C Basic & Clinical Pharmacology & Toxicology 2004, 94, 99–104. Printed in Denmark . All rights reserved Copyright C ISSN 1742-7835 Effects of Silymarin and Vitamins E and C on Liver Damage Induced by Prolonged Biliary Obstruction in the Rat Pablo Muriel and Mario G. Moreno Section of Pharmacology, Cinvestav-I.P.N., Me ´xico 07000, D.F., Me ´xico (Received August 4, 2003, Accepted October 20, 2003) Abstract: Oxidative stress, in particular lipid peroxidation, induces collagen synthesis. Thus, we administered various antioxidants to bile duct-ligated rats for 28 days and lipid peroxidation, glutathione content, fibrosis, necrosis and chol- estasis were evaluated. Extrahepatic cholestasis was induced by double ligation and section of the common bile duct. The study included eight groups (nΩ6), four groups were bile duct-ligated and received either vitamin C (50 mg/kg/day, orally), vitamin E (400 IU/rat/day, orally), silymarin (50 mg/kg/12hr, orally) or vehicles; four groups were sham-operated controls. Collagen content was determined by measuring hydroxyproline in liver samples; malondialdehyde was used to estimate lipid peroxidation levels; reduced and oxidized glutathione were determined fluorometrically; alanine aminotransferase and bilirubins colorimetrically. Bilirubins increased several times, alanine aminotransferase once, reduced/oxidized gluta- thione ratio decreased three times, lipid peroxidation and collagen increased about three-times by biliary obstruction (p0.05). Silymarin, vitamin E or C failed to prevent these effects significantly. It is not possible to clarify the role of oxidative stress in the fibrotic process induced by chronic biliary obstruction with the present results. Therefore, it seems reasonable to propose that a wide mixture of antioxidants, administered by the parenteral route (because cholestasis decreased the absorption of lipophilic compounds), is needed to counteract the oxidant stress produced by cholestasis. Free radicals, oxidative stress and lipid peroxidation are fre- quently associated with several types of liver diseases (Muri- el 1997). However, the question is whether these factors contribute to the development of liver damage or are conse- quences or parallel events of the pathological process. While in some models of liver damage it is clear that oxida- tive stress plays an important role, like in acetaminophen (Muriel et al. 1992) or CCl 4 intoxications (Muriel & Mour- elle 1990a & b; Sotelo-Fe ´lix et al. 2002), in other models it is not so clear. In particular, in liver damage produced by bile flow obstruction the role of oxidative stress and lipid peroxidation processes in the development of liver injury is not fully understood. Several studies have demonstrated that free radicals are present in cholestatic damage. Bile acids enhance the release of reactive oxygen species from activated rat polymorphonuclear cells (Dahm et al. 1998), inflammatory cells which are present in both experimental and human cholestatic liver lesions (Kountaras et al. 1984). High concentrations of plasma lipid peroxides have been observed in humans with cholestasis (Lemonnier et al. 1987) and in bile duct-ligated (BDL) rats (Muriel 1996). A reduced antioxidative capacity in hepatic mitochondria and reduced glutathione (GSH) levels in the liver were reported after 28 days of biliary obstruction in the rat (Krähenbühl et al. 1995). We have shown that lipid peroxidation increased three days after bile duct-ligated rats while liver injury was evident after one day of bile flow obstruction (Muriel & Author for correspondence: Pablo Muriel, Section of Pharma- cology, Cinvestav-I.P.N., P.O. Box 14-740, Me ´xico 07000, D.F., Me ´x- ico (fax π5255 5747-70-95, e-mail pamuriel/mail.cinvestav.mx). Sua ´rez 1994), suggesting that oxidative stress (which actu- ally occurs) is rather a consequence than a cause of liver damage. Moreover, treatment of seven day-bile duct-ligated rats with lipophilic (vitamin E) and/or hydrophilic (trolox) antioxidants completely prevented the increase in lipid per- oxidation and effectively maintained the normal GSH/oxid- ized GSH ratio both in liver and plasma, but failed to pre- vent liver damage measured by histology and by plasmatic enzyme activities (Baro ´ n & Muriel 1999). This strongly sug- gests that oxidative stress does not play an important role in the development of acute liver damage produced by bile duct ligation. However, the role of oxidant stress in chronic cholestatic injury is still a matter of study. Prolonged biliary obstruction for 28 days or more is as- sociated with considerable increases in liver collagen con- tent. It has been postulated that products of lipid peroxi- dation (aldehyde-protein adducts for example) modulate collagen gene expression and may be a link between liver injury and fibrosis (Chojkier et al. 1998). Chojkier et al. (1989) have previously shown that, in cultured cells, en- hanced lipid peroxidation stimulates collagen gene tran- scription whereas basal lipid peroxidation regulates the constitutive collagen gene transcription (Houglum et al. 1991). In the CCl 4 -cirrhosis model, Bedosa et al. (1994) found colocalization of increased collagen a 1 (I) mRNA with lipid peroxidation by means of in situ hybridization and immunohistochemical study for malondialdehyde and 4-hydroxynonenal protein adducts. These experiments sug- gest that hepatocyte lipid peroxidation may play a major role in the regulation of collagen gene expression and that it may be a link between hepatocyte injury and hepatic fi- brosis.