Respiratory carbon nanotubes: Proteins secreted in lung fluid

Health implications of carbon nanotubes

Carbon nanotubes were hailed as a new wonder material upon their discovery in 1991 and their potential is being fulfilled with numerous applications that take advantage of their unique mechanical, electrical and thermal properties. These have been expanded by the ability to coat nanotubes with metal oxides which modify the properties to impart improved catalytic activity, conductivity and energy storage capabilities.

There is no doubt that nanotubes are remarkable materials but their widespread introduction came about without any real risk assessment. One of the key potential problems is related to their structure. CNTs are similar to fibres and there is concern that they might initiate the same kind of health effects as asbestos. In fact, some studies have already shown that rodents develop lung inflammation and pulmonary fibrosis after inhaling them.

Scientists at North Carolina State University in the USA decided to see how CNTs affect the proteins in the bronchoalveolar lavage fluid of mice, the fluid that resides in the airways which can be an indicator of lung disease. Michael Bereman and colleagues exposed mice to two types of CNTs by aspirating a solution into their airways and the results were compared to those of control animals who were treated with the same solution minus the CNTs.

Mouse model of exposure

Pure multiwalled CNTs and those coated on the surface with aluminium oxide were administered to the mice and the bronchoalveolar lavage fluid was collected 24 hours later. After separating the fluid from the CNTs, the proteins in the fluid were treated by conventional digestion and tandem mass spectrometry and measured by a method know as peptide spectral counting.

From a total of 465 individual proteins, those that were present in different amounts between pairs of the three experiments - CNTs, coated CNTs and controls - were identified by searching the constituent peptides against a database of mouse protein sequences.

So, 27 proteins were differentially expressed between the CNTs and the control group, 13 between the coated CNTs and the control group, and 2 between the two CNT groups. In order to ensure that the protein changes were as a result of a true biological response, rather than a physical reaction, the CNTs were added to mouse lung lavage fluid in an in vitro experiment.

From the comparison, only 4 proteins were found to be statistically significant for the in vivo and in vitro studies, so it is safe to assume that the remaining proteins from the in vivo experiment are altered as a result of some biological process.

Immune response activated

Most of the affected proteins were associated with the response to stimuli and the immune response. For instance, myeloperoxidase was raised notably after exposure to both types of CNTs in an immune response. Earlier reports had also found that the same enzyme can degrade CNTs to reduce pulmonary inflammation.

Other proteins that were more abundant after CNT exposure were lactotransferrin and neutrophil gelatinase-associated lipocalin, supporting the induction of an inflammatory response in the lung.

This preliminary study will be supplemented by further work, say the researchers. In particular, "future studies will use protein cleavage isotope dilution mass spectrometry to validate the absolute changes in protein amounts and examine the differences in protein expression across multiple types of multiwalled functionalization."  

The results will be used to see how the intracellular pathways are affected by CNT invasion and the overall aim is to identify a set of biomarkers that can be used in peptide assays to assess the degree of toxicity of CNTs with various coatings. In the meantime, the initial work confirms that exposure to CNTs stimulates proteins that are associated with the immune response and inflammation.

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