title: Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death.
abstract: BACKGROUND
  In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS.
  
  
  OBJECTIVE
  To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity.
  
  
  METHODS
  Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-peroxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE).
  
  
  RESULTS
  Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE.
  
  
  CONCLUSIONS
  Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death.
authors.affiliation: Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Sandra
authors.initials: S
authors.lastname: Dunning
authors.forename: Atta
authors.initials: A
authors.lastname: Ur Rehman
authors.forename: Marjolein H
authors.initials: MH
authors.lastname: Tiebosch
authors.forename: Rebekka A
authors.initials: RA
authors.lastname: Hannivoort
authors.forename: Floris W
authors.initials: FW
authors.lastname: Haijer
authors.forename: Jannes
authors.initials: J
authors.lastname: Woudenberg
authors.forename: Fiona A J
authors.initials: FA
authors.lastname: van den Heuvel
authors.forename: Manon
authors.initials: M
authors.lastname: Buist-Homan
authors.forename: Klaas Nico
authors.initials: KN
authors.lastname: Faber
authors.forename: Han
authors.initials: H
authors.lastname: Moshage
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D005978:Glutathione / Q000378:metabolism*; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male; D009336:Necrosis; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D000099156:Superoxide Dismutase 2
chemical_list: D000975:Antioxidants; D002374:Catalase; D005978:Glutathione; D005979:Glutathione Peroxidase; D006861:Hydrogen Peroxide; D016877:Oxidants; D012333:RNA, Messenger; D013482:Superoxide Dismutase; D000099156:Superoxide Dismutase 2
keywords: 3-amino-1,2,4-triazole; 3AT; BSO; Catalase; Cell death; EDTA; FCS; GCL; GPx; GSH; GSH-MEE; GSH-monoethylester; GSSG; Glutathione peroxidase; HEPES; HO-1; HSC; MS; N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; NADPH-oxidase; Oxidative stress; PBS; PCR; PMSF; PPAR-γ; RNA; ROS; SOD; Superoxide dismutase; TGF-β; dNTP; deoxynucleoside triphosphates; ethylenediaminetetraacetic acid; fetal calf serum; glutamate cysteine ligase; glutathione peroxidase; heme-oxygenase-1; hepatic stellate cell; l-buthionine-sulfoximine; mercaptosuccinic acid; nicotinamide adenine dinucleotide phosphate-oxidase; oxidized glutathione; peroxisome proliferator-activated receptor gamma; phenylmethanesulphonylfluoride; phosphate-buffered saline; polymerase chain reaction; reactive oxygen species; reduced glutathione; ribonucleic acid; super oxide dismutase; transforming growth factor beta; α-SMA; α-smooth muscle actin