In this study, the ICR mice were injected with various doses of nano-anatase TiO2 into abdominal cavity everyday for 14 days. In the 50, 100 and 150 mg/kg body weight nano-anatase TiO2-treated groups, the higher coefficients of the liver were observed (P < 0.05 or P < 0.01). A previous study showed that when a fixed high dose of 5 g/kg body weight of nano-TiO2 suspensions was administrated by a single oral gavage, the coefficients of liver after 2 weeks were significantly increased , demonstrating that nano-TiO2 in higher dose had serious toxicity to the mouse liver. Our studies showed that titanium contents in the liver DNA of mice were gradually elevated with increasing injection doses of nano-anatase TiO2, which were closely related to the coefficients of the liver of mice. Our previous work showed that the order of the titanium accumulation in the organs of mice was liver > kidneys > spleen > lung > brain > heart, the liver function was damaged . The study suggested that, after entering the animals, nano-anatase TiO2 was accumulated in DNA of the mouse liver.
The absorbance decreasing effect can be used as an evidence that there exists an interaction model of binding between metal ions and DNA base pairs or nucleotide, i.e., metal ions can coordinate into DNA base pairs and bind to nucleic acids [19, 20]. The experimental results proved that the π → π* transitions of DNA at 260 nm showed an intensity decrease with increasing doses of nano-anatase TiO2, which supports the notion that there exists an interaction model of binding, i.e., a strong π-stacking interaction between Ti4+ and DNA base pairs [19, 20]. Ti4+ can insert into DNA base pairs and bind to nucleotide. Our results are also consistent with the previous studies on the effects of other heavy metal ions on DNA [21–23].
X-ray absorption spectroscopy (XAS) has been proved to be a very powerful technique to detect the local structure around specific elements. The EXAFS contains information of local atomic arrangement for each absorber atom, as described in theoretical formula based on the single-scattering contribution to XAFS. The X-ray fluorescence excitation XAS warrants detection of low concentrations of transition metals presented in metalloenzyme and DNA systems [22–25]. In order to investigate the direct effects of nano-anatase TiO2 on DNA, we used X-ray absorption technique to study the coordination structure at Ti sites in Ti4+–DNA from the 150 mg/kg body weight nano-anatase TiO2-treated liver of mice. Our data showed that Ti was bound with three oxygen or nitrogen atoms on DNA in its first shell, and the second shell was two phosphorous atoms, proving that nano-anatase TiO2 could be bound with the oxygen or phosphorous atoms of nucleotide, and nitrogen atoms of base pairs in DNA.
To further investigate the evidence for interaction of nano-anatase TiO2 with DNA from the liver of mice, DNA conformation was studied using CD technique. We found that, in the 50, 100 and 150 mg/kg body weight doses of nano-anatase TiO2, the positive bands at 220 and 272 nm increased and red shifted, and the negative bands at 210 and 244 nm decreased and red shifted, indicating that the transformation from A conformation to B conformation was generated with increasing winding of the DNA helix by rotation of the bases, and nano-anatase TiO2 caused the shrink of DNA molecule structure [26, 27] herein produced an obvious change of the secondary structure. It was consistent with absorption spectra with respect to this change. The changes of DNA conformation might interfere with the genetic information transmission of DNA and induced inflammatory response of liver consequently .
By studying the interaction between nano-anatase TiO2 and DNA, many previous in vitro studies proved that indirect interaction is associated with oxidative damage to DNA. Being a proven photocatalyst, nano-TiO2 is capable of undergoing electron transfer reactions under ultraviolet light. For instance, the electron was excitated and transferred then photogenerated electron-holes in nano-TiO2; the electron-holes are reduced when the electron is captured by other molecule, while it is oxidized when itself was captured . In the aqueous environments, nano-TiO2 would produce hydroxy radical, and hydroxy could react with DNA, producing 8-hydroxy guanosine, which resulted in DNA cleavage and oxidative damage under UVA illumination [30, 31]. Dunford et al.  reported that sunlight-illuminated nano-TiO2 catalyzed DNA damage in both in vitro and human cells. They also used nano-TiO2 samples extracted from sunscreens to attack PBII DNA under the ultraviolet light between 300 and 400 nm, and relaxed standards and cleavage were observed . Wamer et al.  irradiated calf thymus DNA in nano-TiO2 solutions with UVA radiation in vitro and found the generation of 8-oxo-7 and 8-dihydro-2 *-deoxyguanosine (8-oxodG) in DNA. Ashikaga et al. indicated that supercoiled pBR 322 DNA was formed to open-circular DNA with 5 J/cm2 of UVA in the presence of TiO2. The studies mentioned above about DNA effects were carried out both in vitro and under light. The present article proved that nano-anatase TiO2 caused the changes of DNA conformation in the liver of mice, and we also clearly observed the DNA ladder in liver by agarose gel electrophoresis from the 150 mg/kg body weight nano-anatase TiO2-treated group, showing that after entering the animals, nano-anatase TiO2 can cause hepatocyte apoptosis in vivo. The previous study used TEM to observe ultrastructure changes of hepatocyte of the mouse liver tissue, presenting significantly hepatocyte tumescent mitochondria, vacuolization and apoptosis body from the 100 and 150 mg/kg body weight nano-anatase TiO2-treated groups . 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 postexposure 2 weeks to the 5 g/kg body weight 80 nm and fine TiO2 particles . Ma et al.  indicated that intraperitoneal injection of higher doses of nano-anatase TiO2 can induce histopathological changes of liver, including congestion of vascellum, prominent vasodilatation, wide-bound basophilia and focal ischemia. The mechanism of DNA cleavage and hepatocyte apoptosis in vivo caused by nano-anatase TiO2 was attributed to the significant accumulation of reactive oxygen species in liver of mice .
Taken together, we speculate that the combination of nano-anatase TiO2 with DNA, which is similar to hepatovirus, might cause the inflammatory cascade of the mouse liver, and the alteration of DNA secondary structure in mice caused by nano-anatase TiO2 might result in the changes of genetic information transmission, and various inflammatory responses, these still need to be confirmed by further study.