Exposure to asbestos and asbestos like fibers may induce chronic respiratory diseases. Fibrous erionite, a zeolite, is a strong mutagen, considered more carcinogenic than asbestos fibers in man and rodents, moreover we have found that even fibers such as epsomite can be dangerous. The principal aim of this reseach is to characterize the nature of fibers through a morphological and chemical point of view and then, their interaction with a cell model in vitro by means of ultrastructural analyses. Different fibers, including minerals already characterized as asbestos fibers (chrysotile, tremolite and amosite), a fibrous silicate not characterized before, an asbeferrite and an erionite one, have been analyzed by means of environmental scanning electron microscopy. Chemical analysis (EDXS Analysis) has been performed on sample aliquots without any preparations under low vacuum conditions (0,80 mbar). Successively, the interaction between fibers and U937 cell membrane has been also investigated. Chemical analysis by inductively coupled plasma-mass spectrometry has been used to determine the trace element composition. The human monocyte U937 cell line, derived from malignant cells obtained from the pleural effusion of a patient with histiocytic lymphoma, has been chosen to verify the internalization and toxicity of erionite into cells, different fiber concentration have been tested on U937 cells (5, 20 and 100nm) with different times of incubation (24, 36 and 48 hours). Cell response after fiber exposure has been investigated by a transmission electron microscope (TEM). Successively, the interaction between selected fibers (epsomite) and macrophages has been also investigated. The human monocyte U937 cell line has been treated with PMA, a phorbol ester used to convert monocytes into macrophages. Then, cells have been incubated with epsomite fibers for 24 and 48 hours at various dosages and processed for ultrastructural analyses. Cell response after fiber exposure has been investigated by a transmission electron microscope (TEM). Ultrastructural observations revealed U937 monocytes ability to internalize fibers, which appear both in the cytoplasm and in the nucleus, depending on the concentration and of exposure time. The literature describes a relevant role of macrophages in the antitumor response induced by exposure to asbestos fibers. However, an abnormal activation or an impairment of these cells leads to release of growth factors that can promote tumor growth and development. The potential toxicity of epsomite, firstly characterized through ESEM analysis, has been investigated by means of ultrastructural approaches in cultured macrophages obtained from U937 monocytes. TEM observations revealed cell ability to internalize epsomite fibers, which can be found near to the cell surface, inside of membrane invaginations or of the cytoplasm, where some fibers appear internalized into lysomal granules. Increasing epsomite dose and time to 48h, large fibers can be observed in the cytoplasm, where cannot be correctly digested. This latter event leads to an impairment of lysosomal system, following by mitochondrial damage, and finally cell death. The epsomite exposure in converted U937 macrophages-like induces cytotoxicity which appears to be essential for the pathogenesis of fiber-induced-inflammatory diseases. The dimensional analysis of the fibers was performed by adapting an official method, used for the calculation of the average diameter of the artificial glass fibers (Rep. Atti n. 211/CSR del 10/11/2016), as the average diameter is a parameter that can be indicative of the danger of the fiber. The adapted protocol was used to determine the mean diameter of the chrysotile, asbeferrite, epsomite and amosite fibers. In the case of erionite, asbeferrite, epsomite and asbestos (chrysotile, tremolite, amosite) we confirmed the expected distribution of the fiber size and of the mean diameter because of their potential carcinogenic risk. Erionite, chrysotile and tremolite are characterized by an extremely fibrous habit, having a diameter about 1 μm and variables lengths, with fibrils of about 0.1 μm diameter. The very small size could favor the deep penetration in the biological system. The morphological and chemical study of asbeferrite, silicate with the presence of iron and magnesium, confirmed the expected distribution of the fiber size and of the mean diameter because of their potential carcinogenic risk. Asbeferrite is characterized by an extremely fibrous habit, having a diameter about 1 μm and variables lengths, with fibrils of about 0.1 μm diameter. The very small size could favor the deep penetration in the biological system, topic that will be the subject of further investigations. The epsomite fibers, a magnesium sulphate with formula MgSO4, have larger diameters (about 4-5 μm), but is characterized by a greater surface reactivity in surface cracking phenomena, and this could be related to the biosolubilization of the fibers in the cellular structures. The formation of fractured crack surfaces could probably be related to hydration reactions with consequent structural variations. The SEM technique is confirmed as a useful tool for the characterization of fibrous material, the next objectives of the project will be the implementation of measures on mineral and synthetic fibers with possible toxic effects, for the purpose of the drafting of a protocol for the determination of the average diameter of the fibers used in the interaction with the cellular matrix and the deepening of the consequent interactions between fibers and cellular models according to the morphological and chemical characteristics. Furthermore, the SEM analysis allows to obtain a protocol of simple implementation for the dimensional characterization of the fibers, a useful protocol when a first and rapid criterion is needed to establish the potential danger of a mineral fiber not yet characterized.

Analisi morfofunzionali dell'interazione tra fibre minerali micro e nanostrutturate con matrici cellulari

Pignataro, Giuseppe
2021

Abstract

Exposure to asbestos and asbestos like fibers may induce chronic respiratory diseases. Fibrous erionite, a zeolite, is a strong mutagen, considered more carcinogenic than asbestos fibers in man and rodents, moreover we have found that even fibers such as epsomite can be dangerous. The principal aim of this reseach is to characterize the nature of fibers through a morphological and chemical point of view and then, their interaction with a cell model in vitro by means of ultrastructural analyses. Different fibers, including minerals already characterized as asbestos fibers (chrysotile, tremolite and amosite), a fibrous silicate not characterized before, an asbeferrite and an erionite one, have been analyzed by means of environmental scanning electron microscopy. Chemical analysis (EDXS Analysis) has been performed on sample aliquots without any preparations under low vacuum conditions (0,80 mbar). Successively, the interaction between fibers and U937 cell membrane has been also investigated. Chemical analysis by inductively coupled plasma-mass spectrometry has been used to determine the trace element composition. The human monocyte U937 cell line, derived from malignant cells obtained from the pleural effusion of a patient with histiocytic lymphoma, has been chosen to verify the internalization and toxicity of erionite into cells, different fiber concentration have been tested on U937 cells (5, 20 and 100nm) with different times of incubation (24, 36 and 48 hours). Cell response after fiber exposure has been investigated by a transmission electron microscope (TEM). Successively, the interaction between selected fibers (epsomite) and macrophages has been also investigated. The human monocyte U937 cell line has been treated with PMA, a phorbol ester used to convert monocytes into macrophages. Then, cells have been incubated with epsomite fibers for 24 and 48 hours at various dosages and processed for ultrastructural analyses. Cell response after fiber exposure has been investigated by a transmission electron microscope (TEM). Ultrastructural observations revealed U937 monocytes ability to internalize fibers, which appear both in the cytoplasm and in the nucleus, depending on the concentration and of exposure time. The literature describes a relevant role of macrophages in the antitumor response induced by exposure to asbestos fibers. However, an abnormal activation or an impairment of these cells leads to release of growth factors that can promote tumor growth and development. The potential toxicity of epsomite, firstly characterized through ESEM analysis, has been investigated by means of ultrastructural approaches in cultured macrophages obtained from U937 monocytes. TEM observations revealed cell ability to internalize epsomite fibers, which can be found near to the cell surface, inside of membrane invaginations or of the cytoplasm, where some fibers appear internalized into lysomal granules. Increasing epsomite dose and time to 48h, large fibers can be observed in the cytoplasm, where cannot be correctly digested. This latter event leads to an impairment of lysosomal system, following by mitochondrial damage, and finally cell death. The epsomite exposure in converted U937 macrophages-like induces cytotoxicity which appears to be essential for the pathogenesis of fiber-induced-inflammatory diseases. The dimensional analysis of the fibers was performed by adapting an official method, used for the calculation of the average diameter of the artificial glass fibers (Rep. Atti n. 211/CSR del 10/11/2016), as the average diameter is a parameter that can be indicative of the danger of the fiber. The adapted protocol was used to determine the mean diameter of the chrysotile, asbeferrite, epsomite and amosite fibers. In the case of erionite, asbeferrite, epsomite and asbestos (chrysotile, tremolite, amosite) we confirmed the expected distribution of the fiber size and of the mean diameter because of their potential carcinogenic risk. Erionite, chrysotile and tremolite are characterized by an extremely fibrous habit, having a diameter about 1 μm and variables lengths, with fibrils of about 0.1 μm diameter. The very small size could favor the deep penetration in the biological system. The morphological and chemical study of asbeferrite, silicate with the presence of iron and magnesium, confirmed the expected distribution of the fiber size and of the mean diameter because of their potential carcinogenic risk. Asbeferrite is characterized by an extremely fibrous habit, having a diameter about 1 μm and variables lengths, with fibrils of about 0.1 μm diameter. The very small size could favor the deep penetration in the biological system, topic that will be the subject of further investigations. The epsomite fibers, a magnesium sulphate with formula MgSO4, have larger diameters (about 4-5 μm), but is characterized by a greater surface reactivity in surface cracking phenomena, and this could be related to the biosolubilization of the fibers in the cellular structures. The formation of fractured crack surfaces could probably be related to hydration reactions with consequent structural variations. The SEM technique is confirmed as a useful tool for the characterization of fibrous material, the next objectives of the project will be the implementation of measures on mineral and synthetic fibers with possible toxic effects, for the purpose of the drafting of a protocol for the determination of the average diameter of the fibers used in the interaction with the cellular matrix and the deepening of the consequent interactions between fibers and cellular models according to the morphological and chemical characteristics. Furthermore, the SEM analysis allows to obtain a protocol of simple implementation for the dimensional characterization of the fibers, a useful protocol when a first and rapid criterion is needed to establish the potential danger of a mineral fiber not yet characterized.
Morphofunctional study of interaction between mineral fibers (nano and microstructured) and cellular matrices
2021
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