Elsevier

Journal of Hepatology

Volume 43, Issue 2, August 2005, Pages 324-332
Journal of Hepatology

Oxidative stress and apoptosis in fetal rat liver induced by maternal cholestasis. Protective effect of ursodeoxycholic acid

https://doi.org/10.1016/j.jhep.2005.02.028Get rights and content

Background/Aims

The sensitivity of fetal rat liver to maternal obstructive cholestasis during pregnancy (OCP), and the effect of ursodeoxycholic acid (UDCA) were investigated.

Methods

UDCA was administered (i.g. 0.6 mg/kg b.wt./day) from day 14 to day 21 of pregnancy after maternal common bile duct ligation.

Results

Impairment in the activity of antioxidant enzymes, levels of total glutathione and GSH/GSSG ratio and the degrees of lipid peroxidation and protein carbonylation were similar in livers of OCP mothers and fetuses at term, despite hypercholanemia was milder in fetuses. Treatment of OCP rats with UDCA reduced maternal and fetal liver oxidative stress. Although maternal hypercholanemia was not corrected, fetal serum concentrations of major bile acids (except UDCA and β-muricholic acid) were reduced. Fetal liver expression of key enzyme in bile acid synthesis, Cyp7a1, Cyp27 and Cyp8b1 was not affected by OCP or UDCA treatment. In OCP fetal livers, the relative expression of Bax-α and Bcl-2 and the activity of caspase-3, but not caspase-8, were increased. These changes were markedly reduced in fetuses of OCP animals treated with UDCA.

Conclusions

OCP induced moderate fetal hypercholanemia but marked liver oxidative stress and apoptosis that were partly prevented by treatment of pregnant rats with UDCA.

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.

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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.

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