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The particles were characterized according to particle size, hydrodynamic size, zeta potential, purity, crystal status, and specific surface area. Figure 1 shows TEM images of the silica NPs and silica MPs. The appearance of silica NPs and silica MPs was approximately spherical. The size of silica NPs and silica MPs were 25.9 ± 3.4 nm and 1087.8 ± 389.6 nm, respectively. Silica NPs tended to aggregate/agglomerate in deionized water, with a hydrodynamic size of 246.6 ± 47.8 nm. The zeta potential of silica NPs and silica MPs were -64.1 mV and -63.0 mV, respectively. The pH value of silica NPs and silica MPs suspensions were 6.7 and 6.4, respectively. The purity of silica NPs and silica MPs were 99.2% and 99.6%, respectively. The crystal structures of silica NPs and silica MPs were both amorphous. The specific surface area of silica NPs and silica MPs were 152.2 m2/g and 4.4 m2/g, respectively.
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The dissolution concentrations of silica NPs and silica MPs were 0.010% ± 0.002% and 0.015% ± 0.006%, respectively, in the simulated gastric solution and 0.420% ± 0.007% and 0.361% ± 0.052%, respectively, in the intestinal solution.
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No mortality or treatment related adverse clinical reactions were found during the study. There were no statistically significant differences in the body weights of male rats between the treatment groups and the control group; however, for low-dose silica NPs in female rats, body weight at the fifth week of exposure was lower than that in the control group (Figure 2A). For middle-dose silica MPs in female rats, food consumption at the eighth week was lower than that in the control group. For low-dose silica NPs in male rats, food consumption at the 11th week was lower than that in the low-dose silica MPs group (Figure 2B).
Figure 2. General observations of subchronic oral toxicity of silica NPs and silica MPs in rats. (A) Mean body weights of rats. (B) Mean food consumption of rats. *Significant difference vs. control group, P < 0.05 (body weight of female rats at the fifth week, food consumption of female rats at the eighth week), #Significant difference vs. silica MPs 166.7 mg/(kg·bw) group, P < 0.05 (food consumption of male rats at the 11th week).
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There were some sporadic, statistically significant changes in hematology and clinical biochemistry parameters (Table 1).
Parameters Control Silica MPs, mg/(kg·bw) Silica NPs, mg/(kg·bw) 166.7 500 1, 500 166.7 500 1, 500 Female MCH (pg) 19.1 ± 1.2 18.3 ± 0.7 18.7 ± 0.6 18.5 ± 0.6 18.3 ± 0.6 17.7 ± 1.0* 18.7 ± 0.2 Cl (mmol/L) 120.7 ± 1.8 118.6 ± 2.0 119.6 ± 1.5 121.6 ± 1.1 120.0 ± 1.8 119.0 ± 2.1 118.1 ± 1.4*# Na (mmol/L) 125.1 ± 2.0 120.2 ± 1.2* 121.0 ± 2.5* 123.8 ± 2.7 122.2 ± 2.3* 122.6 ± 1.0 121.6 ± 1.0* T4 (ng/mL) 28.1 ± 6.6 30.8 ± 7.6 28.5 ± 3.9 27.8 ± 7.7 23.7 ± 7.2 32.1 ± 12.9 39.5 ± 6.4*# Male PT (s) 10.0 ± 0.4 10.0 ± 0.3 9.6 ± 0.4 9.9 ± 0.4 10.5 ± 0.5* 9.5 ± 0.3 9.8 ± 0.2 APTT (s) 59.2 ± 3.0 56.5 ± 2.8 58.4 ± 1.9 60.2 ± 1.8 60.1 ± 2.2# 57.3 ± 1.6 57.0 ± 1.9# TP (g/L) 50.9 ± 2.1 49.5 ± 1.7 55.7 ± 3.4* 54.9 ± 2.6* 55.1 ± 1.1*# 54.0 ± 2.4 52.8 ± 3.2 ALB (g/L) 27.0 ± 1.1 26.0 ± 0.9 27.3 ± 1.3 26.8 ± 1.3 26.8 ± 0.9 26.6 ± 1.1 25.3 ± 1.5* Na (mmol/L) 121.8 ± 2.8 121.6 ± 0.9 122.8 ± 2.0 124.0 ± 3.3 124.9 ± 1.5# 122.6 ± 1.5 121.7 ± 1.2 Note.*P < 0.05 as compared with the control group. #P < 0.05 as compared with the silica MPs group. Table 1. Summary of Statistically Significant Hematology and Clinical Biochemical Parameters for Rats after 90 Days Oral Administration of Silica MPs and Silica NPs
For female rats in the silica NPs middle-dose group, the MCH was significantly lower than that in the control group. For male rats in the silica NPs low-dose group, PT was significantly higher than that in the control group, APTT was significantly higher than that in the silica MPs low-dose group, and for males in the silica NPs high-dose group, APTT was significantly lower than that in the silica MPs high-dose group. For female rats in the silica MPs low-and high-dose groups and the silica NPs low-and middle-dose groups, the level of Na was significantly lower than that in the control group. For females in the silica NPs high-dose group, the level of Cl was significantly lower than that in the control group and silica MPs high-dose group. For male rats in the silica MPs middle-and high-dose group, TP concentration was higher than that in the control group, for males in the silica NPs low-dose group, TP concentration was higher than that in the control and silica MPs low-dose groups, and Na was higher than that in the silica MPs low-dose group. For males in the silica NPs high-dose group, ALB was lower than that in the control group. No significant differences were observed in the serum levels of T3 and TSH. The serum level of T4 in the silica NPs female high-dose group was statistically significantly higher than that in the control group and silica MPs high-dose group.
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There were no remarkable gross pathological alterations in any of the animals. No statistically significant differences in all absolute organ weights and relative organ weights (organ-to-body weight ratios) in the treatment groups of either males or females were found (Table S1 available in www.besjournal.com).
Parameters Control Silica MPs, mg/(kg·bw) Silica NPs, mg/(kg·bw) 166.7 500 1, 500 166.7 500 1, 500 Female Liver (g) 8.23 ± 1.20 8.03 ± 0.74 7.34 ± 0.48 7.52 ± 1.08 7.39 ± 0.55 7.68 ± 0.83 7.24 ± 0.68 Liver (%) 2.82 ± 0.26 2.83 ± 0.19 2.65 ± 0.17 2.67 ± 0.17 2.73 ± 0.12 2.68 ± 0.21 2.69 ± 0.31 Kidneys (g) 2.03 ± 0.30 2.13 ± 0.22 1.96 ± 0.18 2.00 ± 0.18 2.04 ± 0.08 2.14 ± 0.20 1.94 ± 0.12 Kidneys (%) 0.69 ± 0.08 0.75 ± 0.05 0.71 ± 0.06 0.71 ± 0.03 0.76 ± 0.04 0.75 ± 0.05 0.72 ± 0.07 Spleen (g) 0.63 ± 0.04 0.65 ± 0.06 0.63 ± 0.10 0.63 ± 0.07 0.60 ± 0.08 0.67 ± 0.10 0.66 ± 0.07 Spleen (%) 0.22 ± 0.03 0.23 ± 0.02 0.23 ± 0.04 0.22 ± 0.01 0.22 ± 0.03 0.23 ± 0.03 0.24 ± 0.03 Heart (g) 1.15 ± 0.14 1.13 ± 0.09 1.05 ± 0.06 1.11 ± 0.13 1.09 ± 0.05 1.15 ± 0.13 1.05 ± 0.07 Heart (%) 0.42 ± 0.07 0.40 ± 0.03 0.38 ± 0.03 0.39 ± 0.02 0.40 ± 0.02 0.40 ± 0.04 0.39 ± 0.03 Brain (g) 1.83 ± 0.14 1.81 ± 0.08 1.82 ± 0.12 1.83 ± 0.06 1.86 ± 0.09 1.87 ± 0.07 1.86 ± 0.06 Brain (%) 0.63 ± 0.06 0.64 ± 0.04 0.66 ± 0.06 0.66 ± 0.07 0.69 ± 0.07 0.66 ± 0.04 0.69 ± 0.05 Thymus (g) 0.51 ± 0.12 0.57 ± 0.15 0.50 ± 0.09 0.48 ± 0.10 0.45 ± 0.13 0.52 ± 0.10 0.49 ± 0.11 Thymus (%) 0.18 ± 0.04 0.20 ± 0.04 0.18 ± 0.03 0.17 ± 0.03 0.17 ± 0.04 0.18 ± 0.03 0.18 ± 0.04 Ovaries (g) 0.22 ± 0.03 0.21 ± 0.08 0.20 ± 0.04 0.19 ± 0.05 0.19 ± 0.04 0.20 ± 0.03 0.21 ± 0.04 Ovaries (%) 0.08 ± 0.01 0.07 ± 0.03 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.02 0.07 ± 0.01 0.08 ± 0.01 Uterus (g) 0.64 ± 0.13 0.58 ± 0.17 0.59 ± 0.17 0.59 ± 0.16 0.64 ± 0.15 0.68 ± 0.18 0.71 ± 0.16 Uterus (%) 0.22 ± 0.07 0.20 ± 0.06 0.21 ± 0.06 0.21 ± 0.05 0.24 ± 0.06 0.24 ± 0.07 0.27 ± 0.06 Adrenal glands (g) 0.100 ± 0.020 0.104 ± 0.027 0.105 ± 0.039 0.097 ± 0.016 0.101 ± 0.018 0.098 ± 0.016 0.094 ± 0.030 Adrenal glands (%) 0.035 ± 0.008 0.037 ± 0.010 0.038 ± 0.015 0.034 ± 0.004 0.038 ± 0.008 0.034 ± 0.006 0.035 ± 0.011 Thyroid glands (g) 0.022 ± 0.003 0.020 ± 0.002 0.019 ± 0.001 0.022 ± 0.003 0.020 ± 0.001 0.020 ± 0.003 0.021 ± 0.002 Thyroid glands (%) 0.007 ± 0.001 0.007 ± 0.001 0.007 ± 0.001 0.008 ± 0.001 0.007 ± 0.001 0.007 ± 0.001 0.008 ± 0.001 Male Liver (g) 12.60 ± 0.94 12.56 ± 1.11 12.51 ± 1.74 11.45 ± 1.65 11.21 ± 1.88 11.31 ± 1.79 11.20 ± 0.94 Liver (%) 2.71 ± 0.13 2.65 ± 0.14 2.68 ± 0.24 2.60 ± 0.28 2.49 ± 0.22 2.56 ± 0.17 2.50 ± 0.08 Kidneys (g) 3.06 ± 0.20 3.17 ± 0.27 3.11 ± 0.27 3.10 ± 0.29 3.03 ± 0.33 3.02 ± 0.18 2.96 ± 0.25 Kidneys (%) 0.66 ± 0.04 0.67 ± 0.03 0.67 ± 0.06 0.71 ± 0.05 0.68 ± 0.03 0.69 ± 0.06 0.66 ± 0.02 Spleen (g) 0.86 ± 0.13 0.91 ± 0.23 0.81 ± 0.11 0.79 ± 0.09 0.80 ± 0.12 0.83 ± 0.19 0.80 ± 0.15 Spleen (%) 0.18 ± 0.02 0.19 ± 0.04 0.18 ± 0.03 0.18 ± 0.02 0.18 ± 0.02 0.19 ± 0.02 0.18 ± 0.03 Heart (g) 1.66 ± 0.15 1.65 ± 0.19 1.64 ± 0.14 1.58 ± 0.18 1.64 ± 0.15 1.62 ± 0.18 1.64 ± 0.13 Heart (%) 0.36 ± 0.04 0.35 ± 0.02 0.35 ± 0.04 0.36 ± 0.03 0.37 ± 0.04 0.37 ± 0.04 0.37 ± 0.03 Brain (g) 1.92 ± 0.14 2.04 ± 0.03 2.00 ± 0.06 1.98 ± 0.05 1.98 ± 0.14 2.02 ± 0.07 1.99 ± 0.07 Brain (%) 0.42 ± 0.04 0.43 ± 0.02 0.43 ± 0.04 0.45 ± 0.03 0.44 ± 0.04 0.46 ± 0.06 0.45 ± 0.03 Thymus (g) 0.60 ± 0.13 0.68 ± 0.18 0.61 ± 0.18 0.51 ± 0.10 0.49 ± 0.08 0.52 ± 0.18 0.46 ± 0.05 Thymus (%) 0.13 ± 0.02 0.15 ± 0.04 0.13 ± 0.05 0.12 ± 0.02 0.11 ± 0.02 0.12 ± 0.04 0.10 ± 0.01 Testes (g) 3.07 ± 0.49 3.13 ± 0.25 3.10 ± 0.24 3.10 ± 0.19 3.16± 0.21 3.14 ± 0.29 3.05 ± 0.12 Testes (%) 0.66 ± 0.12 0.66 ± 0.05 0.67 ± 0.08 0.71 ± 0.05 0.71± 0.08 0.72 ± 0.08 0.69 ± 0.06 Epididymis (g) 1.28 ± 0.35 1.37 ± 0.14 1.38 ± 0.11 1.38 ± 0.16 1.38 ± 0.17 1.37 ± 0.20 1.40 ± 0.13 Epididymis (%) 0.28 ± 0.07 0.29 ± 0.03 0.30 ± 0.03 0.31 ± 0.05 0.31 ± 0.04 0.31 ± 0.03 0.31 ± 0.03 Adrenal glands (g) 0.080 ± 0.018 0.079 ± 0.025 0.073 ± 0.017 0.078 ± 0.015 0.089 ± 0.020 0.093 ± 0.028 0.079 ± 0.015 Adrenal glands (%) 0.017 ± 0.003 0.017 ± 0.005 0.016 ± 0.004 0.018 ± 0.003 0.020 ± 0.004 0.022 ± 0.007 0.018 ± 0.003 Thyroid glands (g) 0.026 ± 0.002 0.025 ± 0.003 0.024 ± 0.003 0.027 ± 0.003 0.028 ± 0.004 0.026 ± 0.003 0.023 ± 0.002 Thyroid glands (%) 0.006 ± 0.001 0.005 ± 0.001 0.005 ± 0.001 0.006 ± 0.001 0.006 ± 0.001 0.006 ± 0.001 0.005 ± 0.001 Table Table S1. Absolute and Relative Organ Weights for Rats after 90 days Oral Administration of Silica MPs and Silica NPs
The histopathological findings in rats of the control and high-dose groups of silica MPs and silica NPs are summarized in Table 2. The histopathological findings in heart, liver, duodenum, ileum, bladder, and prostate appeared to be incidental, as the frequency and severity of these lesions in silica MPs and silica NPs high-dose groups was comparable with those in the control group. Lesions in these organs included focal myocardial necrosis, mild myocardial fatty degeneration, mild hepatocyte fatty degeneration, hepatocyte spotty necrosis and inflammatory cell infiltrate, epithelial cell autolysis (duodenum and ileum), a small amount of exudate at the mucosal surface of the bladder, interstitial edema, and inflammatory cell infiltrate in the prostate.
Organs Lesions Control Silica MPs Silica NPs Female Male Female Male Female Male Heart Focal myocardial necrosis 1 0 0 0 0 0 Mild myocardial fatty degeneration 1 0 0 0 0 0 Liver Mild hepatocyte fatty degeneration 6 1 3 1 4 0 Spotty necrosis, inflammatory cell infiltrate 0 0 0 1 0 0 Duodenum Epithelial cell autolysis 3 1 0 0 1 0 Lleum Epithelial cell autolysis 0 0 0 1 0 0 Bladder Small amount of exudate at the mucosal surface 0 1 0 0 0 0 Prostate Interstitial edema, inflammatory cell infiltrate - 0 - 1 - 0 Interstitial inflammatory cell infiltrate - 0 - 0 - 1 Table 2. Histopathological Findings in Rats after 90 Days Oral Administration of Silica MPs and Silica NPs [1, 500 mg/(kg·bw)]
Histopathological findings in the lungs of rats in all groups are summarized in Table 3. In the lungs, lesions including mild widening of the alveolar septum, spotty foreign body granulomas, and focal epithelial cell proliferation were found, the frequencies in the high-dose silica groups were higher than those in the control group. Therefore, we conducted a histopathological examination of the lungs in the low-and middle-dose groups of silica MPs and silica NPs. In these lower groups, focal epithelial cell proliferation and foreign body granulomas were also found. The histopathological changes in the lungs are shown in Figure 3.
Figure 3. Histopathological findings in the lungs. (A) Control lung. (B) Foreign body granulomas in the lung, tissues of rats stained with H & E ×100.
Organs Lesions Control Silica MPs Silica NPs Low Middle High Low Middle High F M F M F M F M F M F M F M Lung Mild alveolar septum widening 0 1 0 0 0 0 1 0 0 0 0 1 3 0 Foreign body granulomas 0 0 0 3 3 0 0 3 1 2 1 1 1 0 Focal epithelial cell proliferation 0 0 2 0 2 4 0 3 2 1 1 3 2 1 Note. F, Female; M, Male. Table 3. Histopathological Findings for Lungs of Rats in All Groups after 90 Days Oral Administration of Silica MPs and Silica NPs
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The distribution of Si in the blood, liver, kidneys and testis is shown in Figure 4. In the liver of female rats, the Si concentrations in the low-and high-dose silica MPs groups, and in the low-, middle-, and high-dose silica NPs groups were lower than those in the control group. In the blood of male rats, the Si concentrations in the middle-dose silica MPs group and low-dose silica NPs group were higher than those in the control group, and the Si concentration in the middle-dose silica NPs group was higher than those in the control and middle-dose silica MPs groups.