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. In cultured hepatocytes, colchicine and other vesicular transport inhibitors decreased PM CYPs1A assessed by flow cytometry. Colchicine administration also decreased PM CYPs1A in vivo. Pulse chase experiments with [(35)S]methionine showed that only the newly synthesized CYP molecules are transferred to the PM, whereas microsomal CYP1A2 was stably radiolabeled for several hours. In contrast, radiolabeled CYP1A2 reached the PM and disappeared from the PM with half-lives of less than 30 min. Confocal microscopy, biotinylation, and coimmunoprecipitation experiments showed that PM CYPs1A and CYP reductase are present on the cell surface, and that the reductase is closely associated with PM CYPs. Exposure of whole cells to an anti-CYP1A1/2 antibody at 4 degrees C, before five washes and PM preparation, abolished PM CYPs1A-supported monooxygenase activity, indicating that PM CYPs are mostly located on the external surface. Dihydralazine and other CYPs1A inducers increased PM CYPs1A. In conclusion, newly synthesized CYPs1A follow vesicular flow to the outside of the PM, and NADPH-CYP reductase also is located on the hepatocyte surface. Dihydralazine administration increases PM CYP1A2, its autoimmune target.

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. We also identified CYP-associated steroid 6beta-hydroxylase activity in BEC. CYP and mEH expression was 5- to 20-fold lower in BEC than in autologous hepatocytes, and further differed by a higher ratio of CYP3A5/CYP3A4, and by CYP1A1 predominance over CYP1A2. alphaGST was highly expressed in both hepatocytes and BEC, while piGST was restricted to BEC. In approximately 50% of individuals, muGST was expressed in hepatocytes and at lower levels in BEC. By using the same antibodies as those used in immunoblots, we could show by immunohistochemistry that CYP2E1, CYP3A, mEH, alpha, mu, and piGST immunoreactivities are expressed and display a heterogeneous distribution in the epithelium lining the entire biliary tract except for small intrahepatic bile ducts that were devoid of CYP3A and alphaGST immunoreactivities. In conclusion, BEC contribute to phase II, and although to a lesser extent than hepatocytes, to phase I biotransformation. The distribution of drug-metabolizing enzymes in BEC suggest that they are heterogeneous in their ability to generate and detoxicate reactive metabolites, which may contribute to specific distributions of cholangiopathies.

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. Two activators of macrophages, lipopolysaccharides and zymosan, were found to induce marked increases of IL-1 in the compartment where they had been added: basal if added to the lower compartment and vice versa. In contrast, after a basal stimulation, IL-6 production was mainly increased in the upper compartment that corresponds to a Sertoli cell apical flux. In this system, IL-1 and IL-6 levels were not modified by FSH; they were not also affected by residual bodies and latex beads, probably due to the fact that, in the bicameral system, phagocytosis is restricted to the Sertoli cells situated at the surface of the inner compartment. IL-1beta, but not IL-1alpha, induced IL-6 secretion in the compartment of stimulation. In conclusion, the present study demonstrates that vectorial secretory patterns of IL-1 and IL-6 production greatly differ and that these cytokines are also differently regulated. These results suggest that Sertoli IL-1 and IL-6 have different targets within the testis and that, in normal and pathophysiological conditions, both the tubular and the interstitial compartments may be influenced by the action of these paracrine factors.

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. Therefore, the present study was aimed at identifying IL-1 and -6 expression and production within the human testis, using RT-PCR, bioassays, and enzyme linked immunosorbent assays. We demonstrated that IL-1 and -6 messenger RNA and proteins were produced constitutively in vitro by human Leydig cell- and Sertoli cell-enriched preparations. FSH only stimulated IL-6 production by Sertoli cell-enriched preparations, but increased the release of both IL-1 and -6 in germ cell-depleted Sertoli cell cultures. In addition, lipopolysaccharides and latex beads enhanced the production of both cytokines by Sertoli cell cultures, whereas human chorionic gonadotropin and lipopolysaccharides enhanced the release of both cytokines by Leydig cells. Enzyme linked immunosorbent assays and neutralization experiments revealed that human Sertoli cells produce essentially the alpha form of IL-1, whereas both forms, alpha and beta, are present in Leydig cells. The demonstration that human Leydig and Sertoli cells produce IL-1 and -6 under the control of gonadotropin hormones and exogenous factors, opens the possibility to study the involvement of these cytokines in the control of testis function, in normal and pathological conditions in men.

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. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.

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. Although the liver is the most common target organ for drug candidates in animal toxicity studies, hepatotoxicity rarely leads to cessation of drug development during the preclinical phase. Indeed, contrary to other target organs, liver toxicity is usually reversible and can be monitored in man by sensitive serum enzyme tests. Therefore in many cases a compound found hepatotoxic in an animal species will be tested in man for a definitive assessment of its hepatotoxic potential. Liver toxicity in man may be acceptable when a drug has major therapeutic potential. In this situation mechanistic studies are essential to assess the risk in man and in some cases to identify protective agents. When liver toxicity leads to project termination a secondary screening approach may be envisaged if biologically active analogs are available.

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. Immunoprecipitation, immunoblotting, and flow cytometry experiments were performed with an anti-tienilic acid or an anti-cytochrome P450 2C11 antibody. Cytochrome P450 2C11 was the main microsomal or plasma membrane protein that was alkylated by tienilic acid. Inhibitors of vesicular transport decreased flow cytometric recognition of both unalkylated and tienilic acid-alkylated cytochrome P450 2C11 on the plasma membrane of cultured hepatocytes. Tienilic acid hepatitis sera that were preadsorbed on microsomes from untreated rats (to remove autoantibodies), poorly recognized untreated hepatocytes in flow cytometry experiments, but better recognized tienilic acid-treated hepatocytes. This recognition was decreased by adsorption with tienilic acid or by preexposure to the anti-tienilic acid or the anti-cytochrome P450 2C11 antibody. We conclude that cytochrome P450 2C11 is alkylated by tienilic acid and follows a vesicular route to the plasma membrane. Tienilic acid hepatitis sera contain antibodies against this tienilic acid adduct, in addition to the previously described anticytochrome P450 autoantibodies.

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. We demonstrated the presence of the CYP2E1 apoprotein in microsomes of RLECs by immunoblot analyses, together with chlorzoxazone 6-hydroxylation, an activity known to be mainly catalyzed by CYP2E1. In addition, acetone treatment of these cells resulted in an increase in both CYP2E1 apoprotein and chlorzoxazone 6-hydroxylation activity levels. Finally, we showed the susceptibility of RLECs to N-methyl formamide- and diethylnitrosamine-induced toxicity, suggesting metabolic activation by CYP2E1. Thus, RLECs may cooperate with hepatocytes to CYP2E1-mediated metabolism in the co-culture model. In addition, transfection experiments with a CYP2E1 promoter construct, in which the proximal 539 bp containing the binding site for HNF1alpha were inserted upstream of the chloramphenicol acetyl transferase gene, demonstrated a strong induction upon co-transfection with an HNF1alpha expression plasmid. Thus, RLECs provide a useful tool for studying metabolism and cytotoxicity of CYP2E1 substrates in the absence of other expressed CYPs, and for analyzing CYP2E1 promoter function.

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. METHODS: A monoclonal antibody specific for the rat IL-2 receptor alpha chain was used to label the receptor, whereas effects on cytochrome P450 were determined after 24 hours of culture with human recombinant IL-2 (5000 U/mL). RESULTS: The presence of the IL-2 receptor in hepatocytes was shown by immunoblots, flow cytometry, and scanning confocal microscopy. IL-2 caused a 46% decrease in total cytochrome P450; a 35%, 35%, 36%, 26%, and 56% decrease in immunoreactive cytochrome P4501A1, 2B, 2C11, 2D1, and 3A, respectively; and a marked decrease in cytochrome P4503A2 and 2C11 messenger RNAs. Addition to the culture medium of the anti-receptor antibody or the tyrosine kinase inhibitor genistein prevented the IL-2-mediated decrease in cytochrome P450. CONCLUSIONS: IL-2 down-regulates the expression of cytochrome P450 genes in cultured rat hepatocytes by interacting with its receptor expressed on 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. As a result, it has been suggested that UDCA or one of its conjugates could have a direct protective effect on hepatocyte structure and function. We therefore studied the effects of chenodeoxycholic acid (CDCA) and tauroursodeoxycholic acid (TUDCA) alone and in combination on the viability and certain functions of human hepatocytes in primary culture. TUDCA did not affect intracellular concentrations of CDCA when added concomitantly. In other experiments, CDCA (100 to 500 mumol/L) induced concentration-dependent increases in lactate dehydrogenase (LDH) leakage and decreases in cellular protein synthesis and albumin secretion. Neither TUDCA nor UDCA had similar effects at the same concentrations, nor did they have a protective effect when added concomitantly with CDCA at equimolar or twice-equimolar concentrations. These results suggest that UDCA has no direct cytoprotective effect when the bile acid concentrations to which human hepatocytes are exposed are unchanged. They also suggest that the hepatoprotective effect of UDCA in cholestatic human liver diseases and in the isolated perfused liver loaded with hydrophobic bile acids occurs through its effect on intestinal and hepatocyte transport systems.

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. We determined the route followed by cytochrome P4502B to reach the plasma membrane. METHODS: Rat hepatocytes were cultured for 2 hours after plating with various inhibitors of cellular trafficking. Detached, uncut, nonpermeabilized hepatocytes were then exposed to a monoclonal antibody specific for cytochrome P4502B and studied by flow cytometry and confocal microscopy. RESULTS: The plasma membrane expression of cytochrome P4502B was markedly decreased after 2 hours of culture with cycloheximide (an inhibitor of protein synthesis), caffeine at 20 degrees C (conditions that decrease vesicular transport from the endoplasmic reticulum to the Golgi apparatus), brefeldin A (which redistributes Golgi components back to the endoplasmic reticulum), monensin (an inhibitor of Golgi functions), and colchicine, vinblastine, or nocodazole (three microtubule inhibitors). CONCLUSIONS: Part of cytochrome P4502B follows a microtubule-dependent vesicular route from the endoplasmic reticulum to the plasma membrane in cultured rat hepatocytes.

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, 4.04 +/- 0.33 vs. 6.32 +/- 0.58 mumol/min; P < or = 0.05). The transamination pathway, evaluated by leucine exchanges, was more important in the cirrhotic livers (25.4 vs. 6.8% in controls at 2 mM). The incorporation of alpha-[1-14C]KIC in proteins of cirrhotic liver was increased compared with controls (0.25 +/- 0.04% of alpha-[1-14C]KIC was incorporated in proteins excreted in perfusate vs. 0.20 +/- 0.04 in controls; P < or = 0.05). In addition, a line of evidence suggests that glutamine rather than glutamate is the N donor for leucine synthesis from KIC. The decarboxylation pathway evaluated by beta-hydroxybutyrate production and by 14CO2 release from alpha-[1-14C]KIC was reduced, respectively, by 40-85% (according to KIC dose) and by 24% at 90 min in cirrhotic livers compared with healthy livers. These results indicate a dramatic modification of KIC metabolism in the cirrhotic liver; its uptake by the liver is decreased and its incorporation into proteins is increased via an enhancement of transamination to leucine, probably as a consequence of an inhibition of branched-chain keto acid dehydrogenase.

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. RESULTS: Chenodeoxycholic acid induced an overexpression of MHC class I molecules, whereas ursodeoxycholic acid did not. The level of class I mRNA closely reflected that of the membrane protein. Moreover, cholestasis, induced in the rat by ligation-section of the common bile duct, increased the MHC class I mRNA level. Actinomycin D inhibited bile acid-induced class I transcription of rat hepatocytes in primary culture, whereas cycloheximide did not. Finally, class I mRNA expression was induced in hepatocytes by phorbol myristate acetate and by forskolin. This hyperexpression, as well as that observed with chenodeoxycholic acid, was suppressed by an inhibitor of protein kinase C and protein kinase A. CONCLUSIONS: Taken together, these results suggest that chenodeoxycholic acid, as interferon, activates protein kinase C and protein kinase A, resulting in the induction of MHC class I expression.

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. Results of qualitative and quantitative covalent binding of tienilic acid metabolite(s) to human liver microsomes were then compared to those obtained with two drugs leading to direct toxic hepatitis, namely, acetaminophen and chloroform. Kinetic constants (Km and Vmax) were measured, and the covalent binding profile of the metabolites to human liver microsomal proteins was studied. Tienilic acid had both the lowest Km and the highest covalent binding rate at pharmacological doses. For acetaminophen and chloroform, several microsomal proteins were covalently bound, while covalent binding was highly specific for tienilic acid and dihydralazine, another drug leading to immunoallergic hepatitis. Although low numbers of drugs were tested, these results led us to think that there may exist a relationship between the specificity of covalent binding and the type of hepatotoxicity.

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. F-actin distribution was assessed in the couplets by the ratio of the pericanalicular area fluorescence/total couplet fluorescence (CF/TF). At non-cytotoxic concentrations, TLC (50, 100 microM) and ERY (10, 50, 100 microM) induced a significant accumulation of F-actin around the bile canaliculus as indicated by increased fluorescence in the pericanalicular area and by the increased CF/TF ratio compared with the controls. Electron microscopy studies showed significant alterations in bile canaliculi microvilli in couplets treated with 100 microM TLC. Only a few canaliculi showed an increase in pericanalicular microfilaments after treatment with 100 microM ERY. As assessed by scanning laser cytometry, TUDC prevented changes in F-actin distribution when it was added to the medium with taurolithocholate or erythromycin estolate at equimolar concentrations. However, the morphological changes observed by electron microscopy after treatment with TLC were not modified by co-treatment with TUDC. Taurocholate was ineffective. We conclude that (a) abnormalities of pericanalicular F-actin microfilaments occur in two different models of cholestasis, (b) tauroursodeoxycholate prevents the accumulation of pericanalicular F-actin detected by scanning laser cytometry but not the morphological changes of the canaliculus observed by electronic microscopy. Therefore, in these experimental conditions, the protective effect of TUDC appears to be partial.

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. To test this hypothesis, we used several in vivo and in vitro experimental models. Occlusion of the rat hepatic pedicle in situ for 60 min (normothermic ischemia) induced a significant increase in serum creatine kinase levels relative to those in laparotomized controls (2,530 +/- 530 vs. 389 +/- 64 IU/L, mean +/- SEM; p < 0.005). In the isolated perfused rat liver, 60-min ischemia induced early (< or = 3 min) creatine kinase and AST release (0.87 +/- 0.14 vs. 0.08 +/- 0.01 IU/min/gm liver, respectively). A similar phenomenon was observed after 24-hr or 48-hr hypothermic conservation in University of Wisconsin solution. Electrophoretic analysis and immunoinhibition studies showed that creatine kinase activity comprised creatine kinase-BB (approximately 50%) and mitochondrial creatine kinase. Trypan blue infusion showed a loss of viability in sinusoidal cells, whereas hepatocytes were relatively spared. Finally, murine sinusoidal cells were isolated, cultured and then lysed by a freeze-thaw cycle and sonication. Creatine kinase activity was found in endothelial cells (creatine kinase-BB), Kupffer cells (creatine kinase-BB) and Ito cells (creatine kinase-MM). Creatine kinase-BB was not found in hepatocytes, but mitochondrial creatine kinase was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

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. Treatment with TUDC added concomitantly with CDC or TLC significantly decreased the rise in [Ca2+]i induced by bile acids. These results, obtained with a polarized hepatocyte model that secretes bile, confirmed that cholestatic bile acids increase [Ca2+]i and showed that TUDC inhibits this phenomenon. These data are in agreement with a key role of intracellular calcium in cholestasis.

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. (2) After exposure of uncut, fixed hepatocytes to antibodies, immunofluorescence and immunoperoxidase studies were performed. RESULTS: (1) The specific content of cytochrome P-450 in plasma membrane was 9% of that in microsomes. Plasma membranes showed NADPH-cytochrome c reductase and mono-oxygenase activities; immunoblots showed the presence of cytochromes P-450 1A2, 2C, 2D6, 2E1, and 3A4; cytochromes P-450 1A2, 2D6, and 2C were also recognized by anti-liver microsome and anti-liver/kidney microsome type 1 and type 2 autoantibodies, respectively. (2) Immunofluorescence and immunoperoxidase labeling of the plasma membrane was observed with the three auto-antibodies and with anti-cytochrome P-450 1A2, 2C, 2E1, or 3A4. CONCLUSIONS: It is concluded that cytochromes P-450 are present and functional in the plasma membrane of human hepatocytes and that anti-cytochrome P-450 autoantibodies recognize epitopes expressed on the outer surface.