nanoparticles to mice after intraperitioneal injection

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1 Research Article Received: 8 August 2008, Revised: 3 December 2008, Accepted: 11 December 2008, Published online 20 January 2009 in Wiley Interscience ( DOI /jat.1414 In vivo acute toxicity of titanium dioxide John Wiley & Sons, Ltd. nanoparticles to mice after intraperitioneal injection Jinyuan Chen, a * Xia Dong, a Jing Zhao a and Guping Tang b ABSTRACT: Because of its excellent optical performance and electrical properties, TiO 2 has a wide range of applications in many fields. It is often considered to be physiologically inert to humans. However, some recent studies have reported that nano-sized TiO 2 may generate potential harm to the environment and humans. In this paper the in vivo acute toxicity of nano-sized TiO 2 particles to adult mice was investigated. Mice were injected with different dosages of nano-sized TiO 2 (0, 324, 648, 972, 1296, 1944 or 2592 mg kg 1 ). The effects of particles on serum biochemical levels were evaluated at various time points (24 h, 48 h, 7 days and 14 days). Tissues (spleen, heart, lung, kidney and liver) were collected for titanium content analysis and histopathological examination. Treated mice showed signs of acute toxicity such as passive behavior, loss of appetite, tremor and lethargy. Slightly elevated levels of the enzymes alanine aminotransferase and aspartate aminotransferase were found from the biochemical tests of serum whereas blood urea nitrogen was not significantly affected (P < 0.05). The accumulation of TiO 2 was highest in spleen (P < 0.05). TiO 2 was also deposited in liver, kidney and lung. Histopathological examinations showed that some TiO 2 particles had entered the spleen and caused the lesion of spleen. Thrombosis was found in the pulmonary vascular system, which could be induced by the blocking of blood vessels with TiO 2 particles. Moreover, hepatocellular necrosis and apoptosis, hepatic fibrosis, renal glomerulus swelling and interstitial pneumonia associated with alveolar septal thickening were also observed in high-dose groups. Copyright 2009 John Wiley & Sons, Ltd. Keywords: acute toxicity; nano-sized TiO 2 particle; mice; titanium content; histopathological examination; intraperitoneal injection 330 Introduction Nanomaterials are defined by the National Nanotechnology Initiative as substances that are in at least one dimension on the scale of approximately nm. In 2004, the Royal Society (2004) pointed out that nanomaterials are expected to improve virtually all types of products, and that the commercialization of nano-products that exploit these unique properties is increasing. Widespread application of nanomaterials confers enormous potential for human exposure and environmental release. Previously, nanoparticles have received less attention compared with compounds of same composition but larger size (Banfield and Navrotsky, 2001). Recently, however, scientists and organizations have raised the environmental and other safety concerns about nanotechnology (Dagani, 2003; Warheit, 2004; Service, 2003). Approximately 95% of titanium is used as titanium dioxide (TiO 2 ). TiO 2 is highly insoluble, thermally stable and nonflammable. Because of its excellent optical performance and electrical properties, TiO 2 has a wide range of applications. It was considered to be physiologically inert and to pose little risk to humans. However, TiO 2 may have undesirable or damaging effects on rodents when inhaled. For example, the inhalation of TiO 2 resulted in the development of lung tumor in rats after lifetime exposure to very high concentrations of pigment-grade TiO 2 (Lee et al., 1985). Recently, Wang et al. (2007a) found that ulatrafine (UF)- TiO 2 can induce significant cytotoxicity and genotoxicity in cultured human cells. In addition, TiO 2 has large surface-area-to-weight ratios (Oberdörster et al., 2005) and a high redox activity (Colvin, 2003). These characteristics are common causes of adverse effects on, or intrinsic toxicity to, human health and the environment. Because of these characteristics of TiO 2, a risk assessment of its potential adverse human health and environmental pollution is much needed. A research program to investigate inter-species differences as a result of exposure to TiO 2 and to conduct detailed epidemiological surveys of the major manufacturing sites was initiated by a consortium of TiO 2 manufacturers in Europe (under the European Chemistry Industry Council; CEFIC) and North America (under the American Chemistry Council; ACC). Some detailed results published from these studies showed distinct species differences in lung responses, particle distributions and clearance rates (Bermudez et al., 2002, 2004). Little information on the toxicological effects of TiO 2 has thus far been reported. A long-term study of its accumulation and the detailed mechanisms of its biological effects is clearly needed. In this study, mice were injected i.p. with different doses of TiO 2. The study was focused on where TiO 2 is distributed, how TiO 2 is transported to various organs in vivo and whether TiO 2 induces pathological changes to organs. In addition, the effects of TiO 2 on serum biochemical levels (blood urea nitrogen, alanine * Correspondence to: J. Chen, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, , People s Republic of China. cjy1128@zjut.edu.cn a College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, , People s Republic of China. b Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, , People s Republic of China. Copyright 2009 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2009; 29:

2 In vivo acute toxicity of titanium dioxide nanoparticles Table 1. Doses of nano-tio 2 used for exposure experiments Control Toxicity group Blood group T1 T2 T3 T4 T5 T6 B1 B2 B3 Volume (ml) Dose (mg) PBS Concentration (mg kg 1 ) PBS The concentration of TiO 2 suspension was 64.8 mg ml 1. aminotransferase, aspartate aminotransferase and alkaline phosphatase) were also determined. Materials and Methods Equipment and Chemicals Instruments used were a Rigaku (D/MAX-IIIB) X-ray diffractometer (XRD, Japan), a JEM-2010 (HR) transmission electron microscopy (TEM, Japan), a laser diffraction particle size analyzer (Mastersizer 2000, Malvern, UK), an automated biochemical analyzer (type 7170A, Hitachi, Tokyo) and an inductively coupled plasma-mass spectrometer (ICP-MS, Thermo Elemental X7, Thermo Electron Co.). Chemicals used were polyethylene glycol-4000 (PEG-4000, Shanghai-Pudong GaoNan Chemical Plant), butyl titanate [Ti(OC 4 H 9 ) 4 ], nitric acid, sodium hydroxide and anhydrous ethanol (Hangzhou Changzheng Chemical Plant, China). Preparation of TiO 2 suspension The TiO 2 suspension was prepared by sol gel method under acidic conditions. Ti(OC 4 H 9 ) 4 was used as the starting material. Its hydrolysis and polycondensation are shown below: Ti(OC 4 H 9 ) 4 +4H 2 O Ti(OH) 4 +4C 4 H 9 OH female mice in each group. Various doses of TiO 2 were chosen and labeled as T1, T2, T3, T4, T5, T6 and control, as shown in Table 1. Following exposure, the vital signs and mortality of mice in each group were recorded. Half of the mice in each group were killed on day 7 and the remaining half were killed on day 14. The tissues of heart, liver, spleen, lung and kidney were collected. Fractions of tissues and organs were kept in 10% (v/v) formalin for immediate histopathological examination; the remainder were stored at 65 C for measurement of TiO 2 distribution. Forty mice were used to evaluate 24 and 48 h liver and kidney functions. Mice were divided into four groups, a control group and three experimental groups with five male and five female in each group. Mice were exposed to TiO 2 with doses labeled as B1, B2, B3 and control (Table 1). Blood Biomarker Assay Blood samples were collected via the ocular vein. The serum was obtained by centrifugation of the whole blood at 3000 rpm for 15 min. Liver function was evaluated based on the serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Nephrotoxicity was determined by blood urea nitrogen (BUN). These biochemical parameters were determined by an automated biochemical analyzer. Ti(OH) 4 +Ti(OC 4 H 9 ) 4 2TiO 2 +4C 4 H 9 OH 2Ti(OH) 4 2 TiO 2 +4H2O The concentration of TiO 2 suspension prepared was determined by the constant weight method to be 64.8 mg ml 1. The TiO 2 sample was examined by TEM operating at 200 kv; the morphologies of particles were determined. The crystalline phases, structures and grain sizes of TiO 2 were analyzed by XRD. Animals and Treatments Imprinting Control Region (ICR) mice (about 4 weeks old and 20 ± 2 g in weight) were purchased from the Animal Experiment Center, Zhejiang Academy of Medical Sciences (Hangzhou, China). They were housed by sex in plastic cages with a stainless steel mesh lid in a ventilated room. The room was maintained at 20 ± 2 C and 60 ± 10% relative humidity, with a 12 h light dark cycle. Mice were fed on water and sterilized food. Prior to treatment, mice were kept fasted overnight. Seventy mice were divided into seven groups, i.e. a control group and six experimental groups, with five male and five Titanium Analysis Tissues ( g) were weighed, digested and analyzed for titanium. The digestion was performed overnight in ultrapure nitric acid. After adding 0.5 ml of H 2 O 2, the mixtures were heated on an electric heating plate until the tissues were completely digested and the remaining nitric acid was removed until the solutions were colorless and clear. The solutions were transferred with 5% (v/v) nitric acid and then diluted to 10 ml with ultra pure (ion free) water. Titanium was analyzed by ICP-MS. Indium of 20 ng ml 1 was chosen as the internal standard. The detection limit of titanium was ng ml 1. Histopathological Examination Histological observations were performed according to the standard laboratory procedures. Mice (four mice/treatment group) at the end of day 7 were dissected for histology. A small piece of spleen, lung, kidney or liver 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 light microscopy. 331 J. Appl. Toxicol. 2009; 29: Copyright 2009 John Wiley & Sons, Ltd.

3 J. Chen et al. Statistical Analysis For statistical analysis, each of the experimental values was compared with its corresponding control. Results were expressed as mean ± standard deviation (SD). Multigroup comparisons of the means were carried out by one-way analysis of variance (ANOVA) test. The Dunnett s test was used to compare the differences between the experimental groups and the control group. Statistical significance for all tests was set at P < Results Characterization of TiO 2 Particles The TEM showed that TiO 2 particles had a uniformly scattered situation with a nanocrystalline structure [Fig. 1(A)]. The selected area (electron) diffraction photograph [Fig. 1(B)] shows an obvious electron diffraction ring, which indicated that the nanomaterials were composed of nanocrystalline TiO 2. It suggests that nanosized anatase-tio 2 can be prepared with polyethylene glycol as the dispersant by the sol gel method. The XRD patterns of the TiO 2 in Fig. 2 shows all prominent peaks for the tetragonal crystal structure of anatase-tio 2. The calculation by the Scherrer equation indicates that the average crystal size of synthesized TiO 2 was 36 A or 3.6 nm. The size distribution of the particles indicated that the sizes of TiO 2 particles ranged between 80 and 110 nm, mostly being 100 nm; see Fig. 3. Vital Signs and Mortality The symptoms of exposed mice were observed in skin and fur, eye membranes and respiratory, circulatory, autonomic and Figure 1. TEM photographs of nano-sized TiO 2 particles. (A) TiO 2 particles show a uniform scattered situation. Bar = 50 nm. (B) An obvious electron diffraction ring indicates the nanomaterials were composed of high-purity nanocrystalline particles. 332 Figure 2. XRD pattern of nano-sized TiO 2 particles. Copyright 2009 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2009; 29:

4 In vivo acute toxicity of titanium dioxide nanoparticles central nervous systems, as well as behavior patterns (Wang et al., 2007b). During the first 2 days after exposure, all mice showed obvious signs of passive behavior, loss of appetite, tremor and lethargy as compared with control mice. While these signs for low-dose mice (T1, T2, T3, and T4) thereafter gradually disappeared, high-dose mice (T5 and T6) showed anorexia, diarrhea, lethargy, tremor, body-weight losses, and lusterless skin. Serum Biochemical Parameters Figure 3. Size distribution of nano-sized TiO 2 particle. Table 2 shows the changes in biochemical parameters in the serum of mice induced by TiO 2. The ALT and AST levels were higher while the BUN showed no statistically significant difference between experimental and control groups after 24 h exposure to TiO 2. The 48 h exposure to TiO 2 caused increases in ALT and AST in group B2, but a slight decrease in BUN levels from the control. The ALT showed higher levels in all groups except T4 after 7 days of exposure to TiO 2. We further observed after 14 days of exposure a concentration-dependent trend of increasing serum ALT and AST levels compared with the control group. ALP and BUN levels decreased slightly after exposure to different doses of TiO 2. Our results indicated that TiO 2 resulted in elevated ALT and AST levels (P < 0.05), whereas BUN was not significantly affected. This finding suggested that TiO 2 had a greater impact on liver than on kidneys. Titanium Content The contents of titanium in each tissue (pancreas, heart, lung, kidney, liver) of mice at different times (24 h, 48 h, 7 days and 14 Table 2. Biochemical assay in serum of mice (mean ± SD, n =10) Group ALT (U l 1 ) AST (U l 1 ) ALP (U l 1 ) BUN (mmol l 1 ) 24 h exposure B1 (0.2 ml) ± a,b ± a,b NM 6.41 ± 1.05 B2 (0.4 ml) ± a ± a,b NA 5.68 ± 0.36 B3 (0.6 ml) ± ± a NA 6.54 ± 1.30 Control ± ± NA 6.35 ± h exposure B1 (0.2 ml) ± 7.23 b ± 9.71 a ± ± 0.12 B2 (0.4 ml) ± a ± a NM NM B3 (0.6 ml) ± 9.20 NM NM 5.94 ± 0.78 Control ± ± NA 6.75 ± days exposure T1 (0.1 ml) ± 7.09 NA NM NA T2 (0.2 ml) ± a NA NM 8.07 ± 0.41 T3 (0.3 ml) ± a ± 1.41 a,c NM NM T4 (0.4 ml) ± 3.77 NM NM NM T5 (0.6 ml) ± 6.00 c NA NM NA T6 (0.8 ml) ± a NA NM NM Control ± ± NM 8.22 ± days exposure T1 (0.1 ml) ± ± ± ± 1.28 T2 (0.2 ml) ± a ± a NA 8.49 ± 1.44 T3 (0.3 ml) ± ± c ± ± 0.79 T4 (0.4 ml) ± ± ± a 8.56 ± 1.21 T5 (0.6 ml) ± a,c ± a ± ± 1.11 T6 (0.8 ml) ± a,c ± a NA 8.63 ± 0.58 Control ± ± ± ± 0.27 NA, not available; NM, not measured; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen. a Represents significant difference from the control group (Dunnett s, P <0.05). b Represents significant difference between 24 h and 48 h within treatment (ANOVA, P < 0.05). c Represents significant difference between day 7 and 14 within treatment (ANOVA, P <0.05). 333 J. Appl. Toxicol. 2009; 29: Copyright 2009 John Wiley & Sons, Ltd.

5 J. Chen et al. Figure 4. Contents of titanium in pancreas, heart, lung, kidney and liver of mice 24 h (A), 48 h (B), 7 days (C) and 14 days (D) after exposure to various doses of TiO 2 particles by intraperitoneal injection. a Significant difference from the control group (Dunnett s, P < 0.05). b Significant difference between spleen and other organs (heart, lung, kidneys, liver) within treatment (ANOVA, P < 0.05). 334 days) following an exposure to various doses of TiO 2 are shown in Fig. 4. Titanium mainly accumulated in spleen 24 h after exposure [Fig. 4(A)]. A dose-dependent relationship existed that, the higher the dose of TiO 2 was, the higher the concentration of titanium was. No significant differences were found in other tissues between treated mice and the controls. Titanium content declined with time (48 h) in spleen, although it remained much higher than in other organs [Fig. 4(B)]. Titanium content increased slightly with time in lung, kidney and liver. In heart tissue, no significant difference in titanium existed between dosed mice and the controls. For a longer time (7 days) after exposure, the spleen remained the organ with the highest titanium content [Fig. 4(C)]. In the high-dose group (51.84 mg), in particular, the Ti concentration reached ± ng g 1. Comparatively, Ti also increased in lung, kidney and liver (except heart) to various degrees. In liver, the highest titanium content was ± ng g 1. When time was further increased to 14 days after exposure, the Ti concentrations of the high-dose group (51.84 mg) were lower in lung, kidney and liver [Fig. 4(D)] compared with those in Fig. 4(C). The concentrations with other doses (6.48, 12.96, 19.44, and mg) increased slightly and in most cases exceeded those with the dose of mg. Histopathological Examination Because both female and male mice showed the same pathological changes, these histological photomicrographs are not marked by gender. Some representative images of liver, kidney, spleen and lung sections of mice in high-dose groups (T5, T6) are shown in Figs 5 8. The liver histopathological pictures are illustrated in Fig. 5. Hepatic fibrosis around the central vein where TiO 2 particles attached was extensive and significant [Fig. 5(A)]. Livers showed some loss of sinusoid space and hydropic degeneration with minor fatty change. The occasional necrotic cell, cells with condensed nuclear bodies that have the appearance of apoptotic bodies, and cells showing nuclear division with condensed nuclear material were obvious, mainly at the highest TiO 2 concentration [Fig. 5(B, C)]. Some neutrophilic cells were found [Fig. 5(C)], which indicated that TiO 2 particles induced inflammation in liver tissues. The histopathological changes of kidney are shown in Fig. 6. Slight swelling in the renal glomerulus was observed [Fig. 6(A, B)]. In addition, dilatation and proteinic liquids were found in the renal tubular of mice exposed to TiO 2 particles. The histopathological changes of spleen are shown in Fig. 7. Mice had a severe spleen lesion associated with exposure to TiO 2 particles in high-dose groups (T5, T6). A mass of neutrophilic cells were found [Fig. 7(A)] in spleen tissues, which revealed that inflammation in spleen tissues was very serious. The histopathological pictures [Fig. 7(B, C)] showed that a large number of TiO 2 particles entered spleen tissues. The histopathological changes of lung are shown in Fig. 8. Alveolar septal thickening was found in lung tissues [Fig. 8(A)]. Some neutrophil infiltration was also observed in interstitial pneumonia [Fig. 8(B)]. Figure 8(C) shows thrombosis in the Copyright 2009 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2009; 29:

6 In vivo acute toxicity of titanium dioxide nanoparticles Figure 5. Pathological changes in liver tissue (150 for A; 600 for B and C) in experimental mice 7 days after exposure to TiO 2 particles by intraperitoneal injection. (A) Group T5. Circles show hepatic fibrosis around the central vein. (B) Group T6. Arrow shows necrotic cell. Circles show the hydropic degeneration with minor fatty change. (C) Group T6. Arrows show some cells nuclear division. Circles show the hydropic degeneration with minor fatty change. The section was stained with HE and examined by light microscopy. This figure is available in colour online at Figure 6. Pathological changes in kidney tissue (600 for A; 150 for B) in experimental mice 7 days after exposure to TiO 2 particles by intraperitoneal injection. (A) Group T5; (B) group T6. Arrows show the slight swelling in the glomerulus. Circles show the proteinic liquid in the renal tubule. The section was stained with HE and examined by light microscopy. This figure is available in colour online at Figure 7. Pathological changes in spleens tissue (600 for A and C; 150 for B) in experimental mice 7 days after exposure to TiO 2 particles by intraperitoneal injection. (A) Group T5; (B) group T6; (C) group T6. Circles show many neutrophilic cells. Arrows show that TiO 2 particles have entered the spleen. The section was stained with HE and examined by light microscopy. This figure is available in colour online at pulmonary vascular system in mice from the high-dose group (T6), which could be ascribed to the blockage of blood vessels by TiO 2 particles after intraperitoneal injection. In summary, the lesions of all tissues induced by nano-sized TiO 2 were minor based on the scale of pathological changes. Pathological changes were only found more frequently in highdose groups (T5, T6). There were no evident effects in low-dose groups (data not shown). The lesion was the most severe in the spleen and the lightest in the kidney in most cases. No other significant histopathological changes were found in heart and brain (data not shown). Discussion The acute toxicity of nano-sized TiO 2 particles to mice was investigated in this study. Five mice (two male and three female) died with high doses of TiO 2 (T5 and T6 groups) a week after treatment. Epidemiology research reported that TiO 2 had low toxicity and showed no carcinogenic effect and/or nonmalignant respiratory disease for human (Boffetta et al., 2004; Chen and Fayerweather, 1988). Olmedo et al. (2005) studied the effect of TiO 2 on the oxidative metabolism of alveolar macrophages. They attributed the generation of reactive oxygen species (ROS) to an 335 J. Appl. Toxicol. 2009; 29: Copyright 2009 John Wiley & Sons, Ltd.

7 J. Chen et al. Figure 8. Pathological changes in lung tissue (150 for A and C; 600 for B) in experimental mice 7 days after exposure to TiO 2 particles by intraperitoneal injection. (A) Group T5. Circles show alveolar septal thickening. (B) Group T6. Arrow shows TiO 2 particles have entered blood vessels. Circles show many neutrophilic cells. (C) Group T6. Arrows show thrombosis in pulmonary vascular. Circles show many neutrophilic cells. The section was stained with HE and examined by light microscopy. This figure is available in colour online at adaptive response to TiO 2 particles, because they failed to observe any tissue damage 18 months after injection. The results from the present study indicated that these abnormal signs resulted unambiguously from the particles themselves. First, we observed from the autopsy that a large number of particles had accumulated in the abdominal cavity of mice in high-dose groups (T5, T6) and attached to organs such as intestine and liver. This implies that the death of mice could be correlated with the severe adhesion of TiO 2 particles in the intestine, which resulted in anorexia, lethargy, body-weight loss and finally death. Moreover, some significant pathological changes were found through histopathological examination. Especially for the spleen, a large number of TiO 2 particles entered the tissues, and Fig. 7 shows a severe spleen lesion in the highdose groups (T5, T6). In liver, the large-area hydropic degeneration with fatty change was obvious. Exposure to TiO 2 also caused hepatocellular necrosis and apoptosis, with hepatic fibrosis around the central vein. Some TiO 2 particles were detected in the pulmonary vascular system, leading to the thrombosis generation because of the blockage of blood vessels by TiO 2 particles. Furthermore, renal glomerulus swelling and interstitial pneumonia associated with alveolar septal thickening were also found and evidently caused by exposure to nano-sized TiO 2 particles. For some materials, previous studies showed that the toxicity of inhaled particles increased as particles became smaller and the overall surface area of inhaled material became larger (Maynard and Kuempel, 2005; Oberdörster et al., 2005). Oral administration of polystyrene latex nanoparticles indicated that nanoparticles can be absorbed across the gastrointestinal tract, and pass through the mesentery lymph supply and lymph node to liver and spleen (Jani et al., 1990). The toxic effects observed in this study may be similarly attributed to the processes suggested. However, Chen et al. (2006) reported that the different exposure routes, such as inhalation, dermal contact and different metal salts administration could cause different toxic effects. Therefore, further research on different exposure routes and long-term low level effects caused by nanoscale materials is needed. Blood biochemical parameters (BUN, ALT, AST and ALP) were determined in the present study. When the liver is in dysfunction, the levels of the above enzymes rise (Kellerman, 1995). The BUN was a good indicator of renal function. From Table 2, TiO 2 induced relatively higher ALT and AST levels in treated mice compared with the control, whereas the differences for BUN value between experimental groups and the control group were not evident. It was previously reported that the retention half-time of TiO 2 in vivo was long because of its difficult excretion. Therefore, the difficult clearance of TiO 2 in vivo might directly lead to the particle deposition in liver and result in the hepatic lesion. It also revealed that titanium ions were released into surrounding tissues and reached the internal milieu to be excreted in urine (Jacobs et al., 1991). The International Program on Chemical Safety (1982) shows that most ingested titanium is excreted with urine and not absorbed by organisms. It seems to suggest that nano-tio 2 particles had a stronger toxicity to liver than to kidney. This was further confirmed by the histopathological results that pathological changes in kidney were the lightest compared with other tissues (spleen, lung and liver). Also, the variation in liver-linked enzymatic activity could be associated with enzymatic variation that could be observed on the basis of age, sex and other factors (e.g. diet; Braun et al., 1993). The detailed mechanism needs to be further investigated. Olmedo et al. (2002) reported that 6 months after intraperitoneal injection TiO 2 was deposited in organs such as liver, spleen and lung. The result from Huggins and Froehlich (1966) found that, after intravenous injection of TiO 2 (size μm) of 250 mg/kg to rats, about 69% of the injected TiO 2 at 5 min and 80% at 15 min were accumulated in liver. All these results show that hepatic deposition is the highest among all the selected tissues. Our results show that the initial accumulation of TiO 2 in spleen was the highest and the Ti concentration in spleen was significantly higher than in other tissues (heart, lung, kidney and liver). This was probably due to a large number of TiO 2 particles entering the spleen tissues, as mentioned earlier. Titanium contents in lung, kidney and liver (expect heart) subsequently increased gradually with time to various degrees as well. Previous studies indicated that nanosized particles could cross the small intestine by persorption and further distribute into blood, brain, lung, heart, kidney, spleen, liver, intestine and stomach (Hillyer and Albrecht, 2001). Therefore, we concluded that nano-sized TiO 2 could accumulate in, and transport to, other tissues after intraperitoneal injection. Conclusions On the basis of the results of blood biochemical index examination, a preliminary conclusion could be drawn that some of the particles were excreted from the kidney and nano-tio 2 had a stronger toxicity to liver than to kidney. A distribution experiment showed that TiO 2 particles were mainly retained in spleen, lung, kidney and liver tissues, and the accumulation of TiO 2 particles in spleen was the highest throughout our experimental period. This indicated that nano-tio 2 particles Copyright 2009 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2009; 29:

8 337 In vivo acute toxicity of titanium dioxide nanoparticles could transport to and deposit in other tissues after intraperitoneal injection. TiO 2 particles induced some significant pathological changes. TiO 2 particles entered spleen tissues, leading to serious spleen lesion in high-dose groups. Thrombosis was observed in the pulmonary vascular systemof mice, which could be ascribed to the blocking of blood vessels by TiO 2 particles. From the pathological results, each organ (liver, spleen, lung and kidney) showed varying degrees of lesions. However, the lesions of all tissues induced by nano-sized TiO 2 were minor according to the grading of pathological changes. The lesion was the most severe in the spleen and the lightest in the kidney in most cases. As manufactured commercial nanoparticles, TiO 2 induced significant adverse effects that deserve our attention. With regard to the human health and environmental safety, a risk assessment framework to TiO 2 nanoparticles should be built upon their toxicity studies. Additional work needs to be undertaken to elucidate the mechanisms of damages. Acknowledgements The work was supported by the National Natural Science Foundation of China ( ), the Program of Key Disciplines of Zhejiang Province in Environmental Engineering ( ) and the Key Scientific Research and Society Development Project (2006C23067). References Banfield JF, Navrotsky A Nanoparticles and the Environment. Mineralogical Society of America: Washington, DC. Bermudez E, Mangum JB, Asgharian B, Wong B A, Reverdy EE, Janszen DB, Hext PM, Warheit DB, Everitt JI Long-term pulmonary responses of three laboratory rodent species to subchronic inhalation of pigmentary titanium dioxide particles. Toxicol. 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