Pablo Muriel

Role of free radicals in liver diseases

Reactive oxygen and nitrogen species (ROS and RNS) are produced by metabolism of normal cells. However, in liver diseases, redox is increased thereby damaging the hepatic tissue; the capability of ethanol to increase both ROS/RNS and peroxidation of lipids, DNA, and proteins was demonstrated in a variety of systems, cells, and species, including humans. ROS/RNS can activate hepatic stellate cells, which are characterized by the enhanced production of extracellular matrix and accelerated proliferation. Cross-talk between parenchymal and nonparenchymal cells is one of the most important events in liver injury and fibrogenesis; ROS play an important role in fibrogenesis throughout increasing platelet-derived growth factor. Most hepatocellular carcinomas occur in cirrhotic livers, and the common mechanism for hepatocarcinogenesis is chronic inflammation associated with severe oxidative stress; other risk factors are dietary aflatoxin B1 consumption, cigarette smoking, and heavy drinking. Ischemia–reperfusion injury affects directly on hepatocyte viability, particularly during transplantation and hepatic surgery; ischemia activates Kupffer cells which are the main source of ROS during the reperfusion period. The toxic action mechanism of paracetamol is focused on metabolic activation of the drug, depletion of glutathione, and covalent binding of the reactive metabolite N-acetyl-p-benzoquinone imine to cellular proteins as the main cause of hepatic cell death; intracellular steps critical for cell death include mitochondrial dysfunction and, importantly, the formation of ROS and peroxynitrite. Infection with hepatitis C is associated with increased levels of ROS/RNS and decreased antioxidant levels. As a consequence, antioxidants have been proposed as an adjunct therapy for various liver diseases.

Nitric oxide protection of rat liver from lipid peroxidation, collagen accumulation, and liver damage induced by carbon tetrachloride

The aim of this work was to determine if the inhibition or stimulation of NO synthesis modulates liver damage induced by the chronic administration of CCl4. CCl4 was administered three times a week for 8 weeks to male Wistar rats treated simultaneously with N omega-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg, p.o., twice a day), aminoguanidine (AG, 4 g/L in the drinking water), or L-arginine (500 mg/kg, p.o., twice a day); appropriate controls were performed. Serum NO2- + NO3- increased in the groups treated with CCl4 and/or L-arginine, but the effect was prevented by either L-NAME or AG. In the liver, lipid peroxidation and collagen content increased, while glycogen content decreased in the CCl4-treated group (P < 0.05); L-NAME and AG accentuated these effects. Serum enzyme activities of alanine aminotransferase (ALT), alkaline phosphatase, and gamma-glutamyl transpeptidase (gamma-GTP) and bilirubin content increased about 2-, 3-, 2-, and 6-fold, respectively, after CCl4 intoxication (P < 0.05); L-NAME or AG cotreatment further increased the enzyme activities (P < 0.05). L-Arginine treatment protected the liver partially from the elevation of collagen, bilirubins, and alkaline phosphatase and from glycogen depletion induced by CCl4 intoxication (P < 0.05), but showed no significant effect on ALT, gamma-GTP, or lipid peroxidation. These results suggest that NO protects the liver against oxidative injury, because NO inhibition by L-NAME or AG increased lipid peroxidation and the other markers of liver injury studied herein.

PREVENTION OF CCL4‐INDUCED LIVER CIRRHOSIS BY SILYMARIN

Summary— The efficacy of silymarin treatment in preventing biochemical and histological alterations in CCl4‐induced liver cirrhosis in rats was studied. Four groups of rats were treated with: (1) CCl4; (2) mineral oil; (3) CCl4 + silymarin; and (4) silymarin. All animals were sacrificed 72 h after the end of treatments. The activities of alkaline phosphatase (alk. phosp.), gamma‐glutamyl transpeptidase (GGTP), glutamic pyruvic transaminase (GPT) and glucose‐6‐phosphatase (G6Pase), and bilirubin content were determined in serum. Na+, K+‐ATPase and Ca++‐ATPase activities were measured in isolated plasma membranes. Lipoperoxidation, triglycerides (TG), and glycogen contents were also measured in liver homogenates.

Pirfenidone effectively reverses experimental liver fibrosis

BACKGROUND/AIMS Our group has been involved in searching for different strategies to ameliorate hepatic cirrhosis. The aim of this study was to evaluate the effect of Pirfenidone in the reversion or prevention of cirrhosis experimentally induced in rats by chronic administration of CCl(4) and bile-duct ligation (BDL). METHODS Male cirrhotic Wistar rats (8 weeks of intoxication and then hepatotoxin was discontinued) received either oral saline or Pirfenidone at 500 mg/kg per day. RESULTS High levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase decreased significantly (P<0.001) in animals treated with Pirfenidone (n=11) with regard to saline-administrated animals (n=9). Prothrombin activity and bilirubins were also reduced. Computerized fibrosis index demonstrated a 70% decrease (P<0.001) along with less hydroxyproline content, reduction in activated HSC and higher active cell regeneration. A rearrangement of the parenchyma was also noted and gene expression of collagens I, III and IV, transforming growth factor beta-1, Smad-7, TIMP-1 and PAI-1 decreased considerably in treated animals. Cirrhotic rats in which CCl(4) was not discontinued displayed 40% liver fibrosis reduction. In a different cirrhosis model, 4-week BDL rats treated with the drug showed a significant 50% reduction in hepatic fibrosis (P<0.01). CONCLUSIONS This new drug might be useful in healing human disease.

Evaluation of the effectiveness of Rosmarinus officinalis (Lamiaceae) in the alleviation of carbon tetrachloride-induced acute hepatotoxicity in the rat.

The effect of oral administration of Rosmarinus officinalis L. (Lamiaceae) on CCl(4)-induced acute liver injury was investigated. Rats were daily treated with the plant extract at a dose of 200 mg/kg corresponding to 6.04 mg/kg of carnosol as determined by reverse phase high-performance liquid chromatography. The treatment was initiated 1 h after CCl(4) administration and Rosmarinus officinalis fully prevented CCl(4) effect on hepatic lipid peroxidation after 24 h of CCl(4) administration. The increase in bilirubin level and alanine aminotransferase activity in plasma induced by CCl(4) was completely normalized by Rosmarinus officinalis. The treatment also produced a significant recovery of CCl(4)-induced decrease in liver glycogen content. CCl(4) did not modify the activity of liver cytosolic glutathione S-transferase (GST) compared with that of control groups. However, Rosmarinus officinalis increased liver cytosolic GST activity and produced an additional increment in plasma GST activity in rats treated with CCl(4). Histological evaluation showed that Rosmarinus officinalis partially prevented CCl(4)-induced inflammation, necrosis and vacuolation. Rosmarinus officinalis might exert a dual effect on CCl(4)-induced acute liver injury, acting as an antioxidant and improving GST-dependent detoxification systems.

Pharmacological actions of curcumin in liver diseases or damage.

Since 1900 bc, several therapeutic activities have been attributed to the rhizomes of the plant Curcuma longa for a variety of diseases, including liver disorders. Curcumin, the main active compound obtained from this plant, was first isolated two centuries ago and its structure as diferuloylmethane was determined in 1910. Curcumin has shown anti‐inflammatory, anti‐oxidant, antifungal, antibacterial and anticancer activities. The pharmacological properties of curcumin were reviewed recently and focused mainly on its anticancer properties. However, its beneficial activity on liver diseases (known centuries ago, and demonstrated recently utilizing animal models) has not being reviewed in depth until now. The curcumin ability to inhibit several factors like nuclear factor‐κB, which modulates several pro‐inflammatory and profibrotic cytokines as well as its anti‐oxidant properties, provide a rational molecular basis to use it in hepatic disorders. Curcumin attenuates liver injury induced by ethanol, thioacetamide, iron overdose, cholestasis and acute, subchronic and chronic carbon tetrachloride (CCl4) intoxication; moreover, it reverses CCl4 cirrhosis to some extent. Unfortunately, the number of studies of curcumin on liver diseases is still very low and investigations in this area must be encouraged because hepatic disorders constitute one of the main causes of worldwide mortality.

NF-κB in liver diseases : a target for drug therapy

There are five nuclear factor‐κB (NF‐κB) transcription factors with important roles in innate immunity, liver inflammation, fibrosis and apoptosis prevention. Several inhibitors of NF‐κB, like caffeic acid, captopril, curcumin, pyrrolidine dithiocarbamate, resveratrol, silymarin and thalidomide, have demonstrated antinecrotic, anticholestatic, antifibrotic and anticancer activities in the liver. A link between inflammation and hepatocellular carcinoma through the NF‐κB pathway has been observed, providing ample experimental support for the tumor‐promoting function of NF‐κB in various models of cancer. NF‐κB has been associated with the induction of proinflammatory gene expression and has attracted interest as a target for the treatment of inflammatory disease. However, despite much attention being focused on the deleterious effects of NF‐κB, activation of this factor during the resolution of inflammation is associated with the production of antiinflammatory molecules like interleukin (IL)‐10 and the onset of apoptosis. This suggests that NF‐κB has an antiinflammatory role in vivo involving the regulation of the resolution of inflammation. Also, NF‐κB promotes liver regeneration by upregulating IL‐6 and other molecules like hepatocyte growth factor. It has been postulated that the beneficial properties of NF‐κB are due to p50 homodimers, whose activation prevents cholestatic and chronic liver injury. More basic understanding on the function of the diverse NF‐κB factors is urgently needed in different physiological and pathological conditions, because depending on the subunit composition of the dimmer, the disease and the stage of the illness, inhibition of the factor may result in a beneficial or in a deleterious response. Copyright

Role of glutathione, lipid peroxidation and antioxidants on acute bile-duct obstruction in the rat

The aim of this work was to evaluate the role of lipid peroxidation and glutathione on liver damage induced by 7-day biliary obstruction in the rat. Male Wistar rats were bile-duct-ligated and divided in groups of 10 animals. Groups received vitamin E (400 IU/rat, p.o., daily) or trolox (50 mg/kg, p.o., daily) or both. Lipid peroxidation increased significantly in the livers of bile-duct-ligated rats. Vitamin E and trolox prevented lipid peroxidation. GSH was oxidized in the BDL group and the GSH/GSSG ratio decreased as a consequence. However, total glutathione content increased in liver and blood indicating a possible induction in de novo synthesis of GSH. Antioxidants preserved the normal GSH/GSSG ratio. Despite the observation that antioxidants verted lipid peroxidation and oxidation of GSH, liver injury (as assessed by serum enzyme activities, bilirubin concentration, liver glycogen content and histology) was not affected by the treatments. These results suggest that drugs that inhibit lipid peroxidation and oxidation of glutathione have no effect on conventional biochemical markers of liver injury and on liver histology of bile-duct-ligated rats for 7 days. It seems more likely that the detergent action of bile salts is responsible for solubilization of plasma membranes and cell death, which in turn may lead to oxidative stress, GSH oxidation and lipid peroxidation.

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.

Coffee and liver diseases

Coffee consumption is worldwide spread with few side effects. Interestingly, coffee intake has been inversely related to the serum enzyme activities gamma-glutamyltransferase, and alanine aminotransferase in studies performed in various countries. In addition, epidemiological results, taken together, indicate that coffee consumption is inversely related with hepatic cirrhosis; however, they cannot demonstrate a causative role of coffee with prevention of liver injury. Animal models and cell culture studies indicate that kahweol, diterpenes and cafestol (some coffee compounds) can function as blocking agents by modulating multiple enzymes involved in carcinogenic detoxification; these molecules also alter the xenotoxic metabolism by inducing the enzymes glutathione-S-transferase and inhibiting N-acetyltransferase. Drinking coffee has been associated with reduced risk of hepatic injury and cirrhosis, a major pathogenic step in the process of hepatocarcinogenesis, thus, the benefit that produces coffee consumption on hepatic cancer may be attributed to its inverse relation with cirrhosis, although allowance for clinical history of cirrhosis did not completely account for the inverse association. Therefore, it seems to be a continuum of the beneficial effect of coffee consumption on liver enzymes, cirrhosis and hepatocellular carcinoma. At present, it seems reasonable to propose experiments with animal models of liver damage and to test the effect of coffee, and/or isolated compounds of this beverage, not only to evaluate the possible causative role of coffee but also its action mechanism. Clinical prospective double blind studies are also needed.