Particle and Fibre Toxicology

About the journal

Publisher: BioMed Central
Published: 2004 to the present, England
Language: English
ISSN: 1743-8977 (electronic)
Its record in the NLM Catalog

2020s

Impact of carbon-based fibers morphologies on their carcinogenic potential. A. Wagner, F. Schulz, A. Meyer-Plath, et al. 2026;23(1). PubMed · Publisher · Free full text

Abstract

Background: Carbon based fibers are considered to exhibit a carcinogenic potency when inhaled into the deep lung. Mesotheliomas develop after intraperitoneal application of multi-walled carbon nanotubes (MWCNTs) exceeding a diameter of about 37 nm, whereas carcinogenic potency decreases for diameters below this threshold. While large MWCNT diameters are associated with a rigid fiber geometry, this study examined the effects of MWCNTs with smaller diameters ranging from 10 to 30 nm. Also, a sample of single-walled carbon nanotubes (SWCNTs) exhibiting single fiber diameters significantly below 10 nm and showing a flexible geometry was included since individual SWCNT fibers can aggregate to form bundles that exhibit increased rigidity. Additionally, the carcinogenic effect of pitch-based carbon fiber fragments was investigated. Carbon fibers are industrially produced with diameters larger than 4 µm and are thus not per se respirable. However, pitch-based fibers tend to break along their longitudinal axis, resulting in respirable fragments, partially of critical WHO dimensions. Four CNT samples with a geometric mean diameter (GMD) of 30 nm, 20 nm, 10 nm, and smaller than 10 nm, as well as one fragmented carbon fiber sample (GMD 1.3 µm) were intraperitoneally injected into rats in two dosages (0.1 × 109 and 1 × 109 WHO fibers or WHO-analog nanofibers) and observed for up to 24 months. A long amosite asbestos (GMD 0.37 µm) with known fiber-specific carcinogenic effect served as a positive control (0.1 × 109 WHO fibers).

Results: A small number of mesotheliomas occurred in all fiber types, but not at all dosages. For the carbon fiber material, a possible weak carcinogenic potency is seen at the higher dosage. For the SWCNT fiber, low number of mesotheliomas likewise suggest a weak carcinogenic potency. In the case of the MWCNT fiber with a GMD of 30 nm, very low number of mesotheliomas indicate a possible very weak carcinogenic potency. No clear carcinogenic potency was observed for the MWCNTs with GMDs of 20 nm and 10 nm.

Conclusions: Carbon fiber fragments and thin but bundled MWCNTs showed weak carcinogenic potency. Non-bundled MWCNTs with a diameter below 30 nm did not show clearcarcinogenic potency at a dose up to 1 × 109 WHO-analog nanofibers.

© 2026. The Author(s).
Molecular mechanisms of dust-induced lung disease: exploring the role of physicochemical composition and cellular pathways. Y. Sun, T.D. Waite. 2026;23(1). Review PubMed · Publisher · Free full text

Abstract

Background: Respirable dust and fibres in occupational settings are major contributors to lung dysfunction, imposing a significant societal burden. Following the ban on asbestos in most countries, silica and coal mine dust have emerged as primary concerns due to their abundance and persistence in workplace dust. Exposure to dust containing these elements has been strongly linked to pulmonary fibrosis and lung cancer.

Main Body: Extensive research has explored the molecular mechanisms underlying respirable occupational dust-induced cytotoxicity and immune dysregulation. The toxic effects of respirable occupational dust and fibres are largely induced by oxidative stress, mitochondrial dysfunction, lysosomal damage, inflammasome activation and immune suppression. These exposures can trigger multiple cell death pathways, including ferroptosis, necrosis, and pyroptosis. While sharing common toxicological features, silica, coal dust, and asbestos exhibit distinct toxic effects on various pulmonary cell types involved in fibrosis and cancer progression. The immunological consequences of dust exposure are profound, as dust-induced activation of macrophages and neutrophils, together with natural killer (NK) cell dysfunction, sustains chronic inflammation and disrupts epithelial repair and fibroblast regulation, thereby promoting fibrosis and tumorigenesis. Although no curative treatments exist for pulmonary fibrosis, especially coal workers' pneumoconiosis and silicosis, this review also discusses emerging therapeutic approaches, such as antifibrotic agents and immunotherapy, aimed at mitigating lung fibrosis and lung cancer.

Conclusion: This review summarises recent findings on the toxicological mechanisms underlying respirable occupational dust- and fibre-induced lung diseases, with a focus on crystalline silica, and coal dust and asbestos, especially those bearing reduced iron minerals. By highlighting both cellular toxicity and immunological effects, as well as potential therapeutic strategies, this work provides insights into current trends, challenges, and future research directions in the study of respirable occupational dust- and fibre-induced lung diseases.

© 2026. The Author(s).
Pulmonary toxicity of brake wear particles: effects of composition and surface reactivity. G. Kim, S. Jeon, T.H. Shin, et al. 2026;23(1). PubMed · Publisher · Free full text

Abstract

Background: Airborne brake wear particles (BWPs) are increasingly recognized as important non-exhaust contributors to urban particulate pollution; however, their inhalation toxicity and the key determinants of their pulmonary effects remain poorly defined.

Results: Two types of BWPs were generated using a brake dynamometer equipped with non-asbestos organic (NAO) and low-metallic (LM) brake pads on a cast-iron disc. The ≤ 2.5 μm fractions of BWPs generated from the NAO and LM brake pads were designated NAO2.5 and LM2.5, respectively. SRM 2975 (diesel exhaust particles) and Fe2O3 nanoparticles were included as reference particles. The test particles were characterized for their physicochemical properties, including morphology, size, surface area, crystallinity, colloidal properties, chemical composition, and solubility. The intrinsic oxidative potentials (IOPs) of the test particles, evaluated using a cell-free 2',7'-dichlorodihydrofluorescein diacetate assay, ranked as NAO2.5 > SRM 2975 > LM2.5 > Fe2O3 on a mass basis, and as NAO2.5 > LM2.5 > SRM 2975 > Fe2O3 on a surface area basis. Pulmonary neutrophilic inflammatory responses were evaluated by bronchoalveolar lavage fluid analysis at 24 h after pharyngeal aspiration of 25, 50, and 100 µg/mouse in female BALB/c mice. On a mass basis, the inflammatory response ranked SRM 2975 > NAO2.5 > Fe2O3 > LM2.5 on a mass basis, whereas on a surface area basis, the ranking was NAO2.5 > LM2.5 > SRM 2975 > Fe2O3. Lung burden analysis at days 0, 1, and 28 after a single pharyngeal aspiration of 100 µg/mouse showed relatively higher 28-day retention of NAO2.5 and LM2.5 (approximately 75% and 67%, respectively) compared to that of SRM 2975 (47%).

Conclusions: These data indicate that NAO2.5 is more inflammogenic than LM2.5. Given that BWPs were larger and had substantially lower surface area than the reference particles, their stronger responses under surface area-based comparison suggest greater inflammatory potency per unit surface area. Overall, the BWPs examined in this study exhibited a toxicity profile not fully explained by IOP alone, suggesting an important contribution of particle-specific physicochemical properties.

© 2026. The Author(s).
Copper-enriched automotive brake wear particles perturb human alveolar cellular homeostasis. J.G.H. Parkin, L.S.N. Dean, J.A. Bell, et al. 2025;22(1):4. PubMed · Publisher · Free full text

Abstract

Background: Airborne fine particulate matter with diameter < 2.5 μm (PM2.5), can reach the alveolar regions of the lungs, and is associated with over 4 million premature deaths per year worldwide. However, the source-specific consequences of PM2.5 exposure remain poorly understood. A major, but unregulated source is car brake wear, which exhaust emission reduction measures have not diminished.

Methods: We used an interdisciplinary approach to investigate the consequences of brake-wear PM2.5 exposure upon lung alveolar cellular homeostasis using diesel exhaust PM as a comparator. This involved RNA-Seq to analyse global transcriptomic changes, metabolic analyses to investigate glycolytic reprogramming, mass spectrometry to determine PM composition, and reporter assays to provide mechanistic insight into differential effects.

Results: We identified brake-wear PM from copper-enriched non-asbestos organic, and ceramic brake pads as inducing the greatest oxidative stress, inflammation, and pseudohypoxic HIF activation (a pathway implicated in diseases associated with air pollution exposure, including cancer, and pulmonary fibrosis), as well as perturbation of metabolism, and metal homeostasis compared with brake wear PM from low- or semi-metallic pads, and also, importantly, diesel exhaust PM. Compositional and metal chelator analyses identified that differential effects were driven by copper.

Conclusions: We demonstrate here that brake-wear PM may perturb cellular homeostasis more than diesel exhaust PM. Our findings demonstrate the potential differences in effects, not only for non-exhaust vs exhaust PM, but also amongst different sources of non-exhaust PM. This has implications for our understanding of the potential health effects of road vehicle-associated PM. More broadly, our findings illustrate the importance of PM composition on potential health effects, highlighting the need for targeted legislation to protect public health.

© 2025. The Author(s).
Malignant peritoneal mesotheliomas of rats induced by multiwalled carbon nanotubes and amosite asbestos: transcriptome and epigenetic profiles. S.M. Reamon-Buettner, S. Rittinghausen, A. Klauke, et al. 2024;21(1):3. PubMed · Publisher · Free full text

Abstract

Background: Malignant mesothelioma is an aggressive cancer that often originates in the pleural and peritoneal mesothelium. Exposure to asbestos is a frequent cause. However, studies in rodents have shown that certain multiwalled carbon nanotubes (MWCNTs) can also induce malignant mesothelioma. The exact mechanisms are still unclear. To gain further insights into molecular pathways leading to carcinogenesis, we analyzed tumors in Wistar rats induced by intraperitoneal application of MWCNTs and amosite asbestos. Using transcriptomic and epigenetic approaches, we compared the tumors by inducer (MWCNTs or amosite asbestos) or by tumor type (sarcomatoid, epithelioid, or biphasic).

Results: Genome-wide transcriptome datasets, whether grouped by inducer or tumor type, showed a high number of significant differentially expressed genes (DEGs) relative to control peritoneal tissues. Bioinformatic evaluations using Ingenuity Pathway Analysis (IPA) revealed that while the transcriptome datasets shared commonalities, they also showed differences in DEGs, regulated canonical pathways, and affected molecular functions. In all datasets, among highly- scoring predicted canonical pathways were Phagosome Formation, IL8 Signaling, Integrin Signaling, RAC Signaling, and TREM1 Signaling. Top-scoring activated molecular functions included cell movement, invasion of cells, migration of cells, cell transformation, and metastasis. Notably, we found many genes associated with malignant mesothelioma in humans, which showed similar expression changes in the rat tumor transcriptome datasets. Furthermore, RT-qPCR revealed downregulation of Hrasls, Nr4a1, Fgfr4, and Ret or upregulation of Rnd3 and Gadd45b in all or most of the 36 tumors analyzed. Bisulfite sequencing of Hrasls, Nr4a1, Fgfr4, and Ret revealed heterogeneity in DNA methylation of promoter regions. However, higher methylation percentages were observed in some tumors compared to control tissues. Lastly, global 5mC DNA, m6A RNA and 5mC RNA methylation levels were also higher in tumors than in control tissues.

Conclusions: Our findings may help better understand how exposure to MWCNTs can lead to carcinogenesis. This information is valuable for risk assessment and in the development of safe-by-design strategies.

© 2024. The Author(s).
Synthetic silica fibers of different length, diameter and shape: synthesis and interaction with rat (NR8383) and human (THP-1) macrophages in vitro, including chemotaxis and gene expression profile. N. Białas, N. Rosenkranz, D.G. Weber, et al. 2024;21(1):23. PubMed · Publisher · Free full text

Abstract

Background: Inhalation of biopersistent fibers like asbestos can cause strong chronic inflammatory effects, often resulting in fibrosis or even cancer. The interplay between fiber shape, fiber size and the resulting biological effects is still poorly understood due to the lack of reference materials.

Results: We investigated how length, diameter, aspect ratio, and shape of synthetic silica fibers influence inflammatory effects at doses up to 250 µg cm-2. Silica nanofibers were prepared with different diameter and shape. Straight (length ca. 6 to 8 µm, thickness ca. 0.25 to 0.35 µm, aspect ratio ca. 17:1 to 32:1) and curly fibers (length ca. 9 µm, thickness ca. 0.13 µm, radius of curvature ca. 0.5 µm, aspect ratio ca. 70:1) were dispersed in water with no apparent change in the fiber shape during up to 28 days. Upon immersion in aqueous saline (DPBS), the fibers released about 5 wt% silica after 7 days irrespectively of their shape. The uptake of the fibers by macrophages (human THP-1 and rat NR8383) was studied by scanning electron microscopy and confocal laser scanning microscopy. Some fibers were completely taken up whereas others were only partially internalized, leading to visual damage of the cell wall. The biological effects were assessed by determining cell toxicity, particle-induced chemotaxis, and the induction of gene expression of inflammatory mediators.

Conclusions: Straight fibers were only slightly cytotoxic and caused weak cell migration, regardless of their thickness, while the curly fibers were more toxic and caused significantly stronger chemotaxis. Curly fibers also had the strongest effect on the expression of cytokines and chemokines. This may be due to the different aspect ratio or its twisted shape.

© 2024. The Author(s).
An investigation of the internal morphology of asbestos ferruginous bodies: constraining their role in the onset of malignant mesothelioma. M.L. Avramescu, C. Potiszil, T. Kunihiro, et al. 2023;20(1):19. PubMed · Publisher · Free full text

Abstract

Background: Asbestos is a fibrous mineral that was widely used in the past. However, asbestos inhalation is associated with an aggressive type of cancer known as malignant mesothelioma (MM). After inhalation, an iron-rich coat forms around the asbestos fibres, together the coat and fibre are termed an "asbestos ferruginous body" (AFB). AFBs are the main features associated with asbestos-induced MM. Whilst several studies have investigated the external morphology of AFBs, none have characterised the internal morphology. Here, cross-sections of multiple AFBs from two smokers and two non-smokers are compared to investigate the effects of smoking on the onset and growth of AFBs. Morphological and chemical observations of AFBs were undertaken by transmission electron microscopy, energy dispersive x-ray spectroscopy and selected area diffraction.

Results: The AFBs of all patients were composed of concentric layers of 2-line or 6-line ferrihydrite, with small spherical features being observed on the outside of the AFBs and within the cross-sections. The spherical components are of a similar size to Fe-rich inclusions found within macrophages from mice injected with asbestos fibres in a previous study. As such, the spherical components composing the AFBs may result from the deposition of Fe-rich inclusions during frustrated phagocytosis. The AFBs were also variable in terms of their Fe, P and Ca abundances, with some layers recording higher Fe concentrations (dense layers), whilst others lower Fe concentrations (porous layers). Furthermore, smokers were found to have smaller and overall denser AFBs than non-smokers.

Conclusions: The AFBs of smokers and non-smokers show differences in their morphology, indicating they grew in lung environments that experienced disparate conditions. Both the asbestos fibres of smokers and non-smokers were likely subjected to frustrated phagocytosis and accreted mucopolysaccharides, resulting in Fe accumulation and AFB formation. However, smokers' AFBs experienced a more uniform Fe-supply within the lung environment compared to non-smokers, likely due to Fe complexation from cigarette smoke, yielding denser, smaller and more Fe-rich AFBs. Moreover, the lack of any non-ferrihydrite Fe phases in the AFBs may indicate that the ferritin shell was intact, and that ROS may not be the main driver for the onset of MM.

© 2023. The Author(s).
Difference in carcinogenicities of two different vapor grown carbon fibers with different physicochemical characteristics induced by intratracheal instillation in rats. K. Sato, H. Fukui, Y. Hagiwara, et al. 2023;20(1):37. PubMed · Publisher · Free full text

Abstract

Background: Carbon fibers are high aspect ratio structures with diameters on the submicron scale. Vapor grown carbon fibers are contained within multi-walled carbon tubes, with VGCF™-H commonly applied as a conductive additive in lithium-ion batteries. However, several multi-walled carbon fibers, including MWNT-7, have been reported to induce lung carcinogenicity in rats. This study investigated the carcinogenic potential of VGCF™-H fibers in F344 rats of both sexes with the vapor grown carbon fibers VGCF™-H and MWNT-7 over 2 years. The carbon fibers were administered to rats by intratracheal instillation at doses of 0, 0.016, 0.08, and 0.4 mg/kg (total doses of 0, 0.128, 0.64, and 3.2 mg/kg) once per week for eight weeks and the rats were observed for up to 2 years after the first instillation.

Results: Histopathological examination showed the induction of malignant mesothelioma on the pleural cavity with dose-dependent increases observed at 0, 0.128, 0.64, and 3.2 mg/kg in rats of both sexes that were exposed to MWNT-7. On the other hand, only two cases of pleural malignant mesothelioma were observed in the VGCF™-H groups; both rats that received 3.2 mg/kg in male. The animals in the MWNT-7 groups either died or became moribund earlier than those in the VGCF™-H groups, which is thought related to the development of malignant mesothelioma. The survival rates were higher in the VGCF™-H group, and more carbon fibers were observed in the pleural lavage fluid (PLF) of the MWNT-7 groups. These results suggest that malignant mesothelioma is related to the transfer of carbon fibers into the pleural cavity.

Conclusions: The intratracheal instillation of MWNT-7 clearly led to carcinogenicity in both male and female rats at all doses. The equivocal evidence for carcinogenic potential that was observed in male rats exposed to VGCF™-H was not seen in the females. The differences in the carcinogenicities of the two types of carbon fibers are thought due to differences in the number of carbon fibers reaching the pleural cavity. The results indicate that the carcinogenic activity of VGCF™-H is lower than that of MWNT-7.

© 2023. BioMed Central Ltd., part of Springer Nature.
Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the rat lung after intratracheal instillation: a two-year study. D.M. Saleh, S. Luo, O.H.M. Ahmed, et al. 2022;19(1):30. PubMed · Publisher · Free full text

Abstract

Background: Considering the expanding industrial applications of carbon nanotubes (CNTs), safety assessment of these materials is far less than needed. Very few long-term in vivo studies have been carried out. This is the first 2-year in vivo study to assess the effects of double walled carbon nanotubes (DWCNTs) in the lung and pleura of rats after pulmonary exposure.

Methods: Rats were divided into six groups: untreated, Vehicle, 3 DWCNT groups (0.12 mg/rat, 0.25 mg/rat and 0.5 mg/rat), and MWCNT-7 (0.5 mg/rat). The test materials were administrated by intratracheal-intrapulmonary spraying (TIPS) every other day for 15 days. Rats were observed without further treatment until sacrifice.

Results: DWCNT were biopersistent in the rat lung and induced marked pulmonary inflammation with a significant increase in macrophage count and levels of the chemotactic cytokines CCL2 and CCL3. In addition, the 0.5 mg DWCNT treated rats had significantly higher pulmonary collagen deposition compared to the vehicle controls. The development of carcinomas in the lungs of rats treated with 0.5 mg DWCNT (4/24) was not quite statistically higher (p = 0.0502) than the vehicle control group (0/25), however, the overall incidence of lung tumor development, bronchiolo-alveolar adenoma and bronchiolo-alveolar carcinoma combined, in the lungs of rats treated with 0.5 mg DWCNT (7/24) was statistically higher (p < 0.05) than the vehicle control group (1/25). Notably, two of the rats treated with DWCNT, one in the 0.25 mg group and one in the 0.5 mg group, developed pleural mesotheliomas. However, both of these lesions developed in the visceral pleura, and unlike the rats administered MWCNT-7, rats administered DWCNT did not have elevated levels of HMGB1 in their pleural lavage fluids. This indicates that the mechanism by which the mesotheliomas that developed in the DWCNT treated rats is not relevant to humans.

Conclusions: Our results demonstrate that the DWCNT fibers we tested are biopersistent in the rat lung and induce chronic inflammation. Rats treated with 0.5 mg DWCNT developed pleural fibrosis and lung tumors. These findings demonstrate that the possibility that at least some types of DWCNTs are fibrogenic and tumorigenic cannot be ignored.

© 2022. The Author(s).
Exposure to silicates and systemic autoimmune-related outcomes in rodents: a systematic review. L.M.F. Janssen, M. Ghosh, F. Lemaire, et al. 2022;19(1):4. Systematic review PubMed · Publisher · Free full text

Abstract

Background: Autoimmunity can result from the interplay between genetic background and effects of environmental and/or occupational exposure to hazardous materials. Several compounds, including silica dust, have been linked with systemic autoimmunity and systemic autoimmune diseases, based on epidemiological evidence. For asbestos, a strong link with systemic autoimmune diseases does not yet exist, however, several studies have documented features of autoimmunity following asbestos exposure. Even so, human studies are limited in their ability to identify and examine isolated exposures, making it difficult to demonstrate causation or to assess pathogenic mechanisms. Therefore, this systematic review examines the existing animal evidence regarding autoimmunity and exposure to silicates (silica and asbestos).

Methods: PubMed and EMBASE were systematically searched for peer-reviewed studies examining systemic autoimmune disease-related outcomes after silicate exposure in rodents. Literature databases were searched up to September 2021 for studies written in English and where the full text was available. Search strings were established based on a PECO (Population, Exposure, Comparator, Outcome) format. After title, abstract, and full-text screening, thirty-four studies were identified for further analysis. Quality assessment through ToxR tool and qualitative analysis of the results was performed.

Results: Although there was significant heterogeneity in the included studies in terms of exposure protocol and genetic background of the rodent models used, it was noted that both genetic background and exposure to silicates [(crystalline) silica and asbestos] are highly relevant to the development of (sub-) clinical systemic autoimmune disease.

Conclusion: Parallels were observed between the findings from the animal (this review) and human (epidemiological) studies, arguing that experimental animal models are valuable tools for examining exacerbation or development of autoimmune disease after silicate exposure. However, genetic background and synergism between exposures should be considered in future studies.

© 2022. The Author(s).
Inflammogenic effect of polyacrylic acid in rat lung following intratracheal instillation. C. Nishida, T. Tomonaga, H. Izumi, et al. 2022;19(1):8. PubMed · Publisher · Free full text

Abstract

Background: Some organic chemicals are known to cause allergic disorders such as bronchial asthma and hypersensitivity pneumonitis, and it has been considered that they do not cause irreversible pulmonary fibrosis. It has recently been reported, however, that cross-linked acrylic acid-based polymer, an organic chemical, might cause serious interstitial lung diseases, including pulmonary fibrosis. We investigated whether or not intratracheal instillation exposure to cross-linked polyacrylic acid (CL-PAA) can cause lung disorder in rats.

Methods: Male F344 rats were intratracheally instilled with dispersed CL-PAA at low (0.2 mg/rat) and high (1.0 mg/rat) doses, and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months after exposure to examine inflammatory and fibrotic responses and related gene expressions in the lungs. Rat lungs exposed to crystalline silica, asbestos (chrysotile), and NiO and CeO2 nanoparticles were used as comparators.

Results: Persistent increases in total cell count, neutrophil count and neutrophil percentage, and in the concentration of the cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2 and C-X-C motif chemokine 5 (CXCL5), which correlated with lung tissue gene expression, were observed in bronchoalveolar lavage fluid (BALF) from 3 days until at least 1 month following CL-PAA intratracheal instillation. Persistent increases in heme oxygenase-1 (HO-1) in the lung tissue were also observed from 3 days to 6 months after exposure. Histopathological findings of the lungs demonstrated that extensive inflammation at 3 days was greater than that in exposure to silica, NiO nanoparticles and CeO2 nanoparticles, and equal to or greater than that in asbestos (chrysotile) exposure, and the inflammation continued until 1 month. Fibrotic changes also progressed after 1 month postexposure.

Conclusion: Our results suggested that CL-PAA potentially causes strong neutrophil inflammation in the rat and human lung.

© 2022. The Author(s).
The importance of mineralogical composition for the cytotoxic and pro-inflammatory effects of mineral dust. V.S. Grytting, M. Refsnes, M. Låg, et al. 2022;19(1):46. PubMed · Publisher · Free full text

Abstract

Background: Respirable mineral particles represent a potential health hazard in occupational settings and ambient air. Previous studies show that mineral particles may induce cytotoxicity and inflammatory reactions in vitro and in vivo and that the potency varies between samples of different composition. However, the reason for these differences is largely unknown and the impact of mineralogical composition on the biological effects of mineral dust remains to be determined.

Methods: We have assessed the cytotoxic and pro-inflammatory effects of ten mineral particle samples of different composition in human bronchial epithelial cells (HBEC3-KT) and THP-1-derived macrophages, as well as their membranolytic properties in erythrocytes. Moreover, the results were compiled with the results of recently published experiments on the effects of stone particle exposure and analysed using linear regression models to elucidate which mineral components contribute most to the toxicity of mineral dust.

Results: While all mineral particle samples were more cytotoxic to HBEC3-KT cells than THP-1 macrophages, biotite and quartz were among the most cytotoxic in both cell models. In HBEC3-KT cells, biotite and quartz also appeared to be the most potent inducers of pro-inflammatory cytokines, while the quartz, Ca-feldspar, Na-feldspar and biotite samples were the most potent in THP-1 macrophages. All particle samples except quartz induced low levels of membranolysis. The regression analyses revealed associations between particle bioactivity and the content of quartz, muscovite, plagioclase, biotite, anorthite, albite, microcline, calcite, chlorite, orthopyroxene, actinolite and epidote, depending on the cell model and endpoint. However, muscovite was the only mineral consistently associated with increased cytotoxicity and cytokine release in both cell models.

Conclusions: The present study provides further evidence that mineral particles may induce cytotoxicity and inflammation in cells of the human airways and that particle samples of different mineralogical composition differ in potency. The results show that quartz, while being among the most potent samples, does not fully predict the toxicity of mineral dust, highlighting the importance of other particle constituents. Moreover, the results indicate that the phyllosilicates muscovite and biotite may be more potent than other minerals assessed in the study, suggesting that this group of sheet-like minerals may warrant further attention.

© 2022. The Author(s).
Two-year intermittent exposure of a multiwalled carbon nanotube by intratracheal instillation induces lung tumors and pleural mesotheliomas in F344 rats. M. Hojo, A. Maeno, Y. Sakamoto, et al. 2022;19(1):38. PubMed · Publisher · Free full text

Abstract

Background: A mounting number of studies have been documenting the carcinogenic potential of multiwalled carbon nanotubes (MWCNTs); however, only a few studies have evaluated the pulmonary carcinogenicity of MWCNTs in vivo. A 2-year inhalation study demonstrated that MWNT-7, a widely used MWCNT, was a pulmonary carcinogen in rats. In another 2-year study, rats administered MWNT-7 by intratracheal instillation at the beginning of the experimental period developed pleural mesotheliomas but not lung tumors. To obtain data more comparable with rats exposed to MWNT-7 by inhalation, we administered MWNT-7 to F344 rats by intratracheal instillation once every 4-weeks over the course of 2 years at 0, 0.125, and 0.5 mg/kg body weight, allowing lung burdens of MWNT-7 to increase over the entire experimental period, similar to the inhalation study.

Results: Absolute and relative lung weights were significantly elevated in both MWNT-7-treated groups. Dose- and time-dependent toxic effects in the lung and pleura, such as inflammatory, fibrotic, and hyperplastic lesions, were found in both treated groups. The incidences of lung carcinomas, lung adenomas, and pleural mesotheliomas were significantly increased in the high-dose group compared with the control group. The pleural mesotheliomas developed mainly at the mediastinum. No MWNT-7-related neoplastic lesions were noted in the other organs. Cytological and biochemical parameters of the bronchoalveolar lavage fluid (BALF) were elevated in both treated groups. The lung burden of MWNT-7 was dose- and time-dependent, and at the terminal necropsy, the average value was 0.9 and 3.6 mg/lung in the low-dose and high-dose groups, respectively. The number of fibers in the pleural cavity was also dose- and time-dependent.

Conclusions: Repeated administration of MWNT-7 by intratracheal instillation over the 2 years indicates that MWNT-7 is carcinogenic to both the lung and pleura of rats, which differs from the results of the 2 carcinogenicity tests by inhalation or intratracheal instillation.

© 2022. The Author(s).
Respirable stone particles differ in their ability to induce cytotoxicity and pro-inflammatory responses in cell models of the human airways. V.S. Grytting, M. Refsnes, J. Øvrevik, et al. 2021;18(1):18. PubMed · Publisher · Free full text

Abstract

Background: Respirable stone- and mineral particles may be a major constituent in occupational and ambient air pollution and represent a possible health hazard. However, with exception of quartz and asbestos, little is known about the toxic properties of mineral particles. In the present study, the pro-inflammatory and cytotoxic responses to six stone particle samples of different composition and with diameter below 10 μm were assessed in human bronchial epithelial cells (HBEC3-KT), THP-1 macrophages and a HBEC3-KT/THP-1 co-culture. Moreover, particle-induced lysis of human erythrocytes was assessed to determine the ability of the particles to lyse biological membranes. Finally, the role of the NLRP3 inflammasome was assessed using a NLRP3-specific inhibitor and detection of ASC oligomers and cleaved caspase-1 and IL-1β. A reference sample of pure α-quartz was included for comparison.

Results: Several stone particle samples induced a concentration-dependent increase in cytotoxicity and secretion of the pro-inflammatory cytokines CXCL8, IL-1α, IL-1β and TNFα. In HBEC3-KT, quartzite and anorthosite were the most cytotoxic stone particle samples and induced the highest levels of cytokines. Quartzite and anorthosite were also the most cytotoxic samples in THP-1 macrophages, while anorthosite and hornfels induced the highest cytokine responses. In comparison, few significant differences between particle samples were detected in the co-culture. Adjusting responses for differences in surface area concentrations did not fully account for the differences between particle samples. Moreover, the stone particles had low hemolytic potential, indicating that the effects were not driven by membrane lysis. Pre-incubation with a NLRP3-specific inhibitor reduced stone particle-induced cytokine responses in THP-1 macrophages, but not in HBEC3-KT cells, suggesting that the effects are mediated through different mechanisms in epithelial cells and macrophages. Particle exposure also induced an increase in ASC oligomers and cleaved caspase-1 and IL-1β in THP-1 macrophages, confirming the involvement of the NLRP3 inflammasome.

Conclusions: The present study indicates that stone particles induce cytotoxicity and pro-inflammatory responses in human bronchial epithelial cells and macrophages, acting through NLRP3-independent and -dependent mechanisms, respectively. Moreover, some particle samples induced cytotoxicity and cytokine release to a similar or greater extent than α-quartz. Thus, these minerals warrant further attention in future research.
Rounded atelectasis after exposure to refractory ceramic fibres (RCF). U. Brueckner, A.S. Schulze, D. Walter, et al. 2021;18(1):48. PubMed · Publisher · Free full text

Abstract

Background: Refractory Ceramic fibres (RCF) are man-made mineral fibres used in high performance thermal insulation applications. Analogous to asbestos fibres, RCF are respirable, show a pleural drift and can persist in human lung tissue for more than 20 years after exposure. Pleural changes such as localised or diffuse pleural thickening as well as pleural calcification were reported.

Result: A 45 years old man worked in high performance thermal insulation applications using refractory ceramic fibres (RCF) for almost 20 years. During a occupational medical prophylaxis to ensure early diagnosis of disorders caused by inhalation of aluminium silicate fibres with X-ray including high-resolution computed tomography (HRCT), bilateral pleural thickening was shown and a pleural calcification next to a rounded atelectasis was detected. Asbestos exposure could be excluded. In pulmonary function test a restrictive lung pattern could be revealed. In work samples scanning electron microscopy (SEM) including energy dispersive X-ray analysis (EDX) classified used fibres as aluminium silicate fibres. X-ray powder diffraction (XRD) and transmission electron microscopy (TEM) showed crystalline as well as amorphous fibres.

Conclusions: A comprehensive lung function analysis and in case of restrictive lung disorders additional CT scans are needed in RCF exposed workers in accordance to the guidelines for medical occupational examinations comparable to asbestos exposed workers.

© 2021. The Author(s).
Analysis of particles from hamster lungs following pulmonary talc exposures: implications for pathogenicity. E. Sato, S.A. McDonald, Y. Fan, et al. 2020;17(1):20. PubMed · Publisher · Free full text

Abstract

Background: Talc, a hydrous magnesium silicate, often used for genital hygiene purposes, is associated with ovarian carcinoma in case-control studies. Its potential to cause inflammation, injury, and functional changes in cells has been described. A complication of such studies is that talc preparations may be contaminated with other materials. A previous study by (Beck et al. Toxicol Appl Pharmacol 87:222-34, 1987) used a hamster model to study talc and granite dust exposure effects on various biochemical and cellular inflammatory markers. Our current study accessed key materials used in that 1987 study; we re-analyzed the original talc dust with contemporary scanning electron microscopy and energy dispersive x-ray analysis (SEM/EDX) for contaminants. We also examined the original bronchoalveolar lavage (BAL) cells with polarized light microscopy to quantify cell-associated birefringent particles to gain insight into the talc used.

Results: SEM/EDX analyses showed that asbestos fibers, quartz, and toxic metal particulates were below the limits of detection in the original talc powder. However, fibers with aspect ratios ≥3:1 accounted for 22% of instilled material, mostly as fibrous talc. Talc (based on Mg/Si atomic weight % ratio) was the most abundant chemical signature, and magnesium silicates with various other elements made up the remainder. BAL cell counts confirmed the presence of acute inflammation, which followed intratracheal instillation. Measurements of cell associated birefringent particles phagocytosis revealed significant differences among talc, granite, and control exposures with high initial uptake of talc compared to granite, but over the 14-day experiment, talc phagocytosis by lavaged cells was significantly less than that of granite. Phagocytosis of talc fibers by macrophages was observed, and birefringent particles were found in macrophages, neutrophils, and multinucleate giant cells in lavaged cells from talc-exposed animals.

Conclusion: Our data support the contention that talc, even without asbestos and other known toxic contaminants, may elicit inflammation and contribute to lung disease. Our findings support the conclusions of (Beck et al. Toxicol Appl Pharmacol 87:222-34, 1987) study. By analyzing particulate exposures with polarized light microscopy and SEM/EDX, fibrous talc was identified and a distinctive pattern of impaired particulate ingestion was demonstrated.
Comparative carcinogenicity study of a thick, straight-type and a thin, tangled-type multi-walled carbon nanotube administered by intra-tracheal instillation in the rat. D.M. Saleh, W.T. Alexander, T. Numano, et al. 2020;17(1):48. PubMed · Publisher · Free full text

Abstract

Background: Multi-walled carbon nanotubes can be divided into two general subtypes: tangled and straight. MWCNT-N (60 nm in diameter) and MWCNT-7 (80-90 nm in diameter) are straight-type MWCNTs, and similarly to asbestos, both are carcinogenic to the lung and pleura when administered to rats via the airway. Injection of straight-type MWCNTs into the peritoneal cavity also induces the development of mesothelioma, however, injection of tangled-type MWCNTs into the peritoneal cavity does not induce carcinogenesis. To investigate these effects in the lung we conducted a 2-year comparative study of the potential carcinogenicities of a straight-type MWCNT, MWCNT-A (approximately 150 nm in diameter), and a tangled-type MWCNT, MWCNT-B (7.4 nm in diameter) after administration into the rat lung. Crocidolite asbestos was used as the reference material, and rats administered vehicle were used as the controls. Test materials were administered by intra-Tracheal Intra-Pulmonary Spraying (TIPS) once a week over a 7 week period (8 administrations from day 1 to day 50), followed by a 2-year observation period without further treatment. Rats were administered total doses of 0.5 or 1.0 mg MWCNT-A and MWCNT-B or 1.0 mg asbestos.

Results: There was no difference in survival between any of the groups. The rats administered MWCNT-A or asbestos did not have a significant increase in bronchiolo-alveolar hyperplasia or tumors in the lung. However, the rats administered MWCNT-B did have significantly elevated incidences of bronchiolo-alveolar hyperplasia and tumors in the lung: the incidence of bronchiolo-alveolar hyperplasia was 0/20, 6/20, and 9/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively, and the incidence of adenoma and adenocarcinoma combined was 1/19, 5/20, and 7/20 in the vehicle, 0.5 mg MWCNT-B, and 1.0 mg MWCNT-B groups, respectively. Malignant pleural mesothelioma was not induced in any of the groups.

Conclusions: The results of this initial study indicate that tangled-type MWCNT-B is carcinogenic to the rat lung when administered via the airway, and that straight-type MWCNT-A did not have higher carcinogenic potential in the rat lung than tangled-type MWCNT-B.
Identifying the reactive sites of hydrogen peroxide decomposition and hydroxyl radical formation on chrysotile asbestos surfaces. M. Walter, W.D.C. Schenkeveld, G. Geroldinger, et al. 2020;17(1):3. PubMed · Publisher · Free full text

Abstract

Background: Fibrous chrysotile has been the most commonly applied asbestos mineral in a range of technical applications. However, it is toxic and carcinogenic upon inhalation. The chemical reactivity of chrysotile fiber surfaces contributes to its adverse health effects by catalyzing the formation of highly reactive hydroxyl radicals (HO•) from H2O2. In this Haber-Weiss cycle, Fe on the fiber surface acts as a catalyst: Fe3+ decomposes H2O2 to reductants that reduce surface Fe3+ to Fe2+, which is back-oxidized by H2O2 (Fenton-oxidation) to yield HO•. Chrysotile contains three structural Fe species: ferrous and ferric octahedral Fe and ferric tetrahedral Fe (Fe3+tet). Also, external Fe may adsorb or precipitate onto fiber surfaces. The goal of this study was to identify the Fe species on chrysotile surfaces that catalyze H2O2 decomposition and HO• generation.

Results: We demonstrate that at the physiological pH 7.4 Fe3+tet on chrysotile surfaces substantially contributes to H2O2 decomposition and is the key structural Fe species catalyzing HO• generation. After depleting Fe from fiber surfaces, a remnant fiber-related H2O2 decomposition mode was identified, which may involve magnetite impurities, remnant Fe or substituted redox-active transition metals other than Fe. Fe (hydr)oxide precipitates on chrysotile surfaces also contributed to H2O2 decomposition, but were per mole Fe substantially less efficient than surface Fe3+tet. Fe added to chrysotile fibers increased HO• generation only when it became incorporated and tetrahedrally coordinated into vacancy sites in the Si layer.

Conclusions: Our results suggest that at the physiological pH 7.4, oxidative stress caused by chrysotile fibers largely results from radicals produced in the Haber-Weiss cycle that is catalyzed by Fe3+tet. The catalytic role of Fe3+tet in radical generation may also apply to other pathogenic silicates in which Fe3+tet is substituted, e.g. quartz, amphiboles and zeolites. However, even if these pathogenic minerals do not contain Fe, our results suggest that the mere presence of vacancy sites may pose a risk, as incorporation of external Fe into a tetrahedral coordination environment can lead to HO• generation.

2010s

A novel human 3D lung microtissue model for nanoparticle-induced cell-matrix alterations. P.K. Kabadi, A.L. Rodd, A.E. Simmons, et al. 2019;16(1):15. PubMed · Publisher · Free full text

Abstract

Background: Multi-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm.

Results: As inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins.

Conclusion: 3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.
Carcinogenic effect of potassium octatitanate (POT) fibers in the lung and pleura of male Fischer 344 rats after intrapulmonary administration. M. Abdelgied, A.M. El-Gazzar, W.T. Alexander, et al. 2019;16(1):34. PubMed · Publisher · Free full text

Abstract

Background: Potassium octatitanate fibers (K2O•8TiO2, POT fibers) are used as an asbestos substitute. Their physical characteristics suggest that respirable POT fibers are likely to be carcinogenic in the lung and pleura. However, previous 2-year inhalation studies reported that respired POT fibers had little or no carcinogenic potential. In the present study ten-week old male F344 rats were left untreated or were administered vehicle, 0.25 or 0.5 mg rutile-type nano TiO2 (r-nTiO2), 0.25 or 0.5 mg POT fibers, or 0.5 mg MWCNT-7 by intra-tracheal intra-pulmonary spraying (TIPS), and then observed for 2 years.

Results: There were no differences between the r-nTiO2 and control groups. The incidence of bronchiolo-alveolar cell hyperplasia was significantly increased in the groups treated with 0.50 mg POT and 0.50 mg MWCNT-7. The overall incidence of lung tumors, however, was not increased in either the POT or MWCNT-7 treated groups. Notably, the carcinomas that developed in the POT and MWCNT-7 treated rats were accompanied by proliferative fibrous connective tissue while the carcinomas that developed in the untreated rats and the r-nTiO2 treated rats were not (carcinomas did not develop in the vehicle control rats). In addition, the carcinoma that developed in the rat treated with 0.25 mg POT was a squamous cell carcinoma, a tumor that develops spontaneously in about 1 per 1700 rats. The incidence of mesothelial cell hyperplasia was 4/17, 7/16, and 10/14 and the incidence of malignant mesothelioma was 3/17, 1/16, and 2/14 in the 0.25 mg POT, 0.5 mg POT, and MWCNT-7 treated groups, respectively. Neither mesothelial cell hyperplasia nor mesothelioma developed in control rats or the rats treated with r-nTiO2. Since the incidence of spontaneously occurring malignant mesothelioma in rats is extremely low, approximately 1 per 1000 animals (Japan Bioassay Research Center [JBRC] historical control data), the development of multiple malignant mesotheliomas in the POT and MWCNT-7 treated groups was biologically significant.

Conclusion: The incidence of pleural mesotheliomas in male F344 rats administered POT fibers and MWCNT-7 was significantly higher than the JBRC historical control data, indicating that the incidence of pleural mesothelioma in the groups administered POT fibers and MWCNT-7 fibers via the airway using TIPS was biologically significant. The incidence of type II epithelial cell hyperplasia and the histology of the carcinomas that developed in the POT treated rats also indicates that respirable POT fibers are highly likely to be carcinogenic in the lungs of male F344 rats.
Comment on Shvedova et al. (2016), "gender differences in murine pulmonary responses elicited by cellulose nanocrystals". J.A. Shatkin, G. Oberdörster. 2016;13(1):59. Letter · Comment PubMed · Publisher · Free full text

Abstract

A recent publication in "Particle and Fibre Toxicology" reported on the gender differences in pulmonary toxicity from oro-pharyngeal aspiration of a high dose of cellulose nanocrystals. The study is timely given the growing interest in diverse commercial applications of cellulose nanomaterials, and the need for studies addressing pulmonary toxicity. The results from this study are interesting and can be strengthened with a discussion of how differences in the weights of female and male C57BL/6 mice was accounted for. Without such a discussion, the observed differences could be partially explained by the lower body weights of females, resulting in higher doses than males when standardized to body weight or lung volume. Further, few conclusions can be drawn about the pulmonary toxicity of cellulose nanocrystals given the study design: examination of a single high dose of cellulose nanocrystals, administered as a bolus, without positive or negative controls or low dose comparisons, and at an unphysiological and high dose rate. Simulating the bolus type delivery by inhalation would require a highly unrealistic exposure concentration in the g/m3 range of extremely short duration. A discussion of these limitations is missing in the paper; further speculative comparisons of cellulose nanocrystals toxicity to asbestos and carbon nanotubes in the abstract are both unwarranted and can be misleading, these materials were neither mentioned in the manuscript, nor evaluated in the study.
Gender differences in murine pulmonary responses elicited by cellulose nanocrystals. A.A. Shvedova, E.R. Kisin, N. Yanamala, et al. 2016;13(1):28. PubMed · Publisher · Free full text

Abstract

Background: Cellulose-based materials have been used for centuries to manufacture different goods derived from forestry and agricultural sources. In the growing field of nanocellulose applications, its uniquely engineered properties are instrumental for inventive products coming to competitive markets. Due to their high aspect ratio and stiffness, it is speculated that cellulose nanocrystals (CNC) may cause similar pulmonary toxicity as carbon nanotubes and asbestos, thus posing a potential negative impact on public health and the environment.

Methods: The present study was undertaken to investigate the pulmonary outcomes induced by repeated exposure to respirable CNC. C57BL/6 female and male mice were exposed by pharyngeal aspiration to CNC (40 μg/mouse) 2 times a week for 3 weeks. Several biochemical endpoints and pathophysiological outcomes along with gene expression changes were evaluated and compared in the lungs of male and female mice.

Results: Exposure to respirable CNC caused pulmonary inflammation and damage, induced oxidative stress, elevated TGF-β and collagen levels in lung, and impaired pulmonary functions. Notably, these effects were markedly more pronounced in females compared to male mice. Moreover, sex differences in responses to pulmonary exposure to CNC were also detected at the level of global mRNA expression as well as in inflammatory cytokine/chemokine activity.

Conclusions: Overall, our results indicate that there are considerable differences in responses to respirable CNC based on gender with a higher pulmonary toxicity observed in female mice.
Lung carcinogenicity of inhaled multi-walled carbon nanotube in rats. T. Kasai, Y. Umeda, M. Ohnishi, et al. 2016;13(1):53. PubMed · Publisher · Free full text

Abstract

Background: Multi-walled carbon nanotubes (MWCNTs) constitute one of the most promising types of nanomaterials in industry today. With their increasing use, the potential toxicity and carcinogenicity of MWCNT needs to be evaluated in bioassay studies using rodents. Since humans are mainly exposed to MWCNT by inhalation, we performed a 104-week carcinogenicity study using whole-body inhalation exposure chambers with a fibrous straight type of MWCNT at concentrations of 0, 0.02, 0.2, and 2 mg/m3 using male and female F344 rats.

Results: Lung carcinomas, mainly bronchiolo-alveolar carcinoma, and combined carcinomas and adenomas were significantly increased in males exposed to 0.2 and 2 mg/m3 MWNT-7 and in females exposed to 2 mg/m3 MWNT-7 compared to the clean air control group. However, no development of pleural mesothelioma was observed. Concentration-dependent toxic effects in the lung such as epithelial hyperplasia, granulomatous change, localized fibrosis, and alteration in BALF parameters were found in MWNT-7 treatment groups of both sexes. There were no MWNT-7-specific macroscopic findings in the other organs, including the pleura and peritoneum. Absolute and relative lung weights were significantly elevated in male rats exposed to 0.2 and 2 mg/m3 MWNT-7 and in all exposed female groups. The lung burdens of MWNT-7 were clearly increased in a concentration-dependent as well as a duration-dependent manner.

Conclusion: There is clear evidence that MWNT-7 is carcinogenic to the lungs of male and female F344 rats, however no plural mesothelioma was observed.
Mesothelioma response to carbon nanotubes is associated with an early and selective accumulation of immunosuppressive monocytic cells. F. Huaux, V. d'Ursel de Bousies, M.A. Parent, et al. 2016;13(1):46. PubMed · Publisher · Free full text

Abstract

Background: The asbestos-like toxicity of some engineered carbon nanotubes (CNT), notably their capacity to induce mesothelioma, is a serious cause of concern for public health. Here we show that carcinogenic CNT induce an early and sustained immunosuppressive response characterized by the accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC) that counteract effective immune surveillance of tumor cells.

Methods: Wistar rats and C57BL/6 mice were intraperitoneally injected with carcinogenic multi-walled Mitsui-7 CNT (CNT-7) or crocidolite asbestos. Peritoneal mesothelioma development and immune cell accumulation were assessed until 12 months. Leukocyte sub-populations were identified by recording expression of CD11b/c and His48 by flow cytometry. The immunosuppressive activity on T lymphocytes of purified peritoneal leukocytes was assessed in a co-culture assay with activated spleen cells.

Results: We demonstrate that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like asbestos, an early and selective accumulation of monocytic cells (CD11b/c(int) and His48(hi)) which possess the ability to suppress polyclonal activation of T lymphocytes and correspond to M-MDSC. Peritoneal M-MDSC persisted during the development of peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT (CNT-M, CNT-T) injection. Peritoneal M-MDSC did not accumulate in mice which are resistant to mesothelioma development.

Conclusions: Our data provide new insights into the initial pathogenic events induced by CNT, adding a new component to the adverse outcome pathway leading to mesothelioma development. The specificity of the M-MDSC response after carcinogenic CNT exposure highlights the interest of this response for detecting the ability of new nanomaterials to cause cancer.
Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-β-mediated Akt/GSK-3β/SNAIL-1 signalling pathway. M. Polimeni, G.R. Gulino, E. Gazzano, et al. 2016;13(1):27. PubMed · Publisher · Free full text

Abstract

Background: Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-β/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-β-mediated signalling pathway.

Methods: Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-β production. Experiments with neutralizing anti-TGF-β antibody, specific inhibitors of GSK-3β and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test.

Results: Fully characterized MWCNT (mean length < 5 μm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-β secretion, Akt activation and GSK-3β inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear β-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure.

Conclusions: We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-β and its intracellular effectors Akt/GSK-3β that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.
Persistent effects of Libby amphibole and amosite asbestos following subchronic inhalation in rats. S.H. Gavett, C.U. Parkinson, G.A. Willson, et al. 2016;13:17. PubMed · Publisher · Free full text

Abstract

Background: Human exposure to Libby amphibole (LA) asbestos increases risk of lung cancer, mesothelioma, and non-malignant respiratory disease. This study evaluated potency and time-course effects of LA and positive control amosite (AM) asbestos fibers in male F344 rats following nose-only inhalation exposure.

Methods: Rats were exposed to air, LA (0.5, 3.5, or 25.0 mg/m(3) targets), or AM (3.5 mg/m(3) target) for 10 days and assessed for markers of lung inflammation, injury, and cell proliferation. Short-term results guided concentration levels for a stop-exposure study in which rats were exposed to air, LA (1.0, 3.3, or 10.0 mg/m(3)), or AM (3.3 mg/m(3)) 6 h/day, 5 days/week for 13 weeks, and assessed 1 day, 1, 3, and 18 months post-exposure. Fibers were relatively short; for 10 mg/m(3) LA, mean length of all structures was 3.7 μm and 1% were longer than 20 μm.

Results: Ten days exposure to 25.0 mg/m(3) LA resulted in significantly increased lung inflammation, fibrosis, bronchiolar epithelial cell proliferation and hyperplasia, and inflammatory cytokine gene expression compared to air. Exposure to 3.5 mg/m(3) LA resulted in modestly higher markers of acute lung injury and inflammation compared to AM. Following 13 weeks exposure, lung fiber burdens correlated with exposure mass concentrations, declining gradually over 18 months. LA (3.3 and 10.0 mg/m(3)) and AM produced significantly higher bronchoalveolar lavage markers of inflammation and lung tissue cytokines, Akt, and MAPK/ERK pathway components compared to air control from 1 day to 3 months post-exposure. Histopathology showed alveolar inflammation and interstitial fibrosis in all fiber-exposed groups up to 18 months post-exposure. Positive dose trends for incidence of alveolar epithelial hyperplasia and bronchiolar/alveolar adenoma or carcinoma were observed among LA groups.

Conclusions: Inhalation of relatively short LA fibers produced inflammatory, fibrogenic, and tumorigenic effects in rats which replicate essential attributes of asbestos-related disease in exposed humans. Fiber burden, inflammation, and activation of growth factor pathways may persist and contribute to lung tumorigenesis long after initial LA exposure. Fiber burden data are being used to develop a dosimetry model for LA fibers, which may provide insights on mode of action for hazard assessment.
The NLRP3 inflammasome in pathogenic particle and fibre-associated lung inflammation and diseases. M. Sayan, B.T. Mossman. 2016;13(1):51. Review PubMed · Publisher · Free full text

Abstract

The concept of the inflammasome, a macromolecular complex sensing cell stress or danger signals and initiating inflammation, was first introduced approximately a decade ago. Priming and activation of these intracellular protein platforms trigger the maturation of pro-inflammatory chemokines and cytokines, most notably, interleukin-1β (IL-1β) and IL-18, to promulgate innate immune defenses. Although classically studied in models of gout, Type II diabetes, Alzheimer's disease, and multiple sclerosis, the importance and mechanisms of action of inflammasome priming and activation have recently been elucidated in cells of the respiratory tract where they modulate the responses to a number of inhaled pathogenic particles and fibres. Most notably, inflammasome activation appears to regulate the balance between tissue repair and inflammation after inhalation of pathogenic pollutants such as asbestos, crystalline silica (CS), and airborne particulate matter (PM). Different types of fibres and particles may have distinct mechanisms of inflammasome interaction and outcome. This review summarizes the structure and function of inflammasomes, the interplay between various chemokines and cytokines and cell types of the lung and pleura after inflammasome activation, and the events leading to the development of non-malignant (allergic airway disease and chronic obstructive pulmonary disease (COPD), asbestosis, silicosis) and malignant (mesothelioma, lung cancer) diseases by pathogenic particulates. In addition, it emphasizes the importance of communication between cells of the immune system, target cells of these diseases, and components of the extracellular matrix (ECM) in regulation of inflammasome-mediated events.
An in vitro testing strategy towards mimicking the inhalation of high aspect ratio nanoparticles. C. Endes, O. Schmid, C. Kinnear, et al. 2014;11:40. PubMed · Publisher · Free full text

Abstract

Background: The challenge remains to reliably mimic human exposure to high aspect ratio nanoparticles (HARN) via inhalation. Sophisticated, multi-cellular in vitro models are a particular advantageous solution to this issue, especially when considering the need to provide realistic and efficient alternatives to invasive animal experimentation for HARN hazard assessment. By incorporating a systematic test-bed of material characterisation techniques, a specific air-liquid cell exposure system with real-time monitoring of the cell-delivered HARN dose in addition to key biochemical endpoints, here we demonstrate a successful approach towards investigation of the hazard of HARN aerosols in vitro.

Methods: Cellulose nanocrystals (CNCs) derived from cotton and tunicates, with differing aspect ratios (~9 and ~80), were employed as model HARN samples. Specifically, well-dispersed and characterised CNC suspensions were aerosolised using an "Air Liquid Interface Cell Exposure System" (ALICE) at realistic, cell-delivered concentrations ranging from 0.14 to 1.57 μg/cm2. The biological impact (cytotoxicity, oxidative stress levels and pro-inflammatory effects) of each HARN sample was then assessed using a 3D multi-cellular in vitro model of the human epithelial airway barrier at the air liquid interface (ALI) 24 hours post-exposure. Additionally, the testing strategy was validated using both crystalline quartz (DQ12) as a positive particulate control in the ALICE system and long fibre amosite asbestos (LFA) to confirm the susceptibility of the in vitro model to a fibrous insult.

Results: A rapid (≤ 4 min), controlled nebulisation of CNC suspensions enabled a dose-controlled and spatially homogeneous CNC deposition onto cells cultured under ALI conditions. Real-time monitoring of the cell-delivered CNC dose with a quartz crystal microbalance was accomplished. Independent of CNC aspect ratio, no significant cytotoxicity (p>0.05), induction of oxidative stress, or (pro)-inflammatory responses were observed up to the highest concentration of 1.57 μg/cm2. Both DQ12 and LFA elicited a significant (p<0.05) pro-inflammatory response at sub-lethal concentrations in vitro.

Conclusion: In summary, whilst the present study highlights the benign nature of CNCs, it is the advanced technological and mechanistic approach presented that allows for a state of the art testing strategy to realistically and efficiently determine the in vitro hazard concerning inhalation exposure of HARN.
Asbestos modulates thioredoxin-thioredoxin interacting protein interaction to regulate inflammasome activation. J.K. Thompson, C.M. Westbom, M.B. MacPherson, et al. 2014;11:24. PubMed · Publisher · Free full text

Abstract

Background: Asbestos exposure is related to various diseases including asbestosis and malignant mesothelioma (MM). Among the pathogenic mechanisms proposed by which asbestos can cause diseases involving epithelial and mesothelial cells, the most widely accepted one is the generation of reactive oxygen species and/or depletion of antioxidants like glutathione. It has also been demonstrated that asbestos can induce inflammation, perhaps due to activation of inflammasomes.

Methods: The oxidation state of thioredoxin was analyzed by redox Western blot analysis and ROS generation was assessed spectrophotometrically as a read-out of solubilized formazan produced by the reduction of nitrotetrazolium blue (NTB) by superoxide. Quantitative real time PCR was used to assess changes in gene transcription.

Results: Here we demonstrate that crocidolite asbestos fibers oxidize the pool of the antioxidant, Thioredoxin-1 (Trx1), which results in release of Thioredoxin Interacting Protein (TXNIP) and subsequent activation of inflammasomes in human mesothelial cells. Exposure to crocidolite asbestos resulted in the depletion of reduced Trx1 in human peritoneal mesothelial (LP9/hTERT) cells. Pretreatment with the antioxidant dehydroascorbic acid (a reactive oxygen species (ROS) scavenger) reduced the level of crocidolite asbestos-induced Trx1 oxidation as well as the depletion of reduced Trx1. Increasing Trx1 expression levels using a Trx1 over-expression vector, reduced the extent of Trx1 oxidation and generation of ROS by crocidolite asbestos, and increased cell survival. In addition, knockdown of TXNIP expression by siRNA attenuated crocidolite asbestos-induced activation of the inflammasome.

Conclusion: Our novel findings suggest that extensive Trx1 oxidation and TXNIP dissociation may be one of the mechanisms by which crocidolite asbestos activates the inflammasome and helps in development of MM.

In plain English

This was a laboratory study on human cells grown in dishes, not on people. The researchers exposed human peritoneal mesothelial cells (LP9/hTERT) to crocidolite asbestos, chrysotile asbestos, or glass beads used as a harmless control. Crocidolite oxidized thioredoxin-1 (Trx1), a protective antioxidant protein, which sharply lowered the amount of its active, reduced form. Chrysotile and glass beads did not change the oxidation state of Trx1 at 8 and 24 hours. Crocidolite raised Trx1 gene activity 1.6-fold at 24 hours, lowered total Trx1 protein by about 67%, and increased reactive oxygen species. Cells made to produce extra Trx1 showed a non-significant trend toward lower oxygen species and a trend toward better survival. Lowering the partner protein TXNIP reduced activation of caspase-1, a marker that the inflammasome had switched on. The inflammasome is a protein complex involved in inflammation. In asbestos-exposed cells, blocking caspase-1 beforehand raised cell viability by 18% compared with asbestos alone. The authors concluded that asbestos activates the inflammasome partly through TXNIP, as a consequence of changes in Trx1. They said the caspase-1 cell-death finding still needs confirmation.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.
Free radical scavenging and formation by multi-walled carbon nanotubes in cell free conditions and in human bronchial epithelial cells. P. Nymark, K.A. Jensen, S. Suhonen, et al. 2014;11:4. PubMed · Publisher · Free full text

Abstract

Background: Certain multi-walled carbon nanotubes (MWCNTs) have been shown to elicit asbestos-like toxicological effects. To reduce needs for risk assessment it has been suggested that the physicochemical characteristics or reactivity of nanomaterials could be used to predict their hazard. Fibre-shape and ability to generate reactive oxygen species (ROS) are important indicators of high hazard materials. Asbestos is a known ROS generator, while MWCNTs may either produce or scavenge ROS. However, certain biomolecules, such as albumin - used as dispersants in nanomaterial preparation for toxicological testing in vivo and in vitro - may reduce the surface reactivity of nanomaterials.

Methods: Here, we investigated the effect of bovine serum albumin (BSA) and cell culture medium with and without BEAS 2B cells on radical formation/scavenging by five MWCNTs, Printex 90 carbon black, crocidolite asbestos, and glass wool, using electron spin resonance (ESR) spectroscopy and linked this to cytotoxic effects measured by trypan blue exclusion assay. In addition, the materials were characterized in the exposure medium (e.g. for hydrodynamic size-distribution and sedimentation rate).

Results: The test materials induced highly variable cytotoxic effects which could generally be related to the abundance and characteristics of agglomerates/aggregates and to the rate of sedimentation. All carbon nanomaterials were found to scavenge hydroxyl radicals (•OH) in at least one of the solutions tested. The effect of BSA was different among the materials. Two types of long, needle-like MWCNTs (average diameter >74 and 64.2 nm, average length 5.7 and 4.0 μm, respectively) induced, in addition to a scavenging effect, a dose-dependent formation of a unique, yet unidentified radical in both absence and presence of cells, which also coincided with cytotoxicity.

Conclusions: Culture medium and BSA affects scavenging/production of •OH by MWCNTs, Printex 90 carbon black, asbestos and glass-wool. An unidentified radical is generated by two long, needle-like MWCNTs and these two CNTs were more cytotoxic than the other CNTs tested, suggesting that this radical could be related to the adverse effects of MWCNTs.
In vitro determinants of asbestos fiber toxicity: effect on the relative toxicity of Libby amphibole in primary human airway epithelial cells. K.E. Duncan, P.M. Cook, S.H. Gavett, et al. 2014;11:2. PubMed · Publisher · Free full text

Abstract

Background: An abnormally high incidence of lung disease has been observed in the residents of Libby, Montana, which has been attributed to occupational and environmental exposure to fibrous amphiboles originating from a nearby contaminated vermiculite mine. The composition of Libby amphibole (LA) is complex and minimal toxicity data are available. In this study, we conduct a comparative particle toxicity analysis of LA compared with standard reference asbestiform amphibole samples.

Methods: Primary human airway epithelial cells (HAEC) were exposed to two different LA samples as well as standard amphibole reference samples. Analysis of the samples included a complete particle size distribution analysis, calculation of surface area by electron microscopy and by gas adsorption and quantification of surface-conjugated iron and hydroxyl radical production by the fibers. Interleukin-8 mRNA levels were quantified by qRT-PCR to measure relative pro-inflammatory response induced in HAEC in response to amphibole fiber exposure. The relative contribution of key physicochemical determinants on the observed pro-inflammatory response were also evaluated.

Results: The RTI amosite reference sample contained the longest fibers and demonstrated the greatest potency at increasing IL-8 transcript levels when evaluated on an equal mass basis. The two LA samples and the UICC amosite reference sample consisted of similar particle numbers per milligram as well as similar particle size distributions and induced comparable levels of IL-8 mRNA. A strong correlation was observed between the elongated particle (aspect ratio ≥3:1) dose metrics of length and external surface area. Expression of the IL-8 data with respect to either of these metrics eliminated the differential response between the RTI amosite sample and the other samples that was observed when HAEC were exposed on an equal mass basis.

Conclusions: On an equal mass basis, LA is as potent as the UICC amosite reference sample at inducing a pro-inflammatory response in HAEC but is less potent than the RTI amosite sample. The results of this study show that the particle length and particle surface area are highly correlated metrics that contribute significantly to the toxicological potential of these amphibole samples with respect to the inflammogenic response induced in airway epithelial cells.
Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation. E.M. Rydman, M. Ilves, A.J. Koivisto, et al. 2014;11:48. PubMed · Publisher · Free full text

Abstract

Background: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma.

Methods: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation.

Results: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages.

Conclusions: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.
Nanometer-long Ge-imogolite nanotubes cause sustained lung inflammation and fibrosis in rats. S. van den Brule, E. Beckers, P. Chaurand, et al. 2014;11:67. PubMed · Publisher · Free full text

Abstract

Background: Ge-imogolites are short aluminogermanate tubular nanomaterials with attractive prospected industrial applications. In view of their nano-scale dimensions and high aspect ratio, they should be examined for their potential to cause respiratory toxicity. Here, we evaluated the respiratory biopersistence and lung toxicity of 2 samples of nanometer-long Ge-imogolites.

Methods: Rats were intra-tracheally instilled with single wall (SW, 70 nm length) or double wall (DW, 62 nm length) Ge-imogolites (0.02-2 mg/rat), as well as with crocidolite and the hard metal particles WC-Co, as positive controls. The biopersistence of Ge-imogolites and their localization in the lung were assessed by ICP-MS, X-ray fluorescence, absorption spectroscopy and computed micro-tomography. Acute inflammation and genotoxicity (micronuclei in isolated type II pneumocytes) was assessed 3 d post-exposure; chronic inflammation and fibrosis after 2 m.

Results: Cytotoxic and inflammatory responses were shown in bronchoalveolar lavage 3 d after instillation with Ge-imogolites. Sixty days after exposure, a persistent dose-dependent inflammation was still observed. Total lung collagen, reflected by hydroxyproline lung content, was increased after SW and DW Ge-imogolites. Histology revealed lung fibre reorganization and accumulation in granulomas with epithelioid cells and foamy macrophages and thickening of the alveolar walls. Overall, the inflammatory and fibrotic responses induced by SW and DW Ge-imogolites were more severe (on a mass dose basis) than those induced by crocidolite. A persistent fraction of Ge-imogolites (15% of initial dose) was mostly detected as intact structures in rat lungs 2 m after instillation and was localized in fibrotic alveolar areas. In vivo induction of micronuclei was significantly increased 3 d after SW and DW Ge-imogolite instillation at non-inflammatory doses, indicating the contribution of primary genotoxicity.

Conclusions: We showed that nm-long Ge-imogolites persist in the lung and promote genotoxicity, sustained inflammation and fibrosis, indicating that short high aspect ratio nanomaterials should not be considered as innocuous materials. Our data also suggest that Ge-imogolite structure and external surface determine their toxic activity.
Promotion of lung adenocarcinoma following inhalation exposure to multi-walled carbon nanotubes. L.M. Sargent, D.W. Porter, L.M. Staska, et al. 2014;11:3. PubMed · Publisher · Free full text

Abstract

Background: Engineered carbon nanotubes are currently used in many consumer and industrial products such as paints, sunscreens, cosmetics, toiletries, electronic processes and industrial lubricants. Carbon nanotubes are among the more widely used nanoparticles and come in two major commercial forms, single-walled carbon nanotubes (SWCNT) and the more rigid, multi-walled carbon nanotubes (MWCNT). The low density and small size of these particles makes respiratory exposures likely. Many of the potential health hazards have not been investigated, including their potential for carcinogenicity. We, therefore, utilized a two stage initiation/promotion protocol to determine whether inhaled MWCNT act as a complete carcinogen and/or promote the growth of cells with existing DNA damage. Six week old, male, B6C3F1 mice received a single intraperitoneal (ip) injection of either the initiator methylcholanthrene(MCA, 10 μg/g BW, i.p.), or vehicle (corn oil). One week after i.p. injections, mice were exposed by inhalation to MWCNT (5 mg/m³, 5 hours/day, 5 days/week) or filtered air (controls) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for lung tumor formation.

Results: Twenty-three percent of the filtered air controls, 26.5% of the MWCNT-exposed, and 51.9% of the MCA-exposed mice, had lung bronchiolo-alveolar adenomas and lung adenocarcinomas. The average number of tumors per mouse was 0.25, 0.81 and 0.38 respectively. By contrast, 90.5% of the mice which received MCA followed by MWCNT had bronchiolo-alveolar adenomas and adenocarcinomas with an average of 2.9 tumors per mouse 17 months after exposure. Indeed, 62% of the mice exposed to MCA followed by MWCNT had bronchiolo-alveolar adenocarcinomas compared to 13% of the mice that received filtered air, 22% of the MCA-exposed, or 14% of the MWCNT-exposed. Mice with early morbidity resulting in euthanasia had the highest rate of metastatic disease. Three mice exposed to both MCA and MWCNT that were euthanized early had lung adenocarcinoma with evidence of metastasis (5.5%). Five mice (9%) exposed to MCA and MWCNT and 1 (1.6%) exposed to MCA developed serosal tumors morphologically consistent with sarcomatous mesotheliomas, whereas mice administered MWCNT or air alone did not develop similar neoplasms.

Conclusions: These data demonstrate that some MWCNT exposures promote the growth and neoplastic progression of initiated lung cells in B6C3F1 mice. In this study, the mouse MWCNT lung burden of 31.2 μg/mouse approximates feasible human occupational exposures. Therefore, the results of this study indicate that caution should be used to limit human exposures to MWCNT.
The carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) after intraperitoneal injection in rats. S. Rittinghausen, A. Hackbarth, O. Creutzenberg, et al. 2014;11:59. PubMed · Publisher · Free full text

Abstract

Background: Biological effects of tailor-made multi-walled carbon nanotubes (MWCNTs) without functionalization were investigated in vivo in a two-year carcinogenicity study. In the past, intraperitoneal carcinogenicity studies in rats using biopersistent granular dusts had always been negative, whereas a number of such studies with different asbestos fibers had shown tumor induction. The aim of this study was to identify possible carcinogenic effects of MWCNTs. We compared induced tumors with asbestos-induced mesotheliomas and evaluated their relevance for humans by immunohistochemical methods.

Methods: A total of 500 male Wistar rats (50 per group) were treated once by intraperitoneal injection with 10⁹ or 5 × 10⁹ WHO carbon nanotubes of one of four different MWCNTs suspended in artificial lung medium, which was also used as negative control. Amosite asbestos (10⁸ WHO fibers) served as positive control. Morbid rats were sacrificed and necropsy comprising all organs was performed. Histopathological classification of tumors and, additionally, immunohistochemistry were conducted for podoplanin, pan-cytokeratin, and vimentin to compare induced tumors with malignant mesotheliomas occurring in humans.

Results: Treatments induced tumors in all dose groups, but incidences and times to tumor differed between groups. Most tumors were histologically and immunohistochemically classified as malignant mesotheliomas, revealing a predominantly superficial spread on the serosal surface of the abdominal cavity. Furthermore, most tumors showed invasion of peritoneal organs, especially the diaphragm. All tested MWCNT types caused mesotheliomas. We observed highest frequencies and earliest appearances after treatment with the rather straight MWCNT types A and B. In the MWCNT C groups, first appearances of morbid mesothelioma-bearing rats were only slightly later. Later during the two-year study, we found mesotheliomas also in rats treated with MWCNT D - the most curved type of nanotubes. Malignant mesotheliomas induced by intraperitoneal injection of different MWCNTs and of asbestos were histopathologically and immunohistochemically similar, also compared with mesotheliomas in man, suggesting similar pathogenesis.

Conclusion: We showed a carcinogenic effect for all tested MWCNTs. Besides aspect ratio, curvature seems to be an important parameter influencing the carcinogenicity of MWCNTs.
Asbestos and erionite prime and activate the NLRP3 inflammasome that stimulates autocrine cytokine release in human mesothelial cells. J.M. Hillegass, J.M. Miller, M.B. MacPherson, et al. 2013;10:39. PubMed · Publisher · Free full text

Abstract

Background: Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.

Results: We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model.

Conclusions: These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.
Ken Donaldson: retirement of a young mind. P. Borm, F.R. Cassee. 2013;10:8. Editorial · Historical article PubMed · Publisher · Free full text
Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay. G. Vietti, S. Ibouraadaten, M. Palmai-Pallag, et al. 2013;10:52. PubMed · Publisher · Free full text

Abstract

Background: Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts.

Methods: The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF).

Results: MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis.

Conclusions: In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets and an adaptation of WST-1 as readout was developed. The activity of MWCNT in this test strongly reflects their fibrotic activity in vivo, supporting the predictive value of this in vitro assay in terms of lung fibrosis potential.
A short history of the toxicology of inhaled particles. K. Donaldson, A. Seaton. 2012;9:13. Historical article PubMed · Publisher · Free full text

Abstract

Particle toxicology arose in order to understand the mechanisms of adverse effects of 3 major particle types that had historically exerted the greatest toll of ill-health--quartz, coal and asbestos. By the middle of the last century rat inhalation studies had been carried out and the pathology documented, but true mechanistic particle toxicology did not really take off until the 1970s when cell culture techniques became available. By the 1980s glass fibres were a major focus of interest and attempts to develop a structure-toxicity paradigm centred on biopersistence. In the 1990s environmental particles dominated the particle toxicology agenda and the cardiovascular system emerged as a target for inhaled particles, raising new challenges for particle toxicologists. We are currently in the era of nanotoxicology where a large and diverse range of new nanoparticles types are under scrutiny.
Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos. A.R. Murray, E.R. Kisin, A.V. Tkach, et al. 2012;9:10. PubMed · Publisher · Free full text

Abstract

Background: Carbon nanotubes (CNT) and carbon nanofibers (CNF) are allotropes of carbon featuring fibrous morphology. The dimensions and high aspect ratio of CNT and CNF have prompted the comparison with naturally occurring asbestos fibers which are known to be extremely pathogenic. While the toxicity and hazardous outcomes elicited by airborne exposure to single-walled CNT or asbestos have been widely reported, very limited data are currently available describing adverse effects of respirable CNF.

Results: Here, we assessed pulmonary inflammation, fibrosis, oxidative stress markers and systemic immune responses to respirable CNF in comparison to single-walled CNT (SWCNT) and asbestos. Pulmonary inflammatory and fibrogenic responses to CNF, SWCNT and asbestos varied depending upon the agglomeration state of the particles/fibers. Foci of granulomatous lesions and collagen deposition were associated with dense particle-like SWCNT agglomerates, while no granuloma formation was found following exposure to fiber-like CNF or asbestos. The average thickness of the alveolar connective tissue--a marker of interstitial fibrosis--was increased 28 days post SWCNT, CNF or asbestos exposure. Exposure to SWCNT, CNF or asbestos resulted in oxidative stress evidenced by accumulations of 4-HNE and carbonylated proteins in the lung tissues. Additionally, local inflammatory and fibrogenic responses were accompanied by modified systemic immunity, as documented by decreased proliferation of splenic T cells ex vivo on day 28 post exposure. The accuracies of assessments of effective surface area for asbestos, SWCNT and CNF (based on geometrical analysis of their agglomeration) versus estimates of mass dose and number of particles were compared as predictors of toxicological outcomes.

Conclusions: We provide evidence that effective surface area along with mass dose rather than specific surface area or particle number are significantly correlated with toxicological responses to carbonaceous fibrous nanoparticles. Therefore, they could be useful dose metrics for risk assessment and management.
The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells. F.A. Murphy, A. Schinwald, C.A. Poland, et al. 2012;9:8. PubMed · Publisher · Free full text

Abstract

Carbon nanotubes (CNT) are high aspect ratio nanoparticles with diameters in the nanometre range but lengths extending up to hundreds of microns. The structural similarities between CNT and asbestos have raised concern that they may pose a similar inhalation hazard. Recently CNT have been shown to elicit a length-dependent, asbestos-like inflammatory response in the pleural cavity of mice, where long fibres caused inflammation but short fibres did not. However the cellular mechanisms governing this response have yet to be elucidated. This study examined the in vitro effects of a range of CNT for their ability to stimulate the release of the acute phase cytokines; IL-1β, TNFα, IL-6 and the chemokine, IL-8 from both Met5a mesothelial cells and THP-1 macrophages. Results showed that direct exposure to CNT resulted in significant cytokine release from the macrophages but not mesothelial cells. This pro-inflammatory response was length dependent but modest and was shown to be a result of frustrated phagocytosis. Furthermore the indirect actions of the CNT were examined by treating the mesothelial cells with conditioned media from CNT-treated macrophages. This resulted in a dramatic amplification of the cytokine release from the mesothelial cells, a response which could be attenuated by inhibition of phagocytosis during the initial macrophage CNT treatments. We therefore hypothesise that long fibres elicit an inflammatory response in the pleural cavity via frustrated phagocytosis in pleural macrophages. The activated macrophages then stimulate an amplified pro-inflammatory cytokine response from the adjacent pleural mesothelial cells. This mechanism for producing a pro-inflammatory environment in the pleural space exposed to long CNT has implications for the general understanding of fibre-related pleural disease and design of safe nanofibres.
Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo. A. Schinwald, K. Donaldson. 2012;9:34. PubMed · Publisher · Free full text

Abstract

Background: Frustrated phagocytosis has been stated as an important factor in the initiation of an inflammatory response after fibre exposure. The length of fibrous structures has been linked to the potential of fibres to induce adverse health effects for at least 40 years. However, we only recently reported for the first time the threshold length for fibre-induced inflammation in the pleural space and we implicated frustrated phagocytosis in the pro-inflammatory effects of long fibres. This study extends the examination of the threshold value for frustrated phagocytosis using well-defined length classes of silver nanowires (AgNW) ranging from 3-28 μm and describes in detail the morphology of frustrated phagocytosis using a novel technique and also describes compartmentalisation of fibres in the pleural space.

Methods: A novel technique, backscatter scanning electron microscopy (BSE) was used to study frustrated phagocytosis since it provides high-contrast detection of nanowires, allowing clear discrimination between the nanofibres and other cellular features. A human monocyte-derived macrophage cell line THP-1 was used to investigate cell-nanowire interaction in vitro and the parietal pleura, the site of fibre retention after inhalation exposure was chosen to visualise the cell- fibre interaction in vivo after direct pleural installation of AgNWs.

Results: The length cut-off value for frustrated phagocytosis differs in vitro and in vivo. While in vitro frustrated phagocytosis could be observed with fibres≥14 μm, in vivo studies showed incomplete uptake at a fibre length of ≥10 μm. Recently we showed that inflammation in the pleural space after intrapleural injection of the same nanofibre panel occurs at a length of ≥5 μm. This onset of inflammation does not correlate with the onset of frustrated phagocytosis as shown in this study, leading to the conclusion that intermediate length fibres fully enclosed within macrophages as well as frustrated phagocytosis are associated with a pro-inflammatory state in the pleural space. We further showed that fibres compartmentalise in the mesothelial cells at the parietal pleura as well as in inflammatory cells in the pleural space.

Conclusion: BSE is a useful way to clearly distinguish between fibres that are, or are not, membrane-bounded. Using this method we were able to show differences in the threshold length at which frustrated phagocytosis occurred between in vitro and in vivo models. Visualising nanowires in the pleura demonstrated at least 2 compartments--in leukocyte aggregations and in the mesothelium--which may have consequences for long term pathology in the pleural space including mesothelioma.
A 3-dimensional in vitro model of epithelioid granulomas induced by high aspect ratio nanomaterials. V.C. Sanchez, P. Weston, A. Yan, et al. 2011;8:17. PubMed · Publisher · Free full text

Abstract

Background: The most common causes of granulomatous inflammation are persistent pathogens and poorly-degradable irritating materials. A characteristic pathological reaction to intratracheal instillation, pharyngeal aspiration, or inhalation of carbon nanotubes is formation of epithelioid granulomas accompanied by interstitial fibrosis in the lungs. In the mesothelium, a similar response is induced by high aspect ratio nanomaterials, including asbestos fibers, following intraperitoneal injection. This asbestos-like behaviour of some engineered nanomaterials is a concern for their potential adverse health effects in the lungs and mesothelium. We hypothesize that high aspect ratio nanomaterials will induce epithelioid granulomas in nonadherent macrophages in 3D cultures.

Results: Carbon black particles (Printex 90) and crocidolite asbestos fibers were used as well-characterized reference materials and compared with three commercial samples of multiwalled carbon nanotubes (MWCNTs). Doses were identified in 2D and 3D cultures in order to minimize acute toxicity and to reflect realistic occupational exposures in humans and in previous inhalation studies in rodents. Under serum-free conditions, exposure of nonadherent primary murine bone marrow-derived macrophages to 0.5 μg/ml (0.38 μg/cm2) of crocidolite asbestos fibers or MWCNTs, but not carbon black, induced macrophage differentiation into epithelioid cells and formation of stable aggregates with the characteristic morphology of granulomas. Formation of multinucleated giant cells was also induced by asbestos fibers or MWCNTs in this 3D in vitro model. After 7-14 days, macrophages exposed to high aspect ratio nanomaterials co-expressed proinflammatory (M1) as well as profibrotic (M2) phenotypic markers.

Conclusions: Induction of epithelioid granulomas appears to correlate with high aspect ratio and complex 3D structure of carbon nanotubes, not with their iron content or surface area. This model offers a time- and cost-effective platform to evaluate the potential of engineered high aspect ratio nanomaterials, including carbon nanotubes, nanofibers, nanorods and metallic nanowires, to induce granulomas following inhalation.
Durability and inflammogenic impact of carbon nanotubes compared with asbestos fibres. M.J. Osmond-McLeod, C.A. Poland, F. Murphy, et al. 2011;8:15. PubMed · Publisher · Free full text

Abstract

Background: It has been suggested that carbon nanotubes might conform to the fibre pathogenicity paradigm that explains the toxicities of asbestos and other fibres on a continuum based on length, aspect ratio and biopersistence. Some types of carbon nanotubes satisfy the first two aspects of the fibre paradigm but only recently has their biopersistence begun to be investigated. Biopersistence is complex and requires in vivo testing and analysis. However durability, the chemical mimicking of the process of fibre dissolution using in vitro treatment, is closely related to biopersistence and more readily determined. Here, we describe an experimental process to determine the durability of four types of carbon nanotubes in simulated biological fluid (Gambles solution), and their subsequent pathogenicity in vivo using a mouse model sensitive to inflammogenic effects of fibres. The in vitro and in vivo results were compared with well-characterised glass wool and asbestos fibre controls.

Results: After incubation for up to 24 weeks in Gambles solution, our control fibres were recovered at percentages consistent with their known in vitro durabilities and/or in vivo persistence, and three out of the four types of carbon nanotubes tested (single-walled (CNTSW) and multi-walled (CNTTANG2, CNTSPIN)) showed no, or minimal, loss of mass or change in fibre length or morphology when examined by electron microscopy. However, the fourth type [multi-walled (CNTLONG1)] lost 30% of its original mass within the first three weeks of incubation, after which there was no further loss. Electron microscopy of CNTLONG1 samples incubated for 10 weeks confirmed that the proportion of long fibres had decreased compared to samples briefly exposed to the Gambles solution. This loss of mass and fibre shortening was accompanied by a loss of pathogenicity when injected into the peritoneal cavities of C57Bl/6 mice compared to fibres incubated briefly. CNTSW did not elicit an inflammogenic effect in the peritoneal cavity assay used here.

Conclusions: These results support the view that carbon nanotubes are generally durable but may be subject to bio-modification in a sample-specific manner. They also suggest that pristine carbon nanotubes, either individually or in rope-like aggregates of sufficient length and aspect ratio, can induce asbestos-like responses in mice, but that the effect may be mitigated for certain types that are less durable in biological systems. Results indicate that durable carbon nanotubes that are either short or form tightly bundled aggregates with no isolated long fibres are less inflammogenic in fibre-specific assays.

© 2011 Osmond-McLeod et al; licensee BioMed Central Ltd.
Synchrotron soft X-ray imaging and fluorescence microscopy reveal novel features of asbestos body morphology and composition in human lung tissues. L. Pascolo, A. Gianoncelli, B. Kaulich, et al. 2011;8(1):7. PubMed · Publisher · Free full text

Abstract

Background: Occupational or environmental exposure to asbestos fibres is associated with pleural and parenchymal lung diseases. A histopathologic hallmark of exposure to asbestos is the presence in lung parenchyma of the so-called asbestos bodies. They are the final product of biomineralization processes resulting in deposition of endogenous iron and organic matter (mainly proteins) around the inhaled asbestos fibres. For shedding light on the formation mechanisms of asbestos bodies it is of fundamental importance to characterize at the same length scales not only their structural morphology and chemical composition but also to correlate them to the possible alterations in the local composition of the surrounding tissues. Here we report the first correlative morphological and chemical characterization of untreated paraffinated histological lung tissue samples with asbestos bodies by means of soft X-ray imaging and X-Ray Fluorescence (XRF) microscopy, which reveals new features in the elemental lateral distribution.

Results: The X-ray absorption and phase contrast images and the simultaneously monitored XRF maps of tissue samples have revealed the location, distribution and elemental composition of asbestos bodies and associated nanometric structures. The observed specific morphology and differences in the local Si, Fe, O and Mg content provide distinct fingerprints characteristic for the core asbestos fibre and the ferruginous body. The highest Si content is found in the asbestos fibre, while the shell and ferruginous bodies are characterized by strongly increased content of Mg, Fe and O compared to the adjacent tissue. The XRF and SEM-EDX analyses of the extracted asbestos bodies confirmed an enhanced Mg deposition in the organic asbestos coating.

Conclusions: The present report demonstrates the potential of the advanced synchrotron-based X-ray imaging and microspectroscopy techniques for studying the response of the lung tissue to the presence of asbestos fibres. The new results obtained by simultaneous structural and chemical analysis of tissue specimen have provided clear evidence that Mg, in addition to Fe, is also involved in the formation mechanisms of asbestos bodies. This is the first important step to further thorough investigations that will shed light on the physiopathological role of Mg in tissue response to the asbestos toxicity.
Asbestos, carbon nanotubes and the pleural mesothelium: a review of the hypothesis regarding the role of long fibre retention in the parietal pleura, inflammation and mesothelioma. K. Donaldson, F.A. Murphy, R. Duffin, et al. 2010;7:5. Review PubMed · Publisher · Free full text

Abstract

The unique hazard posed to the pleural mesothelium by asbestos has engendered concern in potential for a similar risk from high aspect ratio nanoparticles (HARN) such as carbon nanotubes. In the course of studying the potential impact of HARN on the pleura we have utilised the existing hypothesis regarding the role of the parietal pleura in the response to long fibres. This review seeks to synthesise our new data with multi-walled carbon nanotubes (CNT) with that hypothesis for the behaviour of long fibres in the lung and their retention in the parietal pleura leading to the initiation of inflammation and pleural pathology such as mesothelioma. We describe evidence that a fraction of all deposited particles reach the pleura and that a mechanism of particle clearance from the pleura exits, through stomata in the parietal pleura. We suggest that these stomata are the site of retention of long fibres which cannot negotiate them leading to inflammation and pleural pathology including mesothelioma. We cite thoracoscopic data to support the contention, as would be anticipated from the preceding, that the parietal pleura is the site of origin of pleural mesothelioma. This mechanism, if it finds support, has important implications for future research into the mesothelioma hazard from HARN and also for our current view of the origins of asbestos-initiated pleural mesothelioma and the common use of lung parenchymal asbestos fibre burden as a correlate of this tumour, which actually arises in the parietal pleura.
Mechanisms of oxidative stress and alterations in gene expression by Libby six-mix in human mesothelial cells. J.M. Hillegass, A. Shukla, M.B. MacPherson, et al. 2010;7:26. PubMed · Publisher · Free full text

Abstract

Background: Exposures to an amphibole fiber in Libby, Montana cause increases in malignant mesothelioma (MM), a tumor of the pleural and peritoneal cavities with a poor prognosis. Affymetrix microarray/GeneSifter analysis was used to determine alterations in gene expression of a human mesothelial cell line (LP9/TERT-1) by a non-toxic concentration (15×10(6) μm2/cm2) of unprocessed Libby six-mix and negative (glass beads) and positive (crocidolite asbestos) controls. Because manganese superoxide dismutase (MnSOD; SOD2) was the only gene upregulated significantly (p < 0.05) at both 8 and 24 h, we measured SOD protein and activity, oxidative stress and glutathione (GSH) levels to better understand oxidative events after exposure to non-toxic (15×10(6) μm2/cm2) and toxic concentrations (75×10(6) μm2/cm2) of Libby six-mix.

Results: Exposure to 15×10(6) μm2/cm2 Libby six-mix elicited significant (p < 0.05) upregulation of one gene (SOD2; 4-fold) at 8 h and 111 gene changes at 24 h, including a 5-fold increase in SOD2. Increased levels of SOD2 mRNA at 24 h were also confirmed in HKNM-2 normal human pleural mesothelial cells by qRT-PCR. SOD2 protein levels were increased at toxic concentrations (75×10(6) μm2/cm2) of Libby six-mix at 24 h. In addition, levels of copper-zinc superoxide dismutase (Cu/ZnSOD; SOD1) protein were increased at 24 h in all mineral groups. A dose-related increase in SOD2 activity was observed, although total SOD activity remained unchanged. Dichlorodihydrofluorescein diacetate (DCFDA) fluorescence staining and flow cytometry revealed a dose- and time-dependent increase in reactive oxygen species (ROS) production by LP9/TERT-1 cells exposed to Libby six-mix. Both Libby six-mix and crocidolite asbestos at 75×10(6) μm2/cm2 caused transient decreases (p < 0.05) in GSH for up to 24 h and increases in gene expression of heme oxygenase 1 (HO-1) in LP9/TERT-1 and HKNM-2 cells.

Conclusions: Libby six-mix causes multiple gene expression changes in LP9/TERT-1 human mesothelial cells, as well as increases in SOD2, increased production of oxidants, and transient decreases in intracellular GSH. These events are not observed at equal surface area concentrations of nontoxic glass beads. Results support a mechanistic basis for the importance of SOD2 in proliferation and apoptosis of mesothelial cells and its potential use as a biomarker of early responses to mesotheliomagenic minerals.

2000s

Mesothelioma: Do asbestos and carbon nanotubes pose the same health risk?. M.C. Jaurand, A. Renier, J. Daubriac. 2009;6:16. PubMed · Publisher · Free full text

Abstract

Carbon nanotubes (CNTs), the product of new technology, may be used in a wide range of applications. Because they present similarities to asbestos fibres in terms of their shape and size, it is legitimate to raise the question of their safety for human health. Recent animal and cellular studies suggest that CNTs elicit tissue and cell responses similar to those observed with asbestos fibres, which increases concern about the adverse biological effects of CNTs. While asbestos fibres' mechanisms of action are not fully understood, sufficient results are available to develop hypotheses about the significant factors underlying their damaging effects. This review will summarize the current state of knowledge about the biological effects of CNTs and will discuss to what extent they present similarities to those of asbestos fibres. Finally, the characteristics of asbestos known to be associated with toxicity will be analyzed to address the possible impact of CNTs.
Particle length-dependent titanium dioxide nanomaterials toxicity and bioactivity. R.F. Hamilton, N. Wu, D. Porter, et al. 2009;6:35. PubMed · Publisher · Free full text

Abstract

Background: Titanium dioxide (TiO(2)) nanomaterials have considerable beneficial uses as photocatalysts and solar cells. It has been established for many years that pigment-grade TiO(2 )(200 nm sphere) is relatively inert when internalized into a biological model system (in vivo or in vitro). For this reason, TiO(2 )nanomaterials are considered an attractive alternative in applications where biological exposures will occur. Unfortunately, metal oxides on the nanoscale (one dimension < 100 nm) may or may not exhibit the same toxic potential as the original material. A further complicating issue is the effect of modifying or engineering of the nanomaterial to be structurally and geometrically different from the original material.

Results: TiO(2 )nanospheres, short (< 5 mum) and long (> 15 mum) nanobelts were synthesized, characterized and tested for biological activity using primary murine alveolar macrophages and in vivo in mice. This study demonstrates that alteration of anatase TiO(2 )nanomaterial into a fibre structure of greater than 15 mum creates a highly toxic particle and initiates an inflammatory response by alveolar macrophages. These fibre-shaped nanomaterials induced inflammasome activation and release of inflammatory cytokines through a cathepsin B-mediated mechanism. Consequently, long TiO(2 )nanobelts interact with lung macrophages in a manner very similar to asbestos or silica.

Conclusions: These observations suggest that any modification of a nanomaterial, resulting in a wire, fibre, belt or tube, be tested for pathogenic potential. As this study demonstrates, toxicity and pathogenic potential change dramatically as the shape of the material is altered into one that a phagocytic cell has difficulty processing, resulting in lysosomal disruption.
The limits of testing particle-mediated oxidative stress in vitro in predicting diverse pathologies; relevance for testing of nanoparticles. K. Donaldson, P.J. Borm, V. Castranova, et al. 2009;6:13. PubMed · Publisher · Free full text

Abstract

In vitro studies with particles are a major staple of particle toxicology, generally used to investigate mechanisms and better understand the molecular events underlying cellular effects. However, there is ethical and financial pressure in nanotoxicology, the new sub-specialty of particle toxicology, to avoid using animals. Therefore an increasing amount of studies are being published using in vitro approaches and such studies require careful interpretation. We point out here that 3 different conventional pathogenic particle types, PM10, asbestos and quartz, which cause diverse pathological effects, have been reported to cause very similar oxidative stress effects in cells in culture. We discuss the likely explanation and implications of this apparent paradox, and its relevance for testing in nanotoxicology.
Mechanisms of action of inhaled fibers, particles and nanoparticles in lung and cardiovascular diseases. B.T. Mossman, P.J. Borm, V. Castranova, et al. 2007;4:4. Editorial PubMed · Publisher · Free full text

Abstract

Background: A symposium on the mechanisms of action of inhaled airborne particulate matter (PM), pathogenic particles and fibers such as silica and asbestos, and nanomaterials, defined as synthetic particles or fibers less than 100 nm in diameter, was held on October 27 and 28, 2005, at the Environmental Protection Agency (EPA) Conference Center in Research Triangle Park, North Carolina. The meeting was the eighth in a series of transatlantic conferences first held in Penarth, Wales, at the Medical Research Council Pneumoconiosis Unit (1979), that have fostered long-standing collaborations between researchers in the fields of mineralogy, cell and molecular biology, pathology, toxicology, and environmental/occupational health.

Results: The goal of this meeting, which was largely supported by a conference grant from the NHLBI, was to assemble a group of clinical and basic research scientists who presented and discussed new data on the mechanistic effects of inhaled particulates on the onset and development of morbidity and mortality in the lung and cardiovascular system. Another outcome of the meeting was the elucidation of a number of host susceptibility factors implicated in adverse health effects associated with inhaled pathogenic particulates.

Conclusion: New models and data presented supported the paradigm that both genetic and environmental (and occupational) factors affect disease outcomes from inhaled particulates as well as cardiopulmonary responses. These future studies are encouraged to allow the design of appropriate strategies for prevention and treatment of particulate-associated morbidity and mortality, especially in susceptible populations.
Exposure and risks from wearing asbestos mitts. J.W. Cherrie, M. Tindall, H. Cowie. 2005;2:5. PubMed · Publisher · Free full text

Abstract

Background: Very high fibre inhalation exposure has been measured while people were wearing personal protective equipment manufactured from chrysotile asbestos. However, there is little data that relates specifically to wearing asbestos gloves or mitts, particularly when used in hot environments such as those found in glass manufacturing. The aim of this study was to assess the likely personal exposure to asbestos fibres when asbestos mitts were used.

Results: Three types of work activity were simulated in a small test room with unused mitts and artificially aged mitts. Neither pair of mitts were treated to suppress the dust emission. The measured respirable fibre exposure levels ranged from <0.06 to 0.55 fibres/ml, with no significant difference in fibre exposure between aged and unused mitts. The use of high localised ventilation to simulate convective airflows from a furnace reduced exposure levels by about a factor of five. Differences between tasks were statistically significant, with simulated "rowing" of molten glass lowest and replacement of side seals on the furnace highest. Estimated lifetime cancer risk from 20 years exposure at the upper end of the exposure range measured during the study is less than 22 per 100,000.

Conclusion: People who wore asbestos mitts were likely to have been exposed to relatively low levels of airborne chrysotile asbestos fibres, certainly much lower than the standards that were accepted in the 1960's and 70's. The cancer risks from this type of use are likely to be very low.
ROS-mediated genotoxicity of asbestos-cement in mammalian lung cells in vitro. E. Dopp, S. Yadav, F.A. Ansari, et al. 2005;2:9. PubMed · Publisher · Free full text

Abstract

Asbestos is a known carcinogen and co-carcinogen. It is a persisting risk in our daily life due to its use in building material as asbestos-cement powder. The present study done on V79-cells (Chinese hamster lung cells) demonstrates the cytotoxic and genotoxic potential of asbestos-cement powder (ACP) in comparison with chrysotile asbestos. A co-exposure of chrysotile and ACP was tested using the cell viability test and the micronucleus assay. The kinetochore analysis had been used to analyse the pathway causing such genotoxic effects. Thiobarbituric acid-reactive substances were determined as evidence for the production of reactive oxygen species. Both, asbestos cement as well as chrysotile formed micronuclei and induced loss of cell viability in a concentration- and time-dependent way. Results of TBARS analysis and iron chelator experiments showed induction of free radicals in ACP- and chrysotile exposed cultures. CaSO4 appeared to be a negligible entity in enhancing the toxic potential of ACP. The co-exposure of both, ACP and chrysotile, showed an additive effect in enhancing the toxicity. The overall study suggests that asbestos-cement is cytotoxic as well as genotoxic in vitro. In comparison to chrysotile the magnitude of the toxicity was less, but co-exposure increased the toxicity of both.

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