Characterization of a Structurally Intact in Situ Lung Model and

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Articles Characterization of a Structurally Intact in Situ Lung Model and Comparison of Naphthalene Protein Adducts Generated in This Model vs Lung Microsomes Ching Yu Lin,*,† Margaret A. Isbell,† Dexter Morin,† Bridget C. Boland,† Michelle R. Salemi,‡ William T. Jewell,‡ Alison J. Weir,§ Michelle V. Fanucchi,§ Gregory L. Baker,§ Charles G. Plopper,§ and Alan R. Buckpitt† Department of Molecular Biosciences, School of Veterinary Medicine, Molecular Structure Facility, and Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, California Received September 9, 2004

Airway epithelial cells are a susceptible site for injury by ambient air toxicants such as naphthalene that undergo P450-dependent metabolic activation. The metabolism of naphthalene in Clara cells to reactive intermediates that bind covalently to proteins correlates with cell toxicity. Although several proteins adducted by reactive naphthalene metabolites were identified in microsomal incubations, new methods that maintain the structural integrity of the lung are needed to examine protein targets. Therefore, we developed a method that involves inflation of the lungs via the trachea with medium containing 14C-naphthalene followed by incubation in situ. The viability of this preparation is supported by maintenance of glutathione levels, rates of naphthalene metabolism, and exclusion of ethidium homodimer-1 from airway epithelium. Following in situ incubation, the levels of adduct per milligram of protein were measured in proteins obtained from bronchoalveolar lavage, epithelial cells, and remaining lung. The levels of adducted proteins obtained in lavage and epithelial cells were similar and were 20-fold higher than those in residual lung tissue. 14C-Labeled adducted proteins were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) and quadrupole-TOF MS/MS. Major adducted proteins include cytoskeletal proteins, proteins involved in folding and translocation, ATP synthase, extracellular proteins, redox proteins, and selenium binding proteins. We conclude that in situ incubation maintains structural integrity of the lung while allowing examination of reactive intermediate activation and interaction with target cell proteins of the lung. The proteins adducted and identified from in situ incubations were not the same proteins identified from microsomal incubations.

Introduction The epithelium of conducting airways is one of the most susceptible sites for acute injury after exposure to a variety of toxicants, including oxidant air pollutants, particulate matter and metabolically activated xenobiotics. The nonciliated bronchiolar (Clara) cell is a prominent epithelial cell type in the airways of rodents (1) and is also the principle site of xenobiotic metabolism catalyzed by the cytochrome P450 monooxygenases in the lung (2, 3). Naphthalene, a compound which undergoes P450 dependent activation to a Clara cell toxicant, is a widespread environmental contaminant to which humans * Corresponding author. Mailing address: Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616. Telephone: 530-752-2534. Fax: 530-752-3394. E-mail: [email protected]. † Department of Molecular Biosciences, School of Veterinary Medicine. ‡ Molecular Structure Facility. § Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine.

are exposed. Previous studies have established that intraperitoneal administration of naphthalene causes bronchiolar epithelial cell necrosis in mice and hamsters and cytotoxicity in the nasal olfactory epithelium of rats, hamsters, and mice (4). The variability in species and regional sensitivity to naphthalene within the respiratory system in rodents underscores the need to understand the biochemical and metabolic mechanisms of naphthalene leading to toxicity. Such data are critically important for estimating the risks of human exposure and for developing biomarkers which are based on a good understanding of the events critical to toxicity. Naphthalene toxicity has been thoroughly characterized in rodents after administration via intraperitoneal and inhalation routes. Intraperitoneal administration of naphthalene to mice causes Clara cell injury in the distal airway at doses of 50-100 mg/kg and, as the dose increases, injury extends into more proximal airways (g300 mg/kg) (4). In contrast, exposure to naphthalene by inhalation causes injury to Clara cells in the proximal

10.1021/tx049746r CCC: $30.25 © 2005 American Chemical Society Published on Web 04/13/2005

Naphthalene Protein Adducts in the Lung

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Figure 1. Schematic diagram showing pathways of naphthalene metabolism which could result in the formation of reactive metabolites that could become bound covalently to proteins.

airways of mice at concentrations as low as 2 ppm. As the dose increases, injury extends into more distal airways (g8.5 ppm) (5). The differences in injury patterns after different routes of administration suggest that susceptibility to naphthalene injury in mice is not determined by airway level cell populations but rather by the concentrations of naphthalene available for in situ metabolism. Previous studies have demonstrated that species and site susceptibility, naphthalene metabolism, total protein covalent binding, and depletion of glutathione (GSH)1 are interrelated events. The rate of in situ naphthalene metabolism to reactive electrophiles appears to be a critical determinant in the region- and species-selective cytotoxicity of naphthalene in rodents (6, 7). In addition, research using mouse pulmonary Clara cells and subcompartments of mouse airway explants have established a close association between the level of total protein binding in airways and lung injury (8). Together, these studies suggest that formation and covalent binding of reactive metabolites may be intimately associated with the processes leading to cytotoxicity. There is currently very little information on the proteins adducted by 1 Abbreviations; GSH, glutathione; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight.

reactive metabolites of naphthalene or on the precursor reactive metabolites that bind. Definitive evidence has been presented for the formation of covalent protein adducts with both the primary 1,2-epoxide and the naphthoquinones (9, 10). The chemical instability of the diepoxide suggests that this may also be a precursor reactive metabolite capable of binding covalently to critical proteins (11). A schematic diagram (Figure 1) indicates the formation of some of the possible reactive metabolites from naphthalene. This study was designed to develop and validate an intact lung in situ incubation method for identifying proteins covalently adducted by bioactivated toxicants in airway epithelium. The method allows intact airway epithelium to be exposed to cytotoxicants and instillation of agarose blocks the parenchyma decreasing the possibility of a confounding influence of metabolites generated in the peripheral lung. The optimal method for identifying protein adducts generated from pulmonary epithelial cell cytotoxicants should maintain the complex architecture of the lung and the key metabolic functions of airway epithelial cells. To validate this approach, several issues needed to be addressed: 1) agarose instilled into the trachea to block access of substrate to the parenchyma should be located below the distal bronchiole

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allowing epithelial cells to be exposed to medium, 2) epithelial cells must remain viable during incubation with medium alone, 3) GSH levels should be maintained at the same level during incubation with medium alone, 4) the rates of naphthalene metabolism should remain constant, and 5) the recovered volume of medium, extracellular wash and protein lysis-lavage from epithelial cells should remain constant after the first and second hours of incubation. These studies take advantage of a recently reported procedure for selective isolation of epithelial cell proteins from the lung (12) to identify those proteins adducted by reactive naphthalene metabolites in target cells for naphthalene toxicity, namely airway epithelial cells.

Methods and Materials Caution: [14C]-Naphthalene is hazardous and should be handled with care. The compound is volatilized easily at room temperature and should be kept in solution to avoid exposure. Wear proper protective clothing while handling this chemical. Radiochemicals, Chemicals, and Reagents. [14C]-Naphthalene (specific activity of 52 mCi/mmol) was obtained from American Radiolabeled Chemicals, St. Louis, MO. The radiochemical purity was >99% as determined by HPLC. The stock solution (specific activity of 1.14 × 105 dpm/nmol at 50 mM) in methanol was added at 10 µL/mL to yield a final substrate concentration of 500 µM for lung microsomal incubations. The stock solution was diluted with methanol to 25 mM and added at 10 µL/mL to yield final substrate concentrations for in situ incubation of 250 µM. Deficient Waymouth’s MB 752/1 medium (without reduced glutathione, L-cysteine, L-cystine, l-glutamine, and L-methionine) was purchased from GibcoBRL Labs (Grand Island, NY). Protease inhibitor cocktail III was obtained from Calbiochem (La Jolla, CA). Duracryl 30 t-2.6 C was obtained from Genomic Solutions (Ann Arbor, MI). All other electrophoresis materials were obtained from Amersham Biosciences (Piscataway, NJ). The fluorochromes ethidium homodimer-1 and YO-PRO-1 were obtained from Molecular Probes (Eugene,OR). 1,2-Dihydroxy1,2-dihydronaphthalene was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). All other chemicals purchased from a commercial vendor were reagent grade or better. Animals. All animal use was approved by the Animal Use and Care Committee at the University of California, Davis. Male NIH Swiss mice (25-35 g) were purchased from Harlan (San Diego, CA). Animals were allowed free access to food and water and were housed in an AAALAC accredited facility in HEPA filtered cage racks at the University of California, Davis, for at least 5 days before use. Lung Microsome Preparation and Incubation. Mouse lung microsomal fractions were prepared by differential centrifugation according to previously published methods (13). Protein concentration of the suspended pellet was determined using the Bradford method (14) with bovine serum albumin as the standard. Incubations contained: 2 mg/mL microsomal protein, NADPH generating system (13), and either 14C-labeled or unlabeled naphthalene. Incubations were done for 20 min at 37 °C in a shaking water bath. Incubations were placed on ice to stop the reaction and were centrifuged at 100,000 g for 60 min. The supernatant was concentrated and desalted using Ultrafree Centrifugal Filter Units (Millipore, Bedford, MA). Lysis solution containing 2 M thiourea, 7 M urea, 4% w/v CHAPS, 0.5% w/v Triton X-100, 1% w/v dithiothreitol, and 2% v/v protease inhibitor cocktail III was added to dissolve concentrated supernatant proteins. The resuspended volume was then removed and stored at -80 °C until isoelectric focusing. Validation of Airway in Situ Incubation. 1. In Situ Incubations of Mouse Airway. Mice were deeply anesthetized

Lin et al. with sodium pentobarbital. The abdomen was opened by midline incision and the animal was exsanguinated by severing the systemic aorta. The animal was hemisected below the diaphragm, the diaphragm was punctured to release the vacuum in the chest and the lungs were left in the thoracic cavity. The trachea was cannulated. Access of incubation medium to the parenchyma was decreased by instillation of low gelling temperature agarose (0.5 mL of 0.75%, 37 °C) in Waymouth’s medium deficient in sulfur-containing amino acids via the trachea as described previously by Wheelock et al. (12). This was followed immediately by instillation of 0.5 mL oxygenated, sulfur amino acid deficient Waymouth’s medium with or without naphthalene at 37 °C. The upper body was placed in a beaker with deficient medium at 4 °C for 10 min to allow the agarose to solidify and then transferred to a new beaker containing deficient Waymouth’s medium and incubated at 37 °C for 1 h. After 1 h, the medium was removed from the airway via the trachea using a syringe and fresh, oxygenated medium was slowly instilled for another hour of incubation at 37 °C. After incubation, the airway medium was removed and the volumes of medium recovered were measured. Lungs from three mice were incubated with deficient medium and examined by confocal microscopy to determine whether viability of epithelial cells was maintained over the time of incubation. Twenty mice were randomly assigned to four groups (n ) 5) and incubated with 250 µM naphthalene or control medium for 1 or 2 h to measure GSH levels in dissected airway subcompartments. Lungs from at least six mice were incubated with 250 µM naphthalene or [14C] naphthalene. Medium recovered from the airways and epithelial proteins removed by lysislavage (12) were used to quantify metabolites and identify protein adducts, respectively. Airway epithelial cell lysate recovered by the lysis-lavage method was used for total protein adduct formation measurements. 2. Histopathologic Examination of Cell Viability of Airway Epithelium. Mouse lungs (n ) 3) were examined for the presence of injured cells by a modification of a method previously described by Van Winkle et al. (15). Briefly, following in situ incubation, the medium was removed and 0.4 mL of ethidium homodimer-1 (5 µM in medium) was instilled into the airway via the trachea. Ten min later, the airways were rinsed three times with phosphate buffered saline and the lungs were fixed with 1% paraformaldehyde and 1% glutaraldehyde in 0.2 M cacodylate buffer via the trachea for at least 1 h. The lungs were removed from the thorax, and the luminal surface of the airway tree of the left lobe was exposed by microdissection and counterstained with the nuclear binding fluorochrome YOPRO-1 (4 µM) for 20 min to label the nuclei. Samples were imaged using a scanning laser confocal microscope (Bio-Rad, Hercules, CA), as described previously (15). 3. GSH Levels in Dissected Airways Following in Situ Incubation. Medium (control) or medium containing naphthalene was removed following incubations at 0-1 or 1-2 h. The lungs were filled with 0.5 mL low-temperature gelling agarose, and the airways were dissected as described in detail earlier (6). Airway subcompartments were placed in 200 mM methane sulfonic acid solution containing 5 mM diethylenetetrapentaacetic acid for determination of GSH levels by previously published HPLC methods with electrochemical detection (16). Precipitated proteins were dissolved in 1 M NaOH and protein content was measured using a modification of the Lowry method (17) using BSA as a standard. 4. Measurement of Water Soluble Naphthalene Metabolites. The medium recovered after the first and second hour incubation with radiolabeled naphthalene was analyzed by reverse phase HPLC with UV detection. Samples were extracted twice with hexane to remove nonmetabolized, volatile radioactive substrate. Earlier studies have shown that this hexane extraction removes variable amounts of 1-naphthol; thus analysis of this phenolic metabolite was not conducted in this work. The remaining aqueous phase was evaporated to dryness in a vacuum centrifuge and 1 mL of 2% acetic acid was added to

Naphthalene Protein Adducts in the Lung adjust the sample to pH 2-3. The solution was applied to a Varian Bond-Elute ENV (1 gm) solid phase extraction column which was prewashed with acetonitrile and conditioned with 2% acetic acid. The column was washed with 2 column volumes of 2% acetic acid, and naphthalene metabolites were eluted with 2 mL of acetonitrile. The acetonitrile was evaporated and the residue reconstituted in water for HPLC analysis as described previously (7). Thioether conjugate standards of naphthalene (glutathione, cysteine, cysteinyl-glycine) were prepared from naphthalene oxide as described previously (18). Fractions of HPLC eluant were collected at 1 min intervals for radioactivity determination. Quantitative measurements of metabolite formation were based on amount of 14C eluting from the column. Proteins Targeted by Reactive Naphthalene Metabolites in Airway Epithelium. 1. Recovery of Proteins from Airway Epithelium by Lysis-Lavage. After mouse airways were incubated with 250 µM naphthalene or 250 µM [14C] naphthalene for 2 h, proteins from airway epithelial cells were recovered for identification of protein adducts using methods described by Wheelock et al. (12). Briefly, the airways were lavaged once with 5% dextrose to remove remaining medium and the airway epithelium was removed using lysis solution as described earlier. Recovered 5% dextrose and protein lysis solution were frozen and stored at -20 °C for protein adduct analysis. 2. Comparison of Protein Covalent Binding Levels in Proteins Recovered by Bronchoalveolar Lavage, LysisLavage and in the Remaining Tissue. Lungs of three mice were filled with 250 µM [14C] naphthalene in situ as described earlier and incubations were conducted for 2 h. Airways were washed with dextrose and airway epithelial cell proteins were removed by lysis-lavage. After removing the airway epithelial proteins, the remaining lung was removed and homogenized with lysis solution for analysis of total protein covalent binding levels. Proteins in the 5% dextrose wash, lysis-lavage solution, and the remaining lung tissue were precipitated by addition of 5 volumes of ice-cold acetone overnight at -20 °C. The protein pellet was washed exhaustively with acetone until DPM counts in the supernatant were not statistically above background. The protein pellets were dried and dissolved in 1 M NaOH for protein measurement using a modification of the Lowry method (17). Additional aliquots were counted to determine the amount of reactive metabolite bound. 3. Separation and Detection of Protein Adducts. IPG buffer (1%), protease inhibitor cocktail III (2%), 1% w/v dithiothreitol, and orange dye were added to protein solutions recovered by lysis-lavage. Three narrow range (pH 3.5-4.5, 4.55.5, 5.5-6.7) 18 cm Immobiline Dry Strips were rehydrated with 800 µg protein each. Isoelectric focusing was conducted at 20 °C gradually increasing the voltage from 50V to 3500V over 7 h and running at 3500V for 19 h to reach a total of 74 kVh. After focusing, the strips were incubated in SDS equilibration buffer (50 mM tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, bromophenol blue) with 0.065 M dithiothreitol. Protein sulfhydryls were alkylated with 0.01 M iodoacetamide in SDS equilibration buffer for 15 min prior to second dimension separation. For proteins separated on pH 6-9 IPG strips, 2-propanol (10%), glycerol (5%), SDS (0.04%), and Triton (1%) were added. Proteins were reduced by tributylphosphine (7 mM) and then alkylated by iodoacetamide (0.01M) before paperbridge loading to IPG stips and isoelectric focusing (Amersham Biosciences’ handbook) to reach a total of 80 kVh. Then, the strips were placed on the top of SDS (Duracryl gels made to 10%T, 2.6%C) (24 cm × 18 cm), and sealed to the gel using 1% IsoGel agarose. A second dimension separation was performed in a Hoefer DALT 2D electrophoresis system at 10 °C overnight until the dye front had migrated 17 cm using manufacturer’s standard protocols. Silver staining for spot visualization and destaining for protein identification were completed according to protocols described by Gharahdaghi et al. (19). Proteins isolated from incubations performed using 14Cnaphthalene were electroblotted to a Sequi-blot PVDF mem-

Chem. Res. Toxicol., Vol. 18, No. 5, 2005 805 brane (0.2 µm, Bio-Rad). Electroblotting was performed in the Hoefer DALT 2D tank at 10 °C for 19 h using 250 mA in continuous mode. Membranes were washed three times with deionized water and once with 60% methanol for 30 min each and dried. The blots were stained with sulforhodamine G to visualize the protein spots, washed, dried and membranes were placed against storage phosphor screens for 45 days with complete lead shielding. The screens were scanned using a Molecular Dynamics Typhoon 8600. The phosphor screens provided an image to guide the selection of protein spots for identification. 4. In-Gel Digestion. The excised protein spots were destained, washed, diced, dried and rehydrated, and alkylated according to established protocols (20). Proteins were digested with sequencing grade, modified trypsin (Promega, Madison, WI) at a final amount of 25 ng per sample at 37 °C for 24 h. Peptides were extracted once with 0.1% TFA in water and once with 5% formic acid in 50% acetonitrile (1:1). The extraction volume was carefully controlled so that it did not exceed 50 µl for mass spectrometric analysis. 5. Peptide Mass Mapping by MALDI-TOF. Tryptic peptides were analyzed with a Bruker Biflex III MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) equipped with a pulsed N2 laser (337 nm), and a delayed extraction ion source. Fractions of tryptic peptides were desalted, mixed with matrix solution (R-cyano-4-hydroxycinnamic acid in 0.1% TFA-50% acetonitrile in water) and applied to the target as described in detail previously (21). The mass spectra were acquired in the reflectron mode. Internal mass calibration was performed with two trypsin autodigestion fragments (842.5 and 2211.1 Da). This procedure typically resulted in mass accuracies of 50 ppm or better. Measured monoisotopic masses of tryptic peptides were used as inputs to search Mus musculus databases or the NCBInr database (all nonredundant GenBank CDS: translations + PDB + SwissProt + PIR + PRF) using the Mascot search engine with a probability based scoring algorithm (http: www.matrixscience.com/). Up to one missed tryptic cleavage was considered in most cases. A mass accuracy of 50 ppm or lower was used for each search. 6. Sequencing of Peptides by Tandem Mass Spectrometry (MS/MS). Aliquots of tryptic peptides were purified and concentrated using POROS R2 resin (Perceptive Biosystems, Framingham, MA) in a microcolumn following the method described in the Protana manual (Protana, Odense, Denmark). Peptides were then eluted into the nanoES capillary using 50% methanol-5% formic acid. Tryptic peptides were analyzed with a hybrid nanospray/ESI-Quadrupole-TOF-MS and MS/MS in a QSTAR mass spectrometer (Applied Biosystems Inc., Foster City, CA). Peptides in methanol/formic acid/H2O were sprayed from the gold coated capillary. Calibration of the QSTAR was performed with a standard peptide mixture, and this calibration yielded mass accuracies of 5 ppm or better. N2 gas was used as the collision gas. More than 10 peptides were selected for fragmentation for each protein. From these spectra, in most cases only the top 2-3 spectra with complete or near complete fragmentation patterns were chosen to facilitate interpretation. Sequences were evaluated using the Analyst de novo sequencing software and manually checked by two individuals and, in all cases, the assignment of all ions in the spectra was attempted. Some MS/MS fragmentation data were used for database searches for further protein identification using the Mascot search engine or MS-Blast search (http:// dove.embl-heidelberg.de/Blast2/msblast.html). Only the highest confidence sequences were reported, and these were often used in corroboration with weaker spectra for confirmation of protein identification. Statistical Analysis. Data were analyzed using SigmaStat (Jandel Corporation, San Rafael, CA) for one way analysis of variance (ANOVA). A p value of