ATP inhibits hydroxyl radical formation and the inflammatory response of stimulated whole blood even under circumstances of severe oxidative stress

We recently reported that Adenosine-5′-triphosphate (ATP) is able to inhibit the inflammatory reaction in stimulated whole blood. Many diseases, in which inflammatory reactions are involved, are associated with oxidative stress. In the present study, we therefore, investigated the effect of ATP on cytokine release in stimulated whole blood under conditions of oxidative stress, as simulated by pre-incubation of blood with hydrogen peroxide (H₂O₂). In the presence of H₂O₂, ATP at concentrations of 100 and 300 μM inhibited Tumour Necrosis factor-alpha (TNF-α) release and stimulated IL-10 release in LPS-PHA stimulated whole blood. Moreover, electron spin resonance (ESR) measurements showed that ATP and its breakdown product Adenosine-5′-diphosphate (ADP) attenuated spin trap-hydroxyl radical adduct formation in the Fenton reaction. Our results demonstrate that even in circumstances of severe oxidative stress, ATP has marked anti-inflammatory properties in stimulated whole blood. Moreover, the inhibition of the hydroxyl radical ESR signal indicates a direct attenuation of oxidative stress by ATP.     

A study on the reaction of human erythrocytes with hydrogen peroxide

Membrane fluidity of human erythrocytes treated with H2O2 (1--20 mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2- or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation. The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (tau c) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes. The ESR spectrum of H2O2-treated erythrocytes at -196 degrees C is characterized by signals of nonheme ferric iron type (g equal to 4.3), low spin ferric iron, and free radical type at g equal to 2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g equal to 2.00 or 4.3. These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.     

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.     

Vanadyl-induced Fenton-like reaction in RNA. An ESR and spin trapping study.

Vanadyl (VO2+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals (.OH). The hydroxyl radicals can be spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2-methyl-2-nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (aN = 1.48 mT, a beta N = 0.25 mT and a beta H = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a beta-nitrogen and beta-hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

Hydrogen peroxide inhibits alveolar macrophage 5-lipoxygenase metabolism in association with depletion of ATP

We have previously shown that the biologically important reactive oxygen metabolite hydrogen peroxide (H2O2) stimulates arachidonic acid (AA) release and thromboxane A2 synthesis in the rat alveolar macrophage. We have now investigated the effects of H2O2 on alveolar macrophage 5-lipoxygenase metabolism. H2O2 failed to stimulate detectable synthesis of leukotriene B4, leukotriene C4, or 5-hydroxyeicosatetraenoic acid (5-HETE) as determined by reverse-phase high performance liquid chromatography (RP-HPLC) and sensitive radioimmunoassays (RIAs). This was not explained by oxidative degradation of leukotrienes by H2O2 at the concentrations used. Moreover, RIA and RP-HPLC analyses demonstrated that H2O2 dose-dependently inhibited synthesis of leukotriene B4, leukotriene C4, and 5-HETE induced by the agonists A23187 (10 microM) and zymosan (100 micrograms/ml), over the same concentration range at which it augmented synthesis of the cyclooxygenase products thromboxane A2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. Four lines of evidence suggested that H2O2 inhibited alveolar macrophage leukotriene and 5-HETE synthesis by depleting cellular ATP, a cofactor for 5-lipoxygenase. 1) H2O2 depleted ATP in A23187- and zymosan-stimulated alveolar macrophages with a dose dependence very similar to that for inhibition of agonist-induced leukotriene synthesis. 2) The time courses of ATP depletion and inhibition of leukotriene B4 synthesis by H2O2 were compatible with a rate-limiting effect of ATP on leukotriene synthesis in H2O2-exposed cultures. 3) Treatment of alveolar macrophages with the electron transport inhibitor antimycin A prior to A23187 stimulation depleted ATP and inhibited leukotriene B4 and C4 synthesis to equivalent degrees, while thromboxane A2 production was spared. 4) Incubation with the ATP precursors inosine plus phosphate attenuated both ATP depletion and inhibition of leukotriene B4 and C4 synthesis in alveolar macrophages stimulated with A23187 in the presence of H2O2. Our results show that H2O2 has the capacity to act both as an agonist for macrophage AA metabolism, and as a selective inhibitor of the 5-lipoxygenase pathway, probably as a result of its ability to deplete ATP. Depletion of cellular energy stores by oxidants generated during inflammation in vivo may be a means by which the inflammatory response is self-limited.     

A long-lived o-semiquinone radical anion is formed from N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial substance

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial peptide, generates hydrogen peroxide (H(2)O(2)) that exerts antitumour activity. We have investigated the precise mechanism of H(2)O(2) production from 5-S-GAD by autoxidation aiming to understand its action toward tumour cells. Using the electron spin resonance (ESR) technique, we detected a strong signal due to radical formation from 5-S-GAD. Surprisingly, the ESR signal of the radical derived from 5-S-GAD appeared after incubation for 30 min at 37 degrees C in the buffer at pH 7.4; the signal was persistently detected for 10 h in the absence of catalytic metal ions. The computer simulation of the observed ESR spectrum together with the theoretical calculation of the spin density of the radical species indicates that an o-semiquinone radical anion was formed from 5-S-GAD. We demonstrated that H(2)O(2) is produced via the formation of superoxide anion O2(.-) by the electron-transfer reduction of molecular oxygen by the 5-S-GAD anion, which is in equilibrium with 5-S-GAD in the aqueous solution. The radical formation and the subsequent H(2)O(2) production were inhibited by superoxide dismutase (SOD), when the antitumour activity of 5-S-GAD was inhibited by SOD. Thus, the formation of the o-semiquinone radical anion would be necessary for the antitumour activity of 5-S-GAD as an intermediate in the production of cytotoxic H(2)O(2).     

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5x10(-7) M) stimulated PMN (6x10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8x10(-6) M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2x10(-5) and 2x10(-4) M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5x10(-6) M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8x10(-6) M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2x10(-6) to 10(-5) M), but N-hexanoyl SPN was less active (from 2x10(-5) to 2x10(-4) M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.     

Hydroxyl radical scavenging action of capsaicin

We recently reported that capsaicin (CAP) is capable of scavenging peroxyl radicals derived from 2,2'-azobis(2,4-dimethylvaleronitrile) as measured by electron spin resonance (ESR) spectroscopy. The present study describes the hydroxyl radical (HO*) scavenging ability of CAP as measured by DNA strand scission assay and by an ESR spin trapping technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The Fenton reaction [Fe(II)+ H(2)O(2) --> Fe(III) + HO* + HO(-)] was used as a source of HO*. The incubation of DNA with a mixture of FeSO(4) and H(2)O(2) caused DNA strand scission. The addition of CAP to the incubation mixture decreased the strand scission in a concentration-dependent manner. To understand the antioxidative mechanism of CAP, we used an ESR spin trapping technique. Kinetic competition studies using different concentrations of DMPO indicated that the decrease of the oxidative DNA damage was mainly due to the scavenging of HO* by CAP, not to the inhibition of the HO* generation system itself. We estimated the second order rate constants in the reaction of CAP and common HO* scavengers with HO* by kinetic competition studies. By comparison with the common HO* scavengers, CAP was found to scavenge HO* more effectively than mannitol, deoxyribose and ethanol, and to be equivalent to DMSO and benzoic acid, demonstrating that CAP is a potent HO* scavenger. The results suggest that CAP may act as an effective HO* scavenger as well as a peroxyl radical scavenger in biological systems.     

Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide

Prior spin trapping studies reported that H(2)O(2) is metabolized by copper,zinc-superoxide dismutase (SOD) to form (.)OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free (.)OH or (.)OH scavenged off the Cu(2+) catalytic site. To distinguish whether (.)OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different (.)OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging (.)OH as the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging (.)OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free (.)OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free (.)OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free (.)OH. EPR studies of the copper center demonstrated that while high H(2)O(2) concentrations induce release of Cu(2+), the magnitude of spin adducts produced by free Cu(2+) was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H(2)O(2) and SOD. Thus, SOD and H(2)O(2) oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound (.)OH not release of (.)OH from the enzyme.     

Magnesium deficiency augments myocardial response to reactive oxygen species

Magnesium (Mg) deficiency and oxidative stress are independently implicated in the etiopathogenesis of various cardiovascular disorders. This study was undertaken to examine the hypothesis that Mg deficiency augments the myocardial response to oxidative stress. Electrically stimulated rat papillary muscle was used for recording the contractile variation. Biochemical variables of energy metabolism (adenosine triphosphate (ATP) and creatine phosphate) and markers of tissue injury (lactate dehydrogenase (LDH) release and lipidperoxidation), which can affect myocardial contractility, were assayed in Langendorff-perfused rat hearts. Hydrogen peroxide (100 micromol/L) was used as the source of reactive oxygen species. The negative inotropic response to H2O2 was significantly higher in Mg deficiency (0.48 mmol Mg/L) than in Mg sufficiency (1.2 mmol Mg/L). Low Mg levels did not affect ATP levels or tissue lipid peroxidation. However, H2O2 induced a decrease in ATP; enhanced lipid peroxidation and the release of LDH were augmented by Mg deficiency. Increased lipid peroxidation associated with a decrease in available energy might be responsible for the augmentation of the negative inotropic response to H2O2 in Mg deficiency. The observations from this study validate the hypothesis that myocardial response to oxidative stress is augmented by Mg deficiency. This observation has significance in ischemia-reperfusion injury, where Mg deficiency can have an additive effect on the debilitating consequences.     

Formation of protein tyrosine ortho-semiquinone radical and nitrotyrosine from cytochrome c-derived tyrosyl radical

Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.     

Hydralazine stimulates production of oxygen free radicals in Eagle's medium and cultured fibroblasts.

The effect of hydralazine on the oxygen free radical production was studied in whole cultured murine liver fibroblasts and mitochondrial and microsomal fractions of the cells by ESR spin trapping with DMPO and measurement of Tiron semiquinone formation. Hydralazine itself was found to generate free radicals in phosphate buffer and especially in Eagle's Minimal Essential Medium. Most of the adduct of the spin trap DMPO was due to its reaction with hydralazine-induced hydroxyl radical. Moreover, this compound stimulated free radical formation in fibroblasts. These data suggest that hydralazine alters the cellular free radical metabolism which may have implications for the biological activity of this drug.     

Spin trapping evidence for the lack of significant hydroxyl radical production during the respiration burst of human phagocytes using a spin adduct resistant to superoxide-mediated destruction

Failure to detect hydroxyl radical (.OH)-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide in electron spin resonance (ESR) spin trapping experiments has been offered as evidence for the lack of the endogenous capacity of stimulated human phagocytes (neutrophils, monocytes, and monocyte-derived macrophages (MDM] to generate .OH. Recent reports that 5,5-dimethyl-1-pyrroline N-oxide spin adducts are unstable in the presence of superoxide-generating systems such as stimulated neutrophils has raised concerns regarding the sensitivity of spin trapping techniques for assessment of phagocyte free radical formation. Consequently, we have employed a new approach that uses the spin trap N-t-butyl-alpha-phenyl-nitrone (PBN) and dimethyl sulfoxide. In the presence of dimethyl sulfoxide and PBN, the formation of .OH via three different mechanisms in air-saturated aqueous solutions all yielded a single nitroxide species whose ESR peak amplitude remained stable in the presence of superoxide (.O2-). This nitroxide, which we have assigned as PBN/.OCH3, appears to be an oxygen-centered radical derived from the spin trapping of the reaction product of O2 and methyl radical. When neutrophils, monocytes, or MDM were stimulated with phorbol 12-myristate 13-acetate or opsonized zymosan in the presence of exogenous iron, catalase-inhibitable PBN/.OCH3 was the sole nitroxide detected. In the absence of exogenous iron, no nitroxide was observed, providing evidence for the lack of the endogenous capacity of neutrophils, monocytes, and MDM to generate .OH.     

In vivo singlet-oxygen generation in blood of chromium(VI)-treated mice

Although it is assumed from in vitro experiments that the generation of reactive oxygen species such as the singlet oxygen (1O2), the hydroxyl radical, and the superoxide anion are responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of 1O2 in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 2,2,6,6-tetramethyl-4-piperidone (TMPD), a specific 1O2 trap, to detect 1O2 in blood. The ESR spectrum of the spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:1:1 intensity pattern of three lines with a hyperfine coupling constant A(N) = 16.08 G and a g-value = 2.0066. The concentration of spin adduct detected in the blood was 1.46 microM (0.1% of total Cr concentration). The adduct production was inhibited by the addition of specific 1O2 scavengers such as 1,4-diazabicyclo[2.2.2]octane and sodium azide to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of 1O2 with TMPD. This is the first report of ESR evidence for the in vivo generation of 1O2 in mammals by Cr(VI).     

A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide

The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)O(2) were calculated using hyperfine coupling constants of a(H2,6) = 1.3 G and a(H3,5) = 7.0 G for the ring and a(beta)(H1) = 16.7 G and a(beta)(H2) = 14.2 G for the methylene protons, and are consistent with a highly constrained, conformationally unstable tyrosyl radical. Spectra obtained at later time points contained a mixture of the 3 s signal and another signal that was insufficiently resolved for simulation. Efficient spin trapping with 3, 5-dibromo-4-nitrosobenzenesulfonic acid was observed only when the spin trap was present at the time of H(2)O(2) addition. Spin trapping experiments with either 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or perdeuterated 2-methyl-2-nitrosopropane (MNP-d(9)), which have been shown to trap tyrosyl radicals, were nearly equally effective when the spin trap was added before or 10 min after the addition of H(2)O(2). The superhyperfine structure of the ESR spectra obtained from Pronase-treated MNP-d(9)/*metMb confirmed the assignment to a tyrosyl radical. Delayed spin trapping experiments with site-directed mutant myoglobins in which either Tyr-103 or Tyr-146 was replaced by phenylalanine indicated that radical adduct formation with either DMPO or MNP-d(9) requires the presence of Tyr-103 at all time points, implicating that residue as the radical site.     

Can nucleotides prevent Cu-induced oxidative damage?

Cu-induced oxidative damage is associated with cancer, diabetes, neurodegenerative and age related diseases. The quest for Cu-chelators as potential antioxidants spans the past decades. Yet, biocompatible Cu-chelators that do not alter the normal metal-ion homeostasis are still lacking. Here, we explored the potential of natural and synthetic nucleotides and inorganic phosphates as inhibitors of the Cu(I)/(II)-induced ()OH formation via either the Fenton or Haber-Weiss mechanisms. For this purpose, we studied by ESR the modulation of Cu-induced ()OH production, from the decomposition of H(2)O(2), by nucleotides and phosphates. ATP inhibited both Cu(I) and Cu(II) catalyzed reactions (IC(50) 0.11 and 0.04mM, respectively). Likewise, adenosine 5'-beta,gamma-methylene triphosphate (AMP-PCP), adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), ADP and tripolyphosphate were identified as good inhibitors. However, AMP and adenosine were poor inhibitors in the Cu(I)-H(2)O(2) system, IC(50) ca. 1.2mM, and radical enhancers in the Cu(II)-H(2)O(2) system. The best antioxidant was adenosine 5'-[beta,gamma-imino] triphosphate (AMP-PNP) (IC(50) 0.05mM at Cu(I)-H(2)O(2) system) which was 15 times more active than the known antioxidant Trolox. ATP and analogues inhibit Cu-induced ()OH formation through an ion chelation rather than a scavenging mechanism. Two phosphate groups are required for making active Fenton-reaction inhibitors. Nucleotides and phosphates triggered a biphasic modulation of the Haber-Weiss reaction, but a monophasic inhibition of the Fenton reaction. We conclude that nucleotides at sub mM concentrations can prevent Cu-induced OH radical formation from H(2)O(2), and hence may possibly prevent oxidative damage.     

Ascorbyl free radical reflects catalytically active iron after intravenous iron saccharate injection

Iron release from intravenous iron formulations can increase both non-transferrin-bound iron (NTBI) and oxidative stress. However, data showing a direct association between these parameters are sparse. The aim of this study was to adapt a recently published electron spin resonance (ESR) method to measure NTBI after iron injection and further to investigate its correlation to levels of oxidative stress markers. Twenty chronic hemodialysis patients were enrolled. NTBI and markers of oxidative stress, ascorbyl free radical (AFR), oxidized LDL, protein carbonyl, total antioxidant capacity, and myeloperoxidase, were measured in blood samples collected before and after intravenous injection of 100 mg iron saccharate. NTBI and all analyzed oxidative stress markers were increased 10 min after iron injection. Specifically, NTBI rose by 375% and AFR by 40%. Significant increases in these parameters were still seen 60 min after the injection. The changes in NTBI and AFR were closely correlated. The close correlation between intravascular release of NTBI and increase in plasma AFR after iv iron injection, as well as the increase in all measured oxidative stress markers, suggests that the iron measured was catalytically active. The ESR method was sufficiently sensitive and robust to measure NTBI also in human plasma.     

Iptakalim protects PC12 cell against H2O2-induced oxidative injury via opening mitochondrial ATP-sensitive potassium channel

The final common pathway in the demise of dopaminergic neurons in Parkinson's disease may involve oxidative stress and excitotoxicity. In this study, we examined the neuroprotective effects of a novel ATP-sensitive potassium channel (K(ATP)) opener, iptakalim (IPT), against H(2)O(2)-induced cytotoxicity in rat dopaminergic PC12 cells. Pretreatment with IPT could attenuate increased extracellular glutamate levels and inhibit calcium influxing induced by H(2)O(2). Moreover, IPT regulated the expressions of bcl-2 and bax which were responsible for inhibiting apoptosis in PC12 cells. These protective effects of IPT were abolished by selective mitoK(ATP) channel blocker 5-hydroxydecanoate. Therefore, IPT can protect PC12 cells against H(2)O(2)-induced oxidative injury via activating mitoK(ATP) channel.     

Mitochondrial ATP-sensitive K+ channel opening decreases reactive oxygen species generation

Mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening was shown previously to slightly increase respiration and decrease the membrane potential by stimulating K(+) cycling across the inner membrane. Here we show that mitoK(ATP) opening reduces reactive oxygen species generation in heart, liver and brain mitochondria. Decreased H(2)O(2) release is observed when mitoK(ATP) is active both with respiration stimulated by oxidative phosphorylation and when ATP synthesis is inhibited. In addition, decreased H(2)O(2) release is observed when mitochondrial Delta pH is enhanced, an effect expected to occur when mitoK(ATP) is open. We conclude that mitoK(ATP) is an effective pathway to trigger mild uncoupling, preventing reactive oxygen species release.