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 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 intraperitoneal injection dose of diethylnitrosamine (DEN) followed by 500 ppm PB in 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 between genes with altered expression compared with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) alterations in mouse hepatocellular adenoma and in liver of CD-1 mice or humanized chimeric mice treated with PB for 7 days were evaluated. With 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 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：

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基于野生型小鼠和人源化嵌合小鼠之间调节基因的分化，负责苯巴比妥促进小鼠肝细胞肿瘤发生的早期关键事件的候选基因


苯巴比妥 (PB) 是一种对啮齿类动物无基因毒性的肝细胞致癌物。 PB 通过激活组成型雄甾烷受体 (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PB促进的肝细胞肿瘤发生，但其分子机制的细节尚不完全清楚。在本研究中，使用微阵列对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，该小鼠肝细胞腺瘤是通过腹腔注射 90 mg/kg 剂量的二乙基亚硝胺 (DEN)，然后在饮食中添加 500 ppm PB 连续 27 周诱导的。在 PB 诱导的小鼠肝细胞腺瘤中，DNA 修饰和数百个基因的表达发生协调改变。其中，基因网络分析显示 CAR 信号传导和肿瘤发展相关基因的改变。通路富集分析显示，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通路，而差异表达的基因主要属于细胞周期。此外，与小鼠肝细胞腺瘤和用PB处理7天的CD-1小鼠或人源化嵌合小鼠的肝脏中的5-甲基胞嘧啶(5mC)和5-羟甲基胞嘧啶(5hmC)改变相比，表达改变的基因之间的重叠进行了评估。通过转录组学和表观遗传学方法的整合，我们检测到了负责 PB 促进的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，这些基因与人源化嵌合小鼠的 PB 处理改变的基因并不重叠，因此表明 PB 对小鼠和人类肝细胞的影响存在物种差异。