Pathogenesis of carbon tetrachloride-induced hepatocyte injury bioactivation of CCI4 by cytochrome P450 and effects on lipid homeostasis

The CCl4-induced development of liver damage was studied in monolayer cultures of primary rat hepatocytes: (1) CCl4 caused accumulation of triglycerides in hepatocytes following cytochrome P450 induction with beta-naphthoflavone or metyrapone. Ethanol or a high dose of insulin plus triiodothyronine had the same effect. (2) CCl4 increased the synthesis of fatty acids and triglycerides and the rate of lipid esterification. Cholesterol and phospholipid synthesis from acetate was also increased. (3) CCl4 reduced beta-oxidation of fatty acids as assessed by CO2-release and ketone body formation. Hydrolysis of triglycerides was also reduced. (4) The content of unsaturated fatty acids in microsomal lipids was decreased by almost 50% after incubation with CCl4, while saturated fatty acids increased slightly. (5) CCl4 exerted a pronounced inhibitory effect on the exocytosis of macromolecules (albumin), but did not affect secretion of bile acids from hepatocytes.     

Effects of retinoic acid on the development of liver fibrosis produced by carbon tetrachloride in mice

The role of retinoic acid (RA) in liver fibrogenesis was previously studied in cultured hepatic stellate cells (HSCs). RA suppresses the expression of alpha2(I) collagen by means of the activities of specific nuclear receptors RARalpha, RXRbeta and their coregulators. In this study, the effects of RA in fibrogenesis were examined in carbon tetrachloride (CCl4) induced liver fibrosis in mice. Mice were treated with CCl4 or RA and CCl4, along side control groups, for 12weeks. RA reduced the amount of histologically detectable fibrosis produced by CCl4. This was accompanied by a attenuation of the CCl4 induced increase in alpha2(I) collagen mRNA and a lower (2-fold versus 3-fold) increase in liver hydroxyproline. Furthermore, RA reduced the levels of 3-nitrotyrosine (3-NT) protein adducts and thiobarbituric acid (TBA) reactive substance (TBARS) in the liver, which are formed as results of oxidative stress induced by CCl4 treatment. These in vivo findings support our previous in vitro studies in cultured HSC of the inhibitory effect of RA on type I collagen expression. The data also provide evidence that RA reduces CCl4 induced oxidative stress in liver, suggesting that the anti-fibrotic role of RA is not limited to the inhibition of type I collagen expression.     

Flk1+间充质干细胞减轻四氯化碳导致的肝纤维化的研究

许多慢性肝脏疾病都会发生肝纤维化,但是目前尚缺乏对肝纤维化切实有效的治疗手段。实验发现,Flk1(fetal liver kinase)阳性间充质干细胞(MSC)能够减轻四氯化碳(CCl4)所致小鼠肝纤维化。取雄性BALB／c小鼠骨髓,分离培养Flk1^ MSC,用CCl4制作雌性小鼠肝纤维化模型,在CCl4损伤后立即或1周后经尾静脉注射Flk1^ MSC,2或5周后检测受体小鼠肝脏的纤维化程度和供体细胞的植入。结果发现,CCl4损伤后立即注射Flk1^ MSC,可以使肝脏损伤程度明显减轻,减少胶原沉积,使肝脏羟脯氨酸含量及血清纤维化指标显下降;而损伤1周后注射细胞则无明显变化。免疫荧光、PCR和荧光原位杂交方法证实,在受体肝脏中有供体细胞植入,呈上皮细胞形态,并表达白蛋白,但是数量很少。因此,Flk1^ MSC具有潜在的植入肝组织的能力,并可能启动肝组织的内源性修复,减轻CCl4导致的肝纤维化。     

Morphological, immunohistochemical and ultrastructural changes in dimethylnitrosamine [correction of dimenthylnitrosamine] induced liver injury. Effect of malotilate

Dimethylnitrosamine (DMN) induced liver injury in rats with cell necrosis, inflammation, hemorrhages, increased collagen type III synthesis and basement membrane component laminin and collagen IV localization in perisinusoidal sites. Malotilate ingestion during DMN treatment abolished inflammation and decreased interstitial collagen deposits and vascularization. It affected clearly less DMN-caused hemorrhage. When malotilate treatment was started subsequently to development of DMN-injury, it also caused decrease in inflammation, though less, as well as in collagen III, BM and fibronectin deposits. We suggest that the mode of the malotilate effect on reducing the DMN-induced fibrosis of the liver is via inhibiting the inflammation, decreased fibronectin deposition possibly also playing a role.     

The collagenolytic effects of the traditional Chinese medicine preparation,Han-Dan-Gan-Le,contribute to reversal of chemical-induced liver fibrosis in rats

Han-Dan-Gan-Le (HDGL), a Chinese herb preparation composed of Stephaniat tetrandra, Salvia miltorrhiza, Radix paeoniae, Astragalus membranaceus, and Ginkgo biloba, has been used to treat human liver fibrosis. This study was designed to examine the therapeutic effect of HDGL on chemical-induced liver fibrosis in adult Wistar rats. Liver fibrosis was produced in rats by carbon tetrachloride (1.2 ml CCl(4)/kg, 2 times/week, after an initial dose of 5.0 ml CCl(4)/kg, sc), plus a diet of 20% fat, 0.05% cholesterol (continuous) and 30% alcohol in the drinking water ad libitum (every other day) for 8 weeks. HDGL (0.5 and 1.0 g/kg, ig, daily for 6 weeks) was administered to rats 72 hrs after the last dose of CCl(4) to examine its therapeutic effects on chemical-induced liver fibrosis. Upon pathological examination, the HDGL treatment had significantly reversed chemical-induced liver fibrosis and other hepatic lesions. Hepatic collagen accumulation induced by CCl(4) was markedly reduced by HDGL treatment, as evidenced by hepatic collagen content and by immunohistochemical analysis of type-I collagen in liver. HDGL appeared to stimulate the collagenolytic process in the liver, as a 30-50% increase in urinary excretion of hydroxyproline was observed with HDGL treatment as compared to rats only given CCl(4). In conclusion, HDGL can effectively reverse chemically induced liver fibrosis, and this appears to be due, at least in part, to the stimulation of hepatic collagenolysis, resulting in a resolution of hepatic fibrosis.     

Antifibrogenic effect in vivo of low doses of insulin-like growth factor-I in cirrhotic rats.

Insulin-like growth factor-I (IGF-I) is produced mainly in the liver and it induces beneficial effects on the nutritional status, the liver function and oxidative hepatic damage in cirrhotic rats. The aim of this work was to analyze the effect of IGF-I on mechanisms of fibrogenesis in cirrhotic rats. Liver cirrhosis was induced by CCl(4) inhalation and phenobarbital in Wistar rats. Ten days after stopping CCl(4) administration (day 0), rats received either IGF-I (2 microg/100 g bw/day) (CI+IGF) or saline (CI) subcutaneously during 14 days. Animals were sacrificed on day 15. As control groups were used: healthy rats (CO) and healthy rats treated with IGF-I (CO+IGF). Liver histopathology, hydroxyproline content, prolyl hydroxylase activity, collagen I and III mRNA expression and the evolution of transformed Ito cells into myofibroblasts were assessed. Among the two control groups (CO+IGF), no differences were found in hydroxyproline content and these levels were lower than those found in the two cirrhotic groups. Compared with untreated cirrhotic rats, the CI+IGF-I animals showed a significant reduction in hydroxyproline content, prolyl hydroxylase activity and collagen alpha 1(I) and alpha1(III) mRNA expression. A higher number of transformed Ito cells (alpha-actin +) was observed in untreated cirrhotic animals as compared to CO and CI+IGF groups. In summary, treatment with IGF-I reduced all of the studied parameters of fibrogenesis. In conclusion, low doses of IGF-I induce in vivo an antifibrogenic effect in cirrhotic rats.     

Susceptibility of DNA to oxidative stressors in young and aging mice

The changes that accompany aging may be a result of oxidative damage to DNA that accumulates as a result of aging and age-related illnesses. Furthermore, a higher susceptibility is thought to be more common among elderly than young individuals. In the present study, we examined the severity of DNA damage caused by carbon tetrachloride (CCl4) and H2O2 in cells from young (2 month old) and older (14 month old) mice using both in vivo and in vitro exposures. CCl(4) is known to generate radical oxidative species (ROS) throughout its biotransformation in the liver. Therefore, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxdGuo) was quantified in liver DNA obtained from young and older mice treated with CCl4. In addition, DNA single-strand breaks were measured by the Comet assay in primary lung fibroblasts cultured from young and older mice and treated in vitro with H2O2. Intracellular ROS production and mitochondrial enzyme activity were determined in parallel. 8-oxodGuo levels were significantly higher in older mouse liver DNA than younger, and increased significantly with CCl4 treatment. When the basal DNA damage was subtracted, the net damage was almost equal for both. In addition, untreated cells cultured from older mice had significantly greater levels of strand breaks than cells derived from young mice. H2O2 increased the level of damage in both cell cultures. Our findings indicate that the DNA damage observed in older animals probably results from the accumulation of endogenous damage with age, perhaps due to insufficient repair, which enhances the injury caused by exposure to the toxic agents.     

Red wine polyphenols affect the collagen composition in the aorta after oxidative damage induced by chronic administration of CCl(4)

Increased amount of collagen type I and decreased amount of type III is described in various pathological processes in the vascular wall. Polyphenols were shown to have protective effect on endothelium, decrease blood pressure and prevent oxidative damage induced by various stimuli. Tetrachlormethane (CCl(4)) is a toxic substance with known negative systemic effects induced by free radicals. Chronic administration of CCl(4) for 12 weeks led to an increase of collagen type I and a decrease of type III in the wall of aorta. Parallel administration of red wine polyphenols significantly reduced the increase of collagen type I, at the same time the content of type III rose to the level above controls. After 4 weeks of spontaneous recovery no changes were observed. If polyphenols were administered during the recovery period, there was a decrease of type I and an increase of type III collagen content in the aorta. It can be concluded that polyphenols have a tendency to lower the amount of type I and to increase the proportion of type III collagen in the wall of the aorta. These changes are significant in prevention or in regression of changes induced by chronic oxidative stress. This effect of polyphenols is most likely the result of their influence on MMP-1 and TIMP activities through which they positively influence the collagen types I and III content ratio in the vascular wall in favor of the type III collagen.     

Antioxidant properties of colchicine in acute carbon tetrachloride induced rat liver injury and its role in the resolution of established cirrhosis

Antioxidant and antifibrotic properties of colchicine were investigated in the carbon tetrachloride (CCl(4)) rat model. (1) The protective effect of colchicine pretreatment on CCl(4) induced oxidant stress was examined in rats subsequently receiving a single lethal dose of CCl(4). Urinary 8-isoprostane, kidney and liver malondialdehyde and kidney glutathione levels increased following CCl(4) treatment, but only the rise in kidney malondialdehyde was significantly inhibited by colchicine pretreatment. Serum total antioxidant levels were significantly higher in the colchicine pretreatment group. (2) The long term effects of colchicine treatment on CCl(4) induced liver damage were investigated using liver histology and biochemical markers (hydroxyproline and type III procollagen peptide). Co-administration of colchicine with sub-lethal doses of CCl(4) over 10 weeks did not prevent progression to cirrhosis. However, rats made cirrhotic with repeated CCl(4) challenge and subsequently treated with colchicine for 12 months, all showed histological regression of cirrhosis. (3) The antioxidant effect of colchicine in vitro was evident only at very high concentrations compared to other plasma antioxidants. In summary, colchicine has only weak antioxidant properties, but does afford some protection against oxidative stress; more importantly, long term treatment with this drug may be of value in producing regression of established cirrhosis.     

Uridine alleviates carbon tetrachloride‐induced liver fibrosis by regulating the activity of liver‐related cells

At present, liver fibrosis is a major challenge of global health. When hepatocyte regeneration cannot compensate for hepatocyte death, it will develop into liver fibrosis in chronic liver disease. Initially, collagen produced by myofibroblasts plays a role in maintaining liver integrity, but excessive collagen accumulation can inhibit the residual liver function, leading to liver failure. At present, many scientists are actively looking for drugs to alleviate liver fibrosis. In the current study, we investigated the potential role of uridine in the treatment of liver fibrosis (uridine is a plant/animal‐derived pyrimidine nucleoside, therefore uridine can also be ingested and absorbed by the body, accompanied by the process of food intake). For this, we systematically studied the effect of uridine on CCl4‐induced liver fibrosis in vitro and in vivo through a series of technologies, such as Western blot, laser confocal scanning microscope, ELISA and immunohistochemistry. The experimental results showed that uridine can effectively reduce the accumulation of collagen in liver. Furthermore, uridine can improve the activity of liver cells and alleviate CCl4‐induced liver injury. Furthermore, uridine can significantly alleviate the risk factors caused by hepatic stellate cell activation, uridine treatment significantly down‐regulated the expression of α‐SMA, collagen type‐I and fibronectin. In conclusion, the current research shows that uridine can alleviate CCl4‐induced liver fibrosis, suggesting that uridine can be used as a potential drug to alleviate liver fibrosis.     

Effect of sodium fluoride and magnesium chloride on different hydroxyproline fractions in rat liver

Sodium fluoride (NaF) is used for prevention of caries in the form of fluoridated drinking water, fluoride tablets etc. In the present study, the effect of NaF-induced alterations in hydroxyproline (Hyp) and collagen was investigated in rat liver. The effect of pretreatment with MgCl2 on NaF-induced changes in liver Hyp and collagen was also studied. The NaF treatment at 5, 10 and 20 mg/kg body wt (reported LD50 of NaF being 24 mg/kg body wt through intraperitoneal route) caused a significant decrease in free Hyp (P < 0.05), when compared to control rats. The rats treated with 20 mg/kg body wt of NaF showed a significant increase in protein-bound Hyp (P < 0.001), as compared to control group, while of NaF treatment at 5 and 10 mg/kg body wt caused no significant change in protein-bound Hyp. All the doses of NaF had no significant effect on peptide-bound and total Hyp and total collagen. Treatment of with MgCl2 alone (30 mg/kg body wt) or with NaF (10 mg/kg body wt) caused a significant decrease in free Hyp (P < 0.05). MgCl2 alone and with NaF caused a significant increase (P < 0.05) in total collagen content. Thus, the present study demonstrated that NaF had no significant effect on total Hyp and collagen, indicating that its use in various products may not interfere with the liver collagen.     

Histophotometric estimation of volume density of collagen as an indication of fibrosis in rat liver

The development of fibrosis in the liver of 16 rats treated for 1, 2, 3 or 4 weeks with CCl4, has been followed with chemical hydroxyproline determination and histophotometric analysis of histological sections stained with Sirius Red F3BA in saturated aqueous picric acid. The readings were taken with a scanning and integrating microphotometer and corrected for picric acid absorbance as a measure for mean protein mass per unit area of the section. It appears that the integrated absorbance readings of Sirius Red absorbing material in the section show a highly significant correlation with the hydroxyproline determinations. It is concluded that picrosirius photometry can be used to give a measure of the volume density of collagen in sections. An advantage of the photometric assay is that measurements are taken on the basis of the microscopic image, so that it is also possible to estimate collagen density in a selected area, e.g. a tumour formation amidst normal tissue, or to exclude necrotic areas.     

Effect of dibutyryl cyclic AMP on collagen synthesis in a clonal osteoblast-like cell line derived from newborn mouse calvaria

The effects of dibutyryl cyclic AMP (DBcAMP) and related compounds on collagen synthesis in a clonal osteoblast-like cell line, MC3T3-E1, were investigated. The addition of DBcAMP to cultures increased the hydroxyproline content of the cells. It also enhanced the incorporation of labeled proline into collagen and elevated the activity of prolyl hydroxylase, an enzyme involved in collagen synthesis. These effects were observed at concentrations of 0.1 to 2 mM DBcAMP. 8-Bromo cyclic AMP also increased the hydroxyproline content of the cells, while sodium butyrate and dibutyryl cyclic GMP had no such effect. These results suggest that the intracellular accumulation of cyclic AMP in osteoblasts leads to their active production of collagen, a major component of the organic matrix of bone.     

Free and small peptide-bound [14C]hydroxyproline synthesis in rat liver in vitro in CCl4-induced hepatic fibrosis

To clarify the process of free and small peptide-bound hydroxyproline synthesis in hepatic fibrogenesis, we measured the in vitro synthesis of [14C]hydroxyproline in the 67% ethanol soluble fraction in rat liver slices, together with hepatic protein-bound [14C]hydroxyproline synthesis. In control rat liver, the amount of free and small peptide-bound [14C]hydroxyproline synthesized was 13.1 +/- 2.6 10(-4) x dpm/g liver/3 hr. In the CCl4-treated rat liver, where the hepatic hydroxyproline content was increased 4.6-fold, the protein-bound [14C]hydroxyproline synthesis was significantly increased 1.5-fold, but free and small peptide-bound [14C]hydroxyproline synthesis was decreased into 70%. There was a significant inverse correlation between free and small peptide-bound [14C]hydroxyproline synthesis, and hepatic hydroxyproline content. These results suggest that the combination of an increase in collagen synthesis and a decrease in free and small peptide-bound [14C]hydroxyproline synthesis contributes to rapid accumulation of collagen in hepatic fibrosis.     

The antioxidant and antifibrogenic effects of the glycosaminoglycans hyaluronic acid and chondroitin-4-sulphate in a subchronic rat model of carbon tetrachloride-induced liver fibrogenesis

Hepatic fibrosis involves the interplay of many factors including reactive oxygen species. Recent reports described antioxidant properties of glycosaminoglycans (GAGs). Since several findings have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) may act as antioxidant molecules, the aim of this research was to evaluate the antioxidant effects of HYA and C4S treatment in a rat model of liver fibrosis. The effect on tissue inhibitors of metalloproteinases (TIMPs) was also studied. Liver fibrosis was induced in rats by eight intraperitoneal injections of CCl4, twice a week for 6 weeks. HYA or C4S alone (25 mg/kg) or HYA and C4S in combination (12.5 + 12.5 mg/kg) were administered daily by the same route during the 6 weeks. At the end of the 6-week treatment period (24 h after the last dose of GAGs), the following parameters were evaluated: (1) serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as index of hepatic cell disruption; (2) hepatic conjugated dienes (CD), as index of lipid peroxidation; (3) hepatic TIMPs activity and expression; (4) hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, as index of endogenous defences; (5) hepatic hydroxyproline, as index of collagen deposition. CCl4-induced liver fibrosis enhanced lipid peroxidation and TIMPs activation, increased ALT and AST, depleted antioxidants SOD and GPx, and caused collagen deposition in liver tissue. Treatment with GAGs, especially when in combination, successfully reduced ALT and AST rise, lipid peroxidation by evaluating conjugated dienes, TIMPs activation and mRNA expression, partially restored SOD and GPx activities, and limited collagen deposition in the hepatic tissue. The data obtained showed that these molecules were able to limit hepatic injury induced by chronic CCl4 intoxication and especially limited liver fibrosis. They also confirm that HYA and C4S may exert antioxidant mechanism, while reduction of TIMPs expression suggests that GAGs may influence MMPs and TIMPs imbalance in liver fibrosis.     

Kupffer cells inhibition prevents hepatic lipid peroxidation and damage induced by carbon tetrachloride

The aim of this work was to determine if the action mechanism of gadolinium on CCl(4)-induced liver damage is by preventing lipid peroxidation (that may be induced by Kupffer cells) and its effects on liver carbohydrate metabolism. Four groups of rats were treated with CCl(4), CCl(4)+GdCl(3), GdCl(3), and vehicles. CCl(4) was given orally (0.4 g 100 g(-1) body wt.) and GdCl(3) (0.20 g 100 g(-1) body wt.) was administered i.p. All the animals were killed 24 h after treatment with CCl(4) or vehicle. Glycogen and lipid peroxidation were measured in liver. Alkaline phosphatase, gamma-glutamyl transpeptidase, alanine amino transferase activities and bilirubins were measured in rat serum. A liver histological analysis was performed. CCl(4) induced significant elevations on enzyme activities and bilirubins; GdCl(3) completely prevented this effect. Liver lipid peroxidation increased 2.5-fold by CCl(4) treatment; this effect was also prevented by GdCl(3). Glycogen stores were depleted by acute intoxication with CCl(4). However, GdCl(3) did not prevent this effect. The present study shows that Kupffer cells may be responsible for liver damage induced by carbon tetrachloride and that lipid peroxidation is produced or stimulated by Kupffer cells, since their inhibition with GdCl(3) prevented both lipid peroxidation and CCl(4)-induced liver injury.     

Carbon tetrachloride toxicity on Escherichia coli exacerbated by superoxide

The effects of carbon tetrachloride (CCl4) and paraquat on the growth of Escherichia coli were investigated. Paraquat at 10 mM caused some inhibition of growth of E. coli in trypticase-soy-yeast extract medium. CCl4 enhanced growth inhibition by paraquat in a concentration-dependent manner. In the absence of paraquat, CCl4 had no effect on growth rate or on surviving cell numbers at stationary phase. CCl4 did not prevent the induction of manganese-superoxide dismutase by paraquat. Under anaerobic conditions, CCl4 and paraquat exhibited no effect on E. coli. In the presence of Mn(II) and paraquat, intracellular superoxide dismutase was markedly induced and protected E. coli against the toxicity of CCl4 and paraquat. The reactive free radical CCl3OO-, which can be formed from the reaction of O2- with CCl4, may cause cell damage. The growth-inhibiting effects of polyhalides in the presence of paraquat followed the order CBrCl3 greater than CCl4 greater than CHCl3 greater than CH2Cl2, which is in accord with that of the reaction rates of these compounds with O2- and with their hepatotoxicities. These results suggest that O2- plays a role in the hepatotoxicity of polyhalides.     

Status of the antioxidant protective system of rat liver exposed to carbon tetrachloride]

The dynamics of enzyme activity of antioxidative protective system of the liver and the content of restored glutathione have been studied in rats poisoned by CCl4 injection. During the first hours followed the injection against the background of maximum accumulation of dienic conjugates and decrease of the restored glutathione level no significant changes in the enzyme activity of the antioxidative protective liver system were observed. At the same time 48 hours later the superoxide dismutase and catalase activity decreased by 38% and 36%, respectively, with relative stability of glutathione-dependent enzymes and a two-fold increase of the restored glutathione level. It is shown that a fall of activity of the cytoplasmic antioxidative liver enzymes is not a result of the immediate inactivating effect of free-radical reactions initiated by CCl4, but is, evidently, caused by the covalent binding of its radical metabolites with corresponding macromolecules.