Protein Targets of Reactive Metabolites of Thiobenzamide in Rat Liver in Vivo

Abstract

Thiobenzamide (TB) is a potent hepatotoxin in rats, causing dose-dependent hyperbilirubinemia, steatosis, and centrolobular necrosis. These effects arise subsequent to and appear to result from the covalent binding of the iminosulfinic acid metabolite of TB to cellular proteins and phosphatidylethanolamine lipids [ Ji et al. ( 2007) Chem. Res. Toxicol. 20, 701- 708 ]. To better understand the relationship between the protein covalent binding and the toxicity of TB, we investigated the chemistry of the adduction process and the identity of the target proteins. Cytosolic and microsomal proteins isolated from the livers of rats treated with a hepatotoxic dose of [ carboxyl- (14)C]TB contained high levels of covalently bound radioactivity (25.6 and 36.8 nmol equiv/mg protein, respectively). These proteins were fractionated by two-dimensional gel electrophoresis, and radioactive spots (154 cytosolic and 118 microsomal) were located by phosphorimaging. Corresponding spots from animals treated with a 1:1 mixture of TB and TB- d 5 were similarly separated, the spots were excised, and the proteins were digested in gel with trypsin. Peptide mass mapping identified 42 cytosolic and 24 microsomal proteins, many of which appeared in more than one spot on the gel; however, only a few spots contained more than one identifiable protein. Eighty-six peptides carrying either a benzoyl or a benzimidoyl adduct on a lysine side chain were clearly recognized by their d 0/ d 5 isotopic signature (sometimes both in the same digest). Because model studies showed that benzoyl adducts do not arise by hydrolysis of benzimidoyl adducts, it was proposed that TB undergoes S-oxidation twice to form iminosulfinic acid 4 [PhC(NH)SO 2H], which either benzimidoylates a lysine side chain or undergoes hydrolysis to 9 [PhC(O)SO 2H] and then benzoylates a lysine side chain. The proteins modified by TB metabolites serve a range of biological functions and form a set that overlaps partly with the sets of proteins known to be modified by several other metabolically activated hepatotoxins. The relationship of the adduction of these target proteins to the cytotoxicity of reactive metabolites is discussed in terms of three currently popular mechanisms of toxicity: inhibition of enzymes important to the maintenance of cellular energy and homeostasis, the unfolded protein response, and interference with kinase-based signaling pathways that affect cell survival.

The Postulated Hepatotoxic Metabolite of Methimazole Causes Mitochondrial Dysfunction and Energy Metabolism Disturbances in Liver

“…40 Sulfonic and sulfenic acids are reactive electrophilic species which are capable of interacting with several intracellular targets such as different proteins. 41,42 In the current study, we found that NMT administration significantly damaged liver mitochondria both in vitro and in vivo. NMT reactive metabolites might also be involved in the mitochondrial injury induced by this chemical in vivo (Figure 7).…”

Role of Reactive Metabolites in Drug-Induced Hepatotoxicity

“…A number of simple chemical compounds such as acetaminophen, bromobenzene, furosemide, methapyrilene (Graham et al 2008), and thiobenzamide (Ikehata et al 2008) produce hepatotoxicity, with damage to extrahepatic tissues in certain cases (bromobenzene is also pneumotoxic and nephrotoxic; Dahl et al 1990), in one or more rodent species after a single dose or a short-term regimen. The reproducibility of these injuries permits detailed mechanistic investigations that are impractical or unachievable in the case of idiosyncratic reactions; however, they may provide a source of crucial insights into the mechanisms of such reactions.…”

“…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).…”

Protein Electrophile-Binding Motifs: Lysine-Rich Proteins Are Preferential Targets of Quinones

“…Antibody-based approaches to adduct detection have revealed a handful of targets for several chemicals, but only in a few cases has adduction been clearly defined at the level of amino acid sequence (Stewart et al, 2007;Ikehata et al, 2008). Thus, identifying specific sites of reactive chemical/metabolite-mediated modification within proteins has been extremely challenging.…”