Dietary lecithin protects against cholestatic liver disease in cholic acid-fed Abcb4- deficient mice.

Mutations in multidrug resistance 3 gene (MDR3 or ABCB4) underlie progressive familial intrahepatic cholestasis type 3 (PFIC3), a severe pediatric liver disease progressing to cirrhosis. Abcb4-/- mice exhibit slowly developing hepatic lesions that can be accelerated by feeding a cholic acid (CA)-supplemented diet. We investigated the beneficial effects of a soybean lecithin (L)-supplemented diet in this model of liver disease. Abcb4-/- mice and wild-type (WT) controls were divided in four groups by the diet they were fed: control (C) diet, L-supplemented diet, CA-supplemented diet, and L- and CA-supplemented (L+CA) diet. After 2 wk on these regimens, liver enzymes and bilirubin were measured in serum with bile flow, total bile acids, and cholesterol (CHOL) and phospholipid (PL) concentrations in bile. Ductular hyperplasia, portal fibroblastic cell proliferation, myofibroblast activation, and hepatic fibrosis were quantified on liver sections. Abcb4-/- mice fed the C diet exhibited mild liver damage. CA produced very high elevations of serum liver enzymes and bilirubin with significant bile duct proliferation, peribiliary fibroblast activation, and fibrosis. The L-supplemented diet dramatically mitigated the hepatic damage in CA-supplemented diet animals. We conclude that L is protective against liver disease in Abcb4-/- mice and suggest that it could offer potential benefit in PFIC3.

The role of mdr2 in manganese-bilirubin induced cholestasis in mice.

The pathogenesis of manganese-bilirubin (Mn-BR) induced cholestasis has only been studied in rats and is associated with alteration in the hepatic homeostasis of cholesterol and phospholipids. Multidrug resistance-2 (mdr2) transporter, which mediates excretion of these lipids, is suggested to be involved in this phenomenon. The present study was undertaken to examine if Mn-BR induced cholestasis is reproducible in mice, then to clarify the role of mdr2 in its pathogenesis, using mice with disrupted mdr2 gene (mdr2 (-/-)). Results showed that Mn-BR combination decreased bile flow in mice. This reduction in bile flow was similar in mdr2 (-/-) and the wild type mdr2 (+/+). Furthermore, the change in biliary lipid excretion was comparable in both genotypes. These data indicate that Mn-BR induced cholestasis is reproducible in mice and provide evidence that mdr2 alteration is not a primary event in this form of cholestasis.

Effect of mdr2 mutation with combined tandem disruption of canalicular glycoprotein transporters by cyclosporine A on bile formation in mice.

UNLABELLED: The inhibition of canalicular glycoprotein transporters has been suggested as the cause of familial intrahepatic cholestasis. Mutations in multidrug resistance 3-glycoprotein (MDR3) gene induce progressive familial intrahepatic cholestasis type 3 (PFIC3). Phenotypically, mutation in mdr2 in mice resembles the disruption of MDR3 in human. Secondly, mutation in the bile salt exporting pump (BSEP)/sister of P-glycoprotein (spgp) gene causes progressive familial intrahepatic cholestasis type 2 in human. However, in spgp knock-out mice only a mild persistent cholestasis occurs. The aim of this study is to evaluate the effects of various P-glycoprotein (Pgp) transporters on bile formation and the canalicular transport of taurocholic acid (TCA) in an attempt to understand the combined role of these transporters in the pathogenesis of familial intrahepatic cholestasis. Total bile acid (TBA) and cholic acid secretion rate were decreased in the mdr2 knock-out mice. However, bile flow (BF) and the secretion of muricholic acids were increased. Secretion of cholesterol was negligible and no phospholipids were detected in bile of mdr2 knock-out mice. Treatment with cyclosporine A (CsA) decreased the BF, and the biliary secretion of bile salts (BS) and phospholipids as compared to wild type mice, but after the injection of TCA+CsA, the BF, and the biliary secretion of BS and lipids were increased as compared to the wild type mice treated with CsA alone. In the mdr2 knock-out mice, CsA treatment decreased the BF and the secretion of BS but after the injection of TCA+CsA, the BF and the biliary secretion of BS were increased and the phospholipid secretion was slightly stimulated as compared to the mdr2 knock-out mice treated with CsA alone. CONCLUSION: Disruption of the mdr2 gene and the inhibition of glycoprotein transporters by CsA induce cholestasis in mice which is characterized by reduced BF, BS and biliary lipid secretion. However, CsA treatment did not significantly increase the cholestatic effect in the mdr2 knock-out mice. The injection of TCA decreased the cholestatic effect in the mdr2 knock-out mice as well as the inhibition of glycoproteins transporters by CsA. These data suggest that mutation in the canalicular mdr2 is an important factor during the development of progressive familial cholestasis.

Phosphatidylcholine-enriched diet prevents gallstone formation in mice susceptible to cholelithiasis.

Cholesterol gallstones affect approximately 10-15% of the adult population in North America. Phosphatidylcholine (PC) is considered to be the main cholesterol solubilizer in bile. This study examined the effect of a PC-enriched diet on gallstone incidence in mice susceptible to cholelithiasis. The result obtained showed that the feeding of a lithogenic (LG) diet for 4 weeks or 8 weeks resulted in cholesterol gallstone incidences of 47% and 89%, respectively. These gallstone incidences were either reduced or prevented when the LG diet was enriched with 2% or 6% PC, respectively. The cholesterol saturation index (CSI) was reduced only in mice fed with LG + 6% PC diet as compared with mice fed the LG diet alone. However, in all groups, the CSI was significantly higher than in mice fed Purina chow diet. The biliary anionic polypeptide fraction (APF) was significantly increased in mice fed the LG + 2% PC diet and was reduced in those fed with LG + 6% PC diet. In conclusion, prevention or delay of gallstone formation was not due to a consistent effect on biliary lipid composition, suggesting a direct effect of PC on cholesterol solubilization and/or the effect of an additional nonlipid biliary component such as APF.

Role of apoptosis in the remodeling of cholestatic liver injury following release of the mechanical stress.

It has been known for a long time that portal fibrosis consecutive to experimental common bile duct ligation is reversible following obstacle removal, but the mechanisms involved remain unknown. We have studied the effect of bilioduodenal anastomosis and of simple biliary decompression on the remodeling of the lesion in bile duct-ligated rats. Rats were subjected to common bile duct ligation for 7 days or 14 days. Bilioduodenal anastomosis was performed after 14 days of bile duct ligation and animals sacrificed at intervals. In other animals, after 7 days or 14 days of ligation, the common bile duct was merely decompressed by bile aspiration and animals sacrificed 24 h later. Collagen deposition, alpha-smooth muscle actin expression and apoptosis were evaluated. Bile was collected and the bile acid profile assessed. After anastomosis, collagen deposition and alpha-smooth muscle actin expression decreased and were back to control values after 7 days. These parameters remained practically unchanged 24 h after biliary decompression. Bile duct ligation by itself induced apoptosis of some fibroblastic and bile ductular cells after 7 days; this was back to normal after 14 days. After anastomosis or decompression, apoptosis of both fibroblastic and bile ductular cells increased greatly and was accompanied by ultrastructural features of extracellular matrix degradation. Total bile acid content decreased after common bile duct ligation, the proportion of dihydroxylated bile acids decreasing and that of trihydroxylated bile acids increasing. Biliary decompression and anastomosis did not modify total concentration and composition of the biliary bile acid pool. In summary, we show that mere biliary decompression, by relieving the mechanical stress, is as effective as bilioduodenal anastomosis to induce apoptosis of portal cells that likely triggers portal fibrosis regression.

Urinary bile acid profile in children with inborn errors of bile acid metabolism and chronic cholestasis; screening technique using electrospray tandem mass-spectrometry (ES/MS/MS).

BACKGROUND: It is well known that urine becomes the major route for bile acid excretion in liver diseases and thus we examined bile acid profile in urine obtained from normal children and children having chronic liver diseases using electrospray tandem mass spectrometry (ES/MS/MS). MATERIAL/METHODS: Bile acid were extracted from 5 ml of urine obtained from five healthy children or from twenty patients with various liver diseases including patients with unknown chronic liver diseases, Zellweger syndrome, peroxisomal bifunctional protein deficiency disease, tyrosinema type 1, biliary atresia, and patients with progressive familial intrahepatic cholestasis (PFIC) of undetermined type. Identification and quantification of bile acids were achieved in 5 minutes using electrospray tandem mass spectrometry (ES/MS/MS). RESULTS: Urinary bile acid excretion increased in liver diseases an average of 100 times as compared to control values. There was a specific profile for different liver disease which confirms the pathology of the disease and could be used for its diagnosis. The results also show that the ions used for the diagnosis of oxo-steroid reductase deficiency disease were present in other chronic liver diseases suggesting that these atypical bile acids may not be a result of an inborn error of bile acid metabolism. CONCLUSIONS: The urinary bile acid profile obtained in this study by ES/MS/MS can be of use for the diagnosis of certain chronic liver diseases.

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats.

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.

Appearance of atypical 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in spgp knockout mice.

Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5 beta-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7 alpha-hydroxylase in male mice only. It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver.

Effects of bile acids on biliary epithelial cells: proliferation, cytotoxicity, and cytokine secretion.

Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependent manner (50-500 microM). Cell viability was significantly decreased by TLC and TCDC at 500 microM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases.

Biliary, fecal and plasma deoxycholic acid in rabbit, hamster, guinea pig, and rat: comparative study and implication in colon cancer.

Bile acids are believed to play a role in the etiology of colorectal cancer, and high fecal excretion of secondary bile acids was correlated with increased incidence of colon cancer. Recently, it was also reported that there is an increase in plasma of the secondary bile acid, deoxycholic acid in men with colorectal adenomas. Since deoxycholic acid is formed in the colon and absorbed into the portal systemic circulation, it was suggested that the blood concentration of this bile acid reflects the level of exposure of colonic cells to deoxycholic acid. The objective of this study was to investigate whether plasma deoxycholic acid level represents the fecal content of this bile acid in several animal species with different bile acid composition and deoxycholic acid contribution to the bile acid pool. Eight rabbits, hamsters, guinea pigs, and rats were used in this study. Blood samples and feces were collected on days 1, 3, 5 and 7. Bile samples were obtained only on day 7. The plasma, fecal and biliary bile acids were analyzed by gas chromatography-mass spectrometry. Bile acid composition and deoxycholic acid content varied greatly between the animal species studied. There was a variation in the concentration of total bile acids in the plasma and feces obtained at different times during the experiments, however, the bile acids profile remained constant throughout the study. The data obtained shows that although plasma bile acid profile was not similar to fecal bile acids profile, however, there was a significant correlation between the level of plasma and fecal deoxycholic acid. Plasma deoxycholic acid concentration might be a reliable biomarker for the degree of exposure of colon cells to this bile acid, and may be useful in further studies on the role of secondary bile acids in colon carcinogenesis.

Is 1-hydroxypyrene a reliable bioindicator of measured dietary polycyclic aromatic hydrocarbon under normal conditions?

Five healthy volunteers consumed similar amounts of identical foods for 5 consecutive days. The concentration of pyrene and of benzo(a)pyrene was determined in each of the 15 meals by a short analytical method that included sample saponification, solvent extraction, and HPLC analysis. The volunteers also provided three daily total volume 8-h urine samples for the duration of the study for the assessment of 1-hydroxypyrene, a biomarker of pyrene and polycyclic aromatic hydrocarbon (PAH) exposure. Mean recoveries were 83 and 75%, respectively, for pyrene and benzo(a)pyrene in food. Daily dietary pyrene doses varied from 0.7 to 3 microg. Excluding two outliers consisting of meals containing charbroiled pork and beef, pyrene content in the meals estimated from the published literature data was correlated to the measured pyrene, but overestimated the actual concentration by ca. 70%. Despite the identical ingested doses of pyrene, there was a 50-76% (coefficient of variation) interindividual variability in the daily-excreted amount of 1-hydroxypyrene. Urinary excretion of this metabolite was not correlated with ingested dose of pyrene under the normal feeding conditions used in this study. Bioavailability, enzymatic polymorphism, and differences in enterohepatic cycling of the metabolite may contribute to the observed variability. It was calculated that dietary pyrene intake accounts for between 87.5 and 99.8% of the sum of dietary and inhalation intake. From the presented data, unless the above-mentioned factors are taken into account, 1-hydroxypyrene might not be a reliable bioindicator of ingested pyrene (PAHs) under normal feeding conditions.

Cellular retinol-binding protein-1 expression and modulation during in vivo and in vitro myofibroblastic differentiation of rat hepatic stellate cells and portal fibroblasts.

Cellular retinol-binding protein-1 (CRBP-1) is involved in vitamin A metabolism because it mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid. CRBP-1 is highly expressed in the liver, particularly in hepatic stellate cells (HSC). In this study, we investigated the liver expression of CRBP-1 during experimental fibrogenesis. We also studied the regulation of CRBP-1 expression in cultured HSC and portal fibroblasts, two fibroblastic cell types involved in liver fibrogenesis. Fibrosis was induced in rats by carbon tetrachloride (CCl(4)) or bile duct ligation. Immunohistochemical staining was performed for CRBP-1 and alpha-smooth muscle (SM) actin, an activation marker of fibrogenic cells. CRBP-1 and alpha-SM actin expression was studied by Western blotting and/or Northern blot in primary cultures of HSC isolated by conventional methods and in portal fibroblasts that were obtained by outgrowth from the biliary tree after enzymatic digestion. In normal liver, contrary to HSC, portal fibroblasts did not express CRBP-1. After CCl(4) injury, CRBP-1 expression was maintained in myofibroblastic alpha-SM actin-positive HSC. After bile duct ligation, portal fibroblasts (which proliferated around ductular structures) acquired expression of both CRBP-1 and alpha-SM actin. During HSC activation in culture, CRBP-1 expression gradually increased until Day 5 when alpha-SM actin expression was obvious. Cultured portal fibroblasts developed both CRBP-1 and alpha-SM actin expression. In both cell populations, transforming growth factor-beta 1 treatment increased CRBP-1 expression. Thus, in normal liver, CRBP-1 expression was different among fibroblastic cells, a finding that adds to the concept of heterogeneity of liver fibrogenic cells. Furthermore, during myofibroblastic differentiation, HSC that lost their stores of retinol maintained a high level of CRBP-1 expression, whereas portal fibroblasts acquired CRBP1 expression. Together, these data suggest a correlation between CRBP-1 expression and myofibroblastic differentiation.

Rapid and improved method for the determination of bile acids in human feces using MS.

A simple method for the determination of bile acids in adult human fecal samples using GC-MS is described. Bile acids are directly extracted from feces by ethanol (95%) containing 0.1 N NaOH. Extracts are purified by passage through a reversed-phase C18 silica cartridge and then analyzed by GC-MS. The present study has shown that lyophilized human feces contain mainly free bile acids, with lithocholic acid (LCA) and deoxycholic acid (DCA) as the major bile acids; however, isomers of LCA and DCA, keto-bile acids, and cholic acid are also present. Any traces of conjugated bile acids are hydrolyzed before the C18 extraction by deconjugating enzymes, which are present in feces and are activated by the addition of water during the homogenization step. Thus, the analysis of fecal bile acids can be performed without the hydrolysis step in less than 4 h in comparison to traditional techniques, which usually require at least 48 h.