RESUMEN
BACKGROUND: Certain multi-walled carbon nanotubes (MWCNTs) have been shown to elicit asbestos-like toxicological effects. To reduce needs for risk assessment it has been suggested that the physicochemical characteristics or reactivity of nanomaterials could be used to predict their hazard. Fibre-shape and ability to generate reactive oxygen species (ROS) are important indicators of high hazard materials. Asbestos is a known ROS generator, while MWCNTs may either produce or scavenge ROS. However, certain biomolecules, such as albumin - used as dispersants in nanomaterial preparation for toxicological testing in vivo and in vitro - may reduce the surface reactivity of nanomaterials. METHODS: Here, we investigated the effect of bovine serum albumin (BSA) and cell culture medium with and without BEAS 2B cells on radical formation/scavenging by five MWCNTs, Printex 90 carbon black, crocidolite asbestos, and glass wool, using electron spin resonance (ESR) spectroscopy and linked this to cytotoxic effects measured by trypan blue exclusion assay. In addition, the materials were characterized in the exposure medium (e.g. for hydrodynamic size-distribution and sedimentation rate). RESULTS: The test materials induced highly variable cytotoxic effects which could generally be related to the abundance and characteristics of agglomerates/aggregates and to the rate of sedimentation. All carbon nanomaterials were found to scavenge hydroxyl radicals (â¢OH) in at least one of the solutions tested. The effect of BSA was different among the materials. Two types of long, needle-like MWCNTs (average diameter >74 and 64.2 nm, average length 5.7 and 4.0 µm, respectively) induced, in addition to a scavenging effect, a dose-dependent formation of a unique, yet unidentified radical in both absence and presence of cells, which also coincided with cytotoxicity. CONCLUSIONS: Culture medium and BSA affects scavenging/production of â¢OH by MWCNTs, Printex 90 carbon black, asbestos and glass-wool. An unidentified radical is generated by two long, needle-like MWCNTs and these two CNTs were more cytotoxic than the other CNTs tested, suggesting that this radical could be related to the adverse effects of MWCNTs.
Asunto(s)
Células Epiteliales/metabolismo , Depuradores de Radicales Libres/metabolismo , Radicales Libres/metabolismo , Nanotubos de Carbono , Asbesto Crocidolita/farmacología , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Supervivencia Celular/efectos de los fármacos , Sistema Libre de Células , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Vidrio , Humanos , Luz , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Dispersión de Radiación , Albúmina Sérica Bovina/farmacología , Hollín/toxicidadRESUMEN
PURPOSE: Malignant mesothelioma is a devastating disease with a need for new treatment strategies. In the present study, we showed the importance of extracellular signal-regulated kinase 5 (ERK5) in malignant mesothelioma tumor growth and treatment. EXPERIMENTAL DESIGN: ERK5 as a target for malignant mesothelioma therapy was verified using mesothelial and mesothelioma cell lines as well as by xenograft severe combined immunodeficient (SCID) mouse models. RESULTS: We first showed that crocidolite asbestos activated ERK5 in LP9 cells and mesothelioma cell lines exhibit constitutive activation of ERK5. Addition of doxorubicin resulted in further activation of ERK5 in malignant mesothelioma cells. ERK5 silencing increased doxorubicin-induced cell death and doxorubicin retention in malignant mesothelioma cells. In addition, shERK5 malignant mesothelioma lines exhibited both attenuated colony formation on soft agar and invasion of malignant mesothelioma cells in vitro that could be related to modulation of gene expression linked to cell proliferation, apoptosis, migration/invasion, and drug resistance as shown by microarray analysis. Most importantly, injection of shERK5 malignant mesothelioma cell lines into SCID mice showed significant reduction in tumor growth using both subcutaneous and intraperitoneal models. Assessment of selected human cytokine profiles in peritoneal lavage fluid from intraperitoneal shERK5 and control tumor-bearing mice showed that ERK5 was critical in regulation of various proinflammatory (RANTES/CCL5, MCP-1) and angiogenesis-related (interleukin-8, VEGF) cytokines. Finally, use of doxorubicin and cisplatin in combination with ERK5 inhibition showed further reduction in tumor weight and volume in the intraperitoneal model of tumor growth. CONCLUSION: ERK5 inhibition in combination with chemotherapeutic drugs is a beneficial strategy for combination therapy in patients with malignant mesothelioma.
Asunto(s)
Mesotelioma/genética , Mesotelioma/terapia , Proteína Quinasa 7 Activada por Mitógenos/genética , Interferencia de ARN , Animales , Antineoplásicos/farmacología , Asbesto Crocidolita/farmacología , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cisplatino/farmacología , Terapia Combinada , Citocinas/genética , Citocinas/metabolismo , Doxorrubicina/farmacología , Activación Enzimática/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Mesotelioma/patología , Ratones , Ratones SCID , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Malignant mesothelioma is a rare cancer caused by exposure to asbestos. Current therapies have limited efficacy and the prognosis is dismal. A better understanding of the underlying mechanism of asbestos-induced malignant transformation will help to identify molecular markers that can be used for diagnosis, prognosis or therapeutic targets. OBJECTIVES: The objectives of this study are (1) to identify altered levels of proteins and phosphoproteins and (2) to establish the interactive network among those proteins in crocidolite-treated benign mesothelial cells and in malignant mesothelial cells. METHODS: Total cellular proteins were extracted from benign mesothelial cells, crocidolite-treated mesothelial cells and malignant mesothelial cells. The expression levels of 112 proteins and phosphoproteins were analyzed using a multiplex immunoblot-based assay followed by computational analysis (Protein Pathway Array). RESULTS: A total of 16 proteins/phosphoproteins (7 down-regulated and 9 up-regulated) were altered after exposure of benign mesothelial cells to crocidolite asbestos and the majority of them are involved in DNA damage repair and cell cycle regulation. In malignant mesothelial cells, 21 proteins/phosphoproteins (5 down-regulated and 16 up-regulated) were dysregulated and majority of them are involved in EGFR/ERK and PI3K/Akt pathways. Within the regulatory network affected by crocidolite, p53 and NF-κB complex are the most important regulators. There was substantial overlap in the regulatory networks between the asbestos-treated cells and malignant mesothelial cells. CONCLUSIONS: Asbestos exposure has extensive effects on regulatory pathways and networks. These altered proteins may be used in the future to identify those with a high risk for developing malignant mesothelioma and as targets for preventing this deadly malignancy.
Asunto(s)
Asbesto Crocidolita/farmacología , Células Epiteliales/metabolismo , Mesotelioma/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Línea Celular , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
Human malignant mesothelioma (HMM), which is strongly related to asbestos exposure, exhibits high resistance to many anticancer drugs. Asbestos fibre deposition in the lung may cause hypoxia and iron chelation at the fibre surface. Hypoxia-inducible factor (HIF)-1alpha, which is upregulated by a decreased availability of oxygen and iron, controls the expression of membrane transporters, such as P-glycoprotein (Pgp), which actively extrude the anticancer drugs. The present study aimed to assess whether asbestos may play a role in the induction of doxorubicin resistance in HMM cells through the activation of HIF-1alpha and an increased expression of Pgp. After 24-h incubation with crocidolite asbestos or with the iron chelator dexrazoxane, or under hypoxia, HMM cells were tested for HIF-1alpha activation, Pgp expression, accumulation of doxorubicin and sensitivity to its toxic effect. Crocidolite, dexrazoxane and hypoxia caused HIF-1alpha activation, Pgp overexpression and increased resistance to doxorubicin accumulation and toxicity. These effects were prevented by the co-incubation with the cell-permeating iron salt ferric nitrilotriacetate, which caused an increase of intracellular iron bioavailability, measured as increased activity of the iron regulatory protein-1. Crocidolite, dexrazoxane and hypoxia induce doxorubicin resistance in human malignant mesothelioma cells by increasing hypoxia-inducible factor-1alpha activity, through an iron-sensitive mechanism.
Asunto(s)
Amianto/toxicidad , Resistencia a Antineoplásicos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Mesotelioma/tratamiento farmacológico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Asbesto Crocidolita/farmacología , Línea Celular Tumoral , Doxorrubicina/farmacología , Humanos , Hipoxia , Hierro/metabolismo , Pulmón/patología , Razoxano/farmacologíaRESUMEN
The impact of crocidolite exposure on the health of former Wittenoom miners and millers (largely male) has been well documented. Less is known about the health outcomes of the 2,968 women and girls who lived (N = 2,552) and worked (N = 416) in the blue asbestos milling and mining town of Wittenoom between 1943 and 1992. Quantitative exposure measurements were derived from dust studies undertaken over the lifetime of the mine and mill and the township. Incident cancers were obtained from the Western Australian (WA) Cancer Registry and the National Cancer Clearing House. Standardized incidence ratios (SIRS) compared Wittenoom females with the WA female population. Exposure-response relationships were examined using a matched case-control study design. There were (47) mesothelioma and (55) lung cancer cases among the 437 cancers in the Wittenoom females over the period 1960-2005. When compared to the WA female population, Wittenoom women and girls had higher rates of mesothelioma and possibly lung cancer. Mesothelioma incidence rates are increasing with the incidence rate of 193 per 100,000 in the period 2000-2005 being more than double that for the period 1995-1999 at 84 per 100,000. A significant exposure-response relationship was present for mesothelioma, but not for lung cancer. Forty years after the asbestos mine and mill at Wittenoom were closed, there is a high toll from cancer among the former female residents of the town and company workers.
Asunto(s)
Asbesto Crocidolita/farmacología , Exposición a Riesgos Ambientales , Neoplasias Pulmonares/epidemiología , Mesotelioma/epidemiología , Exposición Profesional , Adenocarcinoma/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Niño , Estudios de Cohortes , Femenino , Humanos , Incidencia , Persona de Mediana Edad , Minería , Encuestas y Cuestionarios , Australia Occidental/epidemiologíaRESUMEN
Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways.
Asunto(s)
Apoptosis/efectos de los fármacos , Asbesto Crocidolita/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Pulmón/citología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Butionina Sulfoximina/farmacología , Caspasas/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Compuestos Férricos/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Pulmón/efectos de los fármacos , Pulmón/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Laser capture microdissection (LCM) enables the removal of discrete microstructures or cell types from properly prepared histological sections. Extraction of RNA from microdissected tissue followed by quantitative reverse transcriptase-polymerase chain (QRT-PCR) reaction permits the analysis of cell-type or microstructure-specific gene expression changes that occur in response to various stimuli in the environment. In our lab, the combination of LCM and QRT-PCR has proven very useful in the determination of the in vivo gene expression changes that occur in bronchiolar epithelium in response to inhalation of crocidolite asbestos. A detailed description of the preparation of cDNA from bronchiolar epithelial cells obtained by LCM is described in this work.
Asunto(s)
Asbesto Crocidolita/farmacología , Bronquios/citología , Células Epiteliales/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Rayos Láser , Microdisección , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bronquios/fisiología , Células Epiteliales/citología , Humanos , Microdisección/instrumentación , Microdisección/métodos , Micromanipulación/instrumentación , Micromanipulación/métodos , ARN/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
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.
Asunto(s)
Amianto/efectos adversos , Saccharomyces cerevisiae/efectos de los fármacos , Amianto/farmacología , Asbesto Crocidolita/efectos adversos , Asbesto Crocidolita/farmacología , Asbestos Serpentinas/efectos adversos , Asbestos Serpentinas/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cultivo , Fermentación , Microscopía Electrónica de Rastreo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/ultraestructura , Temperatura , Factores de TiempoRESUMEN
Treatment of Beas-2B airway epithelial cells with crocidolite asbestos induced tissue factor (TF) mRNA and TF-dependent procoagulant activity. The mitogen-activated protein kinase (MAPK) inhibitors UO126 and SB203850 decreased TF expression in both naive and crocidolite-treated Beas-2B cells to the same extent. Calphostin, an inhibitor of classical and novel protein kinase C (PKC) isotypes, reduced TF mRNA in both intact and crocidolite-treated Beas-2B cells by about 50%. Conversely, the phosphatidylinositol 3-kinase (PI3 kinase) inhibitor LY294002 and a selective PKCzeta inhibitory peptide decreased TF mRNA expression in asbestos-treated cells to a greater extent than in naive cells, suggesting that signaling via this pathway contributes to asbestos-induced TF expression. These results demonstrate that crocidolite asbestos induces TF expression by Beas-2B cells and suggest that the process involves the PI3 kinase-PKCzeta signaling pathway, representing a newly recognized potential mechanism by which asbestos may contribute to lung remodeling.
Asunto(s)
Asbesto Crocidolita/farmacología , Carcinógenos/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Proteína Quinasa C/fisiología , Tromboplastina/efectos de los fármacos , Células Cultivadas , Factor X/fisiología , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Mucosa Respiratoria/fisiología , Transducción de Señal , Tromboplastina/fisiologíaRESUMEN
OBJECTIVE: To determine the effects of buthionine sulfoximine (BSO) and free radical scavenger, dimethyl sulfoxide (DMSO), on mutation frequency and the formation of 8-hydroxydeoxyganosine (8-OHdG) induced by crocidolite fibers in human-hamster hybrid (A(L)) cells. METHODS: The cytotoxicity and mutagenicity were determined by the formation of colonies. 8-OHdG was examined by immunoperoxidase staining. Non-protein sulfhydryl (NPSH) compound was assayed by modified Tietze's method. RESULTS: The level of NPSH in A(L) cell pretreated with 25 micro mol/L of BSO was decreased to 2 nmol/10(7) cells, only 5% of the control after 24 h. The mutation frequency of CD59 gene of A(L) cell in crocidolite alone treated group was 208 +/- 18 while that in BSO pretreated group (397 +/- 55) was about twice the former (P < 0.05). The mutation frequency of CD59 gene in the group treated with crocidolite and in the presence of DMSO (57 +/- 8) was 72.6% less than that in crocidolite alone treated group. Crocidolite fibers induced a dose-effect relationship in the formation of 8-OHdG in A(L) cells (y = 150 + 20x, r = 0.9621). The level of 8-OHdG in cells was 289 +/- 6 at the dose of 6 micro g/cm(2) crocidolite, which was about twice the control group (137 +/- 9). In the presence of DMSO, 8-OHdG level decreased to 170 +/- 3 at the same dose of crocidolite. CONCLUSION: Free radicals are the important inducer of mutagenesis and DNA damage in A(L) cells caused by crocidolite, which has dose-effect relationship.
Asunto(s)
Asbesto Crocidolita/farmacología , Antígenos CD59/genética , ADN/efectos de los fármacos , Desoxiguanosina/análogos & derivados , Mutación , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Butionina Sulfoximina/farmacología , Cricetinae , ADN/genética , Daño del ADN , Desoxiguanosina/metabolismo , Dimetilsulfóxido/farmacología , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Humanos , Células Híbridas , Técnicas para InmunoenzimasRESUMEN
Asbestos fibers are biopersistent particles that are capable of stimulating chronic inflammatory responses in the pleura of exposed individuals. Exposure of pleural mesothelial cells, the progenitor cell of malignant mesothelioma, to asbestos induces an array of cellular responses. The present studies investigated whether the p38 mitogen-activated protein kinase cascade was induced under asbestos-exposed conditions. p38 plays a vital role in the response to stressful stimuli and enables the cell to enter an inflammatory state characterized by cytokine production. Western blot and in vitro kinase assays showed increases in dual phosphorylation and actual activity of p38 after exposure to fibrous and nonfibrous (milled) crocidolite; in contrast, polystyrene beads and iron (III) oxide had no such effects. In common with other asbestos-induced events, this was shown to be an oxidative stress-sensitive effect, inasmuch as preincubation with N-acetyl-L-cysteine or -tocopherol (vitamin E) ameliorated the effect. The present studies show that p38 activity is important for crocidolite-induced activator protein-1 DNA binding, inasmuch as an inhibitor of p38, SB-203580, reduced this activity. Crocidolite-induced cytotoxicity was also reduced with SB-203580, indicating a role for p38 in asbestos-mediated cell death. Our studies suggest that p38 activity could be a crucial factor in the chronic immune response elicited by asbestos and may represent a target for future pharmacological intervention.
Asunto(s)
Asbesto Crocidolita/farmacología , Carcinógenos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/fisiología , Pleura/efectos de los fármacos , Pleura/enzimología , Animales , Línea Celular , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Epitelio , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Pleura/citología , Piridinas/farmacología , Ratas , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Nitration of proteins by peroxynitrite (ONOO-) has been shown to critically alter protein function in vitro. We have shown previously that asbestos inhalation induced nitrotyrosine formation, a marker of ONOO- production, in the rat lung. To determine whether asbestos-induced protein nitration may affect mitogen-activated protein kinase (MAPK) signaling pathways, lung lysates from crocidolite and chrysotile asbestos-exposed rats and from sham-exposed rats were immunoprecipitated with anti-nitrotyrosine antibody, and captured proteins were subjected to Western blotting with anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies. Both types of asbestos inhalation induced significantly greater phosphorylation of ERK1/2 in rat lung lysates than was noted after sham exposure. Phosphorylated ERK proteins co-immunoprecipitated with nitrotyrosine. Moreover, in MAPK functional assays using Elk-1 substrate, immunoprecipitated phospho-ERK1/2 in lung lysates from both crocidolite-exposed and chrysotile-exposed rats demonstrated significantly greater phosphorylation of Elk-1 than was noted after sham exposure. Asbestos inhalation also induced ERK phosphorylation in bronchoalveolar lavage cells. Lung sections from rats exposed to crocidolite or chrysotile (but not from sham-exposed rats nor from rats exposed to "inert" carbonyl iron particles) demonstrated strong immunoreactivity for nitrotyrosine and phospho-ERK1/2 in alveolar macrophages and bronchiolar epithelium. These findings suggest that asbestos fibers may activate the ERK signaling pathway by generating ONOO- or other nitrating species that induce tyrosine nitration and phosphorylation of critical signaling molecules.
Asunto(s)
Asbesto Crocidolita/farmacología , Asbestos Serpentinas/farmacología , Pulmón/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitratos/metabolismo , Tirosina/metabolismo , Animales , Asbesto Crocidolita/administración & dosificación , Asbestos Serpentinas/administración & dosificación , Activación Enzimática , Inmunohistoquímica , Exposición por Inhalación , Pulmón/enzimología , Masculino , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Pruebas de Precipitina , Ratas , Ratas Endogámicas F344RESUMEN
The cytotoxicity of asbestos has been related to its ability to increase the production of reactive oxygen species (ROS), via the iron-catalyzed reduction of oxygen and/or the activation of NADPH oxidase. The pentose phosphate pathway (PPP) is generally activated by the cell exposure to oxidant molecules. Contrary to our expectations, asbestos (crocidolite) fibers caused a dose- and time-dependent inhibition of PPP and decreased its activation by an oxidative stress in human lung epithelial cells A549. In parallel, the intracellular activity of the PPP rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD), was significantly diminished by crocidolite exposure. This inhibition was selective, as the activity of other PPP and glycolysis enzymes was not modified, and was not attributable to a decreased expression of G6PD. On the opposite, the incubation with glass fibers MMVF10 did not modify PPP and G6PD activity. PPP and G6PD inhibition did not correlate with the increased nitric oxide (NO) production elicited by crocidolite in A549 cells. Experiments with the purified enzyme suggest that crocidolite inhibits G6PD by directly interacting with the protein. We propose here a new mechanism of asbestos-evoked oxidative stress, wherein fibers increase the intracellular ROS levels also by inhibiting the main antioxidant pathway of the cell.
Asunto(s)
Asbesto Crocidolita/farmacología , Células Epiteliales/efectos de los fármacos , Glucosafosfato Deshidrogenasa/metabolismo , Pulmón/efectos de los fármacos , Vía de Pentosa Fosfato/efectos de los fármacos , Western Blotting , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Células Cultivadas/metabolismo , Cartilla de ADN/química , Células Epiteliales/enzimología , Eritrocitos/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Pulmón/enzimología , Nitritos/metabolismo , Oxidación-Reducción , Vía de Pentosa Fosfato/fisiología , Fosfogluconato Deshidrogenasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Crocidolite fibers stimulated nitric oxide synthase (NOS) activity and expression in glial and alveolar murine macrophages: this effect was inhibited by iron supplementation and enhanced by iron chelation. We suggest that in these cells crocidolite stimulates NOS expression by decreasing the iron bioavailability and activating an iron-sensitive transcription factor.
Asunto(s)
Asbesto Crocidolita/farmacología , Deferoxamina/farmacología , Compuestos Férricos/farmacología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/biosíntesis , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/farmacología , Animales , Línea Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Cinética , Ratones , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Óxido Nítrico Sintasa de Tipo IIRESUMEN
We showed previously that both crocidolite and chrysotile asbestos inhalation induced a persistent macrophage inflammatory response within the pleural space of the rat. We postulated that the stimulus for pleural macrophage recruitment after asbestos exposure was the induction of monocyte chemoattractant protein-1 (MCP-1) synthesis by pleural mesothelial cells. To test this hypothesis, rat pleural mesothelial cells (RPMC) were cultured with or without chrysotile or crocidolite asbestos fibers (8 micrograms/cm2) in the presence (50 ng/mL) or absence of either tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). MCP-1 mRNA expression was assessed by RT-PCR in RPMC cultured for 2 to 24 hours, and MCP-1 protein secretion was measured by ELISA in conditioned medium from 24-hour and 48-hour cultures. Crocidolite and chrysotile fibers induced MCP-1 mRNA expression in RPMC which was maximal after 12 hours in the absence of cytokines, but which peaked after 2 hours when RPMC were challenged with asbestos + TNF-alpha or IL-1 beta. Both types of asbestos also significantly increased MCP-1 protein secretion after 24 and 48 hours (P < .0001), an effect that was potentiated by cytokine stimulation. Rats exposed by inhalation to either chrysotile or crocidolite asbestos fibers also had greater amounts of MCP-1 protein in their pleural lavage fluid than did sham-exposed rats. These findings suggest that MCP-1 secretion by RPMC may have a role in the initiation and/or potentiation of asbestos-induced pleural injury.
Asunto(s)
Amianto/farmacología , Quimiocina CCL2/metabolismo , Células Epiteliales/metabolismo , Pleura/citología , Animales , Asbesto Crocidolita/farmacología , Asbestos Serpentinas/farmacología , Técnicas de Cultivo de Célula , Quimiocina CCL2/genética , Citocinas/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Exposición por Inhalación , Masculino , Fibras Minerales , Pleura/química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344RESUMEN
Apoptosis of mesothelial cells has been demonstrated in vitro but not in vivo. To identify apoptotic pleural cells as mesothelial, we used cytokeratin as a marker and found a striking spheroid, aggregated appearance of cytokeratin in apparently apoptotic mesothelial cells. In in vitro studies, we found that the aggregated cytokeratin pattern correlated with apoptosis in primary mesothelial cells from mice, rabbits, and humans and was not seen with necrosis. In in vivo studies in mice, we then used this cytokeratin pattern to identify and quantitate apoptotic mesothelial cells. Apoptotic mesothelial cells were best harvested by pleural lavage, indicating that they were loosely adherent or nonadherent. Instillation of RPMI 1640 medium or wollastonite for 24 h induced apoptosis in 0.1 +/- 0. 1 (SE) and 1.0 +/- 0.7%, respectively, of all mesothelial cells recovered, whereas instillation of known apoptotic stimuli, crocidolite asbestos (25 microg) for 24 h or actinomycin D plus murine tumor necrosis factor-alpha for 12 h, induced apoptosis in 5. 1 +/- 0.5 and 22.4 +/- 4.5%, respectively (significantly greater than in control experiments, P < 0.05). By analysis of cytokeratin staining, mesothelial cell apoptosis has been confirmed in vivo.
Asunto(s)
Apoptosis/fisiología , Queratinas/metabolismo , Pleura/fisiología , Animales , Asbesto Crocidolita/farmacología , Células Cultivadas , Dactinomicina/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Pleura/citología , Pleura/efectos de los fármacos , Pleura/metabolismo , Conejos , Distribución Tisular/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Inhalation of asbestos is associated with pathologic changes in the pleural space, including pleural thickening, pleural plaques, and mesothelioma. These processes are characterized by altered local proteolysis, cellular proliferation, and cell migration, suggesting that the urokinase-type plasminogen activator receptor (uPAR) could be involved in the pathogenesis of asbestos-induced pleural disease. We hypothesized that mesothelial cell uPAR expression is induced by exposure to asbestos. To test this hypothesis, we used complementary techniques in rabbit and human mesothelial cells to determine whether uPAR expression is altered by exposure to asbestos. uPAR expression was induced by chrysotile and crocidolite asbestos, but not by wollastonite, as indicated by binding of radiolabeled urokinase-type plasminogen activator (uPA) to rabbit or human mesothelial cells. uPA was not induced by fiber exposure. Exposure to exogenous uPA increased uPA activity of cells exposed to wollastonite but not asbestos-treated MeT5A cells. uPAR expression increased further when asbestos was preincubated with vitronectin (VN) or serum. Increases in uPAR expression were confirmed by binding of uPA to uPAR in cell membrane preparations and immunofluorescent staining of uPAR at the cell surface, and were associated with increases in steady-state uPAR messenger RNA. Mesothelial cell uPAR expression was also induced by media from monocytes cultured with asbestos incubated with VN and serum. By antibody neutralization, the latter effect appeared to be in part mediated by transforming growth factor-beta. We found that asbestos increases uPAR at the surface of rabbit and human mesothelial cells, suggesting that altered expression of this receptor could be involved in asbestos-induced remodeling of the pleural mesothelium.
Asunto(s)
Asbesto Crocidolita/farmacología , Asbestos Serpentinas/farmacología , Células Epiteliales/efectos de los fármacos , Receptores de Superficie Celular/biosíntesis , Animales , Compuestos de Calcio/farmacología , Membrana Celular/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Células Epiteliales/metabolismo , Femenino , Humanos , Inmunohistoquímica , Radioisótopos de Yodo , Monocitos/efectos de los fármacos , Activadores Plasminogénicos/biosíntesis , Pleura/citología , ARN Mensajero/biosíntesis , Conejos , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Silicatos/farmacología , Organismos Libres de Patógenos Específicos , Regulación hacia Arriba/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c-fos/c-jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR (P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c-fos but not of c-jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c-fos and the development of apoptosis. The ability to block asbestos-induced elevations in c-fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.
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Apoptosis/fisiología , Asbesto Crocidolita/farmacología , Receptores ErbB/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Células Cultivadas , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/metabolismo , Receptores ErbB/genética , Receptores ErbB/fisiología , Regulación de la Expresión Génica/fisiología , Homeostasis/efectos de los fármacos , Fosforilación , Pleura/citología , Pleura/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Transducción de Señal/fisiologíaRESUMEN
This study was designed to assess the effects of in vitro and in vivo asbestos exposure on the adhesion of rat pleural leukocytes (RPLs) labeled with the fluorochrome calcein AM to rat pleural mesothelial cells (RPMCs). Exposure of RPMCs for 24 h to either crocidolite or chrysotile fibers (1.25-10 microgram/cm(2)) increased the adhesion of RPLs to RPMCs in a dose-dependent fashion, an effect that was potentiated by interleukin-1beta. These findings were not observed with nonfibrogenic carbonyl iron particles. Crocidolite and chrysotile plus interleukin-1beta also upregulated vascular cell adhesion molecule-1 mRNA and protein expression in RPMCs, and the binding of RPL to asbestos-treated RPMCs was abrogated by anti-vascular cell adhesion molecule-1 antibody. PRLs exposed by intermittent inhalation to crocidolite for 2 wk manifested significantly greater binding to RPMCs than did RPLs from sham-exposed animals. The ability of asbestos fibers to upregulate RPL adhesion to RPMCs may play a role in the induction and/or potentiation of asbestos-induced pleural injury.
Asunto(s)
Amianto/farmacología , Leucocitos/fisiología , Pleura/fisiología , Molécula 1 de Adhesión Celular Vascular/fisiología , Administración por Inhalación , Animales , Asbesto Crocidolita/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/fisiología , Molécula 1 de Adhesión Intercelular/fisiología , Masculino , Óxido Nítrico/biosíntesis , Óxido Nítrico/fisiología , Pleura/citología , Ratas , Ratas Endogámicas F344RESUMEN
Activation of activator protein (AP-1) by crocidolite asbestos was examined in vitro in a JB6 P+ cell line stably transfected with AP-1-luciferase reporter plasmid and in vivo using AP-1-luciferase reporter transgenic mice. In in vitro studies, crocidolite asbestos caused a dose- and time-dependent induction of AP-1 activation in cultured JB6 cells. The elevated AP-1 activity persisted for at least 48 h. Crocidolite asbestos also induced AP-1 transactivation in the pulmonary and bronchial tissues of transgenic mice. AP-1 activation was observed at 2 days after intratracheal instillation of the mice with asbestos. At 3 days postexposure, AP-1 activation was elevated 10-fold in the lung tissue and 22-fold in bronchiolar tissue as compared with their controls. The induction of AP-1 activity by asbestos appeared to be mediated through the activation of mitogen-activated protein kinase family members, including extracellular signal-regulating protein kinase, Erk1 and Erk2. Aspirin inhibited asbestos-induced AP-1 activity in JB6 cells. Pretreatment of the mice with aspirin also inhibited asbestos-induced AP-1 activation in bronchiolar tissue. The data suggest that further investigation of the role of AP-1 activation in asbestos-induced cell proliferation and carcinogenesis is warranted. In addition, investigation of the potential therapeutic benefits of aspirin in the prevention/amelioration of asbestos-induced cancer is justified.