Introduction

Nanoparticles and materials are being rapidly produced in large quantities throughout the world. Widespread application of nanomaterials confers enormous potential for human exposure and environmental release. Recently, however, scientists and organizations have raised the environmental and other safety concerns about nanotechnology [1–3]. As new types of photo-catalyst, anti-ultraviolet light agents, and photoelectric effect agents, titanium dioxide (TiO2) nanoparticles are used in a variety of consumer products (such as toothpastes, sunscreens, cosmetics, food products) [4], paints and surface coatings [5], and in the environmental decontamination of air, soil, and water [6, 7]. As an ultrafine-sized material, the nanoparticulate TiO2 can enter the human body through various routes such as inhalation (respiratory tract), ingestion (gastrointestinal tract), dermal penetration (skin), and injection (blood circulation) [8, 9].

Although little is known about nanoparticulate TiO2 toxicity, oxidative stress, which elicits a wide variety of cellular events, such as apoptosis, cell cycle arrest, and the induction of antioxidant enzymes, has often been reported as nanoparticulate TiO2- induced toxicity. Numerous previous studies on nanoparticulate TiO2 toxicity, with various animal organ types, such as lung, gill, brain, liver, and kidney, have reported that oxidative stress is one of the most important toxicity mechanisms related to the exposure to nanoparticulate TiO2 [10–14]. Nanoparticulate TiO2 was demonstrated to damage the haemostasis blood system and immune responses in mice [15]. The reduction of immune responses of mice caused by nanoparticulate anatase TiO2 may be due to the spleen damage. Spleen is the largest immune organ in humans, participating in immune response, generating lymphocytes, eliminating aging erythrocytes, and storing blood. However, we need to be focused on whether nanoparticulate TiO2 induces pathological changes to spleen and how nanoparticulate TiO2 damages spleen.

In order to further elucidate the molecular mechanism of nanoparticulate TiO2-induced oxidative stress in the mouse spleen, the evaluation in the toxicity needs to be focused on involving the oxidative stress responding signal transduction pathway and transcription factors caused by nanoparticulate TiO2. The studies of the upstream signaling mechanism responsible for regulating oxidative stress have been focused on the mitogen-activated protein (MAP) kinase cascades, including p38 and c-Jun N-terminal kinase (JNK) [16]. However, the upstream signaling mechanism responsible for regulating the oxidative stress involved in nanoparticulate TiO2 toxicity is rarely reported. The previous studies demonstrated that JNKs and p38 MAP kinases involving MAP kinase cascades are preferentially activated by various stresses, such as X-ray or UV irradiation, heat or osmotic shock, and oxidative or nitrosative stress [17–20]. Moreover, redox-sensitive transcription factors, such as nuclear factor kB (NF-κB) and nuclear factor-E2-related factor-2 (Nrf-2), can also be evaluated as target transcription factors of nanoparticulate TiO2 toxicities. NF-κB, activated by oxidative stress, induces the expression of a variety of proteins that function in the immunological and cellular detoxifying defense systems [21, 22] and has been identified as a transcription factor regulated by the intracellular redox status [23]. When activated by oxidative stress, Nrf-2 breaks free from Kelchlike ECH-associated protein 1 (Keap1) and translocates into the nucleus, where it binds to an antioxidant response factor, a cis-acting enhancer sequence that mediates the transcriptional activation of genes in response to oxidative stress, including heme oxygenase-1 (HO-1) [24]. Heme oxygenases (HO) are rate-limiting enzymes that catalyze the conversion of heme into carbon monoxide and biliverdin [25]. They have antioxidant capacity and therefore act as potent anti-inflammatory proteins whenever oxidative injury takes place [26]. HO-2 is constitutively produced within the brain and testes, whereas HO-1 is produced ubiquitously, but only marginally in the resting state [27]. Rapid induction of HO-1 follows various stresses [28–30]. The previous studies have suggested protective roles of HO-1 in various inflammatory conditions [31, 32]. Many studies have suggested that the transcription factor Nrf-2 plays an essential role in the antioxidant response factor-mediated expression of phase II detoxifying and antioxidant enzymes, as well as other stress-inducible genes, in response to oxidative stress [33–38].

In this article, the spleen pathological changes, the oxidative stress, the expression levels of the oxidative stress genes and their proteins including p38, JNK, NF-κB, Nrf-2, and HO-1 in the mouse spleen were investigated to further understand mechanism of the splenic injury in mice caused by nanoparticulate TiO2.

Materials and Methods

Chemicals and Preparation

TiO2 (100% anatase, CAS #:13463-67-7, VK-TA05) was purchased from Hangzhou Wanjing New Material Co. Ltd. (Hangzhou, China) (the particle characteristics are shown in Table 1) and the particles were used in this experiment.

Table 1 Particle Characteristics of Nanoparticulate TiO2
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A 0.5% hydroxypropyl-methylcellulose K4M (HPMC, K4M) was used as a suspending agent. Each TiO2 powder was dispersed onto the surface of 0.5%, w/v HPMC solution, and then the suspending solutions containing TiO2 particles were treated by ultrasonic for 15–20 min and mechanically vibrated for 2 or 3 min.

Animals and Treatment

Eighty CD-1 (ICR) female mice (22 ± 2 g) were purchased from the Animal Center of Soochow University. Animals were housed in stainless steel cages in a ventilated animal room. Room temperature was maintained at 20 ± 2°C, relative humidity at 60 ± 10%, and a 12-h light/dark cycle. Distilled water and sterilized food for mice were available ad libitum. They were acclimated to this environment for 5 days prior to dosing. All animal procedures were performed in compliance with the regulations and guidelines of the international ethics committee on animal welfare. Animals were randomly divided into four groups: control group (treated with 0.5% HPMC) and three experimental groups (5, 50, and 150 mg/kg BW nano-anatase TiO2). Nanoparticulate anatase TiO2 (5, 50, and 150 mg/kg BW) suspensions were given to mice by intragastric administration every day for 30 days, respectively. The control group was treated with 0.5% HPMC. The symptom and mortality were observed and recorded carefully everyday for 30 days. After 30 days, all animals were first weighed and then sacrificed after being anesthetized by ether. The spleens were excised and weighed.

Histopathological Examination of Spleen

Histological observations were performed according to the standard laboratory procedures. Mice (four mice/control and three treatment groups) at the end of day 30 were dissected for histology. A small piece of spleen, fixed in 10% (v/v) formalin, was embedded in a paraffin block, sliced into 5 μm thicknesses and then placed onto glass slides. The section was stained with hematoxylin–eosin (HE) and examined by optica microscopy (Nikon U-III Multi-point Sensor System: Biodirect-Inc., Nikon, USA), and the identity and analysis of the pathology slides were blind to the pathologist.

ROS and Lipid Peroxidation Assay

ROS Assay

Superoxide ion (O ·–2 ) in spleen tissue was measured as described previously by Oliveira et al. [39], by determining the reduction of 3′-[1-[phenylamino-carbonyl]-3,4- tetrzolium]-bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) in the presence of O ·–2 , with some modifications. The spleen was homogenized with 2 mL of 50 mM Tris–HCl buffer (pH 7.5) and centrifuged at 5,000×g for 10 min. The reaction mixture (1 mL) contained 50 mM Tris–HCl buffer (pH 7.5), 20 μg spleen proteins, and 0.5 mM XTT. The reaction of XTT was determined at 470 nm for 5 min. Corrections were made for the background absorbance in the presence of 50 units of superoxide dismutase (SOD). The production rate of O ·–2 was calculated using an extinction coefficient of 2.16 × 104 M–1 cm–1.

The detection of H2O2 contents in the liver tissues was carried out by the xylenol orange assay [40], with minor modifications. In short, after the preincubation of P2 with mercurials and/or quercetin/catalase (30 min at 25°C), the reaction medium was centrifuged at 17,500×g for 10 min at 4°C and the supernatant was incubated for 30 min in a reaction medium containing 250 mM perchloric acid, 2.5 mM ammonium iron (II) sulfate hexahydrate, and 1 mM xylenol orange. Hydroperoxide levels were determined at 560 nm using a hydrogen peroxide curve as standard.

Lipid Peroxidation

Spleen lipid peroxidation was determined as the concentration of malondialdehyde (MDA) generated by the thiobarbituric acid (TBA) reaction as described by Buege and Aust [41], but with the introduction of an isobutanol extraction step for the removal of interfering compounds. For analysis, a subsample of tissue was thawed, homogenized, and cells lysed using a 4% TBA solution in 0.2M HCl. The reaction mixture was then incubated at 90°C for 45 min. The resulting TBA–MDA adduct was phase-extracted using isobutanol. The isobutanol phase was then read at a wavelength of 535 nm on a UV-3010 spectrophotometer. MDA standard curves were prepared by acid hydrolysis of 1,1,3,3-tetramethoxypropane.

Expression Assay of Oxidative Stress Genes and Proteins

The mRNA expression of regulating the oxidative stress genes, including p38, JNK, NFR2, NF-κB, and HO-1, were determined by real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) [42–44]. Spleens in the same growth period from the three different treatments were used. The right spleens from mice with or without nano-anatase TiO2 treatment were homogenized using QIAzol lysis reagent with a Tissue Ruptor (Roche, Indianapolis, IN). Total RNA from the homogenates was isolated using Tripure Isolation Reagent (Roche) according to the manufacturer’s instructions. The RT reagent (ShineGene: Shanghai Shinegene Co., China) of 30 μL was prepared by mixing 15 μL of 2 × RT buffer, 1 μL random primer in a concentration of 100 pmol·μL−1, 1 μL of RTase, 5 μL RNA, and 8 μL DEPC water together. The reaction condition was 25°C for 10 min, 40°C for 60 min, and 70°C for 10 min.

Synthesized cDNA was used for the real-time PCR. Primers were designed using Primer Express Software according to the software guidelines (Table 2).

Table 2 Real-time PCR Primer Pairs
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All primers were purchased from ShineGene. For the 50-μL PCR reaction, 25 μL 2× PCR buffer, 0.6 μL 2× primers (25 pmol·μL−1), 0.3 μL probe (25 pmol·μL−1), 1 μL cDNA, and 22.8 μL DEPC water (Sigma) were mixed together. The parameters for a two-step PCR were 94°C for 3 min, 94°C for 20 s, 60°C for 20 s, then 72°C for 20 s, 35 cycles.

The gene expression analysis and experimental system evaluation were performed according to the standard curve and quantitation reports.

To determine p38, JNK, NF-κB, Nfr2, and HO-1 levels of the spleen, enzyme-linked immunosorbent assay (ELISA) was performed by using commercial kits that are selective for the mouse spleen p38, JNK, NF-κB, Nfr2, and HO-1 (RD Systems, Minneapolis, USA). The manufacturer’s instruction was followed. The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash, Thermo Electron, Finland), and the p38, JNK, NF-κB, Nfr2, and HO-1 concentration of samples was calculated from a standard curve.

Statistical Analysis

Statistical analyses were done using SPSS11.5 software. Data were expressed as means ± SE. One-way analysis of variance was carried out to compare the differences of means among multigroup data. Dunnett’s test was carried out when each group of experimental data was compared with solvent-control data. Statistical significance for all tests was judged at a probability level of 0.05.

Result

Histopathological Evaluation

The histological photomicrographs of the spleen sections are shown in Fig. 1. No severe damages of spleen tissue were reflected in the 5 mg/kg nanoparticulate anatase TiO2-treated group (Fig. 1b) compared to the control (Fig. 1a), while the congestion of the spleen tissue was showed in the 50 mg/kg nanoparticulate anatase TiO2 group (Fig. 1c) and lymph nodule proliferation was observed in the 150 mg/kg nanoparticulate anatase TiO2-treated group (Fig. 1d). We observed histological photomicrographs of the spleens of four mice of each group, indicating that the 50 and 150 mg/kg nanoparticulate anatase TiO2 caused histological changes in the same manner.

Fig. 1
figure 1

Histopathological observation of spleen caused by intragastric administration with nanoparticulate anatase TiO2 for 30 days (HE, ×original magnification). a Control group (×100) presents no abnormal pathology; b 5 mg/kg nanoparticulate TiO2 group (×200) presents no abnormal pathology; c 50 mg/kg nanoparticulate TiO2 group (×200) presents congestion (arrow); d 150 mg/kg nanoparticulate group (×100) presents lymph nodule proliferation (arrows)

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ROS Production and Lipid Peroxidation

The effects of treatments with various doses of nanoparticulate TiO2 on the accumulation of O2 · −, H2O2, and the MDA content in the mouse spleen are shown in Figs. 2 and 3, respectively. The significant differences were observed the production rate of ROS and the MDA content of the spleen caused by nanoparticulate TiO2 (p < 0.05 or 0.01), e.g. O · −2 was increased by 29.38%, 75.41%, and 145.01%, respectively; H2O2 was promoted by 25.82%, 57.84%, and 109.53%, respectively; MDA was enhanced by 29.36%, 63.65%, and 191.19%, respectively. This phenomenon showed that higher-dose nanoparticulate TiO2 promoted the generation of oxidative stress and caused lipid peroxidation in the mouse spleen.

Fig. 2
figure 2

The ROS accumulation in the mouse spleen after intragastric administration with nanoparticulate TiO2 for 30 days. Bars marked with an asterisk or double asterisk indicate significant difference from the control (no nanoparticules) at the 5% or 1% confidence level, respectively. Values represent means ± SE, n = 5

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Fig. 3
figure 3

The MDA content of spleen of ICR mice after intragastric administration with nanoparticulate TiO2 for 30 days. Bars marked with double asterisk indicate significant difference from the control (no nanoparticules) at the 1% confidence level. Values represent means ± SE, n = 5

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MAP Kinase Signal Transduction and Transcription Factors

The damage happened in the spleen according to the histopathological observations. To confirm expression levels of the oxidative stress genes and their proteins including p38, JNK, NF-κB, Nrf-2, and HO-1 in the nanoparticulate TiO2-induced spleen injury, real-time quantitative RT-PCR and ELISA were used to demonstrate the changes of the oxidative stress genes and their proteins levels in the nanoparticulate TiO2-treated mice (Tables 3 and 4). In Tables 3 and 4, we observed that nanoparticulate TiO2 induced a dramatic increase of p38, JNK, NF-κB, Nrf-2, and HO-1 expression in the treated mouse spleen (P < 0.05 or 0.01). The extents of increase of these genes and their proteins are consistent with the trends on the histological photomicrograph of the spleen sections in the exposed mice.

Table 3 Effect of Nanoparticulate TiO2 on the Amplification of Regulating the Oxidative Stress Gene mRNA of Mouse Spleen by Real-time PCR Analysis After Intragastric Administration with Nanoparticulate TiO2 for 30 days
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Table 4 Effect of Nanoparticulate TiO2 on Regulating the Oxidative Stress-Protein Level of Mouse Spleen by ELISA ANalysis After Intragastric Administration with Nanoparticulate TiO2 for 30 days
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Discussion

Wang et al. [45] observed that the coefficients of the mouse spleen increased only a little, and there are no abnormal pathology changes in the spleen after a fixed large dose of 5 g/kg BW of nanoparticulate TiO2 (25 and 80 nm) was administered by a single oral gavage for 2 weeks. However, Chen et al., by histopathological examinations, observed that nano-TiO2 particles caused a severe lesion of spleen and a mass of neutrophilic cells in spleen tissues by intraperitoneal injection, revealing that inflammation in spleen tissues was very serious [46]. Our researches also observed that the coefficients of the spleen of mice significantly increased by intragastric administration or intraperitoneal injection of higher doses of nanoparticulate TiO2 (5 nm) for 30 or 14 days [15, 47]. The results of this study indicate that the intragastric administration of higher doses of nanoparticulate anatase TiO2 can induce histopathological changes of spleen, including the congestion and lymph nodule proliferation, suggesting an inflammation in spleen tissues (Fig. 1). The present study indicates that the spleen lesion of mice is triggered by nanoparticulate TiO2 oxidative stress generation and activation of the oxidative stress genes that resulted in inflammation of spleen tissue.

To prove spleen oxidative stress of mice, we detected ROS and MDA contents. The studies showed that the obvious production of ROS (such as O .–2 and H2O2) and lipid peroxidation (MDA content increased) occurred in the mouse spleen treated with higher nanoparticulate anatase TiO2 doses (Figs. 2 and 3), indicating that these nanoparticulate anatase TiO2-treated mouse spleen underwent severe oxidative stress. Similarly, nanoparticulate anatase TiO2 was reported to cause oxidative stress in the mouse brain, liver and kidney [12–14]. It has been demonstrated that nanoparticules are mediating their toxicity through production of ROS and that the level of ROS depends on the chemistry and structure of the nanoparticules [4, 9, 10, 48, 49]. The overproduction of ROS would break down the balance of the oxidative/antioxidative system in the spleen, resulting in the lipid peroxidation, which is closely related to the reduction of the antioxidative capacity. In our previous studies, nanoparticulate anatase TiO2 inhibited the activities of SOD, catalase, ascorbic acid peroxidase, and glutathione peroxidase, and decreased ascorbic acid and reduced glutathione in the mouse brain, liver, and kidney [12–14]. In fact, any chemical damage is based on the physical binding interaction. Some intermolecular action mechanisms were also demonstrated to be related to the oxidative stress [50, 51].

To further understand the mechanism of oxidative stress due to exposure to nanoparticulate TiO2, the detection of molecular events, such as MAP kinase signal transduction and subsequent transcription factor activation (Tables 3 and 4), was undertaken. The expressions of p38 and JNK were significantly induced by nanoparticulate TiO2. The overall results for ROS formation and on MAP kinase signaling studies suggested that nanoparticulate TiO2 provokes oxidative stress, and in response to this, mainly the p38 and JNK MAP kinase signaling pathway seems to be activated. As a downstream event of MAP kinase signaling, NF-κB and Nrf-2 transcription factors, which are known to respond to oxidative stress, were examined in the mouse spleen treated with nanoparticulate TiO2 (Tables 3 and 4). Nanoparticulate TiO2 induced the expressions of NF-κB and Nrf-2. To further investigate the cellular consequences of oxidative stress signaling through p38-Nrf-2 activation by nanoparticulate TiO2 exposure, representative antioxidant enzymes, such as HO-1, were examined (Tables 3 and 4). HO-1 is a relatively novel enzyme, with potent anti-inflammatory and cytoprotective antioxidant effects [52–55]. The expression of HO-1 protein was dramatically increased in nanoparticulate TiO2-treated mice. The induction of HO-1 due to exposure to nanoparticulate TiO2 may be mediated through p38 MAP kinase and the Nrf-2 signal transduction pathway. Recently, Lim et al. reported that the cyclopentenone prostaglandin compound stimulates HO-1 expression through the p38 MAP kinase and Nrf-2 pathway in rat vascular smooth muscle cells [56, 57]. Induction of HO-1 can be interpreted as a cellular defense mechanism against oxidative stress; it is well known that HO-1 induction is regulated by Nrf-2 activation [24]. In this study, nanoparticulate TiO2-induced NF-κB activation was not observed, which was unexpected, as NF-κB is the major stress response transcription factor and has been reported to respond to a wide variety of environment stressors.

Conclusion

Overall, the results of experimental studies suggest that nanoparticulate TiO2 may exert their toxicity through oxidative stress. Nanoparticulate TiO2 causes congestion and lymph nodule proliferation of spleen tissue of mice and a significant increase in the spleen ROS productions, and subsequently leads to a strong induction of HO-1 via the p38-Nrf-2 signaling pathway. The tested oxidative stress parameters in this study were rather limited in terms of allowing a full understanding of the oxidative stress and spleen response due to exposure to nanoparticulate TiO2. Further studies on the mechanism by which nanoparticulate TiO2 induce the p38-Nrf-2 signaling pathway to better understand the nanoparticulate TiO2-induced oxidative stress, as well as with concentration– response and time-course analyses, are warranted.