Proc. NatL Acad. Sci. USAVol. 78, No. 2, pp. 893-897, February 1981Biochemistry

Effect of phenobarbital on the level of translatable rat liverepoxide hydrolase mRNA

(drug-metabolizing enzymes/phenobarbital induction/cell-free protein synthesis/immunoprecipitation)

CECIL B. PICKETT* AND ANTHONY Y. H. LutDepartments of *Biochemical Regulation and tAnimal Drug Metabolism and Radiochemistry, Merck Sharp & Dohme Research Laboratories,Rahway, New Jersey 07065

Communicated by P. Roy Vagelos, November 3, 1980

ABSTRACT Liver poly(A)+RNA isolated from untreated andphenobarbital-treated rats has been translated in the rabbit re-ticulocyte cell-free system in order to determine the level of trans-lationally active epoxide hydrolase (EC 3.3.2.3) mRNA. The invitro translation systems were immunoprecipitated with rabbitIgG prepared against purified epoxide hydrolase, and the amountof epoxide hydrolase synthesized by the lysate programmed withcontrol and phenobarbital poly(A)+RNA was quantitated. Thelevel of translatable epoxide hydrolase mRNA is increased 3-foldafter chronic phenobarbital administration. This level of inductioncorrelates well with the 5-fold induction in catalytic activity ofepoxide hydrolase (using styrene 7,8-oxide as substrate) in micro-somes isolated from phenobarbital-treated rats. Therefore, wesuggest that chronic phenobarbital administration increases theamount of functional epoxide hydrolase in rat liver microsomesby way of an increase in the translatable mRNA level encodingfor the enzyme. We do not know whether the increase in mRNAis the result of increased transcription or messenger stability.

Cytochrome P-450 and epoxide hydrolase (EC 3.3.2.3) are in-tegral membrane proteins of the endoplasmic reticulum andfunction in concert in the metabolism of various drugs, muta-gens, and carcinogens (1-5). Although barbituates such as phen-obarbital have been shown to increase the level or activity ofcytochrome P-450 and epoxide hydrolase in rat liver (6-8), itwas unknown whether this increase was related to an increasein the level of translatable mRNA for these proteins. With therecent advances in the purification of multiple species of cy-tochrome P-450, the development of specific antibodies tothese different forms (9-11), and the refinements made in cell-free translation systems, it became possible to examine themolecular basis of induction of cytochrome P-450 by variousxenobiotics. As a result, studies from a number of laboratories(12-16) have demonstrated that the level of functional mRNAfor the phenobarbital-inducible species of cytochrome P-450 inrats is increased by 12-16 hr after a single injection ofphenobarbital.

Despite the recent advances in the purification and charac-terization of rat liver epoxide hydrolase, only recently have anystudies focused on the capacity of mRNA isolated from pheno-barbital-treated rats to direct the synthesis of epoxide hydro-lase (17, 18). Both of these brief communications reported thatthe primary translation product of epoxide hydrolase appearsto be synthesized in cell-free systems as the mature enzyme.These findings suggest that, unlike many secreted proteins andsome membrane proteins, epoxide hydrolase appears not torequire a cleavable signal sequence for insertion into the en-doplasmic reticulum membrane.

In the present investigation we have utilized cell-free proteinsynthesis and specific immunoprecipitation to quantitate the

level of translationally active rat liver epoxide hydrolase mRNAafter chronic phenobarbital administration and have correlatedthe mRNA level with the catalytic activity of the enzyme inmicrosomes isolated from phenobarbital-treated animals. Ourdata indicate that the functional mRNA level and the catalyticactivity of epoxide hydrolase are induced by phenobarbital tosimilar extents.

METHODSPreparation and Characterization of Antibodies Against

Purified Epoxide Hydrolase. Epoxide hydrolase was isolatedfrom microsomes of phenobarbital-treated rats by the methodof Knowles and Burchell (19) as modified by Guengerich et al.(20). Antibody against rat liver epoxide hydrolase was raised inrabbits by intradermal injections (eight sites), 200 ,g per ani-mal, of purified enzyme mixed with Freund's complete adju-vant. Booster injections consisting of 70 ,g of epoxide hydro-lase mixed with Freund's incomplete adjuvant were given atapproximately 7-day intervals. Rabbit IgG was partially purifiedfrom the antiserum by affinity chromatography on DEAE-Affi-Gel blue.

Antibody specificity was determined by Ouchterlony doubleimmunodiffusion (21) and Laurell rocket immunoelectropho-resis (22).RNA Isolations. Male Sprague-Dawley rats (120-170 g)

were used in all experiments. Control animals were given waterad lib, phenobarbital-treated rats were given sodium pheno-barbital in their drinking water (1 mg/ml) for 8 days before sac-rifice. Total liver RNA was isolated by the guanidine thiocya-nate/cesium chloride procedure (23). Livers were rapidlyhomogenized in 4 M guanidine thiocyanate (Tridom, Haup-page, NY)/0.5% sodium N-lauroylsarcosine/25 mM sodium ci-trate, pH 7.0/0.1 M 2-mercaptoethanol. The homogenate waslayered over 1.2 ml of 5.7 M cesium chloride buffered with 0.1M sodium EDTA (pH 7.0) and centrifuged for 20 hr at 40,000rpm and 20°C in a Beckman SW 60 rotor. The RNA pellets wereresuspended in 7.5 M guanidine hydrochloride (Bethesda Re-search Laboratories, Rockville, MD)/5 mM dithiothreitol andbuffered in 0.025 vol of 1 M sodium citrate at pH 7. 0. The RNApellets were heated at 68°C until dissolved and precipitated byaddition of 0.025 vol of 1 M acetic acid and 0.5 voi of absoluteethanol. The precipitated RNA was centrifuged at 10,000 rpmfor 10 min at 10°C in a SS-34 rotor. The final RNA pellet waswashed in absolute ethanol and centrifuged at 10,000 rpm for10 min.The pellet was dried with nitrogen and resuspended in sterile

water at a concentration of 20-30 A260 units/ml. The RNA sus-pension was heated for 2 min at 68°C, rapidly cooled to roomtemperature, and adjusted to (final concentration) 0.5 M in lith-ium chloride, 0.5% in NaDodSO4, 1 mM in EDTA, and 10 mMin Tris'HCI (pH 7.4). The RNA was passed over an oligo(dT)-

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cellulose column (type T-3, Collaborative Research, Waltham,MA) that had been equilibrated with the same buffer.Poly(A)+RNA was eluted from the column with 10 mMTris HCl, pH 7.4. The solution was adjusted to 0.2 M in po-tassium acetate (pH 5.0) and precipitated overnight with 2 volof absolute ethanol. The precipitated poly (A)+RNA was cen-trifuged at 10,000 rpm for 30 min and +50C in a SS-34 rotor.The pellet was lyophilized and resuspended in sterile water.

Cell-Free Protein Synthesis. Total liver poly(A)+RNA wastranslated for 60 min in a micrococcal nuclease-treated rabbitreticulocyte lysate system (24). The reaction mixture (90 p1)contained 30 p.l of lysate, 25 mM Hepes (pH 7.4), 1 mM crea-tine phosphate, 48 mM KC1, 87 mM K acetate, 1.2 mM MgCl2,amino acids (without methionine) at 50 ,uM each, 20 ,uCi of[35S]methionine (1100 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels),and 1.00 pug of poly(A)+RNA. To assess the incorporation of[35S]methionine into protein, 2 ,ul of each translation mixturewas spotted onto Whatman 3 MM filter paper and the paperwas placed into ice-cold 10% trichloroacetic acid. The filter pa-pers were boiled for 15 min in 5% trichloroacetic acid and thenrinsed thoroughly with cold 5% trichloroacetic acid, 100%ethanol, ethanol/ether, 1:1 (vol/vol), and, finally, ether. Thefilter papers were dried and placed in scintillation vials; 500 ,ulof Protosol (New England Nuclear) was added to each vial andthe vials were incubated for 30 min at 55°C. The vials were thencooled to room temperature and 10 ml of Econofluor was addedin preparation for liquid scintillation counting. Aliquots (5-101d) of the translation mixtures were also subjected to Na-DodSO4/polyacrylamide gel electrophoresis on 7.5% gels (25).

Immunoprecipitation and Quantitation of Epoxide Hydro-lase. Epoxide hydrolase was immunoprecipitated from thetranslation mixtures as described (18) with formalin-fixed, heat-inactivated Staphylococcus aureus cells. Immunoprecipitateswere subjected to NaDodSOJpolyacrylamide gel electropho-resis on 7.5% gels. [35S]Methionine-labeled epoxide hydrolasewas detected by fluorography (26). In order to quantitate thelevel of translatable epoxide hydrolase mRNA, the region of thegel corresponding to the position of purified epoxide hydrolasewas excised and dissolved in 60% perchloric acid/30% hydrogenperoxide at 60°C. Aquasol-2 was then added to the solubilizedgel and total radioactivity was determined by liquid scintillationcounting. Background radioactivity was determined by excisinggel regions above and below the radiolabeled epoxide hydrolaseand dissolving the slices as just described. Values obtained werethen subtracted from the radioactivity obtained in the slicescontaining epoxide hydrolase.

Determination of Epoxide Hydrolase Activity. Epoxide hy-drolase activity was measured in liver microsomes isolated fromuntreated and phenobarbital-treated (8 days) rats by the methodof Oesch et al. (27) with [3H]styrene oxide as substrate. Proteinwas determined by the method of Lowry et al. (28) with bovineserum albumin as standard.

RESULTSThe purified epoxide hydrolase preparation utilized in thisstudy for antibody production consisted of a single major bandon NaDodSOJpolyacrylamide gel electrophoresis with an es-timated molecular weight of 50,000 (Fig. 1), which is similarto values reported previously (19, 20, 29-31). The purified en-zyme had a specific activity of 577 nmol/min per mg of proteinwith sytrene 7,8-oxide as substrate. When rabbit anti-epoxidehydrolase antiserum was allowed to react against either the pu-rified enzyme or detergent-solubilized microsomes isolatedfrom untreated or phenobarbital-treated rats, a single immu-noprecipitin band formed after either Ouchterlony double im-

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A B

FIG. 1. NaDodSO4/polyacrylamide gel electrophoresis of purifiedepoxide hydrolase (lane B, 5 ,4g) used in this study for antibody prep-aration and characterization. Lane A, molecular weight markers: bo-vine serum albumin (68,000), ovalbumin (45,000), and carbonic an-hydrase (30,000). The estimated molecular weight of purified epoxidehydrolase is 50,000.

munodiffusion or Laurell rocket immunoelectrophoresis (Fig.2). Ouchterlony double-immunodiffusion gels also revealedlines of identity between the purified enzyme isolated fromphenobarbital-treated rats and detergent-solubilized micro-somes isolated from untreated or phenobarbital-treated rats.These latter data suggest immunochemical identity between thepurified enzyme and the forms present in control and inducedmicrosomes.

Quantitation of the Translatable mRNA Level for Rat LiverEpoxide Hydrolase. As reported (18) poly(A)+RNA isolatedfrom untreated and phenobarbital-treated rats have similartranslational activities in the rabbit reticulocyte lysate system.Incorporation of [3S]methionine into total protein increasedas a function of the amount of RNA. added to the lysate. How-ever, we generally observed a decrease in incorporation at RNAconcentrations >50 pUg/ml.When the translation products directed by poly(A)+RNA

isolated from phenobarbital-treated rats were analyzed byNaDodSOjpolyacrylamide gel electrophoresis and autoradi-ography, we noted an increase in [3S]methionine incorporationinto a polypeptide(s) with a molecular weight of 50,000-52,000(Fig. 3). This molecular weight range corresponds to the sizeof both epoxide hydrolase and the phenobarbital-inducible spe-cies of cytochrome P-450: 50,000 and 52,000, respectively(32-35). We have reported previously (18) that phenobarbitalenhances the synthesis not only of a polypeptide(s) of molecularweight 50,000-52,000 but also of two additional polypeptides

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FIG. 2. Ouchterlony double immunodiffusion performed in anagar gel from Hyland Laboratories, Costa Mesa, CA. Wells 3 and 5,purified epoxide hydrolase; wells 1 and 2, microsomes isolated fromuntreated rats and phenobarbital-treated rats, respectively; centerwell, antiserum against epoxide hydrolase (60 mg/ml); well 4, phos-phate-buffered saline. Incubation was at room temperature for 24 hr.Rat liver microsomes were solubilized in 0.25 M sucrose/0.01 MTris-HCl, pH 7.4/0.2% Emulgen/0.5% sodium cholate. The proteinconcentrations for the purified enzyme and microsomes isolated fromuntreated and phenobarbital-treated rats were 0.77, 9.4, and 4.7 mg/ml, respectively. (B) Rocket immunoelectrophoresis in a 1% agaroseslab gel containing epoxide hydrolase antiserum (1.5 mg/ml) and theGelman high-resolution Tris/barbital/sodium barbital buffer at pH8.8. Seven microliters of purified enzyme or microsomes isolated fromuntreated or phenobarbital-treated rats was placed in the sample wellsand electrophoresis was carried out at 100 V for 22 hr at 15TC. Well 1,purified epoxide hydrolase; wells 2 and 3, detergent-solubilized mi-crosomes isolated from phenobarbital-treated and untreated rats, re-spectively. The microsomes were solubilized in 0.25 M sucrose/0.01 MTris-HCl, pH 7.4/0.2% Emulgen/0.5% sodium cholate. The proteinconcentrations for the purified enzyme and microsomes isolated fromuntreated or phenobarbital-treated rats were 0.77, 9.4, and 4.7 mg/ml, respectively. After electrophoresis the gel was washed with 0.15M NaCl, blotted, washed again in 0.15M NaCl, and dried. The gel wasthen stained with 0.5% Coomassie blue R-250 in ethanol/acetic acid/water, 4.5:1:4.5 (vol/vol).

with molecular weights of approximately 29,000 and 27,000.Our total liver poly(A)+RNA preparations do direct the synthe-sis of polypeptides with molecular weights ranging up to200,000. However, because of the length of exposure of the x-ray film to the radioactive gel, these products cannot be seenin Fig. 3.

In order to quantitate the amount of epoxide hydrolase syn-thesized by the rabbit reticulocyte lysate programmed with totalliver poly(A)+RNA, translation mixtures were subjected to im-munoprecipitation with rabbit anti-epoxide hydrolase IgG. An-tigen-antibody complexes were recovered with formalin-fixed,heat-inactivated S. aureus cells. We found that pretreatment ofthe translation mixtures with an aliquot of the S. aureus cellsbefore immunoprecipitation was absolutely necessary to lowernonspecific binding to the cells.

The immunoprecipitates obtained from control and pheno-barbital poly(A)'RNA translationswere analyzedby NaDodSOJpolyacrylamide gel electrophoresis. Analysis of the fluorogramsof the dried gels revealed a major band of radioactivity asso-ciated with a polypeptide that had an approximate molecularweight of 50,000 (Fig. 4). This polypeptide comigrated exactlywith purified epoxide hydrolase which is in agreement withprevious studies from our laboratory (18) as well as Kasper's lab-oratory (17). When an excess of unlabeled epoxide hydrolasewas added to the translation mixtures before immunoprecipi-tation, no radioactive polypeptide was observed. Furthermore,as found previously (18), when antibody to epoxide hydrolasewas replaced with nonimmune IgG, no radioactive polypeptidecorresponding to epoxide hydrolase was immunoprecipitatedfrom the translation mixtures. The immunoprecipitated epox-

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FIG. 3. NaDodSO4/polyacrylamide gel electrophoresis of[35SImethionine-labeled translation products directed by total liverpoly(A)+RNA isolated from untreated rats (lane B) and phenobarbital-treated rats (lane C). Lane A represents endogenous protein synthesisby the rabbit reticulocyte lysate in the absence of exogenously suppliedpoly(A)+RNA. Equivalent amounts of radioactivity from the transla-tion mixtures programmed with poly(A)+RNA were layered on the gel.The arrow denotes the 50,000-52,000 molecular weight range, wherea major increase in [35Slmethionine incorporation is seen. The molec-ular weight markers were bovine serum albumin (68,000), ovalbumin(45,000), and carbonic anhydrase (30,000).

ide hydrolase could be visualized by fluorography more readilyin translation mixtures directed by liver poly(A)+RNA isolatedfrom phenobarbital-treated rats compared to that from un-treated rats (compare lanes A and B in Fig. 4). However, uponlonger exposure of the x-ray film to the gel, radiolabeled epox-ide hydrolase directed by control poly(A)+RNA became readilydetectable (Fig. 4, lane C).

The level of mRNA specific for epoxide hydrolase was quan-titated by comparing the amount of radioactivity that comi-grated with authentic epoxide hydrolase with the total radio-activity incorporated into protein. It is clear from this type ofanalysis that approximately 3 times more epoxide hydrolase wassynthesized by translation systems programmed withpoly(A)+RNA isolated from phenobarbital-treated rats than bytranslation systems programmed with poly(A)'RNA isolatedfrom untreated rats (Table 1). These data indicate that the levelof functional mRNA for epoxide hydrolase is increased afterchronic phenobarbital administration to rats. In addition, theinduction of epoxide hydrolase mRNA after chronic phenobar-bital administration found in this study corresponds exactly withthe increase observed after acute administration of the drug(17).

In order to correlate the level of translationally active mRNAwith the induction of catalytic activity of epoxide hydrolase, wedetermined the activity of the enzyme in microsomes with sty-rene 7,8-oxide as substrate. The activity was linear over a widerange of protein concentrations (Fig. 5) and was approximately5 times higher in microsomes isolated from phenobarbital-treated (8 days) rats compared to microsomes isolated from un-treated rats (Table 1). Therefore, the increase in catalytic ac-tivity of epoxide hydrolase in isolated microsomes is in goodagreement with the induction we have observed in the func-tional mRNA level for the enzyme in the liver.

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FIG. 4. Fluorogram of NaDodSO4/polyacrylamide gel electropho-resis of immunoprecipitates obtained from translation mixtures pro-grammed with poly(A)+RNA isolated from phenobarbital-treated(lane A) and untreated (lanes B and C) rats. Immunoprecipitationswere performed by using an equivalent amount of input radioactivityfrom each translation assay. An equal aliquot of the immunoprecipi-tate was layered on the 7.5% polyacrylamide gel. Lanes A and B rep-resent a 24-hr fluorogram and lane C represents a 72-hr fluorogramof translation products directed by poly(A)+RNA isolated from un-treated rats. The arrow corresponds to the position of purified epoxidehydrolase in the Coomassie brilliant blue-stained gel from which thefluorograms were made.

DISCUSSIONIn this investigation we found that chronic phenobarbitaladministration results in an increase of translationally active ratliver mRNA for epoxide hydrolase. Furthermore, the relativeincrease in the level of mRNA, approximately 3-fold, correlatesfairly well with the induction of catalytic activity, approximately5-fold, in microsomes isolated from phenobarbital-treated rats.Although we have suggested that the increase in epoxide hy-

Table 1. Induction of rat liver epoxide hydrolasemRNA by phenobarbital

cpm inimmuno-

cpm in precipitatedtotal epoxide

protein,* hydrolase, CatalyticTreatment x 1o-6 x 10-4 activityt

Exp. 1None 12.2 0.45 4.73Phenobarbital 12.2 1.32 23.80

Exp. 2None 11.4 0.37Phenobarbital 11.4 1.22

* Values are corrected for endogenous protein synthesis in the rabbitreticulocyte lysate in the absence of exogenous mRNA.

t Catalytic activity is defined as nmol of styrene glycol formed per minper mg of protein.

Protein, mg

FIG. 5. Catalytic activity of epoxide hydrolase in microsomes iso-lated from untreated (0) and phenobarbital-treated (-) rats. The cat-alytic activity was determined with styrene 7,8-oxide as substrate andis expressed as nmol of styrene glycol formed per minute. An averagevalue for all the protein concentrations is shown in Table 1.

drolase mRNA leads to an increased level of the enzyme, wehave not directly quantitated the amount of epoxide hydrolasein microsomes isolated from untreated and phenobarbital-treated rats. However, studies by Thomas et al. (36) using radialimmunodiffusion to quantitate the amount of epoxide hydrolaseindicate that administration of phenobarbital to rats results ina 3-fold increase in the amount of the enzyme present in mi-crosomes. In addition, they found that the catalytic activity ofepoxide hydrolase (octene oxide as substrate) was increased 3-fold, which correlates well with their immunological quantita-tion. Furthermore, previous studies (37) have demonstratedthat the catalytic activity of epoxide hydrolase is induced byphenobarbital with either octene oxide or styrene oxide as sub-strate to the same extent. Therefore, we think that the increasein activity we noted after chronic phenobarbital administrationis the result of an increase in the amount of enzyme presentin microsomes isolated from phenobarbital-treated rats. Ourfindings also suggest that the increase in epoxide hydrolase in-duced by phenobarbital is probably due not to altered rates ofdegradation of the protein but rather to increased synthesisbrought about by increased levels of mRNA encoding for theprotein.

Although the mechanism by which phenobarbital increasesthe concentration of certain enzymes involved with drug me-tabolism appears to be at the transcriptional or post-transcrip-tional level, no direct evidence has yet been obtained to indicatethe presence of a phenobarbital receptor. However, the spe-cific increases in mRNAs encoding for the drug-metabolizingenzymes after phenobarbital administration are similar to theincrease in transcription of select mRNAs after steroid hormoneadministration (38-40). Therefore, the induction of enzymesby phenobarbital certainly follows the model proposed for ste-roid hormones whereby hormone-receptor complex interactswith chromatin to elicit selective increases in transcription ofspecific mRNAs.

In our current study we do not know if the increase in trans-latable epoxide hydrolase mRNA reflects increased transcrip-tion, increased processing of a putative mRNA precursor into

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a translatable form, or just simply increased message stability.The third possibility has been proposed as a general mechanismaccounting for the increases in mRNA levels induced by pheno-barbital (41, 42). However, if this were the case, the mechanismby which the rate of mRNA degradation is decreased must beselective because only the level of a few mRNAs are affected.Furthermore, Gonzalez and Kasper (17) have found that cor-dycepin, an RNA synthesis chain terminator, can completelyinhibit the increase in epoxide hydrolase mRNA after an acuteadministration of phenobarbital. Therefore, the inhibition ofepoxide hydrolase mRNA by cordycepin and the rapid increasethese authors found in functional epoxide hydrolase mRNA sug-gest that the increase might be due in part to increased tran-scription. However, we would like to stress that the mechanism(increased transcription, message stability, or precursor pro-cessing) responsible for the increase in epoxide hydrolasemRNA may be totally different when animals are treated chron-ically with phenobarbital as opposed to an acute administration.

The use of a translational assay for quantitating mRNA levelsis also subject to certain reservations. For example, differentmRNA species may be translated with different efficiencies inthe in vitro system. As a result, the absolute percentage ofepoxide hydrolase synthesized relative to the total proteinmight not accurately reflect the percentage of mRNA for thatspecific protein in the total RNA preparation. However, we dobelieve that the relative differences in mRNA levels found forepoxide hydrolase in untreated versus phenobarbital-treatedrats is valid. The concentration of mRNA for epoxide hydrolasein the total RNA preparation could be determined more ac-curately if a specific probe for epoxide hydrolase sequenceswere available.The development of a specific probe for epoxide hydrolase

will allow us not only to examine the molecular basis of induc-tion of the enzyme by various xenobiotics but also to understandmore fully the expression of the epoxide hydrolase gene duringdevelopment. In addition, because epoxide hydrolase activityis markedly increased in hyperplastic nodules and in hepatomasby hepatocarcinogens (43, 44), a thorough understanding of theorganization and expression of the epoxide hydrolase gene willalso provide a greater insight into the molecular mechanismsunderlying liver tumoragenesis.The authors thank N. R. Rosenstein and R. L. Jeter for biochemical

assistance and J. Kiliyanski for help in preparation of this manuscript.

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