A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity.

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy. MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport.

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.

NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix.

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.

The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.

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.

The mechanism by which imeglimin inhibits gluconeogenesis in rat liver cells.

Aims: To understand the mechanism by which imeglimin (a new oral hypoglycemic agent whose phase 3 development program in Japan has now been completed) decreases hepatic glucose production. Materials and methods: We compared the effect of imeglimin and metformin on glucose production, ATP/ADP ratio, oxygen consumption rate, mitochondrial redox potential and membrane potential in primary rat hepatocytes. Results: We found that both imeglimin and metformin dose-dependently decreased glucose production and the ATP/ADP ratio. Moreover, they both increased mitochondrial redox potential (assessed by mitochondrial NAD(P)H fluorescence) and decreased membrane potential (assessed by TMRM fluorescence). However, contrary to metformin, which inhibits mitochondrial Complex I, imeglimin did not decrease the oxygen consumption rate in intact cells. By measuring the oxygen consumption of in situ respiratory chain as a function of the concentration of NADH, we observed that imeglimin decreased the affinity of NADH for the respiratory chain but did not affect its Vmax (ie competitive inhibition) whereas metformin decreased both the Vmax and the affinity (ie uncompetitive inhibition). Conclusions: We conclude that imeglimin induces a kinetic constraint on the respiratory chain that does not affect its maximal activity. This kinetic constraint is offset by a decrease in the mitochondrial membrane potential, which induces a thermodynamic constraint on the ATPase responsible for a decrease in the ATP/ADP ratio.

Improved human islets' viability and functionality with mesenchymal stem cells and arg-gly-asp tripeptides supplementation of alginate micro-encapsulated islets in vitro.

INTRODUCTION: The extension of islet transplantation to a wider number of type 1 diabetes patients is compromised by severe adverse events related to the immunosuppressant therapy required for allogenic islet transplantation. In this context, microencapsulation offers the prospects of immunosuppressive-free therapy by physically isolating islets from the immune system. However, current biomaterials need to be optimized to: improve biocompatibility, guaranty the maintenance of graft viability and functionality, and prevent fibrosis overgrowth around the capsule in vivo. Accumulating evidence suggest that mesenchymal stem cells (MSCs) and anchor points consisting of tripeptides arg-gly-asp (RGD) have cytoprotective effects on pancreatic islets. Here, we investigated the effect of supplementing reference M-rich alginate microcapsules with MSCs and RGD-G rich alginate on bioprocessing as well as on human pancreatic islets viability and functionality. METHODS: We characterized the microcapsules components, and then for the new microcapsule composite product: we analyzed the empty capsules biocompatibility and then investigated the benefits of MSCs and RGD-G rich alginate on viability and functionality on the encapsulated human pancreatic islets in vitro. We performed viability tests by confocal microscopy and glucose stimulated insulin secretion (GSIS) test in vitro to assess the functionality of naked and encapsulated islets. RESULTS: Encapsulation in reference M-rich alginate capsules induced a reduction in viability and functionality compared to naked islets. This side-effect of encapsulation was in part counteracted by the presence of MSCs but the restoration was complete with the combination of both MSCs and the RGD-G rich alginate. CONCLUSIONS: The present findings show that bioprocessing a favorable composite environment inside the M-rich alginate capsule with both MSCs and RGD-G rich alginate improves human islets survival and functionality in vitro.

Citrulline stimulates muscle protein synthesis, by reallocating ATP consumption to muscle protein synthesis.

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.

Human mesenchymal stem cells improve rat islet functionality under cytokine stress with combined upregulation of heme oxygenase-1 and ferritin.

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.

Impact of maternal low-level cadmium exposure on glucose and lipid metabolism of the litter at different ages after weaning.

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.

Impact of chronic and low cadmium exposure of rats: sex specific disruption of glucose metabolism.

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.

Protection of PC12 cells from cocaine-induced cell death by inhibiting mitochondrial permeability transition.

Cocaine abuse induces brain injury and neurodegeneration by a mechanism that has not yet been fully elucidated. Mitochondria play a key role in cell death processes, notably through the opening of the permeability transition pore (PTP). In this work, we examined the involvement of the PTP in cocaine-induced toxicity in PC12 cell lines. We used two different PTP inhibitors -i.e. cyclosporin A (CsA) and metformin-to assess their ability to counteract the cocaine induced effects. We first observed that a 48 h exposure to cocaine strongly sensitized cells to calcium overload, as measured by the calcium retention capacity. CsA and metformin significantly decreased the cocaine-induced PTP opening sensitization. We next showed by confocal microscopy that cocaine induced a permanent PTP opening in intact living cells, a phenomenon characterized by the collapse of the mitochondrial membrane potential and the relocation of the NAD(P)H from the mitochondrial matrix to the cytosol. As expected, a cocaine-induced PTP opening was prevented by PTP inhibitors. Finally, a flow cytometry analysis revealed that cocaine induced cell death while CsA and metformin promoted cell survival. Our results demonstrate that cocaine induces PC12 cell death through a mechanism involving permanent PTP opening.

A relevant exposure to a food matrix contaminated environmentally by polychlorinated biphenyls induces liver and brain disruption in rats.

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.

Resveratrol inhibits lipogenesis of 3T3-L1 and SGBS cells by inhibition of insulin signaling and mitochondrial mass increase.

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.

Respective effects of oxygen and energy substrate deprivation on beta cell viability.

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.

Regulation of brain-type creatine kinase by AMP-activated protein kinase: interaction, phosphorylation and ER localization.

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 Ca²âº-pumping.

Expression analysis of ATAD3 isoforms in rodent and human cell lines and tissues.

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, (ATAD3l; 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 peri-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.

Specific Conditions for Resveratrol Neuroprotection against Ethanol-Induced Toxicity.

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 of trans-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 by cis-resveratrol was observed. Conclusion. Protection offered by trans-resveratrol against ethanol-induced neurotoxicity was only effective for low concentrations of this polyphenol.

Influence of vitamin E, sodium selenite, and astrocyte-conditioned medium on neuronal survival after chronic exposure to ethanol.

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.