Cholestasis, altered junctional permeability, and inverse changes in sinusoidal and biliary glutathione release by vasopressin and epinephrine

The mechanism for the vasopressin- and epinephrine-induced decrease in bile formation and increase in sinusoidal efflux of glutathione was investigated in rat livers perfused with recirculating fluorocarbon emulsion. Vasopressin and epinephrine transiently decreased bile flow and excretion of endogenous bile acids and glutathione and increased the bile/perfusate ratio of [14C]sucrose, suggesting an increase in junctional permeability, but had no effect on the bile/perfusate ratio of [3H]polyethylene glycol-900. The decreased biliary glutathione was balanced by an increase in sinusoidal efflux, such that total hepatic release remained unchanged. The adrenergic antagonist dihydroergotamine blocked the effects of epinephrine. To examine whether an increase in junctional permeability per se could account for the changes in glutathione efflux, biliary permeability was increased by either bile duct ligation, lowering of perfusate Ca2+ concentration with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or addition of taurolithocholate, a cholestatic bile acid. All three maneuvers produced a decrease in biliary glutathione excretion and a concomitant increase in sinusoidal glutathione efflux, whereas total glutathione release was largely unaffected. The effects of EGTA were partially reversed if CaCl2 was reintroduced into the perfusate. Because the GSH/GSSG ratio in perfusate could not be measured in this experimental system due to the spontaneous oxidation of GSH to GSSG, additional experiments in the nonrecirculating mode examined the effects of vasopressin and bile duct ligation on sinusoidal release of GSH and GSSG. In control livers there was no detectable GSSG in perfusate (less than 0.5 nmol.min-1.g-1). After vasopressin administration, the additional sinusoidal glutathione was mainly as GSH, although there was also a significant amount of GSSG (1-2 nmol.min-1.g-1). The additional glutathione released into perfusate after bile duct ligation was 47% as GSSG. When vasopressin was administered to livers whose bile duct had been ligated, its ability to enhance sinusoidal glutathione release was diminished, suggesting that the effects of vasopressin and bile duct ligation are not additive. These observations support previous findings that vasopressin and epinephrine can modulate hepatocyte tight junctional permeability and demonstrate that these hormones produce cholestasis and inverse changes in sinusoidal and biliary glutathione efflux. Other maneuvers that increased biliary permeability to [14C]sucrose also produced cholestasis and a redistribution of glutathione efflux from bile to perfusate, suggesting that an increase in junctional permeability may allow biliary glutathione to reflux from bile to plasma.     

The influences of endogenous bile acids on maltase and alkaline phosphatase activities in intrahepatic cholestasis rats

The activities of lysosomal maltase in the serum, bile and liver were determined in intrahepatic cholestasis rats induced by alpha-naphtylisothiocyanate (ANIT, 200 mg/kg, i.p.), and compared with changes in alkaline phosphatase (ALP) activity. Moreover, the influences of endogenous bile acids on the release of maltase activity from the liver in intrahepatic cholestasis rats were studied. The maltase activities in the serum and bile significantly increased from 4 and 8 h after the intraperitoneal administration of ANIT, respectively. Conversely, a significant decrease in liver maltase activity was observed from 4 h after the injection of ANIT. On the other hand, total bile acid concentrations in the serum and bile significantly increased immediately after the treatment of ANIT, when biliary bile acid, exogenous bile acid or Triton X-100 was added to lysosomal fraction in the liver, the maltase activity in the supernate after the reaction significantly increased in proportion to the concentration of each substance added to the liver lysosome. These results suggested that maltase might be released from liver lysosomal membrane by surface active-action of bile acid accumulated in the liver after the administration of ANIT. Moreover, the changes in ALP activities in the serum, bile and liver after the administration of ANIT were almost similar to those in maltase activity.     

No effect of phenobarbital pretreatment of rats on methotrexate pharmacokinetics in the isolated liver perfused in a single-pass way

Pretreatment of the rat with phenobarbital (PB) is known to increase gene expression of the canalicular multispecific organic anion transporter (cMOAT) and hepatobiliary transport of its substrates (glutathione, sulfobromophthalein). To determine the effect of PB on the hepatobiliary transport of methotrexate (MTX, another substrate of cMOAT) and its metabolism to 7-hydroxymethotrexate (7-OHMTX) in the rat, we compared the steady-state pharmacokinetics of MTX in the isolated liver of either PB-pretreated (80 mg/day/kg bw for 4 days, i.p.) or nonpretreated rats. The livers were perfused in a single-pass way at a flow rate of 15 ml/min using a perfusate which consisted of Krebs-Henseleit buffer containing glucose, taurocholate, bovine albumin and erythrocytes. During the perfusion with 50 micromol/l MTX, the steady-state biliary clearance (1.26 +/- 0.24 ml/min) in 7 nonpretreated rats accounted for a major proportion of the hepatic clearance (1.30 +/- 0.33 ml/min), metabolism of MTX to 7- OHMTX was minor (partial metabolic clearance = 0.041 +/- 0.023 ml/min). MTX concentrations in bile surpassed those in the input perfusate by approximately 100-fold. Pretreatment of rats (n = 7) with PB did not change significantly the steady-state hepatic, biliary and partial metabolic clearances of 50 micromol/l MTX. An interesting result is a 38% increase in the hepatic vascular resistance of non-pretreated livers caused by MTX. The results suggest that in rats, pretreatment with PB has no effect on the hepatobiliary transport and hydroxylation of MTX.     

THE INHIBITORY EFFECT OF SA3443, A NOVEL CYCLIC DISULFIDE COMPOUND, ON ALPHA-NAPHTHYL ISOTHIOCYANATE-INDUCED INTRAHEPATIC CHOLESTASIS IN RATS

1. We investigated the effect of SA3443, a novel cyclic disulfide compound, on alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis in rats. 2. SA3443 given orally at doses of 100 or 300 mg/kg, three times at 4h intervals, dosedependently suppressed the elevation of serum total bilirubin, alkaline phosphatase activity and transaminase activities induced by administration of ANIT (80 mg/kg) 3 h before the second dosages of SA3443. 3. SA3443 also significantly inhibited the reduction of bile flow in the ANIT-treated rats. 4. These findings indicated that SA3443 had a markedly inhibitory effect on ANIT-induced intrahepatic cholestasis in rats.     

ANIT-induced disruption of biliary function in rat hepatocyte couplets.

alpha-Naphthylisothiocyanate (ANIT) induces intrahepatic cholestasis in rats, involving damage to biliary epithelial cells; our study aims to investigate whether disruption of biliary function in hepatocytes can contribute to early stages of ANIT-induced intrahepatic cholestasis. Isolated rat hepatocyte couplets were used to investigate biliary function in vitro by canalicular vacuolar accumulation (cVA) of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), within the canalicular vacuole between the two cells. After a 2-h exposure to ANIT, there was a concentration-dependent inhibition of cVA (cVA-IC50; 25 microM), but no cytotoxicity (LDH leakage or [ATP] decline) within this ANIT concentration range. There was no loss of cellular [GSH] at low ANIT concentrations, but, at 50 microM ANIT, a small but significant loss of [GSH] had occurred. Diethylmaleate (DEM) partially depleted cellular [GSH], but addition of 10 microM ANIT had no further effect on GSH depletion. Reduction in cVA was seen in DEM-treated cells; addition of ANIT to these cells reduced cVA further, but the magnitude of this further reduction was no greater than that caused by ANIT alone, indicating that glutathione depletion does not enhance the effect of ANIT. F-actin distribution (by phalloidin-FITC staining) showed an increased frequency of morphological change in the canalicular vacuoles but only a small, non-significant (0.05 < p < 0.1) increase in proportion of the F-actin in the region of the pericanalicular web. The results are in accord with a disruption of hepatocyte canalicular secretion within two h in vitro, at low, non-cytotoxic concentrations of ANIT, and the possible involvement of a thiocabamoyl-GSH conjugate of ANIT (GS-ANIT) in this effect.     

The effect of diethylstilbestrol treatment on the metabolism and biliary excretion of [14C]phenytoin in the isolated perfused rat liver

The metabolism and biliary excretion of [¹⁴C]phenytoin (DPH) were examined in isolated perfused livers taken from Sprague-Dawley rats pretreated with 0.01, 0.05, 0.1, 0.5, and 1.0 mg/day diethylstilbestrol (DES) sc for 6 days. No difference was seen in the rate of disappearance of DPH from the perfusate or in the perfusate levels of its hydroxylated metabolite, 5-phenyl-5-para-hydroxyphenylhydantoin (HPPH). The biliary excretion of HPPH-glucuronide, however, was significantly depressed in livers from DES-treated rats and resulted in a significant increase in the amount of HPPH-glucuronide appearing in the perfusate. A linear relationship existed between the percentage decrease in biliary excretion of HPPH-glucuronide and the log of the pretreatment dose of DES. Bile flow was significantly depressed at all pretreatment doses of DES such that bile flow was 53.7 and 10.9% of bile flow in controls after 0.01 and 1.0 mg/day DES, respecively. The low bile flow appeared to limit secretion of HPPH-glucuronide in the bile since the maximal concentration of HPPH-glucuronide in bile was greater in livers from DES-treated rats than controls and no significant differences were found in the maximal bile/perfusate concentration ratios of HPPH-glucuronide.     

Histological demonstration of wheat germ lectin binding sites in the liver of normal and ANIT treated rats

Wheat-germ lectin peroxidase conjugate was used to stain the liver of normal rats and rats given -naphthylisothiocyanate (ANIT). Changes in patterns in bile duct and canalicular staining were compatible with the hypothesis that cell damage caused by ANIT is essentially restricted to bile ducts.     

Preventive effect of silymarin against taurolithocholate-induced cholestasis in the rat

Increased amounts of monohydroxylated bile salts (BS) have been found in neonatal cholestasis, parenteral nutrition-induced cholestasis and Byler's disease, among others. We analyzed whether the hepatoprotector silymarin (SIL), administered i.p. at the dose of 100mg/kg/day for 5 days, prevents the cholestatic effect induced by a single injection of the model monohydroxylated BS taurolithocholate (TLC, 30 micromol/kg, i.v.) in male Wistar rats. TLC, administered alone, reduced bile flow, total BS output, and biliary output of glutathione and HCO(3)(-) during the peak of cholestasis (-75, -67, -81, and -80%, respectively, P<0.05). SIL prevented partially these alterations, so that the drops of these parameters induced by TLC were of only -41, -25, -60, and -64%, respectively (P<0.05 vs. TLC alone); these differences between control and SIL-treated animals were maintained throughout the whole (120 min) experimental period. Pharmacokinetic studies showed that TLC decreased the intrinsic fractional constant rate for the canalicular transport of both sulfobromophthalein and the radioactive BS [14C]taurocholate by 60 and 68%, respectively (P<0.05), and these decreases were fully and partially prevented by SIL, respectively. SIL increased the hepatic capability to clear out exogenously administered TLC by improving its own biliary excretion (+104%, P<0.01), and by accelerating the formation of its non-cholestatic metabolite, tauromurideoxycholate (+70%, P<0.05). We conclude that SIL counteracts TLC-induced cholestasis by preventing the impairment in both the BS-dependent and -independent fractions of the bile flow. The possible mechanism/s involved in this beneficial effect will be discussed.     

Intrahepatic cholestasis induced by α-naphthylisothiocyanate can cause gut-liver axis disorders

Clinical studies have shown that Intrahepatic cholestasis is closely related to intestinal injury. The gut-liver axis theory suggests that the intestine and liver are closely related, and that bile acids are important mediators linking the intestine and liver. We compared two cholestasis models: a single injection model that received a single subcutaneous ANIT injection (75 mg/kg), and a multiple subcutaneous injection model that received an injection of ANIT (50 mg/kg) every other day for 2 weeks. We used Transmetil (ademetionine 1,4-butanedisulfonate) to relieve intrahepatic cholestasis in the multiple injection group. In the multiple injection group, we found increased hepatic bile duct hyperplasia, increased fibrosis of the liver, increased small intestine inflammation and oxidative damage, increased harmful bile acids, decreased bile acids transporter levels. After treatment with Transmetil, the liver and gut injuries were relieved. These results suggest that intrahepatic cholestasis can cause disorders of the gut-liver axis.     

Modulation of hepatic canalicular or basolateral transport proteins alters hepatobiliary disposition of a model organic anion in the isolated perfused rat liver.

This study examined the impact of hepatic transport protein modulation on the hepatobiliary disposition of a nonmetabolized probe substrate, 5- (and 6)-carboxy-2',7'dichlorofluorescein (CDF) in rat isolated perfused livers (IPLs). In vivo treatment with modulators (100 and 200 mg/kg/day clofibric acid, 80 mg/kg/day phenobarbital, and 25 mg/kg/day dexamethasone) was used to alter the expression of hepatic transport proteins [organic anion transporting polypeptide 1a1, multidrug resistance-associated protein (Mrp) 3, and Mrp2] governing the disposition of CDF. The basolateral and biliary excretion of CDF was measured in single-pass IPLs from control and treated rats. Modulators increased the percentage of CDF eliminated into perfusate of IPLs from treated rats ( approximately 20-35%) compared with controls ( approximately 10%); CDF biliary excretion was decreased in the treated groups. These observations are consistent with modulator-associated increases in the first-order rate constant governing CDF excretion from the hepatocytes into perfusate (k(perfusate)) or decreases in the first-order rate constant governing CDF excretion into bile (k(bile)). Pharmacokinetic modeling of the data and subsequent simulations revealed that the routes of CDF excretion were most sensitive to changes in k(perfusate). In contrast, hepatic accumulation of CDF was most sensitive to k(bile). The differential sensitivity of CDF excretory routes and hepatic accumulation to these rate constants is a function of intrahepatic distribution kinetics, which must be taken into consideration in assessing the potential impact of altered hepatobiliary transport processes.     

1,3-Butanediol pretreatment on the cholestasis induced in rats by manganese-bilirubin combination,taurolithocholic acid,or α-naphthylisothiocyanate

Haloalkane-induced hepatonecrogenesis can be potentiated by the prior administration of ketones (acetone, chlordecone) or ketogenic substances (1,3-butanediol, alloxan). We therefore investigated the possibility that the ketosis induced in rats by 1,3-butanediol (BD) would potentiate cholestasis induced by manganese-bilirubin (Mn-BR) combination, taurolithocholic acid (TLC), and α-naphthylisothiocyanate (ANIT). In the Mn-BR studies, the depression of bile flow was more severe with 15 or 20 mg/kg bilirubin and the threshold dose for manganese was also affected. Furthermore, the protection usually afforded by sulfobromophthalein (BSP) or phenoldibromophthalein disulfonate (DBSP) was lessened in the BD-pretreated group. Potentiation of cholestasis also occurred with TLC, and this was evident in the longer duration of the effect in the pretreated rats. Hyperbilirubinemia was more severe in BD-pretreated rats 24 hr after ANIT (50–200 mg/kg) but not 14 or 18 hr after treatment. Two mechanisms, increased metabolism and increased cell susceptibility, are proposed to explain these effects. It is suggested that ketogenic substances can enhance cholestatic, as well as necrogenic, liver injury.     

Relationship between alpha-naphthylisothiocyanate-induced liver injury and elevations in hepatic non-protein sulfhydryl content.

Acute administration of alpha-naphthylisothiocyanate (ANIT) to rats has been used as a model of intrahepatic cholestasis. The mechanism of toxicity of ANIT is unknown, although recent evidence suggests a causal or permissive role for glutathione (GSH) (Dahm LJ and Roth RA, Biochem Pharmacol 42: 1181-1188, 1991). In these studies, ANIT treatment elevated hepatic non-protein sulfhydryl (NPSH) content, an indicator of GSH content, when liver injury was evident. The purpose of the present study was to characterize the effects of ANIT on hepatic NPSH content and to relate these changes to the development of liver injury. In rats fasted for 24 hr, administration of ANIT (100 mg/kg, per os [p.o.]) did not change hepatic NPSH content, bile flow, or serum measurements of total bilirubin concentration, alanine aminotransferase (ALT) activity, or gamma-glutamyltransferase (GGT) activity by 12 hr post-treatment relative to corn oil vehicle controls. However, by 24 hr after ANIT treatment, rats exhibited cholestasis and elevations in serum markers of liver injury. These markers were associated temporally with an increase in hepatic NPSH content, which consisted entirely of GSH. To determine whether the cholestasis caused by ANIT treatment might have caused the elevation of hepatic NPSH content, an extrahepatic cholestasis in rats was produced by ligation of the common bile duct. Bile duct ligation elevated hepatic NPSH content between 6 and 12 hr after ligation. Administration to rats of a non-hepatotoxic analog of ANIT, beta-naphthylisothiocyanate, also elevated hepatic NPSH content 24 hr after treatment. Taken together, these results indicate that the elevation in hepatic NPSH content after ANIT treatment is associated temporally with the onset of liver injury, but this elevation does not appear to participate causally in the mechanism of injury.     

小叶黑柴胡茎叶总黄酮对ANIT所致肝内胆汁淤积大鼠的影响

目的观察小叶黑柴茎叶胡总黄酮(TFA)对ANIT所致肝内胆汁淤积大鼠的影响。方法采用化学物质α-萘异硫氰酸酯(α-naphthyl isothiocy,ANIT)复制肝内胆汁淤积大鼠模型,灌胃(ig)给药7d后,腹腔注射10%乌拉坦溶液麻醉大鼠,开腹分离总胆管,引流胆汁,记录每小时胆汁流量;观察TFA对大鼠肝内胆汁淤积时血清、肝组织指标变化的影响并与尤思弗胶囊作对照。结果与模型组相比,经TFA预防性治疗后,胆汁淤积大鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、总胆红素(TBIL)、谷酰胺转肽酶(GGT)的活力明显降低(P<0.01),肝组织Na+,K+-ATP酶活性明显提高(P<0.01),TFA能明显提高胆汁淤积大鼠的胆汁流量(P<0.01);TFA高剂量在利胆、提高肝组织Na+,K+-ATP酶活性、降低血清TBIL、ALP活力方面优于尤思弗胶囊组。结论 TFA治疗胆汁淤积疗效确切,具有很好的临床应用前景,能开发成新一代治疗胆汁淤积的中成药。     

Hepatic pharmacokinetics of taurocholate in the normal and cholestatic rat liver

The disposition kinetics of [3H]taurocholate ([3H]TC) in perfused normal and cholestatic rat livers were studied using the multiple indicator dilution technique and several physiologically based pharmacokinetic models. The serum biochemistry levels, the outflow profiles and biliary recovery of [3H]TC were measured in three experimental groups: (i) control; (ii) 17 alpha-ethynylestradiol (EE)-treated (low dose); and (iii) EE-treated (high dose) rats. EE treatment caused cholestasis in a dose-dependent manner. A hepatobiliary TC transport model, which recognizes capillary mixing, active cellular uptake, and active efflux into bile and plasma described the disposition of [3H]TC in the normal and cholestatic livers better than the other pharmacokinetic models. An estimated five- and 18-fold decrease in biliary elimination rate constant, 1.7- and 2.7-fold increase in hepatocyte to plasma efflux rate constant, and 1.8- and 2.8-fold decrease in [3H]TC biliary recovery ratio was found in moderate and severe cholestasis, respectively, relative to normal. There were good correlations between the predicted and observed pharmacokinetic parameters of [3H]TC based on liver pathophysiology (e.g. serum bilirubin level and biliary excretion of [3H]TC). In conclusion, these results show that altered hepatic TC pharmacokinetics in cholestatic rat livers can be correlated with the relevant changes in liver pathophysiology in cholestasis.     

NMR and pattern recognition studies on liver extracts and intact livers from rats treated with alpha-naphthylisothiocyanate

The metabolite profiles from livers of toxin-treated rats were investigated using high resolution 1H NMR spectroscopy of aqueous (acetonitrile/water), lipidic (chloroform/methanol) extracts and magic angle spinning (MAS)-NMR spectroscopy of intact tissue. Rats were treated with the model cholestatic hepatotoxin, alpha-naphthylisothiocyanate (ANIT, 150 mg/kg) and NMR spectra of liver were analysed using principal components analysis (PCA) to extract novel toxicity biomarker information. 1H NMR spectra of control aqueous extracts showed signals from a range of organic acids and bases, amino acids, sugars, and glycogen. Chloroform/methanol extracts showed signals from a range of saturated and unsaturated triglycerides, phospholipids and cholesterol. The MAS 1H NMR spectra of livers showed a composite of signals found in both aqueous and lipophilic extracts. Following ANIT treatment, 1H NMR-PCA of aqueous extracts indicated a progressive reduction in glucose and glycogen, together with increases in bile acid, choline, and phosphocholine signals. 1H NMR-PCA of chloroform/methanol extracts showed elevated triglyceride levels. The 1H MAS-NMR-PCA analysis allowed direct detection of all of the ANIT-induced tissue perturbations revealed by 1H NMR of extracts, enabling metabolic characterisation of the lesion, which included steatosis, bile duct obstruction and altered glucose/glycogen metabolism. MAS-NMR spectroscopy requires minimal sample preparation and, unlike 1H NMR spectroscopy of tissue extracts, does not discriminate metabolites based on their solubility in a particular solvent and so this is a particularly useful exploratory tool in biochemical toxicology.     

Effects of gomisin A on liver functions in hepatotoxic chemicals-treated rats

The effects of gomisin A, which is a lignan component of schizandra fruits, on liver functions in various experimental liver injuries and on bile secretion in CCl4-induced liver injury were studied. Gomisin A weakly accelerated the disappearance of plasma ICG by itself at a high dose (100 mg/kg, i.p.). All of the hepatotoxic chemicals used in this study inhibited the excretion of ICG from plasma. Gomisin A showed a tendency to prevent the delays of the disappearance of plasma ICG induced by CCl4, d-galactosamine and orotic acid, but not that by ANIT. Bile flow and biliary outputs of total bile acids and electrolytes (Na+, K+, Cl- and HCO3-) were decreased in CCl4-treated rats. Gomisin A maintained bile flow and biliary output of each electrolyte nearly to the level of the vehicle-treated group, but did not affect biliary output of total bile acids. These findings suggest that gomisin A possesses a liver function-facilitating property in normal and liver injured rats and that its preventive action on CCl4-induced cholestasis is due to maintaining the function of the bile acids-independent fraction.     

Does paracellular permeability play a role in cholephilic dye-induced cholestasis?

Changes in hepatic paracellular permeability were investigated during the development of cholephilic dye-induced cholestasis in rats. For this purpose, four dyes with different cholestatic potency (phenol red, sulfobromophthalein, bromcresol green and rose bengal) were infused at a high, potentially damaging dose (280 nmol/min per 100 g body wt. i.v.), and changes in paracellular permeability were continuously monitored by measuring the access into bile of the permeability probe [¹⁴C]sucrose. The cholestatic potency of the different dyes was: rose bengal > bromcresol green > sulfobromophthalein > phenol red. All the dyes increased [¹⁴C]sucrose bile-to-plasma ratio, producing a displacement towards curves of higher permeability. The capability of the dyes to increase biliary permeability followed the same order as their respective cholestatic potencies. The possible implications of the present results for cholephilic dye-induced cholestasis are discussed.     

Effect of St John's wort on the disposition of fexofenadine in the isolated perfused rat liver

Objectives This study examined the effects of St John's wort (Hypericum perforatum) on the disposition of fexofenadine, a substrate of P‐glycoprotein/organic anion transporting polypeptide, in the isolated perfused rat liver. Methods Male Sprague‐Dawley rats were given St John's wort, 1000 mg/kg, by intragastric gavage once daily for 14 days. On day 15, livers were isolated surgically and perfused in a recirculating system with fexofenadine (2 μg/ml), either alone or following addition of ciclosporin (0.5 μg/ml) 5 min before the addition of fexofenadine. Perfusate samples and bile were collected for 60 min. Fexofenadine in perfusate, bile and the homogenised livers was measured by HPLC. Key findings Administration of St John's wort significantly increased biliary clearance with respect to perfusate and biliary clearance with respect to the concentration in the liver, by 74% and 71%, respectively. This was reversed by ciclosporin. Conclusions St John's wort enhanced the elimination of fexofenadine into the bile. This could be because it increases the activity of P‐glycoprotein on the canalicular membrane of hepatocytes.     

Potentiation of lithocholic-acid-induced cholestasis by methyl isobutyl ketone.

Methyl isobutyl ketone was found to potentiate intrahepatic cholestasis induced by taurolithocholate and the combination of manganese-bilirubin. The aim of this study was to elucidate the mechanism of this potentiation using the lithocholate-induced cholestasis model. Male rats were given methyl isobutyl ketone 7.5 mumol/kg body wt. daily for 3 days. The effect of this treatment on lithocholate-induced cholestasis, bile formation and taurocholic acid transport was examined. The data showed that methyl isobutyl ketone treatment potentiated lithocholate-induced cholestasis and reduced significantly bile salt, phospholipid and cholesterol secretion rates as well as the transport maximum of taurocholic acid. It is suggested that methyl isobutyl ketone potentiates lithocholate-induced cholestasis by reducing the bile salt pool and interfering with the haptic secretion rate of bile salts.     

Resveratrol effectively attenuates α-naphthyl-isothiocyanate-induced acute cholestasis and liver injury through choleretic and anti-inflammatory mechanisms

Aim:

α-Naphthylisothiocyanate (ANIT) is a well-characterized cholestatic agent for rats. The aim of this study was to examine whether resveratrol could attenuate ANIT-induced acute cholestasis and liver injury in rats.

Methods:

SD rats were treated with resveratrol (15 or 30 mg/kg, ip) or a positive control drug ursodeoxycholic acid (100 mg/kg, po) for 5 consecutive days followed by a single dose of ANIT (60 mg/kg, po). Bile flow, and serum biochemical markers and bile constituents were measured 48 h after ANIT administration. Hepatic levels of oxidative repair enzymes (glutathione peroxidase, catalase and MnSOD), myeloperoxidase activity, TNF-α, IL-6 and ATP content, as well as the expression of liver transporter genes and proteins were assayed.

Results:

ANIT exposure resulted in serious cholestasis and liver injury, as shown by marked neutrophil infiltration in liver, dramatically increased serum levels of ALT, AST, GGT, ALP, TBA, TBIL, IBIL and DBIL, and significantly decreased bile excretion and biliary output of GSH and HCO₃⁻. ANIT significantly increased TNF-α and IL-6 release and myeloperoxidase activity, decreased mitochondrial biogenesis in liver, but had little effect on hepatic oxidative repair enzymes and ATP content. Furthermore, ANIT significantly decreased the expression of Mrp2, FXR and Cyp7a1, markedly increased Mrp3 expression in liver. Pretreatment with resveratrol attenuated ANIT-induced acute cholestasis and liver injury, and other pathological changes. Pretreatment with ursodeoxycholic acid was less effective.

Conclusion:

Resveratrol effectively attenuates ANIT-induced acute cholestasis and liver injury in rats, possibly through suppression of neutrophil infiltration, as well as upregulation of expression of hepatic transporters and enzymes, thus decreasing accumulation of bile acids.