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

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. 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 separate days beginning at ZT0. A two color, reference design experiment in which liver RNA from at least 3 mice per time point were pooled and labeled with Cy3 and hybridized according to Agilent protocols against a reference pool of RNA made up from respective tissue taken from 10 week Mop3fxfx and Mop3fxfxCreAlb mice which was labeled with Cy5.对乙酰氨基酚（acetaminophen, APAP）的肝毒性昼夜节律变化（“时辰毒性”，chronotoxicity）被认为是由受进食或禁食昼夜节律调控的异生物质代谢波动所导致。鉴于APAP与人类中毒的密切相关性，本研究旨在明确视交叉上核（SCN）的中央生物钟以及肝细胞内的自主生物钟在调节APAP时辰毒性过程中的相对贡献。本研究利用Mop3（ArntL, Bmal1）的条件性无效等位基因，可在保留中央生物钟及其他外周生物钟（例如调控进食活动的生物钟）完整的前提下，特异性敲除肝细胞内的生物钟。来自该肝细胞敲除小鼠模型的数据显示：尽管中央生物钟可通过驱动活动模式与进食节律，间接调控部分解毒通路，但肝细胞自主生物钟可不依赖进食节律，控制APAP生物活化的主要环节。本研究使用10~20周龄的Mop3fxfx小鼠，其中一组携带白蛋白启动子驱动的Cre重组酶（Cre-recombinase，阳性组），另一组不携带（阴性组）；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。在两个独立的实验日内，自授时零点（ZT0）起，每4小时处死一批小鼠。实验采用双色参考设计：将每个时间点至少3只小鼠的肝脏RNA混合后用Cy3标记，并按照Agilent实验方案与用Cy5标记的参考RNA池进行杂交；该参考RNA池取自10周龄的Mop3fxfx及Mop3fxfxCreAlb小鼠的相应组织。