Hydrogen Peroxide Induces Ethanol-inducible CYP2E1 via the NF-kB-classical Pathway: CYP2E1 mRNA Levels are not High in Alcoholic Hepatitis.

Abstract

Aims: The aim of the present study is to elucidate the mechanism of CYP2E1 induction as a causative factor of Alcoholic Hepatitis (AH) and its relationship with inflammation.

Background: Chronic alcohol consumption induces CYP2E1, which is involved in the development of Alcoholic Hepatitis (AH). However, the mechanisms underlying the induction of CYP2E1 by alcohol remain unclear. Therefore, we herein investigated the induction of drug-metabolizing enzymes, particularly CYP2E1, by hydrogen peroxide (H₂O₂), the concentration of which is elevated under inflammatory conditions.

Objective: The mechanisms underlying the induction of CYP2E1 by H₂O₂ were examined with a focus on Keap1, a target factor of H₂O₂.

Methods: We assessed changes in the expression of drug-metabolizing enzymes in the human hepatoma cell line, Hep3B, following treatment with H₂O₂, and evaluated changes in the expression of the NF-kB-related factor RelA(p65) after the knockdown of Keap1, a regulator of Nrf2 expression by reactive oxygen species. We also performed a promoter analysis using the upstream region of the CYP2E1 gene. We herein used the GSE89632 series for non-alcoholic hepatitis (NASH) and the GSE28619 series for AH.

Results: The induction of CYP2E1 by H₂O₂ was significantly stronger than that of other drugmetabolizing enzymes. On the other hand, the knockdown of Keap1, a target of H₂O₂, markedly increased RelA(p65), an NFkB factor. Furthermore, the overexpression of RelA(p65) strongly induced the expression of CYP2E1. Four candidate p65-binding sequences were identified upstream of the CYP2E1 gene, and promoter activity assays showed that the third sequence was responsive to the overexpression of RelA(p65). We used the GSE89632 series for NASH and the GSE28619 series for AH in the present study. The expression of CYP2E1 mRNA in the liver was significantly lower in AH patients than in HC patients, but was similar in HC patients and NASH patients.

Conclusion: We herein demonstrated that the expression of CYP2E1 was induced by H₂O₂. The overexpression of RelA(p65) also induced CYP2E1 mRNA expression, whereas H₂O₂ did not after the knockdown of RelA. These results suggest that H₂O₂ acts on Keap1 to upregulate RelA (p65) in the NFkB system. One of the mechanisms underlying the induction of CYP2E1 was dependent on the H₂O₂-Keap1-RelA axis. The results of the database analysis revealed that the expression of CYP2E1 in the liver was significantly lower in AH patients than in NASH patients, suggesting that CYP2E1 is not the main cause of AH; however, CYP2E1 may exacerbate the pathogenesis of AH.