Depletion of Hepatic Glutathione Prevents Death Receptor-Dependent Apoptotic and Necrotic Liver Injury in Mice
2000; Elsevier BV; Volume: 156; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65076-6
ISSN1525-2191
AutoresHannes Hentze, Florian Gantner, Stefan Kolb, Albrecht Wendel,
Tópico(s)Cell death mechanisms and regulation
ResumoThe activation of the death receptors, tumor necrosis factor-receptor-1 (TNF-R1) or CD95, is a hallmark of inflammatory or viral liver disease. In different murine in vivo models, we found that livers depleted of γ-glutamyl-cysteinyl-glycine (GSH) by endogenous enzymatic conjugation after phorone treatment were resistant against death receptor-elicited injury as assessed by transaminase release and histopathology. In apoptotic models initiated by engagement of CD95, or by injection of TNF or lipopolysaccharide into galactosamine-sensitized mice, hepatic caspase-3-like proteases were not activated in the GSH-depleted state. Under GSH depletion, also caspase-independent, TNF-R1-mediated injury (high-dose actinomycin D or α-amanitin), as well as necrotic hepatotoxicity (high-dose lipopolysaccharide) were entirely blocked. In the T-cell-dependent model of concanavalin A-induced hepatotoxicity, GSH depletion resulted in a suppression of interferon-γ release, delay of systemic TNF release, hepatic nuclear factor-κB activation, and an abrogation of sinusoidal endothelial cell detachment as assessed by electron microscopy. When GSH depletion was initiated 3 hours after concanavalin A injection, ie, after the peak of early pro-inflammatory cytokines, livers were still protected. We conclude that sufficient hepatic GSH levels are a prerequisite for the execution of death receptor-mediated hepatocyte demise. The activation of the death receptors, tumor necrosis factor-receptor-1 (TNF-R1) or CD95, is a hallmark of inflammatory or viral liver disease. In different murine in vivo models, we found that livers depleted of γ-glutamyl-cysteinyl-glycine (GSH) by endogenous enzymatic conjugation after phorone treatment were resistant against death receptor-elicited injury as assessed by transaminase release and histopathology. In apoptotic models initiated by engagement of CD95, or by injection of TNF or lipopolysaccharide into galactosamine-sensitized mice, hepatic caspase-3-like proteases were not activated in the GSH-depleted state. Under GSH depletion, also caspase-independent, TNF-R1-mediated injury (high-dose actinomycin D or α-amanitin), as well as necrotic hepatotoxicity (high-dose lipopolysaccharide) were entirely blocked. In the T-cell-dependent model of concanavalin A-induced hepatotoxicity, GSH depletion resulted in a suppression of interferon-γ release, delay of systemic TNF release, hepatic nuclear factor-κB activation, and an abrogation of sinusoidal endothelial cell detachment as assessed by electron microscopy. When GSH depletion was initiated 3 hours after concanavalin A injection, ie, after the peak of early pro-inflammatory cytokines, livers were still protected. We conclude that sufficient hepatic GSH levels are a prerequisite for the execution of death receptor-mediated hepatocyte demise. Apoptosis is an active and highly regulated form of cell death responsible for the cellular default demise of the hepatocyte. This process is thus in charge of tissue homeostasis and maintenance of vital function of the liver.1Galle PR Hofmann WJ Walczak H Schaller H Otto G Stremmel W Krammer PH Runke LL Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage.J Exp Med. 1995; 182: 1223-1230Crossref PubMed Scopus (680) Google Scholar, 2Leist M Gantner F Künstle G Wendel A Cytokine-mediated hepatic apoptosis.Rev Physiol Biochem Pharmacol. 1998; 133: 109-155PubMed Google Scholar, 3Patel T Roberts LR Jones BA Gores GJ Dysregulation of apoptosis as a mechanism of liver disease: an overview.Semin Liver Dis. 1998; 18: 105-114Crossref PubMed Scopus (151) Google Scholar Accordingly, a dysregulation of apoptosis might underlay several pathophysiological disturbances of the liver, eg, hepatitis of viral or autoimmune origin, alcoholic hepatitis, acute hyperinflammatory liver failure, primary biliary cirrhosis, and toxic liver injury.3Patel T Roberts LR Jones BA Gores GJ Dysregulation of apoptosis as a mechanism of liver disease: an overview.Semin Liver Dis. 1998; 18: 105-114Crossref PubMed Scopus (151) Google Scholar, 4Valente M Calabrese F Liver and apoptosis.Ital J Gastroenterol Hepatol. 1999; 31: 73-77PubMed Google Scholar In these cases, a causal involvement of cytokines such as tumor necrosis factor (TNF) or CD95 ligand (CD95L) in the initiation of hepatocyte cell death has been described.1Galle PR Hofmann WJ Walczak H Schaller H Otto G Stremmel W Krammer PH Runke LL Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage.J Exp Med. 1995; 182: 1223-1230Crossref PubMed Scopus (680) Google Scholar, 2Leist M Gantner F Künstle G Wendel A Cytokine-mediated hepatic apoptosis.Rev Physiol Biochem Pharmacol. 1998; 133: 109-155PubMed Google Scholar, 5Galle PR Apoptosis in liver disease.J Hepatol. 1997; 27: 405-412Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 6Bradham CA Plumpe J Manns MP Brenner DA Trautwein C Mechanisms of hepatic toxicity I. TNF-induced liver injury.Am J Physiol. 1998; 275: G387-G392PubMed Google Scholar A better understanding of the detailed modulation of cytokine-mediated destruction of hepatic tissue is therefore the basis for future therapeutic intervention strategies in liver disease. The tripeptide glutathione (GSH, γ-glutamyl-cysteinyl-glycine) represents the major intracellular nonprotein thiol. GSH has a central role in sulfhydryl homeostasis, serves as the major cytosolic antioxidant, and provides defense against xenobiotics as a phase II conjugate substrate.7Jones TW Thor H Orrenius S Cellular defense mechanisms against toxic substances.Arch Toxicol Suppl. 1986; 9: 259-271Crossref PubMed Google Scholar, 8Meister A On the antioxidant effects of ascorbic acid and glutathione.Biochem Pharmacol. 1992; 44: 1905-1915Crossref PubMed Scopus (340) Google Scholar Numerous central cellular functions are controlled by the GSH/glutathione disulfide system, eg, key enzymes of metabolism, cell growth, gene transcription, and apoptosis.9Dröge W Schulze-Osthoff K Mihm S Galter D Schenk H Eck HP Roth S Gmünder H Functions of glutathione and glutathione disulfide in immunology and immunopathology.FASEB J. 1994; 8: 1131-1138Crossref PubMed Scopus (424) Google Scholar, 10Uhlig S Wendel A The physiological consequences of glutathione variations.Life Sci. 1992; 51: 1083-1094Crossref PubMed Scopus (182) Google Scholar, 11Hall AG The role of glutathione in the regulation of apoptosis.Eur J Clin Invest. 1999; 29: 238-245Crossref PubMed Scopus (302) Google Scholar Cells therefore tightly regulate synthesis, utilization, and export of glutathione. Its intracellular concentrations are maintained within the millimolar range under normal conditions.12Anderson ME Glutathione and glutathione delivery compounds.Adv Pharmacol. 1997; 18: 65-78Google Scholar GSH is synthesized by the consecutive ATP-dependent enzymes, γ-glutamylcysteine-synthase and GSH synthase, and glutathione is primarily maintained in its reduced form by glutathione disulfide reductase using NADPH as cosubstrate.12Anderson ME Glutathione and glutathione delivery compounds.Adv Pharmacol. 1997; 18: 65-78Google Scholar The total intracellular GSH concentration varies considerably, especially in the liver, and hepatic GSH can dramatically decrease as a result of drug metabolism,13Mitchell JR Jollow DJ Potter WZ Gillette JR Brodie BB Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione.J Pharmacol Exp Ther. 1973; 187: 211-217PubMed Google Scholar after oxidative stress,10Uhlig S Wendel A The physiological consequences of glutathione variations.Life Sci. 1992; 51: 1083-1094Crossref PubMed Scopus (182) Google Scholar or because of inherited deficiencies in GSH synthesis.10Uhlig S Wendel A The physiological consequences of glutathione variations.Life Sci. 1992; 51: 1083-1094Crossref PubMed Scopus (182) Google Scholar Thus, low GSH levels are observed during sepsis, acetaminophen intoxication, chronic alcohol consumption, and in acute Wilson's disease.10Uhlig S Wendel A The physiological consequences of glutathione variations.Life Sci. 1992; 51: 1083-1094Crossref PubMed Scopus (182) Google Scholar, 13Mitchell JR Jollow DJ Potter WZ Gillette JR Brodie BB Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione.J Pharmacol Exp Ther. 1973; 187: 211-217PubMed Google Scholar, 14Keller GA Barke R Harty JT Humphrey E Simmons RL Decreased hepatic glutathione levels in septic shock. Predisposition of hepatocytes to oxidative stress: an experimental approach.Arch Surg. 1985; 120: 941-945Crossref PubMed Scopus (87) Google Scholar, 15Summer KH Eisenburg J Low content of hepatic reduced glutathione in patients with Wilson's disease.Biochem Med. 1985; 34: 107-111Crossref PubMed Scopus (40) Google Scholar, 16Lieber CS Hepatic and metabolic effects of ethanol: pathogenesis and prevention.Ann Med. 1994; 26: 325-330Crossref PubMed Scopus (76) Google Scholar, 17Videla LA Valenzuela A Alcohol ingestion, liver glutathione and lipoperoxidation: metabolic interrelations and pathological implications.Life Sci. 1982; 31: 2395-2407Crossref PubMed Scopus (219) Google Scholar Moreover, hepatic GSH is subject to pronounced circadian alterations.18Jaeschke H Wendel A Diurnal fluctuation and pharmacological alteration of mouse organ glutathione content.Biochem Pharmacol. 1985; 34: 1029-1033Crossref PubMed Scopus (98) Google Scholar A common experimental approach to create a sustained GSH-deficiency is the inhibition of glutathione synthesis by buthionine-sulfoximine, which affects all organs and requires repetitive treatment of animals.12Anderson ME Glutathione and glutathione delivery compounds.Adv Pharmacol. 1997; 18: 65-78Google Scholar In contrast, α,β-unsaturated carbonyl compounds such as diethyl maleate or phorone preferentially deplete hepatic GSH via enzymatic conjugation by GSH-transferases, followed by biliary excretion of these conjugates.19Plummer JL Smith BR Sies H Bend JR Chemical depletion of glutathione in vivo.Methods Enzymol. 1981; 77: 50-59Crossref PubMed Scopus (247) Google Scholar GSH-depleting compounds do not induce redox-stress in the liver per se.20Mehmetcik G Alptekin N Toker G Uysal M Mitochondrial lipid peroxides and antioxidant enzymes in the liver following phorone-induced glutathione depletion.Res Commun Mol Pathol Pharmacol. 1997; 96: 353-356PubMed Google Scholar However, depletion of GSH predisposes cells to oxidative injury, with the consequence that the liver toxicity of many xenobiotics in man and in animal models is greatly enhanced under this condition. Vice versa, a pharmacological enhancement of hepatic GSH renders the liver less vulnerable and protects against many direct hepatotoxins.7Jones TW Thor H Orrenius S Cellular defense mechanisms against toxic substances.Arch Toxicol Suppl. 1986; 9: 259-271Crossref PubMed Google Scholar, 21Prescott LF Glutathione: a protective mechanism against hepatotoxicity.Biochem Soc Trans. 1982; 10: 84-85PubMed Google Scholar, 22Wendel A Tiegs G Manipulation of liver glutathione status—a double-edged sword.in: Vina J Glutathione: Metabolism and Physiological Functions. CRC Press, Boca Raton1989: 21-28Google Scholar Increasing evidence argues for a dichotomal role of GSH with respect to cellular damage. In some paradigms of cell death where the primary event is apoptosis, a protective (ie, anti-apoptotic), and not an aggravating, effect of GSH depletion was reported. To date, NO-induced apoptosis of macrophages,23Boggs SE McCormick TS Lapetina EG Glutathione levels determine apoptosis in macrophages.Biochem Biophys Res Commun. 1998; 247: 229-233Crossref PubMed Scopus (68) Google Scholar CD95-mediated apoptosis of T cells,24Hampton MB Orrenius S Redox regulation of apoptotic cell death.Biofactors. 1998; 8: 1-5Crossref PubMed Scopus (108) Google Scholar and cytokine-mediated hepatocyte apoptosis in vivo25Hentze H Künstle G Volbracht C Ertel W Wendel A CD95-mediated murine hepatic apoptosis requires an intact glutathione status.Hepatology. 1999; 30: 177-185Crossref PubMed Scopus (61) Google Scholar, 26Jones JJ Fan J Nathens AB Kapus A Shekhman M Marshall JC Parodo J Rotstein OD Redox manipulation using the thiol-oxidizing agent diethyl maleate prevents hepatocellular necrosis and apoptosis in a rodent endotoxemia model.Hepatology. 1999; 30: 714-724Crossref PubMed Scopus (33) Google Scholar were found to depend on a sufficient intracellular GSH level of the respective cells. In these studies, the redox sensitivity of apoptosis-executing caspases, ie, aspartate-specific cysteine proteases,27Thornberry NA Lazebnik Y Caspases: enemies within.Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6222) Google Scholar was hypothesized to be responsible for the observed protection because of decreased GSH levels. However, elevated intracellular GSH levels can also abrogate apoptosis in various cell lines, and GSH export has been reported to be an integral part of death receptor-mediated apoptosis in some experimental systems.11Hall AG The role of glutathione in the regulation of apoptosis.Eur J Clin Invest. 1999; 29: 238-245Crossref PubMed Scopus (302) Google Scholar, 28van den Dobbelsteen DJ Nobel CSI Schlegel J Cotgreave IA Orrenius S Slater FG Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody.J Biol Chem. 1996; 271: 15420-15427Crossref PubMed Scopus (333) Google Scholar, 29Manna SK Kuo MT Aggarwal BB Overexpression of gamma-glutamylcysteine synthetase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappa B and activator protein-1.Oncogene. 1999; 18: 4371-4382Crossref PubMed Scopus (143) Google Scholar Concerning in vivo studies, protection by GSH depletion was reported in models of apoptotic, caspase-dependent liver injury.25Hentze H Künstle G Volbracht C Ertel W Wendel A CD95-mediated murine hepatic apoptosis requires an intact glutathione status.Hepatology. 1999; 30: 177-185Crossref PubMed Scopus (61) Google Scholar, 26Jones JJ Fan J Nathens AB Kapus A Shekhman M Marshall JC Parodo J Rotstein OD Redox manipulation using the thiol-oxidizing agent diethyl maleate prevents hepatocellular necrosis and apoptosis in a rodent endotoxemia model.Hepatology. 1999; 30: 714-724Crossref PubMed Scopus (33) Google Scholar On the contrary, in animal models of primary necrotic hepatotoxicity, this condition always resulted in an enhanced toxicity.7Jones TW Thor H Orrenius S Cellular defense mechanisms against toxic substances.Arch Toxicol Suppl. 1986; 9: 259-271Crossref PubMed Google Scholar, 22Wendel A Tiegs G Manipulation of liver glutathione status—a double-edged sword.in: Vina J Glutathione: Metabolism and Physiological Functions. CRC Press, Boca Raton1989: 21-28Google Scholar It seemed therefore appropriate to comparatively study the influence of GSH depletion on apoptotic and necrotic liver injury models. As a common feature, they all depend on the activation of death receptors, ie, either tumor necrosis factor-receptor-1 (TNF-R1) or CD95.6Bradham CA Plumpe J Manns MP Brenner DA Trautwein C Mechanisms of hepatic toxicity I. TNF-induced liver injury.Am J Physiol. 1998; 275: G387-G392PubMed Google Scholar, 30Leist M Gantner F Künstle G Bohlinger I Tiegs G Bluethmann H Wendel A The 55-kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure.Mol Med. 1996; 2: 109-124Crossref PubMed Google Scholar, 31Schulze-Osthoff K Ferrari D Los M Wesselborg S Peter ME Apoptosis signaling by death receptors.Eur J Biochem. 1998; 254: 439-459Crossref PubMed Scopus (870) Google Scholar Therefore, the following models of acute inflammatory liver injury were studied: 1) in galactosamine-sensitized mice, injection of recombinant TNF (GalN/TNF), or low-dose lipopolysaccharide (GalN/LPS), or injection of activating anti-CD95 antibody (αCD95) in naive mice. These regimens commonly induce hepatocyte apoptosis via the activation of caspases.2Leist M Gantner F Künstle G Wendel A Cytokine-mediated hepatic apoptosis.Rev Physiol Biochem Pharmacol. 1998; 133: 109-155PubMed Google Scholar, 6Bradham CA Plumpe J Manns MP Brenner DA Trautwein C Mechanisms of hepatic toxicity I. TNF-induced liver injury.Am J Physiol. 1998; 275: G387-G392PubMed Google Scholar, 30Leist M Gantner F Künstle G Bohlinger I Tiegs G Bluethmann H Wendel A The 55-kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure.Mol Med. 1996; 2: 109-124Crossref PubMed Google Scholar, 32Leist M Gantner F Bohlinger I Tiegs G Germann PG Wendel A Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models.Am J Pathol. 1995; 146: 1220-1234PubMed Google Scholar, 33Mignon A Rouquet N Fabre M Martin S Pages JC Dhainaut JF Kahn A Briand P Joulin V LPS challenge in D-galactosamine-sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock.Am J Respir Crit Care Med. 1999; 159: 1308-1315Crossref PubMed Scopus (157) Google Scholar 2) High-dose treatment of naive mice with two hepatotoxins of fungal origin, actinomycin-D (ActD) or α-amanitin, ie, with inhibitors of transcription that strongly sensitize hepatocytes toward endogenously produced TNF and induce hepatocyte apoptosis.34Leist M Gantner F Naumann H Bluethmann H Vogt K Brigelius-Flohe R Nicotera P Volk HD Wendel A Tumor necrosis factor-induced apoptosis during the poisoning of mice with hepatotoxins.Gastroenterology. 1997; 112: 923-934Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar 3) Injection of naive mice with concanavalin A (Con A), a plant lectin that activates T cells and thereby induces selective liver injury. In this model, necrotic and apoptotic hepatocyte demise without caspase activation was described.6Bradham CA Plumpe J Manns MP Brenner DA Trautwein C Mechanisms of hepatic toxicity I. TNF-induced liver injury.Am J Physiol. 1998; 275: G387-G392PubMed Google Scholar, 35Tiegs G Hentschel J Wendel A A T cell-dependent experimental liver injury in mice inducible by concanavalin A.J Clin Invest. 1992; 90: 196-203Crossref PubMed Scopus (991) Google Scholar, 36Gantner F Leist M Lohse AW Germann PG Tiegs G Concanavalin A-induced T-cell-mediated hepatic injury in mice: the role of tumor necrosis factor.Hepatology. 1995; 21: 190-198PubMed Google Scholar, 37Künstle G Hentze H Germann PG Tiegs G Meergans T Wendel A Concanavalin A hepatotoxicity in mice: TNF-mediated organ failure independent of caspase-3-like protease activation.Hepatology. 1999; 30: 1241-1251Crossref PubMed Scopus (92) Google Scholar 4) Injection of high-dose LPS (endotoxic shock model). Here, the mode of hepatocyte cell death is regarded to be necrotic, despite its dependence on TNF.33Mignon A Rouquet N Fabre M Martin S Pages JC Dhainaut JF Kahn A Briand P Joulin V LPS challenge in D-galactosamine-sensitized mice accounts for caspase-dependent fulminant hepatitis, not for septic shock.Am J Respir Crit Care Med. 1999; 159: 1308-1315Crossref PubMed Scopus (157) Google Scholar, 38Bohlinger I Leist M Gantner F Angermüller S Tiegs G Wendel A DNA fragmentation in mouse organs during endotoxic shock.Am J Pathol. 1996; 149: 1381-1393PubMed Google Scholar We compared these models with regard to their GSH dependence, the mode of cell death, and the activation of caspases. In the LPS shock models and the Con A models, we also examined the possible role of immunosuppression because cellular GSH levels are known to influence the immune response.9Dröge W Schulze-Osthoff K Mihm S Galter D Schenk H Eck HP Roth S Gmünder H Functions of glutathione and glutathione disulfide in immunology and immunopathology.FASEB J. 1994; 8: 1131-1138Crossref PubMed Scopus (424) Google Scholar, 39Gmünder H Dröge W Differential effects of glutathione depletion on T cell subsets.Cell Immunol. 1991; 138: 229-237Crossref PubMed Scopus (71) Google Scholar, 40Robinson MK Rodrick ML Jacobs DO Rounds JD Collins KH Saporoschetz IB Mannick JA Wilmore DW Glutathione depletion in rats impairs T-cell and macrophage immune function.Arch Surg. 1993; 128: 29-34Crossref PubMed Scopus (74) Google Scholar When GSH was depleted, the onset of liver injury was blocked in all models investigated at the target cell level, ie, the hepatocyte. Additionally, we found that in the Con A model, the structure of sinusoidal endothelial cells was preserved in the GSH-depleted state. Phorone was obtained from Aldrich (Steinheim, Germany), benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (z-VAD-fmk) from Bachem (Heidelberg, Germany), N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (DEVD-afc), Pefablock from Biomol (Hamburg, Germany), galactosamine (GalN) from Roth (Karlsruhe, Germany), 1-cis-chloro-2,4-dinitrobenzene and Epon from Fluka (Buchs, Switzerland), LPS (Salmonella abortus equi) from Metalon (Wusterhausen, Germany), and acetaminophen from EGA (Steinheim, Germany). Interferon-γ (IFN-γ) and recombinant murine TNF were kindly provided by Dr. G. A. Adolf (Bender & Co., Vienna, Austria). All other reagents and recombinant enzymes not further specified were purchased from Sigma (Deisenhofen, Germany). Specific pathogen-free male BALB/c mice (∼25 g, from the in-house animal breeding station of the University of Konstanz) were maintained under controlled conditions (22°C and 55% humidity, constant day/night cycle of 12 hours) and fed a standard laboratory chow. All animals received humane care in concordance with the National Institutes of Health guidelines as well as with the legal requirements in Germany. Mice were starved overnight before the onset of experiments, which generally started at 8 am. Phorone (250 mg/kg) and 1-cis-chloro-2,4-dinitrobenzene (100 mg/kg) were dissolved in 300 μl of vegetable oil and injected intraperitoneally, either before challenge with GalN/TNF, GalN/LPS, acetaminophen, αCD95, or Con A, or delayed 1 hour after challenge to avoid interference of the solvent with LPS, ActD, or α-amanitin at the site of injection. l-buthionin-S,R-sulfoximin (buthionine-sulfoximine, 890 mg/kg), LPS (various doses as indicated), galactosamine (GalN, 700 mg/kg), α-amanitin (3 mg/kg), and ActD (2 mg/kg) were administered intraperitoneally in 300 μl of endotoxin-free saline. Activating anti-CD95 antibody (αCD95, 2 μg per animal), recombinant murine TNF (5 to 10 μg/kg), and IFN-γ (50 μg/kg) were injected intravenously in a volume of 300 μl of saline supplemented with 0.1% human serum albumin. Con A (25 or 50 mg/kg) was given intravenously in 300 μl of endotoxin-free saline. At the time points indicated, mice were euthanized by intravenous injection of 150 mg/kg of pentobarbital plus 0.8 mg/kg of heparin. Blood was withdrawn by cardiac puncture and centrifuged (5 minutes, 14,000 × g, 4°C) to obtain plasma, and the extent of liver damage was assessed by measuring plasma alanine aminotransferase activity with an EPOS 5060 analyzer (Netheler & Hinz, Hamburg, Germany. according to the method of Bergmeyer.41Bergmeyer HU Methods of Enzymatic Analysis. vol 3. Verlag Chemie, Weinheim1983: 1-605Google Scholar Blood samples for the cytokine determinations were obtained either from the tail veins using heparinized syringes, or by cardiac puncture as described above, centrifuged (5 minutes, 14,000 × g, 4°C) and stored at −80°C. To determine further organ parameters, livers were perfused for 10 seconds with a cold perfusion buffer containing 50 mmol/L phosphate, 120 mmol/K NaCl, 10 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4, and subsequently excised. A slice of the large anterior lobe was frozen in liquid nitrogen and stored at −80°C until the measurement of caspase-3-like activity or preparation of nuclear extracts for the nuclear factor-κB (NF-κB) electrophoretic mobility gel shift assay, or was freeze-clamped with liquid nitrogen precooled pliers, and stored at −80°C for the determination of total glutathione (GSx) (GSx = GSH + 2 × glutathione disulfide), that was quantified according to the enzymatic cycling method of Tietze as described in detail.25Hentze H Künstle G Volbracht C Ertel W Wendel A CD95-mediated murine hepatic apoptosis requires an intact glutathione status.Hepatology. 1999; 30: 177-185Crossref PubMed Scopus (61) Google Scholar, 42Tietze F Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues.Anal Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5655) Google Scholar For histology, liver specimens were immediately cut into 1-mm-thick slices and fixed in 2.5% glutaraldehyde in phosphate buffer (0.1 mol/L, pH 7.4) for electron microscopy, or in 4% buffered formalin solution for light microscopy. Additionally, spleen cells were isolated by grinding the spleen through a steel grid (pore diameter, 100 μm) into 5 ml of RPMI 1640 medium. To determine GSx, the cells were centrifuged (5 minutes, 14,000 × g, 4°C) and lysed with 1% sulfosalicylic acid. All enzyme-linked immunosorbent assays were performed on flat-bottomed high-binding polystyrene microtiter plates (Greiner, Nürtingen, Germany). Antibody pairs (specific rat anti-murine mAb) were purchased from Pharmingen (San Diego, CA), except for the TNF enzyme-linked immunosorbent assay (capture: polyclonal ovine anti-mouse TNF antibody, in-house preparation, IgG fraction, 20 mg/ml; detection antibody: polyclonal anti-mouse TNF antibody from Endogen, Boston, MA). Streptavidin-peroxidase was obtained from Jackson Immuno Research (West Grove, PA), and the TMB liquid substrate system was from Sigma (Deisenhofen, Germany). Interleukin (IL)-1β was determined using a commercially available enzyme-linked immunosorbent assay kit (Endogen). The detection limits of the assays were 10 pg/ml for TNF and IFN-γ, 30 pg/ml for IL-2, 10 pg/ml for IL-4, and 15 pg/ml for IL-1β. Cytosolic extracts from liver tissue were prepared by Dounce homogenization in hypotonic extraction buffer (25 mmol/L HEPES, pH 7.5; 5 mmol/L MgCl2; 1 mmol/L EGTA; 1 mmol/L Pefablock; and pepstatin, leupeptin, and aprotinin, 1 μg/ml each), subsequently centrifuged (15 minutes, 14,000 × g, 4°C) and stored at −80°C. The fluorometric DEVD-afc cleavage assay was carried out on microtiter plates (Greiner, Nürtingen, Germany) according to the method originally described by Thornberry.43Thornberry NA Interleukin-1beta converting enzyme.Methods Enzymol. 1994; 244: 615-631Crossref PubMed Scopus (207) Google Scholar Cytosolic extracts (10 μl, ∼1 mg/ml protein) were diluted 1:10 with substrate buffer (55 μmol/L of fluorogenic substrate DEVD-afc in 50 mmol/L HEPES, pH 7.4, 1% sucrose, 0.1% CHAPS, 10 mmol/L dithiothreitol. Blanks contained 10 μl of extraction buffer and 90 μl of substrate buffer. Generation of free 7-amino-4-trifluoromethylcoumarin (afc) at 37°C was kinetically determined by fluorescence measurement (excitation, 385 nm. emission, 505 nm) using the fluorometer plate reader Victor2Leist M Gantner F Künstle G Wendel A Cytokine-mediated hepatic apoptosis.Rev Physiol Biochem Pharmacol. 1998; 133: 109-155PubMed Google Scholar (Wallac Instruments, Turku, Finland). Protein concentrations of the corresponding samples were estimated with the Pierce Assay (Pierce, Rockford, IL), and the activity was calculated using serially diluted standards (0 to 5 μmol/L of afc). Control experiments confirmed that the activity was linear with time and with protein concentration under the conditions described above. Nuclear extracts were prepared from frozen liver sections using a modification of a method by Schreiber et al.44Schreiber E Matthias P Müller MM Schaffner W Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells.Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (4074) Google Scholar Briefly, tissue samples were homogenized in 3 ml of ice-cold hypotonic buffer A (10 mmol/L HEPES pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol, 0.5 mmol/L Pefablock) with a Dounce homogenizer. The homogenate was incubated for 10 minutes on ice and centrifuged (10 minutes, 1,000 × g, 4°C). The cell pellet was suspended in 1.4 ml of ice-cold buffer A, and 90 μl 10. solution of Nonidet P-40 solution was added followed by 10 seconds of vigorous vortexing. The suspension was incubated on ice for 10 minutes and centrifuged (30 seconds, 12,000 × g, 4°C). The supernatant was removed and the nuclear pellet was extracted with 200 μl of hypertonic buffer B (20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L Pefablock) by shaking at 4°C for 30 minutes. The extract was centrifuged (10 minutes, 12,000 × g, 4°C), and the supernatant was stored at −80°C. A double-stranded oligonucleotide probe containing a consensus binding-sequence for NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) (Promega, Heidelberg, Germany) was 5′-end-labeled with γ[hyph]32P-ATP (3000 Ci/mmol, Amersham, Braunschweig, Germany) using T4 polynucleotide kinase (Promega, Heidelberg, Germany). Ten μg of nuclear protein were incubated at room temperature in a 15 μl reaction volume containing 10 mmol/L Tris-HCl pH 7.5, 5 × 104 cpm radiolabeled oligonucleotide probe, 2 μg poly(dIdC), 4% glycerol, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 50 mmol/L NaCl, and 0.5 mmol/L dithiothreitol for 20 minutes. Nucleoprotein-oligonucleotide complexes were resolved by electrophoresis in a 4.5% nondenaturing polyacrylamide gel in 0.25× Tris borate-EDTA at 100 V. The gel was autoradiographed with an intensifying screen at −80°C overnight. The specificity of the DNA-protein complex was confirmed by competition with a 100-fold excess of unlabeled NF-κB sequence (5′-GAT CGA ACT GAC CGC CCG CGG CCC GT-3′, Promega, Heidelberg, Germany). For light microscopy, liver samples were
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