Period 2 Regulates CYP2B10 Expression and Activity in Mouse Liver.

Menglin Chen,Min Chen,Li Zhang,Baojian Wu,Danyi Lu,Zhigang Wang,Yi Wang

doi:10.3389/fphar.2021.764124

Abstract

CYP2B10 is responsible for metabolism and detoxification of many clinical drugs. Here, we aimed to investigate a potential role of Period 2 (PER2) in regulating expression of hepatic CYP2B10. Regulatory effects of PER2 on hepatic expression of CYP2B10 and other enzymes were determined using Per2-deficient mice with exons 4-6 deleted (named Per2 Del4-6 mice). In vitro and in vivo metabolic activities of CYP2B10 were probed using cyclophosphamide (CPA) as a specific substrate. Regulatory mechanism was investigated using luciferase reporter assays. Genotyping and Western blotting demonstrated loss of wild-type Per2 transcript and markedly reduced PER2 protein in Per2 Del4-6 mice. Hepatic expression of a plenty of drug-metabolizing genes (including Cyp2a4/2a5, Cyp2b10, Ugt1a1, Ugt1a9, Ugt2b36, Sult1a1 and Sult1e1) were altered (and majority were down-regulated) in Per2 Del4-6 mice. Of note, Cyp2b10, Ugt1a9 and Sult1a1 were three genes considerably affected with reduced expression. Decreased expression of CYP2B10 was translated to reduced metabolism and altered pharmacokinetics of CPA as well as attenuated CPA hepatotoxicity in Per2 Del4-6 mice. Positive regulation of CYP2B10 by PER2 was further confirmed in both Hepa-1c1c7 and AML-12 cells. Based on luciferase reporter assays, it was shown that PER2 regulated Cyp2b10 transcription in a REV-ERBα-dependent manner. REV-ERBα was negatively regulated by PER2 (increased REV-ERBα expression in Per2 Del4-6 mice) and itself was also a repressor of CYP2B10. In conclusion, PER2 positively regulates CYP2B10 expression and activity in mouse liver through inhibiting its repressor REV-ERBα.

Highlights

Drug metabolism is a main component of pharmacokinetics, profoundly affecting drug efficacy and toxicity (Yan et al, 2018)
Western blotting showed that hepatic Period 2 (PER2) protein was markedly reduced in Per2Del4-6 mice, it was not completely lost (Figure 1D). qPCR assay with primer set 2 targeting non-knockout sequence suggested the presence of a new version of Per2 transcript in Per2Del4-6 mice albeit at a reduced level (Figure 1C)
We have revealed that many hepatic drug-metabolizing enzymes (DMEs) including CYP2B10, UGT1A9 and SULT1A1 are under the control of PER2 in mice (Figures 1–3)

Summary

Introduction

Drug metabolism (biotransformation) is a main component of pharmacokinetics, profoundly affecting drug efficacy and toxicity (Yan et al, 2018). Drug metabolism reactions, mediated by drug-metabolizing enzymes (DMEs), have been historically divided into two types, namely, phase I and phase II reactions. In phase I reactions ( known as functionalization reactions), DMEs such as cytochromes P450 (CYPs) introduce polar chemical moieties to drug molecules. The modified molecules are converted to more hydrophilic and excretable metabolites in phase II reactions catalyzed by conjugating DMEs such as UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs). Human CYP superfamily contains 18 families consisting of 57 members (Elfaki et al, 2018). Human UGTs include 22 isoenzymes and are classified into four families (i.e., UGT1, UGT2, UGT3, and UGT8) (Mano et al, 2018).

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