Period 2 Regulates CYP2B10 Expression and Activity in Mouse Liver

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.


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 (also known as functionalization reactions), DMEs such as cytochromes P450 (CYPs) introduce polar chemical moieties to drug molecules. The modified molecules are then 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). Enzymes from UGT1A and 2B sunfamilies (with a total of 16 members) are the main contributors to drug glucuronidation (Rowland et al., 2013). To date, there are four families of SULT enzymes in humans, namely SULT1, SULT2, SULT4, and SULT6. Enzymes of SULT1 and SULT2 (with a total of 12 members) play a leading role in drug sulfonation (Allali-Hassani et al., 2007).
DMEs such as CYP enzymes are distributed in many tissue and organs, particularly in the drug-metabolizing organs liver, kidney, and small intestine (Renaud et al., 2011). Of CYPs, the members from CYP1 to CYP4 families are of most importance as they metabolize >55% of FDA-approved drugs (Saravanakumar et al., 2019). Mouse CYP2B10 (CYP2B6 in humans) is a member of CYP2B subfamily and primarily expressed in the liver, where it accounts for about 10% of the total microsomal CYP pool (Ekins et al., 1998;Ekins et al., 1999). CYP2B10 is responsible for the metabolism and detoxification of many clinical drugs including cyclophosphamide (CPA) and bupropion (Turpeinen and Zanger, 2012). Expression of CYP2B10 is known to be regulated by a number of nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), glucocorticoid receptors (GR), and vitamin D receptor (VDR) (Beigneux et al., 2002;Pascussi et al., 2004).
Period 2 (PER2) is a central component of the mammalian circadian clock machinery. It acts as a negative regulator of circadian clock through a direct interaction with the transcriptional activator CLOCK/BMAL1 heterodimer (Langmesser et al., 2008). Phosphorylation by casein kinase 1δ/ε (CK1δ/ε) is a key step that determines the stability of the PER2 protein and therefore the period of the circadian rhythms in mammals (Narasimamurthy et al., 2018). A mutation in CK1phosphorylating site of PER2 (S662G) has been identified as a determiner of human familial advanced sleep phase syndrome (Vanselow et al., 2006;Xu et al., 2007). PER2 is also involved in the regulation of many physiological and pathological processes such as neurobiological activities (Kim et al., 2018), metabolic homeostasis (Zani et al., 2013), thermogenesis (Chappuis et al., 2013), and tumourigenesis (Mteyrek et al., 2016). Moreover, it has been found that PER2 regulates the hepatotoxicity of drugs such as acetaminophen in mice probably via modulating the expression of CYP1A2 and CYP2E1 (Kakan et al., 2011;Ge et al., 2021). However, whether and how PER2 regulates CYP2B10 remain unknown.
Interestingly, Per2 −/− mice show decreases in serum triglycerides and free fatty acids coupled with increases in hepatic triglycerides and free fatty acids, indicating that PER2 is involved in lipid metabolism (Kettner et al., 2016). In addition, Heintza's work illustrates that the repression or inhibition of CYP2B (e.g., CYP2B10) may exacerbate metabolic disorders and cause obesity by perturbing fatty acid metabolism, suggesting a role of CYP2B in lipid homeostasis (Heintz et al., 2019). These findings indicate a potential link between PER2 and CYP2B, particularly CYP2B10.
In the present study, we aimed to investigate a potential role of PER2 in regulating expression of hepatic CYP2B10. Regulatory effects of PER2 on hepatic expression of CYP2B10 and other DMEs were determined using Per2-deficient mice with exons 4-6 deleted. mRNAs and proteins were quantified by qPCR and Western blotting, respectively. In vitro and in vivo metabolic activities of CYP2B10 were probed using CPA as a specific substrate. Regulation of CYP2B10 by PER2 was validated in Hepa-1c1c7 and AML-12 cells. Regulatory mechanism was investigated using luciferase reporter assays. We for the first time demonstrated that PER2 positively regulates CYP2B10 expression and activity in mouse liver through inhibiting its repressor REV-ERBα.

Mice
Per2 heterozygotes (on a C57BL/6 background) were obtained from Cyagen Biosciences (Guangzhou, China) (Supplementary Material S1). Homozygotes with exons 4-6 deleted (named Per2 Del4-6 mice) were generated by inter-crossing heterozygous mice. Per2-deficient mice and their wild-type littermates were housed and maintained under a 12 h light/12 h dark cycle (lights on at 6:00 AM ( Zeitgeber time 0/ZT0) and lights off at 6:00 PM ( ZT12)), with free access to food and water. Animal experimental procedures were approved by Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine (Appr. date: 2020-11-19; IACUC Issue No: ZYD-2020-111) and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize suffering and the number of mice used in the experiments.

PCR Genotyping
DNA was extracted from mouse tail (0.5-1 mm). PCR reactions were performed with 400 ng DNA template. Amplification program consisted of an initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 35 s, and extension at 72°C for 35 s. The products were subjected to 2% agarose gel electrophoresis, and bands were imaged using the Omega Lum G imaging system (Aplegen). The primers are listed in Table 1.

Acute Toxicity Study
Per2 Del4-6 and control wild-type mice (8-12 weeks, male, n 5 per group) were injected (i.p.) with CPA at 100 mg/kg or with vehicle at ZT14. Four hours after drug administration, mice were sacrificed to collect plasma and liver samples, followed by biochemical analyses.

Biochemical Analysis
ALT, AST, and GSH were measured with their assay kits according to manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, China).

CYP Microsomal Metabolism Assay
Livers were collected from Per2 Del4-6 and control wild-type mice. Liver microsomes were prepared by sequential ultracentrifugation, first at 9,000 g for 10 min and then at 100,000 g for 1 h as previously described ( Van et al., 1992). CPA was used as a specific substrate to determine the microsomal activity of CYP2B10 (Pass et al., 2005;Zhao et al., 2019). In brief, liver microsomes (4 mg/ml) was incubated with CPA (20 μM), NADPH (1.5 mM), and MgCl 2 (5 mM) in potassium phosphate (50 mM, pH 7.4) at 37°C for 2 h. The reaction was terminated by adding ice-cold acetonitrile (containing an internal standard). To quantify 4-OH-CPA, the resultant mixture was treated with OMHA (1.5 mg/ml) to transform 4-OH-CPA into a stable product OH-CPA O-methyloxime. The reaction mixture was centrifuged at 13,000 g for 15 min and the supernatant was subjected to UPLC-QTOF/MS analysis.

Sulfation Assay
Livers were collected from Per2 Del4-6 and control wild-type mice. Liver S9 fraction was prepared by centrifugation at 9,000 g for 10 min (Richardson et al., 2016). Sulfation activity was determined using an incubation method with liver S9 fraction as previously described (Guo et al., 2018). Briefly, liver S9 fraction (4 mg/ml), PAPS (50 μM) and galangin (6 μM) were incubated in potassium phosphate buffer (50 mM, pH 7.4) at 37°C for 3 h. The reactions were terminated by adding 200 μl ice-cold acetonitrile (containing an internal standard), followed by vortex and centrifugation at 13,000 g for 15 min. The supernatant was subjected to UPCL-QTOF/MS analysis.

qPCR Assay
Total RNA was extracted from mouse liver and cell samples with TRIzol reagent (Accurate Biotech, Hunan, China) following the manufacturer's instructions, and used as a template for cDNA synthesis. qPCR reactions were performed with SYBR Green Master Mix (Vazyme, Nanjing, China) using a Biometra Toptical Thermocycler (Analytik Jena, Goettingen, Germany) as previously described (Yamamura et al., 2010). Amplification reactions consisted of an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Peptidylprolyl isomerase B (Ppib) was used as an internal control. Relative mRNA expression was calculated using the 2 -ΔΔCt method and normalized to the control group. Primer sequences are shown in Table 1.

Western Blotting
Mouse tissues and cell samples were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (HY-K0010, Monmouth Junction, NJ). Protein concentration was detected by a BCA assay kit (Beyotime, Shanghai, China). After denaturing at 95°C for 10 min, protein samples (40 μg) were subjected to SDSpolyacrylamide gel electrophoresis (10% gel) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were sequentially incubated with primary antibody and HRP-conjugated secondary antibody. Protein bands were visualized using the Omega Lum G imaging system (Aplegen), and band density was analyzed by using FlourChem software (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Protein expression was normalized to GAPDH.

Data Analysis
Data are presented as mean ± SD (standard deviation). Sample sizes are provided in figure legends. Student's t-test was used to analyze a statistical difference between two groups. One-way ANOVA followed by Bonferroni post hoc test was used for multiple group comparisons. Statistical analyses were performed with GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA). Pharmacokinetic analysis was performed using WinNonlin software (Pharsight Corp, St. Louis, MO). The level of significance was set at p < 0.05 (*).

Characterization of Per2-Deficient Mice
Per2-deficient mice with exons 4-6 deleted (named Per2 Del4-6 mice) were obtained from Cyagen Biosciences (Guangzhou, China) ( Figure 1A). PCR genotyping of mice was performed using tail biopsies with the primers F1, F2 and R1/2, resulting in a 707 bp fragment for Per2 Del4-6 mice and a 514 bp fragment for wild-type allele ( Figure 1B). qPCR assay with primer set 1 (targeting the knockout sequence) confirmed the absence of wild-type Per2 transcript in mouse liver ( Figure 1C). Western blotting showed that hepatic PER2 protein was markedly reduced in Per2 Del4-6 mice, however, 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 Per2 Del4-6 mice albeit at a reduced level ( Figure 1C). This new transcript may be translated to a PER2-related protein that retains a reactivity with the commercial PER2 antibody, thereby explaining why PER2 protein was detected in Per2 Del4-6 mice ( Figure 1D). Taken together, Per2 Del4-6 mice were characterized with loss of wildtype Per2 transcript and markedly reduced PER2 protein.

Altered Metabolism and Pharmacokinetics of CPA in Per2 Del4-6 Mice
Since CYP2B10 expression was altered in the liver of Per2 Del4-6 mice, we next tried to test whether this affects the metabolism and pharmacokinetics of substrate drugs. To this end, we performed pharmacokinetic experiments of CPA with Per2 Del4-6 and control mice. Per2 Del4-6 mice showed increases in plasma CPA concentrations ( Figure 4A). Accordingly, AUC (area under the curve, a measure of systemic exposure) of CPA was significantly increased in Per2 Del4-6 mice ( Table 2). In the meantime, liver CPA concentrations were increased in Per2 Del4-6 mice ( Figure 4B). By contrast, the plasma and hepatic levels of the metabolite 4-hydroxycyclophosphamide (4-OH-CPA) were lower in Per2 Del4-6 mice, suggesting reduced metabolism of CPA ( Figures 4C,D). Therefore, Per2 ablation altered the pharmacokinetics of CPA in mice by down-regulating drug metabolism. CPA is an anticancer prodrug whose cytotoxic effects depend on metabolic activation to its metabolites such as 4-OH-CPA (Richardson et al., 2016). Our previous study shows that CPA treatment induces hepatotoxicity in mice, which is positively related to formation of 4-OH-CPA (Zhao et al., 2019). We thus examined whether CPA-induced hepatotoxicity is affected in Per2 Del4-6 mice. We found lower levels of ALT and AST activities, but a higher GSH level in the liver of Per2 Del4-6 mice, indicating reduced hepatotoxicity in the genetically modified mice ( Figure 5). This was consistent with reduced metabolism of CPA and lowered formation of 4-OH-CPA (Figure 4). Altogether, CPA hepatotoxicity is reduced in Per2 Del4-6 mice due to decreased metabolism. Per2 Positively Regulates CYP2B10 Expression in Hepa-1c1c7 and AML-12 Cells Next, we assessed the regulatory effects of Per2 on CYP2B10 expression in mouse Hepa-1c1c7 hepatoma cells by performing overexpression and knockdown assays. We confirmed that overexpression plasmid significantly elevated the levels of PER2 mRNA and protein, whereas siRNA decreased PER2 expression, in Hepa-1c1c7 cells ( Figure 6A). Per2 overexpression significantly increased cellular levels of CYP2B10 mRNA and protein ( Figure 6B). By contrast, Per2 knockdown reduced cellular expression of CYP2B10 ( Figure 6B).

PER2 Regulates CYP2B10 Expression Through REV-ERBα
PER2 is an integral component of circadian clock, and functions as a co-repressor to repress the transcriptional activity of the BMAL1/CLOCK heterodimer, a well-known activator of many clock-controlled genes (Langmesser et al., 2008). Thus, it was speculated that PER2 may regulate CYP2B10 expression through an indirect mechanism that involves BMAL1/CLOCK and an intermediate regulator. This intermediate regulator should be a     (Dunlap, 1999;Crumbley and Burris, 2011;Zhang et al., 2018b). We therefore tested whether REV-ERBα mediates PER2 regulation of CYP2B10. We found that REV-ERBα mRNA and protein were significantly increased in the liver of Per2 Del4-6 mice ( Figure 8A). This was expected because PER2 can repress the expression of REV-ERBα by inhibiting the transcriptional activity of BMAL1/CLOCK (Liu et al., 2008). We confirmed that REV-ERBα is a transcriptional repressor of Cyp2b10 in HEK293T cells based on luciferase reporter assays ( Figure 8B). Intriguingly, PER2 dosedependently induced the transcription of Cyp2b10 ( Figure 8C). However, the induction effect of PER2 was attenuated in Rev-erbα silenced cells ( Figure 8D). Moreover, overexpressing Rev-erbα reversed the effect of PER2 on Cyp2b10 transcription ( Figure 8E). Taken together, our findings suggest that PER2 regulates CYP2B10 expression by down-regulating REV-ERBα.

DISCUSSION
In this study, we have revealed that many hepatic DMEs including CYP2B10, UGT1A9 and SULT1A1 are under the control of PER2 in mice (Figures 1-3). We further showed that decreased expression of CYP2B10 was translated to reduced metabolism and altered pharmacokinetics of its substrate drug CPA as well as attenuated CPA hepatotoxicity in Per2-deficient (Per2 Del4-6 ) mice (Figures 4, 5). Positive regulation of CYP2B10 by PER2 was confirmed in both Hepa-1c1c7 and AML-12 cells (Figures 6,  7). Based on luciferase reporter assays, it was shown that PER2 regulated Cyp2b10 transcription in a REV-ERBα-dependent manner ( Figure 8). REV-ERBα is negatively regulated by PER2 (decreased REV-ERBα in Per2 Del4-6 mice, Figure 8) and itself is also a repressor of CYP2B10. Therefore, we propose that PER2 regulates CYP2B10 expression and activity through downregulating REV-ERBα, thereby impacting the metabolism and pharmacokinetics of substrate drugs. CYP2B10/CYP2B6 activity is a critical determinant to the pharmacokinetics and efficacy of many drugs including CPA, ifosfamide, ketamine, pethidine, methadone, nevirapine and efavirenz (Ekins et al., 2008). For instance, CYP2B6*6 allele carriers have been linked to increased ifosfamide plasma levels and exacerbated toxicities (Mo et al., 2009). Induction of CYP2B6 by a selective activator (e.g., CITCO) facilitates the bioactivation of CPA to 4-OH-CPA and improves the therapeutic outcome of CHOP (cyclophosphamide-doxorubicin-vincristine-prednisone) chemotherapy against non-Hodgkin lymphoma (Shu et al., 2017). Therefore, identification of PER2 as a novel pharmacokinetic determinant of CYP2B10 substrates may facilitate an increased understanding of varied pharmacokinetics and possibly varied drug toxicity and efficacy in a circadian fashion.
A previous study has revealed Cyp2b10 as a circadian gene that is under the control of the Clock gene (Zhao et al., 2019). It is noteworthy that Per2 is a circadian clock gene (whose expression oscillates with time of the day) and involved in regulation of circadian rhythms (Yamamura et al., 2010). Therefore, there is a high possibility that Per2 may be involved in regulation of circadian expression of Cyp2b10. However, of clock genes, Rev-erbα seems to be a direct transcriptional regulator of Cyp2b10 and thus a direct key regulator of circadian Cyp2b10 (Zhang et al., 2018a). Circadian regulation of Cyp2b10 by other clock genes such as Per2 and Clock is possible, but is attained through modulating Rev-erbα (Figure 9). In addition, CAR is another direct transcriptional regulator of Cyp2b10 (Kettner et al., 2016). However, CAR should not be involved in PER2 regulation of Cyp2b10 as it positively regulates Cyp2b10 expression (Kettner et al., 2016). Our findings may provide increased understanding of the complex regulatory pathways for CYP2B10, and highlight the role of clock genes in regulating drug metabolism and pharmacokinetics.
It is noteworthy that we detected a new version of Per2 transcript in Per2 Del4-6 mice albeit at a reduced level ( Figure 1C). This new Per2 transcript most likely is a truncated version lacking exons 4-6 transcribed region, and may be translated to a PER2-related protein that retains a reactivity with the commercial PER2 antibody, thereby explaining why PER2 protein was detected in Per2 Del4-6 mice ( Figure 1D). It is acknowledged that whether or not this PER2related protein is functional remains unknown. However, it was of no concern that Per2 Del4-6 mice can be used to determine the regulatory effects of Per2 on hepatic DME in vivo as they were characterized with loss of wild-type Per2 transcript and markedly reduced PER2 protein in the liver (Figure 1).
CPA is a prodrug and bioactivated by CYP2B10 in mice to the hydroxylated metabolite 4-OH-CPA, one active form with cytotoxic effects and hepatotoxicity (Sadagopan et al., 2001). 4-OH-CPA is unstable in biological samples (Sadagopan et al., 2001). To quantify 4-OH-CPA, the samples were treated with OMHA to transform 4-OH-CPA into an equivalent stable product OH-CPA O-methyloxime for mass spectrometric quantification as previously noted (Sadagopan et al., 2001). Alleviated hepatotoxicity of CPA in Per2 Del4-6 mice supported reduced metabolism of CPA to 4-OH-CPA caused by decreased expression of CYP2B10 (Figures 4, 5). Also, reduced CPA metabolism was consistent with decreased systemic clearance (CL/F) according to the pharmacokinetic analysis ( Table 2).
Current study focused on determination of the regulatory effects of PER2 on hepatic CYP2B10 and drug pharmacokinetics. It is noteworthy that CYP2B10 expression in other drugmetabolizing organs such as the kidney and intestine besides the liver may be also under the control of PER2 in mice, however, a potential role of renal and intestinal CYP2B10 in altered CPA pharmacokinetics remains unresolved. In addition, we have provided in vitro and in vivo evidence that hepatic PER2 regulated CYP2B10 in mice to alter pharmacokinetics and drug toxicity. However, whether PER2 regulates CYP2B6mediated pharmacokinetics and drug toxicity in humans as its counterpart does in mouse liver awaits further investigations.
In summary, CYP2B10 was down-regulated at the mRNA, protein, and enzymatic levels in Per2-deficient mice. Consistently, PER2 positively regulated CYP2B10 expression in both Hepa-1c1c7 and AML-12 cells. Moreover, PER2 regulated Cyp2b10 transcription in a REV-ERBα-dependent manner. Data are mean ± SD (n 3 or 6). In panels A/B, *p < 0.05 (t test). In panels (C-E), *p < 0.05 (one-way ANOVA followed by Bonferroni post hoc test). Rel, Relative. Therefore, PER2 regulates CYP2B10 expression and activity through REV-ERBα, impacting the metabolism and pharmacokinetics of substrate drugs.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal experimental procedures were approved by Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine (Appr. date: 2020-11-19; IACUC Issue No: ZYD-2020-111) and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.