Search

In the presence of ciprofloxacin (CPFX), free radical adduct formation was demonstrated in rat cerebral microsomes using a spin trap α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone by electron spin reso...

International audience; 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.

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.

Objectif : Cette etude propose une optimisation du screening toxicologique classique en chromatographie gazeuse couplee a une detection en spectrometrie de masse (CPG-SM), en utilisant un logiciel de deconvolution : Automated Mass Spectral Deconvolution and Identification System (AMDIS) et une detection simultanee en mode SIM et en mode SCAN. Methode s : Les analyses sont effectuees avec un appareil CPG-SM Agilent equipe d'un passeur d'echantillons liquides et associe aux logiciels Retention Time Locking Software (RTL) et AMDIS. Deux bibliotheques commerciales (PMW et NIST) sont utilisees ainsi qu'une bibliotheque propre au laboratoire. Un plasma A surcharge avec 16 molecules et un autre plasma B surcharge avec de la buprenorphine et son metabolite la norbuprenorphine ont ete utilises pour tester respectivement le logiciel AMDIS et la sensibilite de la detection SIM/SCAN. Resultats : Parmi les 16 molecules du plasma A, 11 ont ete mises en evidence par la deconvolution tandis que seulement 5 ont ete identifiees par une recherche en librairie classique. Cinq composes n'ont pas ete reperes pour des problemes chromatographiques ou de sensibilite en mode SCAN. La buprenorphine et son metabolite n'ont ete mis en evidence que grâce au mode SIM/SCAN. Conclusion : La deconvolution permet de detecter des molecules meme lorsqu'aucun pic n'est repere sur le chromatogramme, ceci se verifie au quotidien sur des cas reels. Le mode SIM/SCAN permet de diminuer le nombre d'injections, tout en conservant la meme sensibilite.

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.

Si le role de l'ethanol et des drogues illicites dans l'alteration de l'aptitude au travail est actuellement pris de plus en plus en consideration, il ne faut pas oublier les medicaments psycho-actifs. La prevalence de leur consommation chez les francais, notamment actifs, ne cesse d'augmenter. Quel que soit son niveau hierarchique dans l'entreprise, chacun peut etre soumis a une exigence de performances, le conduisant a utiliser des medicaments pour mieux resister a la fatigue et au stress (dopage social). D'usager occasionnel, le patient peut devenir usager dependant puis abusif, voire veritable «toxicomane» avec son cortege d'effets indesirables ayant une implication directe sur son aptitude au travail. Parmi les effets les plus genants, sont a prendre en compte les risques de somnolence, de diminution de la coordination psychomotrice, de troubles du comportement (modification de la prise de risques), d'atteinte de l'equilibre ou de troubles sensoriels. Si le potentiel d'abus (dependance psychique) peut etre responsable de problemes du comportement dans le milieu du travail, les difficultes se manifesteront par une dependance physique exposant au risque de syndrome de sevrage (exemple des benzodiazepines (BZD)) et par un phenomene de tolerance avec un risque d'exacerbation des effets indesirables toxiques en raison d'une augmentation de la posologie. Parmi les medicaments psycho-actifs a potentiel addictif, il faut en particulier s'interesser aux anxiolytiques, hypnotiques, antidepresseurs, antalgiques majeurs, produits de substitution, psychostimulants, antiparkinsoniens, antihistaminiques, etc... mais egalement a des classes therapeutiques autres comme par exemple celle des steroides anabolisants. Le corps medical se retrouve ainsi confronte a des situations impliquant une conduite addictive medicamenteuse chez des patients ayant une activite professionnelle et pour lesquelles le biologiste toxicologue peut apporter son aide. Citons par exemple, l'evaluation de l'aptitude d'un patient a un poste a risque pour laquelle un depistage de conduite toxicophile est parfois necessaire ; la prise en charge d'un patient toxicomane sous traitement de substitution et pour lequel un suivi biologique peut etre utile ; ou encore l'appreciation de l'imputabilite d'un traitement medicamenteux dans la survenue d'un accident du travail. Dans ce contexte, l'interet de l'analyse des cheveux dans la mise en evidence d'une consommation chronique est rappele.

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.

The nervous system is one of the main targets of ethanol toxicity. Astrocytes might play an important role in ethanol-induced brain toxicity, because their integrity is essential for the normal growth and functioning of neurons. On the other hand, acetaldehyde has been implicated as a mediator in some of the biochemical, pharmacological, and behavioral effects of ethanol. The present study aimed at demonstrating the ability of astrocytes in culture to produce acetaldehyde from ethanol. Significant metabolization of ethanol with production of acetaldehyde was demonstrated in the primary culture of astrocytes. This production was quite low, compared with that usually observed in hepatocytes, but was in the same range as that measured in whole brain homogenates and corresponded to biologically active levels. Such a demonstration could bring new elements for understanding of ethanol neurotoxicity.

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.

En Afrique de l’Ouest, les politiques de post ajustement donnent un role croissant aux organisations paysannes dans divers cadres de concertation ou doivent se negocier les politiques sectorielles et se cogerer les filieres agricoles. A travers le cas la filiere coton au Mali, qui concerne pres du quart de la population et un peu moins de 10 % du PIB, il est mis en balance les nouveaux enjeux auxquels se trouvent confrontees les organisations de producteurs et leurs capacites a les relever, capacites qui sont fortement liees aux diverses conditions de leur emergence. Les OP soit sont initiees par les pouvoirs publics (l’Etat ou la societe cotonniere) soit emergent de la societe civile a l’initiative des paysans ou d’Ong. La perspective d’une gestion interprofessionnelle de la filiere coton en cours de privatisation permet de discuter des options offertes et de leurs implications en termes de reglementation, d’appuis et de recherches.

The aim of this study was to investigate the possible cytotoxic and oxidative stress inducing effects of ciprofloxacin (CPFX) on primary cultures of rat astrocytes. The cultured cells were incubated with various concentrations of CPFX (0.5-300mg/l), and cytotoxicity was determined by neutral red (NR) and MTT assays. Survival profile of cells was biphasic in NR assay: CPFX did not cause any alteration at any concentration for 7h, whereasor =50mg/l concentrations induced significant cell proliferation in incubation periods of 24, 48, 72, and 96h. However, cell proliferation gradually decreased at higher concentrations, and 200 and 300mg/l of CPFX exposure was found to be significantly (p0.05) cytotoxic at all time periods. With MTT assay, no alteration was noted for incubation period of 7h, as observed with NR assay. But, cell viability decreased with approximatelyor =50mg/l CPFX exposure in all other time periods. Cell proliferation was only seen in 24h of incubation with 0.5 and 5mg/l CPFX. Vitamin E pretreatment of cell cultures were found to be providing complete protection against cytotoxicity of 300mg/l CPFX in 96h incubation when measured with both NR and MTT assays. The SOD pretreatment was partially protective with NR assay, but no protection was noted when measured with MTT. A significant enhancement of lipid peroxidation was observed with the cytotoxic concentration of the drug, but total glutathione content and catalase activity of cells did not change. The data obtained in this study suggest that, in accordance with our previous results with fibroblast cells, CPFX-induced cytotoxicity is related to oxidative stress. And the biphasic effect of CPFX possibly resulted from the complex dose-dependent relationships between reactive oxygen species, cell proliferation, and cell viability.

Fluoroquinolones are generally well-tolerated antibiotics in patients. Gastrointestinal, central nervous system, and dermatological adverse events were the most frequent unwanted effects during therapy with these drugs. However, the mechanism of these adverse effects has not yet been elucidated. The aim of this study was to investigate the possible DNA damage-inducing effect of a fluoroquinolone (FQ) antibiotic, ciprofloxacin (CPFX) on primary culture of rat astrocytes. For this purpose, the cultured cells were incubated with various concentrations of CPFX, and DNA damage was monitored by comet assay. Our results showed a concentration-dependent induction of DNA damage by CPFX. Pretreatment of cells with Vitamin E for 4 h provided partial protection against this effect. The data obtained in this study suggest that CPFX-induced DNA damage might be related to oxidative stress and should be considered for further mechanistic studies of central nervous system toxicity of CPFX.

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.

In a previous study, the production of acetaldehyde and free radicals derived from ethanol was characterized in astrocytes in primary culture. In the present study, the effects of chronic exposure on the production of both compounds as well as on the main antioxidant system were compared with those of an acute exposure. This was done to better understand the different ways the brain reacts to these modes of exposure. Under these conditions, both a time-dependent increase in the accumulation of acetaldehyde and a decreased formation of the alpha-hydroxyethyl radical were shown. This was associated with increased activities of catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX) and with decreased glutathione (GSH) content. These effects, which counteract reactive oxygen species (ROS) formation by stimulating the main enzymes of the antioxidant system, were also associated with the reduced amount of radicals derived from ethanol. This could be a beneficial effect, but this was counter-balanced by the increased rate of acetaldehyde accumulation, whose high toxicity is well known. All these effects underline the crucial role played by catalase which, on one hand converts hydrogen peroxide to water and, on the other hand, ethanol to acetaldehyde.

Formation of the alpha-hydroxyethyl radical (CH3 degree CHOH) has already been extensively demonstrated after ethanol metabolism in the liver. Despite favourable conditions, this formation in the brain has remained speculative since there is no direct experimental evidence in intact brain cells. In this preliminary study, the formation of such a radical was observed after exposure of astrocytes and astrocytic C6 glioma cells to ethanol. These cells were studied because astrocyte integrity is essential for normal growth and functioning of neurons. The free radicals were detected by EPR spectroscopy using the spin trapping technique. Astrocytes appeared to be more sensitive than the C6 cells to free radical formation as the intensity of the signal was higher after exposure of the astrocytes and increased with time, a fact not observed after exposure of the C6 cells.

Ethanol evaporation from the culture medium is a potential source of misinterpretation of long-term exposure of cells. Different methods have been proposed to prevent this evaporation, the most effective being the saturation of the atmosphere over the culture medium with ethanol. Unfortunately, no simple predictive method has been devised to determine the appropriate concentration of ethanol in the system avoiding either evaporation or contamination of the culture medium. We present some keys to a solution adapted to the culture of astrocytes, which allow for the first time a direct evaluation of ethanol absorption by these cells. The system described remains compatible with normal growth and viability.

Dextropropoxyphene (DP) is a synthetic analgesic drug structurally related to methadone. In France, DP is not liable to narcotic legal control. This explains its wide use as a substitute in the treatment of drug addicts. DP is mainly metabolized in the liver via a Ndemethylation into norpropoxyphene (NP), with a plasma half-life ranging from 1.6 to 24 hours, whereas NP half-life is longer, ranging from 11.5 to 50 hours.

Morphometric analysis was used to compare the effects of trichloroethylene (Tri) and dichloroacetylene (Dca) on the fibre parameters of the trigeminal nerve. Treated animals were clearly separated from controls according to a discriminant analysis. Furthermore, in the class of nerve fibres defined by a clustering analysis and corresponding to the largest fibres, myelin thickness was significantly decreased in the Dca group, but less so in the Tri group. In the group of the smallest fibres however, the myelin thickness was significantly increased by the treatments, but especially by Tri. Such a variability in the effects of Tri has already been demonstrated. Mechanisms for this are quite unclear although demyelination could be involved as already suggested. Our results thus show the ability of Tri and Dca to alter nerve parameters but probably with different modes of action depending on the size of the fibre.

Free radical formation from ethanol and acetaldehyde was studied in the presence of a spin-trap and a NADPH generating system with a chemical model, Fenton's reagent, or by enzymatic oxidation of these solvents by rat liver and brain microsomes. The free radicals were detected by electron spin resonance spectroscopy (E.S.R.), using the spin-trapping agent, alpha-(4-pyridyl l-oxide)-N-tertbutyl-nitrone (POBN). Under such conditions, the hydroxyethyl radical derived from ethanol was obtained after both incubation in liver and brain microsomes as well as after exposure to the Fenton system. Enzymatic inhibition and activation showed that the mixed function oxidase system plays an important role in the generation of such a radical, even in the brain. Under all the experimental conditions acetaldehyde could also generate a free radical deriving directly from the parent molecule and modified by enzymatic activation or inhibition. A second, longer lasting radical was also observed in the presence of acetaldehyde. On the basis of a comparative study to a known process causing lipoperoxidation, its lipidic origin was suggested.

Free-radical production during the metabolism of various xenobiotics represents a frequent mechanistic explanation for their toxicity. We tested the hypothesis of production of free radicals from two solvents, diethylether and trichloroethylene (TRI), and from two metabolites of TRI, namely trichloroethanol (TCE) and trichloroacetic acid (TCA). The formation of free radicals was detected by electron spin resonance spectroscopy (ESR), using a spin-trapping agent, alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (POBN). Two experimental models were used. The first was a chemical model using Fenton's reagent, a mixture of Fe(II)-chelator and H2O2, for which the normal reaction is OH. production, and the second, a preparation from rat liver and brain microsomes containing NADPH and achieving enzymatic oxidation of the solvents. After addition of diethylether, free-radical production was demonstrated under the two experimental conditions. This free radical probably derived from the parent molecule by hydrogen abstraction. TRI and TCE additions to the Fenton system suppressed normal OH. production whereas this production was increased after TCA addition. The addition of TCE to the microsomal preparations was followed by free-radical production which could derive either from the parent molecule or from other sources, e.g. from membrane degradation, with a preference for the first hypothesis because of the characteristics of the signal. This result was not observed after addition of TRI or TCA. In conclusion, these preliminary results confirm the validity of the hypothesis of production of free radicals from diethylether, but they are less consistent for TRI as this production was observed only after addition of TCE; this result is interesting, however, as TCE is considered to play a major role in the toxicity observed after TRI exposure in humans.

Internuclear Ophthalmoplegia (INO), a dysfunction of the medial lonqitudinal fasciculus, is frequently seen in toxic coma (7 out of 70 cases). INO is most often bilateral and can be associated with different stages of coma. Such an association is a strong argument for the toxic etiology of a coma. But INO has no value in determining the source of intoxication and is no prognostic indicator for the outcome.

Les recommandations européennes en vigueur depuis le deux février 1999 préconisent la pose d'un obstacle médico-légal dans de larges indications qui pourraient être difficiles à mettre en application. Cette étude a pour objectif d'étudier la pratique des médecins en pré-hospitalier, quant à la pose de l'obstacle médico-légal, et de la comparer aux recommandations européennes. Nous avons également cherché à mettre en évidence les facteurs influençant la pose d'un obstacle médico-légal. Enfin, nous nous sommes intéressés à la suite donnée à l'obstacle médico-légal, et notamment la pratique de l'autopsie. Il s'agit d'une enquête descriptive par questionnaire. Elle a été menée au sein de l'équipe médicale du SMUR de Centre Hospitalier d'Annecy en Haute-Savoie, durant 7 mois entre août 2010 et février 2011. Au terme de cette étude, et comparativement aux recommandations européennes en matière d'autopsie, il apparaît que les indications de l'obstacle médico-légal sont moyennement respectées. Les décès par suicide et les décès inattendus du sujet jeune sont insuffisamment signalés. D'autre part, en matière d'autopsie, la pratique française est très éloignée des recommandations européennes.