Relationship between in vivo chlorzoxazone hydroxylation, hepatic cytochrome P450 2E1 content and liver injury in obese non-alcoholic fatty liver disease patients

Hepatology Research 34 (2006) 57–63

Relationship between in vivo chlorzoxazone hydroxylation, hepatic cytochrome P450 2E1 content and liver injury in obese non-alcoholic fatty liver disease patients Myriam Orellana a,∗ , Ram´on Rodrigo a , Nelson Varela a , Julia Araya b , Jaime Poniachik c , Attila Csendes d , Gladys Smok e , Luis A. Videla a a

ICBM, Programa de Farmacolog´ıa Molecular y Cl´ınica, Facultad de Medicina, Universidad de Chile, Casilla 70,000, Santiago 7, Chile b Departamento de Nutrici´ on, Facultad de Medicina, Universidad de Chile, Casilla 70,000, Santiago 7, Chile c Servicio de Gastroenterolog´ıa, Hospital Cl´ınico de la Universidad de Chile, Casilla 5, Santiago 7, Chile d Servicio de Cirug´ıa, Hospital Cl´ınico de la Universidad de Chile, Casilla 5, Santiago 7, Chile e Servicio de Anatom´ıa Patol´ ogica, Hospital Cl´ınico de la Universidad de Chile, Casilla 5, Santiago 7, Chile Received 4 July 2005; received in revised form 26 September 2005; accepted 13 October 2005 Available online 29 November 2005

Abstract The aim of the present study was to test the hypothesis that induction of cytochrome P450 2E1 (CYP2E1) in the liver of patients with non-alcoholic fatty liver disease (NAFLD) is correlated both with the in vivo activity of the cytochrome and with the development of liver injury. For this purpose, the liver content of CYP2E1 was determined by Western blot and the CYP2E1 activity by the in vivo hydroxylation of chlorzoxazone (CLZ). The study groups were obese women with an average body mass index (BMI) of 40.3 kg/m2 , who underwent therapeutic gastroplasty or gastrectomy with a gastro-jejunal anastomosis. Further, the hepatic histology was determined to establish the pathological score grouping the subjects into three categories: control, steatosis and steatohepatitis. The liver CYP2E1 content and the CLZ hydroxylation of obese patients with steatosis and, particularly, with steatohepatitis were significantly higher than controls and correlated positively with both the severity of the liver damage. These data provide evidence that CYP2E1 would be involved in the mechanism of liver injury found in obese NAFLD patients. Also, the correlation between liver CYP2E1 content and in vivo CLZ hydroxylation would validate the latter as a reliable indicator of liver injury in NAFLD, thus providing a simple and not invasive method to study these patients. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Chlorzoxazone; Cytochrome P450 2E1; Non-alcoholic fatty liver disease; Obesity; Steatohepatitis; Steatosis

1. Introduction Hepatic steatosis is the setting for non-alcoholic fatty liver disease (NAFLD), a liver pathological entity observed in patients without a history of significant alcohol consumpAbbreviations: BMI, body mass index; CLZ, chlorzoxazone; CYP2E1, cytochrome P450 2E1; NAFLD, non-alcoholic fatty liver disease; ROS, reactive oxygen species; 6-OH-CLZ, 6-hydroxy-chlorzoxazone ∗ Corresponding author. Tel.: +56 2 9786066; fax: +56 2 7355580. E-mail address: [email protected] (M. Orellana). 1386-6346/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2005.10.001

tion that resembles alcohol-induced liver damage histologically. However, the pathogenic mechanism of liver cell injury, inflammation and hepatic fibrosis in NAFLD is unclear [1–4]. The disorder is commonly associated with obesity, type 2 diabetes, hyperlipidemia and rapid weight loss [2,3,5]. In humans, hepatic steatosis was proposed to be an essential precursor of NAFLD [5,6], but in these patients, the chronic accumulation of fat does not lead to necroinflammatory lesions, as seen in rodent models of genetic obesity, such as obese (ob/ob) mice and fatty (fa/fa) Zucker rats [7,8]. The similarity of hepatic lesions between NAFLD

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and alcoholic steatohepatitis suggests the possibility that the pathogenic mechanism of both conditions share common elements. Numerous studies have emphasized the key role of oxidative stress in the pathophysiological mechanism of alcoholic steatohepatitis [9,10]. Oxidative stress is one of the potential biochemical mechanisms of liver cell injury, inflammation and hepatic fibrosis steatohepatitis [6,9,11]. Evidence of oxidative stress has been found in several experimental models of NAFLD [7,12] and in the liver of humans with alcoholic steatohepatitis [13,14], steatosis and NAFLD [15,16]. Microsomal cytochrome P450 (CYP) enzymes, refers to a group of iron-containing hemoproteins that play a significant role in the biotransformation of xenobiotics and endobiotics. During the oxygen reductase activity of P450, O2 is reduced to superoxide radical (O2 •− ), a precursor species in the formation of hydrogen peroxide (H2 O2 ) and hydroxyl radical (HO• ) of known toxicological potential [17]. This pro-oxidant behavior is particularly important in the P450 isoforms cytochrome P450 2E1 (CYP2E1) and CYP4A, as they exhibit a loose coupling in the operation of the catalytic redox cycle [7,15]. The CYP2E1 is upregulated by ethanol ingestion, in the nutritional states of fasting, diabetes, obesity [14,15] and in other diseases [18]. Induction of CYP2E1 involving higher rates of reactive oxygen species (ROS) generation is associated with an enhancement in lipid peroxidation processes [19–21] and cytotoxicity, considering the abrogation of the hepatotoxic response by the CYP2E1 inhibitor diallylsulfide and the antioxidant Trolox [22]. Oxidative stress occurring in the liver of humans with NAFLD, is a redox imbalance that is developed in patients with steatosis [5] and is further exacerbated in those with steatohepatitis [15,16,23]. In the latter group, increased hepatic CYP2E1 content has been observed [24], a feature that seems to be related to the insulin resistance found in clinical NAFLD by loss of the repressive effect of insulin [6,16,25]. The drug chlorzoxazone (CLZ) has been used extensively as a selective in vivo probe of hepatic CYP2E1 activity [26]. In addition to strong in vitro evidence, the enzyme selectivity of CLZ in vivo is shown by the pronounced reduction in oral CLZ clearance that is observed when a known mechanismbased CYP2E1 inhibitor, disulfiram, is administered [27]. It has been reported that CYP2E1 plays an important role in the pathogenesis of NAFLD in animal models [7,8,12,28] but its role in the pathogenesis of human NAFLD is unclear. The aim of the present study was to test the hypothesis that induction of CYP2E1 in the liver of patients with NAFLD is correlated both with the in vivo activity of the cytochrome and with the development of liver injury. For this purpose, the content of CYP2E1 was determined in the liver of obese NAFLD patients by Western blot analysis, with the concomitant assessment of CYP2E1 activity by the in vivo hydroxylation of CLZ and results were analyzed in the light of the histological features of the liver.

2. Methods 2.1. Patients and laboratory tests Twenty-six obese women with an average body mass index (BMI = weight/height2 ) of 40.3 kg/m2 and an age range of 18–55 years that underwent therapeutic gastroplasty or gastrectomy with a gastro-jejunal anastomosis were included in this study. Liver biopsies were taken for histological diagnosis during the surgery, and a complete clinical history including data on nutrition and alcohol consumption together with anthropometric measurements were obtained. Laboratory tests included liver enzymes, bilirubin, albumin, hepatitis B and C serology and autoantibodies (antinuclear, antimitochondrial and antismooth muscle antibodies). In addition, serum levels of ferritin, transferrin saturation, ceruloplasmin, cholesterol, triglycerides, LDL, HDL and glucose were also determined. The index of insulin resistance was measured by the homeostasis model assessment (HOMA) and calculated as [(fasting insulin × fasting glucose)/22.5] [29]. Subjects who consumed more than 40 g of alcohol per week or who had any blood tests suggesting other specific liver diseases were excluded. Selected patients were subjected to a diet of 25 kcal/kg, with 30% of the calories given as lipids and 15% as proteins, for at least 2 days prior to surgery. Liver biopsies were used for histological diagnosis and to analyze the microsomal parameters. Liver samples were fixed in 10% formaldehyde, paraffin embedded and sections were stained with either hematoxylin–eosin or Van Gieson’s stains. Sections of each liver biopsy were blindly observed and evaluated for histological abnormalities by means of a previously defined code [30]. Three groups of obese individuals were formed: (a) control group (normal liver histology), (b) steatosis (5–100% macrovesicular steatosis) and (c) steatohepatitis (steatosis and occasional to multiple focus of lobular inflammation with hepatocyte ballooning). The degree of steatosis or inflammation was expressed by a semi-quantitative pathology score (PS) where this two features were categorized in the following scales: 0 (absent), 1 (mild), 2 (moderate) and 3 (severe). The Ethic Committee of the University of Chile Clinical Hospital approved the study protocol, according to Helsinki criteria. Informed consent for using part of the blood and liver samples was obtained from all patients. 2.2. In vivo chlorzoxazone hydroxylation CYP2E1 enzymatic activity was assessed in plasma by measuring the chlorzoxazone hydroxylation to 6-hydroxychlorzoxazone (6-OH-CLZ) [26]. Two hours after the ingestion of 500 mg of CLZ, blood samples of each group of patients were received in plastic tubes with EDTA, centrifuged immediately and the serum frozen to −20 ◦ C until use. Then, after de-conjugation with ␤-glucuronidase, the CLZ was separated from 6-OH-CLZ by reverse phase HPLC by using a LiChrospher 100 RP-18 (5 ␮m) column and

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acetonitrile/acetic acid, 30/70 (v/v) at a flow of 1 ml/min, measuring the absorbance at 285 nm [26].

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3. Results 3.1. Pathological and clinical characteristics of NAFLD patients

2.3. Liver cytochrome P450 2E1 content Microsomes were prepared by ultracentrifugation as described elsewhere [31] from at least 1 g of fresh liver biopsies. Microsomal protein was measured by the method of Lowry et al. [32] using bovine serum albumin (BSA) as standard. The total CYP content was measured according to the method of Omura and Sato [33] and the liver CYP2E1 content was determined by Western blot. Western immunoblotting was performed using 10% polyacrylamide-SDS gel as described by Towbin et al. [34] and 20 ␮g of microsomal protein were loaded in each lane. After being transferred to nitrocellulose sheets, blots were developed using polyclonal antibody to hepatic cytochrome P450 2E1 (Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan). Alkaline phosphatase was used as the second antibody and the bands were stained with Nitroblue tetrazolium (NBT)/5bromo-4-cloro-3-indolylphosphate (BCIP) solution. Quantification of CYP2E1 was performed using acetone-treated rat liver microsomes containing cytochrome P450 3E1 (3 pmol/blot) as a standard (Daiichi Pure Chemicals Co. Ltd.).

The obese women in this study presented the following hepatic pathology scores: controls [PS, 0 (n = 6)], steatosis [PS, 1.8 ± 0.30 (n = 10)] and steatohepatitis [PS, 2.6 ± 0.40 (n = 10)]. All subjects presented BMI values above the normal range (36 and 49 kg/m2 ), being the steatohepatitis with a BMI 36% higher than those of both control and steatosis groups. The majority of patients were asymptomatic, with normal or mild alterations of liver function tests and lipid profile. Table 1 shows the main biochemical and clinical characteristics of NAFLD patients. The three groups revealed no significant differences in the clinical parameters studied with the exception of levels of fasting insulin, parameter that was increased significantly in the steatohepatitis group, in spite of the normal values of fasting glucose. Insulin resistance, calculated from the HOMA analysis, was found in the steatosis and steatohepatitis groups, being the values of the last group significantly higher than those of control group.

2.4. Statistical analysis

The hepatic CYP2E1 activity measured as the plasmatic generation of 6-OH-CLZ was quantified as the 6-OHCLZ/CLZ ratio. The 6-OH-CLZ/CLZ ratio increased to 36 and 212% over control values in the steatosis and steatohepatitis groups, respectively (Fig. 1).

Results are expressed as means ± S.E.M. for the number of patients indicated. The sources of variation for multiple comparisons were assessed by one-way analysis of variance (ANOVA), followed by Bonferroni’s multiple comparison test. The differences were considered statistically significant at p < 0.05. To analyze the association between different variables, the Spearman rank order correlation was used.

3.2. In vivo chlorzoxazone hydroxylation

3.3. Liver CYP2E1 content Although the liver microsomal CYP total content was similar in the three groups [0.34 ± 0.03 (n = 6); 0.26 ± 0.01

Table 1 Clinical and biochemical parameters in control obese and patients with non-alcoholic fatty liver disease Parameters (units)

Controls (n = 6)

Age (years) BMI (kg/m2 ) Cholesterol (mg/dl) Triacylglycerols (mg/dl) LDL (mg/dl) HDL (mg/dl) Fasting glucose (mmol/l) Fasting insulin (␮U/ml) HOMA AST (IU/l) ALT (IU/l) AST/ALT ratio ␥-GT (U/l) Bilirubin (mg/dl) Albumin (g/dl)

41 36 155 108 114 50 5.00 14 3.11 26 32 0.81 38 0.60 4.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 1 11 15 9 4 0.4 2 0.8 3 3 0.08 6 0.05 0.1

Steatosis (n = 10) 44 36 193 136 129 45 5.83 24 6.22 36 50 0.72 42 0.56 4.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4 2 13 27 10 3 0.5 4 0.8 7 11 0.13 8 0.06 0.1

Steatohepatitis (n = 10) 40 49 181 187 100 41 5.44 36 8.70 30 47 0.64 38 0.62 4.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 2*C,*S 12 31 8 3 0.8 7*C 0.9*C 3 5 0.08 3 (0) 0.06 0.1

Values represent means ± S.E.M. for the number (n) of subjects indicated. Abbreviations: BMI, body mass index; LDL, low density lipoprotein; HDL, high density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; HOMA, homeostasis model assessment. * Significantly different (p < 0.05) from control (C) or from steatosis (S).

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Fig. 1. Chlorzoxazone (CLZ) hydroxylation in blood of obese controls, steatosis and steatohepatitis patients. Values expressed as 6-OH-CLZ/CLZ ratios are means ± S.E.M. for the number of subjects per group indicated in parenthesis. * Significantly different at p < 0.05 from control (C) or from steatosis (S).

(n = 10) and 0.33 ± 0.02 (n = 10) nmol/mg microsomal protein for control, steatosis and steatohepatitis, respectively], the CYP2E1 content was found increased in the steatosis and steatohepatitis groups. Fig. 2 shows the CYP2E1 content determined in fresh liver biopsies by Western blot, as pmol/mg microsomal protein. The CYP2E1 content increased to 12 and 50% over control in the steatosis and steatohepatitis groups, respectively. 3.4. Liver CYP2E1 content and CLZ hydroxylation as function of the pathology score From 26 obese patients studied, simultaneous measurements of both liver CYP2E1 content and CLZ hydroxylation was possible only in 14. Fig. 3 shows the values of CLZ hydroxylase activity and liver CYP2E1 content. Fig. 3A

Fig. 2. CYP2E1 content determined by Western blot in liver of obese controls, steatosis and steatohepatitis patients (pmol/mg microsomal protein). Values are means ± S.E.M. for the number of subjects per group indicated in parenthesis. * Significantly different at p < 0.05 from control (C) or from steatosis (S).

Fig. 3. Liver CYP2E1 content (A) and chlorzoxazone hydroxylase activity (B) as function of the pathology score assessed by liver histology. Here, both parameters were measured simultaneously in the blood and in the liver of the same 14 patients. r: Spearman rank order correlation.

shows that the liver CYP2E1 content increased with the severity of the morphological alterations assessed by the liver PS (r = 0.62; p < 0.02). Fig. 3B shows that the liver CLZ hydroxylation also increases significantly with the severity of the liver morphological alterations (r = 0.81; p < 0.0005). The correlation between the CLZ hydroxylase activity and liver CYP2E1 content from values obtained from Fig. 3 is shown in Fig. 4. It was found a high correlation (r = 0.54; p < 0.05) between the liver CYP2E1 content and the activity represented by the in vivo CLZ hydroxylation, making evident that both

Fig. 4. Correlation between the chlorzoxazone (CLZ) hydroxylase activity and liver CYP2E1 content as function of the pathology score assessed by liver histology. The data used to calculate the correlation were obtained from those of Fig. 3. r: Spearman rank order correlation.

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parameters increase insofar as the liver injury represented by the pathology score is enhanced.

4. Discussion The results of the present study provide evidence that in obese patients with steatosis and, particularly with steatohepatitis, the CYP2E1 content and the in vivo CLZ hydroxylase activity are enhanced, with a significant correlation between both parameters. Furthermore, both liver CYP2E1 content and activity increase with the severity of the morphological alterations assessed by the pathology score. On other hand, insulin resistance, calculated from the HOMA analysis, was found in the patients with steatosis and, in a major grade, with steatohepatitis. In agreement with earlier observations [4] our patients showed minimal alterations of liver function tests and lipid profile, with elevated BMI over normal values, strengthening the association between obesity and the occurrence of NAFLD [5,30,35,36]. An association of NAFLD with the metabolic syndrome has been suggested [37,38] from the demonstration that those patients who are insulin resistant, have a higher prevalence of severe steatosis. Insulin resistance is believed to lead to accumulation of triglycerides in hepatocyte [39,40]. Furthermore, the presence of steatohepatitis and upregulation of liver CYP2E1 expression may contribute to insulin resistance associated with human NAFLD, as recently shown in the methionine/choline-deficient diet mouse model that induces down-regulation of insulin signaling [41]. It has been reported that CYP2E1 activity is increased in the liver of obese humans as evidenced by increased plasma levels of inorganic fluoride after exposure to halogenated anesthetics and an increase in the clearance of chlorzoxazone, both substrates of CYP2E1 [11,42]. Higher 6-OH-CLZ/CLZ ratios are observed in obese subjects compared with lean controls [30,43], suggesting that obesity itself is associated with CYP2E1 induction and related oxidant stress [23]. Furthermore, Chalasani et al. reported that CLZ hydroxylation and lymphocyte CYP2E1 expression are enhanced in obese subjects with NAFLD [44]. Taken together, these studies indicate that CYP2E1 induction is related to liver damage among obese patients with NAFLD [23,24,28,36,43–45]. The production of oxy-radicals is the basis for the greater capacity for CYP2E1 to initiate NADPH-dependent lipid peroxidation [20,21] in liver of NAFLD patients [24,45]. From the pathogenic point of view, CYP2E1-derived oxidative stress could represent a crucial factor in determining cell injury, particularly when antioxidant defenses are depleted. In a previous study, our group found several changes in parameters of oxidative stress in liver of NAFLD patients, depending upon the severity of the morphological alterations assessed by liver histology [23]. Obese patients with steatosis and steatohepatitis exhibit a pro-oxidant condition in the liver. This pro-oxidant condition occurs concomitantly with a

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significant decrease in the activity of the antioxidant enzyme superoxide dismutase (SOD) and catalase [23,25]. CYP2E1 and CYP4A are two microsomal oxidases involved with fatty acid oxidation being both enzymes the capacity to reduce molecular oxygen to produce pro-oxidant species, which, if not countered efficiently by antioxidants, create oxidative stress [15]. Several of the factors that regulate hepatic CYP2E1 and CYP4A expression have been linked to the pathogenesis of hepatic steatosis and NAFLD [3,6], thus levels of hepatic CYP2E1 increase with type 2 diabetes, insulin resistance, central obesity and fasting [18]. It is known that insulin has a repressive effect on the regulation of CYP2E1 [16] therefore, this factor would be it that leads to increased levels of CYP2E1 in our obese patients, by loss of the repressive effect of this hormone [15]. The finding that CYP2E1 deficiency did not prevent the development of non-alcoholic steatohepatitis in mice subjected to methionine/choline-deficient diet suggests the involvement of a non-CYP2E1 pathway [7]. Under these conditions, CYP4A10 and CYP4A14 were upregulated and microsomal lipid peroxidation was inhibited by anti-mouse CYP4A10 antibody, supporting a role for CYP4A enzymes as alternate initiators of oxidative stress in the liver [7]. However, upregulation of liver CYP4A isoforms in human NAFLD remains to be studied. Unlike alcoholic liver disease in which hepatic CYP2E1 activity subsides within days of discontinuing toxic alcohol intake, CYP2E1 is persistently increased in the livers of patients with NAFLD [24,45]. Furthermore, the distribution of CYP2E1 protein is located in the perivenular (acinar zone 3) region, corresponding to the site of maximal hepatocellular injury in NAFLD [1]. Although obesity increases the activity of CYP2E1 in human liver as shown by studies measuring the pharmacokinetics of chlorzoxazone [36,42] no correlation to liver injury was carried out. The nexus between CYP-induced oxidative stress and cellular injury in the face of fatty acid excess provides a plausible explanation for hepatocellular damage in NAFLD [11,15,46]. Although the inflammatory activity in NAFLD, with its obvious implications for cytokinemediated activation of stellate cells and profibrotic pathways, is important in hepatic fibrogenesis, oxidative stress may also play a role. ROS are increasingly recognized as potential mediators of stellate cell activation [47–49]. This has been partially explored by studies in CYP2E1-overexpressing hepatic stellate cell lines. In the absence of exogenous substrate, CYP2E1-dependent oxidative stress was associated with upregulation of collagen I; this could be further enhanced by GSH depletion and was reversed by antioxidants [48,50]. In conclusion, in obese NAFLD patients, the liver CYP2E1 content and activity increase insofar as increase the morphological alterations assessed by liver histology. These data provide evidence that CYP2E1 would be involved in the mechanism of liver injury found in obese NAFLD patients, suggesting that insulin resistance would be causally linked

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with the activation of this enzyme system, although other mechanism cannot be ruled out. In addition, the correlation between liver CYP2E1 content and in vivo CLZ hydroxylation would validate the latter as a reliable indicator of liver injury in NAFLD, thus providing a simple and not invasive method to study these patients.

Acknowledgements We thank to Drs. Juan Carlos D´ıaz, Patricio Burdiles, Fernado Maluenda, Jorge Rojas and Jorge Contreras for their valuable assistance. This work was supported by grant 1011057 from FONDECYT, Santiago, Chile.

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