Ethanol Liquid Diet Research Articles

Sex differences in ethanol liquid diet consumption in Sprague–Dawley rats

There are reports of sex differences in ethanol intake between different strains of male and female rats; however, ethanol consumption (via an ethanol liquid diet) and blood ethanol levels (BELs) between male and female rats of the Sprague–Dawley strain have not been studied. Therefore, the aims of this study were to examine body growth, ethanol consumption (ml/day and g·kg −1·day −1), and BELs in adult male and female ( n = 6–8 per group; sham-operated and ovariectomized) Sprague–Dawley rats consuming different concentrations (3% to 9% volume/volume) of the Lieber–DeCarli liquid ethanol diet. Throughout the study, male rats weighed significantly more than both female groups, and ovariectomized female rats weighed more than sham female rats. Ethanol diet consumption (ml/day) was significantly greater in male rats than in female rats at higher ethanol concentrations, whereas, when the diet consumption was expressed in grams of ethanol per kilogram of body weight per day, the sham female group was shown to consume significantly more ethanol than the male group. Even though there were differences in ethanol intake, BELs were similar among the groups. The data indicate that, similar to other strains, Sprague–Dawley rats also exhibit sex differences in their pattern of body growth (weight gain) and ethanol intake; however, BELs were similar among the groups.

Analyses of smooth endoplasmic reticulum of cerebellar parallel fibers in aging, ethanol-fed rats

The smooth endoplasmic reticulum (SER), a calcium storage organelle, is essential for normal neuronal function. Dilation of the SER is pathologic and a threat to neuronal calcium homeostasis. Dilation of the SER has been reported within the dendrites of cerebellar Purkinje neurons of aging rats after lengthy ethanol treatment. Ethanol-related alterations of parallel fiber SER have not been investigated despite the fact that such dilation may precede and contribute transsynaptically to SER dilation and degeneration in Purkinje neuron dendrites. Male Fischer 344 rats ( n = 120; age = 12 months old) were randomly divided into three dietary groups (40 rats per group) and fed rat chow, the AIN-93M liquid control diet, or the AIN-93M liquid ethanol diet (without water) for 5, 10, 20, or 40 weeks (30 rats per time point). Sections from posterior vermal lobules were viewed with the electron microscope. Maximum and minimum diameters of parallel fiber SER profiles were measured. Ethanol-related dilation of parallel fiber SER was not found after 5, 10, 20, or 40 weeks of treatment. Age-related dilation of parallel fiber SER profiles did occur. These findings support the suggestions that (1) parallel fiber SER, unlike the SER in Purkinje neurons, is insensitive to ethanol and (2) the mechanisms by which ethanol and aging alter cerebellar function and structure are different.

Prenatal Ethanol Exposure Reduces mGluR5 Receptor Number and Function in the Dentate Gyrus of Adult Offspring

Previous studies in our laboratory indicated that metabotropic glutamate receptor (mGluR)-stimulated phosphoinositide hydrolysis is markedly reduced in the hippocampal formation of adult rat offspring whose mothers drank moderate amounts of ethanol during pregnancy. In the present study, we extended these observations by measuring the impact of prenatal ethanol exposure on proteins associated with the mGluR5 receptor-effector system along with two mGluR5 agonist-mediated responses in dentate gyrus of adult offspring. Sprague-Dawley rat dams consumed one of three diets throughout gestation: (1) a BioServ liquid diet that contained 5% ethanol (v/v), (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet, or (3) lab chow ad libitum. Microdissected slices of dentate gyrus were prepared from adult female offspring from each diet group and used for (1) Western blot analyses of mGluR5, the G-proteins Galphaq and Galpha11, and phospholipase C-beta1; (2) 2-chloro-5-hydroxyphenylglycine (CHPG)-stimulated growth associated protein 43 (GAP-43) phosphorylation; or (3) CHPG potentiation of electrically evoked [H]-D-aspartate (D-ASP) release from dentate gyrus slices. In tissue prepared from untreated control rats, CHPG produced a dose-dependent increase in phosphate incorporation into GAP-43, with maximal agonist stimulation occurring at 20 microM of CHPG. CHPG produced a quantitatively similar dose-dependent increase in the potentiation of electrically evoked D-ASP release from dentate gyrus slices from untreated controls. Fetal ethanol exposure reduced the amount of dentate gyrus mGluR5 receptor protein by 36% compared with the diet control groups. There were no significant differences between diet groups in the two G-proteins or phospholipase C-beta1 protein. Fetal ethanol exposure reduced CHPG-stimulated GAP-43 phosphorylation to approximately one half the amount of CHPG stimulation observed in the control diet groups. Prenatal ethanol exposure also reduced CHPG potentiation of D-ASP release to a similar degree compared with control. These results indicate that prenatal exposure to moderate quantities of ethanol reduces mGluR5 expression in the dentate gyrus of adult offspring. Although the subcellular site(s) for reduced mGluR5 expression cannot be discerned from Western blot data, the quantitatively similar effects of prenatal ethanol exposure on mGluR5 agonist stimulation of presynaptically localized GAP-43 phosphorylation and CHPG potentiation of evoked D-ASP release suggest that the presynaptic nerve terminal is one site where prenatal ethanol exposure has reduced mGluR5 receptor number and function. Furthermore, these data implicate these neurochemical alterations as one factor contributing to the hippocampal synaptic plasticity and behavioral deficits that we have observed previously in prenatal ethanol-exposed offspring.

Chronic Ethanol Treatment Enhances Inflammatory Mediators and Cell Death in the Brain and in Astrocytes

Inflammatory processes and cytokine expression have been implicated in the pathogenesis of several neurodegenerative disorders. Chronic ethanol intake induces brain damage, although the mechanisms involved in this effect are not well understood. We tested the hypothesis that activation of glial cells by ethanol would induce stimulation of signaling pathways and inflammatory mediators in brain, and would cause neurotoxicity. We used cerebral cortex from control and chronic ethanol-fed rats, which received ethanol-liquid diet for 5 months and cultured of astrocytes exposed to 75 mM ethanol for 7 days. Our results demonstrate that chronic ethanol treatment up-regulates iNOS, COX-2 and IL-1beta in rat cerebral cortex and in cultured astrocytes. Under both experimental conditions, up-regulation of these inflammatory mediators and IL-1RI concomitantly occurs with the stimulation of IRAK and MAP kinases, including ERK1/2, p-38 and JNK, which trigger the downstream activation of oxidant-sensitive transcription factors NF-KB and AP-1. These effects were associated with an increased in both caspase-3 and apoptosis in ethanol-fed rats and in astrocytes exposed to ethanol. In conclusion, chronic ethanol treatment stimulates glial cells, up-regulating the production and the expression of inflammatory mediators in the brain, and activating signalling pathways and transcription factors involved in inflammatory damage and cell death.

Residual behavioral and neuroanatomical effects of short-term chronic ethanol consumption in rats

The residual effects of short-term chronic ethanol consumption were investigated in rats maintained on an ethanol liquid diet for 26 consecutive days (mean intake=16.1 g/kg/day). Animals were assessed for spontaneous motor activity (12 days post-ethanol), spatial working memory (17 days post-ethanol), spatial reference memory (184 days post-ethanol), and retention of passive avoidance (201 days post-ethanol). Measurements of brain weights and cortical thickness vertices within the dorsomedial and ventrolateral cortex of eight coronal planes were determined 260 days post-ethanol. Two-dimensional cell profile densities within six coronal planes and within CA1 region of the hippocampus were also obtained, along with the total volumetric measurement of the hippocampus proper. Results indicated between group differences when subjects were assessed on working memory with ethanol-treated animals exhibiting longer escape latencies in a Morris water maze, an effect partially attributed to the perseverance of ethanol-treated animals in exhibiting thigmotaxicity. No other ethanol-related behavioral impairment was noted. Neuroanatomically, ethanol-treated rats had thinner cortical mantles (6.3% and 6.6% reductions) within the frontoparietal cortex and had lower two-dimensional cell profile densities within the most caudal cortical region studied. Interestingly, control animals with thicker cortical mantles tended to perform better on the working memory task, whereas the opposite was true for ethanol-treated subjects. These data led to the conclusion that chronic ethanol consumption of a relatively short duration produces working memory impairments, albeit mild, that are partially related to an inability to abandon ineffectual behavioral strategies, and also produces neuroanatomical alterations within the cortex.

Modification of the Mitochondrial Proteome in Response to the Stress of Ethanol-dependent Hepatotoxicity

Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology.

Alcohol exacerbates murine pulmonary tuberculosis.

Alcohol consumption has been described as a risk factor for infection with Mycobacterium tuberculosis, but its contribution to tuberculosis has been difficult to isolate from other adverse socioeconomic factors. Our objective was to evaluate the impact of alcohol consumption on pulmonary infection with M. tuberculosis in a murine model. BALB/c mice were maintained on the Lieber-DeCarli liquid ethanol diet or a liquid control diet and infected intratracheally with low-dose M. tuberculosis H37Rv. Lung organism burdens, lung and lung-associated lymph node CD4(+)- and CD8(+)- lymphocyte numbers and rates of proliferation, and CD4(+)-lymphocyte cytokine production levels were compared between the groups. The alcohol-consuming mice had significantly higher lung organism burdens than the control mice, and the CD4(+)- and CD8(+)-lymphocyte responses to pulmonary infection with M. tuberculosis were blunted in the alcohol group. Lymphocyte proliferation and production of gamma interferon were decreased in the CD4(+) lymphocytes from the alcohol-consuming mice. Additionally, lung granulomas were significantly smaller in the alcohol-consuming mice. In conclusion, murine alcohol consumption is associated with decreased control of pulmonary infection with M. tuberculosis, which is accompanied by alterations in the region-specific CD4(+)- and CD8(+)-lymphocyte responses and defective lung granuloma formation.

Effects of ethanol consumption on vasopressin and neuropeptide Y immunoreactivity and mRNA expression in peripheral and central areas related to cardiovascular regulation

Results from previous studies have demonstrated that ethanol influences central neural mechanisms involved in the control of blood pressure. We studied the effects of ethanol consumption on vasopressin and neuropeptide Y immunoreactivity and mRNA expression in the nucleus tractus solitarius and paraventricular hypothalamic nucleus, as well as in the petrosal and nodose ganglia of rats. The ethanol-fed rats received liquid diet ad libitum containing 37.5% ethanol-derived calories (6.7% volume/volume), and the pair-fed rats received the same volume of diet containing isocaloric amounts of maltose–dextrin substituted for ethanol for 3 or 28 days. Arterial blood pressure was evaluated in a separate group of rats, which was unchanged by 3 days, but elevated by 21% after 28 days of ethanol consumption. Vasopressin immunoreactivity and mRNA signal were not detected in the ganglia, nor were they changed in the nucleus tractus solitarius and paraventricular hypothalamic nucleus, by 3 days of ethanol consumption. However, after 28 days of ethanol liquid diet consumption, vasopressin-positive terminals were decreased in the nucleus tractus solitarius and vasopressin immunoreactivity cell bodies and mRNA signal were decreased in the paraventricular hypothalamic nucleus. Neuropeptide Y–immunoreactive terminals were increased in the nucleus tractus solitarius only after 28 days of ethanol liquid diet consumption, but they were decreased in the paraventricular hypothalamic nucleus in rats treated with ethanol for 3 or 28 days. We concluded that the levels of both vasopressin and neuropeptide Y neurotransmitters are changed by long-term ethanol consumption in the neuronal pathways related to control of blood pressure.

Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase

Because alcoholic liver disease has been linked to oxidative stress, we investigated the effect of a compromised antioxidant defense system, Cu, Zn-superoxide dismutase (Sod1) deficiency, on alcohol-induced liver injury. C57BL/129SV wild-type (Sod1(+/+)) and Sod1 knockout (Sod1(-/-)) mice were fed dextrose or ethanol (10% of total calories) liquid diets for 3 weeks. Histologic evaluation of liver specimens of Sod1(-/-) mice fed ethanol showed the development of liver injury ranging from mild to extensive centrilobular necrosis and inflammation. Sod1(+/+) mice fed ethanol showed mild steatosis; both Sod1(+/+) and Sod1(-/-) mice fed the dextrose diet had normal histology. Alanine transaminase levels were significantly elevated only in Sod1(-/-) mice fed ethanol. Cytochrome P450 2E1 (CYP2e1) activity was elevated about 2-fold by ethanol in Sod1(+/+) and Sod1(-/-) mice. Ethanol consumption increased levels of protein carbonyls and lipid peroxidation aldehydic products in the liver of Sod1(-/-) mice. Hepatic adenosine triphosphate (ATP) content was reduced dramatically in Sod1(-/-) mice fed ethanol in association with a decrease in the mitochondrial reduced glutathione (GSH) level and activity of MnSOD. Immunohistochemical determination of 3-nitrotyrosine (3NT) residues in liver sections of the Sod1 knockout mice treated with ethanol showed a significant increase of 3NT staining in the centrilobular areas. In conclusion, a rather moderate ethanol consumption promoted oxidative stress in Sod1(-/-) mice, with increased formation of peroxynitrite, protein carbonyls, and lipid peroxidation and decreased mitochondrial GSH and MnSOD. We speculate that the increased oxidative stress causes mitochondrial damage and reduction of ATP content, leading to alcoholic liver injury. This model may be useful in further mechanistic studies on alcohol-induced liver injury.

GABAA receptor alpha 1 and beta 2 subunit null mutant mice: behavioral responses to ethanol.

Mice lacking either the alpha1 or beta 2 subunit of the GABAA receptor were tested for ethanol, saccharin, or quinine consumption, ethanol-conditioned place preference, ethanol-conditioned taste aversion, ethanol-simulated motor activity, and handling-induced seizures following chronic consumption of an ethanol liquid diet. The alpha1 null mutants showed decreased ethanol and saccharin consumption, increased aversion to ethanol, and a marked stimulation of motor activity after injection of ethanol. The beta 2 null mutants showed decreased consumption of saccharin and quinine, but not ethanol. Surprisingly, neither mutant showed marked changes in handling induced seizures before or after withdrawal of ethanol. The unique effects of deletion of these two GABAA receptor subunits on ethanol responses are discussed in terms of the distinct changes in different populations of GABAA receptors.

Freely accessible water does not decrease consumption of ethanol liquid diets

In experimental studies, liquid ethanol diets are usually given as the sole source of nutrition and fluid. Two series of experiments were conducted to examine the effect of freely accessible water on the consumption of ethanol liquid diets in male Long–Evans rats. The consumption of diets and subsequent learning ability of rats were first examined in animals given twice-daily saline injections. One group received diet with no access to water for 12 weeks and was subsequently given free access to water with diets for an additional 12 weeks. A second group was given diet and water ad libitum for 24 weeks. Control animals received an isocaloric sucrose-containing diet (with or without ad libitum access to water). Subsequently, rats were tested for active avoidance learning. In the first 12 weeks, animals with ad libitum access to water drank more diet than did water-restricted animals, and previously water-restricted animals increased their diet consumption when access to water was freely available. All water-restricted animals, in both ethanol- and sucrose-treated groups, showed deficits in active avoidance learning, whereas only ethanol-treated animals in groups with ad libitum access to water showed learning deficits. In the second series of experiments, the effect of saline injections on diet consumption, both in the presence and absence of water, was examined. Although saline injections were associated with decreased diet consumption, there was no effect of free access to water. No differences in blood ethanol concentration were seen among groups. Findings obtained from both series of studies demonstrate that consumption of a Sustacal-based liquid ethanol diet does not decrease if access to water is freely available.

Cardiac structure and function after short-term ethanol consumption in rats

Using an animal model of alcoholism, we investigated whether 4 months of ethanol consumption was associated with a preclinical stage of alcoholic cardiomyopathy (ACM), as well as activation of the renin-angiotensin system and natriuretic peptides (NPs). In other forms of cardiovascular disease, the latter peptide systems have been used as markers of left ventricular (LV) remodeling and dysfunction. Rats were fed either a liquid ethanol or control diet, and serial blood samples were collected at baseline, as well as at 2 and 4 months, for plasma renin activity (PRA), angiotensin II (ANG II), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) levels. Serial echocardiography (echo) was performed at the same time points, and isolated heart studies were performed to assess ex vivo contractility at 4 months. In both groups at 4 months, there were significant and similar increases in end-diastolic and end-systolic echo dimensions and LV mass. At 4 months, however, a significant decrease was found in the relative wall thickness in the ethanol-fed group compared with findings for the control group. In both groups at 4 months, significant and similar time-dependent decreases were shown in BNP and ANP levels compared with baseline values. No differences were found in NP gene expression or tissue levels. In both groups at 4 months, significant and similar increases were found in ANG II levels compared with baseline values. Over time, in both groups, PRA levels were unchanged. In this study, the only cardiac structural feature characteristic of a preclinical ACM was a decrease in relative wall thickness.

Chronic daily ethanol and withdrawal: 5. Diurnal effects on plasma thyroid hormone levels.

We previously demonstrated that chronic daily ethanol consumption and daily withdrawal by male rats in a modified ethanol liquid diet paradigm produced (a) chronically increased adrenal glucocorticoid activity; (b) decreased plasma testosterone; (c) decreased forebrain proopiomelanocortin gene expression; and (d) corresponding alterations in plasma leptin levels-all of which are consistent with reported changes during alcohol abuse and alcoholism. Each of these systems interact with hypothalamo-pituitary-thyroid (HPT) regulation, and links between chronic alcohol abuse and thyroid dysfunction have been suggested by both human and rat studies. Accordingly, we have begun to investigate potential HPT mediation of, or response to, alterations in these systems by investigating plasma thyroid hormone levels in the same chronic daily ethanol/ withdrawal paradigm. Chronic daily episodes of ethanol consumption and withdrawal by male Sprague- Dawley rats decreased plasma levels of free (non-protein- bound) triiodothyronine (T3) (p < 0.01) and free thyroxine (T4) (p < 0.05) in the morning but not in the afternoon, relative to both ad libitum-fed and pair-fed controls (n = 9/treatment). Plasma total T4 levels were likewise suppressed (p < 0.01) in the morning, whereas total T3 levels were increased (p < 0.05) in the afternoon. These changes eliminated normal diurnal patterns (higher in the morning) of plasma free T3, free T4, and total T3 concentrations. Three weeks after cessation of ethanol consumption, morning plasma levels of free and total T3 and T4, as well as plasma thyroid-stimulating hormone (TSH), were all not significantly changed by the prior ethanol consumption or pair-feeding. These results reveal that plasma thyroid hormone concentrations are suppressed in a time of day dependent manner by chronic daily ethanol consumption and daily withdrawal in this model of chronic ethanol abuse. During subsequent long-term "abstinence," these thyroid hormones returned to control levels. These results are consistent with evidence that thyroid function is commonly diminished in alcoholism, with variable reports of recovery during abstinence. Further investigations with this rat model of daily ethanol consumption and daily withdrawal will help resolve interactions and roles of the HPT axis in alcohol abuse.

Dose‐Dependent Effects of Prenatal Ethanol Exposure on Synaptic Plasticity and Learning in Mature Offspring

Background We have observed profound deficits in hippocampal synaptic plasticity and one‐trial learning in offspring whose mothers drank moderate quantities of ethanol during pregnancy. In the present study, we examined the question of whether lower maternal blood ethanol concentrations (BECs) could produce functional deficits in offspring.Methods Rat dams consumed either a 2%, 3%, or 5% ethanol liquid diet throughout gestation. Three other groups of dams were pair‐fed a 0% ethanol liquid diet, and a seventh group consumed lab chow ad libitum. Adult offspring from each diet group were assigned either to studies of evoked [3H]‐D‐aspartate (D‐ASP) release from hippocampal slices or spatial learning studies using the Morris Water Task.Results Consumption of the 2%, 3%, and 5% ethanol liquid diets produced mean peak maternal BECs of 7, 30 and 83 mg/dL, respectively. Consumption of these ethanol diets had no effect on offspring birthweight, litter size or neonatal mortality. Likewise, evoked D‐ASP release from hippocampal slices and performance on a standard version of the Morris Water Task were not affected by prenatal ethanol exposure. By contrast, activity‐dependent potentiation of evoked D‐ASP release from slices and one‐trial learning on a “moving platform” version of the Morris Water Task were markedly reduced in offspring whose mothers consumed the 5% ethanol liquid diet. Intermediate deficits in these two parameters were observed in offspring from the 3% ethanol diet group, whereas offspring from the 2% ethanol diet group were not statistically different than controls.Conclusions We conclude that the threshold for eliciting subtle, yet significant learning deficits in offspring prenatally exposed to ethanol is less than 30 mg/dL. This BEC is roughly equivalent to drinking 1 to 1.5 ounces of ethanol per day.

Effects of maternal ethanol consumption on hematopoietic cells in the rat fetal liver

During development, there are many factors to be considered in studying the efficacy of the hematopoietic system to provide the immune system with adequate numbers of functional cells within the immune repertoire. Hematopoietic cells must be able to develop, proliferate, and emigrate from the hematopoietic fetal liver to other tissues of the body, such as the thymus and bone marrow. There is evidence that ethanol consumption causes immune deficiencies in adults and that maternal ethanol consumption causes immune deficiencies in children, both with correlative effects on cells and cytokinetic regulators. Therefore, the ability of the hematopoietic system to seed the immune system may be jeopardized by maternal ethanol consumption. In this study, test groups included female rats (1) fed a Lieber–DeCarli ethanol liquid diet, (2) pair-fed a control liquid diet, or (3) fed standard laboratory chow. Livers were removed from fetuses 18 and 21 days postconception (dpc) and analyzed by immunophenotyping and flow cytometry. There was a 22% and 38% decrease in fetal body weight and a 25% and 30% decrease in fetal liver weights between ethanol-fed and pair-fed groups at 18 and 21 dpc, respectively. At 18 dpc, fetal body and liver weights of the pair-fed animals were also significantly reduced to those of the chow-fed group. However, by 21 dpc, both body and liver weights of the two control groups were not statistically different. The effects of maternal ethanol consumption on the distribution of hematopoietic cells were characterized by using monoclonal antibodies (mAbs) anti-CD43 (a progenitor hematopoietic cell marker) versus labeling with anti-Vβ8.2 (pre-T cells), anti-B220 (pre-B cells), and anti-NKR-P1A (pre-natural killer cells). With the use of flow cytometric analysis, at 18 dpc ethanol-exposed fetuses showed 40% and 62% decreases in B220 + and Vβ8.2 + cells, respectively, versus findings for pair-fed controls, with no significant change in NKR-P1A + cells. At 21 dpc, ethanol-exposed fetuses showed a 46% decrease in B220 + cells among the CD43 + cells, with a 50% increase in Vβ8.2 + cells. Observed alterations in gross fetal body and liver weights, together with modifications in the percentage of hematopoietic cells in the fetal liver after maternal ethanol consumption, strongly support the suggestion that the ability of the hematopoietic system to impart a repertoire of cells that are functional in the regulatory and effector mechanisms of the immune process may be compromised.

Involvement of platelet-activating factor in hepatic apoptosis and necrosis in chronic ethanol-fed rats given endotoxin.

Platelet-activating factor (PAF)-a potent activator of neutrophils-plays an important role in the pathogenesis of endotoxin-induced tissue injury. However, the role of PAF in hepatic damage during alcoholic hepatitis remains unclear. The aims of the present study were to test whether PAF contributes to hepatic injury in an animal model of alcoholic hepatitis and to investigate the involvement of the Fas-receptor/Fas-ligand system in this process. Male Sprague-Dawley rats were pair-fed with Lieber-DeCarli ethanol liquid diet or isocaloric control diet for 6 weeks. Liver injury was induced by the intravenous (i.v.) injection of lipopolysaccharide (LPS) (1 mg/kg). Rats were pretreated with a specific PAF receptor antagonist (TCV-309; 100 mg/kg i.v.) or vehicle 1 h before LPS treatment. Chronic ethanol administration remarkably sensitized the rats to the effects of LPS, with resultant severe hepatocellular injury, accompanied by significant increases in serum levels of alanine aminotransferase (ALT), tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 (CINC/gro). Histological examination of the damaged livers showed hepatocyte apoptosis and necrosis with extensive infiltration by neutrophils, whereas immunohistochemical studies revealed enhanced Fas-receptor expression on hepatocytes and hepatic accumulation of neutrophils expressing Fas-ligand. Pretreatment with the PAF receptor antagonist protected against hepatic injury, suppressing hepatocyte apoptosis and necrosis, infiltration of neutrophils, expression of Fas-receptor and Fas-ligand, and serum TNF-alpha levels. Our study suggests that PAF is an important mediator of hepatic injury in the ethanol/endotoxin model of alcoholic hepatitis.

Differential display identifies neuroendocrine-specific protein-A (NSP-A) and interferon-inducible protein 10 (IP-10) as ethanol-responsive genes in the fetal rat brain

Fetal alcohol exposure is the most common nonhereditary cause of mental retardation in the western world. Rats prenatally treated with ethanol liquid diet exhibit extensive defects in the brain that accurately model those observed in humans. To analyze the ethanol effects on gene expression during brain development, we performed mRNA differential display and two-dimensional electrophoresis on gestational day (G) 13 and G16 brain from rats treated with ethanol liquid diet. Using mRNA differential display followed by a variety of quantitative analyses, three genes were confirmed to be ethanol-responsive. Among them was Neuroendocrine-Specific Protein-A (NSP-A), which is known to be affected by thyroid hormone in the cortex at this developmental time. However, two additional genes known to be thyroid hormone-responsive were unaffected by ethanol, indicating that interference with thyroid hormone action may not be a predominant pathway by which alcohol induces damage in the fetal brain. The observation that interferon-inducible protein-10 (IP-10) is up-regulated in ethanol-treated fetal brain may indicate the presence of a disease process recruiting CD8+ T-cells capable of interfering with myelination. The result of two-dimensional (2D) electrophoresis and Western analyses demonstrated that few changes in the abundance of individual proteins or the phosphorylation of proteins at threonine and tyrosine were induced by prenatal ethanol exposure. A critical analysis of the approaches used in the present study may be important for future studies in this field.

Hepatoprotective Effects of Arctium lappa Linne on Liver Injuries Induced by Chronic Ethanol Consumption and Potentiated by Carbon Tetrachloride

Arctium lappa Linne (burdock) is a perennial herb which is popularly cultivated as a vegetable. In order to evaluate its hepatoprotective effects, a group of rats (n = 10) was fed a liquid ethanol diet (4 g of absolute ethanol/ 80 ml of liquid basal diet) for 28 days and another group (n = 10) received a single intraperitoneal injection of 0.5 ml/kg carbon tetrachloride (CCl₄) in order to potentiate the liver damage on the 21st day (1 day before the beginning of A. lappa treatment). Control group rats were given a liquid basal diet which did not contain absolute ethanol. When 300 mg/kg A. lappa was administered orally 3 times per day in both the 1-day and 7-day treatment groups, some biochemical and histopathological parameters were significantly altered, both in the ethanol group and the groups receiving ethanol supplemented with CCl₄. A. lappa significantly improved various pathological and biochemical parameters which were worsened by ethanol plus CCl₄-induced liver damage, such as the ethanol plus CCl₄-induced decreases in total cytochrome P-450 content and NADPH-cytochrome c reductase activity, increases in serum triglyceride levels and lipid peroxidation (the deleterious peroxidative and toxic malondialdehyde metabolite may be produced in quantity) and elevation of serum transaminase levels. It could even restore the glutathione content and affect the histopathological lesions. These results tended to imply that the hepatotoxicity induced by ethanol and potentiated by CCl₄ could be alleviated with 1 and 7 days of A. lappa treatment. The hepatoprotective mechanism of A. lappa could be attributed, at least in part, to its antioxidative activity, which decreases the oxidative stress of hepatocytes, or to other unknown protective mechanism(s).

Hepatoprotective effects ofArctium lappa linne on liver injuries induced by chronic ethanol consumption and potentiated by carbon tetrachloride

Arctium lappa Linne (burdock) is a perennial herb which is popularly cultivated as a vegetable. In order to evaluate its hepatoprotective effects, a group of rats (n = 10) was fed a liquid ethanol diet (4 g of absolute ethanol/ 80 ml of liquid basal diet) for 28 days and another group (n = 10) received a single intraperitoneal injection of 0.5 ml/kg carbon tetrachloride (CCl(4)) in order to potentiate the liver damage on the 21st day (1 day before the beginning of A. lappa treatment). Control group rats were given a liquid basal diet which did not contain absolute ethanol. When 300 mg/kg A. lappa was administered orally 3 times per day in both the 1-day and 7-day treatment groups, some biochemical and histopathological parameters were significantly altered, both in the ethanol group and the groups receiving ethanol supplemented with CCl(4). A. lappa significantly improved various pathological and biochemical parameters which were worsened by ethanol plus CCl(4)-induced liver damage, such as the ethanol plus CCl(4)-induced decreases in total cytochrome P-450 content and NADPH-cytochrome c reductase activity, increases in serum triglyceride levels and lipid peroxidation (the deleterious peroxidative and toxic malondialdehyde metabolite may be produced in quantity) and elevation of serum transaminase levels. It could even restore the glutathione content and affect the histopathological lesions. These results tended to imply that the hepatotoxicity induced by ethanol and potentiated by CCl(4) could be alleviated with 1 and 7 days of A. lappa treatment. The hepatoprotective mechanism of A. lappa could be attributed, at least in part, to its antioxidative activity, which decreases the oxidative stress of hepatocytes, or to other unknown protective mechanism(s).

Dietary Mg 2+ supplementation restores impaired vasoactive responses in isolated rat aorta induced by chronic ethanol consumption

Chronic ethanol consumption contributes to cardiovascular dysfunction possibly related to loss of Mg 2+. This study was designed to examine the role of dietary Mg 2+ supplementation on chronic ethanol ingestion-induced vascular alteration. Rats were fed an ethanol liquid diet supplemented with or without Mg 2+ for 12 weeks. The force-generating capacity was examined in thoracic aortic rings. Ethanol-consuming animals exhibited significantly elevated blood pressure. In aorta with intact endothelium, the contractile responses to norepinephrine and KCl were greatly attenuated and potentiated, respectively. Interestingly, the ethanol-induced alterations in blood pressure and vasoconstrictive response were restored by Mg 2+ supplementation. Pretreatment with the β 1-adrenoceptor antagonist atenolol in intact aortic rings abolished the difference in response to norepinephrine between the control and ethanol groups, which implies the involvement of a weakened β 1-adrenoceptor component in vessels from the ethanol-fed rats. The norepinephrine-induced vasoconstriction in intact aorta rings was completely abolished by the α 1-adrenoceptor antagonist prazosin. In endothelium-denuded aorta, the contractile response to norepinephrine or KCl was not significantly different between the ethanol and Mg 2+ groups. Endothelium-dependent vasorelaxation to carbamylcholine chloride was not altered by either ethanol or Mg 2+ supplementation. Sodium nitroprusside-induced vasorelaxation was depressed by ethanol, and restored by Mg 2+, in aorta with or without endothelium. These data suggest that chronic ethanol consumption contributes to alterations of endothelium-dependent and -independent vascular response. These alterations can be compensated by dietary Mg 2+ supplementation.