CYP2E1 potentiates binge alcohol-induced gut leakiness, steatohepatitis, and apoptosis

Abstract

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) contributes to increased oxidative stress and steatosis in chronic alcohol-exposure models. However, its role in binge ethanol-induced gut leakiness and hepatic injury is unclear. This study was aimed at investigating the role of CYP2E1 in binge alcohol-induced gut leakiness and the mechanisms of steatohepatitis. Female wild-type (WT) and Cyp2e1-null mice were treated with three doses of binge ethanol (WT-EtOH or Cyp2e1-null-EtOH) (6g/kg oral gavage at 12-h intervals) or dextrose (negative control). Intestinal histology of only WT-EtOH exhibited epithelial alteration and blebbing of lamina propria, and liver histology obtained at 6h after the last ethanol dose showed elevated steatosis with scattered inflammatory foci. These were accompanied by increased levels of serum endotoxin, hepatic enterobacteria, and triglycerides. All these changes, including the intestinal histology and hepatic apoptosis, determined by TUNEL assay, were significantly reversed when WT-EtOH mice were treated with the specific inhibitor of CYP2E1 chlormethiazole and the antioxidant N-acetylcysteine, both of which suppressed oxidative markers including intestinal CYP2E1. WT-EtOH also exhibited elevated amounts of serum TNF-α, hepatic cytokines, CYP2E1, and lipid peroxidation, with decreased levels of mitochondrial superoxide dismutase and suppressed aldehyde dehydrogenase 2 activity. Increased hepatocyte apoptosis with elevated levels of proapoptotic proteins and decreased levels of active (phosphorylated) p-AKT, p-AMPK, and peroxisome proliferator-activated receptor-α, all of which are involved in fat metabolism and inflammation, were observed in WT-EtOH. These changes were significantly attenuated in the corresponding Cyp2e1-null-EtOH mice. These data indicate that both intestinal and hepatic CYP2E1 induced by binge alcohol seems critical in binge alcohol-mediated increased nitroxidative stress, gut leakage, and endotoxemia; altered fat metabolism; and inflammation contributing to hepatic apoptosis and steatohepatitis.

Keywords: 3-NT; 3-nitrotyrosine; AFLD; ALD; ALDH2; ALT; ASH; Apoptosis; Binge ethanol; CMZ; CYP2E1; Cyp2e1-null–dextrose; Cyp2e1-null–ethanol; Free radicals; Gut leakiness; HAE; HPF; Liver; MDA; N-acetylcysteine; NAC; NAFLD; NASH; Oxidative stress; PPAR-α; RNS; ROS; SOD2; Steatohepatitis; TG; TNF-α; TUNEL; WT; WT-DEX; WT-EtOH; alanine aminotransferase; alcoholic fatty liver disease; alcoholic liver disease; alcoholic steatohepatitis; chlormethiazole; cytochrome P450 2E1; high-power field; hydroxyalkenal; malondialdehyde; mitochondrial aldehyde dehydrogenase 2; mitochondrial superoxide dismutase; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis; null–DEX; null–EtOH; peroxisome proliferator-activated receptor-α; reactive nitrogen species; reactive oxygen species; terminal deoxynucleotidyl transferase dUTP nick-end labeling; triglyceride; tumor necrosis factor-α; wild-type; wild-type–dextrose; wild-type–ethanol..

Fig. 1. Effects of binge ethanol on hepatic steatosis, and inflammation

(A) Representative H&E staining of livers for indicated groups with inflammatory foci (a dotted circle) and (B) hepatic TG levels are shown. (C) Liver/body weight ratios, (D) plasma ALT levels at 6-h post-EtOH, and (E) summary of histochemical detection of neutrophils are presented. Data are expressed as mean ± SEM (n=5 mice/group). *p<0.05, *Significantly different from all other groups; ^#significantly different from the corresponding Cyp2e1-null-DEX control group.

Fig. 2. Evaluation of the levels of serum endotoxin, hepatic enterobacteria content, intestinal histopathological changes and oxidative stress marker proteins

(A) The serum endotoxin levels, (B) hepatic enterobacterial colony formation (n=3/group) and (C) average colony numbers were determined. (D) Representative H&E stainings of lower intestines are presented for indicated groups. WT-EtOH mice showed loss of epithelium (black arrows) and blebbing of lamina propria (dotted black circles) (n=3/group). (E) Equal amounts of intestinal lysates (n=3) were used to determine the levels of: CYP2E1 (top), iNOS (middle), and β-actin as a loading control (bottom). Data are expressed as mean ± SEM (n=4~5/group) unless otherwise indicated. *Significantly different from all other groups; ^#significantly different from the corresponding Cyp2e1-null-EtOH control.

Fig. 3. Effects of CMZ on binge ethanol-mediated hepatic steatosis, CYP2E1 activity, serum endotoxin levels and hepatic enterobacterial contents

(A) Serum endotoxin levels, (B) hepatic enterobacterial colony formation and (C) average colony numbers are presented (n=3/group). (D) Representative H&E staining of liver tissues obtained at 6-h, (E) hepatic CYP2E1 activity and (F) triglyceride levels are shown (n=4/group). Data are expressed as mean ± SEM. *Significantly different from all other groups, #significantly different from Dextrose-control.

Fig. 4. Effects of NAC on binge ethanol-mediated hepatic steatosis and serum endotoxin levels

(A) The serum endotoxin levels and (B) hepatic triglyceride levels are presented. (C) Representative H&E staining of liver tissues obtained at 6-h as indicated. Data are expressed as mean ± SEM (n=3/group). *Significantly different from all other groups, #significantly different from Dextrose-control.

Fig. 5. Changes in the levels of hepatic oxidative parameters

Equal amounts of cytosolic proteins were used to determine: (A) CYP2E1 protein levels (top) and β-actin (bottom); (B) CYP2E1 activity; and (C) hepatic MDA+HAE. Equal amounts of mitochondrial lysates were used to determine the levels of: (D) SOD2 (top), ALDH2 (middle), and ATP-synthase (bottom) as a loading control; (E and F) ALDH2 activity ± pretreatment with 15 mM DTT for 30 min. Data are expressed as mean ± SEM (n=5/group). *Significantly different from all other groups.

Fig. 6. Evaluation of hepatic apoptosis and apoptosis-related proteins

(A) TUNEL-positive apoptotic hepatocytes (arrowheads) in livers of indicated groups. (B) Number of TUNEL-positive hepatocytes in 20 HPFs was calculated. (C) Equal amounts of whole liver lysates were used to determine the levels of Bax, Bim, Fas ligand, and the loading control β-actin by immunoblot analysis. Data are expressed as mean ± SEM (n= 5 mice/group). *Significantly different from all other groups.

Fig. 7. Schematic diagram for the role of CYP2E1 in binge ethanol-induced gut leakiness, hepatic steatosis and apoptosis

The first and second hits for the development of steatohepatitis are presented. Intestinal and hepatic CYP2E1 seem to play a central role in promoting oxidative stress, gut leakiness, and endotoxemia, thereby contributing to the development of hepatic steatosis, inflammation and apoptosis.