RESUMEN

Indole, which is produced by the intestinal microbiota from L-tryptophan, is recovered at millimolar concentrations in the human feces. Indoxyl sulfate (IS), the main indole co-metabolite, can be synthesized by the host tissues. Although indole has been shown to restore intestinal barrier function in experimental colitis, little is known on the effects of indole and IS on colonic epithelial cell metabolism and physiology. In this study, we compared the effects of indole and IS on the human colonic epithelial HT-29 Glc-/+ and Caco-2 cell lines, exposed to these compounds for 1-48 h. Indole, but not IS, was cytotoxic at 5 mM, altering markedly colonocyte proliferation. Both molecules, used up to 2.5 mM, induced a transient oxidative stress in colonocytes, that was detected after 1 h, but not after 48 h exposure. This was associated with the induction after 24 h of the expression of glutathione reductase, heme oxygenase, and cytochrome P450 (CYP)1B1. Indole and IS used at 2.5 mM impaired colonocyte respiration by diminishing mitochondrial oxygen consumption and maximal respiratory capacity. Indole, but not IS, displayed a slight genotoxic effect on colonocytes. Indole, but not IS, increased transepithelial resistance in colonocyte monolayers. Indole and IS used at 2.5 mM, induced a secretion of the pro-inflammatory interleukin-8 after 3 h incubation, and an increase of tumor necrosis factor-α secretion after 48 h. Although our results suggest beneficial effect of indole on epithelial integrity, overall they indicate that indole and IS share adverse effects on colonocyte respiration and production of reactive oxygen species, in association with the induction of enzymes of the antioxidant defense system. This latter process can be viewed as an adaptive response toward oxidative stress. Both compounds increased the production of inflammatory cytokines from colonocytes. However, only indole, but not IS, affected DNA integrity in colonocytes. Since colonocytes little convert indole to IS, the deleterious effects of indole on colonocytes appear to be unrelated to its conversion to IS.

Asunto(s)

Indicán , Triptófano , Humanos , Indicán/metabolismo , Triptófano/metabolismo , Células CACO-2 , Colon/metabolismo , Células Epiteliales/metabolismo , Bacterias , Indoles/farmacología , Indoles/metabolismo

RESUMEN

Reliable and predictive in vitro assays for hazard assessments of manufactured nanomaterials (MNMs) are still limited. Specifically, exposure systems which more realistically recapitulate the physiological conditions in the lung are needed to predict pulmonary toxicity. To this end, air-liquid interface (ALI) systems have been developed in recent years which might be better suited than conventional submerged exposure assays. However, there is still a need for rigorous side-by-side comparisons of the results obtained with the two different exposure methods considering numerous parameters, such as different MNMs, cell culture models and read outs. In this study, human A549 lung epithelial cells and differentiated THP-1 macrophages were exposed under submerged conditions to two abundant types of MNMs i.e., ceria and titania nanoparticles (NPs). Membrane integrity, metabolic activity as well as pro-inflammatory responses were recorded. For comparison, A549 monocultures were also exposed at the ALI to the same MNMs. In the case of titania NPs, genotoxicity was also investigated. In general, cells were more sensitive at the ALI compared to under classical submerged conditions. Whereas ceria NPs triggered only moderate effects, titania NPs clearly initiated cytotoxicity, pro-inflammatory gene expression and genotoxicity. Interestingly, low doses of NPs deposited at the ALI were sufficient to drive adverse outcomes, as also documented in rodent experiments. Therefore, further development of ALI systems seems promising to refine, reduce or even replace acute pulmonary toxicity studies in animals.

RESUMEN

BACKGROUND: 4-hydroxyphenylacetic acid (HO-PAA) is produced by intestinal microbiota from L-tyrosine. High concentrations in human fecal water have been associated with cytotoxicity, urging us to test HO-PAA's effects on human colonocytes. We compared these effects with those of phenylacetic acid (PAA), phenol and acetaldehyde, also issued from amino acids fermentation. METHODS: HT-29 Glc-/+ human colonocytes were exposed for 24 h to metabolites at concentrations between 350 and 1000 µM for HO-PAA and PAA, 250-1500 µM for phenol and 25-500 µM for acetaldehyde. We evaluated metabolites'cytotoxicity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and DNA quantification assays, reactive oxygen species (ROS) production with H2DCF-DA, and DNA damage with the comet assay. We measured cell oxygen consumption and mitochondrial complexes activity by polarography. RESULTS: Although HO-PAA displayed no cytotoxic effect on colonocytes, it decreased mitochondrial complex I activity and oxygen consumption. This was paralleled by an increase in ROS production and DNA alteration. Cells pretreatment with N-acetylcysteine, a ROS scavenger, decreased genotoxic effects of HO-PAA, indicating implication of oxidative stress in HO-PAA's genotoxicity. PAA and phenol did not reproduce these effects, but were cytotoxic towards colonocytes. Last, acetaldehyde displayed no effect in terms of cytotoxicity and mitochondrial metabolic activity, but increased DNA damage. CONCLUSIONS: Several bacterial metabolites produced from amino acids displayed deleterious effects on human colonocytes, in terms of genotoxicity (HO-PAA and acetaldehyde) or cytotoxicity (PAA and phenol). GENERAL SIGNIFICANCE: This study helps understanding the consequences of intestinal microbiota's metabolic activity on the host since amino acids fermentation can lead to the formation of compounds toxic towards colonic epithelial cells.

Asunto(s)

Aminoácidos/metabolismo , Bacterias/metabolismo , Colon/metabolismo , ADN/metabolismo , Estrés Oxidativo , Células HT29 , Humanos , Técnicas In Vitro , Consumo de Oxígeno

RESUMEN

BACKGROUND & AIMS: This review examines to what extent high-protein diets (HPD), which may favor body weight loss and improve metabolic outcomes in overweight and obese individuals, may also impact the gut environment, shaping the microbiota and the host-microbe (co)metabolic pathways and products, possibly affecting large intestine mucosa homeostasis. METHODS: PubMed-referenced publications were analyzed with an emphasis on dietary intervention studies involving human volunteers in order to clarify the beneficial vs. deleterious effects of HPD in terms of both metabolic and gut-related health parameters; taking into account the interactions with the gut microbiota. RESULTS: HPD generally decrease body weight and improve blood metabolic parameters, but also modify the fecal and urinary contents in various bacterial metabolites and co-metabolites. The effects of HPD on the intestinal microbiota composition appear rather heterogeneous depending on the type of dietary intervention. Recently, HPD consumption was shown to modify the expression of genes playing key roles in homeostatic processes in the rectal mucosa, without evidence of intestinal inflammation. Importantly, the effects of HPD on the gut were dependent on the protein source (i.e. from plant or animal sources), a result which should be considered for further investigations. CONCLUSION: Although HPD appear to be efficient for weight loss, the effects of HPD on microbiota-derived metabolites and gene expression in the gut raise new questions on the impact of HPD on the large intestine mucosa homeostasis leading the authors to recommend some caution regarding the utilization of HPD, notably in a recurrent and/or long-term ways.

Asunto(s)

Dieta Rica en Proteínas , Dieta , Microbioma Gastrointestinal , Pérdida de Peso , Peso Corporal/fisiología , Humanos , Mucosa Intestinal/microbiología , Mucosa Intestinal/fisiología , Intestino Grueso/microbiología , Intestino Grueso/fisiología

RESUMEN

BACKGROUND: Classified as carcinogenic to humans by the IARC in 2013, fine air particulate matter (PM2.5) can be inhaled and retained into the lung or reach the systemic circulation. This can cause or exacerbate numerous pathologies to which the elderly are often more sensitive. METHODS: In order to estimate the influence of age on the development of early cellular epigenetic alterations involved in carcinogenesis, peripheral blood mononuclear cells sampled from 90 patients from three age classes (25-30, 50-55 and 75-80 years old) were ex vivo exposed to urban PM2.5. RESULTS: Particles exposure led to variations in telomerase activity and telomeres length in all age groups without any influence of age. Conversely, P16INK4A gene expression increased significantly with age after exposure to PM2.5. Age could enhance MGMT gene expression after exposure to particles, by decreasing the level of promoter methylation in the oldest people. CONCLUSION: Hence, our results demonstrated several tendencies in cells modification depending on age, even if all epigenetic assays were carried out after a limited exposure time allowing only one or two cell cycles. Since lung cancer symptoms appear only at an advanced stage, our results underline the needs for further investigation on the studied biomarkers for early diagnosis of carcinogenesis to improve survival.

Asunto(s)

Envejecimiento , Contaminación del Aire/efectos adversos , Carcinogénesis/inducido químicamente , Epigénesis Genética , Adulto , Anciano , Anciano de 80 o más Años , Contaminantes Atmosféricos/análisis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Marcadores Genéticos , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Material Particulado/efectos adversos , Regiones Promotoras Genéticas , Telomerasa/metabolismo , Acortamiento del Telómero , Proteínas Supresoras de Tumor/genética

RESUMEN

BACKGROUND: High-protein diets (HPD) alter the large intestine microbiota composition in association with a metabolic shift towards protein degradation. Some amino acid-derived metabolites produced by the colon bacteria are beneficial for the mucosa while others are deleterious at high concentrations. The aim of the present work was to define the colonic epithelial response to an HPD. Transcriptome profiling was performed on colonocytes of rats fed an HPD or an isocaloric normal-protein diet (NPD) for 2 weeks. RESULTS: The HPD downregulated the expression of genes notably implicated in pathways related to cellular metabolism, NF-κB signaling, DNA repair, glutathione metabolism and cellular adhesion in colonocytes. In contrast, the HPD upregulated the expression of genes related to cell proliferation and chemical barrier function. These changes at the mRNA level in colonocytes were not associated with detrimental effects of the HPD on DNA integrity (comet assay), epithelium renewal (quantification of proliferation and apoptosis markers by immunohistochemistry and western blot) and colonic barrier integrity (Ussing chamber experiments). CONCLUSION: The modifications of the luminal environment after an HPD were associated with maintenance of the colonic homeostasis that might be the result of adaptive processes in the epithelium related to the observed transcriptional regulations.

Asunto(s)

Colon/metabolismo , Dieta , Proteínas en la Dieta/metabolismo , Mucosa Intestinal/metabolismo , Alimentación Animal , Animales , Análisis por Conglomerados , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glutatión/metabolismo , Masculino , Ratas , Transducción de Señal , Transcriptoma

RESUMEN

Evidence, mostly from experimental models, has accumulated, indicating that modifications of bacterial metabolite concentrations in the large intestine luminal content, notably after changes in the dietary composition, may have important beneficial or deleterious consequences for the colonic epithelial cell metabolism and physiology in terms of mitochondrial energy metabolism, reactive oxygen species production, gene expression, DNA integrity, proliferation, and viability. Recent data suggest that for some bacterial metabolites, like hydrogen sulfide and butyrate, the extent of their oxidation in colonocytes affects their capacity to modulate gene expression in these cells. Modifications of the luminal bacterial metabolite concentrations may, in addition, affect the colonic pH and osmolarity, which are known to affect colonocyte biology per se. Although the colonic epithelium appears able to face, up to some extent, changes in its luminal environment, notably by developing a metabolic adaptive response, some of these modifications may likely affect the homeostatic process of colonic epithelium renewal and the epithelial barrier function. The contribution of major changes in the colonocyte luminal environment in pathological processes, like mucosal inflammation, preneoplasia, and neoplasia, although suggested by several studies, remains to be precisely evaluated, particularly in a long-term perspective.

Asunto(s)

Microambiente Celular , Colon/patología , Células Epiteliales/patología , Animales , Metabolismo Energético , Humanos , Concentración de Iones de Hidrógeno , Metaboloma

RESUMEN

Titanium dioxide nanoparticles (TiO2-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1-50 µg/mL TiO2-NPs, 86% anatase/14% rutile, 24 ± 6 nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO2-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO2-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO2-NPs.

Asunto(s)

Células Epiteliales Alveolares/efectos de los fármacos , Daño del ADN , Mutágenos/toxicidad , Nanopartículas/toxicidad , Titanio/toxicidad , Células A549 , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Micronúcleos , Microscopía Electrónica de Transmisión , Mutágenos/química , Nanopartículas/química , Tamaño de la Partícula , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Titanio/química

RESUMEN

Although the biological effects of titanium dioxide nanoparticles (TiO2-NPs) have been studied for more than two decades, the mechanisms governing their toxicity are still unclear. We applied 2D-gel proteomics analysis on A549 epithelial alveolar cells chronically exposed for 2months to 2.5 or 50µg/mL of deeply characterized TiO2-NPs, in order to obtain comprehensive molecular responses that may reflect functional outcomes. We show that exposure to TiO2-NPs impacts the abundance of 30 protein species, corresponding to 22 gene products. These proteins are involved in glucose metabolism, trafficking, gene expression, mitochondrial function, proteasome activity and DNA damage response. Besides, our results suggest that p53 pathway is activated, slowing down cell cycle progression and reducing cell proliferation rate. Moreover, we report increased content of chaperones-related proteins, which suggests homeostasis re-establishment. Finally, our results highlight that chronic exposure to TiO2-NPs affects the same cellular functions as acute exposure to TiO2-NPs, although lower exposure concentrations and longer exposure times induce more intense cellular response. BIOLOGICAL SIGNIFICANCE: Our results make possible the identification of new mechanisms that explain TiO2-NP toxicity upon long-term, in vitro exposure of A549 cells. It is the first article describing -omics results obtained with this experimental strategy. We show that this long-term exposure modifies the cellular content of proteins involved in functions including mitochondrial activity, intra- and extracellular trafficking, proteasome activity, glucose metabolism, and gene expression. Moreover we observe modification of content of proteins that activate the p53 pathway, which suggest the induction of a DNA damage response. Technically, our results show that exposure of A549 cells to a high concentration of TiO2-NPs leads to the identification of modulations of the same functional categories than exposure to low, more realistic concentrations. Still the intensity differs between these two exposure scenarios. We also show that chronic exposure to TiO2-NPs induces the modulation of cellular functions that have already been reported in the literature as being impacted in acute exposure scenarios. This proves that the exposure protocol in in vitro experiments related to nanoparticle toxicology might be cautiously chosen since inappropriate scenario may lead to inappropriate and/or incomplete conclusions.

Asunto(s)

Células Epiteliales/metabolismo , Nanopartículas , Proteoma/metabolismo , Proteómica , Alveolos Pulmonares/metabolismo , Mucosa Respiratoria/metabolismo , Titanio/farmacología , Línea Celular Tumoral , Humanos , Titanio/química

RESUMEN

Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.

Asunto(s)

Cobre/toxicidad , Macrófagos/metabolismo , Nanopartículas del Metal/toxicidad , Proteómica/métodos , Titanio/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Citocinas/biosíntesis , Roturas del ADN de Doble Cadena/efectos de los fármacos , Dihidroxifenilalanina/farmacología , Electroforesis en Gel Bidimensional , Inducción Enzimática/efectos de los fármacos , Glutatión/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Óxido Nítrico/biosíntesis , Fagocitosis/efectos de los fármacos

RESUMEN

Manufactured nanomaterials (MNMs) have the potential to improve everyday life as they can be utilised in numerous medical applications and day-to-day consumer products. However, this increased use has led to concerns about the potential environmental and human health impacts. The protein p53 is a key transcription factor implicated in cellular defence and reparative responses to various stress factors. Additionally, p53 has been implicated in cellular responses following exposure to some MNMs. Here, the role of the MNM mediated p53 induction and activation and its downstream effects following exposure to five well-characterised materials [namely two types of TiO2, two carbon black (CB), and one single-walled carbon nanotube (SWCNT)] were investigated. MNM internalisation, cellular viability, p53 protein induction and activation, oxidative stress, inflammation and apoptosis were measured in murine cell line and primary pulmonary macrophage models. It was observed that p53 was implicated in the biological responses to MNMs, with oxidative stress associated with p53 activation (only following exposure to the SWCNT). We demonstrate that p53 acted as an antioxidant and anti-inflammatory in macrophage responses to SWCNT and CB NMs. However, p53 was neither involved in MNM-induced cellular toxicity, nor in the apoptosis induced by these MNMs. Moreover, the physicochemical characteristics of MNMs seemed to influence their biological effects-SWCNT the materials with the largest surface area and a fibrous shape were the most cytotoxic in this study and were capable of the induction and activation of p53.

Asunto(s)

Macrófagos Alveolares/efectos de los fármacos , Nanoestructuras/toxicidad , Nanotubos de Carbono/toxicidad , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular , Inflamación/patología , Macrófagos Alveolares/metabolismo , Ratones , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , Titanio/administración & dosificación , Titanio/toxicidad , Proteína p53 Supresora de Tumor/genética

RESUMEN

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations.

Asunto(s)

Nanopartículas del Metal/química , Óxido de Zinc/química , Zinc/química , Animales , Línea Celular , Daño del ADN/efectos de los fármacos , Electroforesis en Gel Bidimensional , Glutatión/metabolismo , Iones/química , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Nanopartículas del Metal/toxicidad , Ratones , Microscopía Electrónica de Transmisión , Estrés Oxidativo/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Proteoma/análisis , Proteoma/efectos de los fármacos

RESUMEN

BACKGROUND: Carbon nanotubes (CNT) are a family of materials featuring a large range of length, diameter, numbers of walls and, quite often metallic impurities coming from the catalyst used for their synthesis. They exhibit unique physical properties, which have already led to an extensive development of CNT for numerous applications. Because of this development and the resulting potential increase of human exposure, an important body of literature has been published with the aim to evaluate the health impact of CNT. However, despite evidences of uptake and long-term persistence of CNT within macrophages and the central role of those cells in the CNT-induced pulmonary inflammatory response, a limited amount of data is available so far on the CNT fate inside macrophages. Therefore, the overall aim of our study was to investigate the fate of pristine single walled CNT (SWCNT) after their internalization by macrophages. METHODS: To achieve our aim, we used a broad range of techniques that aimed at getting a comprehensive characterization of the SWCNT and their catalyst residues before and after exposure of murine macrophages: X-ray diffraction (XRD), High Resolution (HR) Transmission Electron Microscopy (TEM), High Angle Annular Dark Field-Scanning TEM (HAADF-STEM) coupled to Electron Energy Loss Spectroscopy (EELS), as well as micro-X-ray fluorescence mapping (µXRF), using synchrotron radiation. RESULTS: We showed 1) the rapid detachment of part of the iron nanoparticles initially attached to SWCNT which appeared as free iron nanoparticles in the cytoplasm and nucleus of CNT-exposed murine macrophages, and 2) that blockade of intracellular lysosomal acidification prevented iron nanoparticles detachment from CNT bundles and protected cells from CNT downstream toxicity. CONCLUSIONS: The present results, while obtained with pristine SWCNT, could likely be extended to other catalyst-containing nanomaterials and surely open new ways in the interpretation and understanding of CNT toxicity.

Asunto(s)

Compuestos de Hierro/metabolismo , Macrófagos/metabolismo , Nanopartículas del Metal , Nanotubos de Carbono/análisis , Animales , Catepsina B/metabolismo , Línea Celular , Concentración de Iones de Hidrógeno , Compuestos de Hierro/toxicidad , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Macrólidos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Microscopía Electrónica de Transmisión , Nanotubos de Carbono/toxicidad , Espectrometría por Rayos X , Espectroscopía de Pérdida de Energía de Electrones , Sincrotrones , Difracción de Rayos X

RESUMEN

Exposure to titanium dioxide (TiO2) nanoparticles (NPs) is associated with lung remodeling, but the underlying mechanisms are unknown. Matrix metalloprotease (MMP)-1 is an important actor in matrix homeostasis and could therefore participate in TiO2 NP effects. Our aim was to evaluate the effects of TiO2 NPs on MMP-1 expression and activity in lung pulmonary fibroblasts and to understand the underlying mechanisms and assess the importance of the physicochemical characteristics of the particles in these effects. Human pulmonary fibroblasts (MRC-5 cell line and primary cells) were exposed to 10 or 100 µg/cm(2) TiO2 (two anatases, two anatase/rutile mix, one rutile NP, and one micrometric) and carbon black (CB) NPs for 6 to 48 hours. We examined cell viability, MMP-1 expression and activity, and the implication of oxidative stress, transforming growth factor (TGF)-ß, extracellular MMP inducer, and IL-1ß in MMP-1 expression. All TiO2 NPs induced MMP-1 (mRNA and protein expression), repression of procollagen-1, and α-actin expression, but only the two anatase/rutile mix induced MMP-1 activity. Micrometric TiO2 had smaller effects than TiO2 NPs, and CB NPs did not induce MMP-1. MMP-1 induction by TiO2 NPs was not related to TGF-ß, oxidative stress, or EMPRIN expression but was related to IL-1ß expression, which partly drives MMP-1 induction by two TiO2 NPs (one anatase/rutile mix and the rutile one). Taken together, our results show that TiO2 NPs are potent inducers and regulators of MMP-1 expression and activity, partly via an IL-1ß-dependent mechanism. This may explain TiO2 lung remodeling effects.

Asunto(s)

Fibroblastos/efectos de los fármacos , Interleucina-1beta/metabolismo , Pulmón/efectos de los fármacos , Metaloproteinasa 1 de la Matriz/biosíntesis , Nanopartículas del Metal/efectos adversos , Titanio/farmacología , Actinas/genética , Actinas/metabolismo , Basigina/genética , Basigina/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Inducción Enzimática/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-1beta/genética , Pulmón/enzimología , Pulmón/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Procolágeno/genética , Procolágeno/metabolismo , Hollín/farmacología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

BACKGROUND: Titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) have biological effects that could aggravate pulmonary emphysema. The aim of this study was to evaluate whether pulmonary administration of TiO2 or CB NPs in rats could induce and/or aggravate elastase-induced emphysema, and to investigate the underlying molecular mechanisms. METHODS: On day 1, Sprague-Dawley rats were intratracheally instilled with 25 U kg⁻¹ pancreatic porcine elastase or saline. On day 7, they received an intratracheal instillation of TiO2 or CB (at 100 and 500 µg) dispersed in bovine serum albumin or bovine serum albumin alone. Animals were sacrificed at days 8 or 21, and bronchoalveolar lavage (BAL) cellularity, histological analysis of inflammation and emphysema, and lung mRNA expression of heme oxygenase-1 (HO-1), interleukin-1ß (IL-1ß), macrophage inflammatory protein-2, monocyte chemotactic protein-1, and matrix metalloprotease (MMP)-1, and -12 were measured. In addition, pulmonary MMP-12 expression was also analyzed at the protein level by immunohistochemistry. RESULTS: TiO2 NPs per se did not modify the parameters investigated, but CB NPs increased perivascular/peribronchial infiltration, and macrophage MMP-12 expression, without inducing emphysema. Elastase administration increased BAL cellularity, histological inflammation, HO-1, IL-1ß and macrophage MMP-12 expression and induced emphysema. Exposure to TiO2 NPs did not modify pulmonary responses to elastase, but exposure to CB NPs aggravated elastase-induced histological inflammation without aggravating emphysema. CONCLUSIONS: TiO2 and CB NPs did not aggravate elastase-induced emphysema. However, CB NPs induced histological inflammation and MMP-12 mRNA and protein expression in macrophages.

Asunto(s)

Nanopartículas/efectos adversos , Elastasa Pancreática/efectos adversos , Enfisema Pulmonar/inducido químicamente , Enfisema Pulmonar/patología , Hollín/efectos adversos , Titanio/efectos adversos , Animales , Lavado Broncoalveolar , Hemo-Oxigenasa 1/metabolismo , Interleucina-1beta/metabolismo , Intubación Intratraqueal , Masculino , Metaloproteinasa 12 de la Matriz/metabolismo , Modelos Animales , Nanopartículas/administración & dosificación , Neumonía/inducido químicamente , Neumonía/metabolismo , Neumonía/patología , Enfisema Pulmonar/metabolismo , Ratas , Ratas Sprague-Dawley , Hollín/administración & dosificación , Titanio/administración & dosificación

RESUMEN

Several studies suggest that the biological responses induced by manufactured nanoparticles (MNPs) may be linked to their accumulation within cells. However, MNP internalisation has not yet been sufficiently characterised. Therefore, the aim of this study was to compare the intracellular uptake of three different MNPs: two made of carbon black (CB) and one made of titanium dioxide (TiO(2)), in 16HBE bronchial epithelial cells and MRC5 fibroblasts. Transmission electron microscopy was used to evaluate the intracellular accumulation. Different parameters were analysed following a time and dose-relationship: localisation of MNPs in cells, percentage of cells having accumulated MNPs, number of aggregated MNPs in cells, and the size of MNP aggregates in cells. The results showed that MNPs were widely and rapidly accumulated in 16HBE cells and MRC5 fibroblasts. Moreover, MNPs accumulated chiefly as aggregates in cytosolic vesicles and were absent from the mitochondria or nuclei. CB and TiO(2) MNPs had similar accumulation patterns. However, TiO(2) aggregates had a higher size than CB aggregates. Intracellular MNP accumulation was dissociated from cytotoxicity. These results suggest that cellular uptake of MNPs is a common phenomenon occurring in various cell types.

Asunto(s)

Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Nanopartículas , Hollín/metabolismo , Titanio/metabolismo , Línea Celular , Colorantes/metabolismo , Humanos , Pulmón/citología , Microscopía Electrónica de Transmisión , Nanopartículas/ultraestructura , Tamaño de la Partícula

RESUMEN

OBJECTIVE: Imaging of myelin tracts in vivo would greatly improve the monitoring of demyelinating diseases such as multiple sclerosis (MS). To date, no imaging technique specifically targets demyelination and remyelination. Recently, amyloid markers related to Congo red have been shown to bind to central nervous system (CNS) myelin. Here we questioned whether the thioflavine-T derivative 2-(4'-methylaminophenyl)-6-hydroxybenzothiazole (PIB), which also binds to amyloid plaques, could serve as a myelin marker. METHODS: PIB fixation to myelin was studied by fluorescence in the normal and dysmyelinating mouse brain, as well as in the postmortem brain of MS patients. Positron emission tomography (PET) experiments were conducted using [¹¹C]PIB in baboons and in a proof of concept clinical study in 2 MS patients. RESULTS: Applied directly on tissue sections or after intraperitoneal injection, PIB stained CNS myelin, and the decrease in the level of fixation paralleled the amount of myelin loss in a dysmyelinating mutant. In normally myelinated areas of postmortem MS brain, demyelinated and remyelinated lesions were clearly distinguishable by the differential intensity of labeling observed with PIB. PET using intravenously injected radiolabeled [¹¹C]PIB imaged CNS myelin in baboons and humans. In MS patients, the dynamic analysis of PET acquisitions allowed quantitative assessment of demyelination. INTERPRETATION: PIB could be used as an imaging marker to quantify myelin loss and repair in demyelinating diseases.

Asunto(s)

Benzotiazoles , Encéfalo/diagnóstico por imagen , Encéfalo/fisiopatología , Radioisótopos de Carbono , Enfermedades Desmielinizantes/diagnóstico por imagen , Esclerosis Múltiple/diagnóstico por imagen , Esclerosis Múltiple/fisiopatología , Tomografía de Emisión de Positrones , Compuestos de Anilina , Animales , Cadáver , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Regeneración Nerviosa , Papio anubis , Tomografía de Emisión de Positrones/métodos , Tiazoles