Abstract
Characterisation of the mode of action (MOA) of constitutive androstane receptor (CAR)-mediated rodent liver tumours involves measurement 5 key events including activation of the CAR receptor, altered gene expression, hepatocellular proliferation, clonal expansion and increased hepatocellular adenomas/carcinomas. To test whether or not liver 3D microtissues (LiMTs) recapitulate CAR- mediated procarcinogenic key events in response to the prototypical CAR activator phenobarbital (PB) we performed hepatocyte proliferation (LI%) analysis in rat and human LiMTs using a microTMA technology in conjunction with integrated transcriptomics (microarray) and proteomics analysis. The rationale for this approach was that LiMTs containing parenchymal and non-parenchymal cells (NPCs) are more physiologically representative of liver and thus would generate data more relevant to the in vivo situation. Rat and human LiMTs were treated with PB over a range of concentrations (500 uM - 2000 uM) and times (24 h - 96 h) in a dose-response/time-course analysis. 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. ATP levels in the rat and human LiMTs were similar to control in all of the PB treatments. There was also a dose- and time-dependent PB-mediated RNA induction of CAR regulated genes CYP2B6/Cyp2b2, CYP3A7/Cyp3a9 and UGT1A6/Ugt1a6 in human and rat LiMTs, respectively. These CAR regulated genes were also upregulated at the protein level. Ingenuity pathways 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 showed the expected responses at the level of PB-induced hepatocyte proliferation and enzyme induction with rat LiMTs showing significant dose-dependent effects while human LiMTs showed no proliferation response but did show dose-dependent enzyme induction at the RNA and protein levels. In conclusion LiMTs serve as a model to provide mechanistic data for 3 of the 5 key events considered necessary to establish a CAR-mediated MOA for liver tumourigenesis and thus can potentially reduce the use of animals when compiling mechanistic data packages.
Highlights
Liver tumours are a common occurrence in rodent 2 year bioassay tests of drugs and pesticides
Ingenuity pathways analysis (IPA) indicated that there was a significant (Z score > 2.0;-log p value >) activation of constitutive androstane receptor (CAR) by PB in both human and rat liver 3D microtissues (LiMTs). These results indicate that human and rat LiMTs showed the expected responses at the level of PB-induced hepatocyte proliferation and enzyme induction with rat LiMTs showing significant dose-dependent effects while human LiMTs showed no proliferation response but did show dose-dependent enzyme induction at the RNA and protein levels
Hepatocyte growth factor (HGF) treatments caused a significant induction of LI% in both rat and human LiMTs, Fig. 2C and D, respectively
Summary
Liver tumours are a common occurrence in rodent 2 year bioassay tests of drugs and pesticides. 3D liver microtissue models containing primary hepatocytes and non-parenchymal cells, unlike 2D primary hepatocytes, retain 'liver like' gene expression profiles and sustain responsiveness to enzyme inducers such as phenobarbital (PB) for several weeks in culture [11] These models offer the potential to provide an in vitro system that more closely represents in vivo liver responses. To test this hypothesis we utilised novel high throughput histology, transcriptomics and proteomics approaches to investigate three ‘key event’ responses in CAR mediated carcinogenesis, namely CAR activation, enzyme induction and hepatocyte proliferation in a 3D liver microtissue model
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