Ethanol induction of CYP2A5: permissive role for CYP2E1

Abstract

CYP2A5 metabolizes xenobiotics and activates hepatocarcinogens, and induction occurs in response to hepatic damage and cellular stress. We evaluated whether ethanol can elevate CYP2A5 and whether CYP2E1 plays a role in the ethanol induction of CYP2A5. Wild-type (WT), CYP2E1 knockout (KO), and CYP2E1 knockin (KI) mice were fed ethanol for 3 weeks. Ethanol increased CYP2E1 and CYP2A5 protein and activity in WT mice but not in the KO mice. Induction of CYP2A5 (and CYP2E1) was restored in the KI mice. Ethanol induction of CYP2A5 occurred only after CYP2E1 was first induced. Immunohistochemical staining revealed that CYP2E1 and CYP2A5 colocalize to the same zones in the liver. Ethanol also elevated CYP2A5 mRNA levels in WT and KI mice but not in KO mice. Induction of CYP2A5 by cadmium was partially decreased in KO mice compared with WT or KI mice. Ethanol elevated CYP2A4 mRNA levels in all mice although the extent of induction was lowest in the KO mice. In summary, ethanol elevated mouse hepatic CYP2A5 levels, which may be of toxicological significance because CYP2A5 metabolizes nicotine and other drugs and activates hepatocarcinogens. Induction of CYP2A5 by ethanol is potentiated by the induction of CYP2E1. We speculate that ethanol induction of CYP2E1 followed by increases in reactive oxygen species and activation of Nrf2 are important steps in the mechanism by which ethanol induces CYP2A5. The possibility that induction of CYP2E1 is permissive for the induction of CYP2A5 may reflect a new contribution by CYP2E1 to the actions of ethanol.

Fig. 1.

Western blot analysis for the levels of CYP2A5 and CYP 2E1 in liver homogenates from untreated WT, CYP2E1 KO, and CYP2E1 KI mice. Results from three mice in each group are shown. Numbers under the blots refer to the CYP2A5 (lower bands)/β-actin or CYP2E1/β-actin ratio. *, P < 0.05 compared with the CYP2E1 KO group; #, P < 0.05 compared with the CYP2E1 WT group.

Fig. 2.

Chronic ethanol administration elevates CYP2A5 levels in SV/129 WT mice and CYP2E1 KI mice but not in CYP2E1 KO mice. WT, KO, and KI mice (n = 4 in each group) were fed the dextrose or ethanol diets for 3 weeks. A, microsomal oxidation of PNP or coumarin. B, Western blots to assay levels of CYP2E1 and CYP2A5 in liver homogenates. C, immunohistochemical staining of liver slices with anti-CYP2E1 or anti-CYP2A5 IgG to detect CYP2E1 or CYP2A5 in intact liver. *, P < 0.05 compared with WD group; #, P < 0.05 compared with KID group; &, P < 0.05 compared with WE group. WD, wild-type dextrose control; WE, wild-type mice fed ethanol; KOD, knockout mice fed dextrose; KOE, knockout mice fed ethanol; KID, knockin mice fed dextrose; KIE, knockin mice fed ethanol.

Fig. 3.

Time course for the induction of CYP2E1 and CYP2A5 by ethanol. SV/129 WT mice (n = 4 in each group) were fed the ethanol liquid diet for 0, 1, 2, or 3 weeks (w). A, Western blot analysis for levels of CYP2E1 and CYP2A5 protein. B, PNP oxidation as a reflection of CYP2E1 activity and coumarin 7-hydroxlase activity as a reflection of CYP2A5 activity. Results from mice before initiation of the ethanol feeding (0 week) are also shown. *, P < 0.05 compared with the 0-week CYP2E1; #, P < 0.05, compared with the 0-week CYP2A5.

Fig. 4.

Ethanol elevates CYP2A4 (A) and CYP2A5 mRNA (B) levels. WT, KO, and KI mice (n = 4 in each group) were fed the dextrose control or the ethanol liquid diet for 3 weeks. Isolated RNA was reverse-transcribed to produce cDNA, and a real-time quantitative PCR assay for the expression of CYP2A5 and CYP2A4 mRNA was performed as described under Materials and Methods. *, P < 0.05 compared with the WTD group; #, P < 0.05 compared with the KOD group; P < 0.05 compared with the KID group; $, P < 0.05 compared with the KOE group; @, P < 0.05 compared with the WTE group. WTD, wild-type dextrose control; WTE, wild-type mice fed ethanol; KOD, knockout mice fed dextrose; KOE, knockout mice fed ethanol; KID, knockin mice fed dextrose; KIE, knockin mice fed ethanol.

Fig. 5.

Levels of CYP2E1 and CYP2A6 (the human ortholog of mouse CYP2A5) in human livers. Western blots were performed on liver extracts from two patients with alcoholic liver disease (ALD), from four patients with liver cirrhosis, and from a control healthy liver. Diagnoses and liver samples were provided by the Department of Surgical Pathology at Mount Sinai.

Fig. 6.

Induction of CYP2A5 by cadmium in WT, CYP2E1 KO, and CYP2E1 KI mice. Mice were given intraperitoneal injections of cadmium chloride (3 mg/kg) or saline as control, and 18 h later, mice were sacrificed. PNP oxidation as a reflection of CYP2E1 activity and coumarin 7-hydroxlase activity as a reflection of CYP2A5 activity were determined or levels of CYP2E1 or CYP2A5 were determined by immunoblots. A, CYP2A5 but not CYP2E1was induced by cadmium in female and male WT mice (n = 3 in each group). *, P < 0.05 compared with the male control group; #, P < 0.05 compared with the female control group; $, P < 0.05 compared with the male cadmium group. B, Vc and NAC blocked cadmium induction of CYP2A5 in WT mice. WT mice were pretreated with saline (n = 3), Vc (n = 3), or NAC (n = 3) before cadmium treatment as described under Materials and Methods. Controls received saline intraperitoneally (n = 4). Catalytic activities and Western blots are shown. *, P < 0.05 compared with the control group; #, P < 0.05 compared with the cadmium group. C, induction of CYP2A5 by cadmium in female CYP2E1 KO and CYP2E1 KI mice (n = 4 in each group). Treatments are identical to those for the female WT mice in A. Catalytic activities and Western blots are shown. *, P < 0.05 compared with the KO control group; #, P < 0.05 compared with the KI control group; $, P < 0.05 compared with the KO cadmium group. Cont, control; Cd, cadmium.