Multi-walled carbon nanotube instillation impairs pulmonary function in C57BL/6 mice

Abstract

Background: Multi-walled carbon nanotubes (MWCNTs) are widely used in many disciplines due to their unique physical and chemical properties. Therefore, some concerns about the possible human health and environmental impacts of manufactured MWCNTs are rising. We hypothesized that instillation of MWCNTs impairs pulmonary function in C57BL/6 mice due to development of lung inflammation and fibrosis.

Methods: MWCNTs were administered to C57BL/6 mice by oropharyngeal aspiration (1, 2, and 4 mg/kg) and we assessed lung inflammation and fibrosis by inflammatory cell infiltration, collagen content, and histological assessment. Pulmonary function was assessed using a FlexiVent system and levels of Ccl3, Ccl11, Mmp13 and IL-33 were measured by RT-PCR and ELISA.

Results: Mice administered MWCNTs exhibited increased inflammatory cell infiltration, collagen deposition and granuloma formation in lung tissue, which correlated with impaired pulmonary function as assessed by increased resistance, tissue damping, and decreased lung compliance. Pulmonary exposure to MWCNTs induced an inflammatory signature marked by cytokine (IL-33), chemokine (Ccl3 and Ccl11), and protease production (Mmp13) that promoted the inflammatory and fibrotic changes observed within the lung.

Conclusions: These results further highlight the potential adverse health effects that may occur following MWCNT exposure and therefore we suggest these materials may pose a significant risk leading to impaired lung function following environmental and occupational exposures.

Figure 1

Characterization of MWCNT in Dry Powder Form. Characterization of multi-walled carbon nanotubes was determined using electron microscopy and Raman spectroscopy. (A & C) The diameter of the MWNCTs was determined by measuring individual nanotubes visualized by TEM. (B) This SEM image shows a bundle of MWCNTs, with individual nanotubes indicated by arrows. The length of individual nanotubes was determined to be several μm long using SEM. (D) The Raman spectrum of MWCNT obtained using 514.5 nm laser excitation. The presence of strong disorder band (I_D/I_G peak ratio) suggests the existence of structural defects as determined by Raman spectroscopy.

Figure 2

Characterization of MWCNT in Suspension. The size and charge characteristics of the MWCNTs were determined in a suspension of 10% surfactant saline. (A) The size of MWCNT bundles in suspension was determined by dynamic light scattering. (B) The zeta potential and isoelectric point (indicated by the arrow) were determined for the MWCNTs suspended in 10% surfactant saline solution.

Figure 3

Increased collagen content in lung tissue of MWCNT exposed C57BL/6 mice. Collagen content was determined by lung tissue harvested from mice 30 days post-exposure to vehicle control (10% surfactant in saline) or MWCNTs (1, 2, or 4 mg/kg). Mice exposed to the high dose MWCNT (4 mg/kg) displayed significant increase in collagen levels compared to naïve mice. All values are expressed as mean ± SEM (n = 6-11). *p < 0.05 compared to naïve mice and ⁺ p < 0.05 compared between two groups. No significant differences were found between naïve and vehicle treated groups. In addition, differences between vehicle and treatment groups were not significant.

Figure 4

Histopathology of lungs exposed to MWCNT displays granulomatous and fibrotic tissue at 30 days post-exposure. Mice instilled with (A) 10% saline in surfactant vehicle control display normal lung morphology while mice instilled with (B) 4 mg/kg MWCNTs exhibit widely dispersed deposition of MWCNT aggregates within lung tissue. Masson's trichrome staining shows collagen rich granulomas and surrounding fibrotic tissue (blue) in lungs of mice exposed to (D) MWCNTs, but not (C) vehicle control. H&E staining demonstrates granulomatous peribronchioloar foci in lungs of mice exposed to MWCNT (F) but not vehicle (E). Agglomerates of MWCNT within granulomas are indicated by arrows. Images are representative of 4 mice per group with original magnifications of 25 × (A-B), 200 × (C-D) and 400 × (E-F).

Figure 5

Impaired pulmonary function as determined by Snapshot and Quick-prime 3 perturbation after MWCNT instillation. The Snapshot and Quick-prime3 perturbations were performed in tracheotomized C57BL/6 mice instilled with increasing doses of MWCNTs. Since Snapshot perturbation measures the lung as a single compartment, the parameters, R (A), E (B), and C (C), are indicative of the whole respiratory system including lung and chest wall. On the other hand, Quick-prime 3 perturbation measures the lung as multiple compartments. The parameters can differentiate between central airway (Rn (D)) and peripheral lung tissue (G (E) and eta (F)). The mean ± SEM of six mice per group are shown, *p < 0.05 and ** p < 0.01 compared with the naïve mice, ^# p < 0.05 and ^## p < 0.01 compared with the vehicle control (10% surfactant in saline) mice; ⁺ p < 0.05, ⁺⁺ p < 0.01, and ⁺⁺⁺ p < 0.001 compared between two groups. No statistically significant differences were found between naïve and vehicle treated mice.

Figure 6

Impaired pulmonary function as determined by PVr-P perturbation after MWCNT instillation. The PVr-P perturbation was performed in tracheotomized C57BL/6 mice instilled with vehicle (10% surfactant in saline) or increasing doses of MWCNTs. From PV loops, perturbation parameters, (A) A, (B) K, (C) Cst, (D) Est, and (E) Area were determined. The mean ± SEM of six mice per group are shown, *p < 0.05 and ** p < 0.01 compared with the naïve mice; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with the vehicle control mice; ⁺⁺ p < 0.01 compared between two groups. No statistically significant differences were found between naïve and vehicle treated mice.

Figure 7

Induction of Ccl3, Ccl11, and Mmp13 in lungs of C57BL/6 mice exposed to MWCNTs. Real-Time PCR analysis was performed in the left lung of vehicle (10% surfactant in saline), 1 mg/kg, 2 mg/kg, and 4 mg/kg MWCNTs instilled C57BL/6 mice for genes (A) Ccl3, (B) Ccl11, and (C) Mmp13. The mean ± SEM of six mice per group are shown, ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with the vehicle control mice; ⁺ p < 0.05 compared between two groups.

Figure 8

Increased Ccl3, Ccl11, and activity of Mmp13 in BAL fluid after instillation with MWCNTs. ELISA analysis was performed in the BAL fluid of vehicle, 1 mg/kg, 2 mg/kg, and 4 mg/kg MWCNT instilled C57BL/6 mice for chemokines (A) Ccl3 and (B) Ccl11. Collagenase activity in the BAL fluid of vehicle control (10% surfactant in saline) and MWCNT instilled mice was measured using SensoLyte^® Mmp13 Assay Kit. (C) The relative fluorescence units (RFUs) and (D) the reaction velocity of Mmp13 were dose-dependent increased 30 days post-MWCNT instillation. The mean ± SEM of six mice per group are shown, ^# p < 0.05 and ^## p < 0.01 compared with the vehicle control mice.

Figure 9

Induction of IL-33 gene and protein expression in C57BL/6 lung tissue and BALF 30 days post-exposure to MWCNTs. Gene expression of IL-33 (A), determined by Real-Time PCR, demonstrated an approximate three-fold increase in left lung tissue of mice instilled with MWCNTs compared to vehicle control mice. Correspondingly, ELISA protein analysis (B) of IL-33 in BALF of mice exposed to MWCNTs was also significantly increased at all doses (1, 2, and 4 mg/kg) compared to vehicle control (10% surfactant in saline). All values are expressed as mean ± SEM (n = 6-11). ^#p < 0.05 compared to vehicle control mice and ^### p < 0.001 compared with the vehicle control mice.