Proteomic Analysis of Diaminochlorotriazine Adducts in Wister Rat

Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LβT2 cells indicating the formation of DACT prote...
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Proteomic Analysis of Diaminochlorotriazine Adducts in Wister Rat Pituitary Glands and LβT2 Rat Pituitary Cells G. P. Dooley,† K. F. Reardon,‡ J. E. Prenni,§ R. B. Tjalkens,† M. E. Legare,† C. D. Foradori,| J. E. Tessari,† and W. H. Hanneman*,† Department of EnVironmental and Radiological Health Sciences, Department of Chemical and Biological Engineering, Proteomics and Metabolomics Facility, and Department of Biomedical Sciences, Anatomy and Neurobiology Section, Colorado State UniVersity, Fort Collins, Colorado 80523 ReceiVed October 30, 2007

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LβT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LβT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LβT2 cell culture were identified following twodimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Western blots from both exposed rats and LβT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched to concurrently run 2DE gels stained with Sypro Ruby, excised, and in-gel-digested with trypsin. Mass spectrometry analysis of digest peptides resulted in the identification of 19 spots and 8 unique proteins in the rats and 21 spots and 19 unique proteins in LβT2 cells. The identified proteins present in both sample types included proteasome activator complex subunit 1, ubiquitin carboxyl-terminal hydrolase isozyme L1, tropomyosin, ERp57, and RNA-binding proteins. Each of these proteins contains active-site or solvent-exposed cysteine residues, making them viable targets for covalent modification by DACT. Introduction Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) (ATRA) is one of the most commonly used herbicides in the U.S., with 57.4 million pounds applied in 2005 primarily to corn for control of broadleaf and grassy weeds (1). With its widespread use and low sorption to soils, ATRA is frequently detected in surface and drinking waters at levels greater than the 3 µg/L maximum contaminant levels (MCLs) set by the United States Environmental Protection Agency (U.S. EPA) (2–5). The general population is potentially exposed to ATRA from drinking water; however, occupational exposure among agricultural workers is significantly higher as a result of the exposure to spraying and direct contact (6). After oral exposure, ATRA metabolism by P450 enzymes in the liver (7) yields diaminochlorotriazine (DACT), a reactive electrophilic molecule capable of forming covalent adducts with cellular nucleophiles. We have shown that ATRA exposure in rodents results in the formation of a covalent adduct to Cys-34 of albumin and Cys-125 of hemoglobin. Adduct formation was attributed to a nucleophilic aromatic substitution reaction between reduced cysteine residues and DACT. This mechanism was confirmed with in Vitro exposures of hemoglobin and albumin to DACT, * To whom correspondence should be addressed: Colorado State University, 1680 Campus Delivery, 132 Physiology Building, Fort Collins, CO 80523. Telephone: 970-491-8635. Fax: 970-491-7569. E-mail: [email protected]. † Department of Environmental and Radiological Health Sciences. ‡ Department of Chemical and Biological Engineering. § Proteomics and Metabolomics Facility. | Department of Biomedical Sciences.

which resulted in adducts identical to those formed in ViVo (8, 9). Although DACT readily modifies these two proteins, it is unknown whether similar adducts will form in other proteins or if protein modification by DACT plays a role in the endocrine-disrupting effects associated with ATRA exposure (10). In female rats, luteinizing hormone (LH) is necessary to stimulate ovarian follicular development and ovulation, while stimulating the ovaries to produce estrogen and progesterone. Female Sprague–Dawley rats exposed to ATRA have high concentrations of DACT in the plasma and brain shortly after exposure (11), as well as a suppression of the LH surge from the pituitary (10). A dose-dependent suppression of the LH surge in female SD rats was caused by exposure to 30–300 mg/kg ATRA for 5 days with complete blockage of the LH surge at 300 mg/kg. DACT exposure results in similar effects, because total plasma LH and peak LH surge levels have been suppressed by 60 and 58%, respectively, in rats exposed to 300 mg/kg for 5 days (10). A 47% decrease in pituitary release of LH in response to added gonadotropin-releasing hormone was also found in animals treated with 200 mg/kg DACT (10). Currently, there is little known about the mechanism of action of DACT and/or ATRA exposures with relation to the suppression of LH release. Because DACT forms covalent protein adducts with albumin and hemoglobin following ATRA exposure, it is possible that DACT forms adducts with other proteins, some of which impact or are involved in the signal transduction pathway, leading to LH release from the pituitary. Given the critical link between the pituitary gland and LH release, the goal of this research was to identify ATRA-induced protein

10.1021/tx700386f CCC: $40.75  2008 American Chemical Society Published on Web 03/28/2008

Proteomic Analysis of DACT Adducts

adducts formed in the pituitary gland of ATRA-exposed rats. We also used DACT-exposed LβT2 rat pituitary cells to create an in Vitro model of adduct formation and to investigate DACT as an active metabolite following ATRA exposure.

Materials and Methods Chemicals and Materials. ATRA (97.1% purity) and DACT (97.4% purity) were a gift from Syngenta (Research Triangle Park, NC). Acetonitrile, bromophenol blue, Tween-80, and ammonium bicarbonate were purchased from Fisher Chemical Company (Fair Lawn, NJ). Proteomic-grade porcine trypsin, iodoacetamide (IAA), Tris-HCl, glycerol, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), urea, triflouroacetic acid, sodium citrate, hydrogen peroxie, trtiton-X100 agarose, and Pefabloc SC were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol and acetic acid were purchased from Mallinckrodt Baker, Inc. (Paris, KY). Sypro Ruby, acrylamide, sodium dodecyl sulfate (SDS), KCl, MgCl2, dithiothreitol (DTT), Tris-base, mineral oil, and glycine were purchased from Bio-Rad Laboratories (Hercules, CA). The DACT adduct antibody was generated by Strategic Biosolutions (Newark, DE), and the horseradish-peroxidase (HRP)-conjugated antirabbit antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rodent ATRA Exposures. Eight female ovariectomized Wistar rats were allowed to acclimate in ventilated cages for 1 week at the central animal care facility of Colorado State University, which is fully accredited by the American Association for Accreditation for Laboratory Animal Care. The welfare of the animals was maintained following an Animal Care and Use Research Protocol approved by the Colorado State University Animal Care and Use Committee. During the acclimation, they were maintained on a 12 h light/dark cycle at a constant temperature of 25 °C and humidity of 55%. All animals had free access to Teklad NIH-07 rodent diet and tap water. After the acclimation period, rats (four per treatment) were given, via oral gavage, doses of either 200 mg/kg ATRA (97.9% pure) in carboxymethylcellulose or equivalent mg/kg doses of carboxymethylcellulose as the control. These dose levels were chosen to be similar to doses that produced covalent hemoglobin and albumin adducts (8, 9) and LH surge suppression (10) in rats following ATRA exposure. On the 5th day post-exposure, rats were anesthetized with isoflurane and sacrificed via decapitation. The pituitary glands were immediately removed and either snap frozen with liquid nitrogen for two-dimensional electrophoresis (2DE) analysis or fixed in neutral buffered formalin. Immunohistochemistry of Pituitary Sections. Formalinfixed pituitary tissues were embedded in paraffin, sectioned at 10 µm, and mounted on glass slides. Sections were deparaffinized in xylene and hydrated with successive incubations in 100, 95, and 70% ethanol. Sections were boiled in 0.01 M sodium citrate for 10 min for antigen retrieval and endogenous peroxidase activity blocked with 0.6% (v/v) H2O2 in methanol for 2 h. Sections were blocked with 10% horse serum for 1 h at room temperature, washed in phosphate-buffered saline (PBS), incubated with a 1:100 000 dilution of the DACT adduct antibody (Strategic Biosolutions) overnight at 4 °C, and then visualized with a Vectastain Universal ABC kit, following manufacture protocols (Vector Laboratories, Burlingame, CA). Slides were counterstained with hematoxylin and dehydrated in 70, 95, 100% ethanol and xylene. Sections were viewed on a Ziess Axiovert 200 M microscope with a Hammatsu ORCAER cooled charge-coupled device camera.

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LβT2 Cell-Culture DACT Exposures. LβT2 immortalized rat anterior pituitary cells were cultured in a T-150 flask with complete Dulbelco’s modified essential medium (DMEM) containing 4.5 g/L glucose, supplemented with 10% fetal bovine serum (FBS) and 1% Pen/Strep (Media Tech, Herndon, VA). Cultures were grown in a humidified environment with 95% air and 5% CO2 at 37 °C to 80% confluency. They were then exposed to 300 µM DACT in DMEM or dimethylsulfoxide (DMSO) in DMEM (control) for 24 h. After this DACT incubation, the medium was removed and the cells were rinsed with DMEM followed by PBS and then scraped from the flask into a 15 mL conical tube. Tubes were centrifuged for 10 min at 14 000 rpm (16000g) to form a tight cell pellet, snap frozen with liquid nitrogen, and stored at -80 °C until analysis. Immunofluorescence of LβT2 Cell Culture. LβT2 cells were cultured as previously described, and approximately 400 000 cells were plated on FBS-coated glass cover slides. Cells were allowed to adhere for 48 h and then exposed to 300 µM DACT or DMSO for 24 h. Cells were rinsed with PBS, followed by fixation in cold methanol for 5 min. Cells were permeablized with 0.1% Trition X-100 in PBS for 1 min and blocked with 1% BSA in PBS for 30 min. Cells were incubated for 1 h at room temperature with a 1:100 000 dilution of DACT adduct antibody (Strategic Biosolutions, Newark, DE) in PBS, washed, and incubated with a goat antirabbit Alexa Fluor 488 sary antibody (Invitrogen, Eugene, OR) for 1 h. Cells were washed, mounted using Vectashield with DAPI, and viewed at 488 nm for DACT adducts and at 450 nm for DAPI-labeled nucleuses using a Ziess Axiovert 200M microscope with a Hammatsu ORCA-ER cooled charge-coupled device camera. Tissue and Cell Sample Preparations. Pituitary tissue and LβT2 cell samples were both processed with the following procedures. Pooled pituitaries or the LβT2 cell pellets were sonicated in ice-cold lysis buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 10 mM KCl, 0.1% SDS, 0.5 mM DTT, and 0.5 mM Pefabloc SC), incubated on ice for 1 h, and centrifuged at 14 000 rpm (16000g) for 10 min. A total of 100 µL of the supernatant was removed, and proteins were precipitated with the Bio-Rad 2D clean-up kit (Hercules, CA) following the protocols of the manufacturer and dissolved by sonication in 400 µL of rehydration buffer [8 M urea, 0.3% (w/v) DTT, 2% (w/v) CHAPS at pH 3–10 buffer, and bromophenol blue]. Two-Dimensional Electrophoresis. For the first dimension (isoelectric focusing), the 400 µL sample was pipetted into a rehydrating tray, an immobilized pH gradient (IPG) strip at pH 3–10 (Bio-Rad, Hercules, CA) was placed gel side down the sample solution, and mineral oil was pipetted over the IPG strip. The strip was allowed to rehydrate passively for at least 12 h. Using a Multiphor II system (Pharmacia Biotech, Uppsala, Sweden), the protein mixture was focused using the voltage program of an increase from 0 to 500 V over 1 min, from 500 to 3500 over 5 h, and steady exposure to 3500 V for 17.5 h. For the second dimension (SDS-PAGE) separation, the IPG strip was removed from the focusing tray and reduced in 3 mL of 2% (w/v) DTT in equilibration buffer [6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 24 mM Tris-HCl] for 15 min, followed by acetylation in 3 mL of 2.5% (w/v) IAA in equilibration buffer for 5 min. The IPG strip was transferred onto a slab gel (12% polyacrylamide), overlaid with 1 mL of hot 0.5% agarose, and the agarose was allowed to solidify. Electrophoresis was performed in a Protean II cell (Bio-Rad, Hercules, CA) for 2.5 h at 3000 V, 400 W, and 40 ma. Twodimensional gels were either stained with Sypro Ruby for protein detection or subjected to Western blotting for immunodetection

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of DACT-adducted proteins. For protein detection, the gel was fixed for 1 h in an aqueous solution of 10% ethanol/7% acetic acid and then stained in 100 mL of Sypro Ruby stain overnight. The gel was destained in the aqueous ethanol-acetic acid solution for 12 h before imaging. Stained proteins were visualized with 320 nm of UV light using a BioChemi Bioimaging system (UVP, Upland, CA), and the image was saved for a comparison to Western-blotting results. Immunodetection of DACT-Modified Proteins. The following procedures were used for both tissue and cell samples. An unstained 2D gel, a polyvinylidene difluoride (PVDF) membrane, and two extra-thick fiber pads were soaked in transfer buffer (25 mM Tris-HCl, 0.2 M glycine, 0.2% SDS, and 20% methanol) for 15 min. A Western blot filter papermembrane-gel-filter paper sandwich was assembled and then electrophoresed at 15 V for 1 h using a Semi-Dry transfer unit (Bio-Rad, Hercules, CA). After electrophoresis, the membrane was washed in Tris-buffer saline-Tween (TBST) (10 mM TrisHCl, 0.15 M NaCl, 8 mM sodium azide, and 0.05% Tween-20 at pH 8.0) for 30 min and blocked in a 5% nonfat milk-TBST solution for 30 min. The membrane was rinsed in TBST twice for 10 min and incubated with a 1:500 dilution of DACT adduct antibody (Strategic Biosolutions, Newark, DE) in 10 mL of 5% nonfat milk-TBST solution overnight at 4 °C. The membrane was then washed 3 times in TBST and incubated with a HRPconjugated secondary rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:2000 dilution for 1 h at room temperature. The membrane was again washed 3 times with TBST, and the DACT-antibody complex was detected by chemiluminescence with a Immobilon Western Chemiluminescent HRP substrate (Millipore, Billerica, MA) using a BioChemi Bioimaging system (UVP, Upland, CA). The image was saved for comparison to Sypro Ruby-stained gels. Protein spots from the Western blot were matched to spots on Sypro-stained gels using the Delta 2D 3.4 (Decodon, Greifswald, Germany) gel image analysis program. Spot patterns were matched between the gel and Western blot first by automation creating a fusion image of replicates, followed by manual warping to align spots. Protein Identification Using Matrix-Assisted Laser Desorption Ionization-Time of Flight/Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS). Matched spots from pituitary tissue samples were excised from the Sypro Rubystained gel with a One-Touch 2D spot picker (Web Scientific, Crewe, U.K.) and placed in an Eppendorf tube. Gel pieces were washed twice for 15 min (gentle vortex) with 100 µL of 100 mM NH4HCO3/50% acetonitrile (ACN); then 100 µL of 10 mM DTT was added; and the sample was incubated at 60 °C for 30 min. The excess liquid was removed; 100 µL of 55 mM IAA was added; and the mixture was incubated in the dark at room temperature for 30 min to acylate cysteine residues. The excess liquid was again removed; the gel pieces were completely dried; 6 µL of 0.1 µg/µL trypsin solution and 34 µL of 100 mM NH4HCO3 were added; and the mixture was incubated at 37 °C overnight. Peptides were extracted from the gel pieces with 40 µL of 50% ACN with 0.1% trifluoroacetic acid (TFA) under a gentle vortex for 20 min. The extraction was repeated, and the extracts were pooled and concentrated to ∼2 µL. Peptides were further purified with a Zip-Tip (C18 Millipore) following the protocol of the manufacturer. For MS analysis, 1 µL of the tryptic peptide solution was added to 1 µL of R-cyano-4-hydroxycinnamic acid solution (10 mg/mL in 50% ACN and 0.1% TFA) and the mixture was spotted on a MALDI target plate (MTP 384, Bruker Daltonics, Billerica, MA) and allowed to dry. An eight-peptide mixture

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on a spot adjacent to the sample was used for external mass calibration. Analysis was performed with an UltraFlex TOF/ TOF mass spectrometer (Bruker Daltonics, Billerica, MA) in positive-ion reflector mode with a 25 kV acceleration voltage. Data were processed using the SNAP algorithm in the FlexAnalysis software (version 2.4, Bruker Daltonics). A monoisotopic list was generated using a signal-to-noise threshold of 6 for MS spectra and 3 for MS/MS spectra. The combined MS and MS/MS spectra for each sample were searched using the Mascot (version 2.1) database search engine against the Rattus (Taxonomy ID 101144) entries in Swiss-Prot or NCBInr database containing 5446 and 40 152 sequence entries, respectively, on February 27, 2007. Parameters used in the database search were peptide mass tolerance of 0.15 Da, fragment ion mass tolerance of 0.8 Da, trypsin peptides allowing for 1 missed cleavage, and variable modifications of cysteine carbamidomethylation and methionine oxidation. Protein Identification Using Electrospray Ionization (ESI)-Ion Trap. Matched spots from cell culture samples were excised from the Sypro Ruby-stained gel and digested as described for the tissue samples. Concentrated extracts were diluted to 15 µL with 5% ACN and 0.1% acetic acid. For MS analysis, 5 µL of the tryptic peptide solution was injected onto a Zorbax 300SB-C18, 3.5 µm, 150 mm × 75 µm I.D. reversephase column (Agilent Technologies, Santa Clara, CA). Peptides were eluted directly into the mass spectrometer (Thermo Scientific LTQ linear ion trap) using a 40 min linear gradient from 2 to 60% buffer B (80% ACN and 0.1% acetic acid) at a flow rate of 5 µL/min. Spectra were collected over a m/z range of 200–2000 Da using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). Compound lists of the resulting spectra were generated using Bioworks 3.0 software (Thermo Scientific, Waltham, MA) with an intensity threshold of 5000 and 1 scan/group. The compound lists were searched against the NCBInr database using a taxonomy filter Rattus (Taxonomy ID 101144) containing 40 120 sequence entries on March 19, 2007 using the Sequest (Bioworks 3.0, Thermo Scientific, Waltham, MA) database search engine. Parameters used in the database search were average mass, peptide mass tolerance of 1.8 Da, fragment ion mass tolerance of 0.8 Da, tryptic peptides allowing for 1 missed cleavage, and variable modifications of cysteine carbamidomethylation and methionine oxidation.

Results Immunohistochemical Localization of DACT Protein Adducts in the Pituitary and LβT2 Cells. Immunohistochemical analysis of pituitary sections from rats exposed to 200 mg/ kg ATRA showed diffuse granular cytoplasmic staining and scattered nuclear staining in the pars distalis, indicating widespread protein adduct formation in this region (Figure 1A). No significant staining was seen in the pars nervosa or pars intermedia of these sections (data not shown). Control sections showed no staining in the pars distalis (Figure 1B), pars nervosa, or pars intermedia (data not shown). Similar results were seen with the immunofluorescence analysis of the LβT2 cells exposed to DACT (parts B and C of Figure 2), indicating the formation of DACT protein adducts, while no fluorescence was seen in control cells (parts E and F of Figure 2). Detection of DACT-Modified Proteins. The sample preparation and 2DE protocol were effective in separating watersoluble pituitary proteins from Wistar rats exposed to 200 mg/ kg ATRA (Figure 3), with consistent spot patterns and numbers (272 ( 27) observed among replicates (n ) 3). 2DE gels from

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Figure 1. Immunohistochemical detection of DACT-protein adducts in the pars distalis of pituitaries from female Wistar rats treated with 200 mg/kg atrazine (A) or 0 mg/kg atrazine (B).

Figure 2. Immuofluoresence detection of DACT-protein adducts in LβT2 cells treated with DACT (A-C) or vehicle control (D-E). Imaging at 450 nm shows DAPI staining of the nucleus (A and D). Imaging at 488 nm shows DACT adduct staining (B only and no staining in E). C and F show merged images of nuclear morphology and DACT adduct staining.

Figure 3. Two-dimensional electrophoresis of Sypro Ruby-stained water-soluble proteins from the pituitary of a female Wistar rat exposed to 200 mg/kg ATRA for 4 days (Numbering corresponds to protein identifications in Table 1).

unexposed rats had a spot pattern similar to ATRA-exposed rats in both the number of observed spots and relative

abundances (Supplemental Information 1 in the Supporting Information). However, differences in 2DE Western blots with the DACT adduct-specific antibody were clearly evident. A total of 55 spots were detected in animals exposed to ATRA (Figure 4), while no spots were detected in control animals (Supplemental Information 2 in the Supporting Information). Of the 55 spots detected, 44 were matched to spots on the corresponding 2DE gel and were picked for protein identification. Two-dimensional electrophoresis gels of water-soluble pituitary proteins from LβT2 cells exposed to 300 µM DACT showed a representative spot pattern (489 ( 43, n ) 3) seen in Figure 5. Two-dimensional electrophoresis gels from cells exposed to carboxymethylcellulose as a control showed a spot pattern consistent to DACT-exposed cells (Supplemental Information 3 in the Supporting Information). Western blot analysis detected 89 spots in DACT-treated cells (Figure 6) and no spots from control cells (Supplemental Information 4 in the Supporting Information). Of the 89 spots detected, 54 spots were matched to spots on the corresponding 2DE gel and were picked for protein identification. Identification of DACT Antibody-Detected Proteins. In the tissue sample analysis, sufficient protein for successful MS analysis was only obtained from 19 of the 44 matched spots. Proteins detected on 2D Westerns, matched to 2DE gels spots, and identified through MALDI-TOF/TOF MS are listed in Table 1, along with Mascot protein ion scores and cutoffs and

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Figure 4. Two-dimensional electrophoresis Western blot of watersoluble proteins from the pituitary of a female Wistar rat exposed to 200 mg/kg ATRA for 4 days (Numbering corresponds to protein identifications in Table 1).

sequence coverage for each identified protein. Significant protein scores (>23) with two or more matched peptide sequences were obtained for 12 spots, which were identified as six unique proteins. These proteins were serum albumin, protein disulfide isomerase A3 (ERp57), heterogeneous nuclear ribonucleoprotein A2/B1, proteosome acitivator complex subunit 1 (PA28R), ubiquitin carboxy-terminal hydrolase isozyme L1 (UCHL1), and peroxiredoxin 6. Spots 10 and 15 were identified as serum albumin and tropomyosin, respectively, on the basis of a single peptide MS/MS identification but yielded significant protein scores. The remaining five proteins were identified on the basis of significant protein scores from peptide mass fingerprints (PMFs), because the amount of protein was insufficient for MS/ MS analysis. PMF searching resulted in the identification of heterogeneous nuclear ribonuclearprotein H (spot 12), serum albumin (spots 9, 11, and 13), and peroxiredoxin-6 (spot 19). Peptides from tryptic digest of target proteins detected in DACT-exposed LβT2 cells were analyzed with MS/MS. Proteins were identified using a Sequest search of MS/MS data against the Swiss-Prot or NCBInr databases for the Rattus taxonomy. Confident protein identifications were made for 21 of the 54 matched spots based on two or more matched peptides, with Xcorr scores greater than 2 for each peptide. Sequence coverages ranged from 5 to 57%. Table 2 lists 19 unique proteins identified as DACT targets in LβT2 cells from the 21 spots analyzed. A comparison of this list to the DACT-modified proteins in pituitaries of ATRA-exposed rats showed 6 proteins that were detected and identified in both exposure scenarios. These were ERp57, heterogeneous nuclear ribonucleoproteins A2/B1 and H, PA28R, UCHL1, and tropomyosin.

Discussion ATRA is metabolized via successive dealkylations to DACT by P450 enzymes in the liver (2). This metabolic pathway is rapid because ATRA is completely metabolized to DACT within 48 h following exposure (11). We have previously demonstrated in rats exposed to ATRA that DACT is reactive and forms covalent adducts with protein nucleophiles specifically exposed to cystiene residues on hemoglobin and albumin (8, 9). The

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mechanism of adduct formation is through a nucleophilic aromatic substitution with DACT and specific Cys residues and was confirmed in Vitro. Protein adducts could play a central role in the observed toxicity associated with a reactive chemical, such as DACT. Chemical-induced toxicity follows a series of cascading events between ambient exposure and the observation of clinical disease. The formation of a covalent protein adduct is a possible step in a chemical-induced toxicity, which could occur if the adduct disrupts the structure or function of the protein. It has been suggested that covalent binding of a xenobiotic is the initiating event with some target organ toxicity (12). One of the primary toxic affects associated with ATRA exposure in rodents is the suppression of the LH surge from the pituitary (10). This observation has been seen in SD rats exposed separately to ATRA and DACT, as well as in cultured LβT2 cells exposed to DACT (unpublished data). These observations along with high levels of DACT in the brain following ATRA exposure (11) suggested that DACT and not ATRA is responsible for this effect. Currently, there is little known about the mechanism of action of DACT with relation to suppression of LH release. Because DACT is capable of forming covalent protein adducts and brain tissue is exposed to large quantities of DACT following ATRA exposure, specific modifications of proteins within the pituitary could cause aberrant function, leading to the observed suppression of the LH surge. In this study, we identified DACT-modified proteins in both pituitaries of ATRA-exposed Wistar rats and DACTexposed LβT2 cells, providing an initial point to further investigate the potential mechanistic role of proteins adduct with LH suppression in the rodent model. Immunohistochemical analysis of both pituitary sections from ATRA-exposed rats and DACT-exposed LβT2 cells indicated that DACT-modified proteins are widespread in both the cytoplasm and the nucleus of the cells. Using immunochemical detection, more than 50 spots were detected in pituitaries of rats exposed to 200 mg/kg ATRA but only 9 unique proteins could be definitively identified as targets for DACT modification with mass spectrometry. Included in this list was serum albumin, which we previously demonstrated to form a covalent adduct with DACT at Cys-34 (9). The other proteins detected each contain solvent-exposed Cys residues that would provide a clear target for DACT modification. They include ERp57 (Cys-57, -60, -406, and -409) (13), UCHL1 (Cys-90) (14), heterogeneous nuclear ribonucleoprotein A2/B1 (Cys-38) (15), heterogeneous nuclear ribonucleoprotein H (Cys-34) (16), tropomyosin (Cys190) (17), PA28R (Cys-22) (18), and peroxiredoxin 6 (Cys-47) (19). Proteins identified as DACT targets in LβT2 cells can be grouped in several categories based on their physiologic functions, including RNA-binding proteins, cytoskeleton proteins, proteins involved in proteasomal degradation, chaperones, and metabolic enzymes (Table 3). Of particular interest are the proteins that were identified from both in ViVo ATRA-exposed rats and in Vitro DACT-exposed Lβ2 cells. UCHL1 is cystiene protease that catalyzes the removal of ubiquitin from ubiquitin-conjugated proteins by hydrolyzing peptide-ubiquitin bonds (20). Deubiquitination is essential for recycling of the ubiquitin monomers in the ubiquitin-proteasome system. This system serves to degrade ubiquitin-tagged proteins that may be damaged, misfolded, or in excess and is critical for cell growth and differentiation, development, and transcriptional regulation (20). The active site of UCHL1 is a catalytic triad of Cys-90, His-161, and Asp-176 (14). Because these residues are solvent-exposed, the cysteine is a potential target

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Table 1. DACT-Adducted Proteins in the Pituitary of Wistar Rats Exposed to 200 mg/kg ATRA Identified with MALDI-TOF/ TOF MS spot

protein designation

accession number

MASCOT protein ion score (cutoff)a

peptides (MS) (sequence coverage)

peptides (MS/MS)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

serum albumin serum albumin serum albumin serum albumin serum albumin serum albumin ERp57 ERp57 ERp57 serum albumin serum albumin heterogeneous nuclear ribonucleoprotein H serum albumin heterogeneous nuclear ribonucleoprotein A2/B1 tropomyosin 1 R proteasome activator complex subunit 1 ubiquitin carboxyl-terminal hydrolase isozyme L1 peroxiredoxin-6 peroxiredoxin-6

P02770 P02770 P02770 P02770 P02770 P02770 P11598 P11598 P11598 P02770 P02770 120538378 P02770 14043072 P04692 Q63797 Q00981 O35244 O35244

26 (50) 187 (50) 329 (50) 541 (50) 343 (50) 344 (50) 231 (50) 495 (50) 94 (50) 56 (50) 70 (50) 89 (59) 57 (50) 178 (59) 84 (50) 242 (50) 167 (50) 72 (50) 50 (50)

13 (24%) 15 (29%) 16 (34%) 21 (43%) 16 (35%) 3 (30%) 20 (40%) 24 (45%) 13 (26%) 6 (12%) 10 (19%) 7 (23%) 14 (33%) 11 (27%) 7 (17%) 12 (57%) 10 (62%) 7 (37%) 6 (33%)

2 2 4 4 3 3 2 4 1 1 1 1 0 3 1 4 2 2 0

a Protein ion scores greater than the cutoff score are significant (p < 0.05). Supporting Information.

Figure 5. Two-dimensional electrophoresis of Sypro Ruby-stained water-soluble proteins from LβT2 rat pituitary cells exposed to 300 µM DACT for 24 h (Numbering corresponds to protein identifications in Table 2).

for a DACT adduct. Modification of the active-site Cys-90 with a covalent DACT adduct could inactivate the enzyme, resulting in the potential disruption of the ubiquitin-proteasome system. ERp57 is an oxidoreductase found in the endoplasmic reticulum of cells, where its exact function is not completely understood. It has been shown to be involved in the formation of disulphide bonds and folding glycoproteins when associated with calnexin and careticulin (21, 22), as well as in folding major histocompatability complex class I molecules (23). ERp57 contains two redox active Cys-Gly-His-Cys motifs that forms mixed disulfide bonds with the substrate during folding. When these Cys residues are mutated, the reductase activity of ERp57 is completely inhibited (24). These Cys residues are potential targets for DACT, and it is possible that alkylation by DACT could mimic the effects of Cys mutation and inhibit the oxidoreducatse activity of this protein. Interference of normal glycoprotein folding in the ER through the calnexin/calreiculin cycle could result, because it is dependent upon ERp57 binding (25).

b

Individual peptide sequences and ion scores can be found in the

Figure 6. Two-dimensional electrophoresis Western blot of watersoluble proteins from LβT2 rat pituitary cells exposed to 300 µM DACT for 24 h (Numbering corresponds to protein identifications in Table 2).

The proteins hnRNP A2/B1 are hnRNP H2 members of a family of ubiquitously expressed proteins called heterogeneous nuclear ribonucleoproteins (hnRNPs). These proteins bind pre-mRNAs and influence pre-mRNA processing, metabolism, and transport. The hnRNP proteins bind RNA with two repeats of a RNA recognition motif (RRM) (26). Within the first RRM is a Cys-38 (A2/B1) or Cys-34 (H2) that is available to react with DACT (15, 16), but it is unknown if a DACT modification on these residues would influence the RNA-binding capability of this RRM. Tropomyosin can be found in all eukaryotic cell types as a component of the microfilament system. In nonmuscle cells, it binds actin, providing stability, and can influence actin dynamics (17). The tropomyosin unit consists of an R-helical peptide dimer in a coiled-coil structure with one disulphide bond between Cys190 of each peptide. This is the only Cys residue present in the peptide; therefore, it is possible that DACT may react with this Cys and disrupt proper dimerization, leading to alteration in actin binding.

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Table 2. DACT-Adducted Proteins in LβT2 Cells Exposed to 300 µM DACT Identified with LC-MS/MS spot

protein designation

accession number

number of peptides (MS/MS)a

sequence coverage (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

stress-70 protein heterogeneous nuclear ribonucleoprotein 1 26 S protease regulatory subunit 6A heat-shock protein 60 ERp57 ERp57 heterogeneous nuclear ribonucleoprotein H2 R enolase phosphoglycerate kinase 1 predicted: similar to pyridoxal phosphatase heterogeneous nuclear ribonucleoprotein D-like heterogeneous nuclear ribonucleoprotein A2/B1 1 tropomyosin 3 proteasome activator complex subunit 1 proteaseome 26S non-ATPase subunit 9 R ETF ubiquitin carboxyl-terminal hydrolase isozyme L1 flavin reductase triosephosphate isomerase 1 triosephosphate isomerase 1 β-tubulin

116242506 55562839 2492523 51702230 1352384 1352384 62078769 56757324 40254752 109487833 76096336 4043072 438882 18202600 18426882 57527204 61098212 34855391 117935064 117935064 40018568

11 3 9 16 13 8 4 8 12 4 2 7 5 9 3 5 8 2 8 10 3

21 17 25 43 30 18 14 30 38 12 5 24 15 32 14 23 52 22 51 57 10

a Individual peptide sequences and Xcorr scores can be found in the Supporting Information. An Xcorr threshold of 1.5 (+1 ions), 2.0 (+2 ions), and 2.5 (+3 ions) was used for this analysis.

Table 3. Physiologic Functions of DACT-Modified Proteins Identified in LβT2 Cells metabolic enzymes R enolase phosphoglycerate kinase triosephosphate isomerase R-ETF flavin reductase hnRNP D hydrolase pyridoxal phosphatase proteasomal degradation proteasome activator complex-1 proteasome 26S subunit 9 protease regulatory subunit 6a ubiquitin carboxy-terminal hydolase-L1

RNA-binding proteins R enolase hnRNP A1/B1 hnRNP H2 hnRNP L hnRNP D cytoskeletal proteins β-tubulin tropomyosin-3 chaperones stress-70 protein ERp57 heat-shock-60 protein

Proteosome activator complex subunit 1 (PA28R) forms a ring-shaped heteromultimer with PA28β, which binds to and activates the hydrolytic function of the proteasome (27). This protein contains an exposed Cys-22, but it is not located in the proteasome-binding sequence (18). It is unclear what potential effect, if any, DACT modification of this residue may have on the protein stability or function. In summary, we have demonstrated that the ATRA metabolite DACT forms covalent adducts with 8 proteins in the pituitary of rats exposed to ATRA. Also, in Vitro exposure of LβT2 rat pituitary cells to DACT caused 19 proteins to be modified, several of which were common between both exposure scenarios. It is clear from these results that DACT is an active metabolite following ATRA exposure, likely acting as an alkylating agent to Cys residues. Most of the targeted proteins contain exposed Cys residues, which we have previously demonstrated to be modified by DACT (8, 9). The functional effect of these modifications to the proteins is unknown at this time. Further research on the individually identified proteins will be necessary to determine these effects and whether they play a role in the mechanism of LH suppression from the pituitary following ATRA exposure. Acknowledgment. We thank Amanda Ashley for help with the cell culture and Carla Lacerda for help with 2DE. Dr. Foradori was supported by NRSA 5F32NS049892.

Supporting Information Available: 2DE of Sypro Rubystained water-soluble proteins from the pituitary of a female Wistar rat controls for 4 days (Supplemental Information 1), 2DE Western blot of water-soluble proteins from the pituitary of a female Wistar rat controls for 4 days (Supplemental Information 2), 2DE of Sypro Ruby-stained water-soluble proteins from LβT2 rat pituitary cells exposed to DMSO control for 24 h (Supplemental Information 3), 2DE Western blot of water-soluble proteins from LβT2 rat pituitary cells exposed to DMSO control for 24 h (Supplemental Information 4), DACTadducted proteins in the pituitary of Wistar rats exposed to 200 mg/kg ATRA identified with MALDI-TOF/TOF MS (Supplemental Information 5), and DACT-adducted proteins in LβT2 cells exposed to 300 µM DACT identified with LC-MS/MS (Supplemental Data 6). This material is available free of charge via the Internet at http://pubs.acs.org.

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