Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion

The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O2, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.     

Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.

To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.     

Kinetics of the oxidation of reduced Cu,Zn-superoxide dismutase by peroxymonocarbonate

Kinetic evidence is reported for the role of the peroxymonocarbonate, HOOCO(2)(-), as an oxidant for reduced Cu,Zn-superoxide dismutase-Cu(I) (SOD1) during the peroxidase activity of the enzyme. The formation of this reactive oxygen species results from the equilibrium between hydrogen peroxide and bicarbonate. Recently, peroxymonocarbonate has been proposed to be a key substrate for reduced SOD1 and has been shown to oxidize SOD1-Cu(I) to SOD1-Cu(II) much faster than H(2)O(2). We have reinvestigated the kinetics of the reaction between SOD1-Cu(I) and HOOCO(2)(-) by using conventional stopped-flow spectrophotometry and obtained a second-order rate constant of k=1600±100M(-1)s(-1) for SOD1-Cu(I) oxidation by HOOCO(2)(-). Our results demonstrate that peroxymonocarbonate oxidizes SOD1-Cu(I) to SOD1-Cu(II) and is in turn reduced to the carbonate anion radical. It is proposed that the dissociation of His61 from the active site Cu(I) in SOD-Cu(I) contributes to this chemistry by facilitating the binding of larger anions, such as peroxymonocarbonate.     

Peroxynitrite Anion Stimulates Arginine Release from Cultured Rat Astrocytes

The biosynthesis of the physiological messenger nitric oxide (*NO) in neuronal cells is thought to depend on a glial-derived supply of the *NO synthase substrate arginine. To expand our knowledge of the mechanism responsible for this glial-neuronal interaction, we studied the possible roles of peroxynitrite anion (ONOO-), superoxide anion (O2*-), *NO, and H2O2 in L-[3H]arginine release in cultured rat astrocytes. After 5 min of incubation at 37 degrees C, initial concentrations of 0.05-2 mM ONOO- stimulated the release of arginine from astrocytes in a concentration-dependent way; this effect was maximum from 1 mM ONOO- and proved to be approximately 400% as compared with control cells. ONOO(-)-mediated arginine release was prevented by arginine transport inhibitors, such as L-lysine and N(G)-monomethyl-L-arginine, suggesting an involvement of the arginine transporter in the effect of ONOO-. In situ xanthine/xanthine oxidase-generated O2*- (20 nmol/min) stimulated arginine release to a similar extent to that found with 0.1 mM ONOO-, but this effect was not prevented by arginine transport inhibitors. *NO donors, such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, or 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate, and H2O2 did not significantly modify arginine release. As limited arginine availability for neuronal *NO synthase activity may be neurotoxic due to ONOO- formation, our results suggest that ONOO(-)-mediated arginine release from astrocytes may contribute to replenishing neuronal arginine, hence avoiding further generation of ONOO- within these cells.     

The role of hydroxyl radicals in irreversible inactivation of lactoperoxidase by excess H2O2. A spin-trapping/ESR and absorption spectroscopy study

H2O2 is catalytically metabolized by ferric lactoperoxidase (LPO)----compound (cpd) I----cpd II----ferric LPO cycles. An excess of the substrate, however, is degraded by a ferric LPO----cpd I----cpd II----cpd III----ferrous LPO----ferric LPO cycle. This latter pathway leads to the partial or total irreversible inactivation of the enzyme depending on the excess of H2O2 (H. Jenzer, W. Jones, and H. Kohler (1986) J. Biol. Chem. 261, 15550-15556). Spin-trapping/ESR data indicate that in the course of the reaction superoxide (HO2./O2-) and hydroxyl radicals (OH.) are formed. Since many substances known to scavenge radicals, such as a spin trap (e.g., 5,5-dimethyl-1-pyrroline-N-oxide) desferrioxamine, albumin, or mannitol, do not prevent enzyme inactivation, we conclude that OH. generation is a site-specific reaction at or near the active center of LPO where bulky scavenger molecules may not be able to penetrate. We suggest the formation of OH. by a Fenton-like reaction between H2O2 and the intermediate ferrous state of the enzyme, which substitutes for Fe2+ in the Fenton reaction. OH. is a powerful oxidant which in turn may attack rapidly the nearest partner available, either H2O2 to produce HO2. and H2O, or the prosthetic group to give rise to oxidative cleavage of the porphyrin ring structure of the heme moiety of LPO and thus to the liberation of iron.     

Oxidants,antioxidants and carcinogenesis

Reactive oxygen metabolites (ROMs), such as superoxide anions (O2*-) hydrogen peroxide (H2O2), and hydroxyl radical (*OH), malondialdehyde (MDA) and nitric oxide (NO) are directly or indirectly involved in multistage process of carcinogenesis. They are mainly involved in DNA damage leading sometimes to mutations in tumour suppressor genes. They also act as initiator and/or promotor in carcinogenesis. Some of them are mutagenic in mammalian systems. O2*-, H2O2 and *OH are reported to be involved in higher frequencies of sister chromatid exchanges (SCEs) and chromosome breaks and gaps (CBGs). MDA, a bi-product of lipid peroxidation (LPO), is said to be involved in DNA adduct formations, which are believed to be responsible for carcinogenesis. NO, on the other hand, plays a duel role in cancer. At high concentration it kills tumour cells, but at low concentration it promotes tumour growth and metastasis. It causes DNA single and double strand breaks. The metabolites of NO such as peroxynitrite (OONO-) is a potent mutagen that can induce transversion mutations. NO can stimulate O2*-/H2O2/*OH-induced LPO. These deleterious actions of oxidants can be countered by antioxidant defence system in humans. There are first line defense antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). SOD converts O2*- to H2O2, which is further converted to H2O with the help of GPx and CAT. SOD inhibits *OH production. SOD also act as antipoliferative agent, anticarcinogens, and inhibitor at initiation and promotion/transformation stage in carcinogenesis. GPx is another antioxidative enzyme which catalyses to convert H2O2, to H2O. The most potent enzyme is CAT. GPx and CAT are important in the inactivation of many environmental mutagens. CAT is also found to reduce the SCE levels and chromosomal aberrations. Antioxidative vitamins such as vitamin A, E, and C have a number of biological activities such as immune stimulation, inhibition of nitrosamine formation and an alteration of metabolic activations of carcinogens. They can prevent genetic changes by inhibiting DNA damage induced by the ROMs. Therefore, these antioxidants may be helpful in the treatment of human cancer. However, detailed studies are required to draw a definite conclusion.     

Methylglyoxal-induced nitric oxide and peroxynitrite production in vascular smooth muscle cells

Methylglyoxal (MG) is a metabolite of glucose. Our previous study demonstrated an elevated MG level with an increased oxidative stress in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. Whether MG causes the generation of nitric oxide (NO) and superoxide anion (O2*-), leading to peroxynitrite (ONOO-) formation in VSMCs, was investigated in the present study. Cultured rat thoracic aortic SMCs (A-10) were treated with MG or other different agents. Oxidized DCF, reflecting H2O2 and ONOO- production, was significantly increased in a concentration- and time-dependent manner after the treatment of SMCs with MG (3-300 microM) for 45 min-18 h (n = 12). MG-increased oxidized DCF was effectively blocked by reduced glutathione or N-acetyl-l-cysteine, as well as L-NAME (p < 0.05, n = 12). Both O2*- scavenger SOD and NAD(P)H oxidase inhibitor DPI significantly decreased MG-induced oxidized DCF formation. MG significantly and concentration-dependently increased NO and O2*- generation in A-10 cells, which was significantly inhibited by L-NAME and SOD or DPI, respectively. In conclusion, MG induces significant generation of NO and O2*- in rat VSMCs, which in turn causes ONOO- formation. An elevated MG level and the consequential ROS/RNS generation would alter cellular signaling pathways, contributing to the development of different insulin resistance states such as diabetes or hypertension.     

Relationship between protein nitration and oxidation and development of hyperoxic seizures.

Recent studies have implicated nitric oxide (NO*) as a mediator of CNS hyperbaric O2 (HBO2) toxicity. One mechanism by which NO* may contribute to HBO2-induced brain toxicity involves a neurotoxic, pro-oxidative action of NO* via the formation of the potent oxidant peroxynitrite (ONOO-). The present study compares: (a) the formation of protein nitrotyrosine as a marker of ONOO- accumulation and (b) protein oxidation as an indicator of reactive oxygen species production during HBO2 exposure. Rats were exposed to 5 atm 100% O2 to pre-convulsive exposure or until the occurrence of electroencephalographic (EEG) seizures. After exposures, brains were analyzed for protein nitrotyrosine (NT) and protein carbonyl measurement by Western blot and for superoxide dismutase (SOD) activity by NBT assay. The results show a significant increase in protein NT, exceeding control level by several fold. There was only a slow and non-significant increase in the quantity of oxidized proteins during the pre-convulsive phase of HBO2 exposure. Levels of both protein NT and protein carbonyls were significantly (p<0.05) elevated after seizures. Total SOD activity was not changed during preconvulsive exposures, but was significantly (p<0.05) elevated post-seizures. The specific neuronal nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI), significantly reduced the increases in seizure-induced protein NT and protein carbonyl and at the same time very effectively (p<0.05) delayed onset of HBO2 seizures. Pre-seizure increases in protein NT might indicate its role in the mechanism of HBO2-induced brain toxicity. This is supported by the observed capacity of 7-NI to inhibit tyrosine nitration and increase time to seizure.     

Mechanism of hydrogen peroxide-induced Cu,Zn-superoxide dismutase-centered radical formation as explored by immuno-spin trapping: the role of copper- and carbonate radical anion-mediated oxidations

We have reinvestigated the biochemistry of H2O2-induced Cu,Zn-superoxide dismutase (SOD1)-centered radicals, detecting them by immuno-spin trapping. These radicals are involved in H2O2-induced structural and functional damage to SOD1, and their mechanism of generation depends on copper and/or (bi)carbonate (i.e., CO2, CO3(-2), or HCO3-). First, in the absence of DTPA and (bi)carbonate, Cu(II) was partially released and rebound at His, Cys, and Tyr residues in SOD1 with the generation of protein-copper-bound oxidants outside the SOD1 active site by reaction with excess H2O2. These species produced immuno-spin trapping-detectable SOD1-centered radicals associated with H2O2-induced active site ( approximately 5 and approximately 10 kDa fragments) and non-active site (smearing between 3 and 16 kDa) copper-dependent backbone oxidations and subsequent fragmentation of SOD1. Second, in the presence of DTPA, which inhibits H2O2-induced SOD1 non-active site fragmentation, (bi)carbonate scavenged the enzyme-bound oxidant at the SOD1 active site to produce the carbonate radical anion, CO3*-, thus protecting against active site SOD1 fragmentation. CO3*- diffuses and produces side chain oxidations forming DMPO-trappable radical sites outside the enzyme active site. Both mechanisms for generating immuno-spin trapping-detectable SOD1-centered radicals were susceptible to inhibition by cyanide and enhanced at high pH values. In addition, (bi)carbonate enhanced H2O2-induced SOD1 turnover as demonstrated by an enhancement in oxygen evolution and SOD1 inactivation. These results help clarify the free radical chemistry involved in the functional and structural oxidative damage to SOD1 by H2O2 with the intermediacy of copper- and CO3*--mediated oxidations.     

Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation

Red blood cells (RBC) are thought to be well protected against oxidative stress by the antioxidant, cu-pro-zinc enzyme superoxide dismutase (CuZn SOD) which dismutates O2- to H2O2. CuZn SOD, however, is irreversibly inactivated by its product H2O2. Exposure of intact RBC to H2O2 resulted in the inactivation (up to 50%) of endogenous SOD in a concentration-dependent manner. When RBC were exposed to O2- and H2O2, generated by xanthine + xanthine oxidase, an even greater loss of SOD activity (approximately 75%) was observed. Intracellular proteolysis was markedly increased by exposure to these same oxidants; up to a 12-fold increase with H2O2 and a 50-fold increase with xanthine oxidase plus xanthine. When purified SOD was treated with H2O2, inactivation of the enzyme also occurred in a concentration-dependent manner. Accompanying the loss of SOD activity, the binding of the copper ligand to the active site of the enzyme diminished with H2O2 exposure, as evidenced by an increase in accessible copper. Significant direct fragmentation of SOD was evident only under conditions of prolonged exposure (20 h) to relatively high concentrations of H2O2. Gel electrophoresis studies indicated that under most experimental conditions (i.e. 1-h incubation) H2O2, O2-, and H2O2 + O2- treated SOD experienced charge changes and partial denaturation, rather than fragmentation. The proteolytic susceptibility of H2O2-modified SOD, during subsequent incubation with (rabbit, bovine or human) red cell extracts also increased as a function of pretreatment with H2O2. Both enzyme inactivation and altered copper binding appeared to precede the increase in proteolytic susceptibility (whether measured as an effect of H2O2 concentration or as a function of the duration of H2O2 exposure). These results suggest that SOD inactivation and modification of copper binding are prerequisites for increased protein degradation. Proteolytic susceptibility was further enhanced by H2O2 exposure under alkaline conditions, suggesting that the hydroperoxide anion is the damaging species rather than H2O2 itself. In RBC extracts, the proteolysis of H2O2-modified SOD was inhibited by sulfhydryl reagents, serine reagents, transition metal chelators, and ATP; suggesting the existence of an ATP-independent proteolytic pathway of sulfhydryl, serine, and metalloproteases, and peptidases. The proteolytic activity was conserved in a "Fraction II" of both human and rabbit RBC, and was purified from rabbit reticulocytes and erythrocytes to a 670-kDa proteinase complex, for which we have suggested the trivial name macroxyproteinase. In erythrocytes macroxyproteinase may prevent the accumulation of H2O2-modified SOD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory effects of water-soluble cationic manganese porphyrins on peroxynitrite-induced SOS response in Salmonella typhimurium TA4107/pSK1002

We have investigated the protective effects of water-soluble cationic Mn(III) porphyrins against peroxynitrite (ONOO-)-induced DNA damage in the cells of Salmonella typhimurium TA4107/pSK1002 and lipid peroxidation of red blood cell membranes. Mn(III) tetrakis (N-methylpyridinium-4-yl) porphine (TMPyP) and the brominated form, Mn(III) octabromo-tetrakis (N-methylpyridinium-4-yl) porphine (OBTMPyP) effectively reduced the damage and peroxidation induced by N-morpholino sydnonimine (SIN-1), which gradually generates ONOO- from O2*- and *NO produced through hydrolysis. Mn(III)OBTMPyP became 10-fold more active than the non-brominated form. In the presence of authentic ONOO-, the Mn(III) porphyrins were ineffective against damage and strongly enhanced lipid peroxidation, while the coexistence of ascorbic acid inhibited peroxidation. Using a diode array spectrophotometry, the reactions of Mn(III)TMPyP with authentic ONOO- and SIN-1 were measured. Mn(III)TMPyP is known to be catalytic for ONOO- decomposition in the presence of antioxidants. OxoMn(IV)TMPyP with SIN-1 was rapidly reduced back to Mn(III) without adding any oxidants. Further, in the SIN-1 system, the concentration of NO2- and NO3- were colorimetrically determined by Griess reaction based on the two-step diazotization. NO2- increased by addition of Mn(III) porphyrin and the ratio of NO2- to NO3- was 4-7 times higher than that (1.05) of SIN-1 alone. This result suggests that O2*- from SIN-1 acts as a reductant and *NO cogenerated is oxidized to NO2-, a primarily decomposition product of *NO. Under the pathological conditions where biological antioxidants are depleted and ONOO- and O2*- are extensively generated, the Mn(III) porphyrins will effectively cycle ONOO- decomposition using O2*-.     

Bicarbonate enhances the peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical.

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro-gamma-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H(2)O(2)-mediated nitration of gamma-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H(2)O(2)-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H(2)O(2)-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H(2)O(2), suggest that SOD1/H(2)O(2) oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H(2)O(2) by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226-1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H(2)O(2). However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.     

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (50muM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (50muM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.     

Degradation of matrix glycosaminoglycans by peroxynitrite/peroxynitrous acid: evidence for a hydroxyl-radical-like mechanism

The oxidant peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) is generated at sites of inflammation via reaction of O2.- with .NO. Previous studies have shown that these species can oxidize cellular targets, but few data are available on damage to extracellular matrix and its components, despite evidence for matrix modification in a number of pathologies. In the current study we show that reaction of ONOO-/ONOOH with glycosaminoglycans results in extensive polymer fragmentation. Bolus authentic ONOO-/ONOOH modifies hyaluronan, heparin, and chondroitin, dermatan, and heparan sulfates, in a concentration-dependent, but O2-independent, manner. The ONOO-/ONOOH generator 3-(4-morpholinyl)sydnoneimine produces similar time- and concentration-dependent damage. These reactions generate specific polymer fragments via cleavage at disaccharide intervals. Studies at different pH values, and in the presence of bicarbonate, are consistent with ONOOH, rather than the carbonate adduct, CO3.- or ONOO-, being the source of damage. EPR spin trapping experiments have provided evidence for the formation of carbon-centered radicals on glycosaminoglycans and related monosaccharides; the similarity of these spectra to those obtained with authentic HO. is consistent with fragmentation being induced by this oxidant. These data suggest that extracellular matrix fragmentation at sites of inflammation may be due, in part, to the formation and reactions of ONOOH.     

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.     

Involvement of nitric oxide in killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata

In strains of the snail Biomphalaria glabrata (Gastropoda) that are resistant to the parasite Schistosoma mansoni (Trematoda), hemocytes in the hemolymph are responsible for elimination of S. mansoni sporocysts. The defensive role of reactive nitrogen species was investigated in in vitro interactions between hemocytes derived from the resistant 13-16-R1 strain of B. glabrata and the parasite. The nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) and the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide reduced cell-mediated killing of S. mansoni sporocysts. To determine if peroxynitrite (ONOO-) is involved in killing, assays were run in the presence of the ONOO- scavengers uric acid and deferoxamine. These did not influence the rate of parasite killing, indicating that NO is directly responsible for mediating cytotoxicity, but ONOO- is not. The combination of the NOS inhibitor L-NAME and catalase, an enzyme that detoxifies hydrogen peroxide (H2O2), reduced average sporocyst mortality to a greater extent than L-NAME alone. Killing of the sporocysts was, however, not totally inhibited. It is suggested that NO and H2O2 are both involved in hemocyte-mediated toxicity of 13-16-R1 B. glabrata against S. mansoni sporocysts.     

Increased activity of H2O2 in aorta isolated from chronically streptozotocin-diabetic rats: effects of antioxidant enzymes and enzymes inhibitors.

The effects of hydrogen peroxide (H2O2, 1 nM-5 mM) on the tone of the rings of aorta precontracted with phenylephrine (PE) were studied in 4-5 months streptozotocin (STZ)-diabetic rats and their age-matched controls. H2O2 induced brief contraction before relaxation in endothelium-containing rings that was more pronounced in diabetic rats. Removal of the endothelium or pretreatment of rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished H2O2-induced immediate and transient increase in tone, but preincubation with indomethacin (10 microM) had no effect on contractions induced by H2O2 in both group of animals. Pretreatment with L-NAME or indomethacin as well as absence of endothelium produced an inhibition of H2O2-induced relaxation that was more pronounced in diabetic rings. Chronically STZ-diabetes resulted in a significant increase in H2O2-induced maximum relaxation that was largely endothelium-dependent. Decreased sensitivity (pD2) of diabetic vessels to vasorelaxant action of H2O2 was normalized by superoxide dismutase (SOD, 80 U/ml). Pretreatment with SOD had no effect on H2O2-induced maximum relaxations in both group of animals but led to an increase in H2O2-induced contractions in control rats. When the rings pretreated with diethyldithiocarbamate (DETCA, 5 mM), H2O2 produced only contraction in control rats, and H2O2-induced relaxations were markedly depressed in diabetic rats. H2O2 did not affect the tone of intact or endothelium-denuded rings in the presence of catalase (2000 U/ml). Aminotriazole (AT, 10 mM) failed to affect H2O2-induced contractions or relaxations in all rings. Our observations suggest that increased production of oxygen-derived free radicals (OFRs) in diabetic state leads to a decrease in SOD activity resulting an increase in endogenous superoxide anions (O2*-), that is limited cytotoxic actions, and an increase in catalase activity resulting a decrease in both H2O2 concentrations and the production of harmful hydroxyl radical (*OH) in diabetic aorta in long-term. Present results indicate that increased vascular activity of H2O2 may be an important factor in the development of vascular disorders associated with chronically diabetes mellitus. Enhanced formation of *OH, that is a product of exogenous H2O2 and excess O2*, seems to be contribute to increased relaxations to exogenously added H2O2 in chronically diabetic vessels.     

Pindolol is a potent scavenger of reactive nitrogen species

Pindolol is an indolic drug that has been shown to enhance and/or accelerate selective serotonin specific reuptake inhibitors (SSRI)-induced antidepressant (AD) effect, even though the respective mechanism is still unclear. It has been demonstrated that inhibition of nitric oxide (*NO) synthesis in CNS produces anxiolytic and AD-like behavioural effects in a variety of animal paradigms. On the other hand, sustained high levels of *NO may be deleterious to CNS, predominantly due to the formation of peroxynitrite anion (ONOO-), which is generated via reaction of *NO with superoxide radical (O2*-). Therefore, the purpose of the present study was to characterize the putative pindolol scavenging effect on *NO, ONOO-, and O2*-, using in vitro non-cellular systems. The obtained results clearly show that pindolol is a potent scavenger of *NO (IC50 of 449+/-33 microM) and ONOO- (IC50 of 131+/-24 microM). Additionally, the scavenging effect of pindolol increased almost 8 times in the presence of 25 mM NaHCO3 (IC50 of 17+/-3 microM), which indicates that pindolol efficiently scavenges reactive species that are produced from the ONOO-/CO2 reaction such as the nitrogen dioxide radical (*NO2) and the carbonate radical anion (CO3*-). These effects may contribute for the reduction of SSRI antidepressant latency that has been attributed to pindolol and may also constitute an additional value for this drug when depression is associated with pro-oxidant neurodegenerative diseases.     

Inactivation of mitochondrial adenosine triphosphatase from Trypanosoma cruzi by oxygen radicals

Incubation of Trypanosoma cruzi mitochondrial ATPase (Fo-F1) with the xanthine oxidase system (XO), Fenton's reagent (Fe2+ + H2O2) and the ascorbate-Cu system, caused gradual loss of enzyme activity, which increased as a function of incubation time and rate of oxygen radical generation. The essential role of OH. radicals for ATPase inactivation was supported by a) the enzyme protection afforded by superoxide dismutase, catalase and mannitol, when using the XO system; b) the similar effect of mannitol and benzoate with Fenton's reagent; c) the similar effect of catalase, EDTA and histidine with the ascorbate-Cu system; d) the increased rate of ATPase inactivation by 1) the XO system supplemented with chelated iron, and 2) the ascorbate-Cu system supplemented with H2O2. Comparison of oxygen radical generators for their action on membrane-bound (Fo-F1) and soluble F1 revealed that ascorbate-Cu was the most effective one, possibly because of its capability of producing OH. radicals that react preferentially with the enzyme at their formation site.