The Autonomous Hepatocyte Clock Controls Acetaminophen Bioactivation and Chronotoxicity [Kidney]

The diurnal variation in acetaminophen (APAP) hepatotoxicity ("chronotoxicity") is thought to be due to oscillations in xenobiotic metabolism that are influenced by the circadian phases of feeding or fasting. Because of APAP's relevance to human poisoning, we set out to determine the relative contributions of the central clock in the SCN and the autonomous clock in the hepatocyte in modulating the chronotoxicity of APAP. Using a conditional null allele of Mop3 (ArntL, Bmal1) we are able to delete the clock from hepatocytes while keeping the central and other peripheral clocks intact (eg, those controlling food intake). Our data from this hepatocyte-null mouse model suggests that, while the central circadian clock modulates some detoxification pathways indirectly by driving activity patterns and feeding rhythms, the autonomous hepatocyte circadian clock controls major aspects of APAP bioactivation independent of feeding rhythms, possibly through transcriptional regulation of cytochrome p450-oxidoreductase (Por). 10-20 week old Mop3fxfx mice positive or negative for Cre-recombinase driven by the albumin promoter, housed in 12 hour light:12 dark, ad lib feeding and drinking conditions were sacrificed every four hours over two separte days beginning at ZT0. A two color, reference design experiment in which kidney RNA from at least 3 mice per timepoint were pooled and labeled with Cy3 and hybridized according to Agilent protocols against a reference pool of RNA madeup from respective tissue taken from 10 week Mop3fxfx and Mop3fxfxCreAlb mice which was labeled with Cy5.对乙酰氨基酚（acetaminophen，APAP）所致肝毒性的昼夜节律变化（又称“时辰毒性”），被认为源于受进食或禁食昼夜节律调控的外源性物质代谢振荡。鉴于APAP与人类中毒事件密切相关，本研究旨在明确视交叉上核（suprachiasmatic nucleus，SCN）的中枢生物钟与肝细胞自主生物钟，在调控APAP时辰毒性过程中的相对贡献。 本研究使用Mop3（ArntL、Bmal1）的条件性敲除等位基因，可特异性敲除肝细胞中的生物钟，同时保留中枢生物钟与其他外周生物钟的完整性（例如调控进食行为的生物钟）。基于该肝细胞敲除小鼠模型获得的实验数据表明：中枢昼夜生物钟可通过调控活动模式与进食节律，间接调节部分解毒通路；而肝细胞自主昼夜生物钟则不依赖进食节律，直接调控APAP生物活化的主要环节，其机制可能与细胞色素P450氧化还原酶（Por）的转录调控有关。 本研究选取10~20周龄的Mop3fxfx小鼠，其携带或不携带白蛋白启动子驱动的Cre重组酶；所有小鼠均饲养于12小时光照:12小时黑暗的环境中，自由进食饮水。自授时因子时相0（zeitgeber time 0，ZT0）起，连续两天每4小时处死一批小鼠。本实验采用双色参考设计方案：将每个时间点至少3只小鼠的肾脏RNA混合，用Cy3进行标记；以10周龄Mop3fxfx与Mop3fxfxCreAlb小鼠的相应组织提取的RNA混合作为参考池，并用Cy5进行标记，随后按照安捷伦（Agilent）实验流程完成杂交。