Regulation of the Major Detoxication Functions by Phenobarbital and 3-Methylcholanthrene in Co-Cultures of Rat Hepatocytes and Liver Epithelial Cells
Carole Lerche,Carole Lerche,Alain Fautrel,Alain Fautrel,Peter M. Shaw,Denise Glaise,François Ballet,André Guillouzo,Laurent Corcos,Laurent Corcos +9 more
TLDR
The results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.Abstract:
In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.read more
Citations
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Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells
TL;DR: Although the precise mechanisms by which nonparenchymal cells modulate the hepatocyte phenotype remain unelucidated, some new insights on the modes of cell signaling, the extent of cell–cell interaction, and the ratio of cell populations are noted.
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Liver cell models in in vitro toxicology.
TL;DR: There is a need for better culture conditions and differentiated hepatocyte cell lines to overcome the limited availability of human liver tissues and strategies for in vitro analysis of potentially toxic chemicals must be better defined.
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RNA expression in the early characterization of hepatotoxicants in Wistar rats by high-density DNA microarrays.
Steven J. Bulera,Susan M. Eddy,Erika Ferguson,Timothy A. Jatkoe,J. F. Reindel,Michael R. Bleavins,Felix A. De La Iglesia +6 more
TL;DR: This toxico genomic analysis identified multiple genes and groups of genes that were affected by the hepatotoxicants on study, indicating that high‐density microarray expression data are useful to identifygroups of genes involved in toxicity.
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Hepatocyte cell lines: their use, scope and limitations in drug metabolism studies.
TL;DR: The features of liver-derived cell lines, their suitability for drug metabolism studies as well as the state-of-the-art of the strategies pursued in order to generate metabolically competent hepatic cell lines are reviewed.
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Humanized mice with ectopic artificial liver tissues.
Alice A. Chen,David Thomas,Luvena L. Ong,Robert E. Schwartz,Todd R. Golub,Sangeeta N. Bhatia +5 more
TL;DR: M mice with HEALs are used to predict the disproportionate metabolism and toxicity of “major” human metabolites using multiple routes of administration and monitoring to enable manufacturing of reproducible in vivo models for diverse drug development and research applications.
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Christiane Guguen-Guillouzo,Bruno Clément,Georges Baffet,Carole Beaumont,Edith Morel-Chany,Denise Glaise,André Guillouzo +6 more
TL;DR: The results demonstrate for the first time long-term stabilization and reversibility of a specific function (albumin secretion) at high levels by adult hepatocytes cultured in serum-free medium and suggest that both the presence of other liver cell type(s) and the production of an extracellular matrix are needed for the maintenance of specific functions in cultured hepatocytes.