Plummer Simon, Beaumont Bobby, Wallace Stephanie, Ball Graeme, Wright Jayne, McInnes Liz, Currie Richard, Peffer Rich, Cowie David
MicroMatrices Associates Ltd, Dundee, UK.
Dundee University Imaging Facility, Dundee, UK.
Toxicol Rep. 2019 Sep 24;6:998-1005. doi: 10.1016/j.toxrep.2019.09.010. eCollection 2019.
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.
组成型雄烷受体（CAR）介导的啮齿动物肝肿瘤作用模式（MOA）的表征涉及测量5个关键事件，包括CAR受体的激活、基因表达改变、肝细胞增殖、克隆扩增以及肝细胞腺瘤/癌增加。为了测试肝脏三维微组织（LiMTs）是否重现了CAR介导的致癌关键事件以响应原型CAR激活剂苯巴比妥（PB），我们使用微TMA技术结合综合转录组学（微阵列）和蛋白质组学分析，在大鼠和人类LiMTs中进行了肝细胞增殖（LI%）分析。采用这种方法的基本原理是，含有实质细胞和非实质细胞（NPCs）的LiMTs在生理上更能代表肝脏，因此会产生与体内情况更相关的数据。在剂量反应/时间进程分析中，用一系列浓度（500μM - 2000μM）和时间（24小时 - 96小时）的PB处理大鼠和人类LiMTs。大鼠LiMTs中LI%呈剂量依赖性诱导，然而PB对人类LiMTs中的LI%几乎没有影响。在所有PB处理中，大鼠和人类LiMTs中的ATP水平与对照相似。PB还分别在人类和大鼠LiMTs中以剂量和时间依赖性方式诱导CAR调控基因CYP2B6/Cyp2b2、CYP3A7/Cyp3a9和UGT1A6/Ugt1a6的RNA表达。这些CAR调控基因在蛋白质水平也上调。 Ingenuity通路分析（IPA）表明，在人类和大鼠LiMTs中，PB对CAR有显著（Z评分>2.0；-log p值>）激活作用。这些结果表明，人类和大鼠LiMTs在PB诱导的肝细胞增殖和酶诱导水平上表现出预期反应，大鼠LiMTs表现出显著的剂量依赖性效应，而人类LiMTs没有增殖反应，但在RNA和蛋白质水平上表现出剂量依赖性酶诱导。总之，LiMTs可作为一种模型，为建立CAR介导的肝肿瘤发生MOA所需的5个关键事件中的3个提供机制数据，因此在编制机制数据包时可能会减少动物的使用。
