A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets in Rat Liver Cytosol in Vivo

Abstract

Metabolic activation and protein covalent binding are early and apparently obligatory events in the cytotoxicity of many simple organic chemicals including drugs and natural products. Although much has been learned about the chemistry of reactive metabolite formation and reactivity toward protein nucleophiles, progress in identifying specific protein targets for reactive metabolites of various protoxins has been much slower. We previously reported nine microsomal and three cytosolic proteins as targets for reactive metabolites of bromobenzene in rat liver. These results, and contemporary work by others, indicate that protein covalent binding is not totally random in cells. Moreover, as protein targets for other protoxins were identified, little commonality of target proteins became apparent. In the present work, we used two-dimensional gel electrophoresis to separate liver cytosolic proteins from rats treated with 14C-bromobenzene; 110 of the 836 observed spots contained measurable radioactivity that varied over a 600-fold range of adduct density. Of these 110 spots, in-gel digestion coupled with mass spectrometry identified apparently single proteins in 57 spots. A few other spots clearly contained more than one identifiable protein, and in several cases, the same protein was identified in several spots having different apparent molecular masses and/or pI. Altogether, 33 unique new protein targets for bromobenzene metabolites were identified and compared to those known for acetaminophen, naphthalene, butylated hydroxytoluene, benzene, thiobenzamide, and halothane via a target protein database available at http://tpdb.medchem.ku.edu:8080/protein_database/. With increasing numbers of target proteins becoming known, more commonality in targeting by reactive metabolites from diverse chemical agents may be seen. Such commonality may help to separate toxicologically significant covalent binding events from a background of covalent binding that is toxicologically inconsequential.

Filling and mining the reactive metabolite target protein database

“…An unexpected finding in these covalent binding studies was that some proteins would appear in multiple well-separated spots on the 2D gel, usually with the same apparent mass but differing pI values (41, 44, 45). This may be due to differences in post-translational modification such as phosphorylation, but the exact reason is unknown.…”

Role of Reactive Metabolites in Drug-Induced Hepatotoxicity

“…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). Potential targets within individual organelles can now be identified by using model electrophiles in cell fractions (Shin et al 2007;Wong and Liebler 2008).…”

“…The epoxide intermediate may also undergo nonenzymatic isomerisation to 4-bromophenol (Selander et al 1975), which can be further metabolized to yield catechols and quinones, some of which have been associated with hepatotoxicity via redox cycling and oxidative stress (Mizutani and Miyamoto 1999). The postulated mechanisms of bromobenzene-induced hepatotoxicity include GSH depletion, lipid peroxidation, covalent modification of critical cellular proteins, and mitochondrial dysfunction (Casini et al 1985;Fisher et al 1993;Koen et al 2007;Maellaro et al 1990). Modification of rat hepatic proteins by bromobenzene's reactive metabolites has been analyzed in some detail and undoubtedly appears to be a highly selective process at both the molecular (Koen et al 2006) and cellular levels ), although there is partial commonality with the proteins modified by other bioactivated hepatotoxins such as APAP ).…”

The reactive metabolite target protein database (TPDB) – a web-accessible resource

“…It is important to note, however, that covalent binding is a selective process and not a random process that is simply proportional to protein abundance, as discussed in ref. [24]. At present, the mechanistic significance of this commonality remains an open question, and a worthwhile direction for future research.…”