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1.  Utilization of LC-MS/MS Analyses to Identify Site-Specific Chemical Protein Adducts In Vitro 
Biologically reactive intermediates are formed following metabolism of xenobiotics, and during normal oxidative metabolism. These reactive species are electrophilic in nature and are capable of forming stable adducts with target proteins. These covalent protein modifications can initiate processes that lead to acute tissue injury or chronic disease. Recent advancements in mass spectrometry techniques and data analysis has permitted a more detailed investigation of site-specific protein modifications by reactive electrophiles. Knowledge from such analyses will assist in providing a better understanding of how specific classes of electrophiles produce toxicity and disease progression via site-selective protein-specific covalent modification. Hydroquinone (HQ) is a known environmental toxicant, and its quinone–thioether metabolites, formed via the intermediate generation of 1,4-benzoquinone (1,4-BQ), elicit their toxic response via the covalent modification of target proteins and the generation of reactive oxygen species. We have utilized a model protein, cytochrome c, to guide us in identifying 1,4-BQ- and 1,4-BQ-thioether derived site-specific protein modifications. LC-MS/MS analyses reveals that these modifications occur selectively on lysine and glutamic acid residues of the target protein, and that these modifications occur within identifiable “electrophile binding motifs” within the protein. These motifs are found within lysine-rich regions of the protein and appear to be target sites of 1,4-BQ-thioether adduction. These residues also appear to dictate the nature of post-adduction chemistry and the final structure of the adduct. This model system will provide critical insight for in vivo adduct hunting following exposure to 1,4-BQ-thioethers, but the general approaches can also be extended to the identification of protein adducts derived from other classes of reactive electrophiles.
doi:10.1007/978-1-60761-849-2_19
PMCID: PMC4120700  PMID: 20972762
Cytochrome c; 1,4-Benzoquinone; LC-MS/MS; 2-(N-acetylcystein-S-yl)-1,4-benzoquinone; Post-translational modifications; Trypsin solution digest
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.
doi:10.1021/tx3002675
PMCID: PMC3431021  PMID: 22827705
3.  Bioinformatic Analysis of 302 Reactive Metabolite Target Proteins. Which Ones Are Important for Cell Death? 
Toxicological Sciences  2013;135(2):390-401.
Many low molecular weight compounds undergo biotransformation to chemically reactive metabolites (CRMs) that covalently modify cellular proteins. However, the mechanisms by which this covalent binding leads to cytotoxicity are not understood. Prior analyses of lists of target proteins sorted by functional categories or hit frequency have not proven informative. In an attempt to move beyond covalent binding, we hypothesized that xenobiotic posttranslational modification of proteins might disrupt important protein-protein interactions (PPIs) and thereby direct cells from homeostasis into cell death pathways. To test this hypothesis, we analyzed a list of 302 proteins (66% rat, 26% mouse, 5% human) known to be targeted by 41 different cytotoxic CRMs. Human orthologs of rodent proteins were found by blast sequence alignment, and their interacting partners were found using the Human Protein Reference Database. The combined set of target orthologs and partners was sorted into KEGG pathways and Gene Ontology categories. Those most highly ranked based on sorting statistics and toxicological relevance were heavily involved with intracellular signaling pathways, protein folding, unfolded protein response, and regulation of apoptosis. Detailed examination revealed that many of the categories were flagged primarily by partner proteins rather than target proteins and that a majority of these partners interacted with just a small number of proteins in the CRM target set. A similar analysis performed without the partner proteins flagged very few categories as significant. These results support the hypothesis that disruption of important PPIs may be a major mechanism contributing to CRM-induced acute cytotoxicity.
doi:10.1093/toxsci/kft166
PMCID: PMC3807624  PMID: 23897987
reactive metabolites; protein covalent binding; protein-protein interaction; cytotoxicity.
4.  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
5.  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
6.  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
7.  In Vivo Effects of Ozone Exposure on Protein Adduct Formation by 1-Nitronaphthalene in Rat Lung 
The incidence of serious photochemical smog events is steadily growing in urban environments around the world. The electrophilic metabolites of 1-nitronaphthalene (1-NN), a common air pollutant in urban areas, have been shown to bind covalently to proteins. 1-NN specifically targets the airway epithelium, and the toxicity is synergized by prior long-term ozone exposure in rat. In this study we investigated the formation of 1-NN protein adducts in the rat airway epithelium in vivo and examined how prior long-term ozone exposure affects adduct formation. Eight adducted proteins, several involved in cellular antioxidant defense, were identified. The extent of adduction of each protein was calculated, and two proteins, peroxiredoxin 6 and biliverdin reductase, were adducted at high specific activities (0.36–0.70 and 1.0 nmol adduct/nmol protein). Furthermore, the N-terminal region of calreticulin, known as vasostatin, was adducted only in ozone-exposed animals. Although vasostatin was adducted at relatively low specific activity (0.01 nmol adduct/nmol protein), the adduction only in ozone-exposed animals makes it a candidate protein for elucidating the synergistic toxicity between ozone and 1-NN. These studies identified in vivo protein targets for reactive 1-NN metabolites that are potentially associated with the mechanism of 1-NN toxicity and the synergistic effects of ozone.
doi:10.1165/rcmb.2005-0047OC
PMCID: PMC2715307  PMID: 15845863
1-nitronaphthalene; calreticulin; ozone; protein adduct; proteomics
8.  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.
doi:10.1021/tx800093k
PMCID: PMC2493440  PMID: 18547066
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.  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.
doi:10.1021/tx300103j
PMCID: PMC3358484  PMID: 22462724
12.  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
13.  Effects of Fatty Acids on Benzo[a]pyrene Uptake and Metabolism in Human Lung Adenocarcinoma A549 Cells 
PLoS ONE  2014;9(3):e90908.
Dietary supplementation with natural chemoprotective agents is receiving considerable attention because of health benefits and lack of toxicity. In recent in vivo and in vitro experimental studies, diets rich in n-3 polyunsaturated fatty acids have been shown to provide significant anti-tumor action. In this investigation, the effects of control fatty acids (oleic acid (OA), linoleic acid (LA)) and n-3 PUFA, e.g., docosahexaenoic acid (DHA) on the uptake and metabolism of the carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP) was investigated in A549 cells, a human adenocarcinoma alveolar basal epithelial cell line. A549 cells activate BaP through the cytochrome P450 enzyme system to form reactive metabolites, a few of which covalently bind to DNA and proteins. Therefore, multiphoton microscopy spectral analysis combined with linear unmixing was used to identify the parent compound and BaP metabolites formed in cells, in the presence and absence of fatty acids. The relative abundance of select metabolites was associated with altered P450 activity as determined using ethoxyresorufin-O-deethylase activity in cells cultured in the presence of BSA-conjugated fatty acids. In addition, the parent compound within cellular membranes increases significantly in the presence of each of the fatty acids, with the greatest accumulation observed following DHA treatment. DHA treated cells exhibit significantly lower pyrene-like metabolites indicative of lower adducts including DNA adducts compared to control BSA, OA or LA treated cells. Further, DHA reduced the abundance of the proximate carcinogen BaP 7,8-dihydrodiol and the 3-hydroxybenzo[a]pyene metabolites compared to other treatments. The significant changes in BaP metabolites in DHA treated cells may be mediated by the effects on the physicochemical properties of the membrane known to affect enzyme activity related to phase I and phase II metabolism. In summary, DHA is a highly bioactive chemo-protective agent capable of modulating BaP-induced DNA adducts.
doi:10.1371/journal.pone.0090908
PMCID: PMC3961214  PMID: 24651647
14.  Systems Analysis of Protein Modification and Cellular Responses Induced by Electrophile Stress 
Accounts of Chemical Research  2010;43(5):673-683.
Biological electrophiles result from oxidative metabolism of exogenous compounds or endogenous cellular constituents, and they contribute to pathophysiologies such as toxicity and carcinogenicity. The chemical toxicology of electrophiles is dominated by covalent addition to intracellular nucleophiles. Reaction with DNA leads to the production of adducts that block replication or induce mutations. The chemistry and biology of electrophile−DNA reactions have been extensively studied, providing in many cases a detailed understanding of the relation between adduct structure and mutational consequences. By contrast, the linkage between protein modification and cellular response is poorly understood.
In this Account, we describe our efforts to define the chemistry of protein modification and its biological consequences using lipid-derived α,β-unsaturated aldehydes as model electrophiles. In our global approach, two large data sets are analyzed: one represents the identity of proteins modified over a wide range of electrophile concentrations, and the second comprises changes in gene expression observed under similar conditions. Informatics tools show theoretical connections based primarily on transcription factors hypothetically shared between the two data sets, downstream of adducted proteins and upstream of affected genes. This method highlights potential electrophile-sensitive signaling pathways and transcriptional processes for further evaluation.
Peroxidation of cellular phospholipids generates a complex mixture of both membrane-bound and diffusible electrophiles. The latter include reactive species such as malondialdehyde, 4-oxononenal, and 4-hydroxynonenal (HNE). Enriching HNE-adducted proteins for proteomic analysis was a technical challenge, solved with click chemistry that generated biotin-tagged protein adducts. For this purpose, HNE analogues bearing terminal azide or alkyne functionalities were synthesized. Cellular lysates were first exposed to a single type of HNE analogue (azido- or alkynyl-HNE), and then click reactions were performed against the cognate alkynyl- and azido-biotin derivative. The resulting biotin-labeled proteins were captured and enriched over a streptavidin matrix for subsequent mass spectrometric analysis. We thereby identified a multitude of HNE targets. Simultaneous microarray analysis of changes in gene expression triggered by HNE also produced an abundance of data. Functional analysis of both data sets generated the hypothesis that an important pathway of cellular response derives from electrophile modification of protein chaperones, resulting in the release of transcription factors that are their clients. Informatic analysis of the protein modification and microarray data sets identified several transcription factors as potential mediators of the cellular response to HNE-adducted proteins. Among these, heat shock factor 1 (HSF1) was confirmed as a sensitive and robust effector of HNE-induced changes in gene expression. Activation of HSF1 appears, in part, to be mediated by the electrophilic adduction of Hsp70 and Hsp90, which normally maintain HSF1 in an inactive cytosolic complex.
The identification of HSF1 as a mediator of biological effects downstream of HSF1 has provided new opportunities for research, illustrating the potential of our systems-based approach. Accordingly, we characterized HSF1-mediated gene expression in protecting against electrophile-induced toxicity. Among the genes induced by HSF1, Bcl-2- associated athanogene 3 (BAG3) is notable for its actions in promoting cell survival through stabilization of antiapoptotic Bcl-2 proteins, appearing to have a critical role in mediating cellular protection against electrophile-induced death.
doi:10.1021/ar900286y
PMCID: PMC2873822  PMID: 20218676
15.  Degraded protein adducts of cis-2-butene-1,4-dial are urinary and hepatocyte metabolites of furan 
Chemical research in toxicology  2009;22(6):997-1007.
Furan is a liver toxicant and carcinogen in rodents. Based on these observations and the large potential for human exposure, furan has been classified as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. However, the toxicity requires cytochrome P450 catalyzed oxidation of furan. The product of this oxidation, cis-2-butene-1,4-dial (BDA), reacts readily with glutathione, amino acids and DNA and is a bacterial mutagen in Ames assay strain TA104. Characterization of the urinary metabolites of furan is expected to provide information regarding the structure(s) of the reactive metabolite(s). Recently, several urinary metabolites have been identified. We reported the presence of a mono-glutathione-BDA reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine cyclic sulfide. Three additional urinary metabolites of furan were also characterized: R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-L-cysteine and its sulfoxide. It was postulated that these three metabolites are derived from degraded protein adducts. However, the possibility that these metabolites result from reaction of BDA with free lysine and/or cysteine was not ruled out. In this latter case, one might predict that the reaction of thiol-BDA with free lysine would not occur exclusively on the ε-amino group. Reaction of BDA with N-acetylcysteine or GSH in the presence of lysine indicated that both the α- and ε-amino groups of lysine can be modified by thiol-BDA. The N-acetylcysteine-BDA-N-acetyllysine urinary metabolites were solely linked through the ε-amino group of lysine. A GSH-BDA-lysine crosslink was a significant hepatocyte metabolite of furan. In this case, the major product resulted from reaction with the ε-amino group of lysine, however, small amounts of the α-amino reaction product were also observed. Western analysis of liver and hepatocyte protein extracts using anti-GSH antibody indicated that GSH was covalently linked to proteins in tissues or cells exposed to furan. Our data support the hypothesis that GSH-BDA can react with either free lysine or protein lysine groups. These data suggest that there are multiple pathways by which furan can modify cellular nucleophiles. In one pathway, BDA reacts directly with proteins to form cysteine-lysine reaction products. In another, BDA reacts with GSH to form GSH-BDA conjugates which then reacts with cellular nucleophiles like free lysine or lysine moieties in proteins. Both pathways will give rise to N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-L-cysteine. Given the abundance of these metabolites in urine of furan-treated rats, these pathways appear to be major pathways of furan biotransformation in vivo.
doi:10.1021/tx800377v
PMCID: PMC2696637  PMID: 19441776
furan; metabolism; stable isotope methods; capillary LC-MS; urinary metabolites; GSH; hepatocyte metabolites
16.  MOLECULAR MECHANISM OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) INACTIVATION BY α,β-UNSATURATED CARBONYL DERIVATIVES 
Chemical research in toxicology  2011;24(12):2302-2311.
α,β-Unsaturated carbonyls are an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these datagaps, purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was incubated with acrylamide (ACR), acrolein or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration-and time-dependent manner. The rank order of enzyme inhibition (KI); i.e., ACR << MVK < acrolein, was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pKa-lowering microenvironment (pKa = 6.03) and, at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate-state. These data suggest a general cytotoxic mechanism where electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, since chemical derivitization causes loss of protein function.
doi:10.1021/tx200437y
PMCID: PMC3243798  PMID: 22084934
α,β-unsaturated carbonyl chemicals; protein adduct formation; cytotoxicity; electrophile toxicity; nucleophile targets
17.  Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts 
Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5–11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.
doi:10.1016/j.nimb.2009.10.164
PMCID: PMC2864045  PMID: 20454606
Free flow electrophoresis; FFE; AMS; protein separation
18.  Metabolism and Toxicity of Thioacetamide and Thioacetamide S-Oxide in Rat Hepatocytes 
Chemical research in toxicology  2012;25(9):1955-1963.
The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrilobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects TA requires oxidative bioactivation leading first to its S-oxide (TASO) and then to its chemically reactive S,S-dioxide (TASO2) which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis, and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 hr. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole, and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [14C]-TASO is inhibited by unlabeled TA which acts as a “cold-trap” for [14C]-TA and prevents its re-oxidation to [14C]-TASO. This in turn increases the net consumption of [14C]-TASO despite the fact that its oxidation to TASO2 is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification.
doi:10.1021/tx3002719
PMCID: PMC3444651  PMID: 22867114
19.  Nitroimidazole Action in Entamoeba histolytica: A Central Role for Thioredoxin Reductase 
PLoS Biology  2007;5(8):e211.
Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms.
Author Summary
The protist parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia intestinalis grow in environments with low oxygen concentration. Infections with these parasites are commonly treated with metronidazole, a nitroimidazole drug that must be reduced for activation, resulting in several toxic metabolites. We examined the soluble proteome of metronidazole-treated E. histolytica cells for target proteins of these metabolites, applying two-dimensional gel electrophoresis and mass spectrometry. Of about 1,500 proteins visualized, only five formed covalent adducts with metronidazole metabolites, including thioredoxin, thioredoxin reductase, and superoxide dismutase. Metronidazole-bound thioredoxin reductase displayed diminished activity. In addition to these proteins, small thiol molecules, including cysteine, formed adducts with metronidazole. Supplementation with cysteine allowed the cells to survive otherwise lethal metronidazole concentrations. Finally, we discovered that one of the modified proteins, thioredoxin reductase, reduces metronidazole, suggesting a central role for this enzyme with regard to metronidazole toxicity. Taken together, our work reveals a new area of molecular interactions of activated metronidazole with cellular components. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could also occur in other eukaryotic or prokaryotic organisms.
Metronidazole is used for treatment of infections with microaerophilic protist parasites. Here, a new model of metronidazole activation is proposed, with a central role for thioredoxin reductase.
doi:10.1371/journal.pbio.0050211
PMCID: PMC1933457  PMID: 17676992
20.  Proteomic snapshot of the EGF-induced ubiquitin network 
In this work, the authors report the first proteome-wide analysis of EGF-regulated ubiquitination, revealing surprisingly pervasive growth factor-induced ubiquitination across a broad range of cellular systems and signaling pathways.
Epidermal growth factor (EGF) triggers a novel ubiquitin (Ub)-based signaling cascade that appears to intersect both housekeeping and regulatory circuitries of cellular physiology.The EGF-regulated Ubiproteome includes scores ubiquitinating and deubiquitinating enzymes, suggesting that the Ub signal might be rapidly transmitted and amplified through the Ub machinery.The EGF-Ubiproteome overlaps significantly with the EGF-phosphotyrosine proteome, pointing to a possible crosstalk between these two signaling mechanisms.The significant number of biological insights uncovered in our study (among which EphA2 as a novel, downstream ubiquitinated target of EGF receptor) illustrates the general relevance of such proteomic screens and calls for further analysis of the dynamics of the Ubiproteome.
Ubiquitination is a process by which one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins by E3 ligases. Deubiquitinating enzymes (DUBs) revert Ub conjugation, thus ensuring a dynamic equilibrium between pools of ubiquitinated and deubiquitinated proteins (Amerik and Hochstrasser, 2004). Traditionally, ubiquitination has been associated with protein degradation; however, it is now becoming apparent that this post-translation modification is an important signaling mechanism that can modulate the function, localization and protein/protein interaction abilities of targets (Mukhopadhyay and Riezman, 2007; Ravid and Hochstrasser, 2008).
One of the best-characterized signaling pathways involving ubiquitination is the epidermal growth factor (EGF)-induced pathway. Upon EGF stimulation, a variety of proteins are subject to Ub modification. These include the EGF receptor (EGFR), which undergoes both multiple monoubiquitination (Haglund et al, 2003) and K63-linked polyubiquitination (Huang et al, 2006), as well as components of the downstream endocytic machinery, which are modified by monoubiquitination (Polo et al, 2002; Mukhopadhyay and Riezman, 2007). Ubiquitination of the EGFR has been shown to have an impact on receptor internalization, intracellular sorting and metabolic fate (Acconcia et al, 2009). However, little is known about the wider impact of EGF-induced ubiquitination on cellular homeostasis and on the pleiotropic biological functions of the EGFR. In this paper, we attempt to address this issue by characterizing the repertoire of proteins that are ubiquitinated upon EGF stimulation, i.e., the EGF-Ubiproteome.
To achieve this, we employed two different purification procedures (endogenous—based on the purification of proteins modified by endogenous Ub from human cells; tandem affinity purification (TAP)—based on the purification of proteins modified by an ectopically expressed tagged-Ub from mouse cells) with stable isotope labeling with amino acids in cell culture-based MS to obtain both steady-state Ubiproteomes and EGF-induced Ubiproteomes. The steady-state Ubiproteomes consist of 1175 and 582 unambiguously identified proteins for the endogenous and TAP approaches, respectively, which we largely validated. Approximately 15% of the steady-state Ubiproteome was EGF-regulated at 10 min after stimulation; 176 of 1175 in the endogenous approach and 105 of 582 in the TAP approach. Both hyper- and hypoubiquitinated proteins were detected, indicating that EGFR-mediated signaling can modulate the ubiquitin network in both directions. Interestingly, many E2, E3 and DUBs were present in the EGF-Ubiproteome, suggesting that the Ub signal might be rapidly transmitted and amplified through the Ub machinery. Moreover, analysis of Ub-chain topology, performed using mass spectrometry and specific abs, suggested that the K63-linkage was the major Ub-based signal in the EGF-induced pathway.
To obtain a higher-resolution molecular picture of the EGF-regulated Ub network, we performed a network analysis on the non-redundant EGF-Ubiproteome (265 proteins). This analysis revealed that in addition to well-established liaisons with endocytosis-related pathways, the EGF-Ubiproteome intersects many circuitries of intracellular signaling involved in, e.g., DNA damage checkpoint regulation, cell-to-cell adhesion mechanisms and actin remodeling (Figure 5A).
Moreover, the EGF-Ubiproteome was enriched in hubs, proteins that can establish multiple protein/protein interaction and thereby regulate the organization of networks. These results are indicative of a crosstalk between EGFR-activated pathways and other signaling pathways through the Ub-network.
As EGF binding to its receptor also triggers a series of phosphorylation events, we examined whether there was any overlap between our EGF-Ubiproteome and published EGF-induced phosphotyrosine (pY) proteomes (Blagoev et al, 2004; Oyama et al, 2009; Hammond et al, 2010). We observed a significant overlap between ubiquitinated and pY proteins: 23% (61 of 265) of the EGF-Ubiproteome proteins were also tyrosine phosphorylated. Pathway analysis of these 61 Ub/pY-containing proteins revealed a significant enrichment in endocytic and signal-transduction pathways, while ‘hub analysis' revealed that Ub/pY-containing proteins are enriched in highly connected proteins to an even greater extent than Ub-containing proteins alone. These data point to a complex interplay between the Ub and pY networks and suggest that the flow of information from the receptor to downstream signaling molecules is driven by two complementary and interlinked enzymatic cascades: kinases/phosphatases and E3 ligases/DUBs.
Finally, we provided a proof of principle of the biological relevance of our EGF-Ubiproteome. We focused on EphA2, a receptor tyrosine kinase, which is involved in development and is often overexpressed in cancer (Pasquale, 2008). We started from the observation that EphA2 is present in the EGF-Ubiproteome and that proteins of the EGF-Ubiproteome are enriched in the Ephrin receptor signaling pathway(s). We confirmed the MS data by demonstrating that the EphA2 is ubiquitinated upon EGF stimulation. Moreover, EphA2 also undergoes tyrosine phosphorylation, indicating crosstalk between the two receptors. The EGFR kinase domain was essential for these modifications of EphA2, and a partial co-internalization with EGFR upon EGF activation was clearly detectable. Finally, we demonstrated by knockdown of EphA2 in MCF10A cells that this receptor is critically involved in EGFR biological outcomes, such as proliferation and migration (Figure 7).
Overall, our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected and highlight the ‘resource' feature of our EGF-Ubiproteome.
The activity, localization and fate of many cellular proteins are regulated through ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins. While Ub-conjugated and Ub-associated proteomes have been described, we lack a high-resolution picture of the dynamics of ubiquitination in response to signaling. In this study, we describe the epidermal growth factor (EGF)-regulated Ubiproteome, as obtained by two complementary purification strategies coupled to quantitative proteomics. Our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitinating and deubiquitinating enzymes, transporters and proteins involved in translation and transcription. The Ub-based signaling network appears to intersect both housekeeping and regulatory circuitries of cellular physiology. Finally, as proof of principle of the biological relevance of the EGF-Ubiproteome, we demonstrated that EphA2 is a novel, downstream ubiquitinated target of epidermal growth factor receptor (EGFR), critically involved in EGFR biological responses.
doi:10.1038/msb.2010.118
PMCID: PMC3049407  PMID: 21245847
EGF; network; proteomics; signaling; ubiquitin
21.  Electrophilic Adduction of Ubiquitin Activating Enzyme E1 by N,N-Diethyldithiocarbamate Inhibits Ubiquitin Activation and is Accompanied by Striatal Injury in the Rat 
Chemical research in toxicology  2012;25(11):2310-2321.
Previous studies have shown ubiquitin activating enzyme E1 to be sensitive to adduction through both Michael addition and SN2 chemistry in vitro. E1 presents a biologically important putative protein target for adduction due to its role in initiating ubiquitin based protein processing and the involvement of impaired ubiquitin protein processing in two types of familial Parkinson's disease. We tested whether E1 is susceptible to xenobiotic-mediated electrophilic adduction in vivo and explored the potential contribution of E1 adduction to neurodegenerative events in an animal model. N,N-Diethyldithiocarbamate (DEDC) was administered to rats using a protocol that produces covalent cysteine modifications in vivo and brain E1 protein adducts were characterized and mapped using shotgun LC-MS/MS. E1 activity, global and specific protein expression, and protein carbonyls were used to characterize cellular responses and injury in whole brain and dorsal striatal samples. The data demonstrate that DEDC treatment produced S-(ethylaminocarbonyl) adducts on Cys234 and Cys179 residues of E1 and decreased the levels of activated E1 and total ubiquitinated proteins. Proteomic analysis of whole brain samples identified expression changes for proteins involved in myelin structure, antioxidant response and catechol metabolism, systems often disrupted in neurodegenerative disease. Our studies also delineated localized injury within the striatum as indicated by decreased levels of tyrosine hydroxylase, elevated protein carbonyl content, increased antioxidant enzyme and α-synuclein expression, as well as enhanced phosphorylation of tau and tyrosine hydroxylase. These data are consistent with E1 having similar susceptibility to adduction in vivo as previously reported in vitro and support further investigation into environmental agent adduction of E1 as a potential contributing factor to neurodegenerative disease. Additionally this study supports the predictive value of in vitro screens for identifying sensitive protein targets that can be used to guide subsequent in vivo experiments.
doi:10.1021/tx300198h
PMCID: PMC3502677  PMID: 22874009
22.  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
23.  Reactive Intermediates: Molecular and MS-Based Approaches to Assess the Functional Significance of Chemical:Protein Adducts1 
Toxicologic pathology  2012;41(2):315-321.
Biologically reactive intermediates formed as endogenous products of various metabolic processes are considered important factors in a variety of human diseases, including Parkinson’s disease and other neurological disorders, diabetes and complications thereof, and other inflammatory-associated diseases. Chemical-induced toxicities are also frequently mediated via the bioactivation of relatively stable organic molecules to reactive electrophilic metabolites. Indeed, chemical-induced toxicities have long been known to be associated with the ability of electrophilic metabolites to react with a variety of targets within the cell, including their covalent adduction to nucleophilic residues in proteins, and nucleotides within DNA. Although we possess considerable knowledge of the various biochemical mechanisms by which chemicals undergo metabolic bioactivation, we understand far less about the processes that couple bioactivation to toxicity. Identifying specific sites within a protein that are targets for adduction can provide the initial information necessary to determine whether such adventitious post-translational modifications significantly alter either protein structure and/or function. To address this problem we have developed MS-based approaches to identify specific amino acid targets of electrophile adduction (electrophile-binding motifs), coupled with molecular modeling of such adducts, to determine the potential structural and functional consequences. Where appropriate, functional assays are subsequently conducted to assess protein function.
doi:10.1177/0192623312467399
PMCID: PMC4007760  PMID: 23222993
Bioactivation; chemical-induced toxicities; covalent binding; drug metabolism; protein adducts; proteomics; reactive intermediates
24.  CHARACTERIZATION OF BILIARY CONJUGATES OF 4,4'-METHYLENEDIANILINE IN MALE VERSUS FEMALE RATS 
Toxicology and applied pharmacology  2008;232(2):190-202.
4,4’-Methylenedianiline (4,4’-diaminodiphenylmethane; DAPM) is an aromatic diamine used in the production of numerous polyurethane foams and epoxy resins. Previous studies in rats revealed that DAPM initially injures biliary epithelial cells of the liver, that the toxicity is greater in female than in male rats, and that the toxic metabolites of DAPM are excreted into bile. Since male and female rats exhibit differences in the expression of both phase I and phase II enzymes, our hypothesis was that female rats either metabolize DAPM to more toxic metabolites or have a decreased capacity to conjugate metabolites to less toxic intermediates. Our objective was thus to isolate, characterize, and quantify DAPM metabolites excreted into bile in both male and female bile duct-cannulated Sprague Dawley rats. The rats were gavaged with [14C]-DAPM, and the collected bile was subjected to reversed-phase HPLC with radioisotope detection. Peaks eluting from HPLC were collected and analyzed using electrospray MS, NMR and FT-IR spectroscopy. HPLC analysis indicated numerous metabolites in both sexes, but male rats excreted greater amounts of glutathione and glucuronide conjugates than females. Electrospray MS and NMR spectra of HPLC fractions revealed that the most prominent metabolite found in bile of both sexes was a glutathione conjugate of an imine metabolite of a 4’-nitroso-DAPM. Seven other metabolites were identified, including acetylated, cysteinyl-glycine, glutamyl-cysteine, glycine, and glucuronide conjugates. While our prior studies demonstrated increased covalent binding of DAPM in the liver and bile of female compared to male rats, in these studies, SDS-PAGE with autoradiography revealed 4–5 radiolabeled protein bands in the bile of rats treated with [14C]-DAPM. In addition, these bands were much more prominent in female than in male rats. These studies thus suggest that a plausible mechanism for the increased sensitivity of female rats to DAPM toxicity may be decreased conjugation of reactive DAPM metabolites, leading to greater levels of protein adduct formation.
doi:10.1016/j.taap.2008.06.008
PMCID: PMC2614345  PMID: 18692083
Methylene dianiline; bile duct cells; xenobiotic metabolism; sex differences; conjugation; glutathione; biliary excretion
25.  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

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