Abstract

1. Retrorsine (RTS) is a pyrrolizidine alkaloid (distributed in many medicinal plants) that has significant hepatotoxicity. Here, we aimed to determine the daily variations in RTS hepatotoxicity (chronotoxicity) in mice, and to investigate the role of metabolism in generating RTS chronotoxicity.

2. Acute toxicity and pharmacokinetic studies were performed with mice after RTS administration at different times of the day. Hepatotoxicity was assessed by measuring plasma ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels. mRNA and proteins were determined by qPCR and Western blotting, respectively. Time-dependent in vitro metabolism of RTS was assessed by using mouse liver microsomes.

3. We found that RTS toxicity was more severe in the dark phase (zeitgeber time 14 or ZT14 and ZT18) than in the light phase (ZT2 and ZT6). This chronotoxicity was associated with a dosing time difference in the systemic exposures of RTS and a pyrrolic ester metabolite (a cause of hepatotoxicity, measured by the levels of pyrrole-GSH conjugate and pyrrole-protein adducts due to a high chemical reactivity). Moreover, the CYP3A11 (a major enzyme for RTS bioactivation) inhibitor ketoconazole decreased the production of pyrrole-GSH conjugate and abrogated diurnal rhythm in RTS metabolism. In addition, E4bp4 (a circadian regulator of Cyp3a11) ablation abolished the rhythm of CYP3A11 expression and abrogated the dosing time-dependency of RTS toxicity.

4. In conclusion, RTS chronotoxicity in mice was attributed to time-varying hepatic metabolism regulated by the circadian clock. Our findings have implications for reducing pyrrolizidine alkaloid-induced toxicity via a chronotherapeutic approach.

摘要

1. Retrorsine (RTS) 是一种吡咯里西啶生物碱（分布于许多药用植物中），具有显着的肝毒性。在这里，我们旨在确定小鼠 RTS 肝毒性（时间毒性）的日常变化，并研究代谢在产生 RTS 时间毒性中的作用。

2. 在一天中的不同时间对小鼠进行 RTS 给药后的急性毒性和药代动力学研究。通过测量血浆 ALT（丙氨酸氨基转移酶）和 AST（天冬氨酸氨基转移酶）水平来评估肝毒性。mRNA 和蛋白质分别通过 qPCR 和蛋白质印迹法测定。通过使用小鼠肝微粒体评估 RTS 的时间依赖性体外代谢。

3. 我们发现 RTS 毒性在黑暗阶段（zeitgeber time 14 或 ZT14 和 ZT18）比在光明阶段（ZT2 和 ZT6）更严重。这种时间毒性与 RTS 和吡咯酯代谢物全身暴露的给药时间差异有关（肝毒性的一个原因，通过高化学反应性导致的吡咯-GSH 偶联物和吡咯-蛋白质加合物的水平来测量）。此外，CYP3A11（RTS 生物活化的主要酶）抑制剂酮康唑降低了吡咯-GSH 偶联物的产生，并消除了 RTS 代谢中的昼夜节律。此外，E4BP4（的昼夜调节器CYP3A11烧蚀）废除CYP3A11表达的节奏和废止RTS毒性的定量给料的时间依赖性。

4. 总之，小鼠的 RTS 时间毒性归因于由生物钟调节的随时间变化的肝脏代谢。我们的研究结果对通过时间治疗方法减少吡咯里西啶生物碱诱导的毒性具有重要意义。