INTRODUCTION Cattle hepatocyte primary cultures (CHs) have been used as a useful in vitro model for drug metabolism studies. However, they suffer some limitations, such as a difficult adhesion to the substrate following isolation, a limited viability and a rapid loss of differentiated characteristics if compared to ex vivo liver samples (LSs). Therefore, a reliable hepatic in vitro model is actually needed for cattle. As established bovine hepatic cell lines are not available, we characterized the Marby Darby Bovine Kidney (MDBK) cell line transcriptome by using a cDNA microarray approach, and compared it with CHs and ex vivo cattle LSs. MATERIALS AND METHODS LSs coming from preceding studies, CHs isolated by a two-step collagenase isolation method (Giantin et al., 2012) and MDBK cells collected at different passages were used. Overall, the transcriptome of sixteen samples (8 LSs, 4 CHs and 4 MDBK cell splittings) was characterized by using a cDNA microarray technology. The normalization, statistical analyses, data visualization (Hierarchical clustering and Principal Component Analysis) and functional analyses (gene ontology terms, pathway analysis) of microarray data were performed using the GeneSpring software. RESULTS AND CONCLUSIONS When compared with LSs, CHs showed a significant (P < 0.05) down-regulation (fold-changes, FC>2.0) of many cell-mediated, immunity-based and metabolic signaling pathways. About drug metabolism, many cytochromes P450 (CYPs), drug transporters (DTs), glutathione transferases and nuclear receptors (NRs) were down-regulated. Likewise, the MDBK cell line showed a significant (P < 0.05) modulation (FC>2.0) of 5307 genes (of which 2706 were down-regulated). Biotransformation and CYP oxidation were among the altered pathways. This study confirms, to a wider scenario (the whole transcriptome), the known decrease of gene expression occurring in CHs, including CYPs, DTs and NRs. MDBK cells, albeit to a lower extent compared to CHs, constitutively express most of drug metabolizing enzymes and DTs. Prospective studies are needed to better characterize the cell line potential as a useful in vitro model for regulation, induction and functional studies. ACKNOWLEDGEMENTS A project supported by the University of Padua (CPDA109434).
Transcriptomic characterization of Marby Darby Bovine Kidney (MDBK) cell line and its comparison with cattle primary hepatocytes and liver tissue.
ELGENDY, RAMY ELGENDY IBRAHIM MOHAMED;ZANCANELLA, VANESSA;GIANTIN, MERY;DACASTO, MAURO
2015
Abstract
INTRODUCTION Cattle hepatocyte primary cultures (CHs) have been used as a useful in vitro model for drug metabolism studies. However, they suffer some limitations, such as a difficult adhesion to the substrate following isolation, a limited viability and a rapid loss of differentiated characteristics if compared to ex vivo liver samples (LSs). Therefore, a reliable hepatic in vitro model is actually needed for cattle. As established bovine hepatic cell lines are not available, we characterized the Marby Darby Bovine Kidney (MDBK) cell line transcriptome by using a cDNA microarray approach, and compared it with CHs and ex vivo cattle LSs. MATERIALS AND METHODS LSs coming from preceding studies, CHs isolated by a two-step collagenase isolation method (Giantin et al., 2012) and MDBK cells collected at different passages were used. Overall, the transcriptome of sixteen samples (8 LSs, 4 CHs and 4 MDBK cell splittings) was characterized by using a cDNA microarray technology. The normalization, statistical analyses, data visualization (Hierarchical clustering and Principal Component Analysis) and functional analyses (gene ontology terms, pathway analysis) of microarray data were performed using the GeneSpring software. RESULTS AND CONCLUSIONS When compared with LSs, CHs showed a significant (P < 0.05) down-regulation (fold-changes, FC>2.0) of many cell-mediated, immunity-based and metabolic signaling pathways. About drug metabolism, many cytochromes P450 (CYPs), drug transporters (DTs), glutathione transferases and nuclear receptors (NRs) were down-regulated. Likewise, the MDBK cell line showed a significant (P < 0.05) modulation (FC>2.0) of 5307 genes (of which 2706 were down-regulated). Biotransformation and CYP oxidation were among the altered pathways. This study confirms, to a wider scenario (the whole transcriptome), the known decrease of gene expression occurring in CHs, including CYPs, DTs and NRs. MDBK cells, albeit to a lower extent compared to CHs, constitutively express most of drug metabolizing enzymes and DTs. Prospective studies are needed to better characterize the cell line potential as a useful in vitro model for regulation, induction and functional studies. ACKNOWLEDGEMENTS A project supported by the University of Padua (CPDA109434).
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