Alberto Boveris

Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria

Abstract Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O 2 − at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H 2 O 2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H 2 O 2 was essentially produced by disproportionation of O 2 − , which constitutes the precursor of H 2 O 2 . The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O 2 − and 0.63 mol of H 2 O 2 /mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O 2 to O 2 − , since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H 2 O 2 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H 2 O 2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q 1 > Q 0 > Q 2 > Q 6 = Q 10 , in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O 2 − and H 2 O 2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q 0 H 2 ), benzoquinol, duroquinol and menadiol generated O 2 − with k 3 values of 0.1 to 1.4 m − · s −1 and H 2 O 2 with k 4 values of 0.009 to 4.3 m −1 · s −1 .

Production of Nitric Oxide by Mitochondria

The production of NO⋅ by mitochondria was investigated by electron paramagnetic resonance using the spin-trapping technique, and by the oxidation of oxymyoglobin. Percoll-purified rat liver mitochondria exhibited a negligible contamination with other subcellular fractions (1–4%) and high degree of functionality (respiratory control ratio = 5–6). Toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of nitric oxide synthase (NOS) incubated with the spin trapN-methyl-d-glucamine-dithiocarbamate-FeIIproduced a signal ascribed to the NO⋅ spin adduct (g = 2.04; a N = 12.5 G). The intensity of the signal increased with time, protein concentration, andl-Arg, and decreased with the addition of the NOS inhibitorN G-monomethyl-l-arginine. Intact mitochondria, mitochondrial homogenates, and submitochondrial particles produced NO⋅ (followed by the oxidation of oxymyoglobin) at rates of 1.4, 4.9, and 7.1 nmol NO⋅ × (min·mg protein)−1, respectively, with aK m for l-Arg of 5–7 μm. Comparison of the rates of NO⋅ production obtained with homogenates and submitochondrial particles indicated that most of the enzymatic activity was localized in the mitochondrial inner membrane. This study demonstrates that mitochondria are a source of NO⋅, the production of which may effect energy metabolism, O2consumption, and O2 free radical formation.

[57] Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria

Publisher Summary The determination of the rate of O 2 - production is based upon the spectrophotometric measurement of oxidation or reduction reactions in which O 2 - is a reactant. The concentration of the spectrophotometric indicator that reacts with O 2 - is adjusted to compete effectively with the spontaneous dismutation of O 2 - so that nearly all O 2 - produced can be detected. The involvement of O 2 - is ascertained by the use of superoxide dismutase that inhibits the reaction rate specifically due to O 2 - . Cyanide, which is often used as inhibitor of the mitochondrial cytochrome oxidase ( K i about 3 x 10 -5 M) is also an inhibitor of the often used copper-containing superoxide dismutase ( K i about 3 × 10 -4 M). It is possible to use enough cyanide that partially inhibits cytochrome oxidase without inhibiting superoxide dismutase. Alternatively, bacterial or mitochondrial (manganese-containing) superoxide dismutase can be used. Mitochondria have Mn-superoxide dismutase in the matrix space, so to measure the total production of O 2 - by mitochondrial membranes, the dismutase should be inhibited or removed. Because effective inhibitors of Mn-superoxide dismutase are not known, matrical superoxide dismutase is usually removed by the repetitive washing of submitochondrial particles obtained by sonication or by other means. Mitochondrial O 2 - production is pH dependent and increases toward the alkaline region.

Hydroperoxide-initiated chemiluminescence: An assay for oxidative stress in biopsies of heart, liver, and muscle

Hydroperoxide-initiated chemiluminescence was standardized as a microassay to evaluate the occurrence of oxidative stress in human biopsies. Samples of 10 to 50 mg of rat liver or heart were homogenized, diluted in reaction medium, added with tert-butyl hydroperoxide, and assayed for chemiluminescence in a liquid scintillation counter in the out-of-coincidence mode. Optimal conditions for the assay were: 0.3 to 1.2 mg/mL of homogenate protein in 120 mM KCl, 30 mM phosphate buffer (pH 7.4), and 3 mM tert-butyl hydroperoxide at 30 degrees C. In these conditions, maximal chemiluminescence values were 550 +/- 30 and 1100 +/- 40 cps/mg protein, for liver and heart homogenates, respectively. Liver and heart homogenates were subjected to in vitro oxidative stresses such as supplementation with organic hydroperoxide or with enzymatic systems generating superoxide anion or hydrogen peroxide. Chemiluminescence was higher in the poststress samples than in the control ones. The ratio: poststress chemiluminescence/control chemiluminescence (B/A) was about 1.4 or higher for both tissues. Human heart biopsies were utilized to investigate the occurrence of oxidative stress after clinical situations associated to ischemia-reperfusion. B/A ratios were 2.1 +/- 0.4, 1.4 +/- 0.1, and 2.8 +/- 0.4 for human heart, liver, and skeletal muscle, respectively.

Mitochondrial Production of Superoxide Radical and Hydrogen Peroxide

The development and application of sensitive methods for the determination of hydrogen peroxide led, a few years ago in the laboratories of the Johnson Research Foundation, to the recognition, of intact mitochondria as an effective source of H2O2 (Fig. 1; refs. 1–4). Previous observations by Jensen (5) and by Hinkle et al. (6) had indicated that the mitochondrial respiratory chain was capable of producing H2O2. However, these results were taken with caution, in the sense that they might reflect an artificial activity induced by the ultrasonic or alkaline treatment used in the preparation of the submitochondrial particles. In 1971, Chance and Oshino (1) demonstrated variations in the level of the catalase intermediate of the peroxisomal-mitochondrial fraction of rat liver following the addition of mitochondrial substrates and uncouplers. In the same year, Loschen et al. (2) showed H2O2 formation in pigeon heart mitochondria and its relationship to the mitochondrial metabolic state by using the peroxidase-scopoletin method. It was realized that this assay could be easily interfered by endogenous hydrogen donor of the horseradish peroxidase and by exogenous hydrogen donors in the mitochondrial preparations, and consequently, an alternative method was developed.

Mitochondrial production of superoxide anions and its relationship to the antimycin insensitive respiration.

Superoxide anions are produced in the oxidation by molecular oxygen of a multitude of biomolecules [l] . Iron-sulfur flavoproteins like xanthine oxidase [2,3], flavoproteins as flavodoxin [4] , iron-sulfur proteins like spinach and clostridial ferrodoxins [5] , quinols as the reducedt?rm of menadione [4] and some cytostatic agents [6], glutathione [7] and other thiol containing molecules, etc., all of them are effective sources of superoxide radicals. These radical anions dismutate to Hz O2 either nonenzymatically or by the reaction catalyzed by superoxide dismutase, according to the MC Cord Fridovich reaction:

Kinetics of nitric oxide and hydrogen peroxide production and formation of peroxynitrite during the respiratory burst of human neutrophils

Nitric oxide (.NO) release, oxygen uptake and hydrogen peroxide (H2O2) production elicited by increasing phorbol 12‐myristate 13‐acetate (PMA) concentrations were measured in human neutrophils. Half‐maximal activities were sequentially elicited at about 0.0001–0.001 μg (.NO) and 0.001‐0.01 μg (H2O2). At saturated PMA concentrations, .NO production, oxygen uptake and H2O2 release were 0.56 ± 0.04, 3.32 ± 0.52 and 1.19±0.17 nmol · min−1 · 106 cells−1. .NO production accounts for about 30% of the total oxygen uptake. Luminol‐dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half‐maximally activated at about 0.001‐0.01 μg . Preincubation with N G‐monomethyl‐l‐arginine (l‐NMMA) decreased O2 uptake and .NO release but increased H2O2 production, while superoxide dismutase (SOD) increased .NO detection by 30%. Chemiluminescence was also reduced by preincubation with l‐NMMA and/or SOD. The results indicate that .NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of ″NO and O2 .− release favour the formation of other oxidants like peroxynitrite.

Nitric oxide inhibits mitochondrial NADH : ubiquinone reductase activity through peroxynitrite formation

This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.

Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats

Rats fed ethanol (1.74 +/- 0.12 g/day/100 g body wt for 12 weeks) showed a 45% increased microsomal production of O-2 (2.23 +/- 0.14 nmol/min/mg protein) and a 28% increased content of endoplasmic reticulum protein (26.8 +/- 1.4 mg/g liver). This could lead, at substrate saturation, to a 86% increased cytosolic production of O-2 which is not compensated by cytosolic superoxide dismutase levels that remain normal. It is claimed that this unbalance between O-2 production and superoxide dismutase leads to a peroxidative stress in agreement with the 54% increased spontaneous liver chemiluminescence (37 +/- 2 cps/cm2) measured in the ethanol-treated rats. Hydroperoxide-induced chemiluminescence was 57, 43, and 28% higher, respectively, in homogenates, mitochondria, and microsomes isolated from ethanol-treated rats as compared with controls. Vitamins E and A were more effective inhibitors of the hydroperoxide-stimulated chemiluminescence in the liver homogenates from ethanol-treated rats as compared with the effect on the homogenates from control animals. The results are consistent with a peroxidative stress in chronic alcoholism leading to increased lipoperoxidation and decreased levels of antioxidants.

Increased biliary glutathione disulfide release in chronically ethanol-treated rats.

tion was: carbohydrate, 54%; lipid, 193; and protein 27%. Glutathione &sulfide (GSSG) release frc3 liver [I ] has been evaluated as an indicator of oxidative stress, including lipid peroxidation (cf. hydroperoxide metabolism review in [2] )_ At present, the occurrence of enhanced rates of lipid peroxidation or of Ns02 production in the liver of rats c!lronically treated with ethanol is controversial (cf. review in ]3] and references therein) but appears to be a central concept in experimental pathology_ Therefore, we have applied a non-invasive approach and have examined the release of both oxidized and reduced ghrtathione from the liver of rats chronically treated, i.e., for 6 weeks with ethanol, essentially as in [4]. According to the finding that GSSG release occurs into bile [5], measurements were carried out in bile samples obtained from anesthetized rats. The calory percentages of the ingredients in th: final regimen, i.e., basal diet plus ethanol solution, consumed by the animals of the alcohol group were as follows:-ethanol, 41.5%; carbohydrate> 42.6%; lipid, 6.6% and protein, 9.3%. The composition of the diet of the control group was the same except that the ethanol-derived calories were replaced by sucrose. The animals in the ethanol group \lere kept witbhout ethanol for 18 h prior to the experiment but were allowed access to the basal diet and to drinking water.