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The direct photolysis of reversible addition fragmentation chain transfer (RAFT) agents under visible light was demonstrated by electron spin resonance (ESR) using 5,5-dimethyl-1-pyrroline N-oxide as a typical spin trap. The hyperfine coupling lines obtained by ESR spectroscopy showed the successful capture of the carbon-centered and the sulfur-centered radical. Photo-polymerization of vinyl acetate under different wavelengths was performed to verify the effects of wavelength on the process. The effect of the R group of RAFT agents on the photolysis was investigated by spin-trapping experiments using poly (butyl acrylate) and poly (vinyl acetate) as macroRAFT agents. The quantitative experiment showed the yield of photolysis of a xanthate to be only 0.023% under &lambda, &gt, 440 nm.

Skin allergy is a major public health problem. It is the most important immunotoxic reaction in humans. Understanding all mechanisms leading to allergic-inflammatory reactions is essential in order to evaluate the sensitization potential of molecules for risk assessment purposes. Hydroperoxides derived from autoxidation of odorant terpenes used in cosmetics cause skin sensitization. The objective was to study the involvement of radical mechanisms in the formation of the antigenic entities of these compounds. An integrated approach of three actions was proposed: - "in chemico" : reactivity studies with amino acids to establish a complete reactivity profile via radical reactions ; - "in situ" : formation and behavior of radical intermediates in a reconstructed human epidermis 3D model ; - "in cellulo" : ability to activate the innate immune system, namely dendritic cells in their natural keratinocyte context; L’allergie cutanée représente un problème majeur de santé publique. La compréhension de tous les mécanismes conduisant à des réactions allergiques est capitale afin d’évaluer le potentiel de sensibilisation des molécules à risques. Les hydroperoxydes dérivés de l’autoxydation de terpènes utilisés dans les produits cosmétiques sont à l’origine de sensibilisations cutanées. L’objectif a été d’étudier l’intervention de mécanismes radicalaires dans la formation d’entités antigéniques de ces composés. Une approche intégrée de trois actions a été proposée : - "in chemico" : études de réactivité vis-à-vis des acides aminés afin d’établir un profil de réactivité complet suscitant des réactions radicalaires; - "in situ" : étude de la formation et comportement des intermédiaires radicalaires formés dans un modèle 3D d’épiderme humain reconstitué ; - "in cellulo" : étude de leur capacité à activer le système immunitaire inné, à savoir les cellules dendritiques dans leur contexte naturel kératinocytaire.

This paper reports and discusses the beneficial effects on the quality of electrospun polycaprolactone nanofibers brought about by pretreatment of the solvent with non-thermal plasma. Chloroform/dimethylformamide 9:1 (CHCl3:DMF 9:1) and pure chloroform were pretreated by a few minute exposure to the plasma generated by an atmospheric pressure plasma jet (APPJ). Interestingly, when pure chloroform was used, the advantages of plasma pretreatment of the solvent were way less pronounced than found with the CHCl3:DMF 9:1 mixture. The chemical modifications induced by the plasma in the solvents were investigated by means of complementary analytical techniques. GC-MS revealed the formation of solvent-derived volatile products, notably tetrachloroethylene (C2Cl4), 1,1,2,2-tetrachloroethane (C2H2Cl4), pentachloroethane (C2HCl5), hexachloroethane (C2Cl6) and, in the case of the mixed solvent, also N-methylformamide (C2H5NO). The chlorinated volatile products are attributed to reactions of ·Cl and Cl-containing methyl radicals and carbenes formed in the plasma-treated solvents. ·Cl and ·CCl3 radicals were detected and identified by EPR spectroscopy analyses. Ion chromatography revealed the presence of Cl-, NO 3 - , and HCOO- (the latter only in the presence of DMF) in the plasma-treated solvents, thus accounting for the observed increased conductivity and acidification of the solvent after plasma treatment. Mechanisms for the formation of these solvent derived products induced by plasma are proposed and discussed. The major role of radicals and ions in the plasma chemistry of chloroform and of the chloroform/dimethylformamide mixture is highlighted. The results provide insight into the interaction of plasma with organic solvents, a field so far little explored but holding promise for interesting applications.

Skin allergy is a major public health problem. It is the most important immunotoxic reaction in humans. Understanding all mechanisms leading to allergic-inflammatory reactions is essential in order to evaluate the sensitization potential of molecules for risk assessment purposes. Hydroperoxides derived from autoxidation of odorant terpenes used in cosmetics cause skin sensitization. The objective was to study the involvement of radical mechanisms in the formation of the antigenic entities of these compounds. An integrated approach of three actions was proposed: - "in chemico" : reactivity studies with amino acids to establish a complete reactivity profile via radical reactions ; - "in situ" : formation and behavior of radical intermediates in a reconstructed human epidermis 3D model ; - "in cellulo" : ability to activate the innate immune system, namely dendritic cells in their natural keratinocyte context, L’allergie cutanée représente un problème majeur de santé publique. La compréhension de tous les mécanismes conduisant à des réactions allergiques est capitale afin d’évaluer le potentiel de sensibilisation des molécules à risques. Les hydroperoxydes dérivés de l’autoxydation de terpènes utilisés dans les produits cosmétiques sont à l’origine de sensibilisations cutanées. L’objectif a été d’étudier l’intervention de mécanismes radicalaires dans la formation d’entités antigéniques de ces composés. Une approche intégrée de trois actions a été proposée : - "in chemico" : études de réactivité vis-à-vis des acides aminés afin d’établir un profil de réactivité complet suscitant des réactions radicalaires; - "in situ" : étude de la formation et comportement des intermédiaires radicalaires formés dans un modèle 3D d’épiderme humain reconstitué ; - "in cellulo" : étude de leur capacité à activer le système immunitaire inné, à savoir les cellules dendritiques dans leur contexte naturel kératinocytaire.

As superoxide anion is of keen interest in biomedical research, it is highly desirable to have a technique allowing its detection sensitively and specifically in biological media. If electron paramagnetic resonance (EPR) techniques and probes have been individually described in the literature, there is actually no comparison of these techniques in the same conditions that may help guiding researchers for selecting the most appropriate approach. The aim of the present study was to compare different EPR strategies in terms of sensitivity and specificity to detect superoxide (versus hydroxyl radical). Three main classes of EPR probes were used, including paramagnetic superoxide scavengers (such as nitroxides TEMPOL and mitoTEMPO as well as trityl CT-03), a spin trap (DIPPMPO), and diamagnetic superoxide scavengers (such as cyclic hydroxylamines CMH and mitoTEMPO-H). We analyzed the reactivity of the different probes in the presence of a constant production of superoxide or hydroxyl radical in buffers and in cell lysates. We also assessed the performances of the different probes to detect superoxide produced by RAW264.7 macrophages stimulated by phorbol 12-myristate 13-acetate (PMA). In our conditions and models, we found that nitroxides were not specific for superoxide. CT-03 was specific, but the sensitivity of detection was low. Comparatively, we found that nitrone DIPPMPO and cyclic hydroxylamine CMH were good candidates to sensitively and specifically detect superoxide in complex biological media, CMH offering the best sensitivity.

Jilong Wang,1,2,* Hubo Sun,1,2,* Peijun Meng,1,2 Mengmeng Wang,1,2 Mi Tian,3 Yamin Xiong,1,2 Xueying Zhang,1,2 Peili Huang1,2 1School of Public Health, Capital Medical University, 2Beijing Key Laboratory of Environmental Toxicology, 3Medical Experiment and Test Center, Capital Medical University, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: Although quantum dot (QD)-induced toxicity occurs due to free radicals, generation of oxidative stress mediated by reactive oxygen species (ROS) formation is considered an important mechanism. However, free radical mechanisms are essentially difficult to elucidate at the molecular level because most biologically relevant free radicals are highly reactive and short-lived, making them difficult to directly detect, especially in vivo. Antioxidants play an important role in preventing or, in most cases, limiting the damage caused by ROS. Healthy people and animals possess many endogenous antioxidative substances that scavenge free radicals in vivo to maintain the redox balance and genome integrity. The antioxidant capacity of an organism is highly important but seldom studied. In this study, the dose and time effects of CdTe QDs on the antioxidant capacities of the liver and kidneys were investigated in mice using the electron paramagnetic resonance (EPR) spin-trapping technique. We found that the liver and kidneys of healthy mice contain specific antioxidant capacities that scavenge ·OH and ·O2-. Furthermore, oxidative stress markers (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx], glutathione [GSH] and malondialdehyde [MDA]) were examined. In dose course studies, the free radical scavenging efficiencies of the liver and kidneys were found to gradually decrease with increasing concentration of CdTe QD exposure. The activities and levels of SOD, CAT, GPx and MDA were observed to increase in treated groups, whereas those of GSH were reduced. The time course studies revealed that the QD-induced antioxidant efficiency reduction was time dependent with GSH decrease and could recover after a period of time. These experimental results offer new information on QD toxicity in vivo. Specifically, CdTe QDs can deplete GSH to reduce the elimination ability of the liver and kidneys for ·OH and ·O2-, thus inducing oxidative damage to tissues. Keywords: quantum dot, antioxidative capacity, free radical elimination, hydroxyl radical, superoxide anion radical, spin-trapping&nbsp

7 páginas, 7 figuras, Hemoglobin (Hb) from rainbow trout (Oncorhynchus mykiss), Atlantic cod (Gadus morhua), and herring (Clupea harengus) were evaluated in terms of capacity to generate hydroperoxide-derived free radicals in aqueous solution at pH 6.8. Cumene hydroperoxide (CumOOH) was used as a model for preformed lipid hydroperoxides, and free radicals were monitored by stabilizing with the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and further detection by electron spin resonance spectroscopy. The generation of hydroperoxide-derived free radicals was found to be Hb concentration-dependent, and intermediate Hb concentrations corresponding to hydroperoxide/Hb molar ratios of roughly 200 were found most active. The results indicated the following relative activity in the formation of hydroperoxide-derived free radicals under low and medium hydroperoxide levels (hydroperoxide/Hb molar ratios ≤5): cod Hb ≈ herring Hb > trout Hb. This free radical generating-activity was not affected by heating fish hemoglobins at 70 °C for 10 and 45 min in aqueous solution. Cod and herring Hb also exhibited faster autoxidation rates and stronger redox instability in the presence of hydroperoxides compared to trout Hb. According to their ability to generate hydroperoxide-derived free radicals, hemoglobin from cod and herring showed more activity than trout Hb as catalysts of lipid oxidation in lecithin liposomes. These data emphasize the central contribution of the free radicals formed through hydroperoxide-decomposition on the hemoglobin-mediated lipid oxidation, This work was supported by the LMC-Centre for Advance Food Studies and the Spanish Ministry of Science (Grant AGL 2006-12210-C03-01).

Nitrones are known both as therapeutic antioxidants and efficient spin-traps. In this work, the redox behavior of various para-substituted alpha-phenyl-N-tert-butyl nitrones (PBN) was studied by cyclic voltammetry. The polar effect of the substituents was found to correlate with the electrochemical properties of the nitronyl function. Compounds bearing an electron-withdrawing group were more easily reduced than those having an electron-donating group and an opposite trend was observed for the oxidation. Ease of oxidation was also computationally rationalized using DFT approach showing increased ease of oxidation with electron donating functionalities. Since electrochemical properties of nitrones are known to correlate with biological properties, this work provides insights in the design of potent nitrone antioxidants. Using cyclic voltammetry the relative rate of superoxide trapping by nitrones was investigated and compared to the classical antioxidant BHT. The determination of the relative rate of phenyl radical trapping was also carried out but showed no clear correlation with the nature of the substituents. This indicates the absence of a polar effect in agreement with previous data and further supports the intermediate nature, that is, non-or weakly nucleophile, of phenyl radical. On the contrary kinetics of hydroxymethyl radical trapping was found to correlate with the nature of the substituents, demonstrating the nucleophilic nature of its addition onto nitrones.

Nitration of 4-hydroxybenzaldehyde afforded 2,6-dinitro-4-hydroxybenzaldehyde 2, which in the presence of thionyl chloride is converted to 4-chloro-2,6-dinitrobenzaldehyde 3. This compound is very reactive towards nucleophiles, and reacts easily with methoxyamine, affording the intermediate 4-aminomethoxy-2,6-dinitrobenzaldehyde 4. Reaction of 4 with N-t-butylhydroxylamine led to a new spin trap of the nitrone type, namely 4-aminomethoxy-3,5-dinitrophenyl-1-t-butylnitrone 6. The spin-trapping capabilities of the new compound have been tested in a classical system, in which short-lived radicals were generated by irradiation. The new compound 6 contains an acidic proton with a pKa value of 7.8, and removal of this by a base induces a colour change, from yellow to green-blue. In a similar way, a new stable nitronyl-nitroxide radical 8 has been obtained from the intermediate 4.Keywords: Synthesis; free radical; spin-trapping; pH sensor; nitrone

The production of superoxide and nitric oxide induced in U87 glioma treated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was examined by electron spin resonance (ESR) spectroscopy using a newly designed flow-type quartz cuvette without detaching cells from the culture plate. ESR spectra of 2,2,6, 6-tetramethyl-4-hydroxy-1-piperidinyloxy (TEMPOL) with U87 cells on a quartz culture plate were measured at 15 min intervals. The signal intensity of TEMPOL decreased in the presence of U87 cells at the pseudo-first order rate. The signal decay was accelerated in the U87 cells treated with LPS/IFN-gamma for 24 h, and was suppressed in the presence of superoxide dismutase and catalase. By the spin-trapping method, nitric oxide from U87 cells pretreated with LPS/IFN-gamma for 24 h was measured by the ESR, but only a weak signal of nitric oxide adducts was detected. Further, the nitrite and nitrate levels in the medium did not increase for 24 h. By the ESR measurement of cells on culture plates without detachment stress, it was found that the production of superoxide was induced by LPS/IFN-gamma, but that of nitric oxide was not, in U87 glioma cells.

We investigated the influence of the adrenocorticotropic fragment 1-24 [ACTH-(1-24)] on the blood levels of highly-reactive free radicals in a rat model of prolonged asphyxia. Anesthetized animals were endotracheally intubated and mechanically ventilated with room air; after a 10 min stabilization period, the ventilator was turned off to induce asphyxia for 5 min; then, the ventilator was turned back on, and, simultaneously, the rats were intravenously treated with either ACTH-(1-24) (160 microg/kg in a volume of 1 ml/kg) or equivolume saline. Free radicals were detected in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent employed (alpha-phenyl-N-tert-butylnitrone). Arterial pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for the 60 min observation period, or until prior death. At the end of the 5 min period of respiratory arrest, blood levels of free radicals were about four times higher than those of the basal, pre-asphyxia condition, arterial pressure had dramatically decreased, ECG showed marked bradycardia and signs of ischemic damage and the EEG had become isoelectric. Treatment with ACTH-(1-24) produced an immediate normalization of the blood levels of free radicals, associated with a restoration of cardiovascular function and full recovery of EEG within 30-45 min; all the saline-treated rats, on the other hand, died within 6.89 +/- 0.96 min. These results provide direct evidence that in a severe condition of prolonged asphyxia there is a rapid and massive production of highly-reactive free radicals and suggest that the resuscitating effect of adrenocorticotropin fragments in severe hypoxic conditions may be largely due to the inhibition of free radical overproduction during tissue reoxygenation.

International audience; Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

1. The influence of ACTH-(1-24) on the blood levels of highly reactive free radicals in haemorrhagic shock was studied in rats. 2. Volume-controlled haemorrhagic shock was produced in adult rats under general anaesthesia (urethane, 1.25 g kg-1 intraperitoneally) by stepwise bleeding until mean arterial pressure stabilized at 20-23 mmHg. Rats were intravenously (i.v.) treated with either ACTH-(1-24) (160 micrograms kg-1 in a volume of 1 ml kg-1) or equivolume saline. Free radicals were measured in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent (alpha-phenyl-N-tert-butylnitrone). 3. Blood levels of free radicals were 6490 +/- 273 [arbitrary units (a.u.) ml-1 whole blood, before starting bleeding, and 30762 +/- 2650 after bleeding termination (means +/- s.e. mean of the values obtained in all experimental groups). All rats treated with saline died within 30 min, their blood levels of free radicals being 35450 +/- 5450 a.u. ml-1 blood, 15 min after treatment. Treatment with ACTH-(1-24) produced a rapid and sustained restoration of arterial pressure, pulse pressure, heart rate and respiratory function, with 100% survival at the end of the observation period (2 h); this was associated with an impressive reduction in the blood levels of free radicals, that were 12807 +/- 2995, 10462 +/- 2850, 12294 +/- 4120, and 10360 +/- 2080 a.u. ml-1 blood, 15, 30, 60 and 120 min after ACTH-(1-24) administration, respectively. 4. These results provide a direct demonstration that (i) in haemorrhagic shock there is a rapid and massive production of highly reactive free radicals, and that (ii) the sustained restoration of cardiovascular and respiratory functions induced by the i.v. injection of ACTH-(1-24) is associated with a substantial reduction of free radical blood levels. It is suggested that ACTH-(1-24) prevents the burst of free radical generation during blood mobilisation and subsequent tissue reperfusion, and this may be an important component of its mechanism of action in effectively preventing death for haemorrhagic shock.

Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmdeltaP) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting CmdeltaP-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 degrees C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).

The cytotoxicity of some ferrocenium salts and the lack of activity of the corresponding ferrocenes has been already demonstrated. The cytotoxic activity in different conditions of decamethylferrocenium tetrafluoroborate (DEMFc+) in comparison with four other ferrocenium derivatives on MCF-7 cell line is reported. The relative stability in aqueous solutions with different buffering agents is investigated by means of UV-vis spectroscopy and correlated to the cytotoxic properties of the compounds. DEMFc+, the most stable compound, shows the highest efficiency in inhibiting cell growth (IC50 35 M, for 48 h treatment). Relaxation time measurements point out the involvement of water molecules in the degradation process. ESR results confirm the ability of ferrocenium cations to produce oxygen radical species as a consequence of their degradation in water. Oxygen-dependent formation of both hydroxyl and superoxide radicals is established by the spin-trapping technique. A direct evidence of the DEMFc+ radical production into the viable cells is obtained by means of fluorescence-activated cell sorter (FACS) analysis that reveals a dose-dependent growth of 8-oxoguanine, the initial product of the guanine oxidation. This DNA oxidative stress justifies the cytotoxic effect of DEMFc+. Furthermore, the cytotoxic cooperative effect of bleomycin, an iron-dependent antitumor drug, and DEMFc+ has been tested. We have demonstrated the synergic effect between the two drugs, that is explained by the complementary oxidative damage inflicted to DNA as well as by the increasing of bleomycin activation by the iron(II/III) species available in the cell compartment from ferrocenium degradation.

In the field of the design of new free radical scavengers, especially for the ESR study of the role played by oxygen-centered species, such as superoxide and hydroxyl radical, involved in biological processes, the laboratory recently reported the synthesis of a new nitrone, the 2- diethoxyphosphoryl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPMPO). This nitrone was shown to be more efficient than the widely used 2,2-dimethyl-3,4-dihydro-2H-pyrrole-1-oxide (DMPO) : the superoxide spin adduct DEPMPO-OOH has a significantly larger half-life than DMPO-OOH, it does not decompose spontaneously into DEPMPO-OH, and DEPMPO is less sensitive to nucleophilic addition of water than DMPO. Such a behavior is related to the introduction of a -P(O)(OEt)2 group in the 2-position of the ring. In the present work, we tried to investigate the electronic and steric effects of the phosphorus moiety. Then, we prepared 3 new nitrones, the 2-diethoxyphosphorylmethyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (ESP), the 2,2-bisdiethoxyphosphoryl-3,4-dihydro-2H-pyrrole-1-oxide (DPBO) and the 2- ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). In ESP, the introduction of a methylene between the ring and the -P(O)(OEt)2 group leads to a decrease of the electrowithdrawing effect, and the behaviour of this nitrone toward the spin trapping of superoxide is more similar to that of DMPO. For DPBO, the second phosphorylated group does not seem to stabilize the DPBO-OOH adduct. In the case of EMPO, the -P(O)(OEt)2 - group is replaced by -CO 2 Et, which has a lower electrowithdrawing effect. The overall behavior of this nitrone is much closer to that of DEPMPO. The most important difference in the spin trap properties of all these nitrones is the persistance of their superoxide spin adduct. Other nitrones are currently investigated in the laboratory.; Dans la recherche de nouveaux pièges pour identifier par RPE les radicaux libres présents dans les milieux biologiques, particulièrement le superoxyde et le radical hydroxyle, le laboratoire a récemment mis au point une nouvelle nitrone, le 2-diéthoxyphosphoryl-2-méthyl-3,4-dihydro-2H- pyrrole-1-oxyde (DEPMPO), qui présente les avantages suivants par rapport au 2,2- diméthyl-3,4-dihydro-2H-pyrrole-1-oxyde (DMPO) couramment utilisé : meilleure persistance de l'adduit du superoxyde DEPMPO-OOH, absence de décomposition spontanée de cet adduit en l'adduit DEPMPO-OH, moindre sensibilité à l'addition nucléophile de l'eau. Ces caractéristiques sont liées à l'introduction du groupement -P(O)(OEt)2 en position 2 du cycle. L'objectif de notre étude a été de synthétiser et tester plusieurs nitrones pour mieux cerner les effets électroniques et stériques du groupement phosphoré. Nous avons préparé et testé trois nouvelles nitrones, le 2-diéthoxyphosphorylméthyl-2-méthyl-3,4- dihydro-2H-pyrrole-1-oxyde (ESP), le 2,2-bisdiéthoxyphosphoryl-3,4-dihydro-2H-pyrrole- 1-oxyde (DPBO) et le 2-éthoxycarbonyl-2-méthyl-3,4-dihydro-2H-pyrrole-1-oxyde (EMPO). Dans ESP le groupement -P(O)(OEt)2 est éloigné du cycle par un méthylène et ses performances dans le piégeage du superoxyde sont proches de celles de la DMPO. Dans la DPBO, l'introduction d'un second groupement -P(O)(OEt)2 ne semble pas stabiliser l'adduit DPBO-OOH. L'EMPO, où le groupement phosphoré est remplacé par un groupement -CO 2 Et, moins fortement électroattracteur, présente un comportement global plus proche de celui de la DEPMPO. La principale différence observée dans les propriétés de piégeage de toutes ces nitrones concerne la persistance des adduits du superoxyde. D'autres nitrones s'inscrivant dans cette étude sont en cours d'évaluation au laboratoire.

In the field of the design of new free radical scavengers, especially for the ESR study of the role played by oxygen-centered species, such as superoxide and hydroxyl radical, involved in biological processes, the laboratory recently reported the synthesis of a new nitrone, the 2- diethoxyphosphoryl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPMPO). This nitrone was shown to be more efficient than the widely used 2,2-dimethyl-3,4-dihydro-2H-pyrrole-1-oxide (DMPO) : the superoxide spin adduct DEPMPO-OOH has a significantly larger half-life than DMPO-OOH, it does not decompose spontaneously into DEPMPO-OH, and DEPMPO is less sensitive to nucleophilic addition of water than DMPO. Such a behavior is related to the introduction of a -P(O)(OEt)2 group in the 2-position of the ring. In the present work, we tried to investigate the electronic and steric effects of the phosphorus moiety. Then, we prepared 3 new nitrones, the 2-diethoxyphosphorylmethyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (ESP), the 2,2-bisdiethoxyphosphoryl-3,4-dihydro-2H-pyrrole-1-oxide (DPBO) and the 2- ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). In ESP, the introduction of a methylene between the ring and the -P(O)(OEt)2 group leads to a decrease of the electrowithdrawing effect, and the behaviour of this nitrone toward the spin trapping of superoxide is more similar to that of DMPO. For DPBO, the second phosphorylated group does not seem to stabilize the DPBO-OOH adduct. In the case of EMPO, the -P(O)(OEt)2 - group is replaced by -CO 2 Et, which has a lower electrowithdrawing effect. The overall behavior of this nitrone is much closer to that of DEPMPO. The most important difference in the spin trap properties of all these nitrones is the persistance of their superoxide spin adduct. Other nitrones are currently investigated in the laboratory., Dans la recherche de nouveaux pièges pour identifier par RPE les radicaux libres présents dans les milieux biologiques, particulièrement le superoxyde et le radical hydroxyle, le laboratoire a récemment mis au point une nouvelle nitrone, le 2-diéthoxyphosphoryl-2-méthyl-3,4-dihydro-2H- pyrrole-1-oxyde (DEPMPO), qui présente les avantages suivants par rapport au 2,2- diméthyl-3,4-dihydro-2H-pyrrole-1-oxyde (DMPO) couramment utilisé : meilleure persistance de l'adduit du superoxyde DEPMPO-OOH, absence de décomposition spontanée de cet adduit en l'adduit DEPMPO-OH, moindre sensibilité à l'addition nucléophile de l'eau. Ces caractéristiques sont liées à l'introduction du groupement -P(O)(OEt)2 en position 2 du cycle. L'objectif de notre étude a été de synthétiser et tester plusieurs nitrones pour mieux cerner les effets électroniques et stériques du groupement phosphoré. Nous avons préparé et testé trois nouvelles nitrones, le 2-diéthoxyphosphorylméthyl-2-méthyl-3,4- dihydro-2H-pyrrole-1-oxyde (ESP), le 2,2-bisdiéthoxyphosphoryl-3,4-dihydro-2H-pyrrole- 1-oxyde (DPBO) et le 2-éthoxycarbonyl-2-méthyl-3,4-dihydro-2H-pyrrole-1-oxyde (EMPO). Dans ESP le groupement -P(O)(OEt)2 est éloigné du cycle par un méthylène et ses performances dans le piégeage du superoxyde sont proches de celles de la DMPO. Dans la DPBO, l'introduction d'un second groupement -P(O)(OEt)2 ne semble pas stabiliser l'adduit DPBO-OOH. L'EMPO, où le groupement phosphoré est remplacé par un groupement -CO 2 Et, moins fortement électroattracteur, présente un comportement global plus proche de celui de la DEPMPO. La principale différence observée dans les propriétés de piégeage de toutes ces nitrones concerne la persistance des adduits du superoxyde. D'autres nitrones s'inscrivant dans cette étude sont en cours d'évaluation au laboratoire.

In the field of the design of new free radical scavengers, especially for the ESR study of the role played by oxygen-centered species, such as superoxide and hydroxyl radical, involved in biological processes, the laboratory recently reported the synthesis of a new nitrone, the 2- diethoxyphosphoryl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (DEPMPO). This nitrone was shown to be more efficient than the widely used 2,2-dimethyl-3,4-dihydro-2H-pyrrole-1-oxide (DMPO) : the superoxide spin adduct DEPMPO-OOH has a significantly larger half-life than DMPO-OOH, it does not decompose spontaneously into DEPMPO-OH, and DEPMPO is less sensitive to nucleophilic addition of water than DMPO. Such a behavior is related to the introduction of a -P(O)(OEt)2 group in the 2-position of the ring. In the present work, we tried to investigate the electronic and steric effects of the phosphorus moiety. Then, we prepared 3 new nitrones, the 2-diethoxyphosphorylmethyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (ESP), the 2,2-bisdiethoxyphosphoryl-3,4-dihydro-2H-pyrrole-1-oxide (DPBO) and the 2- ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). In ESP, the introduction of a methylene between the ring and the -P(O)(OEt)2 group leads to a decrease of the electrowithdrawing effect, and the behaviour of this nitrone toward the spin trapping of superoxide is more similar to that of DMPO. For DPBO, the second phosphorylated group does not seem to stabilize the DPBO-OOH adduct. In the case of EMPO, the -P(O)(OEt)2 - group is replaced by -CO 2 Et, which has a lower electrowithdrawing effect. The overall behavior of this nitrone is much closer to that of DEPMPO. The most important difference in the spin trap properties of all these nitrones is the persistance of their superoxide spin adduct. Other nitrones are currently investigated in the laboratory., Dans la recherche de nouveaux pièges pour identifier par RPE les radicaux libres présents dans les milieux biologiques, particulièrement le superoxyde et le radical hydroxyle, le laboratoire a récemment mis au point une nouvelle nitrone, le 2-diéthoxyphosphoryl-2-méthyl-3,4-dihydro-2H- pyrrole-1-oxyde (DEPMPO), qui présente les avantages suivants par rapport au 2,2- diméthyl-3,4-dihydro-2H-pyrrole-1-oxyde (DMPO) couramment utilisé : meilleure persistance de l'adduit du superoxyde DEPMPO-OOH, absence de décomposition spontanée de cet adduit en l'adduit DEPMPO-OH, moindre sensibilité à l'addition nucléophile de l'eau. Ces caractéristiques sont liées à l'introduction du groupement -P(O)(OEt)2 en position 2 du cycle. L'objectif de notre étude a été de synthétiser et tester plusieurs nitrones pour mieux cerner les effets électroniques et stériques du groupement phosphoré. Nous avons préparé et testé trois nouvelles nitrones, le 2-diéthoxyphosphorylméthyl-2-méthyl-3,4- dihydro-2H-pyrrole-1-oxyde (ESP), le 2,2-bisdiéthoxyphosphoryl-3,4-dihydro-2H-pyrrole- 1-oxyde (DPBO) et le 2-éthoxycarbonyl-2-méthyl-3,4-dihydro-2H-pyrrole-1-oxyde (EMPO). Dans ESP le groupement -P(O)(OEt)2 est éloigné du cycle par un méthylène et ses performances dans le piégeage du superoxyde sont proches de celles de la DMPO. Dans la DPBO, l'introduction d'un second groupement -P(O)(OEt)2 ne semble pas stabiliser l'adduit DPBO-OOH. L'EMPO, où le groupement phosphoré est remplacé par un groupement -CO 2 Et, moins fortement électroattracteur, présente un comportement global plus proche de celui de la DEPMPO. La principale différence observée dans les propriétés de piégeage de toutes ces nitrones concerne la persistance des adduits du superoxyde. D'autres nitrones s'inscrivant dans cette étude sont en cours d'évaluation au laboratoire.

Recent studies have indicated that activation of A1/A2-receptors may mediate metabolic adaptation of the heart to ischemia/reperfusion stress. This study tests whether pretreatment with A1-selective agonist R(-)-N6-(2-phenylisopropyl) adenosine (R-PIA) might mimic effects of a brief period of coronary occlusion (ischemic preconditioning, IP) on energy metabolism and hydroxyl radical (OH.) formation in canine myocardium following subsequent prolonged ischemia and reperfusion. Anaesthetized dogs were randomized to a control group subjected to 40-min occlusion of a diagonal branch of left anterior descending coronary artery (LAD) followed by 1-h reperfusion, or a preconditioned group (PC) in which the same period of sustained ischemia and reperfusion was preceded by a single cycle of IP (5-min occlusion of the same LAD branch and 10-min reperfusion), or to PIA group in which R-PIA infusion into the same branch of LAD (0.4 microg/kg per min during 5 min) was followed by 10 min of perfusion prior to sustained ischemia-reperfusion. Pretreatment with R-PIA similarly to IP reduced lactate (Lac), creatine (Cr) and inorganic phosphate (Pi) release from myocytes into the interstitial fluid during sustained ischemia compared to these indices in control. By the end of reperfusion, both IP and R-PIA infusion enhanced recovery of myocardial ATP and phosphocreatine (PCr) and attenuated the total creatine (sigmaCr = PCr + Cr) loss, an index of cell membrane damage. A1-receptor activation by R-PIA, as IP, led to a significant reduction in OH. radical generation following reperfusion assessed by a spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) using cardiac microdialysis. R-PIA pretreatment did not affect systemic and cardiac hemodynamic parameters. We conclude that (1) adaptive mechanisms of IP involve A1-receptor activation that contributes to the overall metabolic response and (2) R-PIA acts as a useful preconditioning-mimetic and anti-ischemic agent in dogs.

Experiments were performed on glycerol-extracted muscle fibres prepared from psoas muscle of rabbit in the presence of hydroxyl free radical generating system. Short irradiation of spin-labelled muscle fibres by UV light showed the interaction of probe molecules with oxygen free radicals. The intensity of the EPR signal from maleimide or isothiocyanate spin labels attached to the essential thiol groups decreased following irradiation. Oxygen free radicals affected the rate constant of the transition AM.ADP.Vi→AM.ADP in the ATP hydrolysis cycle. It was found that the essential –SH groups of myosin were involved in the oxidation of sulphydryls by Ce(IV). Ce(IV) complexed to nitrilotriacetic acid in the presence of spin trap produced long-lived free radicals located partly on SH-1 sulphydryls.

A biomimetic model of vanadium-containing bromoperoxidase (BPO) was proposed. The vanadium-containing BPO possesses vanadate ion (+5 oxidation state) as a prosteric group and catalyses the formation of a carbon-bromide bond in the presence of hydrogen peroxide, bromide ion and bromide acceptor. The reaction mechanism is as yet unknown. Several vanadyl (VO2+, +4 oxidation state) complexes with oxalate, glutarate, succinate, malonate and acetate ligands showed similar bromination activity to that of BPO. Especially, the vanadyl-oxalato complex provided the highest BPO-like activity. Both vanadate ion and vanadyl complexes with nitrogen-containing ligands such as EDTA, glycine and serine showed essentially no BPO-like activity. Thus, the vanadyl-oxalato complex is proposed to be not only a possible chemical model of BPO, but a useful compound to elucidate the reaction mechanism of BPO.

Nitric oxide reacts with superoxide to produce peroxynitrite, which may be an important mediator of oxidant-induced cellular injury. Here we report that peroxynitrite is able to oxidize a protein, bovine serum albumin (BSA), to the corresponding protein-thiyl free radical as demonstrated by electron paramagnetic resonance (EPR)-spin-trapping experiments with both α-phenyl-N-tert-butyl nitrone (PBN) and 5,5-dimethyl-1 -pyrroline- N-oxide (DMPO). BSA radical adduct yields increased with pH indicating peroxynitrite anion as its main forming agent. Reaction with peroxynitrite may be another aspect of the antioxidant action of albumin in extracellular fluids.

1. INTRODUCTION The production of superoxide and hydroxyl rad- icals by neutrophils has been shown to occur fol- lowing stimulation with membrane-perturbing agents 111. The production of superoxide radicals appears to be due to a specific activation of a plas- ma membrane-bound NADPH oxidase which is inactive