Protein Targets Of Reactive Metabolites Research Articles

Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes

Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities.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 [(14)C]-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 nonredundant 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 to 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 humans, 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.

Protein targets of reactive metabolites of thiobenzamide in rat liver in vivo.

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

BackgroundThe 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.DescriptionThe 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.ConclusionThe 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

The dark side of a ‘detoxification’ mechanism

Drug metabolism is not always a detoxification; sometimes products are generated that are chemically reactive and can damage cells. In the case of acetaminophen [paracetamol (APAP)], although ‘safe’ metabolites are generated at low doses, once enough drug is ingested the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) can be formed. Fortunately, the cell has an abundant nucleophile, reduced glutathione (GSH), which can act as an antioxidant (forming GSSG in the process) or directly scavenge chemicals such as NAPQI, facilitating their excretion as inactivate conjugates. In addition, glutathione S-transferases (GSTs) accelerate GSH conjugation and thereby usually serve to protect the cell. Unfortunately, in APAP overdose this system is overwhelmed, GSH is depleted, crucial targets are apparently damaged by reactive metabolites 1xIdentification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry. Qui, Y et al. J. Biol. Chem. 1998; 273: 17940–17953Crossref | PubMed | Scopus (185)See all References1 and hepatotoxicity ensues. Although it has been recognized for some time that in certain cases even GS-conjugates can be reactive, Henderson et al. 2xIncreased resistance to acetaminophen toxicity in mice lacking glutathione S-transferase Pi. Henderson, C.J et al. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12741–12745Crossref | PubMed | Scopus (156)See all References2 have recently proposed another way in which having a GST is not necessarily a good thing.Henderson et al. used mice in which both of the GST Pi family enzymes, GST P1 and GST P2, had been knocked out, and showed that this gene deletion did not aggravate APAP toxicity as had been expected; instead, the GstP1/P2−/− mice showed little or no signs of hepatoxicity, whereas the wild-type animals were clearly sensitive to APAP. GSH levels dropped rapidly in both groups but recovered faster in null animals. No significant difference was observed in overall drug–protein adduct formation.So, why was the absence of GST Pi enzymes protective? The enhanced recovery of GSH levels suggested that null mice were better able to maintain GSH levels under oxidative challenge. GST Pi enzymes might preferentially make a reversible ipso GS-adduct, and if the ipso adduct reacted with protein thiols (in addition to GSH), particularly those on crucial targets, three effects could ensue: APAP could be regenerated, GSH could be consumed and specific proteins could be inactivated by the formation of mixed disulfides, all of which could be potentiated by the presence of a catalytic GST. Although there was no evidence of GST Pi mediation of GSH conjugation in this report, other studies have suggested a role for rat and human Pi enzymes in the conjugation of NAPQI. GST Pi has also been identified as a target of adduct formation.Henderson et al. propose an alternative theory, namely that GST Pi, acting as a monomer, blocks activation of JUN by inhibiting a JUN N-terminal kinase (JNK). JUN has been implicated in responses to stress, and APAP has been shown to induce this activity. This highlights a hitherto unexpected role for xenoprotective enzymes: they might be involved in modulating cell signalling. It has been proposed that, under conditions of stress, GST Pi forms dimers and multimers that cannot bind to JNK, thus relieving the inhibition on signalling. It is therefore consistent that a complete absence of GST Pi should potentiate the stress response even further.The challenge now is to determine which, if either, mechanism is responsible for this unexpected effect of the GstP1/P2−/− knockout animal. A key point will be whether this effect is common to other cases of chemical-induced toxicity. Whatever the outcome, it is clear that even the best detoxification mechanisms can show a dark side under the right conditions.

Identification of the Hepatic Protein Targets of Reactive Metabolites of Acetaminophen in Vivoin Mice Using Two-dimensional Gel Electrophoresis and Mass Spectrometry

Liver toxicity following an overdose of acetaminophen is frequently considered a model for drug-induced hepatotoxicity. Extensive studies over many years have established that such toxicity is well correlated with liver protein arylation by acetaminophen metabolites. Identification of protein targets for covalent modifications is a challenging but necessary step in understanding how covalent binding could lead to liver toxicity. Previous approaches suffered from technical limitations, and thus over the last 10 years heroic efforts were required to determine the identity of only a few target proteins. We present a new mass spectrometry-based strategy for identification of all target proteins that now provides a comprehensive survey of the suite of liver proteins modified. After administration of radiolabeled acetaminophen to mice, the proteins in the liver tissue lysate were separated by two-dimensional polyacrylamide gel electrophoresis. In-gel digestion of the radiolabeled gel spots gave a set of tryptic peptides, which were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Interrogation of data bases based on experimentally determined molecular weights of peptides and product ion tags from postsource decay mass spectra was employed for the determination of the identities of modified liver proteins. Using this method, more than 20 new drug-labeled proteins have been identified.

Mechanisms, chemical structures and drug metabolism.

From the studies that have been done by many laboratories over the last 2 decades, it is now clear that the toxicities produced by many drugs are due to their reactive metabolites. It is though that, in many cases, reactive metabolites cause toxicity by binding covalently to tissue proteins. However, until recently it was difficult to identify these protein targets. Due to the development of an immunochemical approach, this problem has been overcome, as is illustrated here by studies that have been conducted on the metabolic basis of the idiosyncratic hepatitis caused by the inhalation anaesthetic halothane. The major problem to solve in the future will be to determine how protein adduct formation leads to toxicity. It is possible that protein adduct formation may alter an important cellular function or may lead to immunopathology, as is thought to occur in the case of halothane hepatitis. If an allergic reaction is suspected, purified protein targets of reactive metabolites can serve as antigens for identifying sensitized individuals. This information can be used to prevent not only an allergic reaction to the drug, but possible cross-reactions to other drugs that are structurally related. Another important application of these studies is the design of safer alternative drugs that will not produce structurally similar toxic reactive metabolites.