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.