The protective effect of hypotaurine and cysteine sulphinic acid on peroxynitrite-mediated oxidative reactions

The protective activity of hypotaurine (HTAU) and cysteine sulphinic acid (CSA) on peroxynitrite-mediated oxidative damage has been assessed by monitoring different target molecules, i.e. tyrosine, dihydrorhodamine-123 (DHR) and glutathione (GSH). The inhibition of tyrosine oxidation exerted by HTAU and CSA both in the presence and the absence of bicarbonate can be ascribed to their ability to scavenge hydroxyl (^•OH) and carbonate (CO₃^•-) radicals. HTAU and CSA also reduce tyrosyl radicals, suggesting that this repair function of sulphinates might operate as an additional inhibiting mechanism of tyrosine oxidation. In the peroxynitrite-dependent oxidation of DHR, the inhibitory effect of HTAU was lower than that of CSA. Moreover, while HTAU and CSA competitively inhibited the direct oxidation of GSH by peroxynitrite, HTAU was again poorly effective against the oxidation of GSH mediated by peroxynitrite-derived radicals. The possible involvement of secondary reactions, which could explain the difference in antioxidant activity of HTAU and CSA, is discussed.     

The protective effect of hypotaurine and cysteine sulphinic acid on peroxynitrite-mediated oxidative reactions

The protective activity of hypotaurine (HTAU) and cysteine sulphinic acid (CSA) on peroxynitrite-mediated oxidative damage has been assessed by monitoring different target molecules, i.e. tyrosine, dihydrorhodamine-123 (DHR) and glutathione (GSH). The inhibition of tyrosine oxidation exerted by HTAU and CSA both in the presence and the absence of bicarbonate can be ascribed to their ability to scavenge hydroxyl (^?OH) and carbonate (CO₃^??) radicals. HTAU and CSA also reduce tyrosyl radicals, suggesting that this repair function of sulphinates might operate as an additional inhibiting mechanism of tyrosine oxidation. In the peroxynitrite-dependent oxidation of DHR, the inhibitory effect of HTAU was lower than that of CSA. Moreover, while HTAU and CSA competitively inhibited the direct oxidation of GSH by peroxynitrite, HTAU was again poorly effective against the oxidation of GSH mediated by peroxynitrite-derived radicals. The possible involvement of secondary reactions, which could explain the difference in antioxidant activity of HTAU and CSA, is discussed.     

Lipoic acid protects efficiently only against a specific form of peroxynitrite-induced damage

The ability of the sulfur-containing compounds glutathione (GSH), glutathione disulphide (GSSG), S-methylglutathione (GSMe), lipoic acid (LA), and dihydrolipoic acid (DHLA) to protect against hypochlorous acid (HOCl)-mediated damage and peroxynitrite (ONOOH)-induced damage has been compared. Protective activity was assessed in competition assays by monitoring several detectors, i.e. dihydrorhodamine-123 (DHR-123) oxidation, alpha(1)-antiproteinase (alpha(1)-AP) inactivation, and glutathione S-transferase P1-1 (GST-P1-1) inactivation. In addition, nitration of tyrosine was measured to assess protection of the sulfur-containing compounds against ONOOH. For protection against HOCl, the efficacy of the antioxidant was controlled by the ratio of the reaction rates of the antioxidant and the detector molecule with the oxidant. The rank order of the activity of the antioxidants (GSH > DHLA approximately LA approximately GSMe > GSSG) appeared to be independent of the detector used. However, the rank order of the antioxidants against ONOOH-induced damage is strongly dependent on the detector. LA was 40 times less active than GSH in the inhibition of ONOOH-induced DHR-123 oxidation, whereas LA was 20 times more active than GSH in preventing the inhibition of GST-P1-1 by ONOOH. This points to different molecular mechanisms of ONOOH damage to DHR-123 compared with ONOOH damage to GST-P1-1. LA is a poor antioxidant in protecting against the form of ONOOH damage involved in DHR-123 oxidation. In the form of ONOOH toxicity involved in GST-P1-1 inhibition, LA is the most potent sulfur-containing antioxidant in our series. It is proposed that an intermediate product in which both sulfur atoms of LA have reacted is involved in the reaction of ONOOH with LA. The high potency of LA to protect GST-P1-1 against ONOOH might be of therapeutic interest.     

Differential effects of peroxynitrite on contractile protein properties in fast- and slow-twitch skeletal muscle fibers of rat

Oxidative modification of contractile proteins is thought to be a key factor in muscle weakness observed in many pathophysiological conditions. In particular, peroxynitrite (ONOO(-)), a potent short-lived oxidant, is a likely candidate responsible for this contractile dysfunction. In this study ONOO(-) or 3-morpholinosydnonimine (Sin-1, a ONOO(-) donor) was applied to rat skinned muscle fibers to characterize the effects on contractile properties. Both ONOO(-) and Sin-1 exposure markedly reduced maximum force in slow-twitch fibers but had much less effect in fast-twitch fibers. The rate of isometric force development was also reduced without change in the number of active cross bridges. Sin-1 exposure caused a disproportionately large decrease in Ca(2+) sensitivity, evidently due to coproduction of superoxide, as it was prevented by Tempol, a superoxide dismutase mimetic. The decline in maximum force with Sin-1 and ONOO(-) treatments could be partially reversed by DTT, provided it was applied before the fiber was activated. Reversal by DTT indicates that the decrease in maximum force was due at least in part to oxidation of cysteine residues. Ascorbate caused similar reversal, further suggesting that the cysteine residues had undergone S-nitrosylation. The reduction in Ca(2+) sensitivity, however, was not reversed by either DTT or ascorbate. Western blot analysis showed cross-linking of myosin heavy chain (MHC) I, appearing as larger protein complexes after ONOO(-) exposure. The findings suggest that ONOO(-) initially decreases maximum force primarily by oxidation of cysteine residues on the myosin heads, and that the accompanying decrease in Ca(2+) sensitivity is likely due to other, less reversible actions of hydroxyl or related radicals.     

Mechanism of peroxynitrite interaction with cytochrome c

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, (.)OH/(.)NO(2) or, in the presence of bicarbonate, CO(3)(-)(.)/(.)NO(2), are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, (.)OH, (.)NO(2) or CO(3)(-)(.), were also studied. The absorbance changes in the Soret band caused by radiolytically produced (.)OH or CO(3)(-)(.) were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. (.)NO(2) produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced (.)OH, (.)NO(2) or CO(3)(-)(.) radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H(2)O(2) with the heme iron of cytochrome c, followed by reaction with the second substrate.     

Taurine is a weak scavenger of peroxynitrite and does not attenuate sodium nitroprusside toxicity to cells in culture

Summary. Many studies have suggested an antioxidant role for taurine, but few studies have directly measured its free radical scavenging activity. The aim of the present study was to directly determine the action of taurine and taurine analogs to inhibit peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR) to rhodamine. Taurine was also tested to determine if it could attenuate the toxicity of sodium nitroprusside (SNP) to neuronal cultures. Taurine at concentrations above 30 mM had a modest ability to inhibit peroxynitrite formation derived from SIN-1. Hypotaurine could inhibit peroxynitrite formation from both SIN-1 (↓75%) and SNP (↓50%) at 10 mM. Other taurine analogs (homotaurine, β-alanine & isethionic acid) slightly potentiated DHR oxidation by SIN-1. Short-term (1-hour) treatment of PC12 cultures with either SNP (1–2 mM) or taurine (20–40 mM) appeared to induce cellular proliferation. In contrast, 24-hour treatment with SNP (1 mM) induced cell death. Combination treatments with taurine and SNP appeared to interact in an additive fashion for both cell proliferation and neurotoxic actions. It appears unlikely that taurine is a major endogenous scavenger of peroxynitrite. Received May 9, 2000 Accepted June 13, 2000     

Carbon dioxide stimulates the production of thiyl, sulfinyl, and disulfide radical anion from thiol oxidation by peroxynitrite

Reaction of peroxynitrite with the biological ubiquitous CO(2) produces about 35% yields of two relatively strong one-electron oxidants, CO(3) and ( small middle dot)NO(2), but the remaining of peroxynitrite is isomerized to the innocuous nitrate. Partial oxidant deactivation may confound interpretation of the effects of HCO3-/CO(2) on the oxidation of targets that react with peroxynitrite by both one- and two-electron mechanisms. Thiols are example of such targets, and previous studies have reported that HCO3-/CO(2) partially inhibits GSH oxidation by peroxynitrite at pH 7.4. To differentiate the effects of HCO3-/CO(2) on two- and one-electron thiol oxidation, we monitored GSH, cysteine, and albumin oxidation by peroxynitrite at pH 5.4 and 7.4 by thiol disappearance, oxygen consumption, fast flow EPR, and EPR spin trapping. Our results demonstrate that HCO3-/CO(2) diverts thiol oxidation by peroxynitrite from two- to one-electron mechanisms particularly at neutral pH. At acid pH values, thiol oxidation to free radicals predominates even in the absence of HCO3-/CO(2). In addition to the previously characterized thiyl radicals (RS.), we also characterized radicals derived from them such as the corresponding sulfinyl (RSO.) and disulfide anion radical (RSSR.-) of both GSH and cysteine. Thiyl, RSO. and RSSR.- are reactive radicals that may contribute to the biodamaging and bioregulatory actions of peroxynitrite.     

Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging

Oxidation of lipids has been implicated in the pathophysiology of atherosclerosis. It has been suggested that scavenging of lipid peroxyl radicals contribute to the antiatherosclerotic effects of naturally occurring compounds such as the isoflavones. This group of polyphenolics includes genistein and is present in relatively high concentrations in food products containing soy. Soy isoflavones are capable of inhibiting lipoprotein oxidation in vitro and suppressing formation of plasma lipid oxidation products in vivo. However, key aspects of the antioxidant mechanisms remain unknown. In this study the antioxidant effects of genistein and other soy isoflavones on lipid peroxidation initiated by mechanistically diverse oxidants was investigated. Although isoflavones inhibited lipid peroxidation stimulated by both metal-dependent and independent processes, the concentration required for these effects were relatively high compared to those found in vivo. Interestingly, however, isoflavones were not consumed and remained in the native state over the time during which inhibition of lipid peroxidation was observed. This was also the case under conditions where synergistic inhibition of LDL oxidation was observed with ascorbate. Furthermore, in an oxidation system driven solely by peroxyl radicals, isoflavones were found to be relatively poor peroxyl radical scavengers. Consistent with the apparent lack of reactivity with lipid-derived oxidants, isoflavones were also relatively resistant to oxidation mediated by the potent oxidant peroxynitrite. The potential antioxidant mechanisms of isoflavones are discussed in the context of possible reactivities of isoflavone-derived phenoxyl radicals.     

Protective effects of n-acetylcysteine on lung injury and red blood cell modification induced by carrageenan in the rat.

Oxidative stress has been suggested as a potential mechanism in the pathogenesis of lung inflammation. The pharmacological profile of n-acetylcysteine (NAC), a free radical scavenger, was evaluated in an experimental model of lung injury (carrageenan-induced pleurisy). Injection of carrageenan into the pleural cavity of rats elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity that contained many neutrophils (PMNs), an infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate, tumor necrosis factor alpha, and interleukin 1beta. All parameters of inflammation were attenuated by NAC treatment. Furthermore, carrageenan induced an up-regulation of the adhesion molecules ICAM-1 and P-selectin, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS), as determined by immunohistochemical analysis of lung tissues. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine, and PARS was reduced by NAC. In vivo NAC treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorhodamine-123, prevented the appearance of DNA damage, an decrease in mitochondrial respiration, and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. A significant alteration in the morphology of red blood cells was observed 24 h after carrageenan administration. NAC treatment has the ability to significantly diminish the red blood cell alteration. Our results clearly demonstrate that NAC treatment exerts a protective effect and clearly indicate that NAC offers a novel therapeutic approach for the management of lung injury where radicals have been postulated to play a role.     

The oxidative and nitrosative chemistry of the nitric oxide/superoxide reaction in the presence of bicarbonate

The primary product of the interaction between nitric oxide (NO) and superoxide () is peroxynitrite (ONOO-), which is capable of either oxidizing or nitrating various biological substrates. However, it has been shown that excess NO or can further react with ONOO- to form species which mediate nitrosation. Subsequently, the controlled equilibrium between nitrosative and oxidative chemistry is critically dependent on the flux of NO and. Since ONOO- reacts not only with NO and but also with CO2, the effects of bicarbonate () on the biphasic oxidation profile of dihydrorhodamine-123 (DHR) and on the nitrosation of both 2,3-diaminonaphthalene and reduced glutathione were examined. Nitric oxide and were formed with DEA/NO [NaEt2NN(O)NO] and xanthine oxidase, respectively. The presence of did not alter either the oxidation profile of DHR with varying radical concentrations or the affinity of DHR for the oxidative species. This suggests that the presence of CO2 does not affect the scavenging of ONOO- by either NO or. However, an increase in the rate of DHR oxidation by ONOO- in the presence of suggests that a CO2-ONOO- adduct does play a role in the interaction of NO or with a product derived from ONOO-. Further examination of the chemistry revealed that the intermediate that reacts with NO is neither ONOO- nor cis-HOONO. It was concluded that NO reacts with both trans-HOONO and a CO2 adduct of ONOO- to form nitrosating species which have similar oxidation chemistry and reactivity with and NO.     

Oxygen-induced radical intermediates in the nNOS oxygenase domain regulated by L-arginine, tetrahydrobiopterin, and thiol

Fully coupled nitric oxide synthase (NOS) catalyzes formation of nitric oxide (NO), l-citrulline, NADP+, and water from l-arginine, NADPH, and oxygen. Uncoupled or partially coupled NOS catalyzes the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite, depending on the availability of cofactor tetrahydrobiopterin (BH4) and l-arginine during catalysis. We identified three distinct oxygen-induced radical intermediates in the ferrous endothelial NOS oxygenase domain (eNOSox) with or without BH4 and/or l-arginine [Berka, V., Wu, G., Yeh, H. C., Palmer, G., and Tsai, A.-L. (2004) J. Biol. Chem. 279, 32243-32251]. The effects of BH4 and l-arginine on the oxygen-induced radical intermediates in the isolated neuronal NOS oxygenase domain (nNOSox) have been similarly investigated by single-turnover stopped-flow and rapid-freeze quench EPR kinetic measurements in the presence or absence of dithiothreitol (DTT). Like for eNOSox, we found different radical intermediates in the reaction of ferrous nNOSox with oxygen. (1) nNOSox (without BH4 or l-Arg) produces superoxide in the presence or absence of DTT. (2) nNOSox (with BH4 and l-Arg) yields a typical BH4 radical in a manner independent of DTT. (3) nNOSox (with BH4 and without l-Arg) yields a new radical. Without DTT, EPR showed a mixture of superoxide and biopterin radicals. With DTT, a new approximately 75 G wide radical EPR was observed, different from the radical formed by eNOSox. (4) The presence of only l-arginine in nNOSox (without BH4 but with l-Arg) caused conversion of approximately 70% of superoxide radical to a novel radical, explaining how l-arginine decreases the level of superoxide production in nNOSox (without BH4 but with l-Arg). The regulatory role of l-arginine in nNOS is thus very different from that in eNOS where substrate was only to decrease the rate of formation of superoxide but not the total amount of radical. The role of DTT is also different. DTT prevents oxidation of BH4 in both isoforms, but in nNOS, DTT also inhibits oxidation of two key cysteines in nNOSox to prevent the loss of substrate binding. This new role of thiol found only for nNOS may be significant in neurodegenerative diseases.     

A Microtiter Plate Assay for Screening Antioxidant Activity in Extracts of Marine Organisms

A novel microtiter plate assay was developed to determine the total peroxyl radical-trapping activity of antioxidants extracted from marine organisms by measuring the inhibition rate of dye-substrate oxidation. We compared use of dihydrorhodamine-123, dihydrofluorescein, and dichlorodihydrofluorescein as reduced substrates for oxidation by peroxyl radicals generated from 2,2-azobis(2-amidinopropane) dihydrochloride. The oxidation products of these highly reactive substrates are intensely colored dyes that absorb maximally in the wavelength region, _max = 489 to 512 nm, and their concentrations were determined photometrically using a 96-well, microtiter plate reader. The microtiter plate method provides for concurrent multisample analysis with automated data storage, regression analyses, and calculation of oxidation inhibition rates. Dihydrorhodamine was selected as the preferred substrate for screening crude extracts, and typical assay results are presented. Novel lead antioxidants are selected from active extracts by chromatographic analysis with electrochemical detection.     

Protection by flavonoids against the peroxynitrite-mediated oxidation of dihydrorhodamine

Peroxynitrite anion is a reactive and short-lived species and its formation in vivo has been implicated in several human diseases. In view of the potential usefulness of compounds that can protect against peroxynitrite or their reactive intermediates, a study focused on flavonoid compounds was carried out. Since the reactivity of peroxynitrite may be modified by Co2/HCO3-, which is an important plasma buffer, the protection of flavonoids against peroxynitrite was evaluated by their ability to inhibit the peroxynitrite-mediated dihydrorhodamine (DHR123) oxidation with or without physiological concentrations of bicarbonate. Flavonoids from different classes were studied to elucidate which structural features are required for an effective protection. The most efficient flavonoids on protecting DHR123 against oxidation by peroxynitrite have their ability diminished in the presence of bicarbonate, but they maintain the hierarchy established in the absence of bicarbonate. The flavones are the most effective flavonoids and their effects depend mainly on the number of hydroxyl groups. These must include either a catechol group in the B-ring or a hydroxyl group at the 3-position. This work also included some isoflavones, flavanones and a flavanol, which enable us to conclude about the importance of another structural feature: the 2,3-double bond. These results indicate that the ability of flavonoids to protect against peroxynitrite depends on some structural features, also important to scavenge oxygen free radicals and to chelate metal ions. The most efficient flavonoids are effective at low concentrations with IC50 of the same magnitude as Ebselen, a selenocompound that has been reported to be excellent at protecting against peroxynitrite. Their effectiveness at low concentrations is an important aspect to take into account when characterizing a compound as an antioxidant with biological interest.     

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.

Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of 相似文献   

Antioxidant activity: what do we measure?

Inhibition of oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by free radicals generated by decomposition of 2,2'-azobis(2-amidopropane) (ABAP) by antioxidants and biological material was studied. A correlation was found between the ability of various substances to delay the onset of ABTS oxidation and their rapid reduction of the ABTS+* cation radical, and between the ability to reduce the maximal rate of ABTS oxidation and slow reduction of ABTS+*. The length of the lag period of ABTS oxidation was found to be independent of ABTS concentration. Similar decrease of peroxynitrite-induced ABTS+* formation by antioxidants was observed when the antioxidants were added before and after peroxynitrite. All these findings indicate that the main effect of antioxidants in this system is reduction of ABTS+* and not prevention of its formation. Reduction of oxidation products rather than inhibition of their formation may be the predominant mode of action of antioxidants in various assays of antioxidant activity.     

Tempol diverts peroxynitrite/carbon dioxide reactivity toward albumin and cells from protein-tyrosine nitration to protein-cysteine nitrosation

Tempol has been shown to protect experimental animals from injuries associated with excessive nitric oxide production. In parallel, tempol decreased the levels of protein-3-nitrotyrosine in the injured tissues, suggesting that it interacted with nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite. Relevantly, a few recent studies have shown that tempol catalytically diverts peroxynitrite/carbon dioxide reactivity toward phenol from nitration to nitrosation. To examine whether this shift occurs in biological environments, we studied the effects of tempol (10-100 microM) on peroxynitrite/carbon dioxide (1 mM/2 mM) reactivity toward proteins, native bovine serum albumin (BSA) (0.5-0.7 cys/mol) and reductively denatured BSA (7-19 cys/mol), and cells (J774 macrophages). Although not a true catalyst, tempol strongly inhibited protein-tyrosine nitration (70-90%) and protein-cysteine oxidation (20-50%) caused by peroxynitrite/carbon dioxide in BSA, denatured BSA, and cells while increasing protein-cysteine nitrosation (200-400%). Tempol consumption was attributed mainly to its reaction with protein-cysteinyl radicals. Most of the tempol, however, reacted with the radicals produced from peroxynitrite/carbon dioxide, that is, nitrogen dioxide and carbonate radical anion. Accordingly, tempol decreased the yields of BSA-cysteinyl and BSA-tyrosyl/tryptophanyl radicals, as well their decay products such as protein-3-nitrotyrosine. The parallel increase in protein-nitrosocysteine yields demonstrated that part of the peroxynitrite is oxidized to nitric oxide by the oxammonium cation produced from tempol oxidation by peroxynitrite/carbon dioxide-derived radicals. Protein-nitrosocysteine formation was shown to occur by radical and nonradical mechanisms in studies with a protein-cysteinyl radical trapper. These studies may contribute to the understanding of the protective effects of tempol in animal models of inflammation.     

The Effects of Different Buffers on the Oxidation of DNA by Thiols and Ferric Iron

Because buffers can act as metal ligands, they can effect several reactions necessary for DNA oxidation by ferric iron and thiols, such as iron reduction. Therefore, these reactions were studied in Hepes and phosphate buffers and unbuffered NaCl. Reduction of Fe³⁺ by dithiothreitol (DTT) and cysteine was observed in either Hepes or NaCl solutions, but not in phosphate buffer. Thiyl radicals were observed in Hepes, but there was much less thiyl radical production in the saline or phosphate solutions. Redox cycling between either DTT or cysteine and Fe³⁺ also resulted in dioxygen consumption in Hepes buffer. Reduction of Fe³⁺ and O₂ resulted in the formation of an oxidant capable of producing 8-hydroxy-2′-deoxyguanosine (8-OHdG) in calf-thymus DNA. The highest levels of 8-OHdG were detected when DTT or cysteine and Fe³⁺ were incubated in Hepes, while much less DNA oxidation was detected when the experiment was done in a saline solution, and almost no DNA oxidation occurred in the phosphate buffer. These results demonstrate that the use of different buffers can greatly affect the ability of thiols to promote iron-dependent oxidations. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 125–132, 1998     

Hydroxyl radical-induced strand break formation of poly(U) in the presence of oxygen: comparison of the rates as determined by conductivity, e.s.r. and rapid-mix experiments with a thiol

The rate of OH radical-induced strand break formation of single-stranded poly(U) in N2O/O2-saturated aqueous solution was studied by measuring the time-dependence of the electrical conductivity following pulse radiolysis. The first half-life of the total conductivity increase depends slightly on pH and the molecular weight and on the dose per pulse. The activation parameters for strand break formation were found to be EA = 52 kJ mol-1 and A = 5 X 10(8) s-1. Similar first half-lives were observed when the decay of peroxyl radicals of poly(U) was measured by e.s.r. under various conditions. This indicates that poly(U)-peroxyl radicals are involved in the rate-determining step of strand break formation. After pulse radiolysis, strand break formation can be inhibited by the addition of dithiothreitol (DTT) in a rapid-mix apparatus. It is postulated that peroxyl radicals of poly(U) react with DTT by formation of hydroperoxides, thereby preventing strand breakage.     

Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3*-) and nitrogen dioxide (*NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3*- and *NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 x 10(9) and 7.6 x 10(6) M(-1) s(-1), respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 x 10(6) M(-1) s(-1). However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with *NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3*- and *NO2 radicals.     

Protection by Flavonoids Against the Peroxynitrite-mediated Oxidation of Dihydrorhodamine

Peroxynitrite anion is a reactive and short-lived species and its formation in vivo has been implicated in several human diseases. In view of the potential usefulness of compounds that can protect against peroxynitrite or their reactive intermediates, a study focused on flavonoid compounds was carried out. Since the reactivity of peroxynitrite may be modified by [Formula: See Text] which is an important plasma buffer, the protection of flavonoids against peroxynitrite was evaluated by their ability to inhibit the peroxynitrite-mediated dihydrorhodamine (DHR123) oxidation with or without physiological concentrations of bicarbonate. Flavonoids from different classes were studied to elucidate which structural features are required for an effective protection. The most efficient flavonoids on protecting DHR123 against oxidation by peroxynitrite have their ability diminished in the presence of bicarbonate, but they maintain the hierarchy established in the absence of bicarbonate. The flavones are the most effective flavonoids and their effects depend mainly on the number of hydroxyl groups. These must include either a catechol group in the B-ring or a hydroxyl group at the 3-position. This work also included some isoflavones, flavanones and a flavanol, which enable us to conclude about the importance of another structural feature: the 2,3-double bond. These results indicate that the ability of flavonoids to protect against peroxynitrite depends on some structural features, also important to scavenge oxygen free radicals and to chelate metal ions. The most efficient flavonoids are effective at low concentrations with IC₅₀ of the same magnitude as Ebselen, a selenocompound that has been reported to be excellent at protecting against peroxynitrite. Their effectiveness at low concentrations is an important aspect to take into account when characterizing a compound as an antioxidant with biological interest.