Alessandro Croce

Continuous Exposure to Chrysotile Asbestos Can Cause Transformation of Human Mesothelial Cells via HMGB1 and TNF-α Signaling

Malignant mesothelioma is strongly associated with asbestos exposure. Among asbestos fibers, crocidolite is considered the most and chrysotile the least oncogenic. Chrysotile accounts for more than 90% of the asbestos used worldwide, but its capacity to induce malignant mesothelioma is still debated. We found that chrysotile and crocidolite exposures have similar effects on human mesothelial cells. Morphological and molecular alterations suggestive of epithelial-mesenchymal transition, such as E-cadherin down-regulation and β-catenin phosphorylation followed by nuclear translocation, were induced by both chrysotile and crocidolite. Gene expression profiling revealed high-mobility group box-1 protein (HMGB1) as a key regulator of the transcriptional alterations induced by both types of asbestos. Crocidolite and chrysotile induced differential expression of 438 out of 28,869 genes interrogated by oligonucleotide microarrays. Out of these 438 genes, 57 were associated with inflammatory and immune response and cancer, and 14 were HMGB1 targeted genes. Crocidolite-induced gene alterations were sustained, whereas chrysotile-induced gene alterations returned to background levels within 5 weeks. Similarly, HMGB1 release in vivo progressively increased for 10 or more weeks after crocidolite exposure, but returned to background levels within 8 weeks after chrysotile exposure. Continuous administration of chrysotile was required for sustained high serum levels of HMGB1. These data support the hypothesis that differences in biopersistence influence the biological activities of these two asbestos fibers.

Asbestos and other fibrous minerals contained in the serpentinites of the Gimigliano-Mount Reventino Unit (Calabria, S-Italy)

Serpentinites are metamorphic rocks with good technological properties and valuable ornamental characteristics, which have been exploited since ancient times. Actually, their use is limited and monitored in several countries worldwide because they can contain fibrous asbestos minerals that may be carcinogenic. Furthermore, certain types of fibrous minerals can be confused with asbestos, and must therefore be carefully investigated. We have investigated the possible presence of the asbestos and non-asbestos fibrous phases contained in serpentinitic rocks in a meta-ophiolitic sequence from the Gimigliano-Mount Reventino Unit (Southern Italy), which had not been previously assessed. The detection and quantification of asbestos and the correct distinction of the fibrous non-asbestos minerals are very important not only from a scientific point of view, but also from a legislative one. This is especially the case for the administrative agencies that have to take decisions with regards to the implementation of public and occupational health protection measures (e.g., in road yards and quarry excavations). As a consequence of this, serpentinitic rock samples have been characterized in detail through X-ray powder diffraction, scanning and transmission electron microscopy combined with energy-dispersive spectrometry, analytical electron microscopy (SEM–EDS and TEM–AEM), differential scanning calorimetry, thermogravimetry and micro-Raman spectroscopy. Two kinds of asbestos and four kinds of non-asbestos fibrous silicates have been detected in the examined samples. In order of decreasing abundance these are polygonal serpentine, chrysotile, fibrous antigorite, tremolite, gedrite and magnesiohornblende. The size, morphology, crystallinity and chemical composition of the fibres were also discussed, in the light of the possible role these properties could play in the carcinogenic effect on human health.

Crystal chemistry of the high temperature product of transformation of cement-asbestos.

In this work, the high-temperature inertization product of a representative batch of samples of cement-asbestos (CA) from different localities in Italy have been characterized with a multidisciplinary approach. All the raw CA samples were heated at 1200°C for 15 min. After firing, they underwent a series of solid state reactions leading to global structural changes of the matrix. Effects of annealing time and temperature on the crystallization kinetics were thoroughly investigated. Both factors acted in favour of equilibrium. Three classes of CA were identified with the aid of phase diagrams and of specific plots relating chemical and mineralogical parameters. This result was considered of importance in view of the potential use of transformed cement-asbestos as a secondary raw material. In principle, the content of CA packages removed from the environment and their corresponding heat-treated products can be classified simply using XRF. This method allows for the selection of appropriate fractions in function of the most suitable recycling solution adopted. Samples belonging to the class called larnite-rich, turned out to be of great interest as possible candidate for substituting a fraction of cement in many building materials and innovative green cement productions.

Study of inorganic particles, fibers, and asbestos bodies by variable pressure scanning electron microscopy with annexed energy dispersive spectroscopy and micro-Raman spectroscopy in thin sections of lung and pleural plaque.

In a previous work it has been demonstrated that micro-Raman spectroscopy is a technique able to recognize crystalline phases on untreated samples. In that case, inorganic particles and uncoated fibers from bronchoalveolar lavage (BAL) of a patient affected by pneumoconiosis were identified and characterized. In this work the technique is applied to asbestos bodies, that is, to coated fibers, and on crystallizations and fibrous phases observed in the plural plaque from patients affected by mesothelioma. From the Raman analysis the abundant fibrous material observed in the pleural area is talc, whereas rounded grains in the pleural tissue show the Raman spectrum of apatite, a calcium phosphate mineral particular to bones. In the pulmonary tissue many asbestos bodies, consisting of the incorporated fibers coated by iron-rich proteins, were observed. Under the 632.8 nm laser beam of the spectrometer, photo-crystallization of hematite in the iron-rich material forming the asbestos bodies can be proposed by the changes in the Raman spectra acquired during subsequent acquisitions. Nevertheless, the identification of the mineral phase constituting the incorporated fiber was possible by analyzing the Raman spectra; the results were confirmed by variable pressure scanning electron microscopy with annexed energy dispersive spectroscopy (VP-SEM-EDS) analyses.

Numerous Iron-Rich Particles Lie on the Surface of Erionite Fibers from Rome (Oregon, USA) and Karlik (Cappadocia, Turkey).

Erionite samples from Rome, Oregon (USA) and Karlik, Cappadocia (Turkey) were analyzed by environmental scanning electron microscopy (E-SEM) coupled with energy-dispersive spectroscopy (EDS) to verify the chemical composition of this mineral phase, and the presence of iron in particular. By means of backscattered electron images, a large number of particles/grains were observed on the surface of the erionite fibers from both locations. The particles were found to be micrometric on samples from Rome and submicrometric on samples from Karlik, and always lighter than the hosting crystal in appearance. In different areas of the same fiber or bundle of fibers, several EDS spectra were recorded. Iron was detected only when a light particle was lying in the path of the electron beam. Iron was never identified in the EDS spectra acquired on the flat erionite surface. The results from E-SEM/EDS were confirmed by micro-Raman spectroscopy, showing bands ascribing to hematite—Fe2O3, goethite—FeO(OH), or jarosite—KFe3(3+)(SO4)2(OH)6 when the laser beam was addressed on the light particles observed on the fiber surface. The evidence that iron is on the surface of erionite fibers, rather than being part of the crystalline structure, may be relevant for the carcinogenic potential of these fibers.

Stability of mineral fibres in contact with human cell cultures. An in situ μXANES, μXRD and XRF iron mapping study

Relevant mineral fibres of social and economic importance (chrysotile UICC, crocidolite UICC and a fibrous erionite from Jersey, Nevada, USA) were put in contact with cultured diploid human non-tumorigenic bronchial epithelial (Beas2B) and pleural transformed mesothelial (MeT5A) cells to test their cytotoxicity. Slides of each sample at different contact times up to 96xa0h were studied in situ using synchrotron XRF, μ-XRD and μ-XAS (I18 beamline, Diamond Light Source, UK) and TEM investigations. XRF maps of samples treated for 96xa0h evidenced that iron is still present within the chrysotile and crocidolite fibres and retained at the surface of the erionite fibres, indicating its null to minor mobilization in contact with cell media; this picture was confirmed by the results of XANES pre-edge analyses. μ-XRD and TEM data indicate greater morphological and crystallinity modifications occurring in chrysotile, whereas crocidolite and erionite show to be resistant in the biological environment. The contact of chrysotile with the cell cultures seems to lead to earlier amorphization, interpreted as the first dissolution step of these fibres. The formation of such silica-rich fibre skeleton may prompt the production of HO in synergy with surface iron species and could indicate that chrysotile may be much more reactive and cytotoxic inxa0vitro in the (very) short term whereas the activity of crocidolite and erionite would be much more sluggish but persistent in the long term.

The concept of ‘end of waste’ and recycling of hazardous materials: in depth characterization of the product of thermal transformation of cement-asbestos

Abstract Selected samples of asbestos-containing material (ACM) with different Ca/Si ratios have been treated thermally at 1200ºC for 15 min to obtain an ‘end of waste geo-inspired material’. Before and after treatment, micro-Raman spectroscopy allowed the investigation of both powdered and massive samples by directing the laser beam onto crystals with elongated morphology, thin fibres and the matrix. In the raw samples, chrysotile and/or crocidolite were detected. After the thermal treatment, no asbestos phases were identified in the Raman spectra collected on fibrous or fibre-like morphologies. The scanning electron microscopy/energy dispersive spectroscopy investigations confirmed the onset of a pseudomorphic process during annealing, leading to the complete transformation of asbestos minerals into non-hazardous magnesium or calcium magnesium silicates such as forsterite, monticellite, åkermanite and merwinite. The identification of such mineral assemblages was inspired by the close inspection of a natural counterpart, the high-temperature contact metamorphic imprint due to the intrusion of a sill into carbonate rocks. The process turned out to occur largely at the solid state and involved substantial mobilization of Ca and Mg to form a spinel phase (namely MgFe2O4) which was recognized in the matrix and within, or close to elongated morphologies.

Preliminary results of the spectroscopic and structural characterization of mesothelioma inducing crocidolite fibers injected in mice

To investigate the structure and microstructure changes of crocidolite asbestos incorporated in biological tissues, fibers of this mineral phase were injected in mice peritoneum. Histological sections of different organs of mice developing mesothelioma after crocidolite inoculation were prepared and analysed by optical microscopy. The tumours developed within the peritoneal cavity, wrapped around the surrounding organs. Many fibres were observed in the fibrotic areas of the peritoneum lining pancreas and spleen. The raw fibers before inoculation and those embedded in mice tissues were characterized using Micro-Raman spectroscopy and in situ synchrotron X-ray diffraction at ESRF- Grenoble. Preliminary results indicate shifts of some bands on the Raman spectra and enlargement of the X-Ray diffraction peaks of the fibers localized in the mice tissue sections. A preliminary structural picture of the fibers incorporated in mice tissues suggests inter-crystalline migration of the iron and sodium ions.

Asbestos fibers in the gallbladder of patients affected by benign biliary tract diseases.

Purpose This exploratory study aimed to evaluate the presence of asbestos fibers in the biliary tract of patients living in an asbestos-polluted area using scanning electron microscopy. Methods Thin gallbladder sections were obtained from five patients who were operated on for gallbladder stones and the bile fluid of one of the patients was analyzed using variable-pressure scanning electron microscopy coupled with energy-dispersive spectroscopy. All patients were from Casale Monferrato, Italy, a well-known asbestos-polluted city, where the Eternit factory had operated since the beginning of the century until 1985. Results All the inorganic phases found in the gallbladder were analyzed for morphology and chemistry. Fibers and particles consistent with minerals defined by law as ‘asbestos’ were detected in three out of five patients. Conclusion These findings suggest that asbestos fibers can be found in the gallbladder of patients exposed to asbestos, although how they reach the biliary tract remains unknown. Further studies to confirm these results are under way.

Environmental Scanning Electron Microscopy Technique to Identify Asbestos Phases Inside Ferruginous Bodies

Ferruginous bodies observed in lungs of patients affected by mesothelioma, asbestosis, and pulmonary carcinoma are important to relate the illness to exposure, environmental or occupational, to asbestos. Identification of the inorganic phase constituting the core of the ferruginous bodies, formed around asbestos but also around phases different from asbestos, is essential for legal purposes. Environmental scanning electron microscopy/energy dispersive spectroscopy was used to identify the fibrous mineral phase in the core of ferruginous bodies observed directly in thin sections of tissue, without digestion of the biological matrix. Spectra were taken with sequential analyses along a line crossing the core of the ferruginous bodies. By comparing the spectra taken near to and far from the core, the chemical elements that make up the core could be identified.