Oxidation of the food preservative 2,6-di-tert-butyl-4-methylphenol (BHT) by mouse lung cytochrome P450 produces electrophilic quinone methides thought to promote lung tumors in mice by covalent binding to critical proteins. Specific pulmonary targets of 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) have not been identified, however. The present work was undertaken to determine if glutathione S-transferase P1-1 (GSTP1-1) is alkylated by BHT-QM, as this protein is overexpressed in tumors and has important roles in protecting cells from electrophiles and oxidants and in regulating stress kinases. This work was conducted with cell lines C10 and E10 derived from mouse lung epithelia and their spontaneous transformants, the tumorigenic cell lines A5 and E9. Cytosolic GSTs were isolated by affinity chromatography and analyzed by ESI-LC/MS. Ion current chromatograms indicated that GSTP1 predominates over the other isoforms, especially in tumorigenic cells. Treatment with BHT-QM inhibited cytosolic GST activity by 28-44%, and inhibition was exacerbated by depleting intracellular GSH. Alkylation of GSTP1 by BHT-QM was investigated by separating cytosolic proteins with two-dimensional SDS-PAGE and detecting adducts by Western blotting with polyclonal antibodies that recognize the BHT group. The identity of GSTP1 comigrating with immunoreactive material was confirmed by in-gel proteolysis and LC/MS/MS analysis. Human GSTP1 was utilized to investigate the specific residues involved in QM binding. The only peptide adduct detected in digests of monoadducted GSTP1 corresponded to Cys101, whereas adducts at Cys14, Cys47, and Cys101 were identified from the trialkylated protein. Losses of transferase activity were most influenced by alkylation at Cys47, but binding to Cys14 appeared to inhibit the activity further. These findings demonstrate that cytosolic GSTP1 may be a target for BHT-QM resulting in decreased cellular protection from electrophiles and oxidants. Alkylation also may interfere with GSTP1 regulation of stress kinases, thereby influencing phosphorylation and cell growth.

Two quinone methide (QM) metabolites of the phenolic antioxidant butylated hydroxytoluene (BHT), 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and the tert-butyl-hydroxylated derivative (BHTOH-QM), are believed to be responsible for promoting lung tumor formation in mice treated with BHT. QMs are strongly electrophilic and undergo Michael type additions with nucleophiles at the exocyclic methylene to form benzylic thioether adducts. Our goal was to identify intracellular protein targets of these QMs in order to gain insight into their effects on tumorigenesis. Cell line E10 of mouse lung epithelial origin and its spontaneous transformant, the tumorigenic E9 cell line, were treated with BHT-QM or BHTOH-QM, and cellular proteins were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Adducted proteins were detected on western blots with polyclonal antibodies developed to a conjugate of BHTOH-QM that recognized adducts of both QMs bound to thiol groups of Cys and side chain amino groups of Lys and His residues. Tryptic digests of immunoreactive proteins were analyzed by HPLC mass spectrometry (LC/MS) and identified by searching protein databases using MS/MS data. In a few cases, adducted peptides in these digests were detected by matrix-assisted laser desorption/ionization time-of-flight MS. A total of 37 immunoreactive proteins were identified including proteins involved in carbohydrate metabolism, nucleic acid synthesis, and RNA and protein processing, in addition to several cytoskeletal and stress-related proteins. About half of the protein adducts were found in both cell lines. Adducts detected only in transformed E9 cells include glutathione S-transferase P1, peroxiredoxin 2, nucleoside diphosphate kinase, and vinculin, whereas several alkylated cytoskeletal proteins such as tubulins, vimentin, calvasculin, and calcyclin were detected exclusively in E10 cells. Several of the proteins modified by BHT-derived QMs have been implicated in various aspects of tumorigenesis and are excellent candidates for further study into the consequences of alkylation on cellular transformation.

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.

Promotion of lung tumors in mice by the food additive butylated hydroxytoluene (BHT) is mediated by electrophilic metabolites produced in the target organ. Identifying the proteins alkylated by these quinone methides (QMs) is a necessary step in understanding the underlying mechanisms. Covalent adducts of the antioxidant enzymes peroxiredoxin 6 and Cu,Zn superoxide dismutase were detected previously in lung cytosols from BALB/c mice injected with BHT, and complimentary in vitro studies demonstrated that QM alkylation causes inactivation and enhances oxidative stress. In the present work, adducts of another protective enzyme, carbonyl reductase (CBR), were detected by Western blotting and mass spectrometry in mitochondria from lungs of mice one day after a single injection of BHT and throughout a 28-day period of weekly injections required to achieve tumor promotion. BHT treatment was accompanied by the accumulation of protein carbonyls in lung cytosol from sustained oxidative stress. Studies in vitro demonstrated that CBR activity in lung homogenates was susceptible to concentration- and time-dependent inhibition by QMs. Recombinant CBR underwent irreversible inhibition during QM exposure, and mass spectrometry was utilized to identify alkylation sites at Cys 51, Lys 17, Lys 189, Lys 201, His 28, and His 204. Except for Lys 17, all of these adducts were eliminated as a cause of enzyme inhibition either by chemical modification (cysteine) or site-directed mutagenesis (lysines and histidines). The data demonstrated that Lys 17 is the critical alkylation target, consistent with the role of this basic residue in NADPH binding. These data support the possibility that CBR inhibition occurs in BHT-treated mice, thereby compromising one pathway for inactivating lipid peroxidation products, particularly 4-oxo-2-nonenal. These data, in concert with previous evidence for the inactivation of antioxidant enzymes, provide a molecular basis to explain lung inflammation leading to tumor promotion in this two-stage model for pulmonary carcinogenesis.