The Acute Toxicity of Mineral Fibres: History
View Latest Version
Please note this is an old version of this entry, which may differ significantly from the current revision.
Subjects: Toxicology
Contributor:
Sonia Scarfì

To gain new insights into the different toxicity mechanisms of carcinogenic mineral fibres, the acute effects of fibrous erionite, crocidolite and chrysotile in the THP-1 M0, M1 and M2 macrophages were investigated. The three mineral fibres apparently act by different toxicity mechanisms. Crocidolite seems to exert its toxicity effects mostly thanks to its biodurability, ROS and cytokine production, and DNA damage. Chrysotile, due to its low biodurability, displays toxic effects related to the release of toxic metals, ROS and cytokine production. Other mechanisms are involved in explaining the toxicity of biodurable fibrous erionite with lower ROS and toxic metal release but exhibiting cation exchange capacity able to alter the intracellular homeostasis of important cations. Their results aim to fill the gap on understanding the fine bio-chemical interactions of different types of carcinogenic fibres inside the cells, hopefully helping in upgrading the existing models of toxicity/carcinogenicity of mineral fibres.

  • mineral fibres
  • asbestos
  • carcinogenicity
  • apoptosis

1. Introduction

Exposure to mineral fibres represents a serious occupational and environmental hazard causing the emergence of fibrotic pulmonary diseases, pneumoconiosis and various types of cancer in exposed subjects [1]. One of the consequences is a high number of casualties and elevated costs for the healthcare systems of both developed and undeveloped countries. Thus, understanding the mechanisms of toxicity and carcinogenicity of inhalable mineral fibres constitutes a fundamental step towards the quantitative classification of the toxicity/carcinogenicity of mineral fibres for preventive medicine and the development of effective treatments for both at-risk workers and the general population.
Macrophages in the lung constitute the first and foremost line of defence against detrimental inhaled stimuli. They comprise a population of myeloid cells that belong to the innate immune system and play a fundamental role in both triggering and maintaining the inflammatory response that develops in the tissues as a consequence of harmful stimuli. In fact, cell damage is a necessary step in triggering the inflammatory response of immune cells. These actors are differentiated mainly by the blood and bone marrow precursors that accumulate in the tissue due to chemokines released from injured cells [2]. Once recruited to the site of inflammation in the specific tissue, macrophages have the capacity to adapt their phenotype in response to different local stimuli, displaying a high degree of plasticity. In the lung, for instance, a limited number of macrophage sentinels, i.e., resident alveolar macrophages [3], rapidly localise to the site of infection or injury and initiate the inflammatory process. Inflammation prompts the release of chemokines with the function of recruiting circulating monocytes and causing their extravasation and maturation in the tissue to non-activated (M0) macrophages. Subsequently, the newly differentiated M0 macrophages are further polarised by local stimuli in the tissue to phenotypes defined as classically activated type 1 (M1) and alternatively activated type (M2) macrophages [4]. Classically activated M1 macrophages are found at the site of damage shortly after injury or infection, with their phenotype developing in the presence of bacterial endotoxin (LPS) and IFN-γ or TNF-α. They release pro-inflammatory cytokines (i.e., TNF-α, IL-1β, IL-6, IL-12 and IL-15) and promote the production of cytotoxic reactive oxygen species (ROS), proteolytic enzymes and bioactive lipids. Conversely, M2 macrophages, which can be polarised by IL-4 and/or IL-13 stimuli, may appear later at the site of inflammation and counteract the pro-inflammatory activity of the M1 phenotype by secreting anti-inflammatory mediators (i.e., IL-10, CCL18 and CCL22) and tissue-repair-promoting factors, such as TGF-β, VEGF and FGF [5]. However, while M1 and M2 cells were initially assumed to be phenotypically and functionally distinct subpopulations with opposite roles in the inflammatory response, it is now recognised that macrophages exist as several subpopulations with different levels of M1/M2 markers and activities [6][7].
A very useful experimental model that can provide insights into the modulation of monocyte and macrophage functions and behaviour is the human monocytic cell line THP-1. These cells can be easily differentiated into the M0, M1 and M2 phenotypes in culture; they can then be co-cultured with a plethora of different cell types to mimic tissue-specific interactions and be challenged by a large variety of stimuli, such as infectious agents, asbestos and pollutants [8]. This in vitro human cell model has previously been used in several studies with the aim of understanding the mechanisms of acute toxicity and the inflammatory response of macrophages towards toxic and carcinogenic mineral fibres [9]. All of the known previous studies always focused on one type of THP-1-derived phenotype, namely, M0 macrophages, which were usually challenged by the most important commercial asbestos fibres: chrysotile [10][11][12][13][14][15] and crocidolite [16][17][18][19][20][21][22]. To date, no comparative data have been obtained from the concomitant analysis of the inflammatory response induced by different mineral fibres in the three macrophage phenotypes, namely, non-activated M0, pro-inflammatory M1 and alternatively activated M2 cells. Indeed, all three phenotypes can be present at the site of injury and contribute to the outcomes of the inflammatory process. Thus, to fill this gap and gain new insights into the different mechanisms of the toxicity of mineral fibres, the three M0, M1 and M2 macrophage phenotypes, obtained from THP-1 cell differentiation, were used to study the acute effects of three types of carcinogenic mineral fibres: fibrous erionite, crocidolite and chrysotile.
Asbestos is a generic term used to identify six mineral fibres of commercial and economic importance: five amphibole species (i.e., amphibole asbestos) and chrysotile (a member of serpentine asbestos) [23][24][25]. The family of amphibole asbestos includes actinolite asbestos, amosite, anthophyllite asbestos, crocidolite and tremolite asbestos. Amphiboles are double-chain silicates with the general formula A0-1B2C5T8O22W2, where A = Na+, K+, Ca2+, Li+ (cation sites with 8- to 12-fold coordination); B = Na+, Li+, Ca2+, Mn2+, Fe2+, Mg2+ (less regular octahedral or 8-fold coordinated cation sites); C = Mg2+, Fe2+, Mn2+, Al3+, Fe3+, Ti3+, Ti4+ (fairly regular octahedral cation sites); and T = Si4+, Al3+ (tetrahedral sites of the silicate chain) [24][26]. The growth of the crystals occurs along the c-axis in amphibole asbestos and is responsible for their fibrous nature. The most relevant mineral fibre of the amphibole asbestos group is crocidolite. Crocidolite, also called blue asbestos, is the fibrous/asbestiform variety of riebeckite, i.e., a sodic amphibole with the ideal formula Na2(Fe2+3Fe3+2)[Si8O22](OH)2. Detailed and up-to-date information on the classification, crystal chemistry and structural characteristics of amphibole asbestos is provided in the literature [26].
Chrysotile is the most common asbestos mineral and belongs to the serpentine group [23][24]. Serpentine minerals are layer silicates composed of Si-centred tetrahedral (T) sheets in a pseudo-hexagonal network joined to Mg-centred octahedral (O) sheets in units with a 1:1 (TO) ratio. Since the TO unit is polar and a misfit exists between the smaller T sheet and the larger O sheet, a differential strain occurs between the two sheets [26]. The strain is released by rolling the TO layer around a preferred axis, leading to the formation of the tubular structure typical of chrysotile fibres [24][26]. The general chemical formula of chrysotile is Mg3(OH)4Si2O5. The most common substitution occurs between Fe2+ and Mg2+ in the octahedral site [26]. Moreover, Al3+ and Fe3+ can replace both Si4+ and Mg2+ in the tetrahedral and octahedral sites. Among the different forms of asbestos, chrysotile is the most extensively used worldwide in the manufacturing of brake and clutch linings, insulation systems, cement boards and roofing tiles, to cite a few [27].
Erionite is a natural zeolite belonging to the so-called ABC-6 family whose structure is composed of columns of cancrinite cages connected by a double six-membered ring of tetrahedra, forming hexagonal prisms [26][28]. The ideal formula of erionite is K2(Na, Ca0.5)7[Al9Si27O72]·28H2O. Erionite is characterised by a large chemical variability, with three different species identified according to the most abundant extra-framework cation: erionite-Na, erionite-K and erionite-Ca.
Asbestos and fibrous erionite are known toxic and pathogenic agents and are included in Group 1, carcinogenic to humans, by the International Agency for Research on Cancer (IARC) [25].

2. Cell–Fibre Interaction Imaging and Fibre Surface Characterisation

In all three types of polarised macrophages (M0, M1 and M2), the mineral fibres were detected inside the cells and identified by micro-Raman analyses after 24 h of incubation. As expected, chrysotile (CHR), crocidolite (CRO) and erionite (ERI) fibres showed different morphologies (Figure 1A).
Ijms 23 02840 g001
Figure 1. (A) Microscope images of M0, M1 and M2 macrophages in the presence of mineral fibres chrysotile, crocidolite and erionite. Panels I, II and III: chrysotile-treated M0, M1 and M2 macrophages, respectively. Panels IV, V and VI: crocidolite-treated M0, M1 and M2 macrophages, respectively. Panels VII, VIII and IX: erionite-treated M0, M1 and M2 macrophages, respectively. Typical morphologies of the fibres are highlighted with yellow arrows. The scale bar represents 30 μm. (B) Raman spectra of the mineral fibres chrysotile (I), crocidolite (II) and erionite (III) in M0 macrophages, acquired in the low-wavenumber spectral range. Photoluminescence bands of Cr3+ emissions are marked with asterisks (*). (C) Raman spectra of the mineral fibres chrysotile (I), crocidolite (II) and erionite (III) in M0 macrophages, acquired in the high-wavenumber spectral range. The Raman bands between ~2800 and 3100 cm−1 are attributed to CH stretching modes due to the cellular signals.
In all three investigated mineral fibres, impurities consisting of non-fibrous micrometric crystals were detected inside the cells along with fibrous phases, as previously reported in the literature (Table 1). In CHR-treated macrophages, bundles of curvilinear chrysotile fibres with splayed/frayed terminations and thinner and shorter single fibres were observed (Figure 1A, panels I, II and III); minor contributions of other minor fibrous and lamellar phases (balangeroite and antigorite, respectively) were also detected in addition to the non-fibrous content. CRO fibres showed a thin and rigid morphology with variable lengths (Figure 1A, panels IV, V and VI). ERI fibres were typically shorter than the other mineral fibres, exhibiting a more uniform size distribution (Figure 1A, panels VII, VIII and IX). The mineral fibres inside the three types of differentiated phagocytic cells were identified by their characteristic Raman spectra, acquired in both the low- and high-wavenumber spectral ranges, and the positions of the main peaks were highlighted (Figure 1B,C). For serpentine minerals, the lattice and internal vibrational modes were similar in different polymorphs, demonstrating that OH stretching signals are distinctive characteristics for the identification of the mineralogical phase in CHR-treated cells [29][30]. In addition to CHR (curve I in Figure 1B,C), the lamellae of antigorite and balangeroite fibres were detected as minor contributions (Table 1) [31]. Additionally, micrometric crystals consisting of impurities were observed in CHR-treated macrophages, highlighting the presence of iron compounds (Table 1) [32]. Furthermore, in the low range, it was possible to detect the photoluminescence bands of Cr3+ emissions at ~671 and 680 nm due to the presence of chromium as a trace element in the CHR fibres (marked with asterisks in curve I in Figure 1B) [33]. In CRO-treated macrophages, CRO was identified by its peculiar Raman spectrum (curve II in Figure 1B,C) [34][35], and minor phases—including iron-bearing micro-crystals—were observed (Table 1) [36][37]. Erionite was the main fibrous mineral phase detected in ERI-treated macrophages, with minor tabular clinoptilolite [38]. Here, iron-bearing impurities—as identified in SEM and TEM analyses in previous studies [28]—were not observed through micro-Raman analyses, as they are generally smaller than the minimum detectable size, considering the spatial resolution of the spectrometer. It is noteworthy that for all three types of polarised macrophages, the Raman signals of cells were always found along with those of the analysed mineral fibres, and no differences in the signals were detected for each mineral fibre in the three types of differentiated macrophages. Cellular signals were especially clear from their CH stretching bands in the spectral range ~2800–3100 cm−1 (Figure 1C).
Table 1. General information on the mineral fibres investigated in this study. Impurities detected in the present work and in [28][32][36][37]. Fibre lengths and widths from Reference [9].
Mineral Fibre Impurities Fibre Length (µm) Fibre Width (µm)
Chrysotile
(Balangero, Turin, Italy)		 Antigorite, balangeroite, calcite, clinochlore, diopside, dolomite, magnetite, microcline, plagioclase, talc, mackinawite, hematite, ilmenite, lepidocrocite, Fe-Ni sulphide, Fe-Mg carbonate * Min: 4.02
Mean: 34.7
** Max: 188		 Min: 0.18
Mean: 0.59
Max: 1.17
Crocidolite
(UICC)		 Hematite, magnetite, quartz, talc, lizardite, calcite, siderite, minnesotaite Min: 2.52
Mean: 16.1
Max: 131		 Min: 0.23
Mean: 0.64
Max: 1.98
Erionite
(Jersey, Nevada, USA)		 Clinoptilolite, iron-rich nanoparticles, iron oxides/hydroxides, nontronite Min: 3.23
Mean: 9.39
Max: 55.0		 Min: 0.25
Mean: 0.55
Max: 6.70

* Minimum; ** Maximum.

3. Acute Toxicity of Mineral Fibres

To analyse the cell viability and the phagocytic ability of M0, M1 and M2 macrophages, the differentiated cells were incubated with the three mineral fibres. After 4 h of incubation, researchers used calcein-AM staining—a green-fluorescent dye used to label live cells—to determine if the phagocytic cells had engaged and internalised the particles and if this had already affected cell viability (Figure 2).
Ijms 23 02840 g002
Figure 2. Qualitative evaluation of short-term cytotoxicity. (A) Visualisation by confocal microscopy analysis (2.5 × digital zoom) in fluorescence mode (panels I, III, V and VII) and in phase contrast (panels II, IV, VI and VIII) of M0 macrophages following 4 h of incubation in the presence or absence of mineral fibres at 50 µg/mL and staining with calcein-AM. Panels I-II: control; panels III-IV: CRO; panel V-VI: CHR; panels VII-VIII: ERI. The white bar represents 40 µm. (B) Visualisation of pro-inflammatory M1 macrophages in the same conditions as cells in panel (A). (C) Visualisation of alternatively activated M2 macrophages in the same conditions as cells in panel (A).
For all three macrophage types, M0 (Figure 2A), M1 (Figure 2B) and M2 (Figure 2C), there were no differences in fluorescence positivity between control cells (panels AI-II, BI-II and CI-II for M0, M1 and M2 macrophages, respectively) and fibre-treated cells (panels III-IV CRO, panels V-VI CHR and panels VII-VIII ERI, respectively). In fact, in all experimental conditions, cells showed intense green cytoplasmic fluorescence, meaning that after 4 h of treatment with the fibres, no signs of cytotoxicity were visible yet. Nonetheless, in all types of macrophages, it was possible to observe the presence of the different types of mineral fibres inside the cytoplasm of the cells when the size of the fibres was short enough to allow their phagocytosis. From a qualitative point of view, M2 macrophages showed a higher number of mineral fibres dispersed at the bottom of the well, especially for CRO (panel C-IV) and ERI (panel C-VIII), compared to M0 and M1 macrophages (panels A and B), which had already captured many fibres or were in direct contact with them. The observed phenomenon may indicate that M2 macrophages are less able to engulf the mineral fibres compared to the other two phenotypes. This is plausible since this type of macrophage polarisation should have a more anti-inflammatory role [7].
Since the qualitative assessment of macrophage viability by calcein staining showed no significant signs of toxicity after 4 h of fibre treatment, the MTT test was used to evaluate the cytotoxic effects of CRO, CHR and ERI (Figure 3, panels A–C, black, grey and white bars, respectively) in macrophages after 24 h. This assay measures mitochondrial enzyme activity by quantifying resultant alterations of energy metabolism and, ultimately, cell death. The viability index showed that there was a dose-dependent toxic effect resulting in a significant cell death rate in both M0 and M1 macrophages after exposure to all three fibres, while CRO exhibited a similar behaviour only in M2 cells. In M0 macrophages (Figure 3A, black bars), CRO caused a dose-dependent cell death rate of 57%, 49% and 22% at 100, 50 and 10 µg/mL, respectively, compared to control cells. Similarly, CHR (grey bars) induced cell death rates of 49%, 46% and 14% at the same fibre concentrations compared to control cells. In M0 macrophages, ERI (white bars) showed slightly lower toxicity compared to asbestos fibres, with significant death rates (45% and 35%) only at the two highest fibre concentrations (100 and 50 µg/mL, respectively) compared to control cells.
Ijms 23 02840 g003
Figure 3. Cell toxicity evaluation and cell lysis assessment after exposure to mineral fibres. (A) M0 macrophage cytotoxicity evaluation by the MTT test at 24 h in the presence of 100, 50 and 10 μg/mL of the mineral fibres CRO (black bars), CHR (grey bars) and ERI (white bars). Results are expressed as cell percentages relative to control cells (striped bar) and are the mean ± SD of three independent experiments performed in quadruplicate. Asterisks indicate the significance in a paired Tukey test (ANOVA, p < 0.0001; Tukey vs. C: * p < 0.05, ** p < 0.005, respectively). (B) Pro-inflammatory M1 macrophage cytotoxicity evaluation in the same conditions as (A). ANOVA, p < 0.000005; Tukey vs. C: * p < 0.05, ** p < 0.005, respectively. (C) Alternatively activated M2 macrophage cytotoxicity evaluation in the same conditions as (A). ANOVA, p < 0.000001; Tukey vs. C: ** p < 0.005. (D) Cell lysis assessment measured by quantification of LDH release in the cell medium at 24 h in M0 macrophages after incubation with 100, 50 and 10 μg/mL of the mineral fibres CRO (black bars), CHR (grey bars) and ERI (white bars). Results are expressed as percentage of cell lysis relative to control cells (striped bar) and are the mean ± SD of three experiments performed in quadruplicate. Asterisks indicate significance in paired Tukey test (ANOVA, p < 0.0005; Tukey vs. C: ** p < 0.005). (E) Cell lysis assessment measured in the same conditions as (D) in pro-inflammatory M1 macrophages. ANOVA, p < 0.01; Tukey vs. C: * p < 0.05, ** p < 0.005, respectively. (F) Cell lysis assessment measured in the same conditions as (D) in alternatively activated M2 macrophages. ANOVA, p < 0.01; Tukey vs. C: * p < 0.05, ** p < 0.005, respectively.
Exposure to the three mineral fibres also induced a relevant cytotoxic effect in M1 macrophages (Figure 3B). The dose-dependent cell death rate after exposure to CRO was higher in M1 cells than in M0 cells (66%, 49% and 14% at 100, 50 and 10 µg/mL, respectively, compared to control cells). CHR at 100 and 50 µg/mL was also more cytotoxic in M1 than in M0, with cell death percentages ranging from 61% to 53%, respectively, compared to control cells. Regarding ERI, a strong cytotoxic effect was observed at 100 and 50 µg/mL (68% and 41%, respectively, compared to control cells).
M2 macrophages (Figure 3C) demonstrated a dose-dependent cytotoxic effect during exposure to CRO, although the cell death rates were lower than those found in M0 and M1 cells, with percentages ranging from 30% to 21% at 100 and 50 µg/mL, respectively, compared to control cells. CHR showed minor but significant cytotoxicity; cell death values were similar at all concentrations and around 14–15% compared to control cells (p < 0.005 vs. C for all). Finally, ERI slightly induced cell death in M2 cells only at 100 and 50 µg/mL, with a 19% reduction in viability at both concentrations compared to control cells (p < 0.05 vs. C for both). Overall, CRO and CHR showed a higher acute toxicity rate, strongly affecting energy metabolism and causing a significant cell death rate, than ERI in M0, M1 and M2 macrophages. In particular, for CRO treatment, their results are in line with a previous work [22], where a similarly high THP-1 macrophage cytotoxicity rate was observed in the first 24 h of exposure. Furthermore, lower fibre-induced cytotoxicity was observed in M2 macrophages compared to the other two phenotypes; this finding can be ascribed to their anti-inflammatory role and consequent lower phagocytic activity, leading to a reduced cell death rate due to frustrated phagocytosis.
To assess the nature of macrophage cell death, two analyses were performed: the lactate dehydrogenase (LDH) assay, which can be considered a cell death marker since the leakage of this cytosolic enzyme in the cell medium occurs following plasma membrane disturbance, and the annexin/propidium iodide positivity assay, measured by confocal microscopy to establish the level of cellular apoptosis. The LDH assay was performed on the three types of differentiated macrophages in contact with the fibres at three concentrations (100, 50 and 10 µg/mL) for 24 h to quantify cell death caused by plasma membrane damage (Figure 3D–F). The assay showed that, for all of the polarised macrophages, the rates of cellular damage observed after exposure to CRO were comparable to the control (Figure 3D–F black bars). Conversely, all types of differentiated macrophages showed a certain degree of plasma membrane cell damage during treatment at all concentrations of CHR (grey bars) and at the two highest concentrations of ERI (100 and 50 µg/mL, white bars). In particular, in M0 cells (Figure 3D), the degree of cellular damage induced by CHR was not dose-dependent, and the relative values of LDH release were 1.56-, 1.68- and 1.56-fold higher than control cells at 100, 50 and 10 µg/mL, respectively, while for ERI, significant dose-dependent plasma membrane cell damage was observed, with values 1.58- and 1.33-fold higher than control cells at the two highest fibre concentrations. The trend was similar in M1 macrophages (Figure 3E), although levels of cell membrane damage were observed to be lower compared to M0 cells. In particular, for CHR treatment, the increase in necrotic events compared to control cells was 1.32-, 1.22- and 1.18-fold at 100, 50 and 10 µg/mL, respectively, while for ERI, it amounted to 1.27- and 1.19-fold compared to control cells at 100 and 50 µg/mL.
The same trend was also evident in M2 cells (Figure 3F), since all tested concentrations of CHR induced an increase in cell lysis in the culture (1.69-, 1.50- and 1.26-fold increase at 100, 50 and 10 µg/mL, respectively), as was also observed with 100 and 50 µg/mL of ERI (1.44- and 1.33-fold for 100 and 50 µg/mL, respectively).
In general, in all differentiated macrophages, exposure to CRO was not able to induce cell lysis, meaning that the high rates of cytotoxicity, measured by the alteration of energy metabolism through the MTT test at 24 h for the same fibre (Figure 3A–C), are the result of other death mechanisms. On the other hand, significant levels of cell lysis were observed after treatment with CHR or ERI. Indeed, in the case of ERI, their results confirm recent findings in the U937 monocyte cell line model, which also demonstrated the direct damage of cell membranes in contact with the fibres [39]. The level was higher for CHR compared to ERI, and this result may point towards multifactorial cytotoxic activity, with cell membrane alteration at least partially capable of inducing cell death. To test this hypothesis, cells were treated with the mineral fibres for 24 h and analysed by confocal microscopy using annexin/propidium iodide staining to both qualitatively (Figure 4) and quantitatively (Figure 5) assess their apoptotic state. Confocal microscopy analysis indicated that in all types of differentiated macrophages (Figure 4A, M0 macrophages; 4B, M1 macrophages; 4C, M2 macrophages), although at different rates, all fibres (Figure 4A–C: panels III-IV CRO, panels V-VI CHR and panels VII-VIII ERI, respectively) caused the significant induction of both early (only green positivity) and late apoptosis (concomitant green/red positivity) compared to control cells (Figure 4A–C: panels I-II in each figure).
Ijms 23 02840 g004
Figure 4. Cell apoptosis assessment by confocal microscopy analysis. (A) Visualisation by confocal microscopy (2.5 × digital zoom) in fluorescence mode (panels I, III, V and VII) and in phase contrast (panels II, IV, VI and VIII) of M0 macrophages following 24 h of incubation in the presence or absence of mineral fibres at 50 µg/mL and staining of annexin-positive (green) and/or propidium iodide–positive (red) cells. Panels I-II: control; panels III-IV: CRO; panels V-VI: CHR; panels VII-VIII: ERI. The white bar represents 40 µm. (B) Visualisation of pro-inflammatory M1 macrophages in the same conditions as cells in panel (A). (C) Visualisation of alternatively activated M2 macrophages in the same conditions as cells in panel (A).
Ijms 23 02840 g005
Figure 5. Cell apoptosis quantification. (A) The quantitative analysis of early (annexin-positive, white bars) and late (both annexin-positive and propidium iodide-positive, black bars) apoptotic M0 THP-1 cells treated with 50 and 10 μg/mL of CRO, CHR and ERI relative to the total number of cells observed by confocal microscopy after 24 h; results are the mean ± SD of counts from five microphotograph. Asterisks indicate significance in Tukey test (ANOVA for white bars p < 0.000001; Tukey vs. C, ** p < 0.005, respectively; ANOVA for black bars p < 0.0005, Tukey vs. C, ## p < 0.005, # p < 0.05, respectively). (B) The same analysis in pro-inflammatory M1 macrophages. ANOVA for white bars p < 0.00005; Tukey vs. C, ** p < 0.005, * p < 0.05, respectively; ANOVA for black bars p < 0.0001, Tukey vs. C, ## p < 0.005, # p < 0.05, respectively. (C) The same analysis in alternatively activated M2 macrophages. ANOVA for white bars p < 0.00001; Tukey vs. C, ** p < 0.005, * p < 0.05, respectively; ANOVA for black bars p < 0.00005, Tukey vs. C, ## p < 0.005, # p < 0.05, respectively.
The quantitative assessment of the apoptotic state, obtained by counting and calculating the mean of only green and red–green cells in five different fields of confocal microscope images for each treatment, is shown in Figure 5. In M0 cells (panel A), CRO induced the highest levels of early signals of apoptosis at both concentrations tested (white bars, 98% and 86% for 10 and 50 μg/mL CRO, respectively, p < 0.005 vs. C, for both bars). Early apoptosis signals after exposure to CHR and ERI were nonetheless significant and dose-dependent, although lower than those after exposure to CRO (white bars, 24% and 84% for 10 and 50 μg/mL CHR, and 41% and 64% for 10 and 50 μg/mL ERI, respectively, p < 0.005 vs. C for all bars). In contrast, regardless of the concentration used, the number of late apoptotic cells after 24h of treatment was similar for all fibres (black bars, from ~20 to ~40%, p < 0.005 vs. C). These data, showing an increase in apoptotic levels for all fibres, are in agreement with the apoptosis assessment in THP-1 M0 cells in their previous work [9], which was performed with a shorter incubation time (8 h), indicating further progression of the apoptotic phenomenon in these cells over time. On the other hand, compared to M0 cells, M1 macrophages (panel B) showed slightly reduced levels of apoptosis. Indeed, in M1 cells, the highest rates of early apoptosis were 31% for both 10 and 50 μg/mL CHR (white bars, p < 0.005 vs. C for all), although all values of early apoptosis were found to be significant compared to control cells (~20% for 10 and 50 μg/mL ERI with p < 0.005 vs. C for both bars, 11% for 10 μg/mL CRO and 29% 50 μg/mL CRO with p < 0.05 and p < 0.005 vs. C, respectively). In addition, all mineral fibres induced late apoptosis to a certain degree (black bars), ranging from ~10% to ~19%, with the highest values measured for 10 and 50 μg/mL ERI and 50 μg/mL CRO (p < 0.05 vs. C for all bars). Finally, M2 macrophages (panel C) showed the highest rates of early apoptosis after CHR treatment (white bars, 37% and 62% for 10 and 50 μg/mL CHR, respectively, p < 0.005 vs. C for both) and a lower rate for CRO (37% and 43% for 10 and 50 μg/mL CRO, respectively, p < 0.005 vs. C for both) and ERI (41% and 26% for 10 and 50 μg/mL ERI, respectively, p < 0.05 vs. C). Regarding late apoptosis, in these cells, it was significantly increased only by the highest concentrations of CHR and CRO compared to control cells (black bars, 37% for 50 μg/mL CHR and 27% for 50 μg/mL CRO, respectively, p < 0.005 vs. C for both). Conversely, both concentrations of ERI appeared to induce significant levels of late apoptosis (24% and 17% for 10 and 50 μg/mL ERI, respectively, p < 0.05 vs. C for both).
Overall, their data indicate that the three types of fibres are able to induce significant damage, resulting in the alteration of energy metabolism, cell membrane lysis and/or apoptosis and likely macrophage activation through phagocytosis. The overall phenomena, together with the incapability of macrophages to completely digest engulfed fibres and the induction of apoptosis resistance in macrophages by the fibres through activation of mitochondrial NOX4 over time [15], lead to a state of prolonged local inflammation and the release of inflammatory cytokines prodromal to the onset of cancer [40][41]. Furthermore, macrophages internalising small fibres may survive in the short term and may act as carriers that transport particles towards the inner lung parenchyma and the mesothelium. The chronic inflammatory state caused by undigested fibres in activated macrophages at these new sites, as well as the direct interaction of the transported fibres with epithelial and mesothelial cells, will then set up a favourable microenvironment for cell transformation and carcinogenesis.

This entry is adapted from the peer-reviewed paper 10.3390/ijms23052840

References

  1. International Programme on Chemical Safety & World Health Organization. Asbestos and Other Natural Mineral Fibres; World Health Organization: Geneva, Switzerland, 1986; Available online: https://apps.who.int/iris/handle/10665/37190 (accessed on 1 February 2022).
  2. Gordon, S. The macrophage: Past, present and future. Eur. J. Immunol. 2007, 37, S9–S17.
  3. Hussell, T.; Bell, T.J. Alveolar macrophages: Plasticity in a tissue-specific context. Nat. Rev. Immunol. 2014, 14, 81–93.
  4. Arora, S.; Dev, K.; Agarwal, B.; Das, P.; Syed, M.A. Macrophages: Their role, activation and polarization in pulmonary diseases. Immunobiology 2018, 223, 383–396.
  5. Martinez, F.O.; Helming, L.; Gordon, S. Alternative activation of macrophages: An immunologic functional perspective. Annu. Rev. Immunol. 2009, 27, 451–483.
  6. Mantovani, A.; Sica, A.; Sozzani, S.; Allavena, P.; Vecchi, A.; Locati, M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004, 25, 677–686.
  7. Laskin, D.L.; Malaviya, R.; Laskin, J.D. Role of macrophages in acute lung injury and chronic fibrosis induced by pulmonary toxicants. Toxicol. Sci. 2019, 168, 287–301.
  8. Chanput, W.; Mes, J.J.; Wichers, H.J. THP-1 cell line: An in vitro cell model for immune modulation approach. Int. Immunopharmacol. 2014, 23, 37–45.
  9. Di Giuseppe, D.; Scarfì, S.; Alessandrini, A.; Bassi, A.M.; Mirata, S.; Almonti, V.; Ragazzini, G.; Mescola, A.; Filaferro, M.; Avallone, R.; et al. Acute cytotoxicity of mineral fibres observed by time-lapse video microscopy. Toxicology 2021, 466, 153081.
  10. Li, M.; Gunter, M.E.; Fukagawa, N.K. Differential activation of the inflammasome in THP-1 cells exposed to chrysotile asbestos and Libby “Six-Mix” amphiboles and subsequent activation of BEAS-2B cells. Cytokine 2012, 60, 718–730.
  11. Morris, G.F.; Danchuk, S.; Wang, Y.; Xu, B.; Rando, R.J.; Brody, A.R.; Shan, B.; Sullivan, D.E. Cigarette smoke represses the innate immune response to asbestos. Physiol. Rep. 2015, 3, e12652.
  12. Boyles, M.S.P.; Poland, C.; Raftis, J.; Duffin, R. Assessment of the physicochemical properties of chrysotile-containing brake debris pertaining to toxicity. Inhal. Toxicol. 2019, 31, 325–342.
  13. Skuland, T.; Maslennikova, T.; Låg, M.; Gatina, E.; Serebryakova, M.K.; Trulioff, A.S.; Kudryavtsev, I.V.; Klebnikova, N.; Kruchinina, I.; Schwarze, P.E.; et al. Synthetic hydrosilicate nanotubes induce low pro-inflammatory and cytotoxic responses compared to natural chrysotile in lung cell cultures. Basic Clin. Pharmacol. Toxicol. 2020, 126, 374–388.
  14. Gualtieri, A.F.; Lusvardi, G.; Pedone, A.; Di Giuseppe, D.; Zoboli, A.; Mucci, A.; Zambon, A.; Filaferro, M.; Vitale, G.; Benassi, M.; et al. Structure model and toxicity of the product of biodissolution of chrysotile asbestos in the lungs. Chem. Res. Toxicol. 2019, 32, 2063–2077.
  15. Larson-Casey, J.L.; Gu, L.; Kang, J.; Dhyani, A.; Carter, A.B. NOX4 regulates macrophage apoptosis resistance to induce fibrotic progression. J. Biol. Chem. 2021, 297, 100810.
  16. Dostert, C.; Pétrilli, V.; Van Bruggen, R.; Steele, C.; Mossman, B.T.; Tschopp, J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 2008, 320, 674–677.
  17. Kanno, S.; Hirano, S.; Chiba, S.; Takeshita, H.; Nagai, T.; Takada, M.; Sakamoto, K.; Mukai, T. The role of Rho-kinases in IL-1β release through phagocytosis of fibrous particles in human monocytes. Arch. Toxicol. 2014, 89, 73–85.
  18. Lee, D.-K.; Jeon, S.; Han, Y.; Kim, S.-H.; Lee, S.; Yu, I.J.; Song, K.S.; Kang, A.; Yun, W.S.; Kang, S.-M.; et al. Threshold Rigidity Values for the asbestos-like pathogenicity of high-aspect-ratio carbon nanotubes in a mouse pleural inflammation model. ACS Nano 2018, 12, 10867–10879.
  19. Munson, P.; Lam, Y.; Dragon, J.; MacPherson, M.; Shukla, A. Exosomes from asbestos-exposed cells modulate gene expression in mesothelial cells. FASEB J. 2018, 32, 4328–4342.
  20. Corti, A.; Bonetti, J.; Dominici, S.; Piaggi, S.; Fierabracci, V.; Foddis, R.; Pompella, A. Induction of gamma-glutamyltransferase activity and consequent pro-oxidant reactions in human macrophages exposed to crocidolite asbestos. Toxicol. Sci. 2019, 177, 476–482.
  21. Ito, F.; Kato, K.; Yanatori, I.; Murohara, T.; Toyokuni, S. Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin. Redox Biol. 2021, 47, 102174.
  22. Ventura, C.; Pereira, J.F.S.; Matos, P.; Marques, B.; Jordan, P.; Sousa-Uva, A.; Silva, M.J. Cytotoxicity and genotoxicity of MWCNT-7 and crocidolite: Assessment in alveolar epithelial cells versus their coculture with monocyte-derived macrophages. Nanotoxicology 2020, 14, 479–503.
  23. Alleman, J.E.; Mossman, B.T. Asbestos revisited. Sci. Am. 1997, 277, 70–75.
  24. Gualtieri, A.F.; Gualtieri, M.L.; Scognamiglio, V.; Di Giuseppe, D. Human health hazards associated with asbestos in building materials. In Ecological and Health Effects of Building Materials; Springer: Cham, Swizerland, 2022; pp. 297–325.
  25. IARC. Arsenic, metals, fibres and dust. In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans Volume 100c; International Agency for Research on Cancer: Lyon, France, 2012.
  26. Ballirano, P.; Bloise, A.; Gualtieri, A.F.; Lezzerini, M.; Pacella, A.; Perchiazzi, N.; Dogan, M.; Dogan, A.U. The crystal structure of mineral fibres. In Mineral Fibres: Crystal Chemistry, Chemical-Physical Properties, Biological Interaction and Toxicity; Gualtieri, A.F., Ed.; European Mineralogical Union-EMU Notes in Mineralogy: London, UK, 2017; pp. 11–53.
  27. Poland, C.; Duffin, R. The toxicology of chrysotile-containing brake debris: Implications for mesothelioma. Crit. Rev. Toxicol. 2019, 49, 11–35.
  28. Gualtieri, A.F.; Gandolfi, N.B.; Pollastri, S.; Pollok, K.; Langenhorst, F. Where is iron in erionite? A multidisciplinary study on fibrous erionite-Na from Jersey (Nevada, USA). Sci. Rep. 2016, 6, 37981.
  29. Petriglieri, J.R.; Salvioli-Mariani, E.; Mantovani, L.; Tribaudino, M.; Lottici, P.P.; Laporte-Magoni, C.; Bersani, D. Micro-Raman mapping of the polymorphs of serpentine. J. Raman Spectrosc. 2015, 46, 953–958.
  30. Rinaudo, C.; Croce, A. Micro-raman spectroscopy, a powerful technique allowing sure identification and complete characterization of asbestiform minerals. Appl. Sci. 2019, 9, 3092.
  31. Groppo, C.; Compagnoni, R. Metamorphic veins from the serpentinites of the Piemonte Zone, western Alps, Italy: A review. Period Miner. 2007, 76, 127–153.
  32. Pollastri, S.; Perchiazzi, N.; Lezzerini, M.; Plaisier, J.R.; Cavallo, A.; Dalconi, M.C.; Gandolfi, N.B.; Gualtieri, A.F. The crystal structure of mineral fibres. 1. Chrysotile. Period Miner. 2016, 85, 249–259.
  33. Bloise, A.; Barca, D.; Gualtieri, A.F.; Pollastri, S.; Belluso, E. Trace elements in hazardous mineral fibres. Environ. Pollut. 2016, 216, 314–323.
  34. Croce, A.; Arrais, A.; Rinaudo, C. Raman Micro-spectroscopy identifies carbonaceous particles lying on the surface of crocidolite, amosite, and chrysotile fibers. Minerals 2018, 8, 249.
  35. Lewis, I.R.; Chaffin, N.C.; Gunter, M.E.; Griffiths, P.R. Vibrational spectroscopic studies of asbestos and comparison of suitability for remote analysis. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 1996, 52, 315–328.
  36. Pacella, A.; Andreozzi, G.B.; Nodari, L.; Ballirano, P. Chemical and structural characterization of UICC crocidolite fibres from Koegas Mine, Northern Cape (South Africa). Period Miner. 2019, 88, 297–306.
  37. Della Ventura, G.; Vigliaturo, R.; Gieré, R.; Pollastri, S.; Gualtieri, A.F.; Iezzi, G. Infra red spectroscopy of the regulated asbestos amphiboles. Minerals 2018, 8, 413.
  38. Tsai, Y.-L.; Huang, E.; Li, Y.-H.; Hung, H.-T.; Jiang, J.-H.; Liu, T.-C.; Fang, J.-N.; Chen, H.-F. Raman spectroscopic characteristics of zeolite group minerals. Minerals 2021, 11, 167.
  39. Cangiotti, M.; Salucci, S.; Battistelli, M.; Falcieri, E.; Mattioli, M.; Giordani, M.; Ottaviani, M.F. EPR, TEM and cell viability study of asbestiform zeolite fibers in cell media. Colloids Surf. B Biointerfaces 2018, 161, 147–155.
  40. D’Arcy, M.S. Cell death: A review of the major forms of apoptosis, necrosis and autophagy. Cell Biol. Int. 2019, 43, 582–592.
  41. Coussens, L.M.; Werb, Z. Inflammation and cancer. Nature 2002, 420, 860–867.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.
This entry is offline, you can click here to edit this entry!
ScholarVision Creations
Feedback