Oxidative stress and apoptosis in fetal rat liver induced by maternal cholestasis. Protective effect of ursodeoxycholic acid☆
Introduction
Several mechanisms may account for the cytotoxicity associated with the most hydrophobic bile acids (BAs) in cholestatic diseases [1]. BAs may disrupt cell membranes through their detergent action on lipid components [2] and can promote the generation of reactive oxygen species that, in turn, oxidatively modify lipids, proteins, and nucleic acids, and eventually cause hepatocyte apoptosis [3]. Additionally, they can activate Kupffer cells to generate reactive oxygen species that may contribute to liver cell insult [4].
Two pathways are involved in triggering hepatocytes apoptosis (for a review see [5]). Toxic BAs can activate Fas death receptors directly [6] and induce oxidative damage that causes mitochondrial dysfunction and apoptosis [7], [8]. The Bcl-2 protein family plays a role in the regulation of the mitochondria-mediated pathway. Two key representative members of this family are the anti-apoptotic Bcl-2 and the pro-apoptotic Bax [9], [10], [11].
Ursodeoxycholic acid (UDCA) has therapeutic usefulness in several cholestatic liver diseases (for a review, see [12]). The major beneficial effects of treatment with UDCA are protection against cytotoxicity due to more toxic BAs [13], stimulation of hepatobiliary secretion [14], antioxidant activity due in part to an enhancement in glutathione levels [15], [16] and inhibition of liver cells apoptosis [7].
Intrahepatic cholestasis of pregnancy (ICP), usually implies a benign condition for the mother. However, in the conceptus it is associated with serious repercussions, including increased fetal distress, premature delivery, and perinatal mortality and morbidity (for a review, see [17]). This is probably due to the higher sensitivity of more fragile fetal developing organs to toxic BAs [18]. In addition, ICP impairs placental functions, reducing the ability of the fetus to eliminate BAs towards the maternal blood [19], which may aggravate the situation.
Like several other cholestatic disorders, ICP has been shown to respond to UDCA treatment with a reduction in maternal pruritus, a normalization of biochemical parameters, including serum bilirubin and transaminases, and a decrease in the number of premature deliveries [20]. Although UDCA administration to pregnant women induces changes in the fetal BA pool [21], several studies have indicated that treatment of ICP with UDCA has no risk for the mother or the fetus [22], [23].
The present study, carried out on an experimental model of complete obstructive cholestasis (OCP) during the last third of pregnancy in the rat, was undertaken to investigate whether maternal cholestasis causes oxidative stress and apoptosis in fetal liver and whether treatment of pregnant rats with UDCA has beneficial effects. Although with a different etiology and degree of impairment in biliary function, the experimental model of OCP shares two important characteristics with human ICP: the presence of marked maternal hypercholanemia, to which the conceptus is exposed, and a reduction in the amount of BAs that reaches the maternal intestine, which limits the absorption of dietary fat and liposoluble vitamins. Other alternative models of cholestasis, such as drugs- or hormones-induced cholestasis, which would be closer to the actual situation of partial cholestasis occurring in ICP, were not selected due to potential placental transfer and interference of cholestatic agents with fetal liver function.
Section snippets
Animals and experimental design
Pregnant Wistar CF rats (University of Salamanca, Spain) were used. The experimental protocols were approved by the Local Ethical Committee for the Use of Laboratory Animals. On day 14 of pregnancy, the rats were anesthetized with ether and a sham operation (Control group) or complete biliary obstruction (OCP group) was performed as previously described [24]. In brief, using a non-absorbable suture, a double ligation separated by 2 mm was carried out. The common bile duct was divided between the
Obstructive cholestasis in pregnant rats
In agreement with previous studies [25], OCP caused a decrease in body weight in both mothers and fetuses, together with a reduction in the number of fetuses per gestation. UDCA treatment partly restored normal maternal body weight gaining and the number of fetuses per pregnancy (Table 1). Changes in several serum biochemical parameters were consistent with typical signs of liver cell injury associated with cholestasis. The repercussions of OCP on fetal biochemical parameters were milder. Serum
Discussion
After bile duct ligation in the rat, biphasic changes in total glutathione levels occur [42], [43]. These are: (i) a transient increase during the first week, which was also observed here in maternal liver during OCP and, (ii) subsequent depletion. Initial accumulation has been explained in terms of the lack of integrity in the biliary pathway for glutahione secretion [43]. This enhanced amount of glutathione was not able to prevent liver oxidative damage in OCP animals. Moreover, in the fetal
Acknowledgements
The authors thank Mrs M.I. Hernandez Rodriguez for her secretarial help, and Mr L. Muñoz de la Pascua and Mr J.F. Martin Martin for caring for the animals. Thanks are also due to Nicholas Skinner for revision of the English text of the manuscript.
References (51)
- et al.
Bile acid-induced liver toxicity: relation to the hydrophobic–hydrophilic balance of bile acids
Med Hypotheses
(1986) - et al.
Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition
Hepatology
(2001) - et al.
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade
Cell
(1997) - et al.
Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited
Hepatology
(2002) - et al.
Hypercholeresis induced by ursodeoxycholic acid and 7-ketolithocholic acid in the rat: possible role of bicarbonate transport
Gastroenterology
(1980) - et al.
Ursodeoxycholic acid protects hepatocytes against oxidative injury via induction of antioxidants
Biochem Biophys Res Commun
(1999) - et al.
Beneficial effect of ursodeoxycholic acid on alterations induced by cholestasis of pregnancy in bile acid transport across the human placenta
J Hepatol
(1998) - et al.
Ursodeoxycholic acid in the treatment of cholestasis of pregnancy: a randomized, double-blind study controlled with placebo
J Hepatol
(1997) - et al.
Ursodeoxycholic acid administration in patients with cholestasis of pregnancy: effects on primary bile acids in babies and mothers
Hepatology
(2001) - et al.
Reversible impairment of neonatal hepatobiliary function by maternal cholestasis
Hepatology
(1996)
Effect of maternal cholestasis on biliary lipid and bile acid secretion in the infant rat
Hepatology
Oxidative damage to proteins: spectrophotometric method for carbonyl assay
Methods Enzymol
Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues
Anal Biochem
Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine
Anal Biochem
Glutathione reductase
Methods Enzymol
Glutathione S-transferases. The first enzymatic step in mercapturic acid formation
J Biol Chem
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples
Anal Biochem
Reduced expression of Bax in ceramide-resistant HL-60 subline
Biochem Biophys Res Commun
Antioxidant defenses in the bile duct-ligated rat
Gastroenterology
Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine
J Hepatol
Antioxidant administration to the mother prevents oxidative stress associated with birth in the neonatal rat
Life Sci
Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation
J Hepatol
Effects of bile salts on the plasma membranes of isolated rat hepatocytes
Biochem J
Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids
Pediatr Res
Effect of deoxycholic acid and ursodeoxycholic acid on lipid peroxidation in cultured macrophages
Gut
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This study was supported in part by the Junta de Castilla y León (Grant SA013/04 and Grant SA017/03) Spain. Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, Spain, alone (CP03/00093) and co-funded by the FEDER-FSE Program of the E.U. (Grant 01/1043). Ministerio de Ciencia y Tecnología, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Grant BFI2003-03208). Dr Maria J. Perez received two Research Fellowships: the ‘Juan Rodés’ Research Fellowship from the Spanish Association for the Study of the Liver (AEEH), and the Research Fellowship from the Fundacion ‘Miguel Casado San Jose’, Salamanca, Spain. The group is member of the Network for Cooperative Research on Hepatitis, Instituto de Salud Carlos III, FIS (Grant G03/015), Spain.