Reactivity of Hg(II) with superoxide: Evidence for the catalytic dismutation of superoxide by HG(II)

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O) and hydrogen peroxide (H₂O₂) production by rat kidney mitochondria, Hg(II) (1–6 μM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H₂O₂ production but an apparent decrease in mitochondrial O production. Hg(II) also inhibited O-dependent cytochrome c reduction (IC₅₀ ≈?2–3 μM) when O was produced from xanthine oxidase. In contrast, Hg(I) did not react with O in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O by Hg(II). Hg(II) also inhibited the reactions of KO₂ (i.e., O) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO₂ in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO₂. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O dismutation, leading to increased production of H₂O₂ by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2- generation in mitochondria respiring on the complex I substrates pyruvate+malate, an effect fully inhibited by rotenone. Antimycin A increased O2- production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2- production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2- formation driven with the complex II substrate succinate. At 0.6 microM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2- formation; however, at 40 microM myxothiazol (which completely inhibits both complexes I and III) eliminated O2- production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2- from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.     

Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: role of mitochondrial superoxide

The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H(2)O(2) and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H(2)O(2)- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the "untargeted" carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.     

Hydrogen peroxide promotes endothelial dysfunction by stimulating multiple sources of superoxide anion radical production and decreasing nitric oxide bioavailability.

Hydrogen peroxide (H(2)O(2)) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H(2)O(2). Exposure of aortic rings to 25 or 50 microM, but not 10 microM, H(2)O(2) for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide ((.)NO) donor sodium nitroprusside. Treatment of PAEC with 50 microM H(2)O(2) significantly decreased ACh-induced accumulation of (.)NO, as measured with a (.)NO-selective electrode, yet such treatment increased nitric oxide synthase activity approximately 3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased (.)NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O(2)(-.)), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O(2)(-.) production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H(2)O(2) decreases the bioavailability of agonist-induced (.)NO as a result of increased production of O(2)(-.) likely derived from xanthine oxidase, NADPH-oxidase and mitochondria. These processes could contribute to H(2)O(2)-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation.     

The O2-dependence of respiration and H2O2 production in the parasitic nematode Ascaridia galli.

1. Respiration in the parasitic nematode worm Ascaridia galli was inhibited at O2 concentrations in excess of 255 microM, and an apparent Km,O2 of 174 microM was determined. 2. Mitochondria-enriched fractions isolated from the tissues of A. galli have much lower apparent Km,O2 values (approx. 5 microM). They produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 3. Antimycin A inhibited respiration in muscle tissue mitochondria by 10%, but had no effect on respiration in gut + reproductive tissue mitochondria; the major portion of respiration in both types of mitochondria could be attributed to an alternative electron-transport pathway. 4. o-Hydroxydiphenyl, an inhibitor of alternative electron-transport pathways, inhibits respiration by 98% and completely inhibits the production of H2O2 in gut-plus-reproductive-tissue mitochondria; respiration and H2O2 production in muscle tissue mitochondria were inhibited by 90 and 86% respectively. 5. Another inhibitor of alternative electron transport, salicylhydroxamic acid, had the same effect as o-hydroxydiphenyl on H2O2 production and respiration in gut-plus-reproductive-tissue mitochondria. However, its effect on muscle tissue mitochondria was complex; a low concentration (0.35 mM) stimulated H2O2 production, whereas 3 mM inhibited respiration by 87% and prevented H2O2 production completely. 6. The similarities between the apparent Km,O2 values for H2O2 production and respiration in muscle mitochondria and in gut-plus-reproductive-tissue mitochondria suggests that the site of H2O2 production on the alternative electron-transport chain is cytochrome 'o'. 7. These results are discussed in relation to potential O2 toxicity in A. galli.     

Manganese superoxide dismutase, MnSOD and its mimics

Increased understanding of the role of mitochondria under physiological and pathological conditions parallels increased exploration of synthetic and natural compounds able to mimic MnSOD - endogenous mitochondrial antioxidant defense essential for the existence of virtually all aerobic organisms from bacteria to humans. This review describes most successful mitochondrially-targeted redox-active compounds, Mn porphyrins and MitoQ(10) in detail, and briefly addresses several other compounds that are either catalysts of O(2)(-) dismutation, or its non-catalytic scavengers, and that reportedly attenuate mitochondrial dysfunction. While not a true catalyst (SOD mimic) of O(2)(-) dismutation, MitoQ(10) oxidizes O(2)(-) to O(2) with a high rate constant. In vivo it is readily reduced to quinol, MitoQH(2), which in turn reduces ONOO(-) to NO(2), producing semiquinone radical that subsequently dismutes to MitoQ(10) and MitoQH(2), completing the "catalytic" cycle. In MitoQ(10), the redox-active unit was coupled via 10-carbon atom alkyl chain to monocationic triphenylphosphonium ion in order to reach the mitochondria. Mn porphyrin-based SOD mimics, however, were designed so that their multiple cationic charge and alkyl chains determine both their remarkable SOD potency and carry them into the mitochondria. Several animal efficacy studies such as skin carcinogenesis and UVB-mediated mtDNA damage, and subcellular distribution studies of Saccharomyces cerevisiae and mouse heart provided unambiguous evidence that Mn porphyrins mimic the site and action of MnSOD, which in turn contributes to their efficacy in numerous in vitro and in vivo models of oxidative stress. Within a class of Mn porphyrins, lipophilic analogs are particularly effective for treating central nervous system injuries where mitochondria play key role. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Mitochondrial Complex I superoxide production is attenuated by uncoupling

Complex I, i.e. proton-pumping NADH:quinone oxidoreductase, is an essential component of the mitochondrial respiratory chain but produces superoxide as a side-reaction. However, conditions for maximum superoxide production or its attenuation are not well understood. Unlike for Complex III, it has not been clear whether a Complex I-derived superoxide generation at forward electron transport is sensitive to membrane potential or protonmotive force. In order to investigate this, we used Amplex Red for H(2)O(2) monitoring, assessing the total mitochondrial superoxide production in isolated rat liver mitochondria respiring at state 4 as well as at state 3, namely with exclusive Complex I substrates or with Complex I substrates plus succinate. We have shown for the first time, that uncoupling diminishes rotenone-induced H(2)O(2) production also in state 3, while similar attenuation was observed in state 4. Moreover, we have found that 5-(N-ethyl-N-isopropyl) amiloride is a real inhibitor of Complex I H(+) pumping (IC(50) of 27 microM) without affecting respiration. It also partially prevented suppression by FCCP of rotenone-induced H(2)O(2) production with Complex I substrates alone (glutamate and malate), but nearly completely with Complexes I and II substrates. Sole 5-(N-ethyl-N-isopropyl) amiloride alone suppressed 20% and 30% of total H(2)O(2) production, respectively, under these conditions. Our data suggest that Complex I mitochondrial superoxide production can be attenuated by uncoupling, which means by acceleration of Complex I H(+) pumping due to the respiratory control. However, when this acceleration is prevented by 5-(N-ethyl-N-isopropyl) amiloride inhibition, no attenuation of superoxide production takes place.     

The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation

The involvement of reactive oxygen species (ROS) in cardiac ischemia-reperfusion injuries is well-established, but the deleterious effects of hydrogen peroxide (H(2)O(2)), hydroxyl radical (HO*) or superoxide anion (O(2)*(-) ) on mitochondrial function are poorly understood. Here, we report that incubation of rat heart mitochondria with each of these three species resulted in a decline of the ADP-stimulated respiratory rate but not substrate-dependent respiration. These three species reduced oxygen consumption induced by an uncoupler without alteration of the respiratory chain complexes, but did not modify mitochondrial membrane permeability. HO* slightly decreased F1F0-ATPase activity and HO* and O(2)*(-) partially inhibited the activity of adenine nucleotide translocase; H(2)O(2) failed to alter these targets. They inhibited NADH production by acting specifically on aconitase for O(2)*(-) and alpha-ketoglutarate dehydrogenase for H(2)O(2) and HO*. Our results show that O(2)*(-), H(2)O(2) and HO* act on different mitochondrial targets to alter ATP synthesis, mostly through inhibition of NADH production.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O(2)*-) and hydrogen peroxide (H(2)O(2)). The electron transfer chain of mitochondria is a well documented source of H(2)O(2); however, the release of O(2)*- from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O(2)*-, elucidating the mechanisms underlying the release of O(2)*- from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O(2)*-. Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochrome c proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O(2)*- release from mitochondria were estimated at approximately 0.04 nmol/min/mg protein, a value increased approximately 8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-based p-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O(2)*- production from mitochondria by approximately 55%, thus suggesting that a large portion of O(2)*- exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O(2)*- in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.     

H(2)O(2) detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system.

Evaluation of the existence of superoxide radicals (O*-(2)), the site of generation and conditions required for one-e(-) transfer to oxygen from biological redox systems is a prerequisite for the understanding of the deregulation of O(2) homeostasis leading to oxidative stress. Mitochondria are increasingly considered the major O*-(2) source in a great variety of diseases and the aging process. Contradictory reports on mitochondrial O*-(2) release prompted us to critically investigate frequently used O*-(2) detection methods for their suitability. Due to the impermeability of the external mitochondrial membrane for most constituents of O*-(2) detection systems we decided to follow the stable dismutation product H(2)O(2). This metabolite was earlier shown to readily permeate into the cytosol. With the exception of tetramethylbenzidine none of the chemical reactants indicating the presence of H(2)O(2) by horseradish peroxidase-catalyzed absorbance change were suited due to solubility problems or low extinction coefficients. Tetramethylbenzidine-dependent H(2)O(2) detection was counteracted by rereduction of the dye through e(-) carriers of the respiratory chain. Although the fluorescent dyes scopoletin and homovanillic acid were found to be suited for the detection of mitochondrial H(2)O(2) release, fluorescence change was strongly affected by mitochondrial protein constituents. The present study has resolved this problem by separating the detection system from H(2)O(2)-producing mitochondria.     

Reactive oxygen species generation is modulated by mitochondrial kinases: correlation with mitochondrial antioxidant peroxidases in rat tissues

Mitochondrial hexokinase (mt-HK) and creatine kinase (mt-CK) activities have been recently proposed to reduce the rate of mitochondrial ROS generation through an ADP re-cycling mechanism. Here, we determined the role of mt-HK and mt-CK activities in regulate mitochondrial ROS generation in rat brain, kidney, heart and liver, relating them to the levels of classical antioxidant enzymes. The activities of both kinases were significantly higher in the brain than in other tissues, whereas the activities of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were higher in both liver and kidney mitochondria. In contrast, manganese superoxide dismutase (Mn-SOD) activity was not significantly different among these tissues. Activation of mitochondrial kinases by addition of their substrates increased the ADP re-cycling and thus the respiration by enhancing the oxidative phosphorylation. Succinate induced hydrogen peroxide (H(2)O(2)) generation was higher in brain than in kidney and heart mitochondria, and the lowest in liver mitochondria. Mitochondrial membrane potential (DeltaPsi(m)) and H(2)O(2) production, decreased with additions of 2-DOG or Cr to respiring brain and kidney mitochondria but not to liver. The inhibition of H(2)O(2) production by 2-DOG and Cr correspond to almost 100% in rat brain and about 70% in kidney mitochondria. Together our data suggest that mitochondrial kinases activities are potent preventive antioxidant mechanism in mitochondria with low peroxidase activities, complementing the classical antioxidant enzymes against oxidative stress.     

Increase of sodium delivery stimulates the mitochondrial respiratory chain H2O2 production in rat renal medullary thick ascending limb

The mitochondria-rich epithelial cells of the renal medullary thick ascending limb (mTAL) reabsorb nearly 25% of filtered sodium (Na(+)) and are a major source of cellular reactive oxygen species. Although we have shown that delivery of Na(+) to the mTAL of rats increases superoxide (O(2)(·-)) production in mTAL, little is known about H(2)O(2) production, given the lack of robust and selective fluorescent indicators for determining changes within the whole cell, specifically in the mitochondria. The present study determined the effect of increased tubular flow and Na(+) delivery to mTAL on the production of mitochondrial H(2)O(2) in mTAL. H(2)O(2) responses were determined in isolated, perfused mTAL of Sprague-Dawley rats using a novel mitochondrial selective fluorescent H(2)O(2) indicator, mitochondria peroxy yellow 1, and a novel, highly sensitive and stable cytosolic-localized H(2)O(2) indicator, peroxyfluor-6 acetoxymethyl ester. The results showed that mitochondrial H(2)O(2) and cellular fluorescent signals increased progressively over a period of 30 min following increased tubular perfusion (5-20 nl/min), reaching levels of statistical significance at ～10-12 min. Responses were inhibited with rotenone or antimycin A (inhibitors of the electron-transport chain), polyethylene glycol-catalase and by reducing Na(+) transport with furosemide or ouabain. Inhibition of membrane NADPH-oxidase with apocynin had no effect on mitochondrial H(2)O(2) production. Cytoplasmic H(2)O(2) (peroxyfluor-6 acetoxymethyl ester) increased in parallel with mitochondrial H(2)O(2) (mitochondria peroxy yellow 1) and was partially attenuated (～65%) by rotenone and completely inhibited by apocynin. The present data provide clear evidence that H(2)O(2) is produced in the mitochondria in response to increased flow and delivery of Na(+) to the mTAL, and that whole cell H(2)O(2) levels are triggered by the mitochondrial reactive oxygen species production. The mitochondrial production of H(2)O(2) may represent an important target for development of more effective antioxidant therapies.     

Characterization by Hg2+ of two different pathways for mitochondrial Ca2+ release

The addition of Hg2+ to loaded kidney mitochondria induces the fast release of the accumulated cation. The Ca2+-efflux reaction exhibits kinetics characteristics that depend on the extent of the binding of Hg2+ to the membrane. At high levels of Hg2+ bound (approx. 11 nmol/mg), Ca2+ efflux rate is highly insensitive to the temperature of incubation, and the efflux seems to be directly related to the internal free Ca2+ concentration. At these levels of bound Hg2+, accumulated Sr2+ is released with characteristics similar to those observed with Ca2+. At lower levels of Hg2+ binding (2.5 nmol/mg), the efflux reaction is highly dependent on the incubation temperature and on the internal free Ca2+ concentration; under these conditions Sr2+ is not released. NAD(P)H oxidation as induced by the low Hg2+ concentration is inhibited at the lower temperatures. Radiolabeled Hg2+ incorporates into two clearly defined regions of membrane proteins separated through sodium dodecyl sulfate gel electrophoresis. One of the regions corresponds to proteins of apparent high molecular mass (i.e., 150 kDa), and the other to proteins with apparent molecular masses of 37-25 kDa. Mitochondria incubated with 2 microM 203Hg2+ incorporate the radionuclide in proteins that have molecular masses of around 41 and 26 kDa. The results indicate that, depending on the amount of Hg2+ bound to the inner membrane, two clearly distinct Ca2+ release mechanisms can be distinguished.     

Protective behavior of tamoxifen against Hg2+-induced toxicity on kidney mitochondria: in vitro and in vivo experiments

Heavy metals are known to induce functional alterations in kidney mitochondria, this damage plays a central role in the mercury-induced acute renal failure. In fact, mercury causes rapid and dramatic changes in the membrane's ionic permeability in such a way that a supra load of mitochondrial Ca(2+) occurs. As a consequence, the phenomenon of permeability transition takes place. In this work we studied in vitro and in vivo the protective effect of the selective estrogen receptor modulator tamoxifen on the deleterious action of mercury-induced nonselective permeability in kidney mitochondria. Added in vitro tamoxifen inhibited membrane nonspecific pore opening, brought about by Hg(2+), as well as the oxidative damage of the enzyme cis-aconitase. In vivo the administration of tamoxifen prevented Hg(2+)-induced poisoning on mitochondrial energy-dependent functions. Permeability transition was analyzed by measuring matrix Ca(2+) retention, mitochondrial swelling, and the build up and maintenance of a transmembrane electric gradient. The pharmacologic action of tamoxifen on mercury poisoning could be ascribed to its cyclosporin-like action.     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.     

Xanthine oxidase and mitochondria contribute to vascular superoxide anion generation in DOCA-salt hypertensive rats

Vascular superoxide anion (O(2)(*-)) levels are increased in DOCA-salt hypertensive rats. We hypothesized that the endothelin (ET)-1-induced generation of ROS in the aorta and resistance arteries of DOCA-salt rats originates partly from xanthine oxidase (XO) and mitochondria. Accordingly, we blocked XO and the mitochondrial oxidative phosphorylation chain to investigate their contribution to ROS production in mesenteric resistance arteries and the aorta from DOCA-salt rats. Systolic blood pressure rose in DOCA-salt rats and was reduced after 3 wk by apocynin [NAD(P)H oxidase inhibitor and/or radical scavenger], allopurinol (XO inhibitor), bosentan (ET(A/B) receptor antagonist), BMS-182874 (BMS; ET(A) receptor antagonist), and hydralazine. Plasma uric acid levels in DOCA-salt rats were similar to control unilaterally nephrectomized (UniNx) rats, reduced with allopurinol and bosentan, and increased with BMS. Levels of thiobarbituric acid-reacting substances were increased in DOCA-salt rats versus UniNx rats, and BMS, bosentan, and hydralazine prevented their increase. Dihydroethidium staining showed reduced O(2)(*-) production in mesenteric arteries and the aorta from BMS- and bosentan-treated DOCA-salt rats compared with untreated DOCA-salt rats. Increased O(2)(*-) derived from XO was reduced or prevented by all treatments in mesenteric arteries, whereas bosentan and BMS had no effect on aortas from DOCA-salt rats. O(2)(*-) generation decreased with in situ treatment by tenoyltrifluoroacetone and CCCP, inhibitors of mitochondrial electron transport complexes II and IV, respectively, whereas rotenone (mitochondrial complex I inhibitor) had no effect. Our findings demonstrate the involvement of ET(A) receptor-modulated O(2)(*-) derived from XO and from mitochondrial oxidative enzymes in arteries from DOCA-salt rats.     

Tumour necrosis factor induced an early release of superoxide and a late mitochondrial membrane depolarization in L929 cells. Increase in the production of superoxide is not sufficient to mimic the action of TNF

Tumour necrosis factor alpha (TNF) cytotoxicity is mediated, at least in part, by oxidative stress. One of the post-receptor events shortly after the addition of TNF is the generation of the superoxide anion (O2-*). In the present study, we attempted to examine the role of O2-* in the regulation of mitochondrial membrane potential (Delta(Psi)m) and the release of cytochrome c (cyto c) in L929 cells after stimulation with TNF. Challenge of cells with TNF (50 ng/ml) resulted in an early (30 min after the addition of TNF) increase in the production of O2-*. The use of mitochondrial electron transport chain inhibitors such as antimycin A and rotenone could, respectively, potentiate or suppress the TNF-mediated release of O2-* and cytotoxicity. TNF also induced a late (>3 h after the addition of TNF) depolarization in the Delta(Psi)m. Reduction in the release of O2-* by rotenone (50 microM) or thenoyltrifluoroacetone (250 microM) suppressed both the TNF-mediated Delta(Psi)m depolarization and cyto c release. However, increase in the production of O2-* by antimycin A (25 microM) only slightly enhanced the TNF effect in altering the Delta(Psi)m and the release of cyto c. Treating cells with antimycin A alone could not induce a reduction in Delta(Psi)m nor a release of cyto c. Taken together, our results indicate that TNF induced damage in mitochondria in L929 cells. Our data also show that an increase in the production of O2-* was important in the TNF cytotoxicity, but was not sufficient to mimic the action of TNF.     

Formation of the Neurotransmitter Glycine from the Anticonvulsant Milacemide Is Mediated by Brain Monoamine Oxidase B

Milacemide (2-n-pentylaminoacetamide) is a secondary monoamine that in the brain is converted to glycinamide and glycine. This oxidative reaction was suspected to involve the reaction of monoamine oxidase (MAO). Using mitochondrial preparations from tissues that contain MAO-A and -B (rat brain and liver), MAO-A (human placenta), and MAO-B (human platelet and bovine adrenal chromaffin cell), it has been established that mitochondria containing MAO-B rather than MAO-A oxidize (H2O2 production and glycinamide formation) milacemide. The apparent Km (30-90 microM) for milacemide oxidation by mitochondrial MAO-B preparations is significantly lower than that for milacemide oxidation by mitochondrial MAO-A (approximately 1,300 microM). In vitro MAO-B (l-deprenyl and AGN 1135) rather than MAO-A (clorgyline) selectively inhibited the oxidation of milacemide. These in vitro data are matched by ex vivo experiments where milacemide oxidation was compared to oxidation of serotonin (MAO-A) and beta-phenylethylamine (MAO-B) by brain mitochondria prepared from rats pretreated with clorgyline (0.5-10 mg/kg) and l-deprenyl (0.5-10 mg/kg). Furthermore, in vivo experiment demonstrated that l-deprenyl selectively increased the urinary excretion of [14C]milacemide and the total radioactivity with a concomitant decrease of [14C]glycinamide. Such changes were not observed after clorgyline treatment, but were evident only at doses beyond clorgyline selectivity. The present data therefore demonstrate that milacemide is a substrate for brain MAO-B, and its conversion to glycinamide, further transformed to the inhibitory neurotransmitter, glycine, mediated by this enzyme may contribute to its pharmacological activities.     

Supplementation of endothelial cells with mitochondria-targeted antioxidants inhibit peroxide-induced mitochondrial iron uptake, oxidative damage, and apoptosis

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H(2)O(2) and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 microM) and MitoVit-E (1 microM) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H(2)O(2)- and lipid peroxide-induced 2',7'-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis.