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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

Aflatoxin B1-induced toxicity in upgraded HepG2 cells. Metabolically competent HepG2 cells (prepared by co-transduction with CYP1A2, CYP2C9 and CYP3A4 adenoviruses) or control HepG2 cells were treated for 24 h with increasing concentrations of Aflatoxin B1. Cell viability (a), nuclear changes indicative of apoptotic death (b), mitochondrial membrane potential (MMP) (c) and intracellular calcium concentration (d) were compared in both cell systems (*p < 0.01). Results are expressed as percentage of untreated cells
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Fig33: Aflatoxin B1-induced toxicity in upgraded HepG2 cells. Metabolically competent HepG2 cells (prepared by co-transduction with CYP1A2, CYP2C9 and CYP3A4 adenoviruses) or control HepG2 cells were treated for 24 h with increasing concentrations of Aflatoxin B1. Cell viability (a), nuclear changes indicative of apoptotic death (b), mitochondrial membrane potential (MMP) (c) and intracellular calcium concentration (d) were compared in both cell systems (*p < 0.01). Results are expressed as percentage of untreated cells

Mentions: As indicated above, simultaneous transfection with a mixture of recombinant CYP adenoviruses results in HepG2 cells with a metabolic competence similar to that of human hepatocytes (Fig. 32). Recently, the procedure of adenovirus coinfection was optimized to cells cultured in 96-well formats with such degree of efficiency and accuracy that the system is amenable to use in high-throughput hepatotoxicity screenings (Tolosa et al. 2011b). As an example, aflatoxin B1, which is bioactivated by several CYPs, was more toxic to upgraded HepG2 cells transiently expressing functional levels of CYP1A2, CYP2C9 and CYP3A4 than to non-metabolically competent HepG2 cells (Fig. 33). By contrast, no appreciable differences in cytotoxicity parameters (cell viability, mitochondrial membrane potential, intracellular calcium concentration and nuclear changes) of non-bioactivated compounds were found between both cell systems.


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)

Aflatoxin B1-induced toxicity in upgraded HepG2 cells. Metabolically competent HepG2 cells (prepared by co-transduction with CYP1A2, CYP2C9 and CYP3A4 adenoviruses) or control HepG2 cells were treated for 24 h with increasing concentrations of Aflatoxin B1. Cell viability (a), nuclear changes indicative of apoptotic death (b), mitochondrial membrane potential (MMP) (c) and intracellular calcium concentration (d) were compared in both cell systems (*p < 0.01). Results are expressed as percentage of untreated cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig33: Aflatoxin B1-induced toxicity in upgraded HepG2 cells. Metabolically competent HepG2 cells (prepared by co-transduction with CYP1A2, CYP2C9 and CYP3A4 adenoviruses) or control HepG2 cells were treated for 24 h with increasing concentrations of Aflatoxin B1. Cell viability (a), nuclear changes indicative of apoptotic death (b), mitochondrial membrane potential (MMP) (c) and intracellular calcium concentration (d) were compared in both cell systems (*p < 0.01). Results are expressed as percentage of untreated cells
Mentions: As indicated above, simultaneous transfection with a mixture of recombinant CYP adenoviruses results in HepG2 cells with a metabolic competence similar to that of human hepatocytes (Fig. 32). Recently, the procedure of adenovirus coinfection was optimized to cells cultured in 96-well formats with such degree of efficiency and accuracy that the system is amenable to use in high-throughput hepatotoxicity screenings (Tolosa et al. 2011b). As an example, aflatoxin B1, which is bioactivated by several CYPs, was more toxic to upgraded HepG2 cells transiently expressing functional levels of CYP1A2, CYP2C9 and CYP3A4 than to non-metabolically competent HepG2 cells (Fig. 33). By contrast, no appreciable differences in cytotoxicity parameters (cell viability, mitochondrial membrane potential, intracellular calcium concentration and nuclear changes) of non-bioactivated compounds were found between both cell systems.

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