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1.  The reactive metabolite target protein database (TPDB) – a web-accessible resource 
BMC Bioinformatics  2007;8:95.
Background
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.
Description
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.
Conclusion
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
doi:10.1186/1471-2105-8-95
PMCID: PMC1832215  PMID: 17367530
2.  Analysis of Naphthalene Adduct Binding Sites in Model Proteins by Tandem Mass Spectrometry 
Chemico-biological interactions  2012;199(2):120-128.
The electrophilic metabolites of the polyaromatic hydrocarbon naphthalene have been shown to bind covalently to proteins and covalent adduct formation correlates with the cytotoxic effects of the chemical in the respiratory system. Although 1,2-naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-napthoquinone have been identified as reactive metabolites of interest, the role of each metabolite in total covalent protein adduction and subsequent cytotoxicity remains to be established. To better understand the target residues associated with the reaction of these metabolites with proteins, mass spectrometry was used to identify adducted residues following 1) incubation of metabolites with actin and protein disulfide isomerase (PDI), and 2) activation of naphthalene in microsomal incubations containing supplemental actin or PDI. All four reactive metabolites bound to Cys, Lys or His residues in actin and PDI. Cys17 of actin was the only residue adducted by all metabolites; there was substantial metabolite selectivity for the majority of adducted residues. Modifications of actin and PDI, following microsomal incubations containing 14C-naphthalene, were detected readily by 2D gel electrophoresis and phosphor imaging. However, target modifications on tryptic peptides from these isolated proteins could not be readily detected by MALDI/TOF/TOF and only three modified peptides were detected using high resolution – selective ion monitoring (HR-SIM). All the reactive metabolites investigated have the potential to modify several residues in a single protein, but even in tissues with very high rates of naphthalene activation, the extent of modification was too low to allow unambiguous identification of a significant number of modified residues in the isolated proteins.
doi:10.1016/j.cbi.2012.05.009
PMCID: PMC3579547  PMID: 22659010
Adducts; mass spectrometry; naphthalene; reactive metabolites; model proteins; microsomal incubations
3.  Protein Targets of Reactive Electrophiles in Human Liver Microsomes 
Chemical research in toxicology  2007;20(6):859-867.
Liver microsomes are widely used to study xenobiotic metabolism in vitro and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB, but not BMCC induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.
doi:10.1021/tx700031r
PMCID: PMC2556149  PMID: 17480101
4.  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.
doi:10.1021/tx3002675
PMCID: PMC3431021  PMID: 22827705
5.  Characterization of Model Peptide Adducts with Reactive Metabolites of Naphthalene by Mass Spectrometry 
PLoS ONE  2012;7(8):e42053.
Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts in vivo as markers of biologic effect.
doi:10.1371/journal.pone.0042053
PMCID: PMC3411726  PMID: 22870282
6.  Evidence for cellular protein covalent binding derived from styrene metabolite 
Chemico-biological interactions  2010;186(3):323-330.
Styrene is one of the most important industrial intermediates consumed in the world. Human exposure to styrene occurs mainly in the reinforced plastics industry, particularly in developing countries. Styrene has been found to be hepatotoxic and pneumotoxic in humans and animals. The biochemical mechanisms of styrene-induced toxicities remain unknown. Albumin and hemoglobin adduction derived from styrene oxide, a major reactive metabolite of styrene, has been reported in blood samples obtained from styrene exposed workers. The objectives of the current study focused on cellular protein covalent binding of styrene metabolite and its correlation with cytotoxicity induced by styrene. We found that radioactivity was bound to cellular proteins obtained from mouse airway trees after incubation with 14C-styrene. Microsomal incubation studies showed that the observed protein covalent binding required the metabolic activation of styrene. The observed radioactivity binding in protein samples obtained from the cultured airways and microsomal incubations were significantly suppressed by co-incubation with disulfiram, a CYP2E1 inhibitor, although disulfiram apparently did not show a protective effect against the cytotoxicity of styrene. A 2-fold increase in radioactivity bound to cellular proteins was detected in cells stably transfected with CYP2E1 compared to the wild-type cells after 14C-styrene exposure. With the polyclonal antibody developed in our lab, we detected cellular protein adduction derived from styrene oxide at cysteinyl residues in cells treated with styrene. Competitive immunoblot studies confirmed the modification of cysteine residues by styrene oxide. Cell culture studies showed that the styrene-induced protein modification and cell death increased with the increasing concentration of styrene exposure. In conclusion, we detected cellular protein covalent modification by styrene oxide in microsomal incubations, cultured cells, and mouse airways after exposure to styrene and found a good correlation between styrene-induced cytotoxicity and styrene oxide-derived cellular protein adduction.
doi:10.1016/j.cbi.2010.05.004
PMCID: PMC3463232  PMID: 20470765
Styrene; Styrene Oxide; Bioactivation; Protein Adduction
7.  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 (http://tpdb.medchem.ku.edu:8080/protein_database/) 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.
doi:10.1016/j.cbi.2008.08.016
PMCID: PMC2669808  PMID: 18823962
8.  Reactive metabolites of phenacetin and acetaminophen: a review 
Phenacetin can be metabolized to reactive metabolites by a variety of mechanisms. (1) Phenacetin can be N-hydroxylated, and the resulting N-hydroxyphenacetin can be sulfated or glucuronidated. Whereas phenacetin N-O sulfate immediately rearranges to form a reactive metabolite which may covalently bind to protein, phenacetin N-O glucuronide slowly rearranges to form reactive metabolites. Incubation of the purified phenacetin N-O glucuronide under a variety of conditions suggests that N-acetyl-p-benzoquinone imine is a reactive metabolite. This metabolite covalently binds to protein, reacts with glutathione to form an acetaminophen-glutathione conjugate, is reduced by ascorbate to acetaminophen or is partially hydrolyzed to acetamide. (2) Phenacetin can be O-deethylated to acetaminophen, and acetaminophen can be converted directly to a reactive metabolite which may be also N-acetyl-p-benzoquinone imine. (3) Phenacetin can be sequentially N-hydroxylated and O-deethylated to N-hydroxyacetaminophen which spontaneously dehydrates to N-acetyl-p-benzoquinone imine. (4) Phenacetin can be 3, 4-epoxidated to form an alkylating and an arylating metabolite. In the presence of glutathione, a S-ethylglutathione conjugate and an acetaminophen-glutathione conjugate are formed. In the absence of glutathione, the alkylating metabolite may bind to protein and the arylating metabolite is completely hydrolyzed to acetamide and another arylating metabolite which may bind to protein. The structures of the alkylating and arylating metabolites are unknown.
Control experiments have shown that in pathway (1) the phenolic oxygen of the acetaminophenglutathione conjugate is derived from water, whereas in pathways (2) and (3) the phenolic oxygen of this metabolite is derived from phenacetin. In pathway (4) the phenolic oxygen was 50% derived from molecular oxygen and 50% from phenacetin. Administration of [p-180]phenacetin to hamsters revealed only a 10% loss of 180 in the acetaminophen mercapturic acid (the further metabolic product of the glutathione conjugate) which suggests that, in the hamster, pathways (2) and/or (3) are the primary mechanism of conversion of phenacetin to reactive metabolites in vivo.
PMCID: PMC1569121  PMID: 6339229
9.  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.
doi:10.1021/tx060142s
PMCID: PMC1661840  PMID: 17112229
10.  Formation of Covalently Bound Protein Adducts from the Cytotoxicant Naphthalene in Nasal Epithelium: Species Comparisons 
Environmental Health Perspectives  2009;118(5):647-652.
Background
Naphthalene is a volatile hydrocarbon that causes dose-, species-, and cell type–dependent cytotoxicity after acute exposure and hyperplasia/neoplasia after lifetime exposures in rodents. Toxicity depends on metabolic activation, and reactive metabolite binding correlates with tissue and site susceptibility.
Objectives
We compared proteins adducted in nasal epithelium from rats and rhesus macaques in vitro.
Methods
Adducted proteins recovered from incubations of nasal epithelium and 14C-naphthalene were separated by two-dimensional (2D) gel electrophoresis and imaged to register radioactive proteins. We identified proteins visualized by silver staining on complementary nonradioactive gels by peptide mass mapping.
Results
The levels of reactive metabolite binding in incubations of rhesus ethmoturbinates and maxilloturbinates are similar to those in incubations of target tissues, including rat septal/olfactory regions and murine dissected airway incubations. We identified 40 adducted spots from 2D gel separations of rat olfactory epithelial proteins; 22 of these were nonredundant. In monkeys, we identified 19 spots by mass spectrometry, yielding three nonredundant identifications. Structural proteins (actin/tubulin) were prominent targets in both species.
Conclusions
In this study we identified potential target proteins that may serve as markers closely associated with toxicity. The large differences in previously reported rates of naphthalene metabolism to water-soluble metabolites in dissected airways from mice and monkeys are not reflected in similar differences in covalent adduct formation in the nose. This raises concerns that downstream metabolic/biochemical events are very similar between the rat, a known target for naphthalene toxicity and tumorigenicity, and the rhesus macaque, a species similar to the human.
doi:10.1289/ehp.0901333
PMCID: PMC2866680  PMID: 20435546
monkey; naphthalene; nasal epithelium; protein adducts; rat; reactive metabolites; species comparisons
11.  Covalent binding of reactive estrogen metabolites to microtubular protein as a possible mechanism of aneuploidy induction and neoplastic cell transformation. 
Neoplastic cell transformation induced by estrogens and some other carcinogens such as benzene appears to involve the induction of mitotic aneuploidy rather than DNA damage and point mutations. As metabolic activation may also play an important role in the mechanism of carcinogenesis of these nongenotoxic compounds, we have studied the interaction of reactive quinone metabolites of various estrogens and of benzene with the major microtubular protein, tubulin, in a cell-free system. Covalent binding of the radioactively labeled metabolites to the alpha- and beta-subunit of tubulin was found to depend on the structure of the metabolite. When the adducted tubulins were tested in vitro for their ability to polymerize to microtubules, inhibition of microtubule assembly was observed in every case, although to varying extents. It is proposed that the formation of covalent tubulin adducts may impair the formation of mitotic spindles and thus contribute to chromosomal nondisjunction and aneuploidy induction.
PMCID: PMC1568022  PMID: 2272306
12.  Profiling the Reactive Metabolites of Xenobiotics Using Metabolomic Technologies 
Chemical research in toxicology  2011;24(5):744-751.
A predominant pathway of xenobiotic-induced toxicity is initiated by bioactivation. Characterizing reactive intermediates will provide information on the structure of reactive species, thereby defining a potential bioactivation mechanism. Because most reactive metabolites are not stable, it is difficult to detect them directly. Reactive metabolites can form adducts with trapping reagents, such as glutathione, which makes the reactive metabolites detectable. However, it is challenging to “fish” these adducts out from a complex biological matrix, especially for adducts generated via uncommon metabolic pathways. In this regard, we developed a novel approach based upon metabolomic technologies to screen trapped reactive metabolites. The bioactivation of pulegone, acetaminophen, and clozapine were reexamined by using this metabolomic approach. In all these cases, a large number of trapped reactive metabolites were readily identified. These data indicate that this metabolomic approach is an efficient tool to profile xenobiotic bioactivation.
doi:10.1021/tx200033v
PMCID: PMC3100669  PMID: 21469730
13.  The p-i Concept: Pharmacological Interaction of Drugs With Immune Receptors 
The immune response in drug hypersensitivity is normally explained by the hapten hypothesis. It postulates that drugs with a molecular weight of less than 1000 D are too small to cause an immune response per se. However, if a chemically reactive drug or drug metabolite binds covalently to a protein and thus forms a so-called hapten-carrier complex, this modified protein can induce an immune response. This concept has recently been supplemented by the p-i concept (or pharmacological interaction with immune receptors), which postulates that some drugs that lack hapten characteristics can bind directly and reversibly (noncovalently) to immune receptors and thereby stimulate the cells. For example, a certain drug may bind to a particular T-cell receptor, and this binding suffices to stimulate the T cell to secrete cytokines, to proliferate, and to exert cytotoxicity. The p-i concept has major implications for our understanding of drug interaction with the specific immune system and for drug hypersensitivity reactions. It is based on extensive investigations of T-cell clones reacting with the drug and recently of hybridoma cells transfected with the drug-specific T-cell receptor for antigen (TCR). It is a highly specific interaction dependent on the expression of a TCR into which the drug can bind with sufficient affinity to cause signaling. Small modification of the drug structure may already abrogate reactivity. Stimulation of T cells occurs within minutes as revealed by rapid Ca++ influx after drug addition to drug-specific T-cell clones or hybridoma cells, thus, before metabolism and processing can occur. As the immune system can only react in an immunologic way, the symptoms arising after drug stimulation of immune receptors imitate an immune response after recognition of a peptide antigen, although it is actually a pharmacological stimulation of some T cells via their TCRs. Clinically, the p-i concept could explain the sometimes rapid appearance of symptoms without previous sensitizations and the sometimes chaotic immune reaction of drug hypersensitivity with participation of different immune mechanisms while normal immune reactions to antigens are highly coordinated. Nevertheless, because the reactions lead to expansion of drug-reactive cells, many features such as skin test reactivity and stronger reactivity upon reexposure are identical to real immune reactions.
doi:10.1097/WOX.0b013e3181778282
PMCID: PMC3651037  PMID: 23282405
p-i concept; drug hypersensitivity; hapten; prohapten; T-cell receptor; T cells
14.  Molecular Targets for 17α-Ethynyl-5-Androstene-3β,7β,17β-Triol, an Anti-Inflammatory Agent Derived from the Human Metabolome 
PLoS ONE  2012;7(2):e32147.
HE3286, 17α-ethynyl-5-androstene-3β, 7β, 17β-triol, is a novel synthetic compound related to the endogenous sterol 5-androstene-3β, 7β, 17β-triol (β-AET), a metabolite of the abundant adrenal steroid dehydroepiandrosterone (DHEA). HE3286 has shown efficacy in clinical studies in impaired glucose tolerance and type 2 diabetes, and in vivo models of types 1 and 2 diabetes, autoimmunity, and inflammation. Proteomic analysis of solid-phase HE3286-bound bead affinity experiments, using extracts from RAW 264.7 mouse macrophage cells, identified 26 binding partners. Network analysis revealed associations of these HE3286 target proteins with nodes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for type 2 diabetes, insulin, adipokine, and adipocyte signaling. Binding partners included low density lipoprotein receptor-related protein (Lrp1), an endocytic receptor; mitogen activated protein kinases 1 and 3 (Mapk1, Mapk3), protein kinases involved in inflammation signaling pathways; ribosomal protein S6 kinase alpha-3 (Rsp6ka3), an intracellular regulatory protein; sirtuin-2 (Sirt2); and 17β-hydroxysteroid dehydrogenase 1 (Hsd17β4), a sterol metabolizing enzyme.
doi:10.1371/journal.pone.0032147
PMCID: PMC3286445  PMID: 22384159
15.  Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate 
Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, β-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA.
doi:10.1165/rcmb.2009-0135OC
PMCID: PMC2933547  PMID: 19843705
thiol oxidation; bronchial epithelial cells; naphthalene; diethyl maleate; proteomics
16.  Screening for Natural Chemoprevention Agents that Modify Human Keap1 
Analytical Biochemistry  2011;421(1):108-114.
Upregulation of cytoprotective enzymes by therapeutic agents to prevent damage by reactive oxygen species and xenobiotic electrophiles is a strategy for cancer chemoprevention. The Kelch-like ECH-associated protein 1 (Keap1) and its binding partner, transcription factor NF-E2-related factor-2 (Nrf2), are chemoprevention targets because of their role in regulating the antioxidant response element (ARE) in response to oxidative stress and exposure to electrophiles. Modification of the sensor protein Keap1 by electrophiles such as the isothiocyanate sulforaphane can direct Nrf2 accumulation in the nucleus and subsequent ARE activation. Since our previous matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS)-based screening method to discover natural products that modify Keap1 does not detect covalent modification of Keap1 by some highly reversible agents such as sulforaphane, a more sensitive screening assay was developed. In this new assay, electrophiles that have reversibly modified Keap1 can be released, trapped and detected as β-mercaptoethanol adducts by mass spectrometry. Isoliquiritigenin and sulforaphane, known ARE activators that target Keap1, were used to validate the assay. To determine the ability of the assay to identify electrophiles in complex matrixes that modify Keap1, sulforaphane was spiked into a cocoa extract, and LC-MS/MS using high resolution mass spectrometry with accurate mass measurement was used to identify β-mercaptoethanol adducts of sulforaphane that had been released from Keap1. This screening assay permits identification of potential chemoprevention agents in complex natural product mixtures that reversibly modify Keap1 but cannot be detected using MALDI-TOF MS.
doi:10.1016/j.ab.2011.10.028
PMCID: PMC3390259  PMID: 22074792
17.  LC-MS-based Metabolomics of Xenobiotic-induced Toxicities 
Xenobiotic exposure, especially high-dose or repeated exposure of xenobiotics, can elicit detrimental effects on biological systems through diverse mechanisms. Changes in metabolic systems, including formation of reactive metabolites and disruption of endogenous metabolism, are not only the common consequences of toxic xenobiotic exposure, but in many cases are the major causes behind development of xenobiotic-induced toxicities (XIT). Therefore, examining the metabolic events associated with XIT generates mechanistic insights into the initiation and progression of XIT, and provides guidance for prevention and treatment. Traditional bioanalytical platforms that target only a few suspected metabolites are capable of validating the expected outcomes of xenobiotic exposure. However, these approaches lack the capacity to define global changes and to identify unexpected events in the metabolic system. Recent developments in high-throughput metabolomics have dramatically expanded the scope and potential of metabolite analysis. Among all analytical techniques adopted for metabolomics, liquid chromatography-mass spectrometry (LC-MS) has been most widely used for metabolomic investigations of XIT due to its versatility and sensitivity in metabolite analysis. In this review, technical platform of LC-MS-based metabolomics, including experimental model, sample preparation, instrumentation, and data analysis, are discussed. Applications of LC-MS-based metabolomics in exploratory and hypothesis-driven investigations of XIT are illustrated by case studies of xenobiotic metabolism and endogenous metabolism associated with xenobiotic exposure.
doi:10.5936/csbj.201301008
PMCID: PMC3962105
metabolomics; LC-MS; xenobiotic; xenobiotic-induced toxicity; biomarker
18.  Protein modification by acrolein: Formation and stability of cysteine adducts 
Chemical research in toxicology  2009;22(4):708-716.
The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues.
doi:10.1021/tx800465m
PMCID: PMC2929760  PMID: 19231900
19.  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.
doi:10.1021/tx600362h
PMCID: PMC2527973  PMID: 17381136
20.  Protein Carbonylation in a Murine Model for Early Alcoholic Liver Disease 
Chemical research in toxicology  2012;25(5):1012-1021.
Hepatic oxidative stress and subsequent lipid peroxidation are well-recognized consequences of sustained ethanol consumption. The covalent adduction of nucleophilic amino acid side-chains by lipid electrophiles is significantly increased in patients with alcoholic liver disease (ALD); a global assessment of in vivo protein targets and the consequences of these modifications, however, has not been conducted. In this report, we describe identification of novel protein targets for covalent adduction in a 6-week murine model for ALD. Ethanol-fed mice displayed a 2-fold increase in hepatic TBARS while immunohistochemical analysis for the reactive aldehydes 4-hydroxynonenal (4-HNE), 4-oxononenal (4-ONE), acrolein (ACR) and malondialdehyde (MDA) revealed a marked increase in the staining of modified proteins in the ethanol-treated mice. Increased protein carbonyl content was confirmed utilizing subcellular fractionation of liver homogenates followed by biotin-tagging through hydrazide chemistry, where approximately a 2-fold increase in modified proteins was observed in microsomal and cytosolic fractions. To determine targets of protein carbonylation, a secondary hydrazide method coupled to a highly sensitive 2-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS or MuDPIT) technique was utilized. Our results have identified 414 protein targets for modification by reactive aldehydes in ALD. The presence of novel in vivo sites of protein modification by 4-HNE (2), 4-ONE (4) and ACR (2) was also confirmed in our data set. While the precise impact of protein carbonylation in ALD remains unknown, a bioinformatic analysis of the data set has revealed key pathways associated with disease progression, including fatty acid metabolism, drug metabolism, oxidative phosphorylation and the TCA cycle. These data suggest a major role for aldehyde adduction in the pathogenesis of ALD.
doi:10.1021/tx300002q
PMCID: PMC3423195  PMID: 22502949
ROS; ALD; 4-HNE; 4-hydroxynonenal; 4-ONE; 4-oxononenal; acrolein; MDA; malondialdehyde; MuDPIT; 2D-LC-MS/MS; DAVID; protein adducts; protein carbonyl; TBARS; lipid peroxidation; electrophile; biotin hydrazide; in vivo adduct; GRP78; bioinformatics; steatosis; fatty liver; ethanol
21.  Oxidation of 4-chlorobiphenyl metabolites to electrophilic species by prostaglandin H synthase 
Hormonally–sensitive tissues, like the prostate, ovary and breast, increasingly studied as targets of environmental chemicals, are sources of an enzyme potentially capable of transforming and activating xenobiotics to highly reactive metabolites. Our study specifically addresses the question of whether prostaglandin H synthase (PGHS) can activate phenolic metabolites of polychlorinated biphenyls (PCBs). We found that human recombinant PGHS-2 catalyzed the oxidation of ortho (2′,3′-, 3′,4′-) and para (2′,5′-) dihydroxy 4-chlorobiphenyl metabolites to their corresponding quinones. These were trapped in situ with N-acetyl cysteine and the reaction products were isolated and characterized by liquid chromatography coupled mass spectrometry and 1H and heteronuclear (1H-13C) nuclear magnetic resonance spectroscopy. Both mono- and di-N-acetyl cysteine Michael addition adducts were identified, with the 2′,3′-, and 2′,5′-dihydroxy metabolites predominantly forming mono-N-acetyl cysteine adducts, while the 3′,4′-dihydroxy predominantly formed di-substituted N-acetyl cysteine adducts. These studies clearly demonstrate that the phenolic metabolites of these environmental pollutants are activated by PGHS, as co-substrates, to highly reactive electrophilic PCB quinones, with a potential for protein and DNA damage, especially in non-hepatic tissues where the enzyme is found.
doi:10.1021/tx800300t
PMCID: PMC2637223  PMID: 19105592
22.  Comparative methods for analysis of protein covalent modification by electrophilic quinoids formed from xenobiotics 
Bioconjugate chemistry  2009;20(4):728-741.
Conjugation of biotin and fluorophore tags is useful for assaying covalent protein modification. Oxidative bioactivation of selective estrogen receptor modulators (SERMs) yields reactive quinoid electrophiles that covalently modify proteins; bioactivation is associated with carcinogenic and chemopreventive effects. Identification of the protein targets of electrophilic metabolites is of general important for xenobiotics. Four methodologies, using SERM derivatives and biotin/fluorophore tags, were compared for purification and quantification: (1) covert oxidatively activated tags (COATags; SERM conjugated to biotin); (2) dansylTags (SERM conjugated to fluorophore); and azidoTags (SERM azide derivatives) in a 2-step conjugation to biotin, either using (3) Staudinger ligation; or (4) click chemistry. All synthetic derivatives retained the estrogen receptor ligand characteristics of the parent SERMs. Model proteins with bioactivation by tyrosinase in buffer or cell lysates and liver proteins with in situ bioactivation in rat primary hepatocytes were studied by immunoassay and fluorescence. Comparison showed: the azidoTag/Staudinger method was sensitive but nonspecific; the azidoTag/click methodology had low sensitivity; and, the dansylTag methodology failed to detect modified proteins in hepatocytes. The COATag methodology was adjudged superior, detecting 5 ng of modified protein in vitro and identifying protein targets in hepatocytes. In metabolism studies in rat liver microsomes, the azide group was metabolically labile, one contributing factor in not selecting an azidoTag methodology in the highly oxidative environments required for bioactivation. For study of the protein targets of electrophilic metabolites formed by in situ oxidative bioactivation, the COATag is both sensitive and specific, and does not appear to suffer from poor cell permeability.
doi:10.1021/bc800435m
PMCID: PMC2827204  PMID: 19301905
xenobiotics; protein modification; biotin tags; quinone electrophiles; proteomics
23.  Mechanism of toxicity of nitro compounds used in the chemotherapy of trichomoniasis. 
The mechanism of the trichomonicidal activity of metronidazole and other 5-nitroimidazoles appears to depend on the ferredoxin-mediated reduction of their nitro group, with generation of a reactive metabolite or metabolites which interact with DNA leading to a subsequent inhibition of nucleic acid and protein synthesis. Redox cycling of these compounds under aerobic conditions appears to be a detoxification reaction by inhibiting net reduction of the drugs, thereby inhibiting their uptake. On the other hand, redox cycling of nitrofurans or other compounds with more positive reduction potential results in formation of high steady-state concentrations of oxygen-derived metabolites that might be of toxicological significance. It seems likely that reduced metabolites of nitroimidazoles (perhaps through covalent binding to tissue macromolecules and/or thiols depletion) are also involved in the nitroimidazoles' toxic effects to animal tissues and in their mutagenic and carcinogenic action.
Images
PMCID: PMC1568619  PMID: 3830698
24.  Role of NAD(P)H:Quinone Oxidoreductase 1 in Clofibrate-Mediated Hepatoprotection from Acetaminophen1 
Toxicology  2006;230(2-3):197-206.
Mice pretreated with the peroxisome proliferator clofibrate (CFB) are resistant to acetaminophen (APAP) hepatotoxicity. Whereas the mechanism of protection is not entirely known, CFB decreases protein adducts formed by the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI). NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme with antioxidant properties that is responsible for the reduction of cellular quinones. We hypothesized that CFB increases NQO1 activity, which in turn enhances the conversion of NAPQI back to the parent APAP. This could explain the decreases in APAP covalent binding and glutathione depletion produced by CFB without affecting APAP bioactivation to NAPQI. Administration of CFB (500 mg/kg, i.p.) to male CD-1 mice for 5 or 10 days increased NQO1 protein and activity levels. To evaluate the capacity of NQO1 to reduce NAPQI back to APAP, we utilized a microsomal activating system. Cytochrome P450 enzymes present in microsomes bioactivate APAP to NAPQI, which binds the electrophile trapping agent, N-acetyl cysteine (NAC). We analyzed the formation of APAP-NAC metabolite in the presence of human recombinant NQO1. Results indicate that NQO1 is capable of reducing NAPQI. The capacity of NQO1 to amelioriate APAP toxicity was then evaluated in primary hepatocytes. Primary hepatocytes isolated from mice dosed with CFB are resistant to APAP toxicity. These hepatocytes were also exposed to ES936, a high affinity, irreversible inhibitor of NQO1 in the presence of APAP. Concentrations of ES936 that resulted in over 94% inhibition of NQO1 activity did not increase the susceptibility of hepatocytes from CFB treated mice to APAP. Whereas NQO1 is mechanistically capable of reducing NAPQI, CFB-mediated hepatoprotection does not appear to be dependent upon enhanced expression of NQO1.
doi:10.1016/j.tox.2006.11.052
PMCID: PMC1885461  PMID: 17188792
acetaminophen; clofibrate; peroxisome proliferators; hepatoprotection; NQO1; NAPQI
25.  Teratogen-Induced Oxidative Stress Targets Glyceraldehyde-3-Phosphate Dehydrogenase in the Organogenesis Stage Mouse Embryo 
Toxicological Sciences  2010;118(2):686-695.
Exposure during the organogenesis stage of the mouse embryo to the model teratogen, hydroxyurea (HU), induces curly tail and limb malformations. Oxidative stress contributes to the developmental toxicity of HU. Reactive oxygen species (ROS) interact with polyunsaturated bilipid membranes to form α,β-unsaturated reactive aldehydes; 4-hydroxy-2-nonenal (4-HNE), one of the most cytotoxic of these aldehydes, covalently adducts with proteins, lipids, and nucleic acids. The goal of the current study is to determine if HU exposure of CD1 mice on gestation day 9 generates region-specific 4-HNE-protein adducts in the embryo and to identify the proteins targeted. The formation of 4-HNE-protein adducts was elevated in the caudal region of control embryos; HU exposure further increased 4-HNE-protein adduct formation in this area. Interestingly, three of the 4-HNE–modified proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamate oxaloacetate transaminase 2, and aldolase 1, A isoform, are involved in energy metabolism. The formation of 4-HNE-GAPDH protein adducts reduced GAPDH enzymatic activity by 20% and attenuated lactate production by 40%. Furthermore, HU exposure induced the nuclear translocation of GAPDH in the caudal region of exposed embryos; this nuclear translocation may be associated with the reactivation of oxidized proteins involved in DNA repair, such as apurinic/apyrimidinic endonuclease-1, and the stimulation of E1A-associated P300 protein/creb-binding protein (p300/CBP) activity, initiating cell death in a p53-dependent pathway. We propose that GAPDH is a redox-sensitive target in the embryo and may play a role in a stress response during development.
doi:10.1093/toxsci/kfq287
PMCID: PMC2984529  PMID: 20889679
4-hydroxy-2-nonenal; protein adducts; glyceraldehyde-3-phosphate dehydrogenase; oxidative stress; nuclear translocation; glycolysis

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