The results of this study indicate that intraperitoneal injection of higher doses of nano-anatase TiO2 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.  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 TiO2 particles, but did not observed significant histopathological change in liver tissues of mice exposed to the 5 g/kg BW 25 nm TiO2 particles. The present study indicates that the hepatitis of mice is triggered by nano-anatase TiO2 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 TiO2 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 TiO2-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 TiO2-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 . Wang et al.  showed that after a single oral gavage of dose of 5 g/kg BW of TiO2 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 TiO2-treated group was higher than those of 150 mg/kg BW bulk-TiO2-treated group (p < 0.05). Compared with bulk TiO2, smaller grain size of nano-anatase TiO2(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-TiO2particles, 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-κB is a critical intracellular mediator of the inflammatory cascade. In quiescent cells, NF-κB is bound to inhibitory proteins called IκBs that prevents NF-κB from migrating to the nucleus and located in the cytoplasm. When an appropriate inducer, such as hepatovirus, affects the cell, IκBs are phosphorylated and degraded, allowing nuclear uptake of NF-κB and initiating gene transcription (such as MIF, the proinflammatory cytokines of TNF-α, IL-6, IL-1β, CRP, and anti-inflammatory cytokines of IL-4 and IL-10) . As an inflammatory factor, MIF also plays a role of inflammatory mediators in various diseases. The liver toxicity caused by nano-TiO2 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 TiO2 can significantly stimulate the mRNA expressions and increase protein levels of several inflammatory cytokines, including NF-κB, MIF, TNF-α, IL-6, IL-1β, 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 TiO2-induced liver toxicity. It had been demonstrated that nano-TiO2 could promote the expression of several cytokines and chemokines in the lung of rat and mice, including placenta growth factor (PlGF), MCP-1, IL-1β, and TNF-α [6, 20], and increase protein level of TNF-α, IL-1β in brain of mice and cause brain inflammation [21, 22]. Here, we speculate that nano-anatase TiO2 particles, which are similar to hepatovirus, can make IκBs phosphorylated and degraded, and then induce NF-κB activation, leading to the expression of the NF-κB-controlled proinflammatory cytokines (such as IL-1β, 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 TiO2 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-TiO2can 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 TiO2particles. Compared with nano-anatase TiO2(5 nm), bulk TiO2, would allow hard entry to mouse cells and its lower surface makes its intake to the liver of mice hard.