CYP2E1 Overexpression in HepG2 Cells Induces Glutathione Synthesis by Transcriptional Activation of γ-Glutamylcysteine Synthetase

Montserrat Marı́,Arthur I Cederbaum

doi:10.1074/jbc.m907022199

Abstract

Induction of CYP2E1 (cytochrome P450 2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. CYP2E1 is a loosely coupled enzyme; formation of reactive oxygen species occurs even in the absence of added substrate. GSH is critical for preserving the proper cellular redox balance and for its role as a cellular protectant. Since cells must maintain optimal GSH levels to cope with a variety of stresses, the goal of this study was to characterize the GSH homeostasis in human hepatocarcinoma cells (HepG2) that overexpress CYP2E1. This study was prompted by the finding that toxicity in CYP2E1-overexpressing cells was markedly enhanced after GSH depletion by buthionine sulfoximine treatment. CYP2E1-overexpressing cells showed a 40-50% increase in intracellular H(2)O(2); a 30% increase in total GSH levels; a 50% increase in the GSH synthesis rate; and a 2-fold increase in gamma-glutamylcysteine synthetase heavy subunit (GCS-HS) mRNA, the rate-limiting enzyme in GSH synthesis. This GCS-HS mRNA increase was due to increased synthesis since nuclear run-on assays showed increased transcription in CYP2E1-expressing cells, and the GCS-HS mRNA decay after actinomycin D treatment was similar in CYP2E1-expressing cells and empty vector-transfected cells. The facts that treatment with GSH ethyl ester almost completely prevented the increase in GCS-HS mRNA and decreased H(2)O(2) levels and that transient transfection with catalase (but not manganese-superoxide dismutase) produced a decrease in GCS-HS mRNA only in CYP2E1-expressing cells suggest a possible role for H(2)O(2) in the induction of GCS-HS gene transcription. In contrast to results with HepG2 cells expressing CYP2E1, no increase in GCS-HS mRNA was found with a HepG2 cell line engineered to express human cytochrome P450 3A4. In summary, CYP2E1 overexpression in HepG2 cells up-regulates the levels of reduced GSH by transcriptional activation of GCS-HS; this may reflect an adaptive mechanism to remove CYP2E1-derived oxidants such as H(2)O(2).

Highlights

GSH, the most abundant antioxidant in cells, plays a major role in the defense against oxidative stress-induced cell injury and is essential in maintenance of intracellular redox balance and the essential thiol status of proteins [1]
CYP2E1overexpressing cells showed a 40 –50% increase in intracellular H2O2; a 30% increase in total GSH levels; a 50% increase in the GSH synthesis rate; and a 2-fold increase in ␥-glutamylcysteine synthetase heavy subunit (GCS-HS) mRNA, the rate-limiting enzyme in GSH synthesis
This GCS-HS mRNA increase was due to increased synthesis since nuclear run-on assays showed increased transcription in CYP2E1-expressing cells, and the GCS-HS mRNA decay after actinomycin D treatment was similar in CYP2E1-expressing cells and empty vector-transfected cells

Summary

Introduction

GSH, the most abundant antioxidant in cells, plays a major role in the defense against oxidative stress-induced cell injury and is essential in maintenance of intracellular redox balance and the essential thiol status of proteins [1]. The cellular GSH level is determined by a balance between the rate of its synthesis and the rates of its utilization (conjugation) and loss (export) [2]. Changes in GCS activity have been reported to result from regulation at the transcriptional (9 –12) and/or post-transcriptional [13] level, and these changes may affect only the heavy or both the heavy and light subunits [13, 14]. Have a slower growth rate than the empty vector-transfected control C34 cells when cellular GSH levels are maintained, but display a loss of cell viability when GSH synthesis is inhibited by treatment with BSO. This suggested that a possible up-regulation of GSH synthesis and/or increased GSH turnover may occur in E47 cells in response to the increased oxidative stress.

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