Role for intestinal CYP2E1 in alcohol-induced circadian gene-mediated intestinal hyperpermeability

Abstract

We have shown that alcohol increases Caco-2 intestinal epithelial cell monolayer permeability in vitro by inducing the expression of redox-sensitive circadian clock proteins CLOCK and PER2 and that these proteins are necessary for alcohol-induced hyperpermeability. We hypothesized that alcohol metabolism by intestinal Cytochrome P450 isoform 2E1 (CYP2E1) could alter circadian gene expression (Clock and Per2), resulting in alcohol-induced hyperpermeability. In vitro Caco-2 intestinal epithelial cells were exposed to alcohol, and CYP2E1 protein, activity, and mRNA were measured. CYP2E1 expression was knocked down via siRNA and alcohol-induced hyperpermeability, and CLOCK and PER2 protein expression were measured. Caco-2 cells were also treated with alcohol or H₂O₂ with or without N-acetylcysteine (NAC) anti-oxidant, and CLOCK and PER2 proteins were measured at 4 or 2 h. In vivo Cyp2e1 protein and mRNA were also measured in colon tissue from alcohol-fed mice. Alcohol increased CYP2E1 protein by 93% and enzyme activity by 69% in intestinal cells in vitro. Alcohol feeding also increased mouse colonic Cyp2e1 protein by 73%. mRNA levels of Cyp2e1 were not changed by alcohol in vitro or in mouse intestine. siRNA knockdown of CYP2E1 in Caco-2 cells prevented alcohol-induced hyperpermeability and induction of CLOCK and PER2 proteins. Alcohol-induced and H₂O₂-induced increases in intestinal cell CLOCK and PER2 were significantly inhibited by treatment with NAC. We concluded that our data support a novel role for intestinal CYP2E1 in alcohol-induced intestinal hyperpermeability via a mechanism involving CYP2E1-dependent induction of oxidative stress and upregulation of circadian clock proteins CLOCK and PER2.

Keywords: clock genes; cytochrome P450 isoform 2E1; ethanol; leaky gut; oxidative stress.

Fig. 1.

Alcohol treatment induces increased Cytochrome P450 isoform 2E1 (CYP2E1) protein in Caco-2 intestinal epithelial cells. Human Caco-2 intestinal epithelial cells were grown to confluence on tissue culture inserts in complete medium (controls) or complete medium containing alcohol (EtOH, 0.2%, 43 mM) as described in materials and methods. At the designated time points over 240 min (4 h), cells were lysed, and CYP2E1 protein expression was assessed by Western blotting. Blots were then stripped and reprobed for actin expression to control for equal loading. A representative blot from 1 of 5 independent experiments is shown. Histogram data are means ± SE of all N = 5 experiments. Calculated means are relative to controls for each time point that are not shown. *P < 0.05 vs. control for that time point.

Fig. 2.

Chronic alcohol feeding induces increased intestinal Cyp2e1 protein in BL/6 mice. C57BL/6 mice were pair fed a complete liquid diet (detailed in materials and methods) for 8 wk containing alcohol (EtOH BL/6; 4.5% vol/vol, 1 M) or control diet with calories matched with dextrose. Cyp2e1 protein expression was analyzed in proximal colon tissue by Western blotting tissue from N = 4 mice for each condition. Representative blot for tissue from 1 mouse for either control or alcohol-fed mice. Blot was stripped and reprobed for actin as a loading control. Histogram data depicts summarized data for all mice N = 4 for each condition. *P < 0.05 vs. control.

Fig. 3.

Alcohol treatment induces increased CYP2E1 activity in Caco-2 intestinal epithelial cells. Caco-2 cells grown on Transwell inserts were treated with complete media containing alcohol (EtOH, 0.2%, 43 mM) or no alcohol (control) for 240 min (4 h). as described for Fig. 1. CYP2E1 activity was measured in Caco-2 cell microsome fractions by the spectrophotometric analysis at 546 nm of the oxidation of p-nitrophenol to p-nitrocatechol in the presence of NADPH and oxygen as described in materials and methods. CYP2E1 activity data are expressed as the means of pmol/mg per minute of oxidized p-nitrophenol/mg protein per minute. Means ± SE for N = 4 experiments. *P < 0.05 vs. control.

Fig. 4.

Alcohol-induced CYP2E1 mRNA expression in Caco-2 cells and mouse colon. A: alcohol-induced CYP2E1 mRNA expression in Caco-2 cells. Caco-2 cells grown on Transwell inserts were treated with alcohol (EtOH, 0.2%, 43 mM) or complete media alone (control) for 4 h as described in Fig. 1 were subjected to mRNA extraction and RT-PCR analysis for CYP2E1 expression as described in materials and methods with expression normalized to β-actin expression using the ΔΔCT method and then expressed as percentage of control. Data are means ± SE for N = 4 experiments. B: chronic alcohol feeding induced Cyp2e1 mRNA expression in proximal colon tissue of BL/6 mice. BL/6 mice were pair fed a complete Nanji (fish oil) liquid diet as described in materials and methods for 8 wk containing either alcohol (EtOH, 4.5% vol/vol, 1 M) or control diet with calories matched with dextrose. Colon tissue stored in RNAlater at −80°C was then analyzed for Cyp2e1 mRNA expression by qRT-PCR as described above for Caco-2 cells. ΔΔCT data relative to β-actin were then expressed as percentage of control for N = 4 mice as means ± SE.

Fig. 5.

siRNA inhibition of CYP2E1 expression prevents alcohol-induced permeability in Caco-2 monolayers. Caco-2 cells grown to confluence on Transwell inserts were treated with control or alcohol containing (EtOH, 0.2%, 43 mM) media and assessed over time (30–240 min) for alcohol-induced paracellular permeability by measurement of transepithelial electrical resistance (TER) changes as described in materials and methods. To investigate the role of CYP12E1, cells were pretreated with either control nontargeting siRNA or siRNA specific for CYP2E1 as described in materials and methods. A: data are TER means (of triplicate wells) ± SE (Ω cm²) for N = 4 separate experiments expressed as percent change from original baseline (set as 100%). B: data are actual TER means (of triplicate wells) ± SE (Ω cm²; with background subtracted) for N = 4 experiments. Cells measured for TER were lysed on membranes at the conclusion of each experiment to provide lysates for the Western blotting analysis shown in Fig. 6. #P < 0.05 vs. control siRNA alone; +P < 0.05 vs. CYP2E1 siRNA + EtOH.

Fig. 6.

siRNA inhibition of CYP2E1 expression prevents alcohol-induced increases in the circadian gene proteins CLOCK and PER2 in Caco-2 cells. The same Caco-2 cells grown on Transwell inserts and tested for alcohol-induced permeability (EtOH, 0.2%, 43 mM) shown in Fig. 5 were lysed at the end of each experiment (N = 4) and used for Western blotting analysis (as described in Fig. 1) for protein expression of CLOCK, PER2, CYP2E1, and β-actin. Representative blots from a single experiment are shown in A. B–D: histograms showing summarized means ± SE for densitometry data compiled from analysis of blots from all 4 separate experiments for expression of PER2, CYP2E1, and CLOCK. *P < 0.05 vs. control siRNA alone; **P < 0.05 vs. control siRNA + EtOH.

Fig. 7.

siRNA inhibition of CYP2E1 dramatically reduces alcohol-induced oxidative stress (3-nitrotyrosine, 3NT) in Caco-2 cells. The same total cell lysates from the SiRNA permeability experiments tested in Fig. 6 above (EtOH, 0.2%, 43 mM) were also analyzed by slot blotting, as described in materials and methods, for 3NT formation with antibody specific for 3NT, a measure of oxidative stress. Top: representative blot (from 1 experiment). Bottom: histogram summarizing blot densitometry means ± SE data from N = 6 complete experiments. *P < 0.05 vs. control siRNA; **P < 0.05 vs. control siRNA + EtOH.

Fig. 8.

N-acetylcysteine (NAC) significantly inhibits alcohol-induced expression of CLOCK and PER2 circadian proteins in Caco-2 intestinal cells. Caco-2 human intestinal cells were grown to confluence on Transwell inserts and stimulated with alcohol (EtOH, 0.2%, 43 mM) for 4 h. Cell lysates were then used to measure CLOCK and PER2 protein expression with Western blotting (30 μg protein/lane). Blots were stripped and reprobed with antibody to β-actin to validate equal lane loading. Blots shown are representative of N = 6 experiments (from 1 experiment), and histogram data are means ± SE for summarized data from all N = 6 experiments. *P < 0.05 vs. control; +P < .05 vs. 0.2% EtOH alone.

Fig. 9.

Stimulation of Caco-2 intestinal cells with H₂O₂ results in increased expression of CLOCK and PER2 circadian proteins. Caco-2 human intestinal cells were grown to confluence on Transwell inserts and stimulated with H₂O₂ (0.0017%, 0.5 mM) for 2 h. Cell lysates were then used to measure CLOCK and PER2 protein expression with Western blotting (30 μg protein/lane). Blots were stripped and reprobed with antibody to β-actin to validate equal lane loading. Blots shown are representative of N = 6 experiments (all from 1 experiment), and histogram data are means ± SE for summarized data from all N = 6 experiments. *P < 0.05 vs. control; +P < 0.05 vs. 0.5 mM H₂O₂ alone.

Fig. 10.

Proposed model for alcohol-mediated oxidative stress stimulation of CLOCK and PER2 circadian proteins and intestinal hyperpermeability. This cartoon summarizes the data in this study using the human intestinal epithelial Caco-2 cell model of intestinal permeability. We show that alcohol induces increased intestinal cell CYP2E1 protein and activity that results in increased oxidative stress and reactive oxygen species (ROS)/reactive nitrogen species (RNS). This oxidative stress can be inhibited by preincubation of cells with NAC. The oxidative stress then promotes increased levels of CLOCK and PER2 circadian clock proteins. The increase in CLOCK and PER2 then stimulates the observed increase in intestinal hyperpermeability, possibly by changes (decreases) in the tight junction proteins that regulate intestinal permeability.