Acute Toxicity of Functionalized Single Wall Carbon Nanotubes: a Biochemical, Histopathologic and Proteomics Approach

Chemico-Biological Interactions 275 (2017) 196e209

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Chemico-Biological Interactions

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Acute toxicity of functionalized single wall carbon nanotubes: A biochemical, histopathologic and proteomics approach

Homa Ahmadi a, Mohammad Ramezani b, Rezvan Yazdian-Robati c, Behzad Behnam d, Kamal Razavi Azarkhiavi a, Azadeh Hashem Nia e, Ahad Mokhtarzadeh f, g, * ** Maryam Matbou Riahi h, Bibi Marjan Razavi i, , Khalil Abnous e, j, a Department od Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b Nanotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran c Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran e Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran f Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran g Department of Biotechnology, Higher Education Institute of Rab-Rashid, Tabriz, Iran h Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran i Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran j Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran article info abstract

Article history: Recently carbon nanotubes (CNTs) showed promising potentials in different biomedical applications but Received 1 January 2017 their safe use in humans and probable toxicities are still challenging. The aim of this study was to Received in revised form determine the acute toxicity of functionalized single walled carbon nanotubes (SWCNTs). In this project, 17 July 2017 PEGylated and Tween functionalized SWCNTs were prepared. BALB/c mice were randomly divided into Accepted 7 August 2017 nine groups, including PEGylated SWCNTs (75,150mg/mouse) and PEG, Tween80 suspended SWCNTs, Available online 12 August 2017 Tween 80 and a control group (intact mice). One or 7 days after intravenous injection, the mice were killed and serum and livers were collected. The oxidative stress markers, biochemical and histopatho- Keywords: Single-walled carbon nanotubes (SWCNTs) logical changes were studied. Subsequently, proteomics approach was used to investigate the alterations fi Acute toxicity of protein expression pro les in the liver. Oxidative stress Results showed that there were not any significant differences in malondealdehyde (MDA), glutathione Proteomics (GSH) levels and biochemical enzymes (ALT and AST) between groups, while the histopathological ob- Tween80 servations of livers showed some injuries. The results of proteomics analysis revealed indolethylamine N- PEG Methyltransferase (INMT), glycine N-Methyltransferase (GNMT), selenium binding protein (Selenbp), thioredoxin peroxidase (TPx), TNF receptor associated protein 1(Trap1), peroxiredoxin-6 (Prdx6), electron transport flavoprotein (Etf-a), regucalcin (Rgn) and ATP5b proteins were differentially expressed in functionalized SWCNTs groups. Western blot analyses confirmed that the changes in Prdx6 were consistent with 2-DE gel analysis. In summary, acute toxicological study on two functionalized SWCNTs did not show any significant toxicity at selected doses. Proteomics analysis also showed that following exposure to functionalized SWCNTs, the expression of some proteins with antioxidant activity and detoxifying properties were increased in liver tissue. © 2017 Elsevier B.V. All rights reserved.

1. Introduction

* Corresponding author. Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Carbon nanotubes (CNTs) are a widely used family of nano- ** Corresponding author. Pharmaceutical and Medicinal Chemistry Research materials. According to the number of graphene layers, these hol- Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, low CNTs are divided to SWCNTs (Single Wall Carbon Nanotube) Iran. and MWCNT (Multi Wall Carbon Nanotube). SWCNTs have E-mail addresses: [email protected] (B.M. Razavi), [email protected] (K. Abnous). remarkable thermal, electronic and optical properties [1,2]. Besides http://dx.doi.org/10.1016/j.cbi.2017.08.004 0009-2797/© 2017 Elsevier B.V. All rights reserved. H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209 197 broad range industrial applications, SWCNs have many potential resulting supernatant was centrifuged at 3000 g for 5 min to medical applications including drug and gene delivery, tissue separate pellets. Then, the supernatant was filtered through Ami- regeneration and artificial implants [3e5]. con filters with 100 kDa and washed 5 times. Finally, 1 mg/mL As pristine carbon nanotubes are intrinsically water insoluble Tween functionalized CNTs solution was prepared and stored at and have a hydrophobic nature, functionalization and surface 2e5 C. modification are required to make them compatible with biomedical applications [6]. There are two approaches for SWCNTs 2.2.2. Covalent functionalization of CNTs functionalization, covalent and noncovalent attachment (physi- In order to introduce carboxylic acid functional groups onto the oadsorption) [7]. By functionalization, CNTs become hydrophilic surface of SWNTs, modified oxidation procedure was used [20]. and ready for linking with drugs or biomolecules (genes, DNA, Briefly, 30 mg SWNT was dispersed in 100 ml HNO3 2.5 M and proteins, enzymes, biosensors, etc.) to deliver them into the target sonicated for 2 min. The mixture was refluxed for 48 h and then cells or organs. Many reports have shown that properly function- sonicated for 30 min. The mixture was refluxed for another 24 h. alized CNTs are valuable nanocarriers for efficient and safe delivery The reaction mixture was filtered using PTFE membrane (200 nm) of biomolecules [8,9]. Previously we showed that proper func- and the filtrate was washed with distilled water several times. The tionalized CNTs can be used as efficient gene delivery vectors with product (SWNT_COOH) was dried at 60 C overnight. The second low cytotoxicity in vitro and in vivo [10e16]. For biomedical ap- step was synthesis of PEG-functionalized SWNT (SWNT-PEG) as plications, various functionalization strategies can be used and each follow, 30 mg of the final product of the oxidation procedure method has its own advantages. Non-covalent functionalization of (SWNT-COOH) was dispersed in 5 ml distilled water and sonicated CNTs with amphiphilic molecules is of great importance, because in for 2 min. Then 191.7 mg EDC and 115.09 mg NHS was added and contrast to covalent functionalization, the graphene-network of the mixture was stirred for 45 min at room temperature. Then, sheets is not disturbed, and their extraordinary physical properties 200 mg NH2-PEG-COOH (MW 3400) was added and the reaction remain intact [17]. In contrast, when a reliable strong link between was completed after 3 days. The reaction mixture was filtered with nanotube and biomolecule is necessary, covalent functionalization PTFE membrane and the supernatant was dialyzed (KD cutoff) for 3 is more valuable. Herein we decided to use two simple and reliable day against 2 L of distilled water and then lyophilized. functionalization methods for SWCNTs. Therefore for covalent functionalization of SWCNTs, Polyethylene glycol (PEG) as a hy- 2.3. Characterization of functionalized carbon nanotubes drophilic polymer with high biocompatibility and dispensability was used [18,19]. On the other hand, Tween 80 as a safe biocom- Size and zeta potentials characterizations were carried out by patible and commercially inexpensive surfactant was used for using Zetasizer (Nano-ZS, Malvern Instruments, UK) [20]. Also noncovalent functionalization of nanotubes. Thermogravimetric analysis (TGA) was done with the TGA 50 in- Many factors including route of administration, type of func- strument (Shimadzu, Japan) by heating the sample at a rate of 10 C tionalization and the nature of tissues strongly affects the toxicity min 1 to 800 C in air. of SWCNTs. It has been shown that a proper functionalization can reduce the toxicity of pristine carbon nanotubes [20]. 2.4. Animals Although several studies were designed to evaluate the toxicity of pristine or functionalized SWCNTs, few data on acute toxicity of All animal experiments were performed in compliance with the intravenously injected SWCNTs have been reported [6,7,9,17e19]. Institutional Ethical Committee and Research Advisory committee In this study we applied proteomics analysis, oxidative stress of Mashhad University of Medical Sciences based on the national and histological examination to assess the potential changes in guidelines from Ministry of health and medicinal education of Iran. response to acute high dose of functionalized SWCNTs in order to Four weeks old BALB/c mice were obtained from Pasteur Insti- provide a more comprehensive understanding of the safety of these tute (Tehran, Iran), and weighted 25e27 g at the time of experi- nanomaterials in biomedical application. ment. They were acclimatized before performing the study with free access to food and water. 2. Materials and methods 2.5. Experimental design 2.1. Materials There were four treatment groups, PEG, PEGylated SWCNTs, A preparation of purified SWNTs, produced by the HiPco method Tween 80, Tween 80 suspended SWCNTs and a control group (0.8e1.2 nm individual diameter and 100e1000 nm length ac- consisted of five untreated mice. Each treatment group was also cording to manufacturer specifications) was purchased from Uni- composed of five animals. Two doses of 75 or 150 mg per mouse dym Inc. (Sunnyvale, USA). IPG strips (pH 3e10, 17 cm), Bio-Lyte were administrated to functionalized SWCNT groups (Tween 80 (pH 3e10), the fluorescent SYPRO R Ruby protein gel stain, and suspended and PEGylated SWCNTs) and 200 mg of PEG or Tween80 protein assay kit were purchased from BioRad (USA). MDA, Acryl- per mouse were administrated to PEG and Tween 80 groups. These mide, SDS, urea, thiourea, Tris-HCl, and glycine were provided by dosages were chosen according to the previous study with some Merck (Germany). Mineral oil, CHAPS, DTT, and iodoac-Glutathione, modification after a pilot study [3]. DTNB, Protease Inhibitor Cocktail, PMSF were purchased from In order to evaluate acute toxicity, mice were sacrificed one or Sigma-Aldrich and ultrapure agarose was obtained from Invitrogen seven days following the exposure. Serum and Liver tissues were (USA). Molecular weight marker was obtained from Fermentase separated. Tissues were stored at 80 C for further experiments. (USA). 2.6. Measuring the MDA in the liver tissue 2.2. Fuctionalization, purification and characterization of SWCNTs In order to measure MDA, as a marker of oxidative stress, the 2.2.1. Noncovalent functionalization of CNTs liver tissues, were homogenized in 1.15% KCl for 2 min at 4 C 1 mg CNTs and 5 mg Tween80 were dispersed in 5 cc deionized (POLYTRON PT 10e35, Kinematic, Switzerland) to provide a 10% water. Then, mixture was sonicated for 1 h in 20e40 C. The homogenate. MDA levels were determined as reported by 198 H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209

Fernandez and coworkers [21]. This procedure is based on the BioRad protein assay kit according to the manufacturer's in- spectrophotometric measurement of the color formed by reaction structions. Rehydration of IPG strips, containing 300 mg protein, of MDA to thiobarbituric acid (TBA). In brief, 1.5 ml of phosphoric was done actively for 12 h at 50 V. Isoelecteric focusing was per- acid (1%) and 0.5 ml TBA (0.6%) were added to 0.25 ml of homog- formed using PROTEAN IEF CELL (BioRad) at 20 C as follows: enate and incubated in a boiling water bath for 45 min. After voltage started at 250 V, then increased linearly to 8000 V and was cooling, 2 ml of n-butanol was added to the mixture and vortex- kept constant at this voltage for 11 h (Volatge linear gradient for mixed for 1 min. After centrifugation for 20 min at 3000 g the 11 h). The current limit was set at 50 mA/gel. After IEF, the 17 cm organic layer was transferred to clean tube and its absorbance (l532 3e10 IPG strips were stabilized in 6 mL of reduction buffer con- nm) was recorded on a spectrophotometer (Jenway 6105 uv/vis, UK). taining 0.375 M Tris-HCl (pH 8.8), 6 M urea, 20% glycerol, 2% SDS, Tissue MDA levels were reported as nmol/g of tissue. and 2% DTT for 15 min. For alkylation, the strips were equilibrated again in 6 mL of solution containing 0.375 M Tris-HCl (pH 8.8), 6 M 2.7. Measuring the reduced GSH in the liver tissue urea, 20% glycerol, 2% SDS, and 2.5% w/v iodoacetamide for 15 min. The strips were placed on the top of 12% SDS-PAGE gels for the The measurement of liver GSH content was made according to second dimension separation. The second dimension of electro- the method of Moron et al. [22]. The liver tissues were homoge- phoresis was carried out using PROTEAN II xi 2-d cell and POWER/ nized in ice cold phosphate buffer saline (PBS, pH 7.4) (10% w/v). PAC 3000(BioRad) at 120 V for 5 h, and then at 150 V up to the Reduced GSH contents were quantified by using 5,50-dithiobis(2- tracking dye reached the bottom of the gel. nitrobenzoic acid) (DTNB), which produced a yellow-colored 5- fl thio-2-nitrobenzoic acid (TNB). Brie y, equal amounts of samples 2.12. Protein staining, image acquisition and analysis and 10% trichloroacetic acid (TCA) were mixed and centrifuged at 3000 g for 5 min. Then, 0.5 mL of 0.04% DTNB reagent was added to After electrophoresis, the gels were stained by using colloidal 0.5 mL of homogenate plus 2 mL PBS (0.1 M, pH 8.0). After that, the coomassie blue protocol so the gels were first washed 5 times by absorbance of yellow colored TNB was measured at 412 nm. Tissue distilled water each time for 10 min, then, incubating the gel using GSH contents were reported as nmol/g of tissue. colloidal coomassie solution containing 0.02% w/v Coomassie Brilliant Blue G-250, 5% w/v aluminum sulfate-(14e18)-hydrate, 2.8. Serum biochemical parameters ethanol (10% v/v), orthophosphoric acid (2% v/v), while shaking for about 10e12 h. The stained gels were scanned by Image Scanner III, Following anesthetization, blood specimens were immediately Lab Scan 6 (Epson J181A, Japan). Protein spots were analyzed using collected using 27 gauge insulin syringes and transferred into Image Master Platinum 6.0 software (GE Healthcare, USA). Five gels microtubes. Each sample was allowed to clot for about 30 min from one each group were analysed. To find the best matches, three (maximum 60 min). After clotting, the sample was centrifuged at of the most appropriate gels in each group were imported to the 750 g for 10 min. The serum then was pipetted from the cellular image master software. Approximately 150 landmarks per gel were elements (erythrocytes, platelets, leucocytes) and transferred to manually set as primary points for the software matching routine. another tube, properly labeled and stored at 4 C until ready for In order to find the correlation among the gels in each group (intra analysis. The activity of alanine aminotransferase (ALT) and aspar- class analysis) and also observing differentially displayed spots tate aminotransferase (AST) in serum was measured through (inter class analysis), the gels were categorized into 5 groups: commercial colorimetric kits from Sigma by auto analyzer. Control, PEGylated SWCNTs and its control (PEG), Tween80 suspended SWCNTs and its control (Tween80). The fold changes of 2.9. Histopathological observations differentially displayed protein spots were calculated and exported as an EXCEL file. Density of these spots was determined after Surgically, the livers of mice were removed under ketamine- normalizing against the total spot volumes on the gel. Spots with xylozin anesthesia and after blood collection. Small pieces of more than 1.5- and less than 0.6 fold change and P value <0.05 were fi livers were immediately xed in 10% neutral buffered formalin, candidates to be identified by for identification by MALDI-TOF/TOF. processed routinely, then embedded into paraffin. Sectioned at 6 mm and colored with hematoxylin and eosin. The slides were examined by an Olympus light microscope (BH-2, Japan). The cell 2.13. In-gel digestion photos were taken with an attached digital camera (Olympus, C- 7070). As previously described protein spots were excised from gels and distained and digested with sequencing grade modified trypsin 2.10. Sample preparation (Promega) [23]. Using Zip Tip C18 (Millipore), all digested samples were desalinated and concentrated. To extract proteins, 0.2 g of frozen liver tissues were homogenized in rehydration buffer containing 6 M urea, 2 M thiourea, 2% 2.14. MS and database searching for the identification of proteins CHAPS, 50 mMDTT, and 0.2% Bio-lyte and complete protease inhibitor cocktail (Sigma P8340) using Polytron Homogenizer The 4800 MALDI-TOF/TOF Analyzer (AB SCIEX, Framingham, M ® (IKA T10, Germany) and sonicated on ice using a probe sonicator A) was used in order to identify and characterize the proteins. (UP100H, Germany), then centrifuged at 14000 g. Total protein MASCOT 2.1 was applied for database searching. The data were concentrations of supernatants were measured using Bradford searched against SwissProt database. In an [PMF þ MS/MS] anal- protein assay kit. ysis, Protein Score was a composition of PMF-type protein score and the total Ion score. Protein or Ion scores MASCOT significance level 2.11. Two - dimensional gel electrophoresis were considered as criteria for diagnosis by MS. If the Ion Score was equal to or greater than the Mascot significance level, calculated in The samples were dissolved in rehydration buffer including 6 M the search, the protein match was regarded to be statistically sig- urea, 2 M thiourea, 2% CHAPS, 50 mM DTT, and 0.2% Biolyte. Protein nificant at the 95% confidence interval (CI). Details have been pre- concentrations of samples were determined using a commercial viously illustrated [23,24]. H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209 199

2.15. Classifications and network interaction maps of protein the analysis of AST and ALT. At both administrated doses, no sig- species nificant changes were observed in AST level neither in PEGylated SWCNTs injected group vs. PEG group (P ¼ 0.08) nor in Tween Biological classification of protein species was performed using suspended SWCNTs injected group vs. Tween80 group (P ¼ 0.073). protein analysis through evolutionary relationships (PANTHER) Moreover, the level of ALT did not significantly change between classification system (http://www.pantherdb.org). Based on the PEGylated SWCNTs and Tween suspended SWCNTs compared with published data and protein function, classifications of Proteins were PEG and Tween80, respectively (P ¼ 0.27 and P ¼ 0.87). reviewed and changes were made. Some of the information about protein roles and locations were also acquired from PANTHER and 3.2.4. Histopathological examination UNIPROT. For enzyme information, STRING (http://string-db.org) There were not any observable pathological changes in control e was applied for the networks and illustrating protein protein group. The histopathological changes of the liver in experimental interaction maps, related to differentially displayed protein species groups revealed some mild pathologic damages such as degener- between the two experimental groups. Furthermore, the data were ative changes and mononuclear inflammatory cell infiltration. It mapped to metabolic pathway maps according to the Kyoto Ency- seems that these pathological changes are more severe in SWCNTs clopedia of Genes and Genomes (KEGG) [25]. UNIPROT number of Tween80 treated group in comparison with PEG functionalized fi each identi ed protein was entered in KEGG database pathway and SWCNTs injected group and also, in rats who were killed 7 days fi converts to KEGG identi er and then searched against KEGG after treatment in comparison with rats who were killed 1 day after pathway. treatment. Indicating a time and dose dependent mild hepatotoxic effect of SWCNTs especially for Tween 80 suspended group (Fig. 2). 2.16. Western blot analysis 3.2.5. Two dimensional gel electrophoresis analysis Peroxiredoxin-6 protein level was relatively evaluated using Five representative 2DE maps from the control, PEGylated primary antibody (#Ab59543, Abcam) by western blotting, as SWCNTs, PEG, Tween80 SWCNTs and Tween80 groups were described previously [26]. demonstrated in Fig. 3A. Five scatter charts were plotted using Image Master Analysis software (Fig. 3B). The correlation coeffi- 2.17. Statistical analysis cient of 86% and 90% among gels in the control group, 84 and 87% among gels in PEGylated SWCNTs group, 86 and 93% among gels in The results are reported as mean ± SD. ANOVA followed by PEG group, 84 and 90% among gels in Tween80 suspended SWCNTs TukeyeKramer test were performed to compare means. P values group and 77 and 87% among gels in Tween80 group, indicated less than 0.05 were considered as significant. good reproducibility of the gels in each group (Fig. 3B). Fifteen differentially displayed protein species were identified by MALDI- 3. Results TOF/TOF. All information about identified proteins, including protein name, entry name, and gene names, Swiss-Prot accession 3.1. Synthesis and characterization of SWNCNTs numbers, theoretical molecular weight, pI of protein, percent of sequence coverage, protein scores (combination of PMF-type pro- The resulted functionalized CNTs were black suspensions of tein score and total ion score), fold changes, numbers of unique nanotubes and were stable without any aggregation during the peptides detected, and the sequence of matched peptide with the study. TGA results showed that nearly 50% weight of the TWEEN highest ion score and CI ¼ 100% and its expectation value, p value, functionalized SWCNTs corresponds to the surfactant Tween 80 and biological function of protein species were listed in Tables 1 and (supplementary materials, Fig. 1). Also 30% of weight of PEGylated 2. Labeled spots are demonstration of the protein species spots that nanotubes was attributed to the presence of PEG polymer were significantly altered after treatments. Also high resolution and (supplementary materials, Fig. 2). Size and Zeta potential data were 3D report of the labeled spots have been shown in Fig. 5. also collected for these nanoparticles and results showed that PEGylated and TWEEN functionalized CNTs had size of 92 nm and 168 nm with the corresponding zeta of 6.8 and 5.7 mV 3.2.6. Pathway analysis and interaction network fi respectively. PANTHER classi cation system was applied in order to show biological classification of differentially displayed protein species 3.2. Effect on oxidative stress markers (Fig. 6a and b). Differentially displayed protein species were clas- sified in four pathways. The identified proteins are involved in 3.2.1. MDA measurement metabolic process, cellular process, immune system process, At any administrated doses, no significant changes were response to stimuli, localization and biological regulation, notably observed in MDA level neither in PEGylated SWCNTs injected group more than 50% of these differentially expressed proteins are related vs. PEG group (P ¼ 0.48) nor in Tween suspended SWCNTs injected to methabolic pathways (55% and 63% in PEGylated and Tween80 group vs. Tween80 group (P ¼ 0.076). There were not also any suspended SWCNTs injected mice). In addition, STRING database significant changes between different groups and intact mice was used to investigate the potential relationship between identi- fi (P>0.05) (Fig. 1 A). ed protein species and recognition of the involved pathways (Figs. 7 and 8). KEGG metabolic pathway revealed that identified 3.2.2. Reduced GSH content measurement proteins are also involved in different metabolic pathways. As shown in Fig. 1B, the level of reduced GSH content did not significantly change between PEGylated SWCNTs and Tween sus- 3.2.7. Validation by western blot analysis pended SWCNTs compared with PEG and Tween80, respectively Western blot analysis was used to confirm the proteomics re- (P ¼ 0.53 and P ¼ 0.17). sults for Prdx6 protein as one of identified differentially expressed proteins. The results showed that the treatment with Tween80 3.2.3. Effect on biochemical enzymes suspended SWCNTs increased Prdx6 protein expression level. This Fig. 1 (C and D), presented the experimental data obtained from was in agreement with the proteomics analysis (Fig. 4). 200 .Amd ta./CeioBooia neatos25(07 196 (2017) 275 Interactions Chemico-Biological / al. et Ahmadi H. e 209

Fig. 1. Effects of functionalized SWCNTs treatment (1 and 7 days) on MDA (A), GSH (B), ALT and AST (C) levels in the liver rat tissues. Data are shown as mean ± SD. TukeyeKramer test, n ¼ 5. H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209 201

Fig. 2. Histopathological changes of BALB/c liver tissues of functionalized CNTs in mice.

4. Discussion 1000mg/mouse groups and remained unchanged in lower doses. There are also reports that GSH can act as a conjugating agent in The increasing use of SWCNTs in biomedical applications high- nanoparticles metabolism [3]. lights the importance of understanding the potential toxic effects of The observed pathological changes might be due to an acute its pristine and functionalized form to human. inflammatory response to carbon nanotubes as a foreign body. The The majority of toxicological studies on SWCNTs are focused on characteristics of this acute response are neutrophil and macro- their pulmonary adverse effects after inhalation exposure, while phage infiltration that peak 1e7 days post exposure. liver is one of the main distributed organs for SWCNTs; there are It has been generally accepted that the key mechanism of the few studies on potential hepatotoxic effects of SWCNTs. In addition, engineered nanomaterials toxicity is the generation of reactive as drug and gene delivery vehicles, SWCNTs are administered via oxygen species (ROS), and consequently the imbalance between intravenous injection, so, this will be a rational to study the toxicity oxidant and antioxidant mechanisms causes oxidative stress which of SWCNTs after intravenous injection in animal models [20]. Thus, may lead to inflammatory response [27,28]. The possible mecha- in the present study, we examined the possible toxic effects of nism of ROS production by PEG or Tween functionalized carbon functionalized SWCNTs in BALB/c mice on biochemical, and nanotubes may be due to their surface oxidizing ability or activa- oxidative stress biomarkers, histopathology of liver and a prote- tion of oxidative enzymatic pathways [29]. omics approach to provide a systematic view of the response to There were some controversies in SWCNTs induced oxidative SWCNTs exposure. Despite some mild histopathological lesions in stress. For example, the evaluation of SWCNTs toxicity in HepG2 the liver of treated groups especially in Tween 80 suspended group, cell lines revealed cytotoxicity, genotoxic and apoptotic effects [30], there were no significant changes in the oxidative stress markers while the long-term toxicity evaluation of Tween 80 suspended (MDA, GSH) and the serum biochemical parameters, neither in SWCNTs, showed a low hepatic toxicity [3]. PEGylated nor in Tween 80 suspended SWCNTs groups. This is in Several studies using different techniques have been accom- support with another study on the cytotoxic effects of intrave- plished to detect toxic effects of SWCNTs in recent years [17]. It has nously injected SWCNTs suspended in tween 80 that showed the been shown that the cells were exposed to phospholipids with PEG serum biochemical parameters only changed at doses of 200 and chains (PL-PEG) functionalized SWCNTs underwent apoptosis or 202 H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209

Fig. 3. (A) 2D gels colloidal coomassie blue of BALB/c liver tissue samples and reproducibility analysis of 2D gels in functionalized SWCNTs-treated group. Gel images were analyzed using the Image Master Platinum 6.0 software. (B) The intensity of each spot in the ﬁrst gel in one group (x-axis) was plotted against its intensity in another gel in the same group (y- axis). Representative reproducibility analysis of 2D gels in the a) SWCNTsþTween 80, b) Tween 80, c) SWCNTsþPEG, d) PEG and e) control.

necrosis and the proliferation rate were decreased [31]. Gambhir enzymes levels among groups. This could be due to the increased and Schipper evaluated toxicity of 4 months intravenous adminis- level of antioxidant enzymes following the phagocytosis of the tration of covalently and non-covalently PEGylated SWCNTs. They SWCNTs by activated macrophages and converting toxic com- concluded that both PEGylated forms of SWCNTs can be safe for pounds into less toxic ones [34]. biological applications [9]. Furthermore, Yang and colleagues sug- In this study, we also identified 14 protein species whose levels gested that the observed toxicity of intravenous administration of were significantly changed in acute administration of PEGylated or high doses of Tween80 SWCNTs was probably due to oxidative Tween80 functionalized SWCNTs. stress caused by the accumulation of SWCNTs in the liver [3]. These protein species were functionally involved in cellular Intraperitoneally injection of high doses (0.25, 0.5 and 0.75 mg/kg) process, metabolic pathways, biological regulation process, im- Tween 80 suspended SWCNTs after 24 h showed increased level of mune system process and localization. serum biochemical parameters. Moreover, the pathological exam- The increase of the expression level of Trap1, Prdx6, INMT and ination revealed the toxicity of this compound due to the oxidative TPx was 210, 390, 370 and 170% in Tween 80 group vs control group stress and clarify the role of functionalization in the toxicity of respectively. While ATP5b and Selenbp1 reduced by 30% and 61%, SWCNTs [32]. respectively. The comparison between PEGylated SWCNTs and ALT and AST are two indicators of hepatic injury. GSH, as a control groups indicated 165%, 214%, 170%,180% and 135% change of critical non-enzymatic antioxidant, and MDA, as one of the Rgn, INMT, ATP5b, GNMT and Etf-a level, and there was a 75% and important products of lipid peroxidation, are two key factors for 50% down regulation in Selenbp1 and Carl, respectively. According investigating oxidative stress. to KEGG pathway, INMT protein mapped to the tryptophan and In this study, we reported that 1 and 7 days following intrave- selenocompound metabolism, GNMT was involved in serine and nous injection of 75 and 150 mg/mouse PEGylated or Tween80 threonine metabolism and Rgn participated in ascorbate, aldarate, suspended SWCNTs, there were not any significant changes in the carbon metabolism and pentose phosphate pathway and finally levels of MDA and GSH, as well as in the levels of ALT and AST. Etf-a was involved in HIPPO signaling pathway and it seems to be However, the histopathological examination of liver tissues showed connected to anti-apoptotic genes. mononuclear inflammatory cell infiltration and mild perivascular Trap1 which increased in Tween 80 SWCNTs administrated that could be considered as signs of mild liver damage, the level of group is known as a heat shock protein. Trap1 can take part in changes were higher in Tween functionalized SWCNTs group after modulating cyclophilin D activity and it can inhibit the opening of 7 days post injection. mitochondrial transition pore in response to cyclophilin D [35].In Liver is a unique organ. It can normally continue its function addition, this protein plays an important role in protecting cells even if it loses more than 60% of its efficiency [33]. In this study, we against oxidative stress and oxygen free radicals apoptosis [35,36]. have not observed significant changes in liver MDA, GSH and Overexpression of Trap1 decreases the levels of ROS, glutathione H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209 203

Fig. 3. (continued). manganese peroxidase and superoxide dismutase [37] and also with control group, are members of a group of proteins with anti- protects mitochondrial proteins from the direct damage caused by oxidant function. The result of western blot analysis conﬁrmed long-term oxidative stress [38]. overexpression of Prdx6 in Tween 80 SWCNTs group. Prx6 is the only member of the Cys1. This protein is unique Overexpression of thioredoxin peroxidase (TPx) leads to the because of its dual functions as phospholipase A2 and peroxidase. protective role against oxidative stress. Activation of this enzyme The peroxidase activity of this protein is about 50 times more than detoxiﬁes hydrogen peroxide. Moreover, it also inhibits lipid per- its phospholipase activity. In addition, Prx6 can limit the lipid oxidation [40]. TPx can be involved directly in the activation of peroxidation reactions and their related damage [39]. macrophages. Macrophages which are treated with recombinant TPx and Prdx6 proteins, which their expression was increased in TPx, could induce Arg1 and secreted large amounts of IL-10 and Tween 80 SWCNTs group, 210 and 390% respectively in comparison prostaglandin E2, and could represent a regulative role in immune 204 H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209

responses [41]. In this study, we observed some mild histopato- logical lesions on the liver that might be due to the activation of macrophages as a result of exposure to functionalized SWCNTs. Moreover, it could be concluded that up regulation of Trap1, Prdx6 and TPx in Tween 80 suspended SWCNTs led to the decrease lipid peroxidation in the liver tissues exposed to Tween 80 suspended SWCNTs and is consistent with our results in the part of evaluation oxidative stress markers. Overexpression of endogenous regucalcin (Rgn) in cells has Fig. 4. The effect of Tween 80 suspended SWCNTs on the protein level of Prdx6 in liver been demonstrated to suppress cell proliferation and apoptotic cell tissue by Western blot analysis.

Table 1 Differentially displayed protein species in liver of BALB/c mouse exposed to Tween 80 suspended SWCNTs as identiﬁed by MALDI-TOF/TOF and MASCOT software.

Protein Name (Entry name)/ Theoretical Calculated Fold-change Protein % Expectation- Biological process or Molecular pathways (PANTHER gene name MW(kDa) pI (Tween80 þ SWCNT/ Score a Sequene valueb classiﬁcation) control) coverage

heat shock protein 75 kDa, 80501 6.25 2.1 59 1 0.035 metabolic process, response to stimulus, mitochondrial precursor (Trap1)/Hsp75 ATP synthase subunit beta, 57976 5.80 0.7 423 12 8.5e-06 localization, metabolic process, ATP synthase, anion mitochondrial (Atp5b) channel, ligand-gated ion channel, DNA binding proteinhydrolase Selenium-binding liver protein 52889 5.97 0.39 109 3 3.1e-07 brown fat cell differentiation, protein transport (Selenbp1)/Lpsb peroxiredoxin-6 (Prdx6)/ 24969 5.71 3.9 258 14 7.2e-05 metabolic process, antioxidant activity, catalytic activity Aop2, Ltw4, Prdx5 indolethylamine N- 30068 6.00 3.7 136 9 0.0022 catalytic activity, metabolic process methyltransferase(Inmt)/ (Temt) thioredoxin peroxidase 22336 1.7 60 0.0043 immune system process, metabolic process, antioxidant (Prdx2)/Tdpx1, Tpx activity, catalytic activity

a Protein score [PMF þ MS/MS] ¼ protein score [PMF] þ total ion score [MS/MS]. It is the Mascot probability based scoring that reports scores as 10 * log10 (P), where P is the probability that the observed match is a random event. The total ion score is calculated by weighting ion scores for all individual peptides matched to the protein that is associated with this peptide and MS/MS spectrum. Ion scores for duplicated matches are excluded in the calculation. b The probability of an incorrect identiﬁcation associated with matched peptide with the highest ion score and C.I. % ¼ 100.

Table 2 Differentially displayed protein species in liver of BALB/c mouse exposed to PEGylated SWCNTs as identiﬁed by MALDI-TOF/TOF and MASCOT software.

Protein Name (Entry name)/ Theoretical pI Fold-change Fold-change Protein % Expectation- Biological process or Molecular function (PANTHER gene name MW(kDa) (PEG þ SWCNT/ (PEG þ SWCNT/ Score a Sequene valueb classiﬁcation) PEG) control) coverage

calreticulin precursor (Calr) 48136 4.33 0.5 e 300 9 1E-06 metabolic process, calcium-binding protein Regucalcin (Rgn)/Smp30 33899 5.15 e 1.65 159 10 0.0002 cellular process, localization, metabolic process, binding, catalytic activity ATP synthase subunit beta, 57976 5.80 1.7 e 73 2 0.0001 localization, metabolic process, ATP synthase, anion mitochondrial (Atp5b) channel, ligand-gated ion channel, DNA binding proteinhydrolase indolethylamine N- 30068 6.00 e 2.14 136 9 0.0022 catalytic activity, tetabolic process methyltransferase(Inmt)/ (Temt) Glycine N- 33110 7.10 1.8 e 202 12 0.015 metabolic process, methylation methyltransferase(Gnmt) Electron Transfer 35360 8.62 1.35 e 179 9 1.4E 05 metabolic process, catalytic activity, Flavoprotein Alpha Polypeptide(ETFa)/Tead2 Selenium-binding liver 52889 5.97 0.39 e 109 3 3.1e-07 brown fat cell differentiation, protein transport protein (Selenbp1)/Lpsb

Fig. 5. High enough resolution and 3D report of some labeled spots in control and functionalized SWCNTs groups. (*) compared with control. (#) compared with PEG or Tween 80. death. In the PEGylated SWCNTs treated groups, upregulation of induced by various signaling factors [42,43]. Rgn was observed. Rgn has a multifunctional role in cell regulation A slight (135%) increase in the protein expression of Etf-a was through a mechanism related to the processes of Ca2þ dependent reported in this study, this protein acts as a receptor for different and independent signaling [30,32]. Rgn is translocated into the electron dehydrogenizes and deﬁciency in Etf-a can lead to the nucleus and regulates nuclear function in cells. glutamic aciduria [44]. Etf-a as an electron carrier is attached to the Rgn can prevent cell proliferation and apoptotic cell death dehydrogenase and involved in fatty acid oxidation [45]. 206 H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209

Fig. 6. Biological process classification of identified protein species by the PANTHER classification system: a) Tween80 suspended SWCNTs. b) PEGylated SWCNTs.

PEGylated SWCNTs also increased the expression of Gnmt. Gnmt The expression of INMT was increased in both Tween80 sus- is a detoxifying enzyme and its expression often is decreased in pended SWCNTs and PEGylated SWCNTs treated groups. Evidence hepatocellular carcinoma [46]. It can regulate SAM (S adenosyl showed that INMT may have a role in xenobiotic detoxification, methionine) levels by consuming SAM to produce sarcosine in the therefore it may be suggested the INMT upregulation is involved in liver [47,48]. Overexpression of this protein is related to decreased the detoxification of compounds and other metabolites such as expression of oncogenes. SWCNTs. Downregulation of Carl was also observed in PEGylated SWCNTs The last differentially expressed protein was Selenbp1 that is a group. It has been implicated that Carl participates in different member of selenoprotein's family. Selenbp1 reduction was cellular processes, including apoptotic cell clearance, cell adhesion, observed in both treatment groups. That may be due to the and cell migration [49e51]. increased GPX1 levels [56]. ATP5B is down regulated in some cancers, in addition, it in- creases the expression of some glycolytic pathway markers 5. Conclusion [52e54]. This enzyme catalyzed oxidation of metabolic substances such as glucose, fatty acids and amino acids The decreased The results of acute toxicological study on two functionalized expression of ATP5B was observed in the Tween80 suspended SWCNTs did not show any significant toxicity at selected doses. SWCNTs treated group while in PEGylated SWCNTs treated group Although some histopathological damages were observed in both ATP5b had an over-expression which can increase the cellular groups especially in Tween 80 suspended SWCNTs which may be content of ATP. It can also suppress gluconeogenesis [55]. Based on due to the activation of immune system. Proteomics analysis also the KEGG pathway, this protein mapped to oxidative phosphory- showed that following exposure to functionalized SWCNTs, the lation pathway and was involved in Parkinson, Huntington and expression of some proteins with antioxidant activity and detoxi- Alzheimer diseases. fying properties were increased in liver tissue. One of these H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209 207

Fig. 7. Proteineprotein interaction maps of Tween 80 suspended SWCNTs associated protein species identiﬁed in this study.

Fig. 8. Proteineprotein interaction maps of PEGylated SWCNTs associated protein species identiﬁed in this study.

proteins whose increased expression in Tween 80 suspended Acknowledgements SWCNTs and confirmed by western blotting, was Prdx6. Financial support of this study was provided by Mashhad Uni- versity of Medical Sciences (grant number: 910683). The results Conflict of interest reported in the present study are part of the M.Sc. thesis of H. Ahmadi. All authors declare that there are no potential conflicts of All authors disclose any actual or potential conflict of interest. interest. 208 H. Ahmadi et al. / Chemico-Biological Interactions 275 (2017) 196e209

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