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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

CP-724,714 was found to completely abolish the activity of bile salt efflux pump (BSEP) in sandwich-cultured primary human hepatocyte cultures compared to vehicle-treated controls. The nuclei were stained with Hoechst. The bile canaliculi were stained by cholyl lysyl fluorescein (CLF), a substrate for BSEP. The clinical development of CP-724,714, a small molecule Her2 inhibitor for oncology indications, was stopped in phase 2 due to jaundice and cholestatic liver damage (for further experimental details, refer to Xu et al. 2012)
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Fig47: CP-724,714 was found to completely abolish the activity of bile salt efflux pump (BSEP) in sandwich-cultured primary human hepatocyte cultures compared to vehicle-treated controls. The nuclei were stained with Hoechst. The bile canaliculi were stained by cholyl lysyl fluorescein (CLF), a substrate for BSEP. The clinical development of CP-724,714, a small molecule Her2 inhibitor for oncology indications, was stopped in phase 2 due to jaundice and cholestatic liver damage (for further experimental details, refer to Xu et al. 2012)

Mentions: Several improvements have been accomplished or envisioned by extending the phenotypic screening concept described above. These include measuring hepatobiliary transport processes, or toxicities requiring inflammatory conditions or extended period of drug exposure. To study the mechanism of inhibition of bile acid transport, a fluorescent bile acid (cholyl lysyl fluorescein or CLF) was employed in the screen. In normal healthy hepatocyte sandwich cultures, CLF is taken up by the hepatocytes and transported to the bile canaliculi space, as shown in the top-right panel in Fig. 47. At 3× clinical Cmax, CP-724,714 completely abolished this process. The inhibition of bile acid transport by CP-724,714 was more potent than erythromycin estolate or cyclosporine A, when compared to their clinical Cmax values (Fig. 47). The clinical development of CP-724,714, a small molecule targeting Her2 expressing tumors, was stopped in phase 2 due to jaundice and cholestatic liver damage. In another study, in order to mimic the effects of inflammatory conditions which may sensitize the liver cell for more drug-induced injury, a mixture of common inflammatory cytokines were added to hepatocyte cultures and their effects on DILI are studied (Cosgrove et al. 2009). The authors screened 90 drugs for cytokine effect in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19 %) synergized with cytokine addition than did the non-hepatotoxic drugs (3 %). Drug–cytokine synergy was observed for trovafloxacin but not levofloxacin, matching similar observation in a lipopolysaccharide-administered rodent model of hepatotoxicity (Shaw et al. 2007). Furthermore, it is now possible to culture and maintain differentiated liver functions up to 3 weeks (Wang et al. 2008b). These longer-term models made it possible to further investigate the mechanism of chronic DILI caused by drugs including nucleoside reverse transcriptase inhibitors (NRTIs) (Kline et al. 2009). It can be expected that with better understanding of mechanisms underlying DILI, further advances in hepatocyte culture systems and image-based or other high-content biomarker measurements, our predictivity toward idiosyncratic DILI can only improve.


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)

CP-724,714 was found to completely abolish the activity of bile salt efflux pump (BSEP) in sandwich-cultured primary human hepatocyte cultures compared to vehicle-treated controls. The nuclei were stained with Hoechst. The bile canaliculi were stained by cholyl lysyl fluorescein (CLF), a substrate for BSEP. The clinical development of CP-724,714, a small molecule Her2 inhibitor for oncology indications, was stopped in phase 2 due to jaundice and cholestatic liver damage (for further experimental details, refer to Xu et al. 2012)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig47: CP-724,714 was found to completely abolish the activity of bile salt efflux pump (BSEP) in sandwich-cultured primary human hepatocyte cultures compared to vehicle-treated controls. The nuclei were stained with Hoechst. The bile canaliculi were stained by cholyl lysyl fluorescein (CLF), a substrate for BSEP. The clinical development of CP-724,714, a small molecule Her2 inhibitor for oncology indications, was stopped in phase 2 due to jaundice and cholestatic liver damage (for further experimental details, refer to Xu et al. 2012)
Mentions: Several improvements have been accomplished or envisioned by extending the phenotypic screening concept described above. These include measuring hepatobiliary transport processes, or toxicities requiring inflammatory conditions or extended period of drug exposure. To study the mechanism of inhibition of bile acid transport, a fluorescent bile acid (cholyl lysyl fluorescein or CLF) was employed in the screen. In normal healthy hepatocyte sandwich cultures, CLF is taken up by the hepatocytes and transported to the bile canaliculi space, as shown in the top-right panel in Fig. 47. At 3× clinical Cmax, CP-724,714 completely abolished this process. The inhibition of bile acid transport by CP-724,714 was more potent than erythromycin estolate or cyclosporine A, when compared to their clinical Cmax values (Fig. 47). The clinical development of CP-724,714, a small molecule targeting Her2 expressing tumors, was stopped in phase 2 due to jaundice and cholestatic liver damage. In another study, in order to mimic the effects of inflammatory conditions which may sensitize the liver cell for more drug-induced injury, a mixture of common inflammatory cytokines were added to hepatocyte cultures and their effects on DILI are studied (Cosgrove et al. 2009). The authors screened 90 drugs for cytokine effect in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19 %) synergized with cytokine addition than did the non-hepatotoxic drugs (3 %). Drug–cytokine synergy was observed for trovafloxacin but not levofloxacin, matching similar observation in a lipopolysaccharide-administered rodent model of hepatotoxicity (Shaw et al. 2007). Furthermore, it is now possible to culture and maintain differentiated liver functions up to 3 weeks (Wang et al. 2008b). These longer-term models made it possible to further investigate the mechanism of chronic DILI caused by drugs including nucleoside reverse transcriptase inhibitors (NRTIs) (Kline et al. 2009). It can be expected that with better understanding of mechanisms underlying DILI, further advances in hepatocyte culture systems and image-based or other high-content biomarker measurements, our predictivity toward idiosyncratic DILI can only improve.

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