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-ditert-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 6 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 9000 g 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 predominance of a mono-adduct 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.

Peroxiredoxin 6 is a molecular target for 1,2-naphthoquinone, an atmospheric electrophile, in human pulmonary epithelial A549 cells

“…Consistent with this, LC-MS analysis identifi ed Cys47, Cys91 and Lys144 as the sites on Prdx6 modifi ed ( Table 1). The S-arylation sites identifi ed on Prdx6 are in agreement with those reported for quinone methides derived from 2,6-ditert-butyl-4-mrthylphenol (Meier et al, 2007). Modifi cation of Prdx6 by 1,2-NQ would affect its catalytic activity.…”

Renoprotective mechanisms of morin in cisplatin-induced kidney injury

“…However, the interaction between cisplatin and the activities of GSH peroxidase and SOD enzymes is not yet understood. It is reported that cisplatin treatment leads to a decline of GSH peroxidase and SOD enzymes in the kidneys, which results from loss of copper and zinc or directly binds to sulfhydryl groups on cysteine [13,14]. Apart from changes in GSH peroxidase and SOD enzyme levels, increased of CYP2E1 levels by cisplatin was decreased by morin (Fig.…”

Quinone Methides and their Prodrugs: A Subtle Equilibrium Between Cancer Promotion, Prevention, and Cure

“…It decreases calpain-II activity, induces ODC expression (a widely recognized marker of tumor progression) and activates the MAPK family member, ERK, in keratinocytes, thus promoting tumor formation [41,50,51]. BHT also binds and inhibits peroxiredoxin 6, Cu-Zn SOD and CR, which are all antioxidant enzymes [49,52,53].…”