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.