Liver Injury In Mice Caused By Nano Titanium Dioxide And Its Molecular Mechanism

Nano titanium dioxide (TiO2) is a noncombustible and odorless white powder,exists in different forms (such as anatase, rutile, and brookite) and is widely used in thecosmetics, pharmaceutical, and paint industries as a white pigment because of its highstability, anticorrosion, and photocatalysis. More nanoparticles (NPs) are released intothe environment with the increasing development of nanotechnology. The small size andlarge surface areas endow them with an active group or intrinsic toxicity. The impacts ofNPs on human and the environment have been concerned recently by some scientistsand organizations. Liver has a rich blood supply and unique morphological structure,which is a metabolic organ (such as sugar, fat, protein, vitamin and hormone substances).Meanwhile, liver has secretion, excretion, biotransformation and other importantfunctions. In the process of heavy metal detoxification, liver also plays a very importantrole. The toxicity effects between liver in mice and nano-anatase TiO2need to be furtherstudied. Therefore, we investigated the toxicic effects and related molecularmechanisms of liver in ICR mice by administration of nano-anatase TiO2, which wasinjected into abdominal cavity for two weeks and stomach for30,60or90consecutivedays. Our findings will be benefit to well understanding of biochemical toxicitiesinduced by NPs especially on human beings, and also arousing of nanomaterialsapplication in biomedicine area.Results are listed as follows:1. ICR mice were injected into abdominal cavity with nano-anatase TiO2(0,5,10,50,100and150mg/kg BW) and with bulk TiO2(150mg/kg) every day for14days.1.1In the experimental groups, the order of the titanium accumulation in theorgans was liver> kidney> spleen> lung> brain> heart (p <0.05), and the titaniumwas mainly accumulated in the liver, kidneys, and spleen. This phenomenon showed that the accumulation of titanium in the organs was closely related to the coefficients oforgans of mice. The serum biochemicalparameters with lower dose of nano-anataseTiO2showed little difference compared with the control mice, while with higherdose of nano-anatase TiO2, the indicators of liver function, such as alkalinephosphatase(ALP), alanine aminotransferase(ALT), leucine acid peptide,pseudocholinesterase(LAP), pseudocholinesterase (PChE), total protein (TP), albumin(ALB) level, were enhanced significantly (P <0.05or P <0.01); the indicators ofkidney function, such as uric acid (UA) and blood urea nitrogen (BUN), were decreased,wheareas creatinine (Cr) was increased(P>0.05); the activities of aspartateaminotransferase (AST), creatine kinase(CK), lactate dehydrogenase (LDH), andalpha-hydroxybutyrate dehydrogenase (-HBDH), were increased (P <0.05or P <0.01). The contents of total cholesterol (TCHO), triglycerides (TG), glucose (GLU), andhigh-density lipoprotein cholesterol (HDL-C) were significantly elevated from100to150mg/kg BW groups than the control group (P <0.05). It was concluded thatnano-anatase TiO2in higher dose had serious toxicity to the liver, kidney, andmyocardium of mice and caused inflammatory response of the liver, kidney, andmyocardium and the metabolism imbalance of blood sugar and lipid.1.2Histopathology of the liver tissue: inflammatory cell infiltration, hepatocyteswelling, vacuolization, congestion of vascellum and prominent vasodilatation,wide-bound eosinophilic and focal ischemia were observed. Ultrastructure of hepatocyte:changes like chromatin pyknosis, mitochondria swelling, vacuolization and apoptoticbody were observed in the liver tissue.1.3The toxicities of titanium in100to150mg/kg BW nano-anatase TiO2(5nm)groups were more severe than bulk TiO2(15μm), such as histopathology, ultrastructure,and alterations of cytokine expression in the liver.1.4We convinced that oxidative stress was one of the toxic mechanisms due tonano-anatase TiO2. Our data showed that over production of reactive oxygen species(ROS)(such as O2and H2O2) occurred in liver of mice with increased nano-TiO2dose,indicating that the liver underwent oxidative stress. Enhancement in malondialdehyde(MDA) levels evidenced peroxidation of liver (P <0.01). The activities of superoxide dismutase (SOD), catalase (CAT), ascorbic acid peroxidase (APx) and glutathioneperoxidase (GSHPx) were significantly inhibited (P <0.05or P <0.01). The ratios ofantioxidants (ascorbate and glutathione) in the50,100and150mg kg BW nano-TiO2groups decreased significantly (P <0.05or P <0.01). Data showed that ROSaccumulation, lipid peroxidation, and decreases of hepatic antioxidative systems had allcontributed to the oxidative damage of liver caused by nano-TiO2.1.5Nano-anatase TiO2could not only enter into the liver of mouse, but alsoaccumulate in liver DNA by inserting itself into DNA base pairs or binding to DNAnucleotide with three oxygen or nitrogen atoms and two phosphorous atoms of DNAwith the Ti–O (N) and Ti–P bond lengths of1.87and2.38, respectively, altering theconformation of DNA as well. And gel electrophoresis showed that higher dose ofnano-anatase TiO2could cause liver DNA cleavage in mice. By various spectralmethods, we demonstrated that nano-TiO2may alter the transduction of geneticinformations and induce liver DNA cleavage.2. Nano-anatase TiO2(0,62.5,125,250mg/kg BW) suspensions were given toICR mice by an intragastric administration every day for30days.We observed that white blood cells (WBC), red blood cells (RBC), haemoglobin(HGB), mean corpuscular haemoglobin concentration (MCHC), thrombocytocrit (PCT)and reticulocytes (Ret) of these nano-TiO2-treated mice decreased, while meancorpuscular volume (MCV), mean corpuscular haemoglobin (MCH), red celldistribution width (RDW), platelets (PLT), hematocrit (HCT) and mean platelet volume(MPV) increased (P <0.05or0.01). The reductions of RBC, RBC, WBC, Ret and HGBsuggested that NPs could affect red blood cell production and lead to anemia. Theincrease of PLT and MPV showed a possible effect of nano-TiO2on blood coagulation.The significant decreases of RBC count and HGB concentration caused by higher dosenano-TiO2could cause a marked decrease in O2content in the blood, which mightdecrease metabolism and immune response of mice. Our data suggested that nano-TiO2decreased the percentage of T lymphocytes (including CD3, CD4and CD8), Blymphocytes and NK lymphocytes, as well as the ratio of CD4to CD8, IL-2activity andIgM of the mouse serum. These results indicated that the damage of liver function may be involved in the reduction of immune response of mice.3. Nano-anatase TiO2(0,5,10,50mg/kg BW) suspensions were given to ICRmice by an intragastric administration every day for60days.Nano-anatase TiO2could activate kupffer cells of liver. NPs increased both mRNAand protein expression levels of several inflammatory cytokines, including nucleicfactor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), macrophage migrationinhibitory factory (MIF), interleukin-6(IL-6), IL-1β, cross-reaction protein (CRP), IL-4,and IL-10. The present study indicates that the hepatitis of mice is triggered by TiO2NPs, as evidenced by alterations of expression levels of the genes and their proteinsinvolved in the signaling pathway (such as TLRs and inflammatory cytokines), and byreduction of immune capacity.4. In this section, we used the whole-genome microarray analysis technique todetermine the gene expression profile in the livers of mice exposed to10mg/kg bodyweight TiO2NPs for90consecutive days.The results showed that more than2.3%(1035genes) of the total genes (45,000genes) were significantly changed following long-term exposure to10mg/kg TiO2NPs.Microarray data showed striking changes in the expression of745genes related tometabolic process, cell communication, immune/inflammatory response, cell cycle,apoptosis, transport, and so on. Of these394genes of metabolic process,254geneswere up-regulated and140genes were down-regulated. The number of differentexpressed genes in lipid metabolism process is96. These genes were included in manymetabolic processes, like “biological process”,“cholesterol biosynthesis”,“endogenoussterols”, and “metabolism of lipids and lipoproteins”. We analyzed the immune genesand found that98genes were significantly altered by exposing to TiO2NPs. Of these98genes,59were up-regulated and39were down-regulated. The obvious reduction in theexpression of Cfd following exposure to TiO2NPs may trigger signaling cascades toactivate inflammatory programs, like the reduction of complement activation andinhibition of innate immunology. In this section, microarray data suggested thatapproximately37genes related to appotosis were significantly changed in the TiO2NP–exposed liver. Of these37genes,28were up-regulated and9were down-regulated. The over expression of Axud1could up-regulate Axin’s expression, which mightactivate the apoptotic process.In conclusion, liver is a central organ to the accumulation and damages ofnano-anatase TiO2NPs, these damages are closely associated with oxidation stress,inflammatory cascade effect, DNA damage, immune dysfunction, and molecularmechanisms of multiple genes working together.