Ethanol induction of CYP2A5: role of CYP2E1-ROS-Nrf2 pathway

Abstract

Chronic ethanol consumption was previously shown to induce CYP2A5 in mice, and this induction of CYP2A5 by ethanol was CYP2E1 dependent. In this study, the mechanisms of CYP2E1-dependent ethanol induction of CYP2A5 were investigated. CYP2E1 was induced by chronic ethanol consumption to the same degree in wild-type (WT) mice and CYP2A5 knockout (Cyp2a5 (-/-)) mice, suggesting that unlike the CYP2E1-dependent ethanol induction of CYP2A5, ethanol induction of CYP2E1 is not CYP2A5 dependent. Microsomal ethanol oxidation was about 25% lower in Cyp2a5 (-/-) mice compared with that in WT mice, suggesting that CYP2A5 can oxidize ethanol although to a lesser extent than CYP2E1 does. CYP2A5 was induced by short-term ethanol consumption in human CYP2E1 transgenic knockin (Cyp2e1 (-/-) KI) mice but not in CYP2E1 knockout (Cyp2e1 (-/-)) mice. The redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) was also induced by acute ethanol in Cyp2e1 (-/-) KI mice but not in Cyp2e1 (-/-) mice. Ethanol induction of CYP2A5 in Nrf2 knockout (Nrf2 (-/-)) mice was lower compared with that in WT mice, whereas CYP2E1 induction by ethanol was comparable in WT and Nrf2 (-/-) mice. Antioxidants (N-acetyl-cysteine and vitamin C), which blocked oxidative stress induced by chronic ethanol in WT mice and acute ethanol in Cyp2e1 (-/-) KI mice, also blunted the induction of CYP2A5 and Nrf2 by ethanol but not the induction of CYP2E1 by ethanol. These results suggest that oxidative stress induced by ethanol via induction of CYP2E1 upregulates Nrf2 activity, which in turn regulates ethanol induction of CYP2A5. Results obtained from primary hepatocytes, mice gavaged with binge ethanol or fed chronic ethanol, show that Nrf2-regulated ethanol induction of CYP2A5 protects against ethanol-induced steatosis.

Fig. 1

Induction of CYP2E1 and CYP2A5 by chronic ethanol feeding in WT, Cyp2e1 ^–/–, and Cyp2a5 ^–/– mice. After 3 weeks of ethanol feeding, induction of CYP2E1 and CYP2A5 was measured as described in Materials and Methods section. (A) Activities of CYP2E1 and CYP2A5 in WT and Cyp2e1 ^–/– mice. (B) Western blotting analyses for CYP2E1 and CYP2A5 in WT and Cyp2e1 ^–/– mice. *p < 0.05, compared with Cont group; #p < 0.05, compared with WT EtOH group. Cont, Control; EtOH, Ethanol. (C) Activities of CYP2E1 and CYP2A5 were inhibited by CMZ. *p < 0.05, compared with Cont group; #p < 0.05, compared to EtOH group. (D) Activities of CYP2E1 and CYP2A5 in WT and Cyp2a5 ^–/– mice. *p < 0.05, compared with Cont group; #p < 0.05, compared with WT EtOH group. (E) Western blotting analyses for CYP2E1 and CYP2A5 in WT and Cyp2a5 ^–/– mice. *p < 0.05, compared with Cont group. (F) Ethanol oxidation in microsomes isolated from WT and Cyp2a5 ^–/– mice fed dextrose or ethanol liquid diet. *p < 0.05, compared with Cont group; #p < 0.05, compared with WT EtOH group.

Fig. 2

Time course of induction of CYP2E1, CYP2A5, and Nrf2 by short-term ethanol feeding in Cyp2e1 ^–/– KI mice. Cyp2e1 ^–/– and Cyp2e1 ^–/– KI mice were fed an ethanol diet for 1, 2, and 3 days. Western blotting analyses for hepatic CYP2E1, CYP2A5, and Nrf2 (A), activities of CYP2E1 (B) and CYP2A5 (C), Western blotting analyses for hepatic nuclear Nrf2 (D), and nuclear Nrf2 binding activities (E) were measured as described in Materials and Methods section. *p < 0.05, compared with 0 day (control group); #p < 0.05, compared with Cyp2e1 ^–/– KI mice.

Fig. 3

. Induction of oxidative stress and Nrf2 by short-term ethanol feeding in Cyp2e1 ^–/– KI mice was blocked by antioxidants. Cyp2e1 ^–/– KI mice were fed ethanol for 2 days, and NAC and Vc were injected the day before initiating ethanol feeding; NAC was injected at 150 mg/kg body weight, ip, once a day; Vc was injected at 125 mg/kg body weight, ip, every 12 h. Hepatic TBARS and GSH (A), Western blotting analyses for nuclear Nrf2 (B), and ELISA for Nrf2 DNA binding activity (C) were measured as described in Materials and Methods section. *p < 0.05, compared with Cont group; #p < 0.05, compared with EtOH group. Cont, Control; EtOH, Ethanol.

Fig. 4

Induction of CYP2A5 but not CYP2E1 by short-term ethanol feeding in Cyp2e1 ^–/– KI mice was blocked by antioxidants. Cyp2e1 ^–/– KI mice were fed ethanol for 2 days, and NAC and Vc were injected the day before initiating ethanol feeding; NAC was injected at 150 mg/kg body weight, ip, once a day; Vc was injected at 125 mg/kg body weight, ip, every 12 h. Hepatic microsomal CYP2A5 and CYP2E1 activities (A) and Western blotting analyses for CYP2A5 and CYP2E1 (B) were measured as described in Materials and Methods section. *p < 0.05, compared with Cont group; #p < 0.05, compared with EtOH group. Cont, Control; EtOH, Ethanol.

Fig. 5

Induction of CYP2A5 but not CYP2E1 by chronic ethanol feeding in WT mice was blocked by antioxidants. WT mice were fed an ethanol diet for 3 weeks; NAC (75 mg/kg body weight) and Vc (125 mg/kg body weight) were injected, ip, once a day. Hepatic microsomal CYP2A5 and CYP2E1 activities (A), Western blotting analyses for CYP2A5, CYP2E1, and Nrf2 (B), and hepatic TBARS and GSH (C) were measured as described in Materials and Methods section. *p < 0.05, compared with Cont group; #p < 0.05, compared with EtOH group. Cont, Control; EtOH, Ethanol.

Fig. 6

Induction of CYP2A5 and CYP2E1 by chronic ethanol feeding in WT and Nrf2 ^–/– mice. WT and Nrf2 ^–/– mice were fed an ethanol diet for 3 weeks, Western blotting analyses for CYP2E1 and CYP2A5 (A) and activities of CYP2A5 (B) and CYP2E1 (C) were measured as described in Materials and Methods section. *p < 0.05, compared with Cont group; #p < 0.05, compared with WT EtOH group. Cont, Control; EtOH, Ethanol.

Fig. 7.

Ethanol-induced steatosis in Cyp2a5 ^–/– mice was more severe compared with that in WT mice. (A, B) WT and Cyp2a5 ^–/– mice were fed ethanol or dextrose liquid Lieber-DeCarli diets for 3 weeks. (A) Hepatic TG levels were increased more by ethanol in Cyp2a5 ^–/– mice than in WT mice. *p < 0.05, compared with Cont group; #p < 0.05, compared with WT EtOH group. Cont, Control; EtOH, Ethanol. (B) Hematoxylin and eosin (H&E) staining shows more lipid droplets in liver sections from ethanol-fed Cyp2a5 ^–/– mice than those from ethanol-fed WT mice. (C, D, E) Primary hepatocytes isolated from WT and Cyp2a5 ^–/– mice were treated with 0–100mM ethanol for 36 h and then were stained with Oil Red O or lysed for TG assay. (C) More lipid droplets were observed in hepatocytes from Cyp2a5 ^–/– mice than those from WT mice. (D) Oil Red O staining was elevated in hepatocytes from Cyp2a5 ^–/– mice than those from WT mice. (E) Higher TG levels were found in ethanol-treated hepatocytes from Cyp2a5 ^–/– mice than those from WT mice. *p < 0.05, compared with WT group. (F, G) WT and Cyp2a5 ^–/– mice were treated by gavage with one dose of ethanol (6 g/kg body weight for 14 h). (F) Hepatic TG levels were increased more by ethanol in Cyp2a5 ^–/– mice than in WT mice. *p < 0.05, compared with Cont group; #p < 0.05, compared with WT EtOH group. Cont, Control; EtOH, Ethanol. (G) H&E staining shows more lipid droplets () in liver sections from Cyp2a5 ^–/– mice than those from WT mice.

Fig. 8.

Ethanol treatment decreases HO-1 protein levels. (A) Western blotting analysis showing that HO-1 protein levels are decreased after ethanol treatment in WT and Cyp2a5 ^–/– hepatocytes. (B) Quantification of Western blotting in (A). *p < 0.05, compared with Cont group. (C) Time course for the decline in HO-1 protein level in livers after binge ethanol treatment in WT mice. (D) Quantification of Western blotting in C. *p < 0.05, compared with 0 h.

Fig. 9.

Scheme of ethanol induction of CYP2A5. Ethanol causes proliferation of SER and upregulates levels of CYP2E1. This leads to increased production of ROS. CYP2E1-mediated ROS activates the redox-sensitive transcription factor Nrf2, which in turn upregulates CYP2A5 expression. For details, please refer to the text.