CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis (original) (raw)
Effects of MCD diet on C57BL6/J mice. Despite a higher food intake relative to body weight, C57BL6/J mice fed the MCD diet lost weight compared with animals fed the control diet (Table 1). This weight loss was apparent immediately after starting the diet, and stabilized after 5 weeks. Despite the extent of weight loss, the general condition of the animals remained good and their behavior appeared normal throughout the experimental period. Furthermore, whereas serum triglyceride levels were decreased, levels of β-hydroxybutyrate remained unchanged in MCD diet–fed mice (Table 1), indicating that the reduction in body weight was not associated with a biochemical effect of fasting.
Body weight and serum biochemical parameters in C57BL6/J mice fed the methionine and choline deficient (MCD) or control diet for 10 weeks
Feeding the MCD diet resulted in a major increase in serum ALT levels compared with controls, reaching 637 ± 128 IU/L after 10 weeks of the dietary regimen (Table 1). At this time, livers revealed lipid droplets as clear macrovacuoles affecting all but zone 1 hepatocytes (periportal hepatocytes) (Figure 1c). Staining with oil red O confirmed the lipid content of these vacuoles (not shown). Consistent with these morphological changes, total hepatic lipid content was increased by a factor of 2, and the triacylglycerol fraction increased 3-fold, compared with controls (Table 2). In addition to hepatic steatosis, there were large areas of mixed inflammatory cell infiltration and hepatocyte necrosis dissecting the liver parenchyma (Figure 1c), together with discrete perivenular and pericellular fibrosis (Figure 1d).
Liver sections from female C57BL6/J mice fed the MCD diet and the control diet for 10 weeks. (a) Control mouse, H&E staining (×100). (b) Control mouse, Sirius red staining (×160). (c) MCD diet–fed mouse, H&E staining (×100). In addition to the macrovesicular steatosis mainly localized in zone 2, large areas of mixed inflammatory infiltrate with lymphocyte and polymorphonuclear neutrophil necroinflammation can be seen throughout the hepatic lobule. (d) MCD diet–fed mouse, Sirius red staining (×160). The collagen fiber deposits (stained red) confirm the discrete perivenular and pericellular fibrosis.
Effects of the methionine and choline deficient (MCD) diet on hepatic lipids and lipid peroxides, and CYP2E1 expression and activity in C57BL6/J mice
Effect of MCD diet on hepatic CYP2E1 expression. To characterize hepatic CYP2E1 expression in C57BL6/J mice fed the MCD or control diet, we determined the levels of liver CYP2E1 mRNA, microsomal protein, and enzyme activity. As shown in Table 2, intake of the MCD diet was associated with pretranslational upregulation of CYP2E1, and the resultant increased level of CYP2E1 protein was catalytically active.
To establish the specificity of upregulation of CYP2E1, the expression of other major cytochromes P450 was examined. The levels of CYP1A and CYP3A proteins were slightly decreased in mice administered the MCD diet, whereas there was no significant change in mRNA levels of the 3 members of the mouse CYP4A subfamily (CYP4A10, CYP4A12, and CYP4A14) (data not shown).
Evidence of lipid peroxidation in C57BL6/J mice with NASH. As indicated by the accumulation of TBARs, total lipid peroxides were dramatically increased (about 100-fold) in the livers of mice fed the MCD diet (Table 2). The possibility that microsomal proteins contributed to this increase was examined by determining NADPH-dependent lipid peroxidation in microsomal fractions. This activity was increased 3-fold in microsomal fractions prepared from MCD diet–fed mice compared with controls (1.52 ± 0.23 μmol and 0.45 ± 0.07 μmol TBARs/mg protein per min, respectively; P < 0.001).
To determine the extent to which CYP2E1 contributed to microsomal NADPH-dependent lipid peroxidation, the effects of chemical and immunological inhibitors of CYP2E1 were studied. DETC was added to microsomal proteins at a concentration (10 μM) that inhibits chlorzoxazone 6-hydroxylase activity by 72% (data not shown), and that has been shown to specifically block CYP2E1-dependent activity (23, 36). DETC significantly inhibited NADPH-dependent lipid peroxidation in microsomes from MCD and control diet–fed mice by 75% and 85%, respectively. Furthermore, addition of anti-rat CYP2E1 antibody inhibited this activity in a dose-dependent manner in both groups (Table 3). In contrast, mouse-specific CYP4A10 and CYP4A12 antibodies failed to modify the rate of formation of lipid peroxides in hepatic microsomes from C57BL6/J mice (Table 3).
Effects of DETC, anti-rat CYP2E1 IgG, anti-mouse CYP4A10 IgG, and anti-mouse CYP4A12 IgG on NADPH-dependent lipid peroxidation in microsomes from C57BL6/J mice fed the methionine and choline deficient (MCD) or the control diet for 10 weeks
Effects of MCD diet in Cyp2e1–/– mice. The previous results indicate that CYP2E1 is responsible for the enhanced lipid peroxidation observed in hepatic microsomes from mice fed the MCD diet. To test the hypothesis that increased expression of this enzyme is involved in the pathogenesis of NASH through the generation of oxidative stress, Cyp2e1–/– mice lacking expression of functional CYP2E1 protein were fed the MCD diet. The results were compared with Cyp2e1+/– mice challenged with the same dietary regimen. Compared with their respective controls, both Cyp2e1+/– and Cyp2e1–/– mice fed the MCD diet lost weight to the same extent (Figure 2a). Cyp2e1–/– mice subjected to the MCD diet also developed histological evidence of gross hepatic steatosis, focal hepatocellular necrosis, and mixed inflammatory infiltrate (Figure 3). The extent and severity of necroinflammatory changes were similar in Cyp2e1–/– and Cyp2e1+/– mice, but Cyp2e1–/– mice exhibited more fat accumulation than did their genetic controls (Figure 2b). Correspondingly, serum ALT levels were significantly higher (P < 0.001) in the MCD diet–fed mice of both genotypes relative to their respective controls (Figure 2c).
Characteristics of Cyp2e1+/– mice (diamonds) and Cyp2e1–/– (squares) mice fed the MCD diet (filled symbols) or the control diet (open symbols) for up to 10 weeks. Changes in (a) body weight, (b) total hepatic lipids, (c) serum ALT levels, and (d) total hepatic lipid peroxides. A_P_ < 0.05 and B_P_ < 0.01 for MCD diet–fed Cyp2e1–/– mice compared with MCD diet–fed Cyp2e1+/– mice.
Liver sections from Cyp2e1+/– mice and Cyp2e1–/– mice fed the MCD diet or the control diet for 10 weeks. (a) Cyp2e1+/– mouse and (b) Cyp2e1–/– mouse fed the control diet. The appearance of the hepatic parenchyma is similar in both genotypes. (c) Cyp2e1+/– mouse and (d) Cyp2e1–/– mouse fed the MCD diet. Administration of the MCD diet to both genotypes produced a severe macrovesicular, panlobular steatosis, hepatic necrosis, and mixed inflammatory infiltrate. H&E staining; ×160.
In both Cyp2e1–/– and Cyp2e1+/– mice fed the MCD diet, there was a major increase of lipid peroxides in the liver (Figure 2d). Likewise, despite the absence of CYP2E1 expression (confirmed by Northern and Western analyses; Figure 4), the basal level of microsomal NADPH-dependent lipid peroxidase activity appeared similar in Cyp2e1–/– and Cyp2e1+/– mice fed the control diet (Table 4). Moreover, administration of the MCD diet significantly increased this activity in both genotypes by a factor of 4.5 (Table 4). As observed in wild-type C57BL6/J mice, addition of DETC or CYP2E1 antiserum to the reaction mixture abrogated NADPH-dependent lipid peroxidation in Cyp2e1+/– mice. However, these reagents had no significant effect in microsomes prepared from Cyp2e1–/– mice (Table 4).
Hepatic CYP2E1 expression in Cyp2e1+/– mice and Cyp2e1–/– mice fed the MCD diet or the control diet for 10 weeks. (a) Immunoblots obtained with anti-rat CYP2E1 antibody. (b) Northern blot analysis using a mouse-specific CYP2E1 riboprobe (see Methods). The signal for 18S rRNA is shown as a control for loading and RNA integrity.
Effects of DETC, anti-rat CYP2E1 IgG, anti-mouse CYP4A10 IgG, and anti-mouse CYP4A12 IgG on hepatic microsomal NADPH-dependent lipid peroxidation in Cyp2e1+/– and _Cyp2e1_–/– mice fed the methionine and choline deficient (MCD) or the control diet
These observations indicated that CYP2E1 is the major enzyme responsible for microsomal lipid peroxidation in mice expressing CYP2E1 protein (Cyp2e1+/– and wild-type C57BL6/J). However, the absence of CYP2E1 in microsomes from Cyp2e1–/– mice did not correlate with a decrease in lipid peroxidase activity. Furthermore, such activity was inducible in the Cyp2e1–/– mice to the same extent as in Cyp2e1+/– mice by feeding the MCD diet. We interpret these data as indicating that in the absence of CYP2E1, an alternative, inducible biochemical pathway operates to mediate microsomal NADPH-dependent lipid peroxidase activity.
Effects of MCD diet on cytochromes P450 in Cyp2e1–/– mice. To test the above assertion, further studies were conducted to characterize MCD-induced changes in hepatic microsomal proteins in Cyp2e1–/– mice. Levels of CYP1A and CYP3A proteins were similar in Cyp2e1–/– mice and Cyp2e1+/– mice fed the control diet, whereas feeding the MCD diet produced an apparent slight (not significant) decrease in both proteins (not shown). The 2 major members of the mouse Cyp4a subfamily are Cyp4a10 and Cyp4a14; expression of Cyp4a12 is barely detectable in female mice. There were no differences in the RNA levels of these 3 genes between Cyp2e1–/– mice and Cyp2e1+/– mice fed the control diet (Figure 5c). However, feeding the MCD diet significantly increased hepatic CYP4A10 and CYP4A14 expression in Cyp2e1–/– mice, by 150% (P = 0.001) and 300% (P < 0.001), respectively (Figure 5, a and c). There was no change in CYP4A12 transcripts (Figure 5, b and c). In contrast, in Cyp2e1+/– mice, the MCD diet produced no significant change in the major forms of CYP4A (CYP4A10 and CYP4A14), although CYP4A12, a minor form, was decreased (Figure 5).
Hepatic levels of CYP4A10, CYP4A12, and CYP4A14 mRNA in Cyp2e1+/– mice and Cyp2e1–/– mice fed the MCD diet or the control diet for 10 weeks. (a) Representative autoradiograph of an RNase protection assay showing the levels of β-actin, CYP4A14, and CYP4A10 mRNA in 2 Cyp2e1+/– and Cyp2e1–/– mice fed the control diet or the MCD diet. (b) Representative autoradiograph of an RNase protection assay showing the levels of β-actin and CYP4A12 mRNA in Cyp2e1+/– mice and Cyp2e1–/– mice fed the control diet or the MCD diet. (c) mRNA levels (mean ± SD; n = 4 in each group) of the 3 mouse CYP4A genes in Cyp2e1+/– mice and Cyp2e1–/– mice fed the control diet (open bars) or the MCD diet (filled bars). Bands were quantified using the PhosphorImager and ImageQuant analysis programs from Molecular Dynamics Inc., with normalization to the β-actin signal (see Methods). Note the different scale used for CYP4A12, which is expressed at very low levels compared with CYP4A10 and CYP4A14. A_P_ < 0.001 and B_P_ < 0.05 compared with controls. Cont, control.
To establish whether CYP4A enzymes might be involved in the generation of lipid peroxides in Cyp2e1–/– mice, we analyzed the effects of anti-mouse CYP4A10 and CYP4A12 antibodies on NADPH-dependent lipid peroxidase activity in microsomal fractions. Significant and substantial immunoinhibition of TBARs production was observed using the anti-CYP4A10 antibody (Table 4). This antibody had only minor effects in microsomes from Cyp2e1+/– mice. No significant effect was noted using the CYP4A12 antiserum (Table 4). Given the lack of information regarding the specificity of immunoinhibition by the antibody, this observation is not conclusive, but is consistent with the low expression level of this isoform and the finding that it was not upregulated by dietary manipulation (Figure 5).