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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cytochrome P450 2E1 (CYP2E1) activates several hepatotoxins and contributes to alcoholic liver damage. Obesity is a growing health problem in the United States. The aim of the present study was to evaluate whether acetone- or pyrazole-mediated induction of CYP2E1 can potentiate liver injury in obesity. CYP2E1 protein and activity were elevated in acetone- or pyrazole-treated obese and lean mice. Acetone or pyrazole induced distinct histological changes in liver and significantly higher aminotransferase enzymes in obese mice compared to obese controls or acetone- or pyrazole-treated lean mice. Higher caspase-3 activity and numerous apoptotic hepatocytes were observed in the acetone- or pyrazole-treated obese mice. Increased protein carbonyls, malondialdehyde, 4-hydroxynonenal-protein adducts, elevated levels of inducible nitric oxide synthase, and higher 3-nitrotyrosine protein adducts were found in livers of acetone- or pyrazole-treated obese animals, suggesting elevated oxidative and nitrosative stress. Liver tumor necrosis factor α levels were higher in pyrazole-treated animals. The CYP2E1 inhibitor chlormethiazole and iNOS inhibitor N-(3-(aminomethyl)-benzyl) acetamidine abrogated the toxicity and the oxidative/nitrosative stress elicited by the induction of CYP2E1. Conclusion: These results show that obesity contributes to oxidative stress and liver injury and that induction of CYP2E1 enhances these effects. (HEPATOLOGY 2007;45:1355–1365.)

Obesity is a potential health issue in the United States.1 Obesity is also considered a causative factor for nonalcoholic fatty liver disease.2 Ethanol-inducible cytochrome P450 2E1 (CYP2E1) is involved in the metabolism of several environmental toxicants to their active forms.3 CYP2E1 produces reactive oxygen species (ROS) and generates oxidative stress, and contributes to the pathogenesis of alcoholic liver injury, including alcoholic steatohepatitis.3, 4 CYP2E1 is elevated in pathophysiological conditions like obesity and diabetes.5, 6

In the present study, we used the ob/ob mouse as a genetic model for obesity, and acetone and pyrazole as inducing agents to elevate CYP2E1 levels in these animals. The homozygous C57BL/6J ob/ob mouse exhibits characteristics associated with human obesity, such as hyperlipidemia, hyperinsulinemia, and mild hyperglycemia,7 and has been used extensively as an animal model to study human obesity.8, 9

Acetone and pyrazole induce CYP2E1 and increase CYP2E1-mediated oxidant stress and subsequent cell injury.10, 11 We have used acetone and pyrazole for long- and short-term induction of CYP2E1, respectively. To elucidate the role of CYP2E1, chlormethiazole (CMZ), which abrogates CYP2E1-mediated liver damage,12, 13 was used. Inducible nitric oxide synthase (iNOS) is involved in alcohol-induced liver injury,14 therefore we used N-(3-(aminomethyl)-benzyl) acetamidine (1400W), a specific inhibitor of iNOS to investigate the role of iNOS in CYP2E1-potentiated liver injury in obese mice. Although evidence for the induction of CYP2E1 in obesity exists, the role of CYP2E1-mediated toxicity in promoting liver injury and the mechanisms involved in the pathogenesis and development of fatty liver need to be studied further. The aim of this study was to study the effects of CYP2E1 induction on promoting oxidative and nitrosative stress and liver injury in ob/ob mice, an experimental model of obesity.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals and Treatment.

Male 8-week-old homozygous obese C57BL/6J ob/ob mice and heterozygous lean littermate C57BL6/J+/? mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The animals were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care and divided into 10 groups, each of which consisted of 4-6 animals. Group 1 and 2 consisted of lean and obese mice, respectively, fed a commercially available high-fat control dextrose diet (Bio-Serv, Frenchtown, NJ) with 42% of calories derived from fat, 16% from protein, and 42% from carbohydrates (dextrin-maltose) ad libitum for 2 weeks. These animals, which served as controls, had access to regular drinking water. Groups 3 and 4 consisted of lean and obese mice, respectively, fed the liquid diet plus 2% acetone in drinking water for the same time period. Groups 5 and 6 consisted of obese and lean mice, respectively, injected intraperitoneally with pyrazole (Sigma), 150 mg/kg body weight, once per day for 2 days. Groups 7 and 8 consisted of obese and lean mice, respectively, injected intraperitoneally with 0.9% saline. Group 9 consisted of obese mice injected with CMZ (Sigma), 75 mg/kg body weight, 2 hours before pyrazole treatment for 2 days. Group 10 consisted of obese mice injected with 1400W (Sigma), 10 mg/kg body weight, 2 hours before pyrazole treatment for 2 days. Mice in groups 5-10 had access to regular drinking water and standard chow ad libitum. Mice were sacrificed with an overdose of pentobarbital.

Liver Pathology.

Liver homogenates were prepared in 8 volumes of ice-cold 50 mM Tris-HCl, pH 7.4, 1.15% KCl, and 1 mM ethylenediamine tetraacetic acid buffer. Liver samples for histology were fixed in 10% formalin, and paraffin-embedded, 5-μm sections were stained with hematoxylin-eosin. The percentage of hepatocytes containing fatty droplets was counted. Degenerative or necrotic changes were observed and were graded as none (0), mild (<25%), moderate (25%-50%), and severe (>75%). Serum ALT and AST levels were measured using a diagnostic kit (Thermo Electron). Triglyceride contents in the liver homogenates were measured by using the Infinity Triglyceride Reagent (Thermo Electron).

Western Blot Analysis.

Levels of CYP2E1, inducible nitric oxide synthase (iNOS), Bcl-xL, Bcl-2, Bax, or Bad protein in 30-50 μg of protein samples from freshly prepared liver homogenates were determined using Western blot analysis with anti-human CYP2E1 polyclonal antibody (1:3,000) (kindly provided by Dr. J.M. Lasker), anti-iNOS antibody (1:1,000), anti-human Bcl-xL polyclonal antibody (1:1,000), anti-human Bcl-2 monoclonal antibody (1:1,000), anti-human Bax polyclonal antibody (1:1,000) or anti-human Bad monoclonal antibody (1:500), respectively, followed by incubation with the appropriate secondary antibody, respectively. All primary antibodies except CYP2E1 were procured from Santa Cruz Biotechnology. Detection by the chemiluminescence reaction was carried out for 1 minute using the ECL kit (Amersham Biosciences) followed by exposure to Kodak Biomax x-ray film (Eastman Kodak Co.). Blots were scanned using the Automated Digitizing System (UN-SCAN-IT gel programs, version 5.1, Silk Scientific Corp., Orem, UT) and results expressed as the protein/β-actin ratio.

CYP2E1 mRNA and Activity.

CYP2E1 mRNA expression was assessed by Northern blot.15 CYP2E1 activity was measured by the rate of oxidation of p-nitrophenol to p-nitrocatechol.16

Lipid Peroxidation.

Lipid peroxidation was measured in liver homogenates using a previously described method.17 Thiobarbituric acid-reactive components were detected at 535 nm, using an extinction coefficient of 1.56 × 105/M/cm to calculate malondialdehyde equivalents.

TUNEL Assay and Caspase-3 Activity Assay.

DNA fragmentation was assessed via terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay using the ApopTag in situ apoptosis detection kit (Chemicon). The quantitative analysis of positive hepatocytes with DNA fragmentation or apoptotic nuclei was performed by counting the average number of apoptotic nuclei per visual field (21 visual fields per sample). Caspase-3 activity was measured in the liver homogenate using the caspase-3 colorimetric assay kit (Sigma).

Protein Carbonyl Adducts and 4-HNE Adducts.

Protein carbonyl adducts were assayed in liver homogenates using 20 μg of protein samples and the OxyBlot Protein Oxidation Detection Kit (Chemicon). Paraffin-embedded liver sections were immunostained for 4-hydroxynonenal (HNE) adducts using the ImmunoCruz Rabbit ABC Staining System Kit (Santa Cruz Biotechnology). The positive staining was detected by dark brown color and was evaluated as negative (−), weakly positive (+), moderately positive (++), and strongly positive (+++).

Immunohistochemistry and Slot Blot for Nitrotyrosine Residues.

Immunohistochemical staining was performed with the ImmunoCruz Rabbit ABC Staining System Kit (Santa Cruz Biotechnology) for 3-nitrotyrosine (3-NT) protein adducts using anti-3-NT antibody (1:100) (Upstate USA Inc). Positive staining was detected by a brownish-yellow color and was graded as negative (−), weakly positive (+), moderately positive (++) and strongly positive (+++). For immunochemical detection of protein nitrotyrosine residues (3NT) in liver homogenate, a slot-blot technique with 0.5 μg of homogenate protein plus polyclonal rabbit anti-3-NT antibody was used.

Tumor Necrosis Factor α Levels.

Tumor necrosis factor α (TNFα) levels in liver homogenates were measured using the mouse TNFα ELISA kit (Pierce Biotechnology, Inc.).

Statistical Analysis.

Analysis of variance (ANOVA) followed by Student-Newman-Keuls post hoc test was employed to calculate the statistical significance between the different groups of treated and untreated mice. Data are presented as the mean ± SE. A P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Acetone or Pyrazole Induce Pathological Changes in the Livers of Obese Mice.

Mice were treated with two different inducers of CYP2E1 to evaluate whether induction of CYP2E1 induces hepatotoxicity in obesity. Hematoxylin& eosin staining showed that the control lean mice did not exhibit any fatty droplets in their liver and acetone or pyrazole treatment of lean mice did not induce distinct pathological changes (Fig. 1A, panels 1, 2, 5, and 6). The livers of control obese mice exhibited 50% steatotic hepatocytes, 35% of hepatocytes exhibiting microvesicular and 15% macrovesicular steatosis in both the pericentral and periportal area (panel 3). Acetone induced extensive fatty changes represented by massive steatosis (60%-85%) with the presence of inflammatory cells; however, mild necrosis (<25%) was observed in the livers of obese mice (Fig. 1A, panel 4). Pyrazole-treated obese mice had pronounced macrovesicular steatosis (75%-90%), inflammatory cells surrounding the steatotic hepatocytes, and multifocal necrosis (>75%), which were not seen in control obese mice (<5%) (Fig. 1A, panels 7, 8). The lipid droplets were located in the cytoplasm of the hepatocytes, pushing the nuclei to the periphery of the cell. All these pathophysiological changes were more pronounced in the pericentral area. Although 50%-65% steatosis was observed in the CMZ- or 1400W-treated obese mice, similar to that observed in control obese mice, necrotic changes were very mild (<20%) (Fig. 1A, panels 9, 10). The triglyceride levels in control obese mice were almost 3-fold higher than the control lean mice (Fig. 1B). Pyrazole treatment caused a small increase in triglyceride content in lean mice but a 2-fold further increase over the elevated triglyceride levels of obese mice. Treatment of the pyrazole-treated obese mice with CMZ or 1400W caused a 2-fold decrease in the triglyceride levels, back to the obese control levels (Fig. 1B).

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Figure 1. (A) Effect of acetone or pyrazole on liver morphology. Photomicrographs of liver samples stained with hematoxylin & eosin: liver section (panels 1 and 5) from control lean mouse shows normal morphology; liver section from acetone-treated lean mouse or pyrazole-treated lean mouse (panels 2 and 6) shows minimal steatotic or necrotic changes; liver section from control obese mouse (panels 3 and 7) shows a mixture of microvesicular and macrovesicular steatosis (50%) as depicted by the presence of scattered lipid droplets in central and periportal areas; liver section from acetone-treated obese mouse (panel 4) shows extensive and severe macrovesicular steatosis (60%–85%) with characteristic large fat droplets, mostly in the pericentral area; liver section from pyrazole-treated obese mouse (panel 8) shows massive steatosis (75%–90%) more pronounced in the pericentral area and inflammatory cells surrounding the steatotic hepatocytes and multifocal necrosis represented by the lighter area containing the damaged cells in the right of the photomicrograph; liver section from CMZ plus pyrazole-treated obese mouse (panel 9) shows almost normal ob/ob morphology with characteristic fatty droplets (50%–65%) and minimal necrosis; liver section from 1400W plus pyrazole-treated obese mouse (panel 10) shows almost normal ob/ob morphology with microvesicular and macrovesicular steatosis (50%–65%). (B) Triglyceride levels. *P < 0.05 for control obese mice versus control lean mice. P < 0.05 for pyrazole-treated lean or obese mice versus control lean or obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole-treated obese mice. Data represent the mean ± SE of six animals/group.

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Acetone treatment did not significantly increase serum ALT or AST levels in the lean mice. Treatment of lean mice with pyrazole resulted in a 50% increase in ALT levels; however, AST levels were not increased significantly (Fig. 2A,B). The control obese mice had higher ALT and AST levels than the control lean mice, and acetone or pyrazole caused further 2- to 4-fold significant increases in ALT and AST levels. Treatment with CMZ or 1400W significantly lowered this increase in ALT and AST levels back to the control obese levels (Fig. 2A,B).

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Figure 2. Acetone or pyrazole increases serum (A) ALT and (B) AST in obese mice. *P < 0.05 for control obese mice versus control lean mice. P < 0.05 for acetone- or pyrazole-treated lean or obese mice versus control lean or obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole-treated obese mice. Data represent the mean ± SE of six animals/group.

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Effect of Acetone or Pyrazole on Caspase-3 Activity and DNA Fragmentation.

Significant increases in caspase-3 activity were observed in the lean mice administered acetone or pyrazole (76% and 98%, respectively), while more substantial increases were observed in the acetone- or pyrazole-treated obese mice (142% and 275% increase in caspase-3 activities, respectively) (Fig. 3A). CMZ or 1400W treatment of pyrazole mice lowered the elevated caspase-3 activity. TUNEL analysis indicated that the control lean mice and the control obese mice exhibited very few cells with the characteristic intense brown staining, although the control obese mice did have higher TUNEL-positive nuclei as compared to control lean mice (Fig. 3B). Treatment of lean and obese mice with either acetone or pyrazole elevated TUNEL-positive staining (Fig. 3B). A 4-fold increase in the number of hepatocytes with positively stained nuclei was observed in the acetone- or pyrazole-treated obese mice as compared to a 2-fold increase in acetone- or pyrazole-treated lean mice (Fig. 3B). CMZ and 1400W lowered the pyrazole-elevated TUNEL back to the control obese levels, nearly completely blunting the pyrazole-induced increase.

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Figure 3. Acetone or pyrazole increases caspase-3 activity (A) and TUNEL-positive hepatocytes (B) in lean and obese mice. (A) *P < 0.05 for control obese mice versus control lean mice. P < 0.05 for acetone- or pyrazole-treated lean or obese mice versus control lean or obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole-treated obese mice. Data represent the mean ± SE of six animals/group.(B) Hepatocytes having apoptotic nuclei and showing positive brown staining (arrows) were detected using TUNEL. The quantitative analysis of positive nuclei with DNA fragmentation was performed by counting the average number of apoptotic nuclei per visual field, which are indicated in parentheses.

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Effect of Acetone or Pyrazole on CYP2E1 mRNA, Protein, and Catalytic Activity.

CYP2E1 mRNA level as normalized to GAPDH was 1.8-fold lower in control obese mice compared to control lean mice (1.74 versus 0.98). Treatment of lean mice with pyrazole caused a 40% decrease in CYP2E1 mRNA levels (1.12 versus 1.74). Pyrazole-treated obese mice had a 55% decrease in CYP2E1 mRNA levels (0.44 versus 0.98). The control lean mice had 2-fold higher basal CYP2E1 protein level compared to the control obese mice (Fig. 4A). Acetone treatment resulted in a 50% increase in CYP2E1 protein in the lean mice and a 150% increase in the obese mice. Pyrazole treatment increased CYP2E1 protein by 32% and a highly significant 270% increase in the lean and obese mice, respectively. CMZ and 1400W caused a 76% and 61% decrease, respectively, in CYP2E1 protein in the pyrazole-treated obese mice. CYP2E1 catalytic activity was higher in lean mice when compared to the obese animals (Fig. 4B) analogous to the higher CYP2E1 protein in the lean mice. The acetone or pyrazole treatment produced about 65% increases in the CYP2E1 activity in lean mice and about 140% increases in obese mice. CMZ and to a lesser extent 1400W decreased the activity of CYP2E1 in pyrazole-treated obese mice. Importantly, CMZ completely blunted the elevation in CYP2E1 activity produced by pyrazole, to the obese control levels. Thus, results assaying content and activity of CYP2E1 show that the acetone or pyrazole treatment increased the lower CYP2E1 level and activity found in the obese mice.

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Figure 4. Acetone or pyrazole increases CYP2E1 protein expression and activity. (A) Top: Western blot showing protein expression of CYP2E1 in liver homogenates from lean or obese mice. Bottom: Densitometric values (du) for CYP2E1/β-actin ratio. Data represent the mean ± SE of three independent experiments. *P < 0.05 for control lean mice versus control obese mice. P < 0.05 for acetone- or pyrazole-treated obese mice versus control obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole-treated obese mice. (B) Microsomal CYP2E1 activity. Data represent the mean ± SE of six animals/group. *P < 0.05 for control lean mice versus control obese mice. P < 0.05 for acetone- or pyrazole-treated lean or obese mice versus control lean or obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole-treated obese mice.

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Effect of Acetone or Pyrazole on Protein Carbonyl, HNE Adduct Formation, and Lipid Peroxidation.

The intensity of the protein carbonyl bands in acetone or pyrazole-treated lean mice was 50% higher than the control lean mice (Fig. 5A). Treatment of the obese mice with acetone or pyrazole increased the formation of the protein carbonyl adducts by about 150% and 106%, respectively. CMZ or 1400W decreased the elevated levels of the protein carbonyl adducts in pyrazole-treated obese mice (Fig. 5A).

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Figure 5. Acetone or pyrazole increases protein carbonyl (A), HNE adduct formation (B), and lipid peroxidation (C) in lean and obese mice. (A) Top: Protein carbonyl bands in liver homogenates from lean or obese mice. Bottom: Densitometric values (du). Data represent the mean ± SE of three independent experiments. *P < 0.05 for control obese mice versus control lean mice. P < 0.05 for acetone- or pyrazole-treated lean or obese mice versus control lean or obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole-treated obese mice. (B) Immunohistochemical detection of HNE protein adducts. Liver sections from control lean and obese mice show minimal HNE adduct formation, while acetone- or pyrazole-treated lean and especially acetone- or pyrazole-treated obese mice show strong positive staining for HNE adducts, mostly in the cytoplasm and in some nuclei in the pericentral area. This strong staining is blunted by CMZ and 1400W in the pyrazole-treated obese mice. (C) Lipid peroxidation. *P < 0.05 for control obese mice versus control lean mice. P < 0.05 for pyrazole-treated lean or obese mice versus control lean or obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole treated obese mice. Data represent the mean ± SE of six animals/group.

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Sections of liver, immunohistochemically stained for HNE adducts showed none or mild staining (−/+) in control lean and obese mice (Fig. 5B), but the acetone- or pyrazole-treated animals showed stronger immunostaining (++/+++), which was highly pronounced in the regions near the central vein. This region contains the highest levels of CYP2E1.4 The acetone- or pyrazole-treated obese mice not only had more intense staining (+++) but also exhibited extensive distribution of HNE adducts when compared to the acetone- or pyrazole-treated lean mice. Positive staining for HNE adduct was seen mainly in the cytoplasm of the hepatocytes. Treatment with CMZ or 1400W strongly lowered HNE adducts in the pyrazole-treated obese mice (+) (Fig. 5B). Control obese mice had higher malondialdehyde (MDA) product formation, likely a reflection of increased lipid peroxidation than the control lean mice (Fig. 5C). Pyrazole treatment caused a 2-fold increase in MDA levels in lean mice and a 3.5-fold increase in MDA levels in obese mice. CMZ or 1400W completely blunted the increase in MDA formation in pyrazole-treated obese mice (Fig. 5C).

Effect of Acetone or Pyrazole on iNOS Protein Expression and 3-NT Adducts.

iNOS levels were similar in the control lean and obese mice (Fig. 6A). Upon acetone administration, the iNOS content in lean mice was not changed; however, there was a 3-fold induction of iNOS expression in the obese mice (Fig. 6A). Pyrazole treatment resulted in 2- or 3-fold increase in iNOS levels in the lean and obese mice, respectively. The formation of 3-NT adducts was examined through immunohistochemical analysis and slot blot. Immunohistochemical staining showed that the livers of control and acetone- or pyrazole-treated lean mice or control obese mice did not have any positive staining for 3-NT (−) (Fig. 6B). However, the obese mice treated with acetone or pyrazole had strong positive staining for 3-NT (++/+++) in the cytoplasm of the hepatocytes, mostly in midcentral and pericentral areas. This intense staining in the pyrazole-treated obese mice was prevented by CMZ or 1400W (+) (Fig. 6B). The results of immunohistochemical staining for 3-NT were quantified by a slot blot technique (Fig. 6C). Treatment with acetone did not change the intensity of the bands significantly in lean mice. The acetone-treated obese mice had a 2.7-fold increase in 3-NT protein adduct staining compared to the control obese mice. Pyrazole treatment caused a 2-fold and 4-fold increase in 3NT levels in lean and obese mice, respectively. CMZ or 1400W decreased the levels of 3NT by 3-fold, back to the control obese levels (Fig. 6C).

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Figure 6. Acetone or pyrazole increase iNOS levels and 3-NT adduct formation in obese mice. (A) Top: Western blot showing the protein expression of iNOS in liver homogenates from lean or obese mice. Bottom: Densitometric values (du) for iNOS. Data represent the mean ± SE of three independent experiments. P < 0.05 for pyrazole-treated lean or obese mice or acetone-treated obese mice versus control lean or obese mice. (B) Photomicrographs of immunohistochemical staining of 3-NT protein adducts in liver sections of control or acetone- or pyrazole-treated lean and obese mice. The strong staining in the pyrazole-treated obese mice livers was prevented by either CMZ or 1400W treatment. (C) Representative slot blot analysis of 3-NT protein adducts in livers of control or acetone- or pyrazole-treated lean and obese mice. Results from three mice in each group are depicted. Densitometric values (du) for 3-NT protein adducts are shown in the bar graphs. Data represent the mean ± SE of three independent experiments. *P < 0.05 for control obese mice versus control lean mice. P < 0.05 for pyrazole-treated lean or obese mice or acetone-treated obese mice versus control lean or obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole-treated obese mice.

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Effect of Acetone on Pro- and Anti-apoptotic Proteins.

The effect of acetone on the expression of various pro- and anti-apoptotic proteins was examined. The basal level of Bcl-2 was similar in both control lean and obese mice and acetone treatment caused a 5- to 6-fold increase in both groups (Fig. 7A,B). The level of Bcl-xL was also not different between the two control groups. Acetone lowered Bcl-xL content in the lean mice about 2-fold; however, acetone treatment of obese animals did not cause any change in Bcl-xL expression (Fig. 7A,B). The control obese mice had a 1.5-fold higher level of Bax than the control lean animals and acetone did not cause significant changes in either the lean or obese mice. The control obese mice also had a 2.3-fold higher level of Bad protein when compared to the lean mice. Acetone treatment resulted in 1.5- and 3-fold increases in the expression of Bad in lean and obese mice, respectively (Fig. 7A,B). The lack of distinct patterns of changes in the regulation of pro- (Bax, Bad) and anti- (Bcl-2) apoptotic mitochondrial proteins in livers of treated obese mice suggests that acetone elicits a mixed response for cell survival and cell death.

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Figure 7. Effect of acetone on pro- and anti-apoptotic proteins. (A) Western blots showing the protein expression of Bcl-2, BCl-xL, Bax, and Bad in liver homogenates from lean or obese mice. (B) Densitometric values (du) of the protein/β-actin ratio for the same blots. Data represent the mean ± SE of three independent experiments. *P < 0.05 for control obese versus control lean and P < 0.05 for acetone-treated lean or obese mice versus control lean or obese mice, respectively.

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Effect of Pyrazole on TNF-α Levels.

Control obese mice had 2.5-fold higher TNF-α levels in liver as compared to the control lean mice (Fig. 8). Pyrazole treatment increased the TNF-α levels 2- to 3-fold in lean and obese mice, with highest TNF-α levels in the pyrazole-treated obese mice. CMZ or 1400W caused a 1.5- to 2-fold decrease in TNF-α levels in pyrazole-treated obese mice.

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Figure 8. Pyrazole increases TNF-α levels in lean and obese mice. *P < 0.05 for control obese mice versus control lean mice. P < 0.05 for pyrazole-treated lean or obese mice versus control lean or obese mice. P < 0.05 for CMZ or 1400W plus pyrazole-treated obese mice versus pyrazole-treated obese mice. Data represent the mean ± SE of six animals/group.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The role of obesity in causing liver damage and the development of steatotic liver in obesity having pathologic manifestations resembling alcoholic liver disease has been well documented in the literature.2, 18 CYP2E1-mediated metabolism of excess free fatty acids in obesity causes oxidative stress and promotes liver injury.19 CYP2E1 is induced by acetone, mainly through protein stabilization.20 Pyrazole affects the metabolism of several drugs by the microsomal mixed-function oxidase system21 and increases CYP2E1.11 The effects of acetone or pyrazole on liver toxicity in obesity have not been studied and was the goal of the current study.

The obese mice having access to acetone showed distinct histological changes in their liver, not observed in the lean mice, which included severe fatty changes, congestion, a marked increase in steatosis, inflammation, and minimal necrosis. Pyrazole-treated mice exhibited pronounced steatosis and inflammatory cells surrounding steatotic hepatocytes and multifocal necrosis. ALT and AST were significantly higher in control obese mice when compared to the lean mice. An increase in ALT and AST was observed in acetone-treated obese, but not in the lean group. Pyrazole increased the serum aminotransferases in both lean and obese mice, though it was significantly higher in the obese group. The increase in the levels of these enzymes and the histopathology in obese mice indicate that acetone or pyrazole promotes liver injury in obese conditions. Acetone or pyrazole induced apoptotic changes in the obese mice which included a high caspase-3 activity, and increased number of hepatocytes having apoptotic nuclei positively stained for TUNEL. All these pathological changes observed with pyrazole-treated animals were abrogated with CMZ or 1400W.

CYP2E1 mRNA expression was lower in the control lean mice when compared to the obese mice. Pyrazole treatment did not increase the mRNA expression as it induces CYP2E1 primarily through protein stabilization.11, 20 CYP2E1 protein expression and activity were lower in the ob/ob mice, and acetone or pyrazole caused a significant induction in both protein and enzyme activity in both lean and especially the obese groups. The increase in CYP2E1 levels was reversed by CMZ or 1400W treatment. In a recent study involving obese patients with nonalcoholic fatty liver disease, increased CYP2E1 protein content and activity correlated with the development of liver injury.22 Constitutive levels of CYP2E1 mRNA, protein, and activity were shown to be decreased in livers of ob/ob mice, and acetone increased CYP2E1 protein and activity levels in both lean and obese mice, but the consequences of this acetone-induced increase in CYP2E1 with respect to liver injury were not studied.23 Binge ethanol increased CYP2E1 levels, apoptosis, and liver injury in obese rats more than in lean controls.24 It is interesting to speculate that the low basal levels of CYP2E1 protein expression in obese mice may account for the absence of severe liver injury despite obesity and could well be a compensatory survival mechanism for these leptin-deficient animals. The decrease in CYP2E1 levels with 1400W is consistent with the findings that adenovirus containing iNOS and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, increased the CYP2E1 expression in iNOS-null hepatocytes.25 Thus, NO may play a role in regulating CYP2E1, although this remains to be further studied.

The pyrazole- or acetone-treated obese mice showed several-fold higher oxidation of protein as assayed by the formation of strong protein carbonyl bands, suggesting that oxidative stress is higher in these animals. The untreated mice, lean and obese, had minimal staining for HNE adducts, and acetone or pyrazole increased the HNE adduct formation in both lean and obese mice. The obese mice displayed a greater distribution of HNE adducts, suggesting that acetone- or pyrazole-mediated induction of CYP2E1 causes increased lipid peroxidation in obesity. This was confirmed by the large increase in the MDA levels found in the pyrazole-treated obese mice. The inhibition of these changes with CMZ confirmed the essential role of CYP2E1 in potentiating liver toxicity and oxidative stress in obesity. Similarly, the iNOS inhibitor 1400W reversed the toxicity observed in these mice.

iNOS plays a critical role in the development of insulin resistance in the liver of obese mice.26 Acetone or pyrazole caused a significant increase in iNOS protein in obese mice. The acetone- or pyrazole-induced increase in CYP2E1, which produces superoxide, and the increase in iNOS in obese mice, which produces NO, could promote peroxynitrate formation and the subsequent formation of 3-NT adducts. Indeed, the acetone- or pyrazole-treated obese mice had higher 3-NT levels. Some increase in nitrosative stress was also observed in the control obese mice as reflected by the formation of 3-NT residues, consistent with previous studies involving obese mice and human subjects.27 The liver samples from acetone- or pyrazole-treated obese mice showed much stronger bands and also a strong positive staining for 3-NT, while these changes were not observed in the acetone-treated lean mice. The use of CMZ or 1400W abrogated all these changes and confirmed the possibility that increased nitrosative stress may play a role in the liver toxicity found in the obese mice treated with pyrazole.

The TNF-α levels were higher in control obese mice as compared to lean mice, consistent with the observations that patients with fatty liver or nonalcoholic steatohepatitis have increased serum TNF-α levels28 and anti-TNF-α antibody improves mitochondrial dysfunction in obese mice.29 Highest levels of TNF-α were found in the livers of the pyrazole-treated obese mice. CMZ or 1400W were effective in abrogating the increase in TNF-α in pyrazole-treated obese mice. The role of TNF-α in the enhanced toxicity found in acetone- or pyrazole-treated obese mice will require further study.

In summary, acetone induces extensive steatosis, pyrazole induces necrotic changes and both these CYP2E1 inducers increased aminotransferase levels in livers of obese mice. The acetone- or pyrazole-treated obese mice also had higher caspase-3 activity and apoptotic hepatocytes. The protein expression and catalytic activity of CYP2E1 was elevated in acetone- or pyrazole-treated lean and obese mice. The obese animals treated with acetone or pyrazole showed formation of more HNE adducts and protein carbonyl bands of a much stronger intensity. Acetone or pyrazole also induced the basal levels of iNOS in obese mice, along with higher 3-NT adduct formation, indicating increased nitrosative stress. Pyrazole also increased TNF-α levels in both lean and obese mice. In conclusion, acetone or pyrazole induces the expression and catalytic activity of CYP2E1 in obese mouse liver, increases oxidative and nitrosative stress, and produces apoptotic liver injury. The inhibitors of CYP2E1-CMZ and iNOS-1400W abrogated the CYP2E1-mediated toxicity induced in obese mice. These results suggest that acetone- or pyrazole-mediated induction of CYP2E1 may synergize with high fat in obesity to promote cell injury.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Acknowledgment: We would like to acknowledge the kind help of Dr. Jingxiang Bai (Mount Sinai School of Medicine) for performing the Northern blot experiment.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References