Synergistic Role of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase and Cholesterol 7{alpha}-Hydroxylase in the Pathogenesis of Manganese-Bilirubin–Induced Cholestasis in Rats

Marie-Yvonne Akoume*,{dagger}, Shahid Perwaiz*,{dagger}, Ibrahim M. Yousef*,{dagger},1 and Gabriel L. Plaa*

* Département de Pharmacologie, Université de Montréal, Montréal, Québec, Canada H3C 3J7; and {dagger} Centre de Recherche de l’Hôpital Sainte Justine, Montréal, Québec, Canada H3T 1C5

Received November 22, 2002; accepted January 21, 2003

ABSTRACT

Manganese (Mn) and bilirubin (BR) induce intrahepatic cholestasis when injected sequentially. It was suggested that accumulation of newly synthesized cholesterol in the canalicular membrane is an initial step in the development of cholestasis. To clarify the involvement of cholesterol in the pathogenesis of Mn-BR-induced cholestasis, we examined biliary secretion and liver subcellular distribution of lipids in the cholestatic conditions (Mn-BR combination). We also examined hepatic metabolism of cholesterol under cholestatic and noncholestatic (Mn or BR given alone) conditions. The Mn-BR combination reduced bile flow by 50%, and bile acid, phospholipids, and cholesterol output by 42, 75, and 90%, respectively. There was a dramatic impairment of cholate excretion but not chenodeoxycholate excretion. Phosphatidylcholine species secreted into bile were unchanged, and microsomal total phospholipid content was significantly increased. Although there was no changes in liver membrane phospholipid content, the cholesterol/phospholipid ratio was increased by more than 50% in the canalicular fraction. Despite the increased concentration of cholesterol in canalicular membrane the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, key enzyme in cholesterol synthesis, and cholesterol 7{alpha}-hydroxylase, key enzyme in cholesterol conversion to bile acids, were significantly reduced. However, the injection of Mn alone significantly increased both enzymes, while BR alone inhibited cholesterol 7{alpha}-hydroxylase by 62%, without affecting HMG-CoA reductase. In conclusion, the critical cholestatic events in Mn-Br-induced cholestasis appear to be mediated through the synergistic effects of Mn and BR acting on synthesis and degradation of cholesterol.

Key Words: cholesterol; phospholipid; cholate; canalicular membranes; HMG-CoA reductase; cholesterol 7{alpha}-hydroxylase.

Intrahepatic cholestasis is defined as a disturbance of bile secretion without anatomic obstruction (Feur and Difonzo, 1992Go). This situation results most often as an adverse consequence of drug therapy (Chan et al., 1998Go; Plaa and Priestly, 1976Go; Stieger et al., 2000Go). However, the pathologic mechanisms underlying cholestatic liver disease in patients remain uncertain. Several hypotheses have emerged from various laboratory models as possible mechanisms leading to intrahepatic cholestasis. Among these hypotheses is the accumulation of cholesterol in the bile canalicular membranes (BCM) (Duguay et al., 1998Go, 2000Go; Yeagle, 1991Go; Yousef et al., 1988Go). Such accumulation of cholesterol in BCM was suggested to result in disappearance of microvilli and disorganization of the canalicular lumen (Yousef et al., 1984Go), leading to a failure of bile secretion and impairment of bile flow. These biochemical and morphologic changes occur in various experimental models of intrahepatic cholestasis such as cholestasis induced by lithocholic acid (Kakis and Yousef, 1978Go), ethinylestradiol (Simon et al., 1980Go), and manganese-bilirubin combination (Plaa et al., 1982Go).

In general, cholesterol synthesis and conversion of cholesterol into bile acids represent key metabolic responses to conserve hepatic cholesterol at normal physiologic levels (Kuipers et al., 1996Go). Cholesterol homeostasis is preserved mainly through the modulation of the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Rodwell et al., 1976Go) and cholesterol 7{alpha}-hydroxylase (Myant and Mitropoulos, 1977Go), two key enzymes involved in synthesis and degradation of cholesterol, respectively. Although the increase in hepatic cholesterol synthesis and accumulation of newly synthesized cholesterol in the canalicular membrane was reported in Mn-BR induced cholestasis, the activity of HMG-CoA reductase was markedly suppressed (Duguay et al., 2000Go).

The aim of the present study was to clarify the metabolic basis associated with the accumulation of the newly synthesized cholesterol in the canalicular membrane in this cholestatic model.

MATERIALS AND METHODS

Chemicals.
Monohydrated manganese sulfate (MnSO4H2O), bilirubin (BR), urethane, mevalonic acid lactone, HMG-CoA, ethylenediaminetetraacetic acid (EDTA), dithiotreitol (DTT), nicotinamide adenine dinucleotide phosphate (reduced form, NADPH), glucose-6-phosphate dehydrogenase (500 U/1 ml) and glucose-6-phosphate were purchased from Sigma Chemical Co. (St Louis, MO). 7{alpha}-Hydroxycholesterol and 6ß-hydroxycholestanol were available from Steraloids Inc. (Wilton, NH). Dimethylethylimidazol was procured from Kogyo Inc. (Tokyo, Japan) and Bond Elut cyanopropyl (CN, 500 mg) from Varian (Harbor City, CA). All other reagents and solvents were of analytical grade.

Animals and experimental protocol.
Male Sprague-Dawley rats (200–250 g) were purchased from Charles River, Inc. (St.-Constant, Québec, Canada) and were kept in animal facilities and maintained on Charles River rat chow diet and water ad libitum for three days prior to their use in the experiments. Animals were anesthetized with urethane (1 g/kg ip). After opening the abdomen, the bile duct was cannulated with polyethylene tubing (PE-10) to allow sampling of bile. Bile was collected in 15-min intervals into preweighed tubes for 120 min, and volumes were determined assuming 1 µl = 1 µg. Body temperature was maintained during the experiment at 37°C using a rectal probe and a thermostatically controlled infrared lamp. Through catheters (PE-10) placed in the femoral vein, animals received freshly prepared aqueous solutions of Mn and BR. MnSO4H2O was dissolved in saline (0.9 %, NaCl) and injected as a single dose of 4.5 mg/kg in 1 min. BR was dissolved in the buffer containing 0.52 g NaCl and 0.52 g Na2CO3 per 100 ml and injected as a single dose of 25 mg/kg in 2 min. To induce cholestasis, both Mn and BR were administered; Mn was given 15 min before BR; the maximum reduction in bile flow was obtained 30 min after BR administration (45 min after Mn injection). At this moment, livers were removed to prepare subcellular fractions.

To investigate hepatic cholesterol metabolism, livers were removed in six different experimental situations: (1) treatment with Mn and BR vehicles (control), (2) 15 min after Mn injection (Mn-15), (3) 45 min after Mn injection (Mn-45), (4) 30 min after BR injection (BR-30), (5) 15 min after sequential compound treatment of Mn and BR (Mn-BR-15), (6) 30 min after sequential compound treatment of Mn and BR (Mn-BR-30).

Preparation of subcellular organelles from rat liver.
Separation of basolateral (SM) and canalicular membranes (BCM) and isolation of subcellular organelles, microsomes, mitochondria, golgi complexes, lysosomes, nuclear fraction, and cytosol, as well as assessment of the purity of these fractions, were performed as previously described from our laboratories (de Lamirande et al., 1981Go; Duguay et al., 1998Go; Yousef and Tuchweber, 1984Go). Liver microsomes, which are used to investigate cholesterol metabolism, were prepared as previously described (Yamashita et al., 1989Go). Briefly, liver was homogenized in 50 mM Tris–HCl buffer (pH 7.4), containing 0.3 M sucrose, 10 mM DTT, 10 mM EDTA, and 50 mM sodium fluoride. The homogenate was centrifuged at 20,000 x g for 15 min, and the supernatant was centrifuged at 100,000 x g for 60 min. The resulting microsomal fraction was suspended in 3 ml of 0.1 M potassium phosphate buffer (pH 7.4), containing 1 mM EDTA and 5 mM DTT. Aliquots were immediately frozen in liquid nitrogen and stored at -80°C until analysis.

Analysis of protein.
Protein determination was performed according to the method of Lowry et al.(1951)Go, using bovine serum albumin as the standard.

Assay of HMG-CoA reductase and cholesterol 7{alpha}-hydroxylase activities.
Microsomal suspensions of 500 µl, containing 5 mg protein, were preincubated for 5 min at 37°C with 450 µl of 0.1 M potassium phosphate buffer (pH 7.4), containing 1 mM EDTA and 12 mM glucose-6-phosphate. The assay was initiated by adding 50 µl of cofactor-substrate solution (0.1 mM HMG-CoA, 3 mM NADPH, and 2 U/ml glucose-6-phosphate dehydrogenase). The incubation was performed at 37°C for 30 min and was terminated by the addition of 50 µl of 1 M NaOH. Purification of reaction products (mevalonolactone and 7{alpha}-hydroxycholesterol) and their analysis by electrospray tandem mass spectrometry were carried out as described by our laboratory (Akoume et al., 2002Go).

Lipid and bile acid analyses.
Lipids were extracted from liver subcellular fractions and bile according to the method of Folch et al.(1957)Go. Total cholesterol was assayed using a commercially available kit for cholesterol determination (Boehringer, Mannheim, Germany), and phospholipids were determined by the colorimetric procedure of Bartlett (1959)Go. Phosphatidylcholine molecular species were identified by electrospray tandem mass spectrometry (Brügger et al., 1997Go).

Total bile acid concentration in bile was determined enzymatically with the 3{alpha}-hydroxysteroid dehydrogenase method (Turley and Dietschy, 1978Go). Electrospray tandem mass spectrometry (ES/MS/MS) and gas chromatography-mass spectrometry (GC/MS) were used for the identification and determination of biliary bile acids (Perwaiz et al., 2001Go). Briefly, for the ES/MS/MS analysis, bile acids were extracted with a C18 (octadecyl) reversed-phase column, and were analyzed by simultaneous monitoring of parent and daughter ions for the identification of glycine and taurine conjugates. For the GC/MS analysis, conjugated bile acids were hydrolyzed in 2.5 M NaOH at 120°C overnight. Bile acids were then extracted, methylated, and acetylated. Identification and quantification of bile acids were achieved using a Hewlett-Packard 5896 gas chromatograph equipped with a Hewlett-Packard 5971A mass selective detector operating in selected-ion monitoring mode (SIM). Quantification was carried out by using a correction factor obtained with 5ß-cholanic acid as internal standard (Perwaiz et al., 2001Go)

Statistical analysis.
Results are expressed as means ± SEM. Statistical analysis of data was performed with the Graph-Pad Prism® computer program and the statistical significance of difference between experimental groups was evaluated by a Student’s t-test. For multiple comparisons to the same group, a one-way ANOVA followed by the Dunnett test was employed. Probability values less than 0.05 were considered significant.

RESULTS

Bile Flow and Biliary Lipid Secretion
Table 1Go depicts bile flow and secretion rates of biliary bile acid, phospholipids, and cholesterol during the cholestatic phase (60- to 75-min interval). In comparison to control values, bile flow and bile acids out put were decreased by 50 and 42%, respectively. However, phospholipids and cholesterol output were dramatically reduced by 75 and 90%, respectively.


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TABLE 1 Bile Flow and Biliary Secretion of Lipids
 
Biliary Bile Acids
The results obtained show that a significant reduction in the biliary excretion of trihydroxylated glycine and taurine conjugated bile acids (Table 2Go). Despite the reduction in bile acid by the Mn-BR treatment, there was no change in the percent contribution of glycine and taurine conjugated bile acids. The percentage of glycine conjugated bile acid was 36.7 in control versus 33.4 in Mn-BR. The percentage of taurine conjugated bile acid averaged 63.7 in control versus 66.4 in Mn-BR. Although there was no change in the amount of dihydroxylated bile acids in Mn-BR as compared to control, there was a significant increase in the percent contribution of the dihydroxylated bile acids conjugated by glycine and taurine (18.6% versus 11.67% for the glycine dihydroxylated bile acid in Mn-BR versus control, and 25.16% versus 16.0% in the taurine conjugated dihydroxylated bile acids for the Mn-BR versus control respectively; Table 2Go).


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TABLE 2 Bile Acid Pattern in Bile
 
Similar changes were observed in the total bile acid profile (glycine, taurine conjugated bile acids, and free bile acids). The contribution of cholic acid was reduced from 78.5 to 58.1% in control and Mn-BR, respectively. However the contribution of the dihydroxylated bile acid chenodeoxycholate and its metabolites {alpha} and ß muricholate, was not significantly changed from the control values (Table 2Go).

Biliary and Microsomal Phosphatidylcholine Species
Analysis of phosphatidylcholine (PC) showed that the molecular species secreted into bile remained unchanged as compared to control; however, there were changes in the intensities of these species as shown by the electrospray mass spectra (Fig. 1Go). The intensities of all PC species increased in microsomes from Mn-BR–treated rats, while they decreased in the bile. The PC species found in the bile were 16:0/18:2, 16:0/20:4 and 16:0/18:0 and 16:0/22:6. The Mn-BR treatment reduced specifically the intensities of 16:0/18:2 and 16:0/20:4 and 16:0/18:0 in bile and increased their intensities in microsomes. The changes in the intensity of 16:0/22:6 species was affected to a lesser extent.



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FIG. 1. Electrospray mass spectra of phosphatidylcholine (PC) obtained from microsomal and biliary lipid extracts. The intensities of PC molecular species (16:0/18:2; 16:0/20:4 and 16:0/18:1; 18:0/18:2; 16:0/22:6; 18:0/20:4; 18:0/22:6), referred by the number beneath each species designation, increased in microsomes and decreased in bile following Mn-BR treatment.

 
Lipid Composition of Hepatic Subcellular Fractions
The subcellular distribution of hepatic protein was similar in control and Mn-BR groups, as shown in Table 3Go. However, the percentage of cholesterol was increased in canalicular and cytosolic fractions, and reduced in the microsomal fraction indicating a redistribution of subcellular cholesterol. The percentage of total phospholipids was increased in microsomal and cytosolic fractions. The cholesterol/phospholipid ratio was significantly increased in the canalicular membrane from 2.02 to 5.64. The lipid distribution in other subcellular fractions was not affected by the Mn-BR treatment.


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TABLE 3 Subcellular Distribution of Hepatic Protein and Lipids
 
Marker Enzymes of SM and BCM Fractions
Table 4Go shows the enzymatic activities for glucose-6-phosphatase (G-6-Pase), leucine aminopeptidase (LAP), and 5'-nucleotidase. The marker enzymes for microsome, canalicular membrane and for liver cell plasma membrane fractions. The G-6-Pase activity was reduced to 15% of that of the homogenate activities in BCM fraction and to 33% in SM fraction in the control group. In Mn-BR, the G-6-Pase activities were reduced by similar percentage, 14% in BCM and 39% in SM. The LAP and 5'-nucleotidase were enriched in both the BCM and SM. However, the LAP activity was much higher in BCM as compared to SM. This indicates that the Mn-Br treatment does not affect the purity or the isolation of the BCM and SM fractions.


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TABLE 4 Marker Enzyme Activities and Relative Enrichment in Homogenate, Bile Canalicular Membrane (BCM), and Sinusoidal Membrane (SM) of Control and Mn-BR Treated Rats
 
Hepatic HMG-CoA Reductase and 7-{alpha} Hydroxylase Activities
The Mn-BR combined treatment affected significantly hepatic cholesterol 7{alpha}-hydroxylase and HMG-CoA reductase activities (Fig. 2Go). Measurement of these enzymes from the moment of BR injection revealed a 55% decrease in cholesterol 7{alpha}-hydroxylase activity at 15 min that progressed and reached 75% at 30 min. The activity of HMG-CoA reductase was inhibited by 62% at 15 min, and by 53% at 30 min. Thirty min after a single administration, BR alone caused the same degree of inhibition for cholesterol 7{alpha}-hydroxylase but exerted no effects on HMG-CoA reductase (Fig. 3Go). In contrast, Mn alone produced a significant increase of both tenzymes, 67 and 63% for HMG CoA reductase and 7-{alpha} hydroxylase respectively 15 min after injection. This enzymatic stimulation was decreased with time 54% for HMG-CoA reductase, and 41% for cholesterol 7{alpha}-hydroxylase, respectively (Fig. 3Go).



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FIG. 2. Changes in microsomal HMG-CoA reductase and 7{alpha}-hydroxylase activities after Mn-BR treatment. Values are mean ± SEM of six rats. **p < 0.01, significantly different from zero time.

 


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FIG. 3. Relative activities of HMG-CoA reductase and 7{alpha}-hydroxylase. Values are mean ± SEM of six rats and are expressed relative to controls. *p < 0.05, **p < 0.01, significantly different from control group.

 
DISCUSSION

Alteration in BCM molecular organization, particularly an increase in the BCM, cholesterol and increase in cholesterol/phospholipid ratio, is associated with defect in bile formation and was suggested to be an important event in the development of certain forms of intrahepatic cholestasis (Kakis and Yousef, 1978Go; Simon et al., 1980Go; Yousef et al., 1984Go), including Mn-BR induced cholestasis (de Lamirande et al., 1981Go; Duguay et al., 1998Go). The present results confirm that the Mn-BR combined treatment increases the cholesterol content in BCM (1.61–4.23% of the total content of hepatocyte) with no significant changes in phospholipids content (Duguay et al., 2000Go). Such accumulation of cholesterol in BCM increases the cholesterol/phospholipids ratio, which is an index of membrane fluidity (Chen et al., 1999Go) and consequently might cause a decrease in 5'-nucleotidase and leucine aminopeptidase activities the marker enzymes of the liver cell membrane fractions.

Previous results showed that the cholesterol accumulated in BCM in the Mn-Br induced cholestasis model was newly synthesized; however, the activity of HMG-CoA reductase, the rate-limiting enzyme for cholesterol synthesis, was not increased (Duguay et al., 1998Go). Consequently, the increase in BCM newly synthesized cholesterol content can only be explained by a reduction in degradation of newly synthesized cholesterol as well as decreased in its biliary secretion. The data obtained in the present study support such hypothesis. Furthermore, the data suggest a synergistic effect for Mn and BR in the development of the cholestasis induced by the Mn-BR combination.

Although extrahepatic cholesterol is an important source of hepatic cholesterol (Oliver, 1990Go), the liver is quantitatively the most important organ for cholesterol synthesis (Steinberg, 1989Go). Furthermore, the liver maintains hepatic cholesterol balance by coordinated regulation of de novo synthesis, degradation to bile acids and direct excretion of cholesterol (Steinberg, 1989Go). The degradation of cholesterol to bile acids occurs by two different pathways leading to the formation of cholic acid, a trihydroxylated bile acid, and chenodeoxycholic acid, a dihydroxylated bile acid, which is precursor of other bile acids such as muricholate (Bandsma et al., 2000Go; Scheibner et al., 1993Go). The production of cholic acid is essentially assured through the main pathway initiated by cholesterol 7{alpha}-hydroxylase (Bandsma et al., 2000Go), which uses preferentially newly synthesized cholesterol (Scheibner et al., 1993Go). The secondary pathway initiated by 27-hydroxylase yields predominantly chenodeoxycholic acid (Bandsma et al., 2000Go), and the extrahepatic pool of cholesterol is the preferred source of cholesterol for this enzyme (Scheibner et al., 1993Go). Thus, analysis of bile acids in bile may be used to indicate the source of cholesterol used in the synthesis of bile acids. This is true in this study as the bile acid pool was depleted for 2 h prior to treatment and thus the majority of bile acids secreted are newly synthesized. In the Mn-BR combined treatment, the secretion of cholic acid was significantly reduced, but there were no changes in the secretion of all other bile acids, including muricholate, another trihydroxylated bile acid. These data suggest that cholic acid synthesis rather than canalicular transport is impaired, as evidenced by a significant decreased in activity of cholesterol 7{alpha}-hydroxylase. Therefore, the newly synthesized cholesterol was not degraded, resulting in its accumulation in the liver. The observation that chenodeoxycholic acid secretion was normal after Mn-BR treatment indicated that the secondary pathway for bile acid synthesis was not affected. Previously, data derived from experiments with differentially radio-labelled cholesterol were interpreted to indicate that the major contribution to cholesterol accumulation in BCM following Mn-BR treatment was newly synthesized cholesterol (Duguay et al., 2000Go). The bile acid results described in the present study, using differential bile acid profiles, are consistent with the hypothesis and further support the importance of newly synthesized cholesterol as the major source of the accumulating cholesterol in the Mn-BR model.

In addition, a direct positive relation was shown to exist between the increase in the detergent effect of bile acids and biliary lipid secretion (Coleman and Rahman, 1992Go). Because the detergent properties of chenodeoxycholic acid are stronger than those of cholic acid (Coleman, 1987Go), the changes induced by the Mn-BR combination in bile acid pattern should theoretically increase the secretion of cholesterol and phospholipids into bile. However, in bile obtained from the Mn-BR treated rats both cholesterol and phospholipids were drastically reduced. This strongly suggests that the Mn-BR combination might affect Mdr2, the transporter of biliary lipids (Smit et al., 1993Go). This possibility is supported by the low excretion of the phosphatidylcholine species 16:0/18:2, 16:0/20:4, and 16:0/18:1, as Mdr2 has been shown to be specificity involved in the transport of these species in bile (Berr et al., 1993Go). However, further work by direct measurement of the Mdr2 protein or activity is needed before this aspect can be clarified. It is also possible that the high amount of phosphatidylcholine species in microsome may not be due to the impairment of Mdr2 alone. Since other species notably 18:0/20:4 and 18:0/22:6, which are increased in microsome, are not secreted into the bile. Some investigators (Bernasconi et al., 2000Go; Kim et al., 2002Go) also showed that an increase in liver cholesterol content increases the proportion of phosphatidylcholine species in microsomes.

An interesting feature of the present study is that only the Mn-BR combination induces cholestasis. Injection of Mn alone leads to significant increase in the activities of cholesterol metabolism key enzymes, HMG-CoA reductase, and cholesterol 7{alpha}-hydroxylase. This enzymatic stimulation is reduced with time. BR injection alone, however, reduced the activity of cholesterol 7{alpha}-hydroxylase with no effect on HMG-CoA reductase. It appears that in the sequential regimen of Mn plus BR, Mn injection increased cholesterol synthesis as well as bile acid synthesis. The BR injection 15 min later decreased 7{alpha}-hydroxylase activity, leading to reduced bile acid synthesis, expressed mainly as a reduction in cholic acid secretion. The time interval between Mn and BR injection is critical for the induction of cholestasis (de Lamirande and Plaa, 1979Go). If one waits too long after Mn to administer the BR, decreased bile flow is not observed (de Lamirande and Plaa, 1979Go). Our results show that the inhibition of cholesterol degradation by BR coincides with the period where the activation of cholesterol synthesis by Mn is higher. Taken together, these data indicate that BR injection preceded by that of Mn, promote the accumulation of newly synthesized cholesterol in liver and favor its redistribution in tissue and specifically in the BCM leading to the cholestasis. This, in turn, may depress hepatic cholesterol synthesis, since it is possible that the intracellular concentration of free cholesterol exerts a negative feedback on HMG-CoA reductase activity in the liver (Hwa et al., 1992Go). In addition, the activity of this enzyme as well as that of cholesterol 7-{alpha} hydroxylase may be affected by the change in the bile acid profile (Naito et al., 1996Go).

Of particular interest from this study are the common features characterized by the disturbance in cholesterol homeostasis and the increased availability of cholesterol in liver and its redistribution in tissue organelle in experimental cholestasis induced by the administration of certain drugs (Andrade et al., 1993Go; Chisholm et al., 1999Go; Ishizaki et al., 1997Go; Simon et al., 1980Go). In all those models, cholesterol predominantly accumulated in canalicular membranes where the proteins involved in the hepatic transport of endogenous and exogenous substances are located. The consequences of such accumulation are a failure in the functions of these transporters, which is involved in bile formation, resulting in cholestasis. These suggest that cholesterol is an important component in the development of cholestasis induced by chemical substances, which is becoming prevalent with the occurrence of multidrug therapy (Chitturi and Farrell, 2001Go).

In conclusion, these results provide evidence that the increase in hepatic availability of newly synthesized cholesterol, which is associated with the pathogenesis of Mn-BR-induced cholestasis, is caused at least in part by the synergistic effects of Mn and BR acting separately on cholesterol synthesis and degradation. These findings may help to further elucidate the biochemical mechanisms of drug-induced cholestasis in human and lead to possible therapeutic maneuvers to prevent or treat the cholestasis induced by drug interactions.

ACKNOWLEDGMENTS

This study was supported by grant from the Canadian Liver Foundation. We thank Diane Mignault for the ES/MS/MS analysis of biliary phospholipids.

NOTES

1 To whom correspondence should be addressed at Université de Montréal, Département de Pharmacologie, C.P. 6128, Succ. Centre-ville, Montréal, Québec, Canada H3C 3J7. Fax: (514) 343-5819. E-mail: ibrahim.yousef{at}umontreal.ca. Back

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