Inactivation of Stress Protein p8 Increases MurineCarbon Tetrachloride Hepatotoxicity via

Preserved CYP2E1 ActivityDavid Taıeb, Cedric Malicet, Stephane Garcia, Palma Rocchi, Christiane Arnaud, Jean-Charles Dagorn,

Juan L. Iovanna, and Sophie Vasseur

The p8 protein is a transcription factor that regulates the expression of genes involved in celldefense against the adverse effects of stress. Its expression is strongly, rapidly, and transientlyinduced in most cells on exposure to various stress agents. This study assessed the role of p8in the response of the liver to CCl4-induced injury. We found that p8 was indeed overex-pressed in the liver after CCl4 administration. Hepatic injury following CCl4 injection wasmonitored in wild-type and p8�/� mice. Serum alanine and aspartate aminotransferaseactivities were higher and peaked earlier in p8�/� mice than in wild-type mice, which is inagreement with the observation of significantly larger areas of necrosis in p8�/� liver. Ab-sence of p8 expression is therefore associated with increased liver sensitivity to CCl4. In fact,CCl4 toxicity is mediated by derivatives generated by its conversion by the enzyme CYP2E1.It is known that CYP2E1 is downregulated in the liver during the first hours following CCl4administration as part of a self-defense mechanism. We found that CYP2E1 downregulationwas significantly delayed in p8�/� liver compared with wild-type liver, allowing increasedproduction of toxic CCl4 derivatives. In conclusion, inactivation of the p8 gene increasesliver sensitivity to CCl4, as it appears to delay the triggering of CYP2E1 downregulation. Thep8 protein is therefore an important element of hepatocyte stress response. (HEPATOLOGY

2005;42:176-182.)

The p8 protein was first identified as a new stress-induced protein that is strongly and rapidly butalso transiently activated in pancreatic acinar cells

during the acute phase of pancreatitis.1 Further experi-ments also demonstrated that p8 mRNA expression isstrongly activated in response to several stresses such assystemic lipopolysaccharide (LPS) administration.2

Moreover, minimal stresses such as routine change of theculture medium also induce p8 gene expression.3 Activa-tion is not restricted to pancreatic cells but occurs also in

the liver, kidney, brain, and intestine.2 Therefore, p8 is aubiquitous protein expressed in response to cellular stressinduction. Several functions—some of which are difficultto reconcile—have been attributed to p8, such as growthpromotion1,4 or inhibition5,6 and promotion of apopto-sis.5 In addition, transforming growth factor �-1 activatesp8 expression, which in turn enhances the Smad-transac-tivating function responsible for transforming growth fac-tor �-1 activity.7 Finally, p8 was also shown to promotetumor growth.8

The messenger RNA (mRNA) of p8 comprises about600 nucleotides and shows a single open reading frameencoding a protein of 80 amino acids. Analysis of p8primary structure showed presence of a canonical bipar-tite nuclear localization signal sequence expected to pro-mote nuclear targeting. The protein was indeed localizedto the nucleus—though not exclusively, because somelabeling was observed in the cytoplasm.4 The p8 proteinshares many features of the HMG-I(Y) protein,9 whichmodulates gene expression by inducing changes in DNAconformation.10-13 On that basis, p8 was defined as a tran-scription factor that regulates gene expression to improvecell resistance to stress.14 More recently, we compared by

Abbreviations: LPS, lipopolysaccharide; mRNA, messenger RNA; ALT, alanineaminotransferase; AST, aspartate aminotransferase; PCNA, proliferative cell nu-clear antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling ; RT-PCR, reverse-transcription polymerase chain reaction.

From INSERM Unite 624, Stress Cellulaire, Parc Scientifique et Technologiquede Luminy, Marseille, France.

Received February 3, 2005; accepted April 20, 2005.Address reprint requests to: Juan Iovanna, Centre de Recherche INSERM, Unite

624, Stress Cellulaire, Parc Scientifique et Technologique de Luminy case 915,13288 Cedex 9 Marseille, France. E-mail: iovanna@marseille.inserm.fr; fax: (33)491826083.

Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20759Potential conflict of interest: Nothing to report.

176

DNA microarray analysis the modifications in the patternof gene expression induced by LPS in the livers of p8�/�

and p8�/� mice. Significant differences were observed forseveral genes involved in many cellular pathways. Suchdifferences probably account for the increased noxious-ness of LPS in p8�/� mice, which underscores the impor-tance of p8 in the defense mechanisms of the liver.15 Theaim of the present study was to check whether such func-tion would extend, beyond LPS aggression, to xenobiotic-induced hepatotoxicity. The well-defined model ofcarbon tetrachloride–induced hepatitis in rodents waschosen.16 Contrary to other hepatotoxic agents, CCl4 isnot toxic per se but through generation by cytochromeP450 of a secondary highly reactive agent (trichloro-methyl radical) responsible for lipid peroxidation andeventual cellular damage.17,18 CCl4 is mainly but not ex-clusively metabolized by the 2E1 isoform of cytochromeP450 (CYP2E1). It was shown that CYP2E1 activity wastransiently decreased during the first hours followingCCl4 administration. This result was interpreted as a self-defense reaction against CCl4 hepatotoxicity, althoughthe mechanism involved remains unknown. We reporthere that inactivation of the p8 gene increases CCl4-in-duced damages to the liver, apparently because transientdownregulation of CYP2E1 is delayed. Therefore, p8seems to be involved in the first line of defense againstCCl4 hepatotoxicity.

Materials and Methods

Animals and CCl4 Treatment. Three month-oldp8�/� and wild-type C57/BL6 mice were used in thisstudy. The p8�/� C57/BL6 mice were generated as pre-viously described.5 After fasting for 18 hours with accessto water ad libitum, CCl4 (Sigma, St. Louis, MO) wasadministered intraperitoneally at 2 �L/g body weight(V/V solution in corn oil) to 64 mice (32 p8�/�, 32 p8�/

�). Controls for each group were injected with the corn oilvehicle only. All studies were performed according to theAmerican National Institute of Health guidelines for an-imal care.

Biochemical Assay and Tissue Samples. Mice weresacrificed at 0, 12, 18, 24, 48, 72, 96, and 120 hoursfollowing CCl4 administration (4 p8�/� mice and 4p8�/� mice at each time). For enzyme analyses, blood wascollected from the abdominal aorta in anesthetized miceand centrifuged at 4°C, and sera were stored at �80°Cuntil use. The liver was removed on ice, weighed, imme-diately frozen in liquid nitrogen, and stored at �80°Cuntil use for RNA and protein extractions—except forthe left lobe, which was used for histological analysis.Blood plasma levels of alanine aminotransferase (ALT)

and aspartate aminotransferase (AST) were measured onthe same run by a multiple-point rate test using a Vitros950AT (Ortho-Clinical Diagnostics) apparatus, accord-ing to the manufacturer’s instructions.

Liver Histology and Immunohistochemical Analy-sis. Liver samples were fixed in 4% formalin and embed-ded in paraffin. Five-micrometer-thick sections werestained with hematoxilin-eosin for standard examination.For necrosis scoring, 10 randomized fields were selected(original magnification �100), and necrotic areas werecontoured manually and measured via image analysis. Re-sults were expressed as a percentage of the total surface.Images were obtained with an Axiophot microscope(Zeiss, Le Pecq, France) and a 3CCD Camera (Sony,Paris, France). They were processed with an image anal-ysis system (SAMBA 2005; Alcatel TITN, Grenoble,France). Immunohistochemical studies on proliferativecell nuclear antigen (PCNA) were performed using amodification of the avidin-biotin complex method.19 Thepurpose of the modification was to reduce the back-ground staining due to the binding of the secondary goatanti-mouse antibody to endogenous immunoglobulins.Briefly, complexes of primary PCNA monoclonal mouseantibody (clone PC10, DakoCytomation) and biotinyl-ated secondary antibodies were first generated. Then, sitesstill available for mouse IgG binding were blocked byincubating the complexes with normal mouse serum be-fore use on tissue sections. Tissue-bound complexes werevisualized using an avidin-biotin detection system. Onlythe PCNA-positive hepatocytes in nonnecrotic areas wereconsidered. For PCNA-positive scoring, 10 randomizedfields were selected (original magnification �400); im-ages were obtained with the Axiophot microscope andprocessed using the SAMBA 2005 image analysis system.The PCNA-positive nuclei index was calculated as thepercentage of PCNA-positive cells per total number ofcells counted.

Terminal Deoxynucleotidyl Transferase-MediateddUTP Nick End Labeling Assay. Apoptotic cells inliver sections were determined via terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling(TUNEL) assay using the Cell Death Detection Kit(Roche Diagnostics, Meylan, France) following the man-ufacturer’s instructions. Briefly, sections were digestedwith proteinase K (20 �g/mL) for 15 minutes at roomtemperature and rinsed with double-distilled water. Slideswere then quenched by 2% H2O2 for 5 minutes at roomtemperature. Slides were then incubated with terminaldeoxynucleotidyl transferase (TdT) buffer (30 mmol/LTrizma base [pH 7.2], 140 mmol/L sodium cacodylate, 1mmol/L cobalt chloride), followed by terminal deoxynu-cleotidyl transferase reaction solution containing terminal

HEPATOLOGY, Vol. 42, No. 1, 2005 TAIEB ET AL. 177

deoxynucleotidyl transferase and dUTP for 90 minutes at37°C, then washed with 2� standard saline citrate to stopthe reaction for 10 minutes at room temperature. Theslides were then washed and incubated with antidigoxige-nin peroxidase for 30 minutes at room temperature.Color was developed using 0.05% 3,3’-dimethyl amino-benzene in 0.01% H2O2 and then lightly counterstainedwith hematoxylin. Sections were then washed, dehy-drated, and mounted. Apoptotic cells were identified by abrown stain over the nuclei, and the apoptotic index wascalculated as the percentage of TUNEL-positive cells pertotal number of cells counted.

Determination of p8 and CYP2E1 mRNA Levels bySemiquantitative Reverse-Transcriptase PolymeraseChain Reaction. Determinations of p8, CYP2E1 (cyto-chrome P450, family 2, subfamily E, polypeptide 1), andcontrol GAPDH mRNA levels were performed on RNAextracted from tissue samples. RNA was extracted usingTrizol (Life Technologies, Cergy Pontoise, France) and a2-mL Lysing Matrix D tube. Total RNA (1 �g) was an-alyzed via semiquantitative reverse-transcriptase polymer-ase chain reaction (RT-PCR) using the SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen, CergyPontoise, France; Life Technologies) and according to themanufacturer’s protocol. For p8, the forward primer was5�-GCCACCTTGCCACCAACAGCC-3� and the re-verse primer was 5�-GCGCCAGGCTTTTTTCC-3�;for CYP2E1, the forward primer was 5�-CAGGACCTT-TCCCAATTCCT-3� and the reverse primer was 5�-TGACTTTTCTGTGGCTTCCA-3�; and for GAPDH,the forward primer was 5�-ACCACAGTCCATGC-CATCAC-3� and the reverse primer was 5�-TCCAC-CACCCTGTTGCTGTA-3�. RT-PCR was performedusing different numbers of cycles to verify that the condi-tions chosen were within the linear range. Reverse tran-scription was carried out for 45 minutes at 45°C followedby 25 (p8), 24 (GAPDH), and 29 (CYP2E1) cycles ofPCR, each cycle consisting in a denaturing step for 10seconds at 95°C, an annealing step for 1 minute at 57°C,and a polymerization step for 1 minute at 72°C. PCRproducts were separated via electrophoresis on 2% aga-rose gels.

Western Blotting. The CYP2E1 protein level was es-timated via Western blotting as previously described.8

Briefly, proteins were extracted in a 2-mL Lysing MatrixD tube containing the following lysis buffer: 0.5% so-dium deoxycholate, 50 mmol/L Tris-HCl (pH 8.0),0.1% SDS, 1% Triton X-100, 150 mmol/L NaCl, and acocktail of protease and phosphatase inhibitors (Sigma).Mouse monoclonal anti-CYP2E1 was applied overnightat a 1:100 concentration (clone 2-106-12, kindly pro-vided by Kristopher Krausz from the National Institutes

of Health,Bethesda, MD). Western blotting was also per-formed for PCNA using the MAb (1:500) (Santa CruzInc., Santa Cruz, CA). ImageJ 1.32 (http://rsbweb.nih.gov/ij/download.html) was used to quantify intensities inWestern blots and RT-PCR.

Statistical Analysis. Statistical evaluation was per-formed using the unpaired Student t test, or ANOVAwhen multiple comparisons were made. A P value lessthan .05 was considered statistically significant.

Results and Discussion

The p8 Protein Is Transiently Induced in the LiverAfter CCl4 Exposure. Following CCl4 injection, theliver goes through several well-characterized stages: necro-sis, inflammatory infiltration, hepatic regeneration, cellproliferation, and deposition of connective tissue (succes-sively). We explored p8 expression in the liver of wild-type mice following CCl4 administration. As shown inFig. 1, p8 mRNA was barely detectable before treatment.Expression was rapidly and strongly increased after CCl4injection, reaching at 12 hours a value more than 20 timeshigher than in control animals. A further increase to 50times the control value was observed at 18 hours. This wasthe climax, because p8 mRNA concentration at 24 hourswas back to values observed at 12 hours and had almostreturned to basal value at 48 hours. Hence, p8 inductionby CCl4 is transient. Because p8 is a transcription factor

Fig. 1. Expression of p8 mRNA is transiently induced in wild-type liversafter exposure to CCl4. (A) Expression of p8 was monitored via RT-PCR onRNA isolated from livers at the indicated time points following CCl4injection; it was detected at 12 hours and peaked at 18 hours. GAPDHwas used as a control. (B) Time course of variations in the p8/GAPDHratio (mean � SD for three experiments).

178 TAIEB ET AL. HEPATOLOGY, July 2005

required to promote hepatic defense against LPS,15 wemade the hypothesis that the early induction of a strongand transient expression of p8 following CCl4 injectionwould also help limit liver injury.

Necrotic Injury Is Increased in p8�/� Mice. Weintended to define the role of the early peak of p8 expres-sion that follows CCl4 injection. A single low (nonlethal)dose of CCl4 that causes significant acute liver injury wasused to monitor the course of liver damage and repair.Serum ALT and AST activities were used as markers ofliver injury (Fig. 2A-B). Maximum levels of serum ALTand AST were observed at 36 hours post–CCl4 injectionin wild-type mice, but at 24 hours in p8�/� mice. Inaddition, those peak values were approximately 1.5-foldhigher in p8�/� mice than in wild-type mice. Unexpect-edly, p8�/� mice showed an additional peak of serumALT and AST at 48 hours, less intense than the first one.This observation might reveal the existence of two con-secutive necrotic processes after CCl4 treatment, whichcould be evidenced in p8�/� livers only because, in thatcase, the first of them is brought forward. In p8�/� livers,the two processes would have similar time courses and

would thus appear as a single process. In p8�/� mice, ALTand AST values declined from 36 hours post–CCl4 injec-tion, reaching at 72 hours the basal value observed in theuntreated animals. However, values at 48 and 60 hoursremained higher in p8�/� than in wild-type mice. Thesedata show that in the absence of p8 activity, CCl4 hepa-totoxicity occurs earlier and is more severe. Livers fromboth groups of animals had similar weights (data notshown). We compared the extent of necrosis in hematox-ylin-eosin–stained liver sections from p8�/� and wild-type mice. In control animals (corn oil–treated), normalliver architecture was observed in wild-type and p8�/�

mice (Fig. 3). The necrotic process started between 12hours and 36 hours post–CCl4 injection, as indicated bythe occurrence of isolated foci of necrotic hepatocytes.Before 36 hours, areas of necrosis represented less than1% of the total and could not be adequately quantified.From 36 hours to 60 hours, necrotic areas progressed.

Fig. 2. p8�/� mice are more sensitive to CCl4-induced hepatotoxicity.Serum levels of (A) ALT and (B) AST were monitored after acute CCl4treatment. Values are expressed as the mean � SD for three animals.Significant differences between p8�/� and p8�/� mice were observed at24 hours (***P � .005) and 48 hours (*P � .05) following CCl4administration. ALT, alanine aminotransferase; AST, aspartateaminotransferase.

C

A B

D

E F

p8+/+ p8-/-

24 h

36 h

60 h

V

VV

V

VV

PTPT

Fig. 3. Histological analysis of p8�/� and p8�/� livers after acuteCCl4 treatment. Paraffin-embedded sections from livers taken 24, 36, or60 hours after acute CCl4 treatment were stained with hematoxylin-eosin.After 24 hours, rare inflammatory cells surrounding necrotic hepatocyteswere randomly scattered in the p8�/� liver (A( (arrow) (original magni-fication �100), whereas in p8�/� livers, foci of necrotic hepatocyteswith inflammatory cells (B) (arrows) were already visible (original mag-nification �100). After 36 hours, necrotic cells observed in the centri-lobular area were more abundant in p8�/� livers (D) than in p8�/� livers(C) (original magnification �200). After 60 hours, necrosis progressivelyconcentrated to the centrilobular area of p8�/� (E) and p8�/� (F) livers(original magnification �100). In panels E and F, a line delimits thenecrotic area. PT, portal tract; V, centrolobular vein.

HEPATOLOGY, Vol. 42, No. 1, 2005 TAIEB ET AL. 179

They exhibited a centrilobular distribution, invading insome cases a large part of the hepatic lobules but alwayssparing periportal areas. From 72 hours to 96 hours, ne-crosis was progressively replaced by inflammatory cells.Liver histology returned to normal at 120 hours in bothgroups. At 36, 48, and 60 hours post–CCl4 injection,p8�/� mice exhibited significantly larger necrotic areascompared with wild-type mice (3.8, 1.8, and 2 times re-spectively) (Fig. 4). At 72 hours, p8�/� livers presentedwith persistent centrilobular lesions, contrary to liversfrom wild-type mice. These data confirm ALT/AST datathat CCl4-induced necrosis is more severe in the absenceof p8. In other words, our findings strongly suggest thatp8 overexpression in damaged liver is involved in tissueprotection. One possibility is that p8 is required for effi-cient apoptosis. In the absence of p8 activity, apoptosiswould be inhibited and the only issue for damaged cellswould be necrosis. However, that possibility was ruled outby experiments showing the extent of apoptosis, as mea-sured via TUNEL, was similar in p8�/� and p8�/� livers(data not shown).

Hepatocytic Proliferation Is Similar in Wild-Typeand p8�/� Mice. Because evaluating an area of necrosisat a given time is a snapshot, it reflects a balance betweendeath and regeneration but does not tell about the relativecontribution of cells dying through necrosis and ofdaughter cells resulting from proliferation. To obtain partof the answer, we compared the expression of PCNA, amarker of liver regeneration, in p8�/� and p8�/� CCl4-treated livers. Both Western blotting (Fig. 5A) and im-munohistochemistry (Fig. 5B) revealed that PCNA

expression was similar in both groups. This observationsuggests that p8 is not involved in hepatic regenerationbut rather in a mechanism that takes place in the earlystages of the disease.

CYP2E1 Downregulation Is Delayed in p8�/�

Mice. The enzyme CYP2E1 is responsible for most ofCCl4 metabolism and, because derivatives generated dur-ing that metabolism are hepatotoxic, it plays a major rolein the modulation of CCl4-induced liver injury.20,21 Forexample, transient downregulation of CYP2E1, alwaysobserved in liver following CCl4 administration, is con-sidered an adaptive mechanism that limits toxicity. If, onthe contrary, CYP2E1 synthesis is increased22 or the en-zyme is stabilized,23 CCl4-induced liver injury is moresevere. That mechanism accounts for ethanol potentia-tion of CCl4 hepatotoxicity.24,25 Finally, CYP2E1 knock-out mice are resistant to hepatoxicity induced by CCl4.21

We asked whether p8 could interfere with the regulationof CYP2E1 gene expression. It does not control basalexpression of the gene, because, in untreated mice,CYP2E1 expression was the same as in wild-type andp8�/� mice. The situation was different after CCl4 treat-ment. Twelve hours after injection, the hepatic level ofCYP2E1 mRNA dropped by 50% in wild-type mice asexpected (Fig. 6A,C). By contrast, CYP2E1 mRNA ex-pression was maintained at 12 hours in p8�/� livers and

Fig. 4. Lack of p8 signaling results in increased liver damage afteracute exposure to CCl4. Liver sections were analyzed by morphometricimage analysis, digitized, and quantified as described in Materials andMethods to calculate the percentage of necrosis in the field. Values areexpressed as the mean � SD for 10 fields. Necrosis was significantlymore important in p8�/� livers (***P � .005) than in p8�/� livers(**P � .01).

Fig. 5. The p8 protein is not required for hepatocyte proliferation andliver repair. (A) Representative Western blot and (B) quantitation ofpositive nuclei for PCNA. No statistical differences were observed be-tween p8�/� and p8�/� mice (three experiments). PCNA, proliferativecell nuclear antigen.

180 TAIEB ET AL. HEPATOLOGY, July 2005

showed only a modest decrease after 18 hours. Similarresults were found when monitoring the level of theCYP2E1 protein (Fig. 6B,D). It is important to note thatin the rodent liver, the CYP2E1 protein has a half-life ofonly 6 to 7 hours.23 Therefore, the CYP2E1 mRNAdownregulation that follows CCl4 treatment is quicklypassed on to the protein level, explaining why the decreasein CYP2E1 protein level is rapid in p8�/� livers and de-layed in p8�/� livers (Fig. 6). As a result, the duration ofsustained CYP2E1 expression is significantly longer in theabsence of p8 activity. A much larger production of nox-ious CCl4 metabolites is therefore expected. We did notmonitor such accumulation but, if confirmed, it couldvery well account for the increased necrosis observed inp8�/� livers. These results indicate that p8 is involved inthe regulation of gene expression that takes place in liverin response to CCl4 administration. Involvement of p8 inprotecting a tissue against necrosis was already reported inthe pancreas during acute pancreatitis.26 However, p8 ex-pression could reduce—but not prevent—the occurrenceof necrosis in liver. Also, it is noteworthy that p8 expres-sion following CCl4 injection and inhibition of CYP2E1are not concomitant, because maximal p8 expression oc-curred when the CYP2E1 level had returned to normalvalues. In addition, CYP2E1 was delayed but not sup-pressed in p8�/� animals, indicating that factors otherthan p8 can downregulate the enzyme. Additional func-

tions of the p8 protein in the mechanisms of liver responseto CCl4 are therefore expected.

In conclusion, the present study demonstrates that p8is involved in the mechanisms protecting the mouse liversubmitted to CCl4 treatment. As in other tissues submit-ted to a stress, p8 is rapidly, strongly, and transientlyactivated in the liver in response to CCl4 injection. Acti-vation of p8 allows rapid downregulation of CYP2E1 ex-pression, which is known to limit the amount ofhepatotoxic CCl4-derived metabolites and consequentlythe extension of liver injury. These findings open up thepossibility that p8-associated pathways are involved in theregulation of other CYP450 isoforms, which, if con-firmed, would provide new therapeutic options for xeno-biotic-induced acute liver injury.27

Acknowledgment: We thank S. Lotersztajn for help-ful discussions and insightful comments.

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