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CYP2E1 overexpression in HepG2 cells induces glutathione synthesis by transcriptional activation of gamma-glutamylcysteine synthetase.
J Biol Chem. 2000 May 19; 275(20):15563-71.JB

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).

Authors+Show Affiliations

Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10748080

Citation

Marí, M, and A I. Cederbaum. "CYP2E1 Overexpression in HepG2 Cells Induces Glutathione Synthesis By Transcriptional Activation of Gamma-glutamylcysteine Synthetase." The Journal of Biological Chemistry, vol. 275, no. 20, 2000, pp. 15563-71.
Marí M, Cederbaum AI. CYP2E1 overexpression in HepG2 cells induces glutathione synthesis by transcriptional activation of gamma-glutamylcysteine synthetase. J Biol Chem. 2000;275(20):15563-71.
Marí, M., & Cederbaum, A. I. (2000). CYP2E1 overexpression in HepG2 cells induces glutathione synthesis by transcriptional activation of gamma-glutamylcysteine synthetase. The Journal of Biological Chemistry, 275(20), 15563-71.
Marí M, Cederbaum AI. CYP2E1 Overexpression in HepG2 Cells Induces Glutathione Synthesis By Transcriptional Activation of Gamma-glutamylcysteine Synthetase. J Biol Chem. 2000 May 19;275(20):15563-71. PubMed PMID: 10748080.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - CYP2E1 overexpression in HepG2 cells induces glutathione synthesis by transcriptional activation of gamma-glutamylcysteine synthetase. AU - Marí,M, AU - Cederbaum,A I, PY - 2000/4/5/pubmed PY - 2000/6/24/medline PY - 2000/4/5/entrez SP - 15563 EP - 71 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 275 IS - 20 N2 - 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). SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/10748080/CYP2E1_overexpression_in_HepG2_cells_induces_glutathione_synthesis_by_transcriptional_activation_of_gamma_glutamylcysteine_synthetase_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=10748080 DB - PRIME DP - Unbound Medicine ER -