Bidirectional Regulation of NAD(P)H Quinone Dehydrogenase 1 Expression in Mouse Hepatic Stellate Cells - Acute versus Long-Term Ethanol Exposure

Abstract

Background: The expression of NAD(P)H quinone dehydrogenase 1 (NQO1) is controlled hierarchically by aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor-2 (Nrf2). This study examined whether ethanol regulates NQO1 expression via the AhR-Nrf2 pathway. Methods: Immortal mouse hepatic stellate cells (MHSCs) were treated with ethanol and/or an AhR ligand and benzo(a)pyrene (BaP). Realtime quantitative reverse transcription polymerase chain reaction assays, western blot analysis, and promoter activity assays were performed to determine the expression and promoter activity of NQO1. Results: Treatment of MHSCs with 25–100 mM ethanol for 6 h elevated NQO1 mRNA and protein in a dose-dependent manner. Exposure of MHSCs to 50 mM ethanol for 6 h reduced whole-cell AhR protein level but increased the following: nuclear AhR protein, wholecell and nuclear Nrf2 protein, and NQO1 promoter activity. Mutation of the Nrf2 binding site in the NQO1 promoter region inhibited the basal and ethanol-induced NQO1 promoter activity, while mutation of the AhR binding site did not affect the NQO1 promoter activity. Knocking down AhR suppressed ethanol-induced Nrf2 and NQO1 expression, while knocking down Nrf2 inhibited both basal and ethanol-induced NQO1 expression. Exposing MHSCs to 50 mM ethanol for 72 h reduced the following: whole cell AhR, Nrf2 and NQO1 protein levels, Nrf2 and NQO1 mRNA levels, and nuclear AhR and Nrf2 protein levels. Treating MHSCs with 10 nM BaP for 6 h increased Nrf2 and NQO1 mRNA and protein levels. Pretreatment of MHSCs with 50 mM ethanol for 72 h diminished the capacity of MHSCs to express NQO1 and Nrf2 upon induction by BaP. Conclusions: Our findings suggest that ethanol upregulates NQO1 expression by activating the AhR-Nrf2 pathway. Long-term ethanol exposure sustains low level of AhR protein and diminishes the inducibility of its target genes Nrf2 and NQO1 by BaP. These findings will contribute to understanding the synergistic toxicity of ethanol and polycyclic aromatic hydrocarbon compounds, such as BaP.