title: Expression and induction potential of cytochromes P450 in human cryopreserved hepatocytes.
abstract: Fresh human hepatocytes are still considered as the "gold standard" to screen in vitro for cytochrome P450 (P450) induction. However, sparse availability of good quality human liver tissue for research purposes and the demand for standardized cell populations, together with the need for proper storage of the cells not immediately required, have resulted in the development of cryopreservation techniques that provide adequate viability and plateability of hepatocytes after thawing. This study aimed at validating cryopreserved human hepatocytes as a model to investigate P450 induction. Cryopreserved cells from four different donors were plated and cultured for 48 h, followed by incubation in the presence of typical P450 inducers. During the experiments, quality of the cultured cells was monitored both physiologically and morphologically. Concomitantly, the activity of CYP1A2, 2B6, 2C9, 2E1, and 3A4 was measured together with their mRNA and protein expression. Determination of CYP1A2, 2B6, 2C9, 2E1, and 3A4 activity in control versus prototypical inducer-treated hepatocytes revealed a maximal significant mean 11.6-, 2.8-, 1.9-, 1.5-, and 9.0-fold induction over their basal expression, respectively. Protein expression analysis of these P450s confirmed these results. Moreover, a mean 44.9-, 3.5-, 3.2-, and 13.8-fold induction of CYP1A2, 2B6, 2C9, and 3A4 mRNA was observed. Our data demonstrate that cryopreserved human hepatocytes are a valuable tool to study the induction of CYP1A2, 2B6, 2C9, 2E1, and 3A4.
authors.affiliation: Preclinical Pharmacokinetics, Johnson & Johnson Pharmaceutical Research & Development, Beerse, Belgium. droymans@tibbe.jnj.com
authors.forename: Dirk
authors.initials: D
authors.lastname: Roymans
authors.forename: Pieter
authors.initials: P
authors.lastname: Annaert
authors.forename: Jos
authors.initials: J
authors.lastname: Van Houdt
authors.forename: Adri
authors.initials: A
authors.lastname: Weygers
authors.forename: Jan
authors.initials: J
authors.lastname: Noukens
authors.forename: Carlo
authors.initials: C
authors.lastname: Sensenhauser
authors.forename: José
authors.initials: J
authors.lastname: Silva
authors.forename: Cis
authors.initials: C
authors.lastname: Van Looveren
authors.forename: Jan
authors.initials: J
authors.lastname: Hendrickx
authors.forename: Geert
authors.initials: G
authors.lastname: Mannens
authors.forename: Willem
authors.initials: W
authors.lastname: Meuldermans
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D015925:Cryopreservation; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans
chemical_list: D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System