Abstract

The mode of action (MOA) of constitutive androstane
receptor (CAR)-mediated rodent liver tumours involves 5 key events including
activation of the CAR receptor, altered gene expression, hepatocellular
proliferation, clonal expansion and increased hepatocellular
adenomas/carcinomas. To test whether liver 3D microtissues (LiMTs)  recapitulate CAR- mediated procarcinogenic
key events in response to phenobarbital (PB) we performed hepatocyte
proliferation (LI%) analysis in rat and human LiMTs using a microTMA technology
integrated with transcriptomics (microarray) and proteomics analysis. LiMTs
containing primary parenchymal and non-parenchymal cells (NPCs) are more
physiologically representative of  liver
than 2D hepatocytes.  Rat and human LiMTs
were treated with PB (500 uM - 2000 uM) for 24 hr - 96 hr. There was a
dose-dependent induction of LI% in rat LiMTs, however there was little or no
effect of PB on LI% in human LiMTs. There was also a dose- and time-dependent
PB-mediated induction of CAR regulated genes in human and rat LiMTs (RNA and
protein). Bioinformatics analysis (IPA) indicated that there was a significant
(Z score >2.0;-log p value >) activation of CAR by PB in both human and rat
LiMTs. These results indicate that human and rat LiMTs gave expected responses in
PB-induced hepatocyte proliferation and enzyme induction with rat LiMTs showing
significant dose-dependent effects while human LiMTs showed no hepatocyte proliferation.
In conclusion LiMTs can provide mechanistic data for 3 of the 5 key events
considered necessary to establish a CAR-mediated MOA for liver tumourigenesis
and thus reduce animal use when compiling mechanistic data packages.