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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

Schematic representation of potential immune cell participation in liver injury induced by hepatotoxic drugs (see Table 8 for details). Upon direct chemical-induced damage, only a small fraction of parenchymal cells (hepatocytes) are killed, releasing death-associated molecular patterns (DAMPs) such as CpG-rich DNA, which are detected by TLR9 expressed in LSEC, HSC and Kupffer cells. In turn, these cells release cytokines (e.g. TNFα, IL-1) which trigger the secretion of chemokines (e.g. Cxcl1) that recruit NK cells and neutrophils. These leukocytes infiltrate the parenchyma at the site of initial injury, where they further extend tissue damage by their cytotoxic arsenal (e.g. IFNγ, Fas-L in NK cells; hypochlorous acid, proteases in neutrophils). Afterward, circulating monocytes are recruited to the site of injury by chemokines (e.g. Cxcl2, RANTES, Mcp-1), where they become infiltrating macrophages (IM). These IM can resolve the cytotoxic immune milieu, by inducing apoptosis of infiltrating neutrophils and by actively removing cell debris. At the same time, HSC become activated and promote tissue repair by deposition of extracellular matrix (collagen-). If there is a single-acute injury, the inflammatory process will regress and the parenchyma will be reconstituted, mainly due to hepatocyte proliferation. However, if the damage is repeated chronically, activated HSC proliferate leading to fibrotic scarring, characterized by extensive collagen I deposition in the parenchyma
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Fig45: Schematic representation of potential immune cell participation in liver injury induced by hepatotoxic drugs (see Table 8 for details). Upon direct chemical-induced damage, only a small fraction of parenchymal cells (hepatocytes) are killed, releasing death-associated molecular patterns (DAMPs) such as CpG-rich DNA, which are detected by TLR9 expressed in LSEC, HSC and Kupffer cells. In turn, these cells release cytokines (e.g. TNFα, IL-1) which trigger the secretion of chemokines (e.g. Cxcl1) that recruit NK cells and neutrophils. These leukocytes infiltrate the parenchyma at the site of initial injury, where they further extend tissue damage by their cytotoxic arsenal (e.g. IFNγ, Fas-L in NK cells; hypochlorous acid, proteases in neutrophils). Afterward, circulating monocytes are recruited to the site of injury by chemokines (e.g. Cxcl2, RANTES, Mcp-1), where they become infiltrating macrophages (IM). These IM can resolve the cytotoxic immune milieu, by inducing apoptosis of infiltrating neutrophils and by actively removing cell debris. At the same time, HSC become activated and promote tissue repair by deposition of extracellular matrix (collagen-). If there is a single-acute injury, the inflammatory process will regress and the parenchyma will be reconstituted, mainly due to hepatocyte proliferation. However, if the damage is repeated chronically, activated HSC proliferate leading to fibrotic scarring, characterized by extensive collagen I deposition in the parenchyma

Mentions: The characteristics and transporter function of a number of NPCs and their contribution to hepatotoxicity are described in section “Non-parenchymal cells and their role in hepatotoxicity.” Here, the role of the immune response and NPCs in drug-induced hepatotoxicity is described. Even considering our understanding of this complex and dynamic process is only partial, some basic principles can clearly be recognized that call for a re-evaluation of in vitro systems aimed to reproduce aspects of liver toxicity in vivo. It is clear that a starting cue comes from chemically induced hepatocyte damage, which results in release of DAMPs that are detected by TLR in LSECs and HSCs. This induces the release of chemokines and cytokines which results in the recruitment of neutrophils and NK cells (Fig. 45). These leukocytes have the potential to exert cytolytic activity against hepatocytes. Neutrophils may release their chemical arsenal in the form of proteases and enzymes that generate ROS. Likewise, NK cells attack hepatocytes by their inherent cytotoxic activity via FasL and release of their granules containing perforin and granzyme (Fig. 45). Although the roles of neutrophils and NK cells have been well validated in several models of liver damage such as ischemia–reperfusion and biliary obstruction (Notas et al. 2009; Jaeschke et al. 2012b), their role in DILI remains controversial (Jaeschke et al. 2012b). This is largely due to off target effects in the experimental approaches such as pre-conditioning the liver by antibody-based neutrophil depletion (Liu et al. 2004; Ishida et al. 2006; Jaeschke and Liu 2007; Jaeschke et al. 2012a, b), or NK cell activation by DMSO used in acetaminophen solutions (Masson et al. 2008; Jaeschke et al. 2012b). Clearly, there is a need for more refined experimental strategies to accurately identify the role of these cells in hepatotoxicity. Current advances in the field of live tissue imaging using genetically labeled fluorescent cells allows direct visualization of immune cells for extensive time periods (McDonald et al. 2010). Such techniques in combination with knockout strains are beginning to shed light in this complex scenario (McDonald et al. 2010). Furthermore, even if neutrophils and NK cells were convincingly demonstrated not to contribute to acetaminophen toxicity, this does not exclude a potential role of the innate immune system in hepatotoxicity induced by other drugs and compounds.


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)

Schematic representation of potential immune cell participation in liver injury induced by hepatotoxic drugs (see Table 8 for details). Upon direct chemical-induced damage, only a small fraction of parenchymal cells (hepatocytes) are killed, releasing death-associated molecular patterns (DAMPs) such as CpG-rich DNA, which are detected by TLR9 expressed in LSEC, HSC and Kupffer cells. In turn, these cells release cytokines (e.g. TNFα, IL-1) which trigger the secretion of chemokines (e.g. Cxcl1) that recruit NK cells and neutrophils. These leukocytes infiltrate the parenchyma at the site of initial injury, where they further extend tissue damage by their cytotoxic arsenal (e.g. IFNγ, Fas-L in NK cells; hypochlorous acid, proteases in neutrophils). Afterward, circulating monocytes are recruited to the site of injury by chemokines (e.g. Cxcl2, RANTES, Mcp-1), where they become infiltrating macrophages (IM). These IM can resolve the cytotoxic immune milieu, by inducing apoptosis of infiltrating neutrophils and by actively removing cell debris. At the same time, HSC become activated and promote tissue repair by deposition of extracellular matrix (collagen-). If there is a single-acute injury, the inflammatory process will regress and the parenchyma will be reconstituted, mainly due to hepatocyte proliferation. However, if the damage is repeated chronically, activated HSC proliferate leading to fibrotic scarring, characterized by extensive collagen I deposition in the parenchyma
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig45: Schematic representation of potential immune cell participation in liver injury induced by hepatotoxic drugs (see Table 8 for details). Upon direct chemical-induced damage, only a small fraction of parenchymal cells (hepatocytes) are killed, releasing death-associated molecular patterns (DAMPs) such as CpG-rich DNA, which are detected by TLR9 expressed in LSEC, HSC and Kupffer cells. In turn, these cells release cytokines (e.g. TNFα, IL-1) which trigger the secretion of chemokines (e.g. Cxcl1) that recruit NK cells and neutrophils. These leukocytes infiltrate the parenchyma at the site of initial injury, where they further extend tissue damage by their cytotoxic arsenal (e.g. IFNγ, Fas-L in NK cells; hypochlorous acid, proteases in neutrophils). Afterward, circulating monocytes are recruited to the site of injury by chemokines (e.g. Cxcl2, RANTES, Mcp-1), where they become infiltrating macrophages (IM). These IM can resolve the cytotoxic immune milieu, by inducing apoptosis of infiltrating neutrophils and by actively removing cell debris. At the same time, HSC become activated and promote tissue repair by deposition of extracellular matrix (collagen-). If there is a single-acute injury, the inflammatory process will regress and the parenchyma will be reconstituted, mainly due to hepatocyte proliferation. However, if the damage is repeated chronically, activated HSC proliferate leading to fibrotic scarring, characterized by extensive collagen I deposition in the parenchyma
Mentions: The characteristics and transporter function of a number of NPCs and their contribution to hepatotoxicity are described in section “Non-parenchymal cells and their role in hepatotoxicity.” Here, the role of the immune response and NPCs in drug-induced hepatotoxicity is described. Even considering our understanding of this complex and dynamic process is only partial, some basic principles can clearly be recognized that call for a re-evaluation of in vitro systems aimed to reproduce aspects of liver toxicity in vivo. It is clear that a starting cue comes from chemically induced hepatocyte damage, which results in release of DAMPs that are detected by TLR in LSECs and HSCs. This induces the release of chemokines and cytokines which results in the recruitment of neutrophils and NK cells (Fig. 45). These leukocytes have the potential to exert cytolytic activity against hepatocytes. Neutrophils may release their chemical arsenal in the form of proteases and enzymes that generate ROS. Likewise, NK cells attack hepatocytes by their inherent cytotoxic activity via FasL and release of their granules containing perforin and granzyme (Fig. 45). Although the roles of neutrophils and NK cells have been well validated in several models of liver damage such as ischemia–reperfusion and biliary obstruction (Notas et al. 2009; Jaeschke et al. 2012b), their role in DILI remains controversial (Jaeschke et al. 2012b). This is largely due to off target effects in the experimental approaches such as pre-conditioning the liver by antibody-based neutrophil depletion (Liu et al. 2004; Ishida et al. 2006; Jaeschke and Liu 2007; Jaeschke et al. 2012a, b), or NK cell activation by DMSO used in acetaminophen solutions (Masson et al. 2008; Jaeschke et al. 2012b). Clearly, there is a need for more refined experimental strategies to accurately identify the role of these cells in hepatotoxicity. Current advances in the field of live tissue imaging using genetically labeled fluorescent cells allows direct visualization of immune cells for extensive time periods (McDonald et al. 2010). Such techniques in combination with knockout strains are beginning to shed light in this complex scenario (McDonald et al. 2010). Furthermore, even if neutrophils and NK cells were convincingly demonstrated not to contribute to acetaminophen toxicity, this does not exclude a potential role of the innate immune system in hepatotoxicity induced by other drugs and compounds.

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