Characterization and protection on acute liver injury of a polysaccharide MP-I from Mytilus coruscus

In this study, we analyzed a water-soluble polysaccharide MP-I isolated from Mytilus coruscus. MP-I was obtained by hot-water extraction, anion-exchange and gel-permeation chromatography. Complete hydrolysis, periodate oxidation, methylation analysis, as well as Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectroscopy were conducted to elucidate its structure. MP-I was subjected to investigate the protective effect on carbon tetrachloride (CCl(4)) induced liver damage in male Kunming mice. Based on the data obtained, MP-I was found to be an alpha-(1-->4)-D-glucan, branched with a single alpha-D-glucose at the C-6 position every eight residue, on average, along the main chain. Based on the calibration with Dextran, the glucan had a molecular weight of about 1.35 x 10(6) Da. Pharmacological studies revealed that MP-I could decrease serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), and hepatic malondialdehyde aldehydes (MDA) levels, increase the hepatic total superoxide dismutase (T-SOD) activity, and improve hepatic damage in the CCl(4) induced liver injury in mice in a dose-dependent manner. The results suggest that the possible mechanism is due to its antioxidant activity of MP-I.     

Plasma amino acids in four models of experimental liver injury in rats

Summary We studied the plasma amino acid profiles in four models of hepatic injury in rats. In partially hepatectomized rats (65% of liver was removed) we observed significant increase of aromatic amino acids (AAA; i.e. tyrosine and phenylalanine), taurine, aspartate, threonine, serine, asparagine, methionine, ornithine and histidine. Branched-chain amino acids (BCAA; i.e. valine, leucine and isoleucine) concentrations were unchanged. In ischemic and carbon tetrachloride acute liver damage we observed extreme elevation of most of amino acids (BCAA included) and very low concentration of arginine. In carbon tetrachloride induced liver cirrhosis we observed increased levels of AAA, aspartate, asparagine, methionine, ornithine and histidine and decrease of BCAA, threonine and cystine. BCAA/AAA ratio decreased significantly in partially hepatectomized and cirrhotic rats and was unchanged in ischemic and acute carbon tetrachloride liver damage. We conclude that a high increase of most of amino acids is characteristic of fulminant hepatic necrosis; decreased BCAA/AAA ratio is characteristic of liver cirrhosis; and decrease of BCAA/AAA ratio may not be used as an indicator of the severity of hepatic parenchymal damage.Abbreviations BCAA branched-chain amino acids (i.e. valine, leucine and isoleucine) - AAA aromatic amino acids (i.e. tyrosine and phenylalanine)     

Hepatoprotective activity of Psidium guajava Linn. leaf extract

The study was designed to evaluate the hepatoprotective activity of P. guajava in acute experimental liver injury induced by carbon tetrachloride, paracetamol or thioacetamide and chronic liver damage induced by carbon tetrachloride. The effects observed were compared with a known hepatoprotective agent, silymarin. In the acute liver damage induced by different hepatotoxins, P. guajava leaf extracts (250 and 500mg/kg, po) significantly reduced the elevated serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and bilirubin. The higher dose of the extract (500 mg/kg, po) prevented the increase in liver weight when compared to hepatoxin treated control, while the lower dose was ineffective except in the paracetamol induced liver damage. In the chronic liver injury induced by carbon tetrachloride, the higher dose (500 mg/kg, po) of P. guajava leaf extract was found to be more effective than the lower dose (250 mg/kg, po). Histological examination of the liver tissues supported the hepatoprotection. It is concluded that the aqueous extract of leaves of guava plant possesses good hepatoprotective activity.     

Branched-chain amino acid-enriched nutrients stimulate antioxidant DNA repair in a rat model of liver injury induced by carbon tetrachloride

Oxidative stress (OS) plays an important role in the progression of chronic liver disease including organ injury and hypoalbuminemia. Long-term oral supplementation with branched-chain amino acids (BCAAs) can inhibit liver dysfunction but their role in the prevention of liver fibrosis and injury to the liver is unclear. The aim of this study was to assess how BCAAs preserve liver function from OS. To investigate how BCAAs specifically prevent OS, we evaluated the effect of oral supplementation with BCAAs on OS using a rat liver cirrhosis model. Liver cirrhosis was induced in ten male Sprague–Dawley rats by administering carbon tetrachloride for 12?weeks. Five of the ten carbon tetrachloride-treated rats were assigned to a control group and five to a BCAA group. BCAA-supplementation significantly preserved plasma albumin concentrations and significantly inhibited the occurrence of organ injury as determined by blood chemistry analysis. Hepatic expression of OGG1 mRNA was increased in the BCAA group compared to the control group. In the BCAA group, increased hepatic levels of OGG1 protein were found by western blot. On the other hand, the number of 8-OHdG-positive cells was significantly higher in liver sections taken 1?month after carbon tetrachloride treatment. Furthermore, OGG1-positive cells were significantly increased in the hepatocytes around the central vein. BCAA was found to reduce OS, which could possibly lead to a decrease in the occurrence of hypoalbuminemia and organ injury. Our results indicate that BCAA-enriched nutrients stimulate antioxidant DNA repair in a rat model of liver injury induced by carbon tetrachloride.     

Fourier-transform infrared spectroscopy: a pharmacotoxicologic tool for in vivo monitoring radical aggression

Among the physico-chemical methods that can be used to investigate induced peroxidation in living cells, Fourier transform infrared (FT-IR) spectroscopy appears to be a valuable technique as it is non-destructive and sensitive for monitoring changes in the vibrational spectra of samples. We examined microsomal fractions from rat liver and brain by FT-IR to study the effect of radical aggression induced in vivo by carbon tetrachloride (CCl4). The length of the acyl chains was increased as a consequence of peroxidation induced by the xenobiotic. Moreover, an enhanced level of cholesterol esters and an increase in phospholipids were observed in the liver and the brain, respectively. The conformational structure of the membrane proteins was changed in both the liver and the brain. In the polysaccharide region, we observed an important loss in glucidic structures, such as a decrease in liver glycogen and in some brain glycolipids. These alterations are probably due to the interactions between cells and CCl4 and the metabolic changes caused by CCl4. Thus, FT-IR spectroscopy appears to be an useful tool and an accurate means for rapidly investigating the in vivo biochemical alterations induced by CCl4 in microsomes, and for correlating them with biochemical and physiological data.     

In vivo effect of diosmin on carrageenan and CCl4-induced lipid peroxidation in rat liver microsomes

The aim of this study was to compare the protective effect of a flavonoid, the 3′5,7-trihydroxy-4′-methoxyflavone 7-rutinoside or diosmin, on liver microsomal lipid peroxidation induced in rats by either carbon tetrachloride or carrageenan. Thirty rats were divided into five groups. Group 1 received no chemical product and was considered as control. Groups 2 and 3 received either an intraperitoneal injection of carrageenan or carbon tetrachloride 48 or 24 hours before killing, respectively. Groups 4 and 5 were treated first with an intraperitoneal injection of diosmin and then by carrageenan (group 4) or carbon tetrachloride (group 5) 48 or 24 hours before killing, respectively. The lipoperoxidant effect of carrageenan and carbon tetrachloride was demonstrated by both significant decreases in polyunsaturated fatty acids, principally 20:4 (n− 6) (p < 0.05) and of vitamin A (p < 0.05) in groups 2 and 3. With diosmin treatment, only thiobarbituric acid reactive substances significantly decreased in group 4, whereas vitamin A level increased. These results could suggest that the effect of diosmin differs with the choice of chemical product used; it seems a better antioxidant against products inducing inflammation. © 1996 John Wiley & Sons, Inc.     

4-羟基苯并恶唑-2-酮对四氯化碳致小鼠急性肝损伤保护作用

日的研究4一羟基苯并恶唑-2-酮（4-hydroxy-2-benzoxazolone,HBOA）对四氯化碳所致小鼠急性肝损伤的保护作用,并探讨其疗效机制。方法采用腹腔注射四氯化碳（carbonte trachloride,cch）制备小鼠急性肝损伤模型,HBOA灌胃给药,检测小鼠血清中的乳酸脱氢酶（LDH）活性以及肝组织中过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）含量,并用免疫组化法观察肿瘤坏死因子（TNF-a）的表达情况。结果HBOA能明显降低CCh致急性肝损伤小鼠血清LDH活性,同时升高肝组织中CAT、GSH-Px的活性并降低肝组织中TNF-a的表达。结论HBOA对CCh所致小鼠急性肝损伤有一定的保护作用。     

Protective effect of Phyllanthus fraternus against carbon tetrachloride-induced mitochondrial dysfunction.

The effect of carbon tetrachloride administration on liver mitochondrial function and the protective effect of an aqueous extract of Phyllanthus fraternus were studied in rats. The following changes were observed in mitochondria due to the administration of carbon tetrachloride. 1) A decrease in the rate of respiration, respiratory control ratio and P/O ratio using glutamate and malate or succinate as substrates. 2) A decrease in the activities of NADH dehydrogenase (35%), succinate dehydrogenase (76%) and cytochrome c oxidase (51%). The rate of electron transfer through site I, site II and site III was studied independently and found to be significantly decreased. 3) A decrease in the content of cytochrome aa3 (34%). 4) A significant decrease in the levels of phospholipids particularly cardiolipin and a significant increase in the lipid peroxide level was observed. The carbon tetrachloride induced toxicity may be partly due to the lipid peroxidation and partly due to the effect on protein synthesis. Administration of rats with an aqueous extract of P. fraternus prior to carbon tetrachloride administration showed significant protection on the carbon tetrachloride induced mitochondrial dysfunction on all the parameters studied.     

Effects of 5-azacytidine and 5-aza-2-deoxycytidine on alphafetoprotein levels in mice.

1. Production of alphafetoprotein in adult C3H mice was monitored by radial immunodiffusion both in controls, and in animals treated with carbon tetrachloride, 5-azacytidine, or 5-aza-2-deoxycytidine, either alone or in combination. 2. Carbon tetrachloride routinely induced alphafetoprotein synthesis in our experiments, but neither of the cytidine analogues showed any effects on the serum levels of this protein when administered alone. 3. Treatment of mice with either cytidine analogue prior to carbon tetrachloride injection markedly reduced the consequent production of alphafetoprotein, whereas if carbon tetrachloride injection was followed by a subsequent injection with either cytidine analogue, a markedly enhanced level of serum alphafetoprotein was detected. 4. It is suggested that carbon tetrachloride induces alphafetoprotein production in adult mice by inducing liver damage, followed by synthesis of the protein in the dividing and differentiating cells during recovery. We also propose that the cytidine analogues ablate this response by a cytotoxic effect on the liver cells when they are administered prior to the CCl4, but enhance the alphafetoprotein levels when administered after the CCl4 because they inhibit the methylation of cytidine residues in the recovery cell population in the liver and thus prevent early cessation of synthesis of the protein.     

FTIR study of five complex beta-lactam molecules.

Five monocyclic 4-benzoyl-4-phenyl-beta-lactam derivatives in carbon tetrachloride solutions were studied by FTIR spectroscopy. The Fourier self-deconvolution method was applied to enhance the resolution of the FTIR spectra. The calculated spectra of these five compounds, which were obtained by quantum mechanical methods, were compared with FTIR data. A complete assignment of the vibrational frequencies in the 4000-400 cm(-1) range was made. Several vibrations were selected as being useful to characterize the beta-lactam ring. Substituents on N1 (p-methoxyphenyl) and C3 (methyl, phenyl, maleimidyl, and phthalimidyl) on the beta-lactam ring increase the amide resonance and the planarity of the ring. The optimized geometry along with the total electric charges on the four atoms of the ring support an antibiotic action mechanism by a nucleophilic attack of the enzymes on the carbonyl carbon atom of the beta-lactam ring.     

Quantitative determination of hydroxy fatty acids as an indicator of in vivo lipid peroxidation: oxidation products of arachidonic and docosapentaenoic acids in rat liver after exposure to carbon tetrachloride.

An improved gas chromatography-mass spectrometry method has been applied to the quantitation of both in vitro and in vivo products of lipid peroxidation in rat liver stimulated with carbon tetrachloride. The method avoids problems of autoxidation of unsaturated fatty acids during sample preparation, and the sensitivity permits assays on as little as 1 mg of tissue. This permits small samples of tissue to be obtained by biopsy from the same organ, thus making it possible to perform in vivo time studies on a single animal. Lipids from whole tissue or cell preparations are simultaneously extracted and reduced by catalytic hydrogenation and then saponified and derivatized to their pentafluorobenzyl esters and trimethylsilyl ethers. Quantitation is accomplished by negative ion chemical ionization gas chromatography-mass spectrometry, using either deuterated compounds or naturally occurring fatty acid metabolites as internal standards. Hydroxy fatty acids which result from reduction of the hydroperoxides of arachidonic and docosapentaenoic acids are found to increase within 20 min after exposure of liver or hepatocyte suspensions to carbon tetrachloride.     

肝再生刺激因子对小鼠实验性急性肝损伤的保护作用

本工作采用雄性初断乳 SD 大鼠肝按 LaBrecque 法提取肝再生刺激因子(HSS),用四氯化碳和半乳糖胺分别损伤小鼠肝来研究 HSS 的保肝作用。结果如后:(1)HSS 可使 CCl_4致肝损伤小鼠的血清 GPT 和 GOT 升高幅度降低,并呈量效关系。(2)肝组织切片表明 HSS 可使 CCl_4损伤肝组织的程度减轻。(3)肝组织化学法表明 HSS 可使 CCl_4损害肝细胞线粒体琥珀酸脱氢酶的活性恢复。(4)胰岛素-胰高糖素可降低半乳糖胺所致的小鼠死亡率,减弱对肝组织的损害和刺激肝细胞增殖,这项实验可作为 HSS 具有保肝作用的证据。     

The stimulatory effects of carbon tetrachloride and other halogenoalkanes on peroxidative reactions in rat liver fractions in vitro. General features of the systems used

1. The general features of the reaction by which carbon tetrachloride stimulates lipid peroxidation have been elucidated in rat liver microsomal suspensions and in mixtures of microsomes plus cell sap. The production of lipid peroxides has been correlated with malonaldehyde production in the systems used. 2. The stimulation of malonaldehyde production by carbon tetrachloride requires a source of reduced NADP(+) and is dependent on the extent of the endogenous peroxidation of the microsomal membranes: if extensive endogenous peroxidation occurs during incubation then no stimulation by carbon tetrachloride is apparent. 3. The stimulation of malonaldehyde production by carbon tetrachloride has been shown to be proportional to the square root of the carbon tetrachloride concentration in the incubation mixture. It is concluded that the stimulation of malonaldehyde production by carbon tetrachloride results from an initiation process that is itself dependent on the homolytic dissociation of carbon tetrachloride to free-radical products. 4. The increased production of malonaldehyde due to carbon tetrachloride is accompanied by a decreased activity of glucose 6-phosphatase in rat liver microsomal suspensions. 5. The relative activities of bromotrichloromethane, fluorotrichloromethane and chloroform have been evaluated in comparison with the effects of carbon tetrachloride in increasing malonaldehyde production and in decreasing glucose 6-phosphatase activity. Bromotrichloromethane was more effective, and fluorotrichloromethane and chloroform were less effective, than carbon tetrachloride in producing these two effects. It is concluded that homolytic bond fission of the halogenomethanes is a requisite for the occurrence of the two effects observed in the endoplasmic reticulum.     

茵陈保肝活性部位的初步筛选研究

目的:筛选茵陈发挥保肝作用的有效部位.方法:用系统溶剂分离法将茵陈分离成极性不同的组分.采用四氯化碳(CCl4)致小鼠急性肝损伤模型,按赖氏法测定小鼠血清中AST,ALT的活性.结果:茵陈的三氯甲烷提取物能明显降低CCl4引起的AST,ALT增高.结论:茵陈三氯甲烷提取部位为其发挥保肝作用的有效部位.     

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.     

Cirrhosis and the synthesis of proline and glycine in rat liver

The relation between availability of free amino acids and development of cirrhosis in rat liver has been experimentally evaluated. Levels of free glycine and proline were found to increase when animals are treated with phenobarbital and carbon tetrachloride. This increase is concomitant with increase in collagen and loss of activity of enzymes responsible for degradation of amino acids. It is concluded that elevated levels of proline observed in the serum of cirrhotic patients may be a consequence, rather than a cause, of collagen accumulation in the liver.     

Hepatic collagenolytic activity in rats after carbon tetrachloride poisoning

1. Collagenolytic activity towards acid-soluble collagen labelled with [(14)C]-proline was assayed in rat liver with and without carbon tetrachloride poisoning. The products of enzymic digestion were found to be free amino acids and peptides. 2. The hepatic collagenolytic activity increased under conditions of single-dose and subacute carbon tetrachloride poisoning, and correlated with hydroxyproline content. The highest activity was found during recovery from subacute poisoning. 3. Under the same experimental conditions, hepatic acid-proteinase activity changed independently of the collagenolytic activity and also of hepatic hydroxyproline content. 4. The increased collagenolytic activity during carbon tetrachloride poisoning was found mainly in the supernatant fraction. 5. The ratio of the collagenolytic activity to hepatic hydroxyproline content increased during recovery from single-dose and subacute poisoning, and decreased during subacute poisoning.     

Activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase in the liver of rats with hepatic injury

The activities of four enzymes catalysing post-translational modifications of the collagen polypeptide chains were assayed in the livers of rats with experimental hepatic injury. The liver injury was induced by injecting carbon tetrachloride twice weekly, and assays of the enzymic activities were carried out 2 and 4 weeks after commencement of administration of carbon tetrachloride. The liver homogenates were preincubated with Triton X-100 before the assays, because such treatment was found to increase the activities of all four enzymes in the supernatants of liver homogenates. The activities of all four enzymes had increased by 2 weeks after commencement of carbon tetrachloride administration. No increase was found in the collagen content of the livers at this stage and thus an increase in all four enzyme activities preceded an increase in the collagen content of the liver. A further slight increase was found in three of the enzyme activities during the subsequent 2 weeks of the experiment, whereas no further increase was found in the collagen galactosyltransferase activity. A statistically significant correlation was found between all four enzyme activities, but the magnitude of the increases varied considerably. The largest increase was found in lysyl hydroxylase activity, and at 4 weeks the magnitude of this was about three times that of the collagen galactosyltransferase activity. The results thus indicate that the increased enzyme activities cannot be explained simply by an increase in the number of collagen-producing cells having similar enzyme activity patterns to those of the cells initially present in the liver.     

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.