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

Abstract

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 with a hepatotoxic dose of [14C]-BB has identified more than 40 target proteins, but no adducted peptides have 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 been attributed to the low level of total covalent binding and to the "dilution" effect of multiple metabolites reacting 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 by use 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 were identified by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whole-molecule mass spectrometry, and peptide mass mapping and found to contain radioactivity corresponding to 0.01-0.05 adduct per molecule of protein. Examination of tryptic digests of these subunits by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization mass spectrometry (ESI-MS) failed to reveal any apparent adducted peptides despite observed sequence coverages up to 87%. However, use of HPLC-linear ion-trap quadrupole Fourier transform mass spectrometry (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 GSTA1 and A2. The identity of these adducts and their location at Cys-111 was confirmed by tandem mass spectrometry (MS-MS). No evidence for the presence of any putative BB-adducts in GST M1, M2, or A3 was obtained. This work highlights the challenges involved in the unambiguous identification of reactive metabolite adducts formed in vivo.

Role of Reactive Metabolites in Drug-Induced Hepatotoxicity

“…Even within a single liver protein there can be selective modification of an amino acid side-chain found repeatedly in the primary structure (Koen et al 2006;Nerland et al 2003). Thus, the microenvironment (pKa, hydrophobicity, etc.)…”

“…Nevertheless, the structure of the metabolite-protein adduct has been determined in only a few cases (Baer et al 2007;Bambal and Hanzlik 1995;Damsten et al 2007;Sleno et al 2007;Yukinaga et al 2007;Zhang et al 2003), and identification of the modified amino acid residue in vivo remains a major analytical challenge (Koen et al 2006). Greater progress has been made in identification of the cellular proteins that are modified in vivo (Druckova et al 2007;Ikehata et al 2008;Koen et al 2007;Qiu et al 1998;Shipkova et al 2004).…”

Idiosyncratic Drug Reactions: Past, Present, and Future

“…It is interesting that the chemical reactivity of reactive metabolites toward small molecules does not predict their in vivo pattern of covalent binding; specifically, binding can be selective for specific thiol groups while at the same time binding can also occur to nucleophilic groups in proteins such as imidazole that would not be expected based on the relative reactivity of the isolated amino acids, that is, cysteine vs histidine (56). Another example is the comparison of the pattern of covalent binding of procainamide, vesnarinone, and clozapine, all of which are associated with a relatively high incidence of agranulocytosis (57).…”

Identification of the Protein Targets of the Reactive Metabolite of Teucrin A in Vivo in the Rat

“…This can be attributed to the dilution effect of the complex peptide mixtures and multiple resultant peptide conjugates, the low level of total covalent binding, and the potential changes in the chromatographic behavior of the modified peptides. Previously reported sites of covalent modification, such as Cys 111 in GST A1 and GST A2 by the benzoquinone metabolite of BB, was possible only after glutathione affinity purification of the GTSs and their chromatographic separation, followed by HPLC linear ion-trap quadrupole Fourier transform mass spectrometry to search for predicted modified tryptic peptides (121). Similarly, Cys 86 modified by acrylonitrile in GST M1 was identified after rigorous GST affinity purification and HPLC separation with the help of radiolabeling (116).…”

“…Additionally, the amount of the adduction by agents such as BB or APAP typically does not exceed one modification per 10 protein molecules (121). Therefore, the in vivo covalent modification of a protein does not necessarily mean a complete loss of its critical function.…”