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J Biol Chem, Vol. 275, Issue 20, 15563-15571, May 19, 2000
From the Department of Biochemistry and Molecular Biology, Mount
Sinai School of Medicine, New York, New York 10029
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
H2O2; a 30% increase in total GSH
levels; a 50% increase in the GSH synthesis rate; and a 2-fold
increase in
CYP2E1 Overexpression in HepG2 Cells Induces Glutathione
Synthesis by Transcriptional Activation of
-Glutamylcysteine
Synthetase*
-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 H2O2
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
H2O2 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
H2O2.
*
This work was supported by United States Public Health
Service Grants AA03312 and AA06610 from the National Institute on
Alcohol Abuse and Alcoholism.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, One Gustave L. Levy Place, P. O. Box 1020, Mount Sinai School of Medicine, New York, NY 10029. Tel.: 212-241-7285;
Fax: 212-996-7214; E-mail: Arthur.Cederbaum@mssm.edu.
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