Inhibition of Glutathione S-Transferase P1-1 in Mouse Lung Epithelial Cells by the Tumor Promoter 2,6-Di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-Quinone Methide): Protein Adducts Inves

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• 作者：Jean-Noë ; l Lemercier ; Brent W. Meier ; Jose D. Gomez ; and John A. Thompson
• 刊名：Chemical Research in Toxicology
• 出版年：2004
• 出版时间：December 2004
• 年：2004
• 卷：17
• 期：12
• 页码：1675 - 1683
• 全文大小：204K
• 年卷期：v.17,no.12(December 2004)
• ISSN：1520-5010

文摘

Oxidation of the food preservative 2,6-di-tert-butyl-4-methylphenol (BHT) by mouse lungcytochrome P450 produces electrophilic quinone methides thought to promote lung tumors inmice 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 presentwork was undertaken to determine if glutathione S-transferase P1-1 (GSTP1-1) is alkylatedby BHT-QM, as this protein is overexpressed in tumors and has important roles in protectingcells from electrophiles and oxidants and in regulating stress kinases. This work was conductedwith cell lines C10 and E10 derived from mouse lung epithelia and their spontaneoustransformants, the tumorigenic cell lines A5 and E9. Cytosolic GSTs were isolated by affinitychromatography and analyzed by ESI-LC/MS. Ion current chromatograms indicated that GSTP1predominates over the other isoforms, especially in tumorigenic cells. Treatment with BHT-QM inhibited cytosolic GST activity by 28-44%, and inhibition was exacerbated by depletingintracellular GSH. Alkylation of GSTP1 by BHT-QM was investigated by separating cytosolicproteins with two-dimensional SDS-PAGE and detecting adducts by Western blotting withpolyclonal antibodies that recognize the BHT group. The identity of GSTP1 comigrating withimmunoreactive material was confirmed by in-gel proteolysis and LC/MS/MS analysis. HumanGSTP1 was utilized to investigate the specific residues involved in QM binding. The only peptideadduct detected in digests of monoadducted GSTP1 corresponded to Cys101, whereas adductsat Cys14, Cys47, and Cys101 were identified from the trialkylated protein. Losses of transferaseactivity were most influenced by alkylation at Cys47, but binding to Cys14 appeared to inhibitthe activity further. These findings demonstrate that cytosolic GSTP1 may be a target forBHT-QM resulting in decreased cellular protection from electrophiles and oxidants. Alkylationalso may interfere with GSTP1 regulation of stress kinases, thereby influencing phosphorylationand cell growth.