European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

Electron Paramagnetic Resonance Spin Trapping (EPR–ST) Technique in Photopolymerization Processes

To face economic issues of the last ten years, free-radical photopolymerization (FRP) has known an impressive enlightenment. Multiple performing photoinitiating systems have been designed to perform photopolymerizations in the visible or near infrared (NIR) range. To fully understand the photochemical mechanisms involved upon light activation and characterize the nature of radicals implied in FRP, electron paramagnetic resonance coupled to the spin trapping (EPR–ST) method represents one of the most valuable techniques. In this context, the principle of EPR–ST and its uses in free-radical photopolymerization are entirely described

Réactions des espèces réactives de l’azote dérivées du monoxyde d’azote avec la mélatonine et quelques indoles apparentés. Implications biologiques.

Nitrogen monoxide (NO), the enzymatic product of NO-synthases in mammals, is an important biological mediator. Its oxidized and reduced derivatives are also thought to play a role in aging, neurodegenerative diseases, inflammation. . . Yet, most of the underlying chemical mechanisms are still unknown. Melatonin, a tryptophan-derived hormone in mammals, is well-known for its scavenging of reactive oxygen species. It was chosen here as a model compound to study the reactions of reactive nitrogen species : peroxynitrite (ONOO-), nitrogen dioxide (NO2), nitroxyl (HNO). . . The reaction of ONOO- with melatonin leads to the formation of mixtures of oxidation products (indol-2-ones, pyrroloindoles, kynuramines), of C-nitration products and of two N-substitution products : 1-nitro- and 1-nitrosomelatonins. Their yields vary according to physicochemical conditions (pH, CO2 concentration). First, peroxynitrite-derived radicals oxidize melatonin and form melatoninyl radical. Then the latter recombines with peroxynitrite-derived ONOO or NO2 radicals. Nitrosation of melatonin is the main transformation observed in aqueous solution with NO2 in the presence of NO2- and with NO or HNO in the presence of O2. 1-nitrosomelatonin is unstable in aqueous solution and behaves as an NO-donor with potential therapeutics applications. This work provided a better understanding of the physicochemical properties and of the biological activity of this compound. Its vasorelaxing and mutagenic properties were expected for an NO-donor. Its antioxidant effect and its ability to increase stimulated release of acetylcholine in the brain may allow a therapeutic approach of Alzheimer's disease.Le monoxyde d'azote (no), synthétisé par voie enzymatique chez les mammifères, est un médiateur biologique important, comme ses dérivés oxydés ou réduits. Or on connait mal les mécanismes chimiques à l'origine de ces propriétés. La mélatonine, hormone dérivée du tryptophane présente chez les mammifères et connue pour sa réaction avec les espèces réactives de l'oxygène, a été choisie comme modèle d'étude des réactions des espèces réactives de l'azote : peroxynitrite (ONOO-), dioxyde d'azote (NO2), nitroxyle (HNO). . . La reaction de ONOO- avec la mélatonine conduit à la formation de produits d'oxydation (indol-2-ones, pyrrolo-indoles, kynuramines), de produits de C-nitration et de deux produits de N-substitution : 1-nitro- et 1-nitrosomelatonine. Les rendements varient en fonction des conditions (pH, concentration en CO2). D'abord a lieu l'oxydation de la mélatonine en radical mélatoninyle par les radicaux issus du peroxynitrite. Puis le radical mélatoninyle se recombine avec les radicaux ONOO ou NO2 issus du peroxynitrite. La nitrosation de la mélatonine est la transformation principale observée en solution aqueuse avec NO2 en presence de NO2- et NO ou HNO en presence de O2. La 1-nitrosomelatonine, instable en solution aqueuse, est un donneur de NO pour lequel une utilisation thérapeutique est envisageable. Ce travail a donc permis d'approfondir les connaissances de ses propriétés physicochimiques et de son activite biologique. Les propriétés vasorelaxantes et mutagènes attendues pour un donneur de NO ont été vérifiées. Ses effets antioxydants et sur la libération de l'acétylcholine dans le cerveau permettent d'envisager une approche thérapeutique de la maladie d'Alzheimer

N-Nitroso products from the reaction of indoles with Angeli's salt

While nitroxyl (HNO) has been shown to engage in oxidation and hydroxylation reactions, little is known about its nitrosating potential. We therefore sought to investigate the kinetics of formation and identity of the reaction products of the classical nitroxyl donor Angeli's salt (AS) with three representative tryptophan derivates (melatonin, indol-3-acetic acid, and N-acetyl-l-tryptophan) in vitro. In the presence of oxygen and at physiological pH, we find that the major products generated are the corresponding N-nitrosoindoles with negligible formation of oxidation and nitration products. A direct comparison of the effects of AS, nitrite, peroxynitrite, aqueous NO* solution, and the NO-donor DEA/NO toward melatonin revealed that nitrite does not participate in the reaction and that peroxynitrite is not an intermediate. Rather, N-nitrosoindole formation appears to proceed via a mechanism that involves electrophilic attack of HNO on the indole nitrogen, followed by a reaction of the intermediary hydroxylamine derivative with oxygen. Further in vivo experiments demonstrated that AS exhibits a unique nitrosation signature which differs from that of DEA/NO inasmuch as substantial amounts of a mercury-resistant nitroso species are generated in the heart, whereas S-nitrosothiols are the major reaction products in plasma. These data are consistent with the notion that the generation of nitroxyl in vivo gives rise to formation of nitrosative post-translational protein modifications in the form of either S- or N-nitroso products, depending on the redox environment. It is intriguing to speculate that the particular efficiency of nitroxyl to form N-nitroso species in the heart may account for the positive inotropic effects observed with AS earlier

New synthetic route to 2,2,6,6-tetraethylpiperidin-4-one: A key-intermediate towards tetraethyl nitroxides

International audienceA new synthetic route to 2,2,6,6-tetraethylpiperidin-4-one and derived aminoxyl (nitroxide) radicals is described. In this preliminary work, 2,2,6,6-tetraethylpiperidin-4-one was obtained from ethyl acetoacetate in 3% yield over eight steps, relying only on common reagents and laboratory equipment for organic synthesis

Réactions des espèces réactives de l'azote dérivées du monoxyde d'azote avec la mélatonine et quelques indoles apparentés (implications biologiques)

LE MONOXYDE D'AZOTE (NO), SYNTHETISE PAR VOIE ENZYMATIQUE CHEZ LES MAMMIFERES, EST UN MEDIATEUR BIOLOGIQUE IMPORTANT, COMME SES DERIVES OXYDES OU REDUITS. OR ON CONNAIT MAL LES MECANISMES CHIMIQUES A L'ORIGINE DE CES PROPRIETES.LA MELATONINE, HORMONE DERIVEE DU TRYPTOPHANE PRESENTE CHEZ LES MAMMIFERES ET CONNUE POUR SA REACTION AVEC LES ESPECES REACTIVES DE L'OXYGENE, A ETE CHOISIE COMME MODELE D'ETUDE DES REACTIONS DES ESPECES REACTIVES DE L'AZOTE : PEROXYNITRITE (ONOO-), DIOXYDE D'AZOTE (NO2), NITROXYLE (HNO)...LA REACTION DE ONOO- AVEC LA MELATONINE CONDUIT A LA FORMATION DE PRODUITS D'OXYDATION (INDOL-2-ONES, PYRROLO-INDOLES, KYNURAMINES), DE PRODUITS DE C-NITRATION ET DE DEUX PRODUITS DE N-SUBSTITUTION : 1-NITRO- ET 1-NITROSOMELATONINE. LES RENDEMENTS VARIENT EN FONCTION DES CONDITIONS (pH, CONCENTRATION EN CO2). D'ABORD A LIEU L'OXYDATION DE LA MELATONINE EN RADICAL MELATONINYLE PAR LES RADICAUX ISSUS DU PEROXYNITRITE. PUIS LE RADICAL MELATONINYLE SE RECOMBINE AVEC LES RADICAUX ONOO OU NO2 ISSUS DU PEROXYNITRITE.LA NITROSATION DE LA MELATONINE EST LA TRANSFORMATION PRINCIPALE OBSERVEE EN SOLUTION AQUEUSE AVEC NO2 EN PRESENCE DE NO2- ET NO OU HNO EN PRESENCE DE O2.LA 1-NITROSOMELATONINE, INSTABLE EN SOLUTION AQUEUSE, EST UN DONNEUR DE NO POUR LEQUEL UNE UTILISATION THERAPEUTIQUE EST ENVISAGEABLE. CE TRAVAIL A DONC PERMIS D'APPROFONDIR LES CONNAISSANCES DE SES PROPRIETES PHYSICOCHIMIQUES ET DE SON ACTIVITE BIOLOGIQUE. LES PROPRIETES VASORELAXANTES ET MUTAGENES ATTENDUES POUR UN DONNEUR DE NO ONT ETE VERIFIEES. SES EFFETS ANTIOXYDANTS ET SUR LA LIBERATION DE L'ACETYLCHOLINE DANS LE CERVEAU PERMETTENT D'ENVISAGER UNE APPROCHE THERAPEUTIQUE DE LA MALADIE D'ALZHEIMER.NITROGEN MONOXIDE (NO), THE ENZYMATIC PRODUCT OF NO-SYNTHASES IN MAMMALS, IS AN IMPORTANT BIOLOGICAL MEDIATOR. ITS OXIDIZED AND REDUCED DERIVATIVES ARE ALSO THOUGHT TO PLAY A ROLE IN AGING, NEURODEGENERATIVE DISEASES, INFLAMMATION... YET, MOST OF THE UNDERLYING CHEMICAL MECHANISMS ARE STILL UNKNOWN.MELATONIN, A TRYPTOPHAN-DERIVED HORMONE IN MAMMALS, IS WELL-KNOWN FOR ITS SCAVENGING OF REACTIVE OXYGEN SPECIES. IT WAS CHOSEN HERE AS A MODEL COMPOUND TO STUDY THE REACTIONS OF REACTIVE NITROGEN SPECIES : PEROXYNITRITE (ONOO-), NITROGEN DIOXIDE (NO2), NITROXYL (HNO)...THE REACTION OF ONOO- WITH MELATONIN LEADS TO THE FORMATION OF MIXTURES OF OXIDATION PRODUCTS (INDOL-2-ONES, PYRROLOINDOLES, KYNURAMINES), OF C-NITRATION PRODUCTS AND OF TWO N-SUBSTITUTION PRODUCTS : 1-NITRO- AND 1-NITROSOMELATONINS. THEIR YIELDS VARY ACCORDING TO PHYSICOCHEMICAL CONDITIONS (pH, CO2 CONCENTRATION). FIRST, PEROXYNITRITE-DERIVED RADICALS OXIDIZE MELATONIN AND FORM MELATONINYL RADICAL. THEN THE LATTER RECOMBINES WITH PEROXYNITRITE-DERIVED ONOO OR NO2 RADICALS.NITROSATION OF MELATONIN IS THE MAIN TRANSFORMATION OBSERVED IN AQUEOUS SOLUTION WITH NO2 IN THE PRESENCE OF NO2- AND WITH NO OR HNO IN THE PRESENCE OF O2.1-NITROSOMELATONIN IS UNSTABLE IN AQUEOUS SOLUTION AND BEHAVES AS AN NO-DONOR WITH POTENTIAL THERAPEUTICS APPLICATIONS. THIS WORK PROVIDED A BETTER UNDERSTANDING OF THE PHYSICOCHEMICAL PROPERTIES AND OF THE BIOLOGICAL ACTIVITY OF THIS COMPOUND. ITS VASORELAXING AND MUTAGENIC PROPERTIES WERE EXPECTED FOR AN NO-DONOR. ITS ANTIOXIDANT EFFECT AND ITS ABILITY TO INCREASE STIMULATED RELEASE OF ACETYLCHOLINE IN THE BRAIN MAY ALLOW A THERAPEUTIC APPROACH OF ALZHEIMER'S DISEASE.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Molecular Probes for Evaluation of Oxidative Stress by In Vivo EPR Spectroscopy and Imaging: State-of-the-Art and Limitations

Oxidative stress, defined as a misbalance between the production of reactive oxygen species and the antioxidant defenses of the cell, appears as a critical factor either in the onset or in the etiology of many pathological conditions. Several methods of detection exist. However, they usually rely on ex vivo evaluation or reports on the status of living tissues only up to a few millimeters in depth, while a whole-body, real-time, non-invasive monitoring technique is required for early diagnosis or as an aid to therapy (to monitor the action of a drug). Methods based on electron paramagnetic resonance (EPR), in association with molecular probes based on aminoxyl radicals (nitroxides) or hydroxylamines especially, have emerged as very promising to meet these standards. The principles involve monitoring the rate of decrease or increase of the EPR signal in vivo after injection of the nitroxide or the hydroxylamine probe, respectively, in a pathological versus a control situation. There have been many successful applications in various rodent models. However, current limitations lie in both the field of the technical development of the spectrometers and the molecular probes. The scope of this review will mainly focus on the latter

In vivo evaluation of different alterations of redox status by studying pharmacokinetics of nitroxides using magnetic resonance techniques

Free radicals, particularly reactive oxygen species (ROS), are involved in various pathologies, injuries related to radiation, ischemia-reperfusion or ageing. Unfortunately, it is virtually impossible to directly detect free radicals in vivo, but the redox status of the whole organism or particular organ can be studied in vivo by using magnetic resonance techniques (EPR and MRI) and paramagnetic stable free radicals – nitroxides. Here we review results obtained in vivo following the pharmacokinetics of nitroxides on experimental animals (and a few in humans) under various conditions. The focus was on conditions where the redox status has been altered by induced diseases or harmful agents, clearly demonstrating that various EPR/MRI/nitroxide combinations can reliably detect metabolically induced changes in the redox status of organs. These findings can improve our understanding of oxidative stress and provide a basis for studying the effectiveness of interventions aimed to modulate oxidative stress. Also, we anticipate that the in vivo EPR/MRI approach in studying the redox status can play a vital role in the clinical management of various pathologies in the years to come providing the development of adequate equipment and probes

Unexpected rapid aerobic transformation of 2,2,6,6-tetraethyl-4-oxo (piperidin-1-yloxyl) radical by cytochrome P450 in the presence of NADPH: Evidence against a simple reduction of the nitroxide moiety to the hydroxylamine

International audienceAminoxyl radicals (nitroxides) are a class of compounds with important biomedical applications, serving as antioxidants, spin labels for proteins, spin probes of oximetry, pH, or redox status in electron paramagnetic resonance (EPR), or as contrast agents in magnetic resonance imaging (MRI). However, the fast reduction of the radical moiety in common tetramethyl-substituted cyclic nitroxides within cells, yielding diamagnetic hydro-xylamines, limits their use in spectroscopic and imaging studies. In vivo half-lives of commonly used tetra-methyl-substituted nitroxides span no more than a few minutes. Therefore, synthetic efforts have focused on enhancing the nitroxide stability towards reduction by varying the electronic and steric environment of the radical. Tetraethyl-substitution at alpha position to the aminoxyl function proved efficient in vitro against reduction by ascorbate or cytosolic extracts. Moreover, 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) radical (TEEPONE) was used successfully for tridimensional EPR and MRI in vivo imaging of mouse head, with a reported half-life of over 80 min. We decided to investigate the stability of tetraethyl-substituted piperidine nitroxides in the presence of hepatic microsomal fractions, since no detailed study of their "metabolic stability" at the molecular level had been reported despite examples of the use of these nitroxides in vivo. In this context, the rapid aerobic transformation of TEEPONE observed in the presence of rat liver microsomal fractions and NADPH was unexpected. Combining EPR, HPLC-HRMS, and DFT studies on a series of piperidine nitroxides-TEEPONE, 4-oxo-2,2,6,6-tetramethyl(piperidin-1-yloxyl) (TEMPONE), and 2,2,6,6-tetraethyl-4-hydroxy(piper-idin-1-yloxyl) (TEEPOL), we propose that the rapid loss in paramagnetic character of TEEPONE is not due to reduction to hydroxylamine but is a consequence of carbon backbone modification initiated by hydrogen radical abstraction in alpha position to the carbonyl by the P450-Fe (V) =O species. Besides, hydrogen radical abstraction by P450 on ethyl substituents, leading to dehydrogenation or hydroxylation products, leaves the aminoxyl function intact but could alter the linewidth of the EPR signal and thus interfere with methods relying on measurement of this parameter (EPR oximetry)