Hepatoprotection by malotilate against carbon tetrachloride-alcohol-induced liver fibrosis

Subchronic treatment of male rats with carbon tetrachloride (CCl₄, twice weekly 0.2 ml/kg p.o) and feeding a 5% alcohol solution instead of drinking water led to a nearly complete liver cirrhosis in all animals within 4 weeks. This was also documented by a three fold increase in hepatic total hydroxyproline content. Steatosis was quantified by enhanced liver triglyceride concentrations and acute necroses by increments of serum enzyme activities (GPT, SDH). Daily oral treatment with malotilate (100 mg/kg) totally prevented the development of liver cirrhosis, hepatic hydroxyproline accumulation and increases in serum enzyme activities induced by CCl₄-alcohol. In cianidanol-treated rats (100 mg/kg p.o.) only portoseptal fibrosis was seen, however hydroxyproline and triglyceride accumulation as well as enhanced serum enzyme activities were not suppressed. D-penicillamine (300 mg/kg p.o.) and colchicine (50 g/kg i.p.) failed to protect rats against CCl₄-alcohol induced fibrosis, necrosis and steatosis in this model.     

Effects of Moderate Alcohol Intake in the Bladder of the Otsuka Long Evans Tokushima Fatty Diabetic Rats

Diabetes is related with a number of cystopathic complications. However, there have been no studies about the influence of alcohol consumption in the bladder of type 2 diabetes. Thus, we investigated the effect of moderate alcohol intake in the bladder of the Otsuka Long Evans Tokushima Fatty (OLETF) diabetic rat. The non-diabetic Long-Evans Tokushima Otsuka (LETO, n=14) and the OLETF control group (n=14) were fed an isocaloric diet; the LETO (n=14) and the OLETF ethanol group (n=14) were fed 36% ethanol 7 g/kg/day. After ten weeks, muscarinic receptors, RhoGEFs, myogenic change, and the level of oxidative stress were evaluated. Moderate alcohol intake significantly decreased excessive muscarinic receptor and Rho kinase expressions in the OLETF rats compared with the LETO rats. In addition, iNOS and collagen expression were not changed in the OLETF rats in spite of alcohol consumption. Superoxide dismutase levels, which is involved in antioxidant defense, in the LETO rats were significantly decreased after alcohol consumption, however those in the OLETF rats were similar. Moderate alcohol consumption reduces the oxidative stress, and may prevent molecular and pathologic changes of the bladder of rats with type 2 diabetes.     

Effects of dithiocarb and (+)-catechin against carbon tetrachloride-alcohol-induced liver fibrosis

Treatment of male rats with carbon tetrachloride (CCl₄, 2 × weekly 0.2 ml/kg p.o.) and a 5% alcohol solution, instead of drinking water, for 4 weeks led to marked increases in serum enzyme activities (GOT, GPT, SDH), hepatic triglyceride and hydroxyproline content. Diethyl dithiocarbamate (dithiocarb, 200 mg/kg p.o.) simultaneously applied with CCl₄ totally suppressed the elevation in serum enzyme activities and hepatic hydroxyproline concentration, and partially suppressed that of the triglyceride content. (+)-Catechin (50–300 mg/kg p.o.). simultaneously applied with CCl₄ had no influence on the enhanced serum enzymes, but depressed the augmented content of both hepatic triglyceride and hydroxyproline in a dose-dependent way. The most effective dose with respect to the reduction of the hydroxyproline concentration was 100 mg/kg (+)-catechin; the highest dose (300 mg/kg), however, enhanced the CCl₄-alcohol-induced hydroxyproline augmentation.     

Role of lipid peroxidation in enhancement of endotoxin hepatotoxicity.

The present study was undertaken in the rats to examine whether endotoxin hepatotoxicity is enhanced by increased lipid peroxidation. The rats were given 10 ml of water, corn oil or heated and oxygenated corn oil per kg body weight by stomach tube twice a day for 14 days, and then they were injected physiological saline solution or endotoxin (2 or 2.5 mg per kg body weight) into the tail vein. In the rats pretreated with water or corn oil, the activity of serum glutamic pyruvic transaminase was within the normal limit, and there was no conspicuous morphological change in the liver, except for accumulation of fine fat droplets in few liver cells. On the other hand, in the rats pretreated with heated and oxygenated corn oil, containing a large amount of lipid peroxides, accumulation of small fat droplets in the liver cells and a slight elevation of serum transaminase activity were induced. The challenge with endotoxin (2.5 mg per kg body weight) caused focal hepatocellular coagulative necrosis and a marked elevation of serum transaminase activity, irrespective of the sorts of pretreatment, and there was no significant difference in the biochemical change and the histopathological damage between the rats pretreated with water, corn oil and heated and oxygenated corn oil. These results suggest that increased lipid peroxidation does not contribute to the enhancement of endotoxin hepatotoxicity, although it is thought that carbon tetrachloride and ethanol enhance endotoxin hepatotoxicity by synergism between endotoxin and the chemicals through lipid peroxidation.     

Endotoxin hepatotoxicity augmented by ethanol

To determine whether alcohol increases endotoxin hepatotoxicity, we administered ethanol (4.8 g/kg body wt in 4 ml of water) to rats through a gastric tube, then immediately injected endotoxin (2, 2.5, or 3 mg/kg body wt). In the rats pretreated with ethanol, the injection of 2 mg/kg body wt of endotoxin induced a slight rise of serum transaminase. However, when 2.5 mg/kg body wt of endotoxin was given, there were no significant histopathological or biochemical differences between the rats pretreated with ethanol and those pretreated with water. Moreover, there was no significant difference in mortality rates between the rats pretreated with ethanol and the controls when 3 mg/kg body wt (LD50) of endotoxin was injected. These results suggest that acute administration of alcohol enhances endotoxin hepatotoxicity when the dose of endotoxin is small, but that the effect of alcohol is masked when larger doses of endotoxin are given.     

Sex differences in the susceptibility of rats to carbon tetrachloride-alcohol-induced liver injury

Treatment of male and female rats with carbon tetrachloride (CCl₄, twice weekly, 0.2 ml/kg p.o.) and a 5% alcohol solution during four weeks evoked strong increments of the serum enzyme activities of the aminotransferases (GOT, GPT) and the sorbitol dehydrogenase (SDH). These occurred earlier and were more pronounced in male compared to female rats. Hepatic triglyceride contens as a measure of fatty infiltration was augmented three-fold both in males and females at the end of the experiment. Hepatic hydroxyproline contents were enhanced seven-fold in males, but only two-fold in females. It is concluded that female rats are less susceptible to CCl₄-alcohol-induced liver damage, especially hydroxyproline accumulation which is explained by the known fact that females are more resistant against CCL₄-hepatotoxicity as a consequence of a minor role of bioactivation in the metabolic degradation of CCO₄ by females.     

Functional heterogeneity of rat hepatic and alveolar macrophages: effects of chronic ethanol administration

Chronic ethanol consumption is associated with increased incidence of hepatic and pulmonary infections. To determine if this is correlated with altered macrophage activity, we analyzed the functional properties of cells isolated sequentially from the liver and lung of rats fed a liquid diet containing ethanol (35% of calories) or malto-dextrin control for 9-12 weeks. Hepatic and alveolar macrophages from control animals were found to exhibit distinct morphologic and functional properties. Thus, hepatic macrophages were highly vacuolated and appeared larger and more irregular in shape than alveolar macrophages. These cells also displayed greater phagocytic activity and random migration. In contrast, lung macrophages produced more superoxide anion and nitric oxide, and exhibited enhanced chemotactic activity toward the complement fragment C5a. Whereas administration of ethanol to rats for 9-12 weeks resulted in decreased chemotaxis and superoxide anion production by alveolar macrophages, cell adhesion molecule expression was reduced in hepatic macrophages. Nitric oxide production and inducible nitric oxide synthase protein expression were decreased in both macrophage populations. These effects were not observed after 3-6 weeks of ethanol administration to rats. Our results suggest that changes in macrophage functioning may play a role in decreased host defense following chronic ethanol exposure.     

Effects of ethanol on microsomal drug metabolism in aging female rats. I. Induction

The purpose of this study was to determine how aging affects the induction by ethanol or acetone of the hepatic microsomal monooxygenase system of female Fischer 344 rats. Young-adult, middle-aged and old rats (4, 14 and 25 months) were fed an ethanol-containing or control liquid diet for 15 days. Cytochrome P-450, cytochrome c reductase, aniline hydroxylase, nitrophenol hydroxylase, nitroanisole O-demethylase and benzphetamine N-demethylase activities were measured in hepatic microsomes. All of the drug metabolism activities except benzphetamine N-demethylase were 20-35% lower in old than in young-adult rats fed the control diet. In addition, the increase in drug metabolism produced by feeding the regular ethanol diet (36% of calories as ethanol) was 50-60% lower in the old rats. However, there was no difference in the magnitude of ethanol induction when ethanol intakes were matched. The effects of chronic acetone consumption (1.2g/day per kg body weight for 15 days) paralleled those of ethanol consumption, except that the extent of induction was greater with acetone. Acetone-induced levels of hepatic microsomal cytochrome P-450, nitrophenol hydroxylase, nitroanisole O-demethylase and aniline hydroxylase were similar in all three age groups. The results of this study indicate that induction of hepatic microsomal drug metabolism by ethanol or acetone is unaffected by the aging process.     

Consumption of alcohol by sows in a choice test

The domestic pig (Sus scrofa domesticus) has been proposed as an animal model for human alcoholism because pigs have been observed to consume alcohol voluntarily to a state of intoxication and to exhibit tolerance and physical dependence. However, it has not been established whether pigs can develop psychological dependence on alcohol. We hypothesised that feed-restricted, stall-housed pregnant sows fed alcohol non-voluntarily for 5 weeks would develop a preference for alcohol and retain this preference after removal of alcohol from the diet. We fed crossbred commercial sows (n=10) 280 ml of 95% ethanol mixed with 0.91 kg of feed and 720 ml of water twice daily for 5 weeks during the first trimester of pregnancy. Control sows (n=7) received dextrose in their feed as a caloric control, and water was added to give the feed a consistency similar to that of the alcohol-treated feed. Immediately before and after 5 weeks of alcohol or dextrose treatment and 3 weeks later, after termination of alcohol or dextrose treatment, we evaluated sow diet preference by comparing the amount of alcohol-supplemented, dextrose-supplemented and plain feed consumed during a 5-min choice test. Contrary to our hypothesis, there was no treatment effect on sow diet preference. Both alcohol-treated and control sows ate less of the alcohol diet than the other two diets in all choice tests. They did not discriminate between the plain and dextrose diets. We conclude that 5 weeks of non-voluntary consumption of alcohol in feed did not produce a preference for alcohol in pregnant sows, either during treatment or after withdrawal, thus providing no evidence for the development of psychological dependence on alcohol under these conditions.     

Effect of paternal alcohol ingestion on fetal growth in rats

To determine the effect of paternal alcohol consumption on fetal growth, male rats were given ethanol in drinking water and rat chow ad libitum (alcohol group), or water and the same amount of rat chow as that consumed by the alcohol group, but the ethanol was substituted isocalorically with corn starch (pair-fed group), or water and rat chow ad libitum (ad libitum control group). The ethanol concentration in the drinking water was 10% (v/v) for the first week, 20% the second week, and 30% during the next four weeks. After six weeks on this regimen the male rats were mated with females of the same strain not treated with alcohol. The rate of fertile matings was not significantly affected by the paternal alcohol intake. On day 21 of gestation no significant differences were seen with respect to fetal and placental weights, litter size or male to female sex ratio of the fetuses among the alcohol, pair-fed and ad libitum control groups. The results indicate that rat fetal growth is not sensitive to the effects of paternal alcohol consumption.     

Ethanol inhibition of compensatory ovarian hypertrophy in unilaterally ovariectomized rats

The purpose of this study was to determine the effect of ethanol consumption on compensatory ovarian hypertrophy (COH) which occurs following unilateral ovariectomy. Holtzman rats, 40 days old, were either unilaterally ovariectomized or sham-ovariectomized. The rats were then placed into subgroups which would receive either an ad libitum chow and water diet, a liquid diet, or a liquid diet containing 5% ethanol. The animals were maintained on their respective diets for 11 days. The rats were killed at 51 days of age, and the ovaries and uteri were removed, weighed, and prepared for histological investigation. The results showed that uteri from ethanol-fed animals failed to develop epithelial glands and exhibited a condensed stroma in comparison to uteri from animals fed ad libitum or pair-fed to ethanol-fed rats. Also, rats that were fed ad libitum had a COH of 82 +/- 16% and rats that were pair-fed a liquid diet had 114 +/- 28% COH; rats that were fed the liquid diet containing ethanol did not experience compensatory ovarian hypertrophy (-3 +/- 10%). Histologically, the ovaries of rats fed ad libitum showed large numbers of corpora lutea and only a few mature ova. The histology of ovaries from pair-fed animals was similar to those from animals fed ad libitum. In contrast, the ovaries from the animals fed the ethanol diet had nearly twice as many mature ova but only one-fourth as many corpora lutea as the number seen in ovaries from the other groups. The data show that ethanol consumption inhibits COH by suppressing ovulation and the subsequent luteal formation.     

Ethanol-induced alterations in the morphology and function of the rat ovary

The purpose of this study was to compare the effects of different dosages of ethanol on ovarian morphology and function. Holtzman rats, 20 days old, were divided into groups as follows: The rats in Group I were autopsied at 20 days of age, and those in Group II were placed on ad libitum chow and water diet; the rats in Groups III and V were fed on a liquid diet containing 2.5% or 5% ethanol respectively; Groups IV and VI were pair-fed controls to Groups III and V, respectively. Rats in Groups II, III, IV, and VI were maintained on the diets for 50–55 days and killed at late proestrus-estrus, while the animals in Group V did not exhibit estrous cycles and were killed on day 55 of treatment. The average increase in body weights of rats in Groups II, III, and IV was significantly greater than the increase in body weights of rats given 5% ethanol or their pair-fed controls. In the rats treated with 5% ethanol, vaginal opening was significantly delayed from the controls, estrous cycles were absent, ovarian weights were similar to those of the 20-day-old rats, ovaries contained corpora lutea of only one estrus, uteri weighed less than controls, and histologically, the uteri and vaginae were similar to those of 20-day-old rats. However, in the rats treated with 2.5% ethanol, all of the parameters were similar to those of the controls. The average serum alcohol level for the rats on the 5% ethanol diet was 249 mg%; the serum alcohol levels were at the lower limit of detection for the rats on the 2.5% ethanol diet. The data show that ovarian function was suppressed in the rats that received the 5% ethanol but not in rats on the 2.5% ethanol diet.     

Characteristics and classification of hippocampal theta rhythm induced by passive translational displacement

Painful peripheral neuropathy induced by chronic ethanol consumption is a major medico-socioeconomical problem. The objective of present investigation was to study the effect of curcumin (20, 40 and 80mg/kg; p.o.) in alcohol-induced neuropathy in rats. Ethanol (35% v/v, 10g/kg; p.o.) was administered for 10 weeks which showed a significant decrease in thermal hyperalgesia, mechanical hyperalgesia, mechanical allodynia and nerve conduction velocity. It caused enhanced malondialdehyde, oxidative-nitrosative stress, total calcium levels, inflammatory mediators (TNF-α and IL-1β levels) along with DNA damage. Co-administration of curcumin and α-tocopherol for 10 weeks significantly and dose-dependently improved nerve functions, biochemical as well as molecular parameters and DNA damage in sciatic nerve of ethanol treated rats. Hence, it was concluded that curcumin is of potent therapeutic value in the amelioration of alcoholic neuropathy in rats and acts by inhibition of pro-inflammatory mediators like TNF-α and IL-1β.     

Protective effect of apocynin in an established alcoholic steatohepatitis rat model

Apocynin is a widely used antioxidant in both basic and clinical research. The current study aimed to investigate the protective effect of apocynin in an established alcoholic steatohepatitis rat model. Healthy SD rats were gastrically fed with ethanol (4.0?g/kg) for 8 weeks to induce alcoholic steatohepatitis. After 8 weeks, rats were fed with ethanol for another 2 weeks with or without a daily intraperitoneal injection of 25?mg/kg apocynin. After sacrificing, serum and liver samples were subjected to hepatic injury measurements. After 8-week ethanol induction, rats exhibited typical alcoholic steatohepatitis features including reduced body weight, hepatic histological changes, elevated serum alanine transaminase (ALT) level, increased levels of oxidative stress and inflammatory markers, NADPH oxidases, and renin-angiotensin system (RAS) marker. Co-treatment of apocynin alleviated the hepatic injury and biochemical parameters induced by alcoholic steatohepatitis. In conclusion, addition of apocynin significantly attenuates hepatic oxidative stress and inflammation induced by alcoholic steatohepatitis. This effect is partly through the inhibition of the RAS system.     

Skeletal development in fetuses of rats consuming alcohol during gestation

Sprague-Dawley female rats were fed either 20% ethanol in drinking water and rat chow ad libitum (alcohol group) or were pair-fed to the alcohol group, with starch substituted isocalorically for ethanol (pair-fed group) or were fed rat chow and tap water ad libitum. After 4 weeks they were bred and the alcohol group was changed to 30% ethanol in water. On day 20 of gestation the fetuses were removed and stained with Alcian Blue and Alizarin Red S. The skeletons were examined for the appearance of ossification centers and the ossification centers in the fore- and hindlimbs were measured. Fetuses from the alcohol group weighed significantly less than those of the two control groups. Ossification centers expected to appear in the skull on day 20 were absent in most of the alcohol group fetuses, but present in the control fetuses. There were fewer ossification centers in the sternum and in the limbs, and the ossification of vertebral centra has progressed less (both anteriorly and posteriorly) in the alcohol fetuses than in the controls. Dimensions of the ossification centers in the limbs were less in the alcohol fetuses than in the controls. No gross malformations were seen in any of the fetuses. It is concluded that by day 20 of gestation skeletal development is retarded by approximately one day in the fetuses of rats given alcohol prior to and throughout gestation.     

The sap of Acer okamotoanum decreases serum alcohol levels after acute ethanol ingestion in rats

In the present study, we examined whether Acer okamotoanum (A. okamotoanum) sap decreased the serum alcohol and acetaldehyde levels after acute ethanol treatment in a rat model. Male rats were orally administered 25, 50 or 100% A. okamotoanum sap 30 min prior to oral challenge with 3 ml of ethanol (15 ml/kg of a 20% ethanol solution in water), and the blood concentrations of alcohol and acetaldehyde were analyzed up to 7 h after the treatment. Pre-treatment with the sap significantly decreased the blood ethanol and acetaldehyde concentrations after 5 h when compared with ethanol treatment alone (a negative control). The expression levels of liver alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) mRNA were increased significantly in animals pre-treated with A. okamotoanum sap when compared with negative and positive controls. The data suggest that sap pre-treatment enhanced the alcohol metabolism rate in the rat liver. To investigate the involvement of mitochondrial regulation in the ethanol-induced hepatocyte apoptosis, we carried out an immunohistochemical analysis of Bax and Bcl-2. Pre-treatment with sap significantly decreased Bax expression and increased Bcl-2 expression 7 h after ethanol administration when compared with the negative control. The data suggest that A. okamotoanum sap pre-treatment may reduce the alcohol-induced oxidative stress in the rat liver.     

Lack of fetal growth retardation in rats consuming alcohol prior to conception but not during gestation

A study was conducted to determine the effect of chronic alcohol consumption by female rats prior to conception but not during gestation upon fetal growth. The rats were divided into two dietary groups. Group 1 (alcohol) received ad libitum a stock diet plus ethanol in the drinking water. The ethanol concentration in the drinking water was 10% for the first week, 20% the second week and 30% during the next three weeks. Group 2 (pair-fed controls) received water ad libitum and the same amount of stock diet as that consumed by the ethanol group, but the ethanol was substituted isocalorically with corn starch. After five weeks on this regimen all animals were mated and the administration of alcohol was discontinued after conception. The maternal body weight gains were similar in the two dietary groups prior to and throughout gestation. No significant differences were seen between the alcohol and control groups with respect to litter size and fetal weight at day 21 of gestation, but the placentas were significantly heavier in the alcohol group. These results indicate that fetal growth retardation, which is characteristic of in utero alcohol exposure, is not induced when the period of alcohol intake is restricted to the time preceding gestation.     

Prevention of hepatotoxic responses to chemicals by glycyrrhizin in rats

To clarify whether glycyrrhizin, the aqueous extract of licorice root and a drug for treatment of chronic active hepatitis, prevents the development of hepatic injury induced by carbon tetrachloride, allyl formate, and endotoxin, the present study was undertaken in rats. The treatment with glycyrrhizin 20 hr before carbon tetrachloride administration protected the development of the pericentral hepatocellular necrosis. Glycyrrhizin treatment 2 hr prior to the administration of allyl formate also inhibited the development of the periportal hepatocellular necrosis. However, glycyrrhizin did not protect the development of endotoxin-induced focal and random hepatocellular necrosis. These experimental results suggest that glycyrrhizin has no protective effect on hepatic injury following sinusoidal circulatory disturbance as seen in the case of endotoxin and that glycyrrhizin can protect against hepatotoxicity induced by the direct action on the hepatocytes due to hepatotoxins, such as carbon tetrachloride and allyl formate.     

Lifelong ethanol consumption enhances the age-related changes in rat sympathetic neurons.

The effects of aging and chronic ethanol administration on the histochemical and morphometric features of rat superior cervical ganglion were studied in a rat strain selected for voluntary alcohol consumption. Ethanol was administered to the experimental group ad libitum (10% v/v in drinking water) from 3 months to 28 months of age, the average ethanol intake being 6.4-5.4 g/kg per day. The sympathetic neurons of the ethanol consuming rats showed several signs of enhanced degeneration, e.g. decreased neuronal packing density, increased amount of age-pigment and decreased intensity of catecholamine histofluorescence and tyrosine hydroxylase immunoreactivity. The results may indicate a selective vulnerability of peripheral sympathetic neurons rather than a universal accelerated aging due to chronic ethanol exposure.     

HEPATOTOXICITY AND ABSORPTION OF EXTRAHEPATIC ACETALDEHYDE IN RATS

Acetaldehyde, the first metabolite of ethanol oxidation, has been proposed as a major initiating factor in ethanol-induced liver injury. The aims of this study were to examine whether acetaldehyde is absorbable from the digestive tract and whether, when delivered chronically in drinking water, it is capable of inducing liver injury in rats. Acetaldehyde concentrations in the rat portal and peripheral blood were measured by head space gas chromatography after intragastric (5 ml) and intracolonic (3 ml) administration of 20 mM acetaldehyde solution. In the hepatotoxicity study, rats were exposed to acetaldehyde (20 and 120 mM ) delivered in drinking water for 11 weeks and histopathological changes in the liver were morphometrically assessed. Peak blood acetaldehyde levels were found at 5 min after acetaldehyde infusion and were 235±11 μμ (mean±SE) after intragastric and 344±83 μμ after intracolonic infusion of 20 mM acetaldehyde solution. The exposure of rats to 120 mM acetaldehyde solution for 11 weeks resulted in the development of fatty liver and inflammatory changes. Morphometric analysis showed significantly more fat accumulation in rats receiving 120 mM acetaldehyde solution (85±2 per cent of hepatocytes occupied by fat) than in rats receiving 20 mM acetaldehyde solution (38±11 per cent) or in controls (36±10 per cent). The dose of extrahepatic acetaldehyde (500 mg/kg per day) producing liver injury corresponds to only around 3 per cent of that derived from hepatic ethanol oxidation in animals receiving an ethanol-containing totally liquid diet (15 g/kg per day). These results indicate that acetaldehyde delivered via the digestive tract can reach the liver by the portal circulation and that acetaldehyde of extrahepatic origin appears to be more hepatotoxic than acetaldehyde formed during ethanol oxidation within the liver.