Drug-induced Intrahepatic Cholestasis Research Articles

Unraveling the mechanisms underlying drug-induced cholestatic liver injury: identifying key genes using machine learning techniques on human in vitro data sets

Drug-induced intrahepatic cholestasis (DIC) is a main type of hepatic toxicity that is challenging to predict in early drug development stages. Preclinical animal studies often fail to detect DIC in humans. In vitro toxicogenomics assays using human liver cells have become a practical approach to predict human-relevant DIC. The present study was set up to identify transcriptomic signatures of DIC by applying machine learning algorithms to the Open TG-GATEs database. A total of nine DIC compounds and nine non-DIC compounds were selected, and supervised classification algorithms were applied to develop prediction models using differentially expressed features. Feature selection techniques identified 13 genes that achieved optimal prediction performance using logistic regression combined with a sequential backward selection method. The internal validation of the best-performing model showed accuracy of 0.958, sensitivity of 0.941, specificity of 0.978, and F1-score of 0.956. Applying the model to an external validation set resulted in an average prediction accuracy of 0.71. The identified genes were mechanistically linked to the adverse outcome pathway network of DIC, providing insights into cellular and molecular processes during response to chemical toxicity. Our findings provide valuable insights into toxicological responses and enhance the predictive accuracy of DIC prediction, thereby advancing the application of transcriptome profiling in designing new approach methodologies for hazard identification.

Quantitative understanding of HepaRG cells during drug-induced intrahepatic cholestasis through changes in bile canaliculi dynamics.

An understanding of the quantitative relationship between bile canaliculus (BC) dynamics and the disruption of tight junctions (TJs) during drug‐induced intrahepatic cholestasis may lead to new strategies aimed at drug development and toxicity testing. To investigate the relationship between BC dynamics and TJ disruption, we retrospectively analyzed the extent of TJ disruption in response to changes in the dynamics of BCs cultured with entacapone (ENT). Three hours after adding ENT, the ZO‐1‐negative BC surface area ratio became significantly higher (4.1‐fold) than those of ZO‐1‐positive BCs. Based on these data, we calculated slopes of surface area changes, m, of each ZO‐1‐positive and ZO‐1‐negative BC. BCs with m ≤ 15 that fell within the 95% confidence interval of ZO‐1‐positive BCs were defined as ZO‐1‐positive. To validate this method, we compared the frequency of ZO‐1‐positive BCs, F Z, with that of BCs with m ≤ 15, F T, in culture using drugs that regulate TJ, or induce intrahepatic cholestasis. F T values were correlated with F Z under all culture conditions (R 2 = .99). Our results indicate that the magnitude of BC surface area changes is a factor affecting TJ disruption, suggesting that maintaining TJ integrity by slowing BC dilation inhibits cell death.

Tight junction stabilization prevents HepaRG cell death in drug-induced intrahepatic cholestasis.

ABSTRACTEntacapone (ENT), a catechol-O-methyltransferase inhibitor, causes liver injury by inducing bile canaliculi (BC) dilation through inhibition of the myosin light kinase pathway. Loss of tight junctions (TJs) induces hepatocyte depolarization, which causes bile secretory failure, leading to liver damage. To understand the influence of TJ structural changes as a consequence of BC dynamics, we compared the datasets of time-lapse and immunofluorescence images for TJ protein ZO-1 in hepatocytes cultured with ENT, forskolin (FOR), ENT/FOR, and those cultured without any drugs. Retrospective analysis revealed that the drastic change in BC behaviors caused TJ disruption and apoptosis in cells cultured with ENT. Exposure to FOR or sodium taurocholate facilitated TJ formation in the cells cultured with ENT and suppressed BC dynamic changes, leading to the inhibition of TJ disruption and apoptosis. Our findings clarify that hepatocyte TJ stabilization protects against cell death induced by BC disruption.

S2399 RAD-140: An Emerging Cause of Drug-Induced Liver Injury

INTRODUCTION: Selective androgen receptor modulators (SARMs) include Ligandrol, enobosarm, and RAD-140. Their tissue-selective activity is thought to promote anabolic actions without significant androgenic effects. Despite lacking FDA approval, these products are widely accessible, and have been gaining popularity as alternatives to traditional androgenic anabolic steroids (AAS). Unlike AAS, only limited human hepatic safety data are available for SARMs. We report a case of drug-induced liver injury (DILI) from RAD-140. CASE DESCRIPTION/METHODS: A healthy 24-year-old Asian male presented to the emergency department with a 2-week history of diffuse abdominal pain, jaundice, scleral icterus and worsening pruritus. He did not take any prescription medications, drink alcohol, or use recreational drugs. His only over the counter medication was RAD-140, which he took for 5 weeks and stopped 2 weeks prior to the presentation due to elevated liver enzymes, noted on routine laboratory tests. Evaluation was incomplete as he left against medical advice. Three weeks later, he was hospitalized with worsening jaundice and pruritus. His mental status remained unchanged. Initial pattern of hepatocellular injury was subsequently followed by a significant cholestasis, with total bilirubin peak around the 11th week after initiating RAD-140 intake. The trend of liver tests and international normalized ratio (INR) are summarized in Table 1. Acute and chronic liver disease evaluations were all unremarkable. Despite mildly elevated alkaline phosphatase, gamma-glutamyl transpeptidase remained normal. Ferritin was significantly elevated at 1523 ng/mL but transferrin saturation was normal. Urine toxicology drug screen was also negative. Liver imaging revealed hepatomegaly and some focal fatty infiltration but excluded biliary or vascular obstruction (Figure 1, A&B). A Liver biopsy revealed blank canalicular cholestasis without remarkable inflammation consistent with drug-induced intrahepatic cholestasis (Figure 1, C&D). Currently, he remains asymptomatic with steady improvement in liver tests after being off RAD-140 for the past 5 months. DISCUSSION: Our patient represents the second reported case of significant DILI due to RAD-140, and the third case of DILI due to SARMs requiring supplement discontinuation. This case raises concerns regarding SARMs advertised selectivity and safety. Until further data is available, these agents should be used with caution.Table 1Figure 1.: A: Contrasted cross sectional imaging showing hepatomegaly and focal fatty infiltration (white arrow). B: Magnetic resonance cholangiopancreatography showing hepatomegaly and patent biliary tree. C: Liver histology showing intrahepatic cholestasis (black arrow). D: Liver histology showing minimal portal inflammation (black arrow).

Switching of cell fate through the regulation of cell growth during drug-induced intrahepatic cholestasis

Understanding the fundamental mechanisms that govern the fate of cells during drug-induced intrahepatic cholestasis provides strategies for the establishment of evaluation methods for drug screening. In the present study, the aggregates of a differentiated human hepatic cell line, HepaRG, were incubated in medium with Y27632 or bosentan to clarify the changes in the behavior of bile canaliculi (BC) with the growth of cells during drug-induced intrahepatic cholestasis. With elapsed exposure time, the aggregates in the culture with bosentan caused the dilation of BC, and the hepatocytes ultimately exhibited apoptotic death after the disruption of BC. Y27632 caused the disruption of BC in the aggregates after dilation. However, there was no change in the number of cells within the aggregates in the culture with Y27632, in spite of its cytotoxicity. After 144h from the start of Y27632 exposure, the aggregates showed the rearrangement of BC. To inhibit cell division, the aggregates exposed to Y27632, which exhibited disruption of BC, were treated with mitomycin C for 2h and continuously exposed to Y27632. The inhibition of cell division could not induce the rearrangement of BC within these aggregates, which was similar to the phenomenon observed in the aggregates exposed to bosentan. These findings indicate that growth is an important factor that influences the switching of cell fate toward survival or death in drug-induced intrahepatic cholestasis process. Thus, the autoregulation of growth is a major contributor to the rearrangement of BC within aggregates.

LCAT protects against Lipoprotein-X formation in a murine model of drug-induced intrahepatic cholestasis.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL‐C levels, low plasma cholesterol esterification, and the formation of Lipoprotein‐X (Lp‐X), an abnormal cholesterol‐rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp‐X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp‐X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp‐X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT‐deficient (KO), wild type (WT), and LCAT‐transgenic (Tg) mice by gavaging mice with alpha‐naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT‐Tg mice, however, prevented the formation of Lp‐X and other plasma lipid abnormalities in WT and LCAT‐KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp‐X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp‐X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp‐X in other diseases besides FLD.

Intrahepatic cholestasis of pregnancy or azithromycin-induced intrahepatic cholestasis

Rationale:Azithromycin-induced liver injury has been rarely reported in adult individuals, let alone in a pregnant woman. Here, we describe the clinical features and outcomes of azithromycin-induced liver injury in a pregnant woman.Patient concerns:A 30-year-old pregnant woman presented with generalized pruritus and elevated serum bile acid level (123.6 μmol/L) on day 4 of azithromycin administration. A diagnosis of intrahepatic cholestasis of pregnancy was made, and cesarean section was performed immediately. Interestingly, the alanine aminotransferase level (ALT) reached 211.2 U/L on day 9 after azithromycin administration.Diagnosis:Therefore, drug-induced intrahepatic cholestasis was considered.Interventions:(1) Azithromycin withdrawal after the patient hospitalized. (2) Termination of pregnancy by cesarean section was performed inmmediately to protect the fetus. (3) Silymarin capsules and bifendate are used to protect the liver after liver enzymes elevation was discovered.Outcomes:The liver enzymes recovered within 4 weeks without any symptoms after treatment with silymarin capsules and bifendate, which helps reduce ALT level and protects the liver from further injury.Lessions:A pregnant woman developed azithromycin-induced intrahepatic cholestasis. Physicians should be aware of this side effect of azithromycin, which is widely prescribed.

Progressive and Preferential Cellular Accumulation of Hydrophobic Bile Acids Induced by Cholestatic Drugs Is Associated with Inhibition of Their Amidation and Sulfation.

Drug-induced intrahepatic cholestasis is characterized by cellular accumulation of bile acids (BAs), whose mechanisms remain poorly understood. The present study aimed to analyze early and progressive alterations of BA profiles induced by cyclosporine A, chlorpromazine, troglitazone, tolcapone, trovafloxacin, and tacrolimus after 4-hour, 24-hour, and 6-day treatments of differentiated HepaRG cells. In BA-free medium, the potent cholestatic drugs cyclosporine A, chlorpromazine, and troglitazone reduced endogenous BA synthesis after 24 hours, whereas the rarely cholestatic drugs tolcapone, trovafloxacin, and tacrolimus reduced BA synthesis only after 6 days. In the presence of physiologic serum BA concentrations, cyclosporine A, chlorpromazine, and troglitazone induced early and preferential cellular accumulation of unconjugated lithocholic, deoxycholic, and chenodeoxycholic acids that increased 8- to 12-fold and 47- to 50-fold after 24 hours and 6 days, respectively. Accumulation of these hydrophobic BAs resulted from strong inhibition of amidation, and in addition, for lithocholic acid reduction of its sulfoconjugation, and was associated with variable alterations of uptake and efflux transporters. Trovafloxacin also caused BA accumulation, especially after 6 days, whereas tolcapone and tacrolimus were still without effect. However, when exogenous BAs were added to the medium at cholestatic serum concentrations, a 6-day treatment with all drugs resulted in cellular BA accumulation with higher folds of chenodeoxycholic and lithocholic acids. At the tested concentration, tolcapone had the lowest effect. These results bring the first demonstration that major cholestatic drugs can cause preferential and progressive in vitro cellular accumulation of unconjugated toxic hydrophobic BAs and bring new insights into mechanisms involved in drug-induced cellular accumulation of toxic BAs.

Early Alterations of Bile Canaliculi Dynamics and the Rho Kinase/Myosin Light Chain Kinase Pathway Are Characteristics of Drug-Induced Intrahepatic Cholestasis.

Intrahepatic cholestasis represents 20%-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na+-taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis.

A Case of Drug-Induced Cholangitis and Cholestasis From Amoxicillin/Clavulanic Acid and Levofloxacin

Introduction: Drug-induced liver injury (DILI) continues to be an important disease in clinical practice. Amoxicillin/clavulanic acid and levofloxacin are commonly used with a good safety profile. We describe a case of drug-induced cholangitis and cholestasis from amoxicillin/clavulanic acid and levofloxacin. Case Report: An 89-year-old male presented with a 1-day history of jaundice, dark urine, and pale stools. He was treated with 2 10-day courses of amoxicillin/clavulanic acid for a urinary tract infection, followed by a 10-day course of levofloxacin for pneumonia 1 week before presentation. He does not drink alcohol and had no personal or family history of liver disease. Physical exam was significant for jaundice. Laboratory evaluation demonstrated a total bilirubin of 11.1 mg/dL, direct bilirubin of 9.1 mg/dL, alkaline phosphatase of 1001 U/L, gamma-glutamyl-transpeptidase of 793 U/L, alanine aminotransferase (ALT) of 343 U/L, aspartate aminotransferase (AST) of 427 U/L, INR of 1.03, and albumin of 3.3 g/dL. Magnetic resonance cholangiopancreatography (MRCP) was negative for extrahepatic and intrahepatic biliary dilation. A liver biopsy revealed inflammation infiltrating the bile ducts and hepatocanalicular cholestasis consistent with drug-induced cholangitis and cholestasis. His liver function tests started to improve gradually within days after stopping levofloxacin. Discussion: DILI continues to be an important disease in clinical practice. Its diagnosis is mainly based on the exclusion of alternative causes of liver damage. Amoxicillin/clavulanic acid is a combination of amoxicillin and the β-lactamase inhibitor clavulanic acid. The latter component seems to be the prime offender for the liver injury. Amoxicillin/clavulanic acid has been reported to cause drug-induced intrahepatic cholestasis due to a metabolic idiosyncrasy or a hypersensitivity mechanism. It has also been reported to cause ductopenia. Levofloxacin is a fluoroquinolone commonly used to treat respiratory infections. Fluoroquinolones have been associated with drug-induced liver injury, ranging from a brief rise in transaminase level or cholestatic jaundice to severe hepatotoxicity. Levofloxacin has been associated with acute hepatitis, fulminant hepatic failure, and death. The mechanism of levofloxacin-induced hepatotoxicity is not known. Treatment for DILI is mainly the cessation of the offending drug, which may lead to recovery in liver enzyme abnormalities. We believe that our patient suffered from amoxicillin/clavulanic acid and levofloxacin-induced cholestasis and cholangitis. Clinicians should be familiar with the potential hepatoxicities from amoxicillin/clavulanic acid and levofloxacin.

Prediction of drug-induced intrahepatic cholestasis: in vitro screening and QSAR analysis ofdrugs inhibiting the human bile salt export pump

Drug-induced intrahepatic cholestasis is one of the major causes of hepatotoxicity, which often occur during the drug discovery and development process. Human ATP-binding cassette transporter ABCB11 (sister of P-glycoprotein/bile salt export pump) mediates the elimination of cytotoxic bile salts from liver cells to bile, and, therefore, plays a critical role in the generation of bile flow. The authors have recently developed in vitro high-speed screening and quantitative structure–activity relationship analysis methods to investigate the interaction of ABCB11 with a variety of compounds. Based on the extent of inhibition of the bile salt export pump, the authors analysed the quantitative structure–activity relationship to identify chemical groups closely associated with the inhibition of ABCB11. This approach provides a new tool to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.

High-Speed Screening and QSAR Analysis of Human ATP-Binding Cassette Transporter ABCB11 (Bile Salt Export Pump) To Predict Drug-Induced Intrahepatic Cholestasis

Human ATP-binding cassette transporter ABCB11 (SPGP/BSEP) mediates the elimination of bile salts from liver cells and thereby plays a critical role in the generation of bile flow. In the present study, we have developed in vitro high-speed screening and quantitative structure-activity relationship (QSAR) analysis methods to investigate the interaction of ABCB11 with a variety of drugs. Plasma membrane vesicles prepared from insect cells overexpressing human ABCB11 were used to measure the ATP-dependent transport of [14C]taurocholate. Over 40 different drugs and natural compounds were tested to evaluate their interaction with ABCB11-mediated taurocholate transport. On the basis of the extent of inhibition, we have analyzed the QSAR to identify one set of chemical fragmentation codes closely associated with the inhibition of ABCB11. This approach can be used to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.

Increased microfilaments in hepatocytes and biliary ductular cells in cholestatic liver diseases.

To assess the extent of microfilaments in cholestatic liver diseases we examined the cytoplasmic microfilaments in intrahepatic and extrahepatic cholestasis in man by electron microscopy. Study subjects were two patients with drug-induced intrahepatic cholestasis, three patients with intrahepatic cholestasis due to viral hepatitis, four patients with extrahepatic cholestasis due to stones of the common bile duct and two patients with primary biliary cirrhosis. Two biopsied specimens from patients without clinical or histological evidence of liver disease served as noncholestatic controls. The microfilaments in hepatocytes and biliary ductular cells were significantly increased in cholestasis compared with those in non-cholestatic controls. Well developed bundles of microfilaments were noted around the pericanalicular ectoplasm and seemed to be parallel to plasma membrane of the hepatocytes in cholestasis. In cholestasis, there were increased bundles of microfilaments around the periluminal region, lateral cell wall, and nucleus of biliary ductular cells. Two patterns of microfilaments bundles (fine microfilamentous network and spindle-shaped dense or clusters of microfilaments) were associated with cholestasis. The clustered form of microfilaments also seemed to be clearly associated with intracytoplasmic vacuoles containing bile salts. In conclusion, the increase of microfilaments in hepatocytes and biliary ductular cells may be the consequence of various forms of cholestasis. Further studies are needed to clarify the functional significance of increased microfilaments in cholestasis.

Effect of budesonide treatment on gestagen-induced hyperbilirubinemia in a patient with alagille syndrome

Multidrug-resistance protein (MRP2), which is involved in hepatobiliary transport of a wide variety of endogenous and exogenous compounds, might play an important role in drug-induced intrahepatic cholestasis. Animal studies have shown that dexamethasone treatment was able to counteract the drug-induced down-regulation of MRP2 expression (Gastroenterology 1999). A patient with Alagille syndrome (female, 30 years old) presented with typical clinical symptoms of hyperbilirubinemia six weeks after starting an oral contraception with levonorgestrel (30 [Jog daily). Although a therapy with ursodeoxycholic acid (15 mg/kg daily) was initiated, total bilirubin and conjugated bilirubin rose up to a maximum of 55.5 mg/dL and 46.4 mg/dL, respectively. Six days later, hyperbilirubinemia was nearly unchanged (total bilirubin: 53.1 mg/dL, conjugated bilirubin: 43.2 mg/dL). At this time point a therapy with budesonide (3x3 mg daily), a corticosteroid having low systemic bioavailability, was started after the patient had given written informed consent. On budesonide, a rapid continuous decrease was observed (six days after starting budesonide treatment: total bilirubin at 32.1 mg/dL, conjugated bilirubin: 23.7 mg/dL). After eight days a slight increase of body weight due to water retention was observed and treated with diuretics. Nevertheless, the patient did not want to continue the therapy with budesonide, which was tapered over six days. Subsequently total bilirubin and conjugated bilirubin did not further drop (37.7 mg/dL and 29.5 mg/dL ten days after reducing budesonide). These results show that gestagen-induced hyperbilirubinemia may be affected by budesonide treatment in vivo. It may be speculated that budesonide like dexamethasone counteracts the drug-induced down-regulation of MRP2 expression.

Intrahepatic cholestasis: a review of biochemical-pathological mechanisms.

Intrahepatic cholestasis involves impaired excretion of bile via the hepatobiliary system as a consequence of one or more lesions within the liver. In humans, intrahepatic cholestasis most often results as a side-effect of drug therapy and the clinical manifestation of this condition, jaundice, has been estimated to account for hospitalization in 2 to 5% of the cases for the general population and approaches as much as 20% in the elderly. With the aging of the population and the common occurrence of poly-drug therapy in geriatric patients, it is to be expected that jaundice due to drug-induced intrahepatic cholestasis will become even more prevalent, and accordingly the need to understand the basic mechanisms of this disease condition will become more urgent. The list of culprit agents implicated in the induction of intrahepatic cholestasis in humans is continually expanding. These include various steroid hormones, bile acids, drugs and other chemicals. Experimentally, a wide spectrum of agents has been shown to precipitate intrahepatic cholestasis. Over the years, a number of hypotheses on the biochemical and pathological mechanisms of intrahepatic cholestasis has emerged, including the following: impaired sinusoidal membrane function; interference with the distribution and binding of cytoplasmic endogenous carrier proteins; interference with mitochondrial energy supply; defects in the canalicular membrane including altered Na+/K+ -ATP-ase activity; impairment of microfilament and microtubule functions; interference with bile secretion involving bile acid dependent and independent fractions, and altered bile acid metabolism due to "hypoactive hypertrophic smooth endoplasmic reticulum". In partial agreement with the latter hypothesis, our studies indicated that impairment of the endoplasmic reticulum might represent one of the early stages in the development of intrahepatic cholestasis. Various experimental conditions that induce intrahepatic cholestasis to different degrees resulted in an interference of the synthesis of microsomal phospholipids and altered microsomal function. The conditions included the administration of various hepatotoxic compounds or steroids, pregnancy, delayed development of the endoplasmic reticulum in neonates, and dietary methyl donor or choline deficiency. This review reports the biochemical-pathological mechanisms postulated to be involved in the genesis of intrahepatic cholestasis with specific reference to experimental models of drug-induced intrahepatic cholestasis. The important practical implications of cholestasis are also briefly surveyed.

A biliary protein identified by immunoblotting stimulates proliferation of peripheral blood T lymphocytes in primary biliary cirrhosis

The source of activation of T lymphocytes in primary biliary cirrhosis (PBC) is undefined. Hence, T-cell-mediated reactivity against a biliary tract antigenic protein from human bile was studied. The bile protein was fractionated by 30-50% saturated ammonium sulphate and gel-chromatography, and analysed by SDS-PAGE and Western immunoblotting using rabbit antisera to the bile protein. The antisera reacted specifically with human bile duct epithelium. Western blotting of bile proteins showed two major bands, the B1 and B2 antigens. B1 stained for sialoglycoprotein but not lipid, but B2 was negative for both. Cell-mediated reactivity was tested by proliferation of peripheral lymphocytes against B1. Taking the upper limit of the normal range for stimulation indices (S.I.) as less than 1.89 (= mean + 2 SD), a mitogenic response was detected in 14 of 16 patients with PBC (S.I.: 11.7 to 2.3), and in 4 of 15 patients with chronic active hepatitis, but in none of 12 patients with drug-induced intrahepatic cholestasis or obstructive jaundice. The B2 protein was non-stimulatory. Lymphocyte proliferation to B1 in PBC was confined to T cell fractions of peripheral blood leucocytes. There was no cross-antigenicity between B1 and the M2 antigens, according to Western blotting using the rabbit antisera and PBC sera with anti-M2 reactivity. Thus, the B1 biliary protein is a possible source of T cell activation in PBC and hence could be an immunological co-factor in the pathogenesis of this disease.

Detection of the cholestatic factor in the liver tissue of patients with acute intrahepatic cholestasis.

A novel lymphokine, which we have designated as cholestatic factor (CF), was produced from peripheral blood lymphocytes of patients with drug-induced allergic intrahepatic cholestasis by stimulation with a causative drug in the presence of the liver soluble fraction containing liver-specific lipoprotein (LSP). Marked reductions in bile flow and bile acid excretion were induced in rats by injecting CF through a mesenteric vein. In order to confirm the presence of CF in the liver tissue of patients, we attempted to detect this lymphokine by using the enzyme-labelled antibody method. As a result, CF was found in the liver tissue of eleven out of thirty-eight patients with acute intrahepatic cholestasis including one with hepatitis A type, one with hepatitis B type, two with hepatitis non-A non-B type, five with drug-induced allergic hepatitis, one with alcoholic hepatitis and one with lupoid hepatitis. In contrast, CF was undetectable in the liver tissue of patients without intrahepatic cholestasis. These results may additionally support our assumption that CF plays an important role in the induction of intrahepatic cholestasis in various liver diseases.

Drug-induced intrahepatic cholestasis: characterization of different pathomechanisms.

The pathogenesis of intrahepatic cholestasis in rats was studied using isolated perfused livers as an experimental model. Three basic mechanisms were differentiated: Permeabilization of the bilio-sinusoidal barrier associated with electron microscopic alterations of the tight junctional complexes was found in livers of rats treated with alpha-naphthylisothiocyanate (ANIT, 250 mg/kg body weight). Consequences of these alterations were: reflux of bile constituents such as taurocholate and sulfobromophthalein and increased access to the biliary space of paracellular markers such as inulin and sucrose. The clear-cut mechanism of ANIT cholestasis was used to distinguish other mechanisms of intrahepatic cholestasis. Inhibition of the basic process of fluid secretion was found to be the primary event in the development of cholestasis induced by estrogens. After 5 days of treating rats with ethinyl estradiol (5 mg/kg/day), bile flow was diminished in isolated livers while the permeability of the biliary tract to sucrose and inulin was not affected. Accordingly, the maximal concentration of taurocholate in bile was increased, indicating that its secretion was sustained. The same effect was observed after 1 week of treatment with the depot estrogen estradiol valerate (1 mg/kg/week). After 3 weeks of treatment, however, the taurocholate concentration in bile was lowered and the clearance of sucrose was increased. Bile flow remained at the same cholestatic level for 20 weeks. These results suggest that estrogens have the potency to increase tight junctional permeability only in a second step in the development of cholestasis, following the inhibition of bile flow. An additional mode of secretory inhibition was induced by lowering the concentration of Ca2+ in the perfusate of isolated liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on drug-induced allergic intrahepatic cholestasis

When peripheral lymphocytes from patients with drug-induced allergic intrahepatic cholestasis were stimulated with a specific drug in vitro in the presence of a liver microsome fraction or soluble liver specific antigen fraction, lympholine production was seen in many cases. By the injection of culture supernatant of stimulated lymphocytes into the mesentery vein of dogs, cholestasis was induced in the liver, chiefly in the central zones of lobules. However, no cholestasis could be observed in dogs administered the supernatants of lymphocyte cultures prepared from normal individuals in the presence of drugs. Moreover, only slight swelling of the hepatocytes was observed in the liver when normal lymphocytes were stimulated with PHA-P and culture supernatant was injected into the mesentery vein of dogs. These results suggest that sensitized lymphocytes may produce a factor (or factors) by stimulation with a specific drug-carrier and this factor (or factors) causes cholestasis in the liver.