Journal of Hazardous Materials 278 (2014) 180–188

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Nano-sized titanium dioxide-induced splenic toxicity: A biological pathway explored using microarray technology Lei Sheng a,1 , Ling Wang b,1 , Xuezi Sang a,1 , Xiaoyang Zhao a,1 , Jie Hong a , Shen Cheng a , Xiaohong Yu a , Dong Liu a , Bingqing Xu a , Renping Hu a , Qingqing Sun a , Jie Cheng a , Zhe Cheng a , Suxin Gui a , Fashui Hong a,∗ a b

Medical College of Soochow University, Suzhou 215123, China Library of Soochow University, Suzhou 215123, China

h i g h l i g h t s • • • •

Exposure to TiO2 Exposure to TiO2 Exposure to TiO2 Exposure to TiO2

a r t i c l e

NPs could be accumulated in the spleen. NPs caused spleen lesions in mice. NPs resulted in immune dysfunction in mice. NPs caused alteration of 1041 genes expression of known function in the spleen.

i n f o

Article history: Received 10 November 2013 Received in revised form 1 June 2014 Accepted 5 June 2014 Available online 12 June 2014 Keywords: Titanium dioxide nanoparticles Spleen injury Splenic dysfunction Gene-expressed profile

a b s t r a c t Titanium dioxide nanoparticles (TiO2 NPs) have been widely used in various areas, and its potential toxicity has gained wide attention. However, the molecular mechanisms of multiple genes working together in the TiO2 NP-induced splenic injury are not well understood. In the present study, 2.5, 5, or 10 mg/kg body weight TiO2 NPs were administered to the mice by intragastric administration for 90 consecutive days, their immune capacity in the spleen as well as the gene-expressed characteristics in the mouse damaged spleen were investigated using microarray assay. The findings showed that with increased dose, TiO2 NP exposure resulted in the increases of spleen indices, immune dysfunction, and severe macrophage infiltration as well as apoptosis in the spleen. Importantly, microarray data showed significant alterations in the expressions of 1041 genes involved in immune/inflammatory responses, apoptosis, oxidative stress, stress responses, metabolic processes, ion transport, signal transduction, cell proliferation/division, cytoskeleton and translation in the 10 mg/kg TiO2 NP-exposed spleen. Specifically, Cyp2e1, Sod3, Mt1, Mt2, Atf4, Chac1, H2-k1, Cxcl13, Ccl24, Cd14, Lbp, Cd80, Cd86, Cd28, Il7r, Il12a, Cfd, and Fcnb may be potential biomarkers of spleen toxicity following exposure to TiO2 NPs. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Nanotechnology has been widely applied in industry, daily life, agriculture, etc. For example, silver nanoparticles can be used for fruit and vegetable preservation [1]; nano-copper particles are used as an additive in lubricants and plastics [2]. Particularly, titanium dioxide nanoparticles (TiO2 NPs), owning excellent photocatalytic properties, anticorrosion and high stability [3,4], are used in paints, sunscreen cosmetics, food additive, drugs, antiseptics, etc.

∗ Corresponding author. Tel.: +86 512 61117563; fax: +86 512 65880103. E-mail address: Hongfsh [email protected] (F. Hong). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jhazmat.2014.06.005 0304-3894/© 2014 Elsevier B.V. All rights reserved.

[5]. However, their applications are also accompanied by questions and worries about the security [6,7]. Workers in the production of TiO2 NP industry are susceptible to lung cancer [8,9]. Furthermore, numerous studies have shown that TiO2 NP exposure resulted in the damages in liver [10–17], kidney [18–21], lung [22–25], brain [26–31], ovary [32,33] and testis [34] of animals, including inflammation and apoptosis. These organic damages may be associated with the immune dysfunction. As we know, the spleen, an organ which interposes in the bloodstream and stands as the body’s largest blood filter, in addition, most important immune reactions about antifungal/antibacterial occur in it. The immune system of spleen is responsible for protecting the organism from invading pathogens, detecting and removing unwanted cells, mechanically damaged, or aberrant cells

L. Sheng et al. / Journal of Hazardous Materials 278 (2014) 180–188

that might lead to tumor formation in the processes of innate and adaptive immunity [35]. Therefore, numerous studies investigated adverse effects of TiO2 NPs on the spleen. For example, TiO2 NP exposure was demonstrated to result in the neutrophilic cell proliferation [36], lymph nodule proliferation [37], splenic apoptosis [38], disperative replication of white pulp, anemia of red pulpin [39], and macrophage infiltration [40,41] in the mouse spleen, leading immune dysfunction. Furthermore, these studies mentioned above suggested that TiO2 NP-induced splenic damages were closely associated with oxidative stress, P38-Nrf-2 signaling pathway [37], mitochondrion-mediated intrinsic apoptotic pathway [38] and NF-␬B-mediated inflammatory pathways as well as MAPKs/PI3-K/Akt signaling pathways [39–41]. Taken together, the mechanisms of splenic damages caused by TiO2 NP exposure are complex and diverse, we hypothesized that TiO2 NP-induced splenic toxicity in animals may have special biomarkers of toxicity. Microarray technology has been used as a screening tool for the identification of molecular mechanisms involved in toxicity. In the present study, we aimed to investigate splenic dysfunction caused by TiO2 NP exposure and alteration in the gene expression profile in the mouse spleen using microarray analysis. The data on the gene expression profile showed significant changes in genes involving immune/inflammatory responses, apoptosis, oxidative stress, stress responses, metabolic processes, ion transport, signal transduction, cell proliferation/division, cytoskeleton and translation in the TiO2 NP-exposed spleen. Our findings will provide further insight into the splenic toxicity and multiple mechanisms of action of TiO2 NPs. 2. Methods 2.1. Chemicals Anatase TiO2 NPs were prepared via controlled hydrolysis of titanium tetrabutoxide. Details of the synthesis and characterization of TiO2 NPs were described in our previous reports [20,42]. The average particle size of powdered TiO2 NPs suspended in 0.5% (w/v) hydroxypropylmethylcellulose (HPMC) K4M (Sigma–Aldrich, St. Louis, MO, USA) solvent that the HPMC solution prepared with deionized and distilled water after 12 h incubation was 5–6 nm and the surface area of the sample was 174.8 m2 /g. The mean hydrodynamic diameter of the TiO2 NPs in HPMC solvent was 294 nm (range, 208–330 nm) and the zeta potential after 12 h incubation was 7.57 [20]. 2.2. Animals and treatment One hundred sixty CD-1 (ICR) female mice (24 ± 2 g) were purchased from the Animal Center of Soochow University (China). The mice were housed in stainless steel cages in a ventilated animal room. The room temperature in the housing facility was maintained at 24 ± 2 ◦ C, with a relative humidity of 60 ± 10% and a 12-h light/dark cycle. Distilled water and sterilized food were available ad libitum. Before treatment, the mice were acclimated to this environment for five days. All the animals were handled in accordance with the guidelines and protocols approved by the Care and Use of Animals Committee of Soochow University (China). TiO2 NP powder was dispersed onto the surface of 0.5% (w/v) HPMC and the suspension containing TiO2 NPs was treated ultrasonically for 30 min and mechanically vibrated for 5 min. The mice were randomly divided into four groups (n = 40 each), including a control group treated with 0.5% (w/v) HPMC and three experimental groups treated with 2.5, 5, or 10 mg/kg TiO2 NPs body weight (BW). For dose selection, we consulted the report of World Health Organization in 1969. According to the report, LD50 of TiO2 for rats

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is >12 g/kg BW after oral administration. In addition, the quantity of TiO2 NP does not exceed 1% by weight of the food according to US Federal Regulations. In the present study, we selected 2.5, 5, or 10 mg/kg TiO2 NPs, which were given to mice by intragastric administration every day. They were equal to about 0.15–0.7 g TiO2 NPs for humans with 60–70 kg body weight for humans with such exposure, which are relatively safe doses. The mice were weighed, volume of TiO2 NP suspensions was calculated for each mouse, and the fresh TiO2 NP suspensions were administered to the mice by intragastric administration every day for 90 days. Any symptom including growth state, eating, drinking and activity, or mortality was observed and recorded carefully everyday during the 90 days. 2.3. Spleen indices After 90 days, mice were weighed and then sacrificed after ether anesthesia. Blood samples were collected from the eye vein by rapidly removing the eyeball. The spleens of all animals were quickly removed and placed on ice. After weighing the body and spleens, the spleen indices (n = 10 each) were calculated as the ratio of spleen (wet weight, mg) to body weight (g). 2.4. Titanium content analysis The frozen spleen tissues (n = 5 each) were thawed and approximately 0.1 g samples were weighed, digested, and analyzed for titanium content using inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7; Thermo Electron Co., Waltham, MA, USA). Indium (20 ng/mL) was chosen as an internal standard element. 2.5. Histopathological evaluation of spleen All histopathological examinations were performed using standard laboratory procedures. Five sets of spleen tissues from mice were embedded in paraffin blocks, sliced to 5-␮m thickness, and placed on separate glass slides (five slices from each spleen). After hematoxylin–eosin staining, the sections were evaluated by a histopathologist unaware of the treatments, using an optical microscope (U-III Multi-point Sensor System; Nikon, Tokyo, Japan). 2.6. Observation of splenocyte ultrastructure The spleens (n = 5 each) were fixed in a fresh solution of 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2 h fixation period at 4 ◦ C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2–7.4). Staining was performed overnight with 0.5% aqueous uranyl acetate. The specimens were dehydrated in a graded series of ethanol (75%, 85%, 95%, and 100%), and embedded in Epon 812. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate, and observed with a HITACHI H600 TEM (Hitachi Co., Japan). Splenocyte apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation). 2.7. Confocal Raman microscopy in spleen sections Raman analysis was performed using backscattering geometry in a confocal configuration at room temperature using a HR-800 Raman microscope system equipped with a 632.817 nm HeNe laser (JY Co., Fort-de-France, Martinique). Reportedly, when the size of TiO2 NPs reached 6 nm, the Raman spectral peak was 148.7 cm−1 [43]. Laser power and resolution were approximately 20 mW and 0.3 cm−1 , respectively, while the integration time was adjusted to 1 s. The spleen specimens (n = 5 each) were embedded in paraffin blocks, sliced into 5-␮m thicknesses, and placed onto glass slides.

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The slides were dewaxed, hydrated, and then scanned using the confocal Raman microscope.

Table 1 Real time PCR primer pairs. PCR primers used in the gene expression analysis. Gene name

Description

Primer sequence

Primer size (bp)

Refer-actin

mactin-F mactin-R

5 -GAGACCTTCAACACCCCAGC-3 5 -ATGTCACGCACGATTTCCC-3

263

Cfd

mCfd-F mCfd-R

5 -AGCAACCGCAGGGACACTT-3 5 -TTTGCCATTGCCACAGACG-3

108

Lbp

mLbp-F mLbp-R

5 -ACTTGACTCCGTCCCATCCTC-3 5 -ACCACAGTGCCCGCTCTTA-3

2.8. Hematological parameters determination Blood samples (n = 5 each) were collected in tubes containing EDTA as anticoagulant. Red blood cells (RBC), lymphocytes (LYMPH), white blood cells (WBC), hemoglobin (HGB), platelets (PLT), and neutrophilic granulocyte (NEUT) were measured using a hematology autoanalyzer (Cell-DYN 3700). 2.9. Cytometric assay of lymphocyte subsets in peripheral blood To identify and quantify lymphocyte subsets, cell suspensions (n = 5 each) were analyzed by flow cytometry. Following red blood cell lysis, cells were stained with anti-mouse monoclonal antibodies against CD3, CD4, CD8, and CD19 (BD Biosciences). Cells were analyzed via four-color flow cytometry on a FACSCaliber (BD Biosciences) in the University of Soochow Immunological Research Center Facility. Lymphocyte subsets, including B cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, double positive thymocytes, and double negative thymocytes, were analyzed. 2.10. Microarray and data assay Gene expression profiles of spleen tissues isolated from mice (n = 5 each) in the control and TiO2 NPs-treated groups were compared by microarray analysis using Illumina BeadChips purchased from Illumina, Inc. (San Diego, CA, USA). Total RNA was isolated using the Ambion Illumina RNA Amplification Kit [44] according to the manufacturer’s protocol, and stored at −80 ◦ C. RNA amplification is the standard method for preparing RNA samples for array analysis [45]. Total RNA was then submitted to Biostar Genechip Inc. (Shanghai, China) for RNA quality analysis using a BioAnalyzer, and cRNA was generated and labeled using the one-cycle target labeling method. cRNA from each mouse was hybridized for 18 h at 55 ◦ C on Illumina Human HT-12 v 3.0 BeadChips containing 45, 200 probes according to the manufacturer’s protocol and subsequently scanned using the Illumina BeadArray Reader 500 to identify differentially expressed genes and establish potential biological significance based on the Gene Ontology Consortium database (http://www.geneontology.org/GO.doc.html). Data analyses were performed with GenomeStudio software version 2009 (Illumina Inc.) by comparing all values obtained at each time point against the 0 h values. Data were normalized with the quantile normalization algorithm and genes were considered as detected if the detection p-value was