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Attenuation of oxidative stress and chromosomal aberrations in cultured macrophages and pulmonary cells following self-sustained high temperature synthesis of asbestos

La Maestra, Sebastiano, Micale, Rosanna T., Ferretti, Maurizio, Izzotti, Alberto, Gaggero, Laura·2020·Scientific Reports
DOI 10.1038/s41598-020-65620-xPMC PMC7244567
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Abstract

Inhalation of asbestos fibres can cause lung and pleural diseases in humans and constitutes a severe public health threat worldwide. The aim of the present study was to assess the biological effects induced in both pulmonary cells (A549) and monocyte/macrophage (RAW 264.7) cell lines by combustion slags obtained from asbestos through a self-sustained high-temperature synthesis (SHS) reaction. The SHS reaction involves rapid thermal treatment and displays great ability to neutralise asbestos. Cytotoxicity, redox status imbalance, lipid peroxide production, DNA strand breaks (comet assay) and chromosomal aberrations (cytokinesis block micronucleus test) were evaluated in cells exposed either to untreated asbestos fibres or to grinded SHS-generated slags of different granulometry, tested in cultured cells at varying doses and for varying exposure times. Our results show that asbestos fibres cause redox status imbalance, especially in monocyte/macrophage cell lines. Moreover, they promote lipid peroxidation and trigger genomic alterations. When the cells were exposed to slag powders, which are the products of SHS asbestos treatment, generation of lipid peroxides and induction of DNA strand breaks still persisted, due to the high content in iron and other metals detected in these samples. However, there was an attenuation of redox status imbalance and an absence of chromosomal aberrations, which probably reflects the loss of the asbestos fibrous structure following SHS reaction, as demonstrated by electron microscopy analyses. In conclusions, SHS-treated asbestos wastes can potentially have deleterious health effects due to the oxidative stress induced by inhaled powders but they loose the asbestos ability to induce chromosomal alterations.

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Figure 1 SEM microphotographs of SHS-treated pellets. (A) Scoriaceous texture. (B) Detail of A. Granular forsterite and wüstite pave the bubble cavities and globular metallic iron drops, suggesting that the Tmelting of iron was attained. (C) Blocky, vesicular texture. (D) Scoriaceous texture showing an outer silicate shell (dark grey), including an inner metallic layer (light grey, arrow on the left). The arrow on the right indicates the inter-growth of globular metallic Fe within olivine. (E) Large globular metallic iron drop. (F) Scoriaceous texture of wüstite + forsterite, including irregularly scattered globular iron drops.
Figure 1 SEM microphotographs of SHS-treated pellets. (A) Scoriaceous texture. (B) Detail of A. Granular forsterite and wüstite pave the bubble cavities and globular metallic iron drops, suggesting that the Tmelting of iron was attained. (C) Blocky, vesicular texture. (D) Scoriaceous texture showing an outer silicate shell (dark grey), including an inner metallic layer (light grey, arrow on the left). The arrow on the right indicates the inter-growth of globular metallic Fe within olivine. (E) Large globular metallic iron drop. (F) Scoriaceous texture of wüstite + forsterite, including irregularly scattered globular iron drops.CC BY · PubMed Central
Figure 2 SEM microphotographs of the three grain-size fractions (<10 μm, <3 μm and <2 μm), referred to as (A–C), respectively. Powder particles were obtained from SHS-treated waste to be tested in A549 and Raw 267.4 cell lines.
Figure 2 SEM microphotographs of the three grain-size fractions (<10 μm, <3 μm and <2 μm), referred to as (A–C), respectively. Powder particles were obtained from SHS-treated waste to be tested in A549 and Raw 267.4 cell lines.CC BY · PubMed Central
Figure 3 (A) Redox status assessed by means of DCF staining in A549 and Raw 267.4 cells. The redox status, expressed as intensity of DCF fluorescence, was evaluated in the two cell lines, either untreated (Ctr) or exposed for 3 h to varying concentrations either of slag powders with varying granulometry (A1–A5, B1–B5, C1–C5) or of untreated asbestos (D1–D5). H2O2 (100 µM) was tested as a positive control. The results are expressed as the average of fluorescence (FU)/number of cells and are the means ± SD of three independent experiments. Statistical analysis: aP < 0.05, bP < 0.01, and cP < 0.001 vs. control. (B,C) Representative microphotographs showing the effect of slag powders in Raw 267.4 cell lines, either unstained (B) or loaded with DCFH-DA (C). (D) TBARS levels, expressed as nmol/mg protein, in A549 and Raw 267.4 cells after contact for 12 h with the highest concentrations either of slag powders (15.62 µg/cm2) of different granulometries (A–C) or of asbestos. (D) The columns report the means + SD of triplicate analyses. Statistical analysis: bP < 0.01 and cP < 0.001 vs. controls.
Figure 3 (A) Redox status assessed by means of DCF staining in A549 and Raw 267.4 cells. The redox status, expressed as intensity of DCF fluorescence, was evaluated in the two cell lines, either untreated (Ctr) or exposed for 3 h to varying concentrations either of slag powders with varying granulometry (A1–A5, B1–B5, C1–C5) or of untreated asbestos (D1–D5). H2O2 (100 µM) was tested as a positive control. The results are expressed as the average of fluorescence (FU)/number of cells and are the means ± SD of three independent experiments. Statistical analysis: aP < 0.05, bP < 0.01, and cP < 0.001 vs. control. (B,C) Representative microphotographs showing the effect of slag powders in Raw 267.4 cell lines, either unstained (B) or loaded with DCFH-DA (C). (D) TBARS levels, expressed as nmol/mg protein, in A549 and Raw 267.4 cells after contact for 12 h with the highest concentrations either of slag powders (15.62 µg/cm2) of different granulometries (A–C) or of asbestos. (D) The columns report the means + SD of triplicate analyses. Statistical analysis: bP < 0.01 and cP < 0.001 vs. controls.CC BY · PubMed Central
Figure 4 Microphotograph showing internalization of SHS- generated powder particles in A549 cells. Magnification: 360x.
Figure 4 Microphotograph showing internalization of SHS- generated powder particles in A549 cells. Magnification: 360x.CC BY · PubMed Central
Figure 5 % Tail DNA, as evaluated by means of the Comet test, in A549 and Raw 267.4 cells after exposure for 12 h to the lowest (0.33 µg/ cm2) and to the highest concentrations (15.62 µg/cm2) either of slag powder particles or of asbestos. The columns report the means + SD of triplicate analyses. Statistical analysis: bP < 0.01 and cP < 0.001 vs. controls.
Figure 5 % Tail DNA, as evaluated by means of the Comet test, in A549 and Raw 267.4 cells after exposure for 12 h to the lowest (0.33 µg/ cm2) and to the highest concentrations (15.62 µg/cm2) either of slag powder particles or of asbestos. The columns report the means + SD of triplicate analyses. Statistical analysis: bP < 0.01 and cP < 0.001 vs. controls.CC BY · PubMed Central

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Metadata

Authors
La Maestra, S., Micale, R. T., Ferretti, M., Izzotti, A., & Gaggero, L.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-05-22
Volume
10
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-020-65620-x
PMC
PMC7244567
Cited by
14
References
0
Language
en
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