The Biotoxicity Of Three Kinds Of Typical Nanomaterials

Background: With the rapid development of nanotechnology and the wide application of nanomaterials, people are increasingly exposed to various kinds of manufactured nanoparticles. However, due to nanomaterials have unique physical and chemical properties that ordinary particles do not have, such as small size effect, surface effect, quantum size effect and macroscopic quantum tunneling effect, they show different characteristics from the conventional materials, for instance heat, light, electricity, magnetic , catalytic and so on. So when they access to the ecological environment or organisms may lead to special biological effects unlike ordinary particles, and then cause negative impact on environment and human health. And not all of the nanomaterials have the same toxicity, the toxicity of nanoparticles are effected by size, shape or structure, surface charge properties, surface area, chemical composition and many undetermined factors, so the macroscopic material safety evaluation results and evaluation methods in the past may be inappropriate for nanomaterials. Studies have shown that nanoparticles can enter the body through a variety of ways, such as respiratory, skin, digestive tract and so on and cause damage on a variety of tissues and organs of experimental animals. They can cause toxic damage on the organisms in a multilevel, such as the overall level, cell level, subcellular level, protein and gene level. Their negative impact may be comprehensive, including genetic mutations, asthma, cardiovascular toxicity, neurotoxicity, skin toxicity, embryos toxicity, emphysema and lung fibrosis. However, the immune toxicity of nanomaterials has few reports. In this study, we adopt bronchial instillation exposure method to study respiratory tract immunotoxicity and overall immunotoxicity on rats induced by silver nanoparticles (Nano-Ag), zinc oxide nanoparticles(Nano-ZnO)and titanium dioxide nanoparticles (Nano-TiO₂). And the preliminary study on rat alveolar macrophages toxic effects of three kinds of nanomaterials were detected. The study results can offer scientific basis for the further mechanism research and evaluation of toxic effects after nanoparticles enter the body through the respiratory tract.Research:(1) The general toxic effects of three nanomaterials. (2) The respiratory tract immunity toxic effects induced by three kinds of nanomaterials.(3) The systemic immunity toxic effects induced by three kinds of nanomaterials.(4) The initial study on toxic effects of alveolar macrophages induced by three kinds of nanomaterials.Methods: Nano-Ag, Nano-TiO₂ and Nano-ZnO were respectively dispersed in calf serum and their size, shape and chemical composition of the basic parameters and the dispersion of nanoparticles were characterized in the transmission electron microscope. In animal study, male wistar rats were divided into the calf serum control group, high dose group (17.5mg.kg-1) and low-dose group (3.5mg.kg-1) of three nanomaterials. The rats were exposed by intratracheal instillation once two days for 5 weeks, and then killed by abdominal aorta bloodletting. The body weight changes and organ coefficients changes were checked. The pathological examinations of rat livers, lung, kidney, spleen and thymus were performed to reflects the substantial changes. The malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione(GSH)in liver and immune organs (spleen and thymus) homogenate were detected , and the MDA, SOD, GSH and nitrogen oxide(NO)in bronchoalveolar lavage fluid (BALF) and peripheral blood serum were also detected to indicate the tissues and organisms oxidative damage and react on the oxidative stress. The interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α) and macrophage inflammatory protein (MIP-2) in BALF and IL-1, TNF-α, interferon-γ(IFN-γ) and the count of cell classification in peripheral blood serum were detected to react on the effects of the immune function of the respiratory tract and the systemic of rats. T lymphocyte subsets in peripheral blood were detected by immunohistochemistry to reflects the effects of the cellular immune function. In vitro experimental study on the toxic effects of nanomaterials on rat alveolar macrophages (AM) , Nano-Ag, Nano-TiO₂ and Nano-ZnO were respectively dispersed in fetal bovine serum (FBS) to prepare the suspension of 200, 100, 50, 25, 10, 5, 2.5μg / ml and the primary culture AM cells of 2 3d were respectively exposed to the suspension, exposure time was 24h, 48h, 72h. The cytotoxicity induced by nanomaterials was measured by cellular morphology observation, WST assay and lactate dehydrogenase (LDH) assay. The effect of AMs phagocytosis induced by nanoparticles were measured by neutral red phagocytosis assay. Results:1.Animal test:(1) General toxicity results: The boby weight of rats in three kinds of nanoparticles groups significantly decreased while the organ coefficients have no significantly changes and the matrix inflammation of liver tissue appeared in the exposure groups. The activities of SOD and the GSH concentrations in liver homogenate in nanoparticles groups significantly decreased while the MDA concentrations significantly increased as compared with control. In addition, the GSH concentrations in serum in nanoparticles groups significantly decreased while the MDA and NO concentrations significantly increased as compared with control. The activities of SOD in serum in three kinds of nanoparticles groups (except for Nano-TiO₂ groups and 3.5mg/kg Nano-Ag subgroup) significantly decreased as compared with control.(2) Respiratory immune toxicity results: Three kinds of nanomaterials can induce lung inflammation, leading to fibrous tubercle, pulmonary alveoli destruction and the matrix inflammation of lung tissue. The oxidative damage result indicate that the activities of SOD (except for Nano-TiO₂ subgroup) and the GSH concentrations in BALF in nanoparticles groups significantly decreased while the MDA and NO concentrations significantly increased as compared with control. In addition, the results of inflammatory cytokines in BALF show that the concentrations of TNF-α(except for low dose Nano-TiO₂ and Nano-Ag subgroup), MIP-2 (except for Nano-TiO₂ subgroup ) in three kinds of nanoparticles groups and the IL-6 concentrations in Nano-Ag groups significantly increased while there is no significantly different in the concentrations of IL-1 as compared with control. The toxic effect comparisions amid three nanomaterials were distinct: the toxicity of Nano-Ag and Nano-ZnO was greater than Nano-TiO₂ .(3) Systemic immune toxicity results: The CD4% in peripheral blood in nanoparticles groups (except for Nano-TiO₂ subgroup) significantly decreased while the CD8% significantly increased and CD4/CD8 (except for Nano-TiO₂ subgroup) significantly decreased as compared with control. The concentrations of IL-1 in peripheral blood serum (except for Nano-TiO₂L and Nano-ZnOL subgroup) significantly increased while the concentrations of IFN-γin Nano-TiO₂H and Nano-AgH subgroup significantly decreased as compared with control. The concentrations of TNF-αin serum in Nano-AgL subgroup significantly increased while the Nano-TiO₂H, Nano-TiO₂L and Nano-ZnOL significantly decreased as compared with control. The activities of SOD in Nano-ZnOL, Nano-ZnOH and Nano-AgH subgroup and the GSH concentrations in Nano-ZnOH and Nano-ZnOL subgroup in spleen homogenate significantly decreased while the MDA concentrations in spleen homogenate significantly increased as compared with control. The GSH concentrations in Nano-ZnOH and Nano-Ag subgroup and the activities of SOD in thymus homogenate in three kinds of nanoparticles groups significantly decreased while the MDA concentrations in Nano-ZnOH , Nano-AgH and Nano-AgL subgroup significantly increased as compared with control. The results of histopathological analysis of spleen and thymus indicated no changes.2 Cell test:(1) AMs morphological changes: The control group cells were normal and the cytoplasm was greater transparency while the exposuer groups AMs result in different degrees of deformation retraction, rounding and floating, full nuclear condensation, forming a number of intracellular vacuoles and the cellular processes and pseudopod significantly reducing or even disappearing. The increased intracellular particles obviously reduced cellular transparency and the distribution of nanoparticles intercellular and cellula surface have a significant impact on the cell growth and metabolism, resulting in significantly reduced number of viable cells.(2) Changes in cell survival: The cell activity in three kinds of nanoparticles in the 5 200μg/ml each dose level(in addition to Nano-TiO₂ subgroup) significantly decreased as compared with control after 24h exposure and its toxicity increased with the dose and exposure time enhanced. After 24h exposure, the cell activity of exposure groups tends to slowly decreased and gradually into the plateau, of which Nano-ZnO subgroup cells most significant decrease in activity, followed by silver nanoparticles. After AMs were exposed for 24h, the result of LDH leakage exhibited a significant cytotoxicity at Nano-ZnO and Nano-TiO₂ concentrations of 25 100μg/ml each dose level while significant cytotoxicity at Nano-Ag concentrations of 10 100μg/ml each dose level , and both showed dose-dependent.(3) Phagocytosis of change: The phagocytosis of neutral red in exposure groups at 10 100μg/ml all dose levels were significantly lower as compared with the control group and decreased as the dose increases. The Differences also emerged amid the materials, AM phagocytosis were from strong to weak: Nano-Ag, Nano-ZnO and Nano-TiO₂.Conclusions:(1) Three kinds of typical nanomaterials can cause oxidative stress and oxidative damage to body and immune system, accordingly bring inflammatory reaction to lungs and liver. The oxidative stress may be one of the main toxic effects which trigger the immunotoxicity induced by nanoparticles.(2) Three kinds of typical nanomaterials can bring immune response, play a role in immune regulation to body immune system of rats and functional disorder of cellular immunity and suppression immunity.(3) Three typical kinds of nanomaterials can lead to decrease activity of AM cells and affect cell membrane integrity and permeability to inhibite cell activity. AM cells phagocytic function were significantly reduced by nanoparticles, weakening the nonspecific defense capability.(4) The toxic effect comparisions amid three kinds of nanomaterials were distinct: the toxicity of Nano-Ag and Nano-ZnO was greater than Nano-TiO₂ and the difference may be attributed to several parameters including particle size, shape, chemical composition and dose.