Pulmonary and systemic responses of highly pure and well-dispersed single-wall carbon nanotubes after intratracheal instillation in rats

Figures & data

Figure 1.  Summary of the experimental design of experiments 1 and 2.

Table 1.  Characterization of bulk SWCNTs and SWCNTs dispersed in the testing solution.

Sample	Characteristic	Value	Measuring method
Bulk SWCNTs	Tube diameter	3.0 ± 1.1 nm^a	Transmission electron microscopy (TEM)
	Maximum bundle length	1200 μm	Micrometer
	BET surface area	1064 ± 37 m^2/g^a	N2 gas adsorption
	D/G ratio	0.14	Raman spectroscopy
	Amorphous carbon content	< 2.3 ± 0.56 %^a	Thermogravimetric analysis (TGA)
	Total metal content	0.05 ± 0.16 %^a
	Each metal content		Inductively coupled plasma− Mass spectrometry (ICP–MS)
	Fe	145 ppm
	Ni	103 ppm
	Cr	34 ppm
	Mn	2 ppm
	Al	12 ppm
SWCNTs dispersed in the testing solution	Bundle diameter	12.0 ± 6.5 nm^a	Atomic force microscopy (AFM)
	Bundle length	0.32 μm (1.76)^b
	D/G ratio	0.19	Raman spectroscopy
	pH	7.2	pH meter

^aValues are expressed as mean ± SD.

^bValues are expressed as geometric mean (geometric standard deviation).

Figure 2.  Morphology of the bulk SWCNTs and SWCNTs dispersed in the testing solution. Panels A–C show transmission electron microscopy (TEM) images of bulk SWCNTs at different magnifications. Panel d shows an atomic electron microscopy (AFM) image of SWCNTs dispersed in the testing solution. Panels e and f show TEM images of SWCNTs dispersed in the solution at different magnifications.

Figure 3.  Distribution of SWCNT bundle diameter (left) and length (right) in 2 mg/mL SWCNT dispersion measured from digital images acquired by atomic force microscopy.

Figure 4.  The resonance Raman scattering spectra of bulk SWCNT samples, and 0.2 and 2 mg/mL of SWCNT dispersions in the frequency regions of 100–3000 cm⁻¹. The inset shows the spectra of the low frequency region. Abbreviations G and D denote G-band and D-band, respectively.

Figure 5.  Absolute left lung weights of SWCNT-exposed rats and corresponding controls at each time point in experiment 1 (left) and 2 (right). Values are represented as the mean ± SD. *Significant increase from vehicle control (p < 0.05).

Figure 6.  Number of neutrophils (a), macrophages (b), lymphocytes(c), and eosinophils (d) in BALF of SWCNT-exposed rats and corresponding controls at each time point in experiments 1 (left column) and 2 (right column). Values are represented as the mean ± SD. *Significant increase from vehicle control (p < 0.05).

Figure 7.  LDH value (a), protein content (b), and IL-1βactivity (c) in BALF of SWCNT-exposed rats and corresponding controls at each time point in experiments 1 (left column) and 2 (right column). Values are represented as the mean ± SD. *Significant increase from vehicle control (p < 0.05).

Table 2.  Pulmonary histopathology severity scores* of rats in experiments 1 and 2.

Findings	Time point	Vehicle control group	SWCNT–exposed group				Silica–exposed group
			0.04 mg/kg	0.2 mg/kg	1.0 mg/kg	2.0 mg/kg
Administered substance accumulation, alveolus	24 h	0	–	1.0	–	3.0	0
	3 d	0	1.0	1.2	2.0	2.6	0
	1 w	0	1.0	1.0	1.4	2.4	0
	1 m	0	0.8	1.0	1.6	1.8	0
	3 m	0	0.8	1.1	1.6	2.0	0
	6 m	0	0.8	1.0	1.5	–	–
Administered substance accumulation, interstitium	24 h	0	–	0	–	1.8	1.8
	3 d	0	0	0.1	1.4	0	0
	1 w	0	0	0.1	1.4	1.2	0
	1 m	0	0	0.1	0.6	1.2	0
	3 m	0	0	0.1	1.4	2.0	1.0
	6 m	0	0	0.8	1.0		–
Inflammatory cell infiltration, alveolus	24 h	1.6	–	1.6	–	1.8	1.8
	3 d	0.1	1.0	0.6	1.2	0	0
	1 w	0	0	0.4	0.4	1.2	0
	1 m	0.2	0	0.2	0.8	1.2	0
	3 m	0	0	0.3	0.2	2.0	1.0
	6 m	0	0	0	0.8	–	–
Macrophage accumulation, alveolus	24 h	1.4	–	1.4	–	1.0	0
	3 d	0.4	1.0	1.2	2.0	0.8	0.2
	1 w	0.1	0.8	1.0	1.4	2.0	0.4
	1 m	0.1	0.8	1.1	1.8	1.6	0
	3 m	0.2	0.8	1.0	2.0	1.8	0.5
	6 m	0	0.8	1.0	1.8	–	–
Foamy macrophage, alveolus	24 h	0	–	0	–	1.0	0
	3 d	0	0	0	0	0.8	0.2
	1 w	0	0	0.1	0	2.0	0.4
	1 m	0	0	0.1	0	1.6	0
	3 m	0	0	0.1	1.6	1.8	0.5
	6 m	0	0	0	1.3	–	–
Macrophage accumulation, interstitium	24 h	0	–	0	–	0	0
	3 d	0.2	0	0.3	1.8	1.6	0
	1 w	0.1	0.2	0.3	1.0	2.4	0
	1 m	0	0	0.1	0.6	1.6	0
	3 m	0	0	0.1	1.4	1.8	0.5
	6 m	0	0	0.8	1.0	–	–
Granuloma	24 h	0	–	0	–	0	0
	3 d	0.2	0.2	0.3	2.0	1.4	0
	1 w	0	0	0	1.0	2.2	0
	1 m	0	0	0	0.4	0.8	0
	3 m	0	0	0	0.6	1.6	0.3
	6 m	0	0	0	0	–	–
Foreign body giant cell	24 h	0	–	0	–	0	0
	3 d	0	0	0	0	0	0
	1 w	0	0	0	0	0	0
	1 m	0	0	0	0	0.6	0
	3 m	0	0	0	0	0.8	0
	6 m	0	0	0	0	–	–
Hypertrophy of alveolar epithelium	24 h	0	–	0	–	0	0
	3 d	0	0	0	0	1.6	0
	1 w	0	0	0	0	1.6	0
	1 m	0	0	0	0	1.0	0
	3 m	0	0	0	0	2.0	0.8
	6 m	0	0	0	0	–	–
Hypertrophy of bronchial epithelium	24 h	0	–	0	–	1.0	0
	3 d	0	0	0	0	0.8	0
	1 w	0	0	0	0	1.6	0
	1 m	0	0	0	0	0	0
	3 m	0	0	0.1	1.0	0.8	0
	6 m	0	0	0	0.5	–	–
Alveolar proteinosis	24 h	0	–	0	–	0	0
	3 d	0	0	0	0	0.6	0
	1 w	0	0	0	0	1.4	0
	1 m	0	0	0	0	1.8	0
	3 m	0	0	0	0	2.4	1.3
	6 m	0	0	0	0	–	–

*: Severity scores given to individual animals from a complete pathological examination are 0, not remarkable; 1, minimal; 2, slight/mild; 3, moderate; and 4, severe; based upon relative evaluation of lesions. Severity scores for each animal within a group (5 or 6 rats) were added, and an average score per animal was calculated, which is shown in the table.

–: Not evaluated.

Table 3.  Summary of the results in experiments 1 and 2.

Endpoint		SWCNT–exposed group				Silica– exposed group
Dose	Mass [mg/kg]	0.04	0.2	1.0	2.0	5.0
	Surface area [m^2/kg]	0.04	0.2	1.0	2.0	0.025
	Number [particle/kg]	1.8 × 10^12	8.8 × 10^12	4.4 × 10^13	8.8 × 10^13	7.3 × 10^8
Clinical sign		–	–	–	–	–
Body weight		–	–	–	–	–
Lung weight		–	+	++	+++	–
BALF	Inflammatory cells	–	+	++	++++	+++
	LDH/protein	–	+	++	++++	+++
	IL–1β	–	+	++	+++	–
Histopathology	Pulmonary inflammation	–	+	++	++++	+++
	Inflammation (other tissues)	–	–	–	–	–

Note: Within the table, ++++ indicates the greatest change, where + indicates the least change.− indicates no significant change compared to the corresponding control.

Figure 8.  Light micrographs of lung tissue sections of rats at 1week after instillation in experiment 1 (H&E stain). No significant changes were observed in the vehicle control group (panel a). Minimal macrophage accumulation was observed in the alveoli of the 0.2 mg/kg SWCNT-exposed group (panel b). Moderate macrophage accumulation accompanied with foamy macrophages and mild inflammatory cell infiltration in the alveoli, and moderate macrophage infiltration and granuloma in the interstitium were observed in the 2.0 mg/kg SWCNT-exposed group (panel c). Minimal macrophage accumulation was observed in the alveoli of the crystalline silica-exposed group (panel d).

Figure 9.  Light micrographs of lung tissue sections of rats at 3 months after instillation in experiment 1 (H&E stain). No significant changes were observed in the vehicle control group (panel a). Minimal macrophage accumulation was observed in the alveoli of the 0.2 mg/kg SWCNT-exposed group (panel b). Moderate macrophage accumulation accompanied with foamy macrophages, mild inflammatory cell infiltration in the alveoli, mild macrophage infiltration and granuloma in the interstitium, and mild hypertrophy of alveolar epithelium were observed in the 2.0 mg/kg SWCNT-exposed group (panel c). Mild macrophage accumulation accompanied with foamy macrophages, minimal inflammatory cell infiltration in the alveoli, and minimal hypertrophy of alveolar epithelium were observed in the crystalline silica-exposed group (panel d).

Figure 10.  Light micrographs of lung tissue sections of rats at 6 months after instillation in experiment 2 (H&E stain). Minimal macrophage accumulation was observed in the alveoli of the vehicle control group. Minimal macrophage accumulation was observed in the alveoli of the 0.04 mg/kg SWCNT-exposed group (panel b). Minimal macrophage accumulation in the alveoli and interstitium were observed in the 0.2 mg/kg SWCNT-exposed group (panel c). Mild macrophage accumulation accompanied with foamy macrophages and minimal inflammatory cell infiltration, minimal macrophage infiltration in the interstitium, and minimal hypertrophy of alveolar epithelium were observed in the 1 mg/kg SWCNT-exposed group (panel d).

Figure 11.  TEM images of SWCNTs deposited in lungs of rats exposed to 2.0 mg/kg SWCNTs at 1 month (panels a–c) and 3 months (panels d–f) after instillation at different magnifications. At any of the time points, SWCNTs deposited in the lungs were typically phagocytosed by alveolar macrophages or macrophages in the interstitial tissues.