2 measurement included the addition of 0.05 to 0.1 mg of mitochondrial protein; 51 µM DCFH-DA; and 64 µL H2O2 buffer containing 145 mM KCl, 30 mM Hepes, 15 mM KH2PO4, 3 mM MgCl2, 0.1 mM EGTA, and 20 U superoxide dismutase (SOD)6 to each well of the microplate. Mitochondria were provided with glutamate or succinate (40 mM) as energy substrates that provide reducing equivalents to the electron transport chain at Complexes I and II, respectively. In a pilot study, the addition of 20 U SOD increased the 2′,7′-dichlorofluorescein (DCF)-induced fluorescence by ∼10%, presumably from increased dismutation of the superoxide anion (O2• −) to H2O2 and oxidation of DCF. Thus, SOD was added to each well to insure all O2• − was converted to H2O2. To demonstrate the specificity of DCF oxidation for H2O2, mitochondria were treated with catalase (900 Sigma units per well) (Figure 1). The final cocktail volume in each well was 124 µL. The microplate was incubated at 37 C and read sequentially at 0, 10, and 30 min by the Cytoflour photofluorimeter. Values of H2O2 were calculated from a standard curve with known amounts of H2O2. Mitochondrial protein concentration was measured with a microprotein determination kit (610-A).6 The final values of H2O2 were corrected for the amount of mitochondrial protein in each well and are expressed as nanomoles per minute per milligram of mitochondrial protein.

Substrate Inhibitor Studies Generation of H2O2 in lung mitochondria was monitored with and without several electron transport chain inhibitors that block electron transfer at specific sites in the respiratory chain as follows: rotenone (Complex I); 4,4,4-Trifluoro-1-[2-thienyl]-1,3-butanedione (TTFA) and malonate (Complex II); myxothiazol (Complex III, Q cycle); antimycin A (which inhibits electron transport within Complex III at cytochrome b562); KCN (cytochrome oxidase, Complex IV); and oligomycin (f1 site of the f1f0ATP synthase, Complex V). Final concentrations used, based on preliminary studies, were rotenone (0.1 mM); myxothiazol, TTFA (1 mM); and KCN (0.04% wt/vol), malonate (0.04 mM), oligomycin (0.01 µL/mg), and Nigericin (0.09 mM) under the reaction conditions mentioned above. Antimycin A (0.1 mM) was also tested in mitochondria in Experiments 1 and 2. To avoid any variability between treatments with regard to background fluorescence, appropriate controls were used for all wells of the microplate, e.g., blanks for mitochondria, all inhibitors, and catalase with both substrates. The final values were corrected with these blanks.

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FIGURE 1. H2O2 production over a 110-min incubation period in incubation medium (Media) alone (open diamond), with mitochondria (Mito) (open triangle), mitochondria with superoxide dismutase (SOD, 20 units per well) (solid square), and mitochondria with catalase (CAT, 900 units per well) (solid circle). Each value represents the mean of three observations. Regression equations for Media, Mito, SOD, and CAT conditions were y = 0.35x + 13.60 (r2 = 0.96), y = 7.99x − 56.79 (r2 = 0.97), y = 8.62 − 40.40 (r2 = 0.99), and y = 0.32x − 1.32 (r2 = 0.98), respectively, and were significant (P < 0.001). *Arbitrary fluorescence units at excitation/emission 480/530 nm).

Statistical Analyses Data are presented as means ± SEM. The data of the first experiment were analyzed using a 2 × 2 factorial design. Selection of birds from each treatment group during each week of the study was randomized. There were no main effects due to week of age or due to replicate in Experiment 1. The data of the second experiment were analyzed using multiple t-tests. All of the above statistical analyses were performed using the general linear models procedure of SAS威 software (SAS Institute, 1996). A probability level of P ≤ 0.05 was considered statistically significant.

RESULTS Electron leakage from mitochondria produces univalent reduction of oxygen to form O2• − that is in turn dismutated by SOD to H2O2 (Chance et al., 1979; Turrens and Boveris, 1980). Supplemental SOD was added to insure that all O2• − would be converted to H2O2 (Figure 1). Evidence that the DCF assay detected primarily H2O2 was confirmed by the low fluorescence that remained after catalase treatment (Figure 1, Table 1). Thus, in this paper the term electron leak and mitochondrial H2O2 generation will be used synonymously. Defects in the electron transport chain can be detected by using various inhibitors; an increase in H2O2 following chemical inhibition indicates that the site of electron leakage is between the site of inhibition and entry of substrate into the electron transport chain (Barja, 1999). Because of similarities in response to the chemical inhibitors in both experiments, the effects of rotenone and myxothiazol are shown in Figure 2, and the effects of other agents on mitochondrial H2O2 production are presented in Table 2.

There were no main effects due to age or replicate in Experiment 1. In lung mitochondria with either energy substrate, Basal H2O2 generation (no inhibitor) was higher in PHS lung mitochondria compared to the other groups (Experiment 1, Figure 2A and B) and in nonselected compared to selected lung mitochondria (Experiment 2, Figure 2C and D). These findings indicate that PHS and nonselected exhibit greater electron leakage compared to control and selected groups. In Experiment 1, high dietary VE attenuated basal H2O2 generation (no inhibitor) in PHS mitochondria (PHS vs. VE-PHS), but had no effect on H2O2 in mitochondria obtained from birds without PHS (control vs. VE). Thus, high dietary VE appears to be beneficial in lowering H2O2 generation in mitochondria obtained from broilers with PHS. Regardless of energy substrate, inhibition of electron transport activity with rotenone (Complex I, NADH Dehydrogenase) and myxothiazol (Complex III, between ubiquinol and the Rieske iron-sulfur protein in the Q cycle (von Jagow et al., 1984) resulted in large increases in H2O2 generation relative to noninhibited mitochondria within each group. These findings suggest that rotenone- and myxothiazol-sensitive sites are likely sites of electron leakage in broiler lung mitochondria. In Experiment 1, rotenone and myxothiazol treatments produced dramatic elevations in H2O2 generation in PHS compared to control mitochondria indicating that electron leakage at Complexes I and III of broiler lung mitochondria is enhanced in broilers with PHS. Although high dietary VE had no effect on rotenone-induced H2O2 generation in mitochondria obtained from healthy broilers (VE vs. control), high dietary VE attenuated lung H2O2 generation in mitochondria obtained from broilers with PHS (VE-PHS vs. PHS). With myxothiazol treatment, VE mitochondria exhibited lower H2O2 levels when metabolizing succinate (Figure 2A), but when glutamate was provided as the energy substrate, H2O2 generation in VE mitochondria was not different from PHS mitochondria (Figure 2B). These findings were unexpected but, as discussed below, could be due in part to a complex interaction between Coenzyme Q (CoQ) and α-tocopherol in the mitochondrial inner membrane. In Experiment 2, H2O2 generation was higher in nonselected than in selected mitochondria regardless of substrate following treatment with rotenone and myxothiazol. These findings are similar to Experiment 1 and provide further evidence that Complexes I and III are likely sites of electron leakage in broiler lung mitochondria. Furthermore, the higher H2O2 generation in nonselected broilers that were healthy (i.e., no symptoms of PHS) indicated that increased radical generation in mitochondria might be an inherent attribute associated with PHS susceptibility and a primary factor involved in the development of PHS. The effects of other electron transport chain inhibitors, as well as oligomycin [adenosine triphosphate (ATP) synthase inhibitor] and Nigericin (dissipates proton gradient across inner mitochondrial membrane) for Experiments 1 and 2 are presented in Table 2. In contrast to the large


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TABLE 1. Non-H2O2 specific residual oxidation of DCF in broiler lung mitochondria remaining following treatment with catalase2 Non-H2O2 specific residual oxidation Experiment 1 Substrates

CON (n = 10)

PHS (n = 8)

Experiment 2

VE (n = 7)

VE-PHS (n = 7)

S (n = 4)

NS (n = 4)

0.2 ± 0.2 0.2 ± 0.2

0.1 ± 0.1 0.1 ± 0.1

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Succinate Glutamate

0.8 ± 0.3 0.2 ± 0.1

0.8 ± 0.5 0.4 ± 0.3

(AFU/min) 0.7 ± 0.5 0.5 ± 0.5 1.2 ± 0.7 0.1 ± 0.1

1 DCF = 2′,7′-dichlorofluorescein; CON = control; PHS = pulmonary hypertension syndrome; VE = vitamin E; VE-PHS = vitamin E-fed PHS birds; S = selected for resistance to PHS; NS = not selected for PHS. 2 All values are the means (number given in parentheses) ± SEM. 3 Arbitrary fluorescent units at excitation/emission 480/530 nm.

increases in H2O2 observed with rotenone and myxothiazol described above, with a few exceptions, all other chemical agents tested in this study had no effect or slightly decreased mitochondrial H2O2 generation (relative to noninhibited values) regardless of energy substrate provided. Antimycin A, inhibitor of electron transfer at cytochrome b562 of Complex III, produced slight but significant elevations in H2O2 in all mitochondria relative to noninhibited mitochondria in both experiments with the exception of PHS mitochondria with succinate (Table 2). The effects of antimycin A on mitochondria metabolizing glutamate were not tested in Experiment 1. Antimycin A-induced increases in H2O2 generation were greater in PHS than in control mitochondria metabolizing succinate

(Experiment 1) and in nonselected than in selected mitochondria (Experiment 2), suggesting a slightly greater leakage of electrons occurring at the antimycin A-sensitive site of Complex III in PHS and nonselected mitochondria relative to controls and PHS-resistant broilers. Slight elevations in H2O2 were observed with Complex II inhibitors (malonate and TTFA) in VE-PHS mitochondria metabolizing succinate, but not glutamate, when compared with no inhibitor (Table 2). Thus, Complex II does not appear to be an important site for electron leakage in lung mitochondria. Inhibition of cytochrome C oxidase (Complex IV) with KCN, or ATP synthase with oligomycin reduced basal lung mitochondrial H2O2 generation (and therefore elec-

FIGURE 2. H2O2 production (nmol/min per mg mitochondrial protein) in mitochondria with no inhibitor (NI), or treated with rotenone (Rot) and myxothiazol (Myx) provided with succinate or glutamate as energy sources in Experiment 1 (A and B) and Experiment 2 (C and D). Treatment groups in Experiment 1 were obtained from control (CON, open bar). Each bar represents the mean ± SE of 7 to 10 observations (Experiment 1) and of 4 observations (Experiment 2). a,b,cValues within a mitochondrial inhibitor group with different letters are different (P < 0.05). *Values differ from NI values within group (P < 0.001).

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IQBAL ET AL. TABLE 2. Effects of different electron transport chain inhibitors on H2O2 generation in lung mitochondria of broilers provided succinate as the energy substrate1 H2O2 2

Experiment 1 Inhibitors

CON (n = 10)

No inhibitor Antimycin A Malonate TTFA KCN Oligomycin Nigericin

3.4 5.0 1.4 1.6 1.9 1.6 2.7

± ± ± ± ± ± ±

0.6b 1.0Xab 0.6Xb 0.5Xb 0.8X 0.4X 0.5

PHS (n = 8) 7.3 6.9 3.1 3.3 1.5 2.1 3.3

± ± ± ± ± ± ±

0.9a 1.8a 0.8Xa 1.0Xa 0.4X 0.6X 0.6X

Experiment 2

VE (n = 7)

VE-PHS (n = 7)

(nmol/min per mg protein) 2.4 ± 0.9b 1.4 ± 0.2c 4.0 ± 0.9Xb 5.0 ± 1.2Xab 2.3 ± 1.5ab 2.4 ± 0.7Xab 2.3 ± 1.0ab 2.3 ± 0.5Xab 0.8 ± 0.4X 1.8 ± 0.7 1.6 ± 0.7X 1.5 ± 0.3 2.6 ± 0.6 2.4 ± 0.4X

S (n = 4) 3.2 7.0 2.8 1.6 1.2 2.2 2.4

± ± ± ± ± ± ±

0.4b 1.0Xb 0.6 0.4Xa 0.9X 0.8X 0.6

NS (n = 4) 4.9 11.7 2.7 2.8 2.1 1.7 3.0

± ± ± ± ± ± ±

0.5a 1.7Xa 0.6X 0.3Xb 0.4X 0.3X 0.5X

Means in the same row and experiment with no common superscript differ significantly (P < 0.05). Inhibitor means are different significantly (P < 0.05) from no inhibitor within a column (same group) in each experiment. 1 All values are the means (number given in parentheses) ± SEM. 2 CON = controls; PHS = pulmonary hypertension syndrome; VE = vitamin E; VE-PHS = vitamin E-fed PHS birds; S = selected for resistance to PHS; NS = not selected for PHS resistance; TTFA = thenoyltrifluroacetone; KCN = potassium cyanide. a–d X

tron leakage) in Experiments 1 and 2 with the exception of VE-PHS group in Experiment 1 with succinate. Dissipation of the proton gradient across the inner mitochondrial membrane with Nigericin lowered H2O2 production relative to no inhibitor values in PHS and nonselected mitochondria, regardless of energy substrate provided (Tables 2 and 3). Other mitochondria in these studies were unaffected by Nigericin treatment with the exception of a slight increase in H2O2 production in VE-PHS mitochondria provided with succinate (Table 2).

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).

Mitochondrial H2O2 Production in PHS The results of this study indicate that broilers with PHS exhibit a higher level of electron leakage in lung mitochondria. This defect in electron transport was attenuated by high dietary vitamin E and was attributed to an inherent defect in electron transport. Thus, electron leakage from lung mitochondria of broilers with PHS does not appear simply to be involved in the sequela of PHS. Based on the effects of chemical inhibitors on H2O2 production, it appears that site-specific defects in the respiratory chain of PHS mitochondria occur at the rotenone and myxothiazol sensitive sites in Complexes I and

DISCUSSION There were no differences in BW between treatment groups, but RV:TV values (means ± SE) were 0.22 ± 0.02, 0.32 ± 0.02, 0.21 ± 0.02, and 0.41 ± 0.02 for control, PHS, VE, and VE-PHS groups, respectively (Iqbal et al., 1999a).

TABLE 3. Effects of different electron transport chain inhibitors on H2O2 generation in lung mitochondria of broilers provided glutamate as the energy substrate1 H2O2 2

Experiment 1 Inhibitor

CON (n = 10)

No inhibitor Antimycin A Malonate TTFA KCN Oligomycin Nigericin

2.7 ... 2.4 3.7 1.9 1.5 3.1

± 0.3b ± ± ± ± ±

0.7b 0.6b 0.9a 0.4Xb 0.6

PHS (n = 8) 7.5 ... 4.5 6.7 0.7 2.7 2.6

± 1.0a ± ± ± ± ±

0.8Xa 1.0a 0.7Xab 0.4Xa 0.7X

VE (n = 8)

Experiment 2 VE-PHS (n = 7)

(nmol/min per mg protein) 2.5 ± 0.9b 2.1 ± 0.8b ... ... 3.0 ± 1.1ab 1.2 ± 0.4c 3.2 ± 0.9bc 2.1 ± 0.6c 0.0 ± 0.0Xb 1.7 ± 0.9a 0.9 ± 0.2Xbc 0.7 ± 0.2Xc 2.7 ± 0.6 2.1 ± 0.7

S (n = 4) 3.0 6.6 2.3 2.9 0.0 1.1 2.0

± ± ± ± ± ± ±

0.6b 1.3Xb 0.5 0.6 0.0X 0.6X 0.5

NS (n = 4) 5.0 9.7 2.2 1.8 0.4 1.2 2.1

± ± ± ± ± ± ±

0.3a 0.6Xa 0.3X 0.4X 0.4X 0.8X 0.5X

Means in the same row and experiment with no common superscript differ significantly (P < 0.05). Inhibitor means are different significantly (P < 0.05) from no inhibitor within a column (same group) in each experiment. 1 All values are the means (number given in parentheses) ± SEM. 2 CON = controls; PHS = pulmonary hypertension syndrome; VE = vitamin E; VE-PHS = vitamin E-fed PHS birds; S = selected for resistance to PHS; NS = not selected for PHS resistance; TTFA = thenoyltrifluroacetone; KCN = potassium cyanide. a–d X


III (CoQ), respectively (Figure 2). Qualitatively similar results were observed in Experiment 1 (control vs. PHS mitochondria) and Experiment 2 (selected vs. nonselected mitochondria) with all chemical agents used in this study. The elevation in H2O2 production with rotenone and myxothiazol was more pronounced in PHS mitochondria than in controls (Experiment 1) and in nonselected mitochondria compared to selected mitochondria (Experiment 2). These observations point to Complexes I and III as being major sites of radical generation in the two broiler lines used in this study. Furthermore, the fact that rotenoneand myxothiazol-induced increases in H2O2 production were lower in lung mitochondria of birds selected for PHS resistance, compared to the nonselected base population in Experiment 2, indicates that electron leakage at Complexes I and III may be genetically linked to PHS susceptibility. The increase in H2O2 production following rotenone inhibition at Complex I in mitochondria metabolizing glutamate concurs with numerous reports in mammals and birds (Takeshige and Minakami, 1979; Turrens and Boveris, 1980; Hansford et al., 1997; Herrero and Barja, 1997, 1998). In mitochondria that are metabolizing succinate, rotenone is added to prevent flux of electrons from succinate back through NADH dehydrogenase (Jones and Lash, 1993). According to Barja (1999), an increase in radical production following inhibition of electron transport chain activity is indicative of increased electron leakage between the site of inhibition and entry of substrate into the electron transport chain. Glutamate and succinate donate electrons at Complexes I and II, respectively. However, rotenone (which inhibits electron transport at Complex I) is added to mitochondria metabolizing succinate to prevent the back flow of electrons from Complex II to Complex I (Jones and Lash, 1993). Thus, the rotenoneinduced increase in H2O2 production in mitochondria metabolizing glutamate or succinate suggests that electron leakage can occur on either side of the rotenone sensitive site (i.e., from electrons flowing down the respiratory chain from glutamate, or from electrons flowing backward from succinate) of NADH dehydrogenase (Complex I). Unlike studies in which myxothiazol treatment inhibited ROS generation in mammals and birds (pigeons, canaries, and parakeets) (Boveris et al., 1976; Turrens et al., 1985; Nohl et al., 1996; Herrero and Barja, 1998), myxothiazol increased H2O2 production in both experiments in the present study. These results may indicate that domesticated broilers have a fundamental difference associated with CoQ of Complex III compared to other birds or mammals. Possibly, these findings could be attributed to a difference in the interaction of myxothiazol with the respiratory chain in broilers. Myxothiazol interferes with the binding of ubiquinone or ubiquinol with the Rieske iron-sulfur protein to block electron flow (von Jagow et al., 1984), maintaining the ubiquinone as ubiquinol (reduced state), and not as the semiquinone that interacts with oxygen to form O2• − and H2O2 (Turrens et al., 1985). Inhibition of binding with the Rieske iron-sulfur protein

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therefore typically decreases H2O2 generation (Boveris et al., 1976; Turrens et al., 1985; Nohl et al., 1996; Herrero and Barja 1998). However, von Jagow et al. (1984) also indicated that under certain conditions, myxothiazol could bind and change the cytochrome b566 heme-ligand environment. This inhibition would be similar to the action of antimycin A that maintains the semiquinone state and enhances O2• − formation (Turrens et al., 1985). Thus, it is plausible that in the broiler lung, there may be a slight difference in cytochrome structure that facilitated the binding of myxothiazol to cytochrome b566. In addition, shuttling of electrons from uqibuinol to cytochrome C1 depends on the ability of the Rieske iron-sulfur protein to move between these sites on the respiratory chain (Crofts et al., 1998; Berry et al., 1999). This movement apparently depends on the integrity of cysteine bonds to the iron group and salt bridges between amino acids of the Rieske iron-sulfur protein that are essential for its function (Iwata et al., 1996, Crofts et al., 1998). Thus, any change in the structure of this protein in broiler lung mitochondria, for example through oxidative damage, may also account for the observations in the present study. Alternatively, effects of myxothiazol in this study might be related to the amount or characteristics of CoQ association with the respiratory chain in broilers. Although the content of CoQ has not been established in broilers, Lass et al. (1997) reported that animals with high CoQ9 levels (rat and mouse) had higher mitochondrial O2• − generation compared to those having more CoQ10 (cow, rabbit, and pig). Some defects in PHS mitochondria might have been overcome as the feeding of CoQ9 has been reported to attenuate PHS mortality in cold-exposed broilers (Nakamura et al. 1996). The amount of mitochondrial O2• − generation may also depend on the relative binding of CoQ to proteins (Lass and Sohal, 1999). Thus, broiler lung mitochondria may have higher amounts of CoQ9 or different protein binding characteristics in mitochondria that would make them more prone to produce O2• −, a process accentuated by myxothiazol. Typically, large increases in H2O2 production are observed following treatment of mitochondria with antimycin A (Boveris et al., 1976; Turrens et al., 1985; Nohl et al., 1996; Herrero and Barja, 1998). Results from antimycin A inhibition of broiler lung mitochondria provided succinate resulted in only modest increases in electron leakage (Table 2). Possibly, these inconsistencies and discrepancy with regard to previous studies may be due to the 100 µM antimycin A concentration that we used. Garcia-Ruiz et al. (1997) reported that antimycin A concentrations greater than 3 nmol/mg protein can cause fluorescence quenching of DCF. Unfortunately, we were not aware of this report until after we had completed our experiments. Antimycin A may be inactivated with storage over time (B. Kristal, personal communication), which might also explain discrepancies observed with antimycin A. Antimycin A also did not alter residual oxygen consumption in liver mitochondria, however (Cawthon et al., 1999).

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Treatment of mitochondria with malonate lowered H2O2 production in PHS mitochondria in Experiment 1, whereas malonate and TTFA lowered H2O2 production significantly in nonselected mitochondria in Experiment 2. Thus, according to Barja (1999) these results would indicate that free radical generator site(s) in PHS and nonselected lung mitochondria would be downstream from Complex II. These results contrast with those of Cawthon et al. (1999), who reported that residual oxygen consumption, indicative of increased electron leakage, was increased in PHS liver mitochondria treated with malonate. Thus, the sites of radical generation in PHS mitochondria may be tissue dependent. This hypothesis is supported by the report that mitochondria ROS production exhibits tissue specificity (Lass et al., 1998). Inhibition of ATP synthase with oligomycin decreased H2O2 production in both experiments. Oligomycin specifically inhibits the f1 function of the f1f0 ATP synthase (Tyler, 1992) thereby indirectly affecting electron transport chain activity by inhibiting proton flux back through the inner mitochondrial membrane via the f0 proton channel. The higher H2O2 production following ATP synthase inhibition in PHS mitochondria compared to controls was observed with glutamate in Experiment 1 (Table 2) and suggests that there is residual ROS generation occurring in mitochondria metabolizing glutamate that is unaffected by oligomycin. Uncoupling of the electron transport chain with Nigericin significantly lowered H2O2 generation in PHS mitochondria in Experiment 1 and in nonselected mitochondria in Experiment 2 (Table 2) indicating that H2O2 generation in PHS or nonselected mitochondria is linked to the establishment of the proton motive force. Dissipation of the proton gradient decreased H2O2 generation in these mitochondria but not in mitochondria obtained from the other groups in the study.

Vitamin E and Mitochondrial H2O2 Production 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). Enkvetchakul et al. (1993) reported that broilers with PHS exhibited a general depletion of endogenous antioxidants and that α-tocopherol decreased at an earlier age than did reduced glutathione or ascorbic acid. A vitamin E implant containing dl-αtocopherol was effective in lowering PHS mortality (Bottje et al., 1995), but feeding high levels of dl-tocopherol acetate had no effect on PHS mortality (Bottje et al., 1997). Because mitochondrial membranes have high levels of α-tocopherol (Bjorneboe et al., 1991), it is of interest to determine the effect of high dietary vitamin E on mitochondrial H2O2 production in broilers. Vitamin E had no effect on basal (uninhibited) H2O2 production in mitochondria obtained from broilers without PHS (VE), whereas vitamin E attenuated mitochondrial H2O2 production in birds that developed PHS (VE-

PHS) (Figure 2). The lower H2O2 generation in VE-PHS compared to PHS mitochondria appears primarily due to attenuation of rotenone- and myxothiazol-induced H2O2 production. A complex interaction between dietary vitamin E, PHS and H2O2 production at the myxothiazol sensitive site is also observed. After myxothiazol treatment, H2O2 production was elevated in VE lung mitochondria metabolizing glutamate, similar to that observed in PHS mitochondria (Figure 2B), but H2O2 production was attenuated in VE mitochondria metabolizing succinate (Figure 2A). An explanation for the interaction between vitamin E, PHS status, and myxothiazol-induced H2O2 production noted above may again lie in the complex nature of CoQ(n). Auto-oxidation of CoQ is a major source of mitochondrial ROS production (Chance et al., 1979; Turrens et al., 1985). Coenzyme Q has been suggested to act as a potent antioxidant in the inner mitochondrial membrane where it can scavenge radicals directly (Takayanagi et al., 1980; Kagan et al., 1990a) and indirectly through regeneration of α-tocopherol from the tocopheroxyl radical (Kagan et al., 1990b; Maguire et al., 1992). However, as indicated previously, the relative binding of CoQ to proteins and amounts of CoQ9 and CoQ10 are important determinates for mitochondrial ROS production as well (Lass and Sohal, 1999). If the interaction between vitamin E and CoQ is facilitated by the amount of protein-bound CoQ, and broilers have a relatively high amount of free CoQ, it may not be able to efficiently interact with α-tocopherol. Addition of high amounts of α-tocopherol in the diet might have caused increased tocopheroxyl radical formation or auto-oxidation of CoQ leading to higher H2O2 formation in VE mitochondria treated with myxothiazol. A shift in the amount of CoQ bound to proteins could be hypothesized to occur in PHS. Following similar logic, if more CoQ is bound to proteins in PHS mitochondria, it may then be able to interact with tocopherol and lower the amount of H2O2 production as observed in VE-PHS mitochondria. The previous statements are of course strictly conjectured at this time. Nevertheless, we do hypothesize that the relative activity and degree of protein binding of CoQn may play an important role in the development of mitochondrial dysfunction in PHS. This hypothesis is indirectly supported by the observation of attenuation of cold temperature-induced incidence of PHS mortality in broilers by feeding of CoQ9 (Nakamura et al., 1996).

Use of DCFH to Detect H202 Generation of H2O2 was determined using the DCFHDA chemical probe that, after deacetylation to DCFH, is oxidized to the highly fluorescent 2′, 7′-dichlorofluorescein (DCF) (Bass et al., 1983). The oxidation of DCFH to DCF has been widely used to determine H2O2 generation by flow cytometry and in indirect visualization techniques (Rothe and Valet, 1990; Dawson et al., 1993; Allen et al., 1997; Garcia-Ruiz et al., 1997). While preparing this manuscript, similar assay conditions were reported in a


recent publication (Wang and Joseph, 1999). To demonstrate the specificity of DCF oxidation for H2O2 generation, mitochondria were treated with catalase (900 Sigma units per well). Amounts of nonspecific DCF oxidation for all treatment groups in Experiments 1 and 2 are presented in Table 1. Fluorescence not quenched by the catalase was subtracted from the final values obtained in substrate-inhibitor studies. In an effort to record maximum free radicals generation, SOD was used in the assays (Chance et al., 1979; Turrens and Boveris, 1980). An approximately 10% increase in free radicals generation has been recorded and was apparently caused by the dismutation of O2• − produced by univalent reduction of electrons leaked from mitochondria to H2O2 (Figure 1). After its first use in neutrophils (Bass et al., 1983), DCF fluorescence detection of H2O2 has been widely used in cells (Hyslop and Sklar, 1984; Rothe and Valet, 1990; Royall and Ischiropoulos, 1993; Carter et al., 1994; Vowells et al., 1995 ) as well as in isolated mitochondria (GarciaRuiz, et al., 1997). Several reports indicate that DCF fluorescence is specific for detection of peroxides (Cathcart et al., 1993; Dawson et al., 1993). Specificity for H2O2 was confirmed in the present study by the low amount of DCF oxidation that remained in catalase-treated mitochondrial samples (Figure 1, Table 1). Discussion of the DCFH assay is warranted, however, because questions about its validity have been raised. In cells, it is assumed that once DCFH-DA enters the cell, the acetyl moiety is cleaved by intracellular esterases followed by oxidation of DCFH to DCF (Bass et al., 1983). Although mitochondria may lack mitochondrial esterase activity (Barja, 1999), spontaneous deacetylation of DCFH-DA was observed in culture medium (Royall and Ischiropoulos 1993) and allowed detection of H2O2 in isolated mitochondria and linear up to 150 µM H2O2 (Garcia-Ruiz et al., 1997). Controversy also exits regarding the effect of SOD on the DCFH assay. Some studies report an inhibitory effect of SOD on the DCFH assay (e.g., Atlante et al., 1997; Rota and Mason, 1999) yet others observed no effect (Yang et al., 1997; Hempel et al., 1999). The observed differences in H2O2 detection with SOD might be due to the different levels of endogenous SOD in different cells from different species or might be based on degree of oxidative stress in the cells. We added SOD to all samples based on the linear relationship of DCF fluorescence over time observed in a pilot study (Figure 1). Because photooxidation of DCFH to DCF (Rota and Mason, 1999; Wang and Joseph, 1999) could lead to over estimation of H2O2 production, measures were taken in the present study to reduce photooxidation by turning the light off during the preparation of the assays and by using equipment with fast light excitation and fast fluorescence capturing (see methods). Finally, appropriate controls were used to correct for oxidation by each chemical inhibitor and from the media. In summary, the higher production of H2O2 observed in PHS lung mitochondria in this study could be physiologically relevant to PHS. Increased H2O2 production in pulmonary cells could cause vaso- or bronchoconstriction

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through the release of leukotrienes and prostanoids (Gurtner et al., 1987; Atzori et al., 1991). A particular prostanoid, thromoboxane, has been shown to cause pulmonary vasoconstriction in broilers (Wideman et al., 1999). Additionally, increased H2O2 generation by lung mitochondria could be a self-perpetuating oxidation due to the formation of toxic compounds such as malondialdehyde and hydroxyalkenals (Esterbauer et al., 1991) that could contribute to further mitochondrial dysfunction (Kristal et al., 1994, 1996; Chen et al., 1995). The results of this study indicate that higher H2O2 production in lung mitochondria isolated from broilers with PHS is related to site-specific defects in the transport of electrons in Complex I (NADH dehydrogenase) and CoQ in Complex III. As similar findings were observed in lung mitochondria obtained from nonselected broilers in the base population compared to lung mitochondria obtained from broilers selected for genetic resistance to PHS, it appears that the site-specific defects may arise from a genetic predisposition to the disease. The defects may also contribute to dysfunction in PHS lung mitochondria that is characterized by oxidative stress and lower respiratory control and ADP:O ratios (Iqbal et al., 1999a,b). High dietary vitamin E was able to attenuate H2O2 generation but did not totally prevent mitochondrial oxidative stress. Increased H2O2 generation in PHS mitochondria could represent an important mechanism in the pathophysiology of PHS that could stimulate pulmonary broncho- and vasoconstriction, thereby bringing about the cardiopulmonary insufficiency that characterizes this metabolic disease. The results of this study may help provide clues for better understanding of the genetic basis of PHS. To our knowledge, the findings presented here and by Cawthon et al. (1999) are the first to report site-specific defects in electron transport within mitochondria associated with PHS.

ACKNOWLEDGMENTS The authors thank N. Rath and D. Horlick (USDA-ARS, University of Arkansas) for assistance in the use of the Cytofluor 2350 used in the detection of DCF fluorescence. Appreciation is also extended to R. McNew (Agriculture Statistics Lab, University of Arkansas) for statistical design and consultation and to Howard French (Hubbard ISA, Walpole, New Hampshire) for providing access to broilers used in the second experiment in this study. Parts of this study were reported at the 6th Annual Oxygen Society meeting in New Orleans (November 18–22, 1999).

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