Spin Trap DMPO Research Articles

Investigation on photogenerated radicals of some carboxylates in solution and in aqueous dispersions of AgBr

The photogenerated free radicals from formate, oxalate and acetate in aqueous solutions without and with dispered AgBr grains under illuminations of 355 nm laser light, natural light and monochromatic red, green or blue light were detected by electron spin resonance with spin-trap DMPO. The results showed that: (a) ·CO2− radicals were produced in the formate solution by all of these illuminations; (b) the signals of ·CO2− radicals were greatly intensified when an AgBr dispersion was present in the formate solution; (c) the signals of ·CO2− radicals in the oxalate solution were also detected and intensified when an AgBr dispersion was present, but only after illumination by the 355 nm laser; (d) ·CH3 radicals, instead of ·CO2−, were weakly detected in the acetate solution, but only in the presence of AgBr under illumination by the 355 nm laser. The photochemical behaviour of these carboxylates, particularly formate and oxalate, perhaps implies their capability for trapping photogenerated holes and their potential for acting as a hole converter to an effective electron carrier.

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 2O 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 2O 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 2O 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 2O 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 2O 2, the classic Fenton system, strongly supports our conclusion that OH is produced by TCBQ and H 2O 2 through a metal-independent mechanism. Metal-independent production of OH from H 2O 2 was also observed with several other halogenated quinones.

The use of the tertiary alkyl tetraoxide–peroxyl equilibrium, ROOOOR ⇌ 2RO2˙, as a clean source of tertiary alkyl peroxylsElectronic supplementary information (ESI) available: second level approximation. See http://www.rsc.org/suppdata/p2/b1/b108805j/

Below 155 K there is a dynamic equilibrium between tertiary alkylperoxyls and their tetraoxide combination products. The system can be recycled repeatedly between 113 and 155 K without loss of radicals. The stationary peroxyl concentration depends solely on the temperature and the initial tetraoxide level. The equilibrium provides a clean source of peroxyls, since no other radicals such as alkoxyls are present. We have used the equilibrium to determine the mechanisms, and to estimate the rate constants for the reactions of tertiary alkylperoxyls with antioxidant and spin trap substrates. The method depends on rapidly mixing cold solutions of the substrates and solutions containing equilibrium concentrations of tertiary peroxyls. The mixing takes place in situ within an EPR cavity and the decay of the peroxyl signal is monitored continuously by EPR. The decay profiles have been kinetically analysed at three levels of approximation with the most accurate rate parameters being obtained by kinetic modelling of the total reaction scheme.The rate parameters for the reaction of peroxyls and the antioxidant, IONOL, are in good agreement with those determined by other kinetic EPR methods (KEPR). Studies of the reaction with the much used spin trap DMPO have shown that a rapid removal of peroxyls does not result in a paramagnetic mono-radical spin adduct, but yields a diamagnetic product probably containing two peroxyls added sequentially or concertedly to the spin trap.

Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase

The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms a superoxide adduct with a half-life of almost 15 min. DEPMPO is very hydrophilic and its use for the detection of radicals in the lipid phase (lipid-derived radicals and superoxide generated in the lipid phase) is therefore limited due to its very low concentration in the lipid phase. For the detection of lipid-derived radicals, three derivatives of DEPMPO with increasing degree of lipid solubility have been investigated: 5-(di-n-propoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DPPMPO), 5-(di-n-butoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DBPMPO), and 5-(bis-(2-ethylhexyloxy)phosphoryl)-5-methyl-1-pyrroline N-oxide (DEHPMPO). As compared with the spin trap DMPO, the half-lives of the respective superoxide adducts were clearly higher in aqueous solutions of the spin traps, which facilitates qualitative ESR measurements. The stability of the superoxide spin adducts formed with the various lipophilic spin traps in aqueous buffer were similar to those observed with DEPMPO (half-life: 7–11 min.). In model experiments using Fe 3+-catalyzed nucleophilic addition of methanol or tert-butanol to the respective spin trap the respective alkoxyl radical adducts were formed in aqueous solution as transient species in the presence of high concentrations of the alcohol. Upon dilution with water the alkoxyl group was substituted by water, giving the respective hydroxyl adduct of the spin trap. Care must therefore be taken when Fenton-type reactions are used for the generation of radicals such as the use of Fe 2+ complexes with phosphate or DTPA or inactivation of iron by addition of “Desferal” (Novarti’s Pharma GmbH, Vienna, Austria) after a short incubation time. Addition of Fe 2+ under anaerobic conditions to an aqueous suspension of linoleic acid hydroperoxide and the spin trap resulted in the detection of three different species: a carbon-centered radical adduct, an acyl radical adduct, and the hydroxyl adduct. In the presence of oxygen a different species was observed with DEPMPO, DPPMPO, and DBPMPO, which was only slightly suppressed upon the addition of SOD, possibly the respective spin adduct of either the alkylperoxyl radical or, in analogy to DMPO, a secondary alkoxyl radical.

Reduction of 1,3-diphenyl-1-triazene by rat hepatic microsomes, by cecal microflora, and in rats generates the phenyl radical metabolite: nn ESR spin-trapping investigation.

An ESR spin-trapping technique was used to determine whether free radical metabolites are formed as a result of the reduction of 1, 3-diphenyl-1-triazene (DPT) in vivo and in vitro by components of the cytochrome P450 (P450) mixed-function oxidase system in microsomes or by gut microflora in anaerobic cecal incubations. The ESR spectrum of the DMPO-phenyl radical adduct was detected in a microsomal incubation containing DPT, DMPO, and NADPH with the following hyperfine coupling constants: a(N) = 15.95 G and = 24.37 G. The amplitude of the spectrum from the phenyl radical adduct generated in microsomal incubations of DPT with DMPO and NADPH was not attenuated by the P450 inhibitor 1-aminobenzotriazole (ABT) or by carbon monoxide, indicating that P450 is not significantly involved in phenyl radical formation. The formation of a DMPO-phenyl radical adduct was also catalyzed by recombinant human cytochrome P450 reductase. Addition of anti-rat P450 reductase antibody led to an attenuation of the signal in incubations containing either microsomes or reductase. Low concentrations of DMPO-phenyl radical adducts were detected by ESR in the toluene extract of cecal contents containing DPT and the spin trap. In the in vivo experiments with rats treated with DPT and the spin trap DMPO, the six-line ESR signal of the DMPO-phenyl radical adduct was readily detected in bile 40-60 min after rats were treated with DPT and DMPO. The results show for the first time that the phenyl radical is formed by the reduction of DPT and may indicate a toxic potential for this chemical.

Physicochemical Modeling of the Role of Free Radicals in Photodynamic Therapy: V. Quantification of Spin-Trapping Kinetics

A new kinetic approach is suggested and experimentally supported for quantification of the spin-trapping procedure. Accordingly, the concentration of the spin adduct formed in the interaction between the spin-trap DMPO and various free radicals (cyanopropylperoxy, cumylperoxy, phenylethylperoxy, and hydroperoxy radicals) generated by the initiated oxidation of the parent molecules is followed by kinetic ESR spectrometry. The initial sections of the corresponding kinetics are linear indicating that during this period the consumption of the adduct is negligible and thus the rate of accumulation (WA) approximates the rate of formation (Wf): WA ∼ Wf = kST[Rad•][DMPO], supported also by the fact that the rate of initiation of oxidation equals WA at high [DMPO]. In addition, the circulatory experimental apparatus enables calculation of the rate of molecular decomposition of the adduct by stopping circulation (Wf becomes negligible) and following the decrease of the ESR signal. Corresponding rate constants are summarized.

Glycoconjugated hypocrellin: photosensitized generation of free radicals (O 2•−, [rad]OH, and GHB •−) and singlet oxygen ( 1O 2)

To improve water solubility and specific affinity for malignant tumors, glycoconjugated hypocrellin B (GHB) has been synthesized. Illumination of deoxygenated DMSO solution containing GHB generates a strong electron paramagnetic resonance (EPR) signal. The EPR signal is assigned to the semiquinone anion radical of GHB (GHB •−) based on a series of experimental results. Spectrophotometric measurements show that the absorption bands at 645 nm and 502 nm (pH 8.0) or 505 nm (pH 11.0) arise from the semiquinone anion radical (GHB •−) and hydroquinone (GHBH 2) of GHB, respectively. GHBH 2 is readily formed via the decay of GHB •− in water-contained solution. The increase of pH value of the reaction media promotes this process. When oxygen is present, superoxide anion radical (O 2 •−) is formed, via the electron transfer from GHB •−, the precursor, to ground state molecular oxygen. Hydroxyl radical can be readily detected by DMPO spin trapping when aerobic aqueous solution containing GHB is irradiated. As compared with the parent compound, hypocrellin B (HB), the efficiency of O 2 •− and OH generation by GHB photosensitization is enhanced significantly. Singlet oxygen ( 1O 2) can be produced via the energy transfer from triplet GHB to ground state oxygen molecules, with a decreased quantum yield, i.e., 0.19. These findings suggest that the new GHB possesses an enhanced type I process and a decreased type II process as compared with hypocrellin B.

Generation of thiyl radical by nitric oxide: a spin trapping study

The free radical intermediates formed during the nitrosation of thiols including glutathione (GSH), L-cysteine (CystSH), captopril (CapSH) and N-acetylpenicillamine (NAPenSH) by NO˙ at physiological pH have been investigated using EPR spectroscopy combined with the spin trap DMPO. We have found that NO˙ in the presence of oxygen reacted with GSH, CystSH and CapSH to generate the corresponding thiyl radicals, which are spin trapped by DMPO to give the spin trapped adducts DMPO-SG, DMPO-SCyst and DMPO-SCap respectively. In the case of NAPenSH no spin trapped adduct is detected. Desferrioxamine, an inhibitor of peroxynitrite (ONOO–), has no effect on the formation of either DMPO-SG or DMPO-SCyst, suggesting that ONOO– is not involved in the formation of GS˙ and Cyst˙. In contrast, desferrioxamine inhibits the detection of DMPO-SCap, pointing to a role for ONOO– in the formation of CapS˙. Our results demonstrate that ˙NO2 is the most likely reactive species in the nitrosation of GSH and CystSH by oxygenated NO˙, thereby generating the corresponding thiyl radicals. Furthermore, our data indicate that the scavenging of NO˙ by thiols may lead to the production of superoxide (O2˙–) via the formation of thiyl radicals.

EPR Characteristics of a dye/colloidal TiO2 system under visible light irradiation

An EPR study is reported of paramagnetic species and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin-trapped adducts formed in a system composed of the ethyl ester of fluorescein (FLEt) and colloidal TiO2 particles under 532 nm pulsed laser irradiation in deaerated and aerated ethanolic media. Following excitation of FLEt, electron injection takes place from the singlet excited state 1FLEt* to the conduction band of TiO2 leading to formation of the radical cations FLEtNsbd+ in oxygen-free dispersions, and whose EPR signals have g = 2.0047. In the presence of oxygen, 532 nm irradiation of the ethanolic FLET/TiO2 dispersion first produced EPR signals with g = 2.0147, tentatively identified with the peroxy radical cation O2FLEtNsbd+, which on further irradiation decreased in intensity and were followed by the concomitant appearance of EPR signals (g = 2.0047) of increasing intensity of the FLEtNsbd+ radicals. Following the addition of the DMPO spin-trap, irradiation of the aerated dispersion gave EPR spectra consistent with formation of superoxide radical anions O2− through scavenging of the electrons on the colloidal TiO2 particles. The mechanistic details that led to changes in the EPR signals and to the generation of O2− are described.

DNA strand-breaking activity and mutagenicity of 2,3-dihydro-3,5-dihydroxy-6-methyl-4 H-pyran-4-one (DDMP), a Maillard reaction product of glucose and glycine

Aqueous solution of glucose and glycine was heated under reflux for 4 h and extracted with ethyl acetate. Reversed phase HPLC of the extract revealed a new DNA strand-breaking substance, which was purified by repeated HPLC and identified as 2,3-dihydro-3,5-dihydroxy-6-methyl-4 H-pyran-4-one (DDMP). DDMP induced DNA strand breaking in a dose- and time-dependent manner. It was active to break DNA strands at pH 7.4 and 9.4. Its pyranone skeleton was destroyed at the pH values. DNA strand breaking by DDMP was inhibited by superoxide dismutase, catalase, scavengers for hydroxyl radical, spin trapping agents and metal chelators, and the breaking was enhanced by Fe(III) ion. A mixture of DDMP and a spin trap DMPO gave electron spin resonance signals of a spin adduct DMPO-OH, indicating generation of hydroxyl radical. DDMP was found to be mutagenic to Salmonella typhimurium TA100 without metabolic activation. These results show DDMP generated active oxygen species to cause DNA strand breaking and mutagenesis.

DMPO Spin Trapping of Superoxide Anion in Strong Alkaline DMSO Solution

Abstract Superoxide anion (O2•−), generated in a strong alkaline DMSO solution and detected by direct 77 K EPR, was trapped with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and the resultant nitroxide radical, DMPO/O2•−, has been found to be sufficiently stable for the subsequent EPR detection.

Detection and Characterization of the Electron Paramagnetic Resonance-Silent Glutathionyl-5,5-dimethyl-1-pyrrolineN-Oxide Adduct Derived from Redox Cycling of Phenoxyl Radicals in Model Systems and HL-60 Cells

The antioxidant function of glutathione includes enzymatic reduction of hydrogen peroxide by glutathione peroxidase and nonenzymatic reduction of organic radicals and reactive oxygen species. The glutathionylS-centered radical, formed by the nonenzymatic reduction process, is a marker of oxidative reactions proceeding by radical mechanisms. Spin-adducts of glutathionyl radicals with the spin trap DMPO, 5,5-dimethyl-1-pyrrolineN-oxide, are not sufficiently stable and can be detected only under steady-state conditions. We developed a novel HPLC method for the detection of an EPR-silent DMPO adduct of glutathionyl radicals in model systems and in cells. We synthesized a sufficient quantity of the adduct for characterization by UV spectrophotometry, ionspray mass spectrometry, and1H NMR spectroscopy. The UV absorption λmaxof the adduct, 258 nm, was indicative of a 2-(S-alkylthiyl)pyrrolineN-oxide chromophore. The molecular mass of the adduct was 418 amu. No signal for the C2proton of the DMPO-derived portion of the adduct was evident in its1H NMR spectrum. The results were consistent with the structure 2-(S-glutathionyl)-5,5-dimethyl-1-pyrrolineN-oxide (GS-DMPO nitrone). We showed that this adduct accumulated in the course of peroxidase-dependent redox cycling of phenol in the presence of glutathione and DMPO as well as in HL-60 cells exposed to a phenol/H2O2/DMPO reaction mixture. The EPR-silent GS-DMPO nitrone was readily assayed by HPLC under conditions incompatible with the detection of the GS-DMPO nitroxide by EPR. This is to our knowledge the first direct experimental evidence for the redox cycling of phenol in this bone marrow-derived cell line. The method may prove useful in the study of radical-driven oxidations of glutathione in various pathophysiological processes associated with radical mechanisms.

Oxygen-Centered Spin Adducts of 5,5-Dimethyl-1-pyrrolineN-oxide (DMPO) and 2H-Imidazole 1-Oxides

The hydroxyl and some alkoxyl spin adducts of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) are difficult to assign due to the remarkable similarity of their EPR spectra. The utility of resolving superhyperfine (SHF) structure followed by computer simulations has been demonstrated to assist in the assignment of EPR spectra with close values of hyperfine splitting constants, e.g., DMPO/·OH and DMPO/·OR. Here,·OR is the alkoxyl radical derived from thermal decomposition of 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH). In addition, two other spin traps, derivatives of 2H-imidazole 1-oxide, namely, 2,2,4-trimethyl-2H-imidazole 1-oxide (TMIO) and 2,2-dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO), have been used in a model study to develop a procedure for distinguishing between oxygen-centered spin adducts. These results are compared with those for DMPO. TMIO and DMPIO spin traps provide more distinguishable individual spectra with·OH and AAPH-derived·OR radicals than the DMPO spin trap. The formation of DMPO/·OR(AAPH) and DMPIO/·OR(AAPH) spin adducts was confirmed by mass spectrometry. The comparison of spin trapping by DMPO and 2H-imidazole 1-oxides using typical biological sources of other oxygen-centered radicals reveals application limits of these spin traps. For example, 2H-imidazole 1-oxides do not form superoxide spin adducts in the xanthine/xanthine oxidase system. Also, for the first time, experimental evidence is presented for SHF structure in spectra of TMIO and DMPIO spin adducts with·OH/·OD and·CH3/·CD3radical species.

Generation Efficiency of the Hydroxyl Radical Adduct of the DMPO Spin Trap in Homogeneous and Heterogeneous Media

AbstractThe generation efficiency of the hydroxyl radical adduct (DMPO-OH) from the spin trap 5,5-dimethyl-1- pyrroline N-oxide (DMPO) has been determined, both in a homogeneous system (H

Identification of Reactive Free Radicals with a New 31P-Labeled DMPO Spin Trap

Identification of Reactive Free Radicals with a New 31P-Labeled DMPO Spin Trap

Reduction of ferrylmyoglobin and ferrylhemoglobin by nitric oxide: a protective mechanism against ferryl hemoprotein-induced oxidations.

The reactions of metmyoglobin (metMb) and methemoglobin (metHb), oxidized to their respective oxoferryl free radical species (.Mb-FeIV = O/.Hb-4FeIV = O) by tert-butyl hydroperoxide (t-BuOOH), with nitric oxide (NO.) were studied by a combination of optical, electron spin resonance (ESR), ionspray mass (MS), fluorescence, and chemiluminescence spectrometries to gain insight into the mechanism by which NO. protects against oxidative injury produced by .Mb-FeIV = O/.Hb-4FeIV = O. Oxidation of metMb/metHb by t-BuOOH in a nitrogen atmosphere proceeded via the formation of two protein electrophilic centers, which were heme oxoferryl and the apoprotein radical centered at tyrosine (for the .Mb-FeIV = O form, the g value was calculated to be 2.0057), and was accompanied by the formation of t-BuOOH-derived tert-butyl(per)oxyl radicals. We hypothesized that NO. may reduce both oxoferryl and apoprotein free radical electrophilic centers of .Mb-FeIV = O/.Hb-4FeIV = O and eliminate tert-butyl(per)oxyl radicals, thus protecting against oxidative damage. We found that NO. reduced .Mb-FeIV = O/.Hb-4FeIV = O to their respective ferric (met) forms and prevented the following: (i) oxidation of cis-parinaric acid (PnA) in liposomes, (ii) oxidation of luminol, and (iii) formation of the tert-butyl(per)oxyl adduct with the spin trap DMPO. NO. eliminated the signals of tyrosyl radical detected by ESR and oxoferryl detected by MS in the reaction of t-BuOOH with metMb. As evidenced by MS of apomyoglobin, this effect was due to the two-electron reduction of .Mb-FeIV = O by NO. at the oxoferryl center rather than to nitrosylation of the tyrosine residues. Results of our in vitro experiments suggest that NO. exhibits a potent, targetable antioxidant effect against oxidative damage produced by oxoferryl Mb/Hb.

Permeabilization of the inner mitochondrial membrane by Ca 2+ ions is stimulated by t-butyl hydroperoxide and mediated by reactive oxygen species generated by mitochondria

The extent of swelling undergone by deenergized mitochondria incubated in KC1/sucrose medium in the presence of Ca 2+ alone or Ca 2+ and t-butyl hydroperoxide decreases by decreasing molecular oxygen concentration in the reaction medium; under anaerobiosis no swelling occurs. This swelling is also inhibited by the presence of exogenous catalase or by the Fe 2+ chelator o-phenanthroline in a time-dependent manner. The production of protein thiol cross-linking that leads to the formation of protein aggregates induced by Ca 2+ and t-butyl hydroperoxide does not occur when mitochondria are incubated in anaerobic medium or in the presence of o-phenanthroline. In addition, it is also shown that the yield of stable methyl radical adducts, obtained from rat liver mitochondria treated with t-butyl hydroperoxide and the spin trap DMPO, is reduced by addition of EGTA and increases by addition of Ca 2+ ions. These data support the hypothesis that Ca 2+ ions stimulate electron leakage from the respiratory chain, increasing the mitochondrial production of reactive oxygen species.

Measurements of Superoxide Anion Radical and Superoxide Anion Scavenging Activity by Electron Spin Resonance Spectroscopy Coupled with DMPO Spin Trapping

Abstract A quantitative analysis of superoxide anion radical (·O2−) and hydroperoxyl radical (·OOH) generated in the hypoxanthine–xanthine oxidase (HPX–XOD) reaction system in the presence of dimethyl sulfoxide (DMSO) was explored by a spin-trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) combined with electron spin resonance spectroscopy (ESR). ·O2− and/or ·OOH was detected by ESR spectra of the spin adduct, DMPO–O2− (or DMPO–OOH). The concentration of DMPO–O2− was increased up to three times by the addition of DMSO. The half-life of DMPO–O2−, which is the time period to reduce to one-half of the initial intensity, also became about 70 times longer than that in the system without DMSO. These results suggest that the short half-life of DMPO–O2− that has been reported is attributable to the partial reaction of hydroxyl radical (·OH) with DMPO–O2−. Consequently quantitative analysis of ·O2− was possible in the presence of DMSO (>0.35 M). Under these conditions, kinetic approaches show that the generation of ·O2− in the HPX-XOD reaction is a first-order reaction and that its rate constant is 6.9 × 10−8 M s−1. Finally, the competitive reaction of DMPO and SOD toward ·O2− was shown to be one unit of superoxide dismutase (Cu/Zn– SOD) scavenging ·O2− by the rate constant of 7.0 × 10−6 M min−1. This method, which can be used for measurement of SOD-like and SOD-minic activity, should be called the superoxide anion scavenging activity method.

Hamamelitannin as a new potent active oxygen scavenger

The active oxygen scavenging activities of hamamelitannin (2′, 5-di- O-galloylhamamelose), which is a major constituent of Hamamelis virginiana, gallic acid, which is thought to be the active moiety of hamamelitannin, and propyl gallate which is a well-known antioxidant, were evaluated. In superoxide anion scavenging, the IC 50 values, which represent the concentration giving 50% inhibition of active oxygens generated, of hamamelitannin (1.31±0.06 μM), gallic acid (1.01±0.03 μM) and propyl gallate (1.41±0.01 μM) were higher than that of ascorbic acid (23.31±2.23 μM). In hydroxyl radical scavenging, hamamelitannin gave the lowest IC 50 value (5.46±0.04 μM) among the compounds tested; the values of gallic acid and propyl gallate were 78.04±11.23 and 86.46±2.31 μM, respectively. In singlet oxygen scavenging, the IC 50 values of hamamelitannin, gallic acid and propyl gallate were 45.51±2.00, 69.81±4.66 and 66.66±2.46 μM, respectively. Hamamelitannin was also found to have antioxidative and scavenging activities against organic radicals like DPPH (1, 1-diphenyl-2-picrylhydrazyl). In a cell culture system, the protective activities of hamamelitannin on cell damage induced by active oxygens, such as superoxide anion radicals, hamamelitannin on cell damage induced by active oxygens, such as superoxide anion radicals, hydroxyl radicals and singlet oxygens, were also evaluated. Against superoxide anion radicals hamamelitannin and propyl gallate protected the cells at over 50 μM, while the protective activity of gallic acid was observed at 500 μM. In contrast, against hydroxyl radicals, hamamelitannin showed protective activity at over 500 μM, while gallic acid and propyl gallate protected at 50 μM. Against singlet oxygens, hamamelitannin indicated survival of 80.6±1.5% at 100 μM, in which the survival of the control was 60.4±6.2%. Gallic acid and propyl gallate showed survivals of 98.6±7.3 and 85.2±4.9% at 500 μM, respectively. DMPO (5,5-dimethyl-1-pyrroline- N-oxide) and PBN (α-phenyl- N-t-butylnitrone) typical ESR spin-trap reagents, were found to protect against cell damage induced by superoxide anion and hydroxyl radicals.

Nitric oxide, superoxide and peroxynitrite: Putative mediators of NMDA-induced cell death in cerebellar granule cells

In this study, we analysed the implication of superoxide ( O 2 . ) and nitric oxide (NO .) free radicals and their resulting product peroxynitrite (ONOO −) in the neuronal death induced by the activation of the glutamatergic receptor of the N- methyl- d-aspartate (NMDA) subtype using cultured cerebellar granule cells. The NO . donor SIN-1 (3-morpholinosydnonimine N-ethylcarbamide), at concentrations which produced a much higher guanylate cyclase activation (i.e. NO . concentration) than NMDA, was not neurotoxic and did not increase the NMDA-induced neuronal death. The absence of involvement of NO . in NMDA-induced neuronal death was confirmed by the ineffectiveness of l -N G - nitroarginine ( l-Narg) as a neuroprotective compound. Electron paramagnetic resonance (EPR) experiments, using 5,5-dimethyl pyrroline 1-oxide (DMPO) as a spin trap, indicated that NMDA receptor stimulation led to the generation of O 2 . from at least 15–30 min. The generation of O 2 . by xanthine (XA)-xanthine oxidase (XO) induced a neuronal death similar to that of NMDA. XA-XO-induced neuronal death was suppressed by addition of either superoxide dismutase (SOD) plus catalase (CAT), or DMPO in the incubation medium. In contrast, NMDA-induced neuronal death was widely blocked by DMPO and other spin trap compounds, but not by SOD ± CAT. XA-XO-induced neuronal death was not potentiated by SIN-1 indicating that ONOO − is not more toxic than O 2 . in our neuronal model.