phalloidine and Cholestasis

Glutathione is a tripeptide comprised by L-glutamate, L-cysteine, and glycine, that serves antioxygenation and deintoxication functions within the cell. Recent study has found that glutathione is the main driving force for bile salt-independent bile flow, impaired biliary excretion of glutathione can lead to cholestasis. This review focuses on hepatobiliary transport of glutathione and its role in cholestasis. Based on the evidence of choleretic effect of glutathione, enhancement of biliary excretion of glutathione may be a good strategy for prevention and treatment of cholestasis.

Topics: Animals; Biological Transport; Cholestasis; Estrogens; Glutathione; Humans; Jaundice, Chronic Idiopathic; Liver; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Mutation; Phalloidine; Ursodeoxycholic Acid

Biliary glutathione is an important generator of the bile-salt independent flow, and is known to be regulated by the hepatic glutathione availability. We investigated, in an acute model of phalloidin-induced cholestasis, biliary glutathione secretion and the role of hepatic glutathione, oxidative stress, and protein kinase c activation, which have been implicated in many hepatotoxin-induced hepatic dysfunctions.. Rats were given a single dose of 80 microg/100 g body weight of phalloidin and the hepatic thiols and glutathione content, redox state and vesicular activity were evaluated during both development of and recovery from cholestasis. The prophylactic effect of N-acetylcysteine (a precursor of glutathione synthesis and an antioxidant) was also examined. In addition, in the isolated perfused rat liver, we studied the prophylactic effect of the PKc inhibitor H7 on phalloidin-induced cholestasis.. In the early stages of cholestasis, phalloidin induced a decline in bile flow, mainly related to a disruption of biliary glutathione secretion. The decline in biliary glutathione content was not associated with increased glutathione degradation, indicated by a parallel decline in biliary non-protein thiols and by the lack of an increase in biliary gamma-glutamyltranspeptidase. There was also no evidence of hepatic depletion of glutathione or of oxidative stress, as measured by the oxidized-to-reduced glutathione ratio. Moreover, phalloidin resulted in inhibition of vascular transcytosis as assessed by horseradish peroxidase labeling. Pre-treatment with N-acetylcysteine did not counteract the decline in biliary glutathione secretion and bile flow produced by phalloidin, supporting the view that the hepatic availability of glutathione and oxidative stress injury are not implicated in the early stages of cholestatic injury. Moreover, treatment with H-7 did not alter the biliary glutathione output, or the decline in bile flow induced by the toxin.. This study suggests that the phalloidin-induced inhibition of bile formation may be attributed to rapid disruption of the hepatocanalicular transport of glutathione.

Topics: Animals; Cholestasis; Disease Models, Animal; Glutathione; Male; Oxidative Stress; Phalloidine; Rats; Rats, Sprague-Dawley

Bile canalicular membrane fluidity is modulated by phospholipid molecular species within membrane lipid bilayers. Thus, organellar membrane lipid composition is a determinant of canalicular function. In this study, the effect of phalloidin-induced cholestasis on bile lipid composition and liver subcellular membrane fraction composition in rats was examined to clarify the relationship between cholestasis and hepatic lipid metabolism.. Each rat received one phalloidin dose (400 microg/kg, i.v.). After the bile was collected, liver microsomes and canalicular membranes were analysed. The bile flow rate decreased by 50% 3.5 h after phalloidin administration. Although the bile acid output remained almost the same, the phospholipid and cholesterol output were significantly decreased (by 40.3+/-5.97% and 76.9+/-5.56%, respectively). Thus, the cholesterol:phospholipid (C:P) ratio in bile was significantly decreased by 80.4+/-10.1%. Phalloidin administration also increased the saturated: unsaturated fatty acid ratio (S:U) in bile for phosphatidylcholine by 25.5+/-3.2%. In the canalicular membrane, the C:P and S:U ratios for phosphatidylcholine were increased (24.8+/-4.2% and 34.4+/-6.9%, respectively), while the S:U for sphingomyelin was decreased by 61.0+/-6.2%. In microsomes, the C:P was decreased by 41.0+/-6.0%, but the S:U for both phosphatidylcholine and sphingomyelin were unaffected. Canalicular membrane fluidity, assayed by 1,6-diphenyl-1,3,5-hexatriene fluorescence depolarization, decreased significantly. Therefore, increased secretion of hydrophobic phosphatidylcholine into bile was associated with more hydrophobic canalicular membrane phosphatidylcholine, while sphingomyelin in the canalicular membrane was less hydrophobic.. These results indicate that phalloidin uncouples secretion of cholesterol and phospholipids, which causes a redistribution of fatty acyl chain species among canalicular membrane phospholipids that alters membrane fluidity. These changes may be a homeostatic response mediated by the phospholipid translocator in the canalicular membrane, although direct evidence for this is unavailable.

Topics: Analysis of Variance; Animals; Bile Acids and Salts; Bile Canaliculi; Cholestasis; Cholesterol; Chromatography; Fatty Acids; Lipid Bilayers; Male; Membrane Fluidity; Phalloidine; Phospholipids; Rats; Rats, Sprague-Dawley

Administration of phalloidin, one of the toxic peptides of the mushroom Amanita phalloides, leads to rapid and sustained cholestasis in rats. Although attributed to the interaction of phalloidin with microfilaments, the events leading to cholestasis are incompletely understood. The adenosine triphosphate (ATP)-dependent, apical conjugate export pump, termed multidrug resistance protein 2 (Mrp2) or canalicular multispecific organic anion transporter, is the major driving force for bile salt-independent bile flow. We investigated the role of Mrp2 in phalloidin-induced cholestasis. Bile flow decreased to 53% and 31% of control at 15 and 30 minutes after phalloidin (0.5 mg/kg), respectively. Mrp2-mediated [3H]leukotriene excretion into bile during the initial 45 minutes was reduced to 44% of control when [3H]LTC4 was injected 15 minutes after phalloidin treatment. Mrp2 was progressively lost from the hepatocyte canalicular membrane and detected predominantly on intracellular membrane structures together with other canalicular proteins including P-glycoproteins, ecto-ATPase, and dipeptidyl-peptidase IV. By contrast, structures involved in intercellular adhesion (zonula occludens, zonula adhaerens, and desmosomes) as well as intermediate filaments of the cytokeratin type appeared largely unaffected within 30 minutes after phalloidin. In line with the immunofluorescence analysis, immunoblots indicated a loss of Mrp2 and P-glycoproteins from the canalicular membrane and a 3- and 4.6-fold increase of these transport proteins in the microsomal fraction, respectively. Our results indicate that phalloidin induces marked alterations of the hepatocyte canalicular architecture and a loss of Mrp2 together with other proteins from the canalicular membrane. The resulting cholestasis can therefore be explained in part by the loss of export pumps, including Mrp2, from the canalicular membrane.

Topics: Animals; Anion Transport Proteins; ATP Binding Cassette Transporter, Subfamily B; Bile; Carrier Proteins; Cholestasis; Female; Fluorescent Antibody Technique; Immunoblotting; Leukotrienes; Liver; Phalloidine; Rats; Rats, Wistar; Tissue Distribution

We investigated sequential changes in bile flow, serum and biliary biochemical parameters in phalloidin-induced cholestasis in rats. Intrahepatic cholestasis was induced by administration with phalloidin (500 microg/kg) for 7 days, and then the animals were allowed to survive for 1, 2, 4, 7, 14 and 28 days after the last treatment. In phalloidin-treated rats, bile flow significantly decreased up to 4 days of recovery, compared with the control animals. In contrast, serum ALP activity, LAP activity, cholesterol concentration and phospholipid concentration exhibited a marked elevation throughout the recovery periods. For biliary parameters, bilirubin excretion rate was unchanged but, cholesterol excretion rate showed a marked decrease throughout the recovery periods. These results demonstrate that some parameters, particularly important indexes of cholestasis (serum ALP, cholesterol, bile flow and so on), continued significant changes at least 4 days after the last administration of phalloidin. These results demonstrate that successive treatment with phalloidin can cause damage in most of serum and biliary parameters at a chronic stage of cholestasis. Thus, our findings may provide useful information for diagnosis of drug-induced cholestasis and help to further elucidate the biochemical mechanisms of drug-induced cholestasis in humans.

Topics: Animals; Bile; Biomarkers; Blood Chemical Analysis; Cholestasis; Liver; Male; Phalloidine; Rats; Rats, Wistar

In this study, the possible role of the hepatic microcirculation in phalloidin-induced cholestasis and hepatotoxicity was examined in isolated perfused rat livers (IPRL). Administration of a phalloidin bolus (1 mg/kg body weight) through the portal vein induced an immediate reduction of bile flow. In 16.9 minutes, bile flow was 50% lower than basal values. Portal pressure was only increased in 60 minutes after phalloidin injection and increased sharply from this time up to the end of perfusion (90 minutes). Under these conditions, phalloidin did not induce liver cell cytolysis, as assessed by aspartate transaminase (AST) and lactate dehydrogenase (LDH) release in the perfusate effluent. Under electron microscopy, hepatocytic vacuolization was mild 15 minutes after phalloidin administration but increased with time. At the end of perfusion, the hepatic architecture was markedly altered; erythrocyte accumulation was observed in both sinusoids and hepatocyte vacuoles. Evaluation by multiple indicator dilution curves showed that extravascular volume (EVV) was significantly affected by phalloidin. It was augmented in 30 minutes after phalloidin administration with values increasing gradually over time. Neither vascular nor cellular volume was altered. The hepatic swelling may be attributed to enlargement of the extravascular space of the liver. These results indicate that changes in the liver microcirculation are not the primary cause of phalloidin-induced cholestasis in the IPRL.

Topics: Animals; Bile; Bile Acids and Salts; Blood Pressure; Cholestasis; Female; Hemodynamics; In Vitro Techniques; Indicator Dilution Techniques; Liver; Liver Circulation; Microcirculation; Perfusion; Phalloidine; Portal System; Rats; Rats, Sprague-Dawley; Tissue Survival

The actions of vasopressin and glucagon, administered alone or together, were assessed on bile flow in perfused livers from rats made cholestatic by the injection of ethynylestradiol and from those allowed to recover from such treatment. Concomitant measurements were made of biliary calcium output as well as changes in the perfusate Ca2+ concentration, glucose output, and oxygen uptake. Experiments were also conducted where cholestasis was induced in vitro in the perfused liver by the infusion of phalloidin. In each case cholestasis was demonstrated to have occurred by a reduction in bile flow by approximately 50%. The data show that the transient increase in bile flow and bile calcium seen in control rat liver soon after the administration of vasopressin, particularly when coadministered with glucagon, is largely absent in cholestasis induced by ethynylestradiol and attenuated in cholestasis induced by phalloidin. At the same time the pattern of perfusate Ca2+ fluxes in ethynylestradiol-induced cholestasis shifts to one reflecting net efflux of the ion from the liver. The responses to glucagon administration alone contrast with those of vasopressin in that in the perfused liver of ethynylestradiol-treated rats, glucagon induces a pronounced and sustained increase in bile flow. In cholestasis induced by both ethynylestradiol and phalloidin, glucagon fails to induce an initial transient decrease in bile flow. The effects of glucagon, including enhancement of vasopressin-stimulated bile flow in control and in ethynylestradiol-treated rats, can be mimicked by dibutyryl cyclic adenosine monophosphate (cAMP). Changes in glucose output and oxygen uptake induced by both hormones are only slightly attenuated. The data show that the modulation of bile flow that occurs rapidly after the administration of vasopressin and glucagon to control perfused rat liver is altered in conditions of cholestasis induced by either ethynylestradiol or phalloidin.

Topics: Animals; Bile; Calcium; Cholestasis; Ethinyl Estradiol; Glucagon; Glucose; Liver; Male; Oxygen Consumption; Perfusion; Phalloidine; Rats; Rats, Wistar; Vasopressins

The mechanism of the protective effect of ursodeoxycholic acid in cholestatic liver diseases remains unclear. Since there is evidence that alterations in the pericanalicular actin microfilament network play a major role in cholestasis, the aims of this study were (a) to determine the effect of the cholestatic agents, taurolithocholate (TLC) and erythromycin estolate (ERY), on F-actin distribution in isolated rat hepatocyte couplets and (b) to assess the effect of tauroursodeoxycholate (TUDC) and taurocholate on the modifications induced by these two compounds. F-actin was stained with fluorescein-isothiocyanate phalloidin and fluorimetric measurements were performed using a scanning laser cytometer ACAS 570. F-actin distribution was assessed in the couplets by the ratio of the pericanalicular area fluorescence/total couplet fluorescence (CF/TF). At non-cytotoxic concentrations, TLC (50, 100 microM) and ERY (10, 50, 100 microM) induced a significant accumulation of F-actin around the bile canaliculus as indicated by increased fluorescence in the pericanalicular area and by the increased CF/TF ratio compared with the controls. Electron microscopy studies showed significant alterations in bile canaliculi microvilli in couplets treated with 100 microM TLC. Only a few canaliculi showed an increase in pericanalicular microfilaments after treatment with 100 microM ERY. As assessed by scanning laser cytometry, TUDC prevented changes in F-actin distribution when it was added to the medium with taurolithocholate or erythromycin estolate at equimolar concentrations. However, the morphological changes observed by electron microscopy after treatment with TLC were not modified by co-treatment with TUDC. Taurocholate was ineffective. We conclude that (a) abnormalities of pericanalicular F-actin microfilaments occur in two different models of cholestasis, (b) tauroursodeoxycholate prevents the accumulation of pericanalicular F-actin detected by scanning laser cytometry but not the morphological changes of the canaliculus observed by electronic microscopy. Therefore, in these experimental conditions, the protective effect of TUDC appears to be partial.

Topics: Actins; Animals; Bile Acids and Salts; Bile Ducts; Cholestasis; Erythromycin Estolate; Fluorescent Dyes; In Vitro Techniques; Isomerism; Liver; Male; Microscopy, Electron; Models, Biological; Phalloidine; Rats; Rats, Sprague-Dawley; Taurodeoxycholic Acid; Taurolithocholic Acid

The architectural framework of the pericanalicular sheath composed of cytokeratin intermediate filaments (IFs) was examined after phalloidin treatment, bile duct ligation, and alcoholic fatty liver in rats to assess the role of IFs in experimental cholestasis. Electron microscopy examination of whole mount unembedded extracted liver slices was employed to visualize the cytoskeleton. Immunofluorescence staining and immunoelectron microscopy of the sheath were also performed using monoclonal antibodies to rat hepatocyte cytokeratins CK49 and CK55. The thickness of the wall and the diameter of the lumens were measured. In the phalloidin-treated rats, the pericanalicular sheath was markedly dilated and thickened. Immunofluorescence staining showed that the CK49 and CK55 IFs were localized in the pericanalicular region, particularly in the pericentral area. Immunoelectron microscopy documented that the IFs at the thickened pericanalicular sheath consisted of both CK49 and CK55, which means that the thickening of the bile canaliculus was in part due to an increase of IFs and not just due to an increase in actin filaments. In the livers where the bile duct was ligated, the pericanalicular sheath was irregularly dilated and some parts of the sheath appeared thinned out or missing. The belt desmosome also appeared absent focally in the pericanalicular sheath. Immunofluorescence studies showed that the staining for CK49 and CK55 was reduced focally in the pericanalicular region. The CK55 antibody stained the cytoplasm of hepatocytes in the periportal area more intensely when compared with the controls. These results indicated that the pericanalicular sheath and the belt desmosome were focally disrupted in response to extrahepatic bile duct obstruction. In the ethanol-fed rats, the pericanalicular sheath was dilated, thickened and tortuous, and appeared focally flattened by large fat droplets. IFs in the cytoplasm were pushed to the cell periphery and were compressed against each other by the fat droplets. CK55 and CK49 appeared increased as indicated by the observed immunofluorescence at the pericanalicular region. Immunoelectron microscopy showed that IFs of the thickened pericanalicular sheath were composed of CK55 and CK49. It is suggested that the pericanalicular sheath functions to mechanically provide a scaffolding for the bile canaliculus which is vulnerable to the different forces involved in cholestasis of different pathogenesis such as focal compression a

Topics: Actins; Animals; Bile Canaliculi; Bile Ducts; Bile Ducts, Intrahepatic; Cholestasis; Cytoskeleton; Fatty Liver, Alcoholic; Fluorescent Antibody Technique; Intermediate Filaments; Keratins; Ligation; Liver; Male; Microscopy, Electron; Phalloidine; Rats; Rats, Inbred Strains

We observed the motility of bile canaliculi in isolated rat hepatocytes obtained from animals that were pretreated with phalloidin (500 micrograms/kg body wt) for 1 and 3 days. Time-lapse cinephotomicrographs were taken in each experiment and in controls for 16.25 h. As we previously reported in normal hepatocytes, active contractions of bile canaliculi were observed. The number of contractions was 127.6 in controls, compared with 58.0 in 1-day phalloidin pretreated and 16.2 in 3-day phalloidin pretreated groups. The contraction process itself was not normal in the experimental groups, the contraction phase being slow and sustained. The altered canalicular motility depended on the dose of phalloidin administered. These results suggest that the integrity of actin filaments is necessary for normal bile canalicular motility. Further, dysfunction of actin microfilaments with altered canalicular motility may be involved in the pathogenesis of canalicular cholestasis.

Topics: Animals; Bile Canaliculi; Cells, Cultured; Cholestasis; Cytoskeleton; Female; Liver; Microscopy, Electron; Muscle Contraction; Muscle, Smooth; Oligopeptides; Phalloidine; Rats; Rats, Inbred Strains; Staining and Labeling; Time Factors

Topics: Adult; Autoimmune Diseases; Cholelithiasis; Cholestasis; Cholestasis, Extrahepatic; Ethanol; Female; Gallbladder Neoplasms; Hepatitis; Humans; Hydrocarbons, Halogenated; Phalloidine

Studies on isolated liver cell plasma membranes enriched in bile canaliculi from male rats treated with phalloidin show marked changes in the membrane polypeptides. Upon examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a protein component identical in mobility to the myosin standard was dramatically reduced, while that corresponding to actin was increased. It is suggested that a myosin-like protein may be necessary for the contractile function of the actin filaments in the liver cells. The observed modifications may be related to the decreased bile secretion and dilatation of bile canaliculi induced by phalloidin.

Topics: Actomyosin; Animals; Cell Membrane; Cholestasis; Cytoskeleton; Liver; Male; Membrane Proteins; Oligopeptides; Phalloidine; Rats

Phalloidin, one of the main toxins of Amanita phalloides, induced hepatotoxicity in female Wistar rats at 0.9 mg/kg dose i.p. Biliary secretion was selectively inhibited after 3h, but was restored after 24 h. Phalloidin also induced a cytolytic lesion, but not a fatty liver, as in alpha-amanitin intoxication. Propranolol pretreatment (30 min prior to phalloidin injection) did not afford protection against hepatotoxicity, but increased alkaline phosphatase, 5'-nucleotidase and aminotransferase activities.

Topics: Animals; Bile; Chemical and Drug Induced Liver Injury; Cholestasis; Female; Lipid Metabolism; Liver; Oligopeptides; Phalloidine; Propranolol; Rats; Transaminases

Rats made cholestatic by bile duct ligation survive phalloidin poisoning. This protection against phalloidin poisoning is correlated with the rate of the toxin uptake by the liver. [3H]demethylphalloin was used as a tracer for uptake studies. This is justified because there is no significant difference in the rate of uptake of the phallotoxins used. Within 4 h after bile duct ligation phalloidin uptake is reduced to about 25% and after 24 h to about 15% of controls. Isolated perfused livers and isolated hepatocytes from cholestatic rats bind less phalloidin than normal controls. Besides morphological changes serum factors may account for the decreased rate of phallotoxin uptake in cholestatic cells.

Topics: Alkaloids; Animals; Cholestasis; In Vitro Techniques; Liver; Male; Oligopeptides; Phalloidine; Rats

Phalloidin, administered to male rats for 7 days (500 microgram per kg/day), increased the mean hepatic content of filamentous actin. Both bile flow and bile acid excretion diminished proportionally, whereas the bile-to-plasma ratios of [3H]inulin and [14C]sucrose increased significantly from 0.08 and 0.16 in controls to 0.37 and 0.69, respectively, in phalloidin-treated animals. Simultaneously, junctional permeability was altered as noted by the free penetration of ionic lanthanum into the zonula occludens and bile canaliculus. Freeze-fracture replicas of the junctional complex revealed rearrangements of the junctional elements and regions in which only a single element separated the canaliculus from the lateral intercellular space. These findings suggest that microfilaments influence the permeability of "tight junctions" between hepatocytes and that bile constituents might reflux from the canaliculus to the intercellular space in phalloidin-induced cholestasis.

Topics: Actins; Animals; Bile; Body Weight; Cholestasis; Cytoskeleton; Intercellular Junctions; Inulin; Liver; Male; Oligopeptides; Permeability; Phalloidine; Rats; Sucrose

Topics: Alkaline Phosphatase; Animals; Bile Ducts; Bilirubin; Cholestasis; gamma-Glutamyltransferase; Male; Oligopeptides; Phalloidine; Rats

The influence of phalloidin, an agent that causes irreversible polymerisation of actin into microfilaments, on bile secretion and hepatocyte ultrastructure was examined in rats. Phalloidin was given intraperitoneally at the dose of 50 microgram per 100 g of body weight per day for 1, 3, or 7 days. The following was observed. (1) Bile flow decreased, as compared to controls, by 19% after 1 day, 34% after 3 days, and 55% after 7 days. Bile acid secretion was also decreased. (2) Electron microscopic examination of the hepatocyte in treated animals revealed an increased thickness of the pericanalicular microfilamentous network and a dilatation of bile canaliculi. Stereological examination revealed an increase in the relative volume of the microfilamentous network (per unit of hepatocyte cytoplasm) of 2.55% after 1 day, 4.06% ater 3 days, and 6.16% after 7 days. (3) [14C]Erythritol biliary clearance, measured after 7 days, decreased in parallel to bile flow, suggesting that the decrease in bile flow was of canalicular origin. [14C]Sucrose biliary clearance increased in treated animals, suggesting an increased permeability of the biliary system to sucrose. There was a predominant decrease in the bile acid independent bile flow. These data provide circumstantial evidence for the hypothesis that microfilament dysfunction can produce cholestasis.

Topics: Animals; Bile; Cholestasis; Erythritol; Injections, Intraperitoneal; Liver; Male; Microscopy, Electron; Oligopeptides; Phalloidine; Rats

Topics: Animals; Bile; Cholestasis; Liver; Phalloidine