Distribution and persistence of pleural penetrations by multi-walled carbon nanotubes

Abstract

Background: Multi-walled carbon nanotubes (MWCNT) are new manufactured nanomaterials with a wide spectrum of commercial applications. The durability and fiber-like dimensions (mean length 3.9 μm long × 49 nm diameter) of MWCNT suggest that these fibers may migrate to and have toxicity within the pleural region. To address whether the pleura received a significant and persistent exposure, C57BL/6J mice were exposed by pharyngeal aspiration to 10, 20, 40 and 80 μg MWCNT or vehicle and the distribution of MWCNT penetrations determined at 1, 7, 28 and 56 days after exposure. Following lung fixation and sectioning, morphometric methods were used to determine the distribution of MWCNT and the number of MWCNT fiber penetrations of three barriers: alveolar epithelium (alveolar penetrations), the alveolar epithelium immediately adjacent to the pleura (subpleural tissue), and visceral pleural surface (intrapleural space).

Results: At 1 day 18%, 81.6% and 0.6% of the MWCNT lung burden was in the airway, the alveolar, and the subpleural regions, respectively. There was an initial, high density of penetrations into the subpleural tissue and the intrapleural space one day following aspiration which appeared to decrease due to clearance by alveolar macrophages and/or lymphatics by day 7. However, the density of penetrations increased to steady state levels in the subpleural tissue and intrapleural from day 28 - 56. At day 56 approximately 1 in every 400 fiber penetrations was in either the subpleural tissue or intrapleural space. Numerous penetrations into macrophages in the alveolar airspaces throughout the lungs were demonstrated at all times but are not included in the counts presented.

Conclusions: The results document that MWCNT penetrations of alveolar macrophages, the alveolar wall, and visceral pleura are both frequent and sustained. In addition, the findings demonstrate the need to investigate the chronic toxicity of MWCNT at these sites.

Figure 1

Light micrograph of MWCNT deposition in alveolar region of lungs. Sirius Red stained micrograph showing the general deposition pattern of MWCNT (arrows) one day after aspiration. A deposit of MWCNT on the epithelium of the terminal bronchiole (TB) near the transition between the airways and the alveolar region is indicated by the asterisk. Smaller deposits near the subpleural tissue region are indicated by double arrows. (dose 80 μg).

Figure 2

Morphometric determination of the initial lung distribution of MWCNT 1 day post aspiration. Results show the distribution of MWCNT fiber burden in airways, alveolar and subpleural tissue regions of the lungs one day after aspiration. As shown by the different shades of bars, MWCNT fiber burden in the airways and alveolar regions were further subdivided into the airspaces of the region, tissue of the region and macrophages. Results are expressed as a percentage of the total lung burden. (Mean ± SE, N = 7).

Figure 3

Light micrograph of fibrotic granulomas 56 days following MWCNT. Micrograph illustrates the extensive collagen network developed within granulomas that developed following a single 20 μg dose of MWCNT. Section stained with Sirius Red show collagen fibers in red.

Figure 4

FESEM of MWCNT penetration of alveolar epithelial cells. As shown in this micrograph, MWCNT were rapidly incorporated into the alveolar epithelium. Micrograph shows two MWCNT passing through an alveolar epithelial cell 1 day after aspiration (80 ug dose). The two fibers are running together on the right side of the cell and appear to separate within the cell as they pass out on the left side of the cell. Another MWCNT penetrates the epithelium in the upper right of micrograph.

Figure 5

Morphometric determination of the number of alveolar epithelial penetrations versus dose 56 days after aspiration. The number of MWCNT penetrations per lung versus aspiration dose of MWCNT demonstrated a sigmoidal dose-response with an ED₅₀ of 15.3 μg. (Mean ± SE, N = 8).

Figure 6

Photomicrograph of lung section showing an intralobular septum. This micrograph shows several MWCNT loaded macrophages (arrows) in the intralobular septum at 56 days post aspiration (40 μg dose).

Figure 7

Representative micrographs of MWCNT in subpleural tissues, visceral pleura and pleural space. In all four panels, the visceral pleural surface runs along the top of each micrograph. The FESEM image in A shows a MWCNT loaded alveolar macrophage in an alveolus immediately beneath the visceral pleura surface. The right side of the image shows a single MWCNT fiber penetrating the alveolar epithelium into the subpleural tissues (80 μg dose, 28 day post-aspiration). Figure 7B shows a dilated subpleural lymphatic vessel which contains a mononuclear inflammatory cell that is penetrated by several MWCNT fibers (80 μg dose, 56 day post-aspiration). A MWCNT penetrating the visceral pleura is shown in the light micrograph of 7C with a MWCNT-loaded alveolar macrophage visible in the left side of the micrograph (80 μg dose, 28 day post-aspiration). A single MWCNT penetrating from the subpleural tissue through the visceral pleura into the pleural space is shown in the FESEM image of 7 D (80 μg dose, 56 day post-aspiration).

Figure 8

Effect of MWCNT dose on MWCNT fiber penetrations into subpleural tissue and intrapleural space 56 days after aspiration. This figure shows the number per lung for fibers which penetrated into the subpleural tissue and for fibers which penetrated into the intrapleural space. As indicated by the asterisks, both the number of subpleural tissue and intrapleural space penetrations at 80 μg were different from the corresponding subpleural tissue and intrapleural space penetrations at doses of 10, 20 and 40 μg. (Mean ± SE, N = 8).

Figure 9

Time course of MWCNT penetrations into the subpleural tissue and intrapleural space. There was a significant number of penetrations into the subpleural tissue and intrapleural space with an initial spike at one day after aspiration with some initial clearance evident by the decline at 7 days. However the number of penetrations in both spaces significantly increased after day 7 and was still elevated 56 days after the initial aspiration. Data from animals given a single aspiration dose of 80 μg. As indicated by the asterisk, the number of subpleural tissue penetrations at 1 day was significantly different from the number of penetrations at 7, 28 and 56 days. (Mean ± SE, N = 8).