Effects of Time Culture and Prototypical Cytochrome P450 3A (CYP3A) Inducers on CYP2B22, CYP2C, CYP3A and Nuclear Receptor (NR) mRNAs in Long-term Cryopreserved Pig Hepatocytes (CPHs)

• Published in: DRUG METABOLISM AND PHARMACOKINETICS 2012, Vol.27 (5), p.495-505
• Main Authors: Giantin, Mery, Zancanella, Vanessa, Lopparelli, Rosa Maria, Granato, Anna, Carletti, Monica, Vilei, Maria Teresa, Muraca, Maurizio, Baratto, Chiara, Dacasto, Mauro
• Format: Article
• Language: English
• Subjects: Animals; Cell Survival - drug effects; Cell Survival - genetics; cryopreserved pig hepatocytes; Cytochrome P-450 Enzyme System - biosynthesis; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Dexamethasone - pharmacology; Down-Regulation - drug effects; Down-Regulation - genetics; drug metabolizing enzymes; Enzyme Induction - drug effects; gene expression; Hepatocytes - cytology; Hepatocytes - drug effects; Hepatocytes - enzymology; Hepatocytes - metabolism; induction; Isoenzymes; long-term cryopreservation; nuclear receptors; Protein Processing, Post-Translational - drug effects; Protein Processing, Post-Translational - genetics; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Retinoid X Receptor alpha - genetics; Retinoid X Receptor alpha - metabolism; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Swine; Transcription, Genetic - drug effects; Transcriptome - drug effects; Up-Regulation - drug effects
• ISSN: 1347-4367; 1880-0920
• DOI: 10.2133/dmpk.DMPK-11-RG-146

In the present study, transcriptional and post-translational effects of culturing time and prototypical cytochrome P450 3A (CYP3A) inducers on principal nuclear receptors (NRs), CYP2B22, 2C and 3A were investigated in long-term stored (~10 years) cryopreserved pig hepatocytes (CPHs). In the time-course study, a crush and rise effect was observed for pregnane X receptor (NR1I2) and constitutive androstane receptor (NR1I3) mRNAs, while a time-dependent increase of retinoid X receptor alpha (NR2B1) was noticed. Cytochrome P450 gene expression profiles were down-regulated as a function of time. In the induction study, an increase of NR1I2, NR1I3 and NR2B1 mRNAs was observed in dexamethasone-exposed CPHs. About CYPs, an overall up-regulation was seen in CPHs exposed to phenobarbital, while dexamethasone and rifampicin up-regulated only CYP3A. In both studies, transcriptional CYP results were confirmed at the post-translational level (immunoblotting and enzyme activities), except for CYP2B immunoblotting in the induction study. The present data demonstrate that long-term stored CPHs may be used to investigate mechanisms involved in CYPs regulation, expression and function; provide further info about NR regulation of CYPs, and confirm species-differences in these mechanisms of regulation; finally, they suggest the usefulness and relevance of gene expression profiling to early detect any modulation of CYP expression and bioactivity.