Luc Barret

Effects of chronic ethanol exposure on acetaldehyde and free radical production by astrocytes in culture

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.

Electron spin resonance study of free radicals produced from ethanol and acetaldehyde after exposure to a Fenton system or to brain and liver microsomes

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, Fentons 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.

There is no simple method to maintain a constant ethanol concentration in long-term cell culture: Keys to a solution applied to the survey of astrocytic ethanol absorption

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.

Ethanol can modify the effects of certain free radical-generating systems on astrocytes.

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.

A Fatal Case of Chlorate Poisoning: Confirmation by Ion Chromatography of Body Fluids

A 49-year-old male chemical industry worker was admitted to intensive care with a 24-hour history of respiratory failure, vomiting, headache, stupor, arterial hypotension, and cyanosed face and limbs. He had acute haemolysis (3.9 g/L plasma haemoglobin concentration) and 30% methaemoglobinaemia. Whereas the search for alcohol, barbiturates and opiates was negative, benzodiazepines and tricyclic antidepressants were present. The patient was in fact being treated with fluvoxamine, amitryptiline, and alprazolam. As the clinical and biological signs suggested chlorate poisoning, chlorate was looked for by using an aniline color reaction. It was found in gastric content and urine. Treatment consisted in mechanical ventilation, vasoactive amines, methylene blue, plasma exchange, exchange transfusion, and haemodialysis. Despite this, the patient had several cardiac arrests and refractory metabolic acidosis. He died 12 h after his admission. Specific ion chromatography was used afterhand to assay the chlorate in various body fluids. The technique was based on a separation on an ion exchange Dionex AS 12A column coupled with conductivity detection. A quantitative estimation was carried out by using external calibration with a four-point calibration curve which was linear between 1 and 15 mg/L. The measured plasma levels of chlorate were 78 and 29 mg/L respectively before and after exchange transfusion. Gastric-lavage liquid contained 1300 mg/L of chlorate and urine 4300 mg/L. Ion chromatography, which is routinely used in environmental studies helped to confirm a massive oral intake of chlorate by measuring the corresponding blood and urine chlorate concentrations, data which had only rarely been reported previously.

In-vitro spin-trapping of free radicals produced during trichloroethylene and diethylether metabolism

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 Fentons 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 in Patients with Toxic Coma Frequency, Prognostic Value, Diagnostic Significance

Internuclear Ophthalmoplegia (INO), a dysfunction of the medial longitudinal 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.