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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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Antiproteolytic effect of acetaldehyde in the single pass perfused rat liver. a Acetaldehyde (5 mmol/l) was infused into the perfused liver for 30 min, and the release of [3H]leucine from pre-labeled rats was taken as a measure of proteolysis. b The resulting inhibition of proteolysis under steady-state conditions at different concentrations of acetaldehyde. Data are from 2 to 4 different experiments and are given as mean ± SEM. From vom Dahl and Häussinger (1998)
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Fig9: Antiproteolytic effect of acetaldehyde in the single pass perfused rat liver. a Acetaldehyde (5 mmol/l) was infused into the perfused liver for 30 min, and the release of [3H]leucine from pre-labeled rats was taken as a measure of proteolysis. b The resulting inhibition of proteolysis under steady-state conditions at different concentrations of acetaldehyde. Data are from 2 to 4 different experiments and are given as mean ± SEM. From vom Dahl and Häussinger (1998)

Mentions: With a constant substrate supply via the portal vein (non-recirculating, single pass perfusion), the concentrations of respective products in the effluent perfusate can be measured. When the equilibrium between substrate concentration and product formation, i.e. the metabolic steady state is reached, the flux rate through a respective pathway can be calculated. Examples are the formation of urea from added NH4C1 or proteolysis inhibition by ethanol and acetaldehyde (Häussinger et al. 1986; Vom Dahl and Häussinger 1998). As shown in Fig. 9, acetaldehyde (5 mmol/l) inhibits proteolysis (measured as 3H-leucine release from in vivo pre-labeled liver proteins) by about 20 %. The effect is fully reversible after withdrawal of acetaldehyde, indicating that the effect is not due to an impaired viability of the perfused liver. Not only the flux through a whole pathway including transport steps across the plasma and mitochondrial membranes, but also the flux through a single enzymatic step can be measured by use of labeled products; for example, flux through pyruvate dehydrogenase may be determined as 14CO2 production from added 1-14C-pyruvate. By combining these techniques with the use of inhibitors, measurements of substrate disappearance and the formation of the various products, complex quantitative metabolic flux maps can be established with identification of sites of metabolic flux control. Recent studies suggest that the effluent perfusate of isolated perfused rat liver can be used to study the liver secretome, i.e. the proteins secreted by the various liver cell types.Fig. 9


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)

Antiproteolytic effect of acetaldehyde in the single pass perfused rat liver. a Acetaldehyde (5 mmol/l) was infused into the perfused liver for 30 min, and the release of [3H]leucine from pre-labeled rats was taken as a measure of proteolysis. b The resulting inhibition of proteolysis under steady-state conditions at different concentrations of acetaldehyde. Data are from 2 to 4 different experiments and are given as mean ± SEM. From vom Dahl and Häussinger (1998)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig9: Antiproteolytic effect of acetaldehyde in the single pass perfused rat liver. a Acetaldehyde (5 mmol/l) was infused into the perfused liver for 30 min, and the release of [3H]leucine from pre-labeled rats was taken as a measure of proteolysis. b The resulting inhibition of proteolysis under steady-state conditions at different concentrations of acetaldehyde. Data are from 2 to 4 different experiments and are given as mean ± SEM. From vom Dahl and Häussinger (1998)
Mentions: With a constant substrate supply via the portal vein (non-recirculating, single pass perfusion), the concentrations of respective products in the effluent perfusate can be measured. When the equilibrium between substrate concentration and product formation, i.e. the metabolic steady state is reached, the flux rate through a respective pathway can be calculated. Examples are the formation of urea from added NH4C1 or proteolysis inhibition by ethanol and acetaldehyde (Häussinger et al. 1986; Vom Dahl and Häussinger 1998). As shown in Fig. 9, acetaldehyde (5 mmol/l) inhibits proteolysis (measured as 3H-leucine release from in vivo pre-labeled liver proteins) by about 20 %. The effect is fully reversible after withdrawal of acetaldehyde, indicating that the effect is not due to an impaired viability of the perfused liver. Not only the flux through a whole pathway including transport steps across the plasma and mitochondrial membranes, but also the flux through a single enzymatic step can be measured by use of labeled products; for example, flux through pyruvate dehydrogenase may be determined as 14CO2 production from added 1-14C-pyruvate. By combining these techniques with the use of inhibitors, measurements of substrate disappearance and the formation of the various products, complex quantitative metabolic flux maps can be established with identification of sites of metabolic flux control. Recent studies suggest that the effluent perfusate of isolated perfused rat liver can be used to study the liver secretome, i.e. the proteins secreted by the various liver cell types.Fig. 9

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