The Molecular and Immune Mechanisms of the Mouse Liver and Spleen Damaged by Exposure to Nanoparticulate TiO₂

As new types of photo-catalyst, anti-ultraviolet light agents, and photoelectric effect agents, titanium dioxide （TiO₂） nanoparticles are used in the fields of paints, waste water treatment, sterilization, cosmetics, food additive, biomedical ceramic and implanted biomaterials largely owing to its high stability, anticorrosion, and photocatalysis. The small size and large surface area endow them with active groups and intrinsic toxicity. Scientists need to address the impact of nanoparticles on human and the environment. Recent studies began to help our understanding in this regard. Many studies have reported that nanoparticulate TiO₂ could cause biological and toxicity effects. But the mechanisms of these effects need to be further studied. So we investigated the toxicity effects and related molecular mechanisms of liver and spleen in mice by administration of nanoparticulate TiO₂. In addition, we also studied the mechanism of interaction of nanoparticulate TiO₂ and LDH in vitro. Our findings will be to benefit the understanding of nanomaterials-induced toxicity on liver and spleen and arouse of nanomaterials application and exposure effects especially on human liver and spllen for long-term and low-dose treatment.Three main aspects are involved:（1） In an effort to examine liver injury, immune response, and other physiological effects in mice caused by intragastric administration of TiO₂ NPs, we assessed T lymphocytes, B lymphocyte and NK lymphocyte counts, hematological indices, biochemical parameters of liver functions, and histopathological changes in TiO₂ NPs-treated mice. Indeed, mice treated with higher dose TiO₂ NPs displayed a reduction in body weight, an increase in coefficients of the liver and histopathological changes in the liver. Specifically, in these TiO₂ NPs-treated mice, interleukin-2 activity, white blood cells, red blood cells, haemoglobin, mean corpuscular haemoglobin concentration, thrombocytes, reticulocytes, T lymphocytes （CD3+, CD4+, CD8+）, NK lymphocytes, B lymphocytes, and the ratio of CD4 to CD8 of mice were decreased, whereas NO level, mean corpuscular volume, mean corpuscular haemoglobin, red （cell） distribution width, platelets, hematocrit, mean platelet volume of mice were increased. Furthermore, liver functions were also disrupted, as evidenced by the enhanced activities of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase and cholinesterase, an increase of the total protein, and the reduction of ratio of albumin to globulin, the total bilirubin, triglycerides, and the total cholesterol levels. These results suggested that the liver function damage observed in mice treated with higher dose TiO₂ NPs is likely associated with the damage of haemostasis blood system and immune response. However, low dose TiO₂ NPs has little influences on haemostasis blood system and immune response in mice.（2） In order to understand the spleen injury induced by intragastric administration of TiO₂ NPs for consecutive 45 days, the spleen pathological changes and splenocyte apoptosis, the oxidative stress, p38 and c-Jun N-terminal kinase （JNK） signaling pathways, along with nuclear factor-kappaB （NF-κB） and nuclear factor-E2-related factor-2 （Nrf-2） as the upstream events of oxidative stress, and apoptosis, and the expression levels of the apoptotic genes and their proteins （ such as caspase-3, -8, -9, Bax, Bcl-2 and Cytc） were investigated in the mouse spleen from exposure to TiO₂ NPs. The results demonstrated that TiO₂ NPs could been accumulation in the mouse spleen, leading to congestion and lymph nodule proliferation of spleen tissue, and splenocyte apoptosis, TiO₂ NPs exposure caused significant accumulations of reactive oxygen species, induced a dramatic increase of p38, JNK, NF-κB, Nrf-2, and HO-1 expression, indicating that oxidative stress in the mosue caused by TiO₂ NPs may be via P38-Nrf-2 signaling pathway. Meanwhile, TiO₂ NPs effectively activated caspase-3 and -9, decreased the Bcl-2 the levels of gene and protein, and increase the levels of Bax, and cytochrome c genes and their protein expression, but didn’tt alter caspase-8 expression. It indicated that TiO₂ NPs-induced apoptosis in the mouse splenocyte may be via mitochondrial-mediated pathway. （3） We examined the interaction between TiO₂ NPs and LDH using spectral methods. Our results showed that LDH activity was greatly increased by low concentration of TiO₂ NPs, while it was decreased by high concentration of TiO₂ NPs. By fluorescence spectral assays, the TiO₂ NPs was determined to be directly bound to LDH with mole ratio of [TiO₂ NPs] to [LDH] was 0.12, and the binding constants of the binding site were 1.77×108 L?mol-1 and 2.15×107 L?mol-1, indicating that each Ti atom was coordinated with five oxygen/nitrogen atoms and a sulfur atoms of amino acid residues with the Ti–O（N） and Ti–S bond lengths of 1.79 and 2.41 ?, respectively, and the binding distance between TiO₂ NPs and the Trp residue of LDH was 4.18 nm, and TiO₂ NPs induced the protein unfolding. We postulated that the bound TiO₂ NPs altered the secondary structure of LDH, created a new metal ion-active site for LDH, and thereby enhanced LDH activity.