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.2020 Feb 21;10(2):379.
doi: 10.3390/nano10020379.

Potential Role of Soluble Metal Impurities in the Acute Lung Inflammogenicity of Multi-Walled Carbon Nanotubes

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Potential Role of Soluble Metal Impurities in the Acute Lung Inflammogenicity of Multi-Walled Carbon Nanotubes

Dong-Keun Lee et al. Nanomaterials (Basel)..

Abstract

Multi-walled carbon nanotubes (MWCNTs) have variable metal impurities, but little is known about the impact of soluble metal impurities on the toxicity of MWCNTs. Here, we evaluated the role of soluble metal impurities to the acute inflammogenic potential of MWCNTs, using five types of high purity MWCNTs (>95%). MWCNTs and their soluble fractions collected at 24 h after incubation in phosphate-buffered saline showed diverse metal impurities with variable concentrations. The fiber-free soluble fractions produced variable levels of reactive oxygen species (ROS), and the iron level was the key determinant for ROS production. The acute inflammation at 24 h after intratracheal instillation of MWCNTs to rats at 0.19, 0.63, and 1.91 mg MWCNT/kg body weight (bw) or fiber-free supernatants from MWCNT suspensions at 1.91 and 7.64 mg MWCNT/kg bw showed that the number of granulocytes, a marker for acute inflammation, was significantly increased with a good dose-dependency. The correlation study showed that neither the levels of iron nor the ROS generation potential of the soluble fractions showed any correlations with the inflammogenic potential. However, the total concentration of transition metals in the soluble fractions showed a good correlation with the acute lung inflammogenic potential. These results implied that metal impurities, especially transitional metals, can contribute to the acute inflammogenic potential of MWCNTs, although the major parameter for the toxicity of MWCNTs is size and shape.

Keywords: inflammation; lung; metal impurities; multi-walled carbon nanotubes; soluble fraction; transitional metals.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
SEM and TEM images of MWCNTs. The large square images are SEM images, and the small insert square images are TEM images. CNT1 (a), CNT2 (b), CNT3 (c), and CNT4 (d) showed tangled form, while CNT5 (e) showed straight form.
Figure 2
Figure 2
The ROS generation potential of MWCNTs or soluble fractions. A cell-free DCFH-DA assay was used to measure the ROS generation potential of (a) MWCNT or (b) the MWCNT-free soluble fractions. Plots of the concentration of (c) iron or (d) transition metals against the ROS generation potentials of the MWCNT-free soluble fractions at 100 µg/mL of MWCNT suspensions. Iron was the highly correlated element for ROS generation (Spearman correlation coefficientr = 0.90). VEH, vehicle control;n = 4 for ROS measurement.
Figure 3
Figure 3
The acute lung inflammation at 24 h after intratracheal instillation of MWCNTs to rats. MWCNTs were instilled at 30, 100, and 300 μg/rat, and cytological and biochemical analysis of BALF were performed. (a) The number of total cells; (b) the number of alveolar macrophages; (c) the number of granulocytes; (d) the number of lymphocytes; (e) the levels of LDH; and (f) the concentration of total protein in BALF.*p < 0.05,**p < 0.01, and***p < 0.001 compared with the vehicle control (VEH);n = 4 per group.
Figure 4
Figure 4
The acute pulmonary inflammation at 24 h after intratracheal instillation of the MWCNT-free soluble fractions to rats. The MWCNT-free soluble fractions were collected by ultracentrifugation of suspensions of MWCNTs at equivalent doses of 0.3 and 1.2 mg/rat, and cytological and biochemical analyses were performed in BALF. (a) The number of total cells; (b) the number of alveolar macrophages; (c) the number of granulocytes; (d) the number of lymphocytes; (e) the levels of LDH; and (f) the concentration of total protein.*p < 0.05,**p < 0.01, and***p < 0.001 compared with the vehicle control (VEH);n = 4 per group.
Figure 5
Figure 5
Correlation plots to evaluate the main parameters of the soluble fractions producing acute pulmonary inflammation. Plots of (a) ROS levels or (b) iron concentration against the number of granulocytes showed a poor correlation, but plots of (c) total metal impurities or (d) total transition metal impurities against the number of granulocytes showed good correlation (Spearmanr = 0.9 and 0.7, respectively). The doses used for plotting of ROS generation potential and lung inflammation were 100 μg/mL and soluble fraction from 7.64 mg MWCNT/kg bw, respectively.
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