Comparative Biochemistry and Physiology, Part C 167 (2015) 183–189
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Analysis of differentially expressed proteins in zebrafish (Danio rerio) embryos exposed to chlorpyrifos Lili Liu a, Yongxue Xu a,b, Lili Xu a, Jian Wang a, Wei Wu a, Lei Xu a, Yanchun Yan a,⁎ a b
Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China Tianjin International Joint Academy of Biomedicine, Tianjin 300457, China
a r t i c l e
i n f o
Article history: Received 31 July 2014 Received in revised form 22 October 2014 Accepted 22 October 2014 Available online 28 October 2014 Keywords: 2D PAGE Chlorpyrifos Danio rerio Mechanism Proteomics Zebrafish embryos
a b s t r a c t In this study, the protein expression profiles of zebrafish embryos under chlorpyrifos (CPF) stress were investigated. Zebrafish embryos were exposed to 0.25 mg/L CPF, and embryo samples were collected until 24 h post-fertilization (hpf). To gain a better understanding of the response of zebrafish embryos to CPF exposure, two-dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with mass spectrometry was employed to carry out a comparative proteomic analysis. Total proteins were extracted from the control and treated samples, separated by 2D PAGE, and visualized by silver staining. A total of 59 protein spots showed reproducible changes compared with the control. Of these 59 spots, 19 were selected and subjected to matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 9 differentially expressed proteins were successfully identified, including 3 up-regulated proteins and 6 down-regulated proteins. The increased expression of 3 proteins associated with detoxification and stress response suggested that the activation of protective proteins was required in zebrafish embryos exposed to CPF. On the other hand, the decreased expression of 6 proteins is mainly involved in cytoskeleton structure, protein translation, signal transduction and lipoprotein metabolism. These data may help us understand the functions and the molecular mechanisms of these proteins in zebrafish embryos' response to CPF exposure. © 2014 Elsevier Inc. All rights reserved.
1. Introduction The insecticide chlorpyrifos (CPF) is a broad-spectrum organophosphate pesticide that has been one of the most widely used insecticides in the world. CPF elicits a number of additional effects, including hepatic dysfunction, hematological and immunological abnormalities, embryotoxicity, teratogenicity and neurotoxicity (Fatma et al., 2010). Of the established environmental neurotoxicants, organophosphate pesticides (OPs) such as chlorpyrifos are potent inhibitors of acetylcholinesterase (AChE), a key enzyme pathway responsible for terminating the transmission of many neuronal cell types across synapses (Yang et al., 2008). However, non-AChE-associated mechanisms of CPF neurotoxicity and behavioral impairments have also been described (Eaton et al., 2008). The zebrafish (Danio rerio) is a small tropical aquarium fish well known to home aquarium enthusiasts. Many of the features have made it become a model system for the examination of embryonic development of vertebrates. It has been observed that early life stages of fish are generally more sensitive to toxic compounds compared with adults and juveniles (Cook et al., 2005), so zebrafish embryos offer a good model to investigate the ecological risk assessments and help ⁎ Corresponding author. Tel.: +86 10 82109685; fax: +86 10 82106609. E-mail address: [email protected] (Y. Yan).
http://dx.doi.org/10.1016/j.cbpc.2014.10.006 1532-0456/© 2014 Elsevier Inc. All rights reserved.
determine the critical neurodevelopmental processes impacted by CPF, in which the mechanisms of neurodevelopment can be more closely analyzed. Global protein expression profile is an excellent approach to visualize the pattern and the level of the proteins expressed under defined environmental conditions. Therefore, proteomics analysis may provide more direct insights into the mechanisms of effects. The current status of proteomic approaches in understanding the toxicity and the mechanisms of exposure to toxicants has been reviewed (Gillardin et al., 2009). Methodologies for proteomic analysis of zebrafish have been developed (Tay et al., 2006). Proteomic techniques in zebrafish embryos have been utilized to observe changes in the proteome over developmental time and a previous study has investigated proteomic changes in zebrafish larval exposure to perfluorooctane sulfonate (Shi et al., 2009). In addition, previous reports on zebrafish proteomics revealed certain expressed proteins as the sensitive stress indicators in zebrafish embryos can reflect the overall fitness of the intact organisms (Shrader et al., 2003). To date, no studies concerning the potential effects of chlorpyrifos on the protein expression profile of developing zebrafish are available. During fish development, early embryonic stages are the most vulnerable stages to environmental stress, like changes in temperature or oxygen content of the water and also to the additional stress due to the increasing pollution (Cook et al., 2005). Thus, the aim of the present
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study is to understand the mechanisms underlying CPF toxicity during zebrafish development. 2D PAGE and peptide mass fingerprinting (PMF) were used to investigate the protein expression profiles of zebrafish embryos that had been exposed to CPF. Proteomic analysis indicated that CPF regulates a set of proteins that are involved in detoxification, cytoskeleton structure, protein translation, signal transduction, lipoprotein metabolism and other proteins. 2. Materials and methods 2.1. Chemicals Chlorpyrifos (CPF, purity N 99%) was purchased from the National Institute of Metrology. The stock solution (5 mg/L) was prepared by dissolving the crystals in HPLC-grade acetone and stored at 4 °C in darkness. All of the chemicals used in this study were of analytical grade, most of which were purchased from GE Healthcare. 2.2. Maintenance of zebrafish and treatments Zebrafish (Tübingen strain) was obtained from the School of Life Science of Peking University of China at the juvenile stage and was cultured in our laboratory. The culture of mature zebrafish (about seven months old) was maintained at 28 ± 0.5 °C with a constant artificial light/dark cycle of 14/10 h in a continuous flow-through system with charcoal filtered water (pH 7.00 ± 0.5 and conductivity 500 ± 50 μS). Fishes were fed with live brine shrimps (Artemia nauplii) twice daily, occasionally supplemented with commercially available artificial diets. Zebrafish embryos were obtained from the spawning adults, males and females in a ratio of 2/1 were placed in breeding chambers the day before embryos were needed. Spawning was induced on the morning when the light was turned on. Zebrafish eggs were collected within 30 min after natural mating, rinsed several times with Holt Buffer (NaCl 3.5 g/L, KCl 0.05 g/L, NaHCO3 0.025 g/L, CaCl2 0.1 g/L). At 2 h post fertilization (hpf), approximately 300 normally developed embryos were randomly distributed into each petri dish and exposed them to 0, and 0.25 mg/L CPF in an incubator set at 28.5 °C. From our pilot experiments we chose the dose of the exposure chemical that did not increase embryonic fatalities or malformations above control rates over the entire exposure time. CPF dilutions or vehicle were changed every 12 h with exposure ending on 24 hpf. At this period, the major organ systems, including the somites, the pronephros, the heart, and the central nervous system, form. The control and treated embryos received 0.01% acetone as a solubilizing agent to assist in stock solution preparation. After 24 hpf, zebrafish embryos were collected and kept at −80 °C until analysis. Three replicates for each treatment concentration were conducted. 2.3. Sample preparation and two-dimensional polyacrylamide gel electrophoresis At 24 hpf, prior to the generation of samples for two-dimensional gel electrophoresis the chorion was removed following the method of Westerfield (2000). The samples were prepared as described in Link et al. (2006). Briefly, approximately 250 deyolked zebrafish embryos were lysed in 150 μL 3% (w/v) SDS solution for 5 min at 90 °C. Then total protein was extracted with the method of methanol–chloroformprecipitation. Samples were lysed in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 2% (w/v) DTT, 0.5% (v/v) IPG buffer pH 4–7, 0.002% (w/v) bromophenol blue). Insoluble particles were removed by centrifugation for 1 h at 12,000 g. Protein concentration was determined using a 2D Quant Kit (GE Healthcare, Piscataway, NJ, USA). Total proteins were frozen in liquid nitrogen or processed directly for 2D gel electrophoresis. The protein solution (350 μg protein on every analytical gel or 1 mg on every preparative gel) was adjusted with a rehydration buffer for a final volume of 450 μL. Based on the procedures for 2D-PAGE described
by Gorg et al. (2004), conditions were optimized with a slight modification. Isoelectric focusing (IEF) was performed in IPG strips (pH 4–7, 24 cm, GE Healthcare) at 50 V for 6.5 h, 150 V for 1.5 h, 300 V for 1 h, 500 V for 4 h 1000 V for 1.5 h, and 10,000 V for 2 h (slowly and deliberately apply pressure to the strip for desalting) and then 10,000 V constant for a total of 85,000 Vh on a Multiphor II system (GE Healthcare). After isoelectric focusing, the IPG strips were equilibrated for 15 min in an equilibration buffer (6 M Urea, 2% (w/v) SDS, 30% (v/v) glycerol, 50 mM Tris–HCl pH 8.8 and a trace of bromophenol blue) containing 20 mg/mL DTT and then alkylated (25 mg/mL iodoacetamide instead of DTT in an equilibration buffer) for 15 min. The SDS-PAGE in the second dimension was carried out in polyacrylamide gels (12.5% T, 2.6% C) with an Ettan DALT II system (GE Healthcare, Piscataway, NJ, USA). Electrophoresis was performed at 1 w/gel for 60 min, followed by separation at 10 w/gel until the dye front had nearly reached the bottom. The protein spots were visualized via silver staining in analytical gels and via coomassie brilliant blue G-250 staining in preparative gels. Three replicates from three independent biological extracts were used for analysis.
2.4. Image acquisition and analysis The 2D gels were scanned with an image scanner (GE Healthcare, Piscataway, NJ 173, USA). The intensity of protein spots was detected and quantified with ImageMaster 2D Platinum (GE Healthcare, Piscataway, NJ, USA) software on the basis of their relative volume. After automated detection and matching, manual editing was carried out. To compensate for subtle differences in sample loading, gel staining and destaining, the intensity of each spot was normalized as a percentage of the total volume of all of the spots present in the gel.
2.5. In-gel digestion and protein identification In-gel digestion was performed according to Chambery et al. (2006). Selected protein spots were manually excised from the CBB-stained gel and destained by washing twice with 100 μL aliquots of water, performing a further washing step with 50% (v/v) acetonitrile. The gel pieces were then vacuum-dried and rehydrated with 10 μL of 50 mM ammonium bicarbonate, followed by the addition of 5 μL of a 70 ng/mL TPCK-treated porcine trypsin solution. Digestion was performed by incubation at 37 °C for 3 h. Further amount of buffer solution without trypsin was added when necessary to keep the gel pieces wet during the digestion. Peptides were extracted in two steps by sequential addition of 1% (v/v) trifluoroacetic acid (TFA) and then of 2% TFA/50% acetonitrile for 5 min in a sonication bath. The combined supernatants were concentrated in the SpeedVac Vacuum for mass spectrometry (MS) analysis. After in situ tryptic digestion, proteins were identified by PMF based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All spectra of proteins were submitted to database searching using the online MASCOT program (http://www. matrixscience.com), against NCBInr, Swiss-Prot, MSDB and EST databases. The classification and functions of the proteins identified were obtained by searching Gene Ontology (http://www.geneontology.org/).
2.6. Statistical analysis In this study, the data of the spot intensities were expressed as mean ± standard deviation (SD). Comparisons were performed by one-way ANOVA and the Least Significant Difference (LSD) test to determine significant differences among group means. A probability value of b0.05 was considered statistically significant. The statistical package SAS, version 9.1 (SAS/STAT Software for PC. SAS Institute Inc., Cary, NC, USA) was used for statistical analysis.
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3. Results and discussion In the present study, 2D PAGE was used to analyze the differentially expressed proteins of zebrafish embryos (24 hpf) in response to CPF stress. The high-resolution 2DE gel pattern in the pI range of 4–7 was detected by silver staining (Fig. 1). More than 1200 protein spots were reproducibly detected in each silver-stained gel (Fig. 1), and CPFinduced changes in protein abundance were found in the appropriate gels (Fig. 1A and B). The treated samples were compared with the controls, 59 differentially expressed distinct spots were found in the treated group. Identical gels were run and used for in-gel tryptic digestion, followed by mass spectrometry. A total of 19 protein spots were selected for protein identification based on spot intensity and spot integrity. These proteins were identified by means of MALDI-TOF-MS/MS. After a PMF search in the NCBI nr database, 9 spots were successfully identified (Fig. 1), of these, 3 protein spots (spots 1,2,6) showed an increase in abundance in the treated samples compared with the controls, while 6 spots (spots 3,4,5,7,8,9) decreased in abundance in the treated samples. Magnified views (Fig. 2A) and relative spot intensity (Fig. 2B) of the differential proteins are given in Fig. 2. Based on the putative functions of the identified proteins, they were further categorized including detoxification, cytoskeleton structure, protein translation, signal transduction, lipoprotein metabolism and other proteins (Table 1). There 4
pI
7
MW (kDa)
A
116.0 1 2
4
3
5 6
7
14.4
8
9
4
pI
7
MW (kDa)
B
116.0
1 2
3
4
5 6
7 14.4
8
9
Fig. 1. Representative 2D maps of 24 hpf zebrafish embryonic proteins exposed to CPF stress. (A) and (B) represent the 2D gel patterns of control and 0.25 mg/L of CPF treated zebrafish embryos, respectively. Total proteins were extracted and separated by 2D PAGE. A total of 350 μg proteins were loaded onto 24 cm pH 4–7 linear IPG strips. SDSPAGE was performed with 12.5% gels. The spots were visualized by silver staining. Arrows indicate the identified and differentially expressed proteins in response to CPF stress.
185
was also a hypothetical protein identified with an unknown function in the database. The field of proteomics is expanding rapidly, which can provide greater volume and quality of protein information to help understand the molecular mechanisms underlying CPF-induced responses in the developing zebrafish embryos. Here, we observed 59 differentially abundant protein spots in response to CPF and 9 of these were successfully identified. Most of these proteins have important biological functions, providing a basis for understanding and annotating their potential roles in response to CPF stress. 3.1. CPF stress-induced up-regulated proteins Among the identified proteins, a total of 3 spots were up-regulated following exposure to CPF stress. These protein spots are 60 kDa heat shock protein (Hsp60), aldehyde dehydrogenase 2 (ALDH2) precursor, and glutathione S-transferase M. These proteins are mainly associated with detoxification and play an important role in cell protection and repair upon stress. Heat shock proteins (Hsps) are ubiquitous, highly conserved proteins that have been found in the cells of all organisms studied thus far, including plants, bacteria, yeast, flies, and vertebrates (Jaattela, 1999). Many previous studies have reported that when organisms are exposed to a variety of stress factors such as cold, heat, heavy metal, and various chemicals, they synthesize a set of Hsps, which usually acts as molecular chaperones, and play diverse roles in transporting, folding, assembling of degraded or misfolded proteins and maintaining normal cellular homeostasis. Particularly Hsp60 and Hsp70 have been strongly implicated in the immune response (Van Eden and Young, 1996). Information about Hsps' expression and function in zebrafish has increased substantially in the past decade, suggesting that zebrafish Hsps play critical roles during normal developmental (Patrick et al., 2003). Hsp60 has been also identified and cloned in the zebrafish, which is required for blastema formation and maintenance (Makino et al., 2005). Thus, the significantly elevated level of Hsp60 (spot 1) in response to CPF exposure is consistent with other researches, which found that the expression levels of various HSPs were increased in fish exposed to environmental contaminants, such as pesticides (Sanders, 1993), and suggests that it might play a vital role in cell protection and repair upon stress. Aldehyde dehydrogenases (ALDHs) form a superfamily of NAD(P)+dependent enzymes that catalyze the oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding carboxylic acids (Vasiliou et al., 1999). In fact, a diversity of metabolic functions has been reported: (i) detoxification, such as the removal of a plethora of aldehydes generated during drug and xenobiotic metabolism, (ii) participation in intermediary metabolism, such as amino acid and retinoic acid metabolism, (iii) protection from osmotic stress by generating osmoprotectants, such as glycine betaine, and (iv) generation of NAD(P)H (Fong et al., 2006). ALDH2 enzyme plays a major role in acetaldehyde oxidation in vivo that has been also reported (Peng et al., 1999). In the present study, a 2.21-fold increase in aldehyde dehydrogenase 2 precursor (spot 2) abundance was observed, which indicated a promising high-level of ALDH2 at 24 hpf zebrafish embryo. Thus, the up-regulation of ALDH2 precursor may be due to the formation of a metabolic product of CPF, in order to reduce oxidative damage. However, whether the increase of ALDH2 displays other functions remains to be elucidated. The expression of glutathione S-transferase M was significantly up-regulated in the zebrafish embryos exposed to CPF. Glutathione S-transferase M (spot 6) belongs to the superfamily of the glutathioneS-transferase (GST) enzymes that form a group of ubiquitous enzymes that catalyze the conjugation between glutathione and several molecules, and play the most important role in the cellular detoxification mechanism of xenobiotic and endogenous compounds (Agianian et al., 2003). The chemical exposure of insects is a classical event that selects
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A
C
1
T
2
1
4
4
7
C
2
C
8
T
3
3
6
6
5
5
7
T
8
9
9
B
Fig. 2. Magnified views. (A) shows the differentially expressed proteins in Fig. 1, and graphical presentation (B) of differentially expressed proteins identified in 24 hpf zebrafish embryos exposed to CPF stress. (C) Control. (T) Treatment with 0.25 mg/L of CPF. Arrows indicate the differential spots between the two treatments. For each treatment, relative abundance ratio of three replicates (±SE) is shown. Different letters indicate a statistically significant difference (p b 0.05) according to the LSD test.
pesticide resistance, and has been related with a high GST activity (Wei et al., 2001). It also has been suggested that the pesticide may conjugate to glutathione by GST and that the compound obtained may therefore act as a detoxification mechanism in arthropods (Wei et al., 2001). In addition, differential expression of this enzyme has also been observed in perfluorooctane sulfonate (PFOS) exposure tilapia (Liu et al., 2007). These results suggest that the increased activity of glutathione S-transferase M might be involved in the activation of the detoxification pathway to protect against cellular damage of zebrafish embryos' exposure to CPF.
3.2. CPF stress-induced down-regulated proteins in zebrafish embryos Among the identified proteins, a total of 6 spots showed decreased levels of expression after treatments with CPF (Table 1). These protein spots are Rplp0 protein, Type I cytokeratin, annexin 5, ApoA protein, lactoylglutathione lyase (LGL), and a hypothetical protein. Ribosomal protein P0 (RpLp0) (spot 3) is a highly conserved protein, which belongs to the L10P family of ribosomal proteins (Zeng et al., 2001). The ribosomal function of P0 is mediated through a pentameric P12·P0·P22 complex, which forms the stalk of the large ribosomal
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Table 1 CPF stress responsive differently expressed proteins in 24 hpf zebrafish embryos identified by MS analysis. Spot IDa
Acc.no.b
Protein namec
Fold change by CPF stressd
1↑
gi| 31044489 gi| 41053732 gi| 29124460 gi| 41388915 gi| 27762270 gi| 47086689 gi| 53733942 gi| 292614682 gi| 47085917
60 kDa heat shock protein, mitochondrial Aldehyde dehydrogenase 2 precursor Rplp0 protein
+2.81
2↑ 3↓ 4↓ 5↓ 6↑ 7↓ 8↓ 9↓ a b c d e f g h i
pIf
Match rateg
SC (%)h
Mowse scorei
Functional description
61.39
5.56
20/47
35%
129
+2.21
57.21
5.93
21/72
39%
141
−1.94
34.55
5.73
18/80
44%
136
Type I cytokeratin, enveloping layer Annexin 5
−2.00
46.53
5.13
18/47
39%
144
−3.51
35.21
5.46
15/50
47%
119
Glutathione S-transferase M
+2.69
26.45
5.58
19/45
48%
115
Molecular chaperones, stressinduced biomarker Oxidize a wide variety of aliphatic and aromatic, aldehyde detoxification Structural constituent of ribosome, translational elongation Intermediate filament, structural molecule activity Signal transduction, anti-apoptosis, transport Detoxification
ApoA protein
−2.81
19.74
5.31
29/70
87%
184
Regulate lipoprotein metabolism
Hypothetical protein LOC325536 Lactoylglutathione lyase
−2.83
345.80
4.78
26/49
10%
65
−2.67
20.40
5.23
18/58
50%
135
MW (kDa)e
Catalyzes the glyoxal pathway, detoxification
Numbering corresponds to the 2DE gel in Fig. 1. Proteins up- (↑) or down- (↓) regulated in response to CPF treatments. Acc. no.: accession number in NCBInr database. All the proteins identified relate to Danio rerio. Fold change: increased (+) or decreased (−) compared with the controls. MW: molecular weight. pI: isoelectric point. Match rate: the percentage of the number of mass values matched to the number of mass values searched. Sequence coverage: the percentage sequence coverage of the hit. Mascot score.
subunit at the GTPase center (Uchiumi and Kominami, 1992). RpLp0 interacts with the elongation factor eEF2 (Justice et al., 1999), and is essential for the ribosomal activity and cell viability in yeast (Santos and Ballesta, 1994). The functions of the RpLp0 are complex and intriguing, and multiple roles have been also reported in many cellular processes, including DNA repair, cell development, apoptosis and carcinogenesis (Alaa et al., 2007). However, to the best of our knowledge, this is the first study indicating an association between CPF and the down-regulation of Rplp0 protein, and a -3.00-fold reduced expression was observed, which strongly suggests that RpLp0 is associated with CPF-induced cytotoxicity. During apoptosis, one of the cellular responses is the regulation of protein translation, which is divided into three distinct phases: initiation, elongation and termination (Holcik and Sonenberg, 2005). Taken together, these results suggest that the decrease of Rplp0 protein in CPF exposure would affect protein translation. Therefore, it might be considered that CPF affects the growth of zebrafish embryos or results in apoptosis. Nevertheless, further investigation is needed to verify this association. This is the first report showing the down-regulation of Type I cytokeratin (spot 4) in zebrafish embryos exposed to CPF. Cytokeratins belong to the gene family of intermediate filaments (IFs), which are the major structural proteins in higher eukaryotic cells and are specifically expressed in epithelial cells of vertebrates (Yoon et al., 2001). Cytokeratins act as a scaffold allowing for the maintenance of cellular architecture, and providing resistance to mechanical and nonmechanical stresses. In addition, other non-structural functions such as cell signaling, protein targeting in polarized epithelia, cell proliferation, and in apoptosis have also been reported (Carlos et al., 2007). A previous study has been reported that type I cytokeratin is expressed in the enveloping layer at early gastrula in zebrafish embryos, and expression increases during development (Charles et al., 2005). Downregulation of type I cytokeratin protein would result in the suppression of proper filament formation and inhibition of growth. In addition, the cytoskeletal integrity might be affected, which reduced the cellular resistance to environmental stress. However, whether the downregulation of type I cytokeratin displays other functions, further studies are still needed.
Annexin 5 (spot 5) is a member of the annexin family of proteins characterized by calcium dependent phospholipid binding and structural similarity (Raynal and Pollard, 1994). It has been suggested that annexins may be implicated in several physiological processes, such as Ca2+ regulated exocytosis, endocytosis, mitogenic signal transduction, differentiation and coagulation (Gerke and Moss, 2002). In the present study, we observed that annexin 5 expression was down-regulated after exposure to 0.25 mg/L CPF. Annexin 5 has been extensively used as an indicator of early apoptosis due to its inherent property of selectively interacting with phosphatidyl serine. Therefore, the significant down-regulation of annexin 5 might be associated with CPF-mediated dysregulation of the cell membrane function, which may result in apoptosis. Apolipoproteins, the protein components of lipoproteins, are key elements with important roles in lipid transport and uptake in vertebrates (Kim et al., 2009). ApoA (spot 7), a member of apolipoprotein family, includes ApoAI, ApoA II and ApoA IV. A previous study has shown that the ApoAI gene is highly expressed during embryonic development (Patrick et al., 1997), which may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway and may protect against atherosclerosis (Kozarsky et al., 1997). In the present study the down-regulation of ApoA was observed, unfortunately, its precise class was not identified. In addition, since most fish utilize lipids as the major energy source (Kondo et al., 2005), lipid metabolism appears much important for homeostasis maintenance in fish. These results suggested that the decrease of ApoA under CPF exposure may affect the lipid metabolism or other transport pathway, disturbing the homeostasis maintenance in zebrafish embryos. However, further studies are needed to explore the actual mechanism of the ApoA induced by CPF. Lactoylglutathione lyase (LGL) (spot 9) is also known as glyoxalase I, which is an enzyme involved in the detoxification of methylglyoxal, a highly toxic electrophilic glycolytic by-product that reacts with and inactivates intracellular macromolecules, including both proteins and nucleic acids (Kalapos, 1999). Separate two-dimensional gel electrophoresis studies demonstrated that LGL of S. mutans was up-regulated in an acidic environment, suggesting that LGL may be linked to the
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acid tolerance (Wilkins et al., 2002). In addition, since LGL was found to be generally up-regulated in Alzheimer's disease brain (Feng et al., 2004) but down-regulated in old age healthy brains (Kuhla et al., 2006); it was thought that the glyoxalase system may limit the carbonyl-induced neurotoxicity and apoptosis (Kikuchi et al., 1999). To date, unfortunately, there has been no study that focuses on the function of LGL in zebrafish embryos. Taken together, the significant downregulation of LGL may result in the accumulation of methylglyoxal that could lead to the inhibition of DNA synthesis, or neurotoxicity and apoptosis. However, further investigation is needed to verify this hypothesis and to know the precise role of LGL and the relation with CPF stress in zebrafish embryos. The drastic down-regulation of spot 8 has also been noticed in zebrafish embryo following exposure to CPF. Then spot 8 was analyzed by MS and then the spectrum was analyzed via BLAST in zebrafish database of NCBI. Unfortunately, there was no matching information. Physiological and biochemical responses of zebrafish embryos in response to CPF stress have been analyzed extensively. However, to the best of our knowledge, the present study represents the first proteome map of zebrafish embryos under CPF stress. In addition, the solvent level of acetone we used was below 0.01% (v/v), which can't cause differential expression of protein (Hallare et al., 2006). In summary, our results show that a broad spectrum of proteins involved in zebrafish embryos' development is affected by CPF exposure. Finally, a total of 9 proteins that are associated with detoxification, cytoskeleton structure, protein translation, signal transduction and lipoprotein metabolism were successfully identified. Thus, profiling of the zebrafish embryo proteome following exposure to CPF enhanced our understanding of the CPF response of zebrafish. For instance, the increased activity of several proteins associated with detoxification, such as heat shock protein, aldehyde dehydrogenase 2, and glutathione S-transferase M, suggests that an increased amount of protective proteins is required to adapt to CPF exposure. Meanwhile the down-regulation of ApoA protein may damage the energy metabolism under CPF stress. These findings provide new insight into the molecular mechanisms underlying CPF toxicity in developing zebrafish. However, the other proteins that showed differential expression and their functional roles yet remain to be identified and hence require further investigation.
Acknowledgments The work is supported by Hi-Tech Research and Development Program of China (No. 2008AA10Z402), the National Natural Science Foundation of China (NSFC, No. 31170119) and the Basic Research Fund of CAAS (No. 0042011006). We are indebted to Professor Bo Zhang, the School of Life Science of Peking University of China for materials and excellent technical assistance and maintenance of zebrafish. We are also thankful to Dr. Md Anisur Rahman and Dr. Kang Li for their help in the manuscript's preparation.
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Evaluation of estrogenic activities and mechanism of action of perfluorinated chemicals determined by vitellogenin induction in primary cultured tilapia hepatocytes. Aquat. Toxicol. 85, 267–277. Makino, S., Whitehead, G.G., Lien, C.L., Kim, S., Jhawar, P., Kono, A., Kawata, Y., Keating, M.T., 2005. Heat-shock protein 60 is required for blastema formation and maintenance during regeneration. Proc. Natl. Acad. Sci. U. S. A. 102, 14599–14604. Patrick, J. Babin, Thisse, Christine, Durliat, Michele, Andre, Michele, Akienko, MarieAndree, Thisse, Bernard, 1997. Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development. Proc. Natl. Acad. Sci. U. S. A. 94, 8622–8627. Patrick, H. Krone, Evans, Tyler G., Blechinger, Scott R., 2003. Heat shock gene expression and function during zebrafish embryogenesis. Semin. Cell Dev. Biol. 14, 267–274. Peng, G.S., Wang, M.F., Chen, C.Y., Luu, S.U., Chou, H.C., Li, T.K., Yin, S.J., 1999. Involvement of acetaldehyde for full protection against alcoholism by homozygosity of the variant allele of mitochondrial aldehyde dehydrogenase gene in Asians. Pharmacogenetics 9, 463–476. Raynal, P., Pollard, H.B., 1994. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium and phospholipid-binding proteins. Biochim. Biophys. Acta 1197, 63–93. Sanders, B.M., 1993. Stress proteins in aquatic organisms: an environmental perspective. Crit. Rev. Toxicol. 23, 49–75. Santos, C., Ballesta, J.P.G., 1994. Ribosomal protein P0, contrary to phosphoproteins P1 and P2, is required for ribosome activity and Saccharomyces cerevisiae viability. J. Biol. Chem. 269, 15689–15696. Shrader, E.A., Henry, T.R., Greeley Jr., M.S., Bradley, B.P., 2003. Proteomics in zebrafish exposed to endocrine disrupting chemicals. Ecotoxicology 12, 485–488. Tay, T.L., Lin, Q., Seow, T.K., Tan, K.H., Hew, C.L., Gong, Z., 2006. 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Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Analysis of differentially expressed proteins in zebrafish (Danio rerio) embryos exposed to chlorpyrifos Lili Liu a, Yongxue Xu a,b, Lili Xu a, Jian Wang a, Wei Wu a, Lei Xu a, Yanchun Yan a,⁎ a b
Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China Tianjin International Joint Academy of Biomedicine, Tianjin 300457, China
a r t i c l e
i n f o
Article history: Received 31 July 2014 Received in revised form 22 October 2014 Accepted 22 October 2014 Available online 28 October 2014 Keywords: 2D PAGE Chlorpyrifos Danio rerio Mechanism Proteomics Zebrafish embryos
a b s t r a c t In this study, the protein expression profiles of zebrafish embryos under chlorpyrifos (CPF) stress were investigated. Zebrafish embryos were exposed to 0.25 mg/L CPF, and embryo samples were collected until 24 h post-fertilization (hpf). To gain a better understanding of the response of zebrafish embryos to CPF exposure, two-dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with mass spectrometry was employed to carry out a comparative proteomic analysis. Total proteins were extracted from the control and treated samples, separated by 2D PAGE, and visualized by silver staining. A total of 59 protein spots showed reproducible changes compared with the control. Of these 59 spots, 19 were selected and subjected to matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 9 differentially expressed proteins were successfully identified, including 3 up-regulated proteins and 6 down-regulated proteins. The increased expression of 3 proteins associated with detoxification and stress response suggested that the activation of protective proteins was required in zebrafish embryos exposed to CPF. On the other hand, the decreased expression of 6 proteins is mainly involved in cytoskeleton structure, protein translation, signal transduction and lipoprotein metabolism. These data may help us understand the functions and the molecular mechanisms of these proteins in zebrafish embryos' response to CPF exposure. © 2014 Elsevier Inc. All rights reserved.
1. Introduction The insecticide chlorpyrifos (CPF) is a broad-spectrum organophosphate pesticide that has been one of the most widely used insecticides in the world. CPF elicits a number of additional effects, including hepatic dysfunction, hematological and immunological abnormalities, embryotoxicity, teratogenicity and neurotoxicity (Fatma et al., 2010). Of the established environmental neurotoxicants, organophosphate pesticides (OPs) such as chlorpyrifos are potent inhibitors of acetylcholinesterase (AChE), a key enzyme pathway responsible for terminating the transmission of many neuronal cell types across synapses (Yang et al., 2008). However, non-AChE-associated mechanisms of CPF neurotoxicity and behavioral impairments have also been described (Eaton et al., 2008). The zebrafish (Danio rerio) is a small tropical aquarium fish well known to home aquarium enthusiasts. Many of the features have made it become a model system for the examination of embryonic development of vertebrates. It has been observed that early life stages of fish are generally more sensitive to toxic compounds compared with adults and juveniles (Cook et al., 2005), so zebrafish embryos offer a good model to investigate the ecological risk assessments and help ⁎ Corresponding author. Tel.: +86 10 82109685; fax: +86 10 82106609. E-mail address: [email protected] (Y. Yan).
http://dx.doi.org/10.1016/j.cbpc.2014.10.006 1532-0456/© 2014 Elsevier Inc. All rights reserved.
determine the critical neurodevelopmental processes impacted by CPF, in which the mechanisms of neurodevelopment can be more closely analyzed. Global protein expression profile is an excellent approach to visualize the pattern and the level of the proteins expressed under defined environmental conditions. Therefore, proteomics analysis may provide more direct insights into the mechanisms of effects. The current status of proteomic approaches in understanding the toxicity and the mechanisms of exposure to toxicants has been reviewed (Gillardin et al., 2009). Methodologies for proteomic analysis of zebrafish have been developed (Tay et al., 2006). Proteomic techniques in zebrafish embryos have been utilized to observe changes in the proteome over developmental time and a previous study has investigated proteomic changes in zebrafish larval exposure to perfluorooctane sulfonate (Shi et al., 2009). In addition, previous reports on zebrafish proteomics revealed certain expressed proteins as the sensitive stress indicators in zebrafish embryos can reflect the overall fitness of the intact organisms (Shrader et al., 2003). To date, no studies concerning the potential effects of chlorpyrifos on the protein expression profile of developing zebrafish are available. During fish development, early embryonic stages are the most vulnerable stages to environmental stress, like changes in temperature or oxygen content of the water and also to the additional stress due to the increasing pollution (Cook et al., 2005). Thus, the aim of the present
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study is to understand the mechanisms underlying CPF toxicity during zebrafish development. 2D PAGE and peptide mass fingerprinting (PMF) were used to investigate the protein expression profiles of zebrafish embryos that had been exposed to CPF. Proteomic analysis indicated that CPF regulates a set of proteins that are involved in detoxification, cytoskeleton structure, protein translation, signal transduction, lipoprotein metabolism and other proteins. 2. Materials and methods 2.1. Chemicals Chlorpyrifos (CPF, purity N 99%) was purchased from the National Institute of Metrology. The stock solution (5 mg/L) was prepared by dissolving the crystals in HPLC-grade acetone and stored at 4 °C in darkness. All of the chemicals used in this study were of analytical grade, most of which were purchased from GE Healthcare. 2.2. Maintenance of zebrafish and treatments Zebrafish (Tübingen strain) was obtained from the School of Life Science of Peking University of China at the juvenile stage and was cultured in our laboratory. The culture of mature zebrafish (about seven months old) was maintained at 28 ± 0.5 °C with a constant artificial light/dark cycle of 14/10 h in a continuous flow-through system with charcoal filtered water (pH 7.00 ± 0.5 and conductivity 500 ± 50 μS). Fishes were fed with live brine shrimps (Artemia nauplii) twice daily, occasionally supplemented with commercially available artificial diets. Zebrafish embryos were obtained from the spawning adults, males and females in a ratio of 2/1 were placed in breeding chambers the day before embryos were needed. Spawning was induced on the morning when the light was turned on. Zebrafish eggs were collected within 30 min after natural mating, rinsed several times with Holt Buffer (NaCl 3.5 g/L, KCl 0.05 g/L, NaHCO3 0.025 g/L, CaCl2 0.1 g/L). At 2 h post fertilization (hpf), approximately 300 normally developed embryos were randomly distributed into each petri dish and exposed them to 0, and 0.25 mg/L CPF in an incubator set at 28.5 °C. From our pilot experiments we chose the dose of the exposure chemical that did not increase embryonic fatalities or malformations above control rates over the entire exposure time. CPF dilutions or vehicle were changed every 12 h with exposure ending on 24 hpf. At this period, the major organ systems, including the somites, the pronephros, the heart, and the central nervous system, form. The control and treated embryos received 0.01% acetone as a solubilizing agent to assist in stock solution preparation. After 24 hpf, zebrafish embryos were collected and kept at −80 °C until analysis. Three replicates for each treatment concentration were conducted. 2.3. Sample preparation and two-dimensional polyacrylamide gel electrophoresis At 24 hpf, prior to the generation of samples for two-dimensional gel electrophoresis the chorion was removed following the method of Westerfield (2000). The samples were prepared as described in Link et al. (2006). Briefly, approximately 250 deyolked zebrafish embryos were lysed in 150 μL 3% (w/v) SDS solution for 5 min at 90 °C. Then total protein was extracted with the method of methanol–chloroformprecipitation. Samples were lysed in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 2% (w/v) DTT, 0.5% (v/v) IPG buffer pH 4–7, 0.002% (w/v) bromophenol blue). Insoluble particles were removed by centrifugation for 1 h at 12,000 g. Protein concentration was determined using a 2D Quant Kit (GE Healthcare, Piscataway, NJ, USA). Total proteins were frozen in liquid nitrogen or processed directly for 2D gel electrophoresis. The protein solution (350 μg protein on every analytical gel or 1 mg on every preparative gel) was adjusted with a rehydration buffer for a final volume of 450 μL. Based on the procedures for 2D-PAGE described
by Gorg et al. (2004), conditions were optimized with a slight modification. Isoelectric focusing (IEF) was performed in IPG strips (pH 4–7, 24 cm, GE Healthcare) at 50 V for 6.5 h, 150 V for 1.5 h, 300 V for 1 h, 500 V for 4 h 1000 V for 1.5 h, and 10,000 V for 2 h (slowly and deliberately apply pressure to the strip for desalting) and then 10,000 V constant for a total of 85,000 Vh on a Multiphor II system (GE Healthcare). After isoelectric focusing, the IPG strips were equilibrated for 15 min in an equilibration buffer (6 M Urea, 2% (w/v) SDS, 30% (v/v) glycerol, 50 mM Tris–HCl pH 8.8 and a trace of bromophenol blue) containing 20 mg/mL DTT and then alkylated (25 mg/mL iodoacetamide instead of DTT in an equilibration buffer) for 15 min. The SDS-PAGE in the second dimension was carried out in polyacrylamide gels (12.5% T, 2.6% C) with an Ettan DALT II system (GE Healthcare, Piscataway, NJ, USA). Electrophoresis was performed at 1 w/gel for 60 min, followed by separation at 10 w/gel until the dye front had nearly reached the bottom. The protein spots were visualized via silver staining in analytical gels and via coomassie brilliant blue G-250 staining in preparative gels. Three replicates from three independent biological extracts were used for analysis.
2.4. Image acquisition and analysis The 2D gels were scanned with an image scanner (GE Healthcare, Piscataway, NJ 173, USA). The intensity of protein spots was detected and quantified with ImageMaster 2D Platinum (GE Healthcare, Piscataway, NJ, USA) software on the basis of their relative volume. After automated detection and matching, manual editing was carried out. To compensate for subtle differences in sample loading, gel staining and destaining, the intensity of each spot was normalized as a percentage of the total volume of all of the spots present in the gel.
2.5. In-gel digestion and protein identification In-gel digestion was performed according to Chambery et al. (2006). Selected protein spots were manually excised from the CBB-stained gel and destained by washing twice with 100 μL aliquots of water, performing a further washing step with 50% (v/v) acetonitrile. The gel pieces were then vacuum-dried and rehydrated with 10 μL of 50 mM ammonium bicarbonate, followed by the addition of 5 μL of a 70 ng/mL TPCK-treated porcine trypsin solution. Digestion was performed by incubation at 37 °C for 3 h. Further amount of buffer solution without trypsin was added when necessary to keep the gel pieces wet during the digestion. Peptides were extracted in two steps by sequential addition of 1% (v/v) trifluoroacetic acid (TFA) and then of 2% TFA/50% acetonitrile for 5 min in a sonication bath. The combined supernatants were concentrated in the SpeedVac Vacuum for mass spectrometry (MS) analysis. After in situ tryptic digestion, proteins were identified by PMF based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All spectra of proteins were submitted to database searching using the online MASCOT program (http://www. matrixscience.com), against NCBInr, Swiss-Prot, MSDB and EST databases. The classification and functions of the proteins identified were obtained by searching Gene Ontology (http://www.geneontology.org/).
2.6. Statistical analysis In this study, the data of the spot intensities were expressed as mean ± standard deviation (SD). Comparisons were performed by one-way ANOVA and the Least Significant Difference (LSD) test to determine significant differences among group means. A probability value of b0.05 was considered statistically significant. The statistical package SAS, version 9.1 (SAS/STAT Software for PC. SAS Institute Inc., Cary, NC, USA) was used for statistical analysis.
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3. Results and discussion In the present study, 2D PAGE was used to analyze the differentially expressed proteins of zebrafish embryos (24 hpf) in response to CPF stress. The high-resolution 2DE gel pattern in the pI range of 4–7 was detected by silver staining (Fig. 1). More than 1200 protein spots were reproducibly detected in each silver-stained gel (Fig. 1), and CPFinduced changes in protein abundance were found in the appropriate gels (Fig. 1A and B). The treated samples were compared with the controls, 59 differentially expressed distinct spots were found in the treated group. Identical gels were run and used for in-gel tryptic digestion, followed by mass spectrometry. A total of 19 protein spots were selected for protein identification based on spot intensity and spot integrity. These proteins were identified by means of MALDI-TOF-MS/MS. After a PMF search in the NCBI nr database, 9 spots were successfully identified (Fig. 1), of these, 3 protein spots (spots 1,2,6) showed an increase in abundance in the treated samples compared with the controls, while 6 spots (spots 3,4,5,7,8,9) decreased in abundance in the treated samples. Magnified views (Fig. 2A) and relative spot intensity (Fig. 2B) of the differential proteins are given in Fig. 2. Based on the putative functions of the identified proteins, they were further categorized including detoxification, cytoskeleton structure, protein translation, signal transduction, lipoprotein metabolism and other proteins (Table 1). There 4
pI
7
MW (kDa)
A
116.0 1 2
4
3
5 6
7
14.4
8
9
4
pI
7
MW (kDa)
B
116.0
1 2
3
4
5 6
7 14.4
8
9
Fig. 1. Representative 2D maps of 24 hpf zebrafish embryonic proteins exposed to CPF stress. (A) and (B) represent the 2D gel patterns of control and 0.25 mg/L of CPF treated zebrafish embryos, respectively. Total proteins were extracted and separated by 2D PAGE. A total of 350 μg proteins were loaded onto 24 cm pH 4–7 linear IPG strips. SDSPAGE was performed with 12.5% gels. The spots were visualized by silver staining. Arrows indicate the identified and differentially expressed proteins in response to CPF stress.
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was also a hypothetical protein identified with an unknown function in the database. The field of proteomics is expanding rapidly, which can provide greater volume and quality of protein information to help understand the molecular mechanisms underlying CPF-induced responses in the developing zebrafish embryos. Here, we observed 59 differentially abundant protein spots in response to CPF and 9 of these were successfully identified. Most of these proteins have important biological functions, providing a basis for understanding and annotating their potential roles in response to CPF stress. 3.1. CPF stress-induced up-regulated proteins Among the identified proteins, a total of 3 spots were up-regulated following exposure to CPF stress. These protein spots are 60 kDa heat shock protein (Hsp60), aldehyde dehydrogenase 2 (ALDH2) precursor, and glutathione S-transferase M. These proteins are mainly associated with detoxification and play an important role in cell protection and repair upon stress. Heat shock proteins (Hsps) are ubiquitous, highly conserved proteins that have been found in the cells of all organisms studied thus far, including plants, bacteria, yeast, flies, and vertebrates (Jaattela, 1999). Many previous studies have reported that when organisms are exposed to a variety of stress factors such as cold, heat, heavy metal, and various chemicals, they synthesize a set of Hsps, which usually acts as molecular chaperones, and play diverse roles in transporting, folding, assembling of degraded or misfolded proteins and maintaining normal cellular homeostasis. Particularly Hsp60 and Hsp70 have been strongly implicated in the immune response (Van Eden and Young, 1996). Information about Hsps' expression and function in zebrafish has increased substantially in the past decade, suggesting that zebrafish Hsps play critical roles during normal developmental (Patrick et al., 2003). Hsp60 has been also identified and cloned in the zebrafish, which is required for blastema formation and maintenance (Makino et al., 2005). Thus, the significantly elevated level of Hsp60 (spot 1) in response to CPF exposure is consistent with other researches, which found that the expression levels of various HSPs were increased in fish exposed to environmental contaminants, such as pesticides (Sanders, 1993), and suggests that it might play a vital role in cell protection and repair upon stress. Aldehyde dehydrogenases (ALDHs) form a superfamily of NAD(P)+dependent enzymes that catalyze the oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding carboxylic acids (Vasiliou et al., 1999). In fact, a diversity of metabolic functions has been reported: (i) detoxification, such as the removal of a plethora of aldehydes generated during drug and xenobiotic metabolism, (ii) participation in intermediary metabolism, such as amino acid and retinoic acid metabolism, (iii) protection from osmotic stress by generating osmoprotectants, such as glycine betaine, and (iv) generation of NAD(P)H (Fong et al., 2006). ALDH2 enzyme plays a major role in acetaldehyde oxidation in vivo that has been also reported (Peng et al., 1999). In the present study, a 2.21-fold increase in aldehyde dehydrogenase 2 precursor (spot 2) abundance was observed, which indicated a promising high-level of ALDH2 at 24 hpf zebrafish embryo. Thus, the up-regulation of ALDH2 precursor may be due to the formation of a metabolic product of CPF, in order to reduce oxidative damage. However, whether the increase of ALDH2 displays other functions remains to be elucidated. The expression of glutathione S-transferase M was significantly up-regulated in the zebrafish embryos exposed to CPF. Glutathione S-transferase M (spot 6) belongs to the superfamily of the glutathioneS-transferase (GST) enzymes that form a group of ubiquitous enzymes that catalyze the conjugation between glutathione and several molecules, and play the most important role in the cellular detoxification mechanism of xenobiotic and endogenous compounds (Agianian et al., 2003). The chemical exposure of insects is a classical event that selects
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A
C
1
T
2
1
4
4
7
C
2
C
8
T
3
3
6
6
5
5
7
T
8
9
9
B
Fig. 2. Magnified views. (A) shows the differentially expressed proteins in Fig. 1, and graphical presentation (B) of differentially expressed proteins identified in 24 hpf zebrafish embryos exposed to CPF stress. (C) Control. (T) Treatment with 0.25 mg/L of CPF. Arrows indicate the differential spots between the two treatments. For each treatment, relative abundance ratio of three replicates (±SE) is shown. Different letters indicate a statistically significant difference (p b 0.05) according to the LSD test.
pesticide resistance, and has been related with a high GST activity (Wei et al., 2001). It also has been suggested that the pesticide may conjugate to glutathione by GST and that the compound obtained may therefore act as a detoxification mechanism in arthropods (Wei et al., 2001). In addition, differential expression of this enzyme has also been observed in perfluorooctane sulfonate (PFOS) exposure tilapia (Liu et al., 2007). These results suggest that the increased activity of glutathione S-transferase M might be involved in the activation of the detoxification pathway to protect against cellular damage of zebrafish embryos' exposure to CPF.
3.2. CPF stress-induced down-regulated proteins in zebrafish embryos Among the identified proteins, a total of 6 spots showed decreased levels of expression after treatments with CPF (Table 1). These protein spots are Rplp0 protein, Type I cytokeratin, annexin 5, ApoA protein, lactoylglutathione lyase (LGL), and a hypothetical protein. Ribosomal protein P0 (RpLp0) (spot 3) is a highly conserved protein, which belongs to the L10P family of ribosomal proteins (Zeng et al., 2001). The ribosomal function of P0 is mediated through a pentameric P12·P0·P22 complex, which forms the stalk of the large ribosomal
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Table 1 CPF stress responsive differently expressed proteins in 24 hpf zebrafish embryos identified by MS analysis. Spot IDa
Acc.no.b
Protein namec
Fold change by CPF stressd
1↑
gi| 31044489 gi| 41053732 gi| 29124460 gi| 41388915 gi| 27762270 gi| 47086689 gi| 53733942 gi| 292614682 gi| 47085917
60 kDa heat shock protein, mitochondrial Aldehyde dehydrogenase 2 precursor Rplp0 protein
+2.81
2↑ 3↓ 4↓ 5↓ 6↑ 7↓ 8↓ 9↓ a b c d e f g h i
pIf
Match rateg
SC (%)h
Mowse scorei
Functional description
61.39
5.56
20/47
35%
129
+2.21
57.21
5.93
21/72
39%
141
−1.94
34.55
5.73
18/80
44%
136
Type I cytokeratin, enveloping layer Annexin 5
−2.00
46.53
5.13
18/47
39%
144
−3.51
35.21
5.46
15/50
47%
119
Glutathione S-transferase M
+2.69
26.45
5.58
19/45
48%
115
Molecular chaperones, stressinduced biomarker Oxidize a wide variety of aliphatic and aromatic, aldehyde detoxification Structural constituent of ribosome, translational elongation Intermediate filament, structural molecule activity Signal transduction, anti-apoptosis, transport Detoxification
ApoA protein
−2.81
19.74
5.31
29/70
87%
184
Regulate lipoprotein metabolism
Hypothetical protein LOC325536 Lactoylglutathione lyase
−2.83
345.80
4.78
26/49
10%
65
−2.67
20.40
5.23
18/58
50%
135
MW (kDa)e
Catalyzes the glyoxal pathway, detoxification
Numbering corresponds to the 2DE gel in Fig. 1. Proteins up- (↑) or down- (↓) regulated in response to CPF treatments. Acc. no.: accession number in NCBInr database. All the proteins identified relate to Danio rerio. Fold change: increased (+) or decreased (−) compared with the controls. MW: molecular weight. pI: isoelectric point. Match rate: the percentage of the number of mass values matched to the number of mass values searched. Sequence coverage: the percentage sequence coverage of the hit. Mascot score.
subunit at the GTPase center (Uchiumi and Kominami, 1992). RpLp0 interacts with the elongation factor eEF2 (Justice et al., 1999), and is essential for the ribosomal activity and cell viability in yeast (Santos and Ballesta, 1994). The functions of the RpLp0 are complex and intriguing, and multiple roles have been also reported in many cellular processes, including DNA repair, cell development, apoptosis and carcinogenesis (Alaa et al., 2007). However, to the best of our knowledge, this is the first study indicating an association between CPF and the down-regulation of Rplp0 protein, and a -3.00-fold reduced expression was observed, which strongly suggests that RpLp0 is associated with CPF-induced cytotoxicity. During apoptosis, one of the cellular responses is the regulation of protein translation, which is divided into three distinct phases: initiation, elongation and termination (Holcik and Sonenberg, 2005). Taken together, these results suggest that the decrease of Rplp0 protein in CPF exposure would affect protein translation. Therefore, it might be considered that CPF affects the growth of zebrafish embryos or results in apoptosis. Nevertheless, further investigation is needed to verify this association. This is the first report showing the down-regulation of Type I cytokeratin (spot 4) in zebrafish embryos exposed to CPF. Cytokeratins belong to the gene family of intermediate filaments (IFs), which are the major structural proteins in higher eukaryotic cells and are specifically expressed in epithelial cells of vertebrates (Yoon et al., 2001). Cytokeratins act as a scaffold allowing for the maintenance of cellular architecture, and providing resistance to mechanical and nonmechanical stresses. In addition, other non-structural functions such as cell signaling, protein targeting in polarized epithelia, cell proliferation, and in apoptosis have also been reported (Carlos et al., 2007). A previous study has been reported that type I cytokeratin is expressed in the enveloping layer at early gastrula in zebrafish embryos, and expression increases during development (Charles et al., 2005). Downregulation of type I cytokeratin protein would result in the suppression of proper filament formation and inhibition of growth. In addition, the cytoskeletal integrity might be affected, which reduced the cellular resistance to environmental stress. However, whether the downregulation of type I cytokeratin displays other functions, further studies are still needed.
Annexin 5 (spot 5) is a member of the annexin family of proteins characterized by calcium dependent phospholipid binding and structural similarity (Raynal and Pollard, 1994). It has been suggested that annexins may be implicated in several physiological processes, such as Ca2+ regulated exocytosis, endocytosis, mitogenic signal transduction, differentiation and coagulation (Gerke and Moss, 2002). In the present study, we observed that annexin 5 expression was down-regulated after exposure to 0.25 mg/L CPF. Annexin 5 has been extensively used as an indicator of early apoptosis due to its inherent property of selectively interacting with phosphatidyl serine. Therefore, the significant down-regulation of annexin 5 might be associated with CPF-mediated dysregulation of the cell membrane function, which may result in apoptosis. Apolipoproteins, the protein components of lipoproteins, are key elements with important roles in lipid transport and uptake in vertebrates (Kim et al., 2009). ApoA (spot 7), a member of apolipoprotein family, includes ApoAI, ApoA II and ApoA IV. A previous study has shown that the ApoAI gene is highly expressed during embryonic development (Patrick et al., 1997), which may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway and may protect against atherosclerosis (Kozarsky et al., 1997). In the present study the down-regulation of ApoA was observed, unfortunately, its precise class was not identified. In addition, since most fish utilize lipids as the major energy source (Kondo et al., 2005), lipid metabolism appears much important for homeostasis maintenance in fish. These results suggested that the decrease of ApoA under CPF exposure may affect the lipid metabolism or other transport pathway, disturbing the homeostasis maintenance in zebrafish embryos. However, further studies are needed to explore the actual mechanism of the ApoA induced by CPF. Lactoylglutathione lyase (LGL) (spot 9) is also known as glyoxalase I, which is an enzyme involved in the detoxification of methylglyoxal, a highly toxic electrophilic glycolytic by-product that reacts with and inactivates intracellular macromolecules, including both proteins and nucleic acids (Kalapos, 1999). Separate two-dimensional gel electrophoresis studies demonstrated that LGL of S. mutans was up-regulated in an acidic environment, suggesting that LGL may be linked to the
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acid tolerance (Wilkins et al., 2002). In addition, since LGL was found to be generally up-regulated in Alzheimer's disease brain (Feng et al., 2004) but down-regulated in old age healthy brains (Kuhla et al., 2006); it was thought that the glyoxalase system may limit the carbonyl-induced neurotoxicity and apoptosis (Kikuchi et al., 1999). To date, unfortunately, there has been no study that focuses on the function of LGL in zebrafish embryos. Taken together, the significant downregulation of LGL may result in the accumulation of methylglyoxal that could lead to the inhibition of DNA synthesis, or neurotoxicity and apoptosis. However, further investigation is needed to verify this hypothesis and to know the precise role of LGL and the relation with CPF stress in zebrafish embryos. The drastic down-regulation of spot 8 has also been noticed in zebrafish embryo following exposure to CPF. Then spot 8 was analyzed by MS and then the spectrum was analyzed via BLAST in zebrafish database of NCBI. Unfortunately, there was no matching information. Physiological and biochemical responses of zebrafish embryos in response to CPF stress have been analyzed extensively. However, to the best of our knowledge, the present study represents the first proteome map of zebrafish embryos under CPF stress. In addition, the solvent level of acetone we used was below 0.01% (v/v), which can't cause differential expression of protein (Hallare et al., 2006). In summary, our results show that a broad spectrum of proteins involved in zebrafish embryos' development is affected by CPF exposure. Finally, a total of 9 proteins that are associated with detoxification, cytoskeleton structure, protein translation, signal transduction and lipoprotein metabolism were successfully identified. Thus, profiling of the zebrafish embryo proteome following exposure to CPF enhanced our understanding of the CPF response of zebrafish. For instance, the increased activity of several proteins associated with detoxification, such as heat shock protein, aldehyde dehydrogenase 2, and glutathione S-transferase M, suggests that an increased amount of protective proteins is required to adapt to CPF exposure. Meanwhile the down-regulation of ApoA protein may damage the energy metabolism under CPF stress. These findings provide new insight into the molecular mechanisms underlying CPF toxicity in developing zebrafish. However, the other proteins that showed differential expression and their functional roles yet remain to be identified and hence require further investigation.
Acknowledgments The work is supported by Hi-Tech Research and Development Program of China (No. 2008AA10Z402), the National Natural Science Foundation of China (NSFC, No. 31170119) and the Basic Research Fund of CAAS (No. 0042011006). We are indebted to Professor Bo Zhang, the School of Life Science of Peking University of China for materials and excellent technical assistance and maintenance of zebrafish. We are also thankful to Dr. Md Anisur Rahman and Dr. Kang Li for their help in the manuscript's preparation.
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