Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice

Our plain-English summary of Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice, by L. Pascolo, G. Zabucchi, A. Gianoncelli, et al (Toxicology Letters, 2016).

The study

• Authors: L. Pascolo, G. Zabucchi, A. Gianoncelli, G. Kourousias, E. Trevisan, E. Pascotto, C. Casarsa, C. Ryan, M. Lucattelli, G. Lungarella, E. Cavarra, B. Bartalesi, M. Zweyer, F. Cammisuli, M. Melato, V. Borelli
• Journal: Toxicology Letters
• Published: 2016;241:111-20
• Cited by: 13 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (μ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, μ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions.

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In plain English

This was an animal experiment in mice. Male C57Bl/6 mice had UICC crocidolite asbestos placed directly into the windpipe. Control mice received saline alone. Their lungs were examined one month later. The authors used synchrotron X-ray fluorescence microscopy at two facilities to map calcium, iron, zinc and other elements in lung sections. Conventional tissue stains showed only a few weakly positive cells for iron and calcium, and no asbestos bodies were seen. In inflamed lung regions of exposed mice, average calcium was around 5 to 6 times higher than in controls and iron more than 10 times higher, while zinc was similar. In 18 small areas chosen as likely fibers, calcium and iron levels were positively correlated (Spearman's coefficient 0.7276, p less than 0.001). Zinc was not correlated with either. Silicon outlined the fibers, and magnesium appeared around them. The authors hypothesized that the first steps of fiber mineralization inside macrophages could involve simultaneous iron and calcium deposition, leading to oxidative stress, chronic inflammation, fibrosis and hyperplasia. They noted that the method underestimates the iron content of single fibers.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal experiment: lung sections from asbestos-exposed and control mice analysed by synchrotron X-ray fluorescence microscopy at two facilities, one using hard (high-energy) X-rays and one using soft (low-energy) X-rays
• Who was studied: Pathogen-free male C57Bl/6 mice aged 6 to 8 weeks (Charles River Laboratories, Calco, Italy), given crocidolite or saline (controls), examined one month later. The number of mice is not stated in this article.
• Exposure: A single intratracheal instillation of UICC reference crocidolite asbestos in sterile saline. The dose is printed as '100 mg', most likely with a micro sign lost in conversion. Control mice received saline alone.
• Main results:
  • In exposed mice, conventional staining (Perls' blue for iron, Von Kossa for calcium) showed only a few weakly positive cells, very likely macrophages
  • No asbestos bodies were observed in the exposed mice
  • In the pure crocidolite standard, iron in single fibers ranged from hundreds to around 25,000 ppm (0.01 to 2.5% by weight). No calcium or zinc was detected in the fibers.
  • In control mouse lung, zinc ranged from 20 to 50 ppm, calcium rarely exceeded 80 ppm, and average iron was around 450 ppm
  • In inflamed lung regions of exposed mice, iron in small fiber-shaped areas reached 15,000 ppm
  • Compared with controls, average calcium was around 5 to 6 times higher in inflamed exposed lung regions, while zinc was similar
  • Iron was more than 10 times higher in inflamed, asbestos-containing lung regions than in controls
  • Across 18 selected possible fiber areas, calcium and iron were positively correlated (Spearman's correlation coefficient 0.7276, p less than 0.001). There was no correlation of calcium with zinc or of iron with zinc.
  • Under soft X-rays, silicon outlined the asbestos fibers. Magnesium was mainly present around the fibers, and iron was also seen just around the fibers.
• The authors' conclusion: The authors hypothesized that the first steps of the bio-mineralization of asbestos fibers inside macrophages could involve simultaneous iron and calcium deposition on the fibers, generating oxidative stress and resulting in chronic inflammation, tissue fibrosis and hyperplasia. They also concluded that, because calcium and iron deposits were proportionally related, they should be triggered by related processes, very likely set off by the interaction between asbestos and cells.
• Limitations the authors state:
  • The hard X-ray system underestimates the iron concentration on single fibers, because the beam spot is larger than the fiber diameter
  • Quantifying elements in the deposited fiber standard may be unreliable, because the real thickness of the fiber aggregates is undefined
  • The low-energy elemental maps give only qualitative and semi-quantitative measurements
  • The authors do not know whether long fibers lying free in the tissue are also surrounded by tissue iron

Topics

The National Library of Medicine indexes this study under subjects our site covers: Asbestosis, Crocidolite.

Medical terms in this study, explained in our Lexicon: Synchrotron X-ray Microscopy, Crocidolite, Alveolar Macrophage, Deposition.

Related studies

• Synchrotron soft X-ray imaging and fluorescence microscopy reveal novel features of asbestos body morphology and composition in human lung tissues — L. Pascolo, A. Gianoncelli, et al. (2011)
• Iron-related toxicity of single-walled carbon nanotubes and crocidolite fibres in human mesothelial cells investigated by Synchrotron XRF microscopy — F. Cammisuli, S. Giordani, et al. (2018)
• The crocidolite fibres interaction with human mesothelial cells as investigated by combining electron microscopy, atomic force and scanning near-field optical microscopy — L. Andolfi, E. Trevisan, et al. (2013)
• Histopathological data of iron and calcium in the mouse lung after asbestos exposure — E. Trevisan, G. Zabucchi, et al. (2016)
• Focused X-Ray Histological Analyses to Reveal Asbestos Fibers and Bodies in Lungs and Pleura of Asbestos-Exposed Subjects — L. Pascolo, A. Gianoncelli, et al. (2016)
• In vivo accumulation of iron on crocidolite is associated with decrements in oxidant generation by the fiber — A.J. Ghio, A. LeFurgey, et al. (1997)

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