• Vinken, Mathieu
• Buyl, Karolien
• Rogiers, Vera
• Vanhaecke, Tamara

Abstract

Recent changes in the European legislation of chemicals have forced the scientific community to urge the search for alternative methods that can replace animal experimentation. In the area of liver-based in vitro modelling, several efforts are hereby focussed on the establishment of systems, particularly primary hepatocyte cultures, which can be used for long-term testing purposes by preventing the loss of the differentiated status. The current study was set up to test a novel strategy in this regard, namely the epigenetic suppression of connexin43, a gap junction component that becomes gradually expressed in primary hepatocyte cultures and that has been associated with the onset of dedifferentiation. For this purpose, a RNA interference protocol, making use of small interfering RNA duplexes targeted towards connexin43 mRNA, was developed. Upon application of this procedure to freshly isolated and cultivated rat hepatocyes, protein amounts of selected phase I (CYP1A1, CYP2B1, CYP3A2) and phase II (UGT1A, UGT2B) xenobiotic biotransformation enzymes and liver-enriched transcription factors (HNF1?) were not affected, whereas the expression levels of other markers (GST?, HNF4?, C/EBP?)became downregulated. These data suggest that the silencing of connexin43 production through RNA interference should not be pursued as a strategy to counteract dedifferentiation in primary hepatocyte cultures.

Topics

• phase