Long-term Ethanol Feeding Research Articles

Ferroptosis contributes to ethanol-induced hepatic cell death via labile iron accumulation and GPx4 inactivation

Alcohol abuse is a significant cause of global morbidity and mortality, with alcoholic liver disease (ALD) being a common consequence. The pathogenesis of ALD involves various cellular processes, including oxidative stress, inflammation, and hepatic cell death. Recently, ferroptosis, an iron-dependent form of programmed cell death, has emerged as a potential mechanism in many diseases. However, the specific involvement and regulatory mechanisms of ferroptosis in ALD remain poorly understood. Here we aimed to investigate the presence and mechanism of alcohol-induced ferroptosis and the involvement of miRNAs in regulating ferroptosis sensitivity. Our findings revealed that long-term ethanol feeding induced ferroptosis in male mice, as evidenced by increased expression of ferroptosis-related genes, lipid peroxidation, and labile iron accumulation in the liver. Furthermore, we identified dysregulation of the methionine cycle and transsulfuration pathway, leading to severe glutathione (GSH) exhaustion and indirect deactivation of glutathione peroxidase 4 (GPx4), a critical enzyme in preventing ferroptosis. Additionally, we identified miR-214 as a ferroptosis regulator in ALD, enhancing hepatocyte ferroptosis by transcriptionally activating the expression of ferroptosis-driver genes. Our study provides novel insights into the involvement and regulatory mechanisms of ferroptosis in ALD, highlighting the potential therapeutic implications of targeting ferroptosis and miRNAs in ALD management.

Early growth response-1 transcription factor promotes hepatic fibrosis and steatosis in long-term ethanol-fed Long-Evans rats.

Previous studies demonstrated that the Long-Evans (LE) rats exhibited liver injury and lipid metabolic abnormalities after 8weeks of ethanol feeding. The goal of this study was to investigate if the LE rats develop more advanced hepatic abnormalities (e.g., fibrosis) after long-term feeding with an ethanol-containing Lieber-DeCarli diet. In addition, the contribution of early growth response-1 (EGR1) transcription factor to these pathological changes was assessed. Long-Evans rats were fed an ethanol-containing or isocaloric control liquid diet for 18months. Livers were processed for histological analyses, studies of fibrosis-related gene expression, cell fractionation and triglyceride measurement. Serum alanine aminotransferase (ALT) levels were assessed. DNA binding activities of p53 and the sterol regulatory element-binding protein-1c (SREBP1c) were analysed. The abundance of EGR1 and enzymes involved in fatty acid synthesis were determined. Chromatin immunoprecipitation was employed to study EGR1 binding to the SREBP1c promoter region. Ethanol feeding generated steatosis, chicken wire fibrosis and ALT elevations in the LE rats. Fibrosis was associated with the upregulation of EGR1 and its downstream target genes. EGR1 upregulation was associated with enhanced p53 activity and an increase in the cellular p66(shc) abundance. Steatosis was linked to the activation of SREBP1c. Importantly, EGR1 upregulation paralleled the expression and transcriptional activity of SREBP1c. Finally, EGR1 was shown to bind to the SREBP1c promoter region. Long-term ethanol feeding promoted steatosis and fibrosis in LE rats via EGR1 activation. The highly abundant EGR1 bound to the SREBP1c promoter and contributed to the steatosis observed in the LE rat model.

A shift in microvillus membrane fucosylation to sialylation by ethanol ingestion in rat intestine

The luminal surface of enterocytes is covered with glycocalyx which is rich in glycoproteins. Ethanol ingestion is shown to induce morphological and biochemical changes in the intestine. In this study, the effect of ethanol ingestion on membrane glycoproteins has been investigated. Chemical analysis of microvillus membranes revealed an increase in hexose and sialic acid contents, but a reduction in fucose levels in ethanol-fed rats compared with controls. The observed changes were apparent in animals fed with ethanol for 35-56 days compared with controls. Lectin-binding assay indicated an increase in Wheat germ agglutinin (affinity for GlcNAc/sialic acid) and a decrease in Aleuria aurantia (affinity for alpha-L: -fucose) reactivity of brush borders in ethanol-fed animals for 4-8 weeks. Western blot analysis using biotin-labeled Wheat germ agglutinin revealed increased binding to proteins of M(r) 66-205 kDa in ethanol-fed rats compared with controls. The binding of Aleuria aurantia to membrane proteins of M(r) 97-185 kDa was reduced in ethanol-fed animals. These findings suggest that long-term ethanol feeding modulates the sialylation and fucosylation processes of microvillus membrane proteins in rat intestine. This could affect the intestinal digestive and absorptive functions in chronic alcoholism.

Liver Gal β1,4GlcNAc α2,6-sialyltransferase is down-regulated in human alcoholics: possible cause for the appearance of asialoconjugates

Gal βl,4GlcNAc α2,6-sialyltransferase (ST6GalI) mediates the glycosylation of proteins and lipids to form functionally important glycoproteins and glycolipids in the Golgi compartment. Our previous work demonstrated that long-term ethanol feeding in rats caused a marked 59% decrease in ST6GalI activity as well as ST6GalI messenger RNA (mRNA) level in the liver that was due to decreased stability of the mRNA. Clinical observations show that down-regulation of ST6GalI gene and consequent impaired activity of ST6GalI seems to be the major cause for the appearance of asialoconjugates in the blood of long-term alcoholics. The plasma carbohydrate-deficient transferrin (CDT) and sialic acid index of plasma apolipoprotein J were also altered in the alcoholic group compared with the nondrinkers. We have now investigated how alcohol affects the gene regulation of ST6GalI and the possible mechanism in postmortem human liver specimens taken from nondrinkers, moderate alcohol drinkers, and heavy alcohol drinkers. Real-time polymerase chain reaction analyses of the liver RNA extract showed that ST6GalI mRNA level was progressively decreased by 49% in moderate drinkers ( P < .01) and by 69% in heavy drinkers ( P < .01) compared with nondrinkers. Western blot analysis showed that liver ST6GalI protein level was negligibly decreased in moderate drinkers but decreased by 30% ( P < .05) in heavy drinkers compared with nondrinkers. We further demonstrated a single ST6GalI mRNA-binding protein complex in the normal human liver extract, which progressively decreased in the liver extracts of moderate and heavy alcohol drinkers. Thus, it is concluded that the appearance of asialoconjugates in alcoholics is possibly due to the down-regulation of ST6GalI gene expression.

Chronic Ethanol Consumption Impairs Cellular Immune Responses Against HCV NS5 Protein Due to Dendritic Cell Dysfunction

Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.

Long-term Ethanol Consumption Alters Pancreatic Gene Expression in Rats

Long-term ethanol consumption does not cause acute pancreatitis but rather sensitizes the pancreas to subsequent insults. The mechanisms responsible for this sensitization are unknown. To determine whether alterations in pancreatic gene expression might participate in ethanol-mediated sensitization, we performed gene-profiling analysis. Animals were fed ethanol-containing Lieber-DeCarli or control diet (pair-fed). After 8 weeks, pancreatic RNA expression was analyzed using Affimetrix GeneChips. Changes in specific genes were verified using quantitative reverse transcriptase-polymerase chain reaction. Long-term ethanol feeding caused a significant alteration of pancreatic gene expression. Selection criteria of changes more than 3-fold and P < 0.05 yielded 114 probe sets. Activating transcription factor 3, heat shock protein 70, heat shock protein 27, and mesotrypsinogen were increased, whereas pancreatitis associate protein, folate carrier, and metallothionein were decreased. Ethanol had a profound effect on pancreatic gene expression. The genes identified as elevated and reduced in this study may contribute to pancreatic sensitivity to stress. This study indicates for the first time the identities of multiple genes whose expression levels are dramatically influenced by long-term ethanol feeding. The identified genes may help explain the relationship between long-term ethanol abuse and pancreatic disease and lead to possible preventative or therapeutic approaches to ethanol-induced pancreatic disease.

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.

Glycine as a Therapeutic Immuno‐Nutrient for Alcoholic Liver Disease

Activation of Kupffer cells by gut-derived endotoxin is an important factor in ethanol hepatotoxicity. Further, it was shown that ethanol modulates both the expression and activity of several intracellular signaling molecules and transcription factors in Kupffer cells and chronic ethanol treatment enhances Kupffer cell sensitivity to endotoxin. These findings suggest that inhibition of Kupffer cell activation is effective for clinical application in alcoholic hepatitis. Recently, accumulating lines of evidence suggest a possibility that glycine is useful as an immuno-modulating amino acid. It has been shown that a diet containing glycine improved survival in endotoxin shock by preventing Kupffer cell activation. Glycine most likely prevents the LPS-induced elevation of intracellular Ca concentration in Kupffer cells, thereby minimizing LPS receptor signaling and cytokine production. Indeed, glycine prevents alcohol-induced liver injury in a long-term enteral ethanol feeding rats (Tsukamoto-French) by decreasing production of TNF-alpha in the liver. Moreover, glycine is protective against apoptosis of sinusoidal endothelial cells (SECs) that is one of the initial events in the development of liver injury. On the other hand, epidemiologic data have identified chronic alcohol consumption as a significant risk factor for carcinogenesis. Interestingly, glycine inhibits growth of tumor in vivo most likely because of the inhibition of angiogenesis. It was shown that the inhibitory effect of glycine on growth and migration of endothelial cells is due to activation of a glycine-gated Cl channel. It is hypothesized that the opening of this anion channel hyperpolarizes the cell membrane, blocks influx of Ca through voltage-dependent Ca channel, thereby blunting growth factor-mediated signaling. Therefore, glycine can be used not only for treatment of alcoholic hepatitis, but also for chemoprevention and treatment of hepatocellular carcinoma in alcoholic cirrhosis. Taken together, it is concluded that glycine is a potent therapeutic immuno-nutrient for various kinds of chronic liver diseases including alcoholic liver disease (ALD).

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.

Mechanism of action of ethanol in the down-regulation of Gal β1, 4GlcNAc α2,6-sialyltransferase messenger RNA in human liver cell lines

Gal βl, 4GlcNAc α2,6-sialyltransferase (2,6-ST) mediates the addition of α2,6-linked sialic acid to glycoproteins in the Golgi compartment. Down-regulation of its gene and consequent impaired activity of 2,6-ST seems to be the major cause for the appearance of asialoconjugates in the blood of long-term alcoholics. Therefore, mechanism(s) involved in the regulation of 2,6-ST gene is important and clinically relevant. Our previous work showed that long-term ethanol feeding in rats caused a marked 59% decrease of 2,6-ST activity as well as 2,6-ST messenger RNA (mRNA) level in liver that were due to the decreased stability of its mRNA. We now mimic these actions of ethanol using ( a) human liver HepG2 cells stably transfected with ethanol-inducible human cytochrome P4502E1 (CYP2E1 cells), or ( b) with high alcohol dehydrogenase (HAD cells) but not in wild-type HepG2 cells lacking either of the above 2 enzymes as models. Incubation of these cells for 72 hours with 100 mmol/L ethanol caused decreases (up to 76%, P < .05) of 2,6-ST mRNA levels in CYP2E1 and HAD cells but not in the wild type. However, incubation of wild-type cells with acetaldehyde at concentrations of 50 and 100 μmol/L showed a dramatic decrease (up to 69%, P < .02) in the 2,6-ST mRNA levels. Furthermore, exposure of CYP2E1 cells to 4-hydroxy-2-nonenal, an endogenous lipid peroxidation product of reactive oxygen species, strongly decreased 2,6-ST mRNA level by 61% ( P < .02). These results demonstrate that 2,6-ST gene is highly sensitive to ethanol action in human liver cells either via its oxidation product, acetaldehyde, or via reactive oxygen species leading to the generation of a more reactive aldehyde such as 4-hydroxy-2-nonenal. Thus, this study assumes major importance and clinical relevance because 2,6-ST gene regulation in a human liver cell model is demonstrated within a few days of ethanol exposure, whereas its in vivo regulation in liver generally takes prolonged period of ethanol exposure.

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.

Pancreatic cholesterol esterase, ES-10, and fatty acid ethyl ester synthase III gene expression are increased in the pancreas and liver but not in the brain or heart with long-term ethanol feeding in rats.

Chronic alcohol consumption predisposes susceptible individuals to both acute and chronic pancreatitis. Our hypothesis was that alcohol increases the risk of pancreatitis by disrupting defense mechanisms and/or enhancing injury-associated pathways through altered gene expression. Hence, we studied the expression of pancreatic genes in rats chronically exposed to ethanol. Male Wistar rats were pair-fed liquid diets without and with ethanol for 4 weeks. Total RNA was extracted from rat pancreas and other organs. The mRNA expression patterns among pancreatic samples from ethanol-fed rats and controls were compared with use of mRNA differential display. The differentially expressed cDNA tags were isolated, cloned, and sequenced. One cDNA tag that was overexpressed in the pancreas showed 99% sequence homology to a rat pancreatic cholesterol esterase mRNA (CEL; Enzyme Commission number [EC] 3.1.1.13). The differential expression was confirmed by realtime PCR. Gene expression was also increased in the liver but not in the heart or brain of the alcohol-fed rats. Because CEL has fatty acid ethyl ester (FAEE)-generating activity and FAEEs play a major role in acute alcoholic pancreatitis, we determined the expression of other genes encoding for FAEE-generating enzymes and showed similar organ-specific expression patterns. Our results demonstrate that chronic ethanol consumption induced expression of FAEE-related genes in the pancreas and liver. This upregulation may be a central mechanism leading to acinar cell injury.

Role of lipopolysaccharide-binding protein in early alcohol-induced liver injury in mice.

Cellular responses to endotoxins are enhanced markedly by LPS-binding protein (LBP). Furthermore, it has been demonstrated that endotoxins and proinflammatory cytokines such as TNF-alpha participate in early alcohol-induced liver injury. Therefore, in this study, a long-term intragastric ethanol feeding model was used to test the hypothesis that LBP is involved in alcoholic hepatitis by comparing LBP knockout and wild-type mice. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. There was no difference in mean urine alcohol concentrations between the groups fed ethanol. Dietary alcohol significantly increased liver to body weight ratios and serum alanine aminotransferase levels in wild-type mice (189 +/- 31 U/L) over high-fat controls (24 +/- 7 U/L), effects which were blunted significantly in LBP knockout mice (60 +/- 17 U/L). Although no significant pathological changes were observed in high-fat controls, 4 wk of dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals as expected (pathology score, 5.9 +/- 0.5). These pathological changes were reduced significantly in LBP knockout mice fed ethanol (score, 2.6 +/- 0.5). Endotoxin levels in the portal vein were increased significantly after 4 wk in both groups fed ethanol. Moreover, ethanol increased TNF-alpha mRNA expression in wild-type, but not in LBP knockout mice. These data are consistent with the hypothesis that LBP plays an important role in early alcohol-induced liver injury by enhancing LPS-induced signal transduction, most likely in Kupffer cells.

Delivery of I[kappa ]B superrepressor gene with adenovirus reduces early alcohol-induced liver injury in rats

Chronic alcohol administration increases gut-derived endotoxin in the portal blood, which activates Kupffer cells through nuclear factor [kappa ]B (NF-[kappa ]B) to produce toxic mediators such as proinflammatory cytokines, leading to liver injury. Therefore, a long-term intragastric ethanol feeding protocol was used here to test the hypothesis that NF-[kappa ]B inhibition would prevent early alcohol-induced liver injury. Adenoviral vectors encoding either the transgene for I[kappa ]B superrepressor (AdI[kappa ]B-SR) or the bacterial [beta ]-galactosidase reporter gene (AdlacZ) were administered intravenously to Wistar rats. Animals were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin (control) for 3 weeks. There was no significant difference in mean urine alcohol concentrations between the groups fed ethanol. I[kappa ]B-SR expression was increased for up to 2 weeks after injection, but was undetectable at 3 weeks. NF-[kappa ]B activation was increased by ethanol and associated with up-regulation of tumor necrosis factor [alpha ] (TNF-[alpha ]). These increases were blunted significantly up to 2 weeks by AdI[kappa ]B-SR. Dietary alcohol significantly increased liver to body weight ratios and serum alanine transaminase (ALT) levels in AdlacZ-treated animals, effects that were blunted significantly in AdI[kappa ]B-SR[ndash ]treated rats. Ethanol caused severe steatosis, inflammation, and focal necrosis in AdlacZ-treated animals. These pathologic changes were significantly decreased by AdI[kappa ]B-SR. The protective effects of I[kappa ]B-SR were significant 2 weeks after injection, but were lost at 3 weeks when I[kappa ]B-SR was no longer expressed. Ethanol increased 4-hydroxynonenal as a maker of oxidative stress in both AdlacZ and AdI[kappa ]B groups. These data support the hypothesis that NF-[kappa ]B inhibition prevents early alcohol-induced liver injury even in the presence of oxidative stress. (HEPATOLOGY 2001;34:1149-1157.)

Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice

Chronic alcohol administration increases gut-derived endotoxin in the portal blood, which activates Kupffer cells and causes liver injury. Mice (C3H/HeJ) with mutations in toll-like receptor 4 (TLR4) are hyporesponsive to endotoxin. To test the hypothesis that TLR4 is involved in early alcohol-induced liver injury, the long-term intragastric ethanol feeding protocol developed by Tsukamoto and French for rats was adapted to mice. Animals with nonfunctional TLR4 and wild-type mice (C3H/HeOuJ) were compared. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 weeks. There was no difference in mean urine alcohol concentrations between the groups. Dietary alcohol significantly increased liver-to-body weight ratios and serum alanine transaminase (ALT) levels in wild-type mice (109 ± 18 U/L) over high-fat controls (40 ± 3 U/L), effects that were blunted significantly in mice with a mutation of TLR4 (55 ± 9 U/L). While no significant pathologic changes were observed in high-fat controls, dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals (pathology score = 5.2 ± 1.2). These pathologic changes were significantly lower in TLR4-deficient mice fed ethanol (score = 2.0 ± 1.3). Endotoxin levels in the portal vein were increased significantly after 4 weeks in both groups fed ethanol. Moreover, ethanol increased tumor necrosis factor α (TNF-α) mRNA expression in wild-type, but not in TLR4-deficient, mice. These data are consistent with the hypothesis that Kupffer cell activation by endotoxin via TLR4 is involved in early alcohol-induced liver injury. (HEPATOLOGY 2001;34:101-108.)

The effect of long-term ethanol feeding on Brucella melitensis infection of rats.

The adverse effects of ethanol on Brucella melitensis have not been studied previously. In this study, a new model of B. melitensis infection was used in the setting of chronic ethanol administration in rats. It was found that the chronically ethanol-receiving rats exposed to B. melitensis infection had significantly greater numbers of B. melitensis in their spleen and liver than the rats in the control group.

Effect of long-term ethanol feeding on brainstem α 2-receptor binding in Wistar–Kyoto and spontaneously hypertensive rats

Our previous studies have shown that ethanol attenuates baroreflex function in Wistar–Kyoto (WKY) but not in spontaneously hypertensive rats (SHRs). The present study determined the effects of chronic ethanol administration on α 2-binding sites in brainstem areas that modulate baroreflexes. In vitro autoradiography was utilized to evaluate the effect of a 3-month ethanol feeding on the density ( B max) and affinity ( K D) of α 2-adrenoceptors in the middle (mNTS) and rostral (rNTS) portions of the nucleus tractus solitarius of SHRs and WKY rats. Autoradiographic examination of brainstem sections preincubated with [ 125I] p-iodoclonidine revealed no inter-strain differences in α 2-binding in control rats. Ethanol feeding caused strain-dependent changes in α 2-binding activity, which comprised significant ( P<0.05) decreases in the density of α 2-binding sites in both areas of the NTS in SHRs versus no effect in WKY rats. These findings do not favor a role for brainstem α 2-adrenoceptors in ethanol-induced attenuation of baroreflexes. Interestingly, the ethanol-evoked reduction in the NTS α 2-receptor density in SHRs may explain reported findings that ethanol abolishes the hypotensive effect of the α 2-adrenoceptor agonist clonidine in this rat model.

Essential role of tumor necrosis factor α in alcohol-induced liver injury in mice

Background & Aims: Tumor necrosis factor (TNF)-α is associated with increased mortality in alcoholics, but its role in early alcohol-induced liver injury is not fully understood. Recently, it was shown that injury induced by the enteral alcohol delivery model of Tsukamoto and French was reduced by antibodies to TNF-α. To obtain clear evidence for or against the hypothesis that TNF-α is involved, we studied TNF receptor 1 (TNF-R1, p55) or 2 (TNF-R2, p75) knockout mice. Methods: Long-term enteral alcohol delivery was modified for male gene–targeted mice lacking TNF-R1 and TNF-R2. Animals were given a high-fat liquid diet continuously with either ethanol or isocaloric maltose-dextrin as a control for 4 weeks. Results: Ethanol elevated serum levels of alanine aminotransferase nearly 3-fold in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice. Likewise, ethanol caused severe liver injury in wild-type mice (pathology score, 5.5 ± 0.6) and TNF-R2 knockout mice (pathology score, 5.0 ± 0.4), but not in TNF-R1 knockout mice (pathology score, 0.8 ± 0.4; P < 0.001). Conclusions: Long-term ethanol feeding caused liver injury in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice, providing solid evidence in support of the hypothesis that TNF-α plays an important role in the development of early alcohol-induced liver injury via the TNF-R1 pathway. Moreover, the long-term enteral ethanol feeding technique we described for the first time for knockout mice provides a useful new tool for alcohol research. GASTROENTEROLOGY 1999;117:942-952

Chronic ethanol consumption causes alterations in the structural integrity of mitochondrial DNA in aged rats.

Chronic ethanol consumption adversely affects the respiratory activity of rat liver mitochondria. It causes increased cellular production of oxygen radical species and selectively decreases mitochondrial glutathione (GSH) levels. Here we show, using Southern hybridization techniques on total rat genomic DNA, that long-term (11-13 months) ethanol feeding, using the Lieber-DeCarli diet, results in a 36% (P <.05; n = 4) decrease in hepatic mitochondrial DNA (mtDNA) levels when compared with paired controls. UV quantitation of mtDNA isolated from hepatic mitochondria showed that chronic ethanol intake (11-13 months) causes a 44% (P <.01; n = 6) decrease in the amount of mtDNA per milligram of mitochondrial protein. No significant decline in mtDNA levels was seen in ethanol-fed animals maintained on the diet for 1 to 5 months. Ethanol feeding caused a 42% (P <.01; n = 4) and a 132% (P <.05; n = 3) increase in 8-hydroxydeoxyguanosine (8-OHdG) formation in mtDNA in animals maintained on the diet for 3 to 6 months and 10 to 11 months, respectively. In addition, agarose gel electrophoresis revealed a 49% increase (P <.05; n = 3) in mtDNA single-strand breaks (SSB) in animals fed ethanol for more than 1 year. These findings suggest that chronic ethanol consumption causes enhanced oxidative damage to mtDNA in older animals along with increased strand breakage, and that this results in its selective removal/degradation by mtDNA repair enzymes.

Detection of circulating antibodies to malondialdehyde-acetaldehyde adducts in ethanol-fed rats

Background & Aims: Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed rats. The aim of this study was to examine the immune response to MAA adducts and other aldehyde adducts during long-term ethanol exposure. Methods: Rats were pair-fed for 7 months with a liquid diet containing either ethanol or isocaloric carbohydrate. Circulating antibody titers against MAA adducts and acetaldehyde adducts were measured and characterized in these animals. Results: A significant increase in antibody titers against MAA-adducted proteins was observed in the ethanol-fed animals. Competitive inhibitions of antibody binding indicated that the circulating antibodies against MAA-modified proteins in the ethanol-fed rats recognized mainly a specific, chemically defined MAA epitope. Antibody titers to reduced and nonreduced acetaldehyde adducts were very low, and no significant differences were observed between ethanol-fed and control animals. Significant plasma immunoreactivity to not only MAA-adducted but also unmodified rat liver proteins (cytosol, microsomes, and especially plasma membrane) were also observed in the ethanol-fed rats. Conclusions: Long-term ethanol feeding generates circulating antibodies not only against MAA epitopes but possibly also against unmodified, native (self) protein epitopes, suggesting that MAA adducts could trigger harmful autoimmune responses. GASTROENTEROLOGY 1998;115:686-692