title: Nrf2 activation involves an oxidative-stress independent pathway in tetrafluoroethylcysteine-induced cytotoxicity.
abstract: Tetrafluoroethylcysteine (TFEC), a metabolite of the industrial gas tetrafluoroethylene, can cause both nephrotoxicity and limited hepatotoxicity in animal models, and this is associated with the covalent modification of specific intramitochondrial proteins including heat shock protein 60 (HSP60), mitochondrial HSP70 (mtHSP70), aspartate aminotransferase (AST), aconitase, and alpha-ketoglutarate dehydrogenase (alphaKGDH). Using the murine TAMH cell line as a useful in vitro model for TFEC toxicity, we demonstrate a rapid and sustained induction of Nrf2, a member of the "cap-and-collar" transcription factor family, following exposure to cytotoxic concentrations of TFEC. A functional correlate was also established with the rapid translocation of cytosolic Nrf2 into the nucleus. In addition, transcriptional and translational upregulation of known Nrf2 regulated genes including glutamate cysteine ligase (GCL), both catalytic and modulatory subunits, heme oxygenase-1, and glutathione S-transferase (GST) isoforms were detected. While Nrf2 activation is often linked to perturbation of cellular thiol status and/or oxidative stress, we were unable to detect any significant depletion of cellular glutathione or oxidation of mitochondrial membrane cardiolipin or increases in reactive oxygen species (ROS). These data suggest Nrf2 activation is likely independent of classical oxidative stress or, at best, a result of a transient, low-level redox stress. Moreover, supporting evidence indicates an early endoplasmic reticular (ER) stress response after TFEC treatment, with a time-dependent upregulation of the ER responsive genes gadd34, gadd45, gadd153, and ndr1 . These findings suggest an alternative pathway for Nrf2 activation, i.e., Nrf2 phosphorylation through ER-mediated protein kinases such as PKR-like endoplasmic reticular kinase (PERK). Overall, the results implicate a role for Nrf2 in the cellular response to TFEC toxicity and suggest a previously unrecognized role for the ER in this model of mitochondrially initiated cytotoxicity.
authors.affiliation: Department of Medicinal Chemistry, Environmental and Occupational Health Sciences, and Pathology, University of Washington, Seattle, Washington 98195, USA. hankiat@u.washington.edu
authors.forename: Han K
authors.initials: HK
authors.lastname: Ho
authors.forename: Collin C
authors.initials: CC
authors.lastname: White
authors.forename: Carolina
authors.initials: C
authors.lastname: Fernandez
authors.forename: Nelson
authors.initials: N
authors.lastname: Fausto
authors.forename: Terrance J
authors.initials: TJ
authors.lastname: Kavanagh
authors.forename: Sidney D
authors.initials: SD
authors.lastname: Nelson
authors.forename: Sam A
authors.initials: SA
authors.lastname: Bruschi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000633:toxicity; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D005978:Glutathione / Q000378:metabolism; D006845:Hydrocarbons, Fluorinated / Q000633:toxicity*; D006861:Hydrogen Peroxide / Q000378:metabolism; D051379:Mice; D051267:NF-E2-Related Factor 2; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000096:biosynthesis*; D005466:Fluorocarbons
chemical_list: D000255:Adenosine Triphosphate; D002118:Calcium; D003545:Cysteine; D004268:DNA-Binding Proteins; D005978:Glutathione; D006845:Hydrocarbons, Fluorinated; D006861:Hydrogen Peroxide; D051267:NF-E2-Related Factor 2; D012333:RNA, Messenger; D015534:Trans-Activators; C495636:Nfe2l2 protein, mouse; C064116:S-(1,1,2,2-tetrafluoroethyl)cysteine; D005466:Fluorocarbons