No abstract

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 uncertainties 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 than 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, neurones entered a death cell process compatible with apoptosis.

No abstract

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 DNA > or = 60 a.u.) generation whereas acetaldehyde rather induced lower breaks (20 < or = tail DNA < or = 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.

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.

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.

No abstract

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.