Role of the CLOCK protein in liver detoxification.
Whether and how circadian clock proteins regulate drug detoxification remain elusive. Here we aimed to clarify the impact of CLOCK (a core circadian clock protein) on drug metabolism and detoxification. The regulatory effects of CLOCK protein on drug-metabolizing enzymes were assessed using Clock knockout (Clock-/- ) mice and Hepa-1c1c7/AML-12 cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Toxicity and pharmacokinetic experiments were performed with Clock-/- and wild-type mice after intraperitoneal injection of coumarin or cyclophosphamide. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays. Clock ablation disrupted hepatic diurnal expressions of a number of drug-metabolizing enzymes in mice. In particular, CYP2A4/5 expressions were markedly downregulated, whereas CYP2B10 was upregulated. Positive regulation of Cyp2a4/5 and negative regulation of Cyp2b10 by CLOCK were confirmed in Hepa-1c1c7 and AML-12 cells. Based on a combination of luciferase reporter, mobility shift and ChIP assays, we found that CLOCK activated Cyp2a4/5 transcription via specific binding to E-box elements in promoter region, and repressed Cyp2b10 transcription through REV-ERBα/β (two target genes of CLOCK and transcriptional repressors of Cyp2b10). Furthermore, Clock ablation sensitized mice to coumarin toxicity by downregulating CYP2A4/5-mediated metabolism (a detoxification pathway), and to cyclophosphamide toxicity by upregulating CYP2B10-mediated metabolism (generating the toxic metabolite 4-hydroxycyclophosphamide). CLOCK protein regulates CYP metabolism and drug detoxification. Our findings have implications for a deep understanding of the crosstalk between circadian clock and drug detoxification.