ToxSci Advance Access originally published online on August 13, 2007
Toxicological Sciences 2007 100(1):156-167; doi:10.1093/toxsci/kfm210
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Proteomic Analysis of Rat Striatal Synaptosomes during Acrylamide Intoxication at a Low Dose Rate
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* Center for Environmental and Human Toxicology, University of Florida, Building 471, Mowry Road, Gainesville, Florida 32611-0885
Protein Chemistry Core, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32611-0885
Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Medical Center, New York 10467
1 To whom correspondence should be addressed at Montefiore Medical Center Moses Research Tower, 7 111 East, 210th Street, Bronx, NY 10467. Fax: (718) 920-5054. E-mail: lopachin{at}aecom.yu.edu.
Received June 6, 2007; accepted August 1, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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We have hypothesized that acrylamide (ACR) intoxication causes cumulative nerve terminal damage by forming adducts with nucleophilic cysteine sulfhydryl groups on critical presynaptic proteins. To determine the cumulative effects of ACR on the cysteine-containing proteome of nerve terminal, we employed cleavable isotope-coded affinity tagging (ICAT) and liquid chromatography-tandem mass spectrometry. ICAT analysis uses a sulfhydryl-specific tag to identify and quantitate cysteine-containing proteins. Synaptosomes were prepared from striatum of ACR-intoxicated rats (21 mg/kg/day x 7, 14, or 21 days) and their age-matched controls. The synaptosomal proteins of each experimental group were labeled with either light (12C9—control) or heavy (13C9—ACR) ICAT reagent. Results show that ACR intoxication caused a progressive reduction in the ICAT labeling of many nerve terminal proteins. A label-free mass spectrometric approach (multidimensional protein identification) was used to show that the observed reductions in ICAT incorporation were not due to general changes in protein abundance and that ACR formed adducts with cysteine residues on peptides which also exhibited reduced ICAT incorporation. The decrease in labeling was temporally correlated to the development of neurological toxicity and confirmed previous findings that cysteine adducts of ACR accumulate as a function of exposure. The accumulation of adduct is consistent with the cumulative neurotoxicity induced by ACR and suggests that cysteine adduct formation is a necessary neuropathogenic step. Furthermore, our analyses identified specific proteins (e.g., v-ATPase, dopamine transporter, N-ethylmaleimide–sensitive factor) that were progressively and significantly adducted by ACR and might, therefore, be neurotoxicologically relevant targets.
Key Words: isotope-coded affinity tag; toxic neuropathy; neurotoxicity; adduct formation; nerve terminal; proteomic analysis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Acrylamide (ACR) is used in multiple industrial settings (e.g., water and wastewater management, ore processing, and dye synthesis) and in scientific laboratories for the electrophoretic separation of macromolecules. ACR is also a food contaminant formed during high-temperature preparation of certain potato- or grain-based products. Exposure to monomeric ACR can produce cognitive changes, ataxia, and skeletal muscle weakness in humans and experimental animal models (reviewed in LoPachin et al., 2003
). Research has shown that the daily ACR dose rate does not determine the magnitude of neurotoxicity achieved in several laboratory species; i.e., a wide range of dose rates (10–50 mg/kg/day) have been shown to cause comparable levels of maximal neurological deficits. Instead, dose rate determined the time to reach maximal neurotoxicity. Thus, at lower ACR dose rates (e.g., 10 mg/kg/day) maximal expression of a neurotoxicological endpoint (e.g., gait changes) required a substantially longer exposure duration (90 days) than the induction of comparable neurotoxicity by higher dose rates (e.g., 11 days at 50 mg/kg/day; see Crofton et al., 1996; Edwards and Parker, 1977; Goldstein and Lowndes, 1979, 1981; Kuperman, 1958; LoPachin et al., 2002b). The observations that ACR neurotoxicity is progressive and that the rate of progression is dose dependent suggests a molecular mechanism that is cumulative (reviewed in LoPachin, 2004; LoPachin and DeCaprio, 2005; LoPachin et al., 2002b).
Evidence now suggests that the cumulative neurotoxicity produced by ACR exposure is linked to nerve terminal damage in the central nervous system and peripheral nervous system (Lehning et al., 1998, 2002a,b, 2003; reviewed in LoPachin et al., 2003). At the molecular level, this presynaptic toxicity appears to be mediated by the formation of sulfhydryl adducts on the cysteine residues of many proteins (Barber and LoPachin, 2004; LoPachin et al., 2004, 2006, 2007a,b). Quantitative (gas chromatography/mass spectrometry) analyses of whole-brain synaptosomes isolated from ACR-intoxicated rats (20–50 mg/kg/day) revealed an accumulation of the cysteine adduct, S-(2-carboxyethyl)-cysteine (CEC) that was closely correlated to the development of neurotoxicity (Barber and LoPachin, 2004). The temporal accumulation of sulfhydryl adducts is consistent with the hypothesis that ACR-induced neurotoxicity is mediated by a cumulative molecular mechanism. However, our previous research provided only global information and did not quantify adduct abundance on specific protein targets. To obtain more specific data regarding the cumulative effects of ACR on the nerve terminal proteome, we used a predigestion labeling strategy known as cleavable isotope-coded affinity tagging (ICAT; Gygi et al., 2002; Schrimpf et al., 2005). The ICAT technique employs a thiol-specific tag and is a useful method to assess toxicant-induced modifications of protein sulfhydryl groups. In the present study, striatal synaptosomes were prepared from control and ACR-intoxicated rats followed by differential isotopic labeling of these samples with either light (12C9) or heavy (13C9) ICAT reagent, respectively (Fig. 1). ACR adduction of cysteine residues during intoxication will block subsequent ICAT labeling and thereby reduce incorporation of the heavy tag. Results of this study have confirmed that cysteine adducts of ACR accumulate as a function of intoxication. In addition, the ICAT method identified specific proteins (e.g., v-ATPase, dopamine transporter, complexin-2) that are likely to be mechanistically relevant targets of ACR.
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MATERIALS AND METHODS |
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Chemicals and materials.
Unless otherwise indicated all reagents were high-performance liquid chromatography (HPLC) grade or better and water was doubly distilled and deionized. ACR (99% purity), Krebs-Henseleit buffer, urea, sodium dodecyl sulfate (SDS), iodoacetamide, and Percoll were purchased from the Sigma/Aldrich Chemical Company (St Louis, MO). Sequencing grade trypsin was from Promega (Madison, WI). C18 ZipTip microcolumns were acquired from Millipore (Bedford, MA). A 15 cm x 75µM i.d. PepMap C18 column was purchased from LC Packings (San Francisco, CA). A 100 mm x 4.6 mm i.d. Polysulfoethyl A strong cation exchange column was purchased from PolyLC, Inc. (Columbia, MD).
Animals and ACR intoxication.
All aspects of this study were in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Montefiore Medical Center Animal Care Committee. Adult male rats (Sprague-Dawley, 300–325 g; Taconic Farms, Germantown, NY) were used in this study. Rats were housed individually in polycarbonate boxes, and drinking water and Purina Rodent Laboratory Chow (Purina Mills, Inc., St Louis, MO) were available ad libitum. The animal room was maintained at approximately 22°C and 50% humidity with a 12-h light/dark cycle. Randomly assigned groups of rats (n = 4–6 per exposure group) were intoxicated with ACR at 21 mg/kg/day (p.o.) for 7, 14, or 21 days (Barber and LoPachin, 2004; LoPachin et al., 2002b, 2004). Body weights and gait scores were determined three times per week as indices of developing neurotoxicity. Gait scoring involved observation of spontaneous open-field locomotion, which included evaluations of ataxia, hopping, rearing, and hindfoot placement (LoPachin et al., 2002b). To assess locomotion, rats were placed in a clear plexiglass box (90 x 90 cm) and were observed for 3 min. Following observations, a gait score was assigned from 1 to 4 where: 1 = a normal gait; 2 = a slightly abnormal gait (slight ataxia, hopping gait, and foot splay); 3 = moderately abnormal gait (obvious ataxia and foot splay with limb abduction during ambulation); 4 = severely abnormal gait (inability to support body weight and foot splay). Corresponding groups of age-matched control rats (n = 4 rats per group) were weighed and gait scores were determined. A trained, blinded observer who was not involved in animal care or ACR exposure performed the testing.
Preparation of striatal synaptosomes.
The striatum was chosen as a source for synaptosomes based on previous research showing that ACR intoxication of rats was associated with structural and functional damage to nerve terminals in this brain region (Lehning et al., 2003; LoPachin et al., 2006). Striatal synaptosomes were isolated from brains of control and ACR-intoxicated rats by the Percoll gradient method of LoPachin et al. (2004). In brief, bilateral striata (100–120 mg wet weight tissue) were rapidly removed from anesthetized (isoflurane inhalation) rats and minced in cold (4°C) buffer containing sucrose 0.32M, EDTA 1mM and dithiothreitol 0.25mM (SED gradient buffer; pH 7.4). Tissue was gently homogenized in SED buffer (10 passes in a Teflon-glass homogenizer; 700 RPM), and the resulting homogenate was centrifuged at 1000 g (10 min, 4°C). The pellet (P1) was washed once and supernatants (S1 and S2) were combined. Protein content of the pooled supernatant was determined by the Bradford assay using bovine serum albumin as standard. The protein concentration of the supernatant was adjusted with SED to 5 mg/ml and then layered on top of a freshly prepared four-step discontinuous Percoll gradient (3, 10, 15, and 23% Percoll in SED, pH 7.4). Gradients were centrifuged at 32,000 g for 6 min, and synaptosomes were collected at the last interface (15%/23%). Synaptosomes were washed twice in Krebs buffer containing NaCl 140mM, KCl 5mM, NaHCO3 5mM, MgCl2 1mM, NaH2PO4 1.2mM, glucose 10mM, and Hepes 10mM (pH 7.4), pelleted, and then resuspended.
ICAT analysis of striatal synaptosomal fractions from control and ACR-intoxicated rats.
To compare the relative abundance of cysteine-containing peptides derived from striatal nerve terminal proteins of ACR-intoxicated rats and their age-matched controls (n = 2–3 per experimental group), ICAT labeling was performed using a modification (Schrimpf et al., 2005) of the original method described by Gygi et al. (2002). Briefly (Fig. 1), total synaptosomal proteins were precipitated by addition of nine volumes of acidified acetone, followed by incubation at – 20°C. Precipitated proteins were collected by centrifugation at 10,000 x g, and the resulting pellet was washed with acetone and air dried. Precipitated proteins were solubilized in denaturing buffer (1% SDS, 200mM Tris/HCl, 6M urea, 5mM EDTA, pH 8.8). Protein content was determined by the Bio-Rad protein assay (Bio-Rad, Richmond, CA), and solubilized proteins (100 µg) from experimental and control samples were reduced by addition of 5mM tris(2-carboxyethyl)phosphine for 30 min at room temperature. Control synaptosomal proteins were reacted with light (12C-labeled) ICAT reagent, whereas ACR-exposed proteins were reacted with heavy (13C-labeled) ICAT reagent for 2 h at room temperature. Labeled protein samples were combined, diluted with 25mM ammonium bicarbonate to a urea concentration below 1M, and digested with trypsin overnight at 37°C. The resulting peptides were desalted by reverse phase chromatography and fractionated by strong cation exchange (SCX) chromatography on an HPLC system using a 1-h linear gradient from 0 to 300mM potassium chloride. Ten SCX fractions were collected, and each SCX fraction was then purified using avidin affinity chromatography according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). The biotin tag was cleaved from the affinity-purified peptides, and samples were concentrated to dryness under reduced pressure. Avidin-purified ICAT-labeled peptides were analyzed by reversed-phase HPLC-tandem mass spectrometry on a hybrid quadrupole time-of-flight instrument (QSTAR XL, Applied Biosystems) equipped with a nano-electrospray source. Solvent delivery at 200 nl/min was provided by an integrated capillary HPLC system (Ultimate, LC Packings, Sunnyvale, CA) in which a 120-min gradient from 3 to 50% acetonitrile in 0.1% acetic acid was employed. Each Information Dependent Acquisition cycle consisted of a survey scan from m/z 400 to 1500 and three MS/MS scans obtained by collision-induced dissociation of ions that demonstrated the largest signal intensity at a given chromatographic time point. Survey and MS/MS scans were accumulated for 1 and 2 s, respectively.
Tandem mass spectrometric data were searched against the IPI rat protein database using the Mascot search algorithm. ICAT modification of cysteine, oxidation of methionine, deamination of asparagine and glutamine, and pyro-glutamate formation from N-terminal glutamine or glutamic acid were included as variable modifications. In general, probability-based MOWSE scores that exceeded the value corresponding to p < 0.05 were considered for protein identification. Scaffold (version Scaffold-01_06_03, Proteome Software Inc., Portland, OR) was used to validate the tandem MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at greater than 90% probability and contained at least one identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on tandem MS analysis alone were grouped to satisfy the principles of parsimony.
Heavy:Light (H:L) ratios of peak areas were calculated using the quantitation algorithm ProICAT v1.1 (Applied Biosystems). This is illustrated in Figure 2 which shows typical mass spectra of ICAT-labeled peptide pairs derived from a control (light) and an ACR-exposed (heavy) mixed synaptosomal sample. Results of ProICAT quantitation were validated manually by evaluating spectra for agreement with identification and integrating extracted ion chromatograms corresponding to all charge sites of the peptide. The overall mean and variance for each peptide was determined from all observations of a given peptide at a specific time point. The probability that the ratios differed from control were determined as described by Cheng et al. (2006). The H/L ratio data for each peptide could be sorted into one of three subgroups based on the respective temporal responses to ACR intoxication. Thus, the data in group I declined progressively during ACR intoxication, whereas the group II H/L ratio data remained relatively close to unity (1.0). Group III H/L ratios were substantially lower by day 7 and did not change much thereafter. To assess the validity of this sorting scheme, linear regression analysis was used to demonstrate that the respective slopes for each peptide in a given group (1–3) were comparable. We then derived a mean slope and corresponding 95% confidence interval (Cheng-Prusoff equation) for each group and compared them by one-tailed Student's t-test (InStatTM, GraphPad Software, San Diego, CA). Consistent with the progressive decline of group I data, analyses showed that the corresponding mean slope (–22 ± 3 x 10–3) was statistically different from the respective data for groups II and III (–6 ± 3 x 10–3 and –3 ± 3 x 10–3).
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Direct MS analysis of ACR adducts in striatal synaptosomal fractions from control and ACR-intoxicated rats.
To confirm that changes in ICAT incorporation were not due to general changes in protein abundance and, to directly, identify ACR adducts, striatal synaptosomal proteins from intoxicated rats (21 mg/kg/day x 21 days) and their age-matched controls were analyzed using label-free Multi-Dimensional Protein Identification (MuDPIT). Proteins were prepared for MS analysis by precipitation (see above) and were solubilized in a Tris-SDS denaturing buffer (1% SDS, 200mM Tris/HCl, 6M urea, 5mM EDTA, pH 8.8). Synaptosomal proteins were reduced, alkylated with iodoacetamide, and digested overnight in sequencing grade trypsin. The resulting peptides were desalted, fractionated by SCX chromatography, and analyzed by LC/MS/MS as described above. Tandem MS data were analyzed using Mascot (Matrix Sciences, London, UK; version 2.0.01) and X! Tandem (www.thegpm.org; version 2006.04.01.2) searching the IPI_rat_20050523 database using trypsin digestion. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.30 Da and a parent ion tolerance of 0.30 Da. ACR modification of cysteine, oxidation of methionine, deamination of asparagine and glutamine, and pyro-glutamate formation from N-terminal glutamine or glutamic acid were included as variable modifications. Scaffold (version Scaffold-01_06_05, Proteome Software Inc.) was used to validate MS/MS-based peptide and protein identifications.
Relative abundance of individual proteins that were common to the control, and treated samples were determined by spectral counting as described by Liu et al. (2004). Briefly, the number of redundant spectra assigned to a given protein by Scaffold analysis relative to the total number of spectra collected for each sample was calculated for each protein identified in control and treated samples. This ratio was calculated by the formula:
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Body Weight and Gait Changes
ACR intoxication at the lower daily dose rate (21 mg/kg) slowed the normal rate of daily weight gain and caused significant increases in gait scores (LoPachin et al., 2002b
). Age-matched control rats for this exposure group had a starting mean body weight of 309 ± 3 g, which increased to 434 ± 5 g by day 21 of the exposure paradigm. This represents a 40% increase in body weight at a rate of approximately 5.9 g per day. In contrast, rats intoxicated at the 21 mg/kg dose rate exhibited a significantly slower rate of weight gain; i.e., rats in this exposure group had a starting weight of 313 ± 4 g, and after 21 days of ACR intoxication body weight rose to 382 ± 3 g. This represents a 22% increase in weight at a daily rate of approximately 3.3 g per day. Age-matched control rats did not exhibit changes in gait during the 21 day experimental period. In contrast, gait scores for ACR-intoxicated rats (21 mg/kg/day) increased from a score of 1.2 ± 0.3 at 7 days to 2.4 ± 0.3 on day 21 of exposure. This final gait score represents a slight level of neurotoxicity.
ICAT Analysis of Striatal Synaptosomes
The purpose of this study was to use the ICAT method to analyze and compare the respective proteomes of synaptosomes isolated from striatum of control and ACR-intoxicated rats. Synaptosomes are pinched-off nerve terminals that contain synaptic vesicles, mitochondria, and are often associated with postsynaptic membrane fragments (Raiteri and Raiteri, 2000). The present ICAT-based survey of the synaptosomal proteome detected specific proteins involved in presynaptic neurotransmitter release, uptake, and vesicular storage, as well as structural proteins and adhesion molecules. In addition, numerous soluble and membrane-associated proteins involved in energy metabolism and postsynaptic signal transduction were identified (Fig. 5; see Schrimpf et al., 2005). Also identified by ICAT analysis were a number nonsynaptic proteins (e.g., serum, glial- and myelin-related proteins), which reflect unavoidable contamination during tissue dissection and preparation (data not shown).
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In the present study, a total of 620 individual synaptosomal proteins were identified, although the number and identity of proteins detected at each time point varied (Fig. 3). Of these 620 proteins, 94 contained peptides that were common to all experimental endpoints, and these peptides were used to compare the longitudinal effects of ACR intoxication. Among these peptides, three basic responses were evident and corresponding proteins were grouped accordingly. Specifically, the majority of affected proteins contained peptides (group I) that exhibited progressive decreases in H:L ratios. Thus, on day 7 the mean (±SEM) H:L ratio for all peptides in this group was 0.89 ± 0.03, and at day 21 the mean ratio was 0.60 ± 0.02. For example (Table 1), the H:L ratio for the synaptotagmin I (IPI00206170) on day 7 (unaffected gait) was 0.92 ± 0.08 and by day 21 (slightly affected gait) this ratio had dropped to 0.60 ± 0.08. Other synaptosomal proteins with peptides exhibiting similar decreases in labeling (Table 1) included complexin-2 (IPI00190397), Na2+-dependent dopamine transporter (IPI00187593), clathrin heavy chain (IPI00193983), the vacuolar ATPase (IPI00199394), and N-ethylmaleimide–sensitive factor (NSF) (IPI00210635). In contrast, a smaller subset of proteins (group II) had peptides that exhibited either no change or relatively small longitudinal decreases in H:L ratios; i.e., on day 7 the mean (±SEM) H:L ratio for this group was 0.93 ± 0.04, and on day 21 the mean ratio was 0.88 ± 0.02. For example, at day 7, the H:L ratio of the
-1 chain precursor for Na/K ATPase (IPI00326305) was 1.10 ± 0.08, and at day 21 the ratio was 1.01 ± 0.08. An additional subpopulation of proteins (group III) contained peptides that exhibited early (day 7) and substantial mean (±SEM) decreases in H:L ratio (0.57 ± 0.06), which remained unchanged throughout the intoxication schedule (e.g., voltage-dependent anion-selective channel 3; Table 1). As illustrated in Figure 5, the proteins altered during ACR intoxication (7–21 days) participate in several broad structure-function categories including neurotransmission, membrane fusion, energy production, endocytosis, and cytoskeletal structure. Finally, our analysis also identified a subset of proteins at days 14 or 21 that exhibited increased H:L ratios, which indicates increased ICAT labeling possibly due to elevated expression or reduced proteolysis (Table 2).
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Quantitation of Protein Levels and Confirmation of Adduct Species
Decreases in H:L ratios could be due to factors other than ACR adduction of free sulfhydryl groups; e.g., unmatched protein samples or toxicant-induced reductions in protein abundance due to reduced anterograde transport or increased proteolysis. Therefore, striatal synaptosomal proteomes from ACR (21 mg/kg/day x 21 days) -intoxicated rats and their age-matched controls were examined using a label-free MuDPIT approach. Using this method, we identified 369 unique proteins with high confidence (data not shown). Spectral counting of the MuDPIT dataset was used to calculate the ratios of proteins identified in control and ACR-intoxicated rats. Using this technique, a value of 0 (log 1) is unity, which indicates complete agreement between the hypothetical control and experimental proteomes; i.e., the treatment did not alter protein abundance relative to control. In our study, the mean log2 ratio of protein abundance determined by MuDPIT analysis was x 0.042 ± 0.003. This indicates very close agreement between the abundance of individual proteins in ACR-exposed and control proteome samples. Of the proteins represented by the 94 peptides that were detected at all three endpoints by ICAT analyses, 48 were present in the spectral counting dataset for day 21. The mean (±SEM) log2 of the relative abundance of these peptides was 0.176 ± 0.034. The slight positive deviation from unity is due to the inclusion of proteins exhibiting elevated abundances (see below). Therefore, generalized decreases in synaptosomal protein abundance did not occur despite significant ACR-induced changes in ICAT labeling. In fact, statistical analysis of the spectral count data identified only eight proteins whose abundance were significantly altered in the ACR-exposed samples (p < 0.01; data not shown). Of interest, one of these was complexin-2 (IPI00190397), the abundance of which increased by a factor of 5 in ACR-intoxicated rats despite a 75% reduction in ICAT incorporation (Table 1; day 21). In contrast to the corresponding label-free measurements, H:L ratios from the same ICAT dataset exhibited a mean log2 value of –0.592 ± 0.001. This negative deviation from zero indicates a significant leftward shift in data dispersion toward markedly lower H:L ratios at the 21-day endpoint. Thus, analysis of the same samples by different techniques; i.e, MuDPIT and ICAT, have provided substantial evidence that the observed reductions in ICAT labeling were not due to changes in synaptosomal protein abundance.
To demonstrate conclusively that the formation of ACR-cysteine adducts was responsible for reduced ICAT labeling, adducts were identified using label-free tandem mass spectrometry of synaptosomal preparations from intoxicated rats. Results showed that ACR formed adducts with cysteine residues on peptides that exhibited reduced ICAT labeling. For example, Figure 4 shows that ACR formed adducts with Cys 73 of complexin-2, the ICAT labeling of which was significantly reduced in ACR-intoxicated rats (Table 1). Our label-free analysis also identified cysteine adducts on other proteins that exhibited decreased ICAT labeling; e.g., voltage-dependent anion channel 3 (IPI00231067), glyceraldehyde-3-phosphate dehydrogenate (IPI00559898) and NSF (IPI00210635l). Together these data indicate that the relative decreases in ICAT labeling were due to the formation of ACR adducts at specific cysteine residues of nerve terminal proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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In the present study, ICAT analysis was used to determine the effects of ACR intoxication (21 mg/kg/day x 7–21 days) on cysteine-containing proteins in the central nervous system nerve terminal proteome. Results indicate a progressive loss of sulfhydryl groups on proteins that participate in a variety of presynaptic processes; e.g., neurotransmission, endocytosis, cellular metabolism, energy production, membrane ion transport, and cytoskeletal architecture. The reduction in ICAT labeling is a direct result of cumulative sulfhydryl adduct formation during ACR intoxication, which subsequently blocks label incorporation. Nonetheless, similar data would be obtained if ACR caused a decrease in the abundance of cysteine-containing proteins in nerve terminals of intoxicated rats. Changes in protein abundance are possible since ACR neurotoxicity has been associated with increased proteolysis, nerve terminal degeneration, and decreased fast anterograde transport (reviewed in LoPachin et al., 2003
; Sickles et al., 2002). Sulfhydryl oxidation as a result of ACR-induced neuronal oxidative stress could also reduce ICAT labeling (Sethuraman et al., 2004). However, several lines of evidence argue against such nonspecific changes in labeling. Using a label-free approach, we positively identified ACR adducts on peptide cysteine residues that coincidentally exhibited reduced ICAT labeling (Fig. 4; see also Barber and LoPachin, 2004; Bordini et al., 2000; Hall et al., 1993). Spectral analysis of the ICAT and label-free datasets also indicated that ACR intoxication did not significantly alter the abundance of synaptosomal proteins. This is consistent with earlier immunoblot analyses that showed no changes in the protein content of nerve terminals from ACR-exposed rats (LoPachin et al., 2004, 2007b) and with extensive studies conducted in this and other laboratories that failed to demonstrate a mechanistically relevant effect of ACR on delivery of proteins by axonal transport (Lehning et al., 1998; LoPachin et al., 2004, 2006; reviewed in LoPachin, 2002, 2004). Although changes in cellular redox status could be a contributing factor, previous studies have found no in vivo evidence to support this suggestion (e.g., Barber and LoPachin, 2004; Johnson and Murphy, 1977; Ku and Billings, 1986; LoPachin et al., 2006). Finally, changes in ICAT labeling were initiated prior to the onsets of both nerve terminal degeneration in the striatum (Lehning et al., 2003; LoPachin et al., 2006) and significant neurological deficits (see LoPachin et al., 2002b). The temporal decrease in sulfhydryl labeling, therefore, does not represent coincidental loss of nerve terminals or nonspecific effects related to overt neurotoxicity. Together, these data indicate that the progressive reduction in ICAT labeling was due to the cumulative formation (see below) of sulfhydryl adducts on cysteine residues of synaptosomal proteins.
The observation that sulfhydryl loss (Table 1) progressed despite a constant exposure rate (i.e., 21 mg/kg/day) suggests that the corresponding protein adducts accumulated. This confirms and extends our previous quantitative findings that the ACR-cysteine adduct (CEC) accumulated in nerve terminals during both subchronic (21 mg/kg/day x 
28 days) and subacute (50 mg/kg/day x 11 days) intoxication (Barber and LoPachin, 2004). In both studies, the accumulation of adduct coincided with the onset and development of neurological toxicity, which suggests a mechanistic role in the cumulative neurotoxicity of ACR (see LoPachin et al., 2002b and references therein). The accumulation of cysteine adducts on certain peptides reflects adduction of nerve terminal proteins with slower turnover rates. That is, adducts formed on these proteins will accumulate since they are replaced slowly. As the accumulation of adducted, presumably dysfunctional proteins surpasses a toxic threshold concentration, nerve terminal defects and cumulative neurotoxicity develop. In contrast, adducted, dysfunctional proteins with short half-lives will not accumulate since they are rapidly replaced and, consequently, will have minimal neurotoxicological impact (reviewed in LoPachin, 2004; LoPachin and DeCaprio, 2005). The present ICAT analysis has identified several nerve terminal proteins including NSF, the dopamine membrane transporter, complexin-2, and the v-ATPase of synaptic vesicles as possible neuropathogenic targets of ACR. These proteins turnover slowly and play critical roles in presynaptic processes (Calakos and Scheller, 1996; Jahn et al., 2003). Thus, our findings suggest that the cumulative neurotoxicity of ACR is mediated by the build-up of adducts on nerve terminal proteins with relatively slow turnover rates (see ahead).
The findings of this study have significant, broad-based implications for molecular sites of ACR action and corresponding mechanisms of nerve terminal dysfunction (Fig. 5). Electrophysiological and neurochemical data have suggested that ACR intoxication impaired neurotransmitter release (Goldstein and Lowndes, 1979, 1981; LoPachin et al., 2004, 2006; reviewed in LoPachin et al., 2002a). Although the corresponding molecular mechanism is unknown, both ICAT analysis (present study) and previous tandem MS determinations (Barber and LoPachin, 2004) have demonstrated that ACR formed adducts with Cys 264 of NSF (IPI00210635). Gene mutational studies have indicated that the ATPase activity of NSF is regulated by the sulfhydryl redox state of this cysteine. Additional evidence suggests that Cys 264 exists in a catalytic triad (Matsushita et al., 2003, 2005). NSF promotes dissolution of the 7S SNARE core complex that mediates membrane fusion and thereby plays a rate-limiting role in neurotransmitter release (Whiteheart et al., 2001). Consistent with this function, we have shown that ACR causes a build-up of the SNARE complex and inhibits synaptosomal neurotransmitter release (Barber and LoPachin, 2004; LoPachin et al., 2004). ICAT analysis also revealed cumulative adduction of complexin-2 at Cys 90 (Table 1; IPI00190397; Fig. 4). Complexin-2 plays a critical role in neurotransmitter release by stabilizing the SNARE core complex (Reim et al., 2001; Tang et al., 2006). If the function of this protein is cysteine dependent (see below), it represents an additional ACR target within the synaptic vesicle cycle. However, our research indicates that ACR inhibition of neurotransmission is more complex than disruption of vesicle cycling. Previous studies have shown that ACR intoxication is also associated with decreased neurotransmitter reuptake and vesicular storage (Barber and LoPachin, 2004; LoPachin et al., 2004, 2006). Correspondingly, the present ICAT data (Table 1) demonstrate that ACR formed adducts with cysteine residues on proteins that mediate these presynaptic processes; i.e., the Na+-dependent dopamine transporter (IPI00187593; Cys 342) and the vacuolar ATPase (IPI00199305; Cys 254), respectively. The formation of these adducts likely has toxicological relevance since the redox state of the respective cysteine residues has been shown to modulate corresponding protein function (Feng and Forgac, 1992; Park et al., 2002).
ICAT analysis of the striatal nerve terminal proteome in intoxicated rats has demonstrated that ACR formed adducts with only a fraction of the available protein cysteine residues (Tables 1). Although the molecular basis of this cysteine selectivity could involve steric factors, the nucleophilic state of the sulfhydryl group is an important consideration. ACR is a soft electrophile that will form Michael-type adducts with soft nucleophiles, which in biological systems are primarily sulfur atoms (Barber and LoPachin, 2004; LoPachin et al., 2004, 2007a,b). Protein sulfhydryl groups exist in the reduced thiol state and the anionic thiolate state. Recent studies (LoPachin et al., 2007b) have shown that ACR preferentially formed adducts with the thiolate state. However, at physiological pH (7.4), this redox state exists primarily in cysteine catalytic triads where proton shuttling through an acid-base pairing of amino acids (e.g., aspartic acid and lysine, respectively) regulates protonation of the sulfhydryl group. It is now recognized that the redox state or nucleophilicity of cysteine sulfhydryl groups within catalytic sites determines the functionality of many nerve terminal proteins. Therefore, these specialized residues represent specific sites for ACR adduction (see detailed discussion in LoPachin and Barber, 2006). Despite this specificity at the molecular level, ICAT analysis has implicated multiple presynaptic protein targets (e.g., SNARE core assembly/dissolution, neurotransmitter uptake, and vesicular storage), which implicates a diffuse pathophysiological mechanism. However, the neurotoxicological relevance of a protein adduct is determined by the functional importance of the adducted residue and the corresponding role of the protein in a given neuronal processes (see detailed discussion in LoPachin and DeCaprio, 2005). With respect to ACR-thiolate adduction, it is important to note that many of the adducted protein sulfhydryl groups are also acceptors for neuronal nitric oxide (NO) signaling (Stamler et al., 2001). NO is a biological electrophile that reversibly forms adducts with thiolate groups (S-nitrosylation) of catalytic triads and, thereby, regulates the activities of many nerve terminal proteins and their respective pathways (reviewed in LoPachin and Barber, 2006). NO specificity is a product of individual signaling modules that act as independent microprocessors for a neuronal pathway. Therefore, the broad-based effects of ACR can be explained by the simultaneous actions of this toxicant at multiple NO-targeted signaling modules. Because ACR forms irreversible adducts with NO-sensitive thiolates, we have hypothesized that NO signaling is blocked and that the loss of reversible, spatially precise neuromodulation produces presynaptic toxicity (LoPachin and Barber, 2006; LoPachin et al., 2006, 2007a).
Delineating the neurotoxic mechanism of ACR could provide a better understanding of human neurodegenerative diseases and environmentally derived neurotoxicities. As an ,ß-unsaturated carbonyl, ACR is a member of a large class of chemicals known as conjugated type-2 alkenes. Our recent studies have suggested that, as a chemical class, type-2 alkenes produce neurotoxicity by damaging nerve terminals (LoPachin et al., 2007a,b). This has significant implications for risk management since the neurotoxic potential of these important industrial chemicals has received limited attention. There is also growing evidence that nerve terminal damage and the oxidative generation of type-2 alkenes (e.g., acrolein, 4-hydroxy-2-nonenal) are involved in the pathogenesis of Alzheimer's disease and other neurodegenerative conditions (Calingasan et al., 1999; Coleman and Yao, 2003). The ICAT method used in the present investigation and resulting adduct data could, therefore, provide a framework for deciphering sites and mechanisms of action for the type-2 alkenes and corresponding involvement in neurodegeneration associated with human diseases and environmental exposures.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Institute of Environmental Health Sciences (NIH RO1 ES03830-20).
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The authors would like to express their sincere thanks to Dr Joseph Ross (Ross Toxicology, Cincinnati, OH) for his helpful comments and criticisms during the preparation of this manuscript.
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