Biliary excretion of taurocholate, organic anions and vinblastine in rats with alpha-naphthylisothiocyanate-induced cholestasis

BACKGROUND AND AIMS: alpha-Naphthylisothiocyanate (ANIT) is known to cause cholestasis due to injury of the bile duct epithelial cells. The aim of the present study was to examine the effect of a single dose of ANIT on the biliary excretion of various cholephilic compounds and on the amount of canalicular transporters. METHODS: Twenty-four hours after the oral administration of ANIT (100 mg/kg), the biliary excretion of taurocholate, leukotriene C(4), pravastatin and vinblastine was studied. The protein levels of the bile salt export pump and multidrug resistance protein 2 and the immunostaining of multidrug resistance protein 2 in the liver were also examined. RESULTS: The ANIT treatment markedly decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C(4), pravastatin and vinblastine. The biliary excretory maximum of taurocholate was also markedly decreased after ANIT treatment. The ANIT treatment had no effect on the protein levels of bile salt export pump and multidrug resistance protein 2 and the immunostaining of multidrug resistance protein 2 in the liver. CONCLUSIONS: These findings support canalicular transporters having little effect on the marked impairment of biliary excretion of cholephilic compounds in ANIT-induced cholestasis.     

Matrine improves 17α-ethinyl estradiol-induced acute cholestasis in rats

Aim:  To explore the effects of matrine (MT) on acute intrahepatic cholestasis induced by 17α-ethinyl estradiol (EE) in rats.
Methods:  Acute intrahepatic cholestasis in rats were induced by EE, and the effects of MT on acute intrahepatic cholestasis were explored and compared with ursodeoxycholic acid (UDCA) by serum biochemical determination and bile excretion experiments.
Results:  The serum biochemical and bile biochemical results indicated that MT and UDCA had notable hepatoprotective effects by counteracting cholestasis induced by EE. The bile flow and the bile excretion of glycocholic acid (GC, a substrate of bile salt export pump [Bsep]), ketoprofen glucuronide (KPG) and rhodamine 123 (Rh123, a substrate of multidrug resistance protein 1 [MDR1]) decreased by EE, were significantly improved after administration of MT.
Conclusion:  MT exhibited potential protection against EE-induced acute intrahepatic cholestasis.     

Effects of urso-deoxycholic acid (UDCA) on alpha-naphthyl-isothiocyanate (ANIT) induced intrahepatic cholestasis in rats]

Using the ANIT induced model of cholestasis in rats, the therapeutic effects of UDCA to the intrahepatic cholestasis were evaluated by changes of serum chemistry and liver histology. ANIT was administered once at a dosage of 40 mg/kg b.w. per os and UDCA was given ad libitum for 7 days by a drinking water containing UDCA at 0.5 and 5.0% solution. In the period of bile duct epithelial degeneration and necrosis, effects of UDCA for jaundice was not detected, but hepato-cellular disturbances were appeared histologically. Moreover, the elevation of serum levels of chenodeoxy-cholic acid, deoxycholic acid and lithocholic acid was accompanied. On the other hand, in the recovery stage of the bile duct epithelium, serum bilirubin was decreased significantly in the UDCA group which seemed to be related with the potent choleretic effect of UDCA. These results may indicate that UDCA is effective for the intrahepatic cholestasis in the case with no bile duct epithelial damage but in the presence of it hepato-cellular injury is introduced by the accumulated toxic bile acids in the blood.     

The effect of bile duct ligation, bile duct cannulation, and hypothermia on alpha-naphthylisothiocyanate-induced hyperbilirubinemia and cholestasis in rats

The hyperbilirubinemic and cholestatic responses to alpha-naphthylisothiocyanate (ANIT) in the rat were altered by subjecting test animals to various environmental and surgical manipulations. Studies utilizing hypo- and hyperthermic conditions showed that a positive correlation exists between the rectal temperature and the effects of ANIT. In addition, it was observed that ANIT produced an apparent poikilothermic response, in that treated rats were unable to maintain normal rectal temperatures. Bile duct ligation inhibited the cholestatic and altered the hypothermic responses to ANIT treatment. Cannulation of the bile duct prior to ANIT administration significantly inhibited the hyperbilirubinemia and cessation of bile flow. These data demonstrate the importance of an intact enterohepatic circulation and normal body temperature in the actions of ANIT. The effect of the various parameters on the ANIT-induced hyperbilirubinemia and cholestasis suggests the involvement of a biotransformation product of ANIT.     

Effects of secretin on bile production in two kinds of cholestatic models by choledocho-caval fistula and bile duct ligation in rats

Secretin is known to stimulate bile flow from the bile duct epithelium. To investigate the effects of secretin in cholestasis, we studied the response of the bile flow and the excretion of biliary components to secretin using two cholestatic models with or without damage to the bile duct epithelium. The model without bile duct epithelial damage was a choledocho-caval (CC) fistula over a 24-hour period, and the model with bile duct damage was a bile duct ligation over a 48-hour period. Secretin was administered by intravenous infusion for 30 minutes and bile was collected for 120 minutes. Controls were given saline similarly. The bile flow and biliary bicarbonate excretion rate were significantly increased after secretin infusion in the CC fistula rats when compared with the control rats, but no stimulation by secretin was observed in the ligated rats. These data indicate that secretin-induced bile production was enhanced under cholestatic conditions with no bile duct epithelial disturbance.     

Effects of secretin on bile production in two kinds of cholestatic models by choledocho-caval fistula and bile duct ligation in rats.

Secretin is known to stimulate bile flow from the bile duct epithelium. To investigate the effects of secretin in cholestasis, we studied the response of the bile flow and the excretion of biliary components to secretin using two cholestatic models with or without damage to the bile duct epithelium. The model without bile duct epithelial damage was a choledocho-caval (CC) fistula over a 24-hour period, and the model with bile duct damage was a bile duct ligation over a 48-hour period. Secretin was administered by intravenous infusion for 30 minutes and bile was collected for 120 minutes. Controls were given saline similarly. The bile flow and biliary bicarbonate excretion rate were significantly increased after secretin infusion in the CC fistula rats when compared with the control rats, but no stimulation by secretin was observed in the ligated rats. These data indicate that secretin-induced bile production was enhanced under cholestatic conditions with no bile duct epithelial disturbance.     

Reduction of alpha-naphthylisothiocyanate-induced hepatotoxicity by recombinant human hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a potent stimulator of DNA synthesis in cultured hepatocytes. To determine whether HGF has any activity in vivo, we have tested HGF in rats in which intrahepatic cholestasis was induced by acute administration of alpha-naphthylisothiocyanate (ANIT). The hepatotoxic effects of a single injection of ANIT were manifested 48 h later as large increases in serum bilirubin, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. These biochemical changes were accompanied by widespread periportal edema, hypertrophy of bile duct epithelium, and randomly scattered areas of liquifaction necrosis in the hepatic parenchyma. The increases in bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were markedly attenuated when HGF was administered 30 min before ANIT and again at 6, 12, 24, 30, and 36 h after ANIT. In addition, this HGF dosing regimen completely prevented the occurrence of parenchymal lesions, although it had no effect on periportal histopathology. The effect of ANIT was dose dependent; a maximal response was observed at 320 micrograms/kg per injection, with an intermediate response at 105 micrograms/kg. Delaying the administration of HGF until 12 h after ANIT was as effective as when administration was begun 30 min before ANIT. Taken together these results show that HGF can prevent some aspects of ANIT hepatotoxicity.     

Specific lectin bindings to oval cells and proliferated bile ductules

Histochemical evaluation of lectins was performed to examine the carbohydrate residues of oval cells induced by administration of alpha-naphthylisothiocyanate (ANIT), 2-acetylaminofluorene (2-AAF) and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) in comparison with those of normal bile ducts and proliferated bile ductules induced by bile duct ligation. The normal bile ducts showed intense binding of Ricinus communis agglutinin, concanavalin A and wheat germ agglutinin, and weak binding of Ulex europaeus agglutinin I (UEA I). A few cells in the portal bile ducts showed binding of peanut agglutinin (PNA). Two different binding patterns were observed in oval cells and proliferated bile ductules. One group showed increased binding of PNA, while the other showed intense binding of UEA I. In both groups, binding of other lectins was similar to those of the normal bile duct. The first group included oval cells induced by 2-AAF and 3'-Me-DAB, and the second included both oval cells induced by ANIT and proliferated bile ductules induced by ligation. These results suggest that oval cells and proliferated bile ductules have their own specific carbohydrate residues and that oval cells induced by the carcinogens might be a cell population different from those induced by non-carcinogens and proliferated bile ductules by ligation.     

The effects of somatostatin and octreotide on the human sphincter of Oddi.

OBJECTIVE: Somatostatin acts at different sites in the human gastrointestinal tract and generally inhibits the release and effects of many gastrointestinal hormones and neuropeptides. Together with its long-acting analogue octreotide, somatostatin is widely used in the treatment of hormone-producing tumours, variceal bleeding, etc., but multi-centre trials have failed to prove a beneficial effect in the treatment of acute pancreatitis or in the prevention of post-ERCP pancreatitis (pancreatitis following endoscopic retrograde cholangiopancreatography). The aim of the present work was to study the effects of somatostatin and octreotide on the human sphincter of Oddi by means of quantitative hepatobiliary scintigraphy (QHBS). METHOD: Fifteen cholecystectomized patients were enrolled in the study, six in the somatostatin group and nine in the octreotide group. QHBS was performed initially with a standard protocol (baseline data), then repeated after 0.1 mg octreotide or a 250 microg bolus + 250 microg/h somatostatin administration. In the 60th min of QHBS, 0.5 mg glyceryl trinitrate (GTN) was administered sublingually. RESULTS: QHBS demonstrated that both somatostatin and octreotide caused a marked impairment in the bile flow: the half-time of excretion (T1/2) over the common bile duct was significantly prolonged compared with baseline data (somatostatin group: common bile duct T1/2 180 min versus 59.7+/-31 min; octreotide group: common bile duct T1/2 140.9+/-60.5 min versus 30.7+/-11.7 min). Glyceryl trinitrate administration accelerated the transpapillary bile flow, with significant decreases in the elevated T1/2 in both groups. CONCLUSION: Increased transpapillary flow induced by glyceryl trinitrate may be beneficial in the treatment of acute or post-ERCP pancreatitis.     

Zinc L-carnosine protects against mucosal injury in portal hypertensive gastropathy through induction of heat shock protein 72

BACKGROUND AND AIMS: Increased susceptibility to gastric mucosal injury is observed in portal hypertensive gastropathy (PHG). In this study, the effects of zinc L-carnosine, an anti-ulcer drug, were evaluated on expression of heat shock protein (hsp) 72 and cytoprotection in gastric mucosa in a rat model of PHG. METHODS: Portal hypertensive gastropathy with liver cirrhosis was induced by bile duct ligation for 4 weeks in male Sprague-Dawley rats. Expression of gastric mucosal hsp72 was evaluated by Western blotting at 6 h after intragastric administration of L-carnosine, zinc sulfate, or zinc L-carnosine. Blood was also collected for determination of serum zinc level. Mucosal protective abilities against hydrochloric acid (HCl) (0.6N) followed by pretreatment with L-carnosine, zinc sulfate or zinc L-carnosine were also studied. RESULTS: L-carnosine, zinc sulfate, and zinc L-carnosine induced hsp72 in gastric mucosa of rats with bile duct ligation. Zinc sulfate and zinc L-carnosine suppressed HCl-induced mucosal injury. However, L-carnosine could not suppress HCl-induced mucosal injury. Serum zinc levels were significantly elevated after zinc L-carnosine administration. Furthermore, pretreatment with zinc L-carnosine (30-300 mg/kg) increased the expression of hsp72 in gastric mucosa and prevented HCl-induced mucosal injury in rats with bile duct ligation in a dose-dependent manner. CONCLUSIONS: Zinc derivatives, especially zinc L-carnosine, protected portal hypertensive gastric mucosa with increased hsp72 expression in cirrhotic rats. It is postulated that zinc L-carnosine may be beneficial to the mucosal protection in PHG as a 'chaperone inducer'.     

Urinary excretion of bile acids in bile duct-ligated rats

Background. In patients with complete bile duct obstruction, the only pathway of bile acid elimination is through the urine. However, the urinary excretion of various bile acid conjugates in the presence of bile duct obstruction has not been clarified. Given this factor, the urinary excretion of various bile acids was compared in rats that were bile duct-ligated for 3 days. Methods. After urinary bladder cannulation, radiolabeled bile acids were intravenously injected, and urine samples were collected every 2 h for 6 h, and radioactivity was counted. Results. Urinary excretion (cumulative percent dose during 6 h) of taurocholate and cholate was similar (19.3% and 16.8%). Urinary excretion of tauroursodeoxycholate, lithocholate, and taurolithocholate-sulfate was less effective (12.7%, 9.8% and 2.1%, respectively). Cholate was mostly conjugated with taurine, and lithocholate was mostly conjugated with taurine and further hydroxylated. Conclusions. These results indicate that unconjugated bile acids were taken up by the liver and excreted into the blood after further biotransformation even under conditions of complete bile duct obstruction. Although bile acid sulfates are the major bile acids in the urine of patients with obstructive jaundice, monohydroxylated bile acids are considered not to be so effectively excreted into the urine, even with conjugation with taurine and sulfate, in rats. Received: October 4, 2002 / Accepted: November 8, 2002 RID="*" ID="*" Reprint requests to: H. Takikawa Acknowledgments. This work was partly supported by a Grant-in-Aid by the Japan Society for the Promotion of Science (C2-14570510).     

Protective effect of melatonin against multistress condition induced lipid peroxidation via measurement of gastric mucosal lesion and plasma malondialdehyde levels in rats

INTRODUCTION It has been accepted that the pathogenesis of ulcer is complex and related to different pathogenic factors. The most common side-effect of indomethacin (NSAID) is gastric mucosal damage. The inhibition of the biosynthesis of gastric prostagla…     

Specific lectin bindings to oval cells and proliferated bile ductules

Histochemical evaluation of lectins was performed to examine the carbohydrate residues of oval cells induced by administration of α-naphthylisothiocyanate (ANIT), 2-acetylaminofluorene (2-AAF) and 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) in comparison with those of normal bile ducts and proliferated bile ductules induced by bile duct ligation. The normal bile ducts showed intense binding of Ricinus communis agglutinin, concanavalin A and wheat germ agglutinin, and weak binding of Ulex europaeus agglutinin I (UEA I). A few cells in the portal bile ducts showed binding of peanut agglutinin (PNA). Two different binding patterns were observed in oval cells and proliferated bile ductules. One group showed increased binding of PNA, while the other showed intense binding of UEA I. In both groups, binding of other lectins was similar to those of the normal bile duct. The first group included oval cells induced by 2-AAF and 3′-Me-DAB, and the second included both oval cells induced by ANIT and proliferated bile ductules induced by ligation. These results suggest that oval cells and proliferated bile ductules have their own specific carbohydrate residues and that oval cells induced by the carcinogens might be a cell population different from those induced by non-carcinogens and proliferated bile ductules by ligation. The authors wish to thank Dr. T. Itoshima and Dr. Y. Okada for their invaluable suggestions, and Mrs. T. Emi for her assistance in preparation of light microscopic examination.     

Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat.

BACKGROUND & AIMS: Cholestasis results in adaptive regulation of bile salt transport proteins in hepatocytes that may limit liver injury. However, it is not known if changes also occur in the expression of bile salt transporters that reside in extrahepatic tissues, particularly the kidney, which might facilitate bile salt excretion during obstructive cholestasis. METHODS: RNA and protein were isolated from liver and kidney 14 days after common bile duct ligation in rats and assessed by RNA protection assays, Western analysis, and tissue immunofluorescence. Sodium-dependent bile salt transport was also measured in brush border membrane vesicles from the kidney. RESULTS: After common bile duct ligation, serum bile salts initially rose and then declined to lower levels after 3 days. In contrast, urinary bile salt excretion rose progressively over the 2-week period. By that time, the ileal sodium-dependent bile salt transporter messenger RNA and protein expression in total liver had increased to 300% and 200% of controls, respectively, while falling to 46% and 37% of controls, respectively, in the kidney. Sodium-dependent uptake of (3)H-taurocholate in renal brush border membrane vesicles was decreased. In contrast, the multidrug resistance-associated protein 2 expression in the kidney was increased 2-fold, even 1 day after ligation. Immunofluorescent studies confirmed the changes in the expression of these transporters in liver and kidney. CONCLUSIONS: These studies show that the molecular expression of bile salt transporters in the kidney and cholangiocytes undergo adaptive regulation after common bile duct obstruction in the rat. These responses may facilitate extrahepatic pathways for bile salt excretion during cholestasis.     

Dose-related effects of dexamethasone on liver damage due to bile duct ligation in rats

INTRODUCTIONObstruction of bile ? ow through the extrahepatic biliary system results in development of oxidant injury, hepatic fibrosis, biliary cirrhosis, and portal hypertension[1,2]. Patients with obstructive jaundice are at significant risk for severe…     

Effect of penicillin G on the biliary excretion of cholephilic compounds in rats

Aim

Penicillin G is reported to increase bile flow by increasing biliary glutathione excretion, as well as the biliary excretion of penicillin G itself. In order to study the effect of penicillin G on the hepatic excretory pathway, the effect of colchicine and genipin on the increase of biliary glutathione excretion induced by penicillin G was studied in rats. The effect of penicillin G on the biliary excretion of sulfobromophthalein and erythromycin was also studied, together with the effect of penicillin G on cholestasis induced by estradiol-17??-glucuronide.

Methods

After bile duct cannulation, penicillin G was administered to rats at the rate of 0.5???mol/min/100?g. The effect was examined of colchicine pretreatment (0.2?mg/100?g) and genipin administration (0.5???mol/min/100?g) on biliary glutathione excretion increased by penicillin G infused at the rate of 0.5???mol/min/100?g. The effect of penicillin G on the biliary excretion of sulfobromophthalein and erythromycin (0.2 and 0.1???mol/min/100?g for 90?min, respectively) was studied, together with the effect of penicillin G on cholestasis induced by estradiol-17??-glucuronide (0.075???mol/min/100?g for 20?min).

Results

Penicillin G increased bile flow and biliary glutathione excretion, which were not inhibited by colchicine or genipin. Biliary penicillin G excretion was markedly reduced in Eisai hyperbilirubinemic rats (EHBR) and Mrp2-deficient rats. Biliary sulfobromophthalein and erythromycin excretion was unchanged by penicillin G. Cholestasis induced by estradiol-17??-glucuronide was not relieved by penicillin G.

Conclusions

It was shown that colchicine-sensitive vesicular transport has no role on the penicillin G-induced insertion of Mrp2 into the canalicular membrane, as has been observed with genipin. Although the choleresis of penicillin G is thought to be due to the increased biliary excretion of glutathione and penicillin G itself by Mrp2, the mechanism of Mrp2 insertion by penicillin G is thought to be partly different from that by genipin.     

Influence of D-galactosamine and alpha-naphthylisothiocyanate on the activities of some intestinal enzymes

The activities of lactase, sucrase, maltase and gamma-glutamyl transferase (gamma-GT) were determined in homogenates of rat jejunal mucosa 24 h after acute administrations of D-galactosamine (GALN) (1.855 mmol/kg; i.p. injection) and alpha-naphthyl-isocyanate (ANIT) (0.540 mmol/kg; given by gastric tube). The animals were fasted either 24 h or 72 h prior to sacrifice. In rats fasted only 24 h, GALN treatment resulted in a pronounced decrease in lactase and in a moderate elevation of sucrase and maltase. ANIT clearly reduced lactase and, to a lesser extent, sucrase, while it increased maltase. Seventy-two hour fasting has a modifying role. All disaccharidase activities tended to decrease, except for maltase in the ANIT treated group, where an increase was recorded. gamma-GT showed no significant changes after either GALN or ANIT treatment in rats fasted 24 h. However, the 72-hour food deprivation diminished it in ANIT intoxication. It is obvious that the intestinal enzymes are influenced by the hepatic damage produced by GALN and ANIT.     

Pressor and renal effects of cross-linked hemoglobin in anesthetized cirrhotic rats

BACKGROUND/AIMS: Cross-linked hemoglobin (XL-Hb), a hemoglobin-based oxygen carrier, is currently under investigation as a blood substitute. In the present study we have evaluated its pressor and renal effects in a rat model of liver cirrhosis by bile duct ligation. METHODS: Experiments were performed 3 weeks after surgery in anesthetized rats In the first protocol, the ability of XL-Hb to recover blood pressure after a hypotensive hemorrhage (0.5 ml/min, 10 min) was analyzed. In the second protocol, the pressor and renal effects produced by the administration of XL-Hb were evaluated during a period of 3 h. RESULTS: After a hypotensive hemorrhage (0.5 ml/min, 10 min), resuscitation with XL-Hb resulted in greater and faster recovery of blood pressure than with the administration of blood. In non-hemorrhaged rats, administration of XL-Hb (5% of blood volume) reversibly increased blood pressure in bile duct ligation and in control rats, but this effect was of longer duration in the control animals. XL-Hb also induced brisk increases in water and sodium excretion in both groups of animals, but the response of the control animals was more intense and sustained than that of the bile duct ligation rats. Glomerular filtration rate and renal blood flow showed slight decreases, but they were well maintained around the baseline levels. All the parameters studied were normalized 3 h later. In additional experiments, the effect of a bolus of L-NAME (10 mg/kg), an inhibitor of nitric oxide synthase, 1 h after the administration of XL-Hb was partially reduced, suggesting that the effect of XL-Hb may be secondary to the disappearance of circulating nitric oxide. CONCLUSIONS: XL-Hb seems to be effective as a resuscitative solution in case of hemorrhage in cirrhotic rats Moreover, this blood substitute only moderately and reversibly elevates blood pressure and does not adversely affects renal function.     

Effect of lipophilic extract of Chlorella vulgaris on alimentary hyperlipidemia in cholesterol-fed rats

The effect of glycolipid (GL) and phospholipid (PL) fractions obtained from Chlorella on serum lipid level and fecal excretion of steroids were examined in cholesterol-fed rats. The increase of the level of serum lipids were inhibited by the feeding of GL, PL and Chlorella powder almost the same degree. Fecal excretion of steroids (mostly of cholesterol, deoxycholic and lithocholic acid) were increased by feeding of GL and PL fractions. It is concluded that the feeding of each fraction inhibits the absorption of exogenous steroids and promotes turnover of bile acids in liver to suppress the increase of serum cholesterol level caused by administration of high cholesterol diet.     

Dexamethasone hypertension in rats

The effects of dexamethasone (DEX) on systolic blood pressure, sodium balance and the renin-angiotensin system were studied in rats. DEX significantly increased systolic blood pressure within three days of its administration, but this effect of DEX on blood pressure was not enhanced by concurrent use of saline solution. In DEX-treated rats, urine volume was significantly increased and urinary sodium excretion showed a tendency toward a slight increase compared to control rats. On the 8th day of DEX administration, plasma renin substrate (PRS) was significantly elevated compared to control rats, whereas plasma aldosterone concentration (PAC) was not significantly different from that of control rats. These results suggest that hypertension induced by DEX may not be dependent on sodium retention or activation of the renin-angiotensin system.