Introduction. The constitutive expression of major drug metabolizing enzymes like cytochromes P450 (CYPs) and related nuclear receptors (NRs) is considered of fundamental importance in drug metabolism studies made in whole-cell systems like hepatocyte primary cultures (HPCs). In fresh and cryopreserved pig HPCs, time-dependent variations of CYP gene expression have been investigated by and large at the post-translational level; furthermore, few data have been published about the effect of time and known CYP3A inducers on NRs mRNAs. In the present study, the transcriptional effects of time and prototypical CYP3A inducers upon CYP2B22, 2C, 3A28 and NR1I2, NR1I3, NR2B1 and NR3C1 were investigated by using cryopreserved pig HPCs. Materials and Methods. To measure time-dependent changes in aforementioned target genes, HPCs were stopped 24, 48, 72 and 96 hrs after plating. As regards the HPCs response to known CYP3A inducers, media containing phenobarbital (PB, 2 mM), pregnenolone 16α-carbonitrile (PCN, 10 μM), rifampicin (RIF, 10 μM), dexamethasone (DEX, 10 μM) and dimethyl sulfoxide (control) were daily added to monolayers from 24 hrs after plating and up to 72 hrs. Additionally, specific reference genes were identified by using geNormPLUS and Normfinder algorithms. Transcriptional effects were measured by using specific qPCR assays. Results. The geometric mean of three reference genes, namely ribosomal protein large P0 (RPLP0), cyclophilin A (PPIA), glyceraldheyde 3-phosphte dehydrogenase and RPLP0, PPIA and β-actin was used to normalize time-course and induction qPCR data, respectively. CYP gene expression was strongly down-regulated as a function of time culture; at 48 hrs, CYP2B22 and CYP3A accounted (%) for 7.14±1.08 and 19.37±11.21 of the RQ value measured at 24 hrs. A low and constant constitutive expression of CYP2C, NR1I2 and NR1I3 was noticed during the whole culturing time, while NR2B1 and NR3C1 mRNA levels were increased (p<0.001 for NR2B1at 96 hrs). Hepatocytes were responsive to model CYP3A inducers; PB significantly increased CYP2B22 (p<0.05), 2C (p<0.01) and 3A (p<0.001) gene expression, while DEX induced CYP3A, NR1I2 (p<0.001) and NR1I3 (p<0.05). Finally, RIF up-regulated only CYP3A mRNA (p<0.05). Conclusions. Data obtained agree with previous published comparative results about time-course and induction in fresh and cryopreserved HPCs. Furthermore, present results would confirm the role played by NRs in CYP expression and regulation phenomena as well as the presence of species-differences in CYP3A drug metabolism. Confirmatory immunoblotting investigations are actually running.

Effects of time culture and prototypical CYP3A inducers on CYP2B22, CYP2C, CYP3A28 and nuclear receptors mRNAs in cryopreserved pig hepatocytes

ZANCANELLA, VANESSA;GIANTIN, MERY;LOPPARELLI, ROSA MARIA;VILEI, MARIA TERESA;MURACA M;DACASTO, MAURO
2011

Abstract

Introduction. The constitutive expression of major drug metabolizing enzymes like cytochromes P450 (CYPs) and related nuclear receptors (NRs) is considered of fundamental importance in drug metabolism studies made in whole-cell systems like hepatocyte primary cultures (HPCs). In fresh and cryopreserved pig HPCs, time-dependent variations of CYP gene expression have been investigated by and large at the post-translational level; furthermore, few data have been published about the effect of time and known CYP3A inducers on NRs mRNAs. In the present study, the transcriptional effects of time and prototypical CYP3A inducers upon CYP2B22, 2C, 3A28 and NR1I2, NR1I3, NR2B1 and NR3C1 were investigated by using cryopreserved pig HPCs. Materials and Methods. To measure time-dependent changes in aforementioned target genes, HPCs were stopped 24, 48, 72 and 96 hrs after plating. As regards the HPCs response to known CYP3A inducers, media containing phenobarbital (PB, 2 mM), pregnenolone 16α-carbonitrile (PCN, 10 μM), rifampicin (RIF, 10 μM), dexamethasone (DEX, 10 μM) and dimethyl sulfoxide (control) were daily added to monolayers from 24 hrs after plating and up to 72 hrs. Additionally, specific reference genes were identified by using geNormPLUS and Normfinder algorithms. Transcriptional effects were measured by using specific qPCR assays. Results. The geometric mean of three reference genes, namely ribosomal protein large P0 (RPLP0), cyclophilin A (PPIA), glyceraldheyde 3-phosphte dehydrogenase and RPLP0, PPIA and β-actin was used to normalize time-course and induction qPCR data, respectively. CYP gene expression was strongly down-regulated as a function of time culture; at 48 hrs, CYP2B22 and CYP3A accounted (%) for 7.14±1.08 and 19.37±11.21 of the RQ value measured at 24 hrs. A low and constant constitutive expression of CYP2C, NR1I2 and NR1I3 was noticed during the whole culturing time, while NR2B1 and NR3C1 mRNA levels were increased (p<0.001 for NR2B1at 96 hrs). Hepatocytes were responsive to model CYP3A inducers; PB significantly increased CYP2B22 (p<0.05), 2C (p<0.01) and 3A (p<0.001) gene expression, while DEX induced CYP3A, NR1I2 (p<0.001) and NR1I3 (p<0.05). Finally, RIF up-regulated only CYP3A mRNA (p<0.05). Conclusions. Data obtained agree with previous published comparative results about time-course and induction in fresh and cryopreserved HPCs. Furthermore, present results would confirm the role played by NRs in CYP expression and regulation phenomena as well as the presence of species-differences in CYP3A drug metabolism. Confirmatory immunoblotting investigations are actually running.
Proceedings of the International Congress CELLTOX 1991-2011 - Twenty years of in vitro toxicology: achievements and future challenges
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