Chem. Res. Toxicol. 2005, 18, 1575-1585
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Immunochemical and Proteomic Analysis of Covalent Adducts Formed by Quinone Methide Tumor Promoters in Mouse Lung Epithelial Cell Lines Brent W. Meier, Jose D. Gomez, Angela Zhou, and John A. Thompson* Department of Pharmaceutical Sciences, Box C238, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received April 21, 2005
Two quinone methide (QM) metabolites of the phenolic antioxidant butylated hydroxytoluene (BHT), 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and the tert-butylhydroxylated derivative (BHTOH-QM), are believed to be responsible for promoting lung tumor formation in mice treated with BHT. QMs are strongly electrophilic and undergo Michael type additions with nucleophiles at the exocyclic methylene to form benzylic thioether adducts. Our goal was to identify intracellular protein targets of these QMs in order to gain insight into their effects on tumorigenesis. Cell line E10 of mouse lung epithelial origin and its spontaneous transformant, the tumorigenic E9 cell line, were treated with BHT-QM or BHTOH-QM, and cellular proteins were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Adducted proteins were detected on western blots with polyclonal antibodies developed to a conjugate of BHTOH-QM that recognized adducts of both QMs bound to thiol groups of Cys and side chain amino groups of Lys and His residues. Tryptic digests of immunoreactive proteins were analyzed by HPLC mass spectrometry (LC/MS) and identified by searching protein databases using MS/MS data. In a few cases, adducted peptides in these digests were detected by matrix-assisted laser desorption/ionization time-of-flight MS. A total of 37 immunoreactive proteins were identified including proteins involved in carbohydrate metabolism, nucleic acid synthesis, and RNA and protein processing, in addition to several cytoskeletal and stress-related proteins. About half of the protein adducts were found in both cell lines. Adducts detected only in transformed E9 cells include glutathione S-transferase P1, peroxiredoxin 2, nucleoside diphosphate kinase, and vinculin, whereas several alkylated cytoskeletal proteins such as tubulins, vimentin, calvasculin, and calcyclin were detected exclusively in E10 cells. Several of the proteins modified by BHT-derived QMs have been implicated in various aspects of tumorigenesis and are excellent candidates for further study into the consequences of alkylation on cellular transformation.
Introduction Cancer is a multistep process involving initiation, promotion, and progression. Promoting agents induce the selective clonal expansion of initiated cells by various mechanisms including direct interactions with specific receptors, inhibition of apoptosis, and generation of reactive oxygen species that alter redox states of kinases and transcription factors (1, 2). A number of antioxidants exhibit cancer chemopreventive properties by acting on this reversible stage of tumorigenesis (3, 4). Despite the well-known radical-quenching properties of 2,6-di-tertbutyl-4-methylphenol (BHT),1 however, this and several structurally related alkylphenols enhance the development of lung tumors in mice when administered in several weekly doses after a single injection of the initiator 3-methylcholanthrene (5). Structure-activity studies and correlations of metabolite formation with species and organ specific tumorigenesis strongly indicate that the promoting effects of BHT result from its conversion to two quinone methides (QMs), BHT-QM and BHTOH-QM, shown in Figure 1A, by pulmonary cyto* To whom correspondence should be addressed. Tel: 303-315-6167. Fax: 303-315-0274. E-mail:
[email protected].
chrome P450 (6-8). Studies in a mouse skin model of multistage carcinogenesis further demonstrated the ability of BHT-QM to enhance tumor formation (9). The BHT-mouse lung system is the most extensively characterized model of multistage pulmonary carcinogenesis and a rare example in which an electrophilic metabolite has been implicated in promotion. A similar role was proposed for the sulfate conjugate of 1′-hydroxysafrole in rat liver tumorigenesis (10). These data suggest that electrophiles are more important contributors to epigenetic aspects of tumorigenesis than is currently recognized (11), although additional knowledge of the specific proteins alkylated in responsive tissues is needed to elucidate critical interactions. 1 Abbreviations: 2-DE, two-dimensional isoelectric focusing SDSPAGE; BHT, 2,6-di-tert-butyl-4-methylphenol; BSA, bovine serum albumin; 4-BT, 4-tert-butyltoluene; DTT, dithiothreitol; EDC, 1-ethyl3-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; GSH, glutathione; GST, glutathione S-transferase; hsp, heat shock protein; KLH, keyhole limpet hemocyanin; MeCN, acetonitrile; MS/MS, tandem MS; MALDITOF, matrix-assisted laser desorption/ionization time-of-flight; MSDB, Mass Spectrometry Database; NAC, N-acetyl-L-cysteine; NAH, NRacetyl-L-histidine; NAL, NR-acetyl-L-lysine; PVDF, poly(vinylidene difluoride); S9, 9000g supernatant fraction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STI, soybean trypsin inhibitor.
10.1021/tx050108y CCC: $30.25 © 2005 American Chemical Society Published on Web 09/07/2005
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Meier et al.
Figure 1. (A) Structures and abbreviations of QMs BHT-QM and BHTOH-QM and their corresponding glutathione (BHT-SG and BHTOH-SG) and N-acetyl cysteine conjugates (BHT-NAC and BHTOH-NAC); (B) the immunogen formed by linking BHTOHNAC to KLH using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC); and (C) glutathione conjugates of 2,4,6-trimethylphenol (TMP-SG) and 4-tert-butyltoluene (4-BT-SG), and the NAL and NAH conjugates of BHT (BHT-NAL and BHT-NAH, respectively).
QMs mediate the covalent binding of BHT to proteins in rat liver and mouse lung (7, 12), but to date, only two liver protein-BHT conjugates have been identified (13). Protein adducts formed in cells and tissues exposed to a variety of electrophilic chemicals and metabolites have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by measuring radioactivity associated with 14C-labeled adducts or by immunoblotting with antibodies developed to conjugates of these electrophiles. For example, antisera to haptens such as BHT (13), hydroquinone (14), estragole (15), and clozapine (16) allowed effective detection of protein adducts in liver homogenates or neutrophils by one-dimensional SDS-PAGE and western blotting. Many more adducts were found in studies employing twodimensional isoelectric focusing SDS-PAGE (2-DE) methodology for separating proteins, for example, in the livers of mice treated with acetaminophen (17), murine pulmonary endothelial cells incubated with monocrotaline pyrrole (18), and epithelial cells isolated from intact murine lungs exposed to naphthalene (19). Radiolabeled compounds were utilized in those studies to detect adducts among gel-separated proteins for subsequent excision, hydrolysis, and identification by MS. Similar
methods were utilized in our work to identify proteins alkylated by BHT-QM and BHTOH-QM in cell lines derived from murine lung, except that adducts were detected by western blotting with polyclonal antibodies to the immunogen derived from BHTOH shown in Figure 1B. The initial objective was to determine the specificity of the antiserum and confirm that it effectively detected adducts of both QMs. Another objective was to investigate protein targets of QMs in E10 cells and their spontaneous transformants, E9 cells (20). The former were derived from murine pulmonary epithelial type 2 cells, display anchoragedependent growth, and are nontumorigenic when injected into mice (21). In contrast, E9 cells grow rapidly in soft agar, are tumorigenic in vivo, and are more resistant to apoptosis than E10 cells after exposure to various chemical agents. The neoplastic characteristics of E9 cells are intermediate between E10 and tumor-derived cell lines, and the E9/E10 sibling pair has been used to model early stages of tumor promotion. In a recent study (22), we determined that the major form of glutathione S-transferase (GST) in these cells, i.e., GSTP1-1 that exhibits both antioxidant and regulatory functions (23), is susceptible to alkylation and inactivation by BHT-QM. We
Quinone Methide Tumor Promoters in Mouse Lung
now provide evidence for additional protein adducts formed in this in vitro model of pulmonary carcinogenesis, including several interactions that merit further investigation for their potential roles in tumor promotion.
Materials and Methods Materials. All chemicals were obtained from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO) unless otherwise noted. Solvents were HPLC grade from Fisher Scientific (Pittsburgh, PA). QMs were prepared by oxidizing the corresponding phenol with silver oxide in acetonitrile (MeCN), and the concentrations were determined by UV analysis as described (8). The conjugates BHT-SG, BHT-N-acetyl-L-cysteine (NAC), BHTOH-SG, TMPSG, and 4-tert-butyltoluene (4-BT)-SG shown in Figure 1A,C have been described (24, 25). BHT-NR-acetyl-L-lysine (NAL) and BHT-NR-acetyl-L-histidine (NAH) were prepared by combining a MeCN solution of BHT-QM with an equal volume of 50 mM sodium phosphate buffer (pH 7.4) containing a 5-fold molar excess of the amino acid. These reactions were conducted for 1 h at 5 °C, MeCN was evaporated, and the products were isolated by solid phase extraction with C18 Sep-Pak cartridges (Millipore, Milford, MA) and recovered by elution with methanol. Conjugates were purified by HPLC on a 4.6 mm × 250 mm Prodigy 5 µ ODS3 column (Phenomenex, Torrance, CA) with a gradient of 30-80% MeCN in water containing 0.1% trifluoroacetic acid over 10 min at a flow rate of 1.0 mL/min and UV detection at 280 nm. Analysis of BHT-NAL by electrospray ionization (ESI) LC/tandem MS (MS/MS) yielded MH+ at m/z 407 Da and fragment ions at 219 (ArCH2+) and 189 Da (NAL + H+). BHT-NAH eluted from the HPLC column in two peaks corresponding to Nπ and Nτ isomers, both yielding MH+ ions at m/z 416 and fragment ions at 219 and 198 Da (NAH + H+). The BHT-NAH utilized in this work was a mixture of isomers. Polyclonal Antibody Preparation and Enzyme-Linked Immunosorbent Assay (ELISA). An antigen was prepared by linking the hapten BHTOH-NAC to amino groups of the carrier protein keyhole limpet hemocyanin (KLH), rabbits were immunized, and antisera titers of 82000-120000 (
