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Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gómez-Lechón MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG - Arch. Toxicol. (2013)

Bottom Line: When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes.One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation.Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139, Dortmund, Germany.

ABSTRACT
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

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Related in: MedlinePlus

Wnt/β-catenin signaling and its role in drug metabolism. In the absence of agonistic Wnt signals, cytosolic β-catenin is phosphorylated by a cytosolic multi-protein complex and subsequently degraded in the proteasome. Wnt binding to FZD receptors impairs β-catenin degradation. As a consequence, the protein accumulates and translocates to the nucleus. Nuclear β-catenin interacts with TCF transcription factors and induces target gene transcription, e.g. nuclear receptors and certain drug-metabolizing enzymes. Cooperative interplay of β-catenin and nuclear receptors also contributes to the induction of drug-metabolizing enzymes, especially from the CYP and GST families
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Fig6: Wnt/β-catenin signaling and its role in drug metabolism. In the absence of agonistic Wnt signals, cytosolic β-catenin is phosphorylated by a cytosolic multi-protein complex and subsequently degraded in the proteasome. Wnt binding to FZD receptors impairs β-catenin degradation. As a consequence, the protein accumulates and translocates to the nucleus. Nuclear β-catenin interacts with TCF transcription factors and induces target gene transcription, e.g. nuclear receptors and certain drug-metabolizing enzymes. Cooperative interplay of β-catenin and nuclear receptors also contributes to the induction of drug-metabolizing enzymes, especially from the CYP and GST families

Mentions: When studying pre-neoplastic liver foci in the rat, it was noted that the expression of a number of drug-metabolizing enzymes was altered in the pre-tumorous cell populations. In particular, a decrease in the expression of different CYP isoforms was observed (Schwarz et al. 1989; Buchmann et al. 1987). However, treatment with phenobarbital, a tumor promoter and CAR agonist was able to induce transiently the expression of CAR target CYPs in these liver foci (Schwarz et al. 1989). Similarly, expression of different drug-metabolizing enzymes, especially CYP isoforms, was observed in murine liver tumors from phenobarbital-treated animals alongside a very low expression of these enzymes in tumors from animals which had not received phenobarbital (Loeppen et al. 2005). In principle, this phenomenon might be caused by the presence of phenobarbital and its acute action on hepatoma cells; however, comparative analyses of the promoted and non-promoted tumors revealed that both tumor populations are characterized by different underlying genetic alterations: in mice injected with the initiator, N-nitroso-diethylamine, at 6 weeks of age without subsequent tumor promotion by phenobarbital, the vast majority of resulting tumors harbored activating mutations in either the Ha-ras or the B-raf proto-oncogene, leading to a constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway (Jaworski et al. 2005). By contrast, tumor promotion by phenobarbital or the phenobarbital-like polychlorinated biphenyl, PCB153, specifically selects for the outgrowth of liver adenomas with activating mutations in Ctnnb1 (Aydinlik et al. 2001; Strathmann et al. 2006), which encodes the transcription factor β-catenin, a crucial player in the canonical Wnt signaling pathway. Detailed information about Wnt signaling and its functions can be found in Barker and Clevers (2000), Huelsken and Behrens (2002), Lustig and Behrens (2003), Polakis (2000), Willert and Nusse (1998) (see also Fig. 6). Constitutive activation of β-catenin signaling is frequently detected in tumors, often caused by activating mutations in Ctnnb1, which lead to a loss of one or more of the N-terminal phosphorylation sites, or by inactivating mutations in Apc, a gene encoding a crucial component of the cytosolic β-catenin destruction complex.Fig. 6


Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gómez-Lechón MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG - Arch. Toxicol. (2013)

Wnt/β-catenin signaling and its role in drug metabolism. In the absence of agonistic Wnt signals, cytosolic β-catenin is phosphorylated by a cytosolic multi-protein complex and subsequently degraded in the proteasome. Wnt binding to FZD receptors impairs β-catenin degradation. As a consequence, the protein accumulates and translocates to the nucleus. Nuclear β-catenin interacts with TCF transcription factors and induces target gene transcription, e.g. nuclear receptors and certain drug-metabolizing enzymes. Cooperative interplay of β-catenin and nuclear receptors also contributes to the induction of drug-metabolizing enzymes, especially from the CYP and GST families
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3753504&req=5

Fig6: Wnt/β-catenin signaling and its role in drug metabolism. In the absence of agonistic Wnt signals, cytosolic β-catenin is phosphorylated by a cytosolic multi-protein complex and subsequently degraded in the proteasome. Wnt binding to FZD receptors impairs β-catenin degradation. As a consequence, the protein accumulates and translocates to the nucleus. Nuclear β-catenin interacts with TCF transcription factors and induces target gene transcription, e.g. nuclear receptors and certain drug-metabolizing enzymes. Cooperative interplay of β-catenin and nuclear receptors also contributes to the induction of drug-metabolizing enzymes, especially from the CYP and GST families
Mentions: When studying pre-neoplastic liver foci in the rat, it was noted that the expression of a number of drug-metabolizing enzymes was altered in the pre-tumorous cell populations. In particular, a decrease in the expression of different CYP isoforms was observed (Schwarz et al. 1989; Buchmann et al. 1987). However, treatment with phenobarbital, a tumor promoter and CAR agonist was able to induce transiently the expression of CAR target CYPs in these liver foci (Schwarz et al. 1989). Similarly, expression of different drug-metabolizing enzymes, especially CYP isoforms, was observed in murine liver tumors from phenobarbital-treated animals alongside a very low expression of these enzymes in tumors from animals which had not received phenobarbital (Loeppen et al. 2005). In principle, this phenomenon might be caused by the presence of phenobarbital and its acute action on hepatoma cells; however, comparative analyses of the promoted and non-promoted tumors revealed that both tumor populations are characterized by different underlying genetic alterations: in mice injected with the initiator, N-nitroso-diethylamine, at 6 weeks of age without subsequent tumor promotion by phenobarbital, the vast majority of resulting tumors harbored activating mutations in either the Ha-ras or the B-raf proto-oncogene, leading to a constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway (Jaworski et al. 2005). By contrast, tumor promotion by phenobarbital or the phenobarbital-like polychlorinated biphenyl, PCB153, specifically selects for the outgrowth of liver adenomas with activating mutations in Ctnnb1 (Aydinlik et al. 2001; Strathmann et al. 2006), which encodes the transcription factor β-catenin, a crucial player in the canonical Wnt signaling pathway. Detailed information about Wnt signaling and its functions can be found in Barker and Clevers (2000), Huelsken and Behrens (2002), Lustig and Behrens (2003), Polakis (2000), Willert and Nusse (1998) (see also Fig. 6). Constitutive activation of β-catenin signaling is frequently detected in tumors, often caused by activating mutations in Ctnnb1, which lead to a loss of one or more of the N-terminal phosphorylation sites, or by inactivating mutations in Apc, a gene encoding a crucial component of the cytosolic β-catenin destruction complex.Fig. 6

Bottom Line: When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes.One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation.Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139, Dortmund, Germany.

ABSTRACT
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Show MeSH
Related in: MedlinePlus