BIOCHEMICAL PHARMACOLOGY
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2023
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IF 5.8
Luomin Lin,Yuwei Huang,Jinyi Wang,Xiaocao Guo,Fangjun Yu,Di He,Chuanbin Wu,Lianxia Guo,Baojian Wu
CYP2A5 , an enzyme responsible for metabolism of diverse drugs, displays circadian rhythms in its expression and activity. However, the underlying mechanisms are not fully established. Here we aimed to investigate a potential role of CRY1/2 (circadian clock modulators) in circadian regulation of hepatic CYP2A5. Regulatory effects of CRY1/2 on CYP2A5 were determined using Cry1 -null and Cry2 -null mice, and validated using AML-12, Hepa1-6 and HepG2 cells. CYP2A5 activities both in vivo and in vitro were assessed using coumarin 7-hydroxylation as a probe reaction. mRNA and protein levels were detected by qPCR and western blotting , respectively. Regulatory mechanism was studied using a combination of luciferase reporter assays, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP). We found that ablation of Cry1 or Cry2 in mice reduced hepatic CYP2A5 expression (at both mRNA and protein levels) and blunted its diurnal rhythms. Consistently, these knockouts showed decreased CYP2A5 activity (characterised by coumarin 7-hydroxylation) and a loss of its time-dependency, as well as exacerbated coumarin-induced hepatotoxicity. Cell-based assays confirmed that CRY1/2 positively regulated CYP2A5 expression and rhythms. Based on combined luciferase reporter, ChIP and Co-IP assays, we unraveled that CRY1/2 interacted with E4BP4 protein to repress its inhibitory effect on Cyp2a5 transcription and expression . In conclusion, CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4. These findings advance our understanding of circadian regulation of drug metabolism and pharmacokinetics .
研究领域:
药理学
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分子生物学
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时间生物学
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PubMed
高影响因子
Oncology Letters
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2016
·Vol. 12
·期 6
·5309-5314
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IF 1.482
Xiao Deng,Qianghong Pu,Erhao Wang,Chao Yu
Human cytochrome P450 (CYP) 2A6 participates in the metabolism of nicotine and precarcinogens, thus the deliberate inhibition of CYP2A6 may reduce cigarette consumption and therefore reduce the risk of developing the types of cancer associated with smoking. The inhibitory effects and mechanisms of celery (Apium graveolens) extract on mouse CYP2A5 and human CYP2A6 activity remain unclear. These effects were investigated in mouse and human liver microsomes using coumarin 7‑hydroxylation in a probe reaction. Celery extract reduced CYP2A5 and CYP2A6 activities in vitro in a dose‑dependent manner. In vivo experiments also showed that celery extract markedly decreased CYP2A5 activity. The inhibition of celery extract on CYP2A5 was time‑ and nicotinamide adenine dinucleotide phosphate (NADPH)‑independent, and was markedly reduced by ultracentrifugation. Additionally, the inhibition of celery extract on CYP2A6 was time and NADPH‑dependent. Levels of inhibition were characterized by a Ki, the measure of the tightness of bonds between the enzyme and its inhibitor, of 266.4 µg/ml for CYP2A5, and a Ki of 1,018 µg/ml and Kinact of 0.3/min for CYP2A6. Kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor. The coumarin derivative 5‑methoxypsoralen present in celery extract did not solely to the inhibition of CYP2A5/6 activity. In conclusion, celery extract inhibited the levels of mouse CYP2A5 and human CYP2A6 activity via different mechanisms: Mixed competitive inhibition for CYP2A5 and mechanism‑based inhibition for CYP2A6.
研究领域:
药理学
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生物化学
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毒理学
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PubMed
高影响因子
DRUG METABOLISM AND DISPOSITION
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2018
·Vol. 46
·期 11
·1538-1545
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IF 3.64
Jiangming Deng,Lianxia Guo,Baojian Wu
Human CYP2A6 (Cyp2a5 in mice) plays an important role in metabolism and detoxification of various drugs and chemicals. Here, we investigated a potential role of peroxisome proliferator-activated receptor γ (Ppar-γ) in circadian regulation of the Cyp2a5 enzyme. We first showed that Cyp2a5 mRNA and protein in mouse liver displayed robust circadian oscillations. Consistent with a circadian protein pattern, Cyp2a5-mediated 7-hydroxylation of coumarin was circadian time–dependent. Formation of 7-hydroxycoumarin was more extensive at a dosing time of Zeitgeber time 2 (ZT2) than that at ZT14. Interestingly, the nuclear receptor Ppar-γ was also a circadian gene. Circadian Ppar-γ protein level was strongly correlated with the Cyp2a5 mRNA level (r = 0.989). Furthermore, Ppar-γ activation (by a selective agonist, rosiglitazone) upregulated Cyp2a5 expression in Hepa-1c1c7 cells, whereas Ppar-γ knockdown downregulated Cyp2a5 expression. Also, Ppar-γ knockdown blunted the rhythmicity of Cyp2a5 mRNA in serum-shocked Hepa-1c1c7 cells. In addition, a combination of promoter truncation analysis, mobility shift, and chromatin immunoprecipitation assays revealed that Ppar-γ directly bound to a PPAR response element (i.e., the −1418- to −1396-bp region) within Cyp2a5 promoter and activated the gene transcription. Taken together, Ppar-γ was a transcriptional activator of Cyp2a5, and its rhythmic expression contributed to circadian expression of Cyp2a5.
研究领域:
药理学
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分子生物学
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昼夜节律生物学
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高影响因子
Expert Opinion on Drug Metabolism & Toxicology
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2025
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IF 3.4
Yongbin Tong,Qi Zhang,Yutong Chen,Zhiqiang Zhao,Jingqi Li,Jinyi Wang,Haishan Xia,Danyi Lu,Yuyu Liu
Introduction Coumarin exhibit various biological activities, including anti-inflammatory, antioxidant and anxiolytic effects. However, its clinical application is restricted due to significant hepatotoxicity observed in multiple animal models. CYP2A5 is the primary enzyme responsible for metabolizing coumarin in the mouse liver, homologous to human CYP2A6.Methods Acute toxicity and pharmacokinetic investigations were conducted on Rev-erbα−/− and wild-type mice following coumarin administration. The regulatory effects of REV-ERBα on CYP2A5 expression were assessed both in vivo and in vitro. Western blotting and qPCR were used to measure the protein and mRNA levels, respectively.Results Firstly, coumarin-induced hepatotoxicity was mitigated in Rev-erbα−/− mice. Secondly, the systemic exposure of coumarin was lowered in Rev-erbα−/− mice compared with wild-type mice. Thirdly, deletion of Rev-erbα resulted in significant upregulation of CYP2A5 mRNA and protein expression in mice. REV-ERBα negatively regulated CYP2A5/2A6 expression in Hepa-1c1c7 and HepG2 cells. Furthermore, luciferase reporter and chromatin immunoprecipitation (ChIP) assays indicated that REV-ERBα modulated Cyp2a5 transcription and expression through the repression of DBP.Conclusions The knockout of Rev-erbα significantly reduces the hepatotoxicity of coumarin by modulating CYP2A5. This research underscores the potential of targeting the REV-ERBα/CYP2A5 axis as a novel strategy for reducing coumarin-induced hepatotoxicity.
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高影响因子
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