Absence of cytochrome P450 2A5 enhances alcohol-induced liver injury in mice

Abstract

Background: Ethanol can induce cytochrome P450 2E1, an active generator of reactive oxygen species, and this cytochrome is considered a risk factor for oxidative liver injury. Recently, we found that in addition to P450 2E1 also cytochrome P450 2A5, another isoform of cytochrome P450, can be induced by ethanol, and that ethanol induction of cytochrome P450 2A5 is P450 2E1-dependent.

Aims: To investigate the role of cytochrome P450 2A5 in alcohol-induced liver injury.

Methods: Cytochrome P450 2A5-knockout mice and wild type mice were fed the Lieber-Decarli ethanol liquid diet to induce liver injury. Controls were fed the Lieber-Decarli control diet.

Results: After 4 weeks of feeding with Lieber-Decarli diet, ethanol-induced liver injury was enhanced in the knockout mice compared with wild type mice, as indicated by serum transaminases, hepatic fat accumulation (steatosis), and necroinflammation observed in liver sections with Haematoxylin & Eosin staining. Ethanol-induced oxidative stress was also higher in the knockout mice than the wild types. Ethanol feeding induced cytochrome P450 2A5 in wild type mice but not in the knockout mice, while induction of cytochrome P450 2E1 was comparable in the knockout and wild type mice.

Conclusion: These results suggest that cytochrome P450 2A5 protects against ethanol-induced oxidative liver injury.

Keywords: Cytochrome P450 2A5; Cytochrome P450 2E1; Ethanol; Liver; Oxidative stress.

Figure 1

Serum alcohol levels and liver major alcohol metabolic enzyme CYP2E1 levels are comparable in cytochrome P450 2A5 knockout and wild type mice. (A) Serum alcohol concentrations. (B) Western blotting analyses for CYP2E1, CYP2A5 and alcohol dehydrogenase in cyp2a5^+/+ and cyp2a5^−/− mice. (C) Western blotting quantification. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. ADH, alcohol dehydrogenase; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 1

Serum alcohol levels and liver major alcohol metabolic enzyme CYP2E1 levels are comparable in cytochrome P450 2A5 knockout and wild type mice. (A) Serum alcohol concentrations. (B) Western blotting analyses for CYP2E1, CYP2A5 and alcohol dehydrogenase in cyp2a5^+/+ and cyp2a5^−/− mice. (C) Western blotting quantification. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. ADH, alcohol dehydrogenase; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 1

Serum alcohol levels and liver major alcohol metabolic enzyme CYP2E1 levels are comparable in cytochrome P450 2A5 knockout and wild type mice. (A) Serum alcohol concentrations. (B) Western blotting analyses for CYP2E1, CYP2A5 and alcohol dehydrogenase in cyp2a5^+/+ and cyp2a5^−/− mice. (C) Western blotting quantification. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. ADH, alcohol dehydrogenase; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 2

Liver injury and steatosis after ethanol feeding are more severe in cytochrome P450 2A5 knockout and wild type mice. (A) Haematoxylin & eosin staining. Black arrows show lipid droplets and white arrows show necroinflammatory foci. (B) necroinflammation and steatosis quantification; (C) Serum alanine aminotransfrerase and aspartate aminotransferase levels; (D) Ratio of Liver/body weight. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (E) Immunohistochemistry staining for collagen I and α-smooth muscle actin. Black arrows show collagen I and red arrows for α-smooth muscle actin. PV, portal vein; CV, central vein. ALT, alanine aminotransfrerase; AST, aspartate aminotransferase; α-SMA, α-smooth muscle actin; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 2

Liver injury and steatosis after ethanol feeding are more severe in cytochrome P450 2A5 knockout and wild type mice. (A) Haematoxylin & eosin staining. Black arrows show lipid droplets and white arrows show necroinflammatory foci. (B) necroinflammation and steatosis quantification; (C) Serum alanine aminotransfrerase and aspartate aminotransferase levels; (D) Ratio of Liver/body weight. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (E) Immunohistochemistry staining for collagen I and α-smooth muscle actin. Black arrows show collagen I and red arrows for α-smooth muscle actin. PV, portal vein; CV, central vein. ALT, alanine aminotransfrerase; AST, aspartate aminotransferase; α-SMA, α-smooth muscle actin; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 2

Liver injury and steatosis after ethanol feeding are more severe in cytochrome P450 2A5 knockout and wild type mice. (A) Haematoxylin & eosin staining. Black arrows show lipid droplets and white arrows show necroinflammatory foci. (B) necroinflammation and steatosis quantification; (C) Serum alanine aminotransfrerase and aspartate aminotransferase levels; (D) Ratio of Liver/body weight. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (E) Immunohistochemistry staining for collagen I and α-smooth muscle actin. Black arrows show collagen I and red arrows for α-smooth muscle actin. PV, portal vein; CV, central vein. ALT, alanine aminotransfrerase; AST, aspartate aminotransferase; α-SMA, α-smooth muscle actin; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 2

Liver injury and steatosis after ethanol feeding are more severe in cytochrome P450 2A5 knockout and wild type mice. (A) Haematoxylin & eosin staining. Black arrows show lipid droplets and white arrows show necroinflammatory foci. (B) necroinflammation and steatosis quantification; (C) Serum alanine aminotransfrerase and aspartate aminotransferase levels; (D) Ratio of Liver/body weight. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (E) Immunohistochemistry staining for collagen I and α-smooth muscle actin. Black arrows show collagen I and red arrows for α-smooth muscle actin. PV, portal vein; CV, central vein. ALT, alanine aminotransfrerase; AST, aspartate aminotransferase; α-SMA, α-smooth muscle actin; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 2

Liver injury and steatosis after ethanol feeding are more severe in cytochrome P450 2A5 knockout and wild type mice. (A) Haematoxylin & eosin staining. Black arrows show lipid droplets and white arrows show necroinflammatory foci. (B) necroinflammation and steatosis quantification; (C) Serum alanine aminotransfrerase and aspartate aminotransferase levels; (D) Ratio of Liver/body weight. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (E) Immunohistochemistry staining for collagen I and α-smooth muscle actin. Black arrows show collagen I and red arrows for α-smooth muscle actin. PV, portal vein; CV, central vein. ALT, alanine aminotransfrerase; AST, aspartate aminotransferase; α-SMA, α-smooth muscle actin; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 3

Fat accumulation in liver fat is more pronounced in cytochrome P450 2A5 knockout mice than cytochrome P450 2A5 wild type mice. (A) Liver triglycerides; (B) Serum triglycerides and glucose. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (C) Western blotting analyses for lipid metabolic enzymes. (D) Western blotting quantification. *P<0.05, compared with WT Control; #P<0.05, compared with KO Control. & P<0.05, compared with WT Ethanol. WT, cyp2a5^+/+ mice; KO, cyp2a5^−/− mice. TG, triglycerides; FAS, fatty acid synthase; AOX, acyl-CoA oxidase; PPARα, peroxisome proliferator-activated receptor α; CPT I, Carnitine palmitoyltransferase I; T-AMPK, total AMPK; p-AMPK, phosphorylated AMP-activated protein kinase; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 3

Fat accumulation in liver fat is more pronounced in cytochrome P450 2A5 knockout mice than cytochrome P450 2A5 wild type mice. (A) Liver triglycerides; (B) Serum triglycerides and glucose. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (C) Western blotting analyses for lipid metabolic enzymes. (D) Western blotting quantification. *P<0.05, compared with WT Control; #P<0.05, compared with KO Control. & P<0.05, compared with WT Ethanol. WT, cyp2a5^+/+ mice; KO, cyp2a5^−/− mice. TG, triglycerides; FAS, fatty acid synthase; AOX, acyl-CoA oxidase; PPARα, peroxisome proliferator-activated receptor α; CPT I, Carnitine palmitoyltransferase I; T-AMPK, total AMPK; p-AMPK, phosphorylated AMP-activated protein kinase; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 3

Fat accumulation in liver fat is more pronounced in cytochrome P450 2A5 knockout mice than cytochrome P450 2A5 wild type mice. (A) Liver triglycerides; (B) Serum triglycerides and glucose. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (C) Western blotting analyses for lipid metabolic enzymes. (D) Western blotting quantification. *P<0.05, compared with WT Control; #P<0.05, compared with KO Control. & P<0.05, compared with WT Ethanol. WT, cyp2a5^+/+ mice; KO, cyp2a5^−/− mice. TG, triglycerides; FAS, fatty acid synthase; AOX, acyl-CoA oxidase; PPARα, peroxisome proliferator-activated receptor α; CPT I, Carnitine palmitoyltransferase I; T-AMPK, total AMPK; p-AMPK, phosphorylated AMP-activated protein kinase; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 3

Fat accumulation in liver fat is more pronounced in cytochrome P450 2A5 knockout mice than cytochrome P450 2A5 wild type mice. (A) Liver triglycerides; (B) Serum triglycerides and glucose. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. (C) Western blotting analyses for lipid metabolic enzymes. (D) Western blotting quantification. *P<0.05, compared with WT Control; #P<0.05, compared with KO Control. & P<0.05, compared with WT Ethanol. WT, cyp2a5^+/+ mice; KO, cyp2a5^−/− mice. TG, triglycerides; FAS, fatty acid synthase; AOX, acyl-CoA oxidase; PPARα, peroxisome proliferator-activated receptor α; CPT I, Carnitine palmitoyltransferase I; T-AMPK, total AMPK; p-AMPK, phosphorylated AMP-activated protein kinase; cyp2a5^−/− mice, cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 4

Ethanol-induced oxidative stress is more severe cytochrome P450 2A5 knockout mice than cytochrome P450 2A5 wild type mice. (A) Hepatic reduced glutathione; (B) 3-nitrotyrosine adduct and (C) 4-hydroxyl-nonenal adduct formation in liver sections detected by Immunohistochemistry staining (D) Hepatic thiobarbituric acid reactive substances. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. PV, portal vein; CV, central vein. GSH, reduced glutathione; TBARS, thiobarbituric acid reactive substances. cyp2a5^−/− mice; cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 4

Ethanol-induced oxidative stress is more severe cytochrome P450 2A5 knockout mice than cytochrome P450 2A5 wild type mice. (A) Hepatic reduced glutathione; (B) 3-nitrotyrosine adduct and (C) 4-hydroxyl-nonenal adduct formation in liver sections detected by Immunohistochemistry staining (D) Hepatic thiobarbituric acid reactive substances. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. PV, portal vein; CV, central vein. GSH, reduced glutathione; TBARS, thiobarbituric acid reactive substances. cyp2a5^−/− mice; cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 4

Ethanol-induced oxidative stress is more severe cytochrome P450 2A5 knockout mice than cytochrome P450 2A5 wild type mice. (A) Hepatic reduced glutathione; (B) 3-nitrotyrosine adduct and (C) 4-hydroxyl-nonenal adduct formation in liver sections detected by Immunohistochemistry staining (D) Hepatic thiobarbituric acid reactive substances. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. PV, portal vein; CV, central vein. GSH, reduced glutathione; TBARS, thiobarbituric acid reactive substances. cyp2a5^−/− mice; cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 4

Ethanol-induced oxidative stress is more severe cytochrome P450 2A5 knockout mice than cytochrome P450 2A5 wild type mice. (A) Hepatic reduced glutathione; (B) 3-nitrotyrosine adduct and (C) 4-hydroxyl-nonenal adduct formation in liver sections detected by Immunohistochemistry staining (D) Hepatic thiobarbituric acid reactive substances. * P<0.05, compared with Control group; # P<0.05, compared with cyp2a5^+/+ Ethanol group. PV, portal vein; CV, central vein. GSH, reduced glutathione; TBARS, thiobarbituric acid reactive substances. cyp2a5^−/− mice; cytochrome P450 2A5 knockout mice; cyp2a5^+/+ mice, cytochrome P450 2A5 wild type mice.

Figure 5

Ethanol-induced liver injury is more severe in peroxisome proliferator-activated receptor α knockout mice than peroxisome proliferator-activated receptor α wild type mice. (A) Serum activity of alanine aminotransferase and aspartate aminotransferase; (B) Serum triglycerides; (C) Liver expression of cytochrome P450 2E1 and cytochrome P450 2A5; (D) Activity of cytochrome P450 2E1 and cytochrome P450 2A5. * P<0.05, compared with Cont group; # P<0.05, compared with pparα^+/+ Ethanol group; &P<0.05, compared with pparα^−/− Control Group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglyceride; pparα^−/−; mice, peroxisome proliferator-activated receptor α knockout mice; pparα^+/+ mice, peroxisome proliferator-activated receptor α wild type mice.

Figure 5

Ethanol-induced liver injury is more severe in peroxisome proliferator-activated receptor α knockout mice than peroxisome proliferator-activated receptor α wild type mice. (A) Serum activity of alanine aminotransferase and aspartate aminotransferase; (B) Serum triglycerides; (C) Liver expression of cytochrome P450 2E1 and cytochrome P450 2A5; (D) Activity of cytochrome P450 2E1 and cytochrome P450 2A5. * P<0.05, compared with Cont group; # P<0.05, compared with pparα^+/+ Ethanol group; &P<0.05, compared with pparα^−/− Control Group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglyceride; pparα^−/−; mice, peroxisome proliferator-activated receptor α knockout mice; pparα^+/+ mice, peroxisome proliferator-activated receptor α wild type mice.

Figure 5

Ethanol-induced liver injury is more severe in peroxisome proliferator-activated receptor α knockout mice than peroxisome proliferator-activated receptor α wild type mice. (A) Serum activity of alanine aminotransferase and aspartate aminotransferase; (B) Serum triglycerides; (C) Liver expression of cytochrome P450 2E1 and cytochrome P450 2A5; (D) Activity of cytochrome P450 2E1 and cytochrome P450 2A5. * P<0.05, compared with Cont group; # P<0.05, compared with pparα^+/+ Ethanol group; &P<0.05, compared with pparα^−/− Control Group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglyceride; pparα^−/−; mice, peroxisome proliferator-activated receptor α knockout mice; pparα^+/+ mice, peroxisome proliferator-activated receptor α wild type mice.

Figure 5

Ethanol-induced liver injury is more severe in peroxisome proliferator-activated receptor α knockout mice than peroxisome proliferator-activated receptor α wild type mice. (A) Serum activity of alanine aminotransferase and aspartate aminotransferase; (B) Serum triglycerides; (C) Liver expression of cytochrome P450 2E1 and cytochrome P450 2A5; (D) Activity of cytochrome P450 2E1 and cytochrome P450 2A5. * P<0.05, compared with Cont group; # P<0.05, compared with pparα^+/+ Ethanol group; &P<0.05, compared with pparα^−/− Control Group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglyceride; pparα^−/−; mice, peroxisome proliferator-activated receptor α knockout mice; pparα^+/+ mice, peroxisome proliferator-activated receptor α wild type mice.