title: Mechanistic basis for inflammation and tumor promotion in lungs of 2,6-di-tert-butyl-4-methylphenol-treated mice: electrophilic metabolites alkylate and inactivate antioxidant enzymes.
abstract: An established model for mechanistic analysis of lung carcinogenesis involves administration of 3-methylcholanthrene to mice followed by several weekly injections of the tumor promoter 2,6-di-tert-butyl-4-methylphenol (BHT). BHT is metabolized to quinone methides (QMs) responsible for promoting tumor formation. QMs are strongly electrophilic and readily form adducts with proteins. The goal of the present study was to identify adducted proteins in the lungs of mice injected with BHT and to assess the potential impact of these modifications on tumorigenesis. Cytosolic proteins from treated mouse lungs were separated by two-dimensional electrophoresis, adducts detected by immunoblotting, and proteins identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eight adducts were detected in the lungs of most, or all, of six experimental groups of BALB mice. Of these adducts, several were structural proteins, but others, namely, peroxiredoxin 6 (Prx6), Cu,Zn-superoxide dismutase (SOD1), carbonyl reductase, and selenium-binding protein 1, have direct or indirect antioxidant functions. When the 9000g supernatant fraction of mouse lung was treated with BHT-QM (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone), substantial lipid peroxidation and increases in hydrogen peroxide and superoxide formation were observed. Studies with human Prx6 and bovine SOD1 demonstrated inhibition of enzyme activity concomitant with adduct formation. LC-MS/MS analysis of digests of adducted Prx6 demonstrated adduction of both Cys 91 and Cys 47; the latter residue is essential for peroxidatic activity. Analysis of QM-treated bovine SOD1 by matrix-assisted laser desorption/ionization time-of-flight MS demonstrated the predominance of a monoadduct at His 78. This study provides evidence that indicates Prx6, SOD1, and possibly other antioxidant enzymes in mouse lung are inhibited by BHT-derived QMs leading to enhanced levels of reactive oxygen species and inflammation and providing a mechanistic basis for the effects of BHT on lung tumorigenesis.
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Box C238, Denver, Colorado 80262, USA.
authors.forename: Brent W
authors.initials: BW
authors.lastname: Meier
authors.forename: Jose D
authors.initials: JD
authors.lastname: Gomez
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Kirichenko
authors.forename: John A
authors.initials: JA
authors.lastname: Thompson
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000478:Alkylation; D000818:Animals; D000975:Antioxidants / Q000737:chemistry* / Q000378:metabolism; D002084:Butylated Hydroxytoluene; D002853:Chromatography, Liquid / Q000379:methods; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D045563:Indolequinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007249:Inflammation / Q000139:chemically induced; D007274:Injections, Intraperitoneal; D008175:Lung Neoplasms / Q000139:chemically induced*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D015394:Molecular Structure; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D054465:Peroxiredoxin VI; D054464:Peroxiredoxins; D010636:Phenols / Q000008:administration & dosage / Q000378:metabolism* / Q000633:toxicity; D017382:Reactive Oxygen Species / Q000378:metabolism; D051137:Selenium-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000975:Antioxidants; D045563:Indolequinones; D010636:Phenols; D017382:Reactive Oxygen Species; D051137:Selenium-Binding Proteins; C068040:quinone methide; D002084:Butylated Hydroxytoluene; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C000614209:AKR7A5 protein, mouse; D010544:Peroxidases; C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI; D054464:Peroxiredoxins; C515983:Prdx6 protein, mouse; D013482:Superoxide Dismutase