Diurnal hepatic CYP3A11 contributes to chronotoxicity of the pyrrolizidine alkaloid retrorsine in mice

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.

Keywords:

• Retrorsine
• chronotoxicity
• CYP3A11
• circadian clock
• E4BP4

Author contributions

LG, MC and BW designed the study; LG, LZ, HX, and MC performed experiments; LG, LZ, HX, PY, ZW, DL and MC collected and analysed data; MC and BW wrote the manuscript.

Disclosure statement

The authors report no declarations of interest.

Additional information

Funding

This work was supported by the Natural Science Foundation of Guangdong Province [2020A1515010682 and 2021A1515011291], the Guangzhou Science and Technology Project (201904010472), the Shenzhen Science and Technology Innovation Commission [No. JCYJ20180302145930274], and the Project of Administration of Traditional Chinese Medicine of Guangdong Province of China [No. 20212047].