Lysiane Richert

Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes.

We evaluated the antioxidant status, namely cellular lipid peroxidation, by measuring thiobarbituric acid reactive substances (TBARS), cellular reduced glutathione (GSH) content, glutathione reductase (GSSG-R), glutathione transferase (GST), glutathione peroxidase (GSH-Px) and catalase activities in rat liver, hepatocytes immediately after isolation and in two-dimensional (2D) culture (on non-coated or collagen-coated dishes, as collagen-collagen or collagen-Matrigel sandwich cultures) or three-dimensional (3D) culture on Matrigel-coated dishes. Microsomal cytochrome P450 (CYP)- and UDP-glucuronosyl transferase (UGT)- dependent activities were also assessed in rat livers and hepatocyte cultures. The overall antioxidant status of rat hepatocytes immediately after isolation was not significantly different from that of rat livers. During culture, GSH was increased in 2D but not in 3D cultures in accordance with morphological observations; that is that matrix-cell interactions involving GSH, important in 2D, are minimal in 3D cultures. While UGT- and GST-dependent activities were equivalent in cultured hepatocytes and in rat livers, both catalase and GSH-Px activities decreased with time in all culture configurations. Constitutive CYP-dependent activities were drastically decreased in hepatocytes after isolation and attachment and did not recover in any culture configuration tested. Our results highlight that, although 2D sandwich cultures and 3D cultures on Matrigel allow longevity of rat hepatocyte cultures and optimal induction of CYPs, an imbalance in phase I/phase II detoxication processes in cultured rat hepatocytes occurs, whatever the culture configuration.

Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating

Isolated primary human hepatocytes are a well accepted system for evaluating pharmacological and toxicological effects in humans. However, questions remain regarding how culturing affects the liver-specific functions of the hepatocytes. In addition, cryopreservation could also potentially affect the differentiation state of the hepatocytes. The first aim of the present study was to compare gene expression in freshly isolated primary hepatocytes to that of the liver of origin and to evaluate the expression changes occurring after cryopreservation/thawing, both when maintained in suspension and after plating. The second aim of the present study was to evaluate gene expression in hepatocytes after cold storage of suspensions up to 24 h compared with freshly isolated hepatocytes in suspension. Our results show that the gene expression in freshly isolated human hepatocytes in suspension after isolation is similar to that of the liver of origin. Furthermore, gene expression in primary human hepatocytes in suspension is not affected by hepatocyte cold storage and cryopreservation. However, the gene expression is profoundly affected in monolayer cultures after plating. Specifically, gene expression changes were observed in cultured relative to suspensions of human hepatocytes that are involved in cellular processes such as phase I/II metabolism, basolateral and canalicular transport systems, fatty acid and lipid metabolism, apoptosis, and proteasomal protein recycling. An oxidative stress response may be partially involved in these changes in gene expression. Taken together, these results may aid in the interpretation of data collected from human hepatocyte experiments and suggest additional utility for cold storage and cryopreservation of hepatocytes.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Qualitative and Quantitative Assessment of Drug-Drug Interaction Potential in Man, Based on Ki, IC50 and Inhibitor Concentration

Strategies used to screen new drug entities as potential inhibitors of CYP450 enzymes are now widely used to select candidates in the drug discovery process. However, the information obtained based on IC50 values are usually more of qualitative nature. The aim of this study was to find out whether a more quantitative assessment of interaction potential could be achieved on the basis of the ratio I/Ki (I corresponds to inhibitor concentration). Ki values, in vivo data, namely plasma exposures under control condition vs in presence of inhibitors, were obtained from literature for 36 compounds. For a quantitative assessment, the following inhibitor concentrations were considered: I max and I in,max (respectively, maximum I in systemic circulation and in portal vein), I max,u and I in,max,u (respectively, maximum unbound I in systemic circulation and in portal vein). The predicted interaction was calculated as AUCinhibitor/AUCcontrol = 1 + I/Ki, where AUCcontrol and AUCinhibitor represent, respectively, the area under curve of the plasma concentration vs time profile under control conditions (ie without inhibitor) and with inhibitor. The use of I/Ki allowed a more quantitative estimation of the interaction potential. In this context, protein binding appeared to be a key parameter to be considered to avoid overestimation of DDI potential. Thus, 60% successful predictions could be achieved based on the ratio I max,u/Ki. Yet, some major deviations between in vivo DDI were obtained with this approach and the observations on the relevance of the inhibitor concentrations and the impact of binding need to be interpreted very cautiously in the absence of information on additional parameters such as fm and fh for example.

Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study

Abstract: Background: The European Center for Validation of Alternative Methods (ECVAM) has funded a prevalidation study in three laboratories (France, USA and UK) on the use of human hepatocyte cultures to predict cytochrome P‐450 induction.

Effects of Andrographis paniculata extract and Andrographolide on hepatic cytochrome P450 mRNA expression and monooxygenase activities after in vivo administration to rats and in vitro in rat and human hepatocyte cultures.

The expression of cytochrome P450 (CYP) is regulated by both endogenous factors and foreign compounds including drugs and natural compounds such as herbs. When herbs are co-administrated with a given drug in modern medicine it can lead to drug-herb interaction that can be clinically significant. The ability of Andrographis paniculata extract (APE) and Andrographolide (AND), the most medicinally active phytochemical in the extract, to modulate hepatic CYP expression was examined in vivo in rats and in vitro in rat and human hepatocyte cultures. After in vivo administration, APE at dose levels of 0.5 g/kg/day (i.e. 5 mg/kg/day AND equivalents) and at 2.5 g/kg/day (i.e. 25 mg/kg/day AND equivalents) and AND at dose levels of 5 and 25 mg/kg/day significantly decreased CYP2C11 activity. In primary cultures of rat and human hepatocytes, treatment with AND 50 microM and APE-containing 50 microM AND also resulted in significant decreases in CYP2C expression and activity. In addition, in human hepatocytes, treatment with APE and AND 50 microM resulted in a decrease in CYP3A expression and activity. In conclusion, this study suggests that AND and APE could cause herb-drug interactions in humans through modulation of CYP2C9 and CYP3A4 expression and activities.

Hepatocyte-based in vitro model for assessment of drug-induced cholestasis

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24-48h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation by hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI≤0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI≤0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling.

Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

Time course of cytochromes P450 decline during rat hepatocyte isolation and culture: effect of L-NAME.

The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte microsomal cytochrome (CYP)-dependent monooxygenase activities: CYP 1A1/2-, 2B1/2-, 3A1/2- and 2E1-dependent activities in microsomes from rat hepatocytes after isolation were about 60% of that of liver microsomes, and CYP 4A1-dependent activity was equivalent to liver microsomes. In contrast, the microsomal protein content of the various CYP isoforms was not affected by hepatocyte isolation. This is in accordance with the hypothesis of CYP inactivation during the process of hepatocyte isolation by collagenase digestion. L-NAME (1 mM) was found unable to protect from the decline of CYP-dependent monooxygenase activities following hepatocyte isolation. It is possible that the decrease in glutathione peroxidase activity observed in the presence of L-NAME, namely depression of defense against peroxynitrite, could counteract the beneficial effect of L-NAME on nitric oxide synthesis inhibition. The present work also shows that L-NAME could not avoid the progressive, isoform-specific, most probably turnover-related, decline of CYP proteins and related monooxygenase activities in cultured hepatocytes. Dysregulations in the mechanisms of CYP expression in rat hepatocyte cultures, presently unknown but nitric oxide independent, thus appear to occur in cultured rat hepatocytes.

Comparison of clearance predictions using primary cultures and suspensions of human hepatocytes

Various incubation conditions of human hepatocytes were compared for their accuracy in predicting the in vivo hepatic clearance (CLH) of model compounds. The test compounds were the highly cleared, low protein bound naloxone (in vivo CLH = 25 ml min−1 kg−1; free fraction = 0.6), the medium clearance, highly protein bound midazolam (CLH = 12 ml min−1 kg−1; free fraction = 0.04) and the low clearance, highly protein bound bosentan (CLH = 3.9 ml min−1 kg−1; free fraction = 0.02). Each compound was tested in three ‘hepatocyte systems’, using resections from three donors, in the presence and absence of human serum. Those hepatocyte systems were: conventional primary cultures, freshly isolated suspensions and cryopreserved suspended hepatocytes. Except for a twofold overestimated CLH for bosentan from conventional primary cultures, and despite variable cryopreservation recoveries, similar predictions of CLH were recorded with all hepatocyte systems. Moreover, the CLH values obtained with cryopreserved suspended hepatocytes were similar to those obtained with freshly isolated suspensions. For midazolam and bosentan, the predicted in vivo CLH was markedly higher in the presence of serum, whereas serum had little influence on the scaled-up CLH of naloxone. In vivo, CLH was properly approached for naloxone and bosentan (particularly from experiments in the presence of serum), but it was strongly underestimated for midazolam (particularly in the absence of serum). Additional compounds need to be investigated to confirm the above findings as well as to assess why the clearances of some highly protein-bound compounds are still considerably underestimated.