Multiwalled carbon nanotubes intratracheally instilled into the rat lung induce development of pleural malignant mesothelioma and lung tumors

Abstract

Multiwalled carbon nanotubes (MWCNT) have a fibrous structure and physical properties similar to asbestos and have been shown to induce malignant mesothelioma of the peritoneum after injection into the scrotum or peritoneal cavity in rats and mice. For human cancer risk assessment, however, data after administration of MWCNT via the airway, the exposure route that is most relevant to humans, is required. The present study was undertaken to investigate the carcinogenicity of MWCNT-N (NIKKISO) after administration to the rat lung. MWCNT-N was fractionated by passing it through a sieve with a pore size of 25 μm. The average lengths of the MWCNT were 4.2 μm before filtration and 2.6 μm in the flow-through fraction; the length of the retained MWCNT could not be determined. For the present study, 10-week-old F344/Crj male rats were divided into five groups: no treatment, vehicle control, MWCNT-N before filtration, MWCNT-N flow-through and MWCNT-N retained groups. Administration was by the trans-tracheal intrapulmonary spraying (TIPS) method. Rats were administered a total of 1 mg/rat during the initial 2 weeks of the experiment and then observed up to 109 weeks. The incidences of malignant mesothelioma and lung tumors (bronchiolo-alveolar adenomas and carcinomas) were 6/38 and 14/38, respectively, in the three groups administered MWCNT and 0/28 and 0/28, respectively, in the control groups. All malignant mesotheliomas were localized in the pericardial pleural cavity. The sieve fractions did not have a significant effect on tumor incidence. In conclusion, administration of MWCNT to the lung in the rat induces malignant mesothelioma and lung tumors.

Keywords: Intratracheal instillation; lung tumors; malignant mesothelioma; multiwalled carbon nanotubes; rat.

Figure 1

Before administration, a portion of the multiwalled carbon nanotube (MWCNT) suspension was fractionated by passing it through a sieve with a pore size of 25 µm. Scanning microscope images of MWCNT‐N in the (a) unfiltered preparation, (b) flow‐through fraction and (c) retained fraction are shown. The mean length of the unfiltered MWCNT‐N in the administration dispersant was 4.2 ± 2.9 µm and that of the MWCNT‐N in the flow‐through fraction was 2.6 ± 1.6 µm. The size of the MWCNT‐N retained by the filtering sieve could not be measured because of the dense agglomerates the retained fibers formed due to the loss of the PF68 dispersant during fractionation.

Figure 2

Accumulation of multiwalled carbon nanotubes (MWCNT) in the lung (a,b) and lymph node (c,d). MWCNT‐N was found in the lung alveoli either in the granulation tissue or macrophages. (a) MWCNT‐N shows granular and needle like shapes. Aggregations of MWCNT‐phagocytosed macrophages can be seen (arrow). The inset shows a polarizing lens image of the aggregation of MWCNT‐phagocytosed macrophages; arrows point to the same macrophage aggregation in the main panel and the inset. MWCNT‐N aggregates that cause polarization can clearly be seen in the inset. Granular agglomerations of MWCNT‐N do not cause polarization. (b) MWCNT‐N also caused thick fibrotic granulation tissue formation (arrow) and calcification (*). The inset shows a polarizing lens image of granulation tissue with deposition of MWCNT‐N. Arrows point to the same lesion in the main panel and the inset. (c) Accumulation of MWCNT‐N in the mediastinal lymph node. The inset shows a polarizing lens image. Arrows point to the same MWCNT‐N accumulations in the main panel and the inset. (d) Accumulation of MWCNT‐N in periaortic connective tissue showing fibrotic thickening. The inset shows a polarizing lens image. (e) MWCNT‐N is present in the periphery of the malignant mesothelioma tissue.

Figure 3

Kaplan–Meier survival plot of the rats in the untreated, vehicle control, unfiltered multiwalled carbon nanotubes (MWCNT), flow‐through MWCNT fraction, and retained MWCNT fraction groups.

Figure 4

Malignant mesotheliomas in the pleural/mediastinal cavity. Malignant mesothelioma showing invasion of the pericardium (a), myocardium (b), periesopahgeal tissue (c) and visceral pleura (d).

Figure 5

Malignant mesothelioma, sarcomatoid type, in the pleural cavity. (a) Tumor cells arranged in a solid manner. Immunostaining of calretinin (b), Wilms tumor protein (WT‐1) (c), podoplanin (Pod) (d), and thyroid transcription factor‐1 (TTF‐1) (e). In the high power magnification images (inset), the cytoplasm of the tumor cells were faintly stained by calretinin and Wilms tumor protein. Nuclei are also faintly stained by Pod. Cytoplasm and nuclei are entirely unstained by TTF‐1.

Figure 6

Bronchiolar epithelium of 109 week old untreated rat. H&E (a) and calretinin (Cal) (b), Wilms tumor protein (WT‐1) (c), podoplanin (d) and thyroid transcription factor‐1 (TTF‐1) (e) staining. The cytoplasm is slightly stained by Cal and WT‐1. Nuclei are clearly positive for TTF‐1.

Figure 7

Gross appearance of lung adenocarcinoma in the pericardial pleural cavity. Histologically, the tumor is composed of packed small epithelial‐like cells forming incomplete glandular structures (see Figure 11a) with invasion of pericardial and diaphragm tissues (arrows). The tumor was positive for TTF‐1. Small tumor nodules of the same histological appearance were found in the lung (circle).

Figure 8

Lung bronchiolo‐alveolar adenoma (adenoma). H&E (a), calretinin (Cal) (b), Wilms tumor protein (WT‐1) (c), podoplanin (d) and thyroid transcription factor‐1 (TTF‐1) (e) staining. The cytoplasm is slightly stained by Cal and WT‐1. Nuclei are clearly positive for TTF‐1.

Figure 9

Bronchiolo‐alveolar carcinoma (adenocarcinoma) of the lung. H&E (a), calretinin (Cal) (b), Wilms tumor protein (c), podoplanin (d) and thyroid transcription factor‐1 (TTF‐1) (e) staining. The cytoplasm is slightly stained by Cal and Wilms tumor protein (WT‐1). Nuclei are clearly positive for TTF‐1.

Figure 10

Adenocarcinoma of the lung forming a large mass in the pleural cavity with invasion of the heart and diaphragm and remote metastasis to the kidney. H&E (a), calretinin (Cal) (b), Wilms tumor protein (c), podoplanin (d) and thyroid transcription factor‐1 (TTF‐1) (e) staining. The cytoplasm is slightly stained by Cal and Wilms tumor protein. Nuclei are clearly positive for TTF‐1.

Figure 11

Adenocarcinoma of the lung forming a large mass in the pleural cavity with invasion of the peribronchial lymphnodes, serosal pericardium and diaphragm (see Fig. 3). Densely proliferating small tumor cells with round nuclei are arranged in an incomplete glandular or solid manner. H&E (a), calretinin (b), Wilms tumor protein (WT‐1) (c), podoplanin (d) and thyroid transcription factor‐1 (TTF‐1) (e) staining. The cytoplasm is slightly stained by WT‐1. All the nuclei are clearly positive for TTF‐1 indicating lung alveolar cell origin. After histological examination, this case was initially diagnosed as an epithelial mesothelioma; however, after staining with TTF‐1, the tumor was re‐diagnosed as a lung adenocarcinoma.