Proteomic analysis of hippocampal proteins of F344 rats exposed to 1-bromopropane

Toxicology and Applied Pharmacology 257 (2011) 93–101

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Proteomic analysis of hippocampal proteins of F344 rats exposed to 1-bromopropane Zhenlie Huang a, b, Sahoko Ichihara c, Shinji Oikawa d, Jie Chang c, Lingyi Zhang a, Masahide Takahashi e, Kaviarasan Subramanian a, Sahabudeen Sheik Mohideen a, Yun Wang e, Gaku Ichihara a,⁎ a

Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan Department of Toxicology, Guangdong Prevention and Treatment Center for Occupational Diseases, Guangzhou 510-300, PR China Graduate School of Regional Innovation Studies, Mie University, Tsu 514-8507, Japan d Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507, Japan e Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan b c

a r t i c l e

i n f o

Article history: Received 27 July 2011 Revised 23 August 2011 Accepted 26 August 2011 Available online 2 September 2011 Keywords: 1-Bromopropane Proteomics Hippocampus Mechanism Neurotoxicity

a b s t r a c t 1-Bromopropane (1-BP) is a compound used as an alternative to ozone-depleting solvents and is neurotoxic both in experimental animals and human. However, the molecular mechanisms of the neurotoxic effects of 1BP are not well known. To identify the molecular mechanisms of 1-BP-induced neurotoxicity, we analyzed quantitatively changes in protein expression in the hippocampus of rats exposed to 1-BP. Male F344 rats were exposed to 1-BP at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks by inhalation. Two-dimensional difference in gel electrophoresis (2D-DIGE) combined with matrix-assisted laser-desorption ionization time-offlight (MALDI-TOF) mass spectrometry (MS) were conducted to detect and identify protein modification. Changes in selected proteins were further confirmed by western blot. 2D-DIGE identified 26 proteins with consistently altered model (increase or decrease after both 1- and 4-week 1-BP exposures) and significant changes in their levels (p b 0.05; fold change ≥ ± 1.2) at least at one exposure level or more compared with the corresponding controls. Of these proteins, 19 were identified by MALDI-TOF-TOF/MS. Linear regression analysis of 1-BP exposure level identified 8 differentially expressed proteins altered in a dose-dependent manner both in 1- and 4-week exposure experiments. The identified proteins could be categorized into diverse functional classes such as nucleocytoplasmic transport, immunity and defense, energy metabolism, ubiquitination-proteasome pathway, neurotransmitter and purine metabolism. Overall, the results suggest that 1BP-induced hippocampal damage involves oxidative stress, loss of ATP production, neurotransmitter dysfunction and inhibition of ubiquitination-proteasome system. © 2011 Elsevier Inc. All rights reserved.

Introduction 1-Bromopropane (1-BP, CAS No. 106-94-5) was introduced as an alternative to ozone-depleting solvents for adhesives, aerosol products, metals, electronics, and precision cleaning (USEPA, 1999, 2000). The global production of 1-BP in 2007 was estimated to be 20,000–30,000 metric tons, while that produced by China alone in 2008 was around 20,000 metric tons; approximately 40% of which was exported. In

Abbreviations: 1-BP, 1-Bromopropane; 2D-DIGE, Two-dimensional difference in gel electrophoresis; CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1 propanesulfonate; CHCA, α-cyano-4-hydrox cinnamic acid; CNS, Central nerve system; DTT, Dithiothreitol; ER, Endoplasmic reticulum; GSH, Glutathione; IEF, First-dimension isoelectric focusing; IPG, Immobilized pH gradient; MALDI-TOF, Laser-desorption ionization time-of-flight; MS, Mass spectrometry; PANTHER, Protein analysis through evolutionary relationships; pI, Isoelectric point; PTMs, Post-translational modifications; PVDF, Polyvinylidene fluoride; UPS, Ubiquitination-proteasome system. ⁎ Corresponding author at: Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Fax: +81 52 744 2126. E-mail address: [email protected] (G. Ichihara). 0041-008X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2011.08.023

2009, the use of 1-BP as a solvent was reported to be 5000 metric tons in the United States and 1100–1200 metric tons in Japan, with an annual growth rate of 15–20% in the United States and Asian countries other than China (HSIA, 2010). Experimental animal studies (Ichihara et al., 2000; Mohideen et al., 2011; Wang et al., 2002, 2003; Yu et al., 2001), as well as investigation on workers (Ichihara et al., 2004a, 2004b; Li et al., 2010) and human cases (CDC, 2008; Harney et al., 2003; Ichihara et al., 2002, 2011; Majersik et al., 2007; Raymond and Ford, 2007; Reh et al., 2002; Sclar, 1999) showed that exposure to 1-BP induces disorders of the central and peripheral nervous systems. However, little is known about the molecular mechanisms underlying 1-BP-induced toxicity of the central nervous system (CNS). The hippocampus, a major region of the brain known to play an important role in learning and memory, is known to be sensitive to neurotoxins (Kim et al., 2009; Park et al., 2010). Symptoms of hippocampal dysfunction, e.g., memory loss and cognitive dysfunction, were found in human cases and workers exposed to 1-BP (Ichihara et al., 2002; Sclar, 1999). Moreover, previous neurological studies identified the hippocampus as a brain region sensitive to 1-BP (Fueta et al.,

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2000, 2002a, 2002b, 2004; Mohideen et al., 2009; Ueno et al., 2007), suggesting that neurochemical changes occur in this region. The rapid development of proteomic techniques has enhanced our understanding of the molecular changes related to chemically-induced neurologic disorders (Kisby et al., 2006). Proteomics is a powerful tool for neurotoxicological investigations, providing quantitative data regarding changes in protein expression as a function of neuronal injury in the case of exposure to neurotoxins (LoPachin et al., 2003), as well as detection and characterization of toxins-induced changes in post-translational modifications (PTMs) (Ficarro et al., 2002). Furthermore, several studies employed hippocampal proteome analysis to determine alteration in protein expression and PTMs in neurodegenerative diseases (Martin et al., 2008; Schonberger et al., 2001). Thus, proteomics-based approaches can potentially provide more insight into the underlying molecular mechanisms of 1-BP-induced neurologic disease. To gain insight into 1-BP-induced neurotoxicity, we analyzed quantitatively changes in protein expression in the hippocampus induced by 1-BP exposure. The purpose of this study was to characterize the molecular mechanisms of 1-BP hippocampal neurotoxicity. For this purpose, we analyzed quantitatively the differential protein expression in the hippocampus of F344 rats exposed to 0, 400, or 1000 ppm of 1-BP for 8 h/day for 1 or 4 weeks, using two-dimensional difference in gel electrophoresis (2DDIGE) combined with matrix-assisted laser-desorption ionization timeof-flight (MALDI-TOF) mass spectrometry (MS). Selected proteins were further confirmed by western blot.

evaporated at room temperature and mixed with a larger volume of clean air to achieve the desired concentration. The vapor concentration of 1-BP was monitored every 5 s by gas chromatography and digitally controlled to be within ±5% of the target concentration by a personal computer. A rectification board with custom-made numerous holes was used for uniform distribution of the 1-BP-rich vapor. The mean concentration of readings measured every 5 s for 8 h was considered the value for a given day. These were then averaged over 1 week or 4 weeks in order to obtain the mean and standard deviation values. Thus, the daily gas concentrations in the three chambers measured were 396 ± 11, 803 ± 15 and 1003 ± 24 ppm in 1-week exposure, or 405 ± 6, 831 ± 12 and 1016± 18 ppm in 4-week exposure, respectively. After exposure, the rats were euthanized by exsanguination through the abdominal aorta under pentobarbital anesthesia. The hippocampus was carefully dissected out en bloc and immediately frozen in liquid nitrogen and stored at −80 °C until use.

Materials and methods

2D-DIGE and image analysis. For 2D-DIGE, we labeled 25 μg each of control, exposure, and internal standard protein sample with cyanine dye (Cy) 3, Cy5 and Cy2 according to the protocol recommended by the manufacturer for CyDye DIGE Fluor minimal dyes (GE Healthcare). The internal standard was a mixture of equal amounts of proteins from 36 samples (n = 6 for each group) and labeled with Cy2. A total of 18 gels were run in parallel (Table 1). Before the first-dimension isoelectric focusing (IEF), equal protein amounts of Cy2, Cy3, and Cy5-labeled samples were mixed and added to an equal volume of 2 × sample buffer [7 M urea, 2 M thiourea, 4% w/v CHAPS, 130 mM dithiothreitol (DTT), 2% IPG buffer (pH 3–11; GE Healthcare), and a cocktail of protease inhibitors]. After incubation on ice for 10 min in the dark, the samples were combined with rehydration buffer [7 M urea, 2 M thiourea, 4% w/v CHAPS, 13 mM DTT, 1% IPG buffer (pH 3–11)] and a trace of bromophenol blue to make 450 μl of total sample volume. In-gel rehydration of the IPG strips (Immobiline DryStrips, 24 cm, non-linear pH 3–11; GE Healthcare) with the samples was performed at room temperature in the dark for 12 h. IEF was run using an Ettan IPGphor II (GE Healthcare) at 500 V for 500 V h, at 1 kV for 1 kV h, and at 8 kV for 99 kV h. After reduction and alkylation with 10 mg/ml DTT and

Chemicals. 1-BP (99.81% purity) was supplied by Tosoh Co. (Japan), and its structure was confirmed with proton nuclear magnetic resonance (NMR). All other chemicals were purchased from commercial sources and were of the highest purity available. Animals. This study was conducted according to the Japanese law concerning the protection and control of animals and the Animal Experimental Guidelines of Nagoya University Graduate School of Medicine. A total of 72 Male F344 rats (specific pathogen free, 9-week-old, body weight 160–180 g) were purchased from Clea, Japan, Inc. (Tokyo, Japan). The rats consisted of two batches of 36 rats each for two experiments, 1- and 4-week exposure experiments. Inbred rats of F344 were chosen to minimize individual variation of genetic background for proteomic analysis. All rats were housed and acclimated to the new environment for 1 week in a temperature (23–25 °C) and humidity (55–60%) controlled room under a 12: 12 h light: dark cycle (lights on at 0900 h and off at 2100 h). Subsequently, the rats were divided at random into 3 groups of 9 animals each in each exposure experiment. Food and water were provided ad libitum. Exposure to 1-BP. Rats of the three groups were exposed to 1-BP for 8 h/day, 7 days/week for 1 week and the other three groups were exposed for 4 weeks at 400 ppm and 1000 ppm, or filtered room air (0 ppm, control). The 1-BP exposure concentrations were selected based on the results of previous studies (Ichihara et al., 2000; Mohideen et al., 2009; Yu et al., 1998): 1) 1-BP dose-dependently altered the mRNA expression levels of hippocampal neurotransmitter receptors (Mohideen et al., 2009), 2) the lowest concentration of 1-BP that induced a decrease in limb grip strength after 12-week exposure was 400 ppm (Ichihara et al., 2000), and 3) exposure to 1-BP at 1000 ppm for 4 weeks did not induce serious debilitation (Yu et al., 1998). We studied exposure by inhalation, because this is the most common route of occupational 1-BP exposure in humans. The inhalation exposure system was similar to that used previously in many of our toxicity studies and is described in detail previously (Ichihara et al., 1997; Takeuchi et al., 1989). In brief, a regulated volume of 1-BP was

Sample preparation. Frozen hippocampus from 9 rats of each group were homogenized individually in a lysis buffer (30 mM Tris–HCl, 7 M urea, 2 M thiourea, 4% w/v CHAPS, and a protease inhibitor mix, pH 8.5) with a PlusOne Sample Grinding Kit (GE Healthcare, Piscataway, NJ) according to procedure supplied by the manufacturer. After incubation for 60 min on ice, the homogenates were centrifuged at 30,000 ×g for 30 min at 4 °C and then the supernatant was collected. The concentration of the protein in the supernatant was determined by the Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA), using bovine serum albumin as a standard.

Table 1 Experimental design of labeling. Gel no. 1 2 3 4 5 6 7 8 9

Cy3

Cy5 a

1 wk-C1 1 wk-C2 1 wk-C3 1 wk-L1 1 wk-L2 1 wk-L3 1 wk-H1 1 wk-H2 1 wk-H3

1 wk-L4 1 wk-H4 1 wk-H5 4 wk-L4 1 wk-C4 1 wk-C5 4 wk-H4 1 wk-C6 1 wk-L5

Cy2 Pool Pool Pool Pool Pool Pool Pool Pool Pool

b

Gel no.

Cy3

Cy5

Cy2

10 11 12 13 14 15 16 17 18

4 wk-C1 4 wk-C2 4 wk-C3 4 wk-L1 4 wk-L2 4 wk-L3 4 wk-H1 4 wk-H2 4 wk-H3

4 wk-L5 4 wk-H5 4 wk-H6 1 wk-L6 4 wk-C4 4 wk-C5 1 wk-H6 4 wk-C6 4 wk-L6

Pool Pool Pool Pool Pool Pool Pool Pool Pool

a Samples from 1-week (1 wk) and 4-week (4 wk) experiments at concentrations of 0 (C1-6), 400 ppm (L1-6) and 1000 ppm (H1-6) of 1-BP exposure were used. b The internal pooled sample was prepared by combining equal amounts from all 36 samples (n = 6, each group) and labeled with Cy2.

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25 mg/ml iodoacetamide, respectively, the second-dimension 12.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was run on an Ettan DALT six large-format vertical system (GE Healthcare). The gels were scanned with a Typhoon 9400 fluorescence scanner (GE Healthcare). Intra-gel matching was performed using DeCyder software version 6.0 (GE Healthcare). Image analysis included the following procedure: spot detection, spot editing, background subtraction and spots matching. The relative quantities of protein spots in each group were calculated by normalization to the internal standard. Protein levels were considered to have changed when the following two criteria were fulfilled: 1) statistically significant change in the protein level (two-tailed Student's t-test, p b 0.05, relative to the control) and 2) the change in protein level was ≥1.2-fold. Identification of proteins. After image analysis, gels containing the additional load of unlabeled proteins from the control group and 1000 ppm group in the 4-week exposure experiment were stained with Colloidal Coomassie Brilliant Blue G (GE Healthcare) and matched to the fluorescent 2D-DIGE images. Selected spots were picked and in-gel digestion of protein samples was performed using the protocol described previously by our group (Oikawa et al., 2009). The extracted peptides were concentrated and the eluent was spotted onto an OptiTOF ™384 Well Insert (123× 81 mm) (Applied Biosystems, Foster City, CA) with 0.5 μl 0.2 mg/ml α-cyano-4-hydrox cinnamic acid (CHCA) as the matrix. The digests were analyzed using the Applied Biosystems 4800 Plus Proteomics Analyzer (MALDI-TOF-TOF/MS) in reflector mode for positive ion detection. Protein identification was carried out using the MS/MS ion search of Paragon (Applied Biosystems) with a detected protein threshold of (0.47) 66.0%. Western blot. To confirm the proteomic results, seven selected proteins were analyzed by western blot. Samples (n = 9 in each group) containing 20 μg hippocampal proteins were separated by 12% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Nonspecific binding was blocked in Tris buffered saline with Tween (TBS-T) containing 5% nonfat milk for 1 h at room temperature. The

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membranes were then washed with 0.1% TBS-T (3× 5 min washes) and incubated with appropriate antibodies overnight at 4 °C. Antibodies for the target proteins were: HSP60 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-59567, 60 kDa), PSMA1 (Santa Cruz Biotechnology, sc166073, 32 kDa), ECH1 (Santa Cruz Biotechnology, sc-133532, 35 kDa) and DJ-1 (Santa Cruz Biotechnology, sc-55572, 23 kDa) at a 1:500 dilution; TPI (Santa Cruz Biotechnology, sc-166785, 30 kDa), Ran (Abcam, Cambridge, UK, ab53775, 26 kDa) and B-CK (Abcam, ab108388, 43 kDa) at a 1:1000 dilution. Following incubation with the primary antibodies, the membranes were washed in 0.1% TBS-T (3 × 10 min washes) and incubated with sheep anti-mouse secondary IgGs (GE Healthcare, NA931V) or donkey anti-rabbit secondary IgGs (GE Healthcare, NA934V) at a dilution of 1:10,000. The formed immunocomplexes were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Cambridge, MA) using Lumivision PRO HSa (AISIN, Japan). Mouse anti-β-actin monoclonal antibody (Sigma, St Louis, MO; A1978, 42 kDa) at a 1:10,000 dilution was used as a loading control. The density of each band was quantified by UNSCAN-IT-gel 6.1 software (Silk Scientific, Inc., Orem, UT). The expression level of each protein was normalized relative to the level of β-actin protein in the same tissue sample. PANTHER analysis. Protein ontology classification was performed by importing proteins into the protein analysis through evolutionary relationships (PANTHER) classification system (http://www.pantherdb. org/, SRI International, Menlo Park, CA). Proteins were grouped accordingly to associate biological processes and molecular functions (Thomas et al., 2003). Statistical analysis. Data are expressed as mean ± SD. A two-tailed Student's t-test was used to determine the differences between paired groups (control and exposure groups). Comparisons among multiple exposure groups and the corresponding control in each exposure experiment were tested using one-way ANOVA followed by Dunnett's test. The relation between the quantity of protein (relative to the internal standard) determined as being altered by 1-BP in 2D-DIGE,

Table 2 Hippocampal proteins whose expression level was modified by 1-BP. Spot no.a

Accession no.b

Proteinc

% Cov.

Peptides (95%)

Fold change up (+)/down (−) 1-BP exposure (ppm) 1 wk-400

1 wk-1000

4 wk-400

4 wk-1000

Up-regulation 540 P06761 800 O08651 831 P63039 1722 P49911 1911 P04904 1926 P48500 1949 P62828 2046 P04906

78 kDa glucose-regulated protein (GRP78) D-3-phosphoglycerate dehydrogenase (PHGDH) 60 kDa heat shock protein, mitochondrial (HSP60) Acidic leucine-rich nuclear phosphoprotein 32 family member A (PHAP1) Glutathione S-transferase alpha-3 (GSTA3) Triosephosphate isomerase (TPI) GTP-binding nuclear protein Ran (Ran) Glutathione S-transferase P (GSTP1)

9.8 13.5 15.7 11.7 17.2 33.3 28.2 39.1

5 2 3 3 5 8 5 6

+ 1.05 + 1.09 + 1.02 + 1.35 + 1.07 + 1.01 + 1.08 + 1.02

+ 1.07 + 1.13 + 1.08d + 1.15 + 1.07 + 1.16d + 1.22d + 1.05

+ 1.09 + 1.04 + 1.05d + 1.22 + 1.09 + 1.23d + 1.35d + 1.01

+ 1.30d + 1.22d + 1.21d + 2.05d + 1.31d + 1.40d + 1.54d + 1.26d

Down-regulation 947 P50554 1095 Q9WTT6 1117 Q03346 1120 P25809 1206 P07335 1395 Q8VHV7 1706 Q62651 1791 P18420 1939 P48500 2036 O88767 2092 P31399

4-aminobutyrate aminotransferase, mitochondrial (ABAT) Guanine deaminase (GDA) Mitochondrial-processing peptidase subunit beta (PMPCB) Creatine kinase U-type, mitochondrial (Mi-CK) Creatine kinase B-type (B-CK) Heterogeneous nuclear ribonucleoprotein H (HNRNPH1) Delta (3,5)-Delta (2,4)-dienoyl-CoA isomerase, mitochondrial (ECH1) Proteasome subunit alpha type-1 (PSMA1) Triosephosphate isomerase (TPI) Protein DJ-1 (DJ-1) ATP synthase subunit d, mitochondrial (ATP5H)

16.8 2.5 13.6 11.5 32.3 2.7 27.8 20.2 37.4 37.6 31.7

5 1 7 4 9 1 5 5 8 6 6

− 1.16d − 1.07 − 1.05 − 1.05 − 1.05 − 1.07 − 1.11 − 1.34 − 1.07 − 1.05 − 1.00

− 1.09 − 1.02 − 1.07 − 1.10 − 1.09 − 1.09 − 1.27d − 1.92d − 1.10d − 1.07d − 1.03

− 1.06 − 1.09 − 1.03 − 1.50d − 1.13d − 1.24d − 1.11d − 1.89d − 1.19d − 1.20d − 1.05

− 1.27d − 1.21d − 1.21d − 1.80d − 1.34d − 1.15 − 1.12d − 2.64d − 1.38d − 1.31d − 1.20d

a b c d

The identities of 8 up-regulated and 11 down-regulated protein spots were determined by MALDI-TOF-TOF/MS. Accession numbers are from Swiss-Prot database (http://www.ebi.ac.uk/uniprot/). Searches were made using the Paragon method with a detection threshold of (0.47) 66.0%. p-value was b 0.05, compared with the corresponding controls in 1-week and 4-week exposure experiments (two-tail Student's t-test). n = 6 for each group.

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and the exposure level was analyzed using linear regression analysis. Statistical analyses were performed using the JMP 8.0 software (SAS Institute Inc., Cary, NC). A probability (p) value b0.05 was considered statistically significant.

400 ppm group and 234 spots in the 1000 ppm group of 4-week exposure compared with the corresponding controls. These results showed that 1-BP exposure modified the hippocampal proteome of F344 rats in dose- and time-response manners.

Results

Identification of differentially expressed proteins in 2D-DIGE gels

Detection of differentially expressed proteins in 2D-DIGE gels

Twenty six protein spots with consistently altered model (increase or decrease after both 1- and 4-week exposure) and significant change in their levels (p b 0.05; fold change ≥ ±1.2) at least at one exposure level or more compared with the corresponding controls, were excised from the gels as candidates for MALDI-TOF-TOF/MS. The latter analysis

Using the DeCyder software version 6.0, 26 differentially expressed protein spots (p b 0.05) were detected in the 400 ppm group and 66 spots in the 1000 ppm group of 1-week exposure, 96 spots in the

Table 3 Coefficients of linear regression analysis for 1-BP exposure level on the differentially expressed proteins identified by 2D-DIGE in the hippocampus. Parameter

1-week 1-BP exposure Coefficient ± SE

GRP78 Intercept Exposure level (× PHGDH Intercept Exposure level (× HSP60 Intercept Exposure level (× PHAP1 Intercept Exposure level (× GSTA3 Intercept Exposure level (× TPI (No.1926) Intercept Exposure level (× Ran Intercept Exposure level (× GSTP1 Intercept Exposure level (× ABAT Intercept Exposure level (× GDA Intercept Exposure level (× PMPCB Intercept Exposure level (× Mi-CK Intercept Exposure level (× B-CK Intercept Exposure level (× HNRNPH1 Intercept Exposure level (× ECH1 Intercept Exposure level (× PSMA1 Intercept Exposure level (× TPI (No.1939) Intercept Exposure level (× DJ-1 Intercept Exposure level (× ATP5H Intercept Exposure level (×

4-week 1-BP exposure p value

Coefficient ± SE

p value

10−5 per ppm)

0.91 ± 0.03 6.2 ± 5.7

b0.0001 0.30

0.84 ± 0.04 25 ± 7

b0.0001 0.0020

10−5 per ppm)

0.95 ± 0.10 12 ± 16

b0.0001 0.46

0.91 ± 0.05 21 ± 7

b0.0001 0.0093

10−5 per ppm)

1.01 ± 0.01 8.7 ± 2.3

b0.0001 0.0017

0.83 ± 0.01 19 ± 2

b0.0001 b0.0001

10−5 per ppm)

1.1 ± 0.2 13 ± 24

b0.0001 0.61

0.77 ± 0.15 88 ± 25

0.0005 0.0070

10−5 per ppm)

1.05 ± 0.04 6.6 ± 6.0

b0.0001 0.29

1.06 ± 0.03 34 ± 5

b0.0001 b0.0001

10−5 per ppm)

0.94 ± 0.03 16 ± 5

b0.0001 0.0047

0.95 ± 0.03 37 ± 5

b0.0001 b 0.0001

10−5 per ppm)

0.87 ± 0.04 21 ± 6

b0.0001 0.0030

0.97 ± 0.04 47 ± 5

b0.0001 b0.0001

10−5 per ppm)

0.90 ± 0.02 4.1 ± 3.6

b0.0001 0.27

1.03 ± 0.04 29 ± 6

b0.0001 0.0003

10−5 per ppm)

0.99 ± 0.04 − 7.5 ± 6.0

b0.0001 0.23

1.00 ± 0.05 − 21 ± 8

b0.0001 0.017

10−5 per ppm)

0.98 ± 0.04 − 1.3 ± 5.9

b0.0001 0.82

1.04 ± 0.04 − 186

b0.0001 0.0071

10−5 per ppm)

1.06 ± 0.02 − 6.0 ± 3.2

b0.0001 0.10

1.13 ± 0.04 − 20 ± 7

b0.0001 0.012

10−5 per ppm)

1.15 ± 0.03 − 11 ± 5

b0.0001 0.073

1.30 ± 0.04 − 60 ± 7

b0.0001 b0.0001

10−5 per ppm)

1.08 ± 0.03 − 9.2 ± 4.1

b0.0001 0.039

1.05 ± 0.02 − 273

b0.0001 b0.0001

10−5 per ppm)

1.07 ± 0.05 − 8.4 ± 7.6

b0.0001 0.29

1.05 ± 0.05 − 12 ± 7

b0.0001 0.13

10−5 per ppm)

1.09 ± 0.04 − 23 ± 5

b0.0001 0.0027

1.09 ± 0.03 − 11 ± 4

b0.0001 0.021

10−5 per ppm)

1.3 ± 0.1 − 65 ± 18

b0.0001 0.0027

1.42 ± 0.06 − 92 ± 10

b0.0001 b0.0001

10−5 per ppm)

1.11 ± 0.02 − 9.9 ± 3.9

b0.0001 0.029

1.18 ± 0.02 − 33 ± 4

b0.0001 b0.0001

10−5 per ppm)

1.14 ± 0.02 − 7.2 ± 2.9

b0.0001 0.023

1.19 ± 0.02 − 28 ± 3

b0.0001 b0.0001

10−5 per ppm)

0.98 ± 0.02 − 2.4 ± 2.5

b0.0001 0.36

1. 03 ± 0.02 − 174

b0.0001 0.0004

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successfully identified 19 spots (Table 2). The identified proteins included 8 up-regulated proteins and 11 down-regulated proteins. Among them, the expression levels of 8 proteins, including HSP60, TPI (No.1926), Ran, B-CK, TPI (No.1939), DJ-1, ECH1 and PSMA1 were altered dose-dependently after both 1- and 4-week exposure, whereas 10 other proteins changed their levels in a dose-dependent manner only after 4-week exposure [GRP78, PHGDH, PHAP1, GSTA3, GSTP1, ABAT, GDA, PMPCB, Mi-CK and ATP5H] (Table 3). With regard to the expression of the former 8 proteins, Ran, TPI (No. 1926), and HSP60 were up-regulated dose-dependently earlier than GRP78, PHGDH, PHAP1, GSTA3 or GSTP1. Similarly, PSMA1, ECH1, TPI (No. 1939), B-CK and DJ-1 were down-regulated dose-dependently earlier than ABAT, GDA, PMPCB, Mi-CK or ATP5H. Interestingly, spots No. 1926 and 1939 in the same gel with similar molecular weight (MW) but different isoelectric point (pI) were identified as TPI, which highlights the PTMs of TPI induced by 1-BP exposure. However, the most common PTMs, for example, phosphorylation, were not detected in TPI using Pro-Q Diamond Phosphoprotein Gel Stain (Molecular Probes, Eugene, OR), even when the maximum 1000 μg hippocampal proteins were loaded (data not shown). Figs. 1A and B show representative 2D-DIGE images and spot relative quantities of the control and 4-week exposure to 1000 ppm, respectively. Western blot HSP60, TPI, Ran, B-CK, ECH1, PSMA1 and DJ-1 antibodies were used to confirm the identity of the above proteins (Fig. 2). These proteins were chosen because of commercial availability of antibodies and the significant dose-dependent changes induced by both 1- and 4-week exposure to 1-BP. Consistent with the proteomic results, the expression of DJ-1, B-CK, ECH1 and PSMA1 in 1-week and/or 4-week 1-BP exposure

Fig. 1. A) Representative 2D-DIGE image of fluorescently labeled proteins from the hippocampuses of male F344 rats from the control and 1000 ppm groups after 4-week exposure to 1-BP. The green spots in red circles or red spots in green circles were found to be down-regulated and up-regulated spots, respectively. Yellow spots represent proteins with unchanged expression level. Molecular weight markers are on the left. The spot numbers correspond to the numbers in Table 2. B) Relative quantities of representative spots shown in (A). Relative fold changes were expressed as mean ± SD. *p b 0.05 and **p b 0.01, compared with the corresponding controls (two-tail Student's t-test). n = 6 rats in each group.

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groups was significantly down-regulated with increased concentrations of 1-BP exposure in the rat hippocampus. In addition, western blot analysis showed significant increases in the expression of Ran in both 1- and 4-week 1-BP exposure samples. However, no significant changes were observed in the expression of wild-type TPI and HSP60 at various concentrations of 1- and 4-week 1-BP exposures. Functional categories of identified proteins To understand the biological impact of 1-BP exposure on the rat hippocampal proteins, the 19 differentially expressed proteins identified by 2D-DIGE analyses combined with MALDI-TOF-TOF/MS were imported into the PANTHER database. The PANTHER classification system revealed that the proteins can be classified into six groups according to their functional properties (Table 4): (1) immunity and defense; (2) energy metabolism; (3) nucleocytoplasmic transport; (4) purine metabolism; (5) neurotransmitter metabolism; (6) ubiquitinationproteasome pathway. Among these dys-regulated proteins, of particular interest are antioxidant proteins and proteins involved in nucleocytoplasmic transport, glucose metabolism and ATP production, as well as proteins associated with neurotransmitter metabolism and ubiquitination-proteasome system (UPS). We expect that oxidative stress, loss of ATP production, neurotransmitter dysfunction and inhibition of UPS play important roles in 1-BP-induced neurotoxicity in the hippocampus. Discussion Our proteomic study showed that exposure of rats to 1-BP results in: 1) modification of hippocampal proteome, 2) differential modification of expression of 19 hippocampal proteins involved in a variety of biological processes; e.g., nucleocytoplasmic transport, immunity and defense, energy metabolism, neurotransmitter metabolism, ubiquitinationproteasome pathway and purine metabolism, 3) dose-dependent and earlier up-regulation of Ran, TPI (No. 1926), and HSP60 compared with those of GRP78, PHGDH, PHAP1, GSTA3 or GSTP1, and 4) dosedependent and earlier down-regulation of PSMA1, ECH1, TPI (No. 1939), B-CK and DJ-1 compared with those of ABAT, GDA, PMPCB, Mi-CK and ATP5H. The results of proteomic analysis were confirmed by western blot, e.g., the altered expression of Ran, B-CK, ECH1, PSMA1 and DJ-1, and lack of such alteration in HSP60 and TPI. Phosphorylation of HSP60 and carbonylation of TPI, which was detected by an on-going experiment (unpublished data) may explain the lack of significant changes observed in western blot. The differences in fold change identified on 2D-DIGE and western blot can be attributed to methodological factors such as the use of fluorescence or chemiluminescence, sample size (n = 6 for 2D-DIGE and 9 for western blot) and/or differences inherent in the technical approach. However, the direction of the change (up or down-regulation) was similar in 2D-DIGE and western blot. These results emphasize the reliability of the proteomic results. The protein that was up-regulated to the greatest extent at an early stage is Ran, which is a member of the Ras family GTPase. Ran forms a complex with RCC1 (regulator of chromosomal condensation), which has critical cellular functions such as DNA replication, cell cycle progression, nuclear structure, RNA processing and exportation, and nuclear protein importation (Bischoff and Ponstingl, 1991; Kadowaki et al., 1993; Matsumoto and Beach, 1991, 1993; Moore and Blobel, 1993; Sazer and Nurse, 1994). In vitro studies showed that Ran expression level negatively controls the nuclear presence of selective transcription factors such as c-Jun and c-Fos of AP-1 (Pham et al., 2010; Qiao et al., 2010). These proteins are reported to play a role in various neurodegenerative diseases (Herdegen and Waetzig, 2001), and c-Fos is also known as a biomarker of neurotoxicity (Ryabinin, 1998). Further studies are needed to understand the role of Ran in neurotoxicity of 1-BP.

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Fig. 2. (A) Western blot confirmed the 2D-DIGE results and (B) relative quantities. Modifications to the relative protein expression levels of HSP60, TPI, DJ-1, B-CK, PSMA1, ECH1 and Ran were investigated by western blot. β-actin served as a loading reference. Relative protein level was expressed as mean ± SD. *p b 0.05 and **p b 0.01, compared with the corresponding controls (one-way ANOVA followed by Dunnett's multiple comparison). n = 9 rats in each group.

The results of the present study lend support to the hypothesis that oxidative stress plays a role in the observed changes in the expression of hippocampal proteins, including HSP60, GRP78, DJ-1, GSTA3 and GSTP1. HSP60 is a mitochondrial matrix protein induced by various kinds of stresses (Calabrese et al., 2006). It is known that the heat shock response provides neuroprotection through inhibition of the NF-κB signaling pathway (Calabrese et al., 2003), which might help explain the link between HSP60 induction in the present study and the previous in vitro finding of 1-BP-induced suppression of NF-κB activity (Yoshida et al., 2007). Consistent with the results of HSP60, GRP78 was also up-regulated by 1-BP exposure, though at a

later stage. GRP78 is an endoplasmic reticulum (ER)-resident molecular chaperone known to protect cultured hippocampal neurons against excitotoxicity and apoptosis by suppression of oxidative stress (Yu et al., 1999). Thus, 1-BP-induced up-regulation of GRP78 seems to indicate ER stress in the hippocampus. DJ-1 is reported to prevent oxidative stress in age-related neurodegeneration (Kahle et al., 2009) and its deficiency in mice is associated with nigrostriatal dopaminergic dysfunction, motor deficits, and hypersensitivity to neurotoxins (Goldberg et al., 2005; Kim et al., 2005). Since DJ-1 was down-regulated after 1 week of exposure, it is possible that such down-regulation results in increased oxidative stress in the hippocampus. GSTA3 and GSTP1

Z. Huang et al. / Toxicology and Applied Pharmacology 257 (2011) 93–101 Table 4 Functional properties of proteins whose expression level was modified by 1-BP. Protein name

Functional description

Immunity and defense DJ-1 Response to stress, immune system process, nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; protein metabolic process, peptidase activity, DNA binding, RNA binding, transcription factor activity GRP78 Response to stress, immune system process, protein metabolic process, anti-apoptosis, protein binding, ATP binding, unfolded protein binding, caspase inhibitor activity HSP60 Response to stress, protein metabolic process, anti-apoptosis GSTA3 Response to toxin, immune system process, muscle contraction, lipid metabolic process, transferase activity GSTP1 Response to toxin, immune system process, transferase activity Energy metabolism Mi-CK Phosphocreatine biosynthetic process, ATP binding, nucleotide binding, transferase activity, kinase activity, creatine metabolic process B-CK Muscle contraction, metabolic process, ATP binding, nucleotide binding, kinase activity, brain development, cellular chloride ion homeostasis, phosphocreatine metabolic process ATP5H Respiratory electron transport chain, cation transport, nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; hydrolase activity, transporter activity, hydrogen ion transmembrane TPI Carbohydrate metabolic process, isomerase activity PMPCB Respiratory electron transport chain, protein metabolic process, oxidoreductase activity, peptidase activity, hydrolase activity, acting on ester bonds PHGDH Carbohydrate metabolic process, cellular amino acid and derivative metabolic process, oxidoreductase activity ECH1 Coenzyme metabolic process, vitamin biosynthetic process, carbohydrate metabolic process, lipid metabolic process, oxidoreductase activity, acyltransferase activity, hydrolase activity, racemase and epimerase activity, ligase activity Nucleocytoplasmic transport PHAP1 Protein binding, RNA metabolic process, intracellular signal transduction, nucleocytoplasmic transport Ran Intracellular protein transport, nuclear transport, cell cycle, intracellular signaling cascade, nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; signal transduction, RNA localization, GTPase activity, protein binding HNRNPH1 Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process, structural constituent of ribosome, nucleic acid binding Neurotransmitter metabolism ABAT Gamma-aminobutyric acid metabolic process, negative regulation of blood pressure, locomotor behavior, transaminase activity, protein homodimerization activity, pyridoxal phosphate binding, succinatesemialdehyde dehydrogenase binding Purine metabolism GDA Purine metabolism, hydrolase activity, metal ion binding Ubiquitination-proteasome pathway PSMA1 Proteolysis involved in cellular protein catabolic process, negative regulation of inflammatory response to antigenic stimulus, protein metabolic process, peptidase activity, lipopolysaccharide binding, threonine-type endopeptidase activity

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The results of proteomic analysis also showed a variety of other biological responses to 1-BP exposure. The observed up-regulation of PHAP1 suggests the effect of 1-BP exposure on nucleocytoplasmic transport. PHAP1, together with CAS and HSP70, is known to promote apoptosome formation (Kim et al., 2008). The observed down-regulation of PSMA1 suggests the effects of 1-BP exposure on the ubiquitinationproteasome system (UPS). The significant changes in Mi-CK, B-CK, ATP5H, TPI, PMPCB, PHGDH and ECH1 levels suggest the effects of 1-BP on glucose metabolism and/or ATP production. Our previous studies also showed that 1-BP significantly down-regulated cerebral B-CK in rats (Wang et al., 2002, 2003). Down-regulation of GDA, which regulates postsynaptic sorting (Firestein et al., 1999) and promotes dendritic branching (Akum et al., 2004), suggests the potential effect of 1-BP on purine metabolism. Changes in ABAT, which is associated with the metabolism of γ-aminobutyric acid (GABA), should be also noticed for understanding the effect of 1-BP on the CNS. In the hippocampus of human brain, GRP78, GSTA3, TPI, GDA and PSMA1 are expressed dominantly in neuronal cells and PHGDH is expressed dominantly in glial cells. HSP60, PHAP1, ABAT, HNRNPH1, ECH1 and DJ-1 are expressed both in the neuronal cells and glial cells without great difference in their expression levels between two different types of cells (The Human Protein Atlas, 2011). Further studies are needed to clarify which cell type is responsible for change in expression of these proteins induced by 1-BP exposure. The present study focused on the hippocampus because previous studies suggested hippocampus is a brain region susceptible to 1-BP exposure (Fueta et al., 2004; Mohideen et al., 2009). Exposure to 1-BP at 700 ppm, 6 h/day, 5 days/week for 12 weeks induced disinhibition in the hippocampal CA1 region and dentate gyrus in rats (Fueta et al., 2004). Exposure to 1-BP at 400, 800 or 1000 ppm induced down-regulation of mRNA expression of dopamine D2 receptor in rat hippocampus, which showed the clearer change in the mRNA expression of neurotransmitter receptors than cerebral cortex, caudate putamen, amygdala, midbrain, cerebellum and pons plus medulla (Mohideen et al., 2009). However, our recent study showed dose-dependent decrease in noradrenergic axons in prefrontal cortex and amygdala, suggesting multiplicity of brain regions susceptible to 1-BP exposure (Mohideen et al., 2011). In conclusion, the present study applied comparative proteomic analysis to identify proteins associated with 1-BP-induced neurotoxicity in the rat hippocampus. 1-BP altered the expression of 19 proteins known to be involved in nucleocytoplasmic transport, immunity and defense, energy metabolism, purine metabolism, neurotransmitter metabolism and ubiquitination-proteasome pathway. The identified proteins probably mediate the effects of 1-BP on oxidative stress, loss of ATP production, GABA dysfunction and inhibition of UPS, at least in the hippocampus. Proteins identified in this study also provide a valuable reference resource for future hypothesis-driven studies on 1-BP-induced neurotoxicity in the CNS. Conflict of interest statement The authors declare no conflict of interest.

were also up-regulated after prolonged 1-BP exposure. The GSTs are a family of detoxification enzymes that protect against oxidative stress (Tchaikovskaya et al., 2005) and neurodegeneration (Castro-Caldas et al., 2009), suggesting their protective role against 1-BP-induced oxidative stress. It has been well documented that 1-BP conjugates with the sulfhydryl functions of proteins or glutathione (GSH), either directly (Barnsley et al., 1966; Jones and Walsh, 1979; Khan and O'Brien, 1991) or indirectly after metabolic oxidization at carbon2 or carbon3 (B'Hymer and Cheever, 2004; Barnsley et al., 1966; Jones and Walsh, 1979). Such conjugation leads to depletion of GSH, which results in promotion of oxidative stress.

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