Cross-species comparison of CAR-mediated procarcinogenic key events in a 3D liver microtissue model

Simon Plummer (Lead / Corresponding author), Bobby Beaumont, Stephanie Wallace, Graeme Ball, Jayne Wright, Liz McInnes, Richard Currie, Rich Peffer, David Cowie

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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.

Original languageEnglish
Pages (from-to)998-1005
Number of pages8
JournalToxicology Reports
Volume6
Early online date24 Sep 2019
DOIs
Publication statusPublished - 2019

Keywords

  • 3D liver microtissues
  • Carcinogenesis
  • Constitutive androstane receptor
  • Cross-species risk assessment
  • Enzyme induction
  • Hepatocyte
  • Key events
  • MicroTMA
  • Mode of action
  • Pathways
  • Proliferation
  • Proteomics
  • Quantitative histopathology
  • Species difference
  • Tissue microarray
  • Transcriptomics

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  • Cite this

    Plummer, S., Beaumont, B., Wallace, S., Ball, G., Wright, J., McInnes, L., Currie, R., Peffer, R., & Cowie, D. (2019). Cross-species comparison of CAR-mediated procarcinogenic key events in a 3D liver microtissue model. Toxicology Reports, 6, 998-1005. https://doi.org/10.1016/j.toxrep.2019.09.010