Identification of Seven Proteins in the Endoplasmic Reticulum as Targets for Reactive Metabolites of Bromobenzene

Abstract

The hepatotoxicity of bromobenzene is strongly correlated with the covalent binding of chemically reactive metabolites to cellular proteins, but up to now relatively few hepatic protein targets of these reactive metabolites have been identified. To identify additional hepatic protein targets we injected an hepatotoxic dose of [14C]bromobenzene to phenobarbital-pretreated male Sprague-Dawley rats ip. After 4 h, their livers were removed and homogenized, and the homogenates fractionated by differential ultracentrifugation. The highest specific radiolabeling (6.1 nmol equiv 14C/mg of protein) was observed in a particulate fraction (P25) sedimented at 25000g from a 6000g supernatant fraction. Proteins in this fraction were separated by two-dimensional electrophoresis and, after transblotting, analyzed for radioactivity by phosphorimaging. More than 20 radiolabeled protein spots were observed in the blots. For 17 of these spots, peptide mass maps were obtained using in-gel digestion with trypsin, followed by MALDI-TOF mass spectrometric analysis of the resulting peptide mixtures. By searching genomic databases, the 17 sets of MS-derived peptide masses were found to match predicted tryptic fragments of just 7 proteins. Spots 1-4 matched with 78 kDa glucose regulated protein (GRP78), protein disulfide isomerase isozyme A1 (PDIA1), endoplasmic reticulum protein ERp29, and PDIA6, respectively. Spots 5 and 6, 7-11, and 12-17 presented as apparent "charge trains" of spots, each of which gave peptide mixtures closely similar to those of other spots within the train. The proteins present in these sets of spots were identified as transthyretin, serum albumin precursor and PDIA3, respectively. The possible relationship of the adduction of these proteins to the toxicological outcome is discussed.

Role of NAD(P)H:quinone oxidoreductase 1 in clofibrate-mediated hepatoprotection from acetaminophen

“…NQO1 is capable of reducing oxidative stress by scavenging superoxide and enhancing endogenous antioxidants (Landi et al, 1997;Siegel et al, 1997;Siegel et al, 2004). Whereas toxicity from carbon tetrachloride and paraquat is primarily associated with oxidative stress due to formation of radicals, APAP and bromobenzene additionally generate reactive quinone metabolites that bind to proteins (Koen and Hanzlik, 2002;Guo et al, 2004). Reduction of these highly reactive quinones by NQO1 may reduce protein adduct formation, thereby decreasing toxicity.…”

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

“…Thus, the toxicological consequence(s) of a reactive TA intermediary may depend on the extent of modification and potential malfunction of disulphide isomerase. Similarly, this protein seems to be a common target for various reactive intermediaries including mycophenolic acid [21], naphthalene [25], halothane [35], bromobenzene [36], monocrotaline [37]. The dnaK-type molecular chaperone Hsc73, also called Hsc70 or Hspa8, was previously shown to be adducted by a reactive intermediary of naphthalene [25].…”

Covalent Modification of Microsomal Lipids by Thiobenzamide Metabolites in Vivo

“…In a separate experiment, a group of six phenobarbital-pretreated rats received an ip dose of [ 14 C]bromobenzene (2.0 mmol/kg) in corn oil (1 mL/kg). Four hours later, the rats were killed and the liver microsomal fraction was isolated as described earlier (33).…”

“…In an earlier study of bromobenzene activation and covalent binding in vivo, we used two-dimensional gel electrophoresis (2DGE) to analyze microsomal proteins from the livers of rats treated with 14 C-bromobenzene (33). In that work, we observed a large streak of radioactive material in the lowpI, low-molecular weight (MW) region of the gel.…”