Daidzin, an Antioxidant Isoflavonoid, Decreases Blood Alcohol Levels and Shortens Sleep Time Induced by Ethanol Intoxication

The extract from an edible vine, Pueraha lebata has been reported to be efficacious in lessening alcohol intoxication. In this study, we have tested the efficacy of one of the major components, daidzin, from this plant extract. When ethanol (40% solution, 3 g/kg body weight) was given to fasted rats intragastrically, blood alcohol concentration (BAC) peaked at 30 min after alcohol ingestion and reached 1.77 ± 0.14 mg/ml (mean values ±sd , n = 6). If daidzin (30 mg/kg) was mixed with the ethanol solution and given to animals intragastrically, BAC was found to peak at 90 min after alcohol ingestion and reached only 1.20 ± 0.30 mg/ml (n = 6) (p < 0.05 vs. controls). The ability of daidzin to delay and decrease peak BAC level after ethanol ingestion was also observed in fed animals, in both fasted and fed rats given alcohol without daidzin, BAC quickly declined after reaching its peak at 30 min. By contrast, BAC levels receded more slowly if daidzin was also fed to the animals. Daidzin showed a chronic effect. Rats fed daidzin for 7 days before ethanol challenge, but not on the day of challenge, also produced lower and later peak BAC levels. Interestingly, daidzin, whether fed to rats only once or chronically for 7 days, did not significantly alter activities of either alcohol dehydrogenase or mitochondrial aldehyde dehydrogenase in the liver. Further experiments demonstrated that daidzin shortened sleep time for rats receiving ethanol intragastrically (7 g/ kg) but not intraperitoneally (2 g/kg). To test whether daidzin delayed stomach-emptying, [Clpolyethylene glycol was mixed with ethanol and fed to rats. It was found that, 30 min after intragastric feeding, more ethanol and [¹⁴C]polyethylene glycol remained in the stomach if rats were also given daidzin. Because daidzin is an isoflavonoid glucoside that possesses strong antioxidant activity, two other antioxidants (i.e., vitamin E and thioctic acid) were tested. Similar to daidzin, these two antioxidants also delayed and suppressed peak BAC, as well as shortened sleep time induced by alcohol ingestion. We conclude that: (1) daidzin is effective in countering alcohol intoxication; (2) suppression of BAC by daidzin is due mainly to delay of stomach-emptying, but not to accelerated clearance of ethanol in circulation by liver enzymes; and (3) the effect of daidzin may in part be due to its antioxidant activity.     

Chronic Alcohol Intoxication Enhances the Expression of CD18 Adhesion Molecules on Rat Neutrophils and Release of a Chemotactic Factor by Kupffer Cells

Chronic alcohol intoxication has been associated with increased migration of inflammatory leukocytes to the liver that may contribute to the development of alcoholic hepatitis in susceptible individuals. Thus, this work was performed to examine the mechanism by which neutrophils [polymorphonuclear neutrophils (PMNs)] are sequestered in the liver during prolonged consumption of alcohol. Male Sprague-Dawley rats were fed with Sustacal supplemented by 36% alcohol, or isocaloric diet for 16 weeks. Circulating blood PMNs were collected and examined for CD18 ( β ₂-integrin) adhesion molecule expression. Monoclonal antibody 1F12, an anti-CD18 antibody and potent neutropenic agent, was used to detect CD18 on PMNs. More than 97% of neutrophils obtained from pair and ethanol-fed rats were positive for the antibody. Fluorescence intensity of fluorescein iso-thiocyanate-1F12 binding to PMNs from ethanol-fed rat was significantly enhanced 2-fold compared with the pair-fed controls. The release of chemoattractant and free radical-generating activity in culture supernatants of Kupffer cells was also examined. Twenty-four hr culture supernatants of Kupffer cells from chronic alcoholic rats enhanced the migration and superoxide anion generation by normal PMNs, compared with those of the pair-fed rats. Antirat interleukin-8 antiserum inhibited chemotactic activity and superoxide generating capacity of culture supernatants. These results suggest that upregulation of adhesion molecules on PMNs and chemotactic factor release from Kupffer cells may contribute, at least in part, to enhanced migration of inflammatory leukocytes to the liver during chronic alcohol intoxication.     

Chronic Ethanol Consumption Results in Atypical Liver Injury in Copper/Zinc Superoxide Dismutase Deficient Mice

Background: Ethanol metabolism increases production of reactive oxygen species, including superoxide () in the liver, resulting in significant oxidative stress, which causes cellular damage. Superoxide dismutase (SOD) is an antioxidant enzyme that converts superoxide to less toxic intermediates, preventing accumulation. Because the absence of SOD would confer less resistance to oxidative stress, we determined whether damage to hepatic proteolytic systems was greater in SOD^?/? than in SOD^+/+ mice after chronic ethanol feeding. Methods: Female wild‐type (SOD^+/+) and Cu/Zn‐SOD knockout (SOD^?/?) mice were pair‐fed ethanol and control liquid diets for 24 days, after which liver injury was assessed. Results: Ethanol‐fed SOD^?/? mice had 4‐fold higher blood ethanol, 2.8‐fold higher alanine aminotransferase levels, 20% higher liver weight, a 1.4‐fold rise in hepatic protein levels, and 35 to 70% higher levels of lipid peroxides than corresponding wild‐type mice. While wild‐type mice exhibited fatty liver after ethanol administration, SOD^?/? mice showed no evidence of ethanol‐induced steatosis, although triglyceride levels were elevated in both groups of knockout mice. Ethanol administration caused no significant change in proteasome activity, but caused lysosomal leakage in livers of SOD^?/? mice but not in wild‐type mice. Alcohol dehydrogenase activity was reduced by 50 to 60% in ethanol‐fed SOD^?/? mice compared with all other groups. Additionally, while ethanol administration induced cytochrome P450 2E1 (CYP2E1) activity in wild‐type mice, it caused no such induction in SOD^?/? mice. Unexpectedly, ethanol feeding significantly elevated total and mitochondrial levels of glutathione in SOD knockout mice compared with wild‐type mice. Conclusion: Ethanol‐fed SOD^?/? mice exhibited lower alcohol dehydrogenase activity and lack of CYP2E1 inducibility, thereby causing decreased ethanol metabolism compared with wild‐type mice. These and other atypical responses to ethanol, including the absence of ethanol‐induced steatosis and enhanced glutathione levels, appear to be linked to enhanced oxidative stress due to lack of antioxidant enzyme capacity.     

Chronic Ethanol Induced Impairment of Hepatic Glycosylation Machinery in Rat Is Independent of Dietary Carbohydrate

In recent years, a number of studies have been reported that have clearly established that hepatic glycosylation machinery is affected by chronic ethanol treatment in rats. We have previously reported that chronic ethanol treatment in rats resulted in decreased glycosylation of transfenin and apolipoprotein E with concomitant decreases in enzymatic activities of Golgi galactosyltransferases and sialyltransferases. In all these studies investigators have invariably used the well-accepted dietary formulation of alcohol diet as proposed by Lieber and DeCarli. However, questions were raised whether the lower carbohydrate content in Lieber's alcohol diet may be responsible for observed effects of ethanol on hepatic glycosylation machinery. Therefore, to verify whether or not the crucial effects of chronic ethanol treatment on hepatic glycosylation machinery as observed in our studies, were truly caused by ethanol, we have extended our studies on protein glycosylation with the inclusion of a third dietary group that was compensated for carbohydrate content. In this investigation, rats were fed with three dietary regimen corresponding to control, ethanol, and carbohydrate compensated ethanol group and studies were done on (i) labeled leucine, galactose and N-acetylmannosamine incorporation into transferrin and apolipoprotein E, and (ii) hepatic galactosyltransferase and sialyttrans-ferase activities in Golgi rich fraction in rat. Our results clearly showed that regardless of the carbohydrate content, marked decreases in the incorporation of labeled sugars into transferrin and the enzymatic activities of galactosyltransferase and sialyltrans-ferase occurred in rats administered chronic ethanol. Thus, it is reasonable to conclude that it is not the carbohydrate content of the diet but ethanol per se, when administered chronically, greatly impairs the glycosylation machinery of rat liver and that the magnitudes of these effects are selectively specific with regard to the type of sugar or the glycosylation enzyme.     

Anti-inflammatory pathways and alcoholic liver disease: Role of an adiponectin/interleukin-10/heme oxygenase-1 pathway

The development of alcoholic liver disease (ALD) is a complex process involving both the parenchymal and non-parenchymal cells in the liver. Enhanced inflammation in the liver during ethanol exposure is an important contributor to injury. Kupffer cells, the resident macrophages in liver, are particularly critical to the onset of ethanol-induced liver injury. Chronic ethanol exposure sensitizes Kupffer cells to activation by lipopolysaccharide via Toll-like receptor 4. This sensitization enhances production ...     

Chronic Alcohol Intoxication Attenuates Human Immunodeficiency Virus-1 Glycoprotein 120-Induced Superoxide Anion Release by Isolated Kupffer Cells

This work tests the hypothesis that chronic alcohol intoxication suppresses the microbicidal activity of Kupffer cells by modulating the expression of cell surface receptors associated with respiratory burst and the release of potent microbicidal agents [i.e., reactive oxygen species (ROS)]. Because alcohol is also a potential risk factor in human immunodeficiency virus-1 (HIV-1) infection, this study examines the effect of HIV-1 glycoprotein 120 (gp120)-induced ROS release by isolated Kupffer cells. After 16 weeks of ethanol feeding, Kupffer cells from male Sprague-Dawley rats were isolated and assayed for HIV-1 gp120-induced superoxide release. Fluorescein iso-thiocyanate (FITC)-HIV-I gp120 binding, NADPH oxidase, and protein kinase C activity in Kupffer cells were measured. Results show that HIV-1 gp120 induced the release of superoxide anion in a dose-dependent manner in normal rats. Mannosylated-bovine serum albumin inhibited FITC-HIV-1 gp120-mediated superoxide release in normal Kupffer cells by 85%. Moreover, 83 ± 6% of Kupffer cells were FITC-HIV 1 gp120-positive, whereas <30% were CD4-positive. In alcohol-fed rats, HIV-1 gp120-induced ROS release was reduced by 70% and FITC-HIV-1 gp120 binding (in terms of fluorescence intensity per 10⁶ Kupffer cells) by 44% in Kupffer cells, without any change in percent positive cells for this ligand. Concomitantly, HIV-1 gp120-induced translocation of NADPH oxidase to the plasma membranes of Kupffer cells in alcohol-fed rats was suppressed by 60%. In contrast, alcohol consumption significantly increased total protein kinase C activity and phorbol ester-induced superoxide release by Kupffer cells. These studies demonstrate that Kupffer cells are likely targets of HIV-1 whose binding sites on macrophages could also include mannose-specific receptors. These observations further suggest that suppression of HIV-1 gp120-mediated ROS production in chronic alcoholics is due to altered cell surface receptor expression for gp120, and defective postreceptor signaling mechanisms, which in turn could lead to attenuated microbicidal activity of hepatic macrophages.     

Chronic Ethanol Consumption Induces the Production of Tumor Necrosis Factor-α and Related Cytokines in Liver and Adipose Tissue

Increases in monocyte/macrophage production of the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), parallel the evolution of liver injury in rats and humans with alcoholic liver disease. However, the possibility that TNF-α expression may be induced in other cell populations before serious liver disease develops has not been evaluated. To clarify this issue, mRNAs and/or protein levels of TNF-α and cytokines [interleukin (IL)-6, IL-10, transforming growth factor-β(TGF)-β, IL-12, and interferon-γ] that regulate its biological activity were measured in sera, liver, and adipose tissues of rats that had developed hepatic steatosis after consuming ethanol-containing diets for 6 weeks. Cytokine expression in the ethanolfed groups was compared with that of pair-fed controls rats that had received isocaloric amounts of a similar, ethanol-free diet for the same time period. Animals were studied both before and after a surgical stress (partial hepatectomy) that is known to provoke cytokine production. Chronic ethanol consumption led to increased serum concentrations of TNF and related cytokines, at least in part, by inducing the overproduction of these factors in the liver and peripheral adipose tissues. Despite the pair-feeding protocol that ensured similar calorie consumption in both groups, adipose tissues in ethanol-fed rats also expressed more leptin, a TNF-α-inducible mRNA that encodes an appetite-suppressing hormone. Thus, white adipose tissue can be an important source of cytokines in nonobese animals and may be a target for ethanol's actions. These data implicate TNF-α as a potential mediator of the nutritional-metabolic ab-berratJons that often accompany chronic alcohol intake, even in the absence of advanced liver disease.     

Acute Ethanol Intoxication Prevents Lipopolysaccharide-Induced Down Regulation of Protein Kinase C in Rat Kupffer Cells

Protein kinase (PK) C has been implicated in a number of cellular events, many of which are also known to be affected by ethanol (ETOH). ETOH intoxication is also known to impair immune function, thereby increasing the host's susceptibility to infection. The purpose of this study was to assess the effect of acute ETOH intoxication on PKC activity and its intracellular distribution in nonparenchymal liver cells following an E. coli lipopolysaccharide (LPS) challenge. The liver was chosen for the study because it is the primary site both for metabolism of ETOH and detoxification of gut derived bacterial products. Catheterized conscious rats were administered saline or ETOH (175 mg/100 g body weight as a bolus followed by a continuous, 7 hr infusion of 28 mg/100 body weight/hr). LPS was injected intravenously (100 micrograms/100 g body weight) 3 hr before the end of the saline or ETOH infusion. Kupffer and endothelial cells were isolated by collagenase-pronase digestion followed by centrifugal elutriation. PKC was assayed after extraction with digitonin containing buffer and partial purification on DE-52 cellulose minicolumns. LPS decreased PKC activity by 69% from control values. Although ETOH infusion alone did not affect PKC activity in Kupffer cells, it completely abrogated the LPS effect. A similar trend was observed for the endothelial cells. No significant differences were observed between groups with respect to the intracellular distribution of PKC. The down-regulation of PKC by LPS may represent a mechanism of functional adaptation of the immunocompetent cells to one of the cytokines, i.e., TNF, whose receptors are down regulated by activation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Ethanol Consumption on Adult Rat Liver Mitochondrial Populations Analyzed by Flow Cytometry

In the present study, the effects of administering ethanol to adult male rats on the distribution of the low fluorescence population (LFP) and high fluorescence population (HFP), and the rhodamine-123 fluorescence intensity of these groups of mitochondria are analyzed by flow cytometry. Our results show that ethanol administration to adult male rats induces a redistribution of the HFP and LFP mitochondrial populations leading to an increase of the less functional HFP mitochondria. In addition, ethanol induced an increase in the mean intensity of green fluorescence of the HFP that is probably related to an increased number of rhodamine-123 binding sites per mitochondria resulting from mitochondria enlargement.     

Free Radical Production during Ethanol Intoxication, Dependence, and Withdrawal

Indices of free radical production and cell damage were examined in male Sprague-Dawley rats chronically exposed to either ethanol (ETOH) or water vapor. In experiment 1, rats experienced either 1 or 11 cycles of ETOH exposure and withdrawal. Brain tissue was harvested 12 hr after ETOH exposure, and 1 hr after being injected with sodium salicylate as a scavenger. Brain tissue was analyzed for the formation of salicylate hydroxylation products as a measure of OH production during withdrawal. Significant group differences for OH production were demonstrated for 2,3- and 2,5-dihydroxybenzoic acid in the single cycle ETOH exposed rats compared with their water cohorts. A significant between group difference for 2,5-dihydroxybenzoic acid, only, was demonstrated for the multiple cycles of ETOH exposure. Spontaneous seizures were shown to correlate with increased production of OH in ETOH exposed rats. In experiment 2, brain tissue was harvested from different groups of rats after removal from the chambers, at 0, 2, 12, 24, 36, and 48 hr after a single exposure cycle. Tissue was analyzed for (1) salicylate hydroxylation (as above), (2) glutamine synthetase activity, (3) whole brain glutamate concentration, and (4) oxidized protein. A multiple regression analysis was conducted on the five dependent variables and found they could be predicted by specific behavioral and neurological ratings. These data suggest that cell damage during withdrawal may have multiple time-dependent components.     

Chronic Ethanol Treatment Reduces the Responsiveness of the Hypothalamic-Pituitary-Thyroid Axis to Central Stimulation

The hypothalamic-pituitary-thyroid (HPT) axis functions abnormally in man and animals chronically exposed to ethanol. The most consistent observation in humans is that the thyrotropin response to thyrotropin-releasing hormone (TRH) is blunted. We have tested the hypothesis that chronic ethanol treatment in rats leads to a diminished responsiveness of the hypothalamus to central stimulation. Animals were maintained on 1 of 3 diets for 4 weeks: (1) laboratory chow and water provided ad libitum (chow-fed), (2) Sustacal chocolate liquid diet with vitamin mixture containing 5% (w/v) ethanol provided ad libitum (ethanol), or (3) Sustacal chocolate liquid diet with vitamin mixture containing sucrose substituted isocalorically (35%) for ethanol and provided in amounts matched to a weight-paired, ethanol-treated animal (pair-fed). At the end of 4 weeks, the animals were evaluated for their response to a single injection of ethanol (3 g/kg, ip) and/or exposure to 5°C. Chronic ethanol treatment produced an increase in TRH mRNA in neurons of the paraventricular nucleus and fully blocked the thyrotropic response to cold exposure. However, chronic ethanol-treated animals did not exhibit altered basal levels of triiodothyronine or thyrotropin, nor did they have an altered response to a single injection of ethanol. These data demonstrate that chronic alcohol exposure alters functioning of the hypo-thalamic-pituitary-thyroid axis at least in part by affecting TRH neurons of the paraventricular nucleus.     

Effect of Ethanol on Maternal and Offspring Characteristics: Comparison of Three Liquid Diet Formulations Fed during Gestation

Maternal blood alcohol levels, weight gain during pregnancy, parturition time, perinatal mortality, and postnatal growth of offspring were compared in groups of pregnant rats fed one of three ethanol-containing liquid diets (Kahn's formula = BSA diet, Revised Wiener's = RA6 diet, and Lieber-DeCarli's high protein 82C diet = LDA diet). The three ethanol diets all contained the same amount of ethanol-derived energy (36% of total energy), but differed in the amount of energy contributed by protein (17, 30, and 25%), fat (36, 24, and 13%), and carbohydrate (12, 10, and 27%), respectively. The experimental design also included dams that were pair-fed isocaloric ethanol-free versions of the three ethanol diets (designated BSP, RP6, and LDA, respectively) and a group of dams fed a pelleted casein-based solid diet (PC diet). All experimental diets were fed ad libitum from gestational day 7 to delivery. The effect of ethanol exposure in utero was most severe in mothers and offspring fed the BSA diet. The feed efficiency ratio (maternal weight gain/total dietary energy consumed) of this low-protein ethanol diet was less than that of RA6 or LDA diets. The feed efficiency ratio calculated for RA6 and LDA diets was not different from that of PC diet. Compared with rats fed RA6 and LDA diets, the rats that were fed BSA diet exhibited deficient maternal weight gain, greater parturition delay, impaired fetal growth, and increased perinatal mortality among the offspring. BSA dams had the highest blood ethanol levels of all groups fed ethanol diets, and exhibited the least difference in blood ethanol concentrations between the day (2 PM) and night (9 PM) periods of the diurnal cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conditioned Taste Aversions Induced by Alcohol and Lithium in Rats Selectively Bred for Ethanol Neurosensitivity

Rats that were selectively bred for differences in alcohol-induced sleep time (alcohol neurosensitivity) were tested for differences in formation and extinction of alcohol- and LiCl-induced conditioned taste aversions. Male rats bred for high, control, or low alcohol sensitivity (HAS, CAS, and LAS rats, respectively) were deprived of water and given daily 30 min access to water for a baseline period of 7 days. Rats were then given a novel 0.125% sodium saccharin solution, followed by an intraperitoneal injection of either saline, 2 g/kg of ethanol (at 10% w/v), or 50.9 mg/kg of LiCl(0.15 M) on 3 conditioning days. Each saccharin exposure was followed by a recovery day of BCCBSS to water. The ethanol-induced saccharin aversion extinguished more rapidly in LAS rats than in CAS or HAS rats ( p < 0.05), but LiCl conditioned equivalent aversions in each group. Also, ethanol injection results in large differences in observed resting behavior in these rats (HAS > CAS > LAS), but LiCl injection produced no reliable group differences in resting. The weaker alcohol-Induced M e aversion in LAS rats accords with their previously measured higher oral consumption of alcohol (Kulkosky et al., Alcoholism 17: M-651, 1993) and the idea that alcohol intake is limited by an expectancy of postingestive consequences. The weaker ethanol induced aversion in LAS rats reflects selective breeding of an alcohol-specific trait and not a general difference In aversive conditioning or chemical neurosensitivity.     

Regulation of Human Monocyte Functions by Acute Ethanol Treatment: Decreased Tumor Necrosis Factorα, Interleukin-lβ and Elevated Interleukin-10, and Transforming Growth Factor-β Production

We and others have previously shown that even acute ethanol exposure has the capacity to modulate immune functions, particularly monocyte functions. Herein, we tested the hypothesis that acute ethanol treatment inhibits inflammatory, while increasing inhibitory cytokine production in human blood monocytes that, in tum, could contribute to the overall immune abnormalities seen after alcohol use. Our data show that in vitro treatment of blood monocytes with a physiologically relevant dose of alcohol (W mM) results in significantly decreased induction of tumor necrosis factor-α (TNFα) and interleukin (IL)-lβ by bacterial stimulation of either Gram-positive [staphylococcal enterotoxin B (SEB), 1 μg/ml of SEB] or Gram-negative [lipopolysaccharide (LPS), 1 μg/ml of LPS] origin both at the protein and mRNA levels. In contrast, acute ethanol treatment induces monocyte production of mediators with immunoinhibitory potential, including transforming growth factor-β and IL-10. We further show that ethanol not only induces monocyte/macrophage (Mø) IL-10 and transforming growth factor-β, but even augments bacterial (both LPS and SEB) stimulation-induced production of both of these cytokines. IL-10 is a potent inhibitor of Mø TNFα production. We found that ethanol-induced elevation in Mø IL-10 levels contributes to the decreased Mø TNFα production to bacterial challenge in eth-anol-exposed Mø. However, mRNA levels for TNFα are downregu-lated as early as 1.5 hr after ethanol treatment, suggesting that ethanol likely has an IL-10 independent, direct effect on early signaling events of TNF α induction.     

Effect of Chronic Dietary Ethanol Consumption on Colonic Cancer in Rats Induced by 1,1-Dimethylhydrazine

Background: The pathogenetic correlation between chronic alcohol consumption and development of colon cancer is not clear. The role of alcohol abuse in the carcinogenic action of 1,1-dimethylhydrazine (DMH), which induces tumors in the colon, was evaluated.
Methods: Twenty male rats were fed liquid diets containing ethanol or carbohydrates for 39 weeks. DMH (20 mg/kg body weight, once a week) was injected subcutaneously from the 5th to the 20th week. Pair feeding was stopped at 10:00 am and DMH was administered at 02:00 pm . Ethanol was not detected in the blood at the time of injection. Liquid diets were provided again at 05:00 pm until 10:00 am next day. The animals were killed at the end of the 39th week, and the colons were removed for examination for the number of aberrant crypt foci (ACF) by methylene blue staining. Tissue sections were stained for histology and cytochrome P4502E1 (CYP2E1) expression.
Results: The number of ACF in colons obtained from ethanol-fed rats with DMH was 24 ( n =5, 4.4±2.5/rat), which was significantly ( p <0.001) more than that of the other treated rats: only 3 ( n =5, 0.6±0.5/rat) in the pair-fed control rats with DMH, and none in the ethanol-fed or control-fed rats without DMH. Cytochrome P4502E1 staining demonstrated marked expression in the colon mucosa from ethanol-fed rats, but not in the pair-fed control rats.
Conclusions: The increased expression of CYP2E1 induced by chronic ethanol consumption promotes the development of DMH-induced colon cancer.