Weeks Of Ethanol Administration Research Articles

Does retigabine affect the development of alcohol dependence?—A pharmaco-EEG study

New antiepileptic drugs have been investigated for their potential role in the treatment of alcohol dependence. One of these drugs is retigabine and this study examines the effect of retigabine co-administered with ethanol on the development of alcohol dependence and the course of acute withdrawal syndrome. A pharmaco-EEG method was used to examine this impact in selected brain structures of rabbits (midbrain reticular formation, hippocampus and frontal cortex). Retigabine was administered p.o. at a dose of 5mg/kg/day with ethanol ad libitum for 6 weeks and then alone for 2 weeks during an abstinence period. Changes in bioelectric activity, which demonstrated the inhibitory effect of alcohol on the brain structures, were already visible after 2 weeks of ethanol administration. In the abstinence period, changes were of a different nature and significant neuronal hyperactivity was observed, particularly in the midbrain reticular formation and the hippocampus. This findings reveal that retigabine decreased ethanol-induced changes during both alcohol administration and abstinence periods. In particular, the modulatory effect of retigabine on the hippocampus may be a significant element of its mechanism of action in alcohol dependence therapy.

Vitamin E and tannic acid improve DNA damage in rats submitted chronic ethanol administration

Purpose – Chronic ethanol consumption induces lipid peroxidation by increasing free radicals or reducing antioxidants and may increase damage to hepatic DNA. Tannins are polyphenolic metabolites present in various plants and one of their effects is antioxidant activity that reduces lipoperoxidation, as is the case for vitamin E. This paper aims to assess the role of tannic acid and vitamin E in lipid peroxidation and in DNA damage in rats receiving ethanol.Design/methodology/approach – A total of 60 Wistar rats were divided into six groups: control+ethanol (0‐24hs), tannic acid+ethanol (0‐24 hs), and vitamin E+ethanol (0‐24 hs). The animals were sacrificed immediately (0 hour) or 24 hours after a period of four weeks of ethanol administration and the following measurements were made: plasma vitamin E and liver glutathione, thiobarbituric acid resistant substances, and α‐tocopherol. The comet test was also applied to hepatocytes.Findings – Ethanol administration led to an increase in DNA damage (148.67±15....

Ethanol‐induced alterations of the antioxidant defense system in rat kidney

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty-two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol-fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long-term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and gamma-glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, gamma-glutamyltranspeptidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose-6-phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long-term ethanol administration.

Functional heterogeneity of rat hepatic and alveolar macrophages: effects of chronic ethanol administration

Chronic ethanol consumption is associated with increased incidence of hepatic and pulmonary infections. To determine if this is correlated with altered macrophage activity, we analyzed the functional properties of cells isolated sequentially from the liver and lung of rats fed a liquid diet containing ethanol (35% of calories) or malto-dextrin control for 9-12 weeks. Hepatic and alveolar macrophages from control animals were found to exhibit distinct morphologic and functional properties. Thus, hepatic macrophages were highly vacuolated and appeared larger and more irregular in shape than alveolar macrophages. These cells also displayed greater phagocytic activity and random migration. In contrast, lung macrophages produced more superoxide anion and nitric oxide, and exhibited enhanced chemotactic activity toward the complement fragment C5a. Whereas administration of ethanol to rats for 9-12 weeks resulted in decreased chemotaxis and superoxide anion production by alveolar macrophages, cell adhesion molecule expression was reduced in hepatic macrophages. Nitric oxide production and inducible nitric oxide synthase protein expression were decreased in both macrophage populations. These effects were not observed after 3-6 weeks of ethanol administration to rats. Our results suggest that changes in macrophage functioning may play a role in decreased host defense following chronic ethanol exposure.

Chronic enteral ethanol treatment causes hypoxia in rat liver tissue in vivo.

It is known that ethanol increases oxygen consumption in in vitro liver models, which could lead to hypoxia. Although it was shown recently that one large dose of ethanol caused hypoxia in rat liver in vivo, whether ethanol produces hypoxia in a clinically relevant chronic model remains unclear. In the present study, therefore, the effect of chronic ethanol on hypoxia was investigated in vivo using the 2-nitroimidazole hypoxia marker, pimonidazole. Male Wistar rats (300-325 g) were exposed to enteral ethanol continuously for 4 weeks. In this model, rats develop steatosis, inflammation, and necrosis characteristic of early stages of clinical alcoholic liver disease in humans. One hour before they were killed, rats were injected with pimonidazole (120 mg/kg intravenously), and livers were surgically isolated, removed, and fixed. Protein-bound pimonidazole adducts were identified on formalin-fixed, paraffin-embedded tissue with immunohistochemistry. Ethanol administration for 4 weeks significantly increased serum aspartate transaminase levels and hepatic pathology scores for steatosis, inflammation, and necrosis, as expected. Ethanol treatment significantly increased both the extent and number of cells that stained positive for pimonidazole compared with control animals given an enteral diet without ethanol. Quantitative image-analysis of pimonidazole binding showed that 4 weeks of ethanol administration nearly doubled the pimonidazole-positive area in tissue. Ethanol also increased pimonidazole binding significantly at 7 days, long before inflammation and necrosis could be detected. These results indicate that chronic ethanol causes hypoxia at the cellular level in rat liver in vivo and lend support to the hypothesis that hypoxia is involved in mechanisms of early alcoholic liver injury.

Adaptation to repeated restraint stress in rats: failure of ethanol-treated rats to adapt in the stress schedule.

Adaptation to a repeated restraint stress schedule was monitored in ethanol-treated and control rats. A single episode of 2 h restraint decreased food intake in both control and ethanol-treated rats. The decreases in control rats were not observed following the 5th daily restraint of 2 h/day, suggesting that adaptation has occurred. Ethanol-treated rats, however, exhibited decreased food intake even after 5th daily restraint of 2 h/day. Ethanol administration decreased weekly but not daily cumulative food intake in unrestrained rats. Food intakes of ethanol-treated and control restrained rats were comparable following 1st-3rd daily restraints, but were smaller in ethanol-treated rats following the 4th and 5th daily restraints. Open-field ambulatory activities monitored 24 h after the 5th daily restraint on the 6th day were comparable in control restrained and unrestrained rats. Ethanol-treated and control unrestrained rats also exhibited comparable ambulation, but ethanol-treated rats exhibited smaller activity than control restrained or ethanol-treated unrestrained rats. Fluid intakes of ethanol and control rats were comparable during the 2 weeks of ethanol administration, but daily restraint schedule decreased ethanol intake. The findings show adaptation to repeated restraint in control rats and inability of ethanol-treated rats to adapt in the stress schedule. These findings imply that excessive alcohol consumption may impair adaptation to stress and thus conceivably precipitate depression.

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.

EVIDENCE THAT CHANGES IN HIPPOCAMPAL EXCITABILITY <italic>IN VITRO</italic> ARE CAUSED BY WITHDRAWAL FROM CHRONIC <italic>IN VIVO</italic> ETHANOL ADMINISTRATION

A complex pattern of changes in the field potentials recorded from mouse hippocampal slices, prepared after chronic ethanol treatment in vivo, has previously been demonstrated in this laboratory. In the present study, recordings from slices prepared immediately after 2 weeks of ethanol treatment, showed only an increase in paired pulse potentiation, compared with controls, whereas recordings made immediately after 16 weeks of ethanol administration showed decreases in the thresholds for single and multiple population spikes, increases in paired pulse potentiation and epileptiform activity. In hippocampal slices prepared after 24 hr withdrawal, following 16 weeks of ethanol treatment, there were no signs of hyperexcitability in the field potentials. Ratings of convulsive behaviour were increased in mice during a 12-hr period after withdrawal from 16 weeks of ethanol treatment. Corresponding behaviour ratings for the mice given ethanol for 2 weeks, or those withdrawn for 24 hr after 16 weeks ethanol treatment, were not significantly different from control values. It was concluded that epileptiform activity seen in hippocampal slices after prolonged ethanol administration may contribute to the ethanol withdrawal hyperexcitability seen in vivo.

Studies of whole blood associated acetaldehyde as a marker for alcohol intake: Effect of gender in mice

A study was undertaken in C57BL mice to evaluate the effect of gender on whole blood associated acetaldehyde following exposure to ethanol in the drinking water (10% v/v). Whole blood associated acetaldehyde (WBAA) was measured from capillary blood samples using a fluorigenic high performance chromatographic assay on days 0, 7, 15 and 27. Ethanol consumption did not impair growth of either male or female mice when compared to controls. Following administration of ethanol, WBAA increased in both male and female mice but marked gender differences were apparent. Female mice consumed more fluid relative to body weight than males (155 +/- 27 S.D. vs. 124 +/- 19 ml/kg/day, p less than 0.001), but had lower mean WBAA levels during the four weeks of ethanol administration (137 +/- 37 vs. 318 +/- 66 nmol/g hemoglobin, p less than 0.001). WBAA levels in male mice were stable over the course of the experiment. Female mice were found to have peak WBAA levels on day seven after which time levels decreased significantly. These experiments emphasize gender differences in ethanol metabolism as well as the need to establish norms based on gender for assays of ethanol consumption which use acetaldehyde adducts with blood proteins.

Membrane tolerance to ethanol is rapidly lost after withdrawal: a model for studies of membrane adaptation.

The structural properties of liver microsomes and erythrocytes obtained from rats that had been chronically administered ethanol were examined by electron spin resonance (ESR) following ethanol withdrawal for 1-10 days. Membranes obtained from control animals exhibited considerable molecular disordering upon the addition of ethanol in vitro (50-100 mM). Conversely, microsomal and erythrocyte membranes from alcoholic animals were resistant to this disordering by ethanol (membrane tolerance). These membrane properties were also apparent in lipid bilayers comprised of either total lipids or phospholipids isolated from the control and alcoholic animals. While several weeks of ethanol administration were required for both erythrocytes and microsomes to develop membrane tolerance, erythrocytes from alcoholic animals were disordered by ethanol in vitro after the animals had been withdrawn from ethanol for only 1 day. The same rapid loss of tolerance was observed in microsomes after 2 days of withdrawal. The same time course for the loss of tolerance was observed in lipid bilayers prepared from the total lipid and phospholipid extracts. No significant differences in the cholesterol/phospholipid ratio were observed between the microsomal or erythrocyte membranes isolated before and after withdrawal. Thus, alterations in the microsomal and erythrocyte phospholipids, and not cholesterol content, were responsible for conveying membrane tolerance. Membrane structural properties can be rapidly adjusted in a mammalian system in response to the withdrawal of the external membrane perturbant ethanol. The withdrawal model, which begins with established membrane tolerance and leads to rapid and complete loss of tolerance, provides a model to analyze the compositional changes responsible for this tolerance to disordering by ethanol.

Memory deficits subsequent to chronic consumption of alcohol in mice: An analysis based on spontaneous alternation behavior

Male mice of the BALB/c strain were given a solution of 15% ethanol as their only source of fluid for periods varying from 5 weeks to 8 months. For behavioral testing, they were compared with control groups which had received either an isocaloric solution of sucrose or tap water. Memory was tested by using spontaneous alternation behavior in a T maze. Each test consisted of two forced trials (acquisition) followed by a free trial (test) given at different acquisition-test intervals (from 30 s to 24 h). Results from two independent experiments showed that after 25 weeks of ethanol administration there was an accelerated rate of decay of spontaneous alternation as a function of the acquisition-test interval. Such a phenomenon persisted after ethanol was omitted from the diet. A third experiment showed that when tested on two successive sessions separated by a 5 h interval, experimental subjects exhibited a decreased ability to perform normally on the second test. Our data are interpreted as showing that long-term ethanol administration results in accelerated forgetting and increased vulnerability to proactive interference and, as such, they are compared to the memory dysfunctions observed in amnesic patients.

An abnormal phospholipid in rat organs after ethanol treatment

Interaction of ethanol with essential fatty acid (EFA) metabolism has been demonstrated in man [ 1,2] and in experimental animals [3,4]. Several laboratories have reported on the effects of ethanol on changed proportions of polyunsaturated fatty acids in cell membranes [5,6,7]. In a series of experiments with ethanol and EFA we found increased concentrations of phospholipids in the brain of rats after 3 weeks of ethanol administration (3 g/kg body weight, daily intraperitoneal injections). This phospholipid increase was due to an increase of the acidic fraction of the phospholipids [4]. Examination of the proportions of individual neutral and acidic lipids by thin-layer chromatography (TLC) revealed the occurrence of an abnormal lipid fraction in organs from rats exposed to ethanol. This article is a report on the appearance of this abnormal lipid in various organs and the relation to the amount and duration of ethanol administration. Our currently available information on the chemical structure of the lipid is also given.

Chronic ethanol treatment affects synaptosomal membrane-bound enzymers

A progressive increase in activity of some brain membrane-bound enzymes is shown after 2 and 4 weeks of ethanol administration. After 4 weeks the activities in brain homogenate of (Na +, K +) ATPase, Ca ++ ATPase, 5′-nucleotidase, acetylcholinesterase and adenylate cyclase increased 150, 200, 140, 125 and 129 percent, respectively. Arrhenius plots of synaptosomal (Na +, K +) ATPase and acetylcholinesterase from alcohol-treated rats showed a lower transition temperature than control rats after two weeks, and this changed to a higher transition temperature after 4 and 8 weeks. Also, when ethanol was added in vitro to the control membranes, the transition temperature was lowered. However, if the alcohol was added to the membranes from alcohol-treated animals, the transition temperature was lowered to a value similar to that of controls. Fluorescence studies with l-anilinonaphthalene-8-sulfonate (ANS) demonstrate that ethanol induces a decrease in the fluorescence of ANS bound to brain synaptic membranes. This decrease in fluorescence is less than when these membranes are derived from chronically ethanol-treated rats. Also, when the synaptosomal enzymes were exposed to exogenous agents such as detergents, the enzyme obtained from alcohol-treated rats was more stable than that from control rats. These findings indicate a protein conformation change, probably due to the alteration of the physical properties of membrane lipids following chronic ethanol administration. These findings also demonstrate that there is a resistance to the effect of ethanol in membranes of animals habituated to ethanol that may be related to the adaptative modifications that underlie tolerance to and physical dependence on alcohol.

Ultrastructure of the rat epiphyseal growth plate following chronic ethanol ingestion.

We have previously demonstrated that ethanol has a direct toxic effect on the rat skeleton characterized by decreased trabecular bone volume. In the present study, we examined the ultrastructure of the distal radial epiphyseal growth plates in these same animals. Eight weeks of ethanol administration to 12 male rats results in serum alcohol levels of 140 mg/dl but did not alter the width or light microscopic appearance of the radial growth plate. Quantitative electron microscopy failed to demonstrate morphologic evidence of toxicity in the skeletal cells. We conclude that although ethanol appears to have a direct effect on rat bone characterized by enhanced resorption, toxicity is not attended by ultrastructural changes in the skeletal cells.

Ultrastructural and biochemical studies of the brain and other organs in rats after chronic ethanol administration: IV. Stereological analysis of mitochondria from rat liver cells after intoxication with various ethanol doses

Ultrastructural and stereological analysis of mitochondria of liver cells from rats was performed after intoxication with various doses of ethanol administered for various time periods in increasing concentrations. The following changes were observed in the cells of the experimental animals as compared with the control ones: -- a decrease of the surface area of mitochondria per unit volume of the hepatocyte, -- a decrease of the mean number of mitochondrial profiles per 100 micrometers 2 surface area of hepatocyte cross section, -- a decrease in the value of the axial ratio of mitochondrial profiles. A diminished volume fraction of the mitochondria was not observed. The above presented results show that, after ethanol poisoning, the mitochondria in hepatic cells become enlarged and rounded and their number seems to diminish. Qualitative studies demonstrated that the structure of mitochondria is frequently damaged, this consisting in injury to their membranes, formation of lamellar structures, and destruction of the system of cristae and matrix. This leads to the presence of degenerated, sometimes monstrously large mitochondria in the cells. The above described changes occurred as early as after 4 weeks of ethanol administration in increasing doses from 2.5 to 10 per cent. An increase of ethanol concentration to 42 per cent and prolongation of the period of treatment with ethanol to 12 weeks did not markedly enhance the changes.

Effect of ethanol on serum cholesterol concentration in dog and man.

The effect of ethanol on serum cholesterol concentration has been studied in normal male dogs subsisting on diets of low cholesterol content. Daily administration of 1.65 Gm. ethanol/Kg, of body weight produced significant increases of serum cholesterol in dogs when fed either a low-fat diet (4 per cent of fat calories) or a high-fat (40 per cent) diet. The mean cholesterol increase after 2 weeks of ethanol administration was 52 mg./100 ml. (SE, 8.2) for 8 dogs on the low-fat diet, and 75 (SE, 6.6) for 24 dogs on the high-fat (lard) diet. No significant difference was found between the mean increases of serum cholesterol produced by ethanol administration, using either the high-fat diet of saturated fat (lard) or that of unsaturated fat (sunflower oil). Serum cholesterol concentration decreases rapidly after discontinuing the administration of ethanol, reaching the prealcohol levels in 2 to 3 weeks. The higher levels of serum cholesterol are maintained as long as the administration of ethanol continues. Sodium acetate, when given at the same molar quantity as ethanol, failed to produce any change of serum cholesterol concentration in the dog. The effect of ethanol administration on serum cholesterol concentration in man was tested in 2 switchback experiments, comparing the effects of alcohol and those of a supplement of syrup of equal calorie value while the men were eating a normal diet containing 38 per cent of fat calories. When 0.45 Gm. ethanol/ Kg./day were given to 59 normal men for 3 weeks, no significant difference of serum cholesterol concentration was observed between the values on syrup and on alcohol. However, the administration of 1.35 Gm./Kg./ day to 14 men produced a mean increase of serum cholesterol concentration of 18 mg./100 ml. (SE, 5.0) within 1 week. The individual increases of serum cholesterol are correlated with the intrinsic cholesterol levels of the subjects (r = + 0.67, p > 0.01). It is concluded that ethanol increases serum cholesterol concentration in the dog and man. The response in the dog is much greater than that observed in the human subjects.