A biochemical and stereological study of neonatal rat hepatocyte subpopulations. Effect of pre- and postnatal exposure to ethanol

Hepatocytes from 12-day-old rats, pre- and post-natally exposed to alcohol, together with those from pair-fed controls, were isolated and subfractionated in six cell subpopulations on Percoll density gradients. These cells were characterized using a combination of biochemical and stereological methods. The low density cells (F2) mainly showed biochemical and stereological features of perivenous hepatocytes, whereas the heavier cells (F6) were primarily periportal hepatocytes. The alcohol-metabolizing enzymes, alcohol dehydrogenase and aldehyde dehydrogenase (high and low Km) showed more activity in the F2 fraction. Alcohol-altered mitochondria and Golgi apparatus occurred mainly in F2 cells, whereas the endoplasmic reticulum and lysosomes appeared to be more altered in the F6 hepatocytes. Alcohol also induced the appearance of some small hepatocytes, with a well-developed rough endoplasmic reticulum and an increased number of mitochondria. Biochemical data indicated that glutamate dehydrogenase and alanine aminotransferase were more affected in F2 cells from alcohol-treated rats, and that the activity of the ethanol-metabolizing enzymes was alos reduced in these hepatocytes. Our results indicate that alcohol exposure during zonal development in the liver could have a selective effect on specific cell components depending on the acinar zone, and that the perivenous hepatocytes appear to be more damaged under these conditions.     

Effect of chronic ethanol or acetaldehyde on hepatic alcohol and aldehyde dehydrogenases, aminotransferases and glutamate dehydrogenase

Ethanol or acetaldehyde orally administered (15% and 2% respectively in drinking water) to male Wistar rats for three months induced alterations in the main liver enzymes responsible for ethanol metabolism, aspartate and alanine aminotransferases and NAD glutamate dehydrogenase. Ethanol produced a significant decrease in the activity of soluble alcohol dehydrogenase, while acetaldehyde induced alterations both in soluble and mitochondrial aldehyde dehydrogenases: soluble activity was significantly higher than in the control and ethanol-treated groups, and mitochondrial activity was significantly diminished. Both soluble aspartate and alanine aminotransferases showed pronounced increases by the chronic effect of acetaldehyde, while mitochondrial activities were practically unchanged by the effect of ethanol or acetaldehyde. Mitochondrial NAD glutamate dehydrogenase showed a rise in its activity both by the effect of chronic ethanol and acetaldehyde consumption. The level of metabolites assayed in liver extracts showed marked differences between ethanol and acetaldehyde treatment which indicates that ethanol produced a remarkable increase in glutamate, aspartate and free ammonia together with marked decrease in pyruvate and 2-oxoglutarate concentrations. Acetaldehyde consumption induced a significant decrease in 2-oxoglutarate and pyruvate concentrations. These observations suggest that ethanol has an important effect on the urea cycle enzymes, while the effect of acetaldehyde contributes to the impairment of the citric acid cycle.     

Effect of ethanol administration on metabolism of lipids in heart and aorta in isoproterenol induced myocardial infarction in rats

Effect of ethanol administration on the severity of myocardial infarction induced by isoproterenol in rats was studied. Even though serum CPK and GOT levels as well as the extent of myocardial damage as revealed by histopathological studies, were similar, the survival rate was higher in rats administered ethanol. Concentration of cholesterol and triglycerides in the serum and heart in rats given ethanol and isoproterenol seems to be the additive effect caused individually by ethanol and isoproterenol. Myocardial alcohol dehydrogenase and aldehyde dehydrogenase both showed increased activity in rats treated with ethanol. The rate of recovery from myocardial infarction however, was slower in rats treated with ethanol as judged from the serum CPK value.     

Serum glutamate dehydrogenase and ornithine carbamyl transferase in Reye's syndrome

Parallel increases in the ornithine carbamyl transferase and glutamate dehydrogenase activities were observed in the serum of 22 Reye's syndrome patients. The increase in the activity of glutamate dehydrogenase was masked by a dialyzable inhibitor. It is proposed that the measurement of serum ornithine carbamyl transferase and glutamate dehydrogenase activities may be useful as an indicator of liver mitochondrial damage in Reye's syndrome.     

Cholestasis following chronic alcohol consumption: Enhancement after an acute dose of chlorpromazine

Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing either 36% of total calories as ethanol or isocaloric carbohydrates (controls) for 4 weeks. Compared to controls, chronic alcohol consumption leads to slightly increased serum activities of various hepatic enzymes including the alkaline phosphatase (AP). Chlorpromazine administered as a single dose 18 hours after ethanol with-drawal resulted within 18 hours in a significant increase of serum AP activity in rats fed ethanol chronically but not in their pair-fed controls. It is concluded that chronic alcohol consumption predisposes to cholestasis due to chlorpromazine.     

Effect of garlic oil extract on serum, liver and kidney enzymes of rats fed on high sucrose and alcohol diets

High levels of alkaline phosphatase and alcohol dehydrogenase were observed in the serum, liver and kidneys of rats fed on high sucrose and high alcohol diets over a period of 75 days. Garlic oil extract fed with any of the diets, significantly lowered the high levels of the two enzymes in the serum, liver and kidneys. This effect may be due to reduced biosynthesis of fatty acids as NADPH, required for the process, is utilised for the metabolism of the oil.     

Changes in activities of some ammonia-metabolizing enzymes in the rat liver and the brain after chronic ethanol administration

The changes in the activities of ammonia-metabolizing enzymes in liver and brain after ethanol intoxication has been investigated in rats. After administration of ethanol 30% (w/v) 6g kg-1 for 4 weeks we found an increase in liver glutamate dehydrogenase and glutaminase activity. In brain tissue the glutaminase activity was significantly higher and glutamate dehydrogenase was significantly lower. Glutamine synthetase activity in liver and brain was practically unchanged. The reasons for these changes in the activities of some ammonia-metabolizing enzymes in liver and brain after ethanol ingestion have been discussed.     

Effect of aspartate and glutamate on experimental myocardial infarction in rats

Cardiac necrosis was produced in rats by administering isoproterenol sulphate (85 mg/kg, sc for 4 days). The myocardial damage was proved by observing the elevated levels of serum aspartate amino-transferase, lactate dehydrogenase and creatine phosphokinase and the changes were confirmed by histopathology of the tissue. Both aspartate and glutamate (100 mg/kg, ip) significantly reduced the elevated levels of these enzymes. The average degree of cardiac necrosis produced in these rats when observed macroscopically and histologically was also found to be significantly reduced on pretreatment with aspartate and glutamate.     

Epoxiconazole‐Induced Degeneration in Rat Placenta and the Effects of Estradiol Supplementation

Epoxiconazole (CAS‐No. 133855‐98‐8) was recently shown to cause both a marked depletion of maternal estradiol blood levels and a significantly increased incidence of late fetal mortality when administered to pregnant rats throughout gestation (GD 7–18 or 21); estradiol supplementation prevented this epoxiconazole effect in rats (Stinchcombe et al., 2013), indicating that epoxiconazole‐mediated estradiol depletion is a critical key event for induction of late fetal resorptions in rats. For further elucidation of the mode of action, the placentas from these modified prenatal developmental toxicity experiments with 23 and 50 mg/kg bw/d epoxiconazole were subjected to a detailed histopathological examination. This revealed dose‐dependent placental degeneration characterized by cystic dilation of maternal sinuses in the labyrinth, leading to rupture of the interhemal membrane. Concomitant degeneration occurred in the trophospongium. Both placentas supporting live fetuses and late fetal resorptions were affected; the highest degree of severity was observed in placentas with late resorptions. Placental degeneration correlated with a severe decline in maternal serum estradiol concentration. Supplementation with 0.5 and 1.0 μg of the synthetic estrogen estradiol cyclopentylpropionate per day reduced the severity of the degeneration in placentas with live fetuses. The present study demonstrates that both the placental degeneration and the increased incidence of late fetal resorptions are due to decreased levels of estrogen, since estrogen supplementation ameliorates the former and abolishes the latter. Birth Defects Res (Part B) 98:208–221, 2013. © 2013 Wiley Periodicals, Inc.     

Adaptation of siblings of female rats given ethanol effect of N-acetyl-L-cysteine

Summary Ethanol administration to female rats before and during pregnancy resulted in decreased number of litters and increased activities of serum GOT, GPT and ALP. The hepatotoxicity of ethanol was indicated by the histological alterations both in the mother and siblings. There was increased levels of tissue lipids in mother and litters born to alcoholic rats. The concentration of TBARS in the liver and kidney were significantly increased in alcohol treated rats and their litters. The activities of the anti-peroxidative enzymes SOD and CAT were decreased on alcohol treatment in female rats. The glutathione content in liver and kidney decreased significantly in litters born to alcoholic rats.We have observed that the treatment with N-acetylcysteine offers protection against the toxic effect of alcohol in female rats during pregnancy and litters born to them. In N-acetylcysteine treated rats the number of litters as well as the average birth weight were close to that of control animals. Nacetylcysteine decreases the activities of serum GOT, GPT and ALP in female rats. We have also observed decreased levels of tissue lipids in mother and litters born to alcoholic rats given N-acetylcysteine when compared to alcoholic rats. The levels of TBARS in liver, kidney were also decreased both in mother and litter born to alcohol + N-acetylcysteine, while the activities of SOD and CAT were increased in liver of alcoholic rats given N-acetylcysteine when compared to alcoholic rats. Histopathological studies also showed the protective effect of N-acetylcysteine in both mother and litter in liver and kidney against alcoholic induced toxicity.     

Cleft palate following prenatal food restriction in mice: association with elevated maternal corticosteroids.

Following food restriction of pregnant CD-1 mice, the maternal serum corticosteroid levels and incidence of skeletal and visceral abnormalities of the progeny were determined. Dams from the groups receiving restricted food on gestation days 6 to 15 had increased resorptions, and the progeny had delayed ossification of the skeleton with cleft palate accompanied by delayed ossification of the presphenoid bone. The incidence and intensity of these changes was correlated with the level of food restriction. The incidence of cleft palate for progeny from dams receiving 4.0 to 2.5 g of feed per day ranged from 6.3% to 100.0%, respectively. Serum corticosteroid levels of pregnant control and food restricted dams were similar on gestation day 10, but the levels of food restricted dams were 6 to 10 times greater than controls on gestation day 15. Thus cleft palate, delayed skeletal ossification, and increased resorptions are predictably produced by food restriction of CD-1 mice and were highly correlated with the elevated endogenous serum corticosteroid levels.     

Hepatoprotective effect of Hibiscus hispidissimus Griffith, ethanolic extract in paracetamol and CCl4 induced hepatotoxicity in Wistar rats

Hibiscus hispidissimus Griff. is used in tribal medicine of Kerala, the southern most state of India, to treat liver diseases. In the present study, the effect of the ethanolic extract of Hibiscus hispidissimus whole plant on paracetamol (PCM)-induced and carbon tetrachloride (CCl4)-induced liver damage in healthy Wistar albino rats was studied. The results showed that significant hepatoprotective effects were obtained against liver damage induced by PCM and CCl4 as evidenced by decreased levels of serum enzymes, glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase (SAKP), serum bilirubin (SB) and an almost normal histological architecture of the liver of the treated groups compared to the toxin controls. The extract also showed significant antilipid peroxidant effects in vitro, besides exhibiting significant activity in quenching 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical, indicating its potent antioxidant effects.     

Hepatotoxicity and cholestasis in rats induced by the sesquiterpene, 9-oxo-10,11-dehydroageraphorone, isolated from Eupatorium adenophorum

Eupatorium adenophorum leaves cause hepatotoxicity and cholestasis in rats. The hepatotoxicant has been characterized as 9-oxo-10,11-dehydroageraphorone (ODA), a cadinene sesquiterpene. Oral administration of ODA, mixed in feed to rats, caused jaundice in 24 h. The liver of the intoxicated animals had focal areas of hepatocellular necrosis, proliferation, and dilation of bile ducts with degenerative changes in the lining epithelium. There was marked increase in the conjugated form of plasma bilirubin and in the activities of the enzymes glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltranspeptidase, glutamate dehydrogenase, and 5'-nucleotidase. The histopathological lesions in liver and biochemical profile of marker enzymes show that ODA induced hepatotoxicity and cholestasis in rats. This is the first report on the toxicity of a cadinene sesquiterpene in rats.     

Parameters of energy and nitrogen metabolism in rats under insulin-induced hypoglycemia

Repeated severe insulin-induced hypoglycemia in rats has led to an increase in aminotransferase, glutaminase, and glutamate dehydrogenase activities in the liver; protease activities in tissues; and in blood serum levels of free fatty acids, urea, and uric acid. These changes are indicative of gluconeogenesis activation in animals exposed to hyperinsulinization. Decreased rates of glycolysis and glycogenolysis, reduced activities of NADP-dependent dehydrogenases, and substantial changes in the activities of enzymes responsible for metabolism of nucleotides and transmitter amino acids have been observed in the brain. All these changes are mainly associated with hypoglycemia and activation of the contrainsular system and can play a significant role in pathogenesis of posthypoglycemic encephalopathy.     

Hepatoprotective and antioxidant effect of tender coconut water on carbon tetrachloride induced liver injury in rats

Hepatoprotective and antioxidant effects of tender coconut water (TCW) were investigated in carbon tetrachloride (CCl4)-intoxicated female rats. Liver damage was evidenced by the increased levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and decreased levels of serum proteins and by histopathological studies in CCl4-intoxicated rats. Increased lipid peroxidation was evidenced by elevated levels of thiobarbituric acid reactive substance (TBARS) viz, malondialdehyde (MDA), hydroperoxides (HP) and conjugated dienes (CD), and also by significant decrease in antioxidant enzymes activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx) and glutathione reductase (GR) and also reduced glutathione (GSH) content in liver. On the other hand, CCl4-intoxicated rats treated with TCW retained almost normal levels of these constituents. Decreased activities of antioxidant enzymes in CCl4-intoxicated rats and their reversal of antioxidant enzyme activities in TCW treated rats, shows the effectiveness of TCW in combating CCl4-induced oxidative stress. Hepatoprotective effect of TCW is also evidenced from the histopathological studies of liver, which did not show any fatty infiltration or necrosis, as observed in CCl4-intoxicated rats.     

Inhibition of cholesterol absorption and synthesis in rats by sesamin

The effects of sesamin, a lignan from sesame oil, on various aspects of cholesterol metabolism were examined in rats maintained on various dietary regimens. When given at a dietary level of 0.5% for 4 weeks, sesamin reduced the concentration of serum and liver cholesterol significantly irrespective of the presence or absence of cholesterol in the diet, except for one experiment in which the purified diet free of cholesterol was given. On feeding sesamin, there was a decrease in lymphatic absorption of cholesterol accompanying an increase in fecal excretion of neutral, but not acidic, steroids, particularly when the cholesterol-enriched diet was given. Sesamin inhibited micellar solubility of cholesterol, but not bile acids, whereas it neither bound taurocholate nor affected the absorption of fatty acids. Only a marginal proportion (ca. 0.15%) of sesamin administered intragastrically was recovered in the lymph. There was a significant reduction in the activity of liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase after feeding sesamin, although the activity of hepatic cholesterol 7 alpha-hydroxylase, drug metabolizing enzymes, and alcohol dehydrogenase remained uninfluenced. Although the weight and phospholipid concentration of the liver increased unequivocally on feeding sesamin, the histological examination by microscopy showed no abnormality, and the activity of serum GOT and GPT remained unchanged. Since sesamin lowered both serum and liver cholesterol levels by inhibiting absorption and synthesis of cholesterol simultaneously, it deserves further study as a possible hypocholesterolemic agent of natural origin.     

Effect of DL-alpha-lipoic acid on mitochondrial enzymes in aged rats.

Mitochondrial dysfunction appears to contribute to some of the loss of function accompanying ageing. Mitochondria from aged tissue use oxygen inefficiently impairing ATP synthesis and results in increased oxidant production. A high flux of oxidants not only damages mitochondria, but other important cell biomolecules as well. In the present investigation, the levels of lipid peroxidation, oxidized glutathione, non-enzymatic antioxidants and the activities of mitochondrial enzymes were measured in liver and kidney mitochondria of young and aged rats before and after lipoic acid supplementation. In both liver and kidney increase in the levels of mitochondrial lipid peroxidation and oxidized glutathione and decrease in the levels of antioxidants and the activities of mitochondrial enzymes were observed in aged rats. DL-alpha-lipoic acid supplemented aged rats showed a decrease in the levels of lipid peroxidation and oxidized glutathione and increase in the levels of reduced glutathione, vitamins C and E and the activities of mitochondrial enzymes like isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, NADH-dehydrogenase and cytochrome-c-oxidase. Thus, lipoic acid reverses the age-associated decline in endogenous low molecular weight antioxidants and mitochondrial enzymes and, therefore, may lower the increased risk of oxidative damage that occurs during ageing. From our results it can be concluded that lipoic acid supplementation enhances the activities of mitochondrial enzymes and antioxidant status and thereby protects mitochondria from ageing.     

Teratogenicity of carbamazepine in rats

The teratogenicity of carbamazepine (CBZ) was investigated in Sprague-Dawley CD rats at doses of 0, 200, 400, and 600 mg/kg administered by gavage in corn oil on days 7-18 of gestation in a dosage volume of 2 ml/kg. The CBZ-600 dose was maternally toxic in that dams in this group weighed 30.6% less than controls by E20. This group had significantly increased resorptions, reduced live fetal weight (51.6% less than controls), and increased skeletal and visceral abnormalities. The CBZ-400 dose also significantly reduced maternal weight gain during gestation to 26.6% less than controls by E20. No significant increase in resorptions occurred in this group; live fetuses weighted 42.9% less than controls and showed an increase in visceral, but not skeletal, abnormalities. The CBZ-200 dose did not significantly affect maternal weight gain or increase resorptions or fetal abnormalities but did reduce fetal body weight (20.3% less than controls). Maternal serum total CBZ concentrations 1 hr after the final dose were 22.9, 27.9, and 34.4 micrograms/ml for the 200, 400, and 600 mg/kg groups, respectively. These levels were little changed 6 h post-treatment. CBZ was 65-70% serum protein bound across dose groups. Human therapeutic levels of CBZ are 4-12 micrograms/ml and the drug is typically 80% serum protein bound. This suggests that abnormalities in rats occur at concentrations well above the human therapeutic range. However, a no-effect level was not found for fetal body weight. Further experiments will be required to determine how much lower doses will need to be in order to find a no-effect level for fetal body weight. Nevertheless, the present data suggest that CBZ is not potent at inducing malformations in rats.     

Attachment of rat hepatocytes to plastic substrata in the absence of serum requires protein synthesis.

Female rats were pair-fed nutritionally adequate liquid diets containing either ethanol (36 % of total cal.) or isocaloric carbohydrates (controls) for 4 weeks. Compared to controls, chronic alcohol consumption led to slightly increased activities of various hepatic enzymes in the serum. Paracetamol administered 18 hours after ethanol withdrawal resulted within 18 hours in a significant increase of serum GOT and GPT activities, which was much more pronounced in rats fed ethanol chronically than in their pair-fed controls. Thus, chronic alcohol consumption predisposes to increased hepatotoxicity due to paracetamol.     

Effects of Buprenorphine on Balance of Oxidant/Antioxidant System in the Different Ages of Male Rat Liver

Our knowledge about a link between buprenorphine and hepatotoxicity is controversial. This study evaluated the effects of buprenorphine on the liver of young, adult, and aged rats. For this reason, young, adult, and aged rats received intraperitoneally 0.25, 0.5, and 1 mg/kg buprenorphine for 30 days. The present results revealed that the normal aging was associated with a significant decrease in the activities of antioxidant enzymes, and an increase in the liver lipid peroxidation, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities in the aged rats. This study also demonstrated that buprenorphine led to a significant increase in the serum activities of ALT, AST, and LDH as well as liver lipid peroxidation content with a decrease in the antioxidant enzymes in the liver of buprenorphine‐treated aged rat versus the aged matched control animals. In conclusion, the present results demonstrate that buprenorphine deteriorated oxidative damage in the aged livers.