Fig 1: Michaelis Menten curves of chlorzoxazone 6-hydroxylation by the COS-7 cell-expressed recombinant wild-type and six variants of CYP2E1 enzymes (each point represents the mean ± SEM of three separate independent experiments).Enzyme kinetic were modeled using a non-linear regression based on the Michaelis-Menten equation.
Fig 2: Detection of CYP2E1 proteins by western-blot analysis.Detection of CYP2E1 proteins by western-blot analysis. (A) Recombinant CYP2E1 variants as well as the wild type (WT) and empty vector (EV) as a negative control were recognized by rabbit-produced anti-CYP2E1 antibody. The loading amount was 10 µg microsomal protein. All lanes are as shown above. 1, WT; 2, c.1263C>T; 3, c.1009C>T; 4, c.517G>A; 5, c.[227G>A;1263C>T]; 6, c.227G>A; 7, c.[517G>A;1263C>T]; 8, EV. (B) Relative expression levels of CYP2E1 variants. The results are expressed as a percentage of the WT. All values are mean +/- S.E.M for three separate independent assays. *P = 0.05,**P = 0.01,***P = 0.001.
Fig 3: Lobular reorganization of metabolizing enzymes and functional consequences. (A) Reorganization of the pericentral enzymes Cyp2e1 and GS and the periportal/midzonal enzyme arginase1 in liver of WD-fed mice. (B) Decrease in Cyp2e1 (pericentral) and the arginase1 (periportal/midzonal) lobular regions and increase of the pericentral GS positive zone after WD feeding. (C) Quantification of the Cyp2e1, arginase1, and GS positive lobular areas in whole slide scans; data represent mean and standard errors of 3–8 mice per time point. *: p < 0.05; ***: p < 0.001 compared to SD week 3, Sidak’s multiple comparisons test; data of individual mice are illustrated by dots. (D,E) Hepatotoxicity of 300 mg/kg APAP in mice fed a SD or a WD for ~50 weeks as evidenced by H&E staining (D) and liver enzymes in blood (E); data represent mean and standard error of 3–4 mice per group. *: p < 0.05; **: p < 0.01 compared to SD, Tukey’s multiple comparisons test; data of individual mice are illustrated by dots. (F–H) Functional consequences of WD feeding (42 weeks) on ammonia (F), glutamine (G), urea and arginine (H) blood concentrations; data represent mean and standard error of 3–4 mice per group. **: p < 0.01; ***: p < 0.001 compared to SD, Sidak’s multiple comparisons test; data of individual mice are illustrated by dots. SD: standard diet; WD: Western diet; GS: glutamine synthetase; ALT: alanine transaminase. Scale bars: 100 µm.
Fig 4: Ahr activation promotes accumulation of toxic acetaminophen metabolites. Female wild-type mice were treated with vehicle or ITE alone, or with vehicle + APAP or ITE + APAP (n = 12 each). Hepatic gene expression of (A) Ahr, (B) Cyp1a2 and (C) Cyp2e1 were analyzed 4 hours post-APAP treatment. Target gene expression is depicted as x- fold expression compared with the vehicle control. (D) Representative stainings of hepatic Cyp1a2 (yellow) and Cyp2e1 (purple) protein expression. Nuclei are stained in blue. Scale bar = 50 µm. Pictures were taken using a Biorevo Keyence BZ-9000 microscope with objective from Nikon (Plan Apo 10x/0,45 8/0.17 WD 4.0) and the Keyence BZ II Viewer and Analyzer software. (E, F) Western blot analysis of Cyp1a2 and Cyp2e1 protein expression (n = 8). (G) Total GSH levels in liver tissue and (H) toxic APAP adducts in plasma 30 min post APAP treatment (n = 10). Two pooled of 3 independent experiments are shown. For statistical analysis, a 1-way analysis of variance followed by Tukey’s multiple comparisons test was used. Results are shown as mean ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001. I, ITE; IA, ITE + APAP; V, vehicle; VA, vehicle + APAP.
Fig 5: Correlation analysis of recombinant protein quantity and enzyme activity for CYP2E1 variant allozymes.The Normalized protein quantities are plotted on the horizontal axis and the Normalized enzyme activities are plotted on the vertical axis. r2 = 0.8833 and p value = 0.0005. Pearson correlations was used to evaluate the associations between the CYP2E1 protein content and enzyme activity, with two-sided t-test.
Supplier Page from MilliporeSigma for Anti-CYP2E1 antibody produced in rabbit