Long-term Ethanol Administration Research Articles

The influence of phenformin on the extracellular matrix of the liver of rats under long-term administration of ethanol [

Experimental and clinical studies have revealed the influence of AMP-activated protein kinase (AMPK) signaling on the development of non-alcoholic liver fibrosis. Currently, the results of experimental studies demonstrate that inhibition of AMPK promotes fibrogenesis, while its activation prevents the development of liver fibrosis. The purpose of this work is to establish the effect of activation of AMP-activated protein kinase by the administration of phenformin on the content of glycosaminoglycans, oxyproline and sialic acids in the liver of rats under the conditions of long-term administration of ethanol. The study was conducted on 24 male Wistar rats. The animals were randomly divided into 4 groups of 6 animals each, on which we modeled ethanol-induced liver damage and administered phenformin hydrochloride at a dose of 10 mg/kg. The experiment lasted 63 days. In the liver of rats, the content of total glycosaminoglycans, the concentration of heparin-heparan, keratan-dermatan and chondroitin fractions of glycosaminoglycans, the content of free oxyproline and sialic acids were studied. Long-term alcoholization leads to a violation of the extracellular matrix of the liver of rats, which is evidenced by a decrease in the concentration of proteoglycans and a redistribution of their fractions in the direction of a decrease in anti-inflammatory and regenerative fractions. Chronic intake of alcohol increases the processes of desialylation of glycoconjugates and the intensity of collagenolysis. Activation of AMP-activated protein kinase by administration of phenformin under the conditions of simulating ethanol-induced liver damage leads to an increase in the concentration of glycosaminoglycans due to the growth of heparin-heparan and chondroitin fractions and reduces the intensity of desialylation of glycoconjugates and collagenolysis in the liver of rats.

Anthocyanin-Rich Extract of Red Cabbage Attenuates Advanced Alcohol Hepatotoxicity in Rats in Association with Mitochondrial Activity Modulation

Abstract Aim The liver is the main target for alcohol-induced injury. The aims of this work were to carry out further research into the mechanisms of liver damage induced by long-term administration of high-dose ethanol to rats and to evaluate the hepatoprotective potential of red cabbage (Brassica oleracea var. capitata f. rubra) anthocyanins (RCE). Material/Methods Male albino Wistar rats were divided into four groups. Group 1 was the control. Groups 2 through 4 received ethanol (4 g/kg body weight, 8 weeks). Group 3 received 11 mg RCE/kg and Group 4 received 22 mg RCE/kg. Dry lyophilised RCE was prepared from fresh red cabbage and analysed. We then evaluated the liver histology, mitochondrial respiration, and biochemical and immunological parameters in these groups of rats. Results The rat intoxication caused steatohepatitis characterised by macro- and microvesicular steatosis, ballooning and fatty liver dystrophies, lymphocytic infiltration, neutral lipid accumulation, and elevations of the serum activities of the liver injury marker enzymes. The treatment of alcohol-administered rats with RCE (mainly, cyanidin-3-diglucoside-5-glucoside, cyanidin-3-coumaroylrutinoside-5-hexoside, cyanidin-3-feruloylrutinoside-5-hexoside, delphinidin-3-feruloylrutinoside-hexoside) dose-dependently alleviated these pathological changes: The sizes of hepatocyte lipid vacuoles and the inflammatory signs were decreased, and the levels of the rat serum biochemical markers of liver injury, proinflammatory cytokines TNFα and IL-6, and the adipokine leptin (and not TGFβ) were significantly reversed. The RCE administration during intoxication completely recovered the changed liver mitochondria respiration rates and the ADP/O coefficient, as well as the phagocytic index and neutrophil metabolic activity in the blood. In experiments in vitro, RCE (13.6 μg/ml) modulated the respiratory parameters of isolated rat liver mitochondria, dissipated mitochondrial membrane potential, and inhibited the Ca2+-induced mitochondrial permeability transition. Conclusion The red cabbage anthocyanins could be useful for treatment of alcoholic liver injury due to their Ca2+-ionophoric/protonophoric activity, influence on Ca2+ homeostasis, and improving mitochondrial functions and inflammatory status.

Ліпополісахаридіндукована системна запальна відповідь обтяжує розвиток окисно-нітрозативного стресу в слинних залозах щурів при їх алкогольному ураженні

We addressed the role of lipopolysaccharide (LPS)-induced systemic inflammatory response (SIR) in the development of oxidative-nitrosative stress in the salivary glands of rats under the influence of alcohol. Ethanol (40%) at the dose of 24 mg/kg was administered intraperitoneally (ip) twice per day for 14 days. SIR was induced by ip administration of LPS (Salmonella typhi) at the dose 0.4 mg/kg for 1 week followed by a weekly LPS administration for 7 weeks. We found that long-term administration of ethanol in the back- ground of LPS-induced SIR increased the circulating level of proinflammatory markers (TNFa, IL-6) and C-reactive protein and this increase exceeded the respective values when LPS and alcohol were administered separately. Under these conditions, in submandibular salivary glands, the superoxide anion production by mitochondria respiratory chain was increased by 25.9 and 30.5%, by microsomal monooxygenases and NO synthase by 19.0 and 27,1%, by phagocyte NADPH-oxidase by 29.5 and 30.0%. The activity of inducible NO-synthase increased by 15.5 and 83.6%, the concentration of peroxynitrites of alkali and alkali-earth metals elevated by 32.5 and 58, 3%, and S- nitrosothiols raised by 20.2 and 22.7%. These changes were accompanied by a decrease in α-amylase activity and the aquaporin-5 concentration that impairs water and protein excretion by salivary glands. We conclude that adminis- tration of ethanol in the background of LPS-induced SIR results in more pronounced development of oxidative- nitrosative stress in the submandibular salivary glands and more marked dysfunction compared to separate use of LPS and alcohol.

The Role of NF-κВ in the Realization of Functions of Various Types of Regeneration-Competent Cells of the Nervous Tissue in Ethanol-Induced Neurodegeneration.

The role of NF-κВ in the realization of the growth potential of neural progenitor cells from the subventricular area of cerebral hemispheres and secretion of neurotrophins by glial elements was studied under conditions of in vitro and in vivo modeled ethanol-induced neurodegeneration. It was found that this transcription factor does not participate in the regulation of mitotic activity of neural stem cells and neuronal-committed progenitors under optimal conditions and under the influence of ethanol in vitro. At the same time, NF-κВ suppresses differentiation/maturation of neural progenitor cells. Long-term peroral administration of ethanol to mice was accompanied by the inhibitory influence of NF-κВ on proliferation of progenitor cells. Blockade of NF-κВ in neural stem cells and committed neuronal precursors in animals with neurodegeneration induced cell cycle progression in these elements. The involvement of NF-κВ in the secretory function of astrocytes and oligodendrogliocytes was established. Inactivation of the nuclear transcription factor reduced the production of neurotrophins, in particular, in the case of ethanol exposure. At the same time, no changes in the function of microglia were noted.

Peculiarities of the Involvement of MAPKS ERK1/2 and р38 in the Implementation of the Functions of Neural Stem Cells and Neuronal Committed Precursors in Ethanol-Induced Neurodegeneration.

We studied the peculiarities of the participation of ERK1/2 and р38 in regulation of various types of progenitor cells of the nervous tissue under conditions of ethanol-induced neurodegeneration modeled in vitro and in vivo. The stimulating role of these signaling molecules in the realization of the growth potential of intact multipotent neural stem cells and committed neuronal precursors (clonogenic PSA-NCAM+ cells) was demonstrated. In vitro exposure to neurotoxic doses of ethanol led to the loss of the specified role of ERK1/2 and p38 in the cell cycle regulation. Inversion of the role of both studied MAP-kinases in determining the proliferation status of neural stem cells after long-term administration of ethanol to experimental animals was revealed. In committed neuronal precursors, this inversion (inhibition of mitotic activity instead of activation) was revealed only for ERK1/2. In mice exposed to chronic alcoholization, ERK1/2 no longer participated in the process of specialization of both types of regeneration-competent cells of the nerve tissue. The revealed fundamental difference between the functions of ERK1/2 and p38 in the cell cycle regulation in neural stem cells and committed neuronal precursors under optimal conditions and during ethanol-induced neurodegeneration does not allow drawing definite conclusions about the prospect of using modifiers of their activity for the therapy for alcohol-related CNS pathologies.

Functional State of Various Types of Regeneration-Competent Cells in the Nervous Tissue in Ethanol-Induced Neurodegeneration.

The in vitro and in vivo models of ethanol-induced neurodegeneration were used to evaluate the content and functional activity of various types of regeneration-competent cells in subventricular zone of the cerebral hemispheres in C57Bl/6JY mice. In nervous tissue culture, ethanol (65 mM) produced no effect on formation of neurospheres. When administered per os in a daily dose of 3 g/kg for 8 weeks, ethanol produced no effect on the number of neural CFU in situ. In both cases, ethanol reduced proliferative activity of neural CFU. Long-term administration of ethanol in vivo suppressed differentiation of neural stem cells and decreased the number of committed precursors (neural cluster-forming units) in the subventricular zone of cerebral hemispheres. In vitro application of ethanol stimulated secretion of humoral growth factors by the cluster-forming neural glial cells. In contrast, in vivo administration of ethanol suppressed this secretion.

The Impact of Zonisamide on the Development and Course of Alcohol Dependence in Rabbits. A pharmaco-EEG study.

Zonisamide is a new anti-epileptic drug whose mechanism of action is associated with neurotransmission systems also involved in the pathogenesis of addiction. Recently, the role of memory processes and the hippocampus (Hp) is underlined in dependence. In our previous study, we determined that zonisamide decreases changes in hippocampal bioelectric activity induced by a single dose of ethanol. This study uses a pharmaco-EEG method to examine the impact of zonisamide on the development and course of alcohol dependence in rabbits. Quantitative changes in EEG were observed in the midbrain reticular formation, Hp and frontal cortex. Zonisamide was administered p.o. once a day at dose of 30 mg/kg/day during the entire experiment. Solutions with increasing concentrations of ethanol were administered for 6 weeks, followed by a 2-week period of abstinence. The long-term administration of ethanol caused characteristic changes in rabbit EEG recordings, which were associated with a shift toward lower frequencies resulting in a depressive effect on the bioelectric activity of selected brain structures. Co-administration of zonisamide and ethanol caused a reduction of ethanol-induced alterations. Changes in EEG recordings were different during period of abstinence and were associated with potent shift toward the high frequencies. Zonisamide significantly decreased encephalographic features of neuronal hyperactivity when administered during the abstinence. Zonisamide decreases ethanol- and abstinence-induced changes in the EEG recordings. These effects may be a significant part of drug's mechanism of action in alcohol addiction therapy. A pharmaco-EEG method was used to determine the influence of a new anti-epileptic drug zonisamide on the development and course of alcohol dependence in rabbits. The drug co-administered with ethanol decreased alcohol-induced changes in selected brain structures. Zonisamide also decreases abstinence-induced changes in the EEG recordings.

EFFECTS OF THE LITHIUM – CONTAINING SORBENT ON TERMS OF BEHAVIORAL REACTIONS UNDER CHRONIC ALCOHOL INTOXICATION MODEL

Lithium preparations are widely used for stabilize mood in case of bipolar affective disorder. Currently neuroprotective and neuroregenerative effects of lithium are of interest as in case of acute brain injury, also in chronic neurodegenerative diseases such as dementia, alcoholism, Alzheimer disease, etc. [1–5]. In clinical practice use of lithium preparations is limited due to difficult adjustment of drug dosage, necessity of monitoring its concentration in blood, side effects development as a result of accumulation of lithium in a body. For the purpose of improvement of pharmacologic properties lithium is combined with other agents (for example modifying sorbent) thus it can produce longer-term and more harmless (less side reactions) effect in the long view. Lithium immobilization on sorption basis will allow to use sorbent as detoxicant and carrying agent of drugs to body. The purpose of the work is studying the effect of the lithium – containing sorbent on terms of behavioral reactions under chronic alcohol intoxication model. Materials and methods. During the work we used nonlinear mice – males, which weight 25–30 g (180 animals). Chronic alcohol intoxication was precipitated via 40% proof spirit injections (oral supplementation in quantity of 3 g/kg during 2 weeks), additionally mice drunk 5% proof spirit from drinking bowl. Each experimental group consisted of 10 animals. Study drugs were inserted inside while ethanol injecting. Control animals were inserted 0,9% salin solution. Emotional state of animals was assessed through forced swim test, short – term memory assessment was performed through conditioned passive avoidance reflex. Effect of chronic alcohol intoxication on the parameters of conditioned reflex activity was measured every 7 days. Results. It was found that the investigated lithium-containing sorbent increases: the number of mice are trained passive avoidance reflex, remembering percent of electric shock animals compared to the negative control , and – the duration of the latent time of immobility in comparison with the negative control. Conclusion. The proposed formulation of lithium (immobilized on a sorbent ) has a complex neurotropic action, showing antitoxic properties against a background of long-term administration of ethanol.

Effects of long-term ethanol administration in a rat total enteral nutrition model of alcoholic liver disease

Male Sprague-Dawley rats were chronically fed a high-unsaturated-fat diet for 130 days by using total enteral nutrition (TEN), or the same diet in which ethanol (EtOH) isocalorically replaced carbohydrate calories. Additional groups were supplemented with the antioxidant N-acetylcysteine (NAC) at 1.7 g·kg(-1)·day(-1). Relative to an ad libitum chow-fed group, the high-fat-fed controls had three- to fourfold greater expression of fatty acid transporter CD36 mRNA and developed mild steatosis but little other hepatic pathology. NAC treatment resulted in increased somatic growth relative to controls (4.0 ± 0.1 vs. 3.1 ± 0.1 g/day) and increased hepatic steatosis score (3.5 ± 0.6 vs. 2.7 ± 1.2), associated with suppression of the triglyceride hydrolyzing protein adiponutrin, but produced no elevation in serum alanine aminotransferase (ALT). Chronic EtOH treatment increased expression of fatty acid transport protein FATP-2 mRNA twofold, resulting in marked hepatic steatosis, oxidative stress, and a twofold elevation in serum ALT. However, no changes in tumor necrosis factor-α or transforming growth factor-β expression were observed. Fibrosis, as measured by Masson's trichrome and picrosirius red staining, and a twofold increase in expression of type I and type III collagen mRNA, was only observed after EtOH treatment. Long-term EtOH treatment increased hepatocyte proliferation but did not modify the hepatic mRNAs for hedgehog pathway ligands or target genes or genes regulating epithelial-to-mesenchymal transition. Although the effects of NAC on EtOH-induced fibrosis could not be fully evaluated, NAC had additive effects on hepatocyte proliferation and prevented EtOH-induced oxidative stress and necrosis, despite a failure to reverse hepatic steatosis.

Long term ethanol consumption leads to lung tissue oxidative stress and injury.

Background: Alcohol abuse is a systemic disorder. The deleterious health effects of alcohol consumption may result in irreversible organ damage. By contrast, there currently is little evidence for the toxicity of chronic alcohol use on lung tissue. Hence, in this study we investigated long-term effects of ethanol in the lung.Results: Though body weight of rats increased significantly with duration of exposure compared to its initial weight, there was no significant change in relative weight (g/100 g body weight) of lung due to ethanol exposure. The levels of thiobarbituric acid reactive substances (TBARS), nitrite, protein carbonyl, oxidized glutathione (GSS G), redox ratio (GSS G/ GSH ) and GST activity elevated; while reduced glutathione (GSH ) level and activities of glutathione reductase (GR), glutathione peroxidase (GPx), catalase, superoxide dismutase (SOD) and Na+K+ATPase reduced significantly with duration of ethanol exposure in the lung homogenate compared to the control group. Total matrix metalloproteinase activity elevated in the lung homogenate with time of ethanol consumption. Histopathologic examination also demonstrated that severity of lung injury enhanced with duration of ethanol exposure.Methods: 16–18 week-old male albino Wistar strain rats weighing 200–220 g were fed with ethanol (1.6 g/kg body weight/day) up to 36 weeks. At the end of the experimental period, blood samples were collected from reteroorbital plexus to determine blood alcohol concentration and the animals were sacrificed. Various oxidative stress-related biochemical parameters, total matrix metalloproteinase activity and histopathologic examinations of the lung tissues were performed.Conclusions: Results of this study indicate that long-term ethanol administration aggravates systemic and local oxidative stress, which may be associated with lung tissue injury.

Oxidative DNA Damage Induced by Ethanol in Mouse Peripheral Leucocytes

It is suggested that reactive oxygen species produced during ethanol intake may induce oxidative DNA damage. The present study, by the use of single cell gel electrophoresis (comet assay), investigated the potential genotoxicity of acute and long-term ethanol administration in mouse peripheral leucocytes. Urinary 8-hydroxy-2'-deoxyguanosine and total antioxidant capacity and reactive oxygen species in whole blood were also detected. Acute or long-term administration of ethanol, at the dose of 2.5 or 5.0 g/kg intraperitoneally, for 30 days, could induce significant DNA damage in peripheral leucocytes, which could be repaired at least 3 days after ethanol withdrawal in long-term ethanol treatment. A significant increase of urinary 8-hydroxy-2'-deoxyguanosine and generations of reactive oxygen species in whole blood after ethanol administration were observed, which demonstrated the ethanol-induced oxidative DNA damage. Interestingly, it was found that the total antioxidant capacity was significantly increased in whole blood after long-term ethanol administration compared to the control group, which suggested enhancement in the activities of the antioxidative defence system against oxidative tissue damage caused by excessive ethanol consumption. Thus, the present studies provide a clear evidence that ethanol induced DNA damage in peripheral leucocytes, which might result from ethanol-induced oxidative stress in the body.

Zinc Supplementation Enhances Hepatic Regeneration by Preserving Hepatocyte Nuclear Factor-4α in Mice Subjected to Long-Term Ethanol Administration

Alcoholic liver disease is associated with sustained liver damage and impaired regeneration, as well as significant zinc deficiency. This study was undertaken to examine whether dietary zinc supplementation could improve liver regeneration by increasing the expression of genes involved in hepatic cellular proliferation in a mouse model of alcoholic liver disease. Adult 129S6 mice fed an ethanol-containing liquid diet for 6 months developed alcoholic liver disease as measured by serum alanine transferase activity and histopathological changes. Zinc supplementation to ethanol-exposed mice enhanced liver regeneration as indicated by increased numbers of proliferation cell nuclear antigen (PCNA)-positive and bromodeoxyuridine (BrdU)-labeled hepatocytes. Zinc-enhanced liver regeneration was associated with an increase in hepatocyte nuclear factor-4alpha (HNF-4alpha), a liver-enriched, zinc-finger transcription factor. Studies using cultured HepG2 cells showed that zinc deficiency suppressed cell proliferation and cell proliferation-related proteins, including hepatocyte growth factor (HGF), insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein 1 (IGFBP1), metallothionein (MT), and cyclin D1, as well as HNF-4alpha. HNF-4alpha gene silencing inhibited cell proliferation in association with decreased protein levels of IGF-I, IGFBP1, MT, and cyclin D1. The present study provides evidence that zinc supplementation enhances liver regeneration at least in part by HNF-4alpha through the up-regulation of cell proliferation-related proteins, suggesting that dietary zinc supplementation may have beneficial effects in alcoholic liver disease.

Hepatic fibrosis after long-term administration of ethanol and moderate vitamin A supplementation in the rat.

Rats were fed up to 9 months diets supplemented with vitamin A in an amount that, by itself, had no apparent adverse effect on the liver. When associated with chronic ethanol administration, vitamin A supplementation strikingly exacerbated ethanol-induced abnormalities: fat accumulation was increased and numerous giant mitochondria were observed. Furthermore, lesions appeared which ethanol alone does not produce in rats, namely necrosis, inflammation, and fibrosis. Vitamin A supplementation increased the number of fat storing cells (lipocytes) which positively correlated with vitamin A accumulation in the liver. However, when vitamin A supplementation was combined with ethanol administration, vitamin A levels in the liver and the number of fat storing cells decreased and numerous myofibroblasts appeared in association with abundant collagen fibers. There was also hepatic inflammation and necrosis, accompanied by a rise in serum glutamate dehydrogenase, SGOT, and SGPT and a decrease in retinol binding protein and vitamin A. We conclude that amounts of vitamin A, which by themselves appear harmless, may produce severe liver lesions when associated with chronic ethanol consumption.

Thymocytes, Pre‐B Cells, and Organ Changes in a Mouse Model of Chronic Ethanol Ingestion—Absence of Subset‐Specific Glucocorticoid‐Induced Immune Cell Loss

The well-known immune deficiency of the chronic alcoholic dictates the need for a long-term rodent ethanol administration model to evaluate the baseline immunologic effects of chronic ethanol abuse, and investigate the genetic determinants of those effects. Much published work with rodents has shown clearly that acute ethanol administration and short-term ethanol-containing liquid diets both cause elevated corticosterone and can cause significant thymocyte, pre-B cell and peripheral lymphocyte losses. Such losses may mask more subtle alterations in immune homeostasis, and in any case are generally short-lived compared with the span of chronic ethanol abuse. Thus, it is important to have a model in which long-term immune alterations can be studied free of corticosteroid-induced cell losses. We have utilized chronic 20% (w/v) ethanol in water administration to several mouse strains for prolonged periods of time and evaluated serum corticosterone, immunologic stress parameters, and other organ changes by standard methods. We now confirm earlier reports that chronic ethanol in water administration to mice does not produce net elevations of corticosterone, although diurnal variation is altered. Importantly, there is neither selective loss of immune cell populations known to be corticosteroid sensitive, CD4+CD8+ thymocytes and pre-B cells, nor are changes observed in the histologic appearance of the thymus. Nonetheless, there are significant chronic ethanol effects in other tissues, including reduced heart weight, mild hepatic steatosis, alterations of gut flora, increased serum peptidoglycan, and as published elsewhere, immune system abnormalities. This model of ethanol administration is convenient, sustainable for up to 1 year, demonstrably feasible in several mouse strains, permits good weight gains in most strains, and results in significant changes in a number of organs. The administration method also will permit modeling of long-term steady abuse punctuated by major binges, and is suitable for supplementation studies using water soluble additives. Overall, the method is useful for a wide range of studies requiring a chronic low-stress method of ethanol administration.

Long-term ethanol feeding enhances susceptibility of the liver to orally administered lipopolysaccharides in rats.

Endotoxin has been implicated in the pathogenesis and progression of alcoholic liver disease. However, it is still unclear how long-term ethanol feeding affects absorption of endotoxin from the intestine and susceptibility of the liver to gut-derived endotoxin. The object of this study was to determine the effect of long-term ethanol feeding on hepatic susceptibility to orally administered endotoxin. Male Wistar rats that weighed approximately 150 g were pair-fed with an ethanol-containing liquid diet or a control diet for 35 days. In some experiments, 0, 10, or 20 mg/kg of lipopolysaccharides (LPS) was added to the liquid diet for 7 days beginning on day 29. On day 36, the animals were killed for blood biochemistry and histologic examination of the liver. We also determined plasma endotoxin levels after 20 mg/kg of LPS administration using a gastric tube. In another set of experiments, we determined intestinal permeability using FD4 (fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4000 D). With 10 mg/kg of LPS, serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were significantly increased in the ethanol-fed rats but not in controls. After 20 mg/kg of LPS administration, more substantial increases in serum ALT and ALP levels were observed in ethanol-fed rats as compared with control diet-fed rats. Plasma endotoxin levels in long-term ethanol-fed rats were higher than those in control rats after intragastric administration of high-dose endotoxin (20 mg/kg). Furthermore, intestinal permeability to FD4 was increased by long-term ethanol administration. Long-term ethanol feeding increases intestinal permeability to and absorption of endotoxin, which can sequentially enhance hepatic susceptibility to orally administered endotoxin. This model has potential as a subclinical experimental model for the study of alcoholic liver disease.

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.

The Effect of Long-Term Ethanol Feeding on Efficacy of Doxycycline plus Rifampicin in the Treatment of Experimental Brucellosis Caused by Brucella melitensis in Rats

A model of Brucella melitensis infection was used in the setting of long-term ethanol administration to study the effects of ethanol on antibiotic therapy of B. melitensis infection. Wistar rats received a liquid diet containing maximally 42.2% of total calories as ethanol. Controls were pair-fed a liquid diet without ethanol. Diets began 15 days pre- and continued post-infection. Rats were infected intraperitoneally with B. melitensis. Doxycycline (10 mg/kg/day) plus rifampicin (6 mg/kg/day) were administered intragastrically starting days 7 to 14 following B. melitensis inoculation. The cure rate was 64.71% in ethanol-fed and 100% in control groups. Although the number of B. melitensis in spleens and livers was reduced, cure was unsuccessful in 6 ethanol-fed rats and this was not explained by the appearance of resistance, since none of the strains isolated following a 7-day course of therapy showed an increase in the minimum inhibitory concentration (MIC) of antibiotics. This study suggests that long-term ethanol ingestion diminishes the efficacy of doxycycline plus rifampicin combination therapy of rat brucellosis in an experimental design.

Inhibition by miltirone of up-regulation of GABA A receptor α 4 subunit mRNA by ethanol withdrawal in hippocampal neurons

Miltirone, a tanshinone isolated from the root of Salvia miltiorrhiza, has been characterized as a low-affinity ligand for central benzodiazepine receptors. We have now shown that this compound bound with low affinity (micromolar range) to central benzodiazepine recognition sites but did not interact with peripheral benzodiazepine receptors. It failed to potentiate Cl − currents induced by γ-aminobutyric acid (GABA) both in Xenopus oocytes expressing recombinant human GABA A receptors and in cultured rat hippocampal pyramidal cells, but it inhibited the ability of diazepam to potentiate the effect of GABA in these systems. Miltirone (1–10 μM) also partially inhibited the increase in the abundance of the mRNA for the α 4 subunit of the GABA A receptor induced by ethanol withdrawal in cultured hippocampal neurons. These results suggest that miltirone might ameliorate the symptoms associated with discontinuation of long-term administration of ethanol or of other positive modulators of the GABA A receptor.

Pathophysiology of alcohol-induced pancreatic injury.

The evidence that ethanol abuse can lead to pancreatitis is overwhelming, but the mechanism(s) by which ethanol causes pancreatic injury and pancreatitis are not known. Many studies have focused on short-term effects of ethanol administration on exocrine pancreatic function, but the results reported have been variable and no clear picture has emerged. Attempts to induce pancreatitis by long-term ethanol administration have, for the most part, failed. We evaluated the effects of ethanol administration on pancreatic secretion of digestive enzymes. These studies indicate that administration of ethanol results in a transient increase in pancreatic amylase output and plasma cholecystokinin (CCK) levels. This phenomenon is mediated by a trypsin-sensitive CCK-releasing factor that is present within the duodenal lumen. These observations lead us to speculate that repeated CCK-mediated, ethanol-induced stimulation of pancreatic digestive enzyme secretion may play a role in the events that link ethanol abuse to the development of pancreatic injury.

Long-Term Ethanol Feeding Enhances Susceptibility of the Liver to Orally Administered Lipopolysaccharides in Rats

Background Endotoxin has been implicated in the pathogenesis and progression of alcoholic liver disease. However, it is still unclear how long-term ethanol feeding affects absorption of endotoxin from the intestine and susceptibility of the liver to gut-derived endotoxin. The object of this study was to determine the effect of long-term ethanol feeding on hepatic susceptibility to orally administered endotoxin. Methods Male Wistar rats that weighed approximately 150 g were pair-fed with an ethanol-containing liquid diet or a control diet for 35 days. In some experiments, 0, 10, or 20 mg/kg of lipopolysaccharides (LPS) was added to the liquid diet for 7 days beginning on day 29. On day 36, the animals were killed for blood biochemistry and histologic examination of the liver. We also determined plasma endotoxin levels after 20 mg/kg of LPS administration using a gastric tube. In another set of experiments, we determined intestinal permeability using FD4 (fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4000 D). Results With 10 mg/kg of LPS, serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were significantly increased in the ethanol-fed rats but not in controls. After 20 mg/kg of LPS administration, more substantial increases in serum ALT and ALP levels were observed in ethanol-fed rats as compared with control diet-fed rats. Plasma endotoxin levels in long-term ethanol-fed rats were higher than those in control rats after intragastric administration of high-dose endotoxin (20 mg/kg). Furthermore, intestinal permeability to FD4 was increased by long-term ethanol administration. Conclusions Long-term ethanol feeding increases intestinal permeability to and absorption of endotoxin, which can sequentially enhance hepatic susceptibility to orally administered endotoxin. This model has potential as a subclinical experimental model for the study of alcoholic liver disease.