Our results show that asbestos continues to be demonstrable in human lungs, that also chrysotile can be identified after many years, and that there is no significant reduction of asbestos fibre concentrations in lung tissues over time after exposure cessation. Unfortunately, there is no way to eliminate asbestos fibers from a person’s lungs completely. 2006, 312: 2633-2640. Contrib Microbiol. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/8/181/prepub. Asbestos-related changes of the lung have been shown to predominantly affect the lower lobe; this will only be reflected adequately when different samples are analysed separately. Interdisciplinary Guideline of the German Respiratory Society and the German Society of Occupational and Environmental Medicine, in German] German Respiratory Society, German Society of Occupational and Environmental Medicine. 1992, 258: 424-429. 1999, 23: 829-840. In contrast, only amphiboles could be detected in cases with pneumoconiosis in the UK , but chrysotile concentrations showed no elevation compared with a control group. Loffler H, Lukas J, Bartek J, Kramer A: Structure meets functionâcentrosomes, genome maintenance and the DNA damage response. Report of the Pneumoconiosis Committee of the College of American Pathologists and the National Institute for Occupational Safety and Health, Asbestos, asbestosis, and cancer, the Helsinki criteria for diagnosis and attribution 2014: recommendations, Zur Dignität von Asbestkörperzählungen im Lungengewebe [On the dignity of asbestos body counts in lung tissue], Guidelines for mineral fibre analyses in biological samples: report of the ERS Working Group. Duesberg P, Li R, Fabarius A, Hehlmann R: Aneuploidy and cancer: from correlation to causation. c) Transmission electron microscopy image of a chrysotile fibre and the corresponding EDX spectra (d).  states that the lung asbestos fibre burden in 23955 patients was analysed to address fibre type and biopersistence; data from 12 patients undergoing two tissue excisions at intervals at least 4 years were considered. Our laboratory has shown that aerosolized chrysotile fibers longer than 16 microns can be deposited in the peripheral lung parenchyma of rats, and the measured clearance rate of these fibers is not significantly different from zero. b) The EDX spectra of the denudated part of the asbestos body with magnesium (Mg), silicon (Si) and iron (Fe). Individual asbestos concentrations in lung tissue remained stable across intervals of 4–21 years between the two tissue excisions. In this context lung dust analysis is considered a valuable tool [5, 6]. By their pin-like/acicular structure the fibres are prone to get caught in the alveoli of the lung parenchyma, so that clearing them from the system cannot be achieved. The traceability of asbestos fibres in human lungs is a matter of discussion especially for chrysotile. Am Rev Respir Dis. At this time the patients' last exposure to asbestos dated back 3–29 years (median 9.5 years). In autopsy cases, analyses were performed in the upper and lower lobes of either side if possible. Freedom to design, conduct, interpret, and publish research is not compromised by this. In interphasic cells the fibers were normally located in perinuclear region, and also in the middle of the nuclei in multinucleated cells. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Additionally an EDX analysis has been done to determine the asbestos fibre type. Three independent experiments were done, and more than 500 nuclei for each treatment and control cells were analyzed, with the use of a 40X objective. Intra-individual longitudinal data display: the asbestos fibre burden in living human lungs is stable over many years http://ow.ly/VtPF30bRETz. The exposure to chrysotile fibers can cause DNA damage, and this damage can be associated with centrosome amplification too. Laser scanning confocal microscope images of HK2 cells treated with chrysotile for 48 h and recovered for 24 h in fiber-free culture medium: A) metaphase cell with multipolar spindle (microtubules in blue, DNA in red, actin filaments in green), presenting small fragments of chrysotile (pink) inside; A') and A") orthogonal projections showing the intracellular localization of chrysotyle fibers; B) 3D reconstructions of optical sections of the same cell; small chrysotile fibers are also seen outside the cell (small pink dots). Privacy In addition, the fibre type can be analysed with energy-dispersive X-ray (EDX) microanalysis . Below are the links to the authorsâ original submitted files for images. The laboratory has had experience in this method since 1987. Shukla A, Gulumian M, Hei TK, Kamp D, Rahman Q, Mossman BT: Multiple roles of oxidants in pathogenesis of asbestos-induced diseases. 15 Issue. Carcinogenesis. The structure of an amphibole is a double chain of silicate tetrahedral which makes it very strong and durable. The macrophages die and form the typical asbestos bodies [7, 33]. AWMF Registernummer 002–038; 2010. The fibers were able to induce structural and numerical chromosomal aberrations in human amniotic fluid cells, increasing the number of hyperdiploid cells in treated cell population . Hut HM, Lemstra W, Blaauw EH, Van Cappellen GW, Kampinga HH, Sibon OC: Centrosomes Split in the Presence of Impaired DNA Integrity during Mitosis. Since genotoxicity is a general prerequisite for the development of malignancy, a great amount of data, collected in several end-point tests, has shown that the exposure to asbestos fibers results in chromosomal aberrations and mutations. However, the present data show that at least small fibers remain associated with cells after the chrysotile-containing medium is changed to normal culture medium. DNA Histograms of control and chrysotile treated cells. The present data show that chrysotile exposure for 48 h can cause centrosome amplification, apoptosis and aneuploid cell formation even when we used long periods of recovery in HK2 cells. In contrast, in control cells the frequency of multi-polar metaphases was around 5%. b) The EDX spectra of the fibre with magnesium (Mg), silicon (Si) and iron (Fe). Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue, in contrast to amphibole exposure, which is related to inflammatory response, granuloma and mild interstitial fibrosis [3, 4]. lungs: new insights into the chrysotile debate” To the Editor: The article by FEDER et al. ArticleÂ (PNG ). After treatment with Triton X-100 0.5% for 10 min and RNAase for 30 min, the nuclei were stained with propidium iodide. The database research generated a collective of 12 patients with longitudinal data of the asbestos fibre burden. Enter multiple addresses on separate lines or separate them with commas. Google ScholarÂ. The cell preparations were analyzed by confocal laser scanning microscopy (Zeiss LSM 510) with the use of a 40X objective. Apart from that, the type and geometry of fibres seem to play a part. "Data suggest that amphiboles are the major cause of mesotheliomas in asbestos workers." 1993, 319: 301-308. Fibers inside the cells in anaphase and late telophase were frequently located between the two daughter nuclei, and could also be associated with chromatin. Amphibole-fibre with the corresponding spectra obtained by energy-dispersive X-ray (EDX) analysis. Instead of obtaining the half-lives of asbestos fibres by approximation and extrapolation [8, 14, 20], the aim of the present study was to present data based on longitudinal intra-individual asbestos quantitation that was performed in the lung tissues of patients with asbestos diseases over intervals of several years. Mutat Res. They are causally related to the development of asbestosis, bronchial cancer, malignant mesothelioma of pleura and peritoneum, and, to a more limited extent, to various gastrointestinal, oropharyngeal and laryngeal cancers [1, 2]. Asbestos concentrations found in the air in the working environment were related to fibre concentrations in lung tissue from autopsies. 2003, 14: 1993-2004. This study is the first to present longitudinal intra-individual data about the asbestos fibre burden in living human lungs. Chrysotile treated cells during cell cycle. Cassiola F, Rogers RA, Kiyohara PK, Joekes I: Yeast cells long-term interaction with asbestos fibers. Cells in different phases of cell cycle presented both long chrysotile fibers and small fragment of fibers internalized. Aberrant mitosis and multi-polar spindles observed in asbestos treated cells could lead to the incorrect chromosome segregation and result in aneuploid cells [9, 10]. Illustration of birth, duration of exposure and time interval between tissue excisions/bronchoalveolar lavage (first fibre analysis from surgery, second fibre analysis from autopsy) in comparison to asbestos consumption in Germany. Thus it is clear that there has been no relevant change in the ratio of amphibole to chrysotile fibres and that the chrysotile concentration has remained stable over the investigation period of 4–21 years. However, cells in metaphase with more than 4 centrosomes and also with 6 centrosomes per cell were observed. The major health hazards associated with asbestos are fibrogenicity and carcinogenicity, with lung cancer and mesothelioma as the main representatives. However, the result of light microscopic counts are a good measure for the overall asbestos fibre burden of the lung tissue under investigation and may well serve for comparative analyses . It is not only the amphibole fibres that can be identified in lung tissue over many years, but also chrysotile fibres, as is typical of pneumoconiosis due to asbestos fibres. Nuclear DNA contents of mononucleated, binucleated and multinucleated control and treated cells were independently quantified by image analysis. Scheme of apoptosis, multinucleation and aneuploid HK2 cells formation after chrysotile exposure. B and C) HK2 cells after treatment with chrysotile for 48 h and additional 24 h in fiber-free medium show high frequency of multinucleated cells (arrows) and apoptosis (arrows tips). It remains less biopersistent than ceramic and special purpose glasses and considerably less biopersistent than amphibole asbestos. The total asbestos fibre burden of the lungs had remained constant across the complete period of investigations. From these 10 patients the asbestos fibre burden of the lung has been determined from tissue both times. Additionally, 3D reconstruction of confocal images, detecting fibers by their own auto-fluorescence, represented an important approach in the study of cell-fibers interaction, and evidenced fibers inside the cytoplasm and closely associated with chromatin. Glaucia M Machado-Santelli. In this study were used human lung cancer cells, which have mutations required for acquisition of malignant phenotypes. 2003, 34: 1117-1129. However ninety to ninety five percent of all asbestos products manufactured have been made from chrysotile asbestos, from 1874 when asbestos was first used for industrial applications to the present. Thus an asbestos-related disease might occur a long time after exposure cessation. BMC Cancer 8, 181 (2008). Fukasawa K: Centrosome amplification, chromosome instability and cancer development. The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. Flow process chart showing how the register's database has been filtered. Takada S, Kelkar A, Theurkauf WE: Drosophila checkpoint kinase 2 couples centrosome function and spindle assembly to genomic integrity. Chrysotile fibre with the corresponding spectra obtained by energy-dispersive X-ray analysis (EDX). Eur Mol Biol Org J. Detached cells were fixed with formaldehyde 3.7% for 30 min, washed with PBSA, and spun down on poly-lysine coated glass slides with the use of a cytological centrifuge (3.104cell/slide, 1000 rpm for 2 min). This issue is of high significance for differential diagnosis, risk assessment and occupational compensation. The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. In our routine examinations great importance is set to keep losses at the lowest possible level when processing the samples and centrifugation is not used. This article has supplementary material available from erj.ersjournals.com. Nevins JR: E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Its clearance from the lung is faster than it is with amphibole fibers, for chrysotile asbestos fails to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Chrysotile, which is rapidly attacked by the acid environment of the macrophage, falls apart in the lung into short fibers and particles, while the amphibole asbestos persist creating a response to the fibrous structure of this mineral. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. ArticleÂ After 24 h in culture, the medium was changed to 2 mL of fresh medium with chrysotile fibers at an approximated final concentration of 0.25 mg/mL. statement and Mol Biol Cell. At the time of the first tissue sampling the last asbestos exposure had ended 3–29 years ago (median 9.5 years). "Calidria chrysotile cleared from the lung with a half-time of 0.3 days, 7 hours, which is faster than any other commercial mineral fiber." Walker C, Everitt J, Barrett JC: Possible cellular and molecular mechanisms of asbestos carcinogenicity. Apoptotic cells could be caused by checkpoint activation due several DNA or cell damage, or by centrosome amplification. The frequency of cells with two or more nuclei increased from about 15% in control cells to 45% in chrysotile-treated cells, reaching approximately 65% after longer recovery time (P < 0.01, Table 1). primarily the Helsinki Criteria [5, 26]. The image stacks were used for 3D reconstruction (B), evidencing the spatial interaction between cell and fiber, where the fiber seems to segment the nucleus. Churg A, Wright JL, Gilks B, DePaoli L: Rapid short-term clearance of chrysotile compared with amosite asbestos in the guinea pig. 1999, 9: 429-432. 2005, 6: 2-9. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. III/01;2001. The lysate was filtered through a 2 µm Millipore nitrocellulose membrane and examined under a light microscope in a meandering pattern using ×200 or ×400 magnification and phase contrast. t: tons. After 48 h chrysotile exposure and 24 h of recovery in normal medium many fibers were found inside the HK2 cells. … Chrysotile treated cells acquired some features rarely observed in control cells (Figure 1A), such as the presence of multinucleated and apoptotic cells (Figure 1B and 1C). Dopp E, Schuler M, Schiffmann D, Eastmond DA: Induction of micronuclei, hyperdiployd and chromosomal breakage affecting the centric/pericentric regions of chromosomes 1 and 9 in human amniotic fluid cells after treatment with asbestos and ceramic fibers. Apoptotic cells, identified by the presence of fragmented chromatin, represented 0.26% of control cells. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. For the whole group there was no relevant decrease in the asbestos fibre burden over the investigated time (p=0.9863, one-sided Wilcoxon signed rank test). In 16.7% asbestosis grade III was diagnosed, but grade I diseases dominated (58.3%) (table 2). Short chrysotile fibres are cleared from the lung rapidly and longer ones at a slower rate. The available data suggest that chrysotile is deposited in the parenchyma but is cleared extremely rapidly, with the vast bulk of fibres removed from human lungs within weeks to months after inhalation; by comparison, amphibole clearance half-lives are of the order of years to decades. The proportion of chrysotile fibres did not correlate with the duration of asbestos exposure (p=0.65; rho –0.24, Spearman's rank correlation) or with the time interval between last asbestos exposure and tissue analyses (p=0.74; rho 0.18 Spearman's rank test). Imaging of chrysotile fiber was performed in a confocal configuration to detect its autofluorescence (excitation with Argon laser at 488 nm, HFT 488 filter; for details see Additional file 1). Supplementary materials ERJ-02534-2016_Supplement. As TEM is considered to be the gold standard for fibre analysis [6, 26], we also applied this method to the available samples confirming the FE-SEM data. According to Roggli and Pratt , 200 asbestos bodies per gram of wet lung tissue correspond to two asbestos bodies in a histological specimen of 2×2 cm at a 5 µm section thickness. 3) The highest asbestos body count had to be above 500 fibres per gram of wet lung tissue. (1992)  this mechanism would involve direct and indirect effects: the physical interaction of fibers with target cells or the free radicals generation from the fiber surface acting directly on DNA and indirectly on inflammatory reactions. In 16.7% where contact with asbestos dated back to between 20 and 29 years, the first lung dust analyses had been performed on BAL samples. They showed elevated asbestos fibre burden that had been confirmed at autopsies carried out 8–13 years later. Short fibres generally exhibit less macrophage toxicity and less release of cytokines than longer ones. Asbestos fibres, therefore, remain in the lung where their sustained presence causes lung disease. The contralateral lobe is available from autopsy tissue, as recommended in the guidelines [6, 26]; also, the lower lobe is available, which commonly contains higher fibre counts. Chrysotile 5R (Quebec Standard) obtained from SAMA MineraÃ§Ã£o de Amianto Ltda (MinaÃ§u, GO, Brazil) were kindly provided by Dr. Flavia M. Cassiola. The cell-fiber interaction was analyzed by confocal microscopy and 3D reconstruction of HK2 cells exposed to chrysotile fibers for 48 h and maintained 24 h in normal medium. Calidria chrysotile has been found to be one of the most rapidly cleared mineral fibers from the lung. However, some fibers interacting with the nucleus were observed. Taking the maximum count from each patient the first analysis produced a median of 1623 (431–9750), and the second analysis of 4269 (714–53 663) asbestos bodies per gram of wet lung tissue (table 1). Cytokinesis failure can also generate cells with abnormal centrosome number, since the centrosome duplication occurs in G1/S  and in this context the cytokinesis failure can be a process involved in centrosome amplification observed in treated cells. Results of fibre analyses at surgery (first tissue excision) and autopsy (second tissue excision) in relation to time from 10 patients. Conclusion. However, the mechanism by which asbestos produce malignancy is unclear at the present moment . California Privacy Statement, Tissue from surgery is of limited size or from the side of the tumour in cases where a tumour is present. Our unique data experimentally confirmed the well-known characteristics of the asbestos fibres (asbestos in ancient Greek ἄσβεστος=imperishable) for individual human lungs in vivo over many years. [FE-SEM Workbook: Determination of Inorganic Fibres in Human Lung Tissue], Assessment of asbestos body formation by high resolution FEG-SEM after exposure of Sprague-Dawley rats to chrysotile, crocidolite, or erionite. Inhalation Toxicology ISSN: 0895-8378 EISSN: 1091-7691 Volume. Our samples are taken from different tissue excisions from the same patient. … Chrysotile fibers in interphasic cells were normally located in perinuclear region, and also in the middle of the nuclei in multinucleated cells (Additional file 2). In Germany at least two asbestos bodies in a specimen of 1×1 cm are required for the histological diagnosis of asbestosis . This ties in very well with earlier reports of latency periods from 10 to 60 years of asbestos-related diseases, which are considered to develop typically while asbestos is present . Correct chromosome segregation depends on the presence of two centrosomes and bipolar spindles. Electron microscopic analyses of all available samples were done (n=6). Chrysotile is considered less harmful to human health than other types of asbestos fibers. 2) Earlier asbestos fibre burden from surgery or bronchoalveolar lavage had to be available. Online ISSN: 1399-3003, Copyright © 2020 by the European Respiratory Society. A) Control HK2 cells were predominantly mononucleated and apoptosis were rarely observed. The differences of frequency of apoptotic, mononucleated, binucleated and multinucleated cells were tested, between the control situation and after the chrysotile treatment, considering the three periods used of recovery in normal medium. ArticleÂ The images of multipolar mitotic cells show small fibers internalized, observed in 3D reconstructions, and some of the small fragments were interacting with the chromatin (Figure 3AâB). Transmission electron microscopy (TEM) may also be used. Twenty-five years of fiber analysis: what have we learned? In contrast, chrysotile has been found post-mortem in smaller amounts than expected in the lungs of asbestos workers" (Mossman 1990, p. 296). The centrosome amplification has been proposed as a mechanism of elimination of damage cells, causing the "mitotic catastrophe" [27â29]. Continuous irritation of the tissue results in chronic inflammation to which the tissue responds by scar formation . Thus, exposure is not a confounder in the present data. Lfg 26. The fibers between 5 and 20μm in length were cleared with a half-time of 7 days. Relatives gave consent for all autopsies. When the nuclear envelop breakdown in early mitosis, chrysotile fibers appear to interact with the chromatin, and could lead to chromatin fragmentation and to cytokinesis failure forming an aneuploid or multinucleated cell with more than one centrosome. The biopersistence of Canadian chrysotile asbestos following inhalation Author(s) Bernstein, DM; Rogers, R; Smith, P Year. Also, loss or gain of chromosomes can be consequence of segregation errors and cytokinesis failure leading to high chromosome instability. Google ScholarÂ. Occupational history is listed in table 2. In contrast to other authors, the analyses reported in this manuscript concerned one and the same patients. 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