Loss of A(1) adenosine receptor attenuates alpha-naphthylisothiocyanate-induced cholestatic liver injury in mice.

Cholestasis has limited therapeutic options and is associated with high morbidity and mortality. The A(1) adenosine receptor (A(1)AR) was postulated to participate in the pathogenesis of hepatic fibrosis induced by experimental extrahepatic cholestasis; however, the contribution of A(1)AR to intrahepatic cholestatic liver injury remains unknown. Here, we found that mice lacking A(1)AR were resistant to alpha-naphthyl isothiocyanate (ANIT)-induced liver injury, as evidenced by lower serum liver enzyme levels and reduced extent of histological necrosis. Bile acid accumulation in liver and serum was markedly diminished in A(1)AR(-/-) mice compared with wild-type (WT) mice. However, biliary and urinary outputs of bile acids were significantly enhanced in A(1)AR(-/-) mice. In the liver, mRNA expression of genes related to bile acid transport (Bsep and Mdr2) and hydroxylation (Cyp3a11) was increased in A(1)AR(-/-) mice. In the kidney, A(1)AR deficiency prevented the decrease of glomerular filtration rate caused by ANIT. Treatment of WT mice with A(1)AR antagonist DPCPX also protected against ANIT hepatotoxicity. Our results indicated that lack of A(1)AR gene protects mice from ANIT-induced cholestasis by enhancing toxic biliary constituents efflux through biliary excretory route and renal elimination system and suggested a potential role of A(1)AR as therapeutic target for the treatment of intrahepatic cholestasis.

[Extrahepatic cholestasia during therapy with Zolmitriptan (AscoTop)]. / Extrahepatische Cholestase unter Therapie mit Zolmitriptan (Asco-Top).

Because of recurrent abdominal pain, jaundice and elevated liver function tests, a sixty-two-year old man had been referred to hospital several times within the preceding six months. By means of ultrasonography, endoscopic retrograde cholangiopancreatography (ERCP) and magnetic resonant cholangiopancreatography, dilated extrahepatic bile ducts were diagnosed. Stones and a tumorous process were excluded. ERCP showed hypermotility of the upper gastrointestinal tract. Quantitative hepatobiliary scintigraphy demonstrated retention of activity in the intra- and extrahepatic bile ducts and delayed transit of activity to the duodenum as signs of papillary dysfunction. Drug anamnesis revealed that the patient had started migraine treatment with Zolmitiptan, a 5-HT (1B/1D)-receptor agonist of the second generation, six months before the beginning of the cholestasia syndrome. Because of the known increase in amplitudes of oesophageal motor waves and of lower oesophageal sphincter tone by Sumatriptan, a 5-HT (1B/1D)-receptor agonist of the first generation, Zolmitriptan treatment was stopped. Thereupon, laboratory findings normalised and the patient has been feeling well for more than one year. Serotonin is an important monamine neurotransmitter that acts both in the CNS and in the gastrointestinal tract on identical receptors and is transported by the same systems. Thus it is not surprising that therapeutic measures which influence the serotoninergic system in the CNS are also effective in the enteric nervous.

Effect of glutathione depletion and hydrophilic bile acids on hepatic acute phase reaction in rats with extrahepatic cholestasis.

BACKGROUND: Extrahepatic cholestasis by biliary obstruction induces an acute phase reaction in the liver. It is a complex process involving cytokines, hormones and growth factors. To determine whether the regulation of acute phase proteins (APP) in cholestasis depends on glutathione (GSH), the effect of buthionine sulfoximine-induced (BSO-induced) GSH depletion on the expression of various APP was studied. In addition, we determined the influence of hepatoprotective bile acids on hepatic APP and underlying cytokine events. METHODS: Liver samples of bile-duct-ligated or sham-operated rats were examined. mRNA expression was quantified by densitometric analysis of Northern blots. RESULTS: Expression of APP increased 2-5-fold in bile-duct-ligated rats as compared to sham-operated controls. This acute phase reaction remained similar independently of whether cholestasis occurred for 5 days or 3 weeks. In contrast to alpha2-macroglobulin and tissue inhibitor of metalloproteinases-1 (TIMP-1), mRNA levels of both beta-fibrinogen and haptoglobin were significantly up-regulated after GSH depletion by BSO in cholestasis. Feeding of ursodeoxycholic and iso-ursodeoxycholic acid markedly down-regulated alpha2-macroglobulin and TIMP-1 expression in cholestasis but did not affect overexpression of beta-fibrinogen and haptoglobin. Cholestasis leads to an increased APP expression accompanied by an increased expression of inflammatory cytokines (IL-6, TNF-alpha). After feeding of hydrophilic bile acids, increases in inflammatory cytokines were abrogated. CONCLUSIONS: We show that GSH is involved in the acute phase reaction during obstructive cholestasis. In addition, bile acids might selectively ameliorate the acute phase response by reducing expression of the APP not affected by GSH depletion (alpha2-macroglobulin and TIMP-1).

The bcl-2 mRNA expression in GCDC-induced obstructive jaundice in rats and its implication in hepatocellular apoptosis.

The modulatory role of bcl-2 gene in hepatocellular apoptosis of rats with glycochenodeoxycholate (GCDC)-induced obstructive jaundice was investigated. The hepatocytes in normal rats and those with bile duct-ligation for 7 days, 14 days and 21 days were isolated and obtained by in situ collagenase perfusion and primary culture. The expression of bcl-2 mRNA in the hepatocytes was detected by RT-PCR. Primary culture was performed on the hepatocytes from normal rats and those with bile duct-ligation for 14 days. 100 mumol/L GCDC was added to the hepatocytes for incubation for 24 h. The hepatocellular apoptotic ratio was measured by using FCM and hepatocellular apoptosis detected in situ by using TUNEL technique. Results showed that the expression of bcl-2 mRNA was not detectable in the hepatocytes of normal rats by RT-PCR technique, while detectable in the hepatocytes of those with bile duct ligation (BDL) for 7, 14 and 21 days. Hepatocellular apoptosis in the BDL group was obviously decreased as compared with normal control group after addition of 100 mumol/L GCDC to the cells for 24 h. It was concluded that the hepatocytes in the BDL rats expressed bcl-2. During obstructive jaundice, expression of bcl-2 from the hepatocytes can inhibit the bile salt-induced hepatocellular apoptosis.

[Paracetamol overdose and experimental cholestasis induced gene expression regulation that codified P-Glycoprotein type 1 in rats]. / La sobredosis de paracetamol y la colestasis experimental inducen la expresión de los genes que codifican la P-glicoprotína tipo 1 en ratas.

Excretion of lipophilic cationic toxic compounds from the interior of the hepatocyte to the bile is mediated by P-Glycoprotein. It is an integral protein located in the bile canaliculi. The present work study the hepatic expression of P-Glycoprotein in different models of experimental liver disease: Acute paracetamol intoxication, extrahepatic cholestasis and cholestasis followed by acute paracetamol intoxication. mRNA was isolated from liver tissue, and the expression of Pg-p was assessed by Northern blot. It is concluded that the hepatic expression of mdr1b is increased in the experimental liver diseases when compared to controls. On the other hand, mdr2 expression was similar between the different groups.

Extrahepatic biliary stenoses after hepatic arterial infusion (HAI) of floxuridine (FUdR) for liver metastases from colorectal cancer.

Hepatic arterial infusion of floxuridine is an effective treatment for unresectable hepatic metastases from colorectal cancer. Despite its pharmacological advantage of higher tumor drug concentration with minimal systemic toxicity, hepatic arterial infusion of floxuridine is characterized by regional toxicity, including hepatobiliary damage resembling idiopathic sclerosing cholangitis (5-29% of treated cases). Unlike previous reports describing biliary damage of both intrahepatic and extrahepatic ducts, a case series of extrahepatic biliary stenosis after hepatic arterial infusion with floxuridine is herein described. Between September 1993 and February 1999, 54 patients received intraarterial hepatic chemotherapy based on continuous infusion of floxuridine (dose escalation 0.15-0.30 mg/kg/day for 14 days every 28 days) plus dexamethasone 28 mg. Twenty-seven patients underwent laparotomy to implant the catheter into the hepatic artery, the other 27 patients receiving a percutaneous catheter into the hepatic artery through a transaxillary access. Five patients (9.2%) developed biliary toxicity with jaundice and cholangitis (3 cases), alterations of liver function tests and radiological features of biliary tract abnormalities. They received from 9 to 19 cycles (mean 14.5 +/- 6.3 cycles) of floxuridine infusion with a total drug delivered dose ranging from 20.3 to 41.02 mg/kg (mean: 31.4 +/- 13.5 mg/kg). Extrahepatic biliary sclerosis was discovered by computed tomography scan and ultrasound, followed by endoscopic retrograde cholangiopancreatography and/or percutaneous cholangiography in 3 cases. Radiological findings included common hepatic duct complete obstruction in 1 case, common hepatic duct stenosis in 2 cases, common bile duct obstruction in 1 case, and intrahepatic bile ducts dilation without a well-recognized obstruction in 1 case. Two patients were treated by sequentially percutaneous biliary drainage and balloon dilation while 1 patient had an endoscopic transpapillary biliary prosthesis placed. Percutaneous or endoscopic procedures obtained the improvement of hepatic function and cholestatic indexes without subsequent jaundice or cholangitis. In two patients suppression of floxuridine infusion allowed the improvement of hepatic function. The present series suggests that in some patients receiving hepatic arterial infusion of floxuridine extrahepatic biliary stenosis may represent the primary event leading to a secondary intrahepatic biliary damage that does not correlate with specific floxuridine toxicity but results from bile stasis and infection, recurrent cholangitis and eventually biliary sclerosis. Aggressive research for extrahepatic biliary sclerosis is advised, since an early nonsurgical treatment of extrahepatic biliary stenosis may prevent an irreversible intrahepatic biliary sclerosis worsening the prognosis of metastatic liver disease.

Differential expression of canalicular membrane Ca2+/Mg(2+)-ecto-ATPase in estrogen-induced and obstructive cholestasis in the rat.

Extracellular adenosine triphosphate (ATP) may regulate hepatocyte and cholangiocyte functions, and under some conditions it may have deleterious effects on bile secretion and cause cholestasis. The canalicular membrane enzyme Ca2+/Mg2+-ecto-ATPase (ecto-ATPase) hydrolyzes ATP/adenosine diphosphate (ATP/ADP) and regulates hepatic extracellular ATP concentration. Changes in liver ecto-ATPase in estrogen-induced cholestasis were examined in male rats receiving 17alpha-ethinylestradiol (E groups) for 1, 3, or 5 days (5 mg/kg/day, sc) and compared with changes in rats subjected to obstructive cholestasis (O groups) for 1, 3, or 8 days. Activity of ecto-ATPase, protein mass in canalicular membranes and bile (estimated by Western blotting), steady state mRNA levels (by Northern blotting), and cellular and acinar distributions of the enzyme (histochemistry and immunocytochemistry) were assessed in these groups. Activity of ecto-ATPase, protein mass in isolated canalicular membranes, and enzyme mRNA levels were significantly increased in E group rats as compared with controls. In contrast, these parameters were markedly decreased in O group rats, and the enzyme protein was undetectable in bile. The ecto-ATPase histochemical reaction was markedly increased in the canalicular membrane of E group rats, extending from acinar zone 2 to zone 1, whereas it decreased in the O group. The ecto-ATPase immunocytochemical reaction was present in the canalicular membrane and pericanalicular vesicles in control and E group hepatocytes, but it decreased in obstructive cholestasis and was localized only to the canalicular membrane. Thus, significant changes in liver ecto-ATPase were apparent in 17alpha-ethinylestradiol-induced cholestasis that were opposite to those observed in obstructive cholestasis. Assuming that the alterations observed in obstructive cholestasis are the result of the cholestatic phenomenon, we conclude that changes in ecto-ATPase in 17alpha-ethinylestradiol-treated rats might be either primary events or part of an adaptive response in 17alpha-ethinylestradiol-induced cholestasis.

Extrapancreatic organ impairment in caerulein induced pancreatitis.

BACKGROUND AND AIMS: Multiorgan function failures are the major fatal complications in acute pancreatitis. In this experiment, we studied 1) the manifestation and time course of extrapancreatic organ damage in an acute pancreatitis model and 2) whether the obstructive liver damage in this model is caused by the obstruction of common biliopancreatic duct compressed by oedematous pancreas. MATERIAL AND METHODS: 80 male Wistar rats were divided into two groups: control and caerulein groups (five subgroups in each group). In the caerulein group, the acute pancreatitis was induced by caerulein intraperitoneal injections. In the controls equal volume of saline was injected. Two subgroups, one in caerulein and one in control groups, had an intrapancreatic bile duct stent inserted transduodenally before the injections. The pancreas, liver, lung and kidney tissues and blood samples were obtained for the measurement or analysis of interstitial oedema, plasma amylase, alanine aminotransferase, bilirubin, urea, creatinine, alkaline phosphatase, lactate dehydrogenase, blood gas and electron microscopy at 1, 6, 12 and 24 hours after the last injection in unstented animals, and at 6 hours in stented animals. RESULTS: Lungs and kidney remained unchanged. Liver damage was found during the first 6-12 hours, manifest as increased plasma alanine aminotransferase and bilirubin and dilatation of bile canaliculi and hepatocyte damage in electron microscopy. The intrapancreatic bile duct stent did not resolve these changes. CONCLUSIONS: The liver may be the first evolved extrapancreatic organ in the early stage in this mild oedematous pancreatitis model and the hepatocyte damage is not caused by the obstruction of common biliopancreatic duct compressed by the oedematous pancreas.

Structure-function correlation of tight junctional impairment after intrahepatic and extrahepatic cholestasis in rat liver.

BACKGROUND & AIMS: Tight junctions, the only barrier between blood and bile, are crucial in bile formation. The aim of this study was to correlate changes in morphology and permeability by comparing structural parameters with marker secretion into normal and cholestatic rat bile. METHODS: Cholestasis was induced by bile duct ligation of 5 and 21 days of ethinylestradiol administration. Quantitated structural parameters induced junctional length, strand number, junctional depth, and spacing of junctional particles. Junctional permeability was probed with horseradish peroxidase and dextrans of increasing sizes. RESULTS: Junctional length was decreased slightly by ethinylestradiol (-16% after 21 days) but increased by ligation (77%). Mean strand number decreased from 4.6 to 3.7 after 21 days of ethinylestradiol and 3.4 after ligation associated with increased junctional depth. The proportions of morphologically horseradish peroxidase-positive junctions increased from 4% to 15% after 21 days of ethinylestradiol and to 56% after ligation. Horseradish peroxidase secretion was increased twofold by ethinylestradiol and 6.5-fold by ligation, paralleled by an increase of dextran size selectivity from 70,000 to 79,000 daltons after ethinylestradiol and to 266,000 daltons after ligation. CONCLUSIONS: Impairment of junctional integrity is paralleled with the degree of cholestasis, whereas correlation of morphological and physiological alterations shows a close structure-function relationship.

Management of biliary complications after heart transplantation.

BACKGROUND: Immunosuppression increases the risk of biliary complications in heart transplant recipients. METHODS: Patients undergoing heart transplantation since 1986 who were at risk for cholelithiasis (n = 60) were retrospectively studied. RESULTS: Cholestatic jaundice developed in all patients after the operation because of biliary obstruction from cholelithiasis, cyclosporine toxicity, Imuran toxicity, or Gilbert's disease. The incidence of cholelithiasis or sludge was 42% (n = 25 of 60). Gallstones developed within 1.8 +/- 1.1 years in 17% of patients (n = 8 of 48) with a normal pretransplantation ultrasonogram. Biliary colic or gallstone pancreatitis developed 2 +/- 1.2 years after transplantation in 58% of patients (n = 7 of 12) with asymptomatic gallstones diagnosed before transplantation. The overall incidence of cholecystectomy or cholecystectomy with Roux-en-Y cystojejunostomy was 40% (n = 24). Both open cholecystectomy (n = 5) and laparoscopic cholecystectomy (n = 19) were performed without significant complications. Recovery is significantly more rapid (p < 0.05) after laparoscopic cholecystectomy versus open cholecystectomy (1 week versus 3 weeks). CONCLUSIONS: This analysis indicates that transplant candidates who have gallstones on pretransplantation evaluation or in whom gallstones develop after transplantation should undergo laparoscopic cholecystectomy at the earliest time in their posttransplantation course (i.e., 3 months) regardless of their symptomatic status. Removal of the diseased gallbladder not only simplifies the evaluation of cholestatic jaundice by eliminating the need for multiple ultrasonograms to exclude acute cholecystitis or choledocholithiasis but also safely minimizes the risk of the development of severe biliary complications.

[Development of biochemical and histopathological parameters in paracetamol overdose in experimental cholestasis]. / Evolución de parámetros bioquímicos e histopatológicos en la sobredosis con paracetamol durante la colestasis experimental.

The aim of the present paper is to determine the effect of Paracetamol (P) acute intoxication in cholestatic rats. Four groups of animals were considered: controls, controls intoxicated with P, rats intoxicated with P and cholestatic rats. Hepatic biochemical tests and liver histology were performed in every group. It is concluded that cholestatic rats intoxicated with P showed less Liver damage than in control groups.

Evolucion de parametros bioquimicos e histopatologicos en la sobredosis con paracetamol durante la colestasis experimental / Development of biochemical and histopathological parameters in paracetamol overdose in experimental cholestasis

En este trabajo se estudia el efecto de la intoxicación aguda con paracetamol (P) en ratas colestáticas. Se tomaron 4 grupos de ratas: controles, controles intoxicadas con P, controles colestáticas y colestáticas intoxicadas con P. Para todos los grupos se realizaron test de bioquímica hepática e histopatología del hígado. Se concluye que las ratas colestáticas sufren menor daño hepático cuando se las somete a intoxicación con P que sus respectivos controles

[Secondary drug-induced cholestasis with bile duct involvement]. / Les cholestases médicamenteuses secondaires à une atteinte canalaire.

This article summarizes our present knowledge concerning drug-induced cholestasis by lesions of the bile ducts. Acute or chronic ductular and ductal cholestasis are induced by a few drugs. The acute form may mimic viral hepatitis or biliary tract obstruction. The outcome is generally good. The mechanism may be immunoallergic. The clinical presentation of the chronic form resembles that of primary biliary cirrhosis or sclerosing cholangitis. Its prognosis is generally good and characterized by a complete recovery. However, an evolution to biliary cirrhosis is sometimes possible. The mechanism could also be immunoallergic. Extrahepatic cholestasis is frequently observed after intra-arterial infusion of floxuridine. The prognosis is generally poor. The mechanism of this sclerosing cholangitis may be ischaemic.

Final outcome of ursodeoxycholic acid treatment in 126 patients with radiolucent gallstones.

One hundred and twenty-six patients with radiolucent gallstones in 'functioning' gallbladders were treated with 8-10 mg ursodeoxycholic acid (UDCA) kg/day and followed to a treatment conclusion. Complete or partial gallstone dissolution was achieved in 74 (59 per cent). However, only 22 achieved complete gallstone dissolution, as judged by two normal oral cholecystograms; ultrasonograms were performed in 16 of these patients, and all were normal. UDCA was stopped in 76 patients: because of cystic duct obstruction (n = 12), severe biliary pain (n = 13), non-response (n = 25) or partial stone dissolution with arrested progress (n = 26). Life-table analysis showed that complete gallstone dissolution rates at four years were 25-30 per cent (two normal oral cholecystograms) and 17-19 per cent (two normal oral cholecystograms plus one ultrasonogram). All patients with complete gallstone dissolution had shown partial stone dissolution at 6-12 months; of those with partial stone dissolution at six months, only 25 per cent went on to complete gallstone dissolution, and then always within two years. Efficacy correlated inversely with stone size but not with age, sex, obesity or on-treatment saturation indices. Acquired surface gallstone calcification developed in 13 patients (life-table analysis 22 +/- 7 per cent at four years); none of these patients achieved complete gallstone dissolution and only five achieved partial stone dissolution. Thus, despite relatively high partial gallstone dissolution rates, the ultimate efficacy of UDCA in achieving complete gallstone dissolution is low.