Effects of duration of phenytoin administration on mRNA expression of cytochrome P450 and P-glycoprotein in the liver and small intestine of rats

Department of Pharmacy,Faculty of Pharmacy,Kindai University,3-4-1 Kowakae,Higashi-osaka,Osaka 577-8502,Japan

Effects of duration of phenytoin administration on mRNA expression of cytochrome P450 and P-glycoprotein in the liver and small intestine of rats

Atsushi Kawase*,Hiroyuki Tanaka,Toru Otori,Kenji Matsuyama, Masahiro Iwaki

Department of Pharmacy,Faculty of Pharmacy,Kindai University,3-4-1 Kowakae,Higashi-osaka,Osaka 577-8502,Japan

A R T I C L EI N F O

Article history:

Received 24 August 2015

Received in revised form 5 April 2016

Accepted 12 April 2016

Available online 9 May 2016

Phenytoin

Cytochromes P450

Microsomes

mRNA

Liver

Phenytoin(5,5-diphenylhydantoin;DPH)induces expression of cytochromes P450(CYPs).Interactions between DPH and tacrolimus suggested that the persistence of CYP induction after discontinuation of DPH is dependent on the history of administration and dosing period of DPH.However,the relationship between the duration of DPH administration and expression of CYPs in the liver and small intestine of rats is not known.Alterations in levels of P-glycoprotein(P-gp;MDR1;ABCB1)as well as CYPs cause drug interactions in the small intestine.We examined the effects of the duration of DPH administration on expression of CYPs and P-gp in the liver and small intestine of rats.Rats were treated with DPH(100 mg/kg, peroral(p.o.)twice a day(b.d.))for 2,4,8,and 16 d.mRNA levels of CYPs and P-gp were examined using the total RNA extracted from the liver and duodenum 2 h and 24 h after the fnal administration of DPH.CYP3A activities were determined using microsomes.DPH administration for 2 d and 4 d markedly increased mRNA levels of CYPs such asCYP3A1,CYP3A2,

CYP2B1,andCYP2B2in the liver.A relatively long duration of DPH administration(8 d and 16 d)resulted in abolition of the induction of hepatic CYP but increased CYP3A activities were maintained.These results suggest that the duration of DPH administration could be an important determinant of hepatic CYP induction.

?2016 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1.Introduction

Cytochromes P450(CYPs)belong to a superfamily of proteins containing a heme cofactor.CYPs are expressed abundantly in the liver and small intestine.CYPs are involved in the metabolism of various endogenous and xenobiotic substrates.CYP inhibition and induction by xenobiotics are the causes of drug–drug interactions and can result in adverse effects.It is known that carbamazepine[1,2],phenobarbital[3–5],and phenytoin(5,5-diphenylhydantoin;DPH)[6,7]are potent inducers of CYPs.DPH also shows competitive inhibition for the hydroxylation oftolbutamide[8].CYP3A4 in humans as well as CYP3A1 and CYP3A2 in rats accounts for≤30%of hepatic CYPs[9].CYP3A is very important in hepatic drug metabolism because CYP3A4 participates in the metabolism of≈50%of marketed drugs[10,11]. Intestinal CYPs are involved in frst-pass metabolism in addition to hepatic CYPs.The major intestinal isoforms of CYP3A in rats are CYP3A9,CYP3A18,and CYP3A62[12].

DPH is an anticonvulsant agent.Interactions between DPH and tacrolimus have been reported[13].Wada et al. demonstrated that the persistence of CYP induction after discontinuation of DPH was dependent on the history of administration and dosing period of DPH[14].Patients treated consecutively with DPH display sustained interactions between DPH and tacrolimus.However,the relationship between the duration of DPH administration and CYP expression in the liver and small intestine of rats is not known.

In addition to the metabolism of drugs by CYP3A in the small intestine,drugs are subject to active effux from cells by adenosine triphosphate-binding cassette transporters such as P-glycoprotein(P-gp;MDR1;ABCB1).Substrates for CYP3A and P-gp overlap[15],showing a concerted barrier function for drug absorption.Identifcation of changes in the expression and function of P-gp during drug therapy is important because P-gp is the cause of drug interactions in the small intestine[16,17]. Therefore,we examined the effects of the duration of DPH administration on P-gp expression in the small intestine.

Expression of CYPs and P-gp are regulated on the transcription level by nuclear receptors such as the pregnane X receptor(PXR)and constitutive androstane receptor(CAR) [18–20].We also determined the effects of the duration of DPH administration on expression of PXR and CAR.

2.Material and methods

2.1.Ethical approval of the study protocol

The study protocol was approved by the Committee for the Care and Use of Laboratory Animals of the Faculty of Pharmacy of Kinki University(Osaka,Japan).

2.2.Compounds and reagents

DPH was purchased from Sigma-Aldrich(Saint Louis,MO,USA). TRIzol and a Bicinchoninic Acid(BCA)Protein Assay kit were obtained from LifeTechnologies(Carlsbad,CA,USA)and Pierce Biotechnology(Rockford,IL,USA),respectively.A P450-Glo CYP3A4 Assay kit and a regeneration system for the reduced form of nicotinamide adenine dinucleotide phosphate(NADPH) were purchased from Promega(Madison,WI,USA).All other chemicals and solvents were of the best purity available.

2.3.Animals and treatments

Six-week-old female Sprague–Dawley rats were purchased from Japan SLC(Shizuoka,Japan).Animals were housed in a temperature-controlled room with free access to standard laboratory chow(MF diet;Oriental Yeast,Tokyo,Japan)and water. Rats were treated with DPH(100 mg/kg,peroral(p.o.)twice a day(b.d.))for 2,4,8,and 16 d[21].At 2 h and 24 h after the fnal administration of DPH,animals were anesthetized with diethyl ether.Then,the liver and the proximal 2–3 cm of the duodenum were perfused with ice-cold physiologic(0.9%)saline and removed.After fash freezing in liquid nitrogen,each sample was preserved at?80°C until used for RNA extraction and microsome preparation.

2.4.Determination of mRNA levels by real-time reverse transcriptase-polymerase chain reaction(RT-PCR)

Total RNA was extracted from samples of liver tissue and small intestine tissue using TRIzol.mRNA expression was measuredusingRT-PCR,asdescribedpreviously[22,23]. Oligonucleotide sequences for each mRNA target are shown in Table 1.Data were analyzed using ABI Prism 7000 SDS(Life Technologies)using the multiplex comparative method.

2.5.Preparation of hepatic microsomes

Liver samples were perfused with ice-cold 0.9%saline and chopped into small pieces.A 25%(w/v)homogenate was made in ice-cold 1.15%KCl solution using a Physcotron Homogenizer(Microtec,Chiba,Japan).The homogenate was centrifuged at 12,000×gfor 20 min at 4°C.The supernatant was centrifuged further at 105,000×gfor 60 min at 4°C to obtain a microsomal pellet.The latter was washed by resuspension in 3 ml of 1.15%KCl.The suspension was centrifuged at 105,000×gfor 30 min at 4°C to obtain the fnal microsomal pellet,which was resuspended in 1.5 ml of 1.15%KCl and stored at?80°C until use.Protein concentrations were determined using a BCA Protein Assay kit.

Table 1–Primer sequences used in PCR assays.

2.6.Measurement of CYP3A activity

CYP3A activity was determined using a P450-Glo CYP3A4 Assay kit.CYP3A1 and CYP3A2 activities in rats and CYP3A4 activity in humans were measured according to manufacturer instructions.Briefy,CYP3A reactions were carried out in a 96-well plate(OptiPlate-96;PerkinElmer,Waltham,MA,USA).An incubation mixture(total volume,50 μl)was prepared that contained 200 mM potassium phosphate buffer(pH 7.4),a NADPH regeneration system,20 μg rat liver microsomes,and 50 μM luciferin 6′benzyl ether(luciferin-BE)as a substrate for CYP3A1 and CYP3A2.After preincubation for 10 min at 37°C,the reaction was initiated by addition of the NADPH regeneration system and then incubated for 30 min at 37°C with constant shaking.The reconstituted luciferin detection reagent(50 μl) was added to terminate the reaction and generate chemiluminescence.Luminescence was measured using FLUOstar Optima(Moritex,Tokyo,Japan).

2.7.Statistical analyses

Statistical analyses were carried out using the Dunnett’s test after analysis of variance.

3.Results and discussion

mRNA levels ofCYP3A1andCYP3A2(Fig.1a)and CYP3A activities(Fig.1b)in the liver after DPH administration were determined.mRNA levels ofCYP3A1andCYP3A2were increased signifcantly 2 h and 24 h after the fnal administration of DPH for 4 d.Longer duration of DPH administration(8 d and 16 d)resulted in recovery to the mRNA levels observed in controls.CYP3A activities were increased signifcantly upon DPH administration for 4,8,and 16 d.At 24 h(but not 2 h)after the fnal DPH administration for 2 d,signifcant increases in CYP3A activities were observed.In preliminary study,the larger variations between individuals were observed in the longer term after last administration of DPH(data not shown).These fndings suggested that 2 h after DPH administration for 2 d was not a suffcient time for increases in levels of protein and activity of CYP3A to be observed.Increased activities of CYP3A showed persistence compared with those ofCYP3A1andCYP3A2mRNA after DPH administration.We primarily examined the mRNA levels and activities but not protein levels of CYP3A.It has been reported that the expression ofCYP1A,CYP2B,CYP2C,andCYP3AmRNA were correlated with those protein levels and metabolic activities in human liver[24,25] and rat and human hepatocytes[26].

We examined the effects of duration of DPH administration on the mRNA levels ofCYP2B1,CYP2B2,andCYP2C12(Fig.2). mRNA levels ofCYP2B1andCYP2B2were increased signifcantly at relatively short durations of DPH administration(2 d and 4 d)and tended to return to control levels after DPH administration for 8 d and 16 d.These results forCYP2B1andCYP2B2were similar to those forCYP3A1andCYP3A2.InCYP2C12,slight changes in mRNA levels were observed upon DPH administration.In control rats,CYP2C is highly expressed in the liver compared with CYP3A,which is differentto the situation in humans[27].Reports of a marked induction of expression of CYP2C12 mRNA upon DPH administration are lacking.

Fig.1–Levels ofCYP3A1andCYP3A2mRNA(a)and CYP3A activities(b)in the liver of rats treated with DPH(p.o.,b.d.)for 2, 4,8,and 16 d.At 2 h and 24 h after the fnal administration of DPH,the liver was excised.Data are the mean±SEM (n=3–6).*p＜0.05,**p＜0.01,and***p＜0.001vscontrol,?p＜0.05 and??p＜0.01vs2 h.

Hepatic mRNA levels ofPXRandCARwere examined(Fig.3). Induction of expression of CYP mRNA by DPH is mediated primarily through activation of CAR(but not PXR)in humans [28–30].At 2 h and 24 h after DPH administration for 2 d and 4 d,mRNA levels ofPXRandCARdid not change,but mRNA levels ofCYP3A1,CYP3A2,CYP2B1,andCYP2B2were increased. The ligand-activated transcriptional activities of PXR and CAR could have increased without affecting alterations in the mRNA levels ofPXRandCAR.Further studies are needed to clarify the precise effects of duration of DPH administration on the activities of nuclear receptors.Interestingly,longer duration of DPH administration(8 d and 16 d)resulted in the reduction of expression ofPXRandCARmRNA.Regulation of negative feedback for CYP3A1,CYP3A2,CYP2B1,and CYP2B2 may have occurred.It has been reported that induction of CYPs is limited by negative feedback[31,32].Studies have shown that DPH is metabolized predominantly by CYP2C9,CYP2C19,and CYP3A4[33–35].In these CYP isoforms,hepatic CYP3A activities were increased in rats treated with DPH,suggesting that DPH is eliminated from the body rapidly by repeat administration of DPH.

To clarify the effects of duration of DPH administration on expression of CYP and P-gp in the small intestine,mRNA levels ofCYP3A9,CYP3A18,CYP3A62,Mdr1a,andMdr1bwere determined(Figs.4 and 5).Slight effects of DPH administration on expression ofCYP3A9,CYP3A18,andCYP3A62were observed compared with those on hepatic expression ofCYP3A1,CYP3A2,CYP2B1,andCYP2B2.CYP3A activities in the small intestine did not change upon DPH administration(data not shown).Some drug interactions are mediatedviaP-gp in the small intestine.mRNA levels ofMdr1aandMdr1bin the small intestine did not change upon DPH administration.In preliminary experiments,mRNA levels ofMdr1aandMdr1bin the liver did not change(data not shown).The protein levels of P-gp showed little correlation with the mRNA levels ofMdr1aandMdr1b[25].Further studies are needed to clarify the effects of DPH administration on P-gp activities.

Fig.2–Levels ofCYP2B1,CYP2B2,andCYP2C12mRNA in the liver of rats treated with DPH(p.o.,b.d.)for 2,4,8,and 16 d.At 2 h and 24 h after the fnal administration of DPH,the liver was excised.Data are the mean±SEM(n=3–4).*p＜0.05,**p＜0.01,and***p＜0.001vscontrol.

4.Conclusion

DPH administration for 2 d and 4 d increased the mRNA levels of hepatic CYPs such asCYP3A1,CYP3A2,CYP2B1,andCYP2B2markedly.Relatively long duration of DPH administration(8 d and 16 d)resulted in transcriptional negative feedback forCYP3A1,CYP3A2,CYP2B1,andCYP2B2.However,the increased activities of CYP3A were maintained.These results suggest that the duration of DPH administration could be an important determinant of CYP induction in the liver.

Fig.3–Levels ofPXRandCARmRNA in the liver of rats treated with DPH(p.o.,b.d.)for 2,4,8,and 16 d.At 2 h and 24 h after the fnal administration of DPH,the liver was excised.Data are the mean±SEM(n=3–4).*p＜0.05,**p＜0.01,and ***p＜0.001vscontrol.

Fig.4–Levels ofCYP3A9,CYP3A18,andCYP3A62mRNA in the duodenum of rats treated with DPH(p.o.,b.d.)for 2,4,8, and 16 d.At 2 h and 24 h after the fnal administration of DPH,the small intestine was excised.Data are the mean±SEM (n=3–4).*p＜0.05vscontrol.

Fig.5–Levels ofMdr1a and Mdr1bmRNA in the duodenum of rats treated with DPH(p.o.,b.d.)for 2,4,8,and 16 d.At 2 h and 24 h after the fnal administration of DPH,the duodenum was excised.Data are the mean±SEM(n=3–4).

Acknowledgments

This work was supported by the“Antiaging”Project for Private Universities,with a matching fund subsidy from the Japanese Ministry of Education,Culture,Sports,Science and Technology(MEXT).This research was also supported in part by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities,2014–2018 (S1411037).

R E F E R E N C E S

[1]Tateishi T,Asoh M,Nakura H,et al.Carbamazepine induces multiple cytochrome P450 subfamilies in rats.Chem Biol Interact 1999;117:257–268.

[2]Yamashita H,Kazawa T,Minatogawa Y,et al.Time-course of hepatic cytochrome p450 subfamily induction by chronic carbamazepine treatment in rats.Int J Neuropsychopharmacol 2002;5:47–52.

[3]Alvares AP,Schilling G,Levin W,et al.Studies on the induction of CO-binding pigments in liver microsomes by phenobarbital and 3-methylcholanthrene.Biochem Biophys Res Commun 1967;29:521–526.

[4]Tsyrlov IB,Zakharova NE,Gromova OA,et al.Possible mechanism of induction of liver microsomal monooxygenases by phenobarbital.Biochim Biophys Acta 1976;421:44–56.

[5]Sidhu JS,Farin FM,Omiecinski CJ.Infuence of extracellular matrix overlay on phenobarbital-mediated induction of CYP2B1,2B2,and 3A1 genes in primary adult rat hepatocyte culture.Arch Biochem Biophys 1993;301:103–113.

[6]Spina E,Pisani F,Perucca E.Clinically signifcant pharmacokinetic drug interactions with carbamazepine. Clin Pharmacokinet 1996;31:198–214.

[7]Yamazaki H,Komatsu T,Takemoto K,et al.Decreases in phenytoin hydroxylation activities catalyzed by liver microsomal cytochrome P450 enzymes in phenytoin-treated rats.Drug Metab Dispos 2001;29:427–434.

[8]Zweers-Zeilmaker WM,Horbach GJ,Witkamp RF. Differential inhibitory effects of phenytoin,diclofenac, phenylbutazone and a series of sulfonamides on hepatic cytochrome P4502C activity in vitro,and correlation with some molecular descriptors in the dwarf goat(Caprus hircus aegagrus).Xenobiotica 1997;27:769–780.

[9]Shimada T,Yamazaki H,Mimura M,et al.Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs,carcinogens and toxic chemicals:studies with liver microsomes of 30 Japanese and 30 Caucasians.J Pharmacol Exp Ther 1994;270:414–423.

[10]Kim RB,Wandel C,Leake B,et al.Interrelationship between substrates and inhibitors of human CYP3A and P-glycoprotein.Pharm Res 1999;16:408–414.

[11]Cummins CL,Jacobsen W,Benet LZ,et al.Unmasking the dynamic interplay between intestinal P-glycoprotein and CYP3A4.J Pharmacol Exp Ther 2002;300:1036–1045.

[12]Matsubara T,Kim HJ,Miyata M,et al.Isolation and characterization of a new major intestinal CYP3A form, CYP3A62,in the rat.J Pharmacol Exp Ther 2004;309:1282–1290.

[13]Thompson PA,Mosley CA.Tacrolimus-phenytoin interaction.Ann Pharmacother 1996;30:544.

[14]Wada K,Takada M,Ueda T,et al.Drug interactions between tacrolimus and phenytoin in Japanese heart transplant recipients:2 case reports.Int J Clin Pharmacol Ther 2007;45:524–528.

[15]Zhang Y,Guo X,Lin ET,et al.Overlapping substrate specifcities of cytochrome P450 3A and P-glycoprotein for a novel cysteine protease inhibitor.Drug Metab Dispos 1998;26:360–366.

[16]Su SF,Huang JD.Inhibition of the intestinal digoxin absorption and exsorption by quinidine.Drug Metab Dispos 1996;24:142–147.

[17]Greiner B,Eichelbaum M,Fritz P,et al.The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J Clin Invest 1999;104:147–153.

[18]Lehmann JM,McKee DD,Watson MA,et al.The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions.J Clin Invest 1998;102:1016–1023.

[19]Geick A,Eichelbaum M,Burk O,et al.Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin.J Biol Chem 2001;276:14581–14587.

[20]Burk O,Arnold KA,Nussler AK,et al.Antimalarial artemisinin drugs induce cytochrome P450 and MDR1 expression by activation of xenosensors pregnane X receptor and constitutive androstane receptor.Mol Pharmacol 2005;67:1954–1965.

[21]Carl GF,Smith DB.The effect of chronic phenytoin treatment on tissue folate concentrations and on the activities of the methyl synthetic enzymes in the rat.J Nutr 1983;113:2368–2374.

[22]Kawase A,Fujii A,Negoro M,et al.Differences in cytochrome P450 and nuclear receptor mRNA levels in liver and small intestine between SD and DA rats.Drug Metab Pharmacokinet 2008;23:196–206.

[23]Kawase A,Yamada A,Gamou Y,et al.Increased effects of ginsenosides on the expression of cholesterol 7 alphahydroxylase but not the bile salt export pump are involved in cholesterol metabolism.J Nat Med 2013;67:545–553.

[24]Watanabe M,Kumai T,Matsumoto N,et al.Expression of CYP3A4 mRNA is correlated with CYP3A4 protein level and metabolic activity in human liver.J Pharmacol Sci 2004;94:459–462.

[25]Ohtsuki S,Schaefer O,Kawakami H,et al.Simultaneous absolute protein quantifcation of transporters,cytochromes P450,and UDP-glucuronosyl transferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities.Drug Metab Dispos 2012;40:83–92.

[26]Richert L,Tuschl G,Abadie C,et al.Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes.Toxicol Appl Pharmacol 2009;235:86–96.

[27]Cheng KC,Schenkman JB.Purifcation and characterization of two constitutive forms of rat liver microsomal cytochrome P-450.J Biol Chem 1982;257:2378–2385.

[28]Wang H,Faucette S,Moore R,et al.Human constitutive androstane receptor mediates induction of CYP2B6 gene expression by phenytoin.J Biol Chem 2004;279:29295–29301.

[29]Jackson JP,Ferguson SS,Moore R,et al.The constitutive active/androstane receptor regulates phenytoin induction of Cyp2c29.Mol Pharmacol 2004;65:1397–1404.

[30]Jackson JP,Ferguson SS,Negishi M,et al.Phenytoin induction of the cyp2c37 gene is mediated by the constitutive androstane receptor.Drug Metab Dispos 2006;34:2003–2010.

[31]Schuetz E,Strom S.Promiscuous regulator of xenobiotic removal.Nat Med 2001;7:536–537.

[32]Bailey I,Gibson GG,Plant K,et al.A PXR-mediated negative feedback loop attenuates the expression of CYP3A in response to the PXR agonist pregnenalone-16α-carbonitrile. PLoS ONE 2011;6:e16703.

[33]Maguire JH.Quantitative estimation of catechol/ methylcatechol pathways in human phenytoin metabolism. Epilepsia 1988;29:753–759.

[34]Szabo GK,Pylilo RJ,Davoudi H,et al.Simultaneous determination ofp-hydroxylated and dihydrodiol metabolites of phenytoin in urine by high-performance liquid chromatography.J Chromatogr 1990;535:279–285.

[35]Komatsu T,Yamazaki H,Asahi S,et al.Formation of a dihydroxy metabolite of phenytoin in human liver microsomes/cytosol:roles of cytochromes P450 2C9,2C19, and 3A4.Drug Metab Dispos 2000;28:1361–1368.

*< class="emphasis_italic">Corresponding author.

.Kindai University,3-4-1 Kowakae,Higashi-osaka,Osaka 577-8502,Japan.Tel.:+81 6 4307 3626;fax:+81 6 6730 1394.E-mail address:kawase@phar.kindai.ac.jp(A.Kawase).

http://dx.doi.org/10.1016/j.ajps.2016.04.003

1818-0876/?2016 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).