Within the June edition of The American Journal of Pathology (vol. 178, No. 6), researchers Murphy et al. from the University of Edinburgh, U.K.¹, have published an interesting article further addressing the issue of carbon nanotubes and pathology occurring in the space between the lung surface and the chest wall known as the pleural space, a site known to be a target for harmful fibres such as asbestos.  

In our second Feature Article for summer 2011, Craig Poland – Research Toxicologist for SAFENANO and co-author on the paper – provides an insightful commentary on the study and its contribution towards investigating the fibre pathogenicity paradigm, and explains the danger of blanket statements such as ‘all carbon nanotubes are like asbestos’.

We hope you find it interesting!

HARN Dimension and Pleural Retention

Craig Poland, SAFENANO Research Toxicologist

An Introduction to the Fibre Pathogenicity Paradigm 

Carbon nanotubes are an interesting form of carbon, and have gained fame as a potential wonder material due to their

Figure 1 - Single Walled Carbon Nanotube (SWCNT)

exceptional structural and electrical properties which are advantageous for various industrial applications from electrical circuitry to high strength composites. Carbon nanotubes can be found in various forms, but are most concisely described as a single graphene sheet with its distinctive hexagonal structure rolled to form a cylinder or single-walled carbon nanotube. A multi-walled carbon nanotube can be described as several graphene cylinders stacked concentrically one inside the other - these were the variety investigated by Murphy et al.

Carbon nanotubes are often described as being a high aspect ratio nanoparticle (HARN; a high length-to-width ratio) but can actually vary greatly in length, diameter, structural properties, and contaminants such as metals left over from the production process. They can be found as highly curled, semi-spherical agglomerates such as those described recently by Pauluhn² or straight/semi-straight high aspect ratio fibres as described by Poland et al.³ meaning that, despite having the same name, two samples of carbon nanotube can vary substantially. 

The fibrous shape of carbon nanotubes has raised suspicions of a possible similarity between their toxicological profile and that of pathogenic fibres such as asbestos⁴. Such concerns often surround new industrially relevant fibres to which there is the potential for human exposure. However, as discussed recently within a review article by several of the authors of the Murphy article, not all fibres are actually dangerous to health and many are benign⁵. Over the last 30 years or so, the investigation of what exactly causes a fibre to be dangerous led to the development of a set of criteria often referred to as the fibre pathogenicity paradigm, which describes the attributes required of a fibre if it is to exert fibre-toxicity. It is worth noting that fibre toxicity represents a degree of added toxicity, thus if a particle is not classified as a fibre, it does not mean that it is harmless, it simply means it won’t exert fibre-toxicity. The paradigm brings together several key attributes which can be summarised as Dose, Dimension, and Durability or the “3 D’s”. 

Dose

When thinking about the fibre paradigm, the issue of dose (quantity of material to which the subject is exposed) is of course the most basic of criteria for all toxic substances; an absence of dose means an absence of effect, since a dose is required to drive an effect. 


Dimension

The importance of dimension firstly relates to the diameter of a fibre, which must be below 3µm in order for it to be able to negotiate the progressively narrowing airways of the lung and deposit in the deep respiratory zone. Fibres which are far thicker than this are simply not respirable and so can never deposit in the deep lung (hence no dose). However, there is an additional issue of length in the fibre paradigm, which has its origin in the hindrance of clearance mechanisms that enable the body to deal with unwanted foreign material. Typically, material depositing in the lung is broadly dealt with in one of two ways depending on where it lands in the lung. In the upper airways, specialist cells with small hair-like projections called cilia, continually waft mucus and trapped foreign material such as bacteria and particles upwards towards the throat where they are swallowed or spat out. A slower method of clearance happens in gas exchange or respiratory zone of the lung. Here the removal of unwanted material is performed by specialist immune cells called macrophages which are mobile and patrol this region, ingesting anything they detect as foreign to the body and clearing it – a process called phagocytosis. The progressively narrowing airways of the lung means that only very small particles (less than ~3µm) can find their way into this region and so these are easily dealt with by macrophages. However fibres, by a quirk of aerodynamics, can align themselves in the airflow and as such present a small diameter but have a length far greater than the 3µm cut-off. 

The problem comes when the length of the fibre exceeds the maximum size a macrophage can realistically deal with (~10–20µm), meaning that the lungs own defence mechanism cannot clear the particle. Macrophages are nothing if not determined and, irrespective of length, will try to clear the particle.  This can result in a situation where the macrophage is struggling to clear a fibre far longer than itself - as shown in the figure below - and is unable to fully enclose it leading to a stressful situation for the cell called 'frustrated phagocytosis'. When thinking of this, I’m often reminded of the iconic image of Laurel and Hardy trying to handle a long plank of wood in the film 'Finishing Touch' and creating havoc with their lack of finesse - a macrophage handling a long fibre is oddly similar.  

	Figure 2 - 'The Finishing Touch' © Hal Roach/MGM

When considering a long fibre in the deep lung unable to be cleared by normal mechanisms, there is only one route of removal – namely the fibre either dissolving or breaking into smaller pieces which can be cleared by macrophages. Therefore, it is easy to see how a fibre that is not durable and rapidly dissolves or breaks (such as certain forms of glass fibre) will not lead to the build up of a large dose so long as exposure/deposition does not exceed the rate of dissolution. However a very durable fibre such as crocidolite asbestos will not easily dissolve in the lung and, as such, every exposure can lead to a further accumulation of fibres until a critical threshold dose is reached and harmful effects begin to manifest.  

The importance of each of these attributes to the overall pathogenicity of a fibre can be seen most simply in an adapted version of the classic Fire or Combustion Triangle (Fig. 3). Rather than Heat, Oxygen and Fuel we have Dose, Dimension and Durability, where removal of any of these ‘ingredients’ results in a loss of pathogenicity, in the same way that removal of heat, oxygen or fuel would extinguish a fire. 

	Figure 3 - The Fibre PathogenicityTriangle

The approach of injecting particles directly into the pleural space may seem, on the face of it, a very non-physiological route of exposure. Indeed one of the only situations where particles are injected into the pleural space of humans is the injection of talc slurry as a therapeutic approach to pleural effusion (build up of liquid in the pleural space) and as such, this is obviously not as a result of environmental exposure. However as discussed in the recent reviews the same authors^5,8, we know from post-mortem studies of people exposed to high dusts concentrations (such as city dwellers) that there is evidence of particle transit from the lungs into the pleural space resulting in characteristic ‘black spots’ on the parietal pleura⁹. 

Indeed two studies have now been published which add further weight to the contention that inhaled carbon nanotubes may pass through into the pleural space from the lungs. The first, published in the journal Nature Nanotechnology by Ryman-Rasmussen et al., showed evidence of deposition of carbon nanotubes after inhalation in the areas directly adjacent to the pleura covering the lung surface and integration of these particles into the lung tissue¹⁰. In a later study, Mercer and colleagues instilled carbon nanotubes into the lungs of mice and observed carbon nanotubes protruding out of the lung into the pleural space and transiting into the pleural space¹¹. These two studies, together with the human evidence of particle transfer from the lung to the pleura, add teeth to the argument that the important question is not ‘do carbon nanotubes reach the pleura?’ but instead ‘are they retained in the pleural space and what do they do when they are there?’. 

The article by Murphy goes some way to answering the questions of what attributes cause nanoparticles to be retained in the pleural space and what this may mean in terms of inflammation and fibrosis. As a concept, the issue of retention and length is not new, but the evidence that it is also relevant to nanoparticles is. Indeed this work builds upon theory put forward by Stanton in the early eighties. Within their seminal paper, Stanton and colleagues over the course of 72 experiments implanted a range of mineral fibres into the pleural space of rats and monitored them over a period of a year or more for signs of tumour formation. The resultant tumours were recorded and the data analysed for correlation between the particle dimensions and the ensuing tumours. Stanton and colleagues concluded that the probability of developing such pleural pathology correlated best with fibres that were 8µm in length and 0.25µm in diameter¹². The particular strength of this study was the large array of particles tested, which allowed them to conclude that the carcinogenicity of fibres depended on its dimensions and durability. The study of Murphy et al. advances the theory of Stanton and shows its relevance to HARN whilst providing evidence for a plausible mechanism for the presentation and retention of dose to the parietal pleura as advanced by Kane and colleagues^13,14.

As discussed at the beginning of this commentary, another important attribute of the fibre paradigm is the issue of biopersistence. While this wasn’t directly dealt with by Murphy and colleagues in this article, they have, in collaboration with the Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO) and the Institute of Occupational Medicine (IOM), recently published an article in the journal Particle and Fibre Toxicology (Osmond-McLeod et al. 2011) looking at this issue with several of the particles used within the Murphy study. They found that of the carbon nanotubes evaluated, most were highly durable but one sample did lose both mass and length indicating that it may not be as durable as the others¹⁵. 

Well, based on both the historical evidence concerning fibres and particles, it seems likely that a small proportion of particles depositing in the lung will pass through the pleural space. The evidence, advanced by Murphy et al., suggests that long, durable fibres that find their way into the pleural space are likely to be retained and cause adverse effects, whilst short, or non-durable fibres are not and this is relevant to both conventional fibres and HARN.

It is important, however, to realise that just as with conventional fibres, not all HARN will meet the criteria of the fibre paradigm - indeed there exists enormous variability within even a single class of HARN. For example, this means that whilst they share the same name, there may be considerable variability between types of carbon nanotube, which in turn may impact significantly on their potential to cause harm. This emphasises the need for careful and reasoned evaluation of potential risks and shows the inappropriateness of blanket statements such as ‘all carbon nanotubes are like asbestos’. Thus when considering the potential risks (particle or fibre) it is important to firstly know what you are working with, perform an adequate risk assessment that identifies, if appropriate, the potential hazards of long fibres if they meet all the criteria of the fibre paradigm.

However it should also be stressed that much work still needs to be done to confirm the generation of disease such as mesothelioma after inhalation exposure of HARN.  Further research should be undertaken, taking heed of the fibre paradigm, to establish this. 

1. Murphy, F., Duffin, R., Poland, C. A., Al-Jamal, K. T., Ali-Boucetta H. Nunes, A., Byrne, F., Prina-Mello, A., Li, S., Mather, S. J., Bianco, A., Prato, M., Macnee, W., Wallace, W. A., Kostarelos, K. & Donaldson, K. 2011 “Length dependent retention of carbon nanotubes in the pleural space of mice initiates sustained inflammation and progressive fibrosis on the parietal pleura”. Am J Path. 178(6):2587-600. 2. Pauluhn, J. 2010, "Subchronic 13-week inhalation exposure of rats to multiwalled carbon nanotubes: toxic effects are determined by density of agglomerate structures, not fibrillar structures", Toxicological Sciences, vol. 113, no. 1, pp. 226-242 3. Poland, C. A., Duffin, R., Kinloch, I. A., Maynard AD, Wallace, W. A., Seaton A, Stone V, Brown, S., MacNee W, & Donaldson K 2008, "Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study", Nature Nanotechnology, vol. doi:10.1038/nnano.2008.111. 4. Service, R. F. 1998, "Chemistry: Nanotubes: The Next Asbestos?", Science, vol. 281, no. 5379, p. 941. 5. Donaldson, K., Murphy, F. A., Duffin, R., & Poland, C. A. 2010, "Asbestos, carbon nanotubes and the pleural mesothelium: a review and the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma", Part Fibre Toxicol., vol. 7, no. 1, p. 5. 6. Craighead JE, Abraham JL, Churg A, Green FH, Kleinerman J, Pratt PC, Seemayer TA, Vallyathan V, Weill H. 1982, “The pathology of asbestos-associated diseases of the lungs and pleural cavities: diagnostic criteria and proposed grading schema. Report of the Pneumoconiosis Committee of the College of American Pathologists and the National Institute for Occupational Safety and Health”. Arch Pathol Lab Med.  8;106(11):544-96. 7. Stahel R. A.,  Weder W.,  Lievens Y.,  Felip E., and On behalf of the ESMO Guidelines Working Group. (2010) “Malignant pleural mesothelioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up” Ann Oncol; 21(suppl 5): v126-v128 8. Donaldson, K., Murphy, F., Schinwald, A., Duffin, R., & Poland, C. A. 2011, "Identifying the pulmonary hazard of high aspect ratio nanoparticles to enable their safety-by-design", Nanomedicine.(Lond), vol. 6, no. 1, pp. 143-156. 9. Mitchev, K., Dumortier, P., & De, V. P. 2002, "'Black Spots' and hyaline pleural plaques on the parietal pleura of 150 urban necropsy cases", Am.J Surg.Pathol., vol. 26, no. 9, pp. 1198-1206. 10. Ryman-Rasmussen, J. P., Cesta, M. F., Brody, A. R., Shipley-Phillips, J. K., Everitt, J. I., Tewksbury, E. W., Moss, O. R., Wong, B. A., Dodd, D. E., Andersen, M. E., & Bonner, J. C. 2009a, "Inhaled carbon nanotubes reach the subpleural tissue in mice", Nat.Nanotechnol., vol. 4, no. 11, pp. 747-751. 11. Mercer, R. R., Hubbs, A. F., Scabilloni, J. F., Wang, L., Battelli, L. A., Schwegler-Berry, D., Castranova, V., & Porter, D. W. 2010, "Distribution and persistence of pleural penetrations by multi-walled carbon nanotubes", Part Fibre Toxicol., vol. 7, p. 28. 12. Stanton, M. F., Layard, M., Tegeris, A., Miller, E., May, M., Morgan, E., & Smith, A. 1981, "Relation of particle dimension to carcinogenicity in amphibole asbestoses and other fibrous minerals", Journal Of The National Cancer Institute, vol. 67, no. 5, pp. 965-975. 13. Kane AB, Macdonald JL, Moalli PA. 1986, “Acute injury and regeneration of mesothelial cells produced by crocidolite asbestos fibers.” Am Rev. Respir Dis, 133:A198 14. Moalli PA, Macdonald JL, Goodglick LA, Kane AB. 1987, “Acute injury and regeneration of the mesothelium in response to asbestos fibers”. Am J Pathol, 128:426–445 15. Osmond-McLeod MJ, Poland CA, Murphy F, Waddington L, Morris H, Hawkins SC, Clark S, Aitken R, McCall MJ, Donaldson K. 2011 “Durability and inflammogenic impact of carbon nanotubes compared with asbestos fibres.” Part Fibre Toxicol. 13;8:15.