Dual Antioxidant Properties and Organic Radical Stabilization in Cellulose Nanocomposite Films Functionalized by In Situ Polymerization of Coniferyl Alcohol.

A new biobased material based on an original strategy using lignin model compounds as natural grafting additive on a nanocellulose surface through in situ polymerization of coniferyl alcohol by the Fenton reaction at two pH values was investigated. The structural and morphological properties of the materials at the nanoscale were characterized by a combination of analytical methods, including Fourier transform infrared spectroscopy, liquid chromatography combined with mass spectrometry, nuclear molecular resonance spectroscopy, electron paramagnetic resonance spectroscopy, water sorption capacity by dynamic vapor sorption, and atomic force microscopy (topography and indentation modulus measurements). Finally, the usage properties, such as antioxidant properties, were evaluated in solution and the nanostructured casted films by radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH•) scavenging tests. We demonstrate the structure-function relationships of these advanced CNC-lignin films and describe their dual functionalities and characteristics, namely, their antioxidant properties and the presence of persistent phenoxy radicals within the material.

Ultrafast multiple paramagnetic species EPR imaging using a total variation based model.

An EPR spectrum or an EPR sinogram for imaging contains information about all the paramagnetic species that are in the analyzed sample. When only one species is present, an image of its spatial repartition can be reconstructed from the sinogram by using the well-known Filtered Back-Projection (FBP). However, in the case of several species, the FBP does not allow the reconstruction of the images of each species from a standard acquisition. One has to use for this spectral-spatial imaging whose acquisition can be very long. A new approach, based on Total Variation minimization, is proposed in order to efficiently extract the spatial repartitions of all the species present in a sample from standard imaging data and therefore drastically reduce the acquisition time. Experiments have been carried out on Tetrathiatriarylmethyl, nitroxide and DPPH.

First combined in vivo X-ray tomography and high-resolution molecular electron paramagnetic resonance (EPR) imaging of the mouse knee joint taking into account the disappearance kinetics of the EPR probe.

Although laboratory data clearly suggest a role for oxidants (dioxygen and free radicals derived from dioxygen) in the pathogenesis of many age-related and degenerative diseases (such as arthrosis and arthritis), methods to image such species in vivo are still very limited. This methodological problem limits physiopathologic studies about the role of those species in vivo, the effects of their regulation using various drugs, and the evaluation of their levels for diagnosis of degenerative diseases. In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are unique, noninvasive methods used to specifically detect and quantify paramagnetic species. However, two problems limit their application: the anatomic location of the EPR image in the animal body and the relative instability of the EPR probes. Our aim is to use EPR imaging to obtain physiologic and pathologic information on the mouse knee joint. This article reports the first in vivo EPR image of a small tissue, the mouse knee joint, with good resolution (≈ 160 µm) after intra-articular injection of a triarylmethyl radical EPR probe. It was obtained by combining EPR and x-ray micro-computed tomography for the first time and by taking into account the disappearance kinetics of the EPR probe during image acquisition to reconstruct the image. This multidisciplinary approach opens the way to high-resolution EPR imaging and local metabolism studies of radical species in vivo in different physiologic and pathologic situations.

Interaction between the reductase Tah18 and highly conserved Fe-S containing Dre2 C-terminus is essential for yeast viability.

Tah18-Dre2 is a recently identified yeast protein complex, which is highly conserved in human and has been implicated in the regulation of oxidative stress induced cell death and in cytosolic Fe-S proteins synthesis. Tah18 is a diflavin oxido-reductase with binding sites for flavin mononucleotide, flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, which is able to transfer electrons to Dre2 Fe-S clusters. In this work we characterized in details the interaction between Tah18 and Dre2, and analysed how it conditions yeast viability. We show that Dre2 C-terminus interacts in vivo and in vitro with the flavin mononucleotide- and flavin adenine dinucleotide-binding sites of Tah18. Neither the absence of the electron donor nicotinamide adenine dinucleotide phosphate-binding domain in purified Tah18 nor the absence of Fe-S in aerobically purified Dre2 prevents the binding in vitro. In vivo, when this interaction is affected in a dre2 mutant, yeast viability is reduced. Conversely, enhancing artificially the interaction between mutated Dre2 and Tah18 restores cellular viability despite still reduced cytosolic Fe-S cluster biosynthesis. We conclude that Tah18-Dre2 interaction in vivo is essential for yeast viability. Our study may provide new insight into the survival/death switch involving this complex in yeast and in human cells.

Deciphering the activation sequence of ferrociphenol anticancer drug candidates.

The complete oxidation sequence of a model for ferrociphenols, a new class of anticancer drug candidate, is reported. Cyclic voltammetry was used to monitor the formation of oxidation intermediates on different timescales, thereby allowing the electrochemical characterization of both the short-lived and stable species obtained from the successive electron-transfer and deprotonation steps. The electrochemical preparation of the ferrocenium intermediate enabled a stepwise voltammetric determination of the stable oxidation compounds obtained upon addition of a base as well as the electron stoichiometry observed for the overall oxidation process. A mechanism has been established from the electrochemical data, which involves a base-promoted intramolecular electron transfer between the phenol and the ferrocenium cation. The resulting species is further oxidized then deprotonated to yield a stable quinone methide. To further characterize the transient species successively formed during the two-electron oxidation of the ferrociphenol to its quinone methide, EPR was used to monitor the fate of the paramagnetic species generated upon addition of imidazole to the electrogenerated ferrocenium. The study revealed the passage from an iron-centered to a carbon-centered radical, which is then oxidized to yield the quinone methide, namely, the species that interacts with proteins and so forth under biological conditions.

Oxidative decarboxylation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes by peroxidases and related hemeproteins: intermediate formation and characterization of the corresponding cations.

Tris(p-carboxyltetrathiaaryl)methyl radicals (TAM*) are good EPR probes for measurement of dioxygen concentration in biological systems and for EPR imaging. It has been previously reported that these radicals are efficiently oxidized by superoxide, O2*(-), or alkylperoxyl radicals, ROO*, and by liver microsomes via an oxidative decarboxylation mechanism leading to the corresponding quinone-methides (QM). This article shows that peroxidases, such as horseradish peroxidase (HRP), lactoperoxidase (LPO) and prostaglandin synthase (PGHS), and other hemeproteins, such as methemoglobin (metHb), metmyoglobin (metMb) and catalase, also efficiently catalyze the oxidation of TAM* radicals to QM by H2O2 or alkylhydroperoxides. These reactions involve the intermediate formation of the corresponding cations TAM(+) that have also been cleanly generated by K2Ir(IV)Cl6 and characterized by UV-Visible spectroscopy and mass spectrometry, and through their reactions with ascorbate or H2O2. Labelling experiments on HRP-catalyzed oxidation of TAM* to QM using H2(18)O or (18)O2 in the presence of glucose and glucose oxidase (GOX) showed that the oxygen atom incorporated into QM came both from O2 and from H2O. Mechanisms for these reactions in agreement with those data were proposed. Oxidative decarboxylation of TAM* to QM is a new reaction catalyzed by peroxidases. Such reactions should be considered when using TAM* as EPR oximetry probes in vivo or in vitro in complex biological media.

Reductive metalation of cyclic and acyclic pseudopeptidic bis-disulfides and back conversion of the resulting diamidato/dithiolato complexes to bis-disulfides.

Cyclic and acyclic pseudopeptidic bis-disulfides built on an o-phenylene diamine scaffold were prepared: (N(2)H(2)S(2))(2), 1a, N(2)H(2)(S-SCH(3))(2), 1b, and N(2)H(2)(S-StBu)(2), 1c. Reductive metalation of these disulfides with (PF(6))[Cu(CH(3)CN)(4)] in the presence of Et(4)NOH as a base, or with (Et(4)N)[Fe(SEt)(4)] and Et(4)NCl, yields the corresponding diamidato/dithiolato copper(III) or iron(III) complex, (Et(4)N)[Cu(N(2)S(2))], 2, or (Et(4)N)(2)[Fe(N(2)S(2))Cl], 5. These complexes display characteristics similar to those previously described in the literature. The mechanism of the metalation with copper has been investigated by X-band electron paramagnetic resonance (EPR) spectroscopy at 10 K. After metalation of the bis-disulfide 1c and deprotonation of the amide nitrogens, the reductive cleavage of the S-S bonds occurs by two one-electron transfers leading to the intermediate formation of a copper(II) complex and a thyil radical. Complexes 2 and 5 can be converted back to the cyclic bis-disulfide 1a with iodine in an 80% yield. Reaction of 5 with iodine in the presence of CH(3)S-SCH(3) affords a 1/1 mixture of the acyclic N(2)H(2)(S-SCH(3))(2) disulfide 1b and cyclic bis-disulfide 1a. From 2, the reaction was monitored by (1)H NMR and gives 1b as major product. While there is no reaction of 2 or 5 with tBuS-StBu and iodine, reaction with an excess of tBuSI affords quantitatively the di-tert-butyl disulfide 1c. To assess the role of the Cu(III) oxidation state, control experiments were carried out under strictly anaerobic conditions with the copper(II) complex, (Et(4)N)(2)[Cu(N(2)S(2))], 6. Complex 6 is oxidized to 2 by iodine, and it reacts with an excess of tBuSI, yielding 1c as final product, through the intermediate formation of complex 2.

Oxidative and reductive metabolism of tris(p-carboxyltetrathiaaryl)methyl radicals by liver microsomes.

Tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals are particularly stable carbon-centered free radicals that are used as contrast agents in NMR imaging and as probes for in vivo oximetry by electron paramagnetic resonance (EPR) imaging. However, nothing is known so far on the metabolism of these persistent radicals in mammals. This article describes the metabolism of two TAM radicals by rat, human, and pig liver microsomes. It shows that microsomal transformation of these free radicals leads to two major metabolites resulting from an oxidation or a reduction of the present compounds. The structures of these metabolites were completely established by 1H and 13C NMR spectroscopy, mass spectrometry, and comparison with authentic compounds. Under aerobic conditions, liver microsomes catalyzed the oxidative decarboxylation of TAM radicals by NADPH and O2 with formation of the corresponding quinone-methide products. This reaction was dependent on cytochromes P450 and cytochrome P450 reductase and greatly implied the involvement of superoxide. Under anaerobic conditions, these enzymes catalyzed the reduction of TAM radicals to the corresponding triarylmethanes. This reduction was strongly inhibited by O2. These metabolic transformations should be considered when using such TAM radicals for pO2 measurement by EPR imaging, especially in tissues in which fast oxidative (inflammation sites) or reductive (hypoxic tissues) metabolism could occur.

Oxidation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes: evidence for their oxidative decarboxylation and molecular origin of their specific ability to react with O2*-.

Tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes are very efficiently oxidized by superoxide and alkylperoxyl radicals with selective formation of quinone-methide products; this should explain the previously reported specific measurement of O2*- using these EPR probes.

Dioxygen activation at a mononuclear Cu(I) center embedded in the calix[6]arene-tren core.

The reaction of a cuprous center coordinated to a calix[6]arene-based aza-cryptand with dioxygen has been studied. In this system, Cu(I) is bound to a tren unit that caps the calixarene core at the level of the small rim. As a result, although protected from the reaction medium by the macrocycle, the metal center presents a labile site accessible to small guest ligands. Indeed, in the presence of O2, it reacts in a very fast and irreversible redox process, leading, ultimately, to Cu(II) species. In the coordinating solvent MeCN, a one electron exchange occurs, yielding the corresponding [CalixtrenCu-MeCN](2+) complex with concomitant release of superoxide in the reaction medium. In a noncoordinating solvent such as CH2Cl2, the dioxygen reaction leads to oxygen insertions into the ligand itself. Both reactions are proposed to proceed through the formation of a superoxide-Cu(II) intermediate that is unstable in the Calixtren environment due to second sphere effects. The transiently formed superoxide ligand either undergoes fast substitution for a guest ligand (in MeCN) or intramolecular redox evolutions toward oxygenation of Calixtren. Interestingly, the latter process was shown to occur twice on the same ligand, thus demonstrating a possible catalytic activation of O2 at a single cuprous center. Altogether, this study illustrates the oxidizing power of a [CuO2](+) adduct and substantiates a mechanism by which copper mono-oxygenases such as DbetaH and PHM activate O2 at the Cu(M) center to produce such an intermediate capable of C-H breaking before the electron input provided by the noncoupled Cu(H) center.

Inhibitory effects of a series of 7-substituted-indazoles toward nitric oxide synthases: particular potency of 1H-indazole-7-carbonitrile.

A series of new 7-monosubstituted and 3,7-disubstituted indazoles have been prepared and evaluated as inhibitors of nitric oxide synthases (NOS). 1H-indazole-7-carbonitrile (6) was found equipotent to 7-nitro-1H-indazole (1) and demonstrated preference for constitutive NOS over inducible NOS. By contrast, 1H-indazole-7-carboxamide (8) was slightly less potent but demonstrated a surprising selectivity for the neuronal NOS. Further substitution of 6 by a Br-atom at carbon-3 of the heterocycle enhanced 10-fold the inhibitory effects. Inhibition of NO formation by 6 appeared to be competitive versus both substrate and the cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B). In close analogies with 1, compound 6 strongly inhibited the NADPH oxidase activity of nNOS and induced a spin state transition of the heme-Fe(III). Our results are explained with the help of the X-ray structures that identified key-features for binding of 1 at the active site of NOS.

mTOR Inhibition via Displacement of Phosphatidic Acid Induces Enhanced Cytotoxicity Specifically in Cancer Cells.

The mTOR is a central regulator of cell growth and is highly activated in cancer cells to allow rapid tumor growth. The use of mTOR inhibitors as anticancer therapy has been approved for some types of tumors, albeit with modest results. We recently reported the synthesis of ICSN3250, a halitulin analogue with enhanced cytotoxicity. We report here that ICSN3250 is a specific mTOR inhibitor that operates through a mechanism distinct from those described for previous mTOR inhibitors. ICSN3250 competed with and displaced phosphatidic acid from the FRB domain in mTOR, thus preventing mTOR activation and leading to cytotoxicity. Docking and molecular dynamics simulations evidenced not only the high conformational plasticity of the FRB domain, but also the specific interactions of both ICSN3250 and phosphatidic acid with the FRB domain in mTOR. Furthermore, ICSN3250 toxicity was shown to act specifically in cancer cells, as noncancer cells showed up to 100-fold less sensitivity to ICSN3250, in contrast to other mTOR inhibitors that did not show selectivity. Thus, our results define ICSN3250 as a new class of mTOR inhibitors that specifically targets cancer cells.Significance: ICSN3250 defines a new class of mTORC1 inhibitors that displaces phosphatidic acid at the FRB domain of mTOR, inducing cell death specifically in cancer cells but not in noncancer cells. Cancer Res; 78(18); 5384-97. ©2018 AACR.

The endogenous neurotransmitter, serotonin, modifies neuronal nitric oxide synthase activities.

Serotonin, an important neurotransmitter, is colocalized with neuronal nitric oxide synthase (nNOS), a homodimeric enzyme which catalyzes the production of nitric oxide (NO(.-)) and/or oxygen species. As many interactions have been reported between the nitrergic and serotoninergic systems, we studied the effect of serotonin on nNOS activities. Our results reveal that nNOS is activated by serotonin as both NADPH consumption and oxyhemoglobin (OxyHb) oxidation were enhanced. The generation of L-citrulline from L-arginine (L-Arg) was not affected by serotonin in the range of 0-200 microM, suggesting an additional production of oxygen-derived species. But 5-hydroxytryptamine (5HT) induced the formation of both O and H(2)O(2) by nNOS, as evidenced by electron paramagnetic resonance (EPR) and by using specific spin traps. Overall, these results demonstrate that serotonin is able to activate nNOS, leading to the generation of reactive oxygen species (ROS) in addition to the NO(.-) production. Such a property must be considered in vivo as various nNOS-derived products mediate different signaling pathways.

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.

In vivo triarylmethyl radical stabilization through encapsulation in Pluronic F-127 hydrogel.

In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are non-invasive technologies used to specifically detect and quantify paramagnetic species. However, the relative instability of spin probes such as triarylmethyl radicals limits their application to conduct oxygen quantification and mapping. In this study we encapsulated tetrathiatriarylmethyl radical (TAM; known as "Finland" probe) in Pluronic F-127 hydrogel (PF-127) in order to limit its degradation and evaluate its in vitro and in vivo EPR properties as a function of oxygen. Our results show that the EPR signal of encapsulated TAM in PF-127 hydrogel is similar to the one in solution. Although it is less sensitive to oxygen, it is suitable for oximetry. We also demonstrated that the incorporation of TAM in PF-127 hydrogel leads to an improved in vivo EPR stability of the radical under anesthesia. This new formulation enables high quality EPR imaging and oximetry and paves the way for the application of TAM radical-based probes in various biomedical fields.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe-4S] protein.

The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe-4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.

Very general formation of tetrahydropterin cation radicals during reaction of iron porphyrins with tetrahydropterins: model for the corresponding NO-synthase reaction.

Electron transfer from tetrahydropterins to iron porphyrins, with formation of intermediate tetrahydropterin cation radicals, is a very general reaction that was shown to occur not only with tetrahydrobiopterin, as originally found in NO-synthases, but also with another important biological cofactor, tetrahydrofolate, and various iron porphyrins, either in their ferric state, or in the Fe(II)O(2) state, as in the first model of the corresponding NO-synthase reaction described in this paper.

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps.

Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H2O2, hydroxyl radical, or peroxynitrite (ONOO(-)). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated ß-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes.

Sub-NOAEL amounts of vinclozolin and xenoestrogens target rat chondrogenesis in vivo.

Several endocrine disrupting compounds (EDC) elicit skeletal dysgenesis at pharmacological doses. We have investigated the impact of doses below the "No Observed Adverse Effect" (NOAEL) for vinclozolin (V), an anti-androgenic fungicide, alone or associated with xenoestrogens (Genistein, G and bisphenol-A, BPA). V, G, BPA and their combinations were administered orally to female Wistar rats during gestation and lactation. F1 and F2 offspring were investigated for skeletal anomalies at post-natal days 30, 110 (d30, d110). Skeletal development was monitored by measuring caudal vertebrae and long bones dimensions by X-ray micro-CT-scan. A significant increase in Inter Transverse Apophysis (ITA) distance at the upper head of caudal vertebrae, associated with a reduction in vertebral body height was observed in treated F1 females, but not males. Histometrical analysis of vertebral body growth plate cartilage was performed on serial sections of caudal vertebrae. F1 females but not males showed a diminution in growth plate thickness, with greater impact on the hypertrophic zone. All effects were maximal at d30. Effects on ITA width persisted until d110 while effects on growth plate disappeared. These effects were essentially vinclozolin or BPA-dependent. F2 animals were not affected. Our data suggest that vinclozolin and xenoestrogens act as cartilage developmental disruptors. We suggest that present NOAEL values for these compounds, and EDC at large, might be reconsidered using gestational exposure models. Finally, micro CT-scan appears a valuable non-invasive technique to detect EDC effects on live fauna.

A S-adenosylmethionine methyltransferase-like domain within the essential, Fe-S-containing yeast protein Dre2.

Yeast Dre2 is an essential Fe-S cluster-containing protein that has been implicated in cytosolic Fe-S protein biogenesis and in cell death regulation in response to oxidative stress. Its absence in yeast can be complemented by the human homologous antiapoptotic protein cytokine-induced apoptosis inhibitor 1 (also known as anamorsin), suggesting at least one common function. Using complementary techniques, we have investigated the biochemical and biophysical properties of Dre2. We show that it contains an N-terminal domain whose structure in solution consists of a stable well-structured monomer with an overall typical S-adenosylmethionine methyltransferase fold lacking two α-helices and a ß-strand. The highly conserved C-terminus of Dre2, containing two Fe-S clusters, influences the flexibility of the N-terminal domain. We discuss the hypotheses that the activity of the N-terminal domain could be modulated by the redox activity of Fe-S clusters containing the C-terminus domain in vivo.