Comparison of human hepatoma HepaRG cells with human and rat hepatocytes in uptake transport assays in order to predict drug induced hepatotoxicity

Human hepatocytes are the gold standard for toxicological studies but they have several drawbacks, like scarce availability, high inter-individual variability, a short lifetime, which limits their applicability. The aim of our investigations was to determine, whether HepaRG cells could replace human hepatocytes in uptake experiments for toxicity studies. HepaRG is a hepatoma cell line with most hepatic functions, including a considerable expression of uptake transporters in contrast to other hepatic immortalized cell lines. We compared the effect of cholestatic drugs (bosentan, cyclosporinA, troglitazone,) and bromosulfophthalein on the uptake of taurocholate and estrone-3-sulfate in human and rat hepatocytes and HepaRG cells. The substrate uptake was significantly slower in HepaRG cells than in human hepatocytes, still, in the presence of drugs we observed a concentration dependent decrease in uptake. In all cell types, the culture time had a significant impact not only on the uptake process but on the inhibitory effect of drugs too. The most significant drug effect was measured at 4 h after seeding. Our report is among the first concerning interactions of the uptake transporters in the HepaRG, at the functional level. Results of the present study clearly show that concerning the inhibition of taurocholate uptake by cholestatic drugs, HepaRG cells are closer to human hepatocytes than rat hepatocytes. In conclusion, we demonstrated that HepaRG cells provide a suitable tool for hepatic uptake studies

Marx, Engels és az írek (könyvismertető)

A recenzált mű: Andrew Boyd: Marx and Engels and the Irish. Socialist History Society Occasional Papers Series, 18. London, 2004. 37 p

Kommunisták Sztálin ellen (könyvismertető)

A recenzált mű: Pierre Broué: Communistes contre Staline. Fayard, Párizs, 2003. 439 p

Gorbacsovról – előítéletek nélkül (könyvismertető)

A recenzált mű: DALOS György: Gorbacsov. Ember és hatalom. Napvilág Kiadó, Budapest, 2011. 259 p

A humán genotoxicitás kutatásának és kimutatásának korszerűsítése = Study of human genotoxicity: An Update and practical considerations

A mutagének metabolikus aktiválására S9 frakciót használnak az Ames tesztben. Ez lehetővé teszi a fázis I enzimek katalizálta metabolikus folyamatok vizsgálatát, de figyelmen kívül hagyja a fázis II enzimek szerepét, ami hamis negatív vagy pozitív eredményre vezethet. Munkánkban a patkány máj S9 frakció humán máj S9 frakcióval, illetőleg in vivo vagy in vitro indukált patkány hepatocitákkal történő helyettesítésének lehetőségét vizsgáltuk. A benzpirén és az aminoantracén (AA) mutagenitását vizsgáltuk az Ames teszttel. Metabolikus aktiváló rendszerként -naftoflavonnal (BNF), fenobarbitállal (PB) vagy -naftoflavon és fenobarbitál kombinációjával (BNF+PB) kezelt patkány máj S9 frakciót alkalmaztunk. Eredmények arra utalnak, hogy a BNF+PB indukció nem biztosít optimális feltételeket minden mutagén kimutatásához. Májsejtek alkalmazása metabolikus aktivátorként lehetőséget nyújt induktorok azonosítására és arra is, hogy a CYP enzimek mellett más enzimek szerepét is vizsgálni lehessen az aktivációs folyamatban. A BNF-nal kezelt állatok májából izolált S9 frakcióval aktivált AA mutagenitását két CYP enzim gátló (α-naftoflavon, benzilimidazol) megnövelte, ami arra utal, hogy az indukált S9 frakciók esetében nem CYP enzimek a AA aktivátorai. Az S9 frakcióval kapott eredményekkel ellentétben a BNF kezelés megnövelte a hepatocitákkal aktivált AA mutagenitását, rámutatva, hogy a mutagén potenciál függhet az Ames tesztben alkalmazott aktiváló rendszertől. | In the Ames test rat S9 fraction is used for activation of mutagens, which allows the investigation of metabolic processes mediated by phase I enzymes, disregarding the role of phase II enzymes resulting in false negative and positive outcome. With the aim of broadening the possibilities of mutagenic assays we studied the replacement of rat hepatic S9 with human hepatic S9 and in vivo or in vitro induced rat hepatocytes. We analysed the mutagenicity of benzpyrene and aminoanthracene (AA) in the Ames test using S9 or hepatocytes from rats treated with -naphthoflavone (BNF), phenobarbital (PB), and a combination of the two (BNF+PB). The results suggest that the BNF+PB induction is inadequate to ensure optimal conditions for all mutagens. The use of hepatocytes for activation provides a useful system to find better inducers, and the effect of other enzymes besides CYPs can also be taken into consideration. Since BNF induces CYP1A enzymes but S9 from BNF treated rats unexpectedly reduced the mutagenicity of 2AA the role of CYPs in the metabolic activation was investigated. CYP inhibitors increased the metabolic activation of AA from BNF- and PB-treated rats suggesting that enzymes other than CYPs might be the main contributors to AA activation. In contrast BNF treatment of rats enhanced the activation of AA by hepatocytes. Our results indicate that identification of mutagenic hazard by use of the Ames test may depend on the metabolic system applied

Statins alter the hepatobiliary transport of unconjugated and conjugated bilirubin in sandwich-cultured rat hepatocytes

Several studies have reported that statins occasionally cause impairment of liver functions characterized by elevated serum bilirubin levels, which might be due to altered function of the multidrug resistanceassociated proteins (Mrp2/3). We aimed to study the modulation of the hepatobiliary transport of bilirubin by four statin derivatives, atorvastatin, fluvastatin, pravastatin, and rosuvastatin in sandwichcultured rat hepatocytes. All statins except pravastatin significantly inhibited the uptake of bilirubin. The biliary efflux of bilirubin conjugates was increased by pravastatin and rosuvastatin concentration dependently. Rosuvastatin stimulated not only the Mrp2 mediated biliary, but the Mrp3 mediated sinusoidal elimination, resulting in decreased intracellular bilirubin accumulation. The significantly induced Mrp2/3 protein levels (ranging from 1.5 to 1.8-fold) accounted for the elevated efflux. Cell polarization, the formation of biliary network was also significantly increased by fluvastatin, pravastatin and rosuvastatin (151%, 216% and 275% of the control, respectively). The simultaneous inhibition of the uptake and the stimulation of the sinusoidal and canalicular elimination may explain, at least in part, the clinical observation of elevated serum bilirubin levels. In conclusion, our results suggest that in spite of the elevated serum bilirubin levels, the altered Mrp2 and Mrp3 functions by statins is probably not associated with hepatotoxic effects

The Janus facet of nanomaterials

Application of nanoscale materials (NMs) displays a rapidly increasing trend in electronics, optics, chemical catalysis, biotechnology, and medicine due to versatile nature of NMs and easily adjustable physical, physicochemical, and chemical properties. However, the increasing abundance of NMs also poses significant new and emerging health and environmental risks. Despite growing efforts, understanding toxicity of NMs does not seem to cope with the demand, because NMs usually act entirely different from those of conventional small molecule drugs. Currently, large-scale application of available safety assessment protocols, as well as their furthering through case-by-case practice, is advisable. We define a standard work-scheme for nanotoxicity evaluation of NMs, comprising thorough characterization of structural, physical, physicochemical, and chemical traits, followed by measuring biodistribution in live tissue and blood combined with investigation of organ-specific effects especially regarding the function of the brain and the liver. We propose a range of biochemical, cellular, and immunological processes to be explored in order to provide information on the early effects of NMs on some basic physiological functions and chemical defense mechanisms. Together, these contributions give an overview with important implications for the understanding of many aspects of nanotoxicity

Comparative study of CYP2B1/2 induction and the transport of bilirubin and taurocholate in rat hepatocyte-mono- and hepatocyte-Kupffer cell co-cultures

Introduction Hepatocyte-Kupffer cell (KC) co-cultures represent a promising approach for in vitro modeling of complex interactions between parenchymal and non-parenchymal cells in the liver, responsible for drug-induced liver injury (DILI). In this study we aimed to compare hepatocyte monocultures with hepatocyte-KC co-cultures regarding some basic liver functions associated with the chemical defense system. These pathways involve transporters and enzymes the function of which is highly sensitive towards hepatotoxic events. Methods CYP2B1/2 induction and the biliary and sinusoidal elimination of bilirubin (B) and taurocholate (TC) were studied in rat hepatocyte sandwich cultures compared with rat hepatocyte-KC sandwich co-cultures of 1:0, 6:1, 2:1 and 1:1 cell combinations representing the physiologic and pathologic conditions of the liver. Results KCs decreased phenobarbital inducibility of CYP2B1/2 in a cell ratio dependent manner and activation of KCs by lipopolisacharide (LPS) amplified this effect. Similarly, KCs decreased the transport of B and its glucuronides (BG) in both sinusoidal and canalicular directions resulting in its intracellular accumulation. In contrast, the uptake and the efflux of TC were greater in the co-cultures than in the hepatocyte monocultures. Immuno-labelling of sodium-dependent taurocholate transporter (Ntcp) revealed increased expression of the transporter in the presence of KCs. Discussion Here we presented that KCs have a direct impact on some hepatocyte functions suggesting that the co-culture model may be more suitable for drug related hepatotoxicity studies than hepatocyte monocultures. Abbreviations B, bilirubin; Bsep, bile salt export pump; CYP, cytochrome P-450; GdCl3, gadolinium(III) chloride; H/KC, hepatocyte-Kupffer-cell co-culture; HBSS, Hanks' balanced salt solution; HPLC, high-performance liquid chromatography; KC, Kupffer-cell; LPS, lipopolisacharide; Mrp, multidrug resistance-associated protein; Ntcp, sodium-dependent taurocholate transporter; PB, phenobarbital; PBS, phosphate-buffered saline; PTX, pentoxyphylline; TC, taurocholat