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Revealing the composition and formation mechanism of carcinogenic asbestos bodies in human lungs

Periodic Reporting for period 1 - BiominAB-3D (Revealing the composition and formation mechanism of carcinogenic asbestos bodies in human lungs)

Reporting period: 2016-05-01 to 2018-04-30

Occupational exposure to asbestos is universally associated with several lung injuries, including respiratory diseases, asbestosis, mesothelioma, and, owing to others co-factors, lung cancer. Pleural malignant mesothelioma, in particular, is highly deadly, the 5-year survival rate being of 5% only (1 over 20). In addition, due to weathering of asbestos-reinforced cement products, asbestos contamination is also becoming of concern for the general population, in particular in urban areas. Asbestos can enter in living organisms by inhalation and, also due to its high bio-persistence, can manifest its toxicity after 20 to 40 years. For these reason asbestos remains a current major worldwide health threat, although, starting from the 1990s, it was banned in most countries and it is almost abolished today, with a few relevant exceptions (Russia, China, Canada, and Brazil). In fact, it is foreseen that the peak of mesothelioma cases in the world will be reached within 2020. At a world scale, the US National Institute of Health (NIH) estimated that 11 million people were exposed to asbestos between 1940 and 1978. These figures push to the conclusion that extensive research on asbestos interaction with host organism can have an impact on a large number of people in Europe and worldwide.
Once penetrated into the lungs of exposed people, asbestos fibers irritate the tissue, causing minerals and proteins to cluster around the foreign fibers in a process known as biomineralization. The resulting clusters are referred to as asbestos bodies. A satisfactory knowledge of the elemental composition and fine structure of the asbestos bodies, and a solid model of their formation mechanism is still lacking, preventing the formulation of the carcinogenic mechanism. The aim of this research project was therefore to obtain a solid morphological, structural, elemental, and chemical characterization of the asbestos bodies in human lung tissue, and to achieve this aim, the project is organized in the following tasks:

- Revealing the morphology and elemental composition of the asbestos bodies with unprecedented level of detail and sensitivity;
- Providing reliable and spatially resolved elemental quantification;
- Detecting possible structural and chemical modifications occurring to the fibers after prolonged stay in the lungs.

These objectives were achieved by combining advanced synchrotron radiation micro-probe tools with electron microscopy techniques and laboratory analyses and the acquired knowledge allowed to propose a model for the formation mechanims of the asbestos bodies.
Lung tissue samples of former workers of cement-asbestos products and textile mill plants, asbestos mine, and steel plant, who have been subjected to prolonged exposure to crocidolite asbestos, were collected after autopsy. All cases were affected by pulmonary asbestosis, pleural plaques, pleural mesothelioma or lung cancer, and one had also silicatosis. The plants where located in NW Italy and workers have been exposed to high levels of asbestos contamination for decades, and are therefore ideal to study the long-term fate of asbestos in human hosts.
Synchrotron radiation techniques were exploited to reveal the elemental composition and spatial distribution of the asbestos bodies (Objectives 1 and 2). Five synchrotron experiments were performed at two synchrotron sources. Transmission electron microscopy (TEM) and laboratory analyses were exploited to detect possible structural and chemical modifications occurring to the fibers after prolonged stay in the lungs (Objective 3). A large part of the experimental work was devoted to the preparation of the samples, as each microprobe technique required a specific method to prepare the samples, and, often, specific protocols had to be developed.
Main dissemination activities focused on the participation as speaker at scientific conferences.
A full description of the project results and dissemination activieties can be found on the official web site of the project (http://biominab3d.altervista.org/). Some results have been published in a scientific paper (Bardelli et al., 2017, 10.1038/srep44862 Open Access), and other papers are planned to be published in the next months.
The main results achieved during the duration of the fellowship are:

1. Spatially resolved elemental composition of the asbestos bodies and of the chemical form of Fe;
2. Better understanding about to what extent previous invasive methods to recover the asbestos bodies for analyses altered their composition;
3. High resolution tridimensional surface and internal morphology of the asbestos bodies;
4. Reliable quantification of the major elements present in the asbestos bodies;
5. A model has been proposed to explain the formation mechanism of the asbestos bodies in biological media (additional experimental work is required to confirm it).

Tomographic techniques have never been applied to study the asbestos bodies before this project, and the same applies to the combination of x-ray fluorescence and phase-contrast techniques. Tomography allowed to reveal also the internal structure of the asbestos bodies, while the combination of x-ray techniques allowed to obtain a reliable elemental quantification of the major elements composing the asbestos bodies. The results of this research added new information, which was not possible to obtain with conventional laboratory analyses. In particular, the use of synchrotron radiation microprobes allowed to calculate for the first time the mass density, the spatial distribution, and the Fe mass of the asbestos bodies in lung tissue.

On the scientific and medical side, the results of this research project added essential pieces of knowledge that can help formulating more solid theories on the carcinogenic mechanism of asbestos. On the society side, the dissemination of the results will contribute to raise the level of awareness of the governments and of the general population on the asbestos danger. An increased awareness can lead to more strict regulations and to more efficient prevention procedures and policies. It also has to be considered that the results and methodologies derived from this research can be applied to other potentially hazardous anthropogenic nanomaterials (carbon or metal-oxide nanotubes, particulate matter of industrial or vehicular origin, metal nanoparticles already used in cosmetic products, or foreseen to be used in future wearable electronic devices), which are becoming of increasing concern for human health.
a) Optical image of an asbestos body. b) and c) distribution of Fe, Si, and P (10.1038/srep44862).
Mass density map (g/cm3) of an asbestos body obtained from tomographic data (unpublished).