Expression and activity of the cytochrome P450 2E1 in patients with nonalcoholic steatosis and...

CL IN ICAL STUDIES

Expressionandactivityofthe cytochromeP4502E1in patientswithnonalcoholic steatosis and steatohepatitisHaithem Chtioui1�, David Semela1�, Monika Ledermann1, Arthur Zimmermann2 and Jean-FrancoisDufour1

1 Institute of Clinical Pharmacology, University of Bern, Bern, Switzerland

2 Department of Pathology, Inselspital, University of Bern, Bern, Switzerland

Keywords

chlorzoxazone – fatty liver –

immunohistochemistry – liver biopsy – stellate

cells

Correspondence

Jean-Francois Dufour, MD, Institute of Clinical

Pharmacology, University of Bern,

Murtenstrasse 35, CH-3010 Bern, Switzerland

Tel: 141 31 632 31 91

Fax: 141 31 632 49 97

e-mail: [email protected]

Received 8 December 2007

accepted 18 March 2007

DOI:10.1111/j.1478-3231.2007.01524.x

AbstractBackground/Aims: Nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty

liver (NAFL) have a different prognosis and should be dealt with differently. The

pathogenesis of NASH implicates the overexpression of cytochrome P450 2E1

(CYP2E1). We investigated whether the noninvasive determination of CYP2E1

activity could replace a liver biopsy in order to differentiate NASH from NAFL.

Method: Forty patients referred for suspicion of NASH underwent liver biopsy. In

these patients, CYP2E1 activity was determined noninvasively by the 6-hydroxy-

chlorzoxazone/chlorzoxazone (CHZ) ratio (CHZ test). Expression of CYP2E1 on

liver slides was assessed by immunohistochemistry, and immunostaining for

smooth muscle actin was used to assess the activation of hepatic stellate cells

(HSC). Results: Thirty patients with NASH were compared with 10 subjects with

NAFL. No statistically significant difference could be identified for the clinical and

biochemical parameters between the two groups. In the histology, steatosis was

more important in NASH than in NAFL (Po 0.0001). There was no difference

either in the activity (CHZ test) or in the expression of CYP2E1 (immunohisto-

chemistry) between patients with NASH and patients with NAFL. The degree of

HSC activation was also comparable between the two groups. A positive and

significant correlation was found between the activity of CYP2E1 and body mass

index (Po 0.001) as well as with the degree of steatosis (P = 0.008). Conclusion:

For patients suspected to have NASH, noninvasive tests including the determination

of the CYP2E1 activity are unable to distinguish them from patients with steatosis.

Nonalcoholic fatty liver diseases comprise nonalco-holic fatty liver (NAFL) and nonalcoholic steatohepa-titis (NASH) (1, 2). While NAFL, which ischaracterized by macrovesicular steatosis follows abenign clinical course, the presence of features ofsteatohepatitis (hepatocyte ballooning, presence ofMallory bodies, lobular inflammation, and fibrosis)in NASH indicates the potential for progressive liverdamage (3, 4), evolving in some cases into cirrhosis(5–7). It is therefore important to make an accuratediagnosis in order to optimize management (8, 9).Although noninvasive diagnostic procedures such asultrasonography or computed tomography (CT) haveenough sensitivity to detect steatosis, it is impossibleto distinguish between NAFL and NASH with presentimaging techniques (1).

Previous studies were able to correlate liver damage inNASH or in alcoholic liver disease with increased cyto-chrome P450 2E1 (CYP2E1) isoenzyme activity (10, 11)or expression (12) and supported the hypothesis of aprivileged role for the CYP2E1 in the pathogenesis ofNASH (10, 11, 13–16). CYP2E1 metabolizes importantxenobiotics such as ethanol and is capable of activatingxenobiotics like nitrosamines into hepatotoxic products(17, 18). CYP2E1 is an inducible enzyme hydroxylatingfatty acids and able to initiate the lipid peroxidationprocess (14, 19). CYP2E1 undergoes a complex regula-tion: besides the CYP2E1 induction in response to ethanolexposure, fatty acids are both substrates and inducers ofCYP2E1 (13, 14, 20). CYP2E1 activity produces freeradicals. Oxidative stress contributes to the process of liverfibrosis by activating hepatic stellate cells (HSC) (10, 15)and thus to the enhancement of collagen gene expressionand to the release of profibrotic cytokines (21–24).�Both authors contributed equally.

Liver International (2007)764 c� 2007 The Authors. Journal compilation c� 2007 Blackwell Munksgaard

Liver International ISSN 1478-3223

CYP2E1 activity can be determined noninvasively.Chlorzoxazone (CHZ), a centrally acting muscle,relaxant is selectively hydroxylated by CYP2E1 to 6-hydroxychlorzoxazone (6-OH-CHZ) in humans (17,18, 25, 26). The plasma concentration ratio of (6-OH-CHZ) to CHZ can thus be used as an index of theCYP2E1 activity (10, 16, 27). The aim of the presentstudy was to test whether the activity and the hepaticexpression of the CYP2E1 in patients with nonalco-holic steatosis is different from patients with NASH.

Materials and methods

Patient population

Forty-three patients referred consecutively to the out-patient clinic of the Institute of Clinical Pharmacologyin Bern (Switzerland) with elevated serum amino-transferase levels and a suspicion of NAFLD under-went a liver biopsy. Definitive inclusion in this studywas achieved after histological confirmation of thediagnosis of steatosis or steatohepatitis according tothe criteria proposed by Brunt (28). Three patientswho did not fulfil the above-mentioned histologicalcriteria were excluded from this study. Inclusion wasbased on the following criteria: 1, persistent abnormalliver biochemistries for more than 6 months; 2, anormal upper abdominal sonography, except for anincreased echogenicity of hepatic parenchyma; and 3,exclusion of other liver diseases such as alcoholic liverdisease, viral hepatitis, autoimmune hepatitis, drug-induced liver disease, primary biliary cirrhosis, biliaryobstruction, haemochromatosis, Wilson’s disease, anda-1 antitrypsin deficiency-associated liver disease. Allpatients were drinking o 40 g of alcohol/week, andthis was confirmed by family members who were inclose contact with the patient. No patient had a historyof bariatric surgery. The exclusion criteria werecontraindication for a liver biopsy (thrombocytecount o 50 000/mL, INR4 1.5, focal lesion in theright hepatic lobe, ascites, or lack of cooperation),pregnancy, breast-feeding, and allergy to CHZ.Informed consent to take part in this study wasobtained from all patients. This study was approvedby the local ethics committee according to the Helsinkicriteria.

Laboratory evaluation

Laboratory evaluation in all patients included serumliver tests [aspartate transaminase (ASAT), alaninetransaminase (ALAT), alkaline phosphatase andg glutamyl-transferase (gGT), cholesterol, triglycerides,and fasting glucose].

Liver biopsy

Percutaneous liver biopsies were performed in allpatients and examined by the same experienced studypathologist (A. Z.), who was blinded to the clinical andlaboratory data. Biopsy specimens were fixed in neu-tral-buffered formalin and embedded in paraffin. Sec-tions were stained with haematoxylin and eosin,chromotrope aniline blue, and Perl’s Prussian bluestain. Haematoxylin and eosin slides were used formorphologic evaluation, assessment of steatosis, neu-trophilic infiltration, and hepatocellular ballooning.Chromotrope aniline blue stain was used for assessmentof fibrosis and Perl’s Prussian blue stain for assessmentof iron deposition. NASH was diagnosed when histolo-gical criteria according to Brunt (3) were fulfilled.

CYP2E1 activity

The enzymatic activity of CYP2E1 was assessed inplasma by the CHZ test, measuring the CHZ hydro-xylation as described by Lucas (17, 29, 30). Briefly, 2 hafter the ingestion of a 500 mg CHZ tablet (LemmonCompany, Sellersville, PA, USA) blood was collected in12 h fasting patients. Measurement of plasma CHZand its metabolite (6-OH-CHZ) concentrations wasperformed by HPLC chromatography. As hydroxyla-tion from CHZ to 6-OH-CHZ is specific to CYP2E1,the 6-OH-CHZ/CHZ ratio reflects the in vivo enzy-matic CYP2E1 activity (and has no units).

Immunostaining of activated HSC

Activated HSC were identified immunohistochemi-cally with a monoclonal antibody raised against thea-smooth muscle isoform of actin (a-SMA) (clone1A4, Sigma Co., St. Louis, MO, USA), dilution 1:1000,using an immunoperoxidase method with 303-diami-nobenzidine (DAB) as a chromagen (Fig. 1). Vascularsmooth muscle cell immunoreactivity was used as aninternal positive control. a-SMA immunoreactiveHSC were semiquantitatively graded: 0 = no cellsstained, 1 = o 1% of HSC stained, 2 = 1–10% of cellsstained, 3 = 10–30% of HSC stained, and 4 = diffusestaining (more than 30% of HSC stained). HSC werescored separately in periportal, intermediate, andperivenular zones in not less than five acini in eachsample. The scores for each zone were added, resultingin an overall HSC index (HSCI) that has no units.

CYP2E1 immunohistochemistry

The evaluation of the expression of CYP2E1 proteinsby immunohistochemistry in liver sections was per-formed according to the method described by

Liver International (2007)c� 2007 The Authors. Journal compilation c� 2007 Blackwell Munksgaard 765

Chtioui et al. Cytochrome P450 2E1 in NAFLD

Ingelman-Sundberg (12, 31). Hepatic sections in par-affin (5–6 mm thick) were mounted on superfrost plusglass slides, deparaffinized with Histo-Clear (NationalDiagnostics, Atlanta, GA, USA) and ethanol, andincubated with H2O2 for 5 min to reduce endogenousperoxidase activity. Thereafter, sections were incu-bated for 2 h with rabbit antihuman CYP2E1 antibody,at a dilution of 1:100 with the addition of goat serum

(1:20) to block nonspecific binding. Sections werethen incubated with an anti-rabbit immunoglobulinG–biotin conjugate (Dako, Dako Cytomation,Glostrup, Denmark), followed by streptavidin perox-idase conjugate (Dako). Peroxidase activity was re-vealed by immersion for 5 min in DAB. Sections werethen counterstained with haematoxylin, dehydrated,and mounted in ultramount (National Diagnostics,

Fig. 1. Immunostaining of activated hepatic stellate cells. (A) Activated HSC with a positive monoclonal antibody raised against thea-smooth muscle isoform of actin (a-SMA). (B) Positive monoclonal antibody (higher magnification).

Fig. 2. Immunohistochemistry for cytochrome P450 2E1. (A, C) Negative controls. The staining was performed in the absence of theprimary antibody. (B, D) Positive staining. The staining was performed in the presence of the primary antibody.


Cytochrome P450 2E1 in NAFLD Chtioui et al.

Atlanta, GA, USA) (Fig. 2). The presence and intensityof the immunoreaction were assessed blindly, and asemiquantitative estimate of CYP2E1 protein levelswas made according to the scale devised by Tsutsumiet al. (32). Thus, 31, 21, 11, and 0 denote respec-tively, intense, moderate, slight, and no specific im-munostaining. There was no immunostaining whenthe primary antibody was omitted from the stainingprocedure.

Statistics

Basic descriptive statistics (means, standard devia-tions, ranges, and percentages) were used to character-ize the study groups. Comparison between the twogroups was made using the Mann–Whitney U-test.Spearman’s rank correlations were used to detect theassociations between the CHZ elimination rate andanthropometric as well as various serum studies. AP value o 0.05 was considered to be statisticallysignificant.

Results

The clinical and biochemical characteristics of patientsare presented in Table 1. According to their histology,patients were divided into two groups: simple steatosis(n = 10) and steatohepatitis (n = 30). There was nosignificant difference in the sex distribution betweenthe steatosis and the NASH groups, female patientsrepresenting 20% and 37% of these groups respec-tively. Patients with NASH were significantly oldercompared with patients with simple steatosis: themean age was 47.2� 9.7 years in the NASH groupand 38.9� 11.7 in the simple steatosis group(Po 0.05). The two groups revealed no significantdifferences in BMI, ASAT, ALAT, alkaline phosphatase,gGT, cholesterol, triglycerides, and fasting glucose.

Histologically, the degree of steatosis (expressed as apercentage of steatosis for each slide) was moreimportant in patients with NASH 38.5� 22.9% thanin patients with NAFL 13.4� 10.9% (Po 0.0001).Quantification of the immunohistochemistry for acti-vated stellate cells (Fig. 1) revealed no significantdifference between the two groups (1.8� 2.7 in pa-tients with NASH vs. 1.1� 1.3 in patients with simplesteatosis; P4 0.05). CYP2E1 immunostaining (Fig. 2),as assessed by the semiquantitative score, was similarin intensity in patients with NASH and in patientswith NAFL, as no statistically significant differencecould be found between the two groups. In bothgroups, the pattern of distribution closely followedthe distribution of steatosis, in some cases extendingbeyond acinar zone 3 into zones 2 and 1.

The CHZ test, which measures noninvasively theactivity of CYP2E1, was not significantly differentbetween the two groups (0.77� 0.41 in patients with

Table 1. Description of the population

Parameter

Mean value� SD

P valueSimple steatosis (n = 10) NASH (n = 30)

Age (years) 38.9� 11.7 47.2� 9.7 0.049BMI (kg/m2) 29.9� 5.5 30.7� 5.7 0.71Female/male 2/8 11/19 NSASAT (U/L) 58.8� 31.5 59.1� 34.6 0.90ALAT (U/L) 108.4� 45.9 83.8� 49.3 0.07Alkaline phosphatase (U/L) 88.6� 29.3 89.2� 31.6 0.73gGT (U/L) 93.2� 80.8 166.2� 213.9 0.36Total cholesterol (mmol/L) 6.5� 1.44 5.9� 1.33 0.35Triglycerides (mmol/L) 2.4� 1.1 1.9� 1.1 0.14Fasting glucose (mmol/L) 5.3� 1.0 5.8� 1.15 0.16

ALAT, alanine transaminase; ASAT, aspartate transaminase; BMI, body mass index; gGT, g glutamyl-transferase; NASH, nonalcoholic steatohepatitis; NS,

not significant; SD, standard deviation.

CY

P2E

1 ac

tivity

6-O

H-C

HZ

/CH

Z

0

1

2

3

Steatosis NASH

Fig. 3. Cytochrome P450 2E1 (CYP2E1) activity as expressed bythe 6-hydroxychlorzoxazone/chlorzoxazone (6-OH-CHZ/CHZ)ratio in patients with NAFL vs. nonalcoholic steatohepatitis(NASH). Whisker plot representing median/10th percentile/25thpercentile.



simple steatosis and 1.07� 0.77 in patients withNASH; P4 0.05) (Fig. 3). A statistically significantcorrelation was found between the CHZ test and thebody mass index (BMI) (Spearman’s correlation coef-ficient: 0.49, Po 0.001; Fig. 4) as well as with thedegree of steatosis (Spearman’s correlation coefficient:0.43, Po 0.008; Fig. 5).

Discussion

Our main finding is that the noninvasive determina-tion of the CYP2E1 activity by the CHZ test does nothelp to single out patients with NASH among those

suspected to suffer from this condition based on theirclinical presentation.

In this study, 40 consecutive patients who had beenreferred to a liver centre for suspicion of NASH wereinvestigated. NASH was confirmed with a liver biopsyin 30 cases, and 10 patients had simple steatosis. Noneof the commonly used clinical parameters couldsegregate the patient with NASH from the patientswith NAFL. Specifically, no significant differences werefound between the two groups for BMI, ASAT, ALAT,ASAT/ALAT ratio, alkaline phosphatase, gGT, glucose,and triglyceride values, although a trend to have high-er BMI values and higher ASAT serum levels wasnoticed in the NASH group. Taken alone, these para-meters are poor predictors for histological lesions ofsteatohepatitis (33–36).

The CHZ test, which measures noninvasively theactivity of CYP2E1, was not significantly differentbetween the two groups. This was confirmed byimmunohistochemistry, which could not detect anincrease in the hepatic expression of CYP2E1 in thepatients with NASH, in comparison with patients withNAFL. These results are not in opposition with pub-lished data reporting an increased expression andactivity of CYP2E1 when patients with NASH werecompared with healthy controls. Chalasani et al. (16)found a significant difference in the CYP2E1 activityassessed by the oral clearance of chlorzoxazone oncomparing 20 patients with NASH with 17 BMI-matched control subjects without liver disease. Orella-na et al. (11) investigated the CHZ test in 26 obesewomen who underwent bariatric surgery. In thisspecial population, the authors found the CHZ test tobe higher in the 10 patients with NASH than in the 10patients with NAFL and the six with normal histology.Our study has a different perspective. Our study testswhether the CHZ test can identify patients with NASHamong patients referred for suspicion of this disease.In this clinically more relevant situation comparingpatients with NASH with patients with NAFL, ourresults show that noninvasive determination of theCYP2E1 activity is not helpful for diagnosis. Obesity isknown to increase the activity of CYP2E1 and consis-tently we observed a positive correlation betweenCYP2E1 activity and the BMI (correlation coeficient:0.49, Po 0.001) as well as a statistically significantcorrelation between the CYP2E1 activity and thedegree of steatosis (correlation coefficient: 0.43,Po 0.008). However, this did not result in signifi-cantly higher CHZ tests in patients with NASH. Ourdata thus confirm the correlation between CYP2E1activity and BMI reported by Emery et al. (37) in apopulation of 16 morbidly obese patients (BMI

BMI (kg/m2)

20 25 30 35 40 45 50 55

CY

P2E

1 ac

tivity

(6-

OH

-CH

Z/C

HZ

)

0

1

2

3

4

Fig. 4. Correlation between cytochrome P450 2E1 (CYP2E1)activity and the body mass index (BMI) (correlation coefficient:0.49, Po 0.001) in patients with nonalcoholic fatty liver (�) andnonalcoholic steatohepatitis(�).

Steatosis (%)

0 20 40 60 80 100

CY

P2E

1 ac

tivity

(6-

OH

-CH

Z/C

HZ

)

0

1

2

3

4

Fig. 5. Correlation between cytochrome P450 2E1 (CYP2E1)activity and the degree of steatosis (correlation coefficient: 0.43,Po 0.008) in patients with nonalcoholic fatty liver (�) andnonalcoholic steatohepatitis (�).



median: 60 kg/m2) and 16 controls with a normalBMI. These authors compared a group with more than50% steatosis with a group with o 50% of steatosisand also compared a group with steatohepatitis with agroup without steatohepatitis (but did not comparesimple steatosis with steatohepatitis). In the study byEmery et al. (37), only one of the obese patients hadelevated liver function tests. Thus, our study confirmsthis correlation between CYP2E1 activity and the BMIin a population without significant differences in theBMI between the steatosis and the steatohepatitisgroups and with a clinically relevant steatosis/steato-hepatitis, as the LFT were increased in all the patients.More importantly, our data suggest that the activity ofthe CYP2E1 is not different in patients with NASHfrom patients with steatosis.

In the liver biopsy, the degree of steatosis wassignificantly more important in patients with NASHthan in patients with NAFL, suggesting that theamount of triglyceride accumulation plays a role inthe pathogenesis of NASH. More surprising is the factthat the degree of activation of the stellate cells was notdifferent in the two groups. Cortez-Pinto et al. (38)reported a greater number of actin-reactive stellatecells in the liver of patients with NASH than in theliver of normal controls. The lack of significant differ-ence in our study in the degree of activation of theHSCs is consistent with the results of Washington et al.(39), who performed immunostaining for activatedHSC in three different groups: normal controls, pa-tients with steatosis (n = 16), and patients with NASH(n = 60), and indicated that most cases (74 or 76) ofsteatosis or NASH showed an increase in a-SMA-positive HSCs, compared with control cases. Theauthors also reported that the mean HSC activationindex was lower in the steatosis group than in theNASH group, but did not comment on the statisticalsignificance or lack of statistical significance of thisdifference. Again, in the present study we comparedpatients with NASH with patients with NAFL and thedifference did not reach statistical significance. It iscommonly viewed that in the context of steatosis, lipidperoxidation is associated with inflammation (9) andwith activation of stellate cells (10, 15, 39). A specificrole of CYP2E1 in the activation of HSC was sup-ported by Nieto et al. (21, 23) in a cell culture model.Niemela et al. (19) compared immunostaining forCYP2E1 between a group of patients with alcoholicliver disease (n = 12) and a control group of patientswith nonalcoholic liver disease (nine nondrinkingpatients and not NASH patients). This control groupwas subdivided into two subgroups: one with fattyliver (irrespective of the stage of the disease, n = 4) and

a second subgroup (n = 5) with nonalcoholic hepatitis,which was composed of three patients with hepatitis C,one with hepatitis B, and one with reactive hepatitis.In the immunostaining, a significant difference wasreported when patients with advanced alcoholic liverdisease were compared with the patients in controlsubgroups or with patients with early alcoholic liverdisease. They observed no significant difference whenthe early alcoholic liver disease group was comparedwith the control subgroups or when the fatty liversubgroup (without stage definition of the disease) wascompared with the hepatitis C/hepatitis B/reactivehepatitis group. We found no difference in the im-munostaining for CYP2E1 between the cases withNASH and the cases with steatosis with a larger andmore homogenous population. In the oxidative stresshypothesis of NASH pathogenesis (40, 41), CYP2E1 isnot the unique provider of reactive oxygen species(ROS), lipid peroxidation, and liver injury. CYP2E1deficiency in CYP2E1�/� mice did not prevent thedevelopment of steatohepatitis when fed with amethionine- and choline-deficient diet (15, 42). Acompensatory increase of the expression of CYP4Awas measured, implying that this isoform contributesto oxidative stress in the absence of CYP2E1 (11, 15,42). However, CYP2E1 has been linked to decreasedhepatocellular sensitivity to apoptosis induced byoxidative stress (43) and impaired insulin signalling(44). In any case, the degree of stellate cell activation inNASH was reported to correlate with the amount offibrosis rather than with the degree of inflammatoryactivity (39).

This study has several limitations. Even if thenumber of patients is larger than in other studieson the same topic (11, 16), it remains relativelysmall. More relevant is the sampling variability, whichshould always be a concern in the histologicaldiagnosis of NASH (45). This is actually one reasonthat motivated us to investigate a functional noninva-sive test to help in the diagnosis of NASH.The morphological and functional coherence of ourdata regarding CYP2E1 assessed by the CHZ test andby immunohistochemistry speaks against samplingerror interfering with our results. Nevertheless,the importance of liver biopsy sampling error shouldbe stressed and the study by Orellana et al. (11)relied presumably on larger intra-operative biopsiesto assess the expression of CYP2E1 by immunoblot-ting. The CHZ test is noninvasive, simple, andsubjected to little variability, but, importantly, it failsto identify patients with NASH from patients withNAFL. Other functional noninvasive tests should beinvestigated.



In conclusion, this study emphasizes the need for aliver biopsy to diagnose NASH in patients suspected tohave this disease. Clinical parameters alone are unableto identify patients with NASH, and the noninvasivedetermination of the CYP2E1 activity by the chlozox-azone test is not useful to distinguish patients withNASH from patients with NAFL.

Acknowledgement

This work was supported by the ‘Stiftung fur die Leberkran-

kheiten’.

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Expression and activity of the cytochrome P450 2E1 in patients with nonalcoholic steatosis and...

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