Effects Of Asbestos Fibers Research Articles

Effect of IL-15 addition on asbestos-induced suppression of human cytotoxic T lymphocyte induction

BackgroundAsbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8+ T cells. Recently, we reported that asbestos-induced suppression of CTL induction is not due to insufficient levels of interleukin-2 (IL-2). In this study, we continue to investigate the mechanism responsible for the effect of asbestos fibers on the differentiation of CTLs and focus on interleukin-15 (IL-15) which is known to be a regulator of T lymphocyte proliferation.MethodsFor MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 μg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8+ T cells with fluorescence-labeled anti-CD3, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CD25, and anti-granzyme B antibodies using flow cytometry. To examine the effect of IL-15 on the expression level of intracellular granzyme B in proliferating and non-proliferating CD8+ lymphocytes, PBMCs were stained using carboxyfluorescein diacetate succinimidyl ester (CFSE) and then washed and used for the MLR.ResultsIL-15 addition partially reversed the decrease in CD3+CD8+ cell numbers and facilitated complete recovery of granzyme B+ cell percentages. IL-15 completely reversed the asbestos-induced decrease in percentage of granzyme B+ cells in both non-proliferating CFSE-positive and proliferating CFSE-negative CD8+ cells. The asbestos-induced decrease in the percentage of CD25+ and CD45RO+ cells in CD8+ lymphocytes was not reversed by IL-15.ConclusionThese findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.

Increased production of matrix metalloproteinase-7 (MMP-7) by asbestos exposure enhances tissue migration of human regulatory T-like cells

The effects of asbestos on immunocompetent cells have been investigated. In particular, attention was paid to regulatory T cell function, which was observed using the HTLV-1 immortalized human polyclonal T cell line MT-2. Exposure to asbestos (approximately more than 25 μg/mL for 1–3 day) induced apoptosis, and we observed an increase in regulatory T cell function and acceleration of the cell cycle with continuous exposure to low concentrations of asbestos (5–10 μg/mL for more than eight months). Furthermore, cDNA microarray analysis in this study revealed that expression of matrix metalloproteinase-7 (MMP-7) was markedly higher in exposed sublines compared to original MT-2 cells. It was determined that MMP-7 had no effect on Treg function, as determined by examination of sublines and by addition of recombinant MMP-7 and neutralizing antibodies or inhibitors of MMP-7. However, when examining melting of the extracellular matrix (an MMP-7-mediated event) or the extent to which the MT-2 parent strain or long-term exposed subline cells pass through a fibronectin-coated filter, more filter passes were observed for the subline. These results suggest that the effect of asbestos fibers on Treg cells results in excessive migration of the tumor microenvironment through hypersecretion of MMP-7 together with an increase in suppressive function and enhancement of cell cycle progression. Therefore, one possible way to prevent the development of asbestos-induced cancer is to reduce the function (including MMP-7 production) or amount of Treg cells by physiologically active substances or food ingredients. Alternatively, it may be possible to invoke immune checkpoint treatments when carcinogenesis occurs.

The Effects of Asbestos Fibers on Human T Cells

Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. The effects of asbestos fibers on immunocompetent cells, however, have not been well studied. Asbestos physically comprises a fibrous substance, which differs from silica particles which are a particulate substance, although chemically it is a mineral silicate. Since silicosis patients previously exposed to silica particles often suffer from lung and autoimmune diseases, it is clear that silica exposure impairs immune tolerance. Similarly, asbestos may alter the immune system in asbestos-exposed individuals. Given that malignant tumors can result following exposure to asbestos, the attenuation of anti-tumor immunity in cases of asbestos exposure is an important area of investigation. We observed the effect of asbestos fibers on T lymphocytes, such as CD8+ cytotoxic T lymphocytes (CTLs), CD4+ helper T (Th), and regulatory T (Treg) cells, and showed that anti-tumor immunity was attenuated, as demonstrated in a system that stimulates fresh cells isolated from peripheral blood in vitro and a system that is continuously exposed to a cell line. In this manuscript, we introduce the experiments and results of studies on CTLs, as well as Th and Treg cells, and discuss how future changes in immunocompetent cells induced by asbestos fibers can be clinically linked.

Enhanced expression of nicotinamide nucleotide transhydrogenase (NNT) and its role in a human T cell line continuously exposed to asbestos

The effects of asbestos fibers on human immune cells have not been well documented. We have developed a continuously exposed cell line model using the human T-lymphotropic virus 1 (HTLV-1)-immortalized human T cell line MT-2. Sublines continuously exposed to chrysotile (CH) or crocidolite (CR) showed acquired resistance to asbestos-induced apoptosis following transient and high-dose re-exposure with fibers. These sublines in addition to other immune cells such as natural killer cells or cytotoxic T lymphocytes exposed to asbestos showed a reduction in anti-tumor immunity. In this study, the expression of genes and molecules related to antioxidative stress was examined. Furthermore, complexes related to oxidative phosphorylation were investigated since the production of reactive oxygen species (ROS) is important when considering the effects of asbestos in carcinogenesis and the mechanisms involved in resistance to asbestos-induced apoptosis. In sublines continuously exposed to CH or CR, the expression of thioredoxin decreased. Interestingly, nicotinamide nucleotide transhydrogenase (NNT) expression was markedly enhanced. Thus, knockdown of NNT was then performed. Although the knockdown clones did not show any changes in proliferation or occurrence of apoptosis, these clones showed recovery of ROS production with returning NADPH/NADP+ ratio that increased with decreased production of ROS in continuously exposed sublines. These results indicated that NNT is a key factor in preventing ROS-induced cytotoxicity in T cells continuously exposed to asbestos. Considering that these sublines showed a reduction in anti-tumor immunity, modification of NNT may contribute to recovery of the anti-tumor effects in asbestos-exposed T cells.

Genotoxicity of carbon nanofibers: Are they potentially more or less dangerous than carbon nanotubes or asbestos?

The production of carbon nanofibers and nanotubes (CNF/CNT) and their composite products is increasing globally. CNF are generating great interest in industrial sectors such as energy production and electronics, where alternative materials may have limited performance or are produced at a much higher cost. However, despite the increasing industrial use of carbon nanofibers, information on their potential adverse health effects is limited. In the current study, we examine the cytotoxic and genotoxic potential of carbon-based nanofibers (Pyrograf®-III) and compare this material with the effects of asbestos fibers (crocidolite) or single-walled carbon nanotubes (SWCNT). The genotoxic effects in the lung fibroblast (V79) cell line were examined using two complementary assays: the comet assay and micronucleus (MN) test. In addition, we utilized fluorescence in situ hybridization to detect the chromatin pan-centromeric signals within the MN indicating their origin by aneugenic (chromosomal malsegregation) or clastogenic (chromosome breakage) mechanisms. Cytotoxicity tests revealed a concentration- and time-dependent loss of V79 cell viability after exposure to all tested materials in the following sequence: asbestos>CNF>SWCNT. Additionally, cellular uptake and generation of oxygen radicals was seen in the murine RAW264.7 macrophages following exposure to CNF or asbestos but not after administration of SWCNT. DNA damage and MN induction were found after exposure to all tested materials with the strongest effect seen for CNF. Finally, we demonstrated that CNF induced predominately centromere-positive MN in primary human small airway epithelial cells (SAEC) indicating aneugenic events. Further investigations are warranted to elucidate the possible mechanisms involved in CNF-induced genotoxicity.

Asbestos and Lung Diseases: Introduction and Epidemiology

Over the last 2 centuries, there has been a growing awareness of occupationally related lung disease resulting from exposure to inorganic particulates that cause chronic and disabling fibrotic lung disease that can be fatal. Fibrotic lung disease, because of dust exposure, is referred to as pneumoconiosis. Asbestos refers to a group of naturally occurring minerals that has been extensively used in diverse industries as the minerals are known for their high tensile strength and flexibility and exhibit high thermal and chemical degradation as well as electrical resistance. 1 Asbestos fibers are either straight or curved and are referred to as amphiboles or serpentine type. Chrysotile, or often called white asbestos, is a serpentine form and widely used in USA in the past. Amphiboles may be more carcinogenic than the serpentine type. In the late 19th century, the industrial import of asbestos to England from Canada grew, and workers’ exposure to asbestos increased, resulting in a gradual increase in asbestos-related morbidity and mortality. Reports of asbestos-related illness and fatal cases started to appear in the lay press in England (1898, 1906), France (1906), and Italy (1908). Many of these reports involved exposure to Canadian chrysotile fiber. From early (1900s) reports of pulmonary fibrosis, referred to as asbestosis, resulting in significant morbidity and mortality, emerged the scientific literature in England, USA, and Europe. Despite the strong evidence regarding the adverse health effects of asbestos fibers, industries had no restriction or regulations in regard to the use of asbestos. 1-3 The studies from Canada were not supportive of asbestos-related lung diseases in the first half of 20th century and they continued to use the fiber. In 1931, regulation regarding asbestos use was established in Britain. Only in 1955, Canada acknowledged the occurrence of asbestosis in the miners and mill workers. 3 The carcinogenicity of asbestos was initially noted in an animal study using mice but the results were not widely accepted. Based on the Saranac research Vorwald reported an increased association of malig

P16 INK4A inactivation mechanisms in non-small-cell lung cancer patients occupationally exposed to asbestos

Epidemiological studies have shown that asbestos fibers constitute the major occupational risk factor and that asbestos acts synergistically with tobacco smoking to induce lung cancer. Although some somatic gene alterations in lung cancer have been linked to tobacco smoke, few data are available on the role of asbestos fibers. P16/ CDKN2A is an important tumor suppressor gene that is frequently altered in lung cancer via promoter 5′-CpG island hypermethylation and homozygous deletion, and rarely via point mutation. Many studies suggest that tobacco smoking produces P16/ CDKN2A promoter hypermethylation in lung cancer, but the status of this gene in relation to asbestos exposure has yet to be determined. The purpose of this study was to investigate the mechanism of P16/ CDKN2A alterations in lung cancer in asbestos-exposed patients. P16/ CDKN2A gene status was studied in 75 human non-small-cell lung cancer (NSCLC) cases with well-defined smoking habits, and detailed assessment of asbestos exposure, based on occupational questionnaire and determination of asbestos bodies in lung tissue. The results of this study confirm published data on the effect of tobacco smoke on P16/ CDKN2A gene alterations, characterized by significantly higher P16/ CDKN2A promoter hypermethylation in heavy smokers (more than 40 pack-years (P-Y)) than in smokers of less than 40 P-Y. These results also demonstrate a higher incidence of loss of heterozygosity and homozygous deletion in asbestos-exposed cases, after adjustment for age and cumulative tobacco consumption, than in unexposed cases ( P = 0.0062). This study suggests that P16/ CDKN2A gene inactivation in asbestos-exposed NSCLC cases mainly occurs via deletion, a feature also found in malignant mesothelioma, a tumor independent of tobacco smoking but associated with asbestos exposure, suggesting a possible relationship with an effect of asbestos fibers.

Genotoxicity Versus Carcinogenicity: Implications from Fiber Toxicity Studies

Although the association between exposure to asbestos fibers and the development of lung cancer and mesothelioma has been well established in humans, the carcinogenic potential of other natural and man-made fibers/particles is not clear. Various in vitro genotoxicity studies have been employed to assess their in vivo carcinogenic potential. Studies using mammalian cell models have suggested that fiber dimensions, surface properties, physical durability, and cell and tissue responses are important criteria for the carcinogenicity of the fibers. Studies using oncogenic transformation as an endpoint have shown that asbestos fibers can induce malignantly transformed foci in certain rodent cells and that oxygen radicals are important in the toxic, oncogenic transforming, and mutagenic effects of asbestos fibers. The mutagenicity of asbestos in mammalian cells have been demonstrated using several model systems that can detect large multilocus deletions. These findings provide a direct link between chromosomal abnormalities that have frequently been demonstrated in fiber-exposed human and rodent cell lines and carcinogenicity in vivo. Although asbestos has not been shown to malignantly transform primary human epithelial cells, it can induce neoplastic conversion of immortalized human bronchial epithelial cells in a stepwise fashion and provides a unique opportunity to assess the molecular alterations associated with each stage of the neoplastic process.

Observations on the Carcinogenidty of Asbestos Fibers

This paper summarizes animal experiments and in vitro data carried out to study the oncogenic effects of asbestos fibers on mesothelial cells. An interpretation of the results is made in light of current statements on the carcinogenicity of asbestos fibers. Experimental results appear to show that the carcinogenicity of mineral fibers is a complex, multiparametric phenomenon. Chromosomal mutations and possibly oxygen derivatives are involved in the genesis of the fiber-induced neoplastic process and may be the result of the intrinsic fiber properties, size, and physicochemistry. The role of fiber solubility is discussed; it is suggested that additional experiments are necessary for a better understanding of the importance of solubility in the concept of biopersistence.

Effects of asbestos fibers on alveolar macrophage-mediated lymphocyte cytostasis

Prolonged asbestos inhalation results in pulmonary inflammation and fibrosis. Since alveolar macrophages are active in regulation of immune responses in lung and appear to be involved in the pathogenesis of asbestosis, we evaluated the effects of asbestos exposure on the ability of these cells to regulate lymphocyte function. Alveolar macrophages obtained by lung lavage from BALB/C mice were treated in vitro with either UICC amosite or chrysotile asbestos and the effects on lymphocyte cytostasis compared with those of macrophages incubated with latex beads or zymosan. Macrophages (10%) incubated either alone or with latex beads for 48 hr effectively inhibited lymphocyte mitogenesis. However, alveolar macrophages incubated with either amosite or chrysotile asbestos did not demonstrate intact cytostatic activity. Decreased viability of chrysotile asbestos-treated macrophages correlated with loss of cytostatic effects, but alveolar macrophages exposed to amosite remained viable. We conclude, therefore, that exposure of alveolar macrophages to asbestos can result in loss of their ability to down-regulate lymphocyte proliferation, a finding which may be important in the pathogenesis of asbestos-related disease.

Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese hamster ovary cells

The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo[a]pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 microns), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possibly be used as an index of the degree of mitotic disturbances induced by mineral fibers.

Asbestos-induced Modulation of Release of Regulatory Molecules from Alveolar and Peritoneal Macrophages

Asbestos inhalation causes several immunologic abnormali ties in exposed individuals and experimental animals, 1 Pernis B Vigliani EC Selikoff JJ Rheumatoid factor in serum of individuals exposed to asbestos. Ann NY Acad Sci. 1965; 132: 112 Crossref PubMed Scopus (52) Google Scholar , 2 Kagan E Solomon A Cochrane JC Bleissner EI Glukman J Rocks PH et al. Immunological studies of patients with asbestosis. Clin Exp Immunol. 1977; 28: 261 PubMed Google Scholar , 3 Bozelka BE Sestini P Gaumer HR Hammad Y Heather CJ Salvaggio JE A murine model of asbestosis. Am J Pathol. 1983; 112: 326 PubMed Google Scholar and it has been suggested that these phenomena have a role in the induction of pulmonary fibrosis and could be related to alterations of macrophage-mediated regulatory activities. 4 Pernis B Vigliani EC The role of macrophages and immunocytes in the pathogenesis of pulmonary diseases due to mineral dusts. Am J Ind Med. 1982; 3: 133 Crossref Scopus (22) Google Scholar To investigate whether asbestos could affect the release of immunoregulatory molecules from macrophages, we have studied the effect of the exposure in vitro of mouse resident alveolar (AMø) and peritoneal macrophages (PMø) to nontoxic concentrations of asbestos on their suppressive capacity on the mitogen-induced proliferation of syngeneic spleen lymphocytes, on their ability to release arachidonic acid metabolites and superoxide anion (O$$) in response to zymosan and on the spontaneous release of interleukin 1 (Il-1). PMø purified by adherence and AMø obtained by bronchoalveolar lavage from 8-16-week-old C3H/HeN mice were cultivated in flat-bottom wells in the presence of control medium, latex beads, or UICC asbestos amosite, 80 µg/ml, for 21 hours. The medium was then carefully aspirated and the Mø further processed. Suppressive capacity was evaluated as previously described 5 Sestini P Bozelka BE deShazo RD Salvaggio JE Murine alveolar macrophage-mediated lymphocyte cytostasis: kinetics and mechanisms. Cell Immunol. 1982; 30: 108 Google Scholar by adding syngeneic spleen cells stimulated with an optimal dose of Con-A and evaluating the incorporation of tritiated thymidine after 72 hours of culture. The release of O$$ was measured by ferricytochrome c reduction after stimulation with opsonized zymosan. Prostaglandin E2 (PCE2) and F2α, (PGF2α), and leukotriene C4 (LTC4) were evaluated by radioimmunoassay after stimulation with zymosan 200 µg/rnl in medium without serum. Il-1 was evaluated by the C3H/HeJ thymocyte proliferation assay. The presence of interleukin-2 (Il-2) activity in the supernatants was assayed by the ability to support the proliferation of the CTLL cell line. In agreement with previous experiments 6 Bozelka BE, Sestini P, Hammad Y, Salvaggio JE. Effects of asbestos fibers on alveolar macrophage-mediated cytostasis. Environ Res (in press) Google Scholar the exposure of AMø and PMø in vitro to amosite caused a dose-dependent decrease in their suppressive capacity, whereas phagocytosis of latex had no effect. This phenomenon was not due to loss of cell viability as judged by LDH release and trypan blue exclusion, and was paralleled by a marked reduction of the ability to produce Ol. Asbestos exposure caused a small increase of spontaneous PGE2 production from both Mø populations; however, it had different effects on the zymosan-induced release of PGE2 and PGF2α from AMø and PMø. In fact, while PMø showed a reduction of the release of these metabolites after exposure to asbestos compared with untreated cells, AMø showed a marked increase. The release of LTC4, however, was decreased in both Mø populations. Latex beads had minimal effects on these activities. In addition, amosite but not latex beads induced the release of Il-1 in cultures of AMø and PMø. Il-1 activity was present in the supernatants during the first 24 hours of culture, and the release continued during further incubation, even in serum-free medium. No Il-2 activity or dialyzable inhibitors were found in the supernatants.

Effect of asbestos fibers on aryl hydrocarbon hydroxylase and aminopyrine N-demethylase activities of rat liver microsomes

Chrysotile asbestos fibers impair the activities of rat liver microsomal aryl hydrocarbon hydroxylase (AHH), aminopyrine (AP) N-demethylase and dimethylnitrosamine (DMN) demethylase in vitro. This inhibition is concentration-dependent. Preincubation of 3-methylcholanthrene (3-MC)-pretreated rat liver microsomes with chrysotile depresses the overall metabolism of [G- 3H] benzo[ a]pyrene (BaP). Various forms of asbestos employed inhibit AHH activity to the same extent. However, other types of asbestos are not as effective as chrysotile in diminishing AP demethylase activity. Chrysotile and crocidolite fibers are not found to significantly change the apparent K m of AHH activity, from 3-MC-pretreated rat liver microsomes, for BaP. Increasing the microsomal protein concentration partially abolishes the inhibition of AHH activity caused by chrysotile fibers. Inhibition of AP demethylase and AHH activities is attenuated by bovine serum albumin (BSA) or ferritin. Depression of AHH activity by crocidolite is significantly reversed by ferritin. Since polymers such as ferritin override enzyme inhibition by chrysotile as well as crocidolite, surface chemical groups of the fibers may be involved in enzyme modification.

Effects of Asbestos Fibers on Mammalian Cells in Culture

In previous reports from this laboratory, we examined the correlation of the toxic effects of several organic pesticides, herbicides and trace metals on the plating efficiency to the ultrastructural changes found in the treated cells in culture. Since there has been a long-term interest in the effects of exposure of humans and experimental animals to asbestos, the current study was undertaken to determine if ultrastructural changes would also be found in mammalian cells grown in vitro after exposure to asbestos. In a series of preliminary experiments, suitable fractionation by low-speed differential centrifugation and concentrations of amphibole asbestos were found in which there was at least 70% to 90% viability depending on the cell type. The viability of the cells incubated in the presence of the asbestos was determined both by growth curves established from direct cell counts and from staining with trypan blue which does not penetrate viable cells.