[42] Use of human hepatocytes to study P450 gene induction

doi:10.1016/S0076-6879(96)72044-X

Methods in Enzymology

Volume 272, 1996, Pages 388-401

https://doi.org/10.1016/S0076-6879(96)72044-X Get rights and content

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The human hepatocyte model system is useful for investigations of interspecies and interindividual differences in P450-related activities, including induction of gene expression in response to xenobiotic exposure and the molecular events controlling gene expression through the transfection of reporter gene constructs. The CYP3A family is the major steroid-inducible cytochrome P450s in rats, rabbits, and humans. No single animal model faithfully predicts the responses observed in human hepatocyte preparations. Hepatocytes can be cryopreserved and reestablished into culture, and the frozen/thawed cells still respond to P450-inducing compounds with enhanced gene transcription and drug-metabolizing capacity. It is crucial to gain a complete understanding of the genetic structure, regulation, and functional activity of human cytochrome P450s because of the relative importance of cytochrome P450s in the biotransformation of a variety of exogenous and endogenous compounds.

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Citation Excerpt :
Demographic information of all donors is shown in Supplemental Table 1. The isolation of human hepatocytes was conducted as described previously [19]. Hepatocytes were plated on collagen-coated T-25 flasks at a density of approximately 106 cells.
The generation of reactive metabolites from therapeutic agents is one of the major mechanisms of drug-induced liver injury (DILI). In order to evaluate metabolism-related toxicity and improve drug efficacy and safety, we generated a battery of HepG2-derived cell lines that express 14 cytochrome P450s (CYPs) (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 3A7) individually using a lentiviral expression system. The expression/production of a specific CYP in each cell line was confirmed by an increased abundance of the CYP at both mRNA and protein levels. Moreover, the enzymatic activities of representative CYPs in the corresponding cell lines were also measured. Using our CYP-expressed HepG2 cells, the toxicity of three drugs that could induce DILI (amiodarone, chlorpromazine and primaquine) was assessed, and all of them showed altered (increased or decreased) toxicity compared to the toxicity in drug-treated wild-type HepG2 cells. CYP-mediated drug toxicity examined in our cell system is consistent with previous reports, demonstrating the potential of these cells for assessing metabolism-related drug toxicity. This cell system provides a practical in vitro approach for drug metabolism screening and for early detection of drug toxicity. It is also a surrogate enzyme source for the enzymatic characterization of a particular CYP that contributes to drug-induced liver toxicity.
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Human hepatocytes were isolated and cultured on collagen type I or EHS matrigel in cell media with or without dexamethasone. The glucocorticoid receptor (GR) antagonist RU486 was used to elucidate the involvement of GR.
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[42] Use of human hepatocytes to study P450 gene induction

Publisher Summary

Arch. Biochem. Biophys.

Methods Enzymol.

Methods Enzymol.

J. Biol. Chem.

Crit. Rev. Toxicol.

Pharmacol. Rev.

JNCI, J. Natl. Cancer Inst.

Hepatology

Drug Metab. Disp.