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Glucose, chief metabolic support for cancer cell survival and growth, is mainly imported into cells by facilitated glucose transporters (GLUTs). The increase in glucose uptake along with tumor progression is due to an increment of facilitative glucose transporters as GLUT1. GLUT1 prevents cell death of cancer cells caused by growth factors deprivation, but there is scarce information about its role on the damage caused by glucose deprivation, which usually occurs within the core of a growing tumor. In prostate cancer (PCa), GLUT1 is found in the most aggressive tumors, and it is regulated by androgens. To study the response of androgen-sensitive and insensitive PCa cells to glucose deprivation and the role of GLUT1 on survival mechanisms, androgen-sensitive LNCaP and castration-resistant LNCaP-R cells were employed. Results demonstrated that glucose deprivation induced a necrotic type of cell death which is prevented by antioxidants. Androgen-sensitive cells show a higher resistance to cell death triggered by glucose deprivation than castration-resistant cells. Glucose removal causes an increment of H2O2, an activation of androgen receptor (AR) and a stimulation of AMP-activated protein kinase activity. In addition, glucose removal increases GLUT1 production in androgen sensitive PCa cells. GLUT1 ectopic overexpression makes PCa cells more resistant to glucose deprivation and oxidative stress-induced cell death. Under glucose deprivation, GLUT1 overexpressing PCa cells sustains mitochondrial SOD2 activity, compromised after glucose removal, and significantly increases reduced glutathione (GSH). In conclusion, androgen-sensitive PCa cells are more resistant to glucose deprivation-induced cell death by a GLUT1 upregulation through an enhancement of reduced glutathione levels. Keywords: Glut1, Prostate cancer, Glucose deprivation, Androgen receptor, Glutathione, Oxidative stress

International audience; Improved methods for the detection and characterization of carried Neisseria meningitidis isolates are needed. We evaluated a multiplex PCR algorithm for the detection of a variety of carriage strains in the meningitis belt. To further improve the sensitivity and specificity of the existing PCR assays, primers for gel-based PCR assays (sodC, H, Z) and primers/probe for real-time quantitative PCR (qPCR) assays (porA, cnl, sodC, H, E, Z) were modified or created using Primer Express software. Optimized multiplex PCR assays were tested on 247 well-characterised carriage isolates from six countries of the African meningitis belt. The PCR algorithm developed enabled the detection of N. meningitidis species using gel-based and real-time multiplex PCR targeting porA, sodC, cnl and characterization of capsule genes through sequential multiplex PCR assays for genogroups (A, W, X, then B, C, Y and finally H, E and Z). Targeting both porA and sodC genes together allowed the detection of meningococci with a sensitivity of 96% and 89% and a specificity of 78% and 67%, for qPCR and gel-based PCR respectively. The sensitivity and specificity ranges for capsular genogrouping of N. meningitidis are 67% - 100% and 98%-100% respectively for gel-based PCR and 90%-100% and 99%-100% for qPCR. We developed a PCR algorithm that allows simple, rapid and systematic detection and characterisation of most major and minor N. meningitidis capsular groups, including uncommon capsular groups (H, E, Z).

International audience; Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.

International audience; The present study was undertaken to determine whether subacute treatment with aqueous extract of carob (Ceratonia siliqua L.) pods (AECPs) protects against ethanol (EtOH)-induced oxidative stress in rat liver. Animals were divided into four groups: control, carob, EtOH and EtOH+carob. Wistar rats were intraperitoneally pretreated with AECP (600mg/kg body weight (bw)) during 7days and intoxicated for 6h by acute oral administration of EtOH (6g/kg bw) 24h after the last injection. We found that acute administration of EtOH leads to hepatotoxicity as monitored by the increase in the levels of hepatic marker aspartate aminotransferase and alanine aminotransferase as well as hepatic tissue injury. EtOH also increased the formation of malondialdehyde in the liver, indicating an increase in lipid peroxidation and depletion of antioxidant enzyme activities as superoxide dismutase, catalase and glutathione peroxidase. Subacute carob pretreatment prevented all the alterations induced by EtOH and returned their levels to near normal. Importantly, we showed that acute alcohol increased hepatic and plasmatic hydrogen peroxide and free iron levels. The carob pretreatment reversed EtOH effects to near control levels. These data suggest that carob could have a beneficial effect in inhibiting the oxidative damage induced by acute EtOH administration and that its mode of action may involve an opposite effect on plasma and tissue-free iron accumulation. Indeed, carob can be offered as a food additive to protect against EtOH-induced oxidative damage.

10, 5 figures, 3 tables, 1 suppl figure and 1 suppl table; International audience; Industrial pollution due to heavy metals such as mercury is a major concern for the environment and public health. Mercury, in particular methylmercury (MeHg), primarily affects brain development and neuronal activity, resulting in neurotoxic effects. Because chemokines can modulate brain functions and are involved in neuroinflammatory and neurodegenerative diseases, we tested the possibility that the neurotoxic effect of MeHg may interfere with the chemokine CCL2. We have used an original protocol in young mice using a MeHg-contaminated fish-based diet for 3 months relevant to human MeHg contamination. We observed that MeHg induced in the mice cortex a decrease in CCL2 concentrations, neuronal cell death, and microglial activation. Knock-out (KO) CCL2 mice fed with a vegetal control food already presented a decrease in cortical neuronal cell density in comparison with wild-type animals under similar diet conditions, suggesting that the presence of CCL2 is required for normal neuronal survival. Moreover, KO CCL2 mice showed a pronounced neuronal cell death in response to MeHg. Using in vitro experiments on pure rat cortical neurons in culture, we observed by blockade of the CCL2/CCR2 neurotransmission an increased neuronal cell death in response to MeHg neurotoxicity. Furthermore, we showed that sod genes are upregulated in brain of wild-type mice fed with MeHg in contrast to KO CCL2 mice and that CCL2 can blunt in vitro the decrease in glutathione levels induced by MeHg. These original findings demonstrate that CCL2 may act as a neuroprotective alarm system in brain deficits due to MeHg intoxication.

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(*-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(*-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

International audience; Iron-induced oxidative stress promotes hepatic injury in hereditary hemochromatosis, which can be influenced by genetic traits affecting antioxidant enzymes. We assessed the influence of Ala16Val-superoxide dismutase 2, Pro198Leu-glutathione peroxidase 1, and -463G/A-myeloperoxidase genotypes (high activity for the Ala, Pro, and G alleles, respectively) on the risks of cirrhosis and hepatocellular carcinoma (HCC) in patients homozygous for the C282Y-hemochromatosis (HFE) gene mutation. Both the 2G-myeloperoxidase genotype and carriage of one or two copies of the Ala-superoxide dismutase 2 allele were more frequent in patients with cirrhosis or HCC. Patients cumulating these two genetic traits had higher rates of cirrhosis and HCC than other patients.

International audience; BACKGROUND & AIMS: Liver resection includes temporal vascular inflow occlusion resulting in ischemia/reperfusion injury in the remnant liver. Here, we developed a rat model of selective lobe occlusion to isolate reperfusion stress from ischemia and to analyze its effect on liver regeneration. METHODS: Left lateral and median lobes of liver were either mobilized or subjected twice for 10min to ischemia followed by 5min reperfusion prior to resection while the regenerative lobes were only subjected to reperfusion. RESULTS: Although intermittent reperfusion stress induced higher levels of serum transaminases, analysis of cell cycle regulators revealed accelerated regenerative response compared to standard partial hepatectomy. The G0/G1 transition occurred before tissue resection, as evidenced by c-fos, junB, and IL-6 induction. Following hepatectomy, Cyclin D1 up-regulation, G1/S transition, and cell division occurred earlier than normal. Unexpectedly, liver mobilization, a component of the clamping procedure, also resulted in earlier G1/S transition. The shortened G1-phase was driven by the c-Jun N-terminal Kinase pathway and was associated with an oxidative stress response as evidenced by the expression of inducible nitric oxide synthase. CONCLUSION: Intermittent selective clamping of lobes to be resected induced reperfusion stress on remnant liver that was beneficial for liver regeneration, suggesting this procedure could be applied in clinical practice.

International audience; G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

International audience; Amyotrophic lateral sclerosis, the most common adult-onset motor neuron disease, leads to death within 3 to 5 years after onset. Beyond progressive motor impairment, patients with amyotrophic lateral sclerosis suffer from major defects in energy metabolism, such as weight loss, which are well correlated with survival. Indeed, nutritional intervention targeting weight loss might improve survival of patients. However, the neural mechanisms underlying metabolic impairment in patients with amyotrophic lateral sclerosis remain elusive, in particular due to the lack of longitudinal studies. Here we took advantage of samples collected during the clinical trial of pioglitazone (GERP-ALS), and characterized longitudinally energy metabolism of patients with amyotrophic lateral sclerosis in response to pioglitazone, a drug with well-characterized metabolic effects. As expected, pioglitazone decreased glycaemia, decreased liver enzymes and increased circulating adiponectin in patients with amyotrophic lateral sclerosis, showing its efficacy in the periphery. However, pioglitazone did not increase body weight of patients with amyotrophic lateral sclerosis independently of bulbar involvement. As pioglitazone increases body weight through a direct inhibition of the hypothalamic melanocortin system, we studied hypothalamic neurons producing proopiomelanocortin (POMC) and the endogenous melanocortin inhibitor agouti-related peptide (AGRP), in mice expressing amyotrophic lateral sclerosis-linked mutant SOD1(G86R). We observed lower Pomc but higher Agrp mRNA levels in the hypothalamus of presymptomatic SOD1(G86R) mice. Consistently, numbers of POMC-positive neurons were decreased, whereas AGRP fibre density was elevated in the hypothalamic arcuate nucleus of SOD1(G86R) mice. Consistent with a defect in the hypothalamic melanocortin system, food intake after short term fasting was increased in SOD1(G86R) mice. Importantly, these findings were replicated in two other amyotrophic lateral sclerosis mouse models based on TDP-43 (Tardbp) and FUS mutations. Finally, we demonstrate that the melanocortin defect is primarily caused by serotonin loss in mutant SOD1(G86R) mice. Altogether, the current study combined clinical evidence and experimental studies in rodents to provide a mechanistic explanation for abnormalities in food intake and weight control observed in patients with amyotrophic lateral sclerosis. Importantly, these results also show that amyotrophic lateral sclerosis progression impairs responsiveness to classical drugs leading to weight gain. This has important implications for pharmacological management of weight loss in amyotrophic lateral sclerosis.

Oxidative stress has been suggested to be an important mediator of dopaminergic cell death in Parkinson's disease (PD). We investigated the neuroprotective potential of Cu/Zn superoxide dismutase (SOD1) overexpression in the rat substantia nigra (SN) following adenovirus-mediated gene transfer. Human dopaminergic SK-N-SH cells were transduced with adenoviral vectors expressing either human SOD1 (Ad-SOD1) or β-galactosidase (Ad-βgal) before exposure to 1 mM of the 1-methyl-4-phenylpyridinium ion (MPP+). A strong neuroprotective effect of SOD1 gene transfer was observed in the SK-N-SH cells exposed to MPP+ compared with controls. Adult rats were then given unilateral injections of either Ad-SOD1 or Ad-βgal into the striatum, and MPP+ was administered 8 days later at the same location. Strong transgene expression was detected in the SN dopaminergic neurons, a consequence of retrograde axonal transport of the adenoviral particles. The amphetamine-induced rotational behavior of the rats was markedly lower in Ad-SOD1-injected rats than in control animals. Also, behavioral recovery significantly correlated with the number of tyrosine hydrolase-expressing neurons in the SN of the treated rats. These results are consistent with oxidative stress contributing to the MPP+-induced neurodegenerative process. They also indicate that SOD1 gene transfer into the nigrostriatal system may be a potential neuroprotective strategy for treating PD. © 2005 Wiley-Liss, Inc.

International audience; The familial form of amyotrophic lateral sclerosis (FALS) has been linked in some cases to dominant mutations in the gene encoding the Cu/Zn superoxide dismutase (SOD1) mutation. Transgenic mice bearing the G93A SOD1 mutation develop clinical symptoms and pathological features similar to those described in the human disease and represent a good model to explore the potential benefit of therapeutic agents. Using this animal model, we tested the efficacy of morphine and tianeptine treatments, separately and in association, on both disease progression and survival. Acute injection of either of them, administered daily and before the onset of the disease, significantly prolonged the survival of the transgenic mice.

International audience; The German Shepherd Dog (GSD) is a popular working and companion breed for which over 50 hereditary diseases have been documented. Herein, SNP profiles for 197 GSDs were generated using the Affymetrix v2 canine SNP array for a genome-wide association study to identify loci associated with four diseases: pituitary dwarfism, degenerative myelopathy (DM), congenital megaesophagus (ME), and pancreatic acinar atrophy (PAA). A locus on Chr 9 is strongly associated with pituitary dwarfism and is proximal to a plausible candidate gene, LHX3. Results for DM confirm a major locus encompassing SOD1, in which an associated point mutation was previously identified, but do not suggest modifier loci. Several SNPs on Chr 12 are associated with ME and a 4.7 Mb haplotype block is present in affected dogs. Analysis of additional ME cases for a SNP within the haplotype provides further support for this association. Results for PAA indicate more complex genetic underpinnings. Several regions on multiple chromosomes reach genome-wide significance. However, no major locus is apparent and only two associated haplotype blocks, on Chrs 7 and 12 are observed. These data suggest that PAA may be governed by multiple loci with small effects, or it may be a heterogeneous disorder.

International audience; This paper deals with the effects of exercise training on oxygen consumption (MO(2)) and ROS metabolism in the red muscle of trained and untrained female silver eels. Their critical swimming speed (U(crit)) was determined before and after a 4-day training (10h of swimming at 70% of U(crit) and 14 h at 50%, every day). The U(crit) of trained eels increased significantly (by about 7%). The in vitro MO(2) and ROS production by the red fibres were higher (not significant) in trained than in untrained eels, but the ROS production/MO(2) ratio was alike in both groups. The antioxidant-enzyme activities and lipoperoxidation index in trained eels were both lower than those of the untrained ones. These biochemical changes related to the increase in U(crit) suggest that such a training session could maintained or even increased aerobic power of the red muscle without deleterious impact by ROS. These regulations could play a role in the eel's swimming performance efficiency.

International audience; Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease which targets motor neurons of the cortex, brainstem and spinal cord. About 5-10% of all amyotrophic lateral sclerosis cases are familial (FALS), and 15-20% of FALS cases are caused by mutations in the zinc-copper superoxide dismutase gene (SOD1). We identified a large family from France with ten members affected with ALS. Linkage was established to the SOD1 locus on chromosome 21 and genomic and cDNA sequencing was performed for the SOD1 gene. This revealed an activated pseudoexon between exons 4 and 5 that was present in two tested members of the family. Translation of this 43 base pair exon results in the introduction of seven amino acids before a stop codon is present, leading to a prematurely truncated SOD1 protein product of 125 amino acids. Sequencing intron 4 in a patient revealed a eterozygous change 304 bp before exon 5 (c.358 - 304C > G), but only 5 bp after the cryptic exon, thus causing this alternative splice product. This mutation segregated in all affected individuals of the family. This adds an additional genetic mechanism for developing OD1-linked ALS and is one which can be more readily targeted by gene therapy.

International audience; OBJECTIVE: To determine the effects of a dried aqueous extract of cinnamon on antioxidant status of people with impaired fasting glucose that are overweight or obese. METHODS: Twenty-two subjects, with impaired fasting blood glucose with BMI ranging from 25 to 45, were enrolled in a double-blind placebo-controlled trial. Subjects were given capsules containing either a placebo or 250 mg of an aqueous extract of cinnamon (Cinnulin PF) two times per day for 12 weeks. Plasma malondialdehyde (MDA) concentrations were assessed using high performance liquid chromatography and plasma antioxidant status was evaluated using ferric reducing antioxidant power (FRAP) assay. Erythrocyte Cu-Zn superoxide (Cu-Zn SOD) activity was measured after hemoglobin precipitation by monitoring the auto-oxidation of pyrogallol and erythrocyte glutathione peroxidase (GPx) activity by established methods. RESULTS: FRAP and plasma thiol (SH) groups increased, while plasma MDA levels decreased in subjects receiving the cinnamon extract. Effects were larger after 12 than 6 weeks. There was also a positive correlation (r = 0.74; p = 0.014) between MDA and plasma glucose. CONCLUSION: This study supports the hypothesis that the inclusion of water soluble cinnamon compounds in the diet could reduce risk factors associated with diabetes and cardiovascular disease.

International audience; The continuous recycling of haem iron following phagocytosis and catabolism of senescent and damaged red blood cells by macrophages is a crucial process in the maintenance of systemic iron homoeostasis. However, little is known about macrophage iron handling in haemolytic states resulting from a deficiency in antioxidant defences. Our observations indicate that the recently described chronic, but moderate regenerative, haemolytic anaemia of aged SOD1 (superoxide dismutase 1)-knockout mice is associated with red blood cell modifications and sensitivity to both intra- and extra-vascular haemolysis. In the present study, we have characterized the molecular pathways of iron turnover in the liver of Sod1-deficient mice. Despite iron accumulation in liver macrophages, namely Kupffer cells, we did not measure any significant change in non-haem liver iron. Interestingly, in Kupffer cells, expression of the rate-limiting enzyme in haem degradation, haem oxygenase-1, and expression of the iron exporter ferroportin were both up-regulated, whereas the hepcidin mRNA level in the liver was decreased in Sod1-/- mice. These results suggest that concerted changes in the hepatic expression of iron- and haem-related genes in response to haemolytic anaemia in Sod1-/- mice act to reduce toxic iron accumulation in the liver and respond to the needs of erythropoiesis.

We read with interest the paper by Mak et al. (1) on asthma in a Hong Kong Chinese population. They reported a protective effect of the catalase (CAT)–262 T allele in non­smoking asthma patients. This result is of importance, as it suggests for the first time a potential role of the CAT–262 polymorphism in the etiology of asthma. At variance with previous publications was the lack of association of erythrocyte catalase activity with the CAT–262 polymorphism within controls and cases. In a cohort of 196 coal miners, we observed lower erythrocyte catalase activities in miners heterozygous and in those homozygous for the CAT–262 T allele, heterozygotes having an intermediate activity (2). Although the difference was not statistically significant, a 28 percent decrease in catalase activity was observed in the non-smoker T carrier controls by Mak et al. (1), similar to the 31 and 33 percent of decrease reported by us (2), and by Ahn et al. (3) in 18 women from a population-based case-control study respectively. As Mak et al. (1) observed a decrease in catalase activity related to CAT–262 polymorphism in non-smokers only, we stratified on smoking status in our coal miner cohort. We found again a significant decrease in catalase activity with the CAT–262 T allele in current and in ex- or non-smokers (see figure), as we found previously in miners with current high and low exposure to coal dust (2), which demonstrates the consistency of the association. The lack of significance of the association in the study by Mak et al. (1) may be due to a low statistical power as 28 subjects were heterozygous and only one was homozygous for the CAT–262 T allele out of the 308 Chinese controls. Figure Catalase activity according to smoking status and to CAT–262 genotype It is interesting to note that Mak et al (1) observed a protective effect of the CAT–262 T allele in non-smoking asthma patients only. Regardless of the disease, an oxidant-rich environment has been found to increase risk only in subjects with the low-activity catalase CAT–262 T allele (4). Factors previously found to be protective, such as an antioxidant-rich dietary environment or a low level of circulating proinflammatory cytokines, seemed to be beneficial only in subjects with the high catalase activity CC genotype (5). Similar to the oxidant-rich environment in our occupational study, asthma may generate reactive oxygen species, and catalase activity significantly increases in asthma patients compared to controls (1). As catalase is involved in the first response to oxidative stimuli, the increase in its activity may be interpreted as a compensatory response to the changes in the oxidant/antioxidant imbalance occuring in asthmatics. In the study of Mak et al. (1), the level of the oxidant state related to asthma may partly mask the a priori deleterious effect of the CAT–262 T allele. Further, the CAT–262 T allele frequency in this Chinese population (1) was five times lower than that observed in Caucasians (mean 26 percent [20–56] from 7 studies), and was similar to the frequency observed in 40 African Americans (6 percent) by Zhou et al. (6). The differences in reported CAT–262 T allele frequency between studies indicates significant variation between populations, and could also partly explain the a priori deleterious effect of the CAT–262 T allele, as patterns of linkage disequilibrium across populations were different. In summary, these results highlight the complexity of the potential role of the CAT–262 polymorphism in the etiology of oxidant-related diseases.

International audience; Eels are submitted to hydrostatic pressure (HP) during their spawning migration (about 6000 Km). Before migration, they change from the yellow to the silver stage (silvering process). The effects of HP in relation to the silvering process have been studied on aerobic metabolism and more precisely on reactive oxygen species (ROS) metabolism. HP acclimatization of yellow eels improves oxidative phosphorylation together with supposed concomitant changes in electron leak and ROS production. Therefore hydroxyl radical (OH*) production, superoxyde dismutase and catalase activities, malondialdehyde content and in parallel oxygen consumption were measured in the red muscle of long-term pressure exposed and control group yellow and silver eels. At atmospheric pressure, yellow eels exhibited significantly higher oxygen consumption and OH* production than silver eels; and significantly lower malondialdehyde content. This could be due to the increase in membrane fluidity induced by the silvering process. Long-term HP exposure decreases yellow eel oxygen consumption which becomes similar to that of the silver stage. In parallel there is a decrease in OH* production and concomitantly antioxidant enzyme activities follow the same tendency. Thus the respiratory chain improvement in pressure acclimatized yellow eels is accompanied by a ROS production decrease which could mean an electron leak decrease.

International audience; Friedreich ataxia (FRDA) results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur protein activity initially ascribed to mitochondrial iron overload. Recent in vitro data suggest that frataxin is necessary for iron incorporation in Fe-S cluster (ISC) and heme biosynthesis. In addition, several reports suggest that continuous oxidative damage resulting from hampered superoxide dismutases (SODs) signaling participates in the mitochondrial deficiency and ultimately the neuronal and cardiac cell death. This has led to the use of antioxidants such as idebenone for FRDA therapy. To further discern the role of oxidative stress in FRDA pathophysiology, we have tested the potential effect of increased antioxidant defense using an MnSOD mimetic (MnTBAP) and Cu,ZnSOD overexpression on the murine FRDA cardiomyopathy. Surprisingly, no positive effect was observed, suggesting that increased superoxide production could not explain by itself the FRDA cardiac pathophysiology. Moreover, we demonstrate that complete frataxin-deficiency neither induces oxidative stress in neuronal tissues nor alters the MnSOD expression and induction in the early step of the pathology (neuronal and cardiac) as previously suggested. We show that cytosolic ISC aconitase activity of iron regulatory protein-1 progressively decreases, whereas its apo-RNA binding form increases despite the absence of oxidative stress, suggesting that in a mammalian system the mitochondrial ISC assembly machinery is essential for cytosolic ISC biogenesis. In conclusion, our data demonstrate that in FRDA, mitochondrial iron accumulation does not induce oxidative stress and we propose that, contrary to the general assumption, FRDA is a neurodegenerative disease not associated with oxidative damage.

Because oxidative stress is involved in arterial hypertension, impairment of hepatic antioxidant defences could develop in the course of this disease. Metallothionein (MT), an antioxidant protein, is present in high rates in the liver. The aim of this study was to investigate the effect of a mineralocorticoid-salt treatment on blood pressure, hepatic antioxidant enzyme activities, and cardiac MT levels in transgenic MT null mice compared with control mice to further clarify the role of MT during the experimental development of arterial hypertension. Control and transgenic MT –/– mice were submitted to an 8-week mineralocorticoid-salt treatment. Hepatic glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase activities and cardiac MT and mineral levels were measured. Mineralocorticoid-salt treatment induced an increase in blood pressure in both transgenic MT –/– and control mice that was associated with an impairment of liver antioxidant status. MT deficiency was associated with modifications of hepatic antioxidant enzyme activities and with a decrease in cardiac iron levels. Adaptive processes of antioxidant systems may explain the absence of an effect of metallothionein deficiency on the development of mineralocorticoid-salt hypertension. The interactions that occur between the in vivo antioxidant systems probably produce a complex regulation of the oxidative balance and consequently prevent antioxidant deficiency.Key words: hepatic antioxidant enzymes, metallothionein, transgenic mice, DOCA-salt hypertension.

International audience; 1. Heat stress (HS) is known to protect the myocardium against ischaemic damage. It has been reported that reactive oxygen species (ROS) are abundantly produced during this stress. Since mechanisms triggering the HS-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective phenomenon. 2. Rats were divided into four groups (n=8 in each group), subjected to either hyperthermia (42 degrees C internal temperature for 15 min) or sham anaesthesia and treated or not with N-2-mercaptopropionyl glycine (MPG), a synthetic antioxidant, 10 min before HS. Twenty-four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30-min occlusion of the left coronary artery followed by 120 min of reperfusion. Myocardial Hsp 27 and 70 expression was assessed by Western blot analysis (n=4). Cardiac activities of antioxidant enzymes (superoxide dismutase and glutathione peroxidase) were also examined (n=4). 3. Infarct-to-risk zone ratio was significantly reduced in HS (17+/-1.3%) compared to Sham (34.3+/-1.7%) hearts. This effect was abolished by MPG pretreatment (40.6+/-1.9% in HS+MPG vs. 39.8+/-2.5% in Sham+MPG hearts). This cardioprotection was associated with an enhanced Hsp 27 and 70 expression, which was not modified by MPG pretreatment. Antioxidant enzyme activities was not modified by heat stress or MPG pretreatment. 4. Free radical production following hyperthermia appears to play a role in the heat stress induced cardioprotection, independently of Hsp levels. Antioxidant enzyme activities do not seem to be implicated in this cardioprotective mechanism.

International audience; Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by selective degeneration of motoneurones. Familial ALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1) is linked to the disease. An animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Recent electrophysiological data emphasised that the striking selective vulnerability of motoneurones might be due to their differential calcium buffering capacities. Therefore we have investigated, using immunohistochemistry, the expression of different calcium binding proteins in brainstem and spinal cord from normal and SOD1 mutated mice. Among the 13 calcium-binding proteins screened, only one, S100A6, a homodimeric calcium-binding protein able to bind four Zn(2+), appeared to be highly expressed in the SOD1 mutated mice. In brainstem, reactive astrocytes, but not motoneurones, from several regions, including nerve 12 root, were highly S100A6-positive. Hypoglossal nucleus was negative for S100A6. In dorsal root, reactive astrocytes from both white matter and anterior horn were highly reactive. If overexpression of S100A6 is specific for ALS, it will be a valuable diagnostic marker for this disease.

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1,25-dihydroxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide synthase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2.-) but also nitric oxide (.NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2.- determined. Conversely, superoxide dismutase (SOD) scavenged the O2.- released and increased the .NO-derived nitrite concentration detected. These data suggested a possible interaction between O2.- and .NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abrogated by SOD and partially inhibited by aminoguanidine or L-NMMA. Since the iNOS inhibitors did not directly scavenge O2.-, LDCL determination in the presence or absence of SOD and/or iNOS inhibitors demonstrated a concomitant production of O2.- and .NO. These radicals induced the formation of a .NO-derived product(s), probably peroxynitrite (ONOO-), which was required to elicit maximal LDCL. Finally, LDCL measurement provided a convenient tool to characterize iNOS triggering and demonstrated an interaction between NADPH oxidase and iNOS products in human macrophagic cells phagocytizing opsonized-zymosan. These findings show that in activated macrophages, iNOS activity can be involved in LDCL and support the debated hypothesis of iNOS participation to the microbicidal activity of human macrophages.

International audience; Hypoxic preconditioning has been shown to induce neuroprotection against a subsequent damaging insult. In order to study the underlying molecular and cellular mechanisms of hypoxic preconditioning, we investigated, in gerbil hippocampus, the effects in vivo of transient exposure to hypoxia (4% O(2) for 6 min followed by either 48 h or 7 days of reoxygenation) (i) on the induction of 72 kDa heat shock protein (HSP72), heme oxygenase-1 (HO-1) and manganese superoxide dismutase (Mn SOD) as assessed by Western immunoblotting and (ii) on the astroglial and microglial activation as detected by both immunohistochemistry and Western immunoblotting for GFAP, and histochemistry for isolectin B4, respectively. Our data show that, although hypoxia and subsequent reoxygenation led to neither neuronal damage nor HSP72 induction in gerbil hippocampus, it induced a progressive and sustained expression of HO-1 and Mn SOD. As expected from the absence of neuronal death, hypoxia was not associated with microglial activation but led to a significant astrocytic activation. These findings demonstrate that transient hypoxia enhances the antioxidative enzymatic defenses of the brain, which are susceptible to increased tolerance against a subsequent damaging insult.

The reasons for the cellular specificity and slow progression of motoneuron diseases such as ALS are still poorly understood. We previously described a motoneuron-specific cell death pathway downstream of the Fas death receptor, in which synthesis of nitric oxide (NO) is an obligate step. Motoneurons from ALS model mice expressing mutant SOD1 showed increased susceptibility to exogenous NO as compared with controls. Here, we report a signaling mechanism whereby NO leads to death of mutant, but not control, motoneurons. Unexpectedly, exogenous NO triggers expression of Fas ligand (FasL) in cultured motoneurons. In mutant SOD1 G93A and SOD1 G85R , but not in control motoneurons, this up-regulation results in activation of Fas, leading through Daxx to phosphorylation of p38 and further NO synthesis. This Fas/NO feedback amplification loop is required for motoneuron death in vitro . In vivo , mutant SOD1 G93A and SOD1 G85R mice show increased numbers of positive motoneurons and Daxx nuclear bodies weeks before disease onset. Moreover, FasL up-regulation is reduced in the presence of transgenic dominant-negative Daxx. We propose that chronic low-level activation of the Fas/NO feedback loop may underlie the motoneuron loss that characterizes familial ALS and may help to explain its slowly progressive nature.