Silvana Capella

Effects of asbestiform antigorite on human alveolar epithelial A549 cells: A morphological and immunohistochemical study

The purpose of the study was to investigate the biological risk of asbestiform antigorite, which is a fibrous variety of antigorite, one of the natural mineral fibres of the serpentine group to which asbestos chrysotile belongs. Asbestiform antigorite is very abundant and commonly found associated with asbestos chrysotile in serpentinites, a kind of rock outcropping present in many geographical locations worldwide. In this study we evaluated the morphological, immunohistochemical and functional effects of antigorite fibres in alveolar epithelial cancer cells (A549), a standardized human cell line currently used as a model to study cytotoxicity induced by pharmacological agents. The antigorite fibres were identified and characterized morphologically and chemically by X-ray powder diffractometry, transmission and scanning electron microscopy, both with annexed energy dispersive spectrometry. The effects of 50 microg/ml of antigorite in A549 lung cells treated at 24 and 48 h resulted in increased synthesis of VEGF, Cdc42 and beta-catenin that represent potential risks for cancer development. Phalloidin labelling showed an irregular distribution of filamentous actin resulting from antigorite contact. Our studies indicate potential cellular toxicity of antigorite in vivo, providing the opportunity to elucidate the effect of asbestos on cancer induction and possible modes of therapy.

Cytotoxicity induced by exposure to natural and synthetic tremolite asbestos: an in vitro pilot study.

Mineral fibers are potential carcinogens to humans. In order to help clarify the etiology of the pathological effects of asbestos, cellular reactions to natural and synthetic asbestos fibers were compared using a lung alveolar cancer cell line (A549 epithelial cells), considered the first target of inhaled micro-environmental contaminants. Natural asbestos tremolite (NAT) fibers were collected from rocks in NW Italy. Synthetic asbestos tremolite (SAT) was iron-free and therefore considered as standard tremolite. Both fibers, subjected to mineralogical characterization by X-ray powder diffractometry, electron microscopy and energy dispersive spectrometry, fell within the definition of respirable and potentially carcinogenic fibers. Several signs of functional and structural cell damage were found after treatment with both fibers, documented by viability, motility, and morphological perturbations. Phalloidin labeling showed irregular distribution of cytoskeletal F-actin, whereas immunohistochemical investigations showed abnormal expression of VEGF, Cdc42, β-catenin, assessed as risks indicators for cancer development. Both fibers caused significant loss of viability, even compared to UICC crocidolite, but, while SAT fibers exerted a more direct cytotoxic effect, survival of damaged cells expressing high VEGF levels was detected after NAT contact. This in vitro pilot study outlines potential health risks of NAT fibers in vivo related to their iron content, which could trigger signaling networks connected with cell proliferation and neoplastic transformation.

New insights on the biomineralisation process developing in human lungs around inhaled asbestos fibres

Once penetrated into the lungs of exposed people, asbestos induces an in vivo biomineralisation process that leads to the formation of a ferruginous coating embedding the fibres. The ensemble of the fibre and the coating is referred to as asbestos body and is believed to be responsible for the high toxicological outcome of asbestos. Lung tissue of two individuals subjected to prolonged occupational exposure to crocidolite asbestos was investigated using synchrotron radiation micro-probe tools. The distribution of K and of elements heavier than Fe (Zn, Cu, As, and Ba) in the asbestos bodies was observed for the first time. Elemental quantification, also reported for the first time, confirmed that the coating is highly enriched in Fe (~20% w/w), and x-ray absorption spectroscopy indicated that Fe is in the 3+ oxidation state and that it is present in the form of ferritin or hemosiderin. Comparison of the results obtained studying the asbestos bodies upon removing the biological tissue by chemical digestion and those embedded in histological sections, allowed unambiguously distinguishing the composition of the asbestos bodies, and understanding to what extent the digestion procedure altered their chemical composition. A speculative model is proposed to explain the observed distribution of Fe.

Environmental exposure to asbestos and other inorganic fibres using animal lung model.

Professional exposure to asbestos fibres is widely recognized as very dangerous to human health and for this reason many countries have banned their commercial uses. People, nevertheless, continue to be exposed to low dose of asbestos from natural and anthropogenic sources still in loco, for which the potential hazard is unknown. The aim of this research is to assess environmental exposure in an area with outcropping serpentinite rocks, which bear asbestos mineralizations, using sentinel animals which are a non-experimental animal model. We studied the burden of inorganic fibres in cattle lungs which come from two areas in Italys Western Alps bearing serpentinitic outcrops: Susa Valley with a heavy anthropization and Lanzo Valleys, with a minor human impact. The identification and quantification of inorganic fibres were performed by Scanning Electron Microscope (SEM) and Energy Dispersive Spectrometer (EDS). In comparison to humans, studies of animals have some advantages, such as no occupational exposure or history of smoking and, in the case of cattle, a sedentary life restricted to one region. Results spotlight that over than 35% of inorganic fibres found both in Susa and Lanzo valleys, belong to asbestos mineralogical species (asbestos tremolite/actinolite, chrysotile s.s., asbestos grunerite, crocidolite). We also observed a higher concentration of artificial fibrous products in Susa samples showing a correlation with the level of anthropization. These results confirm that sentinel animals are an excellent model to assess breathable environmental background because it is possible to eliminate some variables, such as unknown occupational exposure.

In vitro study of biofunctional indicators after exposure to asbestos-like fluoro-edenite fibres.

The in vitro biological response to fluoro-edenite (FE) fibres, an asbestos-like amphibole, was evaluated in lung alveolar epithelial A549, mesothelial MeT-5A and monocyte-macrophage J774 cell lines. The mineral has been found in the vicinity of the town of Biancavilla (Catania, Sicily), where an abnormal incidence of mesothelioma has been documented. Cell motility, distribution of polymerized actin, and synthesis of vascular endothelial growth factor (VEGF) and of beta-catenin, critical parameters for tumour development, progression and survival, were investigated in A549 and MeT-5A cells exposed to 50 microg/ml FE fibres for 24 hr and 48 hr. The levels of cyclooxygenase (COX-2) and prostaglandin (PGE2), two molecules involved in cancer pathogenesis by affecting mitogenesis, cell adhesion, immune surveillance and apoptosis, were measured in J774 cells treated with FE fibres under the same experimental conditions. Finally, FE fibres were studied by SEM and EDS analysis to investigate their chemical composition. Exposure of A549 and MeT-5A cells to FE fibres affected differentially phalloidin-stained cytoplasmic F-actin networks, cell motility and VEGF and beta-catenin expression according to the different sensitivity of the two cell lines. In J774 cells it induced a significant increase in COX-2 expression, as assessed by Western blot analysis, and in the concentration of PGE2, measured in culture media by ELISA. SEM-EDS investigations demonstrated two types of FE fibres, edenite and fluoro-edenite, differing in chemical composition and both recognizable as calcic amphiboles. Fibre width ranged from less than 1 microm (prevalently 0.5 microm) to 2-3 microm (edenite) up to several microm (fluoro-edenite); length ranged from about 6 to 80 microm (edenite) up to some hundred microm (fluoro-edenite). Results provide convincing evidence that FE fibres are capable of inducing in vitro functional modifications in a number of parameters with crucial roles in cancer development and progression. Inhaled FE fibres have the potential to induce mesothelioma, even though their ability to penetrate lung alveoli depends on their aerodynamic diameter.

The wild rat as sentinel animal in the environmental risk assessment of asbestos pollution: A pilot study

Asbestos has been banned in many countries, including Italy. However, sources of exposure may still exist, due to asbestos in-situ or past disposal of asbestos-containing waste. In an urban area with past high environmental exposure, like Casale Monferrato, the lung fiber burden in sentinel animals may be useful to identify such sources. A pilot study was conducted to assess the feasibility of its determination in wild rats, a suitable sentinel species never used before for environmental lung asbestos fiber burden studies. Within the framework of pest control campaigns, 11 adult animals from 3 sites in the urban area of Casale Monferrato and 3 control rats from a different, unexposed town were captured. Further, 3 positive and 3 negative control lung samples were obtained from laboratories involved in breeding programs and conducting experimental studies on rats. Tissue fiber concentration was measured by scanning electron microscopy with energy dispersive spectrometry. Asbestos (chrysotile and crocidolite) was identified in the lungs from rats from Casale Monferrato, but not in control rats and in negative control lung samples. Asbestos grunerite at high concentration was found in positive control lung samples. Measurement of the lung fiber burden in wild rats has proved feasible: it was possible not only to detect, but also to characterize asbestos fibers both qualitatively and quantitatively. The pilot study provides the rationale for using wild rats as sentinels of the soil contamination level in Casale Monferrato, to identify areas with the possible presence of previously unrecognized asbestos sources.

“Rinse and trickle”: a protocol for TEM preparation and investigation of inorganic fibers from biological material

Abstract The purpose of this work is to define a sample preparation protocol that allows inorganic fibers and particulate matter extracted from different biological samples to be characterized morphologically, crystallographically and chemically by transmission electron microscopy-energy dispersive spectroscopy (TEM-EDS). The method does not damage or create artifacts through chemical attacks of the target material. A fairly rapid specimen preparation is applied with the aim of performing as few steps as possible to transfer the withdrawn inorganic matter onto the TEM grid. The biological sample is previously digested chemically by NaClO. The salt is then removed through a series of centrifugation and rinse cycles in deionized water, thus drastically reducing the digestive power of the NaClO and concentrating the fibers for TEM analysis. The concept of equivalent hydrodynamic diameter is introduced to calculate the settling velocity during the centrifugation cycles. This technique is applicable to lung tissues and can be extended to a wide range of organic materials. The procedure does not appear to cause morphological damage to the fibers or modify their chemistry or degree of crystallinity. The extrapolated data can be used in interdisciplinary studies to understand the pathological effects caused by inorganic materials.

Urinary asbestos fibers and inorganic particles in past asbestos workers

ABSTRACT To assess the validity of the procedure as a test of asbestos exposure, we compared urinary asbestos fibers with occupational and environmental exposure data in a random sample of 48 subjects with high past asbestos exposure. Occupational and environmental exposure was estimated on questionnaire, pleural plaques were diagnosed with computed tomography, and inorganic fibers and particles were identified by scanning electron microscope with an energy-dispersive spectrometry. Few urinary asbestos fibers (in 15% of workers and 17% of cases with pleural plaques) and high amount of urinary silicate (particularly nonfibrous particles) were detected. Asbestos undergoes dissolution in lung tissues, but the secondary minerals are largely unknown. These materials, possibly nonfibrous silicates or metals, could be excreted with urine. Therefore, another study including a control group is warranted to discriminate the occupational origin of minerals in the urine.

Diagnosis of Asbestos-Related Diseases: The Mineralogist and Pathologist's Role in Medicolegal Field.

AbstractBecause asbestos diseases represent a complex pattern of legal, social, and political issue, the involvement of the mineralogist and pathologist for a multidisciplinary assessment of its diagnosis helps investigate the relationship between mesothelioma or lung cancer and occupational or environmental asbestos exposure.In the present study, we consider the concentrations of asbestos bodies (ABs) detected by optical microscopy (OM) and scanning electron microscopy (SEM) and the burden of different kinds of mineral fibers (among which is asbestos) identified by SEM combined with an energy dispersive spectrometer (EDS), in 10 lung tissue samples of subjects with occupational and nonoccupational exposure to asbestos.In all subjects with occupational exposure to asbestos, more than 1000 ABs per gram of dry weight were detected both with OM and SEM; this concentration is internationally accepted as suggesting high probability of past occupational exposure to asbestos.In 9 lung samples of the 10 investigated by SEM-EDS different inorganic fibers were found. Asbestos fibers have been identified too, and more than 100,000 fibers per gram of dry weight were detected in subjects with occupational exposure; this concentration is internationally accepted as suggesting high probability of past occupational exposure to asbestos.Instead, when the ABs burden is low or moderate (such as in subjects with absent or probable asbestos exposure), the correlation between ABs concentration determined by OM and those determined by SEM is lost. Therefore, when the ABs value in OM is borderline, the SEM investigation became essential. Furthermore, the mineralogical analysis by SEM-EDS (identification and quantification of inorganic fibers in general and asbestos in particular) of the fibers detected in the lung tissues is very useful, if not necessary, to complete the pathological diagnosis of asbestos-related malignancies in medicolegal field.

Asbestos fibre burden in gallbladder: A case study

The methods conventionally used to determine the burden of asbestos fibres inhaled/incorporated in lung require chemical digestion of the biological matrix before counting/characterising the inorganic fibrous phases under scanning electron microscopy and energy dispersive spectroscopy (SEM/EDS). Asbestos fibres can also be present in extra-pulmonary organs, and we set out to quantify the fibres in gallbladder. Although the standardised procedure requires approximately 5 × 10-1 g of wet tissue, this amount of tissue is not always available. We applied the procedure on about 9 × 10-4 g of gallbladder from a patient with known environmental and workplace exposure to asbestos. The patient died of malignant pleural mesothelioma and was also affected by severe bile-tract problems. The traditional procedure of digesting tissue samples in NaClO and filtering the resulting suspension was carried out. The filter was then examined under SEM/EDS using two methods 1. following the standardised procedure to assess the fibre burden in lung by investigating only 2 mm2 of the filter (660 microscopic fields), and 2. analysing all the microscopic fields in one-quarter of the filter (about 82 mm2). In parallel, histological sections (prepared in the usual way for medical diagnosis) were analysed without digestion or manipulation of the sample using variable pressure SEM/EDS. The fibre counts obtained using the two methods were of the same order of magnitude, i.e., ∼105 fibres/g of wet tissue. We showed that the counting of fibres in human tissue may be successfully carried out even when a limited amount of tissue is available. We also found that, when exposure to asbestos is considerable, the number of asbestos fibres accumulating in the gallbladder may be significant.