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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

Organization of the liver lobule and acinus. Based on the local blood composition, the acinus is roughly divided into three zones, 1 periportal, 2 transitional and 3 perivenous. The periportal zone is close to the portal triad vasculature and supplied by highly oxygenated blood (O2 partial pressure 60–70 mmHg). The perivenous zone is proximal to the central vein and receives poorly oxygenated blood (O2 partial pressure 25–35 mmHg). If no specific zonal mechanisms are active (such as pericentral metabolic activation of many hepatotoxic compounds, because many CYP enzymes are preferentially expressed in the center of the liver lobules), toxicity becomes visible at first in the periportal region, as this is the first zone to filter blood (Allen and Bhatia 2003). Adapted from Bacon et al. (2006)
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Fig2: Organization of the liver lobule and acinus. Based on the local blood composition, the acinus is roughly divided into three zones, 1 periportal, 2 transitional and 3 perivenous. The periportal zone is close to the portal triad vasculature and supplied by highly oxygenated blood (O2 partial pressure 60–70 mmHg). The perivenous zone is proximal to the central vein and receives poorly oxygenated blood (O2 partial pressure 25–35 mmHg). If no specific zonal mechanisms are active (such as pericentral metabolic activation of many hepatotoxic compounds, because many CYP enzymes are preferentially expressed in the center of the liver lobules), toxicity becomes visible at first in the periportal region, as this is the first zone to filter blood (Allen and Bhatia 2003). Adapted from Bacon et al. (2006)

Mentions: The cellular composition of the liver is summarized in Fig. 1a, b and Table S1 (see 10.1007/s00204-013-1078-5). The liver lobule is the histologically well-defined structural unit of the liver (Fig. 2). A lobule has a hexagonal shape, a diameter of approximately 1 mm and the thickness is about 2 mm. In adults, the lobule consists of hepatocyte plates (aka cords), which radiate from a central vein in the center of the hexagon. Adjacent hepatocytes are joined by tight junctions. The tight junctions delimit the bile canaliculi with a diameter of about 1 μm. The hepatocytes in a plate are exposed on both sides to capillaries (sinusoids). The human liver contains about one million lobules. At each vertex of the hexagonal, lobule is a portal triad. A portal triad comprises an artery, a vein and a bile duct bundled by connective tissue. Liver metabolism, oxygenation and extracellular matrix (ECM) distribution are best understood by assuming that the portal triad is the center of symmetry instead of the central vein. Then, the hepatic acinus becomes the smallest liver functional unit and is defined as the population of hepatocytes supplied by one portal triad, i.e. a microcirculatory functional unit. The acinus extends over a roughly elliptical region comprising the hepatocytes from two adjacent lobules. The short axis of the ellipse is the line connecting two portal triads, the long axis connects two central veins (Fig. 2). The length of the long axis is approximately 1 mm. In the acinus area, the hepatocytes are exposed to a spatial biochemical gradient that influences metabolism and gene expression. The gradient is established by the changes in plasma composition and oxygenation occurring downstream to the blood flow in the space between the periportal and the perivenous areas (Jungermann and Kietzmann 1996, 1997; Kietzmann and Jungermann 1997). The blood from the portal vein supplies 80 % of the liver’s blood and contains nutrients that are absorbed from the digestive tract. From the periportal to the perivenous zone, the oxygen concentration drops from about 13 % v/v (equivalent to partial pressure of 60–65 mmHg and to a free concentration of 84–91 μmol/l, periportal) to 9 % v/v (mixed periportal) and finally to 4 % v/v (equivalent to a partial pressure of 30–35 mmHg and to a free concentration of 42–49 μmol/l, perivenous) (Allen and Bhatia 2003; Kietzmann et al. 2006). The oxygen gradient in the acinus regulates the spatial expression of genes encoding carbohydrate-metabolizing enzymes, including pyruvate carboxykinase 1 (mostly expressed in the periportal region), glucokinase and liver pyruvate kinase (both mostly expressed in the perivenous region), through oxygen-responsive transcription factors, such as NRE and HIFs (Kietzmann et al. 2006). This is the so-called metabolic zonation of the liver.Fig. 1


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)

Organization of the liver lobule and acinus. Based on the local blood composition, the acinus is roughly divided into three zones, 1 periportal, 2 transitional and 3 perivenous. The periportal zone is close to the portal triad vasculature and supplied by highly oxygenated blood (O2 partial pressure 60–70 mmHg). The perivenous zone is proximal to the central vein and receives poorly oxygenated blood (O2 partial pressure 25–35 mmHg). If no specific zonal mechanisms are active (such as pericentral metabolic activation of many hepatotoxic compounds, because many CYP enzymes are preferentially expressed in the center of the liver lobules), toxicity becomes visible at first in the periportal region, as this is the first zone to filter blood (Allen and Bhatia 2003). Adapted from Bacon et al. (2006)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Organization of the liver lobule and acinus. Based on the local blood composition, the acinus is roughly divided into three zones, 1 periportal, 2 transitional and 3 perivenous. The periportal zone is close to the portal triad vasculature and supplied by highly oxygenated blood (O2 partial pressure 60–70 mmHg). The perivenous zone is proximal to the central vein and receives poorly oxygenated blood (O2 partial pressure 25–35 mmHg). If no specific zonal mechanisms are active (such as pericentral metabolic activation of many hepatotoxic compounds, because many CYP enzymes are preferentially expressed in the center of the liver lobules), toxicity becomes visible at first in the periportal region, as this is the first zone to filter blood (Allen and Bhatia 2003). Adapted from Bacon et al. (2006)
Mentions: The cellular composition of the liver is summarized in Fig. 1a, b and Table S1 (see 10.1007/s00204-013-1078-5). The liver lobule is the histologically well-defined structural unit of the liver (Fig. 2). A lobule has a hexagonal shape, a diameter of approximately 1 mm and the thickness is about 2 mm. In adults, the lobule consists of hepatocyte plates (aka cords), which radiate from a central vein in the center of the hexagon. Adjacent hepatocytes are joined by tight junctions. The tight junctions delimit the bile canaliculi with a diameter of about 1 μm. The hepatocytes in a plate are exposed on both sides to capillaries (sinusoids). The human liver contains about one million lobules. At each vertex of the hexagonal, lobule is a portal triad. A portal triad comprises an artery, a vein and a bile duct bundled by connective tissue. Liver metabolism, oxygenation and extracellular matrix (ECM) distribution are best understood by assuming that the portal triad is the center of symmetry instead of the central vein. Then, the hepatic acinus becomes the smallest liver functional unit and is defined as the population of hepatocytes supplied by one portal triad, i.e. a microcirculatory functional unit. The acinus extends over a roughly elliptical region comprising the hepatocytes from two adjacent lobules. The short axis of the ellipse is the line connecting two portal triads, the long axis connects two central veins (Fig. 2). The length of the long axis is approximately 1 mm. In the acinus area, the hepatocytes are exposed to a spatial biochemical gradient that influences metabolism and gene expression. The gradient is established by the changes in plasma composition and oxygenation occurring downstream to the blood flow in the space between the periportal and the perivenous areas (Jungermann and Kietzmann 1996, 1997; Kietzmann and Jungermann 1997). The blood from the portal vein supplies 80 % of the liver’s blood and contains nutrients that are absorbed from the digestive tract. From the periportal to the perivenous zone, the oxygen concentration drops from about 13 % v/v (equivalent to partial pressure of 60–65 mmHg and to a free concentration of 84–91 μmol/l, periportal) to 9 % v/v (mixed periportal) and finally to 4 % v/v (equivalent to a partial pressure of 30–35 mmHg and to a free concentration of 42–49 μmol/l, perivenous) (Allen and Bhatia 2003; Kietzmann et al. 2006). The oxygen gradient in the acinus regulates the spatial expression of genes encoding carbohydrate-metabolizing enzymes, including pyruvate carboxykinase 1 (mostly expressed in the periportal region), glucokinase and liver pyruvate kinase (both mostly expressed in the perivenous region), through oxygen-responsive transcription factors, such as NRE and HIFs (Kietzmann et al. 2006). This is the so-called metabolic zonation of the liver.Fig. 1

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