RESUMEN
Human exposures to asbestiform elongate mineral particles (EMP) may lead to diffuse fibrosis, lung cancer, malignant mesothelioma and autoimmune diseases. Cleavage fragments (CF) are chemically identical to asbestiform varieties (or habits) of the parent mineral, but no consensus exists on whether to treat them as asbestos from toxicological and regulatory standpoints. Alveolar macrophages (AM) are the first responders to inhaled particulates, participating in clearance and activating other resident and recruited immunocompetent cells, impacting the long-term outcomes. In this study we address how EMP of asbestiform versus non-asbestiform habit affect AM responses. Max Planck Institute (MPI) cells, a non-transformed mouse line that has an AM phenotype and genotype, were treated with mass-, surface area- (s.a.), and particle number- (p.n.) equivalent concentrations of respirable asbestiform and non-asbestiform riebeckite/tremolite EMP for 24 h. Cytotoxicity, cytokines secretion and transcriptional changes were evaluated. At the equal mass, asbestiform EMP were more cytotoxic, however EMP of both habits induced similar LDH leakage and decrease in viability at s.a. and p.n. equivalent doses. DNA damage assessment and cell cycle analysis revealed differences in the modes of cell death between asbestos and respective CF. There was an increase in chemokines, but not pro-inflammatory cytokines after all EMP treatments. Principal component analysis of the cytokine secretion showed close clustering for the s.a. and p.n. equivalent treatments. There were mineral- and habit-specific patterns of gene expression dysregulation at s.a. equivalent doses. Our study reveals the critical nature of EMP morphometric parameters for exposure assessment and dosing approaches used in toxicity studies.
Asunto(s)
Amianto/efectos adversos , Secreciones Corporales/efectos de los fármacos , Citocinas/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Minerales/efectos adversos , Transcripción Genética/efectos de los fármacos , Contaminantes Ocupacionales del Aire/efectos adversos , Animales , Asbestos Anfíboles/efectos adversos , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/metabolismo , Células Cultivadas , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/metabolismo , Macrófagos Alveolares/metabolismo , Mesotelioma Maligno/inducido químicamente , Mesotelioma Maligno/metabolismo , Ratones , Ratones Endogámicos C57BL , Fibras Minerales/efectos adversos , Exposición Profesional/efectos adversos , Tamaño de la Partícula , Material Particulado/efectos adversosRESUMEN
Cellulose nanocrystals (CNC), also known as nanowhiskers, have recently gained much attention due to their biodegradable nature, advantageous chemical and mechanical properties, economic value and renewability thus making them attractive for a wide range of applications. However, before these materials can be considered for potential uses, investigation of their toxicity is prudent. Although CNC exposures are associated with pulmonary inflammation and damage as well as oxidative stress responses and genotoxicity in vivo, studies evaluating cell transformation or tumorigenic potential of CNC's were not previously conducted. In this study, we aimed to assess the neoplastic-like transformation potential of two forms of CNC derived from wood (powder and gel) in human pulmonary epithelial cells (BEAS-2B) in comparison to fibrous tremolite (TF), known to induce lung cancer. Short-term exposure to CNC or TF induced intracellular ROS increase and DNA damage while long-term exposure resulted in neoplastic-like transformation demonstrated by increased cell proliferation, anchorage-independent growth, migration and invasion. The increased proliferative responses were also in-agreement with observed levels of pro-inflammatory cytokines. Based on the hierarchical clustering analysis (HCA) of the inflammatory cytokine responses, CNC powder was segregated from the control and CNC-gel samples. This suggests that CNC may have the ability to influence neoplastic-like transformation events in pulmonary epithelial cells and that such effects are dependent on the type/form of CNC. Further studies focusing on determining and understanding molecular mechanisms underlying potential CNC cell transformation events and their likelihood to induce tumorigenic effects in vivo are highly warranted.
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Celulosa/toxicidad , Nanopartículas/toxicidad , Celulosa/química , Células Epiteliales/efectos de los fármacos , Humanos , Estudios Longitudinales , Pulmón/efectos de los fármacos , Nanopartículas/química , Estrés Oxidativo/efectos de los fármacos , Pruebas de Toxicidad Crónica , MaderaRESUMEN
Local inflammatory response in the lungs and fi brogenic potential of multi-walled carbon nanotubes were studied in an acute aspiration experiment in mice. The doses were chosen based on the concentration of nanotubes in the air at a workplace of the company-producer. ELISA, fl ow cytometry, enhanced darkfield microscopy, and histological examination showed that multi-walled carbon nanotubes induced local inflammation, oxidative stress, and connective tissue growth (fibrosis). Serum levels of TGF-ß1 and osteopontin proteins can serve as potential exposure biomarkers.
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Fibrosis/inmunología , Nanotubos de Carbono/efectos adversos , Animales , Lavado Broncoalveolar , Fibrosis/inducido químicamente , Inflamación/inducido químicamente , Inflamación/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Osteopontina/sangre , Estrés Oxidativo/efectos de los fármacos , Factor de Crecimiento Transformador beta1/sangreRESUMEN
Nanomaterials are being utilized in an increasing variety of manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNTs) have found numerous applications in the electronics, aerospace, chemical, polymer, and pharmaceutical industries. Previously, we have reported that pharyngeal exposure of C57BL/6 mice to SWCNTs caused dose-dependent formation of granulomatous bronchial interstitial pneumonia, fibrosis, oxidative stress, acute inflammatory/cytokine responses, and a decrease in pulmonary function. In the current study, we used electron spin resonance (ESR) to directly assess whether exposure to respirable SWCNTs caused formation of free radicals in the lungs and in two distant organs, the heart and liver. Here we report that exposure to partially purified SWCNTs (HiPco technique, Carbon Nanotechnologies, Inc., Houston, TX, USA) resulted in the augmentation of oxidative stress as evidenced by ESR detection of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone spin-trapped carbon-centered lipid-derived radicals recorded shortly after the treatment. This was accompanied by a significant depletion of antioxidants and elevated biomarkers of inflammation presented by recruitment of inflammatory cells and an increase in proinflammatory cytokines in the lungs, as well as development of multifocal granulomatous pneumonia, interstitial fibrosis, and suppressed pulmonary function. Moreover, pulmonary exposure to SWCNTs also caused the formation of carbon-centered lipid-derived radicals in the heart and liver at later time points (day 7 postexposure). Additionally, SWCNTs induced a significant accumulation of oxidatively modified proteins, increase in lipid peroxidation products, depletion of antioxidants, and inflammatory response in both the heart and the liver. Furthermore, the iron chelator deferoxamine noticeably reduced lung inflammation and oxidative stress, indicating an important role for metal-catalyzed species in lung injury caused by SWCNTs. Overall, we provide direct evidence that lipid-derived free radicals are a critical contributor to tissue damage induced by SWCNTs not only in the lungs, but also in distant organs.
Asunto(s)
Deferoxamina/farmacología , Radicales Libres/metabolismo , Pulmón/patología , Nanotubos de Carbono/toxicidad , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Líquido del Lavado Bronquioalveolar/química , Citocinas/biosíntesis , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Fibrosis/patología , Corazón , Inflamación/patología , Metabolismo de los Lípidos , Lípidos , Hígado/metabolismo , Cirrosis Hepática/patología , Pulmón/metabolismo , Lesión Pulmonar/patología , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Oxidación-Reducción , Neumonía/patología , Pruebas de Función RespiratoriaRESUMEN
Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 µg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 µg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.
Asunto(s)
Mitosis/efectos de los fármacos , Nanotubos de Carbono/toxicidad , Mucosa Respiratoria/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Aneuploidia , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Mucosa Respiratoria/citologíaRESUMEN
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 predominantly 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.
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Amianto/toxicidad , Supervivencia Celular/genética , Fibroblastos/fisiología , Nanotubos de Carbono/toxicidad , Animales , Amianto/efectos adversos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Cricetulus , Fibroblastos/efectos de los fármacos , Humanos , Pruebas de Mutagenicidad/métodos , Nanotubos de Carbono/efectos adversosRESUMEN
Hard metal or cemented carbide consists of a mixture of tungsten carbide (WC) (85%) and metallic cobalt (Co) (5-15%). WC-Co is considered to be potentially carcinogenic to humans. However, no comparison of the adverse effects of nano-sized WC-Co particles is available to date. In the present study, we compared the ability of nano- and fine-sized WC-Co particles to form free radicals and propensity to activate the transcription factors, AP-1 and NF-kappaB, along with stimulation of mitogen-activated protein kinase (MAPK) signaling pathways in a mouse epidermal cell line (JB6 P(+)). Our results demonstrated that nano-WC-Co generated a higher level of hydroxyl radicals, induced greater oxidative stress, as evidenced by a decrease of GSH levels, and caused faster JB6 P(+) cell growth/proliferation than observed after exposure of cells to fine WC-Co. In addition, nano-WC-Co activated AP-1 and NF-kappaB more efficiently in JB6(+/+) cells as compared to fine WC-Co. Experiments using AP-1-luciferase reporter transgenic mice confirmed the activation of AP-1 by nano-WC-Co. Nano- and fine-sized WC-Co particles also stimulated MAPKs, including ERKs, p38, and JNKs with significantly higher potency of nano-WC-Co. Finally, co-incubation of the JB6(+/+) cells with N-acetyl-cysteine decreased AP-1 activation and phosphorylation of ERKs, p38 kinase, and JNKs, thus suggesting that oxidative stress is involved in WC-Co-induced toxicity and AP-1 activation.
Asunto(s)
Cobalto/toxicidad , Epidermis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Compuestos de Tungsteno/toxicidad , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática/efectos de los fármacos , Células Epidérmicas , Glutatión/metabolismo , Inmunohistoquímica , Indicadores y Reactivos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/biosíntesis , Nanopartículas , Tamaño de la Partícula , Compuestos de Sulfhidrilo/metabolismo , Factor de Transcripción AP-1/biosíntesisRESUMEN
Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus.
Asunto(s)
Aneuploidia , Nanotubos de Carbono , Línea Celular Transformada , Humanos , Hibridación Fluorescente in Situ , Tamaño de la PartículaRESUMEN
Nanotechnology is an emerging science involving manipulation of materials at the nanometer scale. There are several exciting prospects for the application of engineered nanomaterials in medicine. However, concerns over adverse and unanticipated effects on human health have also been raised. In fact, the same properties that make engineered nanomaterials attractive from a technological and biomedical perspective could also make these novel materials harmful to human health and the environment. Carbon nanotubes are cylinders of one or several coaxial graphite layer(s) with a diameter in the order of nanometers, and serve as an instructive example of the Janus-like properties of nanomaterials. Numerous in vitro and in vivo studies have shown that carbon nanotubes and/or associated contaminants or catalytic materials that arise during the production process may induce oxidative stress and prominent pulmonary inflammation. Recent studies also suggest some similarities between the pathogenic properties of multi-walled carbon nanotubes and those of asbestos fibers. On the other hand, carbon nanotubes can be readily functionalized and several studies on the use of carbon nanotubes as versatile excipients for drug delivery and imaging of disease processes have been reported, suggesting that carbon nanotubes may have a place in the armamentarium for treatment and monitoring of cancer, infection, and other disease conditions. Nanomedicine is an emerging field that holds great promise; however, close attention to safety issues is required to ensure that the opportunities that carbon nanotubes and other engineered nanoparticles offer can be translated into feasible and safe constructs for the treatment of human disease.
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Pulmón/efectos de los fármacos , Nanotecnología/métodos , Nanotubos de Carbono/química , Animales , Humanos , Pulmón/patología , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/fisiopatología , Mutágenos/toxicidad , Nanotecnología/legislación & jurisprudencia , Nanotubos de Carbono/toxicidadRESUMEN
Single-walled carbon nanotubes (SWCNT) have been introduced into a large number of new technologies and consumer products. The combination of their exceptional features with very broad applications raised concerns regarding their potential health effects. The prime target for SWCNT toxicity is believed to be the lung where exposure may occur through inhalation, particularly in occupational settings. Our previous work has demonstrated that SWCNT cause robust inflammatory responses in rodents with very early termination of the acute phase and rapid onset of chronic fibrosis. Timely elimination of polymorphonuclear neutrophils (PMNs) through apoptosis and their subsequent clearance by macrophages is a necessary stage in the resolution of pulmonary inflammation whereby NADPH oxidase contributes to control of apoptotic cell death and clearance of PMNs. Thus, we hypothesized that NADPH oxidase may be an important regulator of the transition from the acute inflammation to the chronic fibrotic stage in response to SWCNT. To experimentally address the hypothesis, we employed NADPH oxidase-deficient mice which lack the gp91(phox) subunit of the enzymatic complex. We found that NADPH oxidase null mice responded to SWCNT exposure with a marked accumulation of PMNs and elevated levels of apoptotic cells in the lungs, production of pro-inflammatory cytokines, decreased production of the anti-inflammatory and pro-fibrotic cytokine, TGF-beta, and significantly lower levels of collagen deposition, as compared to C57BL/6 control mice. These results demonstrate a role for NADPH oxidase-derived reactive oxygen species in determining course of pulmonary response to SWCNT.
Asunto(s)
Apoptosis/efectos de los fármacos , Pulmón/efectos de los fármacos , NADPH Oxidasas/metabolismo , Nanotubos de Carbono/toxicidad , Neutrófilos/efectos de los fármacos , Animales , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Fibrosis/etiología , Fibrosis/metabolismo , Inflamación/etiología , Inflamación/patología , Pulmón/patología , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/genética , Neutrófilos/metabolismo , Exposición Profesional/efectos adversos , Factor de Crecimiento Transformador beta/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Organic peroxides, widely used in the chemical and pharmaceutical industries, can act as skin tumor promoters and cause epidermal hyperplasia. They are also known to trigger free radical generation. The present study evaluated the effect of cumene hydroperoxide (Cum-OOH) on the induction of activator protein-1 (AP-1), which is linked to the expression of genes regulating cell proliferation, growth and transformation. Previously, we reported that topical exposure to Cum-OOH caused formation of free radicals and oxidative stress in the skin of vitamin E-deficient mice. The present study used JB6 P+ mouse epidermal cells and AP-1-luciferase reporter transgenic mice to identify whether exposure to Cum-OOH caused activation of AP-1, oxidative stress, depletion of antioxidants and tumor formation during two-stage carcinogenesis. In vitro studies found that exposure to Cum-OOH reduced the level of glutathione (GSH) in mouse epidermal cells (JB6 P+) and caused the induction of AP-1. Mice primed with dimethyl-benz[a]anthracene (DMBA) were topically exposed to Cum-OOH (82.6 micromol) or the positive control, 12-O-tetradecanoylphorbol-13-acetate (TPA, 17 nmol), twice weekly for 29 weeks. Activation of AP-1 in skin was detected as early as 2 weeks following Cum-OOH or TPA exposure. No AP-1 expression was found 19 weeks after initiation. Papilloma formation was observed in both the DMBA-TPA- and DMBA-Cum-OOH-exposed animals, whereas skin carcinomas were found only in the DMBA-Cum-OOH-treated mice. A greater accumulation of peroxidative products (thiobarbituric acid-reactive substances), inflammation and decreased levels of GSH and total antioxidant reserves were also observed in the skin of DMBA-Cum-OOH-exposed mice. These results suggest that Cum-OOH-induced carcinogenesis is accompanied by increased AP-1 activation and changes in antioxidant status.
Asunto(s)
Derivados del Benceno/toxicidad , Transformación Celular Neoplásica/efectos de los fármacos , Estrés Oxidativo , Papiloma/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Factor de Transcripción AP-1/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Ratones , Ratones Transgénicos , Oxidación-Reducción , Papiloma/inducido químicamente , Piel/efectos de los fármacos , Piel/metabolismo , Neoplasias Cutáneas/inducido químicamente , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
Single-walled carbon nanotubes (SWCNT), nano-cylinders with an extremely small diameter (1-2 nm) and high aspect ratio, have unique physico-chemical, electronic and mechanical properties and may exhibit unusual interactions with cells and tissues, thus necessitating studies of their toxicity and health effects. Manufactured SWCNT usually contain significant amounts of iron that may act as a catalyst of oxidative stress. Because macrophages are the primary responders to different particles that initiate and propagate inflammatory reactions and oxidative stress, we utilized two types of SWCNT: (1) iron-rich (non-purified) SWCNT (26 wt.% of iron) and (2) iron-stripped (purified) SWCNT (0.23 wt.% of iron) to study their interactions with RAW 264.7 macrophages. Ultrasonication resulted in predominantly well-dispersed and separated SWCNT strands as evidenced by scanning electron microscopy. Neither purified nor non-purified SWCNT were able to generate intracellular production of superoxide radicals or nitric oxide in RAW 264.7 macrophages as documented by flow-cytometry and fluorescence microscopy. SWCNT with different iron content displayed different redox activity in a cell-free model system as revealed by EPR-detectable formation of ascorbate radicals resulting from ascorbate oxidation. In the presence of zymosan-stimulated RAW 264.7 macrophages, non-purified iron-rich SWCNT were more effective in generating hydroxyl radicals (documented by EPR spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide, DMPO) than purified SWCNT. Similarly, EPR spin-trapping experiments in the presence of zymosan-stimulated RAW 264.7 macrophages showed that non-purified SWCNT more effectively converted superoxide radicals generated by xanthine oxidase/xanthine into hydroxyl radicals as compared to purified SWCNT. Iron-rich SWCNT caused significant loss of intracellular low molecular weight thiols (GSH) and accumulation of lipid hydroperoxides in both zymosan-and PMA-stimulated RAW 264.7 macrophages. Catalase was able to partially protect macrophages against SWCNT induced elevation of biomarkers of oxidative stress (enhancement of lipid peroxidation and GSH depletion). Thus, the presence of iron in SWCNT may be important in determining redox-dependent responses of macrophages.
Asunto(s)
Hierro , Macrófagos Alveolares/efectos de los fármacos , Nanotubos de Carbono/toxicidad , Estrés Oxidativo/efectos de los fármacos , Animales , Línea Celular , Citometría de Flujo , Hierro/química , Macrófagos Alveolares/metabolismo , Ratones , Microscopía Fluorescente , Nanotubos de Carbono/química , Óxido Nítrico/metabolismo , Detección de Spin , Superóxidos/metabolismoRESUMEN
Organic peroxides used in the chemical and pharmaceutical industries have a reputation for being potent skin tumor promoters and inducers of epidermal hyperplasia. Their ability to trigger free radical generation is critical for their carcinogenic properties. Short-term in vivo exposure of mouse skin to cumene hydroperoxide (Cum-OOH) causes severe oxidative stress and formation of spin-trapped radical adducts. The present study was designed to determine the effectiveness of Cum-OOH compared to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the induction of tumor promotion in the mouse skin, to identify the involvement of cyclooxygenase-2 (COX-2) in oxidative metabolism of Cum-OOH in keratinocytes, and to evaluate morphological changes and outcomes of oxidative stress in skin of SENCAR mice throughout a two-stage carcinogenesis protocol. Dimethyl-benz[a]anthracene (DMBA)-initiated mice were treated with Cum-OOH (32.8 micro mol) or TPA (8.5 nmol) twice weekly for 20 weeks to promote papilloma formation. Skin carcinoma formed only in DMBA/Cum-OOH-exposed mice. Higher levels of oxidative stress and inflammation (as indicated by the accumulation of peroxidative products, antioxidant depletion, and edema formation) were evident in the DMBA/Cum-OOH group compared to DMBA/TPA treated mice. Exposure of keratinocytes (HaCaT) to Cum-OOH for 18 h resulted in expression of COX-2 and increased levels of PGE(2). Inhibitors of COX-2 efficiently suppressed oxidative stress and enzyme expression in the cells treated with Cum-OOH. These results suggest that COX-2-dependent oxidative metabolism is at least partially involved in Cum-OOH-induced inflammatory responses and thus tumor promotion.
Asunto(s)
Antioxidantes/metabolismo , Derivados del Benceno/toxicidad , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/metabolismo , Animales , Línea Celular Tumoral , Ciclooxigenasa 2 , Dinoprostona/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Glutatión/metabolismo , Humanos , Inflamación/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Peroxidación de Lípido/efectos de los fármacos , Proteínas de la Membrana , Ratones , Ratones Endogámicos SENCAR , Estrés Oxidativo/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/metabolismo , Neoplasias Cutáneas/patología , Compuestos de Sulfhidrilo/metabolismo , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.
Asunto(s)
Ácido Ascórbico/metabolismo , Cromanos/metabolismo , Radicales Libres/metabolismo , Peroxidasa/metabolismo , Ácido Tióctico/análogos & derivados , Ácido Tióctico/metabolismo , Antioxidantes/metabolismo , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia por Spin del Electrón , Células HL-60 , Humanos , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción , Fenoles/metabolismo , Espectrofotometría , Ciclo del Sustrato/efectos de los fármacosRESUMEN
Organic peroxides are widely used in the chemical industry as initiators of oxidation for the production of polymers and fiber-reinforced plastics, in the manufacture of polyester resin coatings, and pharmaceuticals. Free radical production is considered to be one of the key factors contributing to skin tumor promotion by organic peroxides. In vitro experiments have demonstrated metal-catalyzed formation of alkoxyl, alkyl, and aryl radicals in keratinocytes incubated with cumene hydroperoxide. The present study investigated in vivo free radical generation in lipid extracts of mouse skin exposed to cumene hydroperoxide. The electron spin resonance (ESR) spin-trapping technique was used to detect the formation of alpha-phenyl-N-tert-butylnitrone (PBN) radical adducts, following intradermal injection of 180 mg/kg PBN. It was found that 30 min after topical exposure, cumene hydroperoxide (12 mmol/kg) induced free radical generation in the skin of female Balb/c mice kept for 10 weeks on vitamin E-deficient diets. In contrast, hardly discernible radical adducts were detected when cumene hydroperoxide was applied to the skin of mice fed a vitamin E-sufficient diet. Importantly, total antioxidant reserve and levels of GSH, ascorbate, and vitamin E decreased 34%, 46.5%. 27%, and 98%, respectively, after mice were kept for 10 weeks on vitamin E-deficient diet. PBN adducts detected by ESR in vitamin E-deficient mice provide direct evidence for in vivo free radical generation in the skin after exposure to cumene hydroperoxide.
Asunto(s)
Antioxidantes/metabolismo , Derivados del Benceno/toxicidad , Radicales Libres/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Piel/efectos de los fármacos , Deficiencia de Vitamina E/metabolismo , Administración Cutánea , Animales , Antioxidantes/análisis , Ácido Ascórbico/análisis , Derivados del Benceno/administración & dosificación , Biomarcadores/análisis , Óxidos N-Cíclicos , Femenino , Radicales Libres/análisis , Glutatión/análisis , Ratones , Ratones Endogámicos BALB C , Óxidos de Nitrógeno , Estrés Oxidativo/fisiología , Piel/metabolismo , Marcadores de Spin , Detección de Spin , Compuestos de Sulfhidrilo/análisis , Vitamina E/análisis , Vitamina E/metabolismoRESUMEN
We used myeloperoxidase-containing HL-60 cells to generate phenoxyl radicals from nontoxic concentrations of a vitamin E homologue, 2,2, 5,7,8-pentamethyl-6-hydroxychromane (PMC) to test whether these radicals can induce oxidative stress in a physiological intracellular environment. In the presence of H(2)O(2), we were able to generate steady-state concentrations of PMC phenoxyl radicals readily detectable by EPR in viable HL-60 cells. In HL-60 cells pretreated with succinylacetone, an inhibitor of heme synthesis, a greater than 4-fold decrease in myeloperoxidase activity resulted in a dramatically decreased steady-state concentrations of PMC phenoxyl radicals hardly detectable in EPR spectra. We further conducted sensitive measurements of GSH oxidation and protein sulfhydryl oxidation as well as peroxidation in different classes of membrane phospholipids in HL-60 cells. We found that conditions compatible with the generation and detection of PMC phenoxyl radicals were not associated with either oxidation of GSH, protein SH-groups or phospholipid peroxidation. We conclude that PMC phenoxyl radicals do not induce oxidative stress under physiological conditions in contrast to their ability to cause lipid peroxidation in isolated lipoproteins in vitro.
Asunto(s)
Cromanos/farmacología , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Fenoles , Supervivencia Celular/efectos de los fármacos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Glutatión/metabolismo , Células HL-60 , Humanos , Cinética , Estrés Oxidativo/fisiologíaRESUMEN
Various types of cancer occur in peroxidase-rich target tissues of animals exposed to aryl alcohols and amines. Unlike biotransformation by cytochrome P450 enzymes, peroxidases activate most substrates by one-electron oxidation via radical intermediates. This work analyzed the peroxidase-dependent formation of phenoxyl radicals in HL-60 cells and its contribution to cytotoxicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of glutathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radicals was accompanied by rapid oxidation of glutathione and protein-thiols. Analysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation of protein SH-groups in HL-60 cells incubated in the presence of phenol/H2O2 that was inhibited by cyanide and azide. Additionally, cyanide- and azide-sensitive generation of EPR-detectable ascorbate radicals was observed during incubation of HL-60 cell homogenates in the presence of ascorbate and H2O2. Oxidation of thiols required addition of H2O2 and was inhibited by pretreatment of cells with the inhibitor of heme synthesis, succinylacetone. Radical-driven oxidation of thiols was accompanied by a trend toward increased content of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the DNA of HL-60 cells. Membrane phospholipids were also sensitive to radical-driven oxidation as evidenced by a sensitive fluorescence HPLC-assay based on metabolic labeling of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enhanced H2O2-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid-labeled lipids without addition of H2O2. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H2O2 and was slightly but consistently and significantly higher after exposure to H2O2/phenol. The clonogenicity of HL-60 cells decreased to the same extent after exposure to H2O2 or H2O2/phenol. Treatment of HL-60 cells with either H2O2 or H2O2/phenol at concentrations adequate for lipid peroxidation did not cause a detectable increase in chromosomal breaks. Detection of thiyl radicals as well as rapid oxidation of thiols and phospholipids in viable HL-60 cells provide strong evidence for redox cycling of phenol in this bone marrow-derived cell line.
Asunto(s)
Peroxidación de Lípido/efectos de los fármacos , Peroxidasa/metabolismo , Fenol/metabolismo , Fenol/toxicidad , Compuestos de Sulfhidrilo/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Apoptosis/efectos de los fármacos , Ácido Ascórbico/metabolismo , Daño del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Células HL-60 , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Oxidación-Reducción , Estrés Oxidativo , Fosfolípidos/metabolismo , Cianuro de Potasio/farmacología , Azida Sódica/farmacología , Especificidad por SustratoRESUMEN
Some studies have shown the potential relevance of the oxidation products of 4-hydroxytamoxifen (4OHTAM) in carcinogenesis. Other studies show 4OHTAM has antioxidant properties. We characterized the one-electron oxidative activation reactions of 4OHTAM and three other phenolics, 3-hydroxytamoxifen (3OHTAM), 1-(4-hydroxyphenyl)-1, 2-diphenylethene, and phenol (PhOH), catalyzed by myeloperoxidase (MPx), horseradish peroxidase (HRP), lactoperoxidase, mushroom tyrosinase, and nonenzymatic initiators in vitro under a variety of conditions and in cells. Differences in activation of the phenolics by the enzymes were directly compared using cis-parinaric acid (PnA)-loaded human serum albumin. All phenolics were substrates for the enzymes, but MPx only weakly activated 4OHTAM to its phenoxyl radical. In HL60 cells loaded metabolically with PnA so that effects on phospholipids could be monitored by HPLC with fluorescence detection, PhOH plus H2O2 caused massive oxidation across all phospholipid classes. 4OHTAM dose-dependently protected phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine against both H2O2-induced and normal metabolic oxidation. This suggested 4OHTAM is a poor substrate for intracellular MPx. In rat aorta smooth muscle cells loaded with PnA, 4OHTAM also protected against AMVN-induced peroxidation of those three phospholipids and sphingomyelin, whereas 3OHTAM did not. Spin trapping of glutathionyl radicals (GS*) with DMPO and quantifying the ESR-silent nitrone form of the GS-DMPO adduct by HPLC showed that neither 3OHTAM plus H2O2 nor 4OHTAM plus H2O2 caused a significant level of GSH oxidation with isolated MPx, nor did the latter in HL60 cells, whereas PhOH plus H2O2 was a potent source of GS* in both systems. Both 4OHTAM and 3OHTAM formed the nitrone adduct under cell-free conditions when activated with HRP. The data show that the substrate specificity of a given (myelo)peroxidase determines if a phenolic exerts pro- (through generation of reactive phenoxyl radicals) or antioxidant (through radical scavenging) properties in intracellular environments.
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Antioxidantes/farmacología , Antagonistas de Estrógenos/farmacología , Oxidantes/farmacología , Peroxidasas/metabolismo , Tamoxifeno/análogos & derivados , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ácidos Grasos/química , Glutatión/metabolismo , Células HL-60 , Humanos , Peroxidación de Lípido/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Oxidantes/toxicidad , Oxidación-Reducción , Peroxidasa/metabolismo , Fenol/farmacología , Fosfolípidos/química , Ratas , Albúmina Sérica/química , Tamoxifeno/farmacología , Tamoxifeno/toxicidadRESUMEN
We studied protective effects of NO against tert-butylhydroperoxide (t-BuOOH)-induced oxidations in a subline of human erythroleukemia K562 cells with different intracellular hemoglobin (Hb) concentrations. t-BuOOH-induced formation of oxoferryl-Hb-derived free radical species in cells was demonstrated by low temperature EPR spectroscopy. Intensity of the signals was proportional to Hb concentrations and was correlated with cell viability. Peroxidation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin metabolically labeled with oxidation-sensitive cis-parinaric acid was induced by t-BuOOH. An NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-iu m-1, 2-diolate], produced non-heme iron dinitrosyl complexes and hexa- and pentacoordinated Hb-nitrosyl complexes in the cells. Nitrosylation of non-heme iron centers and Hb-heme protected against t-BuOOH-induced: (a) formation of oxoferryl-Hb-derived free radical species, (b) peroxidation of cis-parinaric acid-labeled phospholipids, and (c) cytotoxicity. Since NO did not inhibit peroxidation induced by an azo-initiator of peroxyl radicals, 2, 2'-azobis(2,4-dimethylvaleronitrile), protective effects of NO were due to formation of iron-nitrosyl complexes whose redox interactions with t-BuOOH prevented generation of oxoferryl-Hb-derived free radical species.
Asunto(s)
Hemo/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Óxido Nítrico/farmacología , Peróxidos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Supervivencia Celular , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Hemoglobinas/metabolismo , Humanos , Cinética , Estrés Oxidativo , Células Tumorales Cultivadas , terc-ButilhidroperóxidoRESUMEN
Blast overpressure (BOP) is a phenomenon that describes the instantaneous rise in atmospheric pressure above ambient, resulting from the firing of large caliber weapons or from military or civilian explosions. Exposure to BOP results in injury to the gas-filled organs, such as the lungs, which exhibit a contusion-type injury. We examined the effects of BOP in rats at 5 and 60 min after exposure to a low-level BOP (62 +/- 3 kPa). The exposure was found to cause oxidative stress in the lung that was characterized by 1) a 3.5-fold decrease in total antioxidant reserves, 2) a depletion of the major water-soluble antioxidants ascorbate and glutathione (GSH) by 50 and 75%, respectively, 3) a depletion of lipid-soluble antioxidant vitamin E by 30%, 4) a 2.5-fold increase of fluorescent end products of lipid peroxidation, and 5) an increased methemoglobin (metHb) content at 60 min after exposure. To elucidate the role of released hemoglobin (Hb) in blast-induced oxidative stress, we studied the interactions of oxyhemoglobin (oxyHb), metHb, and the oxoferryl from of Hb free radical species with two physiologically important reductants, ascorbate and GSH. We found that both ascorbate and GSH were able to convert oxyHb to metHb in a reaction that yielded the one-electron oxidation intermediates semidehydroascorbyl radical and glutathionyl radical, respectively. This reaction did not occur under anaerobic conditions, suggesting that oxyHb-bound O2 acted as the electron acceptor. OxyHb induced peroxidation of cis-parinaric acid in the presence but not absence of ascorbate or GSH. Thus the prooxidant action of water-soluble antioxidants via redox cycling of oxyHb and metHb may promote oxidative stress rather than prevent it.