ArticleLiterature Review

Biomarkers in risk assessment of asbestos exposure

Authors:
  • Patanjali Research Institute
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Abstract

Developments in the field of molecular epidemiology and toxicology have given valuable tools for early detection of impending disease or toxic condition. Morbidity due to respiratory distress, which may be due to environmental and occupational exposure, has drawn attention of researchers worldwide. Among the occupational exposure to respiratory distress factors, fibers and particles have been found to be main culprits in causing diseases like asbestosis, pleural plaques, mesotheliomas and bronchogenic carcinomas. An early detection of the magnitude of exposure or its' effect using molecular end points is of growing importance. The early inflammatory responses like release of the inflammatory cells collected by non-invasive methods give an indication of the unwanted exposure and susceptibility to further complications. Since free radicals like O2-, OH, OOH, NO, NOO, etc. are involved in the progression of asbestos-related diseases and lead to cytogenetic changes, an evaluation of antioxidant states reducing equivalents like GSH and ROS generation can be a good biomarker. The cytogenetic end points like chromosomal aberration, micronucleus formation and sister chromatid exchange give indication of genetic damage, hence they are used as effective biomarkers. New techniques like fluorimetric analysis of DNA unwinding, alkaline elution test, fluorescent in situ hybridization and comet assay are powerful tools for early detection of initiation of disease process and may help in planning strategies for minimizing morbidity related to asbestos fiber exposure. The present review article covers in detail possible biomarkers for risk assessment of morbidity due to fibers/particles in exposed population.

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... Such indicators are vital tools to assess damage to health caused by exposure to certain chemicals. They are associated with biochemical and physiological changes and they also provide information that is used to estimate risk to human health [9][10][11]. ...
... Regarding pneumoconiosis, such changes are associated with chronic inflammatory processes linked to the formation of reactive oxygen species (ROS) in the lower respiratory tract [9,12]. Elevated and continuous ROS production or inadequate ROS removal may overcome the antioxidant defense system and cause oxidative stress, which is an imbalance between antioxidants and oxidants in favor of the latter, leading to cell damage [2,13,14]. ...
... Enzymatic antioxidants include superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST) and catalase (CAT), while glutathione (GSH) is an example of a non-enzymatic antioxidant. Such antioxidants may be considered effective biological indicators, which represent outcomes that cause health side effects [9]. Other biological indicators evaluate cytogenetic alterations caused by exposure to potentially genotoxic agents, and include chromosomal aberrations (CA), sister chromatid exchange, isolated and oncogenic mutations, micronuclei and the comet assay [9]. ...
Article
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Genotoxic effects of occupational workers exposed to asbestos can be evaluated using different biomarkers as oxidative stress enzymes in conjuction with comet assay. This study assessed changes to oxidative stress enzymatic parameters and genotoxic damage in workers occupationally exposed and non-exposed to chrysotile asbestos, who attended the outpatient Clinic of the Center for Worker Health Studies and Human Ecology (CESTEH/ENSP/FIOCRUZ) in Brazil. Chest radiography and spirometry were performed to assess clinical progression of symptoms. The traditional visual score comet assay in peripheral whole blood cells was used to assess DNA damage, and oxidative stress was evaluated by measuring catalase (CAT) and glutathione S-transferase (GST) activities. Respiratory alterations were observed in 53% of workers exposed, as determined by pulmonary function and bronchodilation, and 6 workers were diagnosed with asbestosis. The comet assay was statistically significantly higher in the exposed group and individuals with asbestosis compared to the non-exposed group and individuals without asbestosis, respectively. Linear regression analysis showed that 28,4% and 50,5% of comet assay results were increased by exposure to asbestos and developed asbestosis. The results of CAT and GST were not difference between the groups. These results supports the association of genotoxic damage and the onset of asbestosis by chrysotile asbestos exposure in workers of this study.
... This leads to a feedback loop between ROS generation and increased TNFa expression, resulting in increased DNA damage [81]. Also, other interleukins are released by inflammatory cells [82] upon phagocytosis of fibres and e.g. IL6 has been shown to be up-regulated in airway epithelial cells by NF-jB in response to asbestos-exposure [83]. ...
... EGFR/MAPK/ERK pathway. Asbestosinduced oxidative stress causes activation of the epidermal growth factor receptor (EGFR) by phos- phorylation [82,90,91]. EGFR activates the MAPK/ ERK pathway through phosphorylation of ERK1/2 and ERK5 [92]. ...
Article
Asbestos-exposure is associated with an increased risk of lung cancer, one of the leading causes of cancer deaths worldwide. Asbestos is known to induce DNA and chromosomal damage as well as aberrations in signalling pathways, such as the MAPK and NF-kappaB cascades, crucial for cellular homeostasis. The alterations result from both indirect effects through e.g. reactive oxygen/nitrogen species and direct mechanical disturbances of cellular constituents. This review describes the current knowledge on genomic and pathway aberrations characterizing asbestos-related lung cancer. Specific asbestos-associated molecular signatures can assist the development of early biomarkers, molecular diagnosis, and molecular targeted treatments for asbestos-exposed lung cancer patients.
... Humans continually come into contact with small particles, and they are generally not hurt by being exposed to these particles. However, inhalation of micron-sized quartz particles or of asbestos fibers has proven to be quite harmful to health [2,[336][337][338]. Similarly, nanoparticles resulting from combustion processes such as forest fires or industrial pollution have a detrimental effect [336]. ...
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This review is designed to be a comprehensive source for polymer nanocomposite research, including fundamental structure/property relationships, manufacturing techniques, and applications of polymer nanocomposite materials. In addition to presenting the scientific framework for the advances in polymer nanocomposite research, this review focuses on the scientific principles and mechanisms in relation to the methods of processing and manufacturing with a discussion on commercial applications and health/safety concerns (a critical issue for production and scale-up). Hence, this review offers a comprehensive discussion on technology, modeling, characterization, processing, manufacturing, applications, and health/safety concerns for polymer nanocomposites.
... Asbestos exposure in humans is associated with the development of asbestos-related diseases (ARD) such as pulmonary fibrosis and lung cancer. Although the exact mechanism leading to the progression of ARD has not been fully explained, mounting evidence indicates that reactive oxygen species (ROS), such as the superoxide anion and the hydroxyl radical, plays a significant role (Bhattacharya et al., 2005;Kamp and Weitzman, 1999). Because airways are continuously exposed to high levels of environmental oxidants, they must maintain the proper balance between pro-oxidants and antioxidants to prevent oxidative stress and cellular damage. ...
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The community members of Libby, MT, have experienced significant asbestos exposure and developed numerous asbestos-related diseases including fibrosis and lung cancer due to an asbestos-contaminated vermiculite mine near the community. The form of asbestos in the contaminated vermiculite has been characterized in the amphibole family of fibers. However, the pathogenic effects of these fibers have not been previously characterized. The purpose of this study is to determine the cellular consequences of Libby amphibole exposure in macrophages compared to another well-characterized amphibole fiber; crocidolite asbestos. Our results indicate that Libby asbestos fibers are internalized by macrophages and localize to the cytoplasm and cytoplasmic vacuoles similar to crocidolite fibers. Libby asbestos fiber internalization generates a significant increase in intracellular reactive oxygen species (ROS) as determined by dichlorofluorescein diacetate and dihydroethidine fluorescence indicating that the superoxide anion is the major contributing ROS generated by Libby asbestos. Elevated superoxide levels in macrophages exposed to Libby asbestos coincide with a significant suppression of total superoxide dismutase activity. Both Libby and crocidolite asbestos generate oxidative stress in exposed macrophages by decreasing intracellular glutathione levels. Interestingly crocidolite asbestos, but not Libby asbestos, induces significant DNA damage in macrophages. This study provides evidence that the difference in the level of DNA damage observed between Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure.
... During the Vietnam War an estimated 76,540,964 litres of phenoxylic herbicides, including Agent Orange, were sprayed over approximately 3.6 million hectares of Vietnamese and Laotian land in order to remove forest cover, destroy crops and clear vegetation from around military installations (Stellman et al., 2003). Iannuzzi et al., 2004; Akin et al., 2005; Bhattacharya et al., 2005). The current study was thus performed in an attempt to determine whether New Zealand Vietnam War veterans have incurred any chromosomal disturbances as a consequence of their service in Vietnam. ...
Article
From July 1965 until November 1971, New Zealand Defence Force Personnel fought in the Vietnam War. During this time more than 76,500,000 litres of phenoxylic herbicides were sprayed over parts of Southern Vietnam and Laos, the most common being known as 'Agent Orange'. The current study aimed to ascertain whether or not New Zealand Vietnam War veterans show evidence of genetic disturbance arising as a consequence of their now confirmed exposure to these defoliants. A sample group of 24 New Zealand Vietnam War veterans and 23 control volunteers were compared using an SCE (sister chromatid exchange) analysis. The results from the SCE study show a highly significant difference (P < 0.001) between the mean of the experimental group (11.05) and the mean of a matched control group (8.18). The experimental group also has an exceptionally high proportion of HFCs (cells with high SCE frequencies) above the 95th percentile compared to the controls (11.0 and 0.07%, respectively). We conclude that the New Zealand Vietnam War veterans studied here were exposed to a clastogenic substance(s) which continues to exert an observable genetic effect today, and suggest that this is attributable to their service in Vietnam.
... Fibres also may produce ROS. Moreover, mesothelial cells respond by fibre internalisation according to a phagocytic process associated with oxidative reactions [34,[88][89][90][91][92][93]. ...
Article
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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.
... Furthermore, asbestos-exposed macrophages release various inflammatory cytokines, such interleukin 1 (IL1) and the tumor necrosis factor-alpha (TNF-α). These proteins are assumed to be involved in pulmonary fibrotic processes occurring during asbestosis as they increase the activation of neutrophils and fibroblasts [12,13]. In addition, cytokine and oxidative stress can increase the expression and activity of the inducible nitric oxide synthase (iNOS) in pulmonary alveolar epithelial cells [14]. ...
Article
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Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres.
... Asbestos induced oxidative stress and infl ammation directed pathways are intertwined and complex involving the induced expression of several genes such as TNFα and NF-κB subunits as well as EGFR and MAPK (Figure 7, reviewed in Shukla et al., 2003b). In addition, antioxi-dant enzymes and peptides, such as superoxide dismutase (SOD) and glutathione have been proposed to be involved in the pathogenesis of asbestos-related lung cancer, causing disturbances in the oxidantantioxidant balances and subsequently reducing the cell's capability to protect itself against ROS and RNS (Bhattacharya et al., 2005; Fattman et al., 2006; Pande et al., 2006; Puhakka et al., 2002; Tan et al., 2004; Wang et al., 2006). DNA damage repair pathways are up-regulated in asbestos-treated cells (Upadhyay et al., 2003). ...
Thesis
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Background: Asbestos is a well known cancer-causing mineral fibre, which has a synergistic effect on lung cancer risk in combination with tobacco smoking. Several in vitro and in vivo experiments have demonstrated that asbestos can evoke chromosomal damage and cause alterations as well as gene expression changes. Lung tumours, in general, have very complex karyotypes with several recurrently gained and lost chromosomal regions and this has made it difficult to identify specific molecular changes related primarily to asbestos exposure. The main aim of these studies has been to characterize asbestos-related lung cancer at a molecular level. Methods: Samples from asbestos-exposed and non-exposed lung cancer patients were studied using array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to detect copy number alterations (CNA) as well as microsatellite analysis to detect allelic imbalance (AI). In addition, asbestos-exposed cell lines were studied using gene expression microarrays. Results: Eighteen chromosomal regions showing differential copy number in the lung tumours of asbestos-exposed patients compared to those of non-exposed patients were identified. The most significant differences were detected at 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2 and 19p13.1-p13.3 (p<0.005). The alterations at 2p and 9q were validated and characterized in detail using AI and FISH analysis in a larger study population. Furthermore, in vitro studies were performed to examine the early gene expression changes induced by asbestos in three different lung cell lines. The results revealed specific asbestos-associated gene expression profiles and biological processes as well as chromosomal regions enriched with genes believed to contribute to the common asbestos-related responses in the cell lines. Interestingly, the most significant region enriched with asbestos-response genes was identified at 2p22, close to the previously identified region showing asbestos-related CNA in lung tumours. Additionally, in this thesis, the dysregulated biological processes (Gene Ontology terms) detected in the cell line experiment were compared to dysregulated processes identified in patient samples in a later study (Ruosaari et al., 2008a). Commonly affected processes such as those related to protein ubiquitination, ion transport and surprisingly sensory perception of smell were identified. Conclusions: The identification of specific CNA and dysregulated biological processes shed some light on the underlying genes acting as mediators in asbestos-related lung carcinogenesis. It is postulated that the combination of several asbestos-specific molecular alterations could be used to develop a diagnostic method for the identification of asbestos-related lung cancer. Bakgrund: Asbest är en välkänd cancerframkallande mineralfiber, som i kombination med tobaksrökning ökar risken för lungcancer flerfalt. Flera in vitro och in vivo studier har visat att asbest kan framkalla kromosomskador och förändringar i genexpression. Lungtumörer har i allmänhet mycket komplicerade karyotyper med flera återkommande duplicerade och deleterade kromosomregioner, vilket har gjort det svårt att identifiera specifika molekylära avvikelser som kan associeras med asbestexponering. Det främsta syftet med dessa studier har varit att karaktärisera asbestrelaterad lungcancer på molekylär nivå. Metoder: Prover från asbestexponerade och icke exponerade patienter med lungcancer undersöktes med hjälp av jämförande genomisk hybridisering på mikroarray (aCGH) och fluorescerande in situ hybridisering (FISH) för att identifiera kromosomavvikelser, samt med mikrosatellitanalys för att identifiera allel-obalans (AI). Dessutom utfördes in vitro studier på asbestexponerade cellinjer med hjälp av mikroarrays som mäter enskilda geners expression. Resultat: Aderton kromosomregioner med asbestrelaterade avvikelser identifierades. De mest markanta skillnaderna upptäcktes i 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2 och 19p13.1-p13.3 (p<0,005). Avvikelserna på kromosomarmarna 2p och 9q karakteriserades i detalj och verifierades med hjälp av AI- och FISH-analys på prover från ett ökat antal patienter. In vitro studier genomfördes för att undersöka tidiga förändringar i genexpression som orsakats av asbestexponering i tre olika lungcellinjer. Resultaten avslöjade särskilda asbestrelaterade genexpressionsprofiler och biologiska processer, samt kromosomregioner berikade med gener som antas bidra till de gemensamma asbestrelaterade responserna i cellinjerna. Den mest signifikanta regionen, överrepresenterad med asbestresponsgener, var 2p22 som ligger nära det tidigare identifierade området på 2p med asbestrelaterade kromosomavvikelser i lungtumörer. I denna avhandling jämfördes också de förändrade biologiska processerna (genontologiska termerna) som upptäcktes i cellinjeexperimentet med förändrade processer som identifierats i asbestexponerade och icke exponerade patienters prover, i en senare studie (Ruosaari et al., 2008a). Gemensamt förändrade processer var bl.a. kopplade till protein ubiquitinering, jontransport och överraskande nog, luktförnimmelse. Slutsatser: Kartläggningen av specifika kromosomavvikelser och förändrade biologiska processer kastar ljus över de bakomliggande generna som fungerar som medlare i asbestrelaterad lungkarcinogenes. Det kan antas att en kombination av flera asbestspecifika molekylära förändringar kunde användas för att utveckla en diagnostisk metod, som följaktligen kunde särskilja mellan asbestrelaterad och icke asbestrelaterad lungcancer. TAUSTA: Asbesti on tunnettu syöpää aiheuttava mineraalikuitu, jolla on tupakoinnin yhteydessä synergistinen vaikutus keuhkosyövän riskiin. Useat in vitro- ja in vivo-tutkimukset ovat osoittaneet, että asbesti voi aiheuttaa kromosomivaurioita ja muutoksia geenien ilmentymisessä. Keuhkosyövän karyotyyppi on yleensä hyvin monimutkainen ja toistuvat kromosomialueiden monistumat sekä häviämät ovat yleisiä. Tästä syystä on ollut vaikeaa tunnistaa spesifisiä molekyylitason muutoksia, jotka liittyvät pääasiassa asbestialtistumiseen. Päätavoite näissä tutkimuksissa on ollut asbestiin liittyvän keuhkosyövän tunnistaminen molekyylitasolla. MENETELMÄT: Asbestialtistuneiden ja altistumattomien keuhkosyöpäpotilaiden näytteet tutkittiin käyttäen vertailevaa genomista hybridisaatiota mikrosiruilla (aCGH) ja fluoresenssi in situ hybridisaatiota (FISH), joilla havaitaan kromosomialueiden kopiolukumuutokset, sekä mikrosatelliittianalyysia, jolla havaitaan alleeliepätasapaino (AI). Lisäksi asbestialtistuneita solulinjoja tutkittiin käyttäen geeniekspressiomikrosiruja. TULOKSET: Kahdeksallatoista kromosomialueella osoitettiin kopiolukueroja asbestialtistuneiden ja altistumattomien potilaiden näytteiden välillä. Merkittävimmät erot havaittiin kromosomialueilla 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2 ja 19p13.1-p13.3 (p <0,005). Muutokset 2p ja 9q alueilla karakterisoitiin tarkemmin ja varmennettiin käyttäen AI- ja FISH-analyysejä laajemmassa tutkimusaineistossa. Lisäksi mikrosiruilla tutkittiin muutokset geenien ilmentymisessä asbestialtistuksen jälkeen kolmessa eri keuhkosolulinjassa. Tutkimuksessa tunnistettiin asbestialtistukseen liittyviä geenien ilmentymisprofiileja sekä muuttuneita biologisia prosesseja. Lisäksi havaittiin solulinjoille yhteisten asbestiin liittyvien vastegeenien rikastuttamia kromosomaalisia alueita. Merkittävin asbestivastegeenejä sisältävä alue oli 2p22, joka sijaitsee lähellä aiemmin keuhkosyövissä tunnistettua asbestiin liittyviä kopiolukumuutoksia sisältävää aluetta 2p:ssa. Tässä väitöskirjassa vertailtiin myös asbestialtistuneiden solulinjojen muuttuneita biologisia prosesseja (geeniontologiatermejä) niihin muuttuneisiin prosesseihin, joita myöhemmin havaittiin asbestialtistuneiden potilaiden näytteissä (RUOSAARI et al., 2008a). Yhteiset muuttuneet prosessit liittyivät proteiinien ubikitinaatioon, ionikuljetukseen ja yllättävästi hajuaistimukseen. JOHTOPÄÄTÖKSET: Spesifisten kopiolukumuutosten ja muuttuneiden biologisten prosessien tunnistaminen asbestiin liittyvässä keuhkosyövässä valottaa taustalla olevia geenejä, jotka toimivat välittäjinä asbestin aiheuttamassa keuhkokarsinogeneesissä. Useita asbestiin liittyviä molekyylitason muutoksia voitaisiin käyttää asbestiin liittyvän ja liittymättömän keuhkosyövän erottavien diagnostisten menetelmien kehittämisessä.
... The respiratory droplets contain biomarkers such as those of oxidative stress (8-isoprostane, hydrogen peroxide), inflammation (leukotrienes, prostaglandins), cytokines and chemokines, as well as VOCs [27][28][29]. The precise source of these different biomarkers is probably different for each one and is being actively investigated by several research groups. ...
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Asbestos usage was very common worldwide in the last century and continues in several countries today. Several diseases occur due to asbestos exposure, including malignant tumours such as malignant mesothelioma of the pleura and lung cancer, which have a very poor prognosis. Asbestos inhalation may also result in more benign conditions such as asbestosis (or pulmonary fibrosis due to asbestos), pleural plaques and pleural thickening. It is predicted that asbestos-associated mortality and morbidity will continue to increase, but methods for diagnosing asbestos-related disease are currently invasive and unsuitable for an increasingly elderly population. New non-invasive methods such as analysis of exhaled breath biomarkers e.g. exhaled nitric oxide (F(E)NO), exhaled breath condensate or of exhaled volatile organic compounds could potentially be extremely useful in these conditions. This article reviews the current literature on this topic and suggests areas for their application in the future.
... Similar kind of effect was observed with human lung epithelial and airway epithelial (HEp-2) cell line, when co-treated with antioxidant NAC and Resverartrol against ZnO and CuO nanoparticles respectively [19,21]. Both type of nanoparticles (TiO 2 and MWCNTs) are known to induce Micronuclei (MN) in dose dependent manner, which is well defined biomarker for genotoxicity assay [31]. Our findings of MN MWCNTs + NAC (2 mM). ...
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We study the ameliorative potential of dimetylthiourea (DMTU), an OH• radical trapper and N-acetylcysteine (NAC), a glutathione precursor/H₂O₂ scavenger against titanium dioxide nanoparticles (TiO₂-NPs) and multi-walled carbon nanotubes (MWCNTs) induced cyto-genotoxicity in cultured human lung cancer cells-A549. Cytogenotoxicity was induced by exposing the cells to selected concentrations (10 and 50 µg/ml) of either of TiO₂-NPs or MWCNTs for 24 h. Anti-cytogenotoxicity effects of DMTU and NAC were studied in two groups, i.e., treatment of 30 minutes prior to toxic insult (short term exposure), while the other group received DMTU and NAC treatment during nanoparticles exposure, i.e., 24 h (long term exposure). Investigations were carried out for cell viability, generation of reactive oxygen species (ROS), micronuclei (MN), and expression of markers of oxidative stress (HSP27, CYP2E1), genotoxicity (P⁵³) and CYP2E1 dependent n- nitrosodimethylamine-demethylase (NDMA-d) activity. In general, the treatment of both DMTU and NAC was found to be effective significantly against TiO₂-NPs and MWCNTs induced cytogenotoxicity in A549 cells. Long-term treatment of DMTU and NAC during toxic insults has shown better prevention than short-term pretreatment. Although, cells responded significantly to both DMTU and NAC, but responses were chemical specific. In part, TiO₂-NPs induced toxic responses were mediated through OH• radicals generation and reduction in the antioxidant defense system. While in the case of MWCNTs, adverse effects were primarily due to altering/hampering the enzymatic antioxidant system. Data indicate the applicability of human lung cancer cells-A549 as a pre-screening tool to identify the target specific prophylactic and therapeutic potential of drugs candidate molecules against nanoparticles induced cellular damages.
... L-arginine derived NO production is mediated by activation of nitric oxide synthase (NOS), and there are three isoforms which are endothelial NOS (eNOS), neuronal NOS (nNOS) and [95,96]. NO as a screening tool has been used for various diseases, such as cerebral strokes, asthma and chronic obstructive pulmonary diseases [97][98][99][100]. ...
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Stem cells have been considered for their potential in pharmaceutical research, as well as for stem cell-based therapy for many diseases. Despite the potential for their use, the challenge remains to examine the safety and efficacy of stem cells for their use in therapies. Recently, oxidative stress has been strongly implicated in the functional regulation of cell behavior of stem cells. Therefore, development of rapid and sensitive biomarkers, related to oxidative stress is of growing importance in stem cell-based therapies for treating various diseases. Since stem cells have been implicated as targets for carcinogenesis and might be the origin of "cancer stem cells", understanding of how oxidative stress-induced signaling, known to be involved in the carcinogenic process could lead to potential screening of cancer chemopreventive and chemotherapeutic agents. An evaluation of antioxidant states reducing equivalents like GSH and superoxide dismutase (SOD), as well as reactive oxygen species (ROS) and nitric oxide (NO) generation, can be effective markers in stem cell-based therapies. In addition, oxidative adducts, such as 4-hydroxynonenal, can be reliable markers to detect cellular changes during self-renewal and differentiation of stem cells. This review highlights the biomarker development to monitor oxidative stress response for stem cell-based chemical screening.
... In this context, due to the often unknown environmental exposure to asbestos, low price biomarkers of exposure are needed [56][57][58][59] for the ascertainment of environmental exposure and the early prevention of health effects. From this point of view, a new frontier can be represented by the use of the comet assay [60][61][62][63] since inhaled asbestos may induce oxidative stress and biochemical changes in lipids, proteins, DNA and RNA [18]. ...
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The study describes a cluster of 71 malignant mesothelioma cases among Bari residents without asbestos exposure other than residential exposure. This small cohort, as expected, was composed of a majority of females (56.34%) with a M/F ratio of 0.8, ages ≤ 65 years old (52.11%) and the epithelioid morphological type (78.87%). Sixty-four subjects (90.14%) lived between 10 m and 1000 m from the asbestos cement factory (Fibronit), and the latency length was longer than 55 years for 25 subjects (35.21%). The adjusted risk (adjusted OR) of observing the epithelial form of mesothelioma among subjects living at small distances from Fibronit was high (OR = 1.870 (0.353-9.905)) for people living 550-1000 m from the site and for those living less than 550 m from the site (OR = 1.470 (0.262-8.248)). Additionally, the subjects with a high length of exposure showed a relevant risk of epithelioid mesothelioma both for 21-40 years of exposure (OR = 2.027 (0.521-7.890)) and more than 40 years of exposure (OR = 2.879 (0.651-12.736)). All of the estimates were high but not significant because this transitional study has a typically low power. The adjustment for latency showed the same trend. Using detailed information collected by the regional mesothelioma registry, this study provided evidence of a continuing health impact of the Fibronit asbestos cement factory in Bari on the resident population.
... In their traditional or partially automated formats, the comet and MN assays can be considered low to medium throughput at best, in terms of sample capacity, as well as in terms of sample analysis. The γ-H2AX staining assay (31)(32)(33) and the 'Fluorimetric Detection of Alkaline DNA Unwinding' (FADU) assay (34)(35)(36)(37) are also low to medium throughput assays which have commonly been used to detect and measure the DNA damaging potential of chemical agents, but are now being applied to the determination of the genotoxicity of ENMs (38)(39)(40)(41)(42). All these assays have been successfully applied in genetic toxicology (in vitro and in vivo), population biomonitoring, ecotoxicology, DNA damage and repair research and/or in cancer risk assessment for potential chemical and biological genotoxins. ...
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The rapid development of the engineered nanomaterial (ENM) manufacturing industry has accelerated the incorporation of ENMs into a wide variety of consumer products across the globe. Unintentionally or not, some of these ENMs may be introduced into the environment or come into contact with humans or other organisms resulting in unexpected biological effects. It is thus prudent to have rapid and robust analytical metrology in place that can be used to critically assess and/or predict the cytotoxicity, as well as the potential genotoxicity of these ENMs. Many of the traditional genotoxicity test methods [e.g. unscheduled DNA synthesis assay, bacterial reverse mutation (Ames) test, etc.,] for determining the DNA damaging potential of chemical and biological compounds are not suitable for the evaluation of ENMs, due to a variety of methodological issues ranging from potential assay interferences to problems centered on low sample throughput. Recently, a number of sensitive, high-throughput genotoxicity assays/platforms (CometChip assay, flow cytometry/micronucleus assay, flow cytometry/γ-H2AX assay, automated ‘Fluorimetric Detection of Alkaline DNA Unwinding’ (FADU) assay, ToxTracker reporter assay) have been developed, based on substantial modifications and enhancements of traditional genotoxicity assays. These new assays have been used for the rapid measurement of DNA damage (strand breaks), chromosomal damage (micronuclei) and for detecting upregulated DNA damage signalling pathways resulting from ENM exposures. In this critical review, we describe and discuss the fundamental measurement principles and measurement endpoints of these new assays, as well as the modes of operation, analytical metrics and potential interferences, as applicable to ENM exposures. An unbiased discussion of the major technical advantages and limitations of each assay for evaluating and predicting the genotoxic potential of ENMs is also provided.
... Working conditions that have an adverse impact on health can have long-term effects and cause late-onset occupational illnesses and health problems, even after more than 20 years of exposure, as in the case of asbestos (Bhattacharya et al., 2005;Manning, Vallyathan, & Mossman, 2002). The surveillance of workers' health is a major tool in efforts to prevent such problems. ...
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... Studies in yeast indicate that mitochondrial oxidative stress plays a crucial role in the induction of autophagy [42]. Oxidative stress from H 2 O 2 and hydroxyl radicals (•OH) are prominently implicated in the pathobiology underlying the in vivo and in vitro toxic effects of inhaled asbestos [18,43,45]. Although ROS can induce autophagy through several distinct mechanisms, it is unclear whether asbestos-induced free radical production mediates autophagy in lung epithelial cells [16]. ...
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Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In the present study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells, and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos-induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased A549 cell microtubule-associated protein 2 light chains 3 (LC3-II) expression, an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-P70s6k. Notably, AKT1/AKT2 double knockout (AKT DKO) murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2(-/-) MEFs but not JNK1(-/-) MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, NAC, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of p-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitors 3-methyladenine (3-MA) or ATG5(autophagy-related gene 5) siRNA, indicating that chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.
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Due to the current query whether the predominantly used chrysotile (white) asbestos comprises health risks we performed a literature search including in vitro and animal experiments as well as epidemiological studies.As shown by epidemiological studies chrysotile causes less pleural fibrosis and mesotheliomas when compared with other asbestos types. However, its otherwise inflammatory, toxic, carcinogenic, and fibrosis-inducing effects correspond to those of other occupationally relevant asbestos types. This is based on clinical, animal as well as on in-vitro findings. This means that denying a causal relationship, e. g. in a case with lung fibrosis (= asbestosis) or lung cancer with an asbestos load of 25 fiber-years in the absence of identification of a significant concentration of asbestos fibers or asbestos bodies in the lung (see so-called "hit and run" phenomenon), contradicts the medical-scientific knowledge. © Georg Thieme Verlag KG Stuttgart · New York.
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The present investigations correlate the potentials of the reactive oxygen species (ROS) generation and the cyto-genotoxicity of amphibole asbestos fibers (amosite, crocidolite and tremolite) with their surface iron, under in vitro controlled conditions, using A549 cells (human lung epithelial cell line). The mobilizable surface iron was measured by Atomic Absorption Spectroscopy; the production of ROS was investigated using 2, 7 dichloro-dihydrofluorescein-diacetate (DCFH-DA) dye; for cytotoxicity assessment, the intracellular organelles specific damages were measured, using 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide salt (MTT) assay; and, the genotoxic potential of amphibole fibers was determined by cytokinesis block micronucleus (CBMN) assay. In the study, highest amount of ROS was generated by crocidolite followed by tremolite and minimum with amosite. In MTT assay, the time- and concentration-dependent decrease in percent cell viability was recorded with all the three amphibole fibers, tremolite being most cytotoxic, followed by crocidolite, and then amosite. In genotoxicity assay, an increase in the frequency of micronuclei (MNi) in binucleated (BN) cells was observed, where crocidolite was most genotoxic, followed by tremolite, and amosite the least.The comparison of results depicts a clear trend of cyto-genotoxic potential paralleling the ROS generation, suggesting a definite role of oxidative stress in fiber-induced toxicity. However, amosite contains maximum surface iron (28%), followed by crocidolite (27%), and tremolite carrying least (as contaminant) or no iron, the mobilizable surface iron is maximum in crocidolite followed by amosite and is minimum in tremolite. The mobilizable iron somewhat corresponds with the ROS generation capacity of these fibers. This shows that the surface iron could be mainly responsible for amphibole asbestos-induced ROS toxicity; though it may not be the only factor responsible, other factors like shape and size etc., also play role in amphibole asbestos-induced toxicity.
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Health risk assessment has been used to investigate the cancer and non-cancer risk of Asbestos in the air of Tehran, Iran. This study focused on the risk of lung cancer and mesothelioma on the residents of the region. It presents an overview of Asbestos concentration in 31 samples with the average concentration of 0.01f/ml in different districts in Tehran. Results provided by EPA (IRIS) analysis showed the total lifetime cancer risk of 46.3 × 10−5. Based on the risk calculations presented in EPA (1986a), the average cancer risk value of lung cancer and mesothelioma was calculated as a discrete value for smokers and nonsmokers. Assuming lifetime continuous exposure due to inhalation, the expected incidence is 46 and 152 mesothelioma deaths, and 42 and 13 lung cancer deaths per 100,000 persons for smokers and nonsmokers, respectively. In addition, In accordance with the Air Quality Guidelines of the World Health Organization database, the extra risk of lung cancer between 2.42×10-5 and 1.13×10-3, for smokers and 2.86×10-6 and 1.13×10-3 for nonsmokers was calculated.
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In this study, we evaluated the effect of the antioxidant butylated hydroxytoluene (BHT) on the genotoxicity and cytotoxicity induced by sodium arsenite (NaAsO<sub>2</sub>) in normal adult male SWR/J mouse bone marrow cells. Animals were subjected to intraperitoneal (i.p.) injection of NaAsO<sub>2</sub> at various dose levels (1, 0.5 and 0.25 LD<sub>5</sub>, which corresponds to 9, 4.50 and 2.25 mg kg<sup>-1</sup> b. wt.) and killed 24 h later. Another group of male mice were treated with 30 mg kg<sup>-1</sup> b. wt. of the synthetic antioxidant and hypermethylizing agent butylated hydroxytoluene 1 h prior to NaAsO<sub>2</sub> administration. The three single doses of sodium arsenite significantly (p<0.05) increased the rate of total structural Chromosomal Aberrations (CAs), Sister Chromatid Exchanges (SCEs), micronucleus (MNs) formation, Poly (ADP-ribose) polymerase (PARP) and Lamina-A degradation and apoptosis compared with the negative control. In the combined treatment with BHT, no significant effect was observed in the rate of CAs or SCEs, whereas a significant decrease was observed in the rate of micronucleated polychromatic erythrocytes (MNPCEs) at medium and high doses. The present study has shown that administration of an antioxidant had a negative effect as represented in the rate of CAs, PARP and Lamina-A degradation and apoptosis. On the other hand, the antioxidant had a positive effect as represented in the decreased rate of pulverized chromosomes and MN formation.
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The aim of the present study was to evaluate the cytotoxicity and oxidative stress induced by native and active bentonite particles (BPs) on human B lymphoblast cells using seven assays. Our results showed that the order of cytotoxicity was: active BPs>native BPs>quartz particles (DQ-12)>gypsum, according to the IC50 values in CCK-8 assay and neutral red uptake (NRU) assay. The lactate dehydrogenase (LDH) leakage, the proportions of early apoptotic cells, the reactive oxygen species (ROS) generation, the superoxide dismutase (SOD) inhibition and the malondialdehyde (MDA) release in the native and active BPs groups were significantly higher than those in the gypsum and DQ-12 groups (P<0.05 or P<0.01). Moreover, the cytotoxicity of active BPs with higher adsorption capacity of phenol was higher than that of native BPs with relatively lower adsorption capacity of phenol. The oxidative stress induced by active BPs was significantly higher than that induced by native BPs (P<0.05 or P<0.01). The water-soluble fractions of BPs did not induce the cytotoxicity and ROS generation. These findings indicated that active and native BPs could induce significantly the cytotoxic effects and oxidative stress on human B lymphoblast cells in vitro. The cytotoxic difference between active BPs and native BPs may be associated with the adsorption capacity of BPs and oxidative stress induced by BPs to a certain extent. The insoluble particle fractions may play a main role in the cytotoxic effects and oxidative stress induced by BPs.
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The present study aims to evaluate the effect of antioxidation and hypomethylation on the genotoxicity and apoptosis induced in mice by arsenic trioxide (As<sub>2</sub>O<sub>3</sub>) in normal adult male SWR/J mouse. Animals were treated with intraperitoneally (ip) injected with (2.65, 5.35 or 10.70 mg kg<sup>-1</sup> b.wt. of As<sub>2</sub>O<sub>3</sub> which represent 0.25, 0.50 or 1 of LD<sub>50</sub>, respectively) and killed 24 h later. Another groups were treated with 30 mg kg<sup>-1</sup> b.wt. of antioxidant and hypermethylizing agent butylated hydroxy toluene (BHT) 1 h prior to As<sub>2</sub>O<sub>3</sub> administration. Another different groups were treated with three doses of 5-azacitidine (5-AzaC) 5 mg kg <sup>-1</sup> b.wt. As<sub>2</sub>O<sub>3</sub> administered after 6 days of the last dose. The three single doses of As<sub>2</sub>O<sub>3</sub> significantly (p<0.05) increased the rate of total structural chromosomal aberrations (CAs) compared with the negative control. No significant effect was observed in the combined treatment with BHT or 5-AzaC compared with single treatments. The histopathological analyses of mice liver cells showed significantly (p<0.05) increased in apoptosis markers in all three single doses of As<sub>2</sub>O<sub>3</sub> compared with the negative control and also significantly (p<0.05) increased with combined treatment with BHT at low and high doses compared with single doses. This study showed that administration of As<sub>2</sub>O<sub>3</sub> had a negative effects as represented in CAs test, antioxidant as represented in apoptosis markers and 5-AzaC as represented in rate of pulverized chromosomes, centromeric attenuations, number of polyploidy cells.
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Exposure to a variety of toxins and/or infectious agents leads to disease, degeneration and death, often characterised by circumstances in which cells or tissues do not merely die and cease to function but may be more or less entirely obliterated. It is then legitimate to ask the question as to whether, despite the many kinds of agent involved, there may be at least some unifying mechanisms of such cell death and destruction. I summarise the evidence that in a great many cases, one underlying mechanism, providing major stresses of this type, entails continuing and autocatalytic production (based on positive feedback mechanisms) of hydroxyl radicals via Fenton chemistry involving poorly liganded iron, leading to cell death via apoptosis (probably including via pathways induced by changes in the NF-κB system). While every pathway is in some sense connected to every other one, I highlight the literature evidence suggesting that the degenerative effects of many diseases and toxicological insults converge on iron dysregulation. This highlights specifically the role of iron metabolism, and the detailed speciation of iron, in chemical and other toxicology, and has significant implications for the use of iron chelating substances (probably in partnership with appropriate anti-oxidants) as nutritional or therapeutic agents in inhibiting both the progression of these mainly degenerative diseases and the sequelae of both chronic and acute toxin exposure. The complexity of biochemical networks, especially those involving autocatalytic behaviour and positive feedbacks, means that multiple interventions (e.g. of iron chelators plus antioxidants) are likely to prove most effective. A variety of systems biology approaches, that I summarise, can predict both the mechanisms involved in these cell death pathways and the optimal sites of action for nutritional or pharmacological interventions.
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Background and aim: Asbestosis, is not seen in every person exposed to asbestos therefore the person-specific factors, might play an important role in the pathogenesis. In this study, we aimed to investigate the frequency of superoxide dismutase 2, mitochondrial (SOD2, c.47T>C) and superoxide dismutase 3, extracellular (SOD3, c.172G>A) gene polymorphisms in patients with benign lung disease associated with asbestosis and healthy volunteers with environmental asbestos exposure. Material and method: 48 patients (18 females and 30 males) in whom the benign lung disease that completely develops as a result of exposure to environmental asbestos, and 48 healthy individuals (16 females and 32 males) in whom asbestos-related disease is not detected despite the environmental asbestos exposure were enrolled in the study. Their DNA was isolated. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to determined the frequencies of SOD2 and SOD3 gene polymorphisms. Results: The mean age (58.8 ± 11.5) and the systemic hypertension frequency were statistically significant (p < 0.05) higher in the group suffering from asbestos related disease than those being exposed but developing no disease. According to our PCRRFLP results, the SOD2:c.47 CC genotype (p = 1.000) and SOD3:c.172 AA genotype (p = 0.779) ratios were not statistically significant between groups, and also SOD2 gene c.47 C allele (p = 0.562) and SOD3 gene c.172 polymorphism A allele (p = 1.000) frequencies were determined to be not statistically significant between the two groups. Conclusion: SOD2 and SOD3 polymorphic points might not be involved in development of benign lung disease associated with environmental asbestos exposure.
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Over the last 30 years, the field of biomarkers has greatly expanded as early and specific endpoints for monitoring cellular responses to various disease states and exposures to drugs and chemical agents. They have enjoyed some success as predictors of health outcomes for a number of clinical diseases, but the application to chemical exposure risk assessments has been more limited. Biomarkers may be classified into categories of markers of exposure, effect, and susceptibility. Currently, "omics" biomarkers (i.e., genomic, proteomic, and metabolomic/metabonomic) are the major classes of biomarkers under development. These markers represent a continuum of cellular responses to drug or chemical exposures and provide linkages to mechanisms of cell injury/cell death or carcinogenic transformation. On the other hand, translation and application of these biomarkers for risk assessment has been limited due to validation and interpretation issues that need to be addressed in order for these potentially extremely valuable endpoints to reach their full potential as predictive tools for public health. This short chapter will briefly review these three "omics" biomarker classes and examine some validation/translation aspects needed in order for them to reach their full potential and acceptance as valuable tools for application to risk assessment.
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Dusts are finely divided particles in the solid phase which, when suspended in air, form aerosols. Aerosols may also form from particles in the liquid phase, or mists. Aerosols have a variety of properties and characteristic behavior depending on composition and air movement. Dusts within aerosols behave in air and adjacent surfaces according to their mass, density, size, dimensions, and electrostatic charge. Once inhaled, their fate in the upper respiratory tract and in the lung is determined by their mass, size, shape, and electrostatic charge. Once deposited in the lung, they may persist or be degraded according to their composition and water solubility. Only then does the dust exert any physiological effect it will have and aerosol science and particle deposition studies give way to toxicology. Some dust diseases of the lung may be associated with asthma and immunological responses, such as hypersensitivity pneumonitis, hard metal disease, and beryllium disease, but the majority are characterized by a fibrotic response to the deposition of dust in the alveolus, resulting in a pneumoconiosis. Three are particularly troublesome because of the intensity of the fibrosis and how common they are in the workplace: silicosis, asbestosis, and coal workers' pneumoconiosis.
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This review article focuses on polymer nanocomposites reinforced by layered silicates, graphite, carbon nanotubes, carbon nanofibers and nanoparticles. Particular attention is given to the structure, properties, classification and the procedures used for preparing such nanocomposites. The most common methods used for characterizing these nanocomposites are briefly introduced. Finally, the challenges in processing and production of nanocomposites, their impacts on health and environment as well as their future (potential applications and the market) are discussed.
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Asbestos is the name of a group of minerals with long and thin fibers that originate naturally in the environment. Asbestos mainly affects lungs and the membrane that surrounds the lungs. In general, PCM (phase contrast microscopy) and PLM (polarized light microscopy) have been used to analyze asbestos fibers. However, these methods have often problems to over-estimate number concentration when counting real asbestos fibers. Moreover, there are many difficulties when separating and identifying various asbestos and non-asbestos fibers. In order to determine quantitative information on fibrous particles, source profiles for asbestos and non-asbestos fibers must be initially developed on the basis of their chemical compositions and physical parameters. In our study, a SEM/EDX was used to develop source profiles from known asbestos samples as reference samples. We could make the source profile matrix consisting of 6 types of asbestos fibers and 2 types of non-asbestos fibers by analyzing 380 fibers. Based on these profiles, a rule building expert system was developed by using the visual basic application (VBA). Various fibers were successfully classified by 2 simple rules in the EXCEL environment based on several visual steps such as inserting data, viewing results, and saving results. For a case study to test the expert system, samples from a construction materials and from various indoor environments such as a residental area, a preschool classroom, and an underground store were collected and analyzed. As a result of the survey, a total of 76 individual test fiber particles was well classified into 5 different types of particle classes; 9.3% of chrysotile, 15.4% of amosite, 0.8 of crocidolite, 4.2% of tremolite, 5.8% glass fiber, 21.1% of other fibers, and 43.5% of unknown fibers in terms of number concentration. Even though unknown portion was high, it will be decreased markedly when expanding fiber source profiles.
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The present study aims to evaluate the effect of DNA hypomethylation state on genotoxicity and apoptogenicity induced by sodium arsenite (NaAsO2) in normal adult male SWR/J mouse bone marrow cells. Animals were treated with intraperitoneally (i.p.) injected with (2.25, 4.50 or 9 mg kg(-1) b.wt. of NaAsO2 which represent 0.25, 0.50 or 1 of LD5, respectively) and killed 24 h later. Another different group of male mice was treated with three doses of 5-Azacitidine (5-AzaC), 5 mg kg(-1) b.wt. each dose and 3 h intervals between them. NaAsO2 administered after 6 days of the last dose. The three single doses of sodium arsenite alone significantly (p<0.05) increased the rate of total structural Chromosomal Aberrations (CAs), rate of Sister Chromatid Exchanges (SCEs), micronucleus (MNs) formation, PARP and Lamia-A degradation and apoptosis as compared with the negative control. The combined treatment with hypomethylation agent 5-AzaC significantly increased the rate of SCEs induced by NaAsO2 at low dose. Moreover, this treatment significantly increased the rate of polyploidy at all combined used doses. Furthermore, this treatment induced apoptosis at all used doses. The present study has shown that DNA hypomethylation had a negative effects represented in rate of (CAs), polyploidy, PARP degradation and apoptosis induced by (NaAsO2). On the other hand, DNA hypomethylation had positive effects represented in decreas rate of pulverized chromosomes, centromeric attenuations, (SCEs), (MNs) formation, prevent Lamina-A degradation and apoptosis.
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Inhaled asbestos fibres are known to cause inflammation processes with the result of lung or pleural fibrosis and malignancies. Interleukins (IL), such as IL-1β, IL-6 and IL-10, have various functions in the regulation of the inflammatory response and in proliferative processes after inhalation of silica dust and can, therefore, influence the pathogenesis of asbestos-induced fibrosis and carcinogenesis. Polymorphisms within these genes may be associated with susceptibility to silica and asbestos-induced lung diseases. Thus, IL-1β, IL-6 and IL-10 polymorphisms were examined to determine an association with asbestos or silica-induced fibrosis or malignancies. Association studies were performed in 1180 individuals, using control subjects (n=177), fibrosis patients (n=605), lung cancer (LC) patients (n=364) and malignant mesothelioma (MM) patients (n=34). IL-1β (C-511T; C+3954T), IL-6 (G-174C) as well as IL-10 (G-1082A) polymorphisms were investigated. Compared to a healthy (control) group, a higher risk was seen for malignant mesothelioma patients in all investigated polymorphisms. The IL-6 -174C allele showed a tendency towards a higher risk for fibrosis or asbestos-induced lung cancer (ORasbestosis, 1.338; 95% CI, 0.71-2.53; ORsilicosis, 1.226; 95% CI, 0.54-2.81; ORfibrosis other aetiology, 1.313; 95% CI, 0.58-2.98 and ORLC asbestos, 2.112; 95% CI, 0.75-5.92). The IL-10 -1082A carrier seemed to be at higher risk for silicosis (ORsilicosis, 2.064; 95% CI, 0.78-5.49) but not for asbestosis. In summary, this study did not reveal sufficient evidence for a significant association of the investigated interleukin polymorphisms with asbestos or silica-induced diseases in the population studied.
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Asbestos causes lung fibrosis known as asbestosis as well as cancers such as malignant mesothelioma and lung cancer. Asbestos is a mineral silicate containing iron, magnesium, and calcium with a core of SiO(2). The immunological effect of silica, SiO(2), involves the dysregulation of autoimmunity because of the complications of autoimmune diseases found in silicosis. Asbestos can therefore cause alteration of immunocompetent cells to result in a decline of tumor immunity. Additionally, due to its physical characteristics, asbestos fibers remain in the lung, regional lymph nodes, and the pleural cavity, particularly at the opening sites of lymphatic vessels. Asbestos can induce chronic inflammation in these areas due to the production of reactive oxygen/nitrogen species. As a consequence, immunocompetent cells can have their cellular and molecular features altered by chronic and recurrent encounters with asbestos fibers, and there may be modification by the surrounding inflammation, all of which eventually lead to decreased tumor immunity. In this paper, the brief results of our investigation regarding reduction of tumor immunity of immunocompetent cells exposed to asbestos in vitro are discussed, as are our findings concerned with an investigation of chronic inflammation and analyses of peripheral blood samples derived from patients with pleural plaque and mesothelioma that have been exposed to asbestos.
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The frequency of peripheral blood erythrocyte variants exhibiting allelic loss of glycophorin A (N/M antigen) has been used previously as a biological dosimeter to assess somatic mutations in bone marrow cells from external whole-body irradiation. The aim of the present study was to determine whether this marker could be used as a measure of bone marrow genotoxicity induced by 131I in the treatment of thyroid cancer. Flow cytometry of immunolabeled erythrocytes was performed to enumerate glycophorin A variants before and after eight therapy doses of 131I administered to five patients with differentiated thyroid carcinoma. Bone marrow radiation exposure from each dose was calculated from the integrated retention of 131I in the whole body and in the blood. In addition, the accumulated dose to the bone marrow received from earlier 131I therapy was calculated for each patient. Regression analysis was performed on the frequency of two glycophorin A variant cell types (N/O and N/N) as a function of accumulated dose to the bone marrow. Frequency of N/O variant cells showed a significant dose-related increase with a slope of 10.9 x 10^-6 per sievert. This dose effect is about one-half that previously observed after whole-body external irradiation at high dose rate. This decreased response could be explained by the low dose rate of the radiation to the bone marrow from 131I.
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Asbestos is a commercial term for a group of fibrous minerals often associated with the development of pulmonary interstitial fibrosis (asbestosis), lung cancer, and malignant mesothelioma in occupationally exposed individuals. The pathogenicity of different forms of asbestos varies--long, thin amphibole fibers are most pathogenic, particularly in the induction of mesothelioma. Available data do not support the concept that low-level exposure to asbestos is a health hazard in buildings and schools. The concentration of asbestos fibers in air, type of asbestos, and size of fibers must be considered in evaluation of potential health risks.
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Several in vitro studies suggest the involvement of active oxygen metabolites in cell damage caused by asbestos. To determine if lung injury, inflammation, and asbestosis could be inhibited in vivo in a rapid-onset, inhalation model of disease, a novel method of chronic administration of antioxidant enzymes was developed. In brief, Fischer 344 rats were treated with polyethylene glycol-conjugated (PEG-) superoxide dismutase or catalase in osmotic pumps over a 10-day (5 days/wk for 2 wk) or 20-day (5 days/wk for 2 wk) period of exposure to crocidolite asbestos. Control rats included sham-exposed animals and those exposed to asbestos but receiving chemically inactivated enzymes. After 10 days of exposure to asbestos, lactic dehydrogenase (LDH), alkaline phosphatase, and total protein in bronchoalveolar lavage (BAL) were measured in one group of rats. Total and differnetial cell counts in BAL also were assessed. After 20 days of exposure, lungs of an additional group of rats were evaluated by histopathology and by measurement of hydroxyproline. Asbestos-associated elevations in LDH, protein, and total cell numbers in BAL were reduced in rats receiving PEG-catalase. Decreases in numbers of alveolar macrophages, polymorphonuclear leukocytes, and lymphocytes occurred in these animals. Exposure to asbestos for 20 days caused significant increases in both the amount of hydroxyproline in lung and the severity and extent of fibrotic lesions as determined by histopathology. These indicators of asbestosis were inhibited in a dosage-dependent fashion in rats receiving PEG-catalase. Use of inactivated PEG-catalase failed to boost serum levels of catalase and did not inhibit asbestos-induced elevation of hydroxyproline in lung.(ABSTRACT TRUNCATED AT 250 WORDS)
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Asbestos and other mineral fibers are carcinogenic to humans and animals but differ from many carcinogens in that they do not induce gene mutations. An understanding of these interesting human carcinogens, therefore, is an important problem in cancer research. Asbestos and other fibers induce predominantly two types of cancers: mesotheliomas and bronchogenic carcinomas. Fiber size is an important factor in the carcinogenic activity of these substances as has been shown for mesothelioma induction. For bronchogenic carcinomas, but not for mesotheliomas, a synergistic effect of asbestos exposure and cigarette smoke has been observed in humans. The mechanisms by which fibers alone versus fibers in concert with other carcinogens induce cancers are probably distinct. In addition to fiber dimensions, fiber durability and surface properties of fibers are important properties affecting carcinogenicity. Evidence exists that asbestos is a complete carcinogen, an initiator and a promoter. Multiple mechanisms must be operative to explain the diverse effects of mineral fibers. Although asbestos is inactive as a gene mutagen, there is now clear evidence that it induces chromosomal mutations (aneuploidy and aberrations) in a wide variety of mammalian cells including mesothelial cells. Asbestos also induces transformation of cells in culture including mesothelial cells and fibroblasts. A mechanism for cell transformation, which is dependent on fiber dimension, has been proposed. The fibers are phagocytized by the cells and accumulate in the perinuclear region of the cells. When the cell undergoes mitosis, the physical presence of the fibers interferes with chromosome segregation and results in anaphase abnormalities. The transformed cells show aneuploidy and other chromosome abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)
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Chromosomes can be specifically stained in metaphase spreads and interphase nuclei by in situ hybridization with entire chromosome-specific DNA libraries. Unlabeled human genomic DNA is used to inhibit the hybridization of sequences in the library that bind to multiple chromosomes. The target chromosome can be made at least 20 times brighter per unit length than the others. Trisomy 21 and translocations involving chromosome 4 can be detected in metaphase spreads and interphase nuclei by using this technique.
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We have shown previously that asbestos and other mineral dusts, including glass fibers, induce cell transformation and chromosomal mutations in Syrian hamster embryo cells in culture. In the present study, we observed that both asbestos and glass fibers were phagocytized by these cells and accumulated in the perinuclear region of the cytoplasm. In order to understand the mechanism of fiber length-dependent cellular effects, we examined the phagocytosis and intracellular distribution of glass fibers of differing lengths in cells at various times after treatment. Glass fiber length was decreased by milling with a mortar and pestle. Cells treated with an equal dose of milled glass fibers (on a weight per surface area basis) were exposed to 7-fold more fibers since milling of glass fibers resulted in a 7-fold decrease in length with little change in diameter. However, cells exposed to milled glass fibers phagocytized a similar number of fibers as cells exposed to an equal mass of unmilled glass fibers, indicating that milled fibers were less readily phagocytized. In cells treated with either unmilled or milled glass fibers, the length of the intracellular fibers was more than 2-fold greater than the length of the fibers on the surface, suggesting that cells selectively internalized longer fibers. Fiber length, however, did not appear to affect the migration of intracellular fibers to the perinuclear region of the cytoplasm. Even though cells treated with milled glass fibers contained a similar number of fibers as those treated with unmilled glass fibers, the resulting cytotoxicity, transformation frequency, and frequency of micronuclei were greatly reduced in the cultures treated with milled glass fibers. Thus, fiber length appears to affect the phagocytosis of fibers as well as the ability of intracellular fibers to induce cytogenetic damage and the resultant transformation.
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The cytogenetic effects of chrysotile asbestos on Syrian hamster embryo cells in vitro were investigated at doses which induced morphological and neoplastic transformation but which failed to induce measurable gene mutations in the cells at two genetic loci. Chrysotile asbestos treatment of the cells significantly induced chromosome changes in a dose-dependent manner. Up to 50% of the cells had chromosome abnormalities in number or structure following treatment with asbestos (2.0 micrograms/sq cm) for 48 hr. Numerical chromosome changes were the most pronounced abnormalities although significant increases in metaphases with other chromosome aberrations (breaks, fragments, exchanges, and/or dicentrics) and cells with binuclei or micronuclei were also observed. A linear relationship was observed between the incidences of cells with tetraploid metaphases and binucleated cells, suggesting that binucleation and tetraploidy are related. Cytogenetic effects of other mineral dusts were also tested 48 hr following treatment at a concentration of 2.0 micrograms/sq cm. Crocidolite asbestos was less potent than chrysotile asbestos in its ability to induce cell transformation and cytogenetic damage. Treatment of the cells with thin glass fibers (Code 100) was also able to induce cell transformation and cytogenetic effects, but thick glass fibers (Code 110) were much less potent for both endpoints. Milling of the thin glass fibers decreased the length of the fibers and abolished their ability to induce cell transformation and cytogenetic effects. Nonfibrous alpha-quartz induced neither cell transformation nor cytogenetic effects at the dose of 2.0 micrograms/sq cm. The results indicate that the physical characteristics of the fibers determine their ability to induce cell transformation and their ability to induce chromosome mutations, suggesting a possible mechanistic relationship.
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The assay of bronchoalveolar washings from acutely exposed animals has proven useful as a rapid screen for lung injury from inhaled airborne toxins. The screen is useful for choosing appropriate compounds and exposure levels for subsequent in-depth studies in which complete histopathologic evaluations will be made. An inflammatory response can be detected by the appearance of polymorphonuclear leukocytes and an increase in protein content of lung washings. The release of the cytoplasmic enzyme, lactate dehydrogenase, into the acellular portion of the lavage fluid serves as an indication of cell death or membrane damage. A large increase in some lysosomal enzymes has been found in the bronchoalveolar lavage fluids from animals chronically exposed to insoluble particles. Angiotensin-converting enzyme has been found to be elevated in bronchoalveolar washings from animals with endothelial cell damage in the pulmonary capillaries. The correlation of these cellular and biochemical alterations in the bronchoalveolar lavage fluid with morphological indications of damage has served to validate this method of detecting acute lung injury. Further study is needed to validate indicators of developing chronic disease.
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Studies are summarized demonstrating that the inflammatory cytokines, interleukin IL-6 and IL-8, play a direct role in asbestos lung diseases and are produced by lung epithelial cells in direct response to the fibers. This response is controlled by changes in the cellular oxidative/state induced by iron present in the fiber through Fenton-type chemistry. As a result of this oxidative stress, the redox sensitive transcription factors, NF-κB and NF-IL-6, which help regulate cytokine gene expression, are activated.
Article
Oxidants and inflammatory mediators such as tumour necrosis factor-α (TNF-α) activate nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) transcription factors, and enhance the expression of both pro-inflammatory and protective antioxidant genes. Remodelling of chromatin within the nucleus, controlled by the degree of acetylation/deacetylation of histone residues on the histone core around which DNA is coiled, is important in allowing access for transcription factor DNA binding and hence gene transcription. Unwinding of DNA is important in allowing access for transcription factor DNA binding and hence gene transcription. Nuclear histone acetylation is a reversible process, and is regulated by a group of acetyltransferases (HATs) which promote acetylation, and deacetylases (HDACs) which promote deacetylation. The aim of this study was to determine whether oxidative stress and the pro-inflammatory mediator, TNF-α, altered histone acetylation/deacetylation and the activation of NF-κB and AP-1, leading to the release of the pro-inflammatory cytokine IL-8 in human alveolar epithelial cells (A549). Hydrogen peroxide (H2O2) (100 μM) and TNF-α (10 ng/ml) imposed oxidative stress in A549 cells as shown by depletion of the antioxidant reduced glutathione (GSH) concomitant with increased levels of oxidised glutathione (GSSG). Treatment of A549 cells with H2O2, TNF-α and the HDAC inhibitor, trichostatin A, TSA (100 ng/ml) significantly increased acetylation of histone proteins shown by immunostaining of cells and increased HAT activity, compared to the untreated cells. H2O2, and TNF-α, and TSA all increased NF-κB and AP-1 DNA binding to their consensus sites assessed by the electrophoretic mobility shift assay. TSA treatment potentiated the increased AP-1 and NF-κB binding, produced by H2O2 or TNF-α treatments in A549 cells. Both H2O2 and TNF-α significantly increased IL-8 release, which was further enhanced by pre-treatment of A549 cells with TSA compared to the individual treatments. This study shows that the oxidant H2O2 and the pro-inflammatory mediator, TNF-α induce histone acetylation which is associated with decreased GSH levels and increased AP-1 and NF-κB activation leading to enhanced proinflammatory IL-8 release in alveolar epithelial cells. This indicates a mechanism for the pro-inflammatory effects of oxidative stress.
Article
The systematic development and application of biomarkers in environmental health risk assessment is a relatively new field. At first, the major interest was in biomarkers of exposure, borrowing concepts from pharmacology, then it moved from the external estimates of exposure to internal measures of dose, and ultimately, to markers of target dose. While these markers provide evidence of exposures, they do not provide evidence of that toxicological damage has occurred. For this reason, measurements of DNA adducts and protein adducts are of interest, since they may provide bridges between exposures and disease end-points. In parallel, more quantitative and more sensitive end-points for diseases have been sought. Again, with advancing techniques in cytogenetics, extensive studies were conducted on such markers as chromosomal aberrations, micronuclei and other changes deemed to represent genomic damage. However, these types of end-points are quite unspecific for application to new hazards of uncertain human toxic (carcinogenic) potential. Recent work focusing on more specific early-effect markers such as certain oncogenes and tumour-suppressor genes have substantial promise as shown by work with aflatoxins and vinyl chloride. Such studies have also enhanced mechanistic insight. The advances in molecular genetics have led to an upsurge in interest in most susceptibility factors, and identification of polymorphisms of various enzymes has become possible. Ongoing search for “ultra-high risk” individuals may be fruitful, but probably only relevant to a small segment of potentially exposed populations. Factors associated with a small differential risk, however theoretically or mechanistically important, offer only little practical use.
Article
The aim of this study was to compare the secretion of tumor necrosis factor a (TNFα) and interleukin-1 (IL-1) by alveolar macrophages (AMs) harvested from patients with coal worker's pneumoconiosis (CWP) and control subjects. We observed higher levels of spontaneous TNFα and IL-1 secretion by AMs from patients with CWP than in those from healty controls. We did not find any significant difference between the two groups in the incidence of simple pneumoconiosis and progressive massive fibrosis. In the group of coal miners without radiologic signs of pneumoconiosis, we found high levels of both cytokines in a subgroup of subjects still exposed to the mineral dust but not in the subgroup of subjects removed from exposure. These results indicate that AMs are involved in chronic lung inflammatory reactions to mineral dusts, partly by way of cytokine secretion. Moreover, cytokine secretion by AMs appears to be an early event that is detectable at the moment of mineral dust exposure. The results open new perspec...
Article
Air pollution is a major problem in Calcutta. In this study, exfoliated sputum cytology of the exposed population was done in order to get an insight into the response of the lungs to air pollutants at the individual level. 468 residents from Calcutta and 60 from Sunderban islands, where ambient air pollution is negligible, were studied. Results showed remark-able increase in alveolar macrophage (AM) number in the sputum of the urban group (22.7/hpf) than that of rural controls (2.8/hpf, P < 0.001). Inflammatory cells like neutrophils and eosinophils were also found in increased numbers in the sputum of city dwellers. In the urban group, AM count was highest among residents of north Calcutta where the pollution level was maximum and lowest in the relatively less polluted south Calcutta. Similarly, pollution level and AM count were maximum during the winter, minimum during the monsoon and intermediate in summer. Thus, a close parallelism was observed between the magnitude of air pollution and the AM count in sputum of the exposed persons. Since AM count is simple, non-intrusive and relatively inexpensive the results envisage usefulness of sputum AM count as an indicator of exposure to ambient air pollution especially in large population-based studies in developing countries.
Article
This article reviews the various aspects regarding the carcinogenicity of asbestos and associated reactions catalyzed by iron. Attention is focused on the following: structure of asbestos; physical properties of asbestos involved in carcinogenesis; reactions catalyzed by iron; reactions catalyzed by asbestos; fiber inactivation; physiological effects; and mutations and cancer. 183 refs.
Article
Asbestos exposure causes pulmonary fibrosis and malignant neoplasms by mechanisms that remain uncertain. In this review, we explore the evidence supporting the hypothesis that free radicals and other reactive oxygen species (ROS) are an important mechanism by which asbestos mediates tissue damage. There appears to be at least two principal mechanisms by which asbestos can induce ROS production; one operates in cell-free systems and the other involves mediation by phagocytic cells. Asbestos and other synthetic mineral fibers can generate free radicals in cell-free systems containing atmospheric oxygen. In particular, the hydroxyl radical often appears to be involved, and the iron content of the fibers has an important role in the generation of this reactive radical. However, asbestos also appears to catalyze electron transfer reactions that do not require iron. Iron chelators either inhibit or augment asbestos-catalyzed generation of the hydroxyl radical and/or pathological changes, depending on the chelator and the nature of the asbestos sample used. The second principal mechanism for asbestos-induced ROS generation involves the activation of phagocytic cells. A variety of mineral have been shown to augment the release of reactive oxygen intermediates from phagocytic cells such as neutrophils and alveolar macrophages. The molecular mechanisms involved are unclear but may involve incomplete phagocytosis with subsequent oxidant release, stimulation of the phospholipase C pathway, and/or IgG-fragment receptor activation. Reactive oxygen species are important mediators of asbestos-induced toxicity to a number of pulmonary cells including alveolar macrophages, epithelial cells, mesothelial cells, and endothelial cells. Reactive oxygen species may contribute to the well-known synergistic effects of asbestos and cigarette smoke on the lung, and the reasons for this synergy are discussed. We conclude that there is strong evidence supporting the premise that reactive oxygen species and/or free radicals contribute to asbestos-induced and cigarette smoke/asbestos-induced lung injury and that strategies aimed at reducing the oxidant stress on pulmonary cells may attenuate the deleterious effects of asbestos.
Article
Both the pattern of mediator release during the late-phase response (LPR) and the reduction of the LPR with corticosteroid pretreatment have suggested that basophils, not mast cells, represent the main source of histamine in the late response to nasal antigen challenge. We tested this hypothesis by examining alcian blue-stained cytospin slides of nasal washings obtained before and for 11 hours after nasal antigen challenge in 11 asymptomatic subjects with seasonal allergic rhinitis. In a double-blind manner, subjects received placebo or topical flunisolide (50 μg, each nostril, twice daily) for 1 week before antigen challenge. One month later, the challenge was repeated with the alternate pretreatment. On placebo-treatment days, a twelvefold increase occurred in the number and a threefold increase in the percentage of alcian blue-stained positive cells in nasal washings in the LPR compared to baseline. At least 68% of these alcian blue-stained positive cells were basophils, as determined by light microscopic criteria. Alcian blue-stained cell influx correlated with increases in histamine levels in nasal washes (p < 0.001). Topical steroid pretreatment blocked the influx of alcian blue-stained positive cells, as well as other inflammatory cells, including eosinophils, neutrophils, and mononuclear cells. Symptoms and mediator release were also blocked. These data demonstrate an influx of basophils and suggest that these cells are responsible for the histamine release observed in the LPR. Our findings indicate that pharmacologic control of basophil histamine release may represent a strategy for the treatment of a variety of chronic allergic diseases that are believed to resemble the LPR.
Article
Allergic rhinitis (AR) is part of a systemic disease complex. There is a close relationship between AR and asthma, which has led to the "one airway, one disease" concept. Both conditions share common immunopathology and pathophysiology. In patients with AR, allergen-triggered early and late responses are mediated by a series of inflammatory cells. Within minutes of contact with allergen, IgE-sensitized mast cells degranulate, releasing both preformed and newly synthesized mediators. Immunologic processes in both nasal and bronchial tissue involve T H 2 lymphocytes and eosinophils. Eosinophils are the predominant cell in the chronic inflammatory process characteristic of the late-phase allergic response. Eosinophils release an array of proinflammatory mediators, including cysteinyl leukotrienes, cationic proteins, eosinophil peroxidase, and major basic protein, and might serve as a major source of IL-3, IL-5, GM-CSF, and IL-13. Neuropeptides also appear to contribute to the pathophysiology of AR symptoms. Both AR and asthma exhibit marked day-night variation in symptom severity. Acknowledging both the chronobiology of AR and circadian rhythm-dependent attributes of antiallergy medications might enhance the beneficial effects of allergy therapies.
Article
The release of soluble interleukin 6 receptor (IL-6sR) and interleukin 6 (IL-6) was investigated in polymorphonuclear and whole blood cell cultures from untreated patients with breast cancer. IL-6sR and IL-6 were determined in the culture supernatants by a sensitive enzymoimmunological assay. We have shown a decreased ability of unstimulated and stimulated polymorphonuclear cells (PMNs) and whole blood cells (WBC) of cancer patients to release IL-6sR in comparison to a control group. Simultaneously, we have also found an increased capacity of unstimulated cells from the patients to produce of IL-6. The used stimulators-zymosan and lipopolysaccharide (LPS) resulted in increased IL-6sR secretion by PMNs and WBC of patients but did not influence IL-6 production by the same cells. We have also determined the levels of IL-6sR and IL-6 in the sera of cancer patients. The mean concentrations of IL-6sR and IL-6 in the sera of patients were higher than those in the control group. We have not noticed any correlations in the culture supernatant and serum levels of IL-6sR or IL-6 between control and cancer patient groups.
Article
Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase beta-N-acetylglucosaminidase. Thus macrophage lysosomal enzyem secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both N-acetyl-beta-D-glucosaminidase and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.
Article
Studies carried out jointly with macrophages, fibroblasts and epithelial cells in vitro, have shown that the long fibers of asbestos or glass are incorporated in a special way. Phagocytosis of the long fibers is delayed and/or remains incomplete. Incomplete incorporation of the fiber causes a localized discontinuity in the cell membrane. There results continuous liberation of intra-cellular enzymes, which is compensated for by an increasing glycolytic metabolism. The effect of fibrous dusts cannot be compared with those of granular SiO2-dusts. Fibrous dusts, e.g. asbestos and glass fibers, induce the formation of polykaryotic giant cells by way of fusion; asbestos possibly also causes the interspecific fusion of cells. There are indications that in the process of asbestos induced cell fusioning integrated virus genomes are activated and infectious virus are released. The interaction between the cell and fiber causes a chronic irritation of the cell, which is discussed as a factor for tumour induction. There seems to be a causal relationship between the fibrogenic and carcinogenic effects of anorganic dusts and their shape, i.e. their length and diameter, regardless of their chemical composition. Thus the fibrogenic and carcinogenic effect is limited by a minimal length and a maximal diameter.
Article
Three simple models for the asbestos-smoking interaction on human lung cancer production are considered. In the first model the excess incidence of lung cancer independently due to asbestos and to smoking adds together when both agents are present (additive model). In the second the addition of each one of the two agents produces an effect (increase in lung cancer incidence) which is proportional to the effect of the other (multiplicative model). In the third, asbestos can only increase lung cancer incidence in the presence of smoking. As previously found by other investigators, the additive model appears the least plausible in the light of the data from two published epidemiological studies. A discrimination between the other two models is attempted through a detailed analysis of the five published epidemiological studies today available which provide information on occupational asbestos exposure, smoking habits and lung cancer risk. Although the data do not allow a definitive discrimination, the multiplicative model appears to be more plausible, being also consistent with a multi-stage carcinogenic mechanism and with evidence from animal (rat) experiments. It is relevant both for biology and for public health that in this model asbestos and smoking are regarded as independently capable of producing lung cancer in humans and that they act synergistically when exposure to both occurs.
Article
Alveolar macrophages from rabbits were exposed in a culture medium to asbestos, beryllium sulfate and beryllium oxide. The specific activities of the lysosomal hydrolases, acid phosphatase, α-N-acetylglucosaminidase, β-glucuronidase, and the glycolytic enzyme, phosphohexose isomerase, were determined in the medium, whole cell homogenates, mitochondrial fractions and in the supernatant. These hydrolases increased significantly in the medium but not in the mitochondrial fraction of cells exposed to dust. For the induction of enzyme release in vitro, a higher concentration of beryllium oxide than that of the other dusts was requisite. Very cytotoxic effects to the cells were seen in the culture groups of both asbestos and beryllium sulfate.
Article
Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine-xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O2- in the presence of CaCl2 enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tert-butyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca2+ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.
Article
Although sister-chromatid exchange (SCE) analysis is recognized as an indicator of exposure to DNA-damaging agents, the results of these analyses have been confounded by the use of bromodeoxyuridine (BrdUrd) to differentially label the sister chromatids. Not only does BrdUrd itself induce SCE, it also modulates the frequency of SCE induced by certain DNA-damaging agents. In order to examine this effect of BrdUrd on SCE frequency, an indirect method which lends itself to measurements both with and without BrdUrd was employed. Human teratocarcinoma-derived (P3) cells were exposed to ethyl methanesulfonate (EMS) and cultured with increasing concentrations of BrdUrd for lengths of time corresponding to one, two, and three generations of cell growth. At each time point, the distribution of nuclei among the phases of the cell-cycle and cell growth were evaluated for each concentration and chemical. A statistical model was employed which tested both for the main effects of chemicals and culture times and for interactions between these factors. Both EMS and BrdUrd significantly affected the percentages of nuclei within the cell-cycle. Exposure to EMS resulted in decreases in the percentages of nuclei in G0 + G1 and increases in the G2 + M compartment. Exposure to BrdUrd affected the size of the G0 + G1 compartment as well as the percentage of S-phase nuclei. Cell growth was reduced as a consequence of increasing EMS concentration and as a function of BrdUrd concentration; the effects of these chemicals were more readily apparent at the later time points. Most importantly, for both the cell-cycle kinetics data and the cell growth data, no evidence of an interaction between the effects of EMS and the effects of BrdUrd was detected statistically. These results may be interpreted to mean that while both EMS and BrdUrd affect the induction of SCE, under the conditions of this experiment, the effects are additive rather than interactive.
Article
Under in vitro conditions involving formation of active oxygen species, rat liver mitochondria were found to undergo swelling, peroxidative decomposition of lipids, and distinct disorganization of ultrastructure. Supplementation with free radical scavengers such as superoxide dismutase (SOD), methionine, histidine, and tryptophan accorded considerable protection to the organelle. A possible correlation between oxygen radicals, membrane integrity, and calcium functions is indicated.
Article
The thymidine analog, BrdUrd, induces many biological responses which are of importance to the field of genetic toxicology and related disciplines. These include the induction of SCE, specific-locus mutations, and toxicity, inhibition of cell proliferation, and the expression of fragile sites in the human genome. In early models which addressed the mechanisms of the biological effects of BrdUrd exposure, two pathways were proposed to account for the induction of the biological responses. Incorporation of the enol form of BrdUrd into the nascent DNA strand after pairing with deoxyguanosine was proposed as one pathway, whereas the incorporation of BrdUrd opposite adenosine in place of thymidine was proposed as the second pathway. Many novel and sophisticated techniques have been applied to the study of the mechanism of the induction of biological effects by BrdUrd leading to a substantial increase in our understanding of these mechanisms. However, the experimental evidence clearly supports the contention that BrdUrd exerts its effects on eukaryotic cells through mechanisms similar to those originally proposed to explain the genotoxicity of BrdUrd.
Diffuse interstitial lung disease in asbestos-exposed workers is presumed to represent asbestosis. Among 176 asbestos-exposed persons for whom lung tissue was available, we found nine with clinical features consistent with asbestosis, but histologic sections failed to demonstrate asbestos bodies, the usual requirement for pathologic diagnosis of asbestosis (Group I). These nine were compared by analytic electron microscopy with nine persons with idiopathic pulmonary fibrosis (Group II), and with nine persons with all the criteria of asbestosis (Group III). The three groups did not differ significantly with respect to lung burden of chrysotile or tremolite and actinolite, but Group III had a lung burden of amosite and crocidolite that was three orders of magnitude greater than in Groups I and II, with no overlap. We conclude that (1) the American Thoracic Society criterion of "a reliable history of exposure" is sometimes difficult to define; (2) asbestos bodies are seen in tissue sections only when exposure has been reasonably high, and given the proper clinical setting, the presence of diffuse fibrosis and asbestos bodies in tissue sections are sensitive and specific criteria for a diagnosis of asbestosis; and (3) the prevalence here of 5.1% nonasbestos-induced interstitial lung disease among asbestos-exposed persons is artefactually high because of atypical case selection. However, because asbestosis is a disappearing disease, such cases will become more frequent. The identification of these other diseases is important because therapy and prognosis may differ from that of asbestosis.
Article
Cytogenetic endpoints, conventionally chromosomal aberrations, and later sister chromatid exchanges and micronuclei have long been used to assess exposure of human populations to genotoxic agents. Although the adverse nature of somatic chromosome damage is recognized at the group level, no ill-health manifestations have been causally related to cytogenetic damage at the individual level. In work-related exposures, e.g., ethylene oxide, styrene, benzene, vinyl chloride, and alkylating anticancer agents have been shown to induce somatic chromosomal damage in several studies. For all of these, a carcinogenic risk to humans has also been documented. The possible association of somatic chromosome damage and cancer will be elucidated in a Nordic prospective study. The objective is to find out the significance of a high or low score in any of the cytogenetic parametres to risk of cancer. In the Finnish part of the cohort of 806 individuals, 10 cases of cancer were observed during the first follow-up period. Although the cohort is young and the numbers small, a slightly significant (P = 0.04) trend was observed for individuals with cancer and a score of chromosomal aberrations. No trend was observed for sister chromatid exchanges. The application of cytogenetic surveillance is still not routine methodology, but it is useful and informative in carefully controlled study designs. Special efforts should be directed toward combining different disciplines, i.e., cytogenetics, adduct monitoring, and end-effect epidemiology, in order to reach quantitativeness in risk assessment.
Article
The aim of this study was to compare the secretion of tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) by alveolar macrophages (AMs) harvested from patients with coal worker's pneumoconiosis (CWP) and control subjects. We observed higher levels of spontaneous TNF alpha and IL-1 secretion by AMs from patients with CWP than in those from healthy controls. We did not find any significant difference between the two groups in the incidence of simple pneumoconiosis and progressive massive fibrosis. In the group of coal miners without radiologic signs of pneumoconiosis, we found high levels of both cytokines in a subgroup of subjects still exposed to the mineral dust but not in the subgroup of subjects removed from exposure. These results indicate that AMs are involved in chronic lung inflammatory reactions to mineral dusts, partly by way of cytokine secretion. Moreover, cytokine secretion by AMs appears to be an early event that is detectable at the moment of mineral dust exposure. The results open new perspectives in the study of the mechanisms leading to CWP.
Article
ASBESTOS is a mineral causing much controversy in today's society. Before the passage and enactment of the Occupational Safety and Health Act of 1970, millions of Americans were exposed to relatively high concentrations of airborne asbestos in the work-place. Other citizens — including insulation installers, shipyard workers, and manufacturers of gas masks — encountered asbestos during World Wars I and II, when it was produced at an accelerated rate. Although the importation and use of asbestos have decreased in the United States since 1970, and recently the Environmental Protection Agency (EPA) has proposed a ban on its use, asbestos remains . . .
The cellular and lymphocyte phenotypic composition of bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) from 15 healthy, nonsmoking, asbestos-exposed shipyard workers (AEW) and 10 nonsmoking, age-matched unexposed workers (UEW) were compared. None of the AEW had clinical, radiographic, or physiologic evidence of asbestosis, but six had radiographic evidence of pleural plaques and/or thickening. The mean duration of asbestos exposure was 16.3 +/- 2.3 yr, and the mean period since exposure was 10.8 +/- 0.5 yr. All but three of the AEW and none of the UEW had asbestos bodies detected in the first 20 ml of BAL fluid recovered (0.1 to 35 asbestos bodies/ml). The AEW had a significantly higher mean percentage (19.1 +/- 2.8% versus 9.7 +/- 1.6%) and concentration (31.6 +/- 5.2 x 10(3) cells/ml versus 14.7 +/- 2.5 x 10(3) cells/ml) of BAL lymphocytes compared with that in the UEW, with an increased mean concentration of each phenotype measured. In PB, the mean lymphocyte concentration was also higher in the AEW than in the UEW (2.0 +/- 0.3 x 10(3) cells/ml versus 1.5 +/- 0.3 x 10(3) cells/ml), but the difference was not statistically significant, and there was no increase of any phenotype measured. BAL lymphocytosis did not correlate with exposure history or BAL asbestos body count, but was greater in AEW with pleural abnormality (30.1 +/- 2.9% versus 11.8 +/- 1.6%). BAL concentrations of CD-20, CD-3, and CD-4, but not of CD-8 cells were significantly increased compared with those in the AEW without pleural abnormality. Further longitudinal studies are needed to determine the prognostic significance of these findings.
Glutathione (L-gamma-glutamyl-L-cysteinyl-glycine, GSH), a sulfhydryl-containing tripeptide produced by most mammalian cells, is an efficient scavenger of toxic oxidants, including hydrogen peroxide, an oxidant that plays a major role in the oxidant burden placed on the epithelial surface of the lower respiratory tract in chronic inflammatory states. GSH is present in the epithelial lining fluid of the normal lower respiratory tract, where it is thought to play a major role in providing antioxidant protection to the epithelial cells. In this regard, we hypothesized that the lower respiratory tract of patients with IPF may be chronically depleted of this antioxidant, thus leading to an increased susceptibility of lung epithelial cells to oxidant injury. To evaluate this concept, the concentration of glutathione was determined in the epithelial lining fluid of the lower respiratory tract of 15 patients with IPF and compared to that of 19 normal subjects. Strikingly, whereas ELF glutathione concentrations were high in normal subjects (429 +/- 34 microM), a fourfold decrease was found in patients with IPF (97 +/- 18 microM, p less than 0.001). In the context of the known oxidant burden present in the lower respiratory tract of patients with IPF, these observations of a "GSH deficiency" in IPF ELF suggest that there is a marked oxidant-antioxidant imbalance at the alveolar surface of these persons, thus increasing the susceptibility to the severe epithelial cell damage characteristic of this disease.
Article
Sister chromatid exchange (SCE) reflects an interchange of DNA sequences between helices in a replicating chromosome. This was initially accomplished by Taylor and colleagues (1957) using tritiated thymidine incorporation followed by autoradiography. The development of an elegant technique for differential staining of sister chromatids by incorporating a thymidine analog, 5-bromodeoxyuridine (BrdU) has greatly simplified the detection of SCEs in metaphase chromosomes. In recent years, the analysis of SCE has been considered to be a highly sensitive and additional (i.e., with chromosome aberrations) end point for measuring mutagenic/carcinogenic potential of various environmental agents and is increasingly being used to detect and differentiate among chromosome fragility human diseases that predispose to neoplasia. Attention has been focused to see if the induction of SCEs in lymphocyte cultures can be used as a reliable "biological dosimeter" for genetic risk assessment and to monitor the exposed populations. Several physical or preparatory as well as biological factors that modify the response and formation of SCEs make the monitoring difficult. The purpose of this article is to review and analyze these factors to facilitate an effective development of a standard protocol for SCE testing and for appropriate evaluation of test results. This may also provide clues to understand the yet unknown molecular mechanism(s) and biological significance of SCE formation.
Chronic asbestos exposure is associated with the accumulation of mononuclear phagocytes in the lower respiratory tract. This process can be both protective and injurious, since macrophages can aid in asbestos clearance yet also modulate structural derangements of the alveolar walls. To understand why macrophages accumulate in the lungs of asbestos-exposed persons, 2 possible mechanisms were evaluated using alveolar macrophages from subjects with histories of chronic high exposure to airborne asbestos: enhanced recruitment of blood monocytes to the lung, and an increased rate of replication of macrophages in situ. Monoclonal antibody analysis with antibodies that detect surface antigens on the majority of circulating blood monocytes but only on a minority of mature alveolar macrophages demonstrated that an increased proportion of alveolar macrophages of asbestos workers expressed monocyte lineage antigens, suggesting the presence of "young" newly recruited macrophages and thus enhanced recruitment. Culture of the alveolar macrophages from these subjects with [3H]thymidine followed by autoradiography demonstrated an increased proportion of alveolar macrophages synthesizing DNA, suggesting the macrophages are replicating at an increased rate in situ. These observations are consistent with the concept that both enhanced recruitment of blood monocytes and increased local proliferation of alveolar macrophages contribute to the accumulation mononuclear phagocytes in the lung of persons with chronic asbestos exposure.
Article
The analysis of bronchoalveolar lavage fluid has been used as a probe to detect lung injury in toxicological studies and to diagnose the disease state of the lung in humans. To determine how variable the content of lavage fluid from different species is, bronchoalveolar lavage fluids from normal individuals of four species (hamster, rat, guinea pig, and rabbit) were compared for enzymatic and cellular content as well as total protein and sialic acid. In addition, lavage fluid from young adult rats and hamsters was compared to that from older animals. Finally, the effect of the method of lavage on lavage fluid content was evaluated by comparing lavage fluid obtained from an excised lung with that from a lavage performed in vivo. In general, lavage fluids from the four species were similar. However, lavage fluid from guinea pigs had higher numbers of granulocytes and higher mean beta-glucuronidase activities than fluids from other species. Rats had higher mean alkaline phosphatase activities, reflecting higher serum values of this enzyme. Older hamsters had more protein in their lavage fluid than younger animals, and older rats had lower elastase inhibitory activity than young rats. Performing lavage in vivo, as compared to in vitro, did not greatly alter the lavage fluid except for a trend toward a higher level of sialic acid in fluid taken from the living animal.
Inhalation of asbestos fibers causes a progressive fibrotic lung disease in humans and animals. Pulmonary macrophages are associated with asbestos exposure and have been implicated as significant mediators of the pathogenic process. In previous studies, we showed that macrophages are attracted to sites of asbestos fiber deposition, i.e., alveolar duct bifurcations. We also showed that macrophages accumulated at these sites as the result of asbestos-induced activation of complement proteins on alveolar surfaces, consequently producing C5a, a chemotactic factor for macrophages. In the present study, we have demonstrated the time course of chemotactic factor generation and the corresponding macrophage response in vivo. A complement-dependent chemotactic factor for macrophages was activated during a 3-h exposure to asbestos and reached maximal activity by 3 h postexposure. Macrophage accumulation followed and reached a maximal amount by 24 h postexposure. Rats decomplemented with cobra venom factor exhibited a significant reduction in macrophage accumulation, but the macrophage response ensued when serum complement returned to normal. Approximately 30% of the macrophages lavaged from complement-normal, asbestos-exposed animals contained fibers, whereas only half as many macrophages from decomplemented rats contained asbestos. A small but significant increase in lavaged lung protein was measured in asbestos-exposed animals. Evidence supports the concept that complement proteins on alveolar surfaces are derived from normal transudation of serum components from the pulmonary vasculature. Increased serum transudation could provide a source of alveolar complement that sustains the generation of a chemotactic factor for macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
A quantitative method has been developed for the analysis of 4-aminobiphenyl (4-ABP) covalently bound as the sulfinic acid amide to the 93 beta cysteine of human hemoglobin. The method uses mild basic hydrolysis of hemoglobin to release the parent amine, derivatization to form the pentafluoropropionamide, and capillary gas chromatography with detection by negative-ion chemical ionization mass spectrometry. The method is precise and gives reproducible results on multiple blood samples taken from individuals over 48 h. Application of this method to blood samples from cigarette smokers and nonsmokers revealed consistently higher adduct levels in smokers. The mean value for smokers was 154 pg 4-ABP per g Hb compared to 28 pg/g Hb for nonsmokers, with no overlap of adduct levels between the two groups. Studies on quitting smokers revealed that adduct levels declined over a period of 6-8 weeks to nonsmoker levels. The finding of 4-ABP adducts in all nonsmokers was not anticipated but is consistent with low-level ubiquitous contamination of air, food, or water. In other animals sampled, rats and dogs had measurable adduct levels, but monkeys and fish did not. The hemoglobin adduct of 4-ABP is the product of a series of reactions between the hemoprotein and N-hydroxy-4-ABP. The formation of hydroxylamines from carcinogenic aromatic amines and their subsequent reactions with DNA are generally thought to be critical events in the initiation of bladder tumors. We suggest that the observed hemoglobin adduct levels formed by this proximate carcinogen will reflect the extent to which these steps have occurred. This is the first report of 4-ABP adducts in human blood.
Article
The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.
Article
The fibrogenic response of amosite variety of asbestos was studied in lungs of guinea pigs over a period of 300 days. Histologically there was marked reticulin fibrosis but the maturation into collagen was slow. Biochemical estimation revealed significant increase of hydroxyproline and glycosamine contents. Total lung protein was also found to be higher in the amosite treated animals reaching maximum at 90 days. The significance of these findings have been discussed.
The initial deposition and subsequent translocation of chrysotile asbestos were studied in the lungs of rats exposed for 1 h in inhalation chambers. Using scanning and transmission electron microscopy of tissue fixed by vascular perfusion, we determined that the majority of fibers that pass through the conducting airways deposits at the bifurcations of alveolar ducts. The farther an alveolar duct bifurcation was from its terminal bronchiole, the less asbestos were observed. The amount of asbestos present on the alveolar duct surfaces was significantly decreased 5 h after cessation of the 1-h exposure. Some fibers were taken up by Type I epithelial cells during the first hour of dusting, and this process continued through the 8-day period in which the animals were studied. As early as 24 h after exposure, there was an accumulation of macrophages at the sites of initial asbestos deposition. This may be a significant cellular response in the early pathogenesis of asbestosis.