Search

The oncostatic effects of melatonin correlate with increased reactive oxygen species (ROS) levels, but how melatonin induces this ROS generation is unknown. In the present study, we aimed to elucidate the two seemingly opposing actions of melatonin regarding its relationship with free radicals. We analyzed the effects of melatonin on head and neck squamous cell carcinoma cell lines (Cal-27 and SCC-9), which were treated with 0.5 or 1 mM melatonin. We further examined the potential effects of melatonin to induce ROS and apoptosis in Cal-27 xenograft mice. Here we report that melatonin mediates apoptosis in head and neck cancer by driving mitochondrial reverse electron transport (RET) to induce ROS production. Melatonin-induced changes in tumoral metabolism led to increased mitochondrial activity, which, in turn, induced ROS-dependent mitochondrial uncoupling. Interestingly, mitochondrial complex inhibitors, including rotenone, abolished the ROS elevation indicating that melatonin increased ROS generation via RET. Melatonin also increased membrane potential and CoQ10 H2 /CoQ10 ratio to elevate mitochondrial ROS production, which are essential conditions for RET. We found that genetic manipulation of cancer cells with alternative oxidase, which transfers electrons from QH2 to oxygen, inhibited melatonin-induced ROS generation, and apoptosis. RET restored the melatonin-induced oncostatic effect, highlighting the importance of RET as the site of ROS production. These results illustrate that RET and ROS production are crucial factors in melatonin's effects in cancer cells and establish the dual effect of melatonin in protecting normal cells and inducing apoptosis in cancer cells.

Summary: Background & aims: The main role of the small intestine to absorb the bulk of dietary nutrients, including proteins and amino acids (AA). Enterocytes have long been considered a major player in amino acid homeostasis, especially the glutamine–citrulline–arginine crossroads. In vivo intestinal epithelium cell lines for studies of nitrogen metabolism have now reached their limitations. To push research forward, an interesting approach could be to use the explant incubation model of intestinal tissue, involving in vitro incubation of duodenal tissue biopsy specimens taken from human subjects. Methods: Eight duodenal biopsies were taken during endoscopy from patients without proven intestinal disease. Each biopsy was incubated in complete culture medium for 23 h then weighed, and cell viability was evaluated after 6 and 18 h. We then explored L-citrulline (L-CIT) production (reflecting metabolic activity) and the presence of the main enzymes involved in AA metabolism in the intestine (arginase II, glutaminase I, ornithine aminotransferase, and ornithine carbamoyltransferase). Results: Mean weight of biopsies was 12.36 ± 1.00 mg. Mortality was around 20% after 6 h and 50% after 18 h of incubation. CIT was amply produced by the biopsies, and enzymes implicated in intestinal AA metabolism were expressed in this model. Conclusions: This in vitro explant model of intestinal tissue emerges as a reliable model for conducting ex vivo investigations on AA metabolism. Keywords: Ornithine carbamoyltransferase, Amino acid, Enterocyte, Enzyme

Background Animal studies and clinical data support the interest of citrulline as a promising therapeutic for sarcopenia. Citrulline is known to stimulate muscle protein synthesis, but how it affects energy metabolism to support the highly energy‐dependent protein synthesis machinery is poorly understood. Methods Here, we used myotubes derived from primary culture of mouse myoblasts to study the effect of citrulline on both energy metabolism and protein synthesis under different limiting conditions. Results When serum/amino acid deficiency or energy stress (mild uncoupling) were applied, citrulline stimulated muscle protein synthesis by +22% and +11%, respectively. Importantly, this increase was not associated with enhanced energy status (ATP/ADP ratio) or mitochondrial respiration. We further analysed the share of mitochondrial respiration and thus of generated ATP allocated to different metabolic pathways by using specific inhibitors. Our results indicate that addition of citrulline allocated an increased share of mitochondrially generated ATP to the protein synthesis machinery under conditions of both serum/amino acid deficiency (+28%) and energy stress (+21%). This reallocation was not because of reduced ATP supply to DNA synthesis or activities of sodium and calcium cycling ion pumps. Conclusions Under certain stress conditions, citrulline increases muscle protein synthesis by specifically reallocating mitochondrial fuel to the protein synthesis machinery. Because ATP/ADP ratios and thus Gibbs free energy of ATP hydrolysis remained globally constant, this reallocation may be linked to decreased activation energies of one or several ATP (and GTP)‐consuming reactions involved in muscle protein synthesis.

Cadmium (Cd) is a metal which may participate in the development of type II diabetes even if Cd exposure levels are mild. However, experimental studies focusing on daily environmentally relevant doses are scarce, particularly for glucose metabolism of the offspring of chronically exposed mothers. The aim is to measure the impact of maternal low level Cd exposure on glucose and lipid metabolism of offspring. Female rats were exposed to 0, 50 or 500 μg.kg-1.d-1 of CdCl2, 21 days before mating and during 21 days of gestation and 21 days of lactation. Pups exposure was organized in 3 groups (control, Cd1, Cd2) according to renal dams' Cd burden. Parameters of glucose and lipid metabolisms were measured for the pups on post-natal day 21, 26 and 60. Maternal Cd exposure led to significant amounts of Cd in the liver and kidney of pups. At weaning, insulin secretion upon glucose stimulation was unchanged, but the removal of circulating glucose was slower for pups born from the lowest impregnated dams (Cd1). Five days after, glucose tolerance of all groups was identical. Thus, this loss of insulin sensitivity was reversed, in part by increased adiponectin secretion for the Cd1 group. Furthermore, pups from dams accumulating the highest levels of Cd (Cd2) exhibited a compensatory increased insulin pancreatic secretion, together with increased circulating non-esterified fatty acids, indicating the establishment of insulin resistance, 2 months after birth. This study has demonstrated the influence of maternal exposure to low levels of Cd on glucose homeostasis in the offspring that might increase the risk of developing type II diabetes later in life.

Background NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6. Results We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation. Conclusions NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

Background Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. Results We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. Conclusions These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

Background Islets of Langerhans transplantation is a promising therapy for type 1 diabetes mellitus, but this technique is compromised by transplantation stresses including inflammation. In other tissues, co-transplantation with mesenchymal stem cells has been shown to reduce damage by improving anti-inflammatory and anti-oxidant defences. Therefore, we probed the protection afforded by bone marrow mesenchymal stem cells to islets under pro-inflammatory cytokine stress. Methods In order to evaluate the cytoprotective potential of mesenchymal stem cells on rat islets, co-cultures were exposed to the interleukin-1, tumour necrosis factor α and interferon γ cocktail for 24 h. Islet viability and functionality tests were performed. Reactive oxygen species and malondialdehyde were measured. Expression of stress-inducible genes acting as anti-oxidants and detoxifiers, such as superoxide dismutases 1 and 2, NAD(P)H quinone oxidoreductase 1, heme oxygenase-1 and ferritin H, was compared to non-stressed cells, and the corresponding proteins were measured. Data were analysed by a two-way ANOVA followed by a Holm-Sidak post hoc analysis. Results Exposure of rat islets to cytokines induces a reduction in islet viability and functionality concomitant with an oxidative status shift with an increase of cytosolic ROS production. Mesenchymal stem cells did not significantly increase rat islet viability under exposure to cytokines but protected islets from the loss of insulin secretion. A drastic reduction of the antioxidant factors heme oxygenase-1 and ferritin H protein levels was observed in islets exposed to the cytokine cocktail with a prevention of this effect by the presence of mesenchymal stem cells. Conclusions Our data evidenced that MSCs are able to preserve islet insulin secretion through a modulation of the oxidative imbalance mediated by heme and iron via heme oxygenase-1 and ferritin in a context of cytokine exposure. Electronic supplementary material The online version of this article (10.1186/s13287-019-1190-4) contains supplementary material, which is available to authorized users.

International audience; Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants present in dietary fats. Most studies evaluating PCB effects have been conducted with a single compound or a mixture of PCBs given as a single acute dose. The purpose of this study was to evaluate in vivo PCB toxicity in a realistic model of exposure: a low daily dose of PCBs (twice the tolerable daily intake (TDI)), chronically administered (8 weeks) to rats in contaminated goat milk. Liver and brain PCB toxicities were investigated by evaluating oxidative stress status and mitochondrial function. PCB toxicity in the liver was also estimated by transaminase enzymatic activity. This study shows that even at low doses, chronic PCB exposure resulted in a statistically significant reduction of mitochondrial function in liver and brain. In the liver, oxygen consumption in the condition of adenosine triphosphate (ATP) production (state 3) decreased by 22-29% (p < 0.01), according to the respiratory substrates. In the brain, respiratory chain complexes II and III were reduced by 24% and 39%, respectively (p < 0.005). The exposed rats presented higher lipid peroxidation status (+20%, p < 0.05) and transaminase activity (+30%, p < 0.05) in the blood. Thus, our study showed that exposure of rats to a daily realistic dose of PCBs (twice the TDI in a food complex mixture of environmental origin) resulted in multiple disruptions in the liver and brain.

Background Several epidemiological and animal studies suggest a positive association between cadmium (Cd) exposure and incidence of type 2 diabetes, but the association remains controversial. Besides, the experimental data have mainly been obtained with relatively high levels of Cd, over various periods of time, and with artificial routes of administration. Objectives Do environmental exposures to Cd induce significant disruption of glucose metabolism? Methods Adults Wistar rats were exposed for three months to 0, 5, 50 or 500 μg.kg−1.d−1 of CdCl2 in drinking water. Relevant parameters of glucose homeostasis were measured. Results Cd accumulated in plasma, kidney and liver of rats exposed to 50 and 500 μg.kg−1.d−1, without inducing signs of organ failure. In rats drinking 5 μg.kg−1.d−1 for 3 months, Cd exposure did not lead to any significant increase of Cd in these organs. At 50 and 500 μg.kg−1.d−1 of Cd, glucose and insulin tolerance were unchanged in both sexes. However, females exhibited a significant increase of both fasting and glucose-stimulated plasma insulin that was assigned to impaired hepatic insulin extraction as indicated by unaltered fasting C-peptide plasma levels. Conclusions Glucose homeostasis is sensitive to chronic Cd exposure in a gender-specific way. Moreover, this study proves that an environmental pollutant such as Cd can have, at low concentrations, an impact on the glucose homeostatic system and it highlights the importance of a closer scrutiny of the underlying environmental causes to understand the increased incidence of type 2 diabetes.

Imeglimin (IME) is a novel glucose-lowering agent improving insulin secretion in response to glucose and insulin sensitivity by targeting mitochondrial bioenergetics. IME is currently in phase 3 in Japan. The toxicity of high glucose or fructose concentrations in INS-1 cells was linked to the opening of the permeability transition pore (PTP), a mitochondrial channel involved in cell death. IME has been shown to prevent PTP opening in human endothelial cells under oxidative stress. This study investigated whether IME could also prevent PTP opening and cell death induced by high glucose or fructose concentrations in INS-1 cells and human pancreatic islets. PTP status in intact cells was assessed by confocal microscopy by measuring mitochondrial membrane potential (TMRM) and NAD(P)H (autofluorescence). Cell viability was measured by flow cytometry. In INS-1 cells and human islets, 30 mM Glucose or 2.5 mM Fructose for 24h led to PTP opening (increase in the NAD(P)H/TMRM ratio), this phenomenon being prevented by 100µM IME. Compared to normal glucose concentration (100%), cell viability significantly decreased (p Disclosure S. Lablanche: Other Relationship; Self; Poxel SA. E. Tubbs: None. C. Cottet-Rousselle: None. F. Lamarche: None. A. Moisan: None. V. Persoons: None. P.Y. Benhamou: Consultant; Self; Abbott, Sanofi, Novo Nordisk Inc.. Other Relationship; Self; Eli Lilly and Company, Roche Diabetes Care Health and Digital Solutions. Advisory Panel; Self; Diabeloop S.A.. S. Hallakou-Bozec: None. E. Fontaine: Other Relationship; Self; Poxel SA.

International audience; Deficit in oxygen and energetic substrates delivery is a key factor in islet loss during islet transplantation. Permeability transition pore (PTP) is a mitochondrial channel involved in cell death. We have studied the respective effects of oxygen and energy substrate deprivation on beta cell viability as well as the involvement of oxidative stress and PTP opening. Energy substrate deprivation for 1h followed by incubation in normal conditions led to a cyclosporin A (CsA)-sensitive-PTP-opening in INS-1 cells and human islets. Such a procedure dramatically decreased INS-1 cells viability except when transient removal of energy substrates was performed in anoxia, in the presence of antioxidant N-acetylcysteine (NAC) or when CsA or metformin inhibited PTP opening. Superoxide production increased during removal of energy substrates and increased again when normal energy substrates were restored. NAC, anoxia or metformin prevented the two phases of oxidative stress while CsA prevented the second one only. Hypoxia or anoxia alone did not induce oxidative stress, PTP opening or cell death. In conclusion, energy substrate deprivation leads to an oxidative stress followed by PTP opening, triggering beta cell death. Pharmacological prevention of PTP opening during islet transplantation may be a suitable option to improve islet survival and graft success.

Background: Exercise and citrulline (CIT) are both regulators of muscle protein metabolism. However, the combination of both has been under-studied yet may have synergistic effects on muscle metabolism and performance. Methods: Three-month-old healthy male rats were randomly assigned to be fed ad libitum for 4 weeks with either a citrulline-enriched diet (1 g·kg−1·day−1) (CIT) or an isonitrogenous standard diet (by addition of nonessential amino acid) (Ctrl) and trained (running on treadmill 5 days·week−1) (ex) or not. Maximal endurance activity and body composition were assessed, and muscle protein metabolism (protein synthesis, proteomic approach) and energy metabolism [energy expenditure, mitochondrial metabolism] were explored. Results: Body composition was affected by exercise but not by CIT supplementation. Endurance training was associated with a higher maximal endurance capacity than sedentary groups (P Conclusion: CIT supplementation and endurance training in healthy male rats modulates both muscle protein and energy metabolisms, with synergic effects on an array of parameters, including performance and protein synthesis.

International audience; ATAD3 (ATPase family AAA-Domain containing protein 3) is a mitochondrial inner membrane ATPase with unknown but vital functions. Initial researches have focused essentially on the major p66-ATAD3 isoform, but other proteins and mRNAs are described in the data banks. Using a set of anti-peptide antibodies and by the use of rodent and human cell lines and organs, we tried to detail ATAD3 gene expression profiles and to verify the existence of the various ATAD3 isoforms. In rodent, the single ATAD3 gene is expressed as a major isoform of 67 kDa, (ATAD3I; long), in all cells and organs studied. A second isoform, p57-ATAD3s (small), is expressed specifically throughout brain development and in adult, and overexpressed around the pen-natal period. p57-ATAD3s is also expressed in neuronal and glial rodent cell lines, and during in vitro differentiation of primary cultured rat oligodendrocytes. Other smaller isoforms were also detected in a tissue-specific manner. In human and primates, ATAD3 paralogues are encoded by three genes (ATAD3A, 3B and 3C), each of them presenting several putative variants. Analyzing the expression of ATAD3A and ATAD3B with four specific anti-peptide antibodies, and comparing their expressions with in vitro expressed ATAD3 cDNAs, we were able to observe and define five isoforms. In particular, the previously described p72-ATAD3B is confirmed to be in certain cases a phosphorylated form of ATAD3As. Moreover, we observed that the ATAD3As phosphorylation level is regulated by insulin and serum. Finally, exploring ATAD3 mRNA expression, we confirmed the existence of an alternative splicing in rodent and of several mRNA isoforms in human. Considering these observations, we propose the development of a uniform denomination for ATAD3 isoforms in rodent and human. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.

Introduction et but de l’etude Il est maintenant bien etabli que la citrulline (CIT) plasmatique est un excellent marqueur de la fonctionnalite intestinale, en particulier chez les patients de reanimation ou elle est generalement effondree [1] . Or, les mecanismes impliques ne sont pas connus. Nous proposons que l’hypoxie et l’inflammation observees dans ces situations pourraient impacter negativement la production de CIT. L’objectif de cette etude est donc de determiner, in vitro, si l’hypoxie et l’inflammation ont des effets sur la production de CIT par l’ornithine transcarbamylase (OTC). Ces effets ont ete evalues en utilisant un modele d’incubation d’explant duodenal humain [2] . Le protocole a ete approuve par le comite de protection des personnes du CHU de Grenoble. Materiel et methodes Huit biopsies duodenales ont ete prelevees chez des sujets apres information et obtention d’un consentement ecrit. Les biopsies ont ete incubees dans du milieu de culture complet pendant un temps court (6 h) ou long (18 h) selon quatre conditions : temoin, inflammation (melange de cytokines : IL-1β, 1 μg/L ; TNFα, 20 μg/L ; IFNγ, 10 μg/L), hypoxie (3 % d’O 2 ) et inflammation avec hypoxie. Apres mesure de la viabilite cellulaire, les taux de CIT liberee dans le milieu de culture pendant l’incubation ont ete doses par chromatographie d’echange d’ions. Le niveau d’activite de l’OTC a ete mesure par dosage enzymatique et l’expression proteique de cette enzyme par western blot . Resultats Les pourcentages de mort cellulaire a l’issue de la periode d’incubation ne differaient pas quelles que soient les conditions ( Tableau 1 ). La production intestinale de citrulline etait augmentee dans les conditions hypoxie et hypoxie avec inflammation apres 6 h d’incubation. Ces differences avaient disparu apres 18 h d’incubation. Cependant, cette augmentation de production de citrulline a 6 h ne s’accompagnait ni d’une modification de l’activite ni d’une modification de la quantite de proteine de l’OTC. Conclusion La production de citrulline par les enterocytes apparait augmentee en condition d’hypoxie avec ou sans inflammation sans que cela ne soit explique par une modification de l’activite enzymatique ou de l’expression proteique de l’OTC. Les mecanismes precis restent a determiner.

Ethanol induces brain injury by a mechanism that remains partly unknown. Mitochondria play a key role in cell death processes, notably through the opening of the permeability transition pore (PTP). Here, we tested the effect of ethanol and PTP inhibitors on mitochondrial physiology and cell viability both in vitro and in vivo. Direct addition of ethanol up to 100 mM on isolated mouse brain mitochondria slightly decreased oxygen consumption but did not affect PTP regulation. In comparison, when isolated from ethanol-treated (two doses of 2 g/kg, 2 h apart) 7-day-old mouse pups, brain mitochondria displayed a transient decrease in oxygen consumption but no change in PTP regulation or H2O2 production. Conversely, exposure of primary cultured astrocytes and neurons to 20 mM ethanol for 3 days led to a transient PTP opening in astrocytes without affecting cell viability and to a permanent PTP opening in 10 to 20% neurons with the same percentage of cell death. Ethanol-treated mouse pups displayed a widespread caspase-3 activation in neurons but not in astrocytes and dramatic behavioral alterations. Interestingly, two different PTP inhibitors (namely, cyclosporin A and nortriptyline) prevented both ethanol-induced neuronal death in vivo and ethanol-induced behavioral modifications. We conclude that PTP opening is involved in ethanol-induced neurotoxicity in the mouse.

International audience; Resveratrol is attracting much interest because of its potential to decrease body weight and increase life span, influencing liver and muscle function by increasing mitochondrial mass and energy expenditure. Even though resveratrol was already shown to reduce the adipose tissue mass in animal models, its effects on mitochondrial mass and network structure in adipocytes have not yet been studied. For this purpose, we investigated the effect of resveratrol on mitochondrial mass increase and remodeling during adipogenic differentiation of two in vitro models of adipocyte biology, the murine 3T3-L1 cell line and the human SGBS cell strain. We confirm that resveratrol inhibits lipogenesis in differentiating adipocytes, both mouse and human. We further show that this is linked to inhibition of the normally observed mitochondrial mass increase and mitochondrial remodeling. At the molecular level, the anti-lipogenic effect of resveratrol seems to be mediated by a blunted expression increase and an inhibition of acetyl-CoA carboxylase (ACC). This is one of the consequences of an inhibited insulin-induced signaling via Akt, and maintained signaling via AMP-activated protein kinase. The anti-lipogenic effect of resveratrol is further modulated by expression levels of mitochondrial ATAD3, consistent with the emerging role of this protein as an important regulator of mitochondrial biogenesis and lipogenesis. Our data suggest that resveratrol acts on differentiating preadipocytes by inhibiting insulin signaling, mitochondrial biogenesis, and lipogenesis, and that resveratrol-induced reduction of mitochondrial biogenesis and lipid storage contribute to adipose tissue weight loss in animals and humans.

This work was supported by the French National Institute for Health and Medical Research (INSERM), and by the Joseph-Fourier UniversityWe are grateful to Emily Tubbs for language corrections in this paper; The aim of this study was to characterize the early alterations of the liver mitochondrial function in ZDF (fa/fa) rats that develop diabetes compared to that of their lean counterparts ZDF (fa/+). Liver mitochondrial function was examined in 11‐ and 14‐week‐old ZDF (fa/fa) and ZDF lean (fa/+) rats. Oxygen consumption, H2O2 release, calcium retention capacity (CRC), membrane potential, membrane fluidity, and fatty acid composition were analyzed. State 3 oxygen consumption with palmitoyl‐carnitine increases between 11 and 14 weeks of age in lean but not in diabetic animals. This response was not seen with other substrates, suggesting that the use of fatty acids is impaired in diabetic rats. H2O2 release was lower in 14‐week‐old ZDF (fa/fa) rats as compared to ZDF lean (fa/+). These changes were not associated with differences in enzymatic activities of the respiratory complexes, suggesting regulatory mechanisms independent of their expression levels. Membrane fluidity and composition analyses show only slight effects linked to diabetes progression. The most salient feature was a reduction in CRC in the presence of CsA, an effect reflecting PTP dysregulation. Our data suggest few changes of mitochondrial function in ZDF fa/fa rats. At the age of 11 weeks, liver mitochondria have mainly a reduced effect of CsA on CRC.

Aims: This study aimed at comparing the cerebral cytotoxicity of ethanol and its main metabolite acetaldehyde after acute or chronic exposures of rat astrocytes in primary culture. Methods: Cytotoxicity was evaluated on the cell reduction of viability (MTT reduction test) and on the characterization of DNA damage by single cell gel electrophoresis (or comet assay). Results: Changes in astrocyte survival and in DNA integrity only occurred when the astrocytes were chronically exposed to ethanol (20 mM; 3, 6 or 9 days). On the other hand, viability and DNA integrity were deeply affected by acute exposure to acetaldehyde. Both effects were dependent on the concentration of acetaldehyde. The cytotoxic effect of acetaldehyde was also indirectly evaluated after modifications of the normal ethanol metabolism by the use of different inducers or inhibitors. In presence of ethanol, the concomitant induction of catalase (i.e. by glucose oxidase) and inhibition of aldehyde dehydrogenase (i.e. by methylene blue) led to acetaldehyde accumulation within cells. It was followed by both a reduction in viability and a substantial increase in DNA strand breaks. Conclusions: These data were thus consistent with a possible predominant role of acetaldehyde during brain ethanol metabolism. On the other hand, the effects observed after AMT could also suggest a possible direct ethanol effect and a role for free radical attacks. These data were thus consistent with a possible predominant role of acetaldehyde during brain ethanol metabolism. On the other hand, the effects observed after AMT could also suggest a possible direct ethanol effect and a role for free radical attacks.

Aims: Ethanol can create progressive neuropathological and functional alterations of neurones. However, the influence of exposure duration is still debated. It is difficult to specify the level of alcohol consumption leading to alcohol-induced brain damage. Moreover, the mechanism of toxicity is assumed to combine direct and metabolically induced effects, although numerous uncertain ties remain. Finally, the genotoxic power of ethanol has not fully been investigated in the brain. In the experiment reported herein , primary cultures of neurones were exposed either chronically or acutely to doses of ethanol within the range of blood alcohol levels in intoxicated humans. The impact on the integrity of neurones was assessed by cytotoxicity tests and DNA alterations by single-cell gel electrophoresis (Comet assay) and flow cytometry. Chronic ethanol exposure, even at a low dose, was more harmful to neurones th an acute exposure. Both significant reductions in cell viability and DNA alterations were observed in this condition. On the other hand, DNA repair capacities seemed to be preserved as long as the viability measured by specific tests was not affected. Instead, neu rones entered a death cell process compatible with apoptosis.

International audience; AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca2+-pumping.

Introduction et but de l’etude Les PolyChloroBiphenyles (PCBs) designent une famille de 209 composes lipophiles qui sont presents aujourd’hui dans la plupart des composantes de l’alimentation humaine (et en particulier les produits laitiers, les poissons). A ce jour, la toxicite des PCBs a surtout ete exploree chez le rongeur en utilisant une ou quelques molecules generalement les plus toxiques et a fortes doses. L’objectif de cette etude a ete d’elaborer un modele realiste d’exposition a un melange de PCBs dans le but de tester une eventuelle protection de l’aleurone de ble (AB), une matrice alimentaire issue du son de ble, riche en composes bioactifs. Materiel et methodes Les rats ont ete traites durant 8 semaines avec du lait de chevres contaminees aux PCBs associe ou pas a l’AB. Les quantites de PCBs apportees aux rats correspondent a une dose realiste (2 fois la dose journaliere acceptable). La toxicite des PCBs au niveau du foie et du cerveau a ete etudiee en determinant : l’atteinte de la fonction mitochondriale, le stress oxydant, les transaminases et le metabolisme des acides gras. Resultats et Analyse statistique L’exposition aux PCBs augmente significativement la peroxydation lipidique (TBARs) dans le foie et le cerveau (~20 %) et les transaminases du plasma (de 30 a 50 %). De plus, le rapport n-6/n-3 est significativement augmente de 21 % dans le foie (Tableau 1). Enfin, les PCBs perturbent la fonction mitochondriale dans le foie et le cerveau. Ainsi, par exemple ils reduisent significativement (P De facon surprenante, la consommation de l’AB corrige : – Totalement les augmentations des TBARs, des transaminases et du rapport n-6/n-3 observees lors du l’exposition aux PCBs (Tableau 1). – Partiellement la fonction mitochondriale (tableau page suivante). Conclusion Nos resultats montrent qu’il est possible de prevenir ses atteintes induites par les PCBs par la consommation d’AB. L’utilisation de l’aleurone de cereales comme additif alimentaire pour prevenir la toxicite des polluants organiques persistants a fait l’objet d’un depot de brevet. Tableau 1 : Quelques exemple de l’effet d’une administration de lait contamine aux PCBs avec ou sans prise d’aleurone chez le rat - Communication P015. Controle PCBs PCBs + Aleurone Moyenne SD Moyenne SD Moyenne SD ASAT, UI/L (plasma) 146,4 ± 41,2 A 220,9 ± 32,2 B 154,7 ± 19,0 A TBARs, μmol/g proteine (cerveau) 0,45 ± 0,12 A 0,56 ± 0,09 B 0,48 ± 0,07 A Ratio n-6/n-3 (foie) 5,06 ± 0,59 B 6,11 ± 0,56 A 5,48 ± 0,62 B N =10 par groupe. a. b : les resultats annotes avec des lettres differentes sont significativement differents (p>0,05) one-way ANOVA (analyse de variance a un facteur).

Aims. 3,5,4′-Trihydroxy-trans-stilbene, a natural polyphenolic compound present in wine and grapes and better known as resveratrol, has free radical scavenging properties and is a potent protector against oxidative stress induced by alcohol metabolism. Today, the mechanism by which ethanol exerts its toxicity is still not well understood, but it is generally considered that free radical generation plays an important role in the appearance of structural and functional alterations in cells. The aim of this study was to evaluate the protective action of resveratrol against ethanol-induced brain cell injury.Methods. Primary cultures of rat astrocytes were exposed to ethanol, with or without a pretreatment with resveratrol. We examined the dose-dependent effects of this resveratrol pretreatment on cytotoxicity and genotoxicity induced by ethanol. Cytotoxicity was assessed using the MTT reduction test. Genotoxicity was evidenced using single cell gel electrophoresis. In addition, DNA staining with fluorescent dyes allowed visualization of nuclear damage using confocal microscopy.Results. Cell pretreatment with low concentrations oftrans-resveratrol (0.1–10 μM) slowed down cell death and DNA damage induced by ethanol exposure, while higher concentrations (50–100 μM) enhanced these same effects. No protection bycis-resveratrol was observed.Conclusion. Protection offered bytrans-resveratrol against ethanol-induced neurotoxicity was only effective for low concentrations of this polyphenol.

Ethanol consumption has long been associated with brain damage. However, the mechanism underlying this deleterious effect remains unclear. Among different hypotheses, acetaldehyde is regarded by certain authors as playing a major role in the expression of ethanol toxicity, but there are still some uncertainties about the exact nature of its implication. We therefore tried to characterize the profile of the alterations of neuronal viability and DNA integrity obtained after either a direct exposure to ethanol or to acetaldehyde. Ethanol at concentrations within the range of blood alcohol levels in intoxicated humans (or = 100 mmol/L) induced DNA alterations without any apparent effect on cell viability. Acetaldehyde (or = 1000 micromol/L) can also induce DNA alterations but with a different profile of the DNA cellular alterations. The comparison between the distributions of the comet tail DNA indicated that ethanol induced strong breaks (tail DNAor = 60 a.u.) generation whereas acetaldehyde rather induced lower breaks (20or = tail DNAor = 50 a.u.) formation but affecting a greater number of neurones. Acetaldehyde had thus a different genotoxic potential which may suggest a different mode of action or a different cellular target. Furthermore, when a single 100 mmol/L ethanol exposure did not lead to any loss of cell viability, the addition of an inhibitor of aldehyde dehydrogenase was followed by a significant loss in viability. In contrast, the inhibition of catalase, which suppresses acetaldehyde synthesis, led to no reduced viability in the same exposure conditions. ROS also reduced viability, but this was observed only after both cytochrome P450 stimulation and catalase inhibition. These combined results could suggest that acetaldehyde may play a significant role in the expression of ethanol toxicity in brain.

The central nervous system is vulnerable to oxidative stress, especially when a toxicant can modify the physiological balance between anti- and pro-oxidant mechanisms. Among brain cells, astrocytes seem less vulnerable than neurons, but their impairment can dramatically affect neurons because of their protective role toward neurons. Ethanol is able to stimulate the formation of reactive oxygen species and modify the activity of most of the antioxidant agents. However, ethanol can react with the OH* radical to form the alpha-hydroxyethyl radical, which is considered to be less toxic. Ethanol also can stimulate H2O2 degradation through catalase activation. This study, therefore, sought to determine whether ethanol affected the sensitivity of astrocytes exposed to various free radical-generating systems. The cellular impact of such exposure was assessed by assays exploring cytotoxicity (i.e., NR (neutral red) and MMT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetiazolium bromide) reduction assays) and genotoxicity (comet assay) induced by these treatments. DNA alterations were evaluated by single-cell gel electrophoresis (comet assay), considered a precocious biomarker of intracellular alterations. After concomitant exposure to H2O2 and ethanol, the viability of astrocytes decreased significantly whereas the mean percentage of DNA in the tail increased,reflecting DNA damage (H2O2 was either directly added to the culture medium or endogenously produced from menadione). Ethanol also reduced the loss of viability and DNA alterations after exposure to OH* radicals produced by a Fenton system. The exposure to a xanthine/xanthine oxidase system had the same effect.

Free radicals species generation during ethanol metabolism is implicated in ethanol-induced toxicity. Findings from clinical studies have clearly established the association between alcohol intake and nutritional deficiency. Astrocytes are able to promote neuronal survival against different lethal injuries involved in ethanol-induced toxicity. We therefore studied the ability of hydrosoluble vitamin E (trolox), sodium selenite, and astrocyte-conditioned medium to protect cultured rat neurones against ethanol-induced oxidative stress after chronic exposure to ethanol. When a 6-day exposure to ethanol (20 mM) led to a loss of cell viability, the presence of trolox (10 microM) offered a significant neuroprotection. In the presence of 3-amino-1,2,4-triazole, a catalase inhibitor that created conditions that were favorable to reactive oxygen species accumulation, trolox was able to counteract the deleterious effect of the inhibitor. Moreover, flow cytometric analysis indicated that trolox can maintain the intracellular glutathione content in neurones chronically exposed to ethanol. In these conditions of exposure, the absence of sodium selenite in the culture medium significantly aggravated the exposure-induced effects, whereas sodium selenite (100 nM) offered a significant neuroprotection. Finally, the presence of 25% astrocyte-conditioned medium in the neuronal culture medium induced a neuroprotective effect in the presence of ethanol. Nevertheless, when astrocytes were previously chronically (3 days) exposed to ethanol, their culture medium did not offer a significant protection. These results evidenced that vitamin E and astrocytes can protect neurones from ethanol-induced oxidative stress, notably by contributing to maintaining the intracellular glutathione levels. Selenium, by means of its exogenous addition in the form of sodium selenite, also had an interesting neuroprotective effect.