BILE SALTS MODULATE CHRONIC ETHANOL-INDUCED HEPATOTOXICITY

Anne-Marie Montet, Laurence Oliva, Françoise Beaugé1 and Jean-Claude Montet,*

Laboratoire de Physiopathologie Hépatique, INSERM, 46 Boulevard de la Gaye, 13009, Marseille and
1 Centre de Recherche Pernod-Ricard, Créteil, France

Received 2 March 2001; in revised form 28 July 2001; accepted 6 August 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
— This study tested the hypothesis that chronic ethanol-induced injury in rats may be modified by the hydrophobicity of the bile acid pool. The supplementation to chronic ethanol feeding (28 days) with chenodeoxycholate, a hydrophobic bile salt, aggravated steatosis (accumulation of triacylglycerols and cholesterol esters), lipoperoxidation and cytolysis (expressed as elevations of activities of aspartate aminotransferase and glutamate dehydrogenase), while the addition of ursodeoxycholic acid, a hydrophilic bile salt, alleviated ethanol-induced hepatic alterations. Furthermore, our data show that ursodeoxycholic acid still exerts its beneficial effects in a model of more severe hepatic intoxication induced by the co-administration of ethanol and chenodeoxycholic acid. The hepato-protective effect observed appears to be independent of the choleretic properties of ursodeoxycholic acid and may be due partly to the capacity of the bile acid to preserve mitochondria.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile salts are polar amphiphilic molecules which play a crucial role in bile formation. Variations in the conjugation and in the number, position, and orientation of hydroxyl groups, lead to large variations in the hydrophilic–hydrophobic balance between different bile salts (Armstrong and Carey, 1982Go; Heuman, 1989Go). The more hydrophobic bile salts may be cytotoxic when present in abnormally high concentrations. Thus, deoxycholic acid and chenodeoxycholic acid (CDC), which are the major bile acids in human bile, have a high affinity for phospholipids and may disrupt cell membrane integrity. In contrast, the hydrophilic bile acid, ursodeoxycholic acid (UDC), is essentially non-toxic and has even been shown to prevent or reduce hepatocyte injury and cholestasis induced by high concentrations of hydrophobic bile salts (Heuman et al., 1991aGo,bGo). UDC's beneficial effects have been demonstrated in the treatment of cholestatic diseases (Leuschner et al., 1989Go; Poupon et al., 1991Go; Colombo et al., 1992Go; Galabert et al., 1992Go) and of drug-induced cholestasis (Zhao and Montet, 1990Go; Queneau et al., 1993Go, 1994Go).

On the other hand, UDC has been shown to improve liver-function tests in patients with alcoholic cirrhosis and cholestasis who still continue to consume ethanol (Plevris et al., 1991Go). Moreover, UDC protects Hep G2 cells from ethanol-induced cytotoxicity (Neuman et al., 1995Go) and, similarly, tauroursodeoxycholate (TUDC) counteracts the inhibitory effect of ethanol on bile secretion and vesicular exocytosis in the isolated perfused rat liver (Alvaro et al., 1995Go). We have recently demonstrated that UDC was able to reduce steatosis and lipid peroxidation induced by chronic ethanol feeding in rats (Oliva et al., 1998Go; Tabouy et al., 1998Go). However, the role of bile salts in modulating chronic ethanol cytotoxicity has not yet been explored. The beneficial effect of UDC on steatosis might not occur in the presence of a hydrophobic bile salt pool.

The aim of this study was therefore to compare the effects of CDC and UDC on chronic ethanol hepatotoxicity in rats and to determine whether UDC could be effective in preventing liver damage in rats co-administered ethanol and CDC.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
A nutritionally adequate diet was purchased from UAR Villemoisson, France. UDC was from Sigma Chemical Corporation (St Louis, MO, USA). CDC and taurine were from Calbiochem (Los Angeles, CA, USA). The bile salts used were >98% pure. 3{alpha}-Hydroxysteroid dehydrogenase was from Worthington Biochemical Corporation (Freehold, NJ, USA). Cholylglycine hydrolase was obtained from Sigma. Kits for enzyme activity [aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), and lactate dehydrogenase (LDH)] measurements were obtained from Boehringer (Boehringer Mannheim, Germany). Triglycerides (triacylglycerols) and total and free cholesterol contents were determined by enzymatic procedure with commercial kits from Boehringer (Boehringer Mannheim, Germany). Lipid peroxidation was measured using the LPO-586 assay from Boehringer Ingelheim (Bioproducts, Gagny, France).

Experimental design
Male Sprague–Dawley rats (Iffa-Credo, l’Arbresle, France) weighing 250 ± 5 g (mean ± SEM) were housed at 25°C with 12 h/12 h light/dark cycles. They received humane care and the studies were approved by the Service Vétérinaire de la Santé et de la Protection Animale (no. 001456). Thirty rats were pair-fed liquid diets containing 18% of energy as protein, 35% as fat, 11% as carbohydrate and 36% as either ethanol (ethanol diet) or as an isocaloric maltose–dextrin mixture (control diet), according to Lieber and De Carli (1989). Rats were randomly distributed into five groups of six rats each and received various treatments for 28 days: control liquid diet, ethanol diet, ethanol diet + UDC (100 mg/kg/day), ethanol diet + CDC (80 mg/kg/day), ethanol diet + UDC (100 mg/kg/day) + CDC (80 mg/kg/day). The selected UDC dose (100 mg/kg/day) is rather similar to that previously used to prevent hepatotoxicity induced by various drugs, including ethanol (Zhao and Montet, 1990Go; Queneau et al., 1994Go; Tabouy et al., 1998Go). Various doses of CDC (40–100 mg/kg/day) added to the control liquid diet were tested in order to select the CDC dose that meet two conditions: (1) to markedly enrich the bile acid pool with CDC; (2) to obtain a CDC biliary output lower than the maximum biliary secretion of CDC in the rat (Kitani et al., 1994Go). The CDC dose we selected (80 mg/kg/day) did not induce cholestasis or cytolysis. All diets were supplemented with 0.25% taurine in order to favour tauroconjugation of bile salts. At the end of the treatment, rats were anaesthetized with pentobarbital. Cannulations of the common bile duct and of the femoral vein were performed. Animals were placed in restraining cages. After 30 min equilibration, bile was collected for 1 h to determine bile salt, phospholipid, cholesterol and LDH biliary secretion. An intravenous infusion of 0.9% (w/v) NaCl was maintained at 1.5 ml/h to replenish body fluids. Blood samples were taken for further GLDH, AST and ALT analyses. The peritoneal cavity was then opened, and the liver was perfused through the portal vein with ice-cold lactate physiological serum until most of the blood content of the liver was washed out. The liver was quickly removed and weighed. A sample of ~1 g was homogenized in Tris–HCl buffer (20 mM, pH 7.4) at 4°C with a Potter-Elvehjem homogenizer, in order to determine the rate of lipid peroxidation. Another aliquot of liver was quickly frozen and stored at –25°C until lipid analysis.

Hepatic lipids and peroxidation products
Total liver lipids were extracted from homogenized liver with chloroform/methanol (2/1; v/v) according to Tabouy et al. (1998). Triglyceride, free cholesterol and cholesterol ester concentrations were determined by enzymatic procedure with commercially available test kits. To assess the extent of hepatic lipid peroxidation, the concentrations of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in the liver were measured using the LPO-586 kit. This procedure is based on the reaction of a chromogenic reagent with MDA and 4-HNE to yield a stable chromophore with maximal absorbance at 586 nm.

Biliary lipid secretion
Phospholipid and cholesterol concentrations in bile were analysed as previously described (Zhao and Montet, 1990Go). Total biliary bile salts were determined enzymatically using the 3{alpha}-hydroxysteroid dehydrogenase method (Turley and Dietschy, 1978Go). Individual molecular species of bile salts were determined by gas liquid chromatography after deconjugation by cholylglycine hydrolase (Zhao and Montet, 1990Go). The ratios taurine/glycine-conjugated bile salts were obtained after thin-layer chromatographic separation using butanol/ethyl acetate/acetic acid (50/40/10, by vol) as solvent system, and scanner densitometry. The hydrophobicity index of the bile acid pool was calculated according to Heuman (1989).

Plasma and bile enzymatic activities
The activity of biliary LDH was determined immediately after bile collection. Serum was separated from whole blood by centrifugation (4°C for 10 min at 800 g) and stored at –20°C. GLDH activity was determined by following the oxidation of NADH at 340 nm. AST and ALT activities were measured at 37°C by conventional enzymatic methods with Boehringer kits.

Statistics
Results are expressed as means ± SEM. Statistical differences between groups were assessed by the Kruskal–Wallis test, and the Mann–Whitney test was used for group-by-group comparisons. Differences were regarded as statistically significant if P < 0.05.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver weight and liver lipid concentrations
Table 1Go shows that ethanol-fed rats had a significant 30% increase in liver weight, when compared with control rats. Bile salt feeding modulated liver weight, which was decreased by 17% by UDC supplementation to the ethanol diet, but increased by 10% by CDC supplementation, both in comparison with the liver weight of ethanol-treated rats. When UDC was added to the ethanol + CDC diet, the rise in liver weight was reduced by 15%.


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Table 1. Influence of ursodeoxycholic acid (UDC) and/or chenodeoxycholic acid (CDC) administration on ethanol-induced changes in liver weight and lipids
 
Table 1Go also shows the influence of ethanol and ethanol plus UDC and/or CDC administration on liver lipid accumulation. Ethanol feeding led to a major increase in liver triglycerides and in cholesterol esters. When UDC was co-administered with ethanol, steatosis was partly prevented: liver triglyceride and cholesterol ester levels were decreased by 33% and 31% respectively. By contrast, CDC supplementation to the ethanol diet led to an increase in triglyceride and cholesterol ester contents (+53% and +31% respectively). When UDC was added to the ethanol + CDC diet, steatosis was partly prevented (–47% for triglycerides and –46% for cholesterol esters). Liver lipid concentrations were similar to those of rats receiving the ethanol + UDC diet.

Hepatic lipid peroxidation
As shown in Table 2Go, ethanol promoted lipid peroxidation. Concomitant treatment with UDC during ethanol administration was associated with a significant decrease in lipid peroxidation: –16% for MDA and –20% for MDA + 4-HNE. In contrast, when CDC was co-administered with ethanol, lipid peroxidation was significantly increased by 18% and 16% for MDA and MDA + 4-HNE respectively above the ethanol-only levels. Lipid peroxidation was partially prevented by UDC administration when the ethanol diet was supplemented with CDC. The levels of MDA + 4-HNE were identical with those obtained with the ethanol + UDC diet.


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Table 2. Ursodeoxycholic acid (UDC) and chenodeoxycholic acid (CDC) effects on ethanol-induced lipoperoxidation
 
Biliary lipid secretion and biliary LDH output
Bile flow was unaffected by the different treatments (Table 3Go). Biliary lipid secretion of control rats was not significantly modified by ethanol and ethanol + UDC treatment, but was enhanced by CDC feeding. In the ethanol + UDC + CDC group, secretion of bile salts, phospholipid, and cholesterol was significantly higher than in the ethanol and ethanol + UDC groups. No differences on biliary LDH levels were found between the different groups of rats (Table 3Go).


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Table 3. Bile flow, biliary secretion of lipids and biliary lactate dehydrogenase
 
Bile acid composition and hydrophobicity indices
Taurine supplementation exclusively led to tauroconjugated bile salts. Table 4Go displays the proportions of the different molecular species of bile salts in each group of rats. Chronic ethanol administration did not affect the biliary bile acid profile of control rats. For the groups supplemented with bile salts, the administered bile salt became the predominant biliary bile salt. UDC proportions were 47% for the ethanol + UDC group and 33.8% for the ethanol + UDC + CDC group, whereas the CDC proportions were 49% and 31% for ethanol + CDC and ethanol + UDC + CDC groups, respectively.


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Table 4. Bile acid profile and hydrophobicity index
 
The hydrophobicity index of each bile acid pool, as estimated by reverse-phase high-performance liquid chromatography (Heuman, 1989Go), is also shown in Table 4Go. Control rats and ethanol-treated rats had very similar hydrophobicity indices. UDC supplementation led to a decreased index, whereas CDC feeding led to a positive index. In rats co-administered UDC and CDC, the net hydrophobicity index was slightly negative (–0.09).

Serum parameters
Table 5Go reports the effect of the addition of UDC and/or CDC on ethanol-induced cytotoxicity. Ethanol alone increased ALT, AST and GLDH activities. When ethanol was given concomitantly with UDC, the GLDH activity decreased significantly (by 42%). In contrast, a 2-fold increase in GLDH activity (over the ethanol value) was induced by CDC supplementation to the ethanol diet. This effect was significantly blocked when UDC was added to the ethanol + CDC diet (by 44%). GLDH level was larger in the ethanol + CDC + UDC group than in the ethanol + UDC group, but the difference did not reach statistical significance. CDC administration tended to increase serum AST and ALT activities. When UDC was added to the ethanol and the ethanol + CDC diets, ALT tended to diminish, but AST was significantly decreased.


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Table 5. Ursodeoxycholic acid (UDC) and chenodeoxycholic acid (CDC) effects on serum enzymes
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present investigation indicate that bile salts modulate hepatic disorders induced by chronic ethanol feeding in rats. Supplementation with the hydrophobic bile salt CDC aggravates ethanol-induced damage, whereas the administration of the hydrophilic bile salt UDC has a protective effect. Interestingly, UDC also ameliorates hepatocellular injuries associated with alcohol and CDC co-treatment. Thus, the beneficial action of UDC is maintained when the bile acid pool contains a large fraction of hydrophobic bile salts, a situation which occurs in human bile. Our results also demonstrate that UDC has the capacity to protect hepatocytes in the absence of cholestasis. This is a novel aspect of the positive effect of UDC because, to date, the main target of UDC treatment is cholestasis.

Our observations show that CDC supplementation to the ethanol diet enriched the bile acid pool with CDC without inducing cholestasis. However, this treatment induced hepatic lipid metabolic dysfunctions more severe than those observed with ethanol alone: steatosis was significantly increased (by 53% for triglycerides and 31% for cholesterol ester concentration) and lipid peroxidation level was also increased (by 17%). The additional toxic effects induced by CDC could result from the great capacity of this hydrophobic bile salt to bind to lipid bilayers (Güldütuna et al., 1993Go; Ben Mouaz et al., 2001Go). CDC, at millimolar concentrations, has been shown to increase the fluidity of the apolar core of liver plasma membranes (Güldütuna et al., 1993Go), a fluidizing effect also observed with ethanol (Beaugé et al., 1996Go; Oliva et al., 1998Go). In the presence of ethanol, the toxic threshold of CDC could be lowered and thus both molecules may alter membrane structure under in vivo conditions. On the other hand, the release of the specific mitochondrial enzyme GLDH was significantly increased by CDC supplementation, thus reflecting an alteration of the inner mitochondrial membrane permeability. This is consistent with the studies of Krähenbühl et al. (1994a), who found that hydrophobic bile salts can affect the function of enzyme complexes in the mitochondrial electron transport chain. Also, Spivey et al. (1993) showed that lethal hepatocellular injury by glycochenodeoxycholate was associated with a rapid and significant depletion of cellular ATP followed by a sustained increase in [Ca2+]. Glycochenodeoxycholate impaired state 3, but not state 4, mitochondrial respiration. Similarly, we reported (Tabouy et al., 1998Go) that mitochondria from rats chronically fed ethanol demonstrated an impaired ability to produce energy with a decrease in the ADP-stimulated respiration (V3). Such mitochondrial alterations caused by CDC and/or ethanol may contribute to the development of the steatosis we observed in the present study.

In contrast to the deleterious effect of ethanol + CDC treatment, we showed that exogenous supplementation of the hydrophilic bile salt UDC to ethanol feeding improved ethanol-induced hepatic dysfunction. This UDC beneficial effect may be due to the slightly decreased hydrophobicity index of the bile salt pool, and more specifically to the greater capacity of UDC to protect hepatocytes, than the other two hydrophilic bile acids, cholic and ßMC. Our data confirm and extend previous findings (Alvaro et al., 1995Go; Neuman et al., 1995Go; Oliva et al., 1998Go) on the hepatoprotective effect of UDC. A key point of the present study is that UDC attenuates cytotoxicity of ethanol plus CDC co-administration. Indeed, UDC supplementation reduced hepatomegaly (by 15%), steatosis (by 47% for triglycerides and 46% for cholesterol esters) and lipid peroxidation (by 30%). Moreover, the preventive effect of UDC included an attenuation of the increased serum values of ALT, AST and GLDH produced by ethanol + CDC feeding with a greater effect on those enzymes that reside either partially (AST) or totally (GLDH) in mitochondria. It is noteworthy that in spite of the presence of a high percentage of CDC and of a hydrophobicity index larger than that observed in the ethanol group, UDC administration still exerted beneficial effects when compared with the ethanol group.

The therapeutic effect of UDC in cholestatic diseases has been partly attributed to its ability to induce a choleresis and to displace more hydrophobic bile salts from the hepatocyte. In our intoxication model, these mechanisms cannot explain the protective action of UDC, because no bile flow impairment, and consequently no retention of toxic bile acids in hepatocytes, occurred. Indeed, our data indicate that the biliary output of CDC was rather similar in the ethanol + CDC and ethanol + CDC + UDC groups. Therefore, the efficacy of UDC may be due to its cytoprotective properties. In order to explain this beneficial effect, a direct interaction of UDC with liver membranes has been suggested. First, according to Güldütuna et al. (1993) the mechanism of UDC hepato-protection against hydrophobic bile salts may be linked with its capacity to stabilize plasma membranes. We also found that UDC was an efficient plasma membrane stabilizer against ethanol (Beaugé et al., 1996Go; Oliva et al., 1998Go). Second, recent data indicate a direct protection of liver mitochondrial membranes by UDC both against hydrophobic bile salts (Krähenbühl et al., 1994bGo; Botla et al., 1995Go; Benz et al., 1998Go; Rodrigues et al., 1998Go; Güldütuna et al., 1999Go) and ethanol-induced injury (Neuman et al., 1995Go; Tabouy et al., 1998Go). These two mechanisms are not mutually exclusive. The present results on GLDH release provide additional insights regarding the protective effect of UDC on mitochondria.

In conclusion, the major finding of the current study is that UDC partially protects the liver against the intoxication induced by the co-administration of ethanol and CDC. This beneficial effect appears to be independent of the choleretic properties of UDC and may be attributed, at least in part, to UDC cytoprotection on mitochondria.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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