SpringerProtocols: Abstract: In Vitro Assays for Induction of Drug Metabolism

5. In Vitro Assays for Induction of Drug Metabolism

By: Brian G. Lake¹, Roger J. Price¹, Amanda M. Giddings¹, David G. Walters¹

Abstract

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Hepatic microsomal cytochrome P450 (CYP) forms have a major role in the metabolism of drugs and other chemicals. Primary hepatocyte cultures from humans and experimental animals are a valuable in vitro system for studying the effects of chemicals on CYP forms. This chapter describes methods to evaluate CYP form induction in human and rat hepatocytes cultured in a 96-well plate format. The use of a 96-well plate format permits studies to be performed with relatively small numbers of hepatocytes and obviates the need to harvest cells and prepare subcellular fractions prior to the assay of enzyme activities. The induction of CYP1A and CYP3A forms in human and rat hepatocytes can be determined by measurement of 7-ethoxyresorufin O-deethylase and testosterone 6β-hydroxylase activities, respectively, whereas 7-benzyloxy-4-trifluoromethylcoumarin (BFC) O-debenzylase can be employed to assess both CYP1A and CYP2B form induction in rat hepatocytes. An assay for determining the protein content of hepatocytes cultured in a 96-well plate format is also described.

Affiliation(s): (1) BIBRA International, Woodmansterne Road, Carshalton, Surrey and Centre for Toxicology, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, UK

Book Title: Hepatocyte Transplantation: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 481 | Year: 2009 | Page Range: 1-12 | DOI: 10.1007/978-1-59745-201-4_5

Subject: Molecular Medicine

Key Words: Cytochrome P450 - 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase, enzyme induction - 7-ethoxyresorufin O-deethylase - human hepatocytes, rat hepatocytes - sulphorhodamine B protein assay - testosterone 6β-hydroxylase

References

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