Ecotoxicology and Environmental Safety 108 (2014) 335–339
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Proteomics study of silver nanoparticles toxicity on Oryza sativa L. Fateme Mirzajani a,b,n, Hossein Askari a,n, Sara Hamzelou a, Yvonne Schober d, Andreas Römpp d, Alireza Ghassempour c,nn, Bernhard Spengler d a
Department of Biotechnology, The Faculty of Renewable Energies & New Technologies Engineering (NTE), Shahid Beheshti University, G.C. Evin, Tehran, Iran Department of Nanobiotechnology, Protein Research Institute, Shahid Beheshti University, G.C. Evin, Tehran, Iran c Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C. Evin, Tehran, Iran d Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Schubertstrasse 60, 35392 Giessen, Germany b
art ic l e i nf o
a b s t r a c t
Article history: Received 10 May 2014 Received in revised form 9 July 2014 Accepted 11 July 2014
The increasing use of silver nanoparticles, (AgNPs), will inevitably result in their release into the environment and thereby cause the exposure to plants. It was claimed that using AgNPs is a safe and efficient method to preserve and treat agents of disease in agriculture. This study tries to understand the protein populations and sub-populations and follow up environmental AgNPs stresses. To accomplish these, the action of homemade spherical AgNPs colloidal suspension against Oryza sativa L. was investigated by a proteomic approach (2-DE and NanoLC/FT-ICR MS identification). Twenty-eight responsive (decrement/increment in abundance) proteins were identified. Proteomic results revealed that an exposure of O. sativa L., root with different concentrations of AgNPs resulted in an accumulation of protein precursors, indicative of the dissipation of a proton motive force. The identified proteins are involved in oxidative stress tolerance, Ca2 þ regulation and signaling, transcription and protein degradation, cell wall and DNA/RNA/protein direct damage, cell division and apoptosis. The expression pattern of these proteins and their possible involvement in the nontoxicity mechanisms were discussed. & 2014 Elsevier Inc. All rights reserved.
Keywords: Oryza sativa L. Metal detoxification NanoLC/FT-ICR Oxidative stress Silver nanoparticle Two dimensional electrophoresis
1. Introduction The antimicrobial properties of silver nanoparticles (AgNPs) are being increasingly utilized in consumer products. It has been widely used for the development of many biological and pharmaceutical processes, products, and applications (Chen and Schluesener, 2008; Vidhu and Philip, 2014). In the field of agriculture there are wide varieties of AgNPs as antibacterial agents with different physical properties and with little concern on their side effects (Rahman Nia, 2009). Moreover, there is a risk of enhanced bioavailability of the nanoparticles in the different media (Shrivastava et al., 2007; Asharani et al., 2008) which may have an impact on the beneficial soil flora, plants, animals and humans. Pollution caused by AgNPs, particularly contamination of soil and water resources, has been accelerated as a result of global industrialization and is considered a major risk for communities throughout the world (Navarro et al., 2008a).
n Corresponding authors at: Department of Biotechnology, The Faculty of Renewable Energies & New Technologies Engineering (NTE), Shahid Beheshti University, G.C. Evin, Tehran, Iran. Fax: þ 98 2129903244. nn Corresponding author. Fax: þ 98 2122431783. E-mail addresses: [email protected] (F. Mirzajani), [email protected] (A. Ghassempour).
http://dx.doi.org/10.1016/j.ecoenv.2014.07.013 0147-6513/& 2014 Elsevier Inc. All rights reserved.
In addition to soil ecosystems, plants have been directly exposed to toxic compounds, among which are AgNPs (Krishnaraj et al., 2012; Gubbins et al., 2011). Some of the plants have an ability to adapt to a nano-polluted environment to employ various unique mechanisms, but mostly are vulnerable (Krishnaraj et al., 2012; Pokhrel and Dubey, 2013). It should be noted that the resistance, tolerance or even susceptibility of various plants to nano-pollutions not only depends on their features but also the characteristics of nano-materials and the environmental conditions (Pokhrel and Dubey, 2013; Liu et al., 2014). Navarro et al., have examined the toxicity of ionic silver and silver nanoparticles to photosynthesis in Chlamydomonas reinhardtii using fluorometry. They found that the cysteine abolished the inhibitory effects on photosynthesis and these particles not only show the toxicity on plants but also on algae (Navarro et al., 2008b). Stress response mechanism of plant enables them to survive adverse and mutable conditions in their immediate environments. Various mechanisms have been suggested to responses following the different environmental fluctuations. The stress response in bacteria involves a number of systems that act against an external stimulus. A complex network of global regulatory systems in bacteria certifies that various stress response systems interact with each other and this leads to a coordinated and effective response (Mirzajani et al., 2011, 2013, 2014). Proteomics is the fastest developing field of research and it contributes substantially to our understanding of organisms at the
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cellular level (Pokhrel and Dubey, 2013; Mirzajani et al., 2014; Lok et al., 2006). In addition, this is the study of functions and regulation of biological systems based on analysis of the protein expression profile. Furthermore, this study is the continued compliance with our publication on soil bacterial proteomics. In the case of nanotechnology environment proteomics there is another publication by Li et al. (2011), which used the gel-based proteomics together with other biochemical studies to evaluate the AgNPs action on Staphylococcus aureus. In another research by Vannini et al., the proteomic responses of Eruca sativa exposed to AgNPs and Ag ions were studied. They found that both forms of silver have a significant influence on seedlings and root formation. In the case of rice the same morphological phenomena were reported (Mirzajani et al., 2013). The aim of this study was to evaluate the action of homemade spherical colloidal AgNPs suspension against Oryza sativa L. from proteomic point of view using gel-base combined NanoLC/FT-ICR MSMS method.
were performed at 4 1C. The protein spots in the analytical and preparative gels were visualized by silver nitrate and colloidal CBB G-250, respectively. 2.4. Image and data analysis The analytical gels were immediately scanned using GS-800 calibrated densitometer (BioRad Co., CA, USA) at 600 dpi resolution. The spots of triplicate gels (experimental replicates) of control and experimental groups were detected and matched using Melanie 3 software (GeneBio Co., Switzerland). The molecular mass (kDa) and isoelectric point (pI) of the spots were calculated by standard protein markers (Amersham Pharmacia Biotech) and interpolation of missing values on IPGs, respectively. Quantitative comparison of protein spots was based on their percent volumes. The one-way analysis of variance (ANOVA) and comparison of treatment means were carried out by SAS programs. Only those statistically significant spots (P r0.05) were accepted and they had to be consistently present in all replications. There were four biological replicates and six technical replicates used in the experiments. The accepted spots were filtered based on the average expression level of two-fold or consistent and significant changes in at least three nanoparticle levels. 2.5. Protein digestion and peptide extraction
2. Material and methods 2.1. Silver nanoparticles (AgNPs): synthesis and characterization The homemade silver nanoparticle colloidal suspension (AgNPs) was prepared and characterized according to the previously reported method (Mirzajani et al., 2011). Briefly, colloidal AgNPs with spherical morphology and distinct diffraction peaks of crystalline planes of cubic system, including (111), (200), (220) and (311), have a maximum absorbance (λmax) at 426 nm. Their size was 18.34 nm (X99) with the homogeneity of their size within the range of 0.1–1000 nm.
The spots, showing comparable differences, were cut out manually from a preparatory gel. The spots were washed with water and destained using 10 mM ammonium hydrogen carbonate in 50 percent (v/v) MeCN. They were fully dehydrated in 100 percent (v/v) MeCN and dried using a SpeedVac system (Thermo Fisher Scientific Co., MA, USA) at 20 1C, 14,000g for 15 min. Subsequently, the dried gel pieces were rehydrated with 1–4 mL trypsin (Promega, Madison, WI, USA) solution for 30 min (0.1 mg/mL in 30 mM NH4HCO3 buffer); tryptic digestion was carried out at 37 1C for 20 h. The peptides were extracted from the gel pieces in three steps using MeCN containing five percent formic acid under sonication for 10–15 min. The OMIX C18 10 mL tip (Varian, Inc., Palo Alto, CA, USA) was used to clean up and to pre-concentrate the extracted solution.
2.2. Plant cultivation, treatment and protein extraction 2.6. Mass spectrometry measurement The plant cultivation is based on growth screening results of our previous reports on O. sativa L. (Mirzajani et al., 2013). Seeds of O. sativa L. cv. IR651 were gifted from the Rice and Citrus Research Institute (RCRI), Agricultural Science and Natural Resources University, Sari, Iran. The seedlings were prepared on petri dishes (ID: 7 cm), filled with 10 mL of sterile water, at 27 1C and were kept in dark for seven days. Plant seedlings (ten per bottle/2 L in whole volume) were transferred into rice specific growth cultivation media (Yoshida et al., 1976). They were grown in a phytotron maintained at a thermo-period of 27 1C7 0.5, photoperiod of 16 h, relative humidity of 60 percent, and a photon flux density of 20 mM/ m2s. The water content and the pH were adjusted to 200 mL and 5.0 7 0.1 daily respectively. Every five days of treatment the hydroponic media were renewed. Plants at the age of ten days were irrigated by AgNPs colloidal solution in the concentration of 0 (blank condition), 30 and 60 mg/mL in ten replications. The treatments were continued for twenty days more. Thereafter the plants were washed with water and were kept at 80 1C before analysis. Total protein was isolated from cells by TRIzol reagent and available guidelines (Molecular Research Center, Inc., Cincinnati, OH, USA). Briefly, the plant tissues were ground using mortar in liquid nitrogen and homogenized by TRIzol reagent. 0.1 g was then incubated with chloroform and centrifuged for 15 min at 12,000g at 4 1C. It was mixed with absolute ethanol for 15 s, incubated for 3 min and finally centrifuged at 2000g for 5 min at 4 1C. The proteins in the phenol/ethanol supernatant were precipitated by the addition of absolute acetone and dispersed in the washing agent (0.3 M guanidine hydrochloride in 95 percent ethanol and 2.5 percent glycerol). The latter was repeated three times and followed by washing in ethanol containing 2.5 percent glycerol. The precipitated protein was dissolved in lysis buffer (consisted of 9 M Urea, four percent CHAPS, one percent DTT, one percent pH 3–10 ampholytes, 35 mM Tris base) and stored at 80 1C. Protein concentration was determined according to the Bradford assay kit (BioRad Co., Hercules, CA, USA) in the comparison with BSA as the standard. 2.3. 2-Dimensional gel electrophoresis (2-DE) 2-DE was carried out based on previous report (National Committee of Clinical Laboratory Standards, 2005). IPG strips, 18 cm, pH 4–7, linear (BioRad Co., CA, USA) were loaded with the proteins during the rehydration process for 16 h at room temperature in reswelling tray (Amersham Pharmacia Biotech, Sweden). For analytical and preparative gels, 100 mg and 1 mg of proteins were loaded, respectively. The IPG strips were covered with silicon oil and all IEF separations were performed horizontally at an optimized temperature of 20 1C using a Multiphore II system (Amersham Pharmacia Biotech) for a total of 70,000 Vh. Second-dimension SDS-PAGE separations were performed on a gel (245 180 0.5 mm3; 12.5 percent polyacrylamide) using a PROTEAN II Multi Cell (BioRad Co., CA, USA). Separations
Measurements of the peptides were accomplished on an Ultimate binary nanohigh-performance liquid chromatography (nano-HPLC pump/autosampler) system for HPLC analysis (LCPackings/Dionex Co., Idstein, Germany). Volumes of 5 mL of the sample were pre-focused on a trap column (Dionex Co., C18 PepMap, i.d. 300 mm, length 5 mm) and separated on a fused-silica C18 PepMap100 capillary column (Dionex Co., 3 mm, 100 Å; i.d. 75 mm; length 150 mm). The flow rate was 0.2 mL/min. Solvent A consists of water containing two percent MeCN (v/v) and 0.1 percent formic acid (FA) (v/v). Solvent B was MeCN containing twenty percent water (v/v) and 0.08 percent FA (v/v). Separation was performed as follows: first B was increased from 0 percent to 25 percent in 5 min, increased to 50 percent in 25 min, further increased to 100 percent in 4 min and maintained for 20 min to elute the strongly hydrophobic peptides and to clean the column. The gradient was then ramped down in 1 min to 0 percent solvent B for equilibration for 25 min. The nano-HPLC system was coupled to a nanoelectrospray interface of an LTQ FTICR mass spectrometer (Thermo Scientific GmbH, Bremen, Germany). Mass spectra were acquired on a “Finnigan LTQ FT Ultra” hybrid instrument consisting of a linear quadrupole ion trap and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a 6-T magnet. A nanospray ionization source was used for analysis (capillary temperature 250 1C, capillary voltage 35 V, tube lens 125 V). Nanoelectrospray needles for online measurements, Silica Tips (Tip I.D. 10 μm, New Objective, Woburn, MA, USA) ,were used at a spray voltage of 1.9 kV. Mass resolving power was set to 100,000 at m/z¼ 400, allowing us to record one spectrum per second and thus several spectra per chromatographic peak. Mass accuracy after external calibration was better than 7 1.0 ppm during measurements. The peaks in the survey scan were chosen for fragmentation. The isolation window for the precursor ion was 7 2 u. Instrument specific parameter settings for collisioninduced dissociation (CID) in MS/MS experiments were as follows: activation energy, 30 (normalized; manufacturer-specific units); activation duration, 30 ms; activation Q, 0.25 (manufacturer-specific units); wide scan range, three microscans per spectrum. 2.7. Database search NanoLC/ESI-MS/MS data were searched against the O. sativa L., database using Proteome Discoverer 1.1 (Thermo Fisher Scientific, Bremen, Germany) based on the SEQUEST search algorithm. The mass tolerance for precursor ions was set for 2 ppm, and the mass tolerance for product ions was set to 0.8 u. Two missed cleavages were allowed in order to account for incomplete digestion. Methionine oxidation was allowed as variable modification. Peptides with a “peptide probability” (SEQUEST parameter) of 35 and higher were considered as significant identifications. The false discovery rates, as determined by a reversed database
F. Mirzajani et al. / Ecotoxicology and Environmental Safety 108 (2014) 335–339 search, in all cases were 1.5 percent. Peptide sequences were subjected to BLAST using blast at the National Centre for Biotechnology Information (NCBI: http:// www.ncbi.nlm.nih.gov/BLAST/) and MS BLAST at the European Molecular Biology Laboratory [EMBL: http://dove.embl-heidelberg.de/Blast2/msblast.html].
3. Results 3.1. 2-DE gel electrophoresis and comparison of protein expression optimization From the previous screening of the AgNPs influences on the rice root, it found that the treatment under the 30 mg/mL of AgNPs has a significant influence on root; decrements in growth and increments in branching (Mirzajani et al., 2013). In addition this concentration widely influenced the metabolites production, increments in carotenoids and decrements in total carbohydrate contents. The AgNPs treatment in the concentration of 60 mg/mL has similar but more severe effect. In conclusion of screening results 0 (blank condition), 30 and 60 mg/mL were chosen for the 2-DE proteomics study within a month (Mirzajani et al., 2013). 2-DE pages were then analyzed by silver staining. It was previously reported that 18 cm IPG strips have the capability to load r70– 100 mg/gel of proteins. As a result the 2D-page of 100 mg/gel of proteins (0 of AgNPs mg/mL treatment) was prepared to optimize the quality of the gel. Based on results, it was concluded that for each replication of treatments, 100 mg/gel was prepared (Fig. 1). 2D images were analyzed and volume of spots was estimated and compared across gels. As shown in Fig. 1 the quality of extracted proteins and following gels was highly reduced after the AgNPs treatment. So that, while the blank gels have more than 950 recognizable spots, 230 spots were monitored in the treated gel. The gel quality in the treated sample was not as good as the blank sample (Fig. 1). As can be seen in this figure and what is found in our previous study (Mirzajani et al., 2013), the carotenoids and pigments content was increased following the AgNPs treatment. For the increased gel quality the phenolic compounds cleanup method, introduced by Chatterjee et al., was examined. This method was introduced for phenolic rich roots of chickpea (Cicer arietinum L.) using phenol–SDS buffer extraction with sonication (Chatterjee et al., 2012). The method increased the gel quality in the case of some spots, while some spots cannot be detected and the others had tailing and low resolution. In the case of total protein a long term sonication process can damage proteins. Finally, 170 protein spots were compared based on their density and abundance, and 28 of them showed significant differences in response to AgNPs treatment (Fig. 2). If the density was 0.1 and it at most reaches 0.2, it is classified as an average expression level and if it reaches at least 0.2 it is classified as a significant change in expression level, and both are considered as
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responsive proteins in comparison to control. Protein spots with an average expression level of two-fold or significant changes in at least three AgNPs levels in comparison to the control were considered as responsive proteins (Fig. 2; Table S1, Supplementary material). Comparison of different expressions among AgNPs levels revealed that increase in treatment concentration significantly influences on the abundance of spots (Table S1, Supplementary material). All of the AgNPs-responsive proteins changed in terms of abundance rather than in-gel position and presence or absence. The majority of AgNPsinduced changes was associated with decreased abundance and was observed in the concentration of 60 mg/mL of AgNPs, with eighteen and five proteins increment and decrement in abundance, respectively (Table S1, Supplementary material). In addition some complex patterns were observed in the case of five spots including spot nos. 5, 16, 18, 20, 23 and 24, (Table S1, Supplementary material). 3.2. MS-based protein identification To identify proteins response to AgNPs, the best matching gene product and accession number, molecular weight (MW kDa) and isoelectric point (pI), the number of matching peptides and the score and coverage of peptides with databases were considered. The abundance pattern of each protein in AgNPs concentrations of 0, 30 and 60 mg/mL can be observed in Table S1, Supplementary material. All matching proteins belonged to the O. sativa but there were also some different strains (not included in the table). Responsive proteins (Fig. 2) were analyzed using NanoLC/FTICR after excision from CBB-stained gels and in gel digestion with trypsin. Proteins were identified according to the results of mass spectrometry, their partial sequences and MS–MS in comparison with the rice, Oryza sativa L., database (Table S1, Supplementary material). As can be seen in Table S1, Supplementary material, the proteins were identified based on the two to seventeen peptide sequences of a hydrolyzed protein (the results of identified peptides are mentioned in Supplementary materials). In the case of each protein the constituent digested peptides were identified and compared to previously published results. More than 197 peptides, corresponding to 28 proteins, were identified. The b and y fragments as well as neutral fragments were monitored to identify each peptide (Table S1, Supplementary material). These proteins include several significant responsive proteins. A few proteins with unknown functions or without any match in the database were also observed.
4. Discussions According to the identified proteins different mechanisms involved following the AgNPs treatment were hypothesized. An oxidative response pathway which was represented by superoxide
Fig. 1. Physiological impacts on O. sativa L. exposed to different concentrations of AgNPs (0, 30 and 60 mg/mL). (a) 100 mg, silver nitrate staining, 2D-page analysis of O. sativa L. proteome regarding the treated plant at different concentrations: blank, 0 mg/mL (b); 30 mg/mL (c); and (d) 60 mg/mL.
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Fig. 2. 100 mg of 2D-page protein analysis and AgNPs action on the protein regulation of O. sativa L., and some spots under the AgNPs treatments are marked. (1) Silver nanoparticle toxicity on O. sativa L., (2) expression pattern of proteins in the AgNPs treated O. sativa L., and (3) the mechanism of O. sativa L. toxicity from proteomics point of view.
dismutase –[14], L-ascorbate peroxidase –[9] and Glutathione-sTransferase –[12] is one of them. Some proteins are involved in defense signaling pathways. Among them can be cited the NACtranscription factor – [13], which is one of the mitochondrial retrograde regulation (MRR). Mitochondria signals to the nucleus to steer the expression of responsive genes. Reactive oxygen species and calcium are signaling molecules for MRR. Some calcium-binding messenger proteins (CaM) include calmodulins 1 and 3, 26 and 27. They can transduce calcium signals by binding calcium ions and then modifying its interactions with various target proteins. They also mediate many crucial processes such as inflammation, metabolism, apoptosis and intracellular movement. They are expressed in many cell types and can have different subcellular locations, including the cytoplasm, within organelles, or associated with the plasma or organelle membranes. Furthermore, there are some proteins involved in protein synthesis/degradation processes which were also identified including proteasome subunit α 10 and subunit β 11. They degrade unneeded or damaged proteins by the proteolysis process. Translationally controlled protein (TCP) [21], a protein regulator has roles in cellular processes, most notably in the cell cycle, apoptosis, cell proliferation, growth, stress response, gene regulation and heat shock. TCP [21] is the major regulator of cell growth in animals and fungi. Berkowitz et al. demonstrated that plant TCPs exhibit distinct sequence differences from nonplant homologs but share the key GTPase binding surface. They mentioned that silencing of TCP in Arabidopsis thaliana resulted in slow vegetative growth, leaf expansion is reduced because of smaller cell size, lateral root formation is reduced, and root hair development is impaired. Furthermore, these lines show decreased sensitivity to an exogenously applied auxin analog and have elevated levels of endogenous auxin. These results identify TCP as an important regulator of growth in plants and imply a function of plant TCP similar to that known in nonplant systems. 60S acidic ribosomal
protein –[3] is a phospho-ribosomal protein that contributes to the structural integrity of the ribosome. It also involved in a wide range of cellular processes like the signal recognition which is responsible for targetting proteins to the endoplasmic reticulum and a complex involved in termination of transcription. The detailed mechanism and environmental toxic aspects of AgNPs are not completely understood. There are some hypotheses like cell wall damage, oxidative stress following the ROS production and DNA/protein direct damage (Li et al., 2010). Based on our previous study on S. aureus and Bacillus thuringiensis, it was found that organisms have the ability to adopt and survive at low concentrations of AgNPs (Mirzajani et al., 2011, 2013). It has been previously reported that the ROS production and its role in toxicity and cell death have been studied previously in many researches (Rai et al., 2012). One of the best ways to understand the mechanisms of AgNPs actions is to study the protein expression/ regulation. These phenomena control reactions such as oxidative stress, cell wall damage and DNA/RNA/protein direct interaction following the evidences to confirm or reject the hypothesized mechanisms. AgNPs stress can accelerate the production and detoxification of ROS determining factor of oxidative stress and cell damage. Cellular detoxification enzymes, the expression modification proteins, are produced in response to ROS toxicities. As mentioned in the results an increased abundance of proteins was identified, including superoxide dismutase –[14], L-ascorbate peroxidase –[9] and Glutathione-s-Transferase –[12] which are involved in the detoxification or oxidative stress pathways. The increments in the expression of calcium protein includes calmodulins 1 and 3, [26] and –[27], NAC-transcription factor 13 and translationally controlled protein –[21] implied an occurrence of this toxicity following Ca2 þ regulation. Cheung (1980) suggested that metals effectively substitute for Ca2 þ in calmodulin. Evidence indicates that calmodulin serves as a major intracellular Ca2 þ receptor
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regulating the activity or rate of many key enzymes and cellular processes. Some heavy metals are highly toxic to the cell. It can be hypothesized that metals, here AgNPs or the released ions, interfere with the regulation of cell metabolism by binding to secondmessenger calcium receptors, calcium channels and calcium– sodium ATP pumps (Goyer, 1995). Activation of calmodulin by these metals could upset its normal regulation by the cellular flux of Ca2 þ . Perhaps this could constitute in part a basis for the toxicity of these metals in cell physiology. So the amount of cellular Ca2 þ will increase. It can be hypothesized that with the increase of cellular Ca2 þ following the ROS production, the NACtranscription factor and signaling molecules for MRR increase in abundance. Translationally controlled protein –[21] in addition to Glutathione-s-Transferase –[12] are proteins involved in the apoptosis process which increase in abundance. 5. Conclusion To our knowledge, this is the first report on proteome analysis of O. sativa L. and its response to AgNPs treatment. There are some other reports on the proteomics study in different plants like E. sativa. They reported that Ag in ionic/nanoparticle form causes changes in proteins involved in the redox regulation and in the sulfur metabolism. They claim that the effects of AgNPs are not solely due to the release of Ag þ into the surrounding environment, while the others strongly express that one of the mechanisms of AgNPs toxicity is silver ion release (He et al., 2011; Levard et al., 2012; Yang et al., 2012; Vannini et al. (2013)). So, the mechanism may result in parallel action of the silver nanoform and released ion. As previously mentioned proteins are one of the involved compounds in this mechanism over the direct regulations. Responsive protein changed in terms of its abundance rather than in-gel position or its presence or absence. In conclusion, AgNPs interact with normal cell metabolic processes such as protein synthesis/degradation and apoptosis. Subsequently, it can be hypothesized that the AgNPs enter into the cell and condense the DNA/protein thus inhibiting the normal cell reproduction. This series of studies on the impact of AgNPs suggests that, AgNPs not only have a direct or regulatory impact on the microorganism's proteome (Li et al., 2010; Mirzajani et al., 2013), but also on the plant cells. Furthermore, the increment of detoxification enzymes implies a toxic impact from the production of ROSs and metal toxicity in the presence of AgNPs. Acknowledgments Financial support from the Research Council of Shahid Beheshti University and the Iran Nanotechnology Initiative Council is gratefully acknowledged. This publication represents a component of the doctoral thesis of Fateme Mirzajani at the Medicinal Plants and Drug Research Institute of Shahid Beheshti University, Tehran, Iran. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.07.013.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Proteomics study of silver nanoparticles toxicity on Oryza sativa L. Fateme Mirzajani a,b,n, Hossein Askari a,n, Sara Hamzelou a, Yvonne Schober d, Andreas Römpp d, Alireza Ghassempour c,nn, Bernhard Spengler d a
Department of Biotechnology, The Faculty of Renewable Energies & New Technologies Engineering (NTE), Shahid Beheshti University, G.C. Evin, Tehran, Iran Department of Nanobiotechnology, Protein Research Institute, Shahid Beheshti University, G.C. Evin, Tehran, Iran c Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C. Evin, Tehran, Iran d Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Schubertstrasse 60, 35392 Giessen, Germany b
art ic l e i nf o
a b s t r a c t
Article history: Received 10 May 2014 Received in revised form 9 July 2014 Accepted 11 July 2014
The increasing use of silver nanoparticles, (AgNPs), will inevitably result in their release into the environment and thereby cause the exposure to plants. It was claimed that using AgNPs is a safe and efficient method to preserve and treat agents of disease in agriculture. This study tries to understand the protein populations and sub-populations and follow up environmental AgNPs stresses. To accomplish these, the action of homemade spherical AgNPs colloidal suspension against Oryza sativa L. was investigated by a proteomic approach (2-DE and NanoLC/FT-ICR MS identification). Twenty-eight responsive (decrement/increment in abundance) proteins were identified. Proteomic results revealed that an exposure of O. sativa L., root with different concentrations of AgNPs resulted in an accumulation of protein precursors, indicative of the dissipation of a proton motive force. The identified proteins are involved in oxidative stress tolerance, Ca2 þ regulation and signaling, transcription and protein degradation, cell wall and DNA/RNA/protein direct damage, cell division and apoptosis. The expression pattern of these proteins and their possible involvement in the nontoxicity mechanisms were discussed. & 2014 Elsevier Inc. All rights reserved.
Keywords: Oryza sativa L. Metal detoxification NanoLC/FT-ICR Oxidative stress Silver nanoparticle Two dimensional electrophoresis
1. Introduction The antimicrobial properties of silver nanoparticles (AgNPs) are being increasingly utilized in consumer products. It has been widely used for the development of many biological and pharmaceutical processes, products, and applications (Chen and Schluesener, 2008; Vidhu and Philip, 2014). In the field of agriculture there are wide varieties of AgNPs as antibacterial agents with different physical properties and with little concern on their side effects (Rahman Nia, 2009). Moreover, there is a risk of enhanced bioavailability of the nanoparticles in the different media (Shrivastava et al., 2007; Asharani et al., 2008) which may have an impact on the beneficial soil flora, plants, animals and humans. Pollution caused by AgNPs, particularly contamination of soil and water resources, has been accelerated as a result of global industrialization and is considered a major risk for communities throughout the world (Navarro et al., 2008a).
n Corresponding authors at: Department of Biotechnology, The Faculty of Renewable Energies & New Technologies Engineering (NTE), Shahid Beheshti University, G.C. Evin, Tehran, Iran. Fax: þ 98 2129903244. nn Corresponding author. Fax: þ 98 2122431783. E-mail addresses: [email protected] (F. Mirzajani), [email protected] (A. Ghassempour).
http://dx.doi.org/10.1016/j.ecoenv.2014.07.013 0147-6513/& 2014 Elsevier Inc. All rights reserved.
In addition to soil ecosystems, plants have been directly exposed to toxic compounds, among which are AgNPs (Krishnaraj et al., 2012; Gubbins et al., 2011). Some of the plants have an ability to adapt to a nano-polluted environment to employ various unique mechanisms, but mostly are vulnerable (Krishnaraj et al., 2012; Pokhrel and Dubey, 2013). It should be noted that the resistance, tolerance or even susceptibility of various plants to nano-pollutions not only depends on their features but also the characteristics of nano-materials and the environmental conditions (Pokhrel and Dubey, 2013; Liu et al., 2014). Navarro et al., have examined the toxicity of ionic silver and silver nanoparticles to photosynthesis in Chlamydomonas reinhardtii using fluorometry. They found that the cysteine abolished the inhibitory effects on photosynthesis and these particles not only show the toxicity on plants but also on algae (Navarro et al., 2008b). Stress response mechanism of plant enables them to survive adverse and mutable conditions in their immediate environments. Various mechanisms have been suggested to responses following the different environmental fluctuations. The stress response in bacteria involves a number of systems that act against an external stimulus. A complex network of global regulatory systems in bacteria certifies that various stress response systems interact with each other and this leads to a coordinated and effective response (Mirzajani et al., 2011, 2013, 2014). Proteomics is the fastest developing field of research and it contributes substantially to our understanding of organisms at the
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cellular level (Pokhrel and Dubey, 2013; Mirzajani et al., 2014; Lok et al., 2006). In addition, this is the study of functions and regulation of biological systems based on analysis of the protein expression profile. Furthermore, this study is the continued compliance with our publication on soil bacterial proteomics. In the case of nanotechnology environment proteomics there is another publication by Li et al. (2011), which used the gel-based proteomics together with other biochemical studies to evaluate the AgNPs action on Staphylococcus aureus. In another research by Vannini et al., the proteomic responses of Eruca sativa exposed to AgNPs and Ag ions were studied. They found that both forms of silver have a significant influence on seedlings and root formation. In the case of rice the same morphological phenomena were reported (Mirzajani et al., 2013). The aim of this study was to evaluate the action of homemade spherical colloidal AgNPs suspension against Oryza sativa L. from proteomic point of view using gel-base combined NanoLC/FT-ICR MSMS method.
were performed at 4 1C. The protein spots in the analytical and preparative gels were visualized by silver nitrate and colloidal CBB G-250, respectively. 2.4. Image and data analysis The analytical gels were immediately scanned using GS-800 calibrated densitometer (BioRad Co., CA, USA) at 600 dpi resolution. The spots of triplicate gels (experimental replicates) of control and experimental groups were detected and matched using Melanie 3 software (GeneBio Co., Switzerland). The molecular mass (kDa) and isoelectric point (pI) of the spots were calculated by standard protein markers (Amersham Pharmacia Biotech) and interpolation of missing values on IPGs, respectively. Quantitative comparison of protein spots was based on their percent volumes. The one-way analysis of variance (ANOVA) and comparison of treatment means were carried out by SAS programs. Only those statistically significant spots (P r0.05) were accepted and they had to be consistently present in all replications. There were four biological replicates and six technical replicates used in the experiments. The accepted spots were filtered based on the average expression level of two-fold or consistent and significant changes in at least three nanoparticle levels. 2.5. Protein digestion and peptide extraction
2. Material and methods 2.1. Silver nanoparticles (AgNPs): synthesis and characterization The homemade silver nanoparticle colloidal suspension (AgNPs) was prepared and characterized according to the previously reported method (Mirzajani et al., 2011). Briefly, colloidal AgNPs with spherical morphology and distinct diffraction peaks of crystalline planes of cubic system, including (111), (200), (220) and (311), have a maximum absorbance (λmax) at 426 nm. Their size was 18.34 nm (X99) with the homogeneity of their size within the range of 0.1–1000 nm.
The spots, showing comparable differences, were cut out manually from a preparatory gel. The spots were washed with water and destained using 10 mM ammonium hydrogen carbonate in 50 percent (v/v) MeCN. They were fully dehydrated in 100 percent (v/v) MeCN and dried using a SpeedVac system (Thermo Fisher Scientific Co., MA, USA) at 20 1C, 14,000g for 15 min. Subsequently, the dried gel pieces were rehydrated with 1–4 mL trypsin (Promega, Madison, WI, USA) solution for 30 min (0.1 mg/mL in 30 mM NH4HCO3 buffer); tryptic digestion was carried out at 37 1C for 20 h. The peptides were extracted from the gel pieces in three steps using MeCN containing five percent formic acid under sonication for 10–15 min. The OMIX C18 10 mL tip (Varian, Inc., Palo Alto, CA, USA) was used to clean up and to pre-concentrate the extracted solution.
2.2. Plant cultivation, treatment and protein extraction 2.6. Mass spectrometry measurement The plant cultivation is based on growth screening results of our previous reports on O. sativa L. (Mirzajani et al., 2013). Seeds of O. sativa L. cv. IR651 were gifted from the Rice and Citrus Research Institute (RCRI), Agricultural Science and Natural Resources University, Sari, Iran. The seedlings were prepared on petri dishes (ID: 7 cm), filled with 10 mL of sterile water, at 27 1C and were kept in dark for seven days. Plant seedlings (ten per bottle/2 L in whole volume) were transferred into rice specific growth cultivation media (Yoshida et al., 1976). They were grown in a phytotron maintained at a thermo-period of 27 1C7 0.5, photoperiod of 16 h, relative humidity of 60 percent, and a photon flux density of 20 mM/ m2s. The water content and the pH were adjusted to 200 mL and 5.0 7 0.1 daily respectively. Every five days of treatment the hydroponic media were renewed. Plants at the age of ten days were irrigated by AgNPs colloidal solution in the concentration of 0 (blank condition), 30 and 60 mg/mL in ten replications. The treatments were continued for twenty days more. Thereafter the plants were washed with water and were kept at 80 1C before analysis. Total protein was isolated from cells by TRIzol reagent and available guidelines (Molecular Research Center, Inc., Cincinnati, OH, USA). Briefly, the plant tissues were ground using mortar in liquid nitrogen and homogenized by TRIzol reagent. 0.1 g was then incubated with chloroform and centrifuged for 15 min at 12,000g at 4 1C. It was mixed with absolute ethanol for 15 s, incubated for 3 min and finally centrifuged at 2000g for 5 min at 4 1C. The proteins in the phenol/ethanol supernatant were precipitated by the addition of absolute acetone and dispersed in the washing agent (0.3 M guanidine hydrochloride in 95 percent ethanol and 2.5 percent glycerol). The latter was repeated three times and followed by washing in ethanol containing 2.5 percent glycerol. The precipitated protein was dissolved in lysis buffer (consisted of 9 M Urea, four percent CHAPS, one percent DTT, one percent pH 3–10 ampholytes, 35 mM Tris base) and stored at 80 1C. Protein concentration was determined according to the Bradford assay kit (BioRad Co., Hercules, CA, USA) in the comparison with BSA as the standard. 2.3. 2-Dimensional gel electrophoresis (2-DE) 2-DE was carried out based on previous report (National Committee of Clinical Laboratory Standards, 2005). IPG strips, 18 cm, pH 4–7, linear (BioRad Co., CA, USA) were loaded with the proteins during the rehydration process for 16 h at room temperature in reswelling tray (Amersham Pharmacia Biotech, Sweden). For analytical and preparative gels, 100 mg and 1 mg of proteins were loaded, respectively. The IPG strips were covered with silicon oil and all IEF separations were performed horizontally at an optimized temperature of 20 1C using a Multiphore II system (Amersham Pharmacia Biotech) for a total of 70,000 Vh. Second-dimension SDS-PAGE separations were performed on a gel (245 180 0.5 mm3; 12.5 percent polyacrylamide) using a PROTEAN II Multi Cell (BioRad Co., CA, USA). Separations
Measurements of the peptides were accomplished on an Ultimate binary nanohigh-performance liquid chromatography (nano-HPLC pump/autosampler) system for HPLC analysis (LCPackings/Dionex Co., Idstein, Germany). Volumes of 5 mL of the sample were pre-focused on a trap column (Dionex Co., C18 PepMap, i.d. 300 mm, length 5 mm) and separated on a fused-silica C18 PepMap100 capillary column (Dionex Co., 3 mm, 100 Å; i.d. 75 mm; length 150 mm). The flow rate was 0.2 mL/min. Solvent A consists of water containing two percent MeCN (v/v) and 0.1 percent formic acid (FA) (v/v). Solvent B was MeCN containing twenty percent water (v/v) and 0.08 percent FA (v/v). Separation was performed as follows: first B was increased from 0 percent to 25 percent in 5 min, increased to 50 percent in 25 min, further increased to 100 percent in 4 min and maintained for 20 min to elute the strongly hydrophobic peptides and to clean the column. The gradient was then ramped down in 1 min to 0 percent solvent B for equilibration for 25 min. The nano-HPLC system was coupled to a nanoelectrospray interface of an LTQ FTICR mass spectrometer (Thermo Scientific GmbH, Bremen, Germany). Mass spectra were acquired on a “Finnigan LTQ FT Ultra” hybrid instrument consisting of a linear quadrupole ion trap and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a 6-T magnet. A nanospray ionization source was used for analysis (capillary temperature 250 1C, capillary voltage 35 V, tube lens 125 V). Nanoelectrospray needles for online measurements, Silica Tips (Tip I.D. 10 μm, New Objective, Woburn, MA, USA) ,were used at a spray voltage of 1.9 kV. Mass resolving power was set to 100,000 at m/z¼ 400, allowing us to record one spectrum per second and thus several spectra per chromatographic peak. Mass accuracy after external calibration was better than 7 1.0 ppm during measurements. The peaks in the survey scan were chosen for fragmentation. The isolation window for the precursor ion was 7 2 u. Instrument specific parameter settings for collisioninduced dissociation (CID) in MS/MS experiments were as follows: activation energy, 30 (normalized; manufacturer-specific units); activation duration, 30 ms; activation Q, 0.25 (manufacturer-specific units); wide scan range, three microscans per spectrum. 2.7. Database search NanoLC/ESI-MS/MS data were searched against the O. sativa L., database using Proteome Discoverer 1.1 (Thermo Fisher Scientific, Bremen, Germany) based on the SEQUEST search algorithm. The mass tolerance for precursor ions was set for 2 ppm, and the mass tolerance for product ions was set to 0.8 u. Two missed cleavages were allowed in order to account for incomplete digestion. Methionine oxidation was allowed as variable modification. Peptides with a “peptide probability” (SEQUEST parameter) of 35 and higher were considered as significant identifications. The false discovery rates, as determined by a reversed database
F. Mirzajani et al. / Ecotoxicology and Environmental Safety 108 (2014) 335–339 search, in all cases were 1.5 percent. Peptide sequences were subjected to BLAST using blast at the National Centre for Biotechnology Information (NCBI: http:// www.ncbi.nlm.nih.gov/BLAST/) and MS BLAST at the European Molecular Biology Laboratory [EMBL: http://dove.embl-heidelberg.de/Blast2/msblast.html].
3. Results 3.1. 2-DE gel electrophoresis and comparison of protein expression optimization From the previous screening of the AgNPs influences on the rice root, it found that the treatment under the 30 mg/mL of AgNPs has a significant influence on root; decrements in growth and increments in branching (Mirzajani et al., 2013). In addition this concentration widely influenced the metabolites production, increments in carotenoids and decrements in total carbohydrate contents. The AgNPs treatment in the concentration of 60 mg/mL has similar but more severe effect. In conclusion of screening results 0 (blank condition), 30 and 60 mg/mL were chosen for the 2-DE proteomics study within a month (Mirzajani et al., 2013). 2-DE pages were then analyzed by silver staining. It was previously reported that 18 cm IPG strips have the capability to load r70– 100 mg/gel of proteins. As a result the 2D-page of 100 mg/gel of proteins (0 of AgNPs mg/mL treatment) was prepared to optimize the quality of the gel. Based on results, it was concluded that for each replication of treatments, 100 mg/gel was prepared (Fig. 1). 2D images were analyzed and volume of spots was estimated and compared across gels. As shown in Fig. 1 the quality of extracted proteins and following gels was highly reduced after the AgNPs treatment. So that, while the blank gels have more than 950 recognizable spots, 230 spots were monitored in the treated gel. The gel quality in the treated sample was not as good as the blank sample (Fig. 1). As can be seen in this figure and what is found in our previous study (Mirzajani et al., 2013), the carotenoids and pigments content was increased following the AgNPs treatment. For the increased gel quality the phenolic compounds cleanup method, introduced by Chatterjee et al., was examined. This method was introduced for phenolic rich roots of chickpea (Cicer arietinum L.) using phenol–SDS buffer extraction with sonication (Chatterjee et al., 2012). The method increased the gel quality in the case of some spots, while some spots cannot be detected and the others had tailing and low resolution. In the case of total protein a long term sonication process can damage proteins. Finally, 170 protein spots were compared based on their density and abundance, and 28 of them showed significant differences in response to AgNPs treatment (Fig. 2). If the density was 0.1 and it at most reaches 0.2, it is classified as an average expression level and if it reaches at least 0.2 it is classified as a significant change in expression level, and both are considered as
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responsive proteins in comparison to control. Protein spots with an average expression level of two-fold or significant changes in at least three AgNPs levels in comparison to the control were considered as responsive proteins (Fig. 2; Table S1, Supplementary material). Comparison of different expressions among AgNPs levels revealed that increase in treatment concentration significantly influences on the abundance of spots (Table S1, Supplementary material). All of the AgNPs-responsive proteins changed in terms of abundance rather than in-gel position and presence or absence. The majority of AgNPsinduced changes was associated with decreased abundance and was observed in the concentration of 60 mg/mL of AgNPs, with eighteen and five proteins increment and decrement in abundance, respectively (Table S1, Supplementary material). In addition some complex patterns were observed in the case of five spots including spot nos. 5, 16, 18, 20, 23 and 24, (Table S1, Supplementary material). 3.2. MS-based protein identification To identify proteins response to AgNPs, the best matching gene product and accession number, molecular weight (MW kDa) and isoelectric point (pI), the number of matching peptides and the score and coverage of peptides with databases were considered. The abundance pattern of each protein in AgNPs concentrations of 0, 30 and 60 mg/mL can be observed in Table S1, Supplementary material. All matching proteins belonged to the O. sativa but there were also some different strains (not included in the table). Responsive proteins (Fig. 2) were analyzed using NanoLC/FTICR after excision from CBB-stained gels and in gel digestion with trypsin. Proteins were identified according to the results of mass spectrometry, their partial sequences and MS–MS in comparison with the rice, Oryza sativa L., database (Table S1, Supplementary material). As can be seen in Table S1, Supplementary material, the proteins were identified based on the two to seventeen peptide sequences of a hydrolyzed protein (the results of identified peptides are mentioned in Supplementary materials). In the case of each protein the constituent digested peptides were identified and compared to previously published results. More than 197 peptides, corresponding to 28 proteins, were identified. The b and y fragments as well as neutral fragments were monitored to identify each peptide (Table S1, Supplementary material). These proteins include several significant responsive proteins. A few proteins with unknown functions or without any match in the database were also observed.
4. Discussions According to the identified proteins different mechanisms involved following the AgNPs treatment were hypothesized. An oxidative response pathway which was represented by superoxide
Fig. 1. Physiological impacts on O. sativa L. exposed to different concentrations of AgNPs (0, 30 and 60 mg/mL). (a) 100 mg, silver nitrate staining, 2D-page analysis of O. sativa L. proteome regarding the treated plant at different concentrations: blank, 0 mg/mL (b); 30 mg/mL (c); and (d) 60 mg/mL.
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Fig. 2. 100 mg of 2D-page protein analysis and AgNPs action on the protein regulation of O. sativa L., and some spots under the AgNPs treatments are marked. (1) Silver nanoparticle toxicity on O. sativa L., (2) expression pattern of proteins in the AgNPs treated O. sativa L., and (3) the mechanism of O. sativa L. toxicity from proteomics point of view.
dismutase –[14], L-ascorbate peroxidase –[9] and Glutathione-sTransferase –[12] is one of them. Some proteins are involved in defense signaling pathways. Among them can be cited the NACtranscription factor – [13], which is one of the mitochondrial retrograde regulation (MRR). Mitochondria signals to the nucleus to steer the expression of responsive genes. Reactive oxygen species and calcium are signaling molecules for MRR. Some calcium-binding messenger proteins (CaM) include calmodulins 1 and 3, 26 and 27. They can transduce calcium signals by binding calcium ions and then modifying its interactions with various target proteins. They also mediate many crucial processes such as inflammation, metabolism, apoptosis and intracellular movement. They are expressed in many cell types and can have different subcellular locations, including the cytoplasm, within organelles, or associated with the plasma or organelle membranes. Furthermore, there are some proteins involved in protein synthesis/degradation processes which were also identified including proteasome subunit α 10 and subunit β 11. They degrade unneeded or damaged proteins by the proteolysis process. Translationally controlled protein (TCP) [21], a protein regulator has roles in cellular processes, most notably in the cell cycle, apoptosis, cell proliferation, growth, stress response, gene regulation and heat shock. TCP [21] is the major regulator of cell growth in animals and fungi. Berkowitz et al. demonstrated that plant TCPs exhibit distinct sequence differences from nonplant homologs but share the key GTPase binding surface. They mentioned that silencing of TCP in Arabidopsis thaliana resulted in slow vegetative growth, leaf expansion is reduced because of smaller cell size, lateral root formation is reduced, and root hair development is impaired. Furthermore, these lines show decreased sensitivity to an exogenously applied auxin analog and have elevated levels of endogenous auxin. These results identify TCP as an important regulator of growth in plants and imply a function of plant TCP similar to that known in nonplant systems. 60S acidic ribosomal
protein –[3] is a phospho-ribosomal protein that contributes to the structural integrity of the ribosome. It also involved in a wide range of cellular processes like the signal recognition which is responsible for targetting proteins to the endoplasmic reticulum and a complex involved in termination of transcription. The detailed mechanism and environmental toxic aspects of AgNPs are not completely understood. There are some hypotheses like cell wall damage, oxidative stress following the ROS production and DNA/protein direct damage (Li et al., 2010). Based on our previous study on S. aureus and Bacillus thuringiensis, it was found that organisms have the ability to adopt and survive at low concentrations of AgNPs (Mirzajani et al., 2011, 2013). It has been previously reported that the ROS production and its role in toxicity and cell death have been studied previously in many researches (Rai et al., 2012). One of the best ways to understand the mechanisms of AgNPs actions is to study the protein expression/ regulation. These phenomena control reactions such as oxidative stress, cell wall damage and DNA/RNA/protein direct interaction following the evidences to confirm or reject the hypothesized mechanisms. AgNPs stress can accelerate the production and detoxification of ROS determining factor of oxidative stress and cell damage. Cellular detoxification enzymes, the expression modification proteins, are produced in response to ROS toxicities. As mentioned in the results an increased abundance of proteins was identified, including superoxide dismutase –[14], L-ascorbate peroxidase –[9] and Glutathione-s-Transferase –[12] which are involved in the detoxification or oxidative stress pathways. The increments in the expression of calcium protein includes calmodulins 1 and 3, [26] and –[27], NAC-transcription factor 13 and translationally controlled protein –[21] implied an occurrence of this toxicity following Ca2 þ regulation. Cheung (1980) suggested that metals effectively substitute for Ca2 þ in calmodulin. Evidence indicates that calmodulin serves as a major intracellular Ca2 þ receptor
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regulating the activity or rate of many key enzymes and cellular processes. Some heavy metals are highly toxic to the cell. It can be hypothesized that metals, here AgNPs or the released ions, interfere with the regulation of cell metabolism by binding to secondmessenger calcium receptors, calcium channels and calcium– sodium ATP pumps (Goyer, 1995). Activation of calmodulin by these metals could upset its normal regulation by the cellular flux of Ca2 þ . Perhaps this could constitute in part a basis for the toxicity of these metals in cell physiology. So the amount of cellular Ca2 þ will increase. It can be hypothesized that with the increase of cellular Ca2 þ following the ROS production, the NACtranscription factor and signaling molecules for MRR increase in abundance. Translationally controlled protein –[21] in addition to Glutathione-s-Transferase –[12] are proteins involved in the apoptosis process which increase in abundance. 5. Conclusion To our knowledge, this is the first report on proteome analysis of O. sativa L. and its response to AgNPs treatment. There are some other reports on the proteomics study in different plants like E. sativa. They reported that Ag in ionic/nanoparticle form causes changes in proteins involved in the redox regulation and in the sulfur metabolism. They claim that the effects of AgNPs are not solely due to the release of Ag þ into the surrounding environment, while the others strongly express that one of the mechanisms of AgNPs toxicity is silver ion release (He et al., 2011; Levard et al., 2012; Yang et al., 2012; Vannini et al. (2013)). So, the mechanism may result in parallel action of the silver nanoform and released ion. As previously mentioned proteins are one of the involved compounds in this mechanism over the direct regulations. Responsive protein changed in terms of its abundance rather than in-gel position or its presence or absence. In conclusion, AgNPs interact with normal cell metabolic processes such as protein synthesis/degradation and apoptosis. Subsequently, it can be hypothesized that the AgNPs enter into the cell and condense the DNA/protein thus inhibiting the normal cell reproduction. This series of studies on the impact of AgNPs suggests that, AgNPs not only have a direct or regulatory impact on the microorganism's proteome (Li et al., 2010; Mirzajani et al., 2013), but also on the plant cells. Furthermore, the increment of detoxification enzymes implies a toxic impact from the production of ROSs and metal toxicity in the presence of AgNPs. Acknowledgments Financial support from the Research Council of Shahid Beheshti University and the Iran Nanotechnology Initiative Council is gratefully acknowledged. This publication represents a component of the doctoral thesis of Fateme Mirzajani at the Medicinal Plants and Drug Research Institute of Shahid Beheshti University, Tehran, Iran. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.07.013.
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