The direct instillation of silica into the lungs via the trachea has been employed in many studies as an animal model for silicosis. Inhalation provides a natural route of entry into the host and as such, is preferable for the introduction of silica into the lungs. An intratracheal instillation cannot be further from the physiological condition. The distribution of an instilled material within the respiratory tract will likely differ from the distribution of an inhaled material. The upper respiratory tract (i.e., the nasal passages, oral passages, pharynx, and larynx) can be a potentially important target site for an inhaled test material passed by intratracheal instillation. Especially, another disadvantage of instillation is the introduction of the toxicant being non-physiological and involving invasive delivery usually at a dose and/or dose rate substantially greater than that which would have occurred during inhalation. However, instillation has certain advantages over inhalation, as discussed in detail by Brain et al.. Briefly, with instillation, the actual dose delivered to the lungs of each animal can be essentially assured. The technique is simpler than inhalation exposure procedures and minimizes risks to laboratory workers from highly toxic, carcinogenic, or radioactive materials. Intratracheal instillation permits the introduction of a range of doses to the lungs within a short time, and avoids exposure to the skin and pelt that can occur with inhalation exposure. Furthermore, intratracheal instillation has become sufficiently widely used as a screening tool for determining the approximate dose range that may be appropriate for later inhalation studies, or to determine the ranking of toxicity for a series of structurally similar chemical agents[25-26]. Respiration toxicological information for various particles has reviewed that some biological end points, such as pulmonary inflammation, fibrosis and susceptibility to infection, hypersusceptibility and development of lung cancer, were similar after the rats were exposed to low-solubility by two kind manners of inhalation and instillation. Some researchers have therefore considered that selectively applying intratracheal instillation by investigating toxicological character of dust according to different aims is likely to provide valuable information consistent with inhalation on the base of predominating application rules.
In the pulmonary toxicological study of dust, particle loading into the lungs is an important factor. A number of studies have investigated the relationship that may exist between the pulmonary silica burden and subsequent development of silicosis. The particles exceeding loading do not directly influence pulmonary pathological end point. Over-load of dusts is enough to damage the ability of macrophages to clean particles, then inducing nonspecific inflammation, particles deposition in the pulmonary interstitial or alveolus cavity and epithelia hyperplasia. By contrary, the particles are normally eliminated by all kinds of cleaning mechanisms. A large amount of silica in the lung tissue is closely related to silicosis development. Nagelschmidt summarized the literatures on pathological response of silicosis to multiple silica loads. He considered that there was relationship between increased silica amounts and silicosis pathological classification. In classical silicosis of gold-miners and molder, 1 to 3 g of silica particles was found in their lungs to result in pulmonary fibrosis. This current study referred to a variety of dust dosages/concentrations of experimental silicosis in the animal model[13, 23]. In the trial experiment, every rat was intratracheally instilled with SNs dissolved in saline at 50 mg/mL concentrations via trachea on the premise of established pulmonary fibrosis model induced by the SNs. The rats were successively died at 24 h and they were expected to die of acute pulmonary edema by gross and histopathological inspection of their lungs. Fortunately, five rats that were instilled intratracheally with 50.0 mg microscale SiO2 particles showed no overt abnormal signs at 24 h and no rat died in 7 d, which is an observation period for acute toxicity. These results suggest that the acute pulmonary lesions for the rats exposed to high dosage of SNs are more serious than those from the microscale SiO2 particles group rats at same dosage. These results are in agreement with findings from other investigators. Kaewamatawong reported that mice were intratracheally instilled with 3 mg of 14 nm and 230 nm colloidal silica particles (CSs) and pathologically examined from 30 min to 24 h post exposure. They histopathologically found that the lungs exposed to both sizes of particles showed bronchiolar degeneration and necrosis, neutrophilic inflammation in alveoli with alveolar type Ⅱ cell swelling and particle-laden alveolar macrophages (AMs) accumulation. The 14-nm CSs, however, induces extensive alveolar hemorrhage compared to 230-nm CSs from 30 minutes onwards. The 14-nm CSs also causes more severe bronchiolar epithelial cell necrosis and neutrophil influx in alveoli than 230-nm CSs at 12 and 24 h post exposure. These findings are perhaps in connection with small size effect from the nanoparticles and liability to aggregation. High concentration nanoparticles instilled intratracheally for a comparatively short time aggregate to larger mass compared with macroscale particles, and they also obstruct the airway to a different extent along the bronchus, bronchiole, and terminal bronchioles entering into alveoli. In addition, pulmonary reactivity to SNs is obviously increased because of the large surface area and high bioactivity of the SNs. Therefore, in this study, 25 mg SNs in 1 mL of saline per rat is confirmed highest exposure dose for intratracheal instillation, based on our trial experiment and literature reports.
Sayes et al. reported that rats were exposed to 1 or 5 mg/kg of crystalline silica (Min-U-Sil 5, α-quartz) and precipitated amorphous silica by intratracheal instillation, and BAL fluids from the rats were analyzed at 24 h, 1 week, 1 month, and 3 months post instillation exposure. They found that the crystalline silica particles produced sustained inflammation and cytotoxicity. Precipitated amorphous silica particles produced reversible and transient inflammatory responses. Moreover, results from in vivo pulmonary toxicity studies by Chen et al.demonstrated that the Wistar rats were instilled intratracheally with 20 mg of nanosized SiO2 or 20 mg of microsized SiO2 . The rats were sacrificed at 1 and 2 months after instillation. The lung/body coefficient of nanosized SiO2 groups are significantly lower than those of the microsized SiO2 groups at both 1 and 2 months after instillation (P < 0.05 or P < 0.01), but without significant differences from those of saline control groups. The results from our in vivo pulmonary toxicity studies indicate that the instilled SNs produce little toxicity and the microscale SiO2 particles exposures produced pulmonary fibrosis.
The lung ultrastructural change is the same an intuitionistic index for histomorphological changes. Transmission electron microscopy (TEM) reveals hyperplastic alveolar type Ⅱ cells lining the alveoli, and the occasional clusters of alveolar type Ⅱ cells contained lots of osmiophilic lamellar bodies dissociated in the alveoli cavity. The hypertrophic AMs are activated by silica contained vesicle of phagocytosing silica, lamellar bodies, and lipoproteinosis. Some researchers pointed out hyperplastic alveolar type Ⅱ cells being initial events and hypertrophic AMs being key link to pulmonary fibrosis development in experimental silicosis[34-35]. The ultrastructural changes in the lungs as induced by the SNs and microscale SiO2 particles in this study are in accordance with given literature. The result from the lungs EDS analysis indicate the macrophage engulfed SNs and microscale SiO2 particles.
Bio-distribution and clearance of the nanoparticles are not likely due to the microscale SiO2 particles on account of their small size. Cho et al.prepared ultrafine amorphous silica particles (UFASs) in phosphate buffered saline (PBS) and intratracheally administered to A/J mice at 0, 2, 10, and 50 mg/kg doses. The animals were sacrificed at 24 h, 1, 4, and 14 weeks following exposures. The intratracheal instillation of the UFASs at 2 and 10 mg/kg doses has no effects on the lung/body coefficients gains. In contrast, the instillation of 50 mg/kg UFASs significantly increase the absolute and relative lung weights, while 24 h and 1 week instillation have no effects on the lung/body coefficients gains at 4 or 14 weeks following exposures compared to controls. The histopathological examination reveals that the UFASs induce severe inflammation with neutrophils at an early stage and chronic granulomatous inflammation at the later stage. However, the lung lesions are milder at 4 weeks and have almost recovered at the final time point. These findings perhaps explain that the ultrafine particles may translocate from the site of deposition into the lungs and extrapulmonary organs through the systemic circulation, which may result in the rapid elimination of lung inflammation and injury. However, during the early event after instillation, the severity of injury due to ultrafine particles is more severe than that of fine particles. He et al. investigated the biodistribution and urinary excretion of three types of surface-modified silica nanoparticles with 45 nm size in mice injected intravenously (iv) using an optical imaging method, taking advantage of RuBPY dye doped in the silica matrix as a synchronous fluorescence signal. Results from the in vivo imaging studies show that three types of surface-modified silica nanoparticles can all be cleared from the circulation and present inside organs and partly excreted through the renal route. Several studies corroborat that quartz dust is difficult to eliminate outside the lungs at long exposure periods by organism own clearance system[37-38]. It is an important reason for silicosis development and progress. The current study reveals that the SNs are partly excreted through the renal excretion route. However, this needs further study to assess whether the SNs produce the same pulmonary toxicity as the microscale SiO2 particles. TEM and EDS analysis of urine samples in this study confirm that there is relatively little excretion of small size microscale SiO2 particles through the renal route. In contrast, a large amount of granular substances containe Si in the urine from rats post exposure to SNs at 24 h. The results from the excretion studies confirm that the SNs are in fact excreted through the urine as intact SNs through the renal route. Hence, these findings perhaps explain that the SNs deposition in the lungs are less than microscale SiO2 , and result in slight pulmonary lesion.
Oxidative stress is the result of an imbalance in the pro-oxidant/antioxidant homeostasis. Many reports have been released on the relationship between oxidative stress and inflammation. Oxidative stress may trigger activation of transcription factor, induce mRNA expression of inflammation media, and finally bring up inflammation reaction and related disease. Nitric oxide is an omnipresent signaling molecule produced by a variety of mammalian cells, including vascular endothelium, neurons, smooth muscle cells, macrophages, neutrophils, platelets, and pulmonary epithelium. In pathological condition, nitric oxide formation is increased and it induces inflammation development. Park et al. treated mice with 50 mg/kg silica nanoparticles through intraperitoneal injection. The mice were sacrificed at 12, 24, 48, and 72 h after treatments, respectively. Activated macrophages from the peritoneal cavity of the mice and increased NO release from the cells to the supernatant in a time-dependent manner were observed as a result. At the same time, the reactive oxygen species (ROS) and NO generation were increased and intracellular glutathione (GSH) was decreased in a dose-dependent manner in RAW264.7 cell line which originated from the mouse peritoneal macrophage. Study suggested that silica nanoparticles induced pro-inflammatory responses in vivo/in vitro and pro-inflammatory responses may be triggered by ROS and NO generation. MDA is an end product of lipid peroxidation and superoxide dismutase (SOD) is a cleaner of free radicals. There is negative correlation between MDA and SOD. Yang et al. reported that SiO2 nanoparticles induced inhibition of primary mouse embryo fibroblasts cell viabilities and elevation of ROS and MDA levels in cell medium (at 20-100 μg/mL) in an explicit dose-dependent manner. These results proved that the oxidative stress was probably a key route by which the nanoparticles induced cytotoxicity. Some in vivo experiments directly demonstrated that silicosis was a state of oxidative stress and that increased generation of ROS was associated with enhanced levels of oxidative enzymes and lipid peroxidation[43-44]. Similarly, in our current research study, the level of MDA and NO in the lung homogenate is increased in the SNs group at 12.5 and 25 mg/mL concentration and in the microscale SiO2 particles group at 25 mg/mL concentration. These experimental results reveal that the SNs may result in pulmonary lesion-induced oxidative stress.
Free radical chain reaction was initiated by free crystalline silica inhalation and various bioactive substances, such as TNF-α, TGF-β1, PDGF, and FGF were released by AMs in another study. Besides the AMs, the above cytokines are produced by neutrophils, lymphocytes, and alveolar epithelial cells (type Ⅰ and type Ⅱ) stimulated by the dust. These cytokines promote fibroblasts proliferation, collagen metabolism, eventually leading to pulmonary fibrosis. Park et al.reported in another study that the mRNA expressions of inflammatory-related genes, such as IL-1β, TNF-α, IL-6, iNOS, and COX-2, were upregulated in a time-dependent manner in the macrophages harvested from the mice treated with silica 50 mg/kg nanoparticles. The levels of pro-inflammatory cytokines (IL-1β and TNF-α) released to serum in the treated mice were also elevated after i. p. injection of silica nanoparticles 50 mg/kg. IL-1β reached maximum at 12-24 h after treatment and the level was decreased gradually in a time-dependent manner at 72 h. Also, TNF-α reached maximum at 24 h after treatment and its level decreased gradually in a time-dependent manner. In studies by Choi et al., the histopathological examination revealed that 14nm ultrafine amorphous silica (UFAS), intratracheally administered to A/J mice, induced severe inflammation with neutrophils at an early stage and chronic granulomatous inflammation at the later stage. The mRNA and protein levels of IL-1β, IL-6, IL-8, TNF-α, MCP-1, and MIP-2 in the lung tissues are significantly increased during the early stages, but there are no changes after weeks 1 (TNF-α) or 4 (IL-1β, IL-6, IL-8, MCP-1, and MIP-2). Instillation of UFASs induced transient, but very severe lung inflammation. These results show that the cytokines (IL-1β, IL-6, IL-8, and TNF-α) and chemokines (MCP-1 and MIP-2) play important roles in the inflammation induced by the intratracheal instillation of UFASs. Another study showed that the UFAS induced severe alveolar epithelial thickening and pulmonary fibrosis at 1 week by Gomori's trichrome staining, and almost recovered at 4 and 14 weeks. The mRNA and protein levels of cytokines (IL-4, IL-10, IL-13, and IFN-γ), matrix metalloproteinases (MMP-2, MMP-9, and MMP-10) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in lung tissues are significantly elevated at 24 h and 1 week post-treatment. These levels decrease to near control range at 4 and 14 weeks except for the IFN-γ and MMP-2. These results demonstrate that the UFAS induce pulmonary fibrosis in the same way as crystalline silica. However, the degree of fibrosis observed is transient. This study show that cytokines (IL-4, IL-10, IL-13, and IFN-γ), MMPs (MMP-2, MMP-9, and MMP-10) and TIMP-1 play important roles in the fibrosis induced by the intratracheal instillation of UFAS.
Specific substrate of MMP-2 is Type IV collagen and the later is important component of alveolar wall basement membrane. High expression of MMP-2 induced by nosogenesis plays roles in destruction of alveolar epithelial cells basement membrane and invasion of alveolar fibroblasts to the alveolar space in pulmonary fibrosis. Animal experiments and clinical studies demonstrated elevation of MMP-2 expression in the pulmonary fibrosis[48-49]. The MMP-2 in the pulmonary tissue is mainly from the AMs. Pulmonary interstitial fibroblasts are major cells source for the MMP-2 in early and mid-term pulmonary fibrosis. Therefore, pulmonary interstitial fibroblasts are not simply effective cells in pulmonary fibrosis, as they are concerned with structural damage of the lung basement membrane and initiate the process of pulmonary fibrosis by MMP-2 expression. The increase in collagen synthesis and secretion is an important link to pulmonary fibrosis development under the regulation of AMs. In vitro experiments in another study indicated that the supernatant medium from the AMs exposed to quartz promoted proliferation, collagen synthesis and secretion of fibroblasts[50-51]. According to in vivo study, the lung/body coefficient and hydroxyproline content for the nanosized SiO2 groups are significantly lower than those of the microsized SiO2 groups at both 1 and 2 months after instillation of nanosized SiO2 . The expressions of IL-4 and TGF-β1 in the nanosized SiO2 groups are significantly lower than those in the microsized SiO2 groups, and the effect of fibrogenesis induced by nanosized SiO2 might be milder than that induced by microsized SiO2 at 2 months after instillation.
The Hyp production in the lung tissue from the rats increase in 12.5 and 25 mg/mL SNs in this study, and also in 25 mg/mL microscale SiO2 particles instilled group. The Hyp production in the microscale SiO2 particles group at 25 mg/mL concentration is higher than that in the SNs group at same dosage. The TNF-α, TGF-β1, IL-1β, and MMP-2 expressions are increased in the SNs group at 6.25, 12.5, and 25 mg/mL concentrations and also in the microscale SiO2 particles group at 25 mg/mL concentration when compared with the saline control group. The TNF-α, IL-1β, and MMP-2 expressions in the SNs group and microscale SiO2 particles group at 25 mg/mL concentration are higher than those in the SNs group at 6.25 and 12.5 mg/mL concentrations. Moreover, the TNF-α, IL-1β, and MMP-2 expressions in the microscale SiO2 particles group at 25 mg/mL concentrations are higher than those in the SNs group at same dosage. Cytokine expression in the SNs and microscale SiO2 particles instilled group is mainly located in macrophages and some inflammatory cells, such as neutrophils, lymphocytes and plasma cells. These results demonstrate that the SNs could induce pulmonary fibrosis in the same way as microscale SiO2 particles by stimulating cytokine expression increase in the lung tissue.
The results from the in vivo study are different from that from our previously reported in vitro study. The in vitro investigations reveal that the SNs are more cytotoxic and damaged RAW264.7 cells more severely than the microscale SiO2 particles in same dosage. The study of single 7 mg/kg intravenous infusions of 13 nm SNs in rats, Zhuravskii et al. found mast cell (MC) abundance in the liver and recruitment in the liver preceded fibrosis. The in vivo investigations display that the degree of fibrosis induced by the SNs is milder than that induced by the microsized SiO2 in the rats. On the one hand, these findings are perhaps attributed to discrepancy in the physicochemical properties and biological activity between the SNs and microscale SiO2 particles. Shamsi et al reported that SNs incubated buffalo kidney cystatin (BKC) changed its conformation, reduced cell viability clearly suggesting toxicity of SNs. SNs have a deteriorating effect on BKC thereby causing a decrease in its ability to inhibit papain and hence less functionality. BKC activity decrease can certainly be an implication in many renal diseases highlighting the importance of this important thiol protease inhibitor. Although chemical components of the SNs and microscale SiO2 particles are the same, biological activity for the SNs appear to depend on the size effect due to small size and large surface area of the SNs. The size of the nanoparticles is similar to DNA, proteins, viruses and biological molecules, hence they are likely able to pass through the pulmonary blood barrier and skin barrier, and get into the body by means of simple diffusion and penetration form. These nanoparticles are prone to permeate the pores on the membrane and enter into the cells or organelles like mitochondria, endoplasmic reticulum, lysosomes, Golgi apparatus, cell nucleus, etc, combined with biological macromolecules or catalytic reactions occurring at the same time as well. The normal biological macromolecules and three-dimensional structure of the biological membrane is thus altered, resulting in loss of some important hormones and enzymes activity[55-56]. Some scholars believe therefore that size is an important factor for determining the toxicity of nanoparticles besides dose. Even if they are the same material with same shape and dose, as long as their sizes change, their biological toxicity must be retested and reevaluated. Some experimental results reveal that there is difference of toxicity between microscale and nanoscale materials[5, 7-8, 10, 20]. Lin et al. investigated the cytotoxicity of amorphous (colloidal) SNs (15 and 46 nm) in cultured human alveolar epithelial cells (A549 cells) in another study. Nanoparticles with both sizes of SNs are more cytotoxic than fine quartz particles (Min-USil 5), and there are no significant difference in toxicity for same sized SNs. On the other hand, the effect on fibrogenesis by the SNs may be milder than that of microscale SiO2 particles in rats, potentially resulting from nanoparticles tending to be diffused to pulmonary interstitial, and easily translocating blood circulation and eliminated from the body in the urine, thereby reducing the SNs preservation in the lungs, due to their ultrafine particle size compared to microsized particles. This current study reveal that there are a great deal SNs in the urine of rats instilled intratracheally with the SNs at 24 h. These result support the above discussed view. Research on the SNs has just started and lot of further research needs to be done for the sake of clarifying their toxicity and health effects.