title: Proteomic identification of carbonylated proteins in 1,3-dinitrobenzene neurotoxicity.
abstract: This study demonstrated that 1,3-dinitrobenzene-induced (1,3-DNB) oxidative stress led to the oxidative carbonlyation of specific protein targets in DI TNC1 cells. 1,3-DNB-induced mitochondrial dysfunction, as indicated by loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence, was initially observed at 5h and coincided with peak reactive oxygen species (ROS) production. ROS production was inhibited in cells pre-treated with the mitochondrial permeability transition (MPT) inhibitor, bonkrekic acid (BkA). Pre-incubation with the antioxidant deferoxamine inhibited loss of TMRM fluorescence until 24h after initial exposure to 1,3-DNB. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and subsequent Oxyblot analysis were used to determine if 1,3-DNB exposure led to the formation of protein carbonyls. Exposing DI TNC1 cells to 1,3-DNB led to marked protein carbonylation 45 min following initial exposure. Pre-treatment with deferoxamine or Trolox reduced the intensity of protein carbonylation in DI TNC1 cells exposed to 1mM 1,3-DNB. Tandem MS/MS performed on protein samples isolated from 1,3-DNB-treated cells revealed that specific proteins within the mitochondria, endoplasmic reticulum (ER), and cytosol are targets of protein carbonylation. The results presented in this study are the first to suggest that the molecular mechanism of 1,3-DNB neurotoxicity may occur through selective carbonylation of protein targets found within specific intracellular compartments of susceptible cells.
authors.affiliation: Toxicology Program, School of Public Health, University of Michigan, 1415 Washington Heights, Ann Arbor, MI 48109-2029, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Steiner
authors.forename: Martin A
authors.initials: MA
authors.lastname: Philbert
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D002460:Cell Line; D002839:Chromans / Q000494:pharmacology; D002853:Chromatography, Liquid; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D004136:Dinitrobenzenes / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D015151:Immunoblotting; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D020258:Neurotoxicity Syndromes / Q000209:etiology* / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D050050:Protein Carbonylation / Q000187:drug effects*; D040901:Proteomics / Q000379:methods; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D000975:Antioxidants; D002839:Chromans; D004136:Dinitrobenzenes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009419:Nerve Tissue Proteins; D017382:Reactive Oxygen Species; C017906:3-dinitrobenzene; D003676:Deferoxamine; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid