Tumor Necrosis Factor Increases Hepatocellular Glutathione by Transcriptional Regulation of the Heavy Subunit Chain of γ-Glutamylcysteine Synthetase

Tumor necrosis factor (TNF) is an inflammatory cytokine that causes cell injury by generation of oxidative stress. Since glutathione (GSH) is a key cellular antioxidant that detoxifies reactive oxygen species, the purpose of our work was to examine the regulation of cellular GSH, the expression of heavy subunit chain of γ-glutamylcysteine synthetase (γ-GCS-HS), and control of intracellular generation of reactive oxygen species in cultured rat hepatocytes treated with TNF. Exposure of cells to TNF (10,000 units/ml) resulted in depletion of cellular GSH levels (50–70%) and overproduction of hydrogen peroxide (2–3-fold) and lipid peroxidation. However, cells treated with lower doses of TNF (250–500 units/ml) exhibited increased levels of GSH (60–80% over control). TNF treatment increased (70–100%) the levels of γ-GCS-HS mRNA, the catalytic subunit of the regulating enzyme in GSH biosynthesis. Furthermore, intact nuclei isolated from hepatocytes treated with TNF transcribed the γ-GCS-HS gene to a greater extent than control cells, indicating that TNF regulates γ-GCS-HS at the transcriptional level. The capacity to synthesize GSH de novo determined in cell-free extracts incubated with GSH precursors was greater (50–70%) in hepatocytes that were treated with TNF; however, the activity of GSH synthetase remained unaltered by TNF treatment indicating that TNF selectively increased the activity of γ-GCS. Despite activation of nuclear factor-κB (NF-κB) by TNF, this transcription factor was not required for TNF-induced transcription of γ-GCS-HS as revealed by deletion constructs of the γ-GCS-HS promoter subcloned in a chloramphenicol acetyltransferase reporter vector and transfected into HepG2 cells. In contrast, a construct containing AP-1 like/metal response regulatory elements increased chloramphenicol acetyltransferase activity upon exposure to TNF. Thus, TNF increases hepatocellular GSH levels by transcriptional regulation of γ-GCS-HS gene, probably through AP-1/metal response element-like binding site(s) in its promoter, which may constitute a protective mechanism in the control of oxidative stress induced by inflammatory cytokines. Tumor necrosis factor (TNF) is an inflammatory cytokine that causes cell injury by generation of oxidative stress. Since glutathione (GSH) is a key cellular antioxidant that detoxifies reactive oxygen species, the purpose of our work was to examine the regulation of cellular GSH, the expression of heavy subunit chain of γ-glutamylcysteine synthetase (γ-GCS-HS), and control of intracellular generation of reactive oxygen species in cultured rat hepatocytes treated with TNF. Exposure of cells to TNF (10,000 units/ml) resulted in depletion of cellular GSH levels (50–70%) and overproduction of hydrogen peroxide (2–3-fold) and lipid peroxidation. However, cells treated with lower doses of TNF (250–500 units/ml) exhibited increased levels of GSH (60–80% over control). TNF treatment increased (70–100%) the levels of γ-GCS-HS mRNA, the catalytic subunit of the regulating enzyme in GSH biosynthesis. Furthermore, intact nuclei isolated from hepatocytes treated with TNF transcribed the γ-GCS-HS gene to a greater extent than control cells, indicating that TNF regulates γ-GCS-HS at the transcriptional level. The capacity to synthesize GSH de novo determined in cell-free extracts incubated with GSH precursors was greater (50–70%) in hepatocytes that were treated with TNF; however, the activity of GSH synthetase remained unaltered by TNF treatment indicating that TNF selectively increased the activity of γ-GCS. Despite activation of nuclear factor-κB (NF-κB) by TNF, this transcription factor was not required for TNF-induced transcription of γ-GCS-HS as revealed by deletion constructs of the γ-GCS-HS promoter subcloned in a chloramphenicol acetyltransferase reporter vector and transfected into HepG2 cells. In contrast, a construct containing AP-1 like/metal response regulatory elements increased chloramphenicol acetyltransferase activity upon exposure to TNF. Thus, TNF increases hepatocellular GSH levels by transcriptional regulation of γ-GCS-HS gene, probably through AP-1/metal response element-like binding site(s) in its promoter, which may constitute a protective mechanism in the control of oxidative stress induced by inflammatory cytokines. Tumor necrosis factor-α (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; BSO, buthionine-l-sulfoximine; CAT, chloramphenicol acetyltransferase; DCFDA, 2′,7′-dichlorofluorescin diacetate; DCF, dichlorofluorescein; DHR, dihydrorhodamine; GST, GSHS-transferases; γ-GCS-HS, γ-glutamylcysteine synthetase heavy subunit; HPLC, high pressure liquid chromatography; Mn-SOD, manganese superoxide dismutase; MRE, metal response element; NF-κB, transcription factor κB; PCR, polymerase chain reaction; ROS, reactive oxygen species; bp, base pair(s). 1The abbreviations used are: TNF, tumor necrosis factor; BSO, buthionine-l-sulfoximine; CAT, chloramphenicol acetyltransferase; DCFDA, 2′,7′-dichlorofluorescin diacetate; DCF, dichlorofluorescein; DHR, dihydrorhodamine; GST, GSHS-transferases; γ-GCS-HS, γ-glutamylcysteine synthetase heavy subunit; HPLC, high pressure liquid chromatography; Mn-SOD, manganese superoxide dismutase; MRE, metal response element; NF-κB, transcription factor κB; PCR, polymerase chain reaction; ROS, reactive oxygen species; bp, base pair(s). is a polypeptide that elicits a diversity of cellular reactions, depending upon its concentration and the type of cell where it acts (1Beutler B. Cerami A. Annu. Rev. Biochem. 1988; 57: 505-518Crossref PubMed Scopus (728) Google Scholar, 2Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (999) Google Scholar, 3Fiers W. FEBS Lett. 1991; 285: 199-212Crossref PubMed Scopus (677) Google Scholar). Thus, it has been shown that TNF exerts an important physiological role as a modulator of immune responses by regulating specific genes needed for the host defense against a varied repertoire of agents. TNF appears to play a role in the control of cell cycle as DNA synthesis and cell proliferation increase in cells exposed to TNF, indicating that this cytokine acts as a mitogenic stimuli (4Feingold K. Soued M. Grunfeld C. Biochem. Biophys. Res. Commun. 1988; 153: 576-582Crossref PubMed Scopus (90) Google Scholar). Such regulation of gene expression by TNF is mediated by induction of early responsive genes including c-jun and transcription factors, i.e.NF-κB (5Brach M.A. Gruss H.J. Scott C. Herrmann F. Mol. Cell. Biol. 1993; 13: 4824-4830Crossref Scopus (43) Google Scholar, 6Schutze S. Potthof K. Machleidt T. Berkovic D. Wiegman K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar). Yet, as a proinflammatory cytokine, whose production is increased in a number of stressful and pathological states, TNF promotes cell injury through several mechanisms including the overproduction of ROS (3Fiers W. FEBS Lett. 1991; 285: 199-212Crossref PubMed Scopus (677) Google Scholar, 7Adamson G.H. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Crossref PubMed Scopus (157) Google Scholar, 8Goosens V. Grooten J. Kurt V. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (553) Google Scholar, 9Schulze-Ostholl K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar, 10Schutze S. Machleidt T. Kronke M. Semin. Oncol. 1992; 19: 16-24PubMed Google Scholar).The ability of TNF to kill cells appears to be restricted to tumor and virally infected cells since normal cells are generally insensitive to the toxic effects of TNF. Moreover, cells that normally are insensitive to TNF cytotoxicity can be sensitized by pretreatment with inhibitors of protein and RNA synthesis (11Hill D.B. Schmidt J. Shedlofsky S.I. Cohen D.A. McLain C. Hepatology. 1995; 21: 1114-1119PubMed Google Scholar, 12Wallach D. J. Immunol. 1984; 132: 2464-2469PubMed Google Scholar, 13Pohlman T.H. Harlan J.M. Cell. Immunol. 1989; 119: 41-52Crossref PubMed Scopus (87) Google Scholar). Conversely, it has been shown that sensitive cells can be made resistant to TNF challenge by prior exposure to a sublethal dose of TNF. These findings imply that TNF leads to the induction of genes that confer protective effects on cells. Several protective genes induced by TNF have been reported including plasminogen activator inhibitor type 2, the zinc finger protein A20, and the Bcl-2 related family member A1 (14Dickinson J.L. Bates E.J. Ferrante A. Antalis T.M. J. Biol. Chem. 1995; 270: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 15Kumar S. Baglioni C. J. Biol. Chem. 1991; 266: 20960-20964Abstract Full Text PDF PubMed Google Scholar, 16Opipari A.W. Hu H.M. Yabkowitz R. Dixit V.M. J. Biol. Chem. 1992; 267: 12424-12427Abstract Full Text PDF PubMed Google Scholar, 17Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar).The molecular basis of the cytotoxic action of TNF is not fully understood at present; however, one of the possible mechanisms involved in the toxicity elicited by TNF includes the generation of ROS that may damage critical cellular components such as proteins, lipids, and DNA causing cell injury (7Adamson G.H. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Crossref PubMed Scopus (157) Google Scholar, 8Goosens V. Grooten J. Kurt V. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (553) Google Scholar, 9Schulze-Ostholl K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar). Consistent with the involvement of ROS in mediating the TNF-induced injury, cellular antioxidants attenuate the damaging effects of free radicals, and hence, these compounds may modulate the sensitivity of cells against TNF toxicity (18Wong G.H. Goeddel D.V. Science. 1988; 242: 941-944Crossref PubMed Scopus (833) Google Scholar, 19Wong G.H. Elwell J. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (762) Google Scholar, 20Zimmerman R.J. Marafino B.J. Chan A. Landra P. Winkelhake J.L. J. Immunol. 1989; 142: 1405-1409PubMed Google Scholar). For instance, previous studies indicated that the antioxidant enzyme Mn-SOD determined sensitivity to TNF-induced cell death as TNF treatment up-regulated Mn-SOD gene expression; furthermore, overexpression of Mn-SOD conferred resistance to TNF toxicity in a human kidney embryonal cell line (18Wong G.H. Goeddel D.V. Science. 1988; 242: 941-944Crossref PubMed Scopus (833) Google Scholar, 19Wong G.H. Elwell J. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (762) Google Scholar). However, such a protective role of Mn-SOD may be restricted to specific cell types since studies in hepatocytes revealed that the TNF-induced expression of Mn-SOD was detected only at the mRNA level without increase in the protein level or enzyme activity (21Czaja M.J. Schizky M.L. Xu Y. Schmiedeberg P. Compton A. Ridnour L. Oberley L.W. Am. J. Physiol. 1994; 266: G737-G744PubMed Google Scholar).GSH, the most abundant antioxidant in cells, plays a prominent role in the defense against oxidative stress-induced cell injury. Thus, the GSH redox cycle, where reduced GSH is cofactor of GSH peroxidase, and GST downplay the consequences of a broad range of reactive species (22Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5928) Google Scholar, 23Fernández-Checa J.C. Kaplowitz N. Garcı́a-Ruiz C. Colell A. Marı́ M. Miranda M. Ardite E. Morales A. Am. J. Physiol. 1997; 273: G7-G17Crossref PubMed Google Scholar, 24Garcı́a-Ruiz C. Colell A. Marı́ M. Morales A. Fernández-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). GSH is synthesized from its constituent amino acids in two sequential enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS) and GSH synthetase. The reaction catalyzed by γ-GCS is the rate-limiting step in de novo GSH synthesis; γ-GCS is inhibited by GSH through a feedback mechanism (22Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5928) Google Scholar). Previous studies have shown that manipulation of GSH levels prior to exposure to TNF modulates the cytotoxicity of the cytokine in different cell types (7Adamson G.H. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Crossref PubMed Scopus (157) Google Scholar, 8Goosens V. Grooten J. Kurt V. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (553) Google Scholar). GSH depletion induced by inhibition of GSH reductase with 1,3-bis(chloroethyl)-1-nitrosourea or by incubation with BSO, a specific inhibitor of γ-GCS, prior to the exposure of cells to TNF, results in an increased susceptibility of hepatocytes and fibrosarcoma cells to the cytotoxic effects of TNF (7Adamson G.H. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Crossref PubMed Scopus (157) Google Scholar, 8Goosens V. Grooten J. Kurt V. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (553) Google Scholar). Furthermore, its lethality can be ameliorated by N-acetylcysteine, a GSH precursor, which replenishes cellular GSH by providing intracellular cysteine (20Zimmerman R.J. Marafino B.J. Chan A. Landra P. Winkelhake J.L. J. Immunol. 1989; 142: 1405-1409PubMed Google Scholar).Given the importance of GSH in protecting against the oxidative stress elicited by TNF, the purpose of our work was to examine the regulation of cellular GSH, expression of γ-GCS-HS, and control of intracellular generation of ROS in cultured rat hepatocytes treated with TNF. Our results demonstrate that TNF increases cellular GSH levels, mediated by transcriptional regulation of the γ-GCS-HS gene, which attenuates the generation of hydrogen peroxide and lipid peroxidation. Our findings imply that the up-regulation of cellular GSH may represent an additional protective mechanism to control the consequences of oxidative stress induced by inflammatory cytokines. Tumor necrosis factor-α (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; BSO, buthionine-l-sulfoximine; CAT, chloramphenicol acetyltransferase; DCFDA, 2′,7′-dichlorofluorescin diacetate; DCF, dichlorofluorescein; DHR, dihydrorhodamine; GST, GSHS-transferases; γ-GCS-HS, γ-glutamylcysteine synthetase heavy subunit; HPLC, high pressure liquid chromatography; Mn-SOD, manganese superoxide dismutase; MRE, metal response element; NF-κB, transcription factor κB; PCR, polymerase chain reaction; ROS, reactive oxygen species; bp, base pair(s). 1The abbreviations used are: TNF, tumor necrosis factor; BSO, buthionine-l-sulfoximine; CAT, chloramphenicol acetyltransferase; DCFDA, 2′,7′-dichlorofluorescin diacetate; DCF, dichlorofluorescein; DHR, dihydrorhodamine; GST, GSHS-transferases; γ-GCS-HS, γ-glutamylcysteine synthetase heavy subunit; HPLC, high pressure liquid chromatography; Mn-SOD, manganese superoxide dismutase; MRE, metal response element; NF-κB, transcription factor κB; PCR, polymerase chain reaction; ROS, reactive oxygen species; bp, base pair(s). is a polypeptide that elicits a diversity of cellular reactions, depending upon its concentration and the type of cell where it acts (1Beutler B. Cerami A. Annu. Rev. Biochem. 1988; 57: 505-518Crossref PubMed Scopus (728) Google Scholar, 2Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (999) Google Scholar, 3Fiers W. FEBS Lett. 1991; 285: 199-212Crossref PubMed Scopus (677) Google Scholar). Thus, it has been shown that TNF exerts an important physiological role as a modulator of immune responses by regulating specific genes needed for the host defense against a varied repertoire of agents. TNF appears to play a role in the control of cell cycle as DNA synthesis and cell proliferation increase in cells exposed to TNF, indicating that this cytokine acts as a mitogenic stimuli (4Feingold K. Soued M. Grunfeld C. Biochem. Biophys. Res. Commun. 1988; 153: 576-582Crossref PubMed Scopus (90) Google Scholar). Such regulation of gene expression by TNF is mediated by induction of early responsive genes including c-jun and transcription factors, i.e.NF-κB (5Brach M.A. Gruss H.J. Scott C. Herrmann F. Mol. Cell. Biol. 1993; 13: 4824-4830Crossref Scopus (43) Google Scholar, 6Schutze S. Potthof K. Machleidt T. Berkovic D. Wiegman K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar). Yet, as a proinflammatory cytokine, whose production is increased in a number of stressful and pathological states, TNF promotes cell injury through several mechanisms including the overproduction of ROS (3Fiers W. FEBS Lett. 1991; 285: 199-212Crossref PubMed Scopus (677) Google Scholar, 7Adamson G.H. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Crossref PubMed Scopus (157) Google Scholar, 8Goosens V. Grooten J. Kurt V. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (553) Google Scholar, 9Schulze-Ostholl K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar, 10Schutze S. Machleidt T. Kronke M. Semin. Oncol. 1992; 19: 16-24PubMed Google Scholar). The ability of TNF to kill cells appears to be restricted to tumor and virally infected cells since normal cells are generally insensitive to the toxic effects of TNF. Moreover, cells that normally are insensitive to TNF cytotoxicity can be sensitized by pretreatment with inhibitors of protein and RNA synthesis (11Hill D.B. Schmidt J. Shedlofsky S.I. Cohen D.A. McLain C. Hepatology. 1995; 21: 1114-1119PubMed Google Scholar, 12Wallach D. J. Immunol. 1984; 132: 2464-2469PubMed Google Scholar, 13Pohlman T.H. Harlan J.M. Cell. Immunol. 1989; 119: 41-52Crossref PubMed Scopus (87) Google Scholar). Conversely, it has been shown that sensitive cells can be made resistant to TNF challenge by prior exposure to a sublethal dose of TNF. These findings imply that TNF leads to the induction of genes that confer protective effects on cells. Several protective genes induced by TNF have been reported including plasminogen activator inhibitor type 2, the zinc finger protein A20, and the Bcl-2 related family member A1 (14Dickinson J.L. Bates E.J. Ferrante A. Antalis T.M. J. Biol. Chem. 1995; 270: 27894-27904Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 15Kumar S. Baglioni C. J. Biol. Chem. 1991; 266: 20960-20964Abstract Full Text PDF PubMed Google Scholar, 16Opipari A.W. Hu H.M. Yabkowitz R. Dixit V.M. J. Biol. Chem. 1992; 267: 12424-12427Abstract Full Text PDF PubMed Google Scholar, 17Karsan A. Yee E. Harlan J.M. J. Biol. Chem. 1996; 271: 27201-27204Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The molecular basis of the cytotoxic action of TNF is not fully understood at present; however, one of the possible mechanisms involved in the toxicity elicited by TNF includes the generation of ROS that may damage critical cellular components such as proteins, lipids, and DNA causing cell injury (7Adamson G.H. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Crossref PubMed Scopus (157) Google Scholar, 8Goosens V. Grooten J. Kurt V. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (553) Google Scholar, 9Schulze-Ostholl K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar). Consistent with the involvement of ROS in mediating the TNF-induced injury, cellular antioxidants attenuate the damaging effects of free radicals, and hence, these compounds may modulate the sensitivity of cells against TNF toxicity (18Wong G.H. Goeddel D.V. Science. 1988; 242: 941-944Crossref PubMed Scopus (833) Google Scholar, 19Wong G.H. Elwell J. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (762) Google Scholar, 20Zimmerman R.J. Marafino B.J. Chan A. Landra P. Winkelhake J.L. J. Immunol. 1989; 142: 1405-1409PubMed Google Scholar). For instance, previous studies indicated that the antioxidant enzyme Mn-SOD determined sensitivity to TNF-induced cell death as TNF treatment up-regulated Mn-SOD gene expression; furthermore, overexpression of Mn-SOD conferred resistance to TNF toxicity in a human kidney embryonal cell line (18Wong G.H. Goeddel D.V. Science. 1988; 242: 941-944Crossref PubMed Scopus (833) Google Scholar, 19Wong G.H. Elwell J. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (762) Google Scholar). However, such a protective role of Mn-SOD may be restricted to specific cell types since studies in hepatocytes revealed that the TNF-induced expression of Mn-SOD was detected only at the mRNA level without increase in the protein level or enzyme activity (21Czaja M.J. Schizky M.L. Xu Y. Schmiedeberg P. Compton A. Ridnour L. Oberley L.W. Am. J. Physiol. 1994; 266: G737-G744PubMed Google Scholar). GSH, the most abundant antioxidant in cells, plays a prominent role in the defense against oxidative stress-induced cell injury. Thus, the GSH redox cycle, where reduced GSH is cofactor of GSH peroxidase, and GST downplay the consequences of a broad range of reactive species (22Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5928) Google Scholar, 23Fernández-Checa J.C. Kaplowitz N. Garcı́a-Ruiz C. Colell A. Marı́ M. Miranda M. Ardite E. Morales A. Am. J. Physiol. 1997; 273: G7-G17Crossref PubMed Google Scholar, 24Garcı́a-Ruiz C. Colell A. Marı́ M. Morales A. Fernández-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). GSH is synthesized from its constituent amino acids in two sequential enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS) and GSH synthetase. The reaction catalyzed by γ-GCS is the rate-limiting step in de novo GSH synthesis; γ-GCS is inhibited by GSH through a feedback mechanism (22Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5928) Google Scholar). Previous studies have shown that manipulation of GSH levels prior to exposure to TNF modulates the cytotoxicity of the cytokine in different cell types (7Adamson G.H. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Crossref PubMed Scopus (157) Google Scholar, 8Goosens V. Grooten J. Kurt V. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (553) Google Scholar). GSH depletion induced by inhibition of GSH reductase with 1,3-bis(chloroethyl)-1-nitrosourea or by incubation with BSO, a specific inhibitor of γ-GCS, prior to the exposure of cells to TNF, results in an increased susceptibility of hepatocytes and fibrosarcoma cells to the cytotoxic effects of TNF (7Adamson G.H. Billings R.E. Arch. Biochem. Biophys. 1992; 294: 223-229Crossref PubMed Scopus (157) Google Scholar, 8Goosens V. Grooten J. Kurt V. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (553) Google Scholar). Furthermore, its lethality can be ameliorated by N-acetylcysteine, a GSH precursor, which replenishes cellular GSH by providing intracellular cysteine (20Zimmerman R.J. Marafino B.J. Chan A. Landra P. Winkelhake J.L. J. Immunol. 1989; 142: 1405-1409PubMed Google Scholar). Given the importance of GSH in protecting against the oxidative stress elicited by TNF, the purpose of our work was to examine the regulation of cellular GSH, expression of γ-GCS-HS, and control of intracellular generation of ROS in cultured rat hepatocytes treated with TNF. Our results demonstrate that TNF increases cellular GSH levels, mediated by transcriptional regulation of the γ-GCS-HS gene, which attenuates the generation of hydrogen peroxide and lipid peroxidation. Our findings imply that the up-regulation of cellular GSH may represent an additional protective mechanism to control the consequences of oxidative stress induced by inflammatory cytokines.