Protein thiol oxidation in murine airway epithelial cells in response to naphthalene or diethyl maleate.
Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, beta-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA.
Department of Molecular Biosciences, University of California, Davis, USA. pspiess@uvm.edu
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Spiess
Dexter
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Morin
Chase R
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Williams
Alan R
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Buckpitt
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D008297:Male; D008298:Maleates / Q000494:pharmacology*; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology*; D010084:Oxidation-Reduction; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D008298:Maleates; D009281:Naphthalenes; D013438:Sulfhydryl Compounds; C031721:naphthalene; C014476:diethyl maleate; D005978:Glutathione