RESUMO
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.
Assuntos
Astrócitos/efeitos dos fármacos , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Etanol/farmacologia , Neurônios/metabolismo , Animais , Astrócitos/metabolismo , Neoplasias Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Fígado/efeitos dos fármacos , Fígado/metabolismo , Oxirredução , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
The nervous system is one of the main targets of ethanol toxicity and it has been suggested that astrocytes might play an important role as their integrity is essential for the normal growth and functioning of neurons. Morphological variations of astrocyte cultures were therefore examined after exposure to various doses of ethanol (0.5, 1 and 2%) for different durations (24, 48, 72 and 96 h). The percentage of cell viability and the cell density were calculated and the changes in astrocyte morphology were assessed by an image analysis system (Samba 2005) allowing the characterization of 5 parameters (perimeter, surface, elongation factor, convexity factor and the form factor) of a great number of cells (over 6500). This was necessary because of the high variability in normal cultured astrocyte morphology. A two-way statistical approach (2-factors ANOVA completed by stepwise discriminant analysis) was adopted to emphasize the differences between control and exposed cells. In such conditions, ethanol treated cells became more elongated, less circular and more concave and did not grow like non-exposed cells. The mean pooled values of these parameters tended to be modified as a function of the dose of ethanol. The relationships between parameters clearly separated the groups as a function of the different doses. Finally no significant difference was observed in cell viability and cell density despite lower scores in the groups exposed to the highest dose of ethanol for the longest time. Our results suggest that ethanol might affect astrocytes in two different but probably complementary ways by modifying the cell shape and by altering normal cell development.
Assuntos
Astrócitos/efeitos dos fármacos , Depressores do Sistema Nervoso Central/toxicidade , Etanol/toxicidade , Animais , Astrócitos/patologia , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/análise , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
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.
Assuntos
Astrócitos/metabolismo , Meios de Cultura , Etanol/administração & dosagem , Etanol/metabolismo , Absorção , Animais , Células Cultivadas , Cinética , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Acetaldeído/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Etanol/administração & dosagem , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/metabolismo , Animais , Animais Recém-Nascidos , Antioxidantes/metabolismo , Catalase/antagonistas & inibidores , Catalase/metabolismo , Células Cultivadas , Espectroscopia de Ressonância de Spin Eletrônica , Inibidores Enzimáticos/farmacologia , Radicais Livres , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismoRESUMO
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.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Etanol/farmacologia , Radicais Livres/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , DNA/metabolismo , Embrião de Mamíferos , Feminino , Peróxido de Hidrogênio/farmacologia , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using the monoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.
Assuntos
Sulfatos de Condroitina/análise , Fibras Nervosas/química , Fibras Nervosas/ultraestrutura , Neurônios Aferentes/citologia , Pele/embriologia , Pele/inervação , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Sulfatos de Condroitina/imunologia , Colágeno/análise , Colágeno/imunologia , Células Epiteliais , Epitélio/embriologia , Epitélio/inervação , Gânglios Espinais/química , Gânglios Espinais/citologia , Gânglios Espinais/ultraestrutura , Soros Imunes , Imuno-Histoquímica , Microscopia Eletrônica , Neurônios Aferentes/ultraestrutura , Pele/citologiaRESUMO
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.