Rat hepatocyte cultures and co-cultures in biotransformation studies of xenobiotics

Rogiers, Vera; Vercruysse, Antoine

doi:10.1016/0300-483x(93)02611-j

Cited by 80 publications

(39 citation statements)

References 59 publications

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“…However, in rat hepatocytes, phase II enzymes in general showed a decrease in gene expression level after 72 h compared with gene expression in liver (Kienhuis et al, 2007). For rat hepatocytes, it is known that the phase II enzymes are better preserved in culture than phase I enzymes (Rogiers and Vercruysse, 1993;Kern et al, 1997). Our data confirm these findings, thus showing that mouse hepatocytes are also stable in expression of genes encoding phase II enzymes.…”

Section: Discussionsupporting

confidence: 84%

“…Even within the short period of 24 h after isolation, the expression levels of many P450s decreased tremendously in rat hepatocytes (Boess et al, 2003), whereas in our study gene expression levels showed a slower decrease over time, indicating that mouse hepatocytes remain more stable with respect to P450 gene expression compared with rat hepatocytes but not with human hepatocytes. Because several rat studies suggest that phase II enzyme activities are better preserved in culture than phase I enzyme activities, we decided to only measure enzyme activities of the most important phase I enzymes involved in xenobiotic biotransformation to strengthen our gene expression results (Rogiers and Vercruysse, 1993;Kern et al, 1997). These results show a general decrease in P450 enzyme activity, with only one exception.…”

Section: Discussionmentioning

confidence: 99%

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Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes

Mathijs

Kienhuis²,

Brauers³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, whereas transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles. Primary mouse hepatocytes, isolated using two-step collagenase perfusion, were cultured in a collagen sandwich configuration. Gene expression profiles and the activities of various cytochrome P450 (P450) enzymes were determined after 0, 42, and 90 h in culture. Principal component analysis of gene expression profiles shows that replicates per time point are similar. Gene expression levels of most phase I biotransformation enzymes decrease to approximately 69 and 57% of the original levels at 42 and 90 h, respectively, whereas enzyme activities for most of the studied P450s decrease to 59 and 34%. The decrease for phase II gene expression is only to 96 and 92% of the original levels at 42 and 90 h, respectively. Pathway analysis reveals initial effects at the level of proteins, external signaling pathways, and energy production. Later effects are observed for transcription, translation, membranes, and cell cycle-related gene sets. These results indicate that the sandwich-cultured primary mouse hepatocyte system is robust and seems to maintain its metabolic competence better than that of the rat hepatocyte system.

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes

Mathijs

Kienhuis²,

Brauers³

et al. 2009

Drug Metab Dispos

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“…HSCs are recognized key effectors in the development and progression of hepatic fibrosis (Puche et al, 2013) however, they also help to define the molecular and structural microenvironment of the parenchymal compartment and space of Disse via the production of soluble and insoluble cues, including growth factors, inflammatory cytokines, and deposition of extracellular matrix (ECM) (Friedman, 2008). These microenvironments mediate requisite gene expression patterns for metabolic homeostasis, cellular differentiation, and maturation (Guillouzo et al, 1993;Rogiers and Vercruysse, 1993) and thus modulate the liver's response to both acute and chronic injury. These observations suggest that current in vitro models used to evaluate potential fibrogenic agents lack fundamental cellular components that may moderate or exacerbate hepatocellular injury, an event strongly associated with the initiation of fibrogenesis (Canbay et al, 2004).…”

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confidence: 99%

Editor’s Highlight: Modeling Compound-Induced FibrogenesisIn VitroUsing Three-Dimensional Bioprinted Human Liver Tissues

et al. 2016

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Compound-induced liver injury leading to fibrosis remains a challenge for the development of an Adverse Outcome Pathway useful for human risk assessment. Latency to detection and lack of early, systematically detectable biomarkers make it difficult to characterize the dynamic and complex intercellular interactions that occur during progressive liver injury. Here, we demonstrate the utility of bioprinted tissue constructs comprising primary hepatocytes, hepatic stellate cells, and endothelial cells to model methotrexate-and thioacetamide-induced liver injury leading to fibrosis. Repeated, low-concentration exposure to these compounds enabled the detection and differentiation of multiple modes of liver injury, including hepatocellular damage, and progressive fibrogenesis characterized by the deposition and accumulation of fibrillar collagens in patterns analogous to those described in clinical samples obtained from patients with fibrotic liver injury. Transient cytokine production and upregulation of fibrosis-associated genes ACTA2 and COL1A1 mimics hallmark features of a classic wound-healing response. A surge in proinflammatory cytokines (eg, IL-8, IL-1b) during the early culture time period is followed by concentration-and treatment-dependent alterations in immunomodulatory and chemotactic cytokines such as IL-13, IL-6, and MCP-1. These combined data provide strong proof-of-concept that 3D bioprinted liver tissues can recapitulate drug-, chemical-, and TGF-b1-induced fibrogenesis at the cellular, molecular, and histological levels and underscore the value of the model for further exploration of compound-specific fibrogenic responses. This novel system will enable a more comprehensive characterization of key attributes unique to fibrogenic agents during the onset and progression of liver injury as well as mechanistic insights, thus improving compound risk assessment.Key words: liver fibrosis in vitro; 3D bioprinted liver; compound-induced liver injury.Chronic liver injury progressing to fibrosis and liver failure can result from a wide range of insults including drug or chemical exposure, metabolic disease, alcoholism, or viral infection, and is a major health burden worldwide with 2% of all deaths attributable to liver cirrhosis Murray et al., 2012). Whereas the major precipitating factors underlying drug-and chemicalinduced fibrosis have been gleaned from animal models, the key initiating and series of adaptive events that perpetuate this response, especially in humans, are still not well understood. Regardless of etiology, progressive fibrotic liver injury is

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“…Conventional approaches to maintaining the differentiated properties of isolated hepatocytes in culture include supplementation of the medium with hormones 4,5 ; cofactors such as nicotinamide, pyruvate, dimethyl sulfoxide, and phenobarbital 6,7 ; the application of extracellular matrix components 3,8 ; and coculture with nonparenchymal cell types. [9][10][11] We have adopted a different approach. Instead of using primary cultures of hepatocytes as a model for the study of liver function, we used differentiated pancreatic cells and induced a switch to a hepatocyte phenotype.…”

mentioning

confidence: 99%

Differentiated properties of hepatocytes induced from pancreatic cells

2002

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Transdifferentiation of pancreas to liver is a well-recognized phenomenon and has been described in animal experiments and human pathology. We recently produced an in vitro model for the transdifferentiation (or conversion) of the pancreatic cell line AR42J-B13 to hepatocytes based on culture with dexamethasone (Dex). To determine whether the hepatocytes express markers of hepatic intermediary metabolism and detoxification, we investigated the patterns of expression of glucokinase, cytochrome P450s CYP3A1 and CYP2B1/2, testosterone/4-nitrophenol uridine diphosphate glucuronosyltransferase (UDPGT), and aryl sulfotransferase. All were expressed. We also determined the expression of 2 enzymes involved in ammonia detoxification: carbamoylphosphate synthetase I (CPS I) and glutamine synthetase (GS). These enzymes are normally strictly compartmentalized in liver in a wide periportal pattern and the last downstream perivenous hepatocytes, respectively. Following culture with Dex, CPS I and GS are expressed in 2 different cell populations, suggesting that both periportal and perivenous hepatocytes are induced. We also produced a reporter assay based on the activation of green fluorescent protein (GFP) by the transthyretin (TTR) promoter or glucose-6-phosphatase (G6Pase) promoter. After culture with Dex, transfected cells begin to express GFP, showing that hepatic promoters are activated in concert with the induction of the hepatocyte phenotype. Lastly, we examined the stability of the hepatic phenotype and found that some cells still express liver markers (transferrin or albumin) up to 14 days after removal of Dex. In conclusion, these results suggest that pancreatic hepatocytes produced by this method may offer an alternative model to primary cultures of hepatocytes for the study of liver function. (HEPATOLOGY 2002;36:534-543.) I n vitro cultures of hepatocytes offer a valuable tool in the investigation of liver function. However, the major limitation to the use of cultured hepatocytes is their rapid loss of differentiated properties and gain of a more fetal/neoplastic phenotype. 1-3 For example, the loss of differentiated phenotype is most apparent in the rapid decline of total cytochrome P450 (CYP) after isolation and in culture, particularly the CYP3A1 isoform. 2 Dedifferentiation is reflected not only in decreased liver-specific functions but also in an alteration of morphology; the cells flatten, depolarize, and lose many of the surface characteristics of normal hepatocytes in vivo. The mechanisms responsible for loss of differentiated properties include down-regulation of transcription factors (e.g., C/EBP␣ and HNF1) involved in liver-specific gene expression and a switch from a quiescent to a proliferative mode. 2 It is also difficult to examine the long-term effects of exogenous factors on hepatic gene expression and liver function. Culture conditions that maintain primary rat hepatocytes in a highly differentiated state have recently been developed, but these still have selective expression of hepatic marke...

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Rat hepatocyte cultures and co-cultures in biotransformation studies of xenobiotics

Cited by 80 publications

References 59 publications

Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes

Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes

Editor’s Highlight: Modeling Compound-Induced FibrogenesisIn VitroUsing Three-Dimensional Bioprinted Human Liver Tissues

Differentiated properties of hepatocytes induced from pancreatic cells

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