Protective effects of vitamin E on carbon tetrachloride-induced liver damage in rats

In this study we investigated whether the increase of hepatic vitamin E content by intraperitoneal administration, influences chronic liver damage induced by carbon tetrachloride (CCl(4)) in rats. Thirty adult male Wistar rats were divided into three groups. The first group was used as a control and the rats in the second group were administered CCl(4) in olive oil subcutaneously. Rats in the third group were administered intraperitoneally vitamin E (dl-alpha-tocopherol acetate, 100 mg kg(-1)). This administration was performed three times per week for five weeks. Liver samples were used for the determination of vitamin E levels, glutathione peroxidase (GSHPx) activities and histological examination. Serum levels of alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyltranspeptidase, total and conjugated bilirubin were significantly (p<0.05, p<0.01, p<0.001) higher in animals treated with CCl(4) than in the controls and had returned to normal values by the administration of vitamin E + CCl(4 ). Liver vitamin E levels were significantly (p<0.05) lower in the CCl(4) group than in the control group. However, the liver vitamin E content was significantly (p<0.01, p<0.001) increased in the vitamin E + CCl(4) injected group. On the other hand, liver GSHPx activity was not statistically different among the groups. On histological examination, vitamin E administered animals showed incomplete, but significant, prevention of liver necrosis and cirrhosis induced by CCl(4 ). these data indicate that intraperitoneally administered vitamin E has protective effects against CCl(4)-induced chronic liver damage and cirrhosis as evidenced by biochemical data and conventional histological examination.     

Protective effect of cornuside against carbon tetrachloride-induced acute hepatic injury

This study elucidated the effects of cornuside on carbon tetrachloride (CCl?)-induced hepatotoxicity. Rats were treated intraperitoneally with 0.5 mL/kg of CCl?. Sixteen h after CCl? treatment, the levels of serum aminotransferases, tumor necrosis factor-α (TNF-α), and lipid peroxidation were significantly elevated, whereas the hepatic antioxidative enzyme activities were decreased. These changes were attenuated by cornuside. Histological studies also indicated that cornuside inhibited CCl?-induced liver damage. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were elevated after CCl? treatment, while cytochrome P450 2E1 (CYP2E1) expression was suppressed. Cornuside treatment inhibited the formation of liver nitrite, and reduced the overexpression of iNOS and COX-2 proteins, but restored the liver CYP2E1 content as compared with the CCl?-treated rats. Our data indicate that cornuside protects the liver from CCl?-induced acute hepatotoxicity, perhaps due to its ability to restore the CYP2E1 function and suppress inflammatory responses, in combination with its capacity to reduce oxidative stress.     

肝再生剌激因子对小鼠实验性急性肝损伤的保护作用

A hepatic stimulator substance (HSS) was extracted from the liver of male weanling SD rats according to the method of LaBrecque. The mice were injected with carbon tetrachloride or D-galactosamine to induce hepatic injuries and the protective effect of HSS on thus induced hepatic damage was investigated. The results were as follows: (1) HSS could suppresses the elevation of sGPT and sGOT induced by carbon tetrachloride intoxication in a dose-dependent manner. (2) Hepatic histological findings indicated that the degree of CCl4 or D-galactosamine-induced hepatic lesions could be lessened by HSS. (3) CCl4-induced reduction of hepatic mitochondrial succinic dehydrogenase activity could be restored by HSS. (4) Insulin-glucagon enhanced the survival of D-galactosamine intoxicated mice and stimulated hepatocyte proliferation, thus showing less pronounced hepatic damage.     

Extracts of Phellinus linteus grown on germinated brown rice suppress liver damage induced by carbon tetrachloride in rats

Extracts of Phellinus linteus (EPB), grown on germinated brown rice, protected rats from liver injury induced by carbon tetrachloride (CCl4). Peroxidation products in the liver were decreased to 10% by EPB. Catalase and superoxide dismutase activities were significantly decreased to 55% and 39% by CCl4 administration, but EPB blocked this effect, resulting in enzyme activities at control levels. Expression of cytochromeP450 2E1 (CYP2E1) protein was significantly decreased to 88% in CCl4-treated rats but remained at control levels when EPB was also administered. EPB did not affect the altered fatty acid composition induced by CCl4. The hepatoprotective effect of EPB may be mediated by EPB's prevention of CCl4-induced CYP2E1 degradation.     

Effects of acute carbon tetrachloride poisoning on vitamin D3 metabolism in the rat

To achieve biologic potency, vitamin D must undergo two successive hydroxylations, first, in the liver and then, in the kidney. Carbon tetrachloride is known to cause extensive damage to the liver, but its effect on vitamin D metabolism has not been studied thoroughly. The effect of carbon tetrachloride on renal hydroxylation of 25-hydroxyvitamin D3 has not been studied. To evaluate the acute effect of carbon tetrachloride on vitamin D metabolism in the liver, vitamin D depleted rats received a single intraperitoneal injection of carbon tetrachloride (2.0 mL/kg body weight). After 24 h, they were given 55, 550, or 5050 pmol [3H]vitamin D3 intravenously. Twenty-four hours after injection of [3H]vitamin D3, aliquots of serum and liver were analyzed for [3H]vitamin D3 and its metabolites by high performance liquid chromatography. Sera of carbon tetrachloride treated rats had higher [3H]vitamin D3 and [3H]25-hydroxyvitamin D and lower [3H]1,25-dihydroxyvitamin D3 concentrations than did control sera. Livers of carbon tetrachloride treated rats contained more [3H]vitamin D3, [3H]25-hydroxyvitamin D3, and more fat. Liver histology showed massive centrilobular necrosis in the treated rats. Thus, our experiment in rats given an acute dose of carbon tetrachloride provided no evidence of impairment of vitamin D metabolism by the liver, but offered a suggestion that 25-hydroxyvitamin D3 metabolism by the kidney might be impaired. To determine the acute effect of carbon tetrachloride on metabolism of vitamin D3 by the kidney, we studied hydroxylation of [3H]25-hydroxyvitamin D3 in isolated perfused kidney. Kidneys from the treated rats showed a 66% reduction in [3H]1,25-dihydroxyvitamin D3 production.     

肝细胞生长因子对四氯化碳损伤肝细胞的保护作用及相关基因表达

利用无血清原代培养大鼠肝细胞,观察重组人肝细胞生长因子（ｒｈＨＧＦ）对ＣＣｌ４染毒肝细胞的保护作用。结果表明：（１）ｒｈＨＧＦ（５ｎｇ／ｍｌ）预自理后可显著提高ＣＣｌ４（１５ｍｍｏｌ／Ｌ）染毒肝细胞存活率,降低细胞内丙氨酸氨基转移酶（ＡＬＴ）、Ｋ＾＋的漏出;（２）表皮生长因子（ＥＧＦ,５０ｎｇ／ｍｌ）和ｒｈＨＧＦ（５ｎｇ／ｍｌ）合用预处理肝细胞,ＣＣｌ４染毒后细胞内ＡＬＴ、Ｋ＾＋漏出较ｒｈＨＧＦ和     

Hormonal responsiveness of liver cells during the liver regeneration process induced by carbon tetrachloride administration

In control rats most of the ureagenic effect of adrenaline is mediated through alpha1-adrenoceptors with little participation of beta-adrenoceptors. Administration of carbon tetrachloride to rats induces significant changes in the adrenergic responsiveness of their hepatocytes. In rats intoxicated 3-5 days before the experiments were performed there is a marked increase in the beta-adrenergic and a decrease in the alpha-adrenergic responsiveness of the hepatocytes. The alpha1-adrenergic responsiveness increased with time reaching its basal level 15 days after the administration of carbon tetrachloride; simultaneously, the betal-adrenergic responsiveness was decreased. No change in the responsiveness to vasopressin and angiotensin II was observed in intoxicated animals as compared to the controls. In contrast, the responsiveness to glucagon was increased. Increased ability of local anesthetics to decrease urea production was observed in cells from intoxicated animals. It is suggested that changes at the plasma membrane level (lipids, receptors and transducing proteins) might participate in producing these effects.     

Binding of products originating from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomal membrane.

The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsomes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalently bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro peroxidation of liver microsomes could be detected in the in vivo intoxication with carbon tetrachloride, rats given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisoning, the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.     

Antioxidant activity of Aulosira fertilisima on CCl4 induced hepatotoxicity in rats

Free radicals cause cell injury, when they are generated in excess or when the antioxidant defense is impaired. Carbon tetrachloride (CCl4) is used as a model for liver injury. In this study antioxidant activity of ethanol extract of A. fertilisima (EEA) was investigated using CCl4 intoxicated rat liver as the experimental model. Oral administration of EEA at a dose of 100 mg/kg body weight, for 14 consecutive days, the rate of the production of antioxidant enzymes like super oxide dismutase, catalase, glutathione peroxidase and glutathione transferase in rats compared to the CCl4 treated group without any supporting treatment. Liver damage is detected by the measurement of the activities of serum enzymes like aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transpeptidase and alkaline phosphatase which were released in to the blood from damaged cells. The normalization of these enzymes levels was observed in rats treated with EEA (100 mg/kg body weight) by reducing the leakage of the above enzymes in to the blood. The findings provide a rationale for further studies on isolation of active principles and its pharmacological evaluation. Protection offered by silymarin (standard reference drug) seemed relatively greater.     

Reversal of hepatotoxin-induced pre-fibrogenic events by Emblica officinalis--a histological study

Efficacy of a herbal product of E. officinalis (fruit) (EO) has been evaluated against carbon tetrachloride (CCl4) and thioacetamide (TAA) induced changes in rat liver. Chronic treatment of CCl4 and TAA revealed abnormal histopathology indicative of pre-fibrogenic events. EO reversed such alterations with significant regenerative changes suggestive of its preventive role in prefibrogenesis of liver.     

Experimental study of the use of colchicine in alcoholic liver diseases

Rats kept on a standard diet were subdivided into several experimental groups: group 1, control; group 2, animals receiving ethyl alcohol for 10 days; group 3, animals receiving ethyl alcohol for 3 months; group 4, animals receiving colchicine; group 5, animals receiving alcohol in combination with colchicine; group 6, animals receiving alcohol in combination with carbon tetrachloride (CCl4); and group 7, animals receiving alcohol in combination with CCl4 and colchicine. Electron microscopy of the rat liver has shown that colchicine inhibited significantly the onset of hepatic fibrosis and degenerative changes in hepatocyte organells induced by hepatotoxins (alcohol alone or alcohol in combination with CCl4). Colchicine also inhibited monooxygenase activity in the liver homogenate of experimental rats. Possible mechanisms of hepatoprotective colchicine effect are discussed.     

Increased activities of liver cathepsins T and D in carbon tetrachloride-treated rats

Changes in activities of a new proteinase cathepsin T as well as some other lysosomal acid proteinases and hydrolases were examined in liver homogenate from rats treated with a single hepatotoxic dose of carbon tetrachloride. The most striking changes were several-fold increases of liver cathepsin T and D activities over their levels in untreated rats 3 days after administration of the agent to rats. Increase of cathepsin T was greater than that of cathepsin D at all doses of the hepatotoxin examined. The activities of N alpha-benzoyl-DL-arginine 2-naphthylamide hydrolase, acid phosphatase, beta-galactosidase and beta-glucuronidase in poisoned rat liver were unchanged or only slightly increased. Cathepsin T and D activities were less enhanced in mitochondrial lysosomal fractions than in the homogenate, and were greatly elevated in the supernatant fractions of liver from the treated rats. As judged from the molecular weights, the elevated activities of cathepsins T and D in the treated rat liver could be attributable to the two cathepsins themselves and not to other proteinases. Administration to rats of other hepatotoxic agents, thioacetamide and dimethylnitrosamine, also induced the elevation of the two cathepsin activities in liver, but on partial hepatectomy the activities of liver cathepsins T and D did not show such marked increases. Nonparenchymal liver cell fractions were responsible for almost all the increased activities of liver cathepsins T and D. It is possible that cathepsins T and D play a role in the heterolytic breakdown of hepatocyte molecules following CCl4 poisoning.     

Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver

After 12, 18, and 24 h of oral administration of carbon tetrachloride (as a 1:1 mixture with mineral oil: 4 ml/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group that was given mineral oil (4 ml/kg). In plasma, the activity of caspase-3 was barely detectable in the control rat, but increased significantly 24 h after drug administration along with a dramatic increase in glutamate oxaloacetate transaminase. These results indicate that carbon tetrachloride causes apoptosis in the liver by activating caspase-3, which is released to plasma by secondary necrosis. After 18 and 24 h of carbon tetrachloride administration, the liver concentration of hydrophilic vitamin C was decreased significantly, while that of hydrophobic vitamin E was not affected. The plasma concentration of vitamins C and E was not influenced significantly. These results suggest that carbon tetrachloride induces oxidative stress mainly in the aqueous phase of the liver cell.     

Synthesis of lysyl oxidase in experimental hepatic fibrosis.

The synthesis of lysyl oxidase, which initiates the cross-linking of collagen and elastin, was investigated in carbon tetrachloride (CCl4) induced fibrotic liver of rat. Lysyl oxidase activity of the fibrotic liver was 4 times greater than that of normal liver. mRNAs from the livers of normal and CCl4-treated rats were prepared for in vitro protein synthesis and the products were analyzed by immunoprecipitation with a monoclonal antibody against lysyl oxidase. The mRNAs from the fibrotic liver gave more than 3 times higher level of messenger copies for lysyl oxidase than did mRNAs from normal liver. The molecular weight of the nascent lysyl oxidase was 48,000.     

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.     

Agaricus blazei Murill as an efficient hepatoprotective and antioxidant agent against CCl4-induced liver injury in rats

Agaricus blazei Murill is one of the very popular edible medicinal mushrooms. The present study investigated the protective effect of this biologically active mushroom on the tissue peroxidative damage and abnormal antioxidant levels in carbon tetrachloride induced hepatotoxicity in male albino rats. Male albino rats of Sprague–Dawley strain weighting (120–150 g) were categorized into five groups. The first group served as the normal control, the second and the third groups were treated with Agaricus blazei Mushroom extract and carbon tetrachloride dose, respectively. Fourth group (protective group) was first treated with Agaricus blazei Mushroom extract followed by carbon tetrachloride treatment and fifth (therapeutic group) with carbon tetrachloride first followed by Agaricus blazei Mushroom treatment. The wet fruiting bodies of mushroom Agaricus blazei Murill, crushed and suspended in distilled water was administered orally to the treated groups of male albino rats. The activities of various enzymes (aspartate and alanine transaminase, lactate dehydrogenase, glutathione reductase), levels of non-enzymatic antioxidants (glutathione, vitamin C, vitamin E) and level of lipid peroxidation (malondialdehyde) were determined in the serum of all the experimental animals. Decrease in all the enzymes and non-enzymatic antioxidant, along with an increase in the lipid peroxidative index (malondialdehyde) was found in all the carbon tetrachloride treated rats as compared with normal controls. Also increase level of non-enzymatic antioxidant along with the decrease level in malondialdehyde was found in all experimental animals which were treated with Agaricus blazei Mushroom extract as compared with normal controls. The findings indicate that the extract of Agaricus blazei Murill can protect the liver against carbon tetrachloride induced oxidative damage in rats and is an efficient hepatoprotective and antioxidant agent against carbon tetrachloride induced liver injury.     

The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fractions in vitro. Inhibitory effects of free-radical scavengers and other agents

1. The effects of a number of free-radical scavengers and other agents on the stimulation of malonaldehyde production due to low concentrations of carbon tetrachloride have been studied in rat liver microsome suspensions. 2. Promethazine, propyl gallate and NN′-diphenyl-p-phenylenediamine were extremely active in inhibiting the stimulation of malonaldehyde production due to carbon tetrachloride; inhibitory effects were demonstrable with these agents at 0.1μm. 3. Low concentrations (1–100nm) of vitamin E–polyethylene glycol 1000–succinate increased the stimulation of malonaldehyde production due to carbon tetrachloride, but higher concentrations of the vitamin E preparation decreased both the stimulation due to carbon tetrachloride and the endogenous peroxidation that occurs in the absence of carbon tetrachloride. 4. Other agents tested that were effective in the range 1–20μm in decreasing the stimulation of malonaldehyde production due to carbon tetrachloride were inosine, desferrioxamine and EDTA. Agents tested that were not effective, except at very high concentrations (100μm or greater), were Nupercaine, Cetab and sodium phenobarbitone. 5. The results are discussed in terms of the mechanisms responsible for the observed inhibitions of malonaldehyde production, and of the relevance of the in vitro system to the liver damage produced by carbon tetrachloride in vivo.     

Mitochondrial phospholipid bilayer structure is ruined after liver oxidative injury in vivo

The purpose of this study was to investigate whether, after oxidative injury in vivo, liver mitochondrial phospholipids suffered from structural defects similar to those we have previously observed after either chemical oxidation or respiration state IV incubation of isolated mitochondria in vitro. Oxidative injury of the liver was simulated by endogastric administration of CCl4 to rats in variable amounts for different times, under various conditions. Measurements of the phospholipid bilayer packing order were carried out by electron paramagnetic resonance (EPR) spectrometry of oriented planar samples of phospholipids extracted from liver mitochondria, spin labeled with 5-doxylstearoyl-lecithin. Disordering of the bilayer was revealed by the anisotropy loss of EPR spectra and reached a maximum value 4.5 h after CCl4 administration, vanishing thereafter. The observed disorder also increased with the amount of CCl4 administered, showing distinct dose-dependence, while administration of resveratrol soon after carbon tetrachloride decreased bilayer disordering by 50%. On the contrary, the order parameter S of spin labeled lecithin in isolated mitochondrial membranes from intoxicated rats revealed no change in membrane fluidity after oxidative stress. It is concluded that the phospholipid damage leading to disturbed bilayer geometry after oxidative attack already observed in model membranes and in isolated mitochondria in vitro also occurs in a simulated pathological state in vivo, indicating its possible occurrence also in real oxidative stress-linked pathologies as a contribution to the onset/sustaining of related diseases.     

Effects of ventromedial and lateral hypothalamic stimulation on chemically-induced liver injury in rats

The effects of hypothalamic stimulation on experimental liver injury induced by carbon tetrachloride (CCl4) or dimethylnitrosamine (DMN) were studied in rats, by measuring plasma alanine aminotransferase (ALT) activity as an index of acute liver injury. Electrical stimulation of the ventromedial hypothalamus (VMH) in CCl4-treated rats caused a marked increase in plasma ALT activity, accompanied by a significant decrease in ALT activity in the liver, although CCl4 treatment alone had no significant effect on plasma ALT activity. A similar effect of VMH stimulation on plasma ALT activity was observed in rats treated with DMN, another hepatotoxic chemical. No such exaggerated effect of VMH stimulation on plasma ALT activity was observed after stimulation of the lateral hypothalamic area (LH). Surgical sympathetic denervation of the liver greatly suppressed the increase in plasma ALT activity after CCl4 injection and VMH stimulation. Measurement of regional blood flow indicated that VMH stimulation did not produce a significant change in blood flow to the liver. These results suggest that the VMH is involved in the progress of chemically-induced liver injury through activation of the sympathetic nerve (hepatic nerves), possibly by affecting liver metabolism more than the blood flow change to the liver.