ABSTRACT
BACKGROUND: For certain human cancers, sperm associated antigen 5 (SPAG5) exerts important functions for their development and progression. However, whether RNA interference (RNAi) targeting SPAG5 has antitumor effects has not been determined clinically. RESULTS: The results indicated that Fe-doped chrysotile nanotubes (FeSiNTs) with a relatively uniform outer diameter (15-25 nm) and inner diameter (7-8 nm), and a length of several hundred nanometers, which delivered an siRNA against the SPAG5 oncogene (siSPAG5) efficiently. The nanomaterials were designed to prolong the half-life of siSPAG5 in blood, increase tumor cell-specific uptake, and maximize the efficiency of SPAG5 silencing. In vitro, FeSiNTs carrying siSPAG5 inhibited the growth, migration, and invasion of bladder cancer cells. In vivo, the FeSiNTs inhibited growth and metastasis in three models of bladder tumors (a tail vein injection lung metastatic model, an in-situ bladder cancer model, and a subcutaneous model) with no obvious toxicities. Mechanistically, we showed that FeSiNTs/siSPAG5 repressed PI3K/AKT/mTOR signaling, which suppressed the growth and progression of tumor cells. CONCLUSIONS: The results highlight that FeSiNTs/siSPAG5 caused no activation of the innate immune response nor any systemic toxicity, indicating the possible therapeutic utility of FeSiNTs/siSPAG5 to deliver siSPAG5 to treat bladder cancer.
Subject(s)
Asbestos, Serpentine/pharmacology , Cell Cycle Proteins/genetics , Nanotubes/chemistry , RNA, Small Interfering/chemistry , Urinary Bladder Neoplasms/drug therapy , Animals , Cell Line, Tumor , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Gene Silencing , Genetic Therapy/methods , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Phosphatidylinositol 3-Kinases/metabolism , RNA Interference , Rats , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
AIMS: Asbestos is harmful to human health. However, the pathogenicity of chrysotile is a controversial matter. This study aimed to investigate the apoptosis of a human bronchial epithelial cell line (BEAS-2B) exposed to chrysotile that may function in part through the Fas death receptor pathway. METHODS: Cultured human BEAS-2B cells were treated with chrysotile and cell viability was evaluated by CCK-8 assay. The induction of cell apoptosis was evaluated by FACS analysis. mRNA expression levels of Fas, caspase-3, and caspase-8 were evaluated quantitatively by real-time PCR. The expression of Fas, caspase-3, and caspase-8 proteins were evaluated by Western blot. Meanwhile, cells were preincubated with various concentrations of anti-Fas antibody (CH11) and antagonistic anti-Fas antibody (ZB4). RESULTS: Chrysotile inhibits proliferation, induces apoptosis, and upregulates the expression of Fas, caspase-3, and caspase-8. The role of Fas as a regulator of chrysotile-induced apoptosis in BEAS-2B cells was tested by the prominent increase in and partial blockade of the apoptotic rate with CH11 and ZB4. When CH11 was pretreated, a synergistic effect was apparent on chrysotile-induced apoptosis and the mRNA and protein expression levels of Fas and cleaved caspase-3. CONCLUSION: Chrysotile causes the apoptosis of BEAS-2B cells via the Fas death receptor pathway. The Fas-mediated apoptosis pathway plays an important role in chrysotile-induced apoptosis of BEAS-2B cells in vitro.
Subject(s)
Apoptosis/drug effects , Asbestos, Serpentine/pharmacology , Bronchi/drug effects , Cell Survival/drug effects , Epithelial Cells/drug effects , Asbestos, Serpentine/toxicity , Blotting, Western , Bronchi/cytology , Caspase 3/drug effects , Caspase 3/genetics , Caspase 3/metabolism , Caspase 8/drug effects , Caspase 8/genetics , Caspase 8/metabolism , Cell Line , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Fluorescent Antibody Technique , Humans , Up-Regulation/drug effects , fas Receptor/genetics , fas Receptor/immunology , fas Receptor/metabolismABSTRACT
Exposure to asbestos is a risk for malignant mesothelioma (MM) in humans. Among the commercially used types of asbestos (chrysotile, crocidolite, and amosite), the carcinogenicity of chrysotile is not fully appreciated. Here, we show that all three asbestos types similarly induced MM in the rat peritoneal cavity and that chrysotile caused the earliest mesothelioma development with a high fraction of sarcomatoid histology. The pathogenesis of chrysotile-induced mesothelial carcinogenesis was closely associated with iron overload: repeated administration of an iron chelator, nitrilotriacetic acid, which promotes the Fenton reaction, significantly reduced the period required for carcinogenesis; massive iron deposition was found in the peritoneal organs with high serum ferritin; and homozygous deletion of the CDKN2A/2B/ARF tumour suppressor genes, the most frequent genomic alteration in human MM and in iron-induced rodent carcinogenesis, was observed in 92.6% of the cases studied with array-based comparative genomic hybridization. The induced rat MM cells revealed high expression of mesoderm-specific transcription factors, Dlx5 and Hand1, and showed an iron regulatory profile of active iron uptake and utilization. These data indicate that chrysotile is a strong carcinogen when exposed to mesothelia, acting through the induction of local iron overload. Therefore, an intervention to remove local excess iron might be a strategy to prevent MM after asbestos exposure.
Subject(s)
Asbestos, Serpentine/adverse effects , Biomarkers, Tumor/metabolism , Iron Overload/metabolism , Lung Neoplasms/chemically induced , Lung Neoplasms/metabolism , Mesothelioma/chemically induced , Mesothelioma/metabolism , Peritoneal Neoplasms/chemically induced , Peritoneal Neoplasms/metabolism , Animals , Asbestos, Serpentine/pharmacology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Transformation, Neoplastic/drug effects , DNA Copy Number Variations/drug effects , DNA, Neoplasm/drug effects , Disease Models, Animal , Homeodomain Proteins/metabolism , Iron/metabolism , Lung Neoplasms/pathology , Male , Mesothelioma/pathology , Mesothelioma, Malignant , Nitrilotriacetic Acid/pharmacology , Peritoneal Neoplasms/pathology , Rats , Rats, Inbred BN , Rats, Inbred F344 , Signal Transduction/drug effects , Transcription Factors/metabolismABSTRACT
BACKGROUND: Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. METHODS: The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. RESULTS: HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. CONCLUSION: The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.
Subject(s)
Asbestos, Serpentine/pharmacology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , DNA, Neoplasm/analysis , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Aneuploidy , Apoptosis/drug effects , Asbestos, Serpentine/pharmacokinetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/genetics , DNA, Neoplasm/metabolism , Humans , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Interphase/drug effects , Lung Neoplasms/metabolism , Microscopy, Confocal , Mitosis/drug effects , PhagocytosisABSTRACT
Chrysotile, like other types of asbestos, has been associated with mesothelioma, lung cancer and asbestosis. However, the cellular abnormalities induced by these fibers involved in cancer development have not been elucidated yet. Previous works show that chrysotile fibers induce features of cancer cells, such as aneuploidy, multinucleation and multipolar mitosis. In the present study, normal and cancer derived human cell lines were treated with chrysotile and the cellular and molecular mechanisms related to generation of aneuploid cells was elucidated. The first alteration observed was cytokinesis regression, the main cause of multinucleated cells formation and centrosome amplification. The multinucleated cells formed after cytokinesis regression were able to progress through cell cycle and generated aneuploid cells after abnormal mitosis. To understand the process of cytokinesis regression, localization of cytokinetic proteins was investigated. It was observed mislocalization of Anillin, Aurora B, Septin 9 and Alix in the intercellular bridge, and no determination of secondary constriction and abscission sites. Fiber treatment also led to overexpression of genes related to cancer, cytokinesis and cell cycle. The results show that chrysotile fibers induce cellular and molecular alterations in normal and tumor cells that have been related to cancer initiation and progression, and that tetraploidization and aneuploid cell formation are striking events after fiber internalization, which could generate a favorable context to cancer development.
Subject(s)
Aneuploidy , Asbestos, Serpentine/pharmacology , Lung Neoplasms/pathology , Mitosis/drug effects , Aurora Kinase B/metabolism , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Endosomal Sorting Complexes Required for Transport/metabolism , Humans , Lung Neoplasms/chemically induced , Microfilament Proteins/metabolism , Septins/metabolismABSTRACT
While cases of silicosis are often complicated by various autoimmune disorders, patients with asbestosis develop malignant tumors such as lung cancer and malignant mesothelioma. These differences may derive from different biological effects, particularly on immunological cells, of silica and asbestos. To find differences between silica and asbestos, the early activation antigen, CD69, on T cells was examined because dysregulated and continuous activation of T cells may promote the survival of self-recognizing T cells. After cultivation of peripheral blood mononuclear cells with or without silica or chrysotile-A, an asbestos, only silica induced CD69 expression on the lymphocytes. This induction of CD69 expression was mediated by protein kinase C activation. In addition, cell-cell contact mediated by HLA-DR was more important than soluble factors secreted from silica-phagocytosed cells such as IL-1beta, IL-6, and IL-8, even though IL-6 and IL-8 were produced during the culture of PBMCs with silica and chrysotile-A. It should be examined how these activated, CD69-expressing lymphocytes affect other immune systems as well as alter themselves in terms of cytokine production and cell-cell interaction, leading to autoimmune disorders in silicosis patients.
Subject(s)
Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/immunology , Asbestos, Serpentine/pharmacology , Leukocytes, Mononuclear/drug effects , Antibodies , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Antigens, Differentiation, T-Lymphocyte/genetics , Coculture Techniques , Humans , Lectins, C-Type , Protein Kinase C/antagonists & inhibitorsABSTRACT
Saccharomyces cerevisiae was supported on chrysotile, crocidolite and lixiviated chrysotile. Samples of the supported cells and free cells were observed by confocal laser scanning microscopy. After 30 days, the free cells showed no viability when stored at 30 degrees C, and a viability of 40% when stored at 4 degrees C. Supported cells stored at 30 degrees C were more viable than the free cells at early times, but showed no viability after 30 days. Samples stored at 4 degrees C showed that the adhered cells are more viable than the free cells, up to 30 days. Cells supported on chrysotile and lixiviated chrysotile had 80% viability, and on crocidolite 70% viability. Scanning electron microscopy showed that cells supported on lixiviated chrysotile are fully covered by the support, but crocidolite fibers adhere less, since they are stiffer. Fermentation experiments performed after 3 years storage showed that four from the five lixiviated chrysotile samples and one of the three crocidolite samples were active. In all cases, a delay time for the onset of fermentation was observed indicating a state of latency.
Subject(s)
Asbestos/adverse effects , Saccharomyces cerevisiae/drug effects , Asbestos/pharmacology , Asbestos, Crocidolite/adverse effects , Asbestos, Crocidolite/pharmacology , Asbestos, Serpentine/adverse effects , Asbestos, Serpentine/pharmacology , Cell Survival/drug effects , Cells, Cultured , Culture Techniques , Fermentation , Microscopy, Electron, Scanning , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/ultrastructure , Temperature , Time FactorsABSTRACT
Exposure to chrysotile asbestos exposure is associated with an increased risk of mortality in combination with pulmonary diseases including lung cancer, mesothelioma and asbestosis. Multiple mechanisms by which chrysotile asbestos fibers induce pulmonary disease have been identified, however the role of apoptosis in human lung alveolar epithelial cells (AEC) has not yet been fully explored. Accumulating evidence implicates AEC apoptosis as a crucial event in the development of both idiopathic pulmonary fibrosis and asbestosis. The aim of the present study was to determine whether chrysotile asbestos induces mitochondriaregulated (intrinsic) AEC apoptosis and, if so, whether this induction occurs via the activation of mitogenactivated protein kinases (MAPK). Human A549 bronchoalveolar carcinomaderived cells with alveolar epithelial type IIlike features were used. The present study showed that chrysotile asbestos induced a dose and timedependent decrease in A549 cell viability, which was accompanied by the activation of the MAPK cJun Nterminal kinases (JNK), but not the MAPKs extracellular signalregulated kinase 1/2 and p38. Chrysotile asbestos was also shown to induce intrinsic AEC apoptosis, as evidenced by the upregulation of the proapoptotic genes Bax and Bak, alongside the activation of caspase9, poly (ADPribose) polymerase (PARP), and the release of cytochrome c. Furthermore, the specific JNK inhibitor SP600125 blocked chrysotile asbestosinduced JNK activation and subsequent apoptosis, as assessed by both caspase9 cleavage and PARP activation. The results of the present study demonstrated that chrysotile asbestos induces intrinsic AEC apoptosis by a JNKdependent mechanism, and suggests a potential novel target for the modulation of chrysotile asbestosassociated lung diseases.
Subject(s)
Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/metabolism , Apoptosis/drug effects , Asbestos, Serpentine/pharmacology , JNK Mitogen-Activated Protein Kinases/metabolism , Signal Transduction/drug effects , Caspase 9/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Humans , Poly(ADP-ribose) Polymerases/metabolism , Proteolysis/drug effectsABSTRACT
Silica and related substances such as silicate have been proven to possess "adjuvant effects". We have previously reported a finding of polyclonal human T cell activation induced by silicate as a superantigen in vitro. In this study, we observed activation-induced cell death in human lymphocytes after stimulation with chrysotile, a kind of silicate. Apoptotic cells were detected flow cytometrically using the TUNEL assay, and the maximum appearance of TUNEL positive cells occurred on day 4 of incubation. Simultaneously the manifestation of small-sized cells in the specimens increased implying apoptosis. Fas expression on lymphocytes increased to day 3 of incubation with chrysotile, and then spontaneously decreased on day 4 when remarkable apoptosis could be detected. Based on these results it is conceivable that activation-induced cell death occurred through Fas-Fas ligand interaction in lymphocytes after stimulation with silicate in a concentration with which no acute cytotoxicity has been detected. Whether and how the repeated apoptosis in definite clones of lymphocytes causes the induction of sFas synthesis need clarification.
Subject(s)
Silicates/pharmacology , T-Lymphocytes/immunology , Asbestos, Serpentine/pharmacology , CD3 Complex/biosynthesis , CD3 Complex/drug effects , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/physiology , Carcinogens/administration & dosage , Carcinogens/pharmacology , Cell Death/drug effects , Fas Ligand Protein , Humans , Lipopolysaccharide Receptors/biosynthesis , Lipopolysaccharide Receptors/drug effects , Lymphocyte Activation/drug effects , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/drug effects , Silicates/administration & dosage , Silicates/immunology , Superantigens/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/physiologyABSTRACT
Mononuclear leukocytes were isolated from the peripheral blood of 15 patients with malignant pulmonary diseases, 17 patients with pulmonary infections, 18 patients with chest film abnormalities of non-malignant, non-infectious etiology, and 15 healthy persons. The cells were exposed to zymosan yeast, BCG vaccine, quartz, or chrysotile asbestos, and the subsequent production of reactive oxygen species (ROS) was measured by luminol-dependent chemiluminescence. All the stimulants caused significantly higher ROS production in the patient groups than in the healthy control group, and the asbestos-induced ROS production was significantly more pronounced in the cancer group than in the two non-cancer patient groups combined. After one-year follow-up, 5 of the 15 cancer patients were alive, and these patients had significantly lower mineral dust-induced ROS responses at the time of diagnosis than were found in the patients who died. This result was verified in a subsequent study comprising 19 patients with malignant pulmonary disorders (6 alive after one year). In conclusion, monocytes from patients with malignant diseases seem to be primed for an increased ROS production, and high ROS responses seem to correlate with a poor one-year survival of the patients.
Subject(s)
Dust , Lung Diseases/blood , Lung Neoplasms/blood , Monocytes/metabolism , Reactive Oxygen Species/analysis , Adult , Aged , Asbestos, Serpentine/pharmacology , BCG Vaccine/pharmacology , Cells, Cultured , Follow-Up Studies , Humans , Luminescent Measurements , Lung Diseases/mortality , Lung Neoplasms/mortality , Middle Aged , Monocytes/drug effects , Monocytes/pathology , Quartz/pharmacology , Survival Analysis , Zymosan/pharmacologyABSTRACT
This study has shown, for the first time, that in vitro exposure of rat AMs to either crocidolite (amphibole) or chrysotile (serpentine) asbestos fibers induces the synthesis not only of the O2*- anion, but also of the nitrogen radical, NO*. Furthermore, this asbestos-related effect is enhanced in the presence of interferon-gamma. NO* has been implicated in several pathologic reactions, such as inflammation and immune complex-mediated cell injury. Additionally, NO* may interact with secondary amines to generate nitrosamines, which are potent carcinogens. Our findings could represent a novel type of asbestos-mediated injury, and we propose that the injurious effects of asbestos might be mediated via the interaction of NO* with O2*-, with the generation of ONOO- and other potent toxic free radicals.
Subject(s)
Asbestos, Crocidolite/pharmacology , Asbestos, Serpentine/pharmacology , Interferon-gamma/pharmacology , Macrophages, Alveolar/metabolism , Nitric Oxide/biosynthesis , Animals , Arginine/analogs & derivatives , Arginine/pharmacology , Cells, Cultured , Drug Synergism , Macrophages, Alveolar/drug effects , Nitric Oxide/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolism , omega-N-MethylarginineABSTRACT
The ability of chrysotile asbestos fibers to introduce the exogenous plasmid pUC18 into Escherichia coli JM109 cells was tested. Cells were transformed with pUC18 DNA although the frequency of transformation was quite low: 759+/-301 transformants were obtained per microgram of pUC18. Plasmids were purified from E. coli which had been transformed by mediation with chrysotile asbestos. Following this, the plasmids were confirmed to be pUC18 by Southern hybridization. This asbestos-mediated transformation was optimal within 5 min when 10 mg ml(-1) of asbestos was used. Plasmids up to 7.69 kb were introduced by this method.
Subject(s)
Asbestos, Serpentine/pharmacology , Escherichia coli/genetics , Plasmids , Transformation, Bacterial , Blotting, Southern , Escherichia coli/drug effectsABSTRACT
Effects of pertussis toxin (PTX), cholera toxin (CTX) and an anhydrolyzable GTP analogue, GTP gamma S, on the levels of free intracellular calcium ([Ca2+]i) and the production of reactive oxygen metabolites (ROM) in human polymorphonuclear leukocytes (PMNL) were studied during cell activation. Cells were stimulated by particulate stimuli, quartz or chrysotile, and soluble stimuli, formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). Pretreatment of PMNL with PTX decreased fMLP-induced elevations of [Ca2+]i but not those induced by quartz or chrysotile. CTX, in turn, decreased both quartz- and fMLP-induced elevations of [Ca2+]i. Likewise, PTX inhibited only fMLP-induced production of ROM, whereas CTX inhibited also those induced by quartz, chrysotile or fMLP. PTX or CTX did not, however, have an impact on PMA-induced production of ROM. GTP gamma S alone did not elevate [Ca2+]i or amplify fMLP-, quartz- or chrysotile-induced [Ca2+]i elevation. However, GTP gamma S alone increased the production of ROM and amplified ROM production induced by fMLP and quartz. The present results suggest that a CTX-sensitive G-protein may be involved in quartz-induced PMNL activation whereas an fMLP-induced neutrophil activation may be regulated by G-proteins sensitive to both PTX and CTX. The involvement of G-protein in chrysotile-induced leukocyte activation is not likely. There may be, however, a relationship between G-protein-mediated cell signalling and quartz-induced production of reactive oxygen metabolites in these cells.
Subject(s)
GTP-Binding Proteins/physiology , Neutrophil Activation/physiology , Reactive Oxygen Species/metabolism , Adult , Asbestos, Serpentine/pharmacology , Calcium/metabolism , Cholera Toxin/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Humans , In Vitro Techniques , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/metabolism , Pertussis Toxin , Quartz/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Virulence Factors, Bordetella/pharmacologyABSTRACT
The aim of this study was to investigate whether man-made mineral fibers (MMMF) induce apoptosis of human peripheral blood mononuclear cells (PBMC), as we recently demonstrated for chrysotile B. In vitro cultivation of PBMC with various MMMF as well as chrysotile B clearly produced apoptotic cells. The alteration of the expression for apoptosis related genes at the mRNA level during in vitro cultures of PBMC with various MMMF revealed upregulation of Flice and Apaf-1 genes and down regulation of TNF receptor 1 and Bid genes. These results indicate that MMMF induce apoptosis of PBMC in a similar manner to chrysotile B. However, the process may be mediated not only by the Fas-related apoptotic pathway but also a mitochondrial pathway. Thus, one should be aware that respiratory and immunological abnormalities may occur in workers who are exposed to MMMFs.
Subject(s)
Apoptosis/drug effects , Asbestos, Serpentine/pharmacology , Leukocytes, Mononuclear/drug effects , Mineral Fibers/toxicity , Adult , Antigens, CD/biosynthesis , Antigens, CD/genetics , Apoptosis/genetics , Caspase 8 , Caspase 9 , Caspases/biosynthesis , Caspases/genetics , DNA, Complementary/genetics , Gene Expression Regulation/drug effects , Humans , RNA, Messenger/biosynthesis , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Type I , Reverse Transcriptase Polymerase Chain Reaction , Ubiquitins/biosynthesis , Ubiquitins/geneticsABSTRACT
Epidemiological studies and animal experiments showed that asbestos and cigarette smoking can act synergistically in the development of lung cancer. The mechanism of this synergism is largely unknown. It is well documented that DNA damage and activation of oncogenes play important roles in the development of cancer. The aim of our study was to find out if DNA damage could be increased and c-ras oncogene could be activated when human embryo lung cells were treated with chrysotile (CH) and cigarette smoking solution (CSS) separately or simultaneously. Human embryo lung (HEL) cells were treated with different doses of CH and CSS separately or simultaneously, then DNA strand breaks were detected with single-cell gel electrophoresis assay and the expression of p21 was detected by flow cytometry. Factorial analysis was used to evaluate the combined effect of chrysotile and cigarette smoking solution. The results showed that DNA strand breaks could be increased significantly when HEL cells were exposed to CH and CSS separately for 1 hour and increased in a dose-dependent relationship when cells were exposed to CH and CSS simultaneously for 1 hour. The expression of p21 increased significantly when cells were exposed to CH for 24 hours, but there was no significant increase when cells were exposed to CSS for 24 hours. However, there was an additive effect on the expression of p21 when cells were exposed to CH and CSS simultaneously for 24 hours. When cells were exposed to CH and CSS simultaneously three times (24 hours each time), then passaged for 1 month, the expression of p21 increased synergistically. In conclusion, DNA damage and activation of c-ras may be involved in the process of combined carcinogenesis of CH and CSS.
Subject(s)
Asbestos, Serpentine/pharmacology , DNA Damage , Gene Expression Regulation/drug effects , Proto-Oncogene Proteins p21(ras)/genetics , Smoking , Tars/pharmacology , Cells, Cultured , DNA/drug effects , DNA/metabolism , Flow Cytometry , Humans , Lung/cytology , Lung/embryologyABSTRACT
This hydroponic study addresses the influence of low (0.3) and high (4.0) Ca/Mg molar ratios on Cu resistance of Silene armeria ecotypes from different habitats: a calcareous soil (ecotype Cadriano), a Ni-rich serpentine site (ecotype Prinzera), and an acid Cu-mine spoil soil containing serpentinite (ecotype Vigonzano). Under control conditions, without excess Cu, only Cadriano was negatively affected by the low Ca/Mg ratio. Under both low and high Ca/Mg ratios Cu resistance followed the order Vigonzano more more than Prinzera > Cadriano. More efficient Cu exclusion accounted for enhanced Cu resistance in Prinzera. The low Ca/Mg ratio increased Cu uptake in Prinzera but did not worsen toxicity effects; i.e. the plants had higher internal Cu effect concentrations. In Vigonzano Cu resistance was enhanced by the low Ca/Mg ratio. This was due only in part to better Cu exclusion. Magnesium-induced tolerance to higher Cu tissue concentrations appears to be in ecotypes from serpentine and acid mine spoils, but not in plants from calcareous soil, the exposure to low Ca/Mg ratio favours internal detoxification of Cu by means of more efficient chelation and compartmentation.
Subject(s)
Calcium/pharmacology , Copper/metabolism , Magnesium/pharmacology , Silene/drug effects , Adaptation, Physiological/drug effects , Asbestos, Serpentine/pharmacology , Chelating Agents/pharmacology , Copper/toxicity , Drug Resistance , Nickel/pharmacology , Plant Roots/drug effects , Plant Roots/growth & development , Plant Roots/metabolism , Plant Shoots/drug effects , Plant Shoots/growth & development , Plant Shoots/metabolism , Silene/growth & development , Silene/metabolism , Soil/analysis , Soil Pollutants/metabolism , Soil Pollutants/toxicityABSTRACT
Soot particles, asbestos fibres and irritant gas are common air pollutants which are able to induce lung and airway pulmonary injury. The aim of this study was to investigate the effect of a simultaneous NO2 and particle or fibre exposure on the proinflammatory specific mRNA expression and protein secretion of human alveolar macrophages (AM) in comparison to only particle or fibre exposed AM. AM were simultaneously exposed to FR 101, P 90, TiO2 or Chrysotile B at a concentration of 100 microg/10(6) cells and to NO2 at a concentration of 1.0 ppm for 30 min. Particle or fibre exposure of the AM was continued in humidified air at 5% CO2 and 37 degrees C for an additional hour (harvesting of total RNA) or additional 7 hrs (harvesting of culture supernatant). The mRNA expression of the proinflammatory cytokines IL-1beta, IL-6, IL-8 and TNF-alpha of NO2-particle/fibre co-exposed AM and only particle or fibre exposed AM was detected using specific RT-PCR. IL-1beta-, IL-6-, IL-8- and TNF-alpha-specific protein secretion was measured by ELISA. Cytotoxicity was detected by lactatedehydrogenase quantification in the culture supernatant. We observed an increased IL-1beta-, IL-6-, IL-8- and TNF-alpha-specific mRNA expression of particle or fibre exposed AM, which was decreased after an additional NO2 exposure. Also the particle or fibre exposure induced significant increase in IL-1beta-, IL-6-, IL-8 and TNF-alpha-release of AM which was decreased after an additional NO2 exposure (p <0.031). The relative cytotoxicity of the NO2-particle/fibre co-exposure was higher than the particle or fibre induced cytotoxicity, but mostly <10%. Therefore it is concluded that particle or fibre exposure may result in an increase in proinflammatory cytokine release by AM, which may be decreased by toxic NO2 due to the oxidative potential (e.g. lipidperoxydation) of this irritant gas. Particle, asbestos fibre and irritant gas exposure may induce airway and pulmonary injury by the activation of AM and consecutive proinflammatory cytokine release.
Subject(s)
Air Pollutants/pharmacology , Asbestos, Serpentine/pharmacology , Cytokines/biosynthesis , Gene Expression Regulation/drug effects , Irritants/pharmacology , Macrophages, Alveolar/drug effects , Nitrogen Dioxide/pharmacology , RNA, Messenger/biosynthesis , Titanium/pharmacology , Aged , Air Pollutants/toxicity , Asthma/etiology , Bronchial Neoplasms/pathology , Bronchoalveolar Lavage Fluid , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Small Cell/pathology , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Drug Synergism , Female , Humans , Inflammation , Interleukin-1/biosynthesis , Interleukin-1/genetics , Interleukin-1/metabolism , Interleukin-6/biosynthesis , Interleukin-6/genetics , Interleukin-6/metabolism , Interleukin-8/biosynthesis , Interleukin-8/genetics , Interleukin-8/metabolism , Irritants/toxicity , Lung Neoplasms , Macrophages, Alveolar/metabolism , Male , Middle Aged , Nitrogen Dioxide/toxicity , Particle Size , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
It has been verified that asbestos induce neoplastic transformation of several rodent cell lines in vitro. Very little information is available, however, on asbestos-induced human cell line transformation and oncogene activation in the development of asbestos-induced cell transformation. This study was designed to investigate the induction of cell transformation and transcriptional changes of the c-myc oncogene by chrysotile asbestos fibers (CAF) in human embryo lung (HEL) cells. Surface-modified CAF soaked in aluminium citrate (AC), rare earth compounds (REC) or sodium selenite (SS) solutions were used to observe the effects of surface modification on chrysotile-induced cell transformation and transcriptional changes in the c-myc oncogene. The results showed that the natural CAF caused dose-dependent increases in transformed foci, along with increased transcription of the c-myc oncogene in HEL cells, while the pretreated CAF induced fewer transformed foci and less c-myc oncogene transcription than the natural CAF. The percent inhibition of equivalent AC, REC and SS were 38.9%, 50.0%, 33.3% (for transformation focus), 81.6%, 69.5%, 89.9% (for c-myc oncogene transcription), respectively. These results indicated that CAF may induce cell transformation and transcriptional changes and that the carcinogenicity of CAF might be reduced by pretreatment of CAF with the above-mentioned compounds.
Subject(s)
Asbestos, Serpentine/pharmacology , Carcinogens/pharmacology , Cell Transformation, Neoplastic/drug effects , Genes, myc , Lung/drug effects , Transcription, Genetic/drug effects , Humans , Lung/cytology , Lung/embryologyABSTRACT
We have studied a sample of commercial sepiolite and two samples of commercial vermiculite, which are clay minerals advised to replace asbestos. We have in vitro tested their abilities to produce reacting oxygen species (ROS) after they have been added to suspension of human polymorphonuclear leukocytes and bovine alveolar macrophages. The behaviour of sepiolite and vermiculite have been compared with those of asbestos fibres given by Unione Internationale contre le Cancer (UICC) and with kaolin and illite. Sepiolite was not able to induce ROS production, while vermiculite was able to induce a relevant ROS generation, even if the values were always lower than that obtained from chrysotile. Kaolin was able to generate a high ROS production.
Subject(s)
Aluminum Silicates/pharmacology , Macrophage Activation/drug effects , Macrophages, Alveolar/drug effects , Magnesium Silicates/pharmacology , Neutrophils/drug effects , Reactive Oxygen Species/metabolism , Respiratory Burst/drug effects , Adult , Animals , Asbestos/pharmacology , Asbestos, Crocidolite/pharmacology , Asbestos, Serpentine/pharmacology , Cattle , Cells, Cultured , Humans , Kaolin/pharmacology , Luminescent Measurements , Minerals/pharmacology , Neutrophils/metabolismABSTRACT
Different characteristics of the influence of dusts of chrysotile--asbestos, mordenite, erionite and klinoptilolite on the total generation of the active forms of oxygen by the alveolar macrophages and the generation of O2- and H2O2 by the granulocytes give the possibility to connect some of these characteristics with the peculiarity of biological action these dusts. It is discovered the most strong activating properties has the dust of chrysotile--asbestos, and the most cytotoxic effect has the dust of klinoptilolite.