Effects of alcohol on brain-derived neurotrophic factor mRNA expression in discrete regions of the rat hippocampus and hypothalamus.

Chronic alcohol consumption has adverse effects on the central nervous system, affecting some hippocampal and hypothalamic functions. In this study we tempted to demonstrate that some of these modifications could involve impairment of neurotrophic factors. Three experimental groups of male Sprague Dawley rats were studied: one control group, one chronically treated with alcohol vapor according to a well-established model that induces behavioral dependence, and a third group treated similarly but killed 12 hr after alcohol withdrawal. In all groups, changes in brain-derived neurotrophic factor mRNA expression occurring in the hippocampus and supraoptic nucleus were first analyzed by reverse transcription-polymerase chain reaction and then by in situ hybridization. In parallel, we used ribonuclease protection assay to measure mRNA levels encoding trkB in the two central nervous system regions. We showed that chronic alcohol intoxication decreases brain-derived neurotrophic factor mRNA expression in discrete regions of the rat hippocampus (CA1 region and dentate gyrus) and in the supraoptic nucleus of the hypothalamus. We also showed a global up-regulation of trkB mRNA expression encoding the high-affinity brain-derived neurotrophic factor receptor (TrkB), after applying the same treatment. Following 12 hr of alcohol withdrawal, a significant increase in BDNF mRNA expression was observed in the dentate gyrus and CA3 region of hippocampus and in the hypothalamic supraoptic nucleus. These findings suggest that chronic alcohol intake may modify hippocampal and hypothalamic neuronal functions through modifications in growth factors and its receptors.

Intracerebroventricular injection of antisense oligos to nNOS decreases rat ethanol intake.

Nitric oxide (NO) has been implicated in alcohol drinking behavior using NO synthase (NOS) inhibitors that are nonselective of the different isoforms of NOS. In the brain, there are two constitutive isoforms of NOS, neuronal NOS (nNOS) and endothelial NOS (eNOS). We used an antisense oligodeoxynucleotide directed against nNOS in ethanol dependent male Wistar rats to examine the specific contribution of nNOS in the control of ethanol intake. Rats were subjected to a free-choice situation water/ethanol (10% v/v) after chronic ethanol intoxication by inhalation of ethanol vapor. During the free-choice situation, rats were twice daily for 4 days intracerebroventricularly injected with either saline, or end-capped phosphorothioate-protected antisense or mismatch oligodeoxynucleotide (25 microg/4 microl per injection), or acamprosate (1 mg/kg body weight) as reference product for its anticraving properties. Our results showed that the antisense treatment, but not the mismatch treatment, reduced both ethanol intake and ethanol preference during treatment and posttreatment periods (by 25-30%) without alteration of the body weight gain. The antisense treatment, but not the mismatch treatment, also down-regulated nNOS mRNA levels (by 30%) and NOS activity in the hippocampus. The anticraving drug, acamprosate reduced both ethanol intake (by 58%) and ethanol preference. All these results suggest that nNOS is involved in the regulation of alcohol dependence.

Ursodeoxycholate protects against ethanol-induced liver mitochondrial injury.

The purpose of this work was to examine whether ursodeoxycholate (UDC), a hydrophilic bile salt, could reduce mitochondrial liver injury from chronic ethanol consumption in rats. Animals were pair-fed liquid diets containing 36% of calories as ethanol or isocaloric carbohydrates. They were randomly assigned into 4 groups of 7 rats each and received a specific treatment for 5 weeks: control diet, ethanol diet, control diet + UDC, and ethanol diet + UDC. Respiratory rates of isolated liver mitochondria were measured using a Clark oxygen electrode with sodium succinate as substrate. Mitochondria from rats chronically fed ethanol demonstrated an impaired ability to produce energy. At the fatty liver stage, the ADP-stimulated respiration (V3) was depressed by 33%, the respiratory control ratio (RC) by 25% and the P/O ratio by 15%. In ethanol-fed rats supplemented with UDC, both the rate and efficiency of ATP synthesis via the oxidative phosphorylation were improved: V3 was increased by 35%, P/O by 8%. All the respiratory parameters were similar in control group and control + UDC group. On the other hand, the number and size of mitochondria were assessed by electron microscopy and computer-assisted quantitative analysis. The number of mitochondria from ethanol-treated rats was decreased by 29%, and they were enlarged by 74%. Both parameters were normalized to control values by UDC treatment. These studies demonstrate that UDC has a protective effect against ethanol-induced mitochondrial injury by improving ATP synthesis and preserving liver mitochondrial morphology. These UDC positive effects may contribute to the observed decrease in fat accumulation and may delay the progression of alcoholic injury to more advanced stages.

Ursodeoxycholate alleviates alcoholic fatty liver damage in rats.

The hydrophilic bile salt ursodeoxycholate (UDC) improves cholestasis in several liver diseases and is in vitro an efficient membrane stabilizer. However, its action on chronic ethanol-induced liver damage is not established. We thus sought to determine the effect of UDC on chronic ethanol-induced steatosis and on liver plasma membrane fluidity in rats. Male rats were pair-fed liquid diets containing 36% of calories as ethanol (alcohol diet) or an isocaloric maltose-dextrin mixture (control diet). Four groups of 10 animals received, respectively, during 30 days: the control diet, the control diet + UDC (90 mg/kg/day), the alcohol diet, and the alcohol diet + UDC. Bile was collected for assessment of bile flow, biliary lipids, and individual bile salts. Liver lipid contents and lipid peroxidation were determined. Plasma membrane fluidity was assessed by fluorescence polarization of various probes. Alcohol treatment caused a 4-fold increase in liver triacylglycerol and cholesterol ester levels. UDC supplementation significantly reduced these increases by 50% and 40%, respectively. UDC intake was associated with a marked decrease in alcohol-induced lipid peroxidation. Bile flow, bile salt, and phospholipid secretion were slightly increased by alcohol intake. The addition of UDC-enriched bile with tauroursodeoxycholate (38%) without significantly affecting the biliary parameters. Lastly, UDC treatment almost totally prevented the 20% increase in liver plasma membrane fluidity due to chronic alcohol intake. This study shows that UDC intake, concomitant with alcohol diet, exerts a clear-cut membrane protective effect that might alleviate ethanol-induced lipid disorders.

Involvement of cytochrome P450 2E1 in the (omega-1)-hydroxylation of oleic acid in human and rat liver microsomes.

In vitro techniques have been used to investigate the nature of microsomal cytochrome P450 involved in the metabolism of oleic acid, a physiological monounsaturated fatty acid. Like lauric acid, which is currently used as a model substrate of fatty acid metabolism, the alkyl chain of oleic acid is hydroxylated on its omega and (omega-1) carbons. The identity of these hydroxylated metabolites was ascertained by GC/MS and LC/MS. The omega/omega-1 ratio of oleic acid metabolites (1.22+/-0.01) was found to be similar to that obtained with lauric acid in rat liver microsomes (1.10+/-0.02), while in human liver microsomes this ratio was 0.75+/-0.5 for lauric acid and 5.2+/-2.6 for oleic acid. After treatment of rats with ethanol or clofibrate, inducers of CYP2E1 and CYP4A, respectively, the hydroxylations of oleic acid were shown to be less inducible than those of lauric acid. Five in vitro approaches were used to identify the P450 isoform(s) responsible for the microsomal (omega-1)-hydroxylation of oleic acid: effect of various inducers in rats, correlation studies between specific P450 catalytic activities in a panel of 25 human liver microsomes, chemical inhibitions, immuno-inhibitions and metabolism by cDNA-expressed human P450 enzymes. From the above results, it can be ascertained that P450 2E1 is the main enzyme involved in the (omega-1)-hydroxylation of oleic acid. Furthermore, the omega-hydroxylation of oleic acid was shown to be mainly catalyzed by P450 4A enzymes in human liver microsomes. The turnover number of (omega-1)-hydroxylation of lauric and oleic acids decreased from 7.8 to 1.5 min(-1), respectively, suggesting that the dodecane alkyl chain allows optimal binding to the active site of CYP2E1.

Ethanol intake after chronic intoxication by inhalation of ethanol vapour in rats: behavioural dependence.

In Wistar rats, which practically avoid ethanol when naive, it is possible to induce a large ethanol intake in a free-choice situation after chronic intoxication by ethanol vapour. In this study, we evaluated the ethanol intake of chronically intoxicated and control rats. The ethanol intake was increased in intoxicated animals but the intensity of the response varied according to individuals without any clear relation to the level of the intoxication. The results clearly showed in intoxicated animals two kinds of responders: alcohol-nonpreferring (27/95) and alcohol-preferring rats (68/95). In the alcohol-preferring rats, ethanol intoxication had induced an alcohol drinking-dependent behaviour; about 75% of the animals of this group drank more than 7 g/kg b.wt. per day and could be considered as behaviourally dependent on alcohol. Furthermore, this group presents most of the criteria of alcoholism that an animal model should ideally satisfy.

Regulation of rat neuronal nitric oxide synthase activity by chronic alcoholization.

Rat neuronal nitric oxide (NO) synthase (nNOS) activity was measured in frontal cortex, hippocampus, striatum and cerebellum using the assay of [3H]citrulline, following chronic alcoholization. The Km and Vmax values were significantly increased in the frontal cortex and in the striatum, and were not affected in the cerebellum and hippocampus.

Ethanol potentiates lipopolysaccharide- or interleukin-1 beta-induced nitric oxide generation in RBE4 cells.

Our present study investigated the effects of ethanol treatment on inducible nitric oxide (NO) synthase pathway from lipopolysaccharide- or interleukin-1 beta-treated cultured rat blood-brain barrier cell line (rat brain endothelial 4 cells: RBE4 cells). Cells were lipopolysaccharide- or interleukin-1 beta-treated with or without ethanol (50, 100 or 200 mM) for 16 or 24 h. Inducible NO synthase activity and mRNA expression were measured using Griess reaction and reverse transcription-polymerase chain reaction (RT-PCR) respectively. In the absence of lipopolysaccharide or interleukin-1 beta, ethanol treatments failed to stimulate inducible NO synthase gene expression. Lipopolysaccharide or interleukin-1 beta increased nitrite production and inducible NO synthase mRNA levels, and ethanol potentiated this effect. We concluded that ethanol could aggravate the consequences of NO generation by RBE4 cells after inducible NO synthase induction following inflammation or sepsis. This ethanol action on NO generation could contribute to circulatory failure associated with shock due to sepsis or hemorrhage, and alter blood-brain barrier permeability.

Influence of an n-6 polyunsaturated fatty acid-enriched diet on the development of tolerance during chronic ethanol administration in rats.

This study investigates the effects of n-6 polyunsaturated fatty acids (PUFAs), in the form of dietary Evening Primrose Oil (EPO) and safflower oil, on the development of tolerance to ethanol. The degree of fluorescence polarization of the fluoroprobes DPH, PROP-DPH, and TMA-DPH in isolated cortical synaptosomal membranes was measured. In addition, the development of tolerance, as shown by changes in synaptosomal membrane fluidity after an acute in vitro ethanol challenge, was also determined after 20 weeks of ethanol administration, either alone or together with a PUFA-enriched diet. Although the administration of EPO-enriched diet did not significantly render the inner core of the cortical synaptosomal membrane tolerant to the acute ethanol challenge, concomitant administration of ethanol and EPO was found to increase further the rigidity and tolerance to the acute ethanol challenge in the inner core. Chronic administration of safflower oil, which lacks gamma-linolenic acid (18:3, n-6) but like EPO contains linoleic acid, either alone or together with chronic ethanol had no effect on synaptosomal membrane fluidity after an acute ethanol challenge. The results suggest that gamma-linolenic acid or its metabolites may have an important role to play in the development of tolerance to chronic ethanol.

Cytochrome P-450 2E1 in rat liver, kidney and lung microsomes after chronic administration of ethanol either orally or by inhalation.

In this study, microsomal cytochrome P-450 2E1 (CYP2E1) contents and activities were tested in liver, kidney and lung from Wistar rats after the following treatments (1) oral administration of a 10% ethanol solution for 4 weeks; (2) pair fed controls; (3) oral administration of a 5% acetone solution for 1 week; (4) inhalation of ethanol vapour for 4 weeks. CYP2E1 activity was measured using chlorzoxazone as substrate and CYP2E1 content was measured using Western blot analysis. In addition, the cellular distribution of CYP2E1 was studied in liver, lung and kidney by immunohistochemistry. Basal liver CYP2E1 was 10-20 times lower in lung and kidney than in liver. Inhalation was clearly the most efficient way of inducing CYP2E1, probably due to the continuous and high alcohol exposure. Among the organs tested, lung appeared to be the tissue least sensitive to induction even after ethanol inhalation, suggesting the absence of local induction. After ethanol intoxication, immunostaining was increased in the centrilobular region of the liver, in the alveolar cells of the lung and in the proximal convoluted tube of the kidney. The CYP2E1 activities decreased to control values in the three tissues tested, within 24 h after cessation of intoxication.

Synaptic membrane responses to acute and chronic alcohol intoxication in high alcohol sensitive (HAS) and low alcohol sensitive (LAS) selectively bred rats.

HAS (high alcohol sensitive) and LAS (low alcohol sensitive) lines of rats have been selectively bred based on their differences in ethanol-induced sleep time. In the present study, the two lines were compared to examine another central effect of acute alcohol intoxication: namely, hypothermia. As the disturbances in membrane microorganization have been associated with the nervous system's sensitivity and tolerance to ethanol, the synaptic plasma membrane fluidity and acute sensitivity to ethanol were also evaluated by fluorescence polarization of DPH probes in the HAS and LAS rats. The two lines did not differ in the magnitude of their hypothermic response after acute injection of ethanol (3 g/kg body wt, i.p.). Although membranes in the HAS line were slightly more rigid than in the LAS line, the level of membrane disordering after acute ethanol addition was identical for the two lines in the region examined with the DPH probe. Following the chronic intoxication of the rats, the two lines developed tolerance to the hypnotic and hypothermic effects of ethanol. In the same way, a membrane resistance (i.e. tolerance) to the disordering effect of acute ethanol addition developed in the region probed with DPH. In general, the LAS line showed a more pronounced level of tolerance than the HAS line at both the functional and membrane level. These results suggest that different measures used to assess the depressant action of ethanol, such as sleep time, hypothermia or membrane disordering, may not be related and could indicate different genetic origins.(ABSTRACT TRUNCATED AT 250 WORDS)

Tolerance and dependence in high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats after chronic alcohol intoxication by inhalation.

In rats selected for their differences in sleep time following acute administration of ethanol (high or low alcohol sensitivity: HAS or LAS), alcohol chronic tolerance and behavioural dependence were determined. Tolerance was assessed by calculating the interval between the loss and regain of the righting reflex following an acute administration of ethanol (4 g/kg body wt), after intoxication by inhalation of ethanol vapour for 3 weeks. The importance of behavioural dependence was estimated by measuring the ethanol intake in a free-choice situation water/ethanol (10% v/v), after intoxication by inhalation for 4 weeks. The two HAS and LAS lines did not show any behavioural dependence, while they developed a significant tolerance to the hypnotic effect of ethanol, which was more marked for the LAS line.

Red blood cell deformability and alcohol dependence in humans.

Studies of DPH fluorescence polarization and deformability have shown that alcohol induces rigidification of the red blood cell (RBC) membrane. We investigated a possible link between RBC membrane fluidity and deformability by studying both parameters simultaneously in samples from alcohol-dependent patients (group 1, N = 19), social drinkers (group 2, N = 12) and long-term abstaining alcoholics (group 3, N = 8). The active drinkers showed disturbances of several RBC membrane parameters, including abnormal microorganization of the membrane surface, a decrease in sialic acid content, and resistance to the fluidizing effect of ethanol, that were not completely corrected in the abstinent alcoholics. The RBC transit time was significantly longer in the active drinkers than in the abstainers but not the social drinkers. There were no significant differences between the groups with regard to membrane lipid core fluidity. The main abnormality (fluidization) in RBC from the active alcoholics involved the polar surface of the membrane (probed using TMA-DPH), and correlated with the decrease in sialic acid content but not with RBC deformability.

Rat synaptic membrane fluidity parameters after intermittent exposures to ethanol in vivo.

Differentiated membrane alterations correlate with the development of functional tolerance or dependence during chronic alcohol intoxication in humans as well as in animals. In animal studies, a single period of continuous exposure was generally used. In humans, the consumption can be more episodic with heavier weekend drinking. How a heavy but intermittent alcohol exposure over 4 weeks affects the synaptic membrane fluidity and sensitivity was examined in male and female adult rats. No differences were seen between membranes from males and females. Alterations were found in the negative polar membrane region probed by TMA-DPH and the sensitivity to acute ethanol was significantly reduced in the DPH (lipid core) and TMA-DPH probed membrane regions. Tolerance to the hypothermic effect of ethanol has developed and could be correlated with the resistance of the membrane lipid core to ethanol. Intermittent exposures to ethanol, as continuous ones, can result in development of functional and membrane tolerance and in specific alterations in the fluidity of the polar part of the membrane, probably in relation with dependence.

Influence of dietary n-6/n-3 polyunsaturated fatty acid balance on the development of tolerance during chronic ethanol intoxication in rats.

The present study addresses the possible interacting effects of dietary n-6/n-3 polyunsaturated fatty acid (PUFA) balance and chronic ethanol intoxication on the synaptic membrane responses to ethanol and the development of tolerance in rats. Wistar rats were fed either a standard lab chow or various semi-synthetic diets: rich in PUFA (from soya oil: SO), deficient in linolenate (from sunflower oil: SFO) or rich in long-chain (n-3) PUFA (cod liver oil: CLO). Male adult rats from the second specially fed generation were submitted to a 3-week alcoholization by daily intubation. Functional tolerance was quantified by the hypothermic response to a challenge dose of ethanol. Synaptic fluidity and sensitivity to ethanol (variations after acute ethanol addition) were assessed by fluorescence polarization (FP) of DPH, TMA-DPH or PROP-DPH. Membrane fatty acid composition was determined by GLC. The fatty acid composition of the synaptic membranes was influenced by the diet, but rearrangements among the lipids occurred, resulting in an apparent stability in brain membrane fluidity parameters. Nevertheless, clear-cut differences were noted in response to ethanol intoxication according to the diet. In the same period of time, rats fed SFO or CLO diets were unable to develop tolerance to ethanol at the membrane level as well as functionally, contrarily to the rats fed SO or standard diets. The structurally specific roles of PUFA are suggested by the negative membrane effects of the alpha-linolenate deficient diet (SFO) and the positive ones of a diet (SO) rich and well balanced in (n-3 + n-6) PUFA. Furthermore, the n-6/n-3 PUFA balance in the synaptic membrane needs to be kept within very narrow limits to allow normal development of the adaptive response to ethanol.

Interactive effects of dietary (n-3) polyunsaturated fatty acids and chronic ethanol intoxication on synaptic membrane lipid composition and fluidity in rats.

The influence of dietary polyunsaturated fatty acids on fatty acid composition, cholesterol and phospholipid content as well as 'fluidity' (assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) probes) of brain synaptic plasma membranes (SPM) and their interactions with chronic ethanol effects were studied in rats fed for two generations with diets either devoid of (n-3) fatty acids (sunflower oil diet), rich in alpha-linolenic acid (soya oil diet) or in long chain (n-3) fatty acids (sunflower + cod liver oil diet). Results were compared with rats fed standard lab chow. Sunflower oil led to an increase in the (n-6)/(n-3) ratio in the membranes with an increase of the 'fluidity' at membrane apolar level; sunflower + cod liver oil decreased the (n-6)/(n-3) ratio without affecting membrane 'fluidity' while no difference was seen between the SPM of rats fed soya oil and standard diet. After 3 weeks alcohol intoxication in rat fed the standard diet: oleic alpha-linoleic acids and cholesterol levels were increased, arachidonic acid and the double bond index/saturated fatty acids were decreased and there was a decrease of 'fluidity' in the lipid core of the SPM. Soya oil almost totally abolished these usually observed changes in the SPM fatty acids composition but increased oleic acid and cholesterol without any change in fluidity. Sunflower oil led to the same general alterations of fatty acid as seen with standard diet but to a greater extent, with decrease of the 'fluidity" at the apolar level and in the region probed by TMA-DPH. When sunflower oil was supplemented with cod liver oil, oleic and alpha-linoleic acids were increased while the 'fluidity' of the apolar core of SPM was decreased. So, the small changes in fatty acid pattern seem able to modulate neural properties i.e. the responses to a neurotoxic like ethanol. A structurally specific role of PUFA is demonstrated by the pernicious effects of the alpha-linolenic acid deficient diet which are not totally prevented by the supply of long chain (n-3) PUFA.

Biophysical and biochemical alterations in erythrocyte membranes from chronic alcoholics.

Erythrocyte membranes from healthy controls and alcoholic patients, examined within 24 h of abstinence, were studied for basal membrane fluidity and membrane sensitivity to ethanol by fluorescence polarization of the apolar probe 1,6-diphenyl-1, 3,5-hexatriene (DPH) and its cationic derivative 1,4(trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). The membrane partition (Kp) of ethanol and phenobarbital, and the concentrations of membrane-bound sialic acid and galactose, were also determined. The apolar hydrocarbon region of the membrane (DPH) was less fluid, in the alcoholics than in the controls (p less than 0.005). In the patients this membrane layer, as well as the polar lipid head group region (TMA-DPH), showed reduced fluidizing effect of ethanol (p less than 0.01). This resistance or tolerance to ethanol correlated with a markedly impaired (-59%, p less than 0.025) partition of ethanol into the membrane. The low Kp of ethanol in turn was partly related to reduced concentrations of polar carbohydrates such as sialic acid and galactose (p less than 0.01) at the membrane surface. The Kp of phenobarbital was reduced in the patients (-59%, p less than 0.005) but, apparently unrelated to the carbohydrate changes. These results indicate that in man, chronic alcohol abuse is associated with complex changes of membrane properties at different membrane levels e.g. at the charged surface, in the polar lipid head group region and in the hydrocarbon core. A partial basis for biophysical membrane tolerance to ethanol is suggested, implying that apart from phospholipid alterations, structural changes in membrane-bound glycoconjugates participate in this adaptive process.

Alcohol intoxication and sialic acid in erythrocyte membrane and in serum transferrin.

Microheterogeneity of serum transferrin as well as erythrocyte membrane sialic acid content were examined in alcoholic patients and healthy controls. Both the sialic acid content of erythrocyte membranes and of the circulating transferrin were significantly lower in alcoholic patients than in controls. A moderate daily ethanol intake (less than 80 g) allowed to observe a proportional relationship between alcohol intake and the carbohydrate deficient forms of transferrin, and also a correlation between alcohol intake and the membrane sialic acid content. This supports the hypothesis of ubiquitary alterations of glycosylations in connection to ethanol intoxication. Additional disturbances could explain the absence of correlations between membrane sialic acid, pattern of abnormal forms of serum transferrin, and alcohol intake in heavy alcoholic patients.

Physico-chemical membrane alterations during ethanol intoxication.

The physical state of the synaptic membranes from chronically intoxicated rats was evaluated by determination of "fluidity" parameters. The fluorescence polarizations of DPH and TMA-DPH were used to measure membrane fluidity and sensitivity to ethanol. During the ethanol intoxication, all the animals developed tolerance, and the sensitivities to ethanol in the apolar part as well as in the polar part of the membrane were reduced concurrently with a decreased partitioning of ethanol and changes in the membrane surface glycans. Only some of the animals displayed dependence as well as reduced basal fluidity of the apolar membrane region. Alterations in membrane order and sensitivity to ethanol were similar in erythrocyte membranes and correlated with synaptic alterations. Erythrocyte ghosts from alcoholic patients showed the same pattern of alterations. In this study, two distinct and differentiated disturbances in the physical state of the neuronal and erythrocyte membranes in association with tolerance to or dependence on ethanol were evident. The possible modulation of these membrane correlates could be a basis for therapeutic prospects in alcoholism.