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. 2012;7(3):e30923.
doi: 10.1371/journal.pone.0030923. Epub 2012 Mar 30.

Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-deficient mice

Anna A Shvedova  1 , Alexandr A Kapralov, Wei Hong Feng, Elena R Kisin, Ashley R Murray, Robert R Mercer, Claudette M St Croix, Megan A Lang, Simon C Watkins, Nagarjun V Konduru, Brett L Allen, Jennifer Conroy, Gregg P Kotchey, Bashir M Mohamed, Aidan D Meade, Yuri Volkov, Alexander Star, Bengt Fadeel, Valerian E Kagan
Affiliations

Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-deficient mice

Anna A Shvedova et al. PLoS One. 2012.

Abstract

Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro-inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO - one of the key-orchestrators of inflammatory response - in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characterization of pulmonary inflammatory responses to SWCNT in w/t and MPO k/o mice at day 1 after pharyngeal aspiration exposure.
a–c. Levels of pro-inflammatory cytokines (a - TNF-α; b - IL-6; c – MCP-1) in BAL fluid of w/t and MPO k/o mice. Mean ± SEM (n = 6 mice/group). *p<0.05, vs control PBS-exposed mice. d. Content of PMNs in BAL fluid of w/t and MPO k/o mice. Mean ± SEM (n = 6 mice/group). *p<0.05, vs control PBS-exposed mice. e. Typical microscopic images of inflammatory cells in BAL fluid with SWCNT inclusions (red arrows). f. Content of PMNs with engulfed SWCNT in BAL fluid of w/t and MPO k/o mice. Mean ± SEM (n = 6 mice/group). *p<0.05, vs w/t mice.
Figure 2
Figure 2. Changes in the content of collagen and average thickness of alveolar connective tissue in the lungs of w/t and MPO k/o mice at days 1 and 28 after pharyngeal aspiration of SWCNT.
a. Accumulation of collagen in the lung of w/t or MPO k/o mice. Mean ± SEM (n = 6 mice/group). *p<0.05, vs control PBS-exposed mice, # p<0.05, vs w/t mice 28 days post exposure. b. Morphometric assessments of average thickness of alveolar connective tissue in the lung of w/t or MPO k/o mice. Mean ± SEM (n = 6 mice/group). *p<0.05, vs control PBS-exposed mice, # p<0.05, vs w/t 28 days post exposure.
Figure 3
Figure 3. Assessment of the volume of SWCNT aggregates in the lung tissue sections from w/t and MPO k/o mice.
a. Representative images of the lung tissue sections. Insert - higher magnifigation (2.5× zoom) of a field illustrating the presence of SWCNT (green punctuate spots pointed by white arrows). b. Quantitation of SWCNT aggregates (SWCNT volume/total lung volume) using their specific absorbance (750–850 nm), * p<0.05, vs w/t 1 day post exposure, # p<0.05, vs w/t 28 days post exposure. c. Assessment of SWCNT aggregates - number per microscopic field - using an automated IN Cell Analyser 1000 microscope, * p<0.05, vs w/t 1 day post exposure, # p<0.05, vs w/t 28 days post exposure.
Figure 4
Figure 4. Evaluation of SWCNT size distribution in solubilized lungs of w/t and MPO k/o mice at days 1 and 28 post exposure by t
ransmission electron microscopy. a. Typical TEM images of SWCNT after solubilization of the lung tissue. b. Size distribution of SWCNT present in the lung at days 1 and 28 post exposure. * p<0.05, vs w/t 1 day post exposure, # p<0.05, vs w/t 28 days post exposure. c. Changes in size distribution of SWCNT at day 28 post exposure compared to day 1 post exposure.
Figure 5
Figure 5. Raman spectroscopic evaluation of “oxidative” defects in SWCNT present in the lungs of w/t and MPO k/o mice at days 1 and 28 post exposure using single point Raman spectroscopy or Raman mapping of different areas within the tissue samples.
a. D-band/G- band ratios for single point Raman spectra obtained from samples at days 1 and 28 post exposure. * p<0.05, vs w/t 1 day post exposure, # p<0.05, vs w/t 28 days post exposure. Insert – typical Raman spectra (excitation at 633 nm) of solubilized lungs of w/t mice at days 1 and 28 post exposure. b–d. Raman mapping of SWCNT in different areas of the lung sections. b. Examples of bright-field images with a red box indicating the area where 32×32 Raman spectra were acquired. Note that the sizes of the acquired areas were different at day 1 and day 28 (20 µm×20 µm and 10 µm×10 µm respectively), as significantly smaller SWCNT aggregates were detected at day 28 in w/t mice and the scanned areas were adjusted accordingly. c. Raman maps (excitation at 473 nm excitation) with examples of spectra corresponding to each of the clusters. d. D-band/G-band ratios for Raman spectral maps obtained from the lung of w/t and MPO k/o mice at days 1 and 28 post exposure.
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