Carbonyl Reductase Inactivation May Contribute to ... - ACS Publications

Chem. Res. Toxicol. 2008, 21, 1631–1641

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Carbonyl Reductase Inactivation May Contribute to Mouse Lung Tumor Promotion by Electrophilic Metabolites of Butylated Hydroxytoluene: Protein Alkylation in ViWo and in Vitro Colin T. Shearn, Kristofer S. Fritz, Brent W. Meier,† Oleg V. Kirichenko, and John A. Thompson* Department of Pharmaceutical Sciences, C238-L15, Anschutz Medical Campus, UniVersity of Colorado DenVer, Box 6511, Aurora, Colorado 80045 ReceiVed May 8, 2008

Promotion of lung tumors in mice by the food additive butylated hydroxytoluene (BHT) is mediated by electrophilic metabolites produced in the target organ. Identifying the proteins alkylated by these quinone methides (QMs) is a necessary step in understanding the underlying mechanisms. Covalent adducts of the antioxidant enzymes peroxiredoxin 6 and Cu,Zn superoxide dismutase were detected previously in lung cytosols from BALB/c mice injected with BHT, and complimentary in Vitro studies demonstrated that QM alkylation causes inactivation and enhances oxidative stress. In the present work, adducts of another protective enzyme, carbonyl reductase (CBR), were detected by Western blotting and mass spectrometry in mitochondria from lungs of mice one day after a single injection of BHT and throughout a 28-day period of weekly injections required to achieve tumor promotion. BHT treatment was accompanied by the accumulation of protein carbonyls in lung cytosol from sustained oxidative stress. Studies in Vitro demonstrated that CBR activity in lung homogenates was susceptible to concentration- and time-dependent inhibition by QMs. Recombinant CBR underwent irreversible inhibition during QM exposure, and mass spectrometry was utilized to identify alkylation sites at Cys 51, Lys 17, Lys 189, Lys 201, His 28, and His 204. Except for Lys 17, all of these adducts were eliminated as a cause of enzyme inhibition either by chemical modification (cysteine) or site-directed mutagenesis (lysines and histidines). The data demonstrated that Lys 17 is the critical alkylation target, consistent with the role of this basic residue in NADPH binding. These data support the possibility that CBR inhibition occurs in BHT-treated mice, thereby compromising one pathway for inactivating lipid peroxidation products, particularly 4-oxo-2nonenal. These data, in concert with previous evidence for the inactivation of antioxidant enzymes, provide a molecular basis to explain lung inflammation leading to tumor promotion in this two-stage model for pulmonary carcinogenesis. Introduction A model system for investigating mechanisms of lung tumor promotion involves treating BALB/c mice with an initiator followed by several weekly injections of the food additive butylated hydroxytoluene (BHT)1 (1). BHT is oxidized by pulmonary cytochromes P450 to electrophilic metabolites that mediate 10- to 12-fold increases in tumor multiplicity (1–4). Two QMs, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-QM) and 6-tert-butyl-2-(1′,1′-dimethyl-2′-hydroxy)ethyl4-methylphenol (BHTOH-QM) shown in Scheme 1, are generated (2, 5). These metabolites undergo 1,6-conjugate additions * To whom correspondence should be addressed. Tel: 303-315-6167. Fax: 303-315-0274. E-mail: [email protected]. † Current address: Amgen, Inc., 4000 Nelson Rd., Longmont, CO 80503. 1 Abbreviations: BHT, 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene); BHT-QM, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone; BHTOH, 6-tert-butyl-2-(1′,1′-dimethyl-2′-hydroxy)ethyl-4-methylphenol; BHTOH-QM, 6-tert-butyl-2-(1′,1′-dimethyl-2′-hydroxy)ethyl-4methylene-2,5-cyclohexadienone; CBR, carbonyl reductase; CID, collisioninduced dissociation; 2-DE, two-dimensional SDS-PAGE; ESI, electrospray ionization; HNE, 4-hydroxy-2-nonenal; 4-hydroxymethyl BHT, 2,6-di-tertbutyl-4-hydroxybenzyl alcohol; MALDI, matrix-assisted laser desorption ionization; MeCN, acetonitrile; MSDB, Mass Spectrometry Database; MS/ MS, tandem MS; NEM, N-ethylmaleimide; ONE, 4-oxo-2-nonenal; Prx6, peroxiredoxin 6; PGE2, prostaglandin E2; PVDF, poly(vinylidene difluoride); QM, quinone methide, ROS, reactive oxygen species; SOD1, Cu,Zn superoxide dismutase; TOF, time-of-flight; TOF/TOF, tandem TOF.

Scheme 1. Conversion of BHT to Quinone Methides by Pulmonary Cytochrome P450 and Michael Addition to a Protein Thiol or Amino Group

to a number of proteins in pulmonary epithelial cells treated directly with a QM (6) and in ViVo in the lungs of mice injected with BHT (7). Key antioxidant enzymes peroxiredoxin 6 (Prx6) and Cu,Zn superoxide dismutase (SOD1) were among the alkylation targets detected using immunochemical methods in mice that had received one injection or as many as 4 weekly injections consistent with the protocol for tumor promotion. Complimentary in Vitro studies with purified enzymes, pulmonary epithelial cells and lung homogenates directly exposed to these QMs demonstrated that adduct formation is accompanied by enzyme inactivation and increased levels of hydrogen peroxide, superoxide and products of lipid peroxidation (7, 8). These results suggest that damage to critical antioxidant proteins by QMs contributes to the severe oxidative stress that accompanies BHT administration and supports several other

10.1021/tx800162p CCC: $40.75  2008 American Chemical Society Published on Web 07/03/2008

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studies implicating inflammation as a major factor in this twostage model of pulmonary carcinogenesis (9, 10). Adducts to carbonyl reductase (CBR) were also detected in lungs of mice injected with BHT (7). CBR is a member of the short-chain dehydrogenase superfamily abundant in several tissues including liver and lung (11). This enzyme utilizes NADPH to reduce aldehyde and ketone groups in a wide variety of endogenous and exogenous compounds, including steroids, prostaglandins, quinones and aldehydes formed during lipid peroxidation (11–13). Unlike monomeric rat and human CBRs, the mouse lung enzyme is a homotetramer with a subunit molecular weight of approximately 26 kDa (14). Several lines of evidence implicate CBR as a factor in limiting tumor development. This protein is greatly under-expressed in tumors, for example Lin et al. (15) found that mouse lung adenocarcinomas contain 2 to 6-fold less mRNA and approximately 20fold less protein than surrounding normal tissues and similar differences in protein expression were reported by Ru¨tters et al. (16). In a study comparing mouse lung adenocarcinoma cell lines, the metastatic potential was much greater for cells with barely detectable CBR than for cells with high levels of the protein (17). Low CBR expression was reported for human epithelial ovarian and hepatocellular cancers and was correlated with a poor prognosis in human nonsmall-cell lung cancer (18). The data cited above indicate that CBR is associated with resistance to tumorigenesis and suggest that a loss of functional protein, whether through low expression or covalent modification, is conducive to tumor formation. This enzyme provides downstream protection from the actions of reactive oxygen species (ROS) by reducing and thereby detoxifying reactive aldehydes such as 4-oxo-2-nonenal (ONE) generated during the degradation of polyunsaturated lipids (12, 19). Simultaneous inhibition of CBR, Prx6 and SOD1, therefore, should result in higher levels of ROS and R,β-unsaturated aldehydes capable of modifying proteins and affecting cell signaling as reviewed recently (20, 21). Prostaglandin 9-keto reductase and 15-hydroxy dehydrogenase activities have been associated with CBR (22, 23), so inhibition of the enzyme also may contribute to increased levels of the pro-inflammatory mediator prostaglandin E2 (PGE2) shown to occur in the lungs of mice treated with BHT (24). Links between PGE2, inflammation and tumor promotion are well-known and decreasing the production or increasing the catabolism of PGE2 suppresses tumorigenesis (25, 26). Data outlined here provide the basis for investigating consequences of CBR alkylation by QM metabolites of BHT as they relate to the mechanism of action of this tumor promoter in mouse lung. A goal of the present study was to extend earlier work with CBR adducts in lung cytosol to determine if mitochondrial CBR is alkylated in BHT-treated mice as the protein is located mainly in this organelle (11, 12). Complimentary studies carried out with lung homogenates and recombinant CBR demonstrated that alkylation by either BHT-QM or BHTOH-QM is accompanied by enzyme inhibition. Inhibition was examined in detail by identifying alkylation sites with MS and preparing mutants in which targeted residues of CBR were replaced by residues that do not react with QMs. These data, considered together with earlier results, suggest that alkylation of the protective enzymes CBR, Prx6 and SOD1 in the lungs of BHT-treated mice is a major factor leading to increased levels of ROS and lipid-derived aldehydes capable of altering cell signaling and influencing tumor development.

Shearn et al.

Materials and Methods Chemicals. Chemicals were purchased from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO) unless otherwise noted and HPLC-grade solvents were obtained from Fisher Scientific (Pittsburgh, PA). BHT was purified by recrystallization from hexane and BHTOH and the GSH conjugate of BHT-QM (4S-gluathionylmethyl BHT) were synthesized in this laboratory previously (3, 5, 8). Solutions of QMs in acetonitrile (MeCN) were prepared by oxidizing BHT or BHTOH with freshly prepared silver oxide and the concentrations were measured spectrophotometrically (5). Samples of ONE and 4-hydroxy2-nonenal (HNE) were provided by Dr. D. R. Petersen, University of Colorado Denver. Animals and Treatments. Male BALB/cByJ mice were obtained from Jackson Laboratory (Bar Harbor, ME) at 4-6 weeks of age and acclimated for 1 week. Mice weighing approximately 25 g were randomly distributed into five groups of five animals each (referred to as groups d1, d7, d14, d21 and d28). All mice were injected intraperitoneally with 150 mg of BHT/kg body weight delivered in 100 µL of corn oil. Group d14 was reinjected on day 7, group d21 was reinjected on days 7 and 14 and group d28 was reinjected on days 7, 14, and 21. Group d1 mice were euthanized 24 h after injection and mice in the other groups were euthanized 7 days after the last injection (i.e., on day 7, 14, 21 or 28). Lungs from each group were pooled and homogenized in 50 mM sodium phosphate (pH 7.4) containing 1 mM EDTA and centrifuged at 9,000 g for 20 min to sediment both nuclear and mitochondrial proteins. The supernatant was centrifuged at 100,000 g for 60 min to sediment microsomes and separate cytosolic proteins in the supernatant fraction. Protein concentrations were determined (27) and each fraction was flash frozen in liquid nitrogen for storage at -80 °C until analyzed. Two-Dimensional SDS-PAGE (2-DE) and Immunoblotting. The 2-DE and immunoblotting methodologies for separating and detecting adducted proteins in mouse lung fractions have been described in detail previously (6). In brief, fractions containing 2 mg of protein were concentrated to approximately 50 µL, desalted with a BioSpin 6 gel filtration column (BioRad, Hercules, CA) and exchanged into 1 mM sodium phosphate (pH 7.4). The volume was reduced to approximately 20 µL by lyophilization and divided into two equal samples, and to each was added 600 µL of Destreak rehydration buffer (GE Healthcare, Piscataway, NJ) along with 2 µg of BHT-adducted soybean trypsin inhibitor (positive control). Protein samples were processed in the first dimension by isoelectric focusing on either pH 3-10 or pH 5-8 Ready Strips (17 cm, BioRad) and in the second dimension on 4-15% linear gradient slab gels (18.5 × 20 cm). Proteins were either stained in the gel with Coomassie Brilliant Blue R250 in 40% methanol/10% acetic acid (v/v) overnight or were electroblotted to a poly(vinylidine difluoride) (PVDF) membrane in a semidry transfer apparatus. Membranes were blocked overnight with a solution of 5% nonfat dry milk in TBST buffer (10 mM Tris-HCl, pH 7.4, 140 mM NaCl and 0.1% Tween 20), probed with the primary antiserum (1:500) that recognizes benzylic thioether- and amine-linked BHT and BHTOH groups (6), washed with TBST, probed with antirabbit IgG linked to horseradish peroxidase and visualized by enhanced chemiluminescence (Cell Signaling Technology, Beverly, MA) with exposure of 1 min onto Hyperfilm ECL (GE Healthcare). Protein Identification. Adducts visualized on Western blots were matched to the corresponding spots on Coomassie-stained companion gels with BHT-adducted soybean trypsin inhibitor added as a positive control (6, 7). Adducted proteins were

Mouse Lung Carbonyl Reductase

excised from the gels, hydrolyzed with modified porcine trypsin (Promega, Madison, WI) in 100 µM ammonium bicarbonate and the resulting peptides prepared for analysis by LC-MS as described (7). Peptide samples were introduced via an electrospray (ESI) source into an Esquire HCT ion trap mass spectrometer (Bruker, Billerica, MA) with an Agilent 1100 capillary HPLC system (Palo Alto, CA) and a 1.0 × 150 mm Jupiter Proteo 90 Å column (Phenomenex, Torrance, CA). The flow rate was 50 µL/min of 0.1% formic acid in MeCN (solvent A) and 0.1% aqueous formic acid (solvent B). Mobile-phase composition was held at 3% solvent A for 5 min and then increased to 40% using a linear gradient over 40 min. The instrument was operated under tandem MS (MS/MS) conditions. Peak lists of peptide fragment ions were created using BioTools software (v 2.2, Bruker), exported as Mascot Generic Format files and searched against the comprehensive nonidentical Mass Spectrometry Database (MSDB) obtained from ftp.ncbi.nih.gov/ repository/MSDB via the Mascot search engine (v 2.1.04, www.matrixscience.com). Search parameters were as follows: delta mass 1.0 Da for precursor ions and 0.5 Da for fragment ions allowing one missed cleavage and optional modifications for oxidized methionine, carbamidomethyl cysteine and BHTQM-adducted cysteine, lysine or histidine. Preparation of Recombinant CBR. (i) A sequenced, expressed sequence tag clone (ATCC ID BC012714) containing CBR DNA was purchased from Open Biosystems (Huntsville, AL). Full-length CBR was amplified by PCR with oligonucleotides5′-CCACAAGCTGAATTTCAGTGGCCTGAGG-3′(sense) and 5′-AGGAGGCCAGGTAGCCAGCATCCACC-3′ (antisense) using the vector pCMV-SPORT6-CBR as template. Following amplification, the fragment was TOPO-cloned into His-tagged pET100-TOPO (Invitrogen, Carlsbad, CA), transformed into TOP-10 cells and grown overnight on LB-ampicillin plates (100 µg/mL) according to the manufacturer’s instructions. Colonies were picked and placed into 3-mL LB cultures for 16 h with 100 µg/mL of ampicillin. DNA was subsequently purified using WIZARD Plus SV minipreps (Promega) and sequences were verified at the University of Colorado Cancer Center core facility. (ii) The CBR-pET100 DNA was transformed into BL-21 Star (DE3) Escherichia coli. To express the histidine-tagged CBR, a colony was picked and grown in 3 mL of LB plus 100 µg/ mL ampicillin. After 16 h, 100 µL of the culture was added to 100 mL of fresh LB broth together with ampicillin and the culture was grown until it reached an OD600 of 0.6-0.8. The culture was then induced with 1 mM isopropyl-β-D-thiogalactopyranoside and grown for 4 h at 37 °C. Cultures were sedimented by centrifugation at 6,000 rpm for 20 min and flash frozen. For protein purification, pellets were thawed and cells lysed on ice for 20 min in 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, 10 mM imidazole, 0.5% Triton X-100, together with lysozyme and protease inhibitors (Sigma). This sample was sonicated with three 20 s bursts, followed by centrifugation at 14,000 rpm for 30 min. The supernatant was decanted and 150 µL of 50% immobilized nickel resin (Qiagen, Valencia, CA) in lysis buffer was added and the sample incubated for 4 h at 4 °C on a rotary mixer. Following incubation, the beads were washed five times with 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, containing 20 mM imidazole, then twice with 50 mM imidazole, and finally eluted from the resin with 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 250 mM imidazole and 10% glycerol. The histidine-tagged protein samples were flash frozen in liquid nitrogen and stored at -80 °C until used.

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(iii) The histidine tag was removed as follows. Buffer containing CBR was exchanged using Micro Bio-Spin 6 columns (Bio-Rad, Hercules, CA) for 20 mM Tris (pH 7.4) containing 50 mM NaCl and 2 mM CaCl2. The enzyme was then digested for 16 h at room temperature with 1 U of recombinant enterokinase (Novagen, Madison, WI) per 30 µg of His-tagged CBR. The histidine tag was removed from the solution and the buffer was exchanged to 80 mM potassium phosphate (pH 6.0) with Bio-Spin 6 columns. Protein concentrations were determined with the BCA assay (Pierce, Rockford, IL). Analysis of the resulting protein by electrospray LC-MS (described below in MS analysis of CBR adducts) demonstrated an average molecular mass of 26,453 Da (calc. 26,444) corresponding to the mass of native CBR in which the N-terminal methionine has been replaced by the sequence AspHis-Pro-Phe-His remaining after cleavage of the His tag. LCMS analyses of tryptic digests of CBR (performed as described in Protein identification) confirmed the identity of this protein. (iv) Site-directed CBR mutants were produced as described for the wild type (WT) protein using oligonucleotides listed in Table S1 (Supporting Information) and the QuikChange Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Following digestion with Dpn-1, the nonmethylated DNA was transformed into XL-1 Blue Supercompetent cells and grown overnight on LBampicillin plates (100 µg/mL). Colonies were picked and placed into 3-mL LB cultures for 16 h with 100 µg/mL of ampicillin. The DNA was subsequently purified using QIAprep spin minipreps (Qiagen, Madison WI), the sequences verified at the University of Colorado Cancer Center core facility and protein mobility compared with WT CBR on a 12% SDS PAGE gel. Activity Assays. CBR activity was determined spectrophotometrically in mouse lung cytosol by measuring the oxidation of NADPH at 340 nm (12, 28). A typical assay contained 80 mM potassium phosphate (pH 6.0), 1.0 mg/mL bovine serum albumin, 0.1 mM NADPH, 200 µM menadione and 130 µg of cytosolic protein in a total volume of 1.0 mL incubated at 37 °C in a shaking water bath (14). Quercetrin (140 µM), a specific inhibitor of CBR (12, 29), was added to some incubates and menadione or NADPH was omitted from controls. Some incubations contained dicoumarol (50 µM40), an inhibitor of DT-diaphorase (30), or 1.0 mM NADH instead of NADPH. For studies with recombinant CBR, 9 µg of protein were dissolved in 300 µL of 80 mM potassium phosphate (pH 6.0) containing 1.0 mg/mL bovine serum albumin, 100 µM NADPH and 25 µM menadione. For QM treatment, 27 µg of protein in 50 µL buffer were incubated at 37 °C with BHT-QM or BHTOH-QM for 20 min followed by transfer of 17 µL into 283 µL of buffer containing menadione and NADPH (as above) and NADPH oxidation measured as described for cytosolic CBR except that a microtiter plate reader was employed (Molecular Devices, Sunnyvale, CA). MS Analysis of CBR Adducts. Recombinant CBR (25 µg) in 50 µL of 20 mM aqueous ammonium bicarbonate (pH 7.4) was treated with BHT-QM or BHTOH-QM in MeCN to achieve QM concentrations noted in the text while maintaining the solvent content at or below 10% (v/v). The intact proteins were incubated at 37 °C for 30 min and analyzed by LC-MS. Samples were introduced with an Agilent 1100 capillary HPLC system utilizing a 1.0 × 150 mm Jupiter C4, 300 Å column (Phenomenex) maintained at 40 °C and a mobile phase of 0.01% aqueous trifluoroacetic acid (solvent A) and 0.01% trifluoroacetic acid in MeCN (solvent B) with a 20-min linear gradient from 40% to 80% solvent B. Spectra were recorded on an Agilent SL ion

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trap mass spectrometer and the envelopes of multiply charged ions for each protein were deconvoluted utilizing Agilent processing software, DataAnalysis for LC/MSD Trap v 3.2, with the ion abundance cutoff set at 10% and a minimum of 8 peaks for each compound. Adducted CBR was also digested with trypsin and prepared for analysis as described above in Protein identification. Digests were added to C18 Zip-Tips (Millipore, Billerica, MA) and peptides eluted with 10 µL of 0.1% trifluoroacetic acid in 60% MeCN. Each sample (0.5 µL) was combined with an equal volume of R-cyano-4-hydroxycinnamic acid in the same solvent (10 mg/mL), spotted onto an OptiTOF plate and analyzed with an ABI 4800 plus matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF/TOF) MS (Applied Biosystems, Foster City, CA). The instrument was operated under standard conditions for peptides at laser power 3000-3300 with external mass calibrations performed using a ProteoMass Calibration Kit (Sigma). The MS/ MS spectra were obtained with the collision gas air, collision energy 1 kV, precursor mass window of resolution 300 and laser power 4000-4300. Data analysis was performed with Applied Biosystems 4000 Series Explorer v 3.5. Protein Carbonyl Measurements. Protein carbonyls were measured spectrophotometrically in mouse lung cytosol as described (31) with minor modifications. Briefly, samples containing 0.5 mg of protein (determined by the BCA assay) were obtained from mice treated with BHT as described for group d28, but euthanized on day 24. Controls were treated identically except that mice were injected with the vehicle only. All samples were incubated for 2 h at 37 °C with 10 mM 2,4dinitrophenylhydrazine in 2 M HCl, the proteins were precipitated by treatment with 10% trichloroacetic acid on ice for 10 min followed by centrifugation at 1000 g for 10 min. The supernatants were discarded and pellets washed with three 1-mL portions of 1:1 ethanol/ethyl acetate, followed by centrifugation for 10 min at 1000 g. The resulting pellets were solubilized in 100 mM Tris-HCl (pH 6.8) containing 2% SDS. The 2,4dinitrophenylhydrazone concentrations were determined on a microtiter plate reader at 370 nm utilizing an extinction coefficient of 22,000 M-1 cm-1. Carbonyl groups were analyzed in the same samples after conversion to biotin-linked hydrazones (32) by incubating 20 µg of protein for 2 h at 25 °C with 2.5 mM EZ-Link Biotin Hydrazide (Pierce) in 20 mM sodium phosphate (pH 7.4) and 100 mM NaCl, followed by the addition of 10 µL of 5X SDS loading buffer and heating for 4 min at 95 °C. Proteins were subsequently separated on a 12% SDS-PAGE gel and electroblotted to a PVDF membrane. Once transfer was complete, membranes were blocked for 30 min in 5% nonfat dry milk and 0.5% TBST. This treatment was followed by incubation for 1 h with horseradish peroxidase-conjugated goat polyclonal antibiotin 1:5000 in 0.5% TBST (GeneTex, San Antonio, TX). Samples were washed three times with TBST and visualized by chemiluminescence (Pierce, Rockford, IL). To verify equal protein loading, the membranes were stripped for 10 min in Restore Plus Western Blot Stripping Buffer (Pierce). Membranes were blocked again for 30 min in 5% nonfat dry milk and 0.5% TBST, probed with mouse monoclonal antiactin (Sigma) for 1 h at 25 °C and washed three times in TBST. Membranes were then incubated for 1 h with donkey antimouse IgG (1:5000) (Santa Cruz Biotechnology, Santa Cruz, CA), washed three times in TBST and visualized by chemiluminescence.

Shearn et al.

Figure 1. Western blots of protein adducts formed with BHT-QM. (A) Blots from 2-DE separations of nuclear/mitochondrial fractions from the lungs of BHT-treated mice from group d21 (left) and untreated mice (right). Immunoreactive spots were excised from companion gels and identified by LC-MS/MS as described under Materials and Methods. The numbers correspond to spot 1, albumin; spot 2, γ-actin; spot 3, tropomyosin 5; and spot 4, histone H2B. STI refers to soybean trypsin inhibitor (added as a positive control). (B) Blots of purified, recombinant CBR treated with 0, 1, 5, or 10 mol equiv of BHT-QM.

Table 1. LC-MS/MS Identification of Mitochondrial CBR Detected on 2-D Gels by Immunoblotting with Antibodies to QM Adducts experimental groupa d1 d7 d14 d21 d28

peptides matched

coverage (%)

Mowse scoreb

3 43 17 20 35

11 62 36 36 43

73 693 484 479 286

a Mice were euthanized on day 1, 7, 14, 21, or 28 (groups d1 through d28, respectively), following an initial injection of BHT. Groups d1 and d7 received one injection. Additional injections were given to d14 mice on day 7, d21 mice on days 7 and 14, and d28 mice on days 7, 14, and 21. Lung samples were prepared as described in Materials and Methods. b Score from Mascot probability-based matching of MS/MS fragment ions to the MSDB. A Mowse score of 64 is considered a reliable match (www.mascot.com).

Results CBR Adducts and Protein Damage. Lungs of BHT-treated mice were examined for the presence of CBR adducts in mitochondria by 2-DE and Western blotting using polyclonal antibodies with affinity for both BHT and BHTOH residues bound via benzylic thioether or amine linkages (6). Representative Western blots from untreated and BHT-treated mice are shown in Figure 1A. Immunoreactive proteins were excised from parallel gels, digested with trypsin and analyzed by ESI LCMS/MS. The results (Table 1) demonstrate that CBR was identified with a high degree of confidence in lungs from all five groups of mice from Mascot searches of the MSDB with MS/MS data. These adducts were detected in lung tissue within 24 h after a single injection and persisted for at least 7 days after each injection. Alkylation was confirmed in Vitro by directly treating purified, recombinant CBR with increasing quantities of BHT-QM. Immunoblots shown in Figure 1B demonstrate the lack of nonspecific binding and show a maximal antibody response from treatment with a 5-fold excess of the electrophile. Lung damage accompanying BHT exposure was assessed by measuring total protein carbonyls generated through direct oxidations by ROS and conjugate additions of R,β-unsaturated aldehydes from lipid peroxidation (33, 34). Colorimetric


Figure 2. Western blots of biotinylated protein carbonyls. Lung proteins from mice administered 4 weekly injections of BHT (+) or the vehicle only (-) were treated with biotin hydrazide, separated by SDS-PAGE, and analyzed by Western blotting with antibiotin polyclonal antibodies. To verify equal protein loads, membranes were stripped and reprobed with mouse monoclonal anti-β-Actin.

measurements of 2,4-dinitrophenylhydrazones demonstrated a 45% increase among cytosolic proteins after four weekly injections of BHT as compared with control mice receiving the vehicle only (8.80 ( 0.59 versus 6.07 ( 0.74 nmol/mg protein, means ( SD of 3 determinations, p < 0.01). In addition, protein carbonyls were converted to biotin-linked hydrazones and analyzed by SDS-PAGE with Western blotting using antibiotin polyclonal antibodies. Blots shown in Figure 2 demonstrate substantial increases in protein damage after 4 weekly injections relative to controls. Effects of QMs on CBR Activity in Vitro. CBR activity was investigated in lung homogenates from untreated mice utilizing the substrate menadione. The data shown in Figure 3A are consistent with CBR activity rather than that of another cytosolic reductase. Reactions were substrate- and cofactordependent and NADPH was the preferred cofactor. In addition the specific CBR inhibitor quercetrin prevented this reaction whereas dicoumarol, an inhibitor of the cytosolic quinone reductase DT-diaphorase, had no effect on activity. Data in Figures 3B and 3C demonstrate, respectively, time- and concentration-dependent decreases in CBR activity after exposures to BHT-QM or BHTOH-QM. A similar degree of inhibition occurred for 60 µM BHT-QM as for 20 µM BHTOHQM (40-45% decrease) and inhibition by 200 µM BHT-QM was comparable to that of 50 µM BHTOH-QM (75-80% decrease). Complete inactivation is likely with further increases in QM concentrations. The higher potency of BHTOH-QM is consistent with the greater reactivity of this electrophile determined in earlier work (5). CBR inhibition was confirmed by treating the recombinant protein with either of these QMs (Figure 3D). Additional studies were conducted to assess the contributions of contaminants and degradation products to enzyme inhibition, including BHT from incomplete chemical oxidation, 4-hydroxymethyl BHT from competitive hydration of the QM in buffer and 4-S-gluthationlymethyl BHT formed in competition with protein binding in cytosol (8). As shown in Table 2, none of these compounds affected CBR at concentrations relevant to the current work. Additionally, inhibition produced with QMtreated CBR was unaffected by gel filtration to remove residual small molecules. These results are consistent with the covalent modification of one or more protein residues essential for activity. Mechanism of Inhibition. An ESI mass spectrum of recombinant CBR (Figure 4A) exhibited an envelope of ions


representing all charge states in the range from +23 to +32 and deconvolution yielded a singly charged species at the expected mass of 26,453 Da. Treatment with a 15-fold molar excess of BHT-QM sufficient to inhibit >70% of enzyme activity generated a mixture of adducts eluting from the column after the native protein. Deconvolution of the complex spectrum shown in Figure 4B revealed a mixture of adducts containing from one to four QM groups corresponding to an increase of 218 Da for each. A 5-fold excess of BHT-QM generated a mixture of unmodified, monoadducted and doubly adducted forms of CBR (Figure 1S, Supporting Information). Both cysteine residues of CBR, Cys 51 and Cys 58, are likely alkylation targets due to their accessibility near the protein’s surface (PDB entry 1CYD) and the preference of QMs for thiol relative to amine binding (35, 36). The influence of cysteine modification on enzyme activity, therefore, was investigated by treating CBR with the thiol-selective acylating agent Nethylmaleimide (NEM). The molecular mass increased by 250 Da to 26,703 Da corresponding to efficient modification of both residues, however this treatment did not significantly influence enzyme activity (Table 2) indicating that inhibition was due to alkylation at one or more lysine or histidine residues. The susceptibility of CBR to lysine modification was demonstrated in an earlier study utilizing 2,4,6-trinitrobenzene-1-sulfonic acid to inhibit activity (37). CBR treated with a 15-fold molar excess of either BHT-QM or BHTOH-QM was digested with trypsin and the adducts shown in Table 3 were detected by MALDI TOF/TOF MS (confirmed by LC-MS/MS, not shown). The MH+ for each peptide matched its theoretical mass plus an increment of 218 Da for BHT-QM or 234 Da for BHTOH-QM and the predominant daughter ion formed by CID corresponded to the native peptide in each case. Previous work demonstrated that neutral losses of these QMs are more facile than cleavage of the amide backbone (38). The MS data for P1 and P2 adducts of both QMs are shown in Figure 5 and spectra of the other adducts are presented as Supporting Information (Figure S2). The only adduct yielding sufficient information from CID to pinpoint the adducted residue was P6. Several low intensity fragment ions demonstrated that alkylation occurred at Cys 51 rather than Cys 58 (Figure S2). The rationale for assigning alkylation sites to specific histidine or lysine residues in P1-P5 was based on the absence of an alternative nucleophile capable of QM binding as arginines are not sufficiently reactive (35, 36). Tryptic peptides P1-P3 contain an internal lysine resulting from missed cleavage as expected from QM binding to the -amino group (7, 38) and the same reasoning implicates binding at His 28 of P4 rather than the C-terminal lysine. The contributions of these targeted amino acids to enzyme activity were investigated by site-directed mutagenesis. Except for K17R, each CBR variant shown in Table 4 was nearly as active or more active than the WT enzyme and each was inhibited by BHT-QM to a similar degree as the WT. In contrast, the mutant generated by replacing Lys 17 with arginine was only 34% as active as the WT and QM treatment did not further lower activity. These findings indicate that adduct formation with Lys 17 is the principal reason for CBR inhibition; this conclusion is consistent with the prominent role of this lysine in NADPH binding (39, 40). Additional evidence was provided by adding NADPH to the protein before exposure to BHT-QM; as shown in Table 2, the coenzyme protected CBR from inactivation and controls demonstrated no direct reaction between these compounds in the absence of protein. Adducts P1 and P2 were detected by MALDI TOF MS even at low ratios

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Figure 3. Effects of various treatments on cytosolic and recombinant mouse lung CBR. (A) Activity [(µmol NADPH oxidized) (min)-1 (mg protein)-1] measured in cytosol with added NADPH (100 µM) and menadione (200 µM). Incubations and assays were conducted as described in Materials and Methods. (B) Activity in the complete cytosolic system after preincubations for varying times with 100 µM BHT-QM or 50 µM BHTOH-QM. (C) Effects of the indicated concentrations of BHT-QM or BHTOH-QM on cytosolic CBR activity assayed after 20-min preincubations with a QM. (D) Activity [(µmol NADPH oxidized) (min)-1 (µg protein)-1] of purified, recombinant CBR after 20-min preincubations with indicated concentrations of BHT-QM or BHTOH-QM.

Table 2. Effects of Various Treatments on the Activity of Recombinant CBR activitya

CBR sample

% of untreated activity

b

CBR incubated with: no addition BHT 4-hydroxymethyl BHT 4-S-glutathionlymethyl BHT CBR pretreated with:c BHT-QM BHT-QM + GF GF only NEM NADPH + BHT-QMd

1.98 ( 0.18 1.90 ( 0.10 2.06 ( 0.23 1.90 ( 0.03

100 96 104 96

0.55 ( 0.11 0.60 ( 0.05 2.00 ( 0.02 1.83 ( 0.06 1.91 ( 0.02

28 30 101 92 96

a Results are the means ( SD of 3-4 determinations reported as (mmol NADPH oxidized) (min)-1 (mg)-1 protein. b Activity with menadione and NADPH as described in the text. Some incubations contained BHT (100 µM), 4-hydroxymethyl BHT (200 µM), or 4-S-glutathionylmethyl BHT (100 µM). c Recombinant CBR was subjected to one or more of the following treatments prior to measuring activity: incubation for 20 min with BHT-QM (200 µM), gel filtration (GF), incubation with a 10-fold molar excess of NEM, or addition of NADPH (100 µM) prior to BHT-QM (200 µM). d No interaction was detected spectrophotometrically between NADPH and BHT-QM in the absence of CBR.

of QM:protein. As shown in Figure S3 (Supporting Information), these adducts produced weak ions after reducing the ratio from 15:1 (QM:protein) to 5:1 but were not detected at a ratio of

1:1. On the other hand, a 5-fold excess of BHTOH-QM produced stronger signals than BHT-QM for both adducts. The P2 adduct of BHTOH-QM was detectable even when the QM: protein ratio was reduced to 1:1 coinciding with the greater reactivity and inhibition potency of this electrophile relative to BHT-QM. Reduction of ONE by Recombinant CBR. In order to assess possible consequences of CBR inactivation, we examined the ability of the mouse lung enzyme to metabolize ONE and HNE. An earlier study established that the former is an excellent substrate for the human liver form of CBR, but HNE is not reduced by this enzyme (19). Data summarized in Figure 6 demonstrate similar results for our recombinant CBR with activity toward ONE but not HNE. We also confirmed that BHT-QM exposure inhibits CBR activity with ONE as the substrate.

Discussion The results of this and earlier work (7) establish CBR, Prx6 and SOD1 as prominent targets of QM alkylation in the lungs of BHT-treated mice. Adducts were detected immunochemically in several different treatment groups demonstrating their rapid formation within 24 h and persistence for at least 7 days after BHT administration. Several lines of evidence support a causal relationship between BHT-induced pulmonary inflammation and



Figure 4. LC-MS analyses of recombinant CBR before and after treatment with BHT-QM. (A) Base peak chromatogram (BPC) of the untreated protein utilizing conditions described in Materials and Methods. MS peaks corresponding to all charge states from +23 to +32 were observed. Deconvolution software predicts a singly charged protein with mass 26,453 Da. (B) Data utilizing the same analytical conditions after treating CBR with a 15-fold molar excess of BHT-QM. The average spectrum over the range from 10.0 to 13.4 min demonstrates a complex mixture of multiply charged species. Deconvolution utilizing Agilent data analysis software with the ion abundance cutoff at 10% revealed four protein adducts of masses 26,670, 26,890, 27,107, and 27,326 Da corresponding to CBR adducted with 1, 2, 3, or 4 molecules of BHT-QM, respectively.

Table 3. Peptide Adducts Detected by MALDI TOF/TOF MS in Digests of BHT-QM-Treated CBRa MH+ observed peptide

sequence numbers

P1 P2 P3 P4 P5 P6

10-21 189-198 200-203 26-33 202-207 50-69

+

b

amino acid composition

MH calculated (m/z)

precursor (m/z)

product (m/z)

(R)ALVTGAGK17GIGR(D) (K)K189VSADPEFAR(K) (K)LK201ER(H) (K)ALH28ASGAK(V) (K)H204PLR(K) (K)EC51PGIEPVCVDLGDWD-ATEK(A)

1099.66 1119.58 545.34 754.42 522.31 2232.98c

1317.78 1337.74 779.50 972.57 740.45 2451.15

1099.62 1119.52 545.36 754.36 522.28 2233.02

a Each peptide was also detected as an adduct from BHTOH-QM-treated CBR with a mass increment of 234 Da. parentheses, and the sites of adduction are in bold font. c Includes one carbamidomethyl group.

Table 4. Effects of Mutations and BHT-QM on CBR Activity activitya - QM

mutation WT H28A H204A K17R K189R K201R

1.95 1.55 2.00 0.67 2.63 2.44

( ( ( ( ( (

0.09 0.21 0.10 0.11 0.11 0.12

+ QM (79)c (102) (34) (139) (129)

1.07 0.85 1.22 0.54 1.31 1.47

( ( ( ( ( (

0.17 0.17 0.05 0.15 0.02 0.13

inhibitionb 45d 45d 39d 19e 50d 40d

Results are the means ( SD of 3-4 determinations reported as (mmol NADPH oxidized) (min)-1 (mg)-1 protein. b Percentage decrease in activity of the mutant protein after treatment with 200 µM BHT-QM. c Percentage of WT activity in parentheses. d QM-treated significantly different from untreated, P < 0.01 in each case except H28A (P ) 0.03). e QM-treated not different from untreated (P ) 0.50). a

tumor promotion (9, 10) and our data suggest a mechanism for the pro-oxidant effects of this food additive and its role in carcinogenesis. Several factors indicate that adduct formation causes a significant degree of enzyme inhibition in ViVo. The treatment regimen involves multiple high doses of BHT and

b

Flanking residues are in

two electrophilic metabolites are generated in the target organ; the rapid formation and persistence of adducts suggests that substantial proportions of these proteins become alkylated; and irreversible inhibition of protective enzymes and oxidative stress from QM exposure was demonstrated in Vitro in lung homogenates and epithelial cells. BHT-induced oxidative stress also was demonstrated in mice by the accumulation of protein carbonyls reflecting attacks by ROS and conjugate additions of lipid peroxidation products (32–34). A strong relationship between low CBR expression and enhanced carcinogenesis has been demonstrated by several investigators (15–18). Presumably, losses of functional enzyme due to covalent modification would have a similar effect, particularly in combination with muted expression in tumor cells. The mechanism by which CBR suppresses tumor development has not been established; however, this enzyme protects tissues from oxidant-induced damage by metabolizing endogenous aldehydes. CBR is only one of many enzymes capable of reducing carbonyl groups (41), nevertheless this protein is abundant in normal lung tissue and is known to reduce the keto

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Shearn et al.

Figure 5. MALDI TOF MS of adducted peptides P1 and P2 in digests of recombinant CBR treated with a 15-fold molar excess QM. Mass spectra of P1 and P2 alkylated by (A) BHT-QM or (B) BHTOH-QM and the corresponding CID spectra of each adduct. (C) Spectrum of the same mass range from a digest of untreated CBR. The reference peak at m/z 1308.7 in each spectrum is a nonadducted peptide present in these digests.

group of ONE to form HNE (Figure 6 and ref 19). Both of these aldehydes are important products of lipid peroxidation (21, 42), however the former is more reactive in forming protein adducts and cross-links (43). Damage to CBR, therefore, may disrupt cell homeostasis during oxidative stress occurring in the lungs of BHT-treated mice. In a recent study comparing the sources of protein carbonyls, Yuan et al. (44) concluded that conjugate additions of R,β-unsaturated aldehydes are quantitatively more important than direct oxidations by ROS. The accumulation of protein damage accompanying BHT administration in the present work may be due in part to ONE from a combination of CBR inhibition and increased lipid peroxidation.

The mechanism of inhibition was investigated by mass spectrometry and site-directed mutagenesis. After treatment of recombinant CBR with excess BHT-QM, MS data of intact protein demonstrated multiple alkylation sites that were subsequently identified by MALDI TOF analyses of tryptic digests. Cysteine residues were determined not to be critical sites as NEM derivatization of these thiols had no effect on activity. Two histidine and three lysine resides located on or near the solvent surface (Figure 7A) were alkylated by both BHT-QM and BHTOH-QM; however, adducts of the latter were detected at lower QM:protein ratios consistent with the higher potency of this electrophile for enzyme inhibition and tumor promotion,


Figure 6. CBR activity with R,β-unsaturated aldehydes. Incubations were conducted and activity measured as described in Materials and Methods and the legend to Figure 3, except that ONE or HNE was substituted for menadione. In one experiment, CBR was incubated with 200 µM BHT-QM prior to measuring activity. Results are the means ( SD of 3 determinations.

Figure 7. Chain A of mouse lung CBR (Protein Databank entry 1CYD) displayed with the Visual Molecular Dynamics viewer from www.ks.uiuc.edu/research/vmd. (A) Ribbon representation of the protein backbone, CPK representations of the targeted residues, and a stick representation (blue) of bound NADPH. (B) Lysines 17 and 189 in CPK format and bound NADPH (red) shown in relation to hydrophobic surfaces within 5 Å (green). These surfaces are transparent and actually enclose portions of each lysine and the coenzyme.

and its greater chemical reactivity (3, 5, 7, 35). The contributions of these adducts to enzyme inhibition were assessed by preparing five mutants in which the nucleophilic amino acid was replaced by alanine or arginine using site-directed mutagenesis. The mutant K17R was the only protein exhibiting significantly lower catalytic activity than WT CBR. Lys 17, Arg 38 and Lys 153 are known to be involved in NADPH binding (39). Nakanishi et al. (40) also demonstrated that the K17R mutant retains


activity, indicating that the basic side chain of Arg 17 is capable of mimicking the -amino groups of Lys 17 for this interaction. Unlike lysine, however, arginine does not form QM adducts and consequently the K17R mutant was resistant to inactivation by BHT-QM. Lys 153 is located inside the binding pocket and is not alkylated, whereas Lys 17 at the entrance to the NADPH binding site is considerably more accessible. Both Lys 17 and Lys 189 appear to be preferred targets as these were the only adducts detected at very low QM to protein ratios (Figure S3). Selectivity may be due to the presence of hydrophobic amino acids within 5 Å of these residues (Figure 7B) and within the NADPH binding pocket which is believed to accommodate hydrocarbon tails of fatty acids such as arachidonic acid (40). The QMs are lipophilic and are expected to accumulate at accessible hydrophobic regions of the protein resulting in high local concentrations and an opportunity to react with nearby nucleophiles. Other examples of QM additions to amine sidechains include reactions of BHT-QM with bovine SOD1 (7) and the para-QM from 2-tert-butyl-4,6-dimethylphenol with myoglobin (45). Monocrotoline pyrrole, a toxic electrophile formed by P450-catalyzed oxidation of monocrotoline, was initially thought to react only with cysteines but later found to bind with proteins that do not contain cysteine (46). These results support earlier conclusions concerning the importance of local environments within proteins for influencing adduct formation (36, 47). Electrophiles may potentially affect cell signaling by a variety of pathways, for example by direct binding to regulatory proteins such as Keap1 or enzymes involved in phosphorylation or dephosphorylation (21, 48). To date, such adducts have not been detected in mouse lung; however, an adduct of glutathione S-transferase P1-1 was identified in transformed pulmonary epithelial cells incubated with BHT-QM (38). This protein has regulatory functions through complex formation with c-Jun N-terminal kinase (49) that may potentially be compromised by alkylation. The failure to detect this adduct in ViVo may reflect the low expression of glutathione S-transferase P1-1 in normal cells relative to tumor cells (50). The bulk of our data, however, support an indirect role for QMs in tumor promotion; we hypothesize that these metabolites cause oxidative stress during chronic BHT treatment due to lowered GSH levels from conjugate formation and increased levels of ROS and products of lipid peroxidation capable of altering cellular homeostasis and affecting cell growth and/or apoptosis (8, 20, 21). Our data suggest a role for CBR inhibition and increased levels of ONE (and possibly other reactive aldehydes) capable of modifying proteins and DNA. The 2.5-fold increase in PGE2 measured in BHT-treated mice (24) may be due in part to CBR inhibition as this pro-inflammatory mediator is a substrate for the enzyme (22, 23). In our hands PGE2 was a poor substrate for the mouse lung form of CBR (data not shown); however, activity was completely destroyed by BHT-QM exposure. The results of this investigation support previously published data implicating chronic inflammation as the primary cause of enhanced tumorigenesis in this model system (9, 10) and provide plausible mechanisms involving the cumulative effects of damaging several protective enzymes by adduct formation. Acknowledgment. We thank Mr. James Roede for assistance with protein carbonyl measurements and Dr. Dennis Peterson for gifts of ONE and HNE. This work was supported by USPHS grant RO1 CA41248 from the National Cancer Institute. Supporting Information Available: Table S1 of oligonucleotides for the preparation of CBR mutants, Figure S1 with LC-

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MS data of intact CBR treated with a 5-fold excess of BHTQM, and Figures S2 and S3 with MALDI TOF data for several peptide adducts. This material is available free of charge via the Internet at http://pubs.acs.org.

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