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Role of the CLOCK protein in liver detoxification.

ABSTRACT: BACKGROUND AND PURPOSE:Whether and how circadian clock proteins regulate drug detoxification are not known. Here, we have assessed the effects of CLOCK (a core circadian clock protein) on drug metabolism and detoxification. EXPERIMENTAL APPROACH:Regulation by CLOCK protein of drug-metabolizing enzymes was 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. KEY RESULTS:Clock deletion disrupted hepatic diurnal expressions of a number of drug-metabolizing enzymes in mice. In particular, CYP2A4/5 expressions were markedly down-regulated, whereas CYP2B10 was up-regulated. 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-ERB?/? (two target genes of CLOCK and transcriptional repressors of Cyp2b10). Furthermore, Clock ablation sensitized mice to coumarin toxicity by down-regulating CYP2A4/5-mediated metabolism (a detoxification pathway) and to cyclophosphamide toxicity by up-regulating CYP2B10-mediated metabolism (generating the toxic metabolite 4-hydroxycyclophosphamide). CONCLUSION AND IMPLICATIONS:CLOCK protein regulates metabolism by the cytochrome P450 family and drug detoxification. The findings improve our understanding of the crosstalk between circadian clock and drug detoxification.

SUBMITTER: Zhao M

PROVIDER: S-EPMC6965678 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Role of the CLOCK protein in liver detoxification.

Zhao Mengjing M Zhao Huan H Deng Jiangming J Guo Lianxia L Wu Baojian B

British journal of pharmacology 20191228 24

<h4>Background and purpose</h4>Whether and how circadian clock proteins regulate drug detoxification are not known. Here, we have assessed the effects of CLOCK (a core circadian clock protein) on drug metabolism and detoxification.<h4>Experimental approach</h4>Regulation by CLOCK protein of drug-metabolizing enzymes was assessed using Clock knockout (Clock<sup>-/-</sup> ) mice and Hepa-1c1c7/AML-12 cells. The relative mRNA and protein levels were determined by qPCR and Western blotting respectiv ...[more]

PMID: 31404943

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Project description:Controlling bilirubin to a low level is necessary in physiology because of its severe neurotoxicity. Therefore, it is of great interest to understand the regulatory mechanisms for bilirubin homeostasis. In this study, we uncover a critical role for circadian clock in regulation of bilirubin detoxification and homeostasis. Methods: The mRNA and protein levels of Bmal1 (a core clock gene), metabolic enzymes and transporters were measured by qPCR and Western blotting, respectively. Luciferase reporter, mobility shift and chromatin immunoprecipitation were used to investigate transcriptional gene regulation. Experimental hyperbilirubinemia was induced by injection of bilirubin or phenylhydrazine. Unconjugated bilirubin (UCB) and conjugated bilirubin were assessed by ELISA. Results: We first demonstrated diurnal variations in plasma UCB levels and in main bilirubin-detoxifying genes Ugt1a1 and Mrp2. Of note, the circadian UCB levels were antiphase to the circadian expressions of Ugt1a1 and Mrp2. Bmal1 ablation abrogated the circadian rhythms of UCB and bilirubin-induced hepatotoxicity in mice. Bmal1 ablation also decreased mRNA and protein expressions of both Ugt1a1 and Mrp2 in mouse livers, and blunted their circadian rhythms. A combination of luciferase reporter, mobility shift, and chromatin immunoprecipitation assays revealed that Bmal1 trans-activated Ugt1a1 and Mrp2 through specific binding to the E-boxes in the promoter region. Further, Bmal1 ablation caused a loss of circadian time-dependency in bilirubin clearance and sensitized mice to chemical induced-hyperbilirubinemia. Moreover, bilirubin stimulated Bmal1 expression through antagonism of Rev-erbα, constituting a feedback mechanism in bilirubin detoxification. Conclusion: These data supported a dual role for circadian clock in regulation of bilirubin detoxification, generating circadian variations in bilirubin level via direct transactivation of detoxifying genes Ugt1a1 and Mrp2, and defending the body against hyperbilirubinemia via Rev-erbα antagonism. Thereby, our study provided a potential mechanism for management of bilirubin related diseases.

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Role of the CLOCK protein in liver detoxification.

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Role of the CLOCK protein in liver detoxification.

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