Phenobarbital Induces Cell Cycle Transcriptional Responses in Mouse Liver Humanized for Constitutive Androstane and Pregnane X Receptors (mRNA)

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcino- genesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CARKO -PXRKO ), double humanized CAR and PXR (CARh - PXRh), and wild-type C57BL/6 mice. Wild-type and CARh-PXRh mouse livers exhibited temporally and quantitatively similar tran- scriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were ob- served with peak expression occurring between 1 and 7 days PB ex- posure. All these transcriptional responses were absent in CARKO- PXRKO mouse livers and largely reversible in wild-type and CARh - PXRh mouse livers following 91 days of PB exposure and a subse- quent 4-week recovery period. Furthermore, PB-mediated upregu- lation of the noncoding RNA Meg3, which has recently been associ- ated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CARh-PXRh mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB. 13-week oral (drinking water) investigative phenobarbital study in male C57BL/6 wild type, CAR/PXR knockout and humanized CAR/PXR mice with a 4 week recovery period组成型雄烷受体（constitutive androstane receptor, CAR）与孕烷X受体（pregnane X receptor, PXR）是一类密切相关的核受体，参与药物代谢过程，在苯巴比妥（phenobarbital, PB）诱导的啮齿类非遗传毒性肝癌发生机制中发挥重要作用。本研究采用人源化CAR/PXR小鼠模型，探究苯巴比妥暴露后肝脏组织受体依赖型分子应答的潜在物种差异。我们对双敲除CAR和PXR（CAR^KO-PXR^KO）、双人源化CAR和PXR（CAR^h-PXR^h）以及野生型C57BL/6小鼠的肝脏组织，开展了持续性苯巴比妥暴露后的早期与晚期转录组响应分析。野生型与CAR^h-PXR^h小鼠肝脏在91天苯巴比妥暴露期间，呈现出时间模式与定量水平均相似的转录应答，包括异生物质应答基因Cyp2b10、Wnt信号通路抑制剂Wisp1以及Dlk1-Dio3基因座来源的非编码RNA生物标志物的持续性诱导表达。在苯巴比妥暴露1~7天期间，可观察到DNA复制相关基因（Hells、Mcm6与Esco2）、有丝分裂基因（Ccnb2、Cdc20与Cdk1）以及增殖相关核抗原Mki67的瞬时诱导表达，且表达量达到峰值。上述所有转录应答在CAR^KO-PXR^KO小鼠肝脏中均未出现；而在野生型与CAR^h-PXR^h小鼠中，经91天苯巴比妥暴露并继之以4周恢复期后，这些转录应答基本可逆转。此外，苯巴比妥介导的非编码RNA Meg3（近期被发现与细胞多能性相关）的上调效应，在野生型与CAR^h-PXR^h小鼠中呈现出相似的剂量响应特征与肝静脉周肝细胞特异性定位模式。综上，共表达人源CAR与PXR的小鼠肝脏，在苯巴比妥暴露后可同时介导异生物质代谢相关与增殖相关的转录应答。本研究为针对雄性C57BL/6野生型、CAR/PXR敲除及人源化CAR/PXR小鼠的13周口服（饮水给药）苯巴比妥探索性研究，设置了4周恢复期。