FXR: Big fish or small fry for drug-induced liver injury?

By integrating network analysis and molecular modeling, a "system pharmacology" approach identified FXR as a potential off-target protein mediating non-steroidal anti-inflammatory drugs (NSAID)-induced liver injury. In vitro assays showed that NSAID are potent FXR antagonists that inhibit FXR transcriptional activity. Given the role of FXR in bile acid homeostasis, liver inflammation and cell proliferation, the data suggest that FXR antagonism could mediate, at least in part, NSAID-induced liver injury.

Preventing Drug-Induced Liver Injury: How Useful Are Animal Models?

Drug-induced liver injury (DILI) is the most common organ toxicity encountered in regulatory animal toxicology studies required prior to the clinical development of new drug candidates. Very few reports have evaluated the value of these studies for predicting DILI in humans. Indeed, compounds inducing liver toxicity in regulatory toxicology studies are not always correlated with a risk of DILI in humans.

Back to basics for idiosyncratic drug-induced liver injury: dose and metabolism make the poison.

Two studies show that the risk of drug-induced liver injury (DILI) is increased when the daily dose of a drug given by oral route is higher than 10mg per day and/or when the drug undergoes a significant hepatic metabolism. If confirmed, these data suggest that developing drugs with high potency and low hepatic metabolism will reduce the risk of idiosyncratic DILI in man.

Vesicular transport of newly synthesized cytochromes P4501A to the outside of rat hepatocyte plasma membranes.

Anti-cytochrome P450 (CYP)1A2 autoantibodies are found in dihydralazine-induced hepatitis, and CYPs2B and 2C have been shown to follow vesicular flow to the plasma membrane (PM). However, it is unknown whether other CYPs follow this route, whether NADPH-CYP reductase is present on the hepatocyte surface, and whether autoimmune hepatitis-inducing drugs increase PM CYPs. In this study, we determined the transmembrane topology and transport of CYPs1A in rat hepatocytes.

Phase I and phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium.

Tissue expression of drug-metabolizing enzymes influences susceptibility to drugs and carcinogens. Because the biliary epithelium, exposed to bile-borne chemicals, may give rise to drug-induced cholangiopathies and to cholangiocarcinomas, we determined the pattern of expression of drug-metabolizing enzymes in this epithelium. We first demonstrated by blot analyses that biliary epithelial cells (BEC) isolated from human gallbladders display cytochrome P450 (CYP) 1A, 2E1, and 3A, microsomal epoxide hydrolase (mEH), alpha, mu, and pi glutathione S-transferase (GST), transcripts and proteins.

Vectorial production of interleukin 1 and interleukin 6 by rat Sertoli cells cultured in a dual culture compartment system.

The bidirectional production of interleukin-1 (IL-1) and IL-6 by Sertoli cells and its regulation by inflammatory and physiological stimuli has been studied using a dual compartment culture system allowing the study of Sertoli cell apical and basal secretory activities. Another Sertoli cell activity, the vectorial transferrin production was also studied in all culture conditions. A low constitutive IL-1 production appeared equally distributed between both poles, while IL-6 and transferrin constitutive production was predominantly directed apically.

Human Leydig cells and Sertoli cells are producers of interleukins-1 and -6.

Interleukins (IL)-1 and -6 have been shown to be produced by several categories of cells in the rat testis and involved in the paracrine control of testicular function. Evidence of high amounts of IL-1 have been shown in the human testis, but nothing is known about its cellular origin. Furthermore, to our knowledge, the presence of IL-6 in the human testis has not yet been investigated.

Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells.

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities.

Hepatotoxicity in drug development: detection, significance and solutions.

Despite considerable progress in the understanding of the mechanism of liver toxicity we are not yet able to design non-hepatotoxic molecules rationally. Also, there is no "universal" in vitro primary screening approach for early identification of hepatotoxic molecules. In most cases hepatotoxicity is detected at later stages of drug development in animal toxicity studies or clinical trials.

Antigenic targets in tienilic acid hepatitis. Both cytochrome P450 2C11 and 2C11-tienilic acid adducts are transported to the plasma membrane of rat hepatocytes and recognized by human sera.

Patients with tienilic acid hepatitis exhibit autoantibodies that recognize unalkylated cytochrome P450 2C9 in humans but recognize 2C11 in rats. Our aim was to determine whether the immune reaction is also directed against neoantigens. Rats were treated with tienilic acid and hepatocytes were isolated.

Rat liver epithelial cells express functional cytochrome P450 2E1.

Rat liver epithelial cells (RLECs) isolated by trypsinization of the livers of normal 10 day old rats are largely used in co-culture with primary hepatocytes. The aim of the present study was to investigate the expression of biotransformation enzyme-encoding genes in three preparations of RLEC lines. Although no expression of cytochrome P450 1A1/2 (CYP1A1/2), CYP2B1/2, CYP2C6 or CYP3A mRNAs could be detected, we found that all of the different preparations of RLECs expressed a high level of CYP2E1 mRNA.

The interleukin-2 receptor down-regulates the expression of cytochrome P450 in cultured rat hepatocytes.

Background & Aims: Interleukin (IL) 2 is used in advanced cancers, but its effects on cytochrome P450 remain unknown. Other cytokines down-regulate hepatic cytochrome P450, but it is not known whether this involves cytokine receptors. The aim of this study was to determine whether the IL-2 receptor is expressed on hepatocytes and whether its activation by IL-2 depresses cytochrome P450 in cultured rat hepatocytes.

Effects of ursodeoxycholic acid and chenodeoxycholic acid on human hepatocytes in primary culture.

Hepatic bile acid concentrations are elevated in chronic cholestasis because of reduced canalicular excretion and active ileal absorption of the fraction eliminated in the gut. Ursodeoxycholic acid (UDCA) reduces the intestinal absorption of endogenous bile acids, thereby diminishing the concentrations to which liver cells are exposed. In the isolated perfused liver (in which vectorial bile acid transport is maintained), UDCA reduces the cytotoxic and cholestatic effects of endogenous bile acids.

Cytochrome P4502B follows a vesicular route to the plasma membrane in cultured rat hepatocytes.

Background/aims: Autoantibodies against cytochrome P450 are found in some forms of autoimmune hepatitis. Cytochrome P450 is synthesized and mainly located in the endoplasmic reticulum but may also be expressed on the plasma membrane of hepatocytes. Vesicles migrate from the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane along microtubules.

Metabolism of alpha-ketoisocaproic acid in isolated perfused liver of cirrhotic rats.

To determine the hepatic fate of alpha-ketoisocaproate (KIC) in cirrhosis, six groups of isolated rat livers were perfused with 0, 0.5, 1 (with or without alpha-[1-14C]KIC), 2, and 5 mM KIC; control livers from healthy rats were studied in parallel under similar conditions. KIC was rapidly removed by the normal livers, whereas uptake was lower in the cirrhotic livers at all concentrations tested (at 2 mM, 4.

Effects of bile acids and cholestasis on major histocompatibility complex class I in human and rat hepatocytes.

Background/aims: Major histocompatibility complex (MHC) class I molecules, which are normally poorly expressed on the surface of hepatocytes, are overexpressed during cholestasis. The mechanisms responsible for this overexpression were examined.

Methods: The expression of class I molecules, assessed by flow cytofluorimetry, and the class I messenger RNA (mRNA) transcripts, assessed by Northern blot analysis, were measured on normal human hepatocytes in primary culture.

Specificity of in vitro covalent binding of tienilic acid metabolites to human liver microsomes in relationship to the type of hepatotoxicity: comparison with two directly hepatotoxic drugs.

In order to better understand the first steps leading to drug-induced immunoallergic hepatitis, we studied the target of anti-LKM2 autoantibodies appearing in tienilic acid-induced hepatitis, and the target of tienilic acid-reactive metabolites. It was identified as cytochrome P450 2C9, (P450 2C9): indeed, anti-LKM2 specifically recognized P450 2C9, but none of the other P450s tested (including other 2C subfamily members, 2C8 and 2C18). Tienilic acid-reactive metabolite(s) specifically bound to P450 2C9, and experiments with yeast expressing active isolated P450s showed that P450 2C9 was responsible for tienilic acid-reactive metabolite(s) production.

Effect of tauroursodeoxycholate on actin filament alteration induced by cholestatic agents. A study in isolated rat hepatocyte couplets.

The mechanism of the protective effect of ursodeoxycholic acid in cholestatic liver diseases remains unclear. Since there is evidence that alterations in the pericanalicular actin microfilament network play a major role in cholestasis, the aims of this study were (a) to determine the effect of the cholestatic agents, taurolithocholate (TLC) and erythromycin estolate (ERY), on F-actin distribution in isolated rat hepatocyte couplets and (b) to assess the effect of tauroursodeoxycholate (TUDC) and taurocholate on the modifications induced by these two compounds. F-actin was stained with fluorescein-isothiocyanate phalloidin and fluorimetric measurements were performed using a scanning laser cytometer ACAS 570.

Creatine kinase-BB: a marker of liver sinusoidal damage in ischemia-reperfusion.

Cell damage within the sinusoidal lining of human liver grafts during transplantation is an early event that is critical in ischemia-reperfusion injury and probably plays a key role in primary liver dysfunction after transplantation. No simple biochemical marker for sinusoidal injury is currently available. Because creatine kinase activity has been described in heart endothelial cells, we hypothesized that release of this enzyme might serve as an index of sinusoidal injury.

Effect of bile acids on intracellular calcium in isolated rat hepatocyte couplets.

The effects of bile acids on cytosolic free calcium ([Ca2+]i) were studied in single isolated rat hepatocyte couplets using a scanning laser cytometer and the fluorescent dye, indo-1. Cholestatic bile acids, chenodeoxycholate (CDC) and taurolithocholate (TLC) increased [Ca2+]i in a dose-dependent manner. Choleretic bile acids, tauroursodeoxycholate (TUDC) and taurocholate (TC), did not induce any change in [Ca2+]i except TC at very high doses.

Cytochromes P-450 in human hepatocyte plasma membrane: recognition by several autoantibodies.

Background: Anti-cytochrome P-450 autoantibodies are present in several forms of autoimmune hepatitis. The possibility that cytochromes P-450 are present in the plasma membrane of human hepatocytes was examined.

Methods: (1) Plasma membranes with microsomal contamination < 1%, as judged from the activities of glucose-6-phosphatase and NADH-cytochrome c reductase, were prepared.

Effect of recombinant human interleukin 1β (rhIL-1β) on amino acid flux in the isolated perfused rat liver.

We studied the effect of recombinant human IL-1β (rhIL-1β) on hepatic amino acid (AA) flux in the isolated perfused rat liver model. Two experimental groups were used - a control group (n = 5) and a rhIL-1β-treated group (n = 5). IL-1 was added to the perfusate in two successive boluses of 0.

Branched-chain amino acid metabolism in isolated perfused liver of cirrhotic rats.

We examined the possible contribution of the liver to the alterations in branched-chain amino acid (BCAA) metabolism in cirrhosis. The livers of male Sprague-Dawley rats with CCl4-induced cirrhosis were removed and placed in a recirculating perfusion system. Net amino acid uptake and release were determined over 55 min.

Intrasplenic hepatocellular transplantation corrects hepatic encephalopathy in portacaval-shunted rats.

The aim of this work was to evaluate the effect of intrasplenic hepatocellular transplantation on hepatic encephalopathy in an experimental model of chronic liver failure induced by end-to-side portacaval shunt in the rat. Inbred male Wistar Furth rats were divided into three groups: rats subjected to portacaval shunt (n = 10), rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10) and sham-operated rats (n = 10). Behavior tests were performed in a blind fashion at 3 wk, at 2 mo and at 3 mo after surgery.