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1.  Covalent Modification of Lipids and Proteins in Rat Hepatocytes, and In Vitro, by Thioacetamide Metabolites 
Chemical research in toxicology  2012;25(9):1868-1877.
Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO2). To explore possible parallels between the metabolism, covalent binding and toxicity of TA and thiobenzamide (TB) we exposed freshly isolated rat hepatocytes to [14C]-TASO or [13C2D3]-TASO. TLC analysis of the cellular lipids showed a single major spot of radioactivity that mass spectral analysis showed to consist of N-acetimidoyl PE lipids having the same side chain composition as the PE fraction from untreated cells; no carbons or hydrogens from TASO were incorporated into the fatty acyl chains. Many cellular proteins contained N-acetyl- or N-acetimidoyl lysine residues in a 3:1 ratio (details to be reported separately). We also oxidized TASO with hydrogen peroxide in the presence of dipalmitoyl phosphatidylenthanolamine (DPPE) or lysozyme. Lysozyme was covalently modified at five of its six lysine side chains; only acetamide-type adducts were formed. DPPE in liposomes also gave only amide-type adducts, even when the reaction was carried out in tetrahydrofuran with only 10% water added. The exclusive formation of N-acetimidoyl PE in hepatocytes means that the concentration or activity of water must be extremely low in the region where TASO2 is formed, whereas at least some of the TASO2 can hydrolyze to acetylsulfinic acid before it reacts with cellular proteins. The requirement for two sequential oxidations to produce a reactive metabolite is unusual, but it is even more unusual that a reactive metabolite would react with water to form a new compound that retains a high degree of chemical reactivity toward biological nucleophiles. The possible contribution of lipid modification to the hepatotoxicity of TA/TASO remains to be determined.
PMCID: PMC3444623  PMID: 22667464
2.  Liver Protein Targets of Hepatotoxic 4-Bromophenol Metabolites 
Chemical research in toxicology  2012;25(8):1777-1786.
The hepatotoxicity of bromobenzene (BB) is directly related to the covalent binding of both initially formed epoxide and secondary quinone metabolites to at least 45 different liver proteins. 4-Bromophenol (4BP) is a significant BB metabolite and a precursor to reactive quinone metabolites, yet when administered exogenously it has negligible hepatotoxicity compared to BB. The protein adducts of 4BP were thus labeled as non-toxic (Monks, T. J.; Hinson, J. A.; Gillette, J. R. (1982) Life Sci. 30, 841–848). To help identify which BB-derived adducts might be related to its cytotoxicity, we sought to identify the supposedly non-toxic adducts of 4BP and eliminate them from the BB target protein list. Administration of [14C]-4BP to phenobarbital-induced rats resulted in covalent binding of 0.25, 0.33 and 0.42 nmol-eq 4BP/mg protein in the mitochondrial, microsomal and cytosolic fractions, respectively. These values may be compared to published values of 3–6 nmol/mg protein from a comparable dose of [14C]-BB. After subcellular fractionation and 2D electrophoresis, 47 radioactive spots on 2D gels of the mitochondrial, microsomal and cytosolic fractions were excised, digested and analyzed by LC-MS/MS. Twenty nine of these spots contained apparently single proteins, of which 14 were non-redundant. Nine of the 14 are known BB targets. Incubating freshly-isolated rat hepatocytes with 4BP (0.1–0.5 mM) produced time- and concentration-dependent increases in lactate dehydrogenase release and changes in cellular morphology. LC-MS/MS analysis of the cell culture medium revealed rapid and extensive sulfation and glucuronidation of 4BP as well as formation of a quinone-derived glutathione conjugate. Studies with 7-hydroxycoumarin (7HC), (−)-borneol or D-(+)-galactosamine (DGN) showed that inhibiting the glucuronidation/sulfation of 4BP increased the formation of a GSH-bromoquinone adduct, increased covalent binding of 4BP to hepatocyte proteins and potentiated its cytotoxicity. Taken together, our data demonstrate that protein adduction by 4BP metabolites can be toxicologically consequential, and provide a mechanistic explanation for the failure of exogenously administered 4BP to cause hepatotoxicity. Thus the probable reason for the low toxicity of 4BP in vivo is that rapid conjugation limits its oxidation and covalent binding and thus its toxicity.
PMCID: PMC3431021  PMID: 22827705
3.  Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes 
Chemical Research in Toxicology  2012;25(5):1145-1154.
Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [14C]-TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 non-redundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2–4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver (Methogo, R., Dansette, P. and Klarskov, K. (2007) Int. J. Mass Spectrom., 268, 284–295), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e. rat vs. human), and still another may be the method of detection of adducted proteins (i.e. Western blot vs. C-14). Knowledge of human target proteins is very limited. Of more than 350 known protein targets of reactive metabolites, only 42 are known from human and only 21 of these are known to be targets for more than one chemical. Nevertheless, the demonstration that human target proteins can be identified using isolated hepatocytes in vitro should enable the question of species differences to be addressed more fully in the future.
PMCID: PMC3358484  PMID: 22462724
4.  Filling and Mining the Reactive Metabolite Target Protein Database 
Chemico-biological interactions  2008;179(1):38-44.
The post-translational modification of proteins is a well-known endogenous mechanism for regulating protein function and activity. Cellular proteins are also susceptible to post-translational modification by xenobiotic agents that possess, or whose metabolites possess, significant electrophilic character. Such non-physiological modifications to endogenous proteins are sometimes benign, but in other cases they are strongly associated with, and are presumed to cause, lethal cytotoxic consequences via necrosis and/or apoptosis. The Reactive Metabolite Target Protein Database (TPDB) is a searchable, freely web-accessible ( resource that attempts to provide a comprehensive, up-to-date listing of known reactive metabolite target proteins. In this report we characterize the TPDB by reviewing briefly how the information it contains came to be known. We also compare its information to that provided by other types of “-omics” studies relevant to toxicology, and we illustrate how bioinformatic analysis of target proteins may help to elucidate mechanisms of cytotoxic responses to reactive metabolites.
PMCID: PMC2669808  PMID: 18823962
5.  Protein Targets of Reactive Metabolites of Thiobenzamide in Rat Liver In Vivo 
Chemical research in toxicology  2008;21(7):1432-1442.
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., Chem. Res. Toxicol. 2007, 20, 701−708). To understand better the relationship between the protein covalent binding and 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−14C]-TB contained high levels of covalently bound radioactivity (25.6 and 36.8 nmol eq./mg protein, respectively). These proteins were fractionated by 2-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-d5 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 d0/d5 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 iminosulfonic 4 (PhC(=NH)SO2H) which either benzimidoylates a lysine side chain or undergoes hydrolysis to 9 (PhC(=O)SO2H) 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.
PMCID: PMC2493440  PMID: 18547066
6.  Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners 
Protein covalent binding by reactive metabolites of drugs, chemicals and natural products can lead to acute cytotoxicity. Recent rapid progress in reactive metabolite target protein identification has shown that adduction is surprisingly selective and inspired the hope that analysis of target proteins might reveal protein factors that differentiate target- vs. non-target proteins and illuminate mechanisms connecting covalent binding to cytotoxicity.
Sorting 171 known reactive metabolite target proteins revealed a number of GO categories and KEGG pathways to be significantly enriched in targets, but in most cases the classes were too large, and the "percent coverage" too small, to allow meaningful conclusions about mechanisms of toxicity. However, a similar analysis of the directlyinteracting partners of 28 common targets of multiple reactive metabolites revealed highly significant enrichments in terms likely to be highly relevant to cytotoxicity (e.g., MAP kinase pathways, apoptosis, response to unfolded protein). Machine learning was used to rank the contribution of 211 computed protein features to determining protein susceptibility to adduction. Protein lysine (but not cysteine) content and protein instability index (i.e., rate of turnover in vivo) were among the features most important to determining susceptibility.
As yet there is no good explanation for why some low-abundance proteins become heavily adducted while some abundant proteins become only lightly adducted in vivo. Analyzing the directly interacting partners of target proteins appears to yield greater insight into mechanisms of toxicity than analyzing target proteins per se. The insights provided can readily be formulated as hypotheses to test in future experimental studies.
PMCID: PMC2711050  PMID: 19523227
7.  Covalent Modification of Microsomal Lipids by Thiobenzamide Metabolites in Vivo 
Chemical research in toxicology  2007;20(4):701-708.
Thiobenzamide (TB) is hepatotoxic in rats causing centrolobular necrosis, steatosis, cholestasis and hyperbilirubinemia. It serves as a model compound for a number of thiocarbonyl compounds that undergo oxidative bioactivation to chemically reactive metabolites. The hepatotoxicity of TB is strongly dependent on the electronic character of substituents in the meta- and para- positions, with Hammett rho values ranging from −4 to −2. On the other hand ortho substituents which hinder nucleophilic addition to the benzylic carbon of S-oxidized TB metabolites abrogate the toxicity and protein covalent binding of TB. This strong linkage between the chemistry of TB and its metabolites and their toxicity suggests that this model is a good one for probing the overall mechanism of chemically-induced biological responses. While investigating the protein covalent binding of TB metabolites we noticed an unusually large amount of radioactivity associated with the lipid fraction of rat liver microsomes. Thin layer chromatography showed that most of the radioactivity was contained in a single spot more polar than the neutral lipids but less polar than the phospholipid fractions. Mass spectral analyses aided by the use of synthetic standards identified the material as N-benzimidoyl derivatives of typical microsomal phosphatidylethanolamine (PE) lipids. Quantitative analysis indicated that up to 25% of total microsomal PE became modified within 5 h after a hepatotoxic dose of TB. Further studies will be required to determine the contribution of lipid modification to the hepatotoxicity of thiobenzamide.
PMCID: PMC2527973  PMID: 17381136
8.  Site-specific Arylation of Rat Glutathione S-Transferase A1 and A2 by Bromobenzene Metabolites in vivo 
Chemical research in toxicology  2006;19(11):1426-1434.
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 an 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 transferase, a well known and abundant BB-target protein, was isolated from liver cytosol of rats treated with 14C-BB using a GSH-agarose affinity column and further resolved by reverse phase HPLC into subunits M1, M2, A1, A2 and A3. The subunits were identified by a combination of 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 MALDI-TOF and ESI-MS again failed to reveal any apparent adducted peptides despite observed sequence coverages up to 87%. However, using HPLC-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 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.
PMCID: PMC1661840  PMID: 17112229
9.  The reactive metabolite target protein database (TPDB) – a web-accessible resource 
BMC Bioinformatics  2007;8:95.
The toxic effects of many simple organic compounds stem from their biotransformation to chemically reactive metabolites which bind covalently to cellular proteins. To understand the mechanisms of cytotoxic responses it may be important to know which proteins become adducted and whether some may be common targets of multiple toxins. The literature of this field is widely scattered but expanding rapidly, suggesting the need for a comprehensive, searchable database of reactive metabolite target proteins.
The Reactive Metabolite Target Protein Database (TPDB) is a comprehensive, curated, searchable, documented compilation of publicly available information on the protein targets of reactive metabolites of 18 well-studied chemicals and drugs of known toxicity. TPDB software enables i) string searches for author names and proteins names/synonyms, ii) more complex searches by selecting chemical compound, animal species, target tissue and protein names/synonyms from pull-down menus, and iii) commonality searches over multiple chemicals. Tabulated search results provide information, references and links to other databases.
The TPDB is a unique on-line compilation of information on the covalent modification of cellular proteins by reactive metabolites of chemicals and drugs. Its comprehensiveness and searchability should facilitate the elucidation of mechanisms of reactive metabolite toxicity. The database is freely available at
PMCID: PMC1832215  PMID: 17367530

Results 1-9 (9)