Growth and liver morphology after long-term ethanol consumption of rats

Ethanol was administered to female and male Wistar rats by mixing it with their drinking water. Ethanol concentrations were gradually increased up to either 8% or 15%. Female rats receiving 8% ethanol in their drinking water consumed 5-13 g, males 4-10 g daily. The ethanol/total food caloric intake percentages were 13 to 20% and 9 to 15% for female and male rats, respectively. There was no difference in body weight and relative liver weight between treated rats and their controls. Female and male rats receiving 15% of ethanol in their drinking water consumed 8-14 g ethanol per kg body weight per day. The percentages of ethanol/total food caloric intake were stabilized at about 25% for both sexes. Growth of the rats differed only slightly from controls; a tendency for a higher increase of body weight of the control rats was found. No difference in relative liver weight between ethanol-treated and control rats was observed. Microscopic examinations revealed that the ethanol treatment resulted in fat accumulation in the liver cells. A proliferation of the Smooth Endoplasmic Reticulum (SER) was more marked in the 15% dosed rats than in the 8% dosed rats and more distinct in female rats than in male rats in both dosage groups.     

In vivo inhibition of liver alcohol dehydrogenase by ethanol administration

The activity of liver alcohol dehydrogenase (LADH) from rats sacrificed two hours after the administration of ethanol 3, 4 or 5 g/kg intraperitoneally was significantly inhibited compared to the activity of LADH from control rats. LADH activity was inversely related to the dose of ethanol administered, to the concentration of ethanol in the liver, and to the concentration of ethanol in the blood. The clearance of blood ethanol in rats was dose-dependent and was inversely related to the dose administered. The half-life of ethanol elimination increased as the dose of ethanol increased. These results suggest that ethanol-induced inhibition of LADH can occur in vivo and that the level of activity of this enzyme determines the rate of oxidation of ethanol.     

Ethanol metabolism in guinea pig: In vivo ethanol elimination,alcohol dehydrogenase distribution,and subcellular localization of acetaldehyde dehydrogenase in liver

Guinea pig ethanol metabolism as well as distribution and activities of ethanol metabolizing enzymes were studied. Alcohol dehydrogenase (ADH; EC 1.1.1.1) is almost exclusively present in liver except for minor activities in the cecum. All other organ tissues tested (skeletal muscle, heart, brain, stomach, and testes) contained only negligible enzyme activities. In fed livers, ADH could only be demonstrated in the cytosolic fraction (2.94 μmol/g liver/min at 38 °C) and its apparent K_m value of 0.42 mm for ethanol as substrate is similar to the average K_m of the human enzymes. Acetaldehyde dehydrogenase (ALDH; EC 1.2.1.3) of guinea pig liver was measured at low (0.05 mm) and high (10 mm) acetaldehyde concentrations and its subcellular localization was found to be mainly mitochondrial. The total acetaldehyde activity in liver amounts to 3.56 μmol/g/ min. Fed and fasted animals showed similar zero-order alcohol elimination rates after intraperitoneal injection of 1.7 or 3.0 g ethanol/kg body wt. The ethanol elimination rate of fed animals after 1.7 g ethanol/kg body wt (2.59 μmol/g liver/min) was inhibited by 80% after intraperitoneal injection of 4-methylpyrazole. Average ethanol elimination rates in vivo after 1.7 g/kg ethanol commanded only 88% of the totally available ADH activity in fed guinea pig livers. Catalase (EC 1.11.1.6), an enzyme previously implicated in ethanol metabolism, is of 3.4-fold higher activity in guinea pig (10,400 U/g liver) than in rat livers (3,100 U/g liver), but 98% inhibition by 3-amino-1,2,4-triazole did not significantly alter ethanol elimination rates. After ethanol injection, fed and fasted guinea pigs reacted with prolonged hyperglycemia.     

Mechanism of hypocalcemia induced by intraperitoneal, intragastric, and intravenous administration of ethanol to the rat

Acute hypocalcemic effects of intraperitoneal administration of 3 and 5 g ethanol/kg body weight; intragastric administration of 3, 5, and 7 g ethanol/kg body weight; and intravenous administration of 2.5 a ethanol/kg body weight were investigated in 20 h fasted female Wistar rats. Dose-dependent hypocalcemia was similarly induced by intraperitoneal and intragastric routes of administration. Net calcium efflux from plasma, as indicated by the plasma 45Ca activity, was unaffected by 3 g ethanol/kg body weight but was delayed at higher doses of ethanol. Intragastric, but not intraperitoneal, administration of ethanol increased the gastrointestinal luminal calcium content partly by enhancing calcium secretion. Significantly increased tissue 45Ca content 30 min after ethanol administration was evident in the duodenum (31%), jejunum (27%), and colon (33%) in the intragastric ethanol-treated group and in the duodenum (40%), jejunum (38%), ileum (45%), colon (39%), and liver (25%) in the intraperitoneal ethanol-treated group. Thus, the hypocalcemia induced by both intraperitoneal and intragastric administration of ethanol could be partly accounted for by the suppression of calcium efflux from some soft tissues. In contrast, intravenous administration of ethanol was found to enhance the calcium efflux from plasma without affecting the net 45Ca content in the soft tissues. The mechanism(s) by which ethanol affects calcium transport has yet to be elucidated.     

Effect of age increase on metabolism and toxicity of ethanol in female rats

Age-dependent change in the effects of acute ethanol administration on female rat liver was investigated. Female Sprague-Dawley rats, each aged 4, 12, or 50 weeks, received ethanol (2 g/kg) via a catheter inserted into a jugular vein. Ethanol elimination rate (EER), most rapid in the 4 weeks old rats, was decreased as the age advanced. Hepatic alcohol dehydrogenase activity was not altered by age, but microsomal p-nitrophenol hydroxylase activity was significantly greater in the 4 weeks old rats. Relative liver weight decreased with age increase in proportion to reduction of EER. Hepatic triglyceride and malondialdehyde concentrations increased spontaneously in the 50 weeks old nai;ve rats. Ethanol administration (3 g/kg, ip) elevated malondialdehyde and triglyceride contents only in the 4 and the 12 weeks old rats. Hepatic glutathione concentration was increasingly reduced by ethanol with age increase. Ethanol decreased cysteine concentration in the 4 weeks old rats, but elevated it significantly in the older rats. Inhibition of gamma-glutamylcysteine synthetase activity by ethanol was greater with age increase, which appeared to be responsible for the increase in hepatic cysteine. The results indicate that age does not affect the ethanol metabolizing capacity of female rat liver, but the overall ethanol metabolism is decreased in accordance with the reduction of relative liver size. Accordingly induction of acute alcoholic fatty liver is less significant in the old rats. However, progressively greater depletion of glutathione by ethanol in older rats suggests that susceptibility of liver to oxidative damage would be increased as animals grow old.     

Effect of pyrazole on ethanol metabolism in ethanol-tolerant rats.

Adult male rats were pair-fed liquid diets, providing 37% of calories as ethanol or sucrose, for 1 month. Alcohol dehydrogenase (ADH) activity in the cytosol fractions of liver homogenates from the two groups did not differ with respect to total activity per 100 g body weight, Km for ethanol, or Ki for pyrazole. Other rats, fed in the same way, were fasted for 18-24 H, then given an intraperitoneal injection of pyrazole followed 1 h later by an injection of ethanol, 3g/kg. Blood alcohol curves showed an unexplained slower rise to maximum level in the chronic alcohol group. Both groups showed a period of several hours in which the blood alcohol stayed at the respective maximum concentrations, which were higher in the control group. After 7-8h the alcohol concentration began to fall in both groups, significantly more rapidly in the chronic alcohol-fed animals. A kinetic analysis shows that the results are adequately explained by the known effects of pyrazole on the ADH-mitochondrial system. The results are interpreted as evidence against the function of any microsomal ethanol oxidizing system in vivo.     

Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration

The effect of a single administration of ethanol (2 g/kg body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.     

Sex differences in total body water in adolescent rhesus macaques estimated by ethanol dilution

Abstract: Non‐human primates are widely used in research, yet relatively few studies have addressed potential pharmacokinetic differences between males and females. The present study examined the relationship between total body water, sex, age, and weight in the rhesus macaque (Macaca mulatta). Ethanol‐naïve, adolescent rhesus macaques (n = 119) were administered ethanol (males, 2.1 g/kg; females, 2.0 g/kg) intravenously, and blood samples for blood ethanol concentration obtained at 5, 10, and 60 minutes following the end of the infusion. Non‐linear regression was used to compare and contrast a series of pharmacokinetic models examining the relationship between weight, sex, age, V_d and zero‐order elimination rate. V_d (mean ± SEM) for male rhesus was 0.771 ± 0.008 l/kg and for females was 0.730 ± 0.008 l/kg, different at P < 0.00001. There were no sex differences in the rate of zero‐order ethanol elimination, estimated to be 0.0032 ± 0.0004 g/kg/minute. The data reported here may be useful in designing and interpreting pharmacokinetic studies using rhesus monkeys.     

Hepatic lipids of intact or thyroidectomized rats after administration of tetracycline or ethanol]

The purpose of this work was to study the quantitative modifications of the hepatic lipids in adult thyroidectomized rats after administration of tetracycline or ethanol (acute dose or prolonged ingestion). 1. - Thyroidectomy did not inhibit the accumulation of fat in the liver of fed euthyroid or hypothyroid rats after intraperitoneal infusion of tetracycline (320 mg/body weight in 2 injections at an interval of 16 h, the diet containing 6% of lipids). 2. - Sixteen hours after the oral administration of a single large dose of ethanol (5 g/kg body weight), there were only found some small modifications of the lipid composition of the liver in fasting euthyroid or thyroidectomized rats, receiving a diet with 6% of lipids before the experiment; on the contrary, when the diet contained 19% of lipids, a fatty liver occurred in the intact rat, but not in the thyroidectomized rat. 3. - The prolonged ethanol intake (in a 20% solution in water) for 5 months with a diet containing 19% of lipids did not induce a fatty liver in intact rats but produced a decrease of hepatic non-phosphorus lipid and an increase of the cholesterol amounts. After the administration of L-thyroxin (10 mug/100 g body weight per day) to these alcoholic thyroidectomized rats during 2 weeks, it was found an increase of the hepatic non phosphorus lipids till an higher amount than in the euthyroid rats. 4. - The hepatic phospholipid amounts were relatively constant in the different experiments. These results accounting for this differential effects were discussed.     

The effects of pregnancy on ethanol clearance

We have studied the effects of pregnancy on ethanol clearance rates and on blood and urine ethanol concentrations (BECs and UECs) in adult Sprague-Dawley rats infused with ethanol intragastrically. Pregnant rats had greater ethanol clearance following an intragastric or intravenous ethanol bolus (3 or 0.75 g/kg, respectively) relative to non-pregnant rats (p<0.05). Pregnant rats infused with ethanol-containing diets for several days had lower (p<0.05) UECs than non-pregnant rats when given the same dose of ethanol. Non-pregnant rats infused ethanol-containing diets at two levels of calories (the higher caloric intake required by pregnant rats [220 kca/kg75/d] or the normal calories required for non-pregnant rats [187 kcal/kg75/d]) had statistically equal UECs, suggesting that increased caloric intake was not responsible for the effect of pregnancy. While the activity of hepatic alcohol dehydrogenase (ADH) did not differ with pregnancy, gastric ADH activity was increased (p<0.001). Furthermore, total hepatic aldehyde dehydrogenase (ALDH) and hepatic mitrochrondrial protein were increased (p<0.05) and hepatic CYP2E1 activity was suppressed (p<0.05). The results suggest that pregnancy increases ethanol elimination in pregnant rats by: 1) induction of gastric ADH; 2) elevated hepatic ALDH activity; and 3) increased mitochondrial respiration. The greater ethanol clearance results in lower tissue ethanol concentrations achieved during pregnancy for a given dose, and this may have clinical significance as a mechanism to protect the growing fetus from ethanol toxicity.     

The effect of acute ethanol treatment on rates of oxygen uptake, ethanol oxidation and gluconeogenesis in isolated rat hepatocytes.

In hepatocytes isolated from fed rats, acute ethanol pretreatment (at a dose of 5.0 g/kg body wt.) did not change rates of O2 uptake. In cells from starved animals, acute ethanol pretreatment increased O2 uptake by 17-29%. The increased O2 uptake in hepatocytes from starved rats was not accompanied by increased rates of ethanol oxidation, but was accompanied by increased rates of gluconeogenesis under some conditions. The provision of ethanol (10 mM) as a substrate to cells from fed or starved rats decreased O2 uptake in the absence of other substrates or in the presence of lactate, and increased it in the presence of pyruvate or lactate and pyruvate. The results of this study show that the acute effects of ethanol on liver O2 uptake are dependent on the physiological state of the liver. Previously reported large (2-fold) increases in O2 uptake after acute ethanol pretreatment may have been an artefact owing to low control uptake rates (approximately 1.8 micromol/min per g wet wt. of cells) in the liver preparation used. The ATP contents (2.4-2.6 micromol/g wet wt. of cells) and rates of O2 uptake (2.5-5.0 micromol/min per g wet wt. of cells) of cells used in the present study were the same as values reported under conditions close to those in vivo. Therefore the increase in O2 uptake in cells from starved rats after acute ethanol pretreatment is likely to be of physiological significance.     

Alcohol and acetaldehyde in rat's milk following ethanol administration

Alcohol and acetaldehyde were measured in milk and peripheral blood in chronic alcoholic rats, at 5 and 15 days of lactation. Ethanol in blood increased throughout lactation and the levels of acetaldehyde were much higher than in nonlactating alcoholic rats. The concentration of acetaldehyde in milk was always ca. 50% of that in blood, whereas that of ethanol varied within the range of 44-80% of the blood levels. Blood alcohol levels in the corresponding sucking pups were much lower than in maternal blood and increased throughout lactation. The time course of ethanol and acetaldehyde concentration in blood and milk were determined in normal lactating rats after cyanamide (40 mg/kg) and ethanol administration (2 or 4 g/kg). Milk alcohol reached higher concentrations than in blood within the first hour of ethanol administration, decreasing and remaining constant thereafter at ca. 65% of those in blood. Acetaldehyde levels in milk were always 35-45% lower than in blood. No alcohol dehydrogenase activity was found in homogenates of mammary tissue; however there was some aldehyde dehydrogenase activity. A significant decrease in mammary tissue aldehyde dehydrogenase was found in chronic alcoholic rats. The role of this enzyme is discussed.     

Mechanisms for the effects of ethanol on hepatic phosphatidate phosphohydrolase.

1. The effects of the intramuscular administration of glycerol and dihydroxyacetone (40mmol per kg body wt.), sorbitol and glucose (20mmol per kg body wt.) or NaCl (1.5mmol per kg body wt. in 10ml of water per kg body wt.) were investigated on soluble phosphatidate phosphohydrolase and certain metabolites in rat liver. 2. The effects of ethanol and glycerol on phosphatidate phosphohydrolase were also studied in isolated perfused livers. 3. The administration of glycerol, sorbitol and dihydroxyacetone in vivo increased hepatic phosphatidate phosphohydrolase activity by 137, 63 and 32% respectively in 4h. 4. A significant positive correlation was found between the hepatic sn-glycerol 3-phosphate concentration and phosphatidate phosphohydrolase after the administration of various substrates in vivo. 5. The soluble phosphatidate phosphohydrolase activity tended to increase during perfusions of isolated rat livers without added substrates, and neither ethanol nor glycerol produced additional effects. 6. The activity of soluble phosphatidate phosphohydrolase was 2.5 times higher in the livers of hyperthyroid rats than in normal rats. This activity was not influenced by intragastric ethanol or glycerol administration, nor was the concentration of sn-glycerol 3-phosphate changed by these compounds. 7. It is concluded that the ethanol-induced increase in hepatic phosphatidate phosphohydrolase may at least in part be mediated by the hepatic concentration of metabolites, probably by the concentration of sn-glycerol 3-phosphate.     

Stress-induced consumption of ethanol by rats

The natural aversion of rats to ethanol was overcome by subjecting rats to immobilization stress for a two-week period during which increasing concentrations of ethanol were offered in the drinking water. The rats subjected to this regimen consumed 47% of total calories as ethanol, indefinitely, following removal of the stress. Ethanol was consumed at a rate of 17.1 g/kg body weight along with sufficient stock diet to assure adequate nutrition in the absence of ethanol.     

Stimulation of hepatic phosphatidate phosphohydrolase activity by a single dose of ethanol;.

An acute ethanol load (5 g per kg body wt) given by gastric intubation to fasted rats caused a significant increase in phosphatidate phosphohydrolase activity in the soluble fraction of the liver. The activity was two-fold at 8 hours and three-fold at 16 hours after the ethanol administration and decreased to the control level a few hours after the disappearance of ethanol from the blood. Results from in vivo experiments with intraportally injected [³H]glycerol showed an ethanol-induced cross-over point between glycerol incorporation into phosphatidic acid and neutral glycerolipids. This cross-over could be observed only when the phosphatidate phosphohydrolase activity was increased.     

Liver necrosis induced by acute intraperitoneal ethanol administration in aged rats

It is generally agreed that the deleterious pathophysiological effects of ethanol are caused, at least partially by an increase in free radical production. However, little attention has been directed to the effects of ethanol upon elderly organisms. Male Wistar rats at ages 3, 6, 12, 18 and 24 months were treated either with a single i.p. dose of 35% ethanol (v/v) at 3 g ethanol/kg body weight or an isovolumetric amount of 0.9% saline solution. We then assessed the plasma levels of transaminases and hepatic levels of oxidative stress-related parameters, followed by liver histological evaluation. The younger rats (3 months old) were not affected by the treatment with ethanol with respect to any of the studied parameters except for a lowering of total hepatic GSH and an increase in hepatic thiobarbituric acid reactants (TBARS) formation, while animals older than 3 months were increasingly more affected by the treatment. Acute ethanol treatment elicited the similar responses to those in the 3 months-old group, plus a decrease in the hepatic and plasma levels of beta-carotene and the plasma level of alpha-tocopherol, as well as an increase in the activity of plasma transaminases. In the 12,18 and 24 months old groups, there was increasing liver necrosis. These findings suggest that liver damage induced by acute ethanol administration in elderly rats may involve a lack of antioxidants.     

The contribution of the stomach to ethanol oxidation in the rat

To estimate the amount of ethanol that can be oxidized in the stomach, steady-state conditions were created in a group of fed rats by giving a loading dose of ethanol (2 g/kg body wt I.V.) followed by continuous infusion either intravenously or intragastrically. The rate of ethanol oxidation was calculated from the rate of infusion required to maintain steady blood levels of approximately 30 mM for at least 3 hours. Gastrointestinal ethanol concentrations and total contents also remained steady. The rate of ethanol oxidation was 19.3% faster during intragastric than during intravenous infusion (p less than 0.01). When measured at the prevailing luminal ethanol concentration (145 mM) and expressed per body weight, the gastric ADH activity represented 14% of the hepatic activity at 30 mM ethanol, suggesting that gastric ADH activity could account for most of the increased rate of oxidation when ethanol is given intragastrically. Thus, gastric ethanol oxidation by a high Km ADH in the rat represents a significant fraction of the total rate of ethanol oxidation and it is therefore one of the factors which determines the bioavailability of orally administered ethanol.     

System of ethanol and acetaldehyde metabolism in the rat liver during the development of ethanol tolerance

Following daily intraperitoneal injections of ethanol at a dose of 3.5 g/kg rats developed tolerance to its hypnotic action, which was manifested in a drastic decrease of ethanol--induced sleep time and the number of animals sleeping more than 60 min. In the liver, this process was characterized by an elevated alcohol dehydrogenase activity and a decreased aldehyde dehydrogenase one, whereas that of MEOS remained unchanged.     

The effect of ethanol on 5'-nucleotidase of rat liver plasma membranes

The activity of 5'-nucleotidase of rat liver plasma membranes has been investigated in normal and acutely ethanol-intoxicated rats (7 g ethanol/Kg body wt). Ethanol was also added to the incubation mixture for 5'-nucleotidase assay. The alcohol modified the Km of the enzyme when added to plasma membranes of normal rats; moreover, it increased the activation energy of the reaction. The treatment with the alcohol in vivo lowered the Vmax, but no modifications of Km could be detected in this case, upon further addition of the toxic in vitro. It is concluded that ethanol is able to act by itself on 5'-nucleotidase activity of rat liver plasma membranes; however, ethanol produces other effects in vivo, probably due to its metabolism.     

Hepato-protective potential of carotenoid meso-zeaxanthin against paracetamol, CCl4 and ethanol induced toxicity

Hepato-protective potential of carotenoid meso-zeaxanthin [(3R, 3'S)-beta, beta-carotene-3, 3'-diol] was studied using in vivo rat models. Paracetamol (3 g/kg body wt, orally), 20% ethanol (7.5 g/kg body wt, orally) and CCl4 (2.5 ml /kg, ip) were used as hepato toxins. Levels of marker enzymes of hepatic injury such as serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase and alkaline phosphatase, and serum bilirubin, which were drastically elevated by these hepato toxins were significantly decreased by meso-zeaxanthin pretreatment in a dose-dependent manner. Oxidative stress markers, tissue lipid peroxidation, conjugated dienes and tissue hydroperoxides, were high in the paracetamol treated control group animals, which were lowered by meso-zeaxanthin administration. Level of glutathione and antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, in liver tissue was increased by meso-zeaxanthin pretreatment compared to control group during alcohol and CCl4 induced hepatotoxicity. Hydroxyproline, an indicator of fibrosis in liver tissue, decreased remarkably by meso-zeaxanthin administration despite its notable elevation in ethanol treated rats. Histopathological analysis of liver tissue showed the hepatoprotective potential of meso-zeaxanthin.