Promotion of lung adenocarcinoma following inhalation exposure to multi-walled carbon nanotubes

Abstract

Background: Engineered carbon nanotubes are currently used in many consumer and industrial products such as paints, sunscreens, cosmetics, toiletries, electronic processes and industrial lubricants. Carbon nanotubes are among the more widely used nanoparticles and come in two major commercial forms, single-walled carbon nanotubes (SWCNT) and the more rigid, multi-walled carbon nanotubes (MWCNT). The low density and small size of these particles makes respiratory exposures likely. Many of the potential health hazards have not been investigated, including their potential for carcinogenicity. We, therefore, utilized a two stage initiation/promotion protocol to determine whether inhaled MWCNT act as a complete carcinogen and/or promote the growth of cells with existing DNA damage. Six week old, male, B6C3F1 mice received a single intraperitoneal (ip) injection of either the initiator methylcholanthrene(MCA, 10 μg/g BW, i.p.), or vehicle (corn oil). One week after i.p. injections, mice were exposed by inhalation to MWCNT (5 mg/m³, 5 hours/day, 5 days/week) or filtered air (controls) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for lung tumor formation.

Results: Twenty-three percent of the filtered air controls, 26.5% of the MWCNT-exposed, and 51.9% of the MCA-exposed mice, had lung bronchiolo-alveolar adenomas and lung adenocarcinomas. The average number of tumors per mouse was 0.25, 0.81 and 0.38 respectively. By contrast, 90.5% of the mice which received MCA followed by MWCNT had bronchiolo-alveolar adenomas and adenocarcinomas with an average of 2.9 tumors per mouse 17 months after exposure. Indeed, 62% of the mice exposed to MCA followed by MWCNT had bronchiolo-alveolar adenocarcinomas compared to 13% of the mice that received filtered air, 22% of the MCA-exposed, or 14% of the MWCNT-exposed. Mice with early morbidity resulting in euthanasia had the highest rate of metastatic disease. Three mice exposed to both MCA and MWCNT that were euthanized early had lung adenocarcinoma with evidence of metastasis (5.5%). Five mice (9%) exposed to MCA and MWCNT and 1 (1.6%) exposed to MCA developed serosal tumors morphologically consistent with sarcomatous mesotheliomas, whereas mice administered MWCNT or air alone did not develop similar neoplasms.

Conclusions: These data demonstrate that some MWCNT exposures promote the growth and neoplastic progression of initiated lung cells in B6C3F1 mice. In this study, the mouse MWCNT lung burden of 31.2 μg/mouse approximates feasible human occupational exposures. Therefore, the results of this study indicate that caution should be used to limit human exposures to MWCNT.

Figure 1

Foreign material observed by light microscopy (A) and polarized light (B). A. The figure is a low magnification image of MWCNT deposition in the lungs. Macrophages in alveolar spaces of the right apical lobe of the lung contain intracytoplasmic, black, elongate to finely granular particulate material (presumptive MWCNT). (40x). B. Using polarized light, the low magnification image demonstrates presumptive MWCNT are seen in macrophages in the right apical lung lobe of mouse treated with MWCNT. The material in the macrophages are birefringent. (40x).

Figure 2

Cytoviva imaging. A. The figure is an example of typical images from light and enhanced darkfield imaging of MWCNT in lungs and diaphragm. The light micrograph of H&E stained section demonstrates MWCNT (black fibers) in the alveolar interstitium of a MWCNT Air exposed animal 17 months following inhalation exposure. This micrograph shows an example of MWCNT present within the alveolar interstium. Light microscope image using 100x oil immersion objective. Magnification bar is 10 microns. B. The micrograph is an enhanced darkfield image from the lung of an animal 17 months following exposure to MWCNT. The central area of the micrograph shows a region of alveolar wall with numerous MWCNT fibers present. The MWCNT are bright white due to imaging of scattered light over a broad range of wavelengths by this nanomaterial. Lung tissue, which does not significantly scatter light, is brown-to-orange and airspaces are black. The enhanced darkfield microscope image was photographed using a 100x oil immersion objective. The magnification bar is 20 microns. C. An enhanced darkfield imagine showing MWCNT in the diaphragm. MWCNT (indicated by the upper two arrows) are bright white. Nuclei are brown-to-orange, muscle cells are green and red blood cells are yellow. The parietal pleural border is indicated by the arrow in the lower part of the figure. Enhanced darkfield microscope image using 100x oil immersion objective. Magnification bar is 20 microns.

Figure 3

Hyperplasia. A Focal adenomatous hyperplasia in a mouse exposed to i.p corn oil and inhaled MWCNT. A. At low magnification, a focus of bronchoalveolar hyperplasia forms a discrete, hypercellular focus that retains normal alveolar architecture (bar = 200 microns). B. At higher magnification, the hypercellularity is attributable to a population of hypertrophied epithelial cells characterized by moderate anisokaryosis and mild karyomegaly. To distinguish these foci from foci of reactive hyperplasia and because of morphologic similarities to atypical adenomatous hyperplasia in the human lung, we have used the term focal adenomatous hyperplasia for these foci. MWCNT were sometimes seen within or near these foci (bar = 50 microns).

Figure 4

A, B and C Lung adenoma, 20× and 40×. A. The figure is a representative pulmonary bronchio-alveolar adenoma from a male B6C3F1 mouse treated with MCA + MWCNT. The mass is composed of relatively uniform cells that compressed the surrounding lung tissue. The photo with taken with a 4x objective. The magnification bar is 200 microns. B. Figure 4B is a photomicrograph of a pulmonary bronchiolo-alveolar adenoma from a MCA + MWCNT treated mouse. The focal, moderately cellular mass involved a bronchiole. The mass was composed of relatively uniform cells that distorted and replaced alveolar architecture. The image was photographed using a 20x objective. The magnification bar is 50 microns. C. The light micrograph is a higher magnification of the H&E stained section demonstrating MWCNT (black fibers) in the tissue surrounding the bronchiolo-alveolar adenoma (arrows 1, 2 and 3). The light microscope image was taken using 100x oil immersion objective. The magnification bar is 20 microns.

Figure 5

Pulmonary bronchiolo-alveolar adenocarcinomas in MCA + MWCNT treated mice. A. The photomicrograph shows a right cardiac lung lobe of a MCA + MWCNT-treated mouse that contains a bronchiolo-alveolar adenocarcinoma (white arrow). A bronchiolo-alveolar adenoma (black arrow) is in the adjacent lung lobe. (2x). The magnification bar is 500 microns. B. The figure is a photomicrograph of a bronchiolo-alveolar adenocarcinoma in the right cardiac lobe of a mouse lung treated with MCA + MWCNT (20x). This infiltrative adenocarcinoma filled ~85% of the lobe on histologic cross section. The scale bar is 200 microns. C. The photomicrograph is a higher magnification of the bronchiolo-alveolar adenocarcinoma of the right cardiac lobe in figure B showing heterogeneous growth and pleomorphic cytologic features (40x). The scale bar is 50 microns. D. The photomicrograph shows a metastasis of the bronchiolo-alveolar adenocarcinoma in the right cardiac lobe of a mouse lung 17 months following treatment with MCA + MWCNT (40×). The arrow demonstrates a metastasis in a pulmonary vein. The scale bar is 50 microns.

Figure 6

Volume of lung tumors 17 months after exposure to MWCNT. The figure demonstrates the volume of the lung occupied by tumor per individual mouse. The volume of the lung occupied by tumor was greater in the MCA + MWCNT (361 mm³+/-18.72) compared to the mice treated with MCA (202.61 mm³+/-36.75). The MWCNT group had an average tumor volume of 107.88 mm³+/- 28.70 compared to the negative control, Air group, with a volume of 22.29 mm³ +/- 3.93. *Indicates a significant difference compared to the respective air control group at p < .0001.

Figure 7

Life table of mice treated with one dose of corn oil (Air), MC (MCA), MWCNT only MWCNT or MCA followed by MWCNT (MC + MWCNT). The life table in Figure 7 shows the percent of mice alive in each treatment group at 30 weeks after exposure to the time of sacrifice 70 weeks after exposure. The MCA + MWCNT treated animals had a greater number of early deaths than the MWCNT, the MCA or the Air treated mice.

Figure 8

Malignant serosal tumors in the lung (A) and the diaphragm (B) in mice exposed to MC followed by MWCNT. A. The photomicrograph shows a pulmonary vein in the right cardiac lung lobe that contained variably-sized polygonal to spindloid cells similar to those on the diaphragm (Figure 8B). The arrow indicates a metastasis of the malignant serosal tumor (20×). The magnification bar is 50 microns. B. The skeletal muscle of the diaphragm in the photomicrograph is infiltrated by a nodular mass composed of variably-sized polygonal to spindloid cells (malignant serosal tumors). (20×). The magnification bar is 50 microns.

Figure 9

Immunofluorescence staining for podoplanin (red) and cytokeratins (green) in malignant serosal tumors morphologically consistent with sarcomatous mesothelioma. In A, the lining mesothelium (solid arrows) stained positively for podoplanin in this double label immunofluorescent image of diaphragm. The cells beneath the mesothelial lining are also red due to expression of podoplanin and these are cells of a malignant serosal tumor. In B, only the single label green fluorescence is shown to demonstrate weak expression of cytokeratins in the lining mesothelium (solid arrow), while staining of the subjacent tumor for cytokeratins is equivocal. In C, a double label immunofluorescent image demonstrates a malignant serosal tumor between the liver and gall bladder that is lined by reactive mesothelium (solid arrow) which stains red for podoplanin as well as green for cytokeratins. The cells of the subjacent malignant serosal tumor stain weakly red for podoplanin. In D, the photomicrograph shows this same tumor but only the green fluorescence for cytokeratins. The reactive mesothelium lining the malignant serosal tumor (solid arrow) stains green for cytokeratins while the serosal tumor has no evidence of cytokeratin expression. The normal mesothelium lining the liver is weakly positive for cytokeratins (dashed arrow). The epithelium lining the gall bladder (open arrow) strongly expresses cytokeratins. Magnification bar is 50 μm.