L. Vereczkey

ABCC2/Abcc2: A multispecific transporter with dominant excretory functions

ABCC2/Abcc2 (MRP2/Mrp2) is expressed at major physiological barriers, such as the canalicular membrane of liver cells, kidney proximal tubule epithelial cells, enterocytes of the small and large intestine, and syncytiotrophoblast of the placenta. ABCC2/Abcc2 always localizes in the apical membranes. Although ABCC2/Abcc2 transports a variety of amphiphilic anions that belong to different classes of molecules, such as endogenous compounds (e.g., bilirubin-glucuronides), drugs, toxic chemicals, nutraceuticals, and their conjugates, it displays a preference for phase II conjugates. Phenotypically, the most obvious consequence of mutations in ABCC2 that lead to Dubin-Johnson syndrome is conjugate hyperbilirubinemia. ABCC2/Abcc2 harbors multiple binding sites and displays complex transport kinetics.

Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes

Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24h of APAP treatment starting at 24h after seeding (EC50: 3.8mM, 7.6mM, and 28.2mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC50 values were 6.0mM, 12.5mM, and 18.8mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.

Contribution of High Basolateral Bile Salt Efflux to the Lack of Hepatotoxicity in Rat in Response to Drugs Inducing Cholestasis in Human

Intrahepatic bile acid accumulation due to inhibition of the bile salt export pump (BSEP) has been proposed as a mechanism for drug-induced cholestasis. Many cholestatic drugs do not initiate hepatotoxicity in rats, although they inhibit rat Bsep and cause elevated serum bile acid concentration. In this study, we examined changes in the taurocholate (TC) transport in response to cholestatic drug treatments in human and rat sandwich-cultured hepatocytes. Our experimental setup allows studying the basolateral and canalicular efflux simultaneously, thus comparing drug-induced changes in the vectorial efflux of TC. We found that TC elimination highly differs in human and rat hepatocytes. In human hepatocytes, an equal fraction of TC(uptake) was eliminated by basolateral (34.8%) and canalicular (34.4%) transporters and remained in the cells (30.5%), while in the case of rats, the basolateral transport was dominant (71.7%) and intracellular TC accumulation was negligible (6.9%). The inhibition of BSEP/Bsep resulted in significantly higher intracellular TC(conc) in humans than in rats. The 15-fold difference in intracellular TC(conc) of control in human versus rat hepatocytes was increased 25-fold by troglitazone treatment. MK571 and indomethacin decreased the basolateral efflux and significantly increased the intracellular TC(conc) in rats. In rat hepatocytes, the highest intracellular TC(conc) was observed with cyclosporine A and glibenclamide, which inhibited TC elimination in both directions. Nevertheless, the basolateral transport remained dominant. We conclude that in rats, the higher rate of basolateral bile salt efflux represents an additional protective mechanism in cholestasis, which contributes to species differences in response to hepatotoxic drugs.

The effect of synthetic glucocorticoid, dexamethasone on CYP1A1 inducibility in adult rat and human hepatocytes

Glucocorticoids act synergistically with polycyclic aromatic hydrocarbons in increasing mRNA and protein levels of CYP1A1 in rat liver. The action of dexamethasone to modify CYP1A1 expression has been investigated in adult human hepatocytes. The effect of dexamethasone on the induction of CYP1A1 by 3‐methylcholanthrene is different in rat and human liver cells. Dexamethasone potentiates the induction of CYP1A1 about 3‐ to 4‐fold in rat cells. In human hepatocytes, it reduces CYP1A1 induction by 50–60% at enzyme protein level, while it does not have an effect on CYP1A1 mRNA amount.

Biliary efflux transporters involved in the clearance of rosuvastatin in sandwich culture of primary rat hepatocytes

Rosuvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) has been shown to be excreted mostly unchanged into the bile; interactions on the level of hepatic apical efflux transporters may represent a risk of liver toxicity. So far, controversial and insufficient data are available concerning transporters involved in the elimination process. This study was designed to elucidate, which transporters take part in the biliary clearance of rosuvastatin using sandwich-cultured primary rat hepatocytes. The canalicular efflux of rosuvastatin was measured in the presence of inhibitors: Ko 134, mitoxanthrone, novobiocin for breast cancer resistance protein (Bcrp); verapamil for multidrug resistance protein (Mdr1); benzbromarone, sulfasalazine, probenecid for multidrug resistance associated protein (Mrp 2); and cyclosporine A, glibenclamide, troglitazone for bile salt export pump (Bsep). Mrp2 inhibitors decreased the biliary efflux of rosuvastatin most potently by 78.9%, 35%, 54.1%; benzbromarone, probenecid, sulfasalazine, respectively, while Bcrp and Bsep inhibitors showed much less effect (29.1%, 23.0% ,30.0%; Ko 134, mitoxanthrone, novobiocin, respectively, and 32.6%, 29.3%, 20.6%, glibenclamide, cyclosporine A, troglitazone, respectively). The marked decline of canalicular transport by Mrp2 inhibitors suggests major role of Mrp2 in this process; however, Bcrp and Bsep might also contribute to the biliary elimination of rosuvatatin in sandwich-cultured rat hepatocytes.

Some aspects of interindividual variations in the metabolism of xenobiotics

Differences in drug metabolism among individuals are caused by numerous factors: differences in production and stability of mRNA of xenobiotic metabolizing enzymes, differences in the rate of enzyme synthesis and degradation, or enzyme inhibition. One of the most important reasons is genetic polymorphism of cytochrome P450 genes or cyto-chrome P450 regulatory factors. Nuclear receptors play great role in the regulation of these genes. The presence of the ligand induces the nuclear receptor to bind to the dimerisation partner and as a hetero/homodimer it can activate the DNA responsive element. In addition, several co-activators, co-repressors and other factors can modulate the effect of nuclear receptors. Hepatic levels of cytochrome P450 enzymes are reduced in multiple models of inflammation or infection.Cytochrome P450 enzymes of four families (CYP1-CYP4) are known to be involved in xenobiotic metabolism. Their genetic polymorphism and regulation are discussed in this review.

Modulation of sinusoidal and canalicular elimination of bilirubin‐glucuronides by rifampicin and other cholestatic drugs in a sandwich culture of rat hepatocytes

Aim:  Drug‐induced hyperbilirubinemia has been shown to often be derived from modulation of the expression and activity of hepatobiliary transporters. In this study we examined the interactions of some therapeutic agents, which have been shown to cause cholestasis, with the elimination of bilirubin‐glucuronides, in order to clarify whether these drugs modify the activity of Mrp2 and Mrp3 directly.

Ipriflavone as an inhibitor of human cytochrome P450 enzymes

Reduction of theophylline metabolism and elimination were observed in a theophylline‐treated patient during ipriflavone administration. After withdrawal of ipriflavone, the serum theophylline level decreased to an extent similar to that found before administration of ipriflavone. The effects of ipriflavone and its major metabolites 7‐hydroxy‐isoflavone and 7‐(1‐carboxy‐ethoxy)‐isoflavone on cytochrome P450 activities were studied in vitro in human liver microsomes from three donors. Ipriflavone and 7‐hydroxy‐isoflavone competitively inhibited phenacetin O‐deethylase and tolbutamide hydroxylase activity. The parent compound and its dealkylated metabolite were strong inhibitors exhibiting Ki values around 10–20 μM, while 7‐(1‐carboxy‐ethoxy)‐isoflavone had no effect on the cytochrome P450 activities investigated. 7‐Hydroxy‐isoflavone is the only one that influenced nifedipine oxidase activity. It competitively inhibited this activity with a Ki value of 129.5 μM. The steady state concentrations of ipriflavone and 7‐hydroxy‐isoflavone in plasma of patients receiving 3×200 mg daily doses of ipriflavone for 48 weeks were found to be 0.33±0.32 μM and 1.44±0.77 μM, respectively. The results indicate that the decrease in theophylline metabolism observed in a patient treated with ipriflavone may be due to a competitive interaction of ipriflavone or its metabolite, 7‐hydroxy‐isoflavone with CYP1A2. On the other hand, our in vitro findings predict some more interaction with CYP2C9.

Canalicular and sinusoidal disposition of bilirubin mono- and diglucuronides in sandwich-cultured human and rat primary hepatocytes

Due to cholestasis or adverse drug effects, the excretion of bilirubin conjugates can decrease; therefore, the level of bilirubin (B) and bilirubin glucuronides (BGs) increases in the serum with the concomitant shift of bilirubin diversus monoglucuronide (BDG/BMG) equilibrium. The aim of this study was to utilize the collagen-sandwich culture of hepatocytes as an in vitro model for studying B conjugation and canalicular versus sinusoidal disposition of BGs. Canalicular and sinusoidal efflux of BMG and BDG obtained in sandwich-cultured rat primary hepatocytes was compared with that measured in human hepatocyte cultures. The BMG and BDG were separated by high-performance liquid chromatography and identified by mass spectrometry. The biliary excretion index (BEI) was estimated by measuring disposition of BGs into standard and Ca2+, Mg2+-free medium. Significantly more BGs were excreted into the canalicular networks than into the medium in 96-h sandwich culture of both human and rat hepatocytes (BEI, 62.5 and 80.6, respectively). The BDG/BMG ratio in the medium versus that in the canalicular networks was 0.55/1.48, which is similar to the serum/bile values (0.6/1.5) observed in vivo by Mesa et al. [Mesa VA, De Vos R, and Fevery J (1997) J Hepatol 27:912–916]. In contrast, the BEI for p-nitrophenol glucuronide was 5.2. The low BEI value is in agreement with empirical observations, which suggest that molecules with low molecular weight are preferably excreted by the kidney. In conclusion, sandwich-cultured primary hepatocytes provide a useful in vitro method to differentiate between sinusoidal and canalicular disposition of BGs. Since the normal BDG/BMG ratio changes in hyperbilirubinemia, this model could be used to predict drug effects leading to hyperbilirubinemia.

Metabolism of mesocarb in the rat

1. Rats treated orally with [14C]mesocarb (I; 3-(1-methyl-2-phenyl[2-(14C]ethyl)-N-(phenylaminocarbonyl)sydnone imine) (50 mg/kg) excrete 35% of the radioactivity in 24 h urine and 51% in 48 h urine. 2. Only traces of unchanged drug were found in urine. Hydroxy-mesocarb (II), dihydroxy-mesocarb (III), amphetamine (VII) and the conjugates of II and III account for 86% of the urinary radioactivity. 3. Cannulated male rats excrete about 40% of the radioactivity in 30 h in bile, mainly as conjugates of II and III.