TY - JOUR
T1 - 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)
AU - Giantin, Mery
AU - Zancanella, Vanessa
AU - Lopparelli, Rosa Maria
AU - Granato, Anna
AU - Carletti, Monica
AU - Vilei, Maria Teresa
AU - Muraca, Maurizio
AU - Baratto, Chiara
AU - Dacasto, Mauro
PY - 2012
Y1 - 2012
N2 - 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.
AB - 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.
KW - Cryopreserved pig hepatocytes
KW - Drug metabolizing enzymes
KW - Gene expression
KW - Induction
KW - Long-term cryopreservation
KW - Nuclear receptors
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U2 - 10.2133/dmpk.DMPK-11-RG-146
DO - 10.2133/dmpk.DMPK-11-RG-146
M3 - Article
C2 - 22447117
AN - SCOPUS:84869140362
VL - 27
SP - 495
EP - 505
JO - Drug Metabolism and Pharmacokinetics
JF - Drug Metabolism and Pharmacokinetics
SN - 1347-4367
IS - 5
ER -