| Silica nanoparticles, which took up the highest proportion of nanopowder production in industrial in China and even in the whole world, have been widely used in chemical production, construction materials, cosmetics, biochemical and other fields. With the growing commercialization and application of silica nanoparticles, human exposure to silica nanoparticles is increasing. Lung is the most prime and major target organ for particles. So it is essential to study the pulmonary toxicity of silica nanoparticles.Some studies indicated that the smaller the size of a particle is, the larger surface area it has, and the higher biological activity it exhibits. Some in vitro studies revealed that the biological effects of nanoparticles may be correlated with the size of the particle. But the informations from in vivo studies are still scarcely reported. In the study, we used the silica particles (600 nm) as control particles. Comparatively study of pulmonary toxicity and the related mechanism induced by multi-scale silica nanoparticles (90nm,60nm,30nm) could provide the experimental evidence for safety evaluation of nanomaterials exposing in the respiratory track.Male Wistar rats were exposed by intratracheal instillation once every two days for totally 16 times. The control group was intratracheally instilled only with saline solution instead.Three dosages were used in this experiment:2 mg/kg bw was chosen as a low dose exposure,5 mg/kg.bw for a median dose exposure,10 mg/kg bw represented a high one. Characterization of silica nanoparticles were determined by transmission electron microscopy (TEM) and dynamic light scattering particle size analyzer (DLS); general toxicity of the rats exposed to silica nanoparticle were evaluated by body weight, liver and kidney organ coefficient, biochemical indexes in peripheral blood, the histopathological examination of liver and kidney; the silicon content was quantified by inductively coupled plasma optical emission spectrometer (ICP-OES); morphological changes in lung tissues were detected by HE staining; ultrastructural changes of lung tissues were observed by using transmission electron microscope (TEM); hydroxyproline contents in lung and total protein (TP), lactate dehydrogenase (LDH), total antioxidant capacity (T-AOC), superoxide dismutase (SOD) levels in bronchoalveolar lavage fluid were measured by biochemical assay; the levels of reactive oxygen species (ROS), interleukin-1 (IL-1), interleukin-6 (1L-6), tumor necrosis factor-a (TNF-a) in the bronchoalveolar lavage fluid were determined by using the enzyme linked immunosorbent assay (ELISA); expression of NF-лB, TGF-β1, Fas, FasL, caspase-8, caspase-3 in lung were determined using immunohistochemistry.1. Characterization of silica particlesOur results showed that silica particles, in four scales (600nm,90nm,60nm,30nm), were mostly spherical with uniform size and well dispersed in high purity water and normal saline. Si600, Nano-Si90, Nano-Si60 maintained a good dispersion in normal saline, but Nano-Si30 exhibited a slight agglomeration. Nano-Si90, Nano-Si60, Nano-Si30 were spherical with uniform but slightly increased size in tissues, which were still in nanoscale range.2. Effects of silica nanoparticles on the general toxicity of ratsThere were no significant changes in body weight, the organ coefficient of liver and kidney in silica particles exposure groups. HE staining results showed that there were no obviouse changes in liver and kidney of the silica nanoparticle exposed groups. The results of biochemical indexes showed that silica nanoparticles could cause some adverse effects on liver and kindey.3. Effects of silica nanoparticles on the morphology of lungThe histopathological examination of lungs in the silica nanoparticle exposed group showed infiltration with mononuclear, macrophages, lymphocytes and epithelial cells exfoliation in alveolar space. Alveoliar wall congestion, thickened alveolar septum, lymph nodes around bronchi enlargement and cell nodules were observed in the lung. The alveolar septum became wider as the particle size decreased, Binucleated and multinucleated cells of cytoplasmic positive cells were observed in the pulmonary alveoli in silica nanoparticle exposed groups. The frequency of binucleated and multinucleated cells increased in response to the increase of dosage and the decrease of the particle size.4. Effects of silica nanoparticles on the ultrastructure of lungThe TEM examination of lungs in the silica nanoparticle exposed group showed decreased microvilli, cavitation nuclear matrix, slightly expansion of the endoplasmic reticulum, vacuolation of lamellar bodies in alveolar type II cells. The vacuoles in lamellar bodies within the cytoplasm significantly increased and enlarged as the particle size decreased; increased lysosomes and swelling mitochondrial were observed in the macrophage cytoplasm. Nanoparticles were observed in the alveolar typeⅡcells and macrophage cytoplasm in Nano-Si60 and Nano-Si30 groups. Nanoparticles were also observed in Nano-Si90 group, but only in the macrophage cytoplasm. Silica nanoparticles in cytoplasm existed in cluster or scattered pattern. Some of the nanoparticles were found in endocytic vesicles and lysosomes, others scattered in the cytoplasm without membrane binding.5. Effects of silica nanoparticles on hydroxyproline contents in the lungOur results revealed that the lung hydroxyproline contents increased with the increase of doseage and decrease of nanoparticle size. There were significant difference between Nano-Si90, Nano-Si60, Nano-Si30 groups in high dosage level and control group, respectively (P<0.05). The lung hydroxyproline contents of Nano-Si90, Nano-Si60, Nano-Si30 groups were significantly higher than those of Si600 group in the high dosage level (P<0.05).6. Effects of silica nanoparticles on silicon contents in the lungThe silicon content of Si600, Nano-Si90, Nano-Si60 and Nano-Si30 groups in the lung were significantly higher than those of control group (P<0.05), Our results revealed that the silicon contents in lung increased with the increase of dose, but there were no significant differences among Si600, Nano-Si90, Nano-Si60, Nano-Si30 groups (P>0.05).7. Effects of silica nanoparticles on pro-inflammatory cytokines levels in BALFIn Si600 and Nano-Si90 groups, IL-6 and TNF-αlevels increased with the increase of dosage (P<0.05). IL-6 levels in all dosage levels were significantly higher than those of control group, TNF-αlevels in high dosage level group were significantly higher than those of control group (P<0.05); In Nano-Si60 and Nano-Si30 groups, IL-1, IL-6, TNF-αlevels increased with the increase of dosage (P<0.05), IL-6 levels in all dosage levels were significantly higher than those of control group (P<0.05); TNF-αand IL-1 levels of Nano-Si60 were significantly higher than those of control group in high dosage level (P <0.05); IL-1 levels of Nano-Si30 group in the median and high dosage levels were significantly higher than those of control group (P<0.05) TNF-αlevels of Nano-Si30 group in low and high dosage levels were significantly higher than those of control group (P<0.05). In the low dosage level, TNF-αlevels of Nano-Si30 group were significantly higher than those of Nano-Si90 and Si600 groups (P<0.05); in the high dosage level, TNF-α, IL-1 levels of Nano-Si30 group were significantly higher than those of Si600 group (P<0.05), IL-6 levels of Nano-Si30 group were significantly higher than those of Nano-Si60, Nano-Si90, Si600 groups (P<0.05). 8. Effects of silica nanoparticles on the oxidative stress levels in BALFROS levels were significantly higher than those of control group in all exposure (P <0.05). In Si600 group, there were significant differences between LDH and SOD levels of Si600 group and control group in the median and high dosage levels, respectively (P<0.05), T-AOC levels were significantly higher than those of control group in the high dosage level (P<0.05). In Nano-Si90 group, LDH levels in the high dosage level were significantly higher than those of control group (P<0.05), SOD and T-AOC levels in all dosage levels were significantly lower than those of control group (P<0.05). In Nano-Si60 and Nano-Si30 groups, there were significant differences between LDH, SOD, T-AOC levels of Nano-Si60, Nano-Si30 groups in all dosage levels and control group, respectively (P<0.05). In the median dosage level, T-AOC levels of Nano-Si30 group were significantly lower than those of Si600 group (P<0.05). In the high dosage level, SOD levels of Si600 group was significantly lower than those of Nano-Si60 and Nano-Si30 groups (P<0.05), ROS levels of Nano-Si30 group was significantly higher than that of Si600 group (P<0.05).9. Effects of silica nanoparticles on the lung apoptosisOur results revealed that the apoptotic rate increased with the increase of dosage and decrease of nanoparticle size. In Si600 and Nano-Si90 groups, the apoptotic rate in median and high dosage levels were significantly higher than those of control group (P<0.05), In Nano-Si60 and Nano-Si30 groups, the apoptotic rate in all dosage levels were significantly higher than those of control group (P<0.05). In the low dosage level, the apoptotic rate of Si600 group was significantly lower than that of Nano-Si30 group (P<0.05); In the high dosage level, the apoptotic rate of Si600 group was significantly lower than that of Nano-Si30 Nano-Si60, Nano-Si90 groups (P<0.05).10. Effects of silica nanoparticles on the expression of the lung injury related proteinIn Si600 group, the average optical density of TGF-pi, NF-κB, Fas, FasL, Caspase-8, Caspase-3 in the median and high dosage levels were significantly higher than those of control group (P<0.05). In Nano-Si90 group, the average optical density of TGF-β,NF-κB, Fas, Caspase-8 in the median and high dosage levels were significantly higher than those of control group (P<0.05), the average optical density of FasL and Caspase-3 in all dosage levels were significantly higher than those of control group (P<0.05). In Nano-Si60 group, the average optical density of TGF-β,NF-κB in the median and high dosage levels were significantly higher than those of control group (P<0.05), the average optical density of Fas, FasL, Caspase-8, Caspase-3 in all dosage levels were significantly higher than those of control group (P<0.05). In Nano-Si30 group, the average optical density of TGF-β1, NF-κB, Fas, FasL, Caspase-8, Caspase-3 in the all dosage levels were significantly higher than those of control group (P<0.05). In the low dosage level, the average optical density of TGF-β1 and Caspase-3 of Si600 group were significantly lower than those of Nano-Si30 group (P <0.05), the average optical density of Fas and Caspase-8 of Si600 group were significantly lower than those of Nano-Si60 and Nano-Si30 groups (P<0.05), the average optical density of Fas of Nano-Si90 and Nano-Si60 groups were significantly lower than that of Nano-Si30 group (P<0.05), the average optical density of NF-κB and FasL of Si600 group were significantly lower than those of Nano-Si90, Nano-Si60 and Nano-Si30 groups (P<0.05), the average optical density of FasL and Caspase-3 were significantly lower than those of Nano-Si30 group; In the median dosage level, the average optical density of NF-κB of Si600 group was significantly lower than that of Nano-Si30 group (P<0.05), the average optical density of TGF-β1 and FasL of Nano-Si30 group were significantly higher than those of Si600, Nano-Si90 and Nano-Si60 groups (P<0.05), the average optical density of Fas of Si600 and Nano-Si90 groups were significantly lower than those of Nano-Si60 and Nano-Si30 groups (P<0.05), the average optical density of Caspase-3 of Si600 and Nano-Si90 groups were significantly lower than that of Nano-Si30 group (P<0.05). In the median dosage level, the average optical density of NF-κB of Si600 group was significantly lower than that of Nano-Si30 group (P<0.05), the average optical density of TGF-β1 and FasL of Nano-Si30 group were significantly higher than those of Si600, Nano-Si90 and Nano-Si60 groups (P<0.05), the average optical density of Fas of Si600 and Nano-Si90 groups were significantly lower than those of Nano-Si60 and Nano-Si30 groups (P<0.05), the average optical density of Caspase-3 of Si600 and Nano-Si90 groups were significantly lower than that of Nano-Si30 group (P<0.05). In the high dosage level, the average optical density of TGF-β1 and Fas of Si600 group were significantly lower than those of Nano-Si90, Nano-Si60 and Nano-Si30 groups (P<0.05), the average optical density of NF-κB of Si600 group was significantly lower than that of Nano-Si60 and Nano-Si30 groups (P<0.05), the average optical density of NF-κB of Nano-90 group was significantly lower than that of Nano-Si30 group (P<0.05), the average optical density of Fas of Si600 and Nano-Si90 groups were significantly lower than those of Nano-Si30 group (P<0.05), the average optical density of FasL of Si600 and Nano-Si90 groups were significantly lower than those of Nano-60 and Nano-Si30 groups (P<0.05), the average optical density of Caspase-8 and Caspase-3 of Nano-Si30 group were significantly higher than those of Si600, Nano-Si90 and Nano-Si60 groups (P<0.05).In summary, our results indicated that silica nanoparticles could cause some adverse effects on the lung, which may be correlated with the size of the nanoparticles. The possible routes may be oxidative damages, inflammatory reactions and apoptosis. The low dosage silica nanoparticles exposure level in our study, which is the maximum limit value of PM2.524 provided by WHO air quality guidelines, performed significant pulmonary toxical effects, our results indicated that the pulmonary toxicity of silica nanoparticles could be stronger than that of PM2.524, and has beyond the compensatory ability of the respiratory system in rats.
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