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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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CYP activities in HepG2 cells simultaneously transduced with a mix of adenoviruses encoding CYP1A2, CYP2C9 and CYP3A4. HepG2 cells were co-transfected with a mixture of adenoviral CYP constructs (6 moi CYP1A2 + 90 moi CYP2C9 + 66 moi CYP3A4). CYP1A2 (phenacetin O-deethylation), CYP2C9 (diclofenac 4′-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation) were determined 48 h later in the cells and compared to those in control HepG2 cells and human hepatocytes (HH) in primary culture. Activity values are expressed as pmol of the corresponding metabolite formed per minute and per mg of cell protein
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Fig32: CYP activities in HepG2 cells simultaneously transduced with a mix of adenoviruses encoding CYP1A2, CYP2C9 and CYP3A4. HepG2 cells were co-transfected with a mixture of adenoviral CYP constructs (6 moi CYP1A2 + 90 moi CYP2C9 + 66 moi CYP3A4). CYP1A2 (phenacetin O-deethylation), CYP2C9 (diclofenac 4′-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation) were determined 48 h later in the cells and compared to those in control HepG2 cells and human hepatocytes (HH) in primary culture. Activity values are expressed as pmol of the corresponding metabolite formed per minute and per mg of cell protein

Mentions: The adenovirus vector strategy has been used for introducing target genes into liver-derived cells with very high efficiencies (reviewed by Wickham (2000), and cell transfer is not dependent on active cell division. Recombinant adenoviruses rapidly and efficiently infect hepatic cell lines, and almost 100 % of the cells can express functional levels of the transgene after a short exposure to the virus (Castell et al. 1997). This technology has facilitated the generation of transient metabolically competent cells after transduction with recombinant-defective adenoviral vectors encoding for CYP genes (Castell et al. 1997; Bai and Cederbaum 2004; Naiki et al. 2004; Hosomi et al. 2011; Donato et al. 2010). A major characteristic of this approach is that functional levels of CYP are easily modulated as a function of the number of infecting virus particles (Fig. 31). Among several hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7 and Fa2N4 cells) efficiently infected with adenovirus vector harboring CYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and corresponding testosterone 6β-hydroxylase activity (Hosomi et al. 2011). The use of adenoviral strategy to confer CYP activities to HepG2 cells has been extensively reported (Vignati et al. 2005; Donato et al. 2010; Aoyama et al. 2009). This cell system has been proposed as a new in vitro tool for metabolism-mediated toxicity and clearance prediction of drugs metabolized by CYPs (Vignati et al. 2005; Donato et al. 2010). Recently, it has been shown that HepG2 cells can be co-transduced with multiple CYPs (Tolosa et al. 2011b). By selecting an appropriated mixture of recombinant CYP adenoviruses, transgene expression in HepG2 cells is comparable to that of human hepatocytes as shown by the enzymatic activity assessed with specific substrates (Fig. 32). Important advantageous properties of adenoviral vectors include: feasible production of high-titer vector preparations, high efficiency in transducing both quiescent and actively dividing cells, high levels of controllable gene expression and co-transduction with multiple CYPs which can reproduce the in vivo relative abundance of each enzyme and a lack of mutagenic properties associated with integrating vectors. However, a limitation of adenovirus transduction is that expression of the enzyme of interest is transient, and a new transfection is required for each experiment.Fig. 32


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)

CYP activities in HepG2 cells simultaneously transduced with a mix of adenoviruses encoding CYP1A2, CYP2C9 and CYP3A4. HepG2 cells were co-transfected with a mixture of adenoviral CYP constructs (6 moi CYP1A2 + 90 moi CYP2C9 + 66 moi CYP3A4). CYP1A2 (phenacetin O-deethylation), CYP2C9 (diclofenac 4′-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation) were determined 48 h later in the cells and compared to those in control HepG2 cells and human hepatocytes (HH) in primary culture. Activity values are expressed as pmol of the corresponding metabolite formed per minute and per mg of cell protein
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig32: CYP activities in HepG2 cells simultaneously transduced with a mix of adenoviruses encoding CYP1A2, CYP2C9 and CYP3A4. HepG2 cells were co-transfected with a mixture of adenoviral CYP constructs (6 moi CYP1A2 + 90 moi CYP2C9 + 66 moi CYP3A4). CYP1A2 (phenacetin O-deethylation), CYP2C9 (diclofenac 4′-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation) were determined 48 h later in the cells and compared to those in control HepG2 cells and human hepatocytes (HH) in primary culture. Activity values are expressed as pmol of the corresponding metabolite formed per minute and per mg of cell protein
Mentions: The adenovirus vector strategy has been used for introducing target genes into liver-derived cells with very high efficiencies (reviewed by Wickham (2000), and cell transfer is not dependent on active cell division. Recombinant adenoviruses rapidly and efficiently infect hepatic cell lines, and almost 100 % of the cells can express functional levels of the transgene after a short exposure to the virus (Castell et al. 1997). This technology has facilitated the generation of transient metabolically competent cells after transduction with recombinant-defective adenoviral vectors encoding for CYP genes (Castell et al. 1997; Bai and Cederbaum 2004; Naiki et al. 2004; Hosomi et al. 2011; Donato et al. 2010). A major characteristic of this approach is that functional levels of CYP are easily modulated as a function of the number of infecting virus particles (Fig. 31). Among several hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7 and Fa2N4 cells) efficiently infected with adenovirus vector harboring CYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and corresponding testosterone 6β-hydroxylase activity (Hosomi et al. 2011). The use of adenoviral strategy to confer CYP activities to HepG2 cells has been extensively reported (Vignati et al. 2005; Donato et al. 2010; Aoyama et al. 2009). This cell system has been proposed as a new in vitro tool for metabolism-mediated toxicity and clearance prediction of drugs metabolized by CYPs (Vignati et al. 2005; Donato et al. 2010). Recently, it has been shown that HepG2 cells can be co-transduced with multiple CYPs (Tolosa et al. 2011b). By selecting an appropriated mixture of recombinant CYP adenoviruses, transgene expression in HepG2 cells is comparable to that of human hepatocytes as shown by the enzymatic activity assessed with specific substrates (Fig. 32). Important advantageous properties of adenoviral vectors include: feasible production of high-titer vector preparations, high efficiency in transducing both quiescent and actively dividing cells, high levels of controllable gene expression and co-transduction with multiple CYPs which can reproduce the in vivo relative abundance of each enzyme and a lack of mutagenic properties associated with integrating vectors. However, a limitation of adenovirus transduction is that expression of the enzyme of interest is transient, and a new transfection is required for each experiment.Fig. 32

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