Picroliv, picroside-I and kutkoside from Picrorhiza kurrooa are scavengers of superoxide anions.

Picroliv, the active principle of Picrorhiza kurrooa, and its main components which are a mixture of the iridoid glycosides, picroside-I and kutkoside, were studied in vitro as potential scavengers of oxygen free radicals. The superoxide (O2-) anions generated in a xanthine-xanthine oxidase system, as measured in terms of uric acid formed and the reduction of nitroblue tetrazolium were shown to be suppressed by picroliv, picroside-I and kutkoside. Picroliv as well as both glycosides inhibited the non-enzymic generation of O2- anions in a phenazine methosulphate NADH system. Malonaldehyde (MDA) generation in rat liver microsomes as stimulated by both the ascorbate-Fe2+ and NADPH-ADP-Fe2+ systems was shown to be inhibited by the Picroliv glycosides. Known antioxidants tocopherol (vitamin E) and butylated hydroxyanisole (BHA) were also compared with regard to their antioxidant actions in the above system. It was found that BHA afforded protection against ascorbate-Fe(2+)-induced MDA formation in microsomes but did not interfere with enzymic or non-enzymic O2- anion generation; and tocopherol inhibited lipid peroxidation in microsomes by both prooxidant systems and the generation of O2- anions in the non-enzymic system but did not interfere with xanthine oxidase activity. The present study shows that picroliv, picroside-I and kutkoside possess the properties of antioxidants which appear to be mediated through activity like that of superoxide dismutase, metal ion chelators and xanthine oxidase inhibitors.     

Selenocarbamates are effective superoxide anion scavengers in vitro.

We investigated the superoxide anion-scavenging effects of six selenocarbamates and four thiocarbamates, using a highly sensitive quantitative chemiluminescence method. At 333 nM, six selenocarbamates and four thiocarbamates scavenged in the range of 2.9-68.7% of O(2)*-. Se-methyl N-phenylselenocarbamate and Se-methyl N-(4-methylphenyl)selenocarbamate exhibited the strongest superoxide anion-scavenging activity among the Se-selenocarbamates. In contrast, the corresponding S-thiocarbamates had moderate inhibitory effect. The 50% inhibitory concentrations (IC(50)) of Se-methyl-N-phenylselenocarbamate and Se-methyl-N-(4-methylphenyl)selenocarbamate were determined to be 140 nM and 162 nM, respectively. Thus, these compounds acted in vitro as effective and potentially useful O(2)*- scavengers.     

Tertiary selenoamide compounds are useful superoxide radical scavengers in vitro.

We investigated the scavenging effects of tertiary selenoamide compounds for super oxide radicals using a highly sensitive and quantitative chemiluminescence method. At 333 nM, tertiary selenoamide compounds scavenged 25.8-81.6% of O(2)(-). N-(Phenylselenocarbonyl) piperidine was the most effective scavenger of superoxide radicals. While N,N-diethyl-2-selenonaphthylamide and N,N-diethyl-4-chloroselenobenzamide was a moderately effective scavenger of superoxide radicals. The IC(50) of N-(phenylselenocarbonyl) piperidine and N,N-diethyl-2-selenonaphthylamide were determined to be 110 and 182 nM, respectively. The results suggest that tertiary selenoamide compounds are useful scavengers of superoxide radicals.     

Superoxide anions,free-radical scavengers,and nitrergic neurotransmission

• 1.1. There is now strong evidence that the L-arginine/nitric oxide (NO) pathway generates the transmitter released from certain nonadrenergic, noncholinergic nerves that mediate smooth-muscle relaxation in the respiratory, gastrointestinal, and urogenital tracts. In particular, nitric oxide synthase (NOS) has been detected in these nitrergic nerves, and nerve-induced relaxation can be prevented by NOS inhibitors. Thus, free-radical NO has been considered the putative transmitter candidate.
• 2.2. Despite such evidence, a number of superoxide anion-generating compounds and direct NO scavengers have been found to abolish relaxations to exogenous NO, but to have very little effect on relaxations in response to nitrergic field stimulation. A number of hypotheses have been put forward to explain this paradox: first, that the NO generated within the nerve is attached to a carrier molecule (such as a thiol) to form an adduct, that is released into the junctional gap and that is resistant to superoxide anions and other scavengers; second, that over short distances (up to 200 μm) the rapid diffusion characteristics of NO render it resistant to inhibition by scavengers; third, that NO is indeed released as a free radical, but that it is protected from radical scavengers by other substances present in the junctional region.
• 3.3. Recent experimental evidence supports the third explanation, because nitrergic relaxations, normally resistant to inhibition by superoxide anions, become sensitive following inactivation of copper/ zinc superoxide dismutase (Cu/Zn SOD); the inhibition can be reversed by adding exogenous Cu/Zn SOD (or ascorbate). In addition, the ability of two NO-scavenger compounds, hydroquinone and carboxy-PTIO, to inhibit relaxations to exogenous NO is prevented by certain physiological antioxidants (ascorbate and reduced glutathione in the case of hydroquinone, and ascorbate and α-tocopherol in the case of carboxy-PTIO).
• 4.4. Thus, it is possible that the presence of integrated antioxidant mechanisms within the tissue protects neuronally- released NO from attack by scavenging molecules; exogenous NO would be vulnerable before reaching the protection of the tissue, thus explaining the paradoxical effects mentioned. Organ antioxidant status may therefore be very important in preserving the potency of nitrergic transmission and in preventing NO from reacting with other compounds to produce cytotoxic metabolites (eg., with superoxide anions to form peroxynitrite).

     

The influence of calcium channel blockers on superoxide anions

Four calcium channel blockers were tested from the point of view of their influence on enzymic lipid oxidation and on generation of superoxide anions. All the compounds were found to be antioxidants as tested by the inhibition of NADPH-stimulated malonaldehyde formation from lipids. IC50 values were 60 microM for nifedipine; 1.1 microM for verapamil; 1.4 microM for fendiline and 20.6 microM for diltiazem. Only nifedipine scavenged superoxide anions both in an enzymic (xanthine:xanthine oxidase) and non-enzymic (phenazine methosulphate:NADH) generating system. IC50 values for this inhibition were about 2.5 times higher than for inhibition of formation of malonaldehyde. Nifedipine inhibited also xanthine oxidase-mediated formation of uric acid.     

Flavonoids as peroxynitrite scavengers: the role of the hydroxyl groups.

It has been reported that flavonoids efficiently protect against peroxynitrite toxicity. Two pharmacophores have been identified in flavonoids, namely the catechol group in ring B and the hydroxyl (OH) group at the 3-position. In this study, this structure-activity relationship was further examined. It was found that catechol (1,2-dihydroxybenzene) is a potent peroxynitrite scavenger, whereas phenol (hydroxybenzene) is not. Of the flavonols tested without a catechol group in ring B, kaempferol (OH groups at positions 3,5,7,4') and galangin (OH groups at positions 3,5,7) are also potent scavengers, whereas apigenin (OH groups at positions 5,7,4') and chrysin (OH groups at positions 5,7) are not. This confirms the importance of the OH group at the 3-position. However, the synthetic flavonol TUM 9761 and 3-hydroxyflavone (OH group only at position 3) are poor scavengers. Based on these results, the structure-activity relationship on the peroxynitrite scavenging activity of flavonols was refined. The catechol in ring B remains important. Also the 3-OH group remains important, but the activity of this pharmacophore is influenced by the substituents at position 5 and at position 7.     

High density lipoprotein is a scavenger of superoxide anions

Present work describes a new property of HDL to act as a scavenger of O2- free radicals in vitro. This lipoprotein prevents both enzymic and non-enzymic generation of O2- anions as evidenced by inhibition of xanthine oxidase, peroxidase, peroxidation of pyrogallol and phenazine methosulphate-NADH reaction. Ascorbate stimulated MDA formation in microsomes has been shown to be suppressed by HDL and these effects are comparable with that of BHA.     

Phenolic compounds and an analog as superoxide anion scavengers and antioxidants.

Five phenolic compounds and pyridoxine were studied for their activities as both scavengers of superoxide anions and inhibitors of lipid peroxidation. The superoxide anions were generated in a phenazin methosulfate-NADH system and were assayed by the reduction of nitroblue tetrazolium. The superoxide anion scavenging activities of verbascoside and alizarin yellow R were the strongest, followed by those of caffeic acid and phloridzin; vanillin and pyridoxine exhibited the weakest activity. The concentration values yielding 50% inhibition of lipid peroxidation in mouse liver microsomes were 10(-5) M for verbascoside, 10(-4) M for alizarin yellow R and caffeic acid, and 10(-3) M for phloridzin; vanillin and pyridoxine had almost no antioxidative activity. The inhibition of lipid peroxidation by these individual compounds was much weaker than by butylated hydroxyanisole. The results showed that phenolic compounds and pyridoxine have more than one mechanism of action for free radicals and are able to suppress free radical processes at two stages: the formation of superoxide anions and the production of lipid peroxides.     

Role of superoxide dismutase enzymes and ascorbate in protection of nitrergic relaxation against superoxide anions in mouse duodenum

Aim: The aim of this study was to investigate whether superoxide dismutase (SOD) enzymes and ascorbate play a role in the protection of the nitrergic relaxation against superoxide anion inhibition in the mouse duodenum. Methods: The effects of exogenous SOD, N , N '-bis(salicylidene) ethylenediamine chloride (EUK-8; a synthetic cell-permeable mimetic of the manganese SOD [Mn SOD] and ascorbate on relaxant responses induced by nitrergic nerve stimulation), exogenous nitric oxide (NO), and nitroglycerin were investigated in isolated mouse duodenum tissues. Results: Diethyldithiocarbamate (DETCA) inhibited the relaxation to exogenous NO and nitroglycerin, but not relaxation to electrical field stimulation (EFS). SOD and ascorbate partially prevented the inhibitory effect of DETCA on relaxation to NO, abut not to nitroglycerin. The DETCA-induced inhibition on nitroglycerin was prevented by EUK-8. Hemoglobin, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolinel-oxyl-3-oxide, and hydroxo-cobalamin inhibited the relaxation to NO, but not to EFS and nitroglycerin in the presence of DETCA. Pyrogallol and hydroquinone inhibited the relaxation to NO, but not to EFS and nitroglycerin. This inhibition was prevented by exogenous SOD and ascorbate, but was not prevented by EUK-8. Pyrogallol and hy-droquinone did not inhibit the EFS-induced relaxation in the presence of DETCA. Duroquinone and 6-anilino-5.8-quinolinedione inhibited the relaxation to EFS, NO, and nitroglycerin, and this inhibition was prevented by EUK-8. Conclusion: These results suggest that the nitrergic neurotransmission in the mouse duodenum is protected by endogenous tissue antioxidants against superoxide anions, and Mn SOD, in addition to copper/zinc SOD, can protect NO from attack from superoxide anion generators intracellularly. Also, the possibility that the endogenous neurotransmitter may not be the free NO but a NO-containing or NO-generating molecule in the mouse duodenum remains open.     

Participation of superoxide anions at the prostaglandin phase of carrageenan foot-oedema.

Bovine Superoxide dismutase (SOD: 0.5–2.0 mg/kg) administered intravenously to rats, completely suppressed the prostaglandin phase swelling (2–4 hr) of carrageenan foot-oedema, but had no effect on the histamine and serotonin phase ($\text{1}\text{2}\text{–1}\text{1}\text{2}\text{hrs}$.). Heat-inactivated SOD, bovine serum albumin and catalase and high doses of the hydroxyl radical scavengers, sodium benzoate and d-mannitol or the oxygen scavenger, 1.3-diphenylisobenzofuran had no effect on the swelling. Mepylamine plus methysergide did not influence the inhibitory action of SOD. Carrageenan foot-oedema of agranulocyte rats, induced by methotrexate injections, was more susceptible than that of normal rats to SOD inhibition. Even at 1 hr, about 70 per cent inhibition of swelling was observed suggesting the importance of macrophages in this inflammation model. Indomethacin and oxyphenbutazone were also examined for comparison with the effect of SOD. The role of Superoxide anions in inflammation is discussed in connection with macrophage emigration, releases of lysosomal enzymes and prostaglandin biosynthesis.     

Involvement of superoxide anions in ozone-induced airway hyperresponsiveness in unanesthetized guinea pigs

To evaluate the involvement of superoxide in ozone (O(3))-induced airway hyperresponsiveness, we studied the effects of superoxide dismutase (SOD), a scavenger of superoxide anion, and apocynin, an inhibitor of superoxide anion-generating NADPH oxidase in phagocytes, on the airway responses induced by O(3) in unanesthetized guinea pigs. Airway responsiveness was measured by PC(200)Mch, the concentration required to produce a doubling in the baseline specific airway resistance to an inhaled methacholine aerosol, in spontaneously breathing animals. Before exposure to 3 ppm O(3) for 30 min, animals inhaled either SOD (5000 U/ml) or vehicle for 5 min. Although SOD did not affect PC(200)Mch in the air control group, this agent reduced the O(3)-induced airway hyperresponsiveness. Repeated administration of apocynin (12 mg/kg for 4 days) also attenuated the O(3)-induced airway hyperresponsiveness. These data suggest that superoxide may be involved in the pathogenesis of O(3)-induced airway hyperresponsiveness, possibly through the stimulation of superoxide anions release from bronchoalveolar phagocytes. The data also suggest a potential therapeutic role for antioxidants in oxidant injury by air pollutants.     

Activity of polyphenolic crude extracts as scavengers of superoxide radicals and inhibitors of xanthine oxidase.

In view of the pharmacological interest in phenolic substances, we have determined the total amount of anthocyanins and polyphenols present in the berries of several cultivars of Ribes, Rubus, and Vaccinium genera. The in vitro antiradical activity of the crude extracts on chemically-generated superoxide radicals as well as the inhibitory activity towards the enzyme xanthine oxidase were studied. All the crude extracts examined showed a remarkably high activity towards chemically-generated superoxide radicals. The activities were greater than those expected on the basis of the quantities of anthocyanins and polyphenols present in the samples. Furthermore, the extracts showed a certain inhibitory activity towards xanthine oxidase. Ribes nigrum extracts exhibit the highest activity, being the richest in both anthocyanins and polyphenols. On the other hand, Ribes rubrum extracts seem to contain more active substances than the other crude extracts.     

Streptonigrin-induced deoxyribose degradation: inhibition by superoxide dismutase, hydroxyl radical scavengers and iron chelators

The aminoquinone antitumour antibiotic streptonigrin stimulates deoxyribose degradation in cell-free systems. This degradation is dependent both on reduction of the drug to a semiquinone and on traces of molecular oxygen in the reaction. Inhibition by a variety of hydroxyl radical scavengers and by catalase implicates a radical species with properties similar to the hydroxyl radical. Iron salts appear to play some part in radical formation as DETAPAC and desferrioxamine partly inhibit. Deoxyribose degradation under conditions of low oxygen concentration is strongly inhibited by superoxide dismutase.     

Differential role of extra- and intracellular superoxide anions for nitric oxide-mediated apoptosis induction

Nitric oxide (NO) has recently been shown to mediate apoptosis induction selectively in transformed fibroblasts, in contrast to their nontransformed parental cells. Here we show that NO-mediated apoptosis induction in transformed fibroblasts can be divided into two major phases. During phase 1, peroxynitrite is generated by the interaction of extracellular superoxide anions with NO and the intracellular glutathione level is subsequently lowered. This defines the beginning of phase 2, in which NO-mediated signaling depends on intracellular superoxide anions exclusively. The resultant peroxynitrite seems to activate the mitochondrial permeability transition pore and thus triggers execution of apoptosis. Experimental depletion of intracellular glutathione causes a drastic decrease in the length of phase 1 in transformed cells and renders nontransformed cells sensitive to NO-mediated apoptosis induction. These findings allow the prediction that either induction of superoxide anion generation or glutathione depletion may render cells sensitive to NO-mediated apoptosis induction.     

Iron complexes of deferiprone and dietary plant catechols as cytoprotective superoxide radical scavengers(1).

Superoxide radicals have been implicated in the pathogenesis of aging, cataract, ischemia-reperfusion, cancer and inflammatory diseases. In the present work, we found that deferiprone (L1), an iron-chelating drug, and dietary dihydroxycinnamic acids (catechols) were much more effective at protecting isolated rat hepatocytes against hypoxia-reoxygenation injury if complexed with Fe(3+). Furthermore, the 2:1 catechol-metal complexes with Cu(2+), Fe(2+), and Fe(3+) were also more effective than uncomplexed catechols in scavenging superoxide radicals generated enzymically (xanthine oxidase/hypoxanthine). The 2:1 deferiprone:Fe(3+) complex was less effective at scavenging enzymically generated superoxide radicals even though it was effective at preventing hepatocyte hypoxia-reoxygenation injury. On the other hand, the 1:1 deferoxamine:Fe(3+) complex, another iron-chelating drug, did not prevent hepatocyte hypoxia-reoxygenation injury and did not scavenge enzymically generated superoxide radicals. Furthermore, hepatocytes readily reduced the 2:1 deferiprone:Fe(3+) complex but not the deferoxamine:Fe(3+) complex. These results suggest that the initial step in superoxide radical scavenging (SRS) activity is the formation of a redox complex between Fe(3+) and deferiprone or catechols. The [deferiprone:Fe(3+)] complex was more cytoprotective than would be expected from its SRS activity. This suggests that [deferiprone:Fe(3+)] complex is reduced by a ferrireductase present on the hepatocyte membrane to form [deferiprone:Fe(2+)] complex, which then scavenges superoxide radicals. Therefore, the clinically used deferiprone (L1) may have therapeutic advantages over deferoxamine in having a double role therapeutically: (a) it chelates iron to alleviate iron overload pathology, and (b) the readily formed iron complex protects hepatocytes from superoxide radical-mediated hypoxia-reoxygenation injury.     

The application of ultrasounds for detection of scavenging of superoxide anions by drugs.

In aqueous solutions ultrasounds are known to generate oxygen free radicals. Here we report that ferricytochrome c and nitroblue tetrazolium when sonificated in 1% ethanolic buffered solutions are reduced predominantly in a SOD-inhibitable manner. Aqueous solutions of adrenaline undergo oxidation when exposed to ultrasounds. Therefore, it seems that in the presence of ethanol or adrenaline ultrasounds generate substantial amounts of superoxide anions along with other free radicals. We have tried to adapt ultrasound technique for detection of scavenging of superoxide anions by a metabolite of molsidomine, SIN-1 and flavonoids. In the presence of SIN-1 reduction of indicators by superoxide anions generated by ultrasounds was prevented. In the case of quercetin we failed to detect this property because ultrasounds were found to transform native flavonoids into oxidized derivatives.     

Scavenging of NADPH oxidase-derived superoxide anions improves depressed baroreflex sensitivity in spontaneously hypertensive rats

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Cell metabolic changes of porphyrins and superoxide anions by anthracene and benzo(a)pyrene

The aim of this work was to evaluate the induction of protoporphyrins IX (PpIX) activity and superoxide anions (SO) in human leukocytes exposed to anthracene (ANT) and benzo(a)pyrene (B(a)P). The leukocyte LC(50)s for both hydrocarbons and the PpIX accumulation and SO overproduction were measured. The LC(50)s were 0.35 and 3.23μM for ANT and B(a)P, respectively. A linear relationship (r=0.97, p<0.01) between PpIX and ANT concentration was obtained. The induced accumulation of PpIX was proportional (r=0.63, p<0.01) to B(a)P concentration. SO overproduction showed a linear relationship (r=0.83, p<0.05) with ANT concentrations. The linear regression analysis of the effect of B(a)P on the superoxide anion overproduction showed a good coefficient (r=0.97, p<0.01), showed that ANT and B(a)P exposure induces PpIX accumulation, probably by disruption of the haem biosynthesis. ANT and B(a)P induce SO overproduction, perhaps through a process of redox cycling.