A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets

Chem. Res. Toxicol. 2007, 20, 511-519

511

A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets in Rat Liver Cytosol in Vivo Yakov M. Koen,† Natalia V. Gogichaeva,‡ Michail A. Alterman,‡,§ and Robert P. Hanzlik*,† Department of Medicinal Chemistry and Analytical Proteomics Laboratory, UniVersity of Kansas, 1251 Wescoe Hall DriVe, Lawrence, Kansas 66045-7582 ReceiVed NoVember 10, 2006

Metabolic activation and protein covalent binding are early and apparently obligatory events in the cytotoxicity of many simple organic chemicals including drugs and natural products. Although much has been learned about the chemistry of reactive metabolite formation and reactivity toward protein nucleophiles, progress in identifying specific protein targets for reactive metabolites of various protoxins has been much slower. We previously reported nine microsomal and three cytosolic proteins as targets for reactive metabolites of bromobenzene in rat liver. These results, and contemporary work by others, indicate that protein covalent binding is not totally random in cells. Moreover, as protein targets for other protoxins were identified, little commonality of target proteins became apparent. In the present work, we used two-dimensional gel electrophoresis to separate liver cytosolic proteins from rats treated with 14C-bromobenzene; 110 of the 836 observed spots contained measurable radioactivity that varied over a 600-fold range of adduct density. Of these 110 spots, in-gel digestion coupled with mass spectrometry identified apparently single proteins in 57 spots. A few other spots clearly contained more than one identifiable protein, and in several cases, the same protein was identified in several spots having different apparent molecular masses and/or pI. Altogether, 33 unique new protein targets for bromobenzene metabolites were identified and compared to those known for acetaminophen, naphthalene, butylated hydroxytoluene, benzene, thiobenzamide, and halothane via a target protein database available at http:// tpdb.medchem.ku.edu:8080/protein_database/. With increasing numbers of target proteins becoming known, more commonality in targeting by reactive metabolites from diverse chemical agents may be seen. Such commonality may help to separate toxicologically significant covalent binding events from a background of covalent binding that is toxicologically inconsequential. Introduction Many simple organic compounds that have no obvious complementarity to pharmacological receptors and no obvious ability to inhibit the active sites of enzymes critical to cell survival are nevertheless able to cause significant tissue damage in an acute and organ-specific manner. The occurrence of this phenomenon is a significant obstacle in drug discovery and development (1-3) and a potential concern for individuals who become exposed to such chemicals (4-6). A significant step in understanding the mechanism of such tissue damage occurred in the early 1970s with the discovery that the hepatotoxicity associated with bromobenzene and acetaminophen depends on their metabolic conversion to chemically reactive intermediates capable of covalently binding to hepatocellular proteins (711). Liver, kidney, and lungs are the tissues most commonly affected by organotropic cytotoxic chemicals, and it is no coincidence that these tissues contain high levels of xenobioticmetabolizing enzymes, particularly cytochromes P450. While organ damage is a complex process that may involve several cell types within a given tissue (4, 12-15), it is equally clear that metabolism-dependent chemical cytotoxicity can be ob* To whom correspondence should be addressed. Fax: 785-864-5326. E-mail: [email protected]. † Department of Medicinal Chemistry. ‡ Analytical Proteomics Laboratory. § Current address: Center for Biologics Evaluation and Research, FDA, NIH Building 29A, Room 2D12, HFM-735, 8800 Rockville Pike, Bethesda, MD 20892.

served in isolated cell preparations (16-19) and on a time scale too short to invoke normal apoptotic responses (20, 21). Several types of chemically reactive metabolites have been implicated in protein covalent binding and the resulting cytotoxicity (3, 22). Oxidative enzymes, especially cytochromes P450, can generate epoxides, quinones, and Michael acceptors from relatively unreactive precursors. These alkylating intermediates often react preferentially with protein sulfhydryl groups, but alkylations of protein N-terminal amino groups and the nucleophilic side chains of lysine, histidine, methionine, tryptophan, and glutamate/aspartate are also known. On the other hand, acylating metabolites with a strong preference for lysine side chains are generated in some P450-catalyzed O-dealkylations, by sequential S-oxidation of thioamides and thioureas, and in the β-lyase-induced fragmentation of certain S-(haloalkyl)cysteines. Such diversity of electrophiles, coupled with the large number of individual proteins having multiple potential target sites, means that “protein covalent binding” usually results in a rather complicated mixture, the totality of which, for many compounds, correlates anatomically, histologically, temporally, and quantitatively with cell/tissue damage. While it is not hard to imagine that such covalent binding events constitute “microlesions” that collectively damage individual cells and hence tissues, it is also not hard to imagine that some such events might have no detectable toxic consequences for the cell. Indeed, a number of compounds are known whose metabolism leads to significant levels of total covalent binding without causing apparent cytotoxicity (23-25).

10.1021/tx6003166 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/17/2007

512 Chem. Res. Toxicol., Vol. 20, No. 3, 2007

A significant question that arises in this light is “for a given chemical toxicant, which adducts are toxicologically significant?” Our laboratory has long studied the metabolism and covalent binding of the model hepatotoxin bromobenzene (2628). As a step toward identifying toxicologically relevant adducts, we recently reported the identification of nine microsomal (29, 30) and three cytosolic (31) proteins that become adducted by bromobenzene metabolites in vivo. In this manuscript, we extend this work and report the identification of another 33 cytosolic protein targets of bromobenzene metabolites and show that protein targeting is remarkably selective among liver proteins. We also compare bromobenzene target proteins to those reported as targets for six other metabolically activated cytotoxic chemicals with a view toward finding a common subset of toxicologically significant target proteins for electrophilic metabolites.

Experimental Procedures [14C]Bromobenzene

Materials. (5.17 Ci/mol) was prepared in our laboratory, stored, and handled as described previously (29, 32). Trypsin, sequencing grade, from bovine pancreas was obtained from Roche (Nutley, NJ). Sequenal grade urea and CHAPS were obtained from Pierce (Rockford, IL). Dithiothreitol (DTT) and phenylmethylsulfonylfluoride (PMSF) were obtained from Sigma (St. Louis, MO). 4-Vinylpyridine was obtained from Sigma and was distilled and stored under nitrogen at -20 °C. Tris, SDS, glycine, Sequi-blot PVDF membranes (0.2 µm), Bradford reagent, broad range IEF Standards, and Precision Protein Standards were obtained from Bio-Rad (Hercules, CA). All other electrophoresis supplies were obtained from Amersham Biosciences (Uppsala, Sweden). HPLC grade solvents and analytical grade inorganic salts were obtained from Fisher (Fair Lawn, NJ). Deionized water (resistivity 18.2 MΩ/cm) was used for preparation of all buffers. Treatment of Animals and Preparation of Subcellular Fractions. Male Sprague Dawley rats (150-170 g, Charles River Laboratories, Wilmington, MA) were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle and ad libitum access to food and water. After acclimating for at least 3 days, animals were given three daily ip injections of sodium phenobarbital (80 mg/kg) in 0.9% saline (4.0 mL/kg). After the third dose, the animals weighed 183-195 g. Food was withheld overnight, and the next morning, the rats were ip injected with [14C]bromobenzene (2 mmol/kg) in corn oil (1 mL/kg) and 4 h later killed by decapitation under CO2 narcosis. The livers were removed, and subcellular fractions were isolated as previously described (29). In brief, the livers were chilled in ice-cold solution, containing 50 mM potassium phosphate, pH 7.4, 0.15 M KCl, 5 mM EDTA, and 1 mM PMSF (buffer A), minced on an ice-cold glass plate, pooled, and homogenized in ice-cold buffer A (3 mL/g tissue) using a motor-driven Teflon glass homogenizer. All subsequent steps were carried out at 4 °C. The homogenate was successively centrifuged at 6500gave (10 min), 25000gave (20 min), and 100000gmax (60 min). The 100000g supernatant (cytosol) was clarified by recentrifugation at 100000g and further dialyzed against 20 mM potassium phosphate, pH 7.4, containing 0.5 mM DTT (4 × 60 volumes), to remove reversibly bound radioactivity. The dialyzed cytosol was aliquoted and stored at -70 °C. The six rats (total body weight, 1106 g) furnished a total of 43 g of liver, from which 69 mL of final clear cytosol containing 18.7 mg protein/mL was obtained. Determination of Covalently Bound Radiolabel. Four 100 µL aliquots of the dialyzed cytosol were each diluted to 500 µL with buffer A. The diluted samples were treated with cold 20% trichloroacetic acid (500 µL), and the precipitates were successively washed with cold acetone (800 µL), methanol/water (80:20 v/v, 1 mL; three times), acetone (1 mL), and diethyl ether (1 mL). They were then dried by rotary evaporation, dissolved in 1 mL of 1 M NaOH, and neutralized with an equal volume of 1 M HCl. After this, the radioactivity was measured in duplicate by scintillation counting and protein was determined by Bradford assay (Bio-Rad).

Koen et al. Two-Dimensional Electrophoresis (2DE) and Phosphorimaging. Electrophoretic separations were performed using a flatbed Multiphor II unit equipped with programmable Power Supply EPS 3501 and IEF Kit. Immobiline DryStrips pH 3-10 NL, 18 cm, were used for the first dimension, and ExcelGel XL SDS 1214, 24.5 cm × 18 cm, were for the second dimension. Electrophoresis was performed according to the manufacturer’s instructions with the following modifications. Aliquots of the cytosolic fraction (55 µL, 1 mg protein) were mixed with 330 µL of Destreak Rehydration Solution each, and the mixture was incubated at ambient temperature for 60 min, after which 350 µL of the resulting solubilizate (935 µg protein) was applied to the entire IPG strip by placing the sample with the strip in a rehydration tray for 16 h at ambient temperature. Isoelectrofocusing was conducted at 20 °C using the following voltage gradient: 0-300 V, 1 min; 300 V, 3 h; 300-1000 V, 2 h; 1000-3500 V, 1 h; and 3500 V, 17 h (total 64 kV h). After focusing, the strips were incubated for 20 min with standard SDS-PAGE equilibration solution (50 mM Tris-HCl, pH 6.8, 6 M urea, 30% glycerol, and 2% SDS) supplemented with 130 mM DTT, followed by incubation for 20 min with the same solution containing 200 mM 4-vinylpyridine and 0.005% bromophenol blue in place of DTT. The strips were then applied to the flat-bed SDS gel, and the proteins were further separated by SDS-PAGE. The separation was carried out at 15 °C at the following conditions: 20 mA, 45 min; 40 mA, 5 min; and 50 mA, 2 h or until the dyefront reached the anode buffer strip. Proteins were visualized by staining with 0.025% Coomassie R250, 30% methanol, and 0.5% acetic acid for 2 h, followed by destaining with 30% methanol. In all separation experiments, broad range pI markers and broad range molecular mass markers (Bio-Rad) were used for calibration of the first and second dimension gels, respectively. The 2DE-separated proteins were transferred from the nonstained SDS gel to a Sequi-blot PVDF membrane (0.2 µm) by electroblotting in semidry conditions using Multiphor II with NovaBlot Kit according to the manufacturer’s instructions. The blots were successively stained with 0.1% Coomassie R250, 50% methanol, and 7% acetic acid, destained with 50% methanol and 7% acetic acid, rinsed with H2O, air-dried, and subjected to phosphorimaging using a Molecular Dynamics storage phosphor screen and Bio-Rad Molecular Imager FX scanning unit and software. The integrated image densities of the radioactive spots on the phosphorimage and of the corresponding spots on the Coomassie-stained PVDF blot were then used for estimation of the radiolabel and protein contents of individual spots as follows. Radioactivity was calculated based on a calibration curve prepared using radioactive standards exposed to the same phosphor storage screen along with the experimental blot. The mol equiv amount of protein-bound 14C was then calculated based on the specific radioactivity of 14C-BB. For protein content, the integrated image density of a Coomassie-stained spot was taken as a fraction of total density of all spots detected on the blot. Then, assuming that the total density corresponds to the total amount of protein applied on the IPG strip, the calculated fractions were converted into µg of protein and then into nmol of protein using molecular mass (MM) values estimated from the 2DE blot. Finally, the average specific labeling density of each spot was expressed as molecules of adduct per 1000 molecules of protein (see Table 1). Altogether, seven replicate 2D gels were run. In-Gel Digestion. Aliquots of the cytosolic fraction (1 mg of protein) were separated by 2DE as described above, and individual protein spots corresponding to radioactive spots on the blot were excised along with a few apparently unlabeled abundant protein spots. To collect a sufficient amount of protein, identical spots from three to five different gels were pooled. The protein was then reduced with DTT, alkylated with 4-vinylpyridine, and in-gel digested with trypsin as described previously (31). The resulting peptide mixtures were collected and stored at -20 °C. As a control, protein-free gel fragments of approximately the same size were processed identically. Mass Spectrometry of Tryptic Digests. The digest samples were analyzed by matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) MS and tandem MS/MS on a Proteomics

Bromobenzene Metabolite Target Proteome

Chem. Res. Toxicol., Vol. 20, No. 3, 2007 513

Table 1. Liver Cytosolic Proteins Identified as Targets of Bromobenzene by In-Gel Digestion and MALDI-TOF MS spot no.a

adduct densityb

1 2 3 4 11 16 17 20 23 23.1 29 30 30.1 31 31.1 37 37.1e 40 41 43 44 45 46 47 48 49 50 50.1 51 51.1 52 53 53.1 54 55 58 61 62 63 63.1 64 64.1h

41.8 5.5 62.7 8.6 0.5 3.1 32.8 15.8 9.0 0.4 4.1 2.1 ND 4.0 0.7 5.6 ND 1.5 14.2 9.4 1.3 16.3 3.5 18.5 40.8 18.0 17.6 3.3 4.9 7.9 1.5 1.1 3.2 4.3 0.8 0.2 16.6 1.8 27.0 7.0 30.9 3.0

65 65.1h

4.3 16.9

66 66.1 67h

4.7 0.3 2.3

68 70h

9.7 2.8

71 72h

1.9 0.9

73 74 75 75.1 76 77h

1.9 5.9 1.6 ND 13.7 11.6

80 81 83 83.1 84 85 86h

12.3 6.8 1.9 4.2 4.0 11.2 25.4

88

4.3

protein name

accession no.

MM (kDa) calcd obsd

thioredoxin thioredoxin fatty acid-binding protein, brain ribonuclease UK114 phosphatidylethanolamine-binding protein proteasome activator complex subunit 1 regucalcin (SMP-30, RC) tropomyosin R-chain, brain-3 26S protease regulatory subunit 6B 26S protease regulatory subunit 6A heat shock cognate 71 kDa protein heat shock cognate 71 kDa protein heat shock cognate 71 kDa protein D-dopachrome decarboxylase D-dopachrome decarboxylase protein DJ-1 (CAP1 protein) peroxiredoxin 6 (1-Cys PRX) guanidinoacetate N-methyltransferase triosephosphate isomerase (TIM) R-enolase (enolase 1) R-enolase (enolase 1) aldehyde dehydrogenase 9A1 aldehyde dehydrogenase 9A1 aldehyde dehydrogenase, mitochondrialf selenium-binding protein 2 selenium-binding protein 2 serum albumin, precursor (608 aa)g serum albumin, precursor (608 aa)g serum albumin, precursor (608 aa)g serum albumin, precursor (608 aa)g serum albumin, precursor (608 aa)g serum albumin, precursor (608 aa)g serum albumin, precursor (608 aa)g macrophage migration inhibitory factor fatty acid-binding protein, liver hemoglobin β-chain, major form triosephosphate isomerase (TIM) glutathione S-transferase µ2 triosephosphate isomerase (TIM) glutathione S-transferase µ2 glutathione S-transferase µ2 glutathione S-transferase µ2; carbonic anhydrase 3; glutathione S-transferase µ1 glutathione S-transferase µ2 glutathione S-transferase µ2; glutathione S-transferase µ1 3-R-hydroxysteroid dehydrogenase carbonic anhydrase 3 glycerol-3-phosphate dehydrogenase [NAD+]; similar to RIKEN cDNA 4931406C07 3-R-hydroxysteroid dehydrogenase glycine N-methyltransferase; sulfotransferase 1B1 sulfotransferase 1B1 sulfotransferase 1B1; glycine N-methyltransferase arginase 1 3-R-hydroxysteroid dehydrogenase 3-R-hydroxysteroid dehydrogenase arginase 1 3-R-hydroxysteroid dehydrogenase carbonic anhydrase 3; 3-R-hydroxysteroid dehydrogenase β-ureidopropionase isocitrate dehydrogenase [NADP] β-ureidopropionase fumarylacetoacetate hydrolase phosphoglycerate kinase 1 selenium-binding protein 2 UDP-glucose 6-dehydrogenase; aldehyde dehydrogenase 1A1; kinesin light chain 3 aldehyde dehydrogenase, cytosolic 1

P11232 P11232 P55051 P52759 P31044 Q63797 Q03336 P04692-5 Q63570 Q63569 P63018 P63018 P63018 P80254 P80254 O88767 O35244 P10868 P48500 P04764 P04764 Q9JLJ3 Q9JLJ3 P11884 Q8VIF7 Q8VIF7 P02770 P02770 P02770 P02770 P02770 P02770 P02770 P30904 P02692 P02091 P48500 P08010 P48500 P08010 P08010 P08010 P14141 P04905 P08010 P08010 P04905 P23457 P14141 O35077 55249800 P23457 P13255 P52847 P52847 P52847 P13255 P07824 P23457 P23457 P07824 P23457 P14141 P23457 Q03248 P41562 Q03248 P25093 P16617 Q8VIF7 O70199 P51647 Q9ESH7 P13601

11.7 11.7 14.9 14.3 20.8 28.6 33.4 32.7 47.4 49.1 70.9 70.9 70.9 13.0 13.0 20.0 24.8 26.4 26.9 47.1 47.1 53.7 53.7 56.5 52.5 52.5 68.7 68.7 68.7 68.7 68.7 68.7 68.7 12.3 14.3 15.8 26.9 25.6 26.9 25.7 25.7 25.7 29.4 25.9 25.7 25.7 25.9 37.0 29.4 37.4 35.0 37.0 32.5 34.8 34.8 34.8 32.5 34.9 37.0 37.0 35.0 37.0 29.4 37.0 44.0 46.7 44.0 45.9 44.6 52.5 54.9 54.5 55.9 54.6

12.6 12.3 13.7 14.0 23.0 28.2 30.6 34.0 48.7 48.1 74.6 71.9 70.6 12.2 12.5 21.5 20.9 25.7 24.9 46.6 46.4 50.6 50.1 50.6 51.3 50.6 68.1 66.6 65.7 64.1 63.9 64.6 65.7 11.2 12.2 12.7 25.0 23.7 24.4 23.5 23.5 23.5 23.5 23.9 32.7 23.4 31.5 32.7 29.9 29.8 29.6 33.8 32.0 32.3 33.6 31.8 32.0 41.7 42.4 40.6 39.4 40.1 50.4 49.8 49.5

pI calcd

obsd

matches

% coveragec

MOWSE scored

4.8 4.8 5.5 7.8 5.5 5.8 5.4 4.7 5.1 5.1 5.4 5.4 5.4 6.1 6.1 6.3 5.6 5.7 6.4 6.2 6.2 6.6 6.6 6.6 6.1 6.1 6.1 6.1 6.1 6.1 6.1 6.1 6.1 7.3 7.8 8.0 6.4 7.3 6.4 6.9 6.9 6.9 6.9 8.3 6.9 6.9 8.3 6.7 6.9 6.3 6.2 6.7 7.1 8.2 8.2 8.2 7.1 6.8 6.7 6.7 6.8 6.7 6.9 6.7 6.5 6.5 6.5 6.7 7.5 6.1 7.5 7.9 6.2 7.1

4.3 4.4 4.6 4.7 5.2 5.2 4.8 4.5 4.9 4.9 5.0 5.1 5.1 5.6 5.6 5.7 5.7 5.9 6.2 5.8 6.0 5.5 5.7 5.9 6.0 6.1 5.6 5.7 5.8 5.8 5.9 6.0 6.1 6.7 6.6 7.5 6.6 6.9 6.9 6.9 7.0 7.0

4 8 15 9 13 13 10 23 22 26 16 36 21 4 7 11 9 18 20 20 24 6 12 5 12 8 16 18 38 19 43 29 35 7 9 10 18 17 18 28 13 10 8 7 15 29 9 11 10 8 12 15 12 9 19 12 8 14 16 13 9 16 12 11 23 19 23 10 11 11 22 17 4 8

39 60 66 61 82 42 32 65 39 50 29 43 34 41 83 42 40 68 70 45 54 11 27 11 23 15 27 29 57 31 60 44 53 30 55 65 62 54 60 67 48 29 26 30 54 67 37 38 56 21 25 41 35 21 44 27 31 43 52 32 20 41 57 34 54 43 55 24 30 23 42 35 11 15

4.94E+01 1.11E+04 2.34E+07 9.86E+04 1.82E+05 7.66E+06 5.57E+05 1.44E+06 1.46E+12 6.54E+16 1.73E+09 2.50E+18 8.28E+10 2.34E+03 5.99E+05 3.27E+06 1.78E+04 1.10E+14 3.41E+10 2.73E+14 1.35E+13 2.37E+02 2.23E+07 6.63E+03 5.81E+05 2.86E+05 4.48E+06 1.90E+08 7.07E+21 2.78E+10 3.57E+24 1.87E+16 7.04E+20 7.23E+02 5.87E+05 8.41E+07 2.49E+10 4.40E+10 1.38E+10 7.19E+16 2.06E+06 3.20E+06 9.34E+04 1.40E+04 3.88E+09 1.88E+17 3.75E+05 2.86E+07 4.97E+09 1.78E+04 7.91E+05 8.59E+09 7.95E+07 2.48E+04 4.12E+09 3.90E+05 4.61E+03 1.48E+08 5.73E+10 1.77E+06 3.96E+04 2.10E+08 2.02E+08 7.23E+06 1.27E+12 2.71E+09 1.18E+11 2.89E+05 2.84E+06 6.89E+06 2.20E+08 1.29E+07 6.00E+01 1.12E+04

7.3 7.5 6.3 7.1 6.4 6.6 6.9 7.0 7.1 6.7 6.8 6.9 6.9 7.0 7.1 6.5 6.5 6.8 6.7 7.1 6.3 6.9 7.0


Koen et al.

Table 1 (Continued) spot no.a

adduct densityb

89h

3.3

91e 92e 93 94 95 96

ND ND ND 0.9 ND 0.9

protein name carbonic anhydrase 3; aldehyde dehydrogenase, cytosolic 1 superoxide dismutase [Cu-Zn]i superoxide dismutase [Cu-Zn]i R-enolase (enolase 1) glutathione S-transferase µ1 carbonic anhydrase 3 thioredoxin

accession no.

MM (kDa) calcd obsd

P14141 P13601 P07632 P07632 P04764 P04905 P14141 P11232

29.4 54.6 15.9 15.9 47.1 25.9 29.4 11.7

48.9 16.8 17.9 44.7 22.8 100.8 12.3

pI calcd

obsd

matches

% coveragec

MOWSE scored

6.9 7.1 5.9 5.9 6.2 8.3 6.9 4.8

7.1

11 6 11 12 12 35 14 10

52 13 54 63 25 82 58 60

2.30E+08 9.88E+02 1.95E+07 2.06E+07 1.91E+08 1.24E+18 4.06E+09 2.22E+05

6.0 6.0 6.3 9.1 7.2 4.5

a 2DE-separated protein spots are numbered as in Figure 1. b Number of adducts per 1000 molecules of protein; ND, below detection limit (0.2 mmol equiv/mol protein; for details, see Table S1 in the Supporting Information). c The sequence coverage was calculated as the percent of total amino acids in a protein precursor covered by matched peptides. d Calculated using MS-Fit. e Nontarget spots selected as controls for reference use (see text). f Databanks give several entries with MM values varying from 53.3 to 56.5 and pI values from 5.7 to 7.6. g Processed (mature) rat albumin contains 584 aa and has MM/pI values of 65.9 kDa and 5.8, respectively. h Different proteins comigrated to form a single radioactive spot so that the adducted species is unknown. i Databanks give several entries with MM values varying from 15.6 to 17 kDa.

Analyzer 4700 (Applied Biosystems, Foster City, CA). The samples were desalted and concentrated on C18 ZipTips (Millipore, Bedford, MA), eluted with 2 µL of a saturated solution of R-cyanohydroxycinnamic acid in aqueous 50% acetonitrile/0.1% trifluoroacetic acid directly onto a sample plate, and allowed to crystallize. Mass spectra were acquired in positive ion reflectron mode. Peptide mass acquisition was performed over the m/z range 700-3000. Mass spectra were externally calibrated using a standard mixture of known peptides covering the entire mass range, which typically resulted in mass accuracy within 50 ppm or better. Where possible, the calibration was verified using internal m/z peaks arising from trypsin autolysis. Fragmentation of major precursor peptide ions (usually 3-5) was performed using air as the collision gas. Protein Database Searching. The observed monoisotopic peptide masses, along with fragment ion masses, were exported to Mascot files using Applied Biosystems software and compared to respective theoretical masses for all proteins available from SWISSPROT and TrEMBL databases (http://us.expasy.org) or the NCBInr database (http://www.ncbi.nlm.nih.gov) using the Mascot searching engine with a probability-based scoring algorithm (http://www.matrixscience.com). Pyridylethylation and alkylation of cysteine residues by acrylamide as well as the presence of methionine sulfoxide were considered, and one missed cleavage was allowed in the search. Where available, the database information on specific posttranslational modifications (e.g., N-terminal acetylation) was taken into consideration. The identification was considered positive when the highest-scoring protein entry showed a MOWSE score higher than 56 (p < 0.05). Typically, the most probable candidates showed scores >80 (p < 0.005), with more than seven peptides matching at a mass error within 20-30 ppm and calculated protein MM differing from the electrophoretically estimated MM within 2-3 kDa. The results of the automated search were then checked by visual inspection of the observed mass spectra and verified by searching the protein databases with the aid of MS-Fit (http:// prospector.ucsf.edu/) and, in a few cases, ProFound (http:// 129.85.19.192/profound_bin/WebProFound.exe), using the observed major monoisotopic peptide masses as inputs. Also, for select peptide molecular ions, the observed MS/MS data were compared to calculated fragment ion patterns of peptides of all known proteins using MS-Tag (http://prospector.ucsf.edu/) or were compared to theoretical fragmentation patterns for putative matching peptides using fragment ion calculator (Applied Biosystems). The match was considered positive if at least 70% of predicted immonium ions and y or b ions were observed with a mass error e0.1 u. Finally, the identification of a protein spot was considered positive if the highest-ranking protein entry showed the MS-Fit MOWSE score of at least three orders higher than that for the next-ranking candidate or appeared as the only probable entry with at least four peptides matching major MS-derived masses within 50 ppm and at least one peptide fragment ion pattern matched.

Results Separation of Radiolabeled Cytosolic Proteins. The pooled cytosolic fraction isolated from the livers of six rats 4 h after ip injection of a hepatotoxic dose of 14C-BB contained 54 µCi of radioactivity, corresponding to 19% of the radioactivity observed in the whole liver homogenate and 0.43% of the original dose. Upon exhaustive dialysis of the cytosol, 41% of its initial radioactivity was removed. Of the remaining radiolabel, 86% remained covalently bound after precipitation of protein with trichloroacetic acid and washing with organic solvents. This corresponds to an average labeling density of 3.9 nmol equiv of 14C-BB/mg of protein, which is within the range typically observed for protein adduction by BB and similar hepatotoxins (2). For a protein of 30 kDa, this level of labeling corresponds to one adduct moiety for every 8-9 molecules of protein. The dialyzed cytosol was submitted to 2DE, followed by electroblotting and phosphorimaging to locate radiolabeled proteins. Seven replicate separations showed only minor intergel or inter-run variations in the resulting 2DE protein patterns. The results of a typical separation experiment are presented in Figure 1A. A total of 836 distinct protein spots of various abundance were detected on the PVDF blot stained with Coomassie R250. Of these, only 110 spots contained detectable levels of radioactivity (Figure 1B), the apparent labeling density varying from 0.3 to 62.7 mmol equiv BB/mol of protein among identified protein spots (Table 1) and from 0.2 to 128.8 mmol equiv BB/mol of protein (i.e., 600-fold) among all detected radioactive spots (Supporting Information, Table S1). The protein in the radioactive spots accounted for approximately 40% of the total protein detected on the blot (Supporting Information, Table S1). A majority of the radioactive spots appeared to be minor, low abundance proteins (for example, spots 1, 32-35, 39, 41, and 61), while many relatively abundant proteins contained negligible radioactivity (for example, spots 57, 58, and 91-93). These results clearly indicate that protein covalent binding of BB metabolites in liver cytosol in vivo is highly selective. Identification of Protein Targets. Radiolabeled cytosolic proteins were separated by 2DE, and after visualization by Coomassie staining, all 110 radioactive spots were excised from the gel. Because different gels showed virtually identical 2DE protein patterns, we were able to match protein spots in a gel to corresponding radioactive spots on the blot. This enabled us to pool identical protein spots from several gels, which was necessary in order to obtain sufficient material for subsequent MS analysis of many less abundant target proteins. The excised protein spots were digested in-gel with trypsin, and the resulting



Figure 1. 2DE separation and phosphorimaging of liver cytosolic proteins from rats treated with a hepatotoxic dose of 14C-bromobenzene. Top, Coomassie-stained blot; bottom, phosphorimage of the same blot.

peptide mixtures were analyzed by MALDI-TOF MS, followed by tandem MS/MS of 3-5 of the major peptides. The data were then matched against the complete Swiss-Prot database using an automated search with the aid of the Mascot peptide mass fingerprinting program and Applied Biosystems software. The results were then checked and verified manually using MS-Fit, MS-Tag, and ProFound. As controls, a few abundant but apparently nonradioactive protein spots, as well as protein-free areas of gel the size of an average protein spot, were processed and analyzed identically. A summary of the results of protein identification is presented in Table 1; full details can be found in the Supporting Information (Table S2). The proteins in a total of 65 radioactive spots and seven apparently unlabeled spots were identified with a high degree of confidence. For example, all major observed peptide masses were matched to corresponding theoretical peptide masses within 20-30 ppm, with an average of 15 matches per spot and sequence coverages ranging from 15 to 82% (median 42%). However, for a few heavily radiolabeled

but low abundance spots, we only observed 4-6 matching peptides. As a result, we obtained relatively low identification scores in these cases (e.g., Table 1, spots 1, 31, and 54). This was due in part to the extremely small amounts of sample available. In addition, the matching proteins in these spots are relatively small molecules (11-13 kDa) and possess few tryptic cleavage sites. For example, the total number of tryptic peptides (700-3000 u) possible after complete hydrolysis of the proteins identified in spots 1, 31, and 54 is only 7, 9, and 5, respectively. Nevertheless, by carefully comparing the results obtained using several search algorithms, along with visual inspection of the mass spectra, we found that (i) the matching proteins reported were the only probable candidates selected from the databases, (ii) all major MS peaks were matched with high mass accuracy, and (iii) the observed MS-MS fragment ion patterns of 1-2 peptides were in good agreement with the putative matches. Therefore, we consider these protein spots identified to a relatively high degree of confidence. Similar considerations were taken into account in the identification of moderately radioactive


spots 45 and 47 (Table 1). Another 10 spots, including the five most intensely radiolabeled ones (spots 32-35.1), were not identified because the mass spectra of their digests showed only a limited number of relatively weak peaks, which resulted in multiple low-scoring matches (results not shown). Multiple Proteins in Single Spots vs Multiple Spots for Single Proteins. Among the 65 radioactive protein spots identified, eight spots clearly contained more than one molecular species per spot, while the remaining 57 spots each appeared to contain just a single protein (Table 1). These 57 identifications comprised only 33 unique protein targets because, as observed previously with microsomal targets of bromobenzene metabolites (29), some individual proteins appeared in more than one spot on the 2DE gel (see below). Of the eight composite radioactive spots, six contained two protein species (spots 65.1, 67, 70, 72, 77, and 89) and two contained three proteins (spots 64.1 and 86; for details, see Supporting Information, Table S2). Comigration of different proteins as a single spot is not uncommon for highly complex mixtures in which several proteins may have similar molecular masses and pI values, as do the proteins in spot 86, for example. Likewise, spot 65.1 showed high-scoring matches for both glutathione S-transferase (GST) M1 and M2 (Table 1). Although these two proteins are very similar (25.6 vs 25.8 kDa; 78% identity and 91% similarity), visual inspection of the mass spectrum of the digest revealed the presence of masses matching subunit-specific peptides of both proteins. We do not know which of the two proteins in this particular spot actually bears the observed radioactivity. However, GSTM2 was also identified as a single protein in several other radioactive spots (e.g., spots 62, 63.1, 64, and 65). Likewise, GSTM1 was identified as a single protein in radioactive spot 94. Thus, GSTM1 and -M2 are clearly both targets for bromobenzene metabolites. This is in a good agreement with the results of a separate experiment showing that five major liver GST isoforms, including M1 and M2, isolated from bromobenzene-treated rats by the use of affinity chromatography and HPLC, all are adducted by bromobenzene metabolites (33). In a similar fashion, sulfotransferase 1B1, present in a mixture with another protein in spots 70 and 72 (Table 1), was identified also as a single protein in radioactive spot 71. Because this protein, like the two GST isoforms, appears both in a composite spot and as a single protein in another radioactive spot, it is clearly a target for bromobenzene metabolites. In contrast, a few radioactive spots (e.g., 67 and 86) each contained two or three proteins, which were not found as single species in any of the other analyzed spots. Therefore, we cannot claim them as targets for bromobenzene metabolites. Carbonic anhydrase-3 (CA3), a major cytoplasmic protein, was identified in several discrete protein spots having similar pI values but different MMs, either as a major component (i.e., spots 64.1, 77, and 89) or as the sole constituent (spots 66.1 and 95; see Table 1 and Figure 1). All five of these spots were consistently observed throughout a series of 2DE gels, and four of the five spots showed the presence of some radioactivity. Because at least one of the radioactive spots (spot 66.1) does not seem to contain proteins other than CA3, we consider CA3 to be a BB target protein. Qiu et al. (34) observed two different MM forms of CA3 among targets for acetaminophen in mouse liver. On the basis of the positions of the relevant spots on their published blot, the CA3 forms reported by Qiu et al. appear to correspond to our spots 66.1 and 89 (Figure 1). It is surprising that five separable molecular species of quite different apparent MM would all share a large enough portion of sequence to allow

Koen et al.

their identification as the same protein, especially when the overall coverages are as high as those observed here (Table 1). Because we did not observe any peptides indicating the presence of polyubiquitinated CA3 in these spots and because there are no reports suggesting that CA3 is a glycoprotein, there is no obvious explanation for this phenomenon. In other cases, two or more distinct radioactive protein spots with similar MMs but different apparent pI values were matched to a single protein. For example, seven spots numbered 5053.1 were each identified as serum albumin precursor, while spots 66, 68, 74, 75, 75.1, and 76 were all matched to 3-Rhydroxysteroid dehydrogenase. In these and several other cases, the spots appear to form a “charge train” on the gel. Furthermore, within such series, the radioactivity was not always distributed uniformly. For example, among the three spots identified as R-enolase (Table 1, spots 43, 44, and 93), the most acidic but least abundant protein spot shows the highest level of radioactivity, while the least acidic but most abundant spot shows no detectable radiolabel (Figure 1). In another series of spots (29, 30, and 30.1, all identified as heat shock cognate 71 kDa protein), spot 30.1 appears to be unlabeled, while the others are moderately radioactive. This putative charge train phenomenon has also been observed among proteins targeted by other reactive metabolites [e.g., acetaminophen (34) and thioacetamide (35)], but the origins of this phenomenon are unclear. Because reactive metabolites can modify ionizable protein nucleophiles such as lysine, histidine, and even carboxylate groups, it is theoretically possible that adduction could shift the apparent pI of the targeted protein. It is well-known that treatment of proteins with high concentrations of formic acid or urea (containing ammonium cyanate as an impurity) can lead to formylation or carbamoylation of basic lysine side chains causing the appearance of charge trains upon 2DE. However, because the fractional adduction of target proteins by chemically reactive metabolic intermediates is on average quite low (e13%; see Table 1), it seems most unlikely that the charge trains observed here result from adduction of ionizable groups by reactive metabolites. Furthermore, a number of apparently “normal” proteins are also known to show charge trains on 2DE; examples include liver fatty acid binding protein (36), protein disulfide isomerase (35, 37, 38), Grp78 (39), and a plant lectin protein viscumin (40). Isotopic Signatures for Bromine-Containing Peptides. Because at least some chemically reactive BB metabolites retain Br in their structure, one might expect to observe at least a few Br-containing peptides in the mass spectra of the digests. Although a high degree of sequence coverage was observed for many of the proteins that we identified, visual inspection of the mass spectra of even the most heavily labeled proteins did not reveal any peaks showing a clear bromine isotopic signature. This may be due to the low ratio of modified to unmodified protein and to the extremely low concentration of each specifically adducted peptide as discussed in detail elsewhere (33, 41).

Discussion The identification of proteins targeted by chemically reactive metabolites has long been regarded as an important step in elucidating the mechanism(s) by which reactive metabolites elicit cytotoxic effects (1, 3, 20, 42). In previous work with bromobenzene as a representative hepatotoxin, we identified nine rat liver microsomal proteins that experience significant covalent binding by reactive metabolites within 4 h after a cytotoxic dose (29, 30). In the present work, we have identified


an additional 33 cytosolic proteins that are also targeted by reactive metabolites of bromobenzene in vivo (Table 1). Although it requires additional effort, searching for protein targets in isolated subcellular fractions, as opposed to whole tissue homogenates, seems to be advantageous. It allows one to remove a majority of loosely bound radioactive species through washing and dialysis steps, and it enriches samples with fraction-specific proteins, thereby facilitating the detection and identification of less-abundant proteins. In some cases, lowabundance proteins are actually among the major in vivo targets in terms of their level of adduction (mol adduct/mol protein). For example, thioredoxin (spots 1 and 2) is a cytosolic protein having a relatively low abundance (Figure 1), but it has the highest overall level of specific adduction (Table 1). Likewise, transthyretin is a low-abundance microsomal protein (ultimately destined for secretion into plasma) that is heavily adducted by bromobenzene metabolites in vivo (29). As mentioned in the Results section, identifying two or more proteins in a single radioactive spot creates an ambiguity over which protein(s) actually are adducted. Of equal concern, however, is the possiblity that the adducted protein in a given spot may not be any of those identified but instead may be a highly labeled, low-abundance protein that comigrates with the identified major protein(s) in the spot. As noted above, we observed a few cases of highly labeled, low-abundance proteins but only a few. While we cannot exclude the potential misidentification of a protein as a target protein through such coincidences, it seems unlikely that this problem would be a frequent one. The identification of a particular protein as a true target protein might be made with greater certainty if one or more adducted peptides could be observed. The question then becomes one of obtaining sufficient information to support the claim that a particular ion truly represents an adducted peptide. Such claims are often supported by observing a predicted mass shift relative to the mass of the unadducted peptide, but the veracity of such claims depends on the mass accuracy with which this shift is measured (for example, see ref 33). Isotopic signatures can potentially confirm that a putative adduct is actually derived from the precursor of interest, but for larger peptides, the envelope of natural isotopes can obscure artificial isotope signatures. Finally, peptide sequence information derived from MS/MS experiments can add confidence to the identification of an adducted peptide. Unfortunately, not all peptides bearing post-translational modifications, whether endogenous or xenobiotic in origin, behave well enough to support this approach. Thus, observing modified peptides from proteins adducted in vivo under toxicologically relevant conditions remains a significant technological challenge, especially when large numbers of samples are involved (33, 42, 5052). Some highly adducted bromobenzene target proteins including glutathione S-transferases, protein disulfide isomerases, and liver fatty acid-binding protein are also relatively abundant in hepatocytes. Their high degree of adduction may be due simply to their relatively greater abundance and availability for interaction with reactive intermediates in classical bimolecular processes. On the other hand, a large number of cytosolic proteins (about 90% of the spots observed on 2DE; see Figure 1), including many highly abundant ones, experience little or no adduction, and even among the adducted proteins, there is a 600-fold variation in the extent of adduction. This strongly indicates a relatively high selectivity of protein alkylation by BB metabolites, at least in vivo, which is in good agreement with the observations reported for some other well-studied


cytotoxic chemicals such as acetaminophen (34), BHT (2,6-ditert-butyl-4-hydroxytoluene) (43), and naphthalene (44) under whole cell, in situ, or in vivo conditions. Factors other than simple abundance that probably also influence the relative adduction of various proteins under competitive conditions would include the degree to which they possess the type(s) of nucleophilic residues preferred by a given reactive metabolite and the local environment around those nucleophiles. For example, a hydrophobic patch or pocket adjacent to a protein nucleophile can increase the probability of its alkylation by means of noncovalent preassociation of a hydrophobic electrophile (33, 45-47). On the other hand, protonation or hydrogen bonding can decrease the reactivity of nucleophiles even if they are otherwise exposed to electrophiles in solution (48, 49). Protein covalent binding has long been considered an obligatory step in the production of cytotoxic effects by reactive metabolites derived from many small organic compounds including drugs (3, 53). Given that protein covalent binding is at least somewhat selective, one might therefore expect some commonality in the targeting of various proteins by different toxic agents, especially among those agents that generate a similar type of electrophilic reactive intermediate. Until the late 1990s, however, only a limited number of individual proteins had been identified as in vivo targets for hepatotoxic compounds, mainly acetaminophen and halothane, and no such commonality was apparent. With the implementation of systematic proteomic approaches based on 2DE, in-gel proteolysis, and mass spectrometry, the list of known target proteins started to grow rapidly. Currently, it encompasses >120 individual proteins (54). For example, 32 hepatic proteins are now known as targets for acetaminophen, while about 30 lung proteins have been recently identified as cellular targets for BHT (43) and another 17 for naphthalene (44). Like these latter cytotoxins, bromobenzene also targets a large group (g44) of functionally diverse hepatic proteins that participate in virtually all major processes of cell physiology, including protein synthesis and degradation, metabolism of carbohydrates, lipids, amino acids, and steroids, thiol-disulfide exchange, stress response, and others (see Table 1 and ref 29). To facilitate comparisons of the target proteomes for common cytotoxic chemicals and drugs, we recently designed an annotated, web accessible Target Protein Database (54). This database catalogs in a convenient, searchable fashion all of the publicly available information about the identities of mammalian proteins that become covalently adducted by chemically reactive metabolic intermediates of xenobiotics in vivo or in cell culture. Currently, the TPDB library contains information on 121 distinct well-identified proteins targeted by metabolites of 16 different drugs and chemicals. Analysis of this information using the Commonality Matrix tool contained in the TPDB software shows that there is relatively little commonality of protein targets between different toxic chemicals. An example of this using just the seven best-studied cytotoxic agents is shown in Table 2. At first, the relatively low commonality of protein targets among so many chemically similar agents was surprising. However, this apparent low commonality may be artifactual, resulting largely from sparse data and statistical chance. This hypothesis is supported by the observation that the largest number of common target proteins is found between the two agents for which the largest number of target proteins are known, bromobenzene and thiobenzamide. In fact, the number of target proteins common to any two of the chemicals in Table 2


Koen et al.

Table 2. Numbers of Identified Protein Targets for Reactive Metabolites of Representative Cytotoxic Chemicalsa alkylators (epoxides and/or quinones)

BB APAP BHT NAPH BENZ TB HAL

acylators

BB

APAP

BHT

NAPH

BENZ

TB

HAL

rat liver 44b 7 4 5 3 28 4

mouse liver

mouse lung

mouse lung

mouse liver

rat liver

rat liver

32 2 3 3 6 2

28 3 2 6 0

17 2 7 1

15 2 1

known to undergo protein binding in vivo (24). For some other cytotoxins such as molinate, diclofenac, and menadione, only one or two targets are presently known (54). To determine whether there exists a subset of protein targets common to diverse cytotoxic small chemicals and whether their covalent modification is causally related to cytotoxicity will require much more effort to expand the target proteomes of these chemicals. Acknowledgment. This work was supported by NIH Grant GM-21784.

71c 4

9

a

Numbers on the diagonal are for individual compounds. The offdiagonal numbers indicate the number of common targets for any pair of chemicals. These seven best-studied agents have led to 216 target identifications, among which there is some redundancy as indicated in the matrix; the total number of nonredundant proteins is 148. Data source: http:// tpdb.medchem.ku.edu:8080/protein_database/. Compound abbreviations: BB, bromobenzene; APAP, acetaminophen; BHT, 2,6-di-tert-butyl-4hydroxytoluene; NAPH, naphthalene; BENZ, benzene; TB, thiobenzamide; and HAL, halothane. b Includes 33 target proteins reported in this manuscript. c Unpublished data from our laboratory (manuscript in preparation).

increases as the total number of known targets for each chemical increases. Because different animal species are often used in studying different toxins, it is of course necessary to make such comparisons based on functionally orthologous proteins having similar rather than identical structures. It seems likely that as larger numbers of targets are identified for metabolites of a given chemical, the observed commonality of protein targeting will increase. The main question then will become “what is the significance of this commonality?” Is it simply a product of the relative abundance and number of nucleophilic groups of various proteins, or does it have some underlying mechanistic significance in regard to cytotoxicity? The accumulated data on protein covalent binding (54) indicate that at least a few proteins are fairly common to a number of cytotoxic chemicals. For example, protein disulfide isomerase A1 (PDI A1), a multifunctional protein involved in the processes of protein maturation in the cell, appears to be the most common target. It is targeted by reactive metabolites of at least five different cytotoxic chemicals, including acetaminophen, bromobenzene, naphthalene, thiobenzamide, and benzene. Four of these chemicals also target PDI A3, a functionally related enzyme of protein folding/refolding. Among the other common targets are two selenium-binding proteins with unknown physiological function, SBP1 and SBP2, each targeted by at least three different chemicals. Determining specifically whether these proteins are or are not targeted by other chemicals for which they are not yet reported to be targets should be a high priority for future research. While some of the common target proteins clearly have important cellular functions, the significance of their modification by reactive intermediates is much less clear. For example, reactive metabolites from p-bromophenol (55), 3′-hydroxyacetanilide (25, 56-58), and trans-stilbene (23) covalently bind to liver proteins without causing significant hepatotoxicity. In addition, several abundant protein targets for acetaminophen metabolites are also known to be adducted by its nontoxic regioisomer 3′-hydroxyacetanilide (59). This may indicate the irrelevance of the adduction of these proteins by these particular metabolites to toxic outcomes, but it could equally well indicate that different modifications of the same protein are perceived differently by the cell. In this regard, it would be interesting to compare side-by-side the protein targeting by bromobenzene and its major nontoxic metabolite, p-bromophenol, which is also

Supporting Information Available: Table of protein and radioactivity contents of individual protein spots obtained by 2DE of liver cytosol from bromobenzene-treated rats and table of search results (MS-Fit) for cytosolic proteins targeted by bromobenzene. This material is available free of charge via the Internet at http:// pubs.acs.org.

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A Proteomic Analysis of Bromobenzene Reactive Metabolite Targets

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