Direct observation of singlet oxygen production by merocyanine 540 associated with phosphatidylcholine liposomes

The production of singlet molecular oxygen (1O2) by the photosensitizing dye merocyanine 540 (MC540) bound to phosphatidylcholine liposomes has been demonstrated by direct detection of 1O2 luminescence at 1268 nm. 1O2 phosphorescence emission was enhanced in deuterated buffer and upon saturation of the sample with oxygen and could be quenched by the addition of sodium azide to the external medium. No 1O2 luminescence was detected in nitrogen-saturated samples, in the absence of dye, or with MC540 in aqueous solution. Photobleaching of liposome-bound MC540 was also observed to be dependent on oxygen concentration. These studies are consistent with 1O2 intermediacy in the mechanism of MC540-mediated photosensitization.     

A comparison of cobalt(II) and iron(II) hydroxyl and superoxide free radical formation

We have employed the electron spin resonance spin-trapping technique to study the reaction of Co(II) with hydrogen peroxide in a chemical system and in a microsomal system. In both cases, we employed the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and were able to detect the formation of DMPO/.OH and DMPO/.OOH. DMPO/.OOH was the predominant radical adduct formed in the chemical system, while the two adducts were of similar concentrations in the microsomal system. The formation of both of these adducts in either reaction system was inhibited by the addition of superoxide dismutase or catalase, and by chelating the cobalt with either ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The incorporation of the hydroxyl radical scavengers ethanol, formate, benzoate, or mannitol inhibited the formation of DMPO/.OH in both systems. We also repeated the study using Fe(II) in place of Co(II). In contrast to the Co(II) results, Fe(II) reacted with hydrogen peroxide to yield only DMPO/.OH, and this adduct formation was relatively insensitive to the presence of added superoxide dismutase. In addition, Fe(II)-mediated DMPO/.OH formation increased when the iron was chelated to either EDTA or DTPA rather than being inhibited as for Co(II). Thus, we propose that Co(II) does not react with hydrogen peroxide by the classical Fenton reaction at physiological pH values.     

Metal-independent production of hydroxyl radicals by halogenated quinones and hydrogen peroxide: an ESR spin trapping study

The metal-independent production of hydroxyl radicals (*OH) from H(2)O(2) and tetrachloro-1,4-benzoquinone (TCBQ), a carcinogenic metabolite of the widely used wood-preservative pentachlorophenol, was studied by electron spin resonance methods. When incubated with the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), TCBQ and H(2)O(2) produced the DMPO/*OH adduct. The formation of DMPO/*OH was markedly inhibited by the *OH scavenging agents dimethyl sulfoxide (DMSO), ethanol, formate, and azide, with the concomitant formation of the characteristic DMPO spin trapping adducts with *CH(3), *CH(CH(3))OH, *COO(-), and *N(3), respectively. The formation of DMPO/*OH and DMPO/*CH(3) from TCBQ and H(2)O(2) in the absence and presence, respectively, of DMSO was inhibited by the trihydroxamate compound desferrioxamine, accompanied by the formation of the desferrioxamine-nitroxide radical. In contrast, DMPO/*OH and DMPO/*CH(3) formation from TCBQ and H(2)O(2) was not affected by the nonhydroxamate iron chelators bathophenanthroline disulfonate, ferrozine, and ferene, as well as the copper-specific chelator bathocuproine disulfonate. A comparative study with ferrous iron and H(2)O(2), the classic Fenton system, strongly supports our conclusion that *OH is produced by TCBQ and H(2)O(2) through a metal-independent mechanism. Metal-independent production of *OH from H(2)O(2) was also observed with several other halogenated quinones.     

Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+.

Ringer's solution containing salicylic acid (5 nmol/microliters/min) was infused directly through an intracranial microdialysis probe to detect the generation of hydroxyl radicals (.OH) reflected by the formation of dihydroxybenzoic acids (DHBA) in the caudate nucleus of anesthetized rats. Brain dialysate was assayed for dopamine, 2,3-, and 2,5-DHBA by a high-pressure liquid chromatography-electrochemical (HPLC-EC) procedure. 1-Methyl-4-phenylpyridinium ions (MPP+, 0 to 150 nmol) increased dose-dependently the release of dopamine and the formation of DHBA. A positive linear correlation between the release of dopamine and the formation of 2,3- or 2,5-DHBA was observed (R2 = .98). The present results demonstrate the validity of the use of not only 2,3-DHBA but also 2,5-DHBA as an in vivo index of oxidative damage generated by reactive .OH radicals. In conclusion, the present study demonstrates a novel use of intracranial microdialysis of salicylic acid to assess the oxidative damage elicited by .OH in living brain.     

Dopaminergic denervation enhances susceptibility to hydroxyl radicals in rat neostriatum

To determine if greater amounts of hydroxyl radical (*OH) are formed by dopamine (DA) denervation and treatment with L-dihydroxyphenylalanine (L-DOPA), the neostriatum was DA denervated (99% reduction in DA content) by 6-hydroxydopamine treatment (134microg icv, desipramine pretreatment) of neonatal rats. At 10 weeks the peripherally restricted dopa decarboxylase inhibitor carbidopa (12.5mg/kg i.p.) was administered 30min before vehicle, L-DOPA (60mg/kg i.p.), or the known generator of reactive oxygen species, 6-hydroxydopa (6-OHDOPA) (60mg/kg i.p.); and this was followed 30min later (and 15 min before termination) by the spin trap, salicylic acid (8micromoles icv). By means of a high performance liquid chromatographic method with electrochemical detection, we found a 4-fold increase in the non-enzymatically formed spin trap product, 2,3-dihydroxybenzoic acid (2,3-DHBA), with neither L-DOPA nor 6-OHDOPA having an effect on 2,3-DHBA content of the neostriatum. Basal content of 2,5-DHBA, the enzymatically formed spin trap product, was 4-fold higher vs. 2,3-DHBA in the neostriatum of untreated rats, while L-DOPA and 6-OHDOPA each reduced formation of 2,5-DHBA. We conclude that DA innervation normally suppresses *OH formation, and that the antiparkinsonian drug L-DOPA has no effect (2,3-DHBA) or slightly reduces (2,5-DHBA) *OH formation in the neostriatum, probably by virtue of its bathing the system of newly formed *OH.     

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

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

Oxygen-based free radical generation by ferrous ions and deferoxamine

Deferoxamine accelerates the autooxidation of iron as measured by the rapid disappearance of Fe2+, the associated appearance of Fe3+, and the uptake of oxygen. Protons are released in the reaction. The formation of H2O2 was detected by the horseradish peroxidase-catalyzed oxidation of scopoletin, and the formation of hydroxyl radicals (OH.) was suggested by the formation of the OH. spin trap adduct (DMPO/OH). with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the generation of the methyl radical adduct on the further addition of dimethyl sulfoxide. (DMPO/OH). adduct formation was inhibited by catalase but not by superoxide dismutase. The oxidant formed converted iodide to a trichloroacetic acid-precipitable form (iodination) and was bactericidal to logarithmic phase Escherichia coli. Both iodination and bactericidal activity was inhibited by catalase and by OH. scavengers, but not by superoxide dismutase. Iodination was optimal in 5 x 10(-4) M acetate buffer, pH 5.0, and when the Fe2+ and deferoxamine concentrations were equimolar at 10(-4) M. Fe2+ could not be replaced by Fe3+, Co2+, Zn2+, Ca2+, Mg2+, or Mn2+, or deferoxamine by EDTA, diethylenetriaminepentaacetic acid, or bathophenanthroline. These findings indicate that Fe2+ and deferoxamine can act as an oxygen radical generating system, which may contribute to its biological effects in vitro and in vivo.     

Impaired phosphatidylcholine biosynthesis and ascorbic acid depletion in lung during lipopolysaccharide-induced endotoxaemia in guinea pigs

Recently there has been a moderate resurgence in the use of flax-seed in a variety of ways including bread. The scientific basis of its use is very limited. There is some claim for beneficial effects in cancer and lupus nephritis. These claims could be due to its ability to scavenge oxygen radicals. However, its antioxidant activity is not known. Recently a method has been developed to isolate secoisolariciresinol diglucoside (SDG) from defatted flax-seed in large quantity (patent pending). We investigated the ability of SDG to scavenge úOH using high pressure liquid chromatography (HPLC) method. úOH was generated by photolysis of H2O2 (1.25-10.0 \sgmaelig;moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce úOH-adduct products 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. H2O2 produced a concentration-dependent úOH as estimated by 2,3-DHBA and 2,5-DHBA. A standard curve was constructed for known concentrations of 2,3-DHBA and 2,5-DHBA against corresponding area under the peaks which then was used for measurement of 2,3-DHBA and 2,5-DHBA generated by UV irradiation of H2O2 in the presence of salicylic acid. SDG in the concentration range of 25, 50, 100, 250, 500, 750, 1000 and 2000 \sgmaelig;g/ml (36.4, 72.8, 145.6, 364.0, 728.0, 1092.0, 1456.0 and 2912.0 \sgmaelig;M respectively) produced a concentration-dependent decrease in the formation of 2,3-DHBA and 2,5-DHBA, the inhibition being 4 and 4.65% respectively with 25 \sgmaelig;g/ml (36.4 \sgmaelig;M) and 82 and 74% respectively with 2000 \sgmaelig;g/ml (2912.0 \sgmaelig;M). The decrease in úOH-adduct products was due to scavenging of úOH not and by scavenging of formed 2,3-DHBA and 2,5-DHBA. SDG prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner in the concentration range from 319.3-2554.4 \sgmaelig;M. These results suggest that SDG scavenges úOH and therefore has an antioxidant activity.     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.     

Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping.

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) gives the PBN.OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH, implying the intermediacy of H2O2 and O2 in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH. already implicated in the degradation of DNA.     

Reaction of the carbonate radical with the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide in chemical and cellular systems: pulse radiolysis, electron paramagnetic resonance, and kinetic-competition studies

Carbonate radicals (CO3-) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite-carbon dioxide adduct (ONOOCO2-), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO3(-) to yield transient species to yield finally the DMPO-OH spin adduct. In this study, the kinetics of reaction of CO3(-) with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 x 10(6) M(-1) s(-1). A Fenton system, composed of Fe(II)-DTPA plus H2O2, generated OH that was trapped by DMPO; the presence of 50-500 mM bicarbonate, expected to convert OH to CO3(-), markedly inhibited DMPO-OH formation. This was demonstrated to be due mainly to a fast reaction of CO3(-) with FeII-DTPA (k=6.1 x 10(8) M(-1) s(-1)), supported by kinetic analysis. Generation of CO3(-) by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3'-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO3(-). Similarly, the formation of CO3(-) by CuZnSOD/H(2)O(2)/bicarbonate and peroxynitrite-carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO3(-) with DMPO to yield DMPO-OH was shown in peroxynitrite-forming macrophages. In conclusion, CO3(-) reacts quite rapidly with DMPO and may contribute to DMPO-OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO3(-) will be sensitive to the presence of DMPO.     

Effect of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) on human neutrophil function: novel inhibition of neutrophil stimulus-response coupling?

Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.     

Thiol-induced hydroxyl radical formation and scavenger effect of thiocarbamides on hydroxyl radicals

The effects of thiols and thiocarbamides on hydroxyl radical (.OH) formation by the hypoxanthine(HYP)-xanthine oxidase(XOD)-Fe3+ .EDTA system were investigated in the range of 0.5-5 mM by colorimetrically measuring salicylate hydroxylation. Thiocarbamides powerfully inhibited the hydroxylation while thiols showed a paradoxical effect, enhancing it at low concentrations, but inhibiting it at high ones. Thiols in the presence of Fe3+ .EDTA generated superoxide anions (O2-.) and .OH during the oxidation, but thiocarbamides did not. A study of the effect of ergothioneine, a thiocarbamide present in mammals, on the .OH spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by EPR spectrometry showed that it effectively decreased the .OH spin adduct without causing the appearance of other signals. Reaction mechanisms are proposed for the O2-. evolution and .OH formation by the thiols themselves in the presence of Fe3+ .EDTA and .OH with thiols and thiocarbamides.     

When are metal ion-dependent hydroxyl and alkoxyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide artifacts?

The formation of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/.OH adduct of the spin trap DMPO has been reported to occur through nucleophilic addition of water in the presence of aqueous ferric chloride (K. Makino, T. Hagiwara, A. Hagi, M. Nishi, and A. Murakami, 1990, Biochem. Biophys. Res. Commun. 172, 1073-1080). Due to the serious implications of these findings with respect to many spin trapping studies, the suitability of DMPO as a hydroxyl radical spin trap was studied in typical Fenton systems. Using 17O-enriched water, we show conclusively that nucleophilic addition of water occurs at the nitrone carbon (or C-2 position) of DMPO in the presence of either Fe or Cu ions. Furthermore, our results demonstrate that this nucleophilic reaction is a major pathway to the DMPO/.OH adduct, even during the reaction of Fe(II) or Cu(I) with hydrogen peroxide. Primary alkoxyl adducts of DMPO also form in aqueous solution through nucleophilic addition in the presence of both Fe(III) and Cu(II). Attempts to obtain secondary and tertiary alkoxyl adducts by this mechanism were unsuccessful, possibly due to steric effects. When the reaction is carried out in various buffers, however, or in the presence of metal ion chelators, nucleophilic addition to DMPO from Fe(III) is effectively suppressed. Chelators also suppress the reaction with Cu(II). Hence, under most common experimental conditions in biochemical free radical research, nucleophilic addition to DMPO should not be of major concern.     

Activated oxygen species produced by photoexcited furocoumarin derivatives

Furocoumarin derivatives (FCD) are investigated in order to determine their ability to photosensitize the production of activated oxygen species. Using the method based on the specific singlet oxygen (1O2) oxydation of cholesterol, all FCD except bergaten appeared to be able to produce 1O2 with various efficiencies. EPR spin trapping experiments show that photoexcited FCD produce hydroxyl radicals as detected by the formation of a DMPO-OH signal which can be abolished when the photosensitization reaction is carried out in the presence of specific OH scavengers. Moreover, the photo-ejection of hydrated electron (e-) by FCD is demonstrated by the loss of paramagnetic absorption of nitroxide free radicals as e- trap.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Production of hydroxyl free radical in the xanthine oxidase system with addition of 1-methyl-3-nitro-1-nitrosoguanidine

We have examined the mechanism of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG)-induced gastric cancer with respect to the production of hydroxyl free radical (OH). Nucleophilic attack by H2O2 on the nitroso group of MNNG produces 1-methyl-3-nitroguanidine (MNG) and the intermediate peroxynitric acid (ONOOH), which splits into hydroxyl free radical (OH) and nitrogen dioxide leading to the formation of nitric and nitrate ions in water. Xanthine oxidase (XO) induces the production of O2.- or H2O2 from molecular oxygen, depending on the overall level of enzyme reduction. In this study, we examined OH production by the reaction of MNNG with H2O2 derived from the XO-HX system containing XO and the purine substrate hypoxanthine by ESR using the spin trapping reagent 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). OH was produced in the XO-HX-DMPO system with addition of MNNG (the MNNG-XO-HX-DMPO system) under aerobic conditions, but was not in the XO-HX-DMPO system, and production of OH was inhibited by catalase but not by superoxide dismutase, suggesting that OH was produced by the reaction of MNNG with H2O2 derived from the XO-HX system. The production of OH was significantly increased with increase in the reducing activity of XO, though that of O2.- was not, also suggesting the O2(.-)-independent .OH production. The productions of nitrite ion and MNG in the MNNG-XO-HX system were determined by the colorimetric method and HPLC, respectively. Based on these findings, we conclude that .OH was produced by homolytic split of the intermediate ONOOH formed by nucleophilic attack of H2O2 derived from the XO-HX system on MNNG.     

Antioxidant activity of allicin,an active principle in garlic

Garlic has been claimed to be effective against diseases, in the pathophysiology of which oxygen free radicals (OFRs) have been implicated. Effectiveness of garlic could be due to its ability to scavenge OFRs. However, its antioxidant activity is not known. We investigated the ability of allicin (active ingredient of garlic) contained in the commercial preparation Garlicin to scavenge hydroxyl radicals (·OH) using high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce ·OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced a concentration-dependent ·OH as estimated by ·OH adduct products 2,3-DHBA and 2,5-DHBA. Allicin equivalent in Garlicin (1.8, 3.6, 7.2, 14.4, 21.6, 28.8 and 36 g) produced concentration-dependent decreases in the formation of 2,3-DHBA and 2,5-DHBA. The inhibition of formation of 2,3-DHBA and 2,5-DHBA with 1.8 g/ml was 32.36% and 43.2% respectively while with 36.0 g/ml the inhibition was approximately 94.0% and 90.0% respectively. The decrease in ·OH adduct products was due to scavenging of ·OH and not by scavenging of formed ·OH adduct products. Allicin prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner. These results suggest that allicin scavenges ·OH and Garlicin has antioxidant activity.     

Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase

We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes.