Phenobarbital (PB) is a nongenotoxic hepatocellular carcinogen in rodents. PB induces hepatocellular tumors by activating the constitutive androstane receptor (CAR). Some previous research has suggested the possible involvement of epigenetic regulation in PB-promoted hepatocellular tumorigenesis, but the details of its molecular mechanism are not fully understood. In the present study, comprehensive analyses of DNA methylation, hydroxymethylation and gene expression using microarrays were performed in mouse hepatocellular adenomas induced by a single 90 mg kg⁻¹ intraperitoneal injection dose of diethylnitrosamine (DEN) followed by 500 ppm PB in the diet for 27 weeks. DNA modification and expression of hundreds of genes are coordinately altered in PB-induced mouse hepatocellular adenomas. Of these, gene network analysis showed alterations of CAR signaling and tumor development-related genes. Pathway enrichment analysis revealed that differentially methylated or hydroxymethylated genes belong mainly to pathways involved in development, immune response and cancer cells in contrast to differentially expressed genes belonging primarily to the cell cycle. Furthermore, overlap was evaluated between the genes with altered expression levels with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) alterations in mouse hepatocellular adenoma induced by DEN/PB and the genes with altered expression levels in the liver of CD-1 mice or humanized chimeric mice treated with PB for 7 days. With the integration of transcriptomic and epigenetic approaches, we detected candidate genes responsible for early key events of PB-promoted mouse hepatocellular tumorigenesis. Interestingly, these genes did not overlap with genes altered by the PB treatment of humanized chimeric mice, thus suggesting a species difference between the effects of PB in mouse and human hepatocytes.

中文翻译：

---

基于野生型小鼠和人源化嵌合小鼠之间调控基因的差异，负责苯巴比妥促进小鼠肝细胞癌发生的早期关键事件的候选基因

苯巴比妥（PB）是啮齿类动物的一种非遗传毒性肝细胞致癌物。PB通过激活组成型雄激素受体（CAR）诱导肝细胞肿瘤。先前的一些研究表明表观遗传调控可能参与了PB促进的肝细胞肿瘤发生，但是其分子机制的细节尚未完全了解。在本研究中，使用单芯片对90 mg kg ^-1诱导的小鼠肝细胞腺瘤进行了DNA甲基化，羟甲基化和基因表达的综合分析。腹膜内注射二乙基亚硝胺（DEN），然后在饮食中添加500 ppm PB，持续27周。在PB诱导的小鼠肝细胞腺瘤中，DNA修饰和数百种基因的表达均发生了协同变化。其中，基因网络分析显示了CAR信号转导和肿瘤发展相关基因的改变。途径富集分析显示差异甲基化或羟甲基化基因主要属于参与发育，免疫反应和癌细胞的途径，而差异表达基因则主要属于细胞周期。此外，评估了DEN / PB诱导的小鼠肝细胞腺瘤中具有5-甲基胞嘧啶（5mC）和5-羟甲基胞嘧啶（5hmC）改变表达水平的基因与CD-1小鼠肝脏中表达水平改变的基因之间的重叠PB处理7天的人源化嵌合小鼠。通过转录组和表观遗传学方法的整合，我们检测了PB促进小鼠肝细胞肿瘤发生的早期关键事件的候选基因。有趣的是，这些基因不与通过人源化嵌合小鼠的PB处理改变的基因重叠，因此表明PB在小鼠和人肝细胞中的作用之间存在物种差异。我们检测了负责PB促进小鼠肝细胞肿瘤发生的早期关键事件的候选基因。有趣的是，这些基因不与通过人源化嵌合小鼠的PB处理改变的基因重叠，因此表明PB在小鼠和人肝细胞中的作用之间存在物种差异。我们检测了负责PB促进小鼠肝细胞肿瘤发生的早期关键事件的候选基因。有趣的是，这些基因不与通过人源化嵌合小鼠的PB处理改变的基因重叠，因此表明PB在小鼠和人肝细胞中的作用之间存在物种差异。