Site-Specific Arylation of Rat Glutathione S-Transferase A1 and A2 by Bromobenzene Metabolites in Vivo

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• 作者：Yakov M. Koen ; Weimin Yue ; Nadezhda A. Galeva ; Todd D. Williams ; Robert P. Hanzlik
• 刊名：Chemical Research in Toxicology
• 出版年：2006
• 出版时间：November 2006
• 年：2006
• 卷：19
• 期：11
• 页码：1426 - 1434
• 全文大小：400K
• 年卷期：v.19,no.11(November 2006)
• ISSN：1520-5010

文摘

The hepatotoxicity of bromobenzene (BB) derives from its reactive metabolites (epoxides and quinones),which arylate cellular proteins. Application of proteomic methods to liver proteins from rats treated witha hepatotoxic dose of [¹⁴C]-BB has identified more than 40 target proteins, but no adducted peptideshave yet been observed. Because such proteins are known to contain bromophenyl- and bromodihydroxyphenyl derivatives of cysteine, histidine, and lysine, the failure to observe modified peptides has beenattributed to the low level of total covalent binding and to the "dilution" effect of multiple metabolitesreacting at multiple sites on multiple proteins. In this work glutathione S-transferase (GST), a well-known and abundant BB-target protein, was isolated from liver cytosol of rats treated with ¹⁴C-BB byuse of a glutathione (GSH)-agarose affinity column and further resolved by reverse-phase high-performance liquid chromatography (HPLC) into subunits M1, M2, A1, A2 and A3. The subunits wereidentified by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),whole-molecule mass spectrometry, and peptide mass mapping and found to contain radioactivitycorresponding to 0.01-0.05 adduct per molecule of protein. Examination of tryptic digests of these subunitsby matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionizationmass spectrometry (ESI-MS) failed to reveal any apparent adducted peptides despite observed sequencecoverages up to 87%. However, use of HPLC-linear ion-trap quadrupole Fourier transform massspectrometry (LTQ-FTMS) to search for predicted modified tryptic peptides revealed peaks corresponding,with a high degree of mass accuracy, to a bromobenzoquinone adduct of peptide 89-119 in both GSTA1and A2. The identity of these adducts and their location at Cys-111 was confirmed by tandem massspectrometry (MS-MS). No evidence for the presence of any putative BB-adducts in GST M1, M2, orA3 was obtained. This work highlights the challenges involved in the unambiguous identification ofreactive metabolite adducts formed in vivo.