The Acute Liver Injury in Mice Caused by Nano-Anatase TiO2

Although it is known that nano-TiO2or other nanoparticles can induce liver toxicities, the mechanisms and the molecular pathogenesis are still unclear. In this study, nano-anatase TiO2(5 nm) was injected into the abdominal cavity of ICR mice for consecutive 14 days, and the inflammatory responses of liver of mice was investigated. The results showed the obvious titanium accumulation in liver DNA, histopathological changes and hepatocytes apoptosis of mice liver, and the liver function damaged by higher doses nano-anatase TiO2. The real-time quantitative RT-PCR and ELISA analyses showed that nano-anatase TiO2can significantly alter the mRNA and protein expressions of several inflammatory cytokines, including nucleic factor-κB, macrophage migration inhibitory factor, tumor necrosis factor-α, interleukin-6, interleukin-1β, cross-reaction protein, interleukin-4, and interleukin-10. Our results also implied that the inflammatory responses and liver injury may be involved in nano-anatase TiO2-induced liver toxicity.


Introduction
Titanium dioxide nanoparticles (nano-TiO 2 ) (\100 nm) are widely used in the cosmetics, pharmaceutical, and paint industries as a coloring material because of its high stability, anticorrosion, and photocatalysis. More and more nanoparticles are brought into the environment with the increasing development and application of nanotechnology. With the small size and large surface area, nanoparticles can be an active group or exert intrinsic toxicity. It is therefore important to clarify the effects of various nanoparticles on organs health as well as the pathogenic mechanisms involved.
This information may have important clinical implications regarding the safety issue, as nano-TiO 2 are widely used in the different spheres. Extra caution should therefore be taken in the handling of higher dose nano-TiO 2 . Many in vivo studies showed that nanoparticles can be accumulated in the liver, kidney, spleen, lung, heart, and brain, whereby generating various inflammatory responses [1][2][3][4][5][6][7][8]. For instance, nanoparticles can promote enzymatic activities and the mRNA expression of cytokines during proinflammatory responses in rats and mice [5][6][7][8] and in human dermal fibroblasts and human lung epithelial cells [9]. In the study of toxicity of nano-TiO 2 to rats by intratracheal instillation, Afaq et al. [10] found that the number of alveolar macrophage increased, the activities of glutathione peroxidase, glutathione reductase, 6-phosphate glucose dehydrogenase, and glutathione S-transferase were significantly elevated. However, the production of lipid peroxidation and hydrogen peroxide radicals was not altered with increased activities of these enzymes, suggesting that nano-TiO 2 (\30 nm) could induce the generation of antioxidant enzymes in animals [10]. Oberdörster et al. [11] showed that the nano-TiO 2 (20 nm) induced the increase of the total protein of bronchoalveolar lavage fluid and the activities of lactate dehydrogenase and acid-glucosidase in rats and mice. The toxic effects of nano-TiO 2 in adult mice have been accomplished, suggesting that higher dose nano-TiO 2 (25 and 80 nm) increased the ratio of alanine aminotransferase to aspartate aminotransferase, the activity of lactate dehydrogenase and the liver weight, and caused the hepatocyte necrosis [8]. Our previous reports indicated higher dose nano-anatase TiO 2 (5 nm) could damage liver function [12] and induced an oxidative attack in liver of mice [13].
It is well known that overexpression and activation of nucleic factor-jB (NF-jB) may contribute to the pathogenesis of hepatitis in animals [14]. Although it is known that nano-TiO 2 or other nanoparticles can induce serious liver toxicities, the mechanisms and the molecular pathogenesis are still unclear. For example, can nano-TiO 2 particles, which are similar to hepatovirus, bind to DNA and cause the inflammatory cascade after accumulation in liver? When nano-TiO 2 particles stimulate hepatocytes, can they induce inhibitory proteins such as IjBs phosphorylated and degraded, and then NF-jB activation, leading to the gene transcription of the proinflammatory cytokines and anti-inflammatory cytokines in the mouse liver?
Zhu et al. [15] proved that nano-TiO 2 of different size and types showed different extents of cytotoxicity on CHO cells and 293T cells with the sequence as 10-20 nm anatase [ 50-60 nm anatase [ 50-60 nm rutile. TiO 2 can be classified into three types: anatase, rutile, and amorphous. The photoactivity of anatase-type TiO 2 was greater than that of rutile, whereas amorphous do not show photocatalytic activity. Because anatase-type TiO 2 has the greatest toxicity to cells among the three types, we need further research to investigate its toxicity in mouse liver.
In this study, we investigate the effect of nano-anatase TiO 2 on the induction of liver toxicity and inflammatory response, its mechanisms, and the molecular pathogenesis. Our findings will provide an important theoretical basis for evaluating the toxicity underlying effects of nanoparticles on animals and human.

Chemicals and Preparation
Nano-anatase TiO 2 was prepared via controlled hydrolysis of titanium tetrabutoxide. The details of the synthesis are as follows [16]: Colloidal titanium dioxide was prepared via controlled hydrolysis of titanium tetrabutoxide. In a typical experiment, 1 mL of Ti(OC 4 H 9 ) 4 dissolved in 20 mL of anhydrous isopropanol was added dropwise to 50 mL of double distilled water adjusted to pH 1.5 with nitric acid under vigorous stirring at room temperature. Then, the temperature was raised to 60°C and kept 6 h for better crystallization of nano-TiO 2 particles. The resulting translucent colloidal suspension was evaporated using a rotary evaporator yielding a nanocrystalline powder. The obtained powder was washed three times with isopropanol and dried at 50°C until complete evaporation of the solvent. The average grain size calculated from broadening of the (101) X-ray diffraction peak of anatase ( Fig. 1) using Scherrer's equation was approximately 5 nm. The Ti 2? content in the nano-anatase was measured by inductively coupled plasma mass spectroscopy (ICP-MS), and O, C, and H contents in the nano-anatase were assayed by Elementar Analysensysteme Gmbh, showing that Ti, O, C, and H contents in the nano-anatase were 58.114, 40.683, 0.232, and 0.136%, respectively. Bulk TiO 2 (rutile) was purchased from Shanghai Chem., Co., and the average grain size was 10-15 lm.
A 0.5% hydroxypropylmethylcellulose K4M (HPMC, K4M) was used as a suspending agent. Nano-anatase TiO 2 and bulk TiO 2 powder was dispersed onto the surface of 0.5%, w/v HPMC, and then the suspending solutions containing nano-TiO 2 and bulk TiO 2 particles were treated by ultrasonic for 30 min and mechanically vibrated for 5 min.

Animals and Treatment
CD-1 (ICR) mice of 70 females (20 ± 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, with 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 Fig. 1 The average grain size calculated from broadening of the (101) XRD peak of anatase using Scherrer's equation 5 days prior to dosing. All procedures used in animal experiments were in compliance with the local ethics committee. Animals were randomly divided into seven groups: control group (treated with 0.5% HPMC) and six experimental groups. Experimental groups were injected into abdominal cavity with nano-anatase TiO 2 (5, 10, 50, 100, and 150 mg/kg BW) and with bulk TiO 2 (150 mg/kg BW) everyday for 14 days, respectively. The control group was treated with 0.5% HPMC. The symptom and mortality were observed and recorded carefully everyday for 14 days. After 14 days, the body weight of all animals were weighed accurately and sacrificed after being anesthetized by ether. Blood samples were collected from the eye vein by removing the eyeball quickly. Serum was collected by centrifuging blood at 2,500 rpm for 10 min. The tissues and organs, such as liver, spleen, kidneys, lung, heart, and brain, were excised and washed carefully using 95% saline, then weighed accurately.

Coefficients of Liver
After weighing the body and tissues, the coefficients of liver to body weight were calculated as the ratio of tissues (wet weight, mg) to body weight (g).

Preparation of DNA Samples from Mouse Liver
The DNA was extracted from the liver and purified as described by the manual of DNA kits (Takara company), A260/A280 ([1.8) indicated that the DNA was sufficiently free of protein. The purified DNA was resuspended in Tris-HCl buffer (pH 7.2).
Titanium Content Analysis of Liver and Liver DNA Tissues were taken out and thawed. About 0.1-0.3 g of each liver tissue and 0.5 mg of liver DNA from various treated mice were weighed, digested, and analyzed for titanium content. Briefly, prior to elemental analysis, the tissues of interest were digested in nitric acid (ultrapure grade) overnight. After adding 0.5 mL of H 2 O 2 , the mixed solutions were heated at about 160°C using high-pressure reaction container in an oven chamber until the samples were completely digested. Then, the solutions were heated at 120°C to remove the remaining nitric acid until the solutions were colorless and clear. At last, the remaining solutions were diluted to 3 mL with 2% nitric acid. ICP-MS (Thermo Elemental X7, Thermo Electron Co.) was used to analyze the titanium concentration in the samples. Of indium, 20 ng/mL was chosen as an internal standard element. The detection limit of titanium was 0.076 ng/mL. Data are expressed as nanograms per gram of fresh tissue.

Histopathological Examination
For pathological studies, all histopathological tests were performed using standard laboratory procedures. The tissues were embedded in paraffin blocks, then sliced into 5 lm in thickness and placed onto glass slides. After hematoxylin-eosin (HE) staining, the slides were observed, and the photos were taken using optical microscope (Nikon U-III Multi-point Sensor System, USA), and the identity and analysis of the pathology slides were blind to the pathologist.
Observation of Hepatocyte Ultrastructure by TEM Liver was fixed by 2.5% glutaraldehyde in 0.1 mol/dm 3 cacodylate buffer for 2 h, washed three times with 0.1 mol dm cacodylate buffer (pH 7.2-7.4) and post-fixed for 1 h in 1% osmium tetroxide. The specimens were dehydrated by 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 JEOL 1010 transmission electron microscope.

Expression Amount and Concentration Assay of Inflammatory Cytokines
The mRNA expression of nucleic factor-jB (NF-jB), macrophage migration inhibitory factor (MIF), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), interleukin-1b (IL-1b), cross-reaction protein (CRP), interleukin-4 (IL-4), and interleukin-10 (IL-10) were determined by realtime quantitative RT polymerase chain reaction (RT-PCR) [17][18][19]. Liver in the same growth period from the three different treatments were used. The right livers from mice with or without nano-anatase TiO 2 treatment were homogenized using QIAzol lysis reagent with a Tissue Ruptor (Roche). Total RNA from the homogenates was isolated using Tripure Isolation Reagent (Roche) according to the manufacturer's instructions. The RT reagent (Shinegene, China) of 30 lL was prepared by mixing 15 lL of 29 RT buffer, 1 lL random primer in a concentration of 100 pmol lL -1 , 1 lL of RTase, 5 lL RNA, and 8 lL 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.
The gene expression analysis and experimental system evaluation were performed according to the standard curve and quantitation reports.

Statistical Analysis
Statistical analyses were done using SPSS11.5 software. Data were expressed as means ± SD. One-way analysis of variance (ANOVA) was carried out to compare the differences of means among multi-group 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.

Results
The Enhancement of Body Weight and the Coefficients of Liver After 14 days, the mice were weighed, various organs were collected and they were also weighed. Table 1 shows the coefficients of the liver to body weight which were expressed as milligrams (wet weight of tissues)/grams (body weight). No obvious differences were found in the body weight of seven groups. The significant differences were not observed in the coefficient of the liver in the 5 and 10 mg/kg BW nanoanatase TiO 2 -treated groups (p [ 0.05). However, the coefficients of the liver in the 50, 100, and 150 mg/kg BW nano-anatase TiO 2 -treated groups and 150 mg/kg BW bulk TiO 2 -treated group were significantly higher (p \ 0.05 or 0.01) than the control, suggesting that higher dose nanoanatase TiO 2 and bulk TiO 2 might cause the damage of the liver of mice.

Titanium Contents in Liver and Liver DNA
The contents of titanium in liver and the purified DNA from liver of mice during 14 days daily injection of various doses nano-anatase TiO 2 and 150 mg/kg BW bulk TiO 2 are shown in Fig. 2. With increasing injection dose of nano-anatase Ranks marked with an asterisk or double asterisks means it is significantly different from the control (no nano-anatase or bulk TiO 2 ) at the 5 or 1% confidence level, respectively. Values represent means ± SE, n = 10 TiO 2 , the titanium contents in the liver and DNA were significantly elevated, this observation suggested that the accumulation of titanium in the liver and DNA was closely related to the coefficients of the liver of mice, suggesting that, after entering the animals, nano-TiO 2 combine with the biomolecules such as DNA. However, the contents of titanium of the liver and DNA in 150 mg/kg BW bulk TiO 2 -treated group were lower than those of 150 mg/kg BW nano-anatase TiO 2 -treated group (p \ 0.05), suggesting that nano-anatase TiO 2 entered the liver of mice and combined with the DNA more easily than the bulk TiO 2 did.

Assay of Liver Function
The serum biochemical parameters were assayed to further evaluate the toxicity of nano-anatase TiO 2 on the liver of mice. PChE, and LAP, and the decrease of ratio of ALB to GLB, TBIL levels which are important indicators of the hepatic injury, demonstrated that nano-anatase TiO 2 induced hepatic injury. Furthermore, the increase of enzyme activity and the decrease of ratio of ALB to GLB, TBIL levels are dose-dependent, inferring that the induced hepatic injury is dose-dependent. TG, TCHO, and HDL-C from 100 to 150 mg/kg BW-treated groups were higher than the control group (p \ 0.05). LDL-C contents from 100 to 150 mg/kg BW nano-anatase TiO 2 treated groups were lower than the control group (p \ 0.05). In the 150 mg/kg BW bulk-TiO 2treated group, the contents of THCO and HDL-C were higher than the control group (p \ 0.05), but the contents of TG and LDL-C were not significantly different from the control group (p [ 0.05). These results indicate that nanoanatase TiO 2 in higher dose caused metabolism imbalance of lipids HDL-C and low-density LDL-C in mice liver.

Liver Histopathological Evaluation
The histological photomicrographs of the liver sections are shown in Fig. 2. In the 5 mg/kg BW nano-anatase TiO 2treated group, the liver tissue had no abnormal pathology changes compared with the control. In the 100, 150 mg/kg BW nano-anatase TiO 2 -treated groups and 150 mg/kg BW bulk TiO 2 -treated group, however, the significant histopathological changes were observed in the liver tissue, for example, congestion of vascellum and prominent vasodilatation were observed in 100 mg/kg BW nano-anatase TiO 2 -treated group (Fig. 2c), and wide-bound basophilia and focal ischemia occurred in 150 mg/kg BW nanoanatase TiO 2 -treated group (Fig. 2d, e), and congestion of central veins was showed in 150 mg/kg BW bulk TiO 2treated group (Fig. 2f).

Hepatocyte Evaluation
Ultrastructure of hepatocyte in female mice is shown in Fig. 3. It was observed that the ultrastructure of hepatocyte from 5 mg/kg BW nano-anatase TiO 2 -treated group was similar to the control, but from 100 mg/kg BW nanoanatase TiO 2 -treated group turned tumescent mitochondria and vacuolization, and 150 mg/kg BW nano-anatase TiO 2treated group indicated apoptotic body. The results suggested that nano-anatase TiO 2 could damage the structure of hepatocyte of mice.

Inflammatory Cytokines in Nano-Anatase TiO 2 -Treated Mice Liver Tissues
The inflammation happened in liver according to the histopathological and hepatocyte ultrastructure observations. To confirm the role of inflammatory cytokine pathway in Fig. 2 The contents of titanium in liver tissue and liver DNA of female mouse after intraperitoneal injection with nano-anatase TiO 2 suspensions for consecutive 14 days. Values represent means ± SE, n = 5. Columns marked with asterisk or double asterisks means it is significantly different from the control (no nano-anatase or bulk-TiO 2 ) at the 5 or 1% confidence level, respectively Table 2 The changes of biochemical parameters in the blood serum of mouse liver after intraperitoneal injection with nano-anatase TiO  nano-anatase TiO 2 -induced liver injury, real-time quantitative RT-PCR, and ELISA were used to demonstrate inflammatory cytokines (such as NF-jB, MIF, IL-6, IL-1b, CRP, TNF-a, IL-4, and IL-10) induction in nano-anatase TiO 2 -treated mice. Real-time quantitative RT-PCR analysis showed that NF-jB, MIF, IL-1b, IL-6, CRP, TNF-a, IL-4, and IL-10 were significantly up-regulated in the liver tissues of mice treated with nano-anatase TiO 2 for consecutive 14 days (p \ 0.05 or 0.01). The 150 mg/kg BW bulk TiO 2 microparticles (micro TiO 2 ) had less effect on induction of these genes compared with 150 mg/kg BW nano-anatase TiO 2 particles ( Table 3).
The nano-anatase TiO 2 -induced inflammatory cytokine expression was also examined at the protein level after intraperitoneal injection with various doses of nano-anatase TiO 2 suspensions for consecutive 14 days (Fig. 4). ELISA analysis showed that nano-anatase TiO 2 caused significant induction of serum levels of NF-jB, MIF, IL-6, IL-1b, CRP, TNF-a, IL-4, and IL-10 protein in a dose-dependent manner ( Table 4, p \ 0.05 or 0.01), which might be produced mainly by infiltrating macrophages and some liver epithelial cells.
The results mentioned earlier are consistent with that the histological photomicrograph and hepatocyte ultrastructure of the liver sections was observed in the treated mice. The inflammation is able to induce an increase of the expression level of inflammatory cytokines by nano-anatase TiO 2 .

Discussion
The results of this study indicate that intraperitoneal injection of higher doses of nano-anatase TiO 2 can increase coefficients of the liver, and its significant accumulation in the mouse liver can induce histopathological changes of liver, including congestion of vascellum, prominent vasodilatation, wide-bound basophilia and focal ischemia, hepatocyte tumescent mitochondria, vacuolization and apoptosis, thus leading to the damage of liver function. Wang et al. [8] observed that the hydropic degeneration around the central vein was prominent and the spotty necrosis of hepatocyte in the liver tissue of female mice post-exposure 2 weeks to the 5 g/kg BW 80 nm and fine TiO 2 particles, but did not observed significant histopathological change in liver tissues of mice exposed to the 5 g/ kg BW 25 nm TiO 2 particles. The present study indicates that the hepatitis of mice is triggered by nano-anatase TiO 2 activation of inflammatory cytokines that resulted in disruption of liver tissue, and hepatocyte injury and apoptosis.
Alkaline phosphatase is mainly distributed in the liver, bone, and in bile duct, and ALT and AST exist in the liver, heart, and other organs. When the organs injured, the activities of ALP, ALT, and AST in serum would increase. It is well known that LDH is an important isoenzyme in glycolysis and glyconeogenesis and widely exists in the heart, liver, lung, and many other tissues. When the tissues are subjected to injury, LDH would leak into the serum of blood from organs or cells, which resulted in the increase of LDH activity and its isoenzyme in the corresponding organs. Pseudocholinesterase (PChE, acylcholine acyl hydrolase) has been found in many animal tissues, and it may function in the metabolism of lipids and low-density lipoprotein. When the liver is subjected to injury, PChE activity is significantly elevated, thus leading to the damage of the metabolism of lipids and low-density lipoprotein. In order to further study the biochemical mechanism of nano-anatase TiO 2 particles, the parameters for the damages of the liver function, and lipid contents in the blood were determined. The results showed that, in the 50, 100, and 150 mg/kg BW nano-anatase TiO 2 -treated groups, the parameters for hepatic function including ALT, ALP, AST, LDH, LAP, PChE, TP, ALB, GLB, TBIL, TG, TCHO, and HDL-C increased greatly and LDL-C decreased significantly in blood (p \ 0.05 or 0.01). However, the parameters mentioned earlier from the 5 and 10 mg/kg BW nano-anatase Ratio of p-10/actin 0.065 ± 0.003 0.084 ± 0.004* 0.102 ± 0.005** 0.107 ± 0.005** 0.082 ± 0.004* Ranks marked with an asterisk or double asterisks means it is significantly different from the control (no nano-anatase or bulk TiO 2 ) at the 5 or 1% confidence level, respectively. Values represent mean ± SE, n = 5 TiO 2 -treated groups were not significantly different from the control group. The results are consistent with histopathological changes of liver and the damage of hepatocyte substructure and with our previous report [12]. Wang et al. [8] showed that after a single oral gavage of dose of 5 g/kg BW of TiO 2 suspensions (25 and 80 nm), ALT, LDH, and TBIL in serum had statistical significance compared with the control mice.
Our studies showed that the obvious titanium accumulation in the liver and liver DNA of mice was observed. The accumulation of titanium is consistent with the coefficients of liver and the liver injury of mice. In addition, the accumulation of titanium of the organs in 150 mg/kg BW nano-anatase TiO 2 -treated group was higher than those of 150 mg/kg BW bulk-TiO 2 -treated group (p \ 0.05). Compared with bulk TiO 2 , smaller grain size of nano-  Table 4 Effects of nano-Tio 2 on the cytokine protein level of mice by ELISA analysis after intraperitoneal injection with nano-anatase TiO 2 suspensions for consecutive 14 days anatase TiO 2 (5 nm) would allow easier entry to mouse cells and its higher surface makes its intake to the liver and bound to liver DNA of mice easier. Combination of both resulted in the enhancement of the titanium in the liver and DNA. It implies that nano-TiO 2 particles, which are similar to hepatovirus, can enter liver cells or nuclei and bind to DNA, thus might cause the changes of genetic information transfer and the inflammatory cascade.
It is well known that the hepatitis pathogenesy is that hepatocytes generate various immunopathogenesis injuries, including cellular and humoral immunity. However, hepatovirus itself does not directly damage hepatocytes, some cytokines induced by hepatovirus play important roles in inflammatory responses. Transcription factor NF-jB is a critical intracellular mediator of the inflammatory cascade. In quiescent cells, NF-jB is bound to inhibitory proteins called IjBs that prevents NF-jB from migrating to the nucleus and located in the cytoplasm. When an appropriate inducer, such as hepatovirus, affects the cell, IjBs are phosphorylated and degraded, allowing nuclear uptake of NF-jB and initiating gene transcription (such as MIF, the proinflammatory cytokines of TNF-a, IL-6, IL-1b, CRP, and anti-inflammatory cytokines of IL-4 and IL-10) [14]. As an inflammatory factor, MIF also plays a role of inflammatory mediators in various diseases. The liver toxicity caused by nano-TiO 2 has been reported [8,12], but its molecular pathogenesis is not known. In this study, the real-time quantitative RT-PCR and ELISA analysis showed that nano-anatase TiO 2 can significantly stimulate the mRNA expressions and increase protein levels of several inflammatory cytokines, including NF-jB, MIF, TNF-a, IL-6, IL-1b, CRP, IL-4, and IL-10. The obvious increase of these cytokines mRNA expression and protein levels indicated that the inflammatory responses and hepatocyte apoptosis may be involved in nano-anatase TiO 2 -induced liver toxicity. It had been demonstrated that nano-TiO 2 could promote the expression of several cytokines and chemokines in the lung of rat and mice, including placenta growth factor (PlGF), MCP-1, IL-1b, and TNF-a [6,20], and increase protein level of TNF-a, IL-1b in brain of mice and cause brain inflammation [21,22]. Here, we speculate that nano-anatase TiO 2 particles, which are similar to hepatovirus, can make IjBs phosphorylated and degraded, and then induce NF-jB activation, leading to the expression of the NF-jB-controlled proinflammatory cytokines (such as IL-1b, IL-6) in liver of mice and the inflammatory response of liver, but the molecular mechanism of inflammatory response of mouse liver caused by nano-anatase TiO 2 needs to be studied in future. Further investigations are needed to elucidate the potential liver toxicity of different nanoparticles and their pathogenesis.
The present article also demonstrated that bulk-TiO 2 can elevate coefficients of the liver, be accumulated in liver and liver DNA of mice, cause histopathological changes of liver, damage liver function and induce inflammatory response of liver, but it has less toxicity compared with 150 mg/kg BW nano-anatase TiO 2 particles. Compared with nano-anatase TiO 2 (5 nm), bulk TiO 2 , would allow hard entry to mouse cells and its lower surface makes its intake to the liver of mice hard.

Conclusion
The results of this study add our understanding of nanoanatase TiO 2 -induced liver toxicity and inflammatory responses in liver of mice. Both are complicated multifactorial disease processes. We suggest that inflammatory cytokines cascade may cause inflammatory cell chemotaxis, and apoptosis, resulting in serious liver injury.