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Table 3 In vivo studies on nanosilica particles (SNPs) toxicity

From: The nanosilica hazard: another variable entity

Silica
form
Size
(primary)
Material characterization Exposure model Test Biological endpoints and findings Ref
Quartz 10-20 nm (average size: 12), 30-65 (average size: 50), 300 nm - 2 μm • Synthesis
• Surface area
• Crystallinity
• Metal impurities
Rats instilled intratracheally with various particle types (1 or 5 mg/kg), sacrificed at 24 h, 1 week, 1 month, and 3 months post-exposure • Bronchoalveolar lavage (BAL) fluid analysis: cell counts, differentials, and pulmonary biomarkers (Lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and lavage fluid protein)
• Cell proliferation
• Morphological/Histopathology examination
• Hemolytic Potential of particles
Exposures to the various quartz particles produced differential degrees of pulmonary inflammation and cytotoxicity, which were not consistent with particle size but correlated with surface activity, particularly hemolytic potential. [148]
Silica dust 10 ± 5 nm; and 0.5-10 μm (80% of the particles
1-5 μm)
• Composition uknown
• Surface area
Rats instilled intratracheally (20 mg), sacrificed 1 and 2 months after dosing • The changes of lung/body coefficient and hydroxyproline content
• Pathologic examination
• Immunohistochemical staining for IL-4 and TGF-beta1
One month after instillation cellular nodules (Stage I silicosis) were found in the nanosized SiO2 group, while in microsized SiO2 group Stage II, II+ of silicotic nodules were observed.
Two months after instillation, still only Stage I silicotic nodules in nanosilica group were found, while in the micro-silica group the disease progressed and Stage II+, and III silicotic nodules were found.
The experiment revealed that in rats the effect of fibrogenesis of nano-SiO2 might be milder than that of micro-SiO2.
[147]
Ludox colloidal silica - • Mass median aerodynamic diameter (2.9, 3.3 and 3.7 μm)
• Chamber Ludox concentration
Rats Inhalation (nose-only) for 2 or 4 weeks at concentrations 10, 50 and 150 mg/m3.
Additional groups of rats exposed for 4 weeks were given a 3-month recovery period
• Lung silica analysis
• BAL analysis: cell differential counts and biochemical assay (LDH, ALP, lavage fluid protein)
• Pulmonary macrophage cell culture and phagocytosis assay
• SEM ananlysis
• Additional groups of animals were processed for cell labeling studies or lung deposition studies.
The inflammatory responses, mainly seen as increased numbers of neutrophils in BALF, following the 2 and/or 4 weeks of exposure was evident at 50 mg/m3 (or higher) group. Three months after exposure most biochemical parameters returned to control values.
Results showed that exposures to 150 mg/m3 Ludox for 2 or 4 weeks produced pulmonary inflammation along with increases in BAL protein, LDH, and alkaline phosphatase values (p less than 0.05) and reduced macrophage phagocytosis.
Autoradiographic studies demonstrated that the labeling indices of terminal bronchiolar and lung parenchymal cells were generally increased in the 50 and 150 mg/m3 groups after 2 and 4 weeks of exposure but, with one exception, returned to normal levels following a 3-month postexposure period.
[143]
Aerosol containing colloidal silica Average size: 22 nm • Mass median aerodynamic diameter (2.9, 3.3 and 3.7 μm)
• Chamber Ludox concentration
Rats inhalation (from 10 to 150 mg/m3), 6 h/day, 5 days/week for 4 weeks; 3 months postexposure • Lung silica determination
• Body weights and clinical observations
• Clinical pathology (urine and blood samples)
• Histopathology
No effects after exposure to the lowest concentration
Lung weights were increased significantly after 4 exposure to 50 and 150 mg/m3.
A dose dependent alveolar macrophage response, polymorphonuclear leukocytic infiltration, and Type II pneumocyte hyperplasia in alveolar duct regions was reported.
Lung-deposited nanosilica cleared rapidly from the lungs with half-times of approximately 40 and 50 days for the 50 and 150 mg/m3 groups, respectively. The lungs did not show fibrotic scar tissue formation or alveolar bronchiolarization.
[52]
Colloidal silica (UFCSs, average size of 14 nm)
fine colloidal silica particles (FCSs; average size of 213 nm)
• Size distribution
• Surface area
• Metal composition
Mice instilled intratracheally (3 mg) and sacrificed 0.5, 2, 6,12 and 24 h after dosing • Histopathology
• Immunohistochemistry
• Electron microscopy
Histopathological examination revealed for both sizes bronchiolar degeneration, necrosis, neutrophilic inflammation, alveolar type II cell swelling and alveolar macrophage accumulation.
UFCs induced extensive alveolar hemorrhage, a more severe bronchiolar epithelial cell necrosis and neutrophil influx in alveoli compared to FCSs.
Electron microscopy demonstrated UFCSs and FCSs on bronchiolar and alveolar wall surface as well as in the cytoplasm of alveolar epithelial cells, alveolar macrophages and neutrophils.
The findings suggest that UFCSs (possibly linked to larger surface area) have greater ability to induce lung inflammation and tissue damages than FCSs.
[98]
Colloidal silica average size: 14 nm • Size distribution
• Surface area
• Metal composition
Mice instilled intratracheally (0.3,3,10,30 or 100 μg) and sacrificed 3 days after dosing; 1 to 30 days postexposure • BAL analysis: cells quantification, viability and differentiation, total protein concentration
• Histopathology
• Immunohistochemistry
• Apoptosis (TUNEL assay)
Exposure up to 100 μg of UFCSs produced moderate to severe pulmonary inflammation and tissue injury 3 days post exposure.
Mice instilled with 30 μg of UFCSs and sacrificed at intervals from 1 to 30 days post-exposure showed moderate pulmonary inflammation and injury on BALF indices at acute period; however, these changes gradually regressed with time. Histopathological and immunohistochemical examination correlated to BALF data.
A significant increase of the apoptotic index (TUNEL) in lung parenchyma at all observation times was reported.
The findings suggest that instillation of a small dose of UFCSs caused an acute, but transient, lung inflammation and tissue damage in which oxidative stress and apoptosis may be involved.
[146]
Amorphous silica 14 nm • Endotoxins content Mice instilled intratracheally (2,10 and 50 mg/kg) and sacrificed 24 h, 1,4 and 14 weeks after dosing • BAL analysis: total protein and endotoxin concentration, cell differential counts
• Histopathology
• Real-time PCR
• Immunohistochemistry
Significantly increased lung weights, total BAL cells and proteins were observed until 1 week after treatment.
Particles induced acute inflammation (with neutrophils) at an early stage and chronic granulomatous inflammation at the later stage.
The significant up-regulation of cytokines (IL-1β, IL-6, IL-8, and TNF-α) and chemokines (MCP-1 and MIP-2) was observed during the early stages, but there were no changes after week 1.
In conclusion, Instillation of nanoparticles induced transient but very severe lung inflammation.
[144]
Amorphous silica 37.9 ± 3.3 nm • Size distribution
• Surface area
• Particle number
Rats inhalation (24.1 mg/m3, 40 min/day, 4 weeks
The age factor involved 3 levels (young/
adult/old)
• Electrocardiography
• BAL analysis
• Hemorheological analysis
• Serum biomarker assay
• Pathology
Inhalation of SNP under identical conditions caused the strongest pulmonary and cardi ovascular alterations in old rats, yet less change in young and adult rats.
Observed changes included pulmonary inflammation, myocardial ischemic damage, atrio-ventricular blockage, and increase in fibrinogen concentration and blood viscosity.
Old individuals were more sensitive to nanoparticle exposure
than the young and adult rats. The risk of causing pulmonary
damages was: old > young > adult. The risk of cardiovascular disorder was observed only in old age.
[145]
Amorphous silica 37 nm and 83 nm • The generation of nanosilica aerosol
• Size distribution
Rats inhalation
(3.7 × 107 or 1.8 × 108 particles/cm3), 6h/day, for 1- or 3-days
several post-exposure time points (up to 2 months)
• Bal analysis: cell counts, differentials, enzymatic activity of LDH,and ALP
• Genotoxicity endpoints (micronuclei induction)
One- or three-day aerosol exposure produced no significant pulmonary inflammatory, genotoxic, or adverse lung histopathological effects in rats exposed to very high particle numbers corresponding to a range of mass concentrations (1.8 or 86 mg/m3). [149]
Amorphous silica 14 nm o Daily mean mass median aerodynamic diameter (2.1 ± 0.1 μm) Rats inhalation
(head/nose only; 26.9 ± 3 mg/m3), 6h/day during 6 days);
Challenging the animals by inhalation to a minimally irritating concentration of allergen trimellitic anhydride (TMA)
• Breathing parameters
• Cellular and biochemical changes in BAL
• Histopathological airway changes
Exposure to SNPs alone resulted in transient changes in breathing parameters during exposure, and in nasal and alveolar inflammation with neutrophils and macrophages.
Exposure to particles before a single TMA challenge resulted in only a slightly irregular breathing pattern during TMA challenge. Pre-exposure to particles also diminished the effect of TMA on tidal volume, laryngeal ulceration, laryngeal inflammation, and the number of BAL eosinophils in most animals.
When the additional group of animals was exposed to nanosilica before a second challenge to TMA, the pulmonary eosinophilic infiltrate and edema induced by a second TMA challenge in control animals was diminished by the preceding silica exposure, but the number of lymphocytes in BAL was increased.
[153]
Amorphous silica ~30 nm and ~30 μm • Size distribution Feeding of mice for 10 weeks (total fed amount of 140 g/kg mice) • Blood analysis
• Cytological analysis of lungs and liver tissue sections
• Analysis of silicon in organs
The nano-sized silica particle dieted group showed higher value of ALT (alanine aminotransferase) than normal and micron-sized silica dieted groups.
H&E staining of the liver of the nano-sized particle dieted group indicated some fatty liver pattern. The contents of Si in the livers of the groups were almost the same.
[154]
Amorphous silica (organically modified) 20-25 nm • Synthesis
• Conjugation with fluorophore
• Radiolabelling
Mice injected intravenously with SPN
(2.0 mg/kg body weight)
• Fluorescence imaging (CRi)
• MicroPET imaging
• Histological Analysis
Greater acummulation of nanoparticles in liver, spleen and stomach that in kidney, heart and lungs.
Almost 100% of the injected nanoparticles were effectively cleared out of the animals over a period of 15 days via the hepatobiliary excretion.
No signs of organs toxicity were observed.
[155]
Amorphous (mesoporous) silica 150 nm, 800 nm and 4 μm
(pore sizes of 3 nm, 7 nm
and 16 nm)
• Synthesis
• Size
• Endotoxins content
Rats injected subcutaneously (30 mg per rat), Mice injected intraperitoneally and intravenously • Hematoxylin and eosin staining and histological examination When the particles were injected subcutaneously, the amount of residual material decreased progressively over 3 months, with good biocompatibility on histology at all time points.
Intra-peritoneal and intra-venous injections in mice resulted in death or euthanasia. No toxicity was seen with subcutaneous injection of the same particles in mice.
Microscopic analysis of the lung tissue of the mice indicated that death may be due to thrombosis.
[156]
Amorphous silica 75, 311 and 830 nm Not specified Mice injected intravenously (10-100 mg/kg) • H&E staining; histological analysis of the liver, kidney, spleen and lung
• Biochemical assays
• Gadolinium chloride, cyclophosphamide and hepatic hydroxyproline assay
70 nm SNP induced liver injury at 30 mg/kg body weight, while SP300 or 1000 had no effect even at 100 mg/kg.
Administration of 70 nm SNP dose-dependently increased serum markers of liver injury, serum aminotransferase and inflammatory cytokines.
Repeated administration of 70 nm SNP twice a week for 4 weeks, even at 10 mg/kg, caused hepatic fibrosis.
[157]
Amorphous silica 50, 100 and 200 nm • Synthesis
• Fluorescence labeling
Mice injected intravenously (50 mg/kg) • Confocal laser scanning microscopy
• Immunofluorescence staining
• Fluorescence microplate readings
Significant increase of inflammation in the liver at 12 h for the 100 and 200 nm silica nanoparticles treatment groups.
The tissue distribution and excretion of the injected particles were different depending on particle size. As particle sizes increased, more particles were trapped by macrophages in the liver and spleen. All particles were cleared via urine and bile; however, the 50 nm silica nanoparticles excreted faster than the other two particles.
[158]