Les effets de l’Angiopoietin-like 2 sur la voie cytoprotectrice antioxydante Nrf2

L’angiopoietin-like 2 (angptl2) est une glycoprotéine de 64 kDa pro-inflammatoire et pro-athérogénique associée à divers maladies inflammatoires chroniques. Il est probable que l’angptl2 possède également un effet pro-oxydant, puisqu’elle stimule la production aigüe de dérivés réactifs oxygénés (DRO). Le facteur de transcription « nuclear factor (erythroidderived 2)-like 2 » (Nrf2) fait partie d’un mécanisme antioxydant majeur permettant de maintenir l’équilibre redox via l’induction de plusieurs gènes de l’élément de réponse antioxydante (ERA). En présence de DRO, la protéine Keap-1 se dissocie de Nrf2 et cesse de promouvoir sa dégradation protéasomale. Cette dissociation est stimulée par la protéine DJ-1 qui favorise la translocation nucléaire de Nrf2. La p38MAPK peut phosphoryler Nrf2 et promouvoir son interaction avec Keap-1. Nous avons posé l’hypothèse selon laquelle l’angptl2 causait un stress oxydant chronique, d’abord en stimulant en aigu la production de DRO, puis en inhibant la voie antioxydante Nrf2. Nous avons étudié, par immunobuvardage de type Western sur des cellules endothéliales en culture (HUVEC), les effets de l’angptl2 recombinante (100 nM) sur les niveaux protéiques nucléaires de Nrf2, les niveaux de Keap-1 dans le cytosol et de DJ-1 dans le noyau, en absence et en présence de l’antioxydant NAC (10 μM). Nous avons également étudié l’activation de la p38MAPK. Les niveaux nucléaires de Nrf2 n’ont pas été affectés par la stimulation aigüe (10 min) à l’angptl2 recombinante, mais ont été diminués par la stimulation chronique (24 h). L’ajout d’un agent antioxydant n’a pas altéré l’effet chronique, indiquant que les DRO ne sont pas directement impliqués. Les niveaux protéiques cytosoliques de Keap-1, protéine inhibitrice de Nrf2, et les niveaux nucléaires de DJ-1, protéine stabilisatrice de Nrf2, n’ont pas été affectés par l’angptl2 de manière significative. La phosphorylation de la p38MAPK n’a pas été non plus affectée par la stimulation aigüe ou chronique à l’angptl2. Ces données suggèrent que l’angptl2 n’a pas d’effet aigu, mais a un effet chronique inhibiteur sur la voie Nrf2, qui n’est pas associé à un effet sur Keap-1, DJ-1 ou p38MAPK.

The co-catalytic effect of chlorpromazine on peroxidase-mediated oxidation of melatonin: enhanced production of N-1-acetyl-N (2)-formyl-5-methoxykynuramine

Accumulating evidence points to relationships between increased production of reactive oxygen or decreased antioxidant protection in schizophrenic patients. Chlorpromazine (CPZ), which remains a benchmark treatment for people with schizophrenia, has been described as a pro-oxidant compound. Because the antioxidant compound melatonin exerts protective effects against CPZ-induced liver disease in rats, in this investigation, our main objective was to study the effect of CPZ as a co-catalyst of peroxidase-mediated oxidation of melatonin. We found that melatonin was an excellent reductor agent of preformed CPZ cation radical (CPZ(center dot+)). The addition of CPZ during the horseradish peroxidase (HRP)-catalyzed oxidation of melatonin provoked a significant increase in the rate of oxidation and production of N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK). Similar results were obtained using myeloperoxidase. The effect of CPZ on melatonin oxidation was rather higher at alkaline pH. At pH 9.0, the efficiency of oxidation of melatonin was 15 times higher and the production of AFMK was 30 times higher as compared with the assays in the absence of CPZ. We suggest that CPZ is able to exacerbate the rate of oxidation of melatonin by an electron transfer mechanism where CPZ(center dot+), generated during the peroxidase-catalyzed oxidation, is able to efficiently oxidize melatonin.

Effects of acute creatine supplementation on iron homeostasis and uric acid-based antioxidant capacity of plasma after wingate test

Background: Dietary creatine has been largely used as an ergogenic aid to improve strength and athletic performance, especially in short-term and high energy-demanding anaerobic exercise. Recent findings have also suggested a possible antioxidant role for creatine in muscle tissues during exercise. Here we evaluate the effects of a 1-week regimen of 20 g/day creatine supplementation on the plasma antioxidant capacity, free and heme iron content, and uric acid and lipid peroxidation levels of young subjects (23.1 +/- 5.8 years old) immediately before and 5 and 60 min after the exhaustive Wingate test. Results: Maximum anaerobic power was improved by acute creatine supplementation (10.5 %), but it was accompanied by a 2.4-fold increase in pro-oxidant free iron ions in the plasma. However, potential iron-driven oxidative insult was adequately counterbalanced by proportional increases in antioxidant ferric-reducing activity in plasma (FRAP), leading to unaltered lipid peroxidation levels. Interestingly, the FRAP index, found to be highly dependent on uric acid levels in the placebo group, also had an additional contribution from other circulating metabolites in creatine-fed subjects. Conclusions: Our data suggest that acute creatine supplementation improved the anaerobic performance of athletes and limited short-term oxidative insults, since creatine-induced iron overload was efficiently circumvented by acquired FRAP capacity attributed to: overproduction of uric acid in energy-depleted muscles (as an end-product of purine metabolism and a powerful iron chelating agent) and inherent antioxidant activity of creatine.

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

Reduction of the in vitro pro-inflammatory response by macrophages to poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

This study evaluates the pro-inflammatory response to the thermoplastic biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) through the analysis of cellular responses in vitro. The murine macrophage RAW264.7 cell line was cultured on solvent cast PHBV films, which was found to induce pro-inflammatory activity that required direct contact between the material and the macrophages. The identity of the pro-inflammatory stimulus was determined by culturing bone marrow-derived macrophages from bacterial lipopolysaccharide (LPS) hyporesponsive C3H/HeJ mice and CpG non-responsive TLR9-/- mice on PHBV. The lack of a pro-inflammatory response by the C3H/HeJ cells indicates that the pro-inflammatory agent present within PHBV is predominately LPS while the TLR9-/- macrophages confirmed that CpG-containing bacterial DNA is unlikely to contribute to the activity. A series of purification procedures was evaluated and one procedure was developed that utilized hydrogen peroxide treatment in solution. The optimized purification was found to substantially reduce the pro-inflammatory response to PHBV without adversely affecting either the molecular structure or molecular weight of the material thereby rendering it more amenable for use as a biomaterial in vivo. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.

Time controlled photo-oxidation of polyolefins by polymer bound additives

A variety of iron compounds containing vinyl or thiol functional groups (used as photoactivators) have been synthesised and some of these were successfully bound to both polyethylene and polypropylene backbones during processing in the presence of peroxide and interlinking agent. Concentrates (masterbatches) of the photoactivators in PP and PE were prepared and the pro-oxidant effect of the diluted masterbatches in absence and presence of an antioxidant was evaluated. An antioxidant photoactivator (FeDNC ) was found to sensitise the photoactivity of pro-oxidants (Metone A / Metone M) whereas an antioxidant (ZnDNC) was found to stabilise the polymer (PP and PE) containing both of these combinations. It was observed that the lower concentration of FeDNC sensitises the stability of the polymer containing very small concentration of NiDNC whereas higher concentration of FeDNC stabilises the polymer (LDPE) containing same amount of NiDNC compared to FeDNC alone. The photostability of unstabilised PP containing FeAc could be varied by varying the concentration of ZnDEC. Both the induction period and the UV - life time of the polymer increased by increasing concentration of ZnDEC. It is suggested that ligand exchange reaction may take place between FeAc and ZnDNC. A polymer bound UV stabiliser (HAEB) and a thermal stabiliser (DBBA) were used with a non extractable photoactivator (FeAc) in PP. Small concentrations of the stabilisers (HAEB and DBBA) in combination with the photoactivator (FeAc) sensitise the polymer. The antioxidant present in commercial polymer (LDPE and PP) was found to be of a hindered phenol type, which was found to antagonise with ZnDNC when used in combination with the photoactivators.

Multifaceted roles of curcumin: two sides of a coin!

Introduction: Curcumin has been a front-line topic of mainstream scientific research for a variety of diseases from cancer to Alzheimer's to infectious diseases. Curcumin suppresses the type 1 immune response, which might lead to alleviation of type 1 immune response disorders. However, the inhibition of type 1 immune response might invite infections with opportunistic pathogens. Considering its low bioavailability, several curcumin derivatives have been designed to improve its functionality. Areas covered: This is a consolidated review which aims to compare and contrast diverse aspects of curcumin in variety of diseases. The intricate underlying mechanisms and the functional determinants of curcumin are discussed. Expert opinion: Curcumin being considered as a spicy panacea, is not a remedy for all diseases. However, its ability to act differentially as an antioxidant or pro-oxidant akin to that of a double-edged sword/friend turning foe can be either beneficial or harmful for the host. It exhibits antioxidant properties at concentrations achievable in the body, making the host vulnerable to infections due to the suppression of innate immune responses. With the increase in knowledge of its functional groups, production of analogues of curcumin is underway to enhance its bioavailability and hence its therapeutic potency.

Molecular biomarkers and toxic consequences of impact by organic pollution in aquatic organisms

Organic contaminants are readily bioaccumulated by aquatic organisms. Exposure to and toxic effects of contaminants can be measured in terms of the biochemical responses of the organisms (i.e. molecular biomarkers). The hepatic biotransformation enzyme cytochrome P4501A (CYP1A) in vertebrates is specifically induced by organic contaminants such as aromatic hydrocarbons, PCBs and dioxins, and is involved in chemical carcinogenesis via catalysis of the covalent binding of organic contaminants to DNA (DNA-adducts). Hepatic CYP1A induction has been used extensively and successfully as a biomarker of organic contaminant exposure in fish. Fewer but equally encouraging studies in fish have used hepatic bulky, hydrophobic DNA-adducts as biomarkers of organic contaminant damage. Much less is known of the situation in marine invertebrates, but a CYPlA-like enzyme with limited inducibility and some potential for biomarker application is indicated. Stimulation of reactive oxygen species (ROS) production is another potential mechanism of organic contaminant-mediated DNA and other damage in aquatic organisms. A combination of antioxidant (enzymes, scavengers) and pro-oxidant (oxidised DNA bases, lipid peroxidation) measurements may have potential as a biomarker of organic contaminant exposure (particularly those chemicals which do not induce CYP1A) and/or oxidative stress, but more studies are required. Both CYP1A- and ROS-mediated toxicity are indicated to result in higher order deleterious effects, including cancer and other aspects of animal fitness.

Sphingomyelinase D/Ceramide 1-Phosphate in Cell Survival and Inflammation

Sphingolipids are major constituents of biological membranes of eukaryotic cells. Many studies have shown that sphingomyelin (SM) is a major phospholipid in cell bilayers and is mainly localized to the plasma membrane of cells, where it serves both as a building block for cell architecture and as a precursor of bioactive sphingolipids. In particular, upregulation of (C-type) sphingomyelinases will produce ceramide, which regulates many physiological functions including apoptosis, senescence, or cell differentiation. Interestingly, the venom of some arthropodes including spiders of the genus Loxosceles, or the toxins of some bacteria such as Corynebacterium tuberculosis, or Vibrio damsela possess high levels of D-type sphingomyelinase (SMase D). This enzyme catalyzes the hydrolysis of SM to yield ceramide 1-phosphate (C1P), which promotes cell growth and survival and is a potent pro-inflammatory agent in different cell types. In particular, C1P stimulates cytosolic phospholipase A2 leading to arachidonic acid release and the subsequent formation of eicosanoids, actions that are all associated to the promotion of inflammation. In addition, C1P potently stimulates macrophage migration, which has also been associated to inflammatory responses. Interestingly, this action required the interaction of C1P with a specific plasma membrane receptor, whereas accumulation of intracellular C1P failed to stimulate chemotaxis. The C1P receptor is coupled to Gi proteins and activates of the PI3K/Akt and MEK/ERK1-2 pathways upon ligation with C1P. The proposed review will address novel aspects on the control of inflammatory responses by C1P and will highlight the molecular mechanisms whereby C1P exerts these actions.

Concentrations of Cadmium and Zinc in Seawater of Bohai Bay and Their Effects on Biomarker Responses in the Bivalve Chlamys farreri

Both in-field chemical investigation and in the laboratory toxic tests were carried out to systematically understand the pollution status of cadmium (Cd) and zinc (Zn) in Bohai Bay. Samples collected from surface seawater were determined to describe the distributions of Cd and Zn in Bohai Bay. The average values in our study of Cd and Zn were 0.15 mu g/L and 19.68 mu g/L, respectively. Both of them were lower than the first class limit of seawater quality standard in China. In the laboratory, antioxidant enzymes [SOD (Cu/Zn-SOD, Mn-SOD), CAT], lipid peroxidation (MDA), phase I and phase II enzymes (CYP4501A and GST) were investigated in the bivalves Chlamys farreri exposed to Cd and Zn at the concentration levels of Bohai Bay seawater, which were obtained from our in-field investigation. The reduced SOD, CAT, and EROD (7-ethoxyresorufin-O-deethylase) activities (with the inhibitory rate of 16.8%, 31.5%, and 51.6%, respectively) in Cd treatment were observed and resulted in obvious lipid peroxidation damage. However, treatment of Zn showed elevations in SOD and GST by 13.3% and 29.9%, respectively, and with no influence on lipid peroxidation. In summary, seawater quality in Bohai Bay seawater was ranked as good in general, but it seemed that Cd might possess a potential environmental risk by effecting pro-oxidant/antioxidant balance and phase I detoxification in C. farreri.

A Dual Role for Lecithin:Cholesterol Acyltransferase (EC 2.3.1.43) in Lipoprotein Oxidation

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme involved in lipoprotein metabolism. It mediates the transesterification of free cholesterol to cholesteryl ester in an apoprotein A-I-dependent process. We have isolated purified LCAT from human plasma using anion-exchange chromatography and characterized the extracted LCAT in terms of its molecular weight, molar absorption coefficient, and enzymatic activity. The participation of LCAT in the oxidation of very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) was examined by supplementing lipoproteins with exogenous LCAT over a range of protein concentrations. LCAT-depleted lipoproteins were also prepared and their oxidation kinetics examined. Our results provide evidence for a dual role for LCAT in lipoprotein oxidation, whereby it acts in a dose-responsive manner as a potent pro-oxidant during VLDL oxidation, but as an antioxidant during LDL oxidation. We believe this novel pro-oxidant effect may be attributable to the LCAT-mediated formation of oxidized cholesteryl ester in VLDL, whereas the antioxidant effect is similar to that of chain-breaking antioxidants. Thus, we have demonstrated that the high-density lipoprotein-associated enzyme LCAT may have a significant role to play in lipoprotein modification and hence atherogenesis. (C) 2007 Elsevier Inc. All rights reserved.

Adrenomedullin Gene Delivery is Cardio-protective in a Model of Chronic Nitric Oxide Deficiency Combining Pressure Overload, Oxidative Stress and Cardiomyocyte Hypertrophy

BACKGROUND/AIMS:
Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo.

METHODS:
The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling.

RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension.

CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

AGEs, RAGE, and Diabetic Retinopathy

Diabetic retinopathy is a major diabetic complication with a highly complex etiology. Although there are many pathways involved, it has become established that chronic exposure of the retina to hyperglycemia gives rise to accumulation of advanced glycation end products (AGEs) that play an important role in retinopathy. In addition, the receptor for AGEs (RAGE) is ubiquitously expressed in various retinal cells and is upregulated in the retinas of diabetic patients, resulting in activation of pro-oxidant and proinflammatory signaling pathways. This AGE-RAGE axis appears to play a central role in the sustained inflammation, neurodegeneration, and retinal microvascular dysfunction occurring during diabetic retinopathy. The nature of AGE formation and RAGE signaling bring forward possibilities for therapeutic intervention. The multiple components of the AGE-RAGE axis, including signal transduction, formation of ligands, and the end-point effectors, may be promising targets for strategies to treat diabetic retinopathy.

Pigment epithelium-derived factor mitigates inflammation and oxidative stress in retinal pericytes exposed to oxidized low-density lipoprotein

Oxidized and/or glycated low-density lipoprotein (LDL) may mediate capillary injury in diabetic retinopathy. The mechanisms may involve pro-inflammatory and pro-oxidant effects on retinal capillary pericytes. In this study, these effects, and the protective effects of pigment epithelium-derived factor (PEDF), were defined in a primary human pericyte model. Human retinal pericytes were exposed to 100 microg/ml native LDL (N-LDL) or heavily oxidized glycated LDL (HOG-LDL) with or without PEDF at 10-160 nM for 24 h. To assess pro-inflammatory effects, monocyte chemoattractant protein-1 (MCP-1) secretion was measured by ELISA, and nuclear factor-kappaB (NF-kappaB) activation was detected by immunocytochemistry. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS), peroxynitrite (ONOO(-)) formation, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) production. The results showed that MCP-1 was significantly increased by HOG-LDL, and the effect was attenuated by PEDF in a dose-dependent manner. PEDF also attenuated the HOG-LDL-induced NF-kappaB activation, suggesting that the inhibitory effect of PEDF on MCP-1 was at least partially through the blockade of NF-kappaB activation. Further studies demonstrated that HOG-LDL, but not N-LDL, significantly increased ONOO(-) formation, NO production, and iNOS expression. These changes were also alleviated by PEDF. Moreover, PEDF significantly ameliorated HOG-LDL-induced ROS generation through up-regulation of superoxide dismutase 1 expression. Taken together, these results demonstrate pro-inflammatory and pro-oxidant effects of HOG-LDL on retinal pericytes, which were effectively ameliorated by PEDF. Suppressing MCP-1 production and thus inhibiting macrophage recruitment may represent a new mechanism for the salutary effect of PEDF in diabetic retinopathy and warrants more studies in future.