Genomics and proteomics analysis of cultured primary rat hepatocytes

Beigel, Juergen; Fella, Kerstin; Kramer, Peter-Jürgen; Kroeger, Michaela; Hewitt, Philip

doi:10.1016/j.tiv.2007.06.019

Cited by 49 publications

(39 citation statements)

References 42 publications

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“…Nevertheless, their routine implementation in preclinical safety evaluation, particularly for long-term studies, is hampered by the progressive deterioration of liver-specific features (Elaut et al 2006; Vinken et al 2006a). Altered mRNA and protein levels of phase I and phase II biotransformation enzymes and drug transporters are well-known hallmarks of hepatocyte dedifferentiation (Baker et al 2001; Elaut et al 2006; Beigel et al 2008; Rowe et al 2010). In fact, the entire physiological repertoire in hepatocytes collapses as a function of culture time.…”

Section: Hepatocyte Dedifferentiationmentioning

confidence: 99%

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

Hewitt²,

et al. 2013

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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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

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Section: Hepatocyte Dedifferentiationmentioning

confidence: 99%

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

Hewitt²,

et al. 2013

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“…Cultured cells are convenient to work with and facilitate the investigation of toxic mechanisms at a level of detail often not possible in vivo . However, the expression and activity of drug metabolizing enzymes in cultured rodent hepatocytes decrease significantly beyond 24 h (172–173) and although expression of some cytochromes P450 is maintained longer in human hepatocytes, eventually these cells also lose their drug-metabolizing capabilities (174). In addition, variation in drug metabolism across donors and the difficulty in obtaining tissue can make the use of primary human hepatocytes difficult or impractical.…”

Section: Considerations For Cell Culture Experimentsmentioning

confidence: 99%

Metabolism and Disposition of Acetaminophen: Recent Advances in Relation to Hepatotoxicity and Diagnosis

2013

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Acetaminophen (APAP) is one of the most widely used drugs. Though safe at therapeutic doses, overdose causes mitochondrial dysfunction and centrilobular necrosis in the liver. The first studies of APAP metabolism and activation were published more than forty years ago. Most of the drug is eliminated by glucuronidation and sulfation. These reactions are catalyzed by UDP-glucuronosyltransferases (UGT1A1 and 1A6) and sulfotransferases (SULT1A1, 1A3/4, and 1E1), respectively. However, some is converted by CYP2E1 and other cytochrome P450 enzymes to a reactive intermediate that can bind to sulfhydryl groups. The metabolite can deplete liver glutathione (GSH) and modify cellular proteins. GSH binding occurs spontaneously, but may also involve GSH-S-transferases. Protein binding leads to oxidative stress and mitochondrial damage. The glucuronide, sulfate, and GSH conjugates are excreted by transporters in the canalicular (Mrp2 and Bcrp) and basolateral (Mrp3 and Mrp4) hepatocyte membranes. Conditions that interfere with metabolism and metabolic activation can alter the hepatotoxicity of the drug. Recent data providing novel insights into these processes, particularly in humans, are reviewed in the context of earlier work, and the effects of altered metabolism and reactive metabolite formation are discussed. Recent advances in the diagnostic use of serum adducts are covered.

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“…Hepatocyte culture models demonstrating physiologically localized efflux transporters in canalicular surfaces require the cells to be maintained in culture for at least 4 -7 days (48). This is confounded by the well-documented dedifferentiation and loss of hepatocyte-specific function and metabolic enzyme activity that occurs over days in nonflow-based culture models (1,2,4,10). As a result, it is rare to find in vitro hepatocyte systems that sustain in vivo morphological and physiological features simultaneously and stably over a prolonged period of time.…”

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Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro

Dash

Simmers

Deering

et al. 2013

American Journal of Physiology-Cell Physiology

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Dash A, Simmers MB, Deering TG, Berry DJ, Feaver RE, Hastings NE, Pruett TL, LeCluyse EL, Blackman BR, Wamhoff BR. Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro. Am J Physiol Cell Physiol 304: C1053-C1063, 2013. First published March 13. 2013 doi:10.1152/ajpcell.00331.2012.-In vitro primary hepatocyte systems typically elicit drug induction and toxicity responses at concentrations much higher than corresponding in vivo or clinical plasma Cmax levels, contributing to poor in vitro-in vivo correlations. This may be partly due to the absence of physiological parameters that maintain metabolic phenotype in vivo. We hypothesized that restoring hemodynamics and media transport would improve hepatocyte architecture and metabolic function in vitro compared with nonflow cultures. Rat hepatocytes were cultured for 2 wk either in nonflow collagen gel sandwiches with 48-h media changes or under controlled hemodynamics mimicking sinusoidal circulation within a perfused Transwell device. Phenotypic, functional, and metabolic parameters were assessed at multiple times. Hepatocytes in the devices exhibited polarized morphology, retention of differentiation markers [E-cadherin and hepatocyte nuclear factor-4␣ (HNF-4␣)], the canalicular transporter [multidrug-resistant protein-2 (Mrp-2)], and significantly higher levels of liver function compared with nonflow cultures over 2 wk (albumin ϳ4-fold and urea ϳ5-fold). Gene expression of cytochrome P450 (CYP) enzymes was significantly higher (fold increase over nonflow: CYP1A1: 53.5 Ϯ 10.3; CYP1A2: 64.0 Ϯ 15.1; CYP2B1: 15.2 Ϯ 2.9; CYP2B2: 2.7 Ϯ 0.8; CYP3A2: 4.0 Ϯ 1.4) and translated to significantly higher basal enzyme activity (device vs. nonflow: CYP1A: 6.26 Ϯ 2.41 vs. 0.42 Ϯ 0.015; CYP1B: 3.47 Ϯ 1.66 vs. 0.4 Ϯ 0.09; CYP3A: 11.65 Ϯ 4.70 vs. 2.43 Ϯ 0.56) while retaining inducibility by 3-methylcholanthrene and dexamethasone (fold increase over DMSO: CYP1A ϭ 27.33 and CYP3A ϭ 4.94). These responses were observed at concentrations closer to plasma levels documented in vivo in rats. The retention of in vivo-like hepatocyte phenotype and metabolic function coupled with drug response at more physiological concentrations emphasizes the importance of restoring in vivo physiological transport parameters in vitro.hemodynamics; hepatocyte; metabolism; organotype; phenotype HEPATOTOXICITY AND BIOAVAILABILITY issues comprise over 60% of drug failures during clinical trials (45) and are a major cause of postmarketing withdrawal (23), pointing to the need to develop more efficient and predictive preclinical test systems. Simple cellular and subcellular assays used to screen compound libraries offer the advantage of higher throughput but are often unable to capture complex biological effects that may require a physiological context for drug interactions with cells. Primary in vitro hepatocyte models widely used to study liver disease, drug metabolism, and toxicity are extensively reviewed in the literature (16,42). The ability to test the metabolic f...

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Genomics and proteomics analysis of cultured primary rat hepatocytes

Cited by 49 publications

References 42 publications

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

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

Metabolism and Disposition of Acetaminophen: Recent Advances in Relation to Hepatotoxicity and Diagnosis

Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro

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