Extrapulmonary transport of MWCNT following inhalation exposure

Abstract

Background: Inhalation exposure studies of mice were conducted to determine if multi-walled carbon nanotubes (MWCNT) distribute to the tracheobronchial lymphatics, parietal pleura, respiratory musculature and/or extrapulmonary organs. Male C57BL/6 J mice were exposed in a whole-body inhalation system to a 5 mg/m3 MWCNT aerosol for 5 hours/day for 12 days (4 times/week for 3 weeks, lung burden 28.1 ug/lung). At 1 day and 336 days after the 12 day exposure period, mice were anesthetized and lungs, lymph nodes and extrapulmonary tissues were preserved by whole body vascular perfusion of paraformaldehyde while the lungs were inflated with air. Separate, clean-air control groups were studied at 1 day and 336 days post-exposure. Sirius Red stained sections from lung, tracheobronchial lymph nodes, diaphragm, chest wall, heart, brain, kidney and liver were analyzed. Enhanced darkfield microscopy and morphometric methods were used to detect and count MWCNT in tissue sections. Counts in tissue sections were expressed as number of MWCNT per g of tissue and as a percentage of total lung burden (Mean ± S.E., N = 8 mice per group). MWCNT burden in tracheobronchial lymph nodes was determined separately based on the volume density in the lymph nodes relative to the volume density in the lungs. Field emission scanning electron microscopy (FESEM) was used to examine MWCNT structure in the various tissues.

Results: Tracheobronchial lymph nodes were found to contain 1.08 and 7.34 percent of the lung burden at 1 day and 336 days post-exposure, respectively. Although agglomerates account for approximately 54% of lung burden, only singlet MWCNT were observed in the diaphragm, chest wall, liver, kidney, heart and brain. At one day post exposure, the average length of singlet MWCNT in liver and kidney, was comparable to that of singlet MWCNT in the lungs 8.2 ± 0.3 versus 7.5 ± 0.4 um, respectively. On average, there were 15,371 and 109,885 fibers per gram in liver, kidney, heart and brain at 1 day and 336 days post-exposure, respectively. The burden of singlet MWCNT in the lymph nodes, diaphragm, chest wall and extrapulmonary organs at 336 days post-exposure was significantly higher than at 1 day post-exposure.

Conclusions: Inhaled MWCNT, which deposit in the lungs, are transported to the parietal pleura, the respiratory musculature, liver, kidney, heart and brain in a singlet form and accumulate with time following exposure. The tracheobronchial lymph nodes contain high levels of MWCNT following exposure and further accumulate over nearly a year to levels that are a significant fraction of the lung burden 1 day post-exposure.

Figure 1

FESEM examples of MWCNT in lung and kidney tissue sections after MWCNT inhalation exposure. FESEM image of alveolar macrophages 1 day post-exposure in Figure 1A shows typical examples of MWCNT fibers protruding from the surface of alveolar macrophages (black arrow multiple fibers, white arrows singlets). Examples of MWCNT found in the alveolar interstitial space are shown in Figure 1B (336 day post-exposure). The image of a kidney section in Figure 1C shows a typical example of the single MWCNT found in extrapulmonary organs (336 day post-exposure). The location of this MWCNT singlet within Bowman’s capsule of the kidney is shown by the black rectangle given in the low magnification insert in the lower left of the figure.

Figure 2

Enhanced dark-field images of tissue sections from tracheobronchial lymph nodes 1 and 336 days after MWCNT inhalation exposure. As typified by the micrograph, at 1 day post-exposure singlet MWCNT fibers were observed scattered throughout sections of lymph nodes. However, 336 days post-exposure, numerous dense concentrations of MWCNT fibers were found within the lymph nodes. MWCNT fibers are bright white in these enhanced darkfield images due to scattering of light by MWCNT, while cell nuclei are brownish red and other tissue elements are green.

Figure 3

Enhanced darkfield images of MWCNT fibers in the diaphragm, kidney and brain at 1 day and 336 days after inhalation exposure. MWCNT fibers in these figures are bright white, cell nuclei are brownish red and other tissue elements are green. With rare exceptions, MWCNT fibers detected in extrapulmonary organs were singlets. Normal (transmitted) light was blended into the fields and contrast adjusted to make the tissue histology of the organs visible in these photographs.

Figure 4

Light and enhanced darkfield micrographs of MWCNTs detected in lavage of pleural space. The figure shows a comparison of the light and enhanced darkfield image of a singlet MWCNT in lavage of the pleural space in mice at 336 days post-exposure.

Figure 5

Percentage of 1 day post-exposure lung burden detected in tracheobronchial lymph nodes, extrapulmonary organs, diaphragm and chest wall. By 336 days post-exposure there was a 7-fold increase in MWCNT in extrapulmonary organs and diaphragm over that measured at 1 day post-exposure. Categories of extrapulmonary organs are ordered relative to MWCNT concentration in the respective tissue. Asterisks indicate significantly different between day 1 and 336 days post-exposure, p < 0.05.