Weeks Of Ethanol Treatment Research Articles

Ginger extract attenuates ethanol-induced pulmonary histological changes and oxidative stress in rats.

Chronic ethanol consumption is associated with changes in the function and structure of the lungs. The aim of this study was to investigate the effect of chronic ethanol exposure on the lungs and whether ginger extract mitigated pulmonary abnormalities induced by ethanol in rats. Male Wistar rats were divided into the control group, the ethanol group, and the ethanol plus ginger extract group. Six weeks of ethanol treatment increased the proliferation of lung cells, and induced fibrosis, inflammation and leukocyte infiltration. A significant rise in the level of 8-hydroxydeoxyguanosine, NADPH oxidase, and oxidized low-density lipoprotein was also observed. Ginger extract significantly ameliorated the above changes. These findings indicate that ethanol induces abnormalities in the lungs by oxidative DNA damage and oxidative stress, and that these effects can be alleviated by ginger, which may function as an antioxidant and anti-inflammatory agent.

Effects of Chronic Ethanol Consumption on Biochemical Parameters and Oxidative Stress on Rat

Background: The biochemical effect of chronic alcohol consumption has been rarely studied although, most of the human population drinks moderate ethanol. The aims of this study was described some biochemical effects induced by moderate ethanol ingestion on rats in four and 12 weeks duration. Methods: Eighteen adult male albino Wistar rats (175 to 210 g) were fed with ethanol (1.6 ml/kg body weight/day) for 4 and 12 weeks. Animals randomly divided into 3 groups (1 to 3) comprised 6 animals each as following: Control group as group (1): Received orally drinking water. Group II: Treated orally with 1.6 g diluted ethanol/ kg body weight daily for 28 successive days. Group III:Treated orally with 1.6 g diluted ethanol/ kg body weight daily for 84 successive days. Blood samples were collected from retero-orbital plexus of animals before to start the ethanol administration (0 week), and at the end of 4 and 12 weeks of ethanol treatment. The serum was used for biochemical tests such as Blood Urea Nitrogen (BUN), Creatinine, Total protein, Albumin, Alanine Amino transferases (ALT), Aspartate Amino transferases (AST), Alkaline Phosphatases (ALP), Glutamyl transpeptidase (GGT) and Nitrite concentration. Reduced Glutathione (GSH), Mallondialdehyde (MDA) content, Catalase, Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPH) activities were estimated on homogenized samples. For Analysis of data paired t-student test was used. Results: Ethanol exposure initially affects liver function followed by renal function. Chronic ethanol ingestion increased hepatocellular enzyme markers such as ALT, AST, ALP, GGT and MDA and nitrate concentration; while decreased total protein, albumin, GSH, ascorbic acid contents and SOD and catalase activities compare to control group. Conclusion: Long-term ethanol exposure induced toxicity which revealed by increase insystemic, local oxidative stress markers and hepatic enzyme indicators.

Ethanol-Induced Alterations in Purkinje Neuron Dendrites in Adult and Aging Rats: a Review.

Uncomplicated alcoholics suffer from discrete motor dysfunctions that become more pronounced with age. These deficits involve the structure and function of Purkinje neurons (PN), the sole output neurons from the cerebellar cortex. This review focuses on alterations to the PN dendritic arbor in the adult and aging Fischer 344 rat following lengthy alcohol consumption. It describes seminal studies using the Golgi-Cox method which proposed a model for ethanol-induced dendritic regression. Subsequent ultrastructural studies of PN dendrites showed dilation of the extensive smooth endoplasmic reticulum (SER) which preceded and accompanied dendritic regression. The component of the SER that was most affected by ethanol was the sarco/endoplasmic reticulum Ca(2+) ATPase pump (SERCA) responsible for resequestration of calcium into the SER. Ethanol-induced decreases in SERCA pump levels, similar to the finding of SER dilation, preceded and occurred concomitantly with dendritic regression. Discrete ethanol-induced deficits in balance also accompanied these decreases. Ethanol-induced ER stress within the SER of PN dendrites was proposed as an underlying cause of dendritic regression. It was recently shown that increased activation of caspase 12, inherent to the ER, occurred in PN of acute slices in ethanol-fed rats and was most pronounced following 40 weeks of ethanol treatment. These findings shed new light into alcohol-induced disruption in PN dendrites providing a new model for the discrete but critical changes in motor function in aging, adult alcoholics.

14-Deoxyandrographolide alleviates ethanol-induced hepatosteatosis through stimulation of AMP-activated protein kinase activity in rats

Andrographis paniculata (AP) is a traditional medicinal plant of Ayurveda. It grows widely in Asia and is prescribed in the treatment of liver diseases. Here we have investigated the beneficial role of 14-deoxyandrographolide (14-DAG), a bioactive diterpenoid from AP, against alcoholic steatosis in rats. 14-DAG was extracted from aerial parts (leaves and stems) of AP. Rats were fed with ethanol for 8 weeks. Animals were treated with 14-DAG during the last 4 weeks of ethanol treatment. In vitro studies were undertaken in a human hepatocellular liver carcinoma cell line culture. Hepatosteatosis was assessed from histopathological studies of liver sections. Acetyl-CoA, malonyl-CoA, and triglyceride contents were determined using commercially available kits. Fatty acid synthesis was evaluated from incorporation of 1-14C acetate. Regulation of fatty acid oxidation and lipogenesis were monitored with immunoblotting and immunoprecipitation studies. Ethanol exposure led to hepatotoxicity, as evident from the marked enhancement in the levels of AST and ALT. The values decreased almost to control levels in response to 14-DAG treatment. Results showed that ethanol feeding induced deactivation of AMP-activated protein kinase (AMPK) that led to enhanced lipid synthesis and decreased fatty acid oxidation, culminating in hepatic fat accumulation. Treatment with 14-DAG activated AMPK through induction of cyclic AMP-protein kinase A pathway. Activation of AMPK was followed by down-regulation of sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, leading to suppression of lipogenesis. This was associated with up-regulation of sirtuin 1 and depletion of malonyl-CoA, in favor of increased fatty acid oxidation. 14-DAG controlled ethanol-induced hepatosteatosis by interfering with dysregulation of lipid metabolism. In conclusion, our results indicated that 14-DAG was capable of preventing the development of fatty liver through AMPK-mediated regulation of lipid metabolism. This finding supported the hepatoprotective role of 14-DAG, which might serve as a therapeutic option to alleviate hepatosteatosis in chronic alcoholism.

Time course of SERCA 2b and calreticulin expression in Purkinje neurons of ethanol-fed rats with behavioral correlates.

Chronic ethanol consumption for 40 weeks in adult rats results in dilation of the extensive smooth endoplasmic reticulum (SER), a major component of the calcium homeostatic system within Purkinje neuron (PN) dendrites. The aim of the present study was to determine whether chronic ethanol consumption results in alterations of the sarco/endoplasmic reticulum Ca(2+) ATPase pump (SERCA) on the SER membrane of PN dendrites. The density of calreticulin, a calcium chaperone, was also investigated in the PN along with balancing ability. Ninety 8-month-old rats were exposed to rat chow, the AIN-93 M liquid control or ethanol diets (30/diet) for a duration of 10, 20 or 40 weeks (30/duration). Age changes relative to the rat chow controls were assessed with 3-month-old control rats (n = 10). Balance was assessed prior to euthanasia. Quantitative immunocytochemistry was used to determine the density of SERCA 2b + dendrites and calreticulin + PN soma and nuclei. Molecular layer volumes were also determined. Following 40 weeks of ethanol treatment, there were ethanol-induced decreases in SERCA 2b densities within the dendritic arbor and decreased balancing ability on the more difficult round rod balance test. There were no ethanol-induced changes in calreticulin densities. It can be concluded that ethanol-induced decreases in the SERCA pump accompany SER dilation and contribute to previously reported ethanol-induced dendritic regression in PN. Ethanol-induced changes in balance also occurred. Chronic ethanol consumption does not alter calreticulin expression in PN.

Alcohol Consumption and Its Effect on Testicular Structure and on Sperm Count and Motility in Parent Mice and Their Offspring

Introduction: The goal of the present study was to examine the effect of alcohol consumption on sperm count and motility and the morphological changes in the seminiferous tubules of parent mice and their offspring. Methods: Animals were divided into two groups, Group 1 (alcohol group) of twelve male and twelve female mice, were given a daily dose of (3 g/kg body weight as 25%, v/v) ethanol by gastric gavage for four and eight weeks. Group 2 (control group) also of twelve male and twelve female mice; received normal access of food and water. After four weeks of treatment, the males and females in each group were allowed to mate, and ethanol treatment continued for up to another four weeks. Twelve male offspring from group 1 and twelve male offspring from group 2 were selected randomly and allowed to become mature. Male parent mice were killed at the 4th and 8th weeks of treatment, and their male offsprings were killed when they reached maturity age. Results: Physiological examination of the sperm solution showed that there was a significant decrease in sperm count and motility after 4 and 8 weeks of ethanol treatment in parent male mice, but this decrease was not significant in their adult offspring. Furthermore, histological investigations indicated testicular lesions in the parent male mice and their adult male offspring. Conclusion: Alcohol abuse has deleterious effects on the testes structure and on the sperm count and motility of the epididymal spermatozoa of both parent mice and their offspring.

Honokiol reverses alcoholic fatty liver by inhibiting the maturation of sterol regulatory element binding protein-1c and the expression of its downstream lipogenesis genes

Ethanol induces hepatic steatosis via a complex mechanism that is not well understood. Among the variety of molecules that have been proposed to participate in this mechanism, the sterol regulatory element (SRE)-binding proteins (SREBPs) have been identified as attractive targets for therapeutic intervention. In the present study, we evaluated the effects of honokiol on alcoholic steatosis and investigated its possible effect on the inhibition of SREBP-1c maturation. In in vitro studies, H4IIEC3 rat hepatoma cells developed increased lipid droplets when exposed to ethanol, but co-treatment with honokiol reversed this effect. Honokiol inhibited the maturation of SREBP-1c and its translocation to the nucleus, the binding of nSREBP-1c to SRE or SRE-related sequences of its lipogenic target genes, and the expression of genes for fatty acid synthesis. In contrast, magnolol, a structural isomer of honokiol, had no effect on nSREBP-1c levels. Male Wistar rats fed with a standard Lieber–DeCarli ethanol diet for 4 weeks exhibited increased hepatic triglyceride and decreased hepatic glutathione levels, with concomitantly increased serum alanine aminotransferase and TNF-α levels. Daily administration of honokiol (10 mg/kg body weight) by gavage during the final 2 weeks of ethanol treatment completely reversed these effects on hepatotoxicity markers, including hepatic triglyceride, hepatic glutathione, and serum TNF-α, with efficacious abrogation of fat accumulation in the liver. Inhibition of SREBP-1c protein maturation and of the expression of Srebf1c and its target genes for hepatic lipogenesis were also observed in vivo. A chromatin immunoprecipitation assay demonstrated inhibition of specific binding of SREBP-1c to the Fas promoter by honokiol in vivo. These results demonstrate that honokiol has the potential to ameliorate alcoholic steatosis by blocking fatty acid synthesis regulated by SREBP-1c.

Increased Circulating Vasopressin May Account for Ethanol-induced Hypertension in Rats

Long-term ethanol intake has been reported to evoke both hypertension and increase of systemic vasopressin levels in rats. In this work, we investigated the involvement of systemic vasopressin in the hypertension evoked in rats by long-term ethanol (20% vol/vol) intake for 2 weeks, by systemic treatment with the V1-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP (50 microg/kg). Moreover, plasma arginine-vasopressin (AVP) content was quantified using an AVP radioimmunoassay and the expression of vasopressin mRNA in the supraoptic (SON) and paraventricular (PVN) nuclei was measured using real-time PCR. Mild hypertension was observed after 2 weeks of ethanol treatment when compared with control animals. Moreover, an increase in both the expression of vasopressin mRNA and the vasopressin blood content was observed in ethanol-treated rats in comparison to the control group. Basal blood pressure levels of ethanol-treated animals were significantly reduced by IV treatment with the V1-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP. However, dTyr(CH2)5(Me)AVP had no effect on the blood pressure of control animals. The results indicate that mild hypertension is already observed at an early phase of ethanol consumption in rats. Because the content of circulating vasopressin was increased in ethanol-treated rats and their basal blood pressure returned to control levels after IV treatment with a V1-vasopressin receptor antagonist, it is proposed that increased circulating vasopressin content may mediate the hypertension observed in ethanol-treated rats.

Oral Metformin-Ascorbic Acid Co-Administration Ameliorates Alcohol-Induced Hepatotoxicity In Rats

Alcoholic liver disease remains a major cause of liver failure worldwide with no available curative or prophylactic therapy as at present. High dose metformin is reported to ameliorate liver injuries in both human and animal models of acute and chronic alcoholic liver injuries. The aim of the present in vivo animal study was to determine whether metformin-ascorbic acid co-administration also prevents alcoholic hepatotoxicity in chronic alcohol exposure. In the present study, ameliorating effect of 200 mg/ kg/day of ascorbic acid (Asc), 500 mg/kg/day of metformin (Met) and their co-administration (Met-Asc) were investigated in 5 groups of 50% ethanol-treated male Wistar rats for 2 weeks of the experiment. The body weight of each rat was taken on days 1, 7, and 14 of the experiment, respectively. On day 15, fasted blood samples for plasma lipids and liver enzyme markers were collected via cardiac puncture from the rats under diethyl ether anaesthesia. Results showed that administration of graded oral doses of 50% ethanol for 14 days significantly (p<0.001) elevated the plasma liver enzymes--aspartate aminotransferase (AST), alanine aminotansferase (ALT) and alkaline phosphatase (ALP). Two weeks of ethanol treatment also induced alterations in the plasma triglycerides (PTG), total cholesterol (PTC), high density lipoprotein (HDL-c), and low density lipoprotein (LDL-c). However, these elevations were significantly (p<0.05) attenuated by Asc, Met, and Met-Asc after 14 days of oral treatment, with Met-Asc having higher significant (p<0.001) ameliorating effect than Asc alone but with comparative effect to that of Met alone. High dose metformin-ascorbic acid co-administration protected the liver against the deleterious effects of chronic high dose alcohol and the hepatoprotective effect of Met-Asc appeared to be due mainly to the metformin molecule of the drug combination. However, further studies would be required to evaluate the mechanisms underlying the observed effects.

MGlu5 receptor and protein kinase C implicated in the development and induction of neuropathic pain following chronic ethanol consumption

The central mechanisms of neuropathic pain following chronic ethanol consumption are poorly understood. We previously reported that the levels of metabotropic glutamate 5 (mGlu5) receptor and phosphorylated-protein kinase C (PKC) were significantly increased in the spinal cord following chronic ethanol consumption. The aim of this study was to investigate whether mGlu5 receptor and PKC inhibitors directly attenuate the neuropathic pain-like state induced by chronic ethanol treatment in rats. A significant decrease in the mechanical nociceptive threshold was observed 5 weeks of chronic ethanol consumption. This hyperalgesia was significantly attenuated by repeated i.p. injection of ( S)-2,6-diamino- N-[[1-(oxotridecyl)-2-piperidinyl]methyl] hexanamide dihydrochloride (NPC15437), a selective PKC inhibitor, once a day for a week after 4 weeks of ethanol treatment. Furthermore, this hyperalgesia was also significantly attenuated by repeated i.p. injection of 6-methyl-2-[phenylethynyl]-pyridine (MPEP), a selective mGlu5 receptor inhibitor, once a day for a week after 4 weeks of ethanol treatment. Furthermore, the hyperalgesia that developed after 5 weeks of ethanol treatment was significantly suppressed by a single i.p. post-injection with either NPC15437 or MPEP. These findings constitute direct evidence that spinal mGlu5 receptor and PKC play substantial roles in the development and maintenance of an ethanol-dependent neuropathic pain-like state in rats.

Effects of chronic ethanol administration on brain protein levels: A proteomic investigation using 2-D DIGE system

The effects of chronic ethanol treatment on the brain proteome were investigated in the long-fin striped strain of zebrafish Danio rerio. Prolonged exposure to 0.5% (v/v) ethanol resulted in the development of tolerance to the ethanol-induced disruption of normal swimming behavior. This behavioral tolerance was manifested after two weeks of continuous treatment and was maintained for an additional three weeks. After four weeks of ethanol treatment, zebrafish brains were divided into 40,000 g supernatant and pellet fractions, and an Ettan 2-D fluorescence difference gel electrophoresis (DIGE) system was used to detect ethanol-induced alterations in the level of protein expression. Protein identification was carried out using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and the Mascot and ProFound search engines. In the present study, we have identified some novel protein targets as well as substantiated some putative previous targets of chronic ethanol exposure.

Thalidomide prevents alcoholic liver injury in rats through suppression of Kupffer cell sensitization and TNF-α production

Background & Aims: Sensitization of Kupffer cells (KCs) to lipopolysaccharide (LPS) and overproduction of tumor necrosis factor (TNF) α are critical for progression of alcoholic liver injury. Thalidomide has been shown to suppress TNF-α production from macrophages. Accordingly, the purpose of this study was to determine whether thalidomide could prevent alcohol-induced liver injury. Methods: Rats were given ethanol (5 g/kg body wt) and thalidomide (5 mg/kg) once every 24 hours intragastrically. To assess the sensitization of Kupffer cells, LPS (5 mg/kg intravenously) was administered and liver histology was evaluated 24 hours later. KCs were isolated after 4 weeks of ethanol treatment and intracellular Ca2+ ([Ca2+]i) was measured using fura-2, whereas TNF-α was evaluated by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. CD14 was determined by Western and fluorescence staining. Results: Treatment with ethanol for 8 weeks caused marked steatosis, necrosis, and inflammation in the liver. These pathologic parameters were diminished markedly by treatment with thalidomide. In the 4-week ethanol group, the LPS-induced liver damage was aggravated and KCs were sensitized to LPS. Coadministration of thalidomide with ethanol prevented the KC sensitization completely. Furthermore, thalidomide abolished the LPS-induced increase in CD14 expression and [Ca2+]i elevation in KCs. Gut permeability was increased about 10-fold after 4 weeks of ethanol exposure, which was not affected by thalidomide. Moreover, thalidomide reduced the LPS-induced TNF-α production by KCs by decreasing TNF-α messenger RNA. Conclusions: These results collectively indicate that thalidomide prevents alcoholic liver injury through suppression of TNF-α production and abolishment of KC sensitization.GASTROENTEROLOGY 2002;123:291-300

Kupffer cell sensitization by alcohol involves increased permeability to gut-derived endotoxin.

Studies with gut sterilization and Kupffer cell inactivation support the hypothesis that endotoxin and Kupffer cells are involved in mechanisms of alcohol-induced liver injury. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hr later. Moreover, we established a new, simple animal model of ethanol hepatotoxicity based on Kupffer cell sensitization. The purpose of this study was to determine the mechanisms by which alcohol sensitizes Kupffer cells to lipopolysaccharide (LPS). Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hr intragastrically, and ethanol concentration, ethanol elimination, and portal vein endotoxin were measured. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated 24 hr after ethanol administration in vivo and were cultured in RPMI 1640 with 10% fetal bovine serum. After the addition of LPS, intracellular Ca2+ was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and tumor necrosis factor (TNF)-alpha was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western analysis. Ethanol levels exhibited a cyclic pattern in ethanol-treated rats. Similar results were obtained in groups given ethanol and antibiotics for 4 weeks. Rates of alcohol elimination were around 3.5 mmol/kg/hr in control rats. After 4 weeks of ethanol treatment with or without antibiotics, elimination rates were not changed. Translocation of horseradish peroxidase was increased about 3-fold in gut segments by treatment with ethanol. This increase was not altered by treatment with antibiotics. Moreover, portal vein endotoxin levels were increased from nearly undetectable levels to 80 pg/ml in plasma of rats treated with ethanol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells from rats treated with ethanol for 4 weeks, CD14, LPS-induced intracellular Ca2+, and TNF-alpha all were increased. These phenomena were blocked by antibiotics. Kupffer cells isolated from rats treated with ethanol for 4 weeks exhibit sensitization to LPS. It is likely that increased permeability of the gut is a prominent event that leads to alcoholic liver injury.

Kupffer Cell Sensitization by Alcohol Involves Increased Permeability to Gut‐Derived Endotoxin

Background: Studies with gut sterilization and Kupffer cell inactivation support the hypothesis that endotoxin and Kupffer cells are involved in mechanisms of alcohol‐induced liver injury. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hr later. Moreover, we established a new, simple animal model of ethanol hepatotoxicity based on Kupffer cell sensitization. The purpose of this study was to determine the mechanisms by which alcohol sensitizes Kupffer cells to lipopolysaccharide (LPS).Methods: Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hr intragastrically, and ethanol concentration, ethanol elimination, and portal vein endotoxin were measured. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated 24 hr after ethanol administration in vivo and were cultured in RPMI 1640 with 10% fetal bovine serum. After the addition of LPS, intracellular Ca2+ was measured by using a microspectrofluorometer with the fluorescent indicator fura‐2, and tumor necrosis factor (TNF)‐α was measured by enzyme‐linked immunosorbent assay. CD14 was evaluated by Western analysis.Results: Ethanol levels exhibited a cyclic pattern in ethanol‐treated rats. Similar results were obtained in groups given ethanol and antibiotics for 4 weeks. Rates of alcohol elimination were around 3.5 mmol/kg/hr in control rats. After 4 weeks of ethanol treatment with or without antibiotics, elimination rates were not changed. Translocation of horseradish peroxidase was increased about 3‐fold in gut segments by treatment with ethanol. This increase was not altered by treatment with antibiotics. Moreover, portal vein endotoxin levels were increased from nearly undetectable levels to 80 pg/ml in plasma of rats treated with ethanol. As expected, this increase was prevented (&lt;20 pg/ml) by antibiotics. In isolated Kupffer cells from rats treated with ethanol for 4 weeks, CD14, LPS‐induced intracellular Ca2+, and TNF‐α all were increased. These phenomena were blocked by antibiotics.Conclusions: Kupffer cells isolated from rats treated with ethanol for 4 weeks exhibit sensitization to LPS. It is likely that increased permeability of the gut is a prominent event that leads to alcoholic liver injury.

Short-term ethanol exposure increases the expression of Kupffer cell CD14 receptor and lipopolysaccharide binding protein in rat liver.

Gut-derived endotoxins (lipopolysaccharide, LPS) complexed to LPS-binding protein (LBP) activate liver Kupffer cells via their CD14 receptor. Pro-inflammatory cytokines are released and this is postulated to promote liver injury. We previously demonstrated enhanced expression of CD14 endotoxin receptor after 2 weeks of alcohol administration. A similar result, based on 6 weeks of ethanol treatment, was recently reported and suggested to correlate with alcohol-induced liver injury. To establish whether this occurs prior to or after the initiation of damage, we investigated the temporal effect of continuous ethanol exposure on the expression of CD14 and the associated LBP. In addition, we studied the effect of treatment with gadolinium chloride (GdCl3) that inactivates Kupffer cells and alleviates alcohol-induced liver damage. The amount of CD14 and LBP mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR), was unchanged 4-8 h after intragastric ethanol administration. However, after 24-48 h of repeated ethanol administration, CD14 and LBP mRNA both increased significantly and reached a level similar to that observed after 6 weeks of ethanol exposure by liquid diet. Immunostaining experiments with ED2 antibody demonstrated that GdCl3 efficiently inactivated Kupffer cells. However, there was no concomitant reduction in the expression of CD14 mRNA, suggesting that compensatory infiltration by ED2-negative, but CD14-positive, macrophages had occurred. Our results demonstrate that soon after the initiation of ethanol exposure, i.e. within 24-48 h, the hepatic expression of both the CD14 receptor and LBP is increased. This suggests that these increases could contribute to the initiation of alcoholic damage rather than being a consequence of the injury.

The Number of Granule Cells and Spine Density on Purkinje Cells in Aged, Ethanol-fed Rats

The purpose of this study was to determine whether chronic intake of ethanol by aged F344 rats was associated with a reduction in parallel fiber input to cerebellar Purkinje neurons (PN). Previous results from this laboratory provided direct evidence that synaptic density in PN dendritic arbors was significantly decreased and indirect evidence that terminal dendritic segments of PN were deleted during chronic ethanol treatment. From these results, it was hypothesized that an ethanol-related deletion of PN terminal dendritic segments might result from 1) a reduction in parallel fiber input to PN from cerebellar granule neurons or 2) a reduction in dendritic spines, the postsynaptic sites for parallel fiber input to PN dendrites. Measurements of the total number of cerebellar granule neurons (GN) and the volume of the GN layer, and measurements of the density of spines on PN terminal dendritic segments were made in separate groups of aged, ethanol-treated and control rats. There were no significant ethanol-related changes in these parameters after 40–48 weeks of ethanol treatment.

The reaction of astrocytes and neurons in the hippocampus of adult rats during chronic ethanol treatment and correlations to behavioral impairments

Chronic ethanol treatment of Wistar rats to 10% (v/v) ethanol over a period of 4, 12, and 36 weeks produced distinct alterations of the glial fibrillary acidic protein immunoreactivity (GFAP-IR) of dorsal hippocampal astrocytes. Ethanol consumption over a period of 4 weeks caused an increase in the total GFAP-IR of the astrocytes. Down-regulation of the total GFAP-IR was measured in all examined brain regions after 36 weeks of ethanol treatment. Prolonged ethanol treatment induced a significant loss of the total number of hippocampal pyramidal and dentate gyrus granule cells. Regional differences in the vulnerability to the neurotoxic effects of chronic ethanol intake over 36 weeks were found: CA3 > CA1 + CA2 ⪢ CA4 > GD. In agreement with the degree of neuronal cell loss, ethanol-induced behavioral impairments were found. The acquisition of maze performance using a complex elevated labyrinth was deteriorated after 36 weeks of ethanol treatment, suggesting a deficit in learning and memory. These findings illustrate the importance of time-response analysis when determining the structural and functional changes produced by chronic ethanol treatment.

Impaired adaptation of the hypothalamic-pituitary-adrenal axis to chronic ethanol stress in aged rats.

Acute injection of ethanol in young rats stimulates corticosterone secretion, and repeated daily ethanol injection results in tolerance to this response. This study examined the tolerance development of the corticosterone response to 14 daily ethanol injections (2 g/kg i.p.) in young (3-month-old) and aged (24-month-old) male rats. By day 7 the young rats exhibited complete tolerance to the stimulatory effects of ethanol on corticosterone levels, as measured 1 h after injection. In contrast, the aged rats displayed a fairly large corticosterone response to ethanol on day 7, and they still exhibited a significant corticosterone response to ethanol on day 14. There was no difference between young and aged rats in blood ethanol levels at any time point examined, and blood ethanol levels did not change across days of treatment, indicating that neither the tolerance development, nor the age-related difference in tolerance development could be explained by time- and age-related differences in ethanol metabolism. There also was little evidence for the impaired tolerance development in the aged rats to be secondary to a generalized dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis following 2 weeks of ethanol treatment. Thus, there was no difference in basal corticosterone levels between young and aged rats on each test day, and there was no difference between young and aged rats in the corticosterone response to an acute restraint stress challenge when administered 24 h after the last day of ethanol treatment. This study suggests that in aged rats there are changes in the adaptive mechanisms contributing to tolerance of the HPA axis response to ethanol.

Glycine prevents alcohol-induced liver injury by decreasing alcohol in the rat stomach

BACKGROUND & AIMS: Inactivation of Kupffer cells prevents alcohol- induced liver injury, and hypoxia subsequent to a hypermetabolic state caused by activated Kupffer cells probably is involved in the mechanism. Glycine is known to prevent hepatic reperfusion injury. The purpose of this study was to determine whether glycine prevents alcohol- induced liver injury in vivo. METHODS: Male Wistar rats were exposed to ethanol (10-12 g.kg-1.day-1) continuously for up to 4 weeks via an intragastric feeding protocol. The effect of glycine on the first-pass metabolism of ethanol was also examined in vivo, and the effect on alcohol metabolism was estimated specifically in perfused liver. RESULTS: Glycine decreased ethanol concentrations precipitously in urine, breath, peripheral blood, portal blood, feces, and stomach contents. Serum aspartate amino-transferase levels were elevated to 183 U/L after 4 weeks of ethanol-treatment. In contrast, values were significantly lower in rats given glycine along with ethanol. Hepatic steatosis and necrosis also were reduced significantly by glycine. Glycine dramatically increased the first-pass elimination of ethanol in vivo but had no effect on alcohol metabolism in the perfused liver. CONCLUSIONS: Glycine minimizes alcohol-induced liver injury in vivo by preventing ethanol from reaching the liver by activating first-pass metabolism in the stomach. (Gastroenterology 1996 May;110(5):1536-42)

Site-specificity of ethanol-induced dephosphorylation of rat hepatocyte keratins 8 and 18: A 31P NMR study.

Chronic feeding of ethanol to rats results in disorganization of the keratin intermediate filament network within hepatocytes. Previous studies from this laboratory have shown that intermediate filament organization in cultured cells is related to the phosphorylation state of the proteins. Therefore, we have examined the phosphorylation state of hepatocyte keratins from control and ethanol-fed rats. Feeding ethanol to rats results in dephosphorylation of one site on keratin 8 and one site on keratin 18 at all time points beginning with 6 weeks of ethanol treatment. Dephosphorylation was detected by phosphate analysis and by two-dimensional electrophoresis in which a change in isoelectric point of keratins from ethanol-fed rats was observed. These observations indicate that dephosphorylation of keratins in ethanol-fed animals may be an early step in alcoholic hepatitis which has occurred by 6 weeks of ethanol treatment. To further characterize keratin dephosphorylation in ethanol-fed rats, we used 31P NMR spectroscopy to classify the dephosphorylation site(s). Hepatocyte keratins were purified and solubilized in 9.5 M urea, 10 mM Tris-Cl, pH 8.1. 31P NMR spectra were obtained at 109 MHz, in 10 mm tubes at 30 degrees C. Samples of hepatocyte keratins were phosphorylated with A-kinase, protein kinase C, casein kinase II or Ca/CAM kinase and these samples were analyzed by 31P NMR spectroscopy. The resulting spectra were used as standards to compare the 31P chemical shifts of the resonances produced by these kinases with the phosphorus resonances of control and experimental samples. The 31P NMR spectrum of control hepatocyte keratins shows three resonances at 0.7, 4 and 5 ppm. In vitro phosphorylation by A-kinase produces a resonance at 4 ppm which is distinctly different from the resonance produced by each of the other kinases. In hepatocyte keratins from ethanol-fed animals, the resonance at 4 ppm was missing from the spectrum. These observations indicate that the keratin site that is dephosphorylated in ethanol-fed rats is characterized by the same 31P chemical shift as the keratin site that is phosphorylated by A-kinase.