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1.
Part Fibre Toxicol ; 14(1): 39, 2017 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-28969663

RESUMEN

BACKGROUND: Toxicity of airborne particulate matter (PM) is difficult to assess because PM composition is complex and variable due to source contribution and atmospheric transformation. In this study, we used an in vitro toxicoproteomic approach to identify the toxicity mechanisms associated with different subfractions of Ottawa urban dust (EHC-93). METHODS: A549 human lung epithelial cells were exposed to 0, 60, 140 and 200 µg/cm2 doses of EHC-93 (total), its insoluble and soluble fractions for 24 h. Multiple cytotoxicity assays and proteomic analyses were used to assess particle toxicity in the exposed cells. RESULTS: The cytotoxicity data based on cellular ATP, BrdU incorporation and LDH leakage indicated that the insoluble, but not the soluble, fraction is responsible for the toxicity of EHC-93 in A549 cells. Two-dimensional gel electrophoresis results revealed that the expressions of 206 protein spots were significantly altered after particle exposures, where 154 were identified by MALDI-TOF-TOF-MS/MS. The results from cytotoxicity assays and proteomic analyses converged to a similar finding that the effects of the total and insoluble fraction may be alike, but their effects were distinguishable, and their effects were significantly different from the soluble fraction. Furthermore, the toxic potency of EHC-93 total is not equal to the sum of its insoluble and soluble fractions, implying inter-component interactions between insoluble and soluble materials resulting in synergistic or antagonistic cytotoxic effects. Pathway analysis based on the low toxicity dose (60 µg/cm2) indicated that the two subfractions can alter the expression of those proteins involved in pathways including cell death, cell proliferation and inflammatory response in a distinguishable manner. For example, the insoluble and soluble fractions differentially affected the secretion of pro-inflammatory cytokines such as MCP-1 and IL-8 and distinctly altered the expression of those proteins (e.g., TREM1, PDIA3 and ENO1) involved in an inflammatory response pathway in A549 cells. CONCLUSIONS: This study demonstrated the impact of different fractions of urban air particles constituted of various chemical species on different mechanistic pathways and thus on cytotoxicity effects. In vitro toxicoproteomics can be a valuable tool in mapping these differences in air pollutant exposure-related toxicity mechanisms.


Asunto(s)
Pulmón/efectos de los fármacos , Material Particulado/toxicidad , Proteómica/métodos , Solventes/química , Toxicología/métodos , Agua/química , Células A549 , Supervivencia Celular/efectos de los fármacos , Quimiocina CCL2/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-8/metabolismo , Pulmón/metabolismo , Pulmón/patología , Material Particulado/química , Medición de Riesgo , Transducción de Señal/efectos de los fármacos , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Factor A de Crecimiento Endotelial Vascular/metabolismo
2.
J Appl Toxicol ; 37(6): 721-731, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-27917503

RESUMEN

In this study, we used cytotoxicity assays, proteomic and gene expression analyses to examine the difference in response of A549 cells to two silica particles that differ in physical properties, namely cristobalite (CR) and α-quartz (Min-U-Sil 5, MI). Cytotoxicity assays such as lactate dehydrogenase release, 5-bromo-2'-deoxyuridine incorporation and cellular ATP showed that both silica particles could cause cell death, decreased cell proliferation and metabolism in the A549 human lung epithelial cells. While cytotoxicity assays revealed little difference between CR and MI exposures, proteomic and gene expression analyses unveiled both similar and unique molecular changes in A549 cells. For instance, two-dimensional gel electrophoresis data indicated that the expression of proteins in the cell death (e.g., ALDH1A1, HTRA2 and PRDX6) and cell proliferation (e.g., FSCN1, HNRNPAB and PGK1) pathways were significantly different between the two silica particles. Reverse transcription-polymerase chain reaction data provided additional evidence supporting the proteomic findings. Preliminary assessment of the physical differences between CR and MI suggested that the extent of surface interaction between particles and cells could explain some of the observed biological effects. However, the differential dose-response curves for some other genes and proteins suggest that other physical attributes of particulate matter can also contribute to particulate matter-related cellular toxicity. Our results demonstrated that toxicoproteomic and gene expression analyses are sensitive in distinguishing subtle toxicity differences associated with silica particles of varying physical properties compared to traditional cytotoxicity endpoints. Copyright © 2016 Her Majesty the Queen in Right of Canada. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.


Asunto(s)
Células Epiteliales/efectos de los fármacos , Material Particulado/toxicidad , Proteoma/efectos de los fármacos , Dióxido de Silicio/toxicidad , Transcriptoma/efectos de los fármacos , Células A549 , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Electroforesis en Gel Bidimensional , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Material Particulado/química , Proteómica/métodos , Cuarzo/química , Cuarzo/toxicidad , Sensibilidad y Especificidad , Dióxido de Silicio/química , Propiedades de Superficie
3.
Part Fibre Toxicol ; 12: 28, 2015 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-26376633

RESUMEN

BACKGROUND: While exposure to ambient air contaminants is clearly associated with adverse health outcomes, disentangling mechanisms of pollutant interactions remains a challenge. OBJECTIVES: We aimed at characterizing free radical pathways and the endothelinergic system in rats after inhalation of urban particulate matter, ozone, and a combination of particles plus ozone to gain insight into pollutant-specific toxicity mechanisms and any effect modification due to air pollutant mixtures. METHODS: Fischer 344 rats were exposed for 4 h to a 3 × 3 concentration matrix of ozone (0, 0.4, 0.8 ppm) and EHC-93 particles (0, 5, 50 mg/m(3)). Bronchoalveolar lavage fluid (BALF), BAL cells, blood and plasma were analysed for biomarkers of effects immediately and 24 h post-exposure. RESULTS: Inhalation of ozone increased (p < 0.05) lipid oxidation products in BAL cells immediately post-exposure, and increased (p < 0.05) total protein, neutrophils and mature macrophages in the BALF 24 h post-exposure. Ozone increased (p < 0.05) the formation of reactive oxygen species (ROS), assessed by m-, p-, o-tyrosines in BALF (Ozone main effects, p < 0.05), while formation of reactive nitrogen species (RNS), indicated by 3-nitrotyrosine, correlated with dose of urban particles (EHC-93 main effects or EHC-93 × Ozone interactions, p < 0.05). Carboxyhemoglobin levels in blood exhibited particle exposure-related increase (p < 0.05) 24 h post recovery. Plasma 3-nitrotyrosine and o-tyrosine were increased (p < 0.05) after inhalation of particles; the effect on 3-nitrotyrosine was abrogated after exposure to ozone plus particles (EHC-93 × Ozone, p < 0.05). Big endothelin-1 (BET-1) and ET-1 were increased in plasma after inhalation of particles or ozone alone, but the effects appeared to be attenuated by co-exposure to contaminants (EHC-93 × Ozone, p < 0.05). Plasma ET levels were positively correlated (p < 0.05) with BALF m- and o-tyrosine levels. CONCLUSIONS: Pollutant-specific changes can be amplified or abrogated following multi-pollutant exposures. Oxidative and nitrative stress in the lung compartment may contribute to secondary extra-pulmonary ROS/RNS formation. Nitrative stress and endothelinergic imbalance emerge as potential key pathways of air pollutant health effects, notably of ambient particulate matter.


Asunto(s)
Endotelinas/sangre , Nitratos/metabolismo , Estrés Oxidativo , Ozono/toxicidad , Animales , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar , Exposición por Inhalación , Ratas , Ratas Endogámicas F344
4.
Chemosphere ; 296: 133933, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35157883

RESUMEN

Short-term increases in particulate matter (PM) are associated with heightened morbidity and mortality from cardiovascular causes. Inhalation of PM is known to increase endothelin (ET)-1 levels. Yet, less is known about particle composition-related changes at the molecular level including the endothelinergic system and relationship with cardiovascular function changes. In this work, adult Wistar male rats were exposed for 4 h by nose-only inhalation to clean air, Ottawa urban particles (EHC-93, 48 mg/m3) and water-leached (EHC-93L, 49 mg/m3) particles, to examine the effect of particle compositional changes on oxidative stress, circulating ETs, blood pressure, and heart electrophysiology. Particle deposition in the respiratory compartment was estimated at 85 µg (25 ng/cm2). Lung cell proliferation was low in both treatment groups, indicating absence of acute injury. Inhalation of EHC-93 caused statistically significant elevations (p < 0.05) of oxidative stress markers, ET-1, ET-3, blood pressure, and a decrease of ST-segment duration in the ECG at 1.5 days post-exposure. Leached particles (EHC-93L) caused rapid but transient elevation (p < 0.05) of oxidative stress, ET-1, ET-2, and ET-3 at earlier time points, with no changes in blood pressure or ST-segment. These results demonstrate that inhalation of urban particles at an internal dose inadequate to cause acute lung injury can induce oxidative stress, enhance vasoactive endothelins, leading to vasopressor response, affecting cardiac electrophysiology in Wistar rats, consistent with the cardiovascular impacts of ambient particles in human populations. Change in particle potency after removal of soluble species, notably cadmium, zinc and polar organics suggests that the toxicodynamics of cardiovascular effects can be modified by physicochemical properties of particles.


Asunto(s)
Contaminantes Atmosféricos , Material Particulado , Contaminantes Atmosféricos/análisis , Animales , Presión Sanguínea , Endotelina-1/farmacología , Exposición por Inhalación/efectos adversos , Pulmón , Masculino , Estrés Oxidativo , Tamaño de la Partícula , Material Particulado/farmacología , Ratas , Ratas Wistar
5.
J Proteomics ; 149: 53-63, 2016 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-27084686

RESUMEN

This study combined cytotoxicity assays with proteomic analysis to characterize the unique biological responses of the A549 human lung epithelial cell line to two physicochemically distinct respirable particles titanium dioxide (TiO2) and carbon black (CB). Cellular LDH, ATP, BrdU incorporation and resazurin reduction indicated that CB was more potent than TiO2. Proteomic analysis was done using 2D-GE and MALDI-TOF-TOF-MS. Proteomic changes reflected common and particle-specific responses. Particle-specific proteomic responses were associated with cell death (necrosis and apoptosis), viability and proliferation pathways. Our results suggested that these pathways were consistent with the cytotoxicity data. For instance, increased expressions of anti-proliferative proteins LMNA and PA2G4 were in agreement with the decreased BrdU incorporation in A549 cells after exposure to CB. Similarly, increased expression of HSPA5 that is associated with ATPase activity was consistent with decreased cellular ATP levels in these cells. These findings reveal that proteomic changes can explain the cellular cytotoxicity characteristics of the particles. In essence, our results demonstrate that the in vitro toxicoproteomic approach is a promising tool to gain insight into molecular mechanisms underlying particle exposure-specific cytotoxicity. BIOLOGICAL SIGNIFICANCE: In this study we have shown that toxicoproteomics is a sensitive and informative method to resolve the toxicity characteristics of particles with different physicochemical properties. This approach can be useful in the investigation of molecular mechanisms underpinning cellular cytotoxic responses elicited by particle exposures. Thus, the toxicoproteomic approach can be valuable in assessing the risk associated with particle exposures in vitro.


Asunto(s)
Células Epiteliales Alveolares/efectos de los fármacos , Proteínas/análisis , Proteómica/métodos , Hollín/toxicidad , Protectores Solares/toxicidad , Titanio/toxicidad , Células A549 , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Epiteliales Alveolares/metabolismo , Análisis de Varianza , Supervivencia Celular/efectos de los fármacos , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/metabolismo , Humanos , Lamina Tipo A/análisis , Lamina Tipo A/metabolismo , Tamaño de la Partícula , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Pruebas de Toxicidad
6.
Data Brief ; 8: 687-91, 2016 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-27508218

RESUMEN

Here, we have described the dataset relevant to the A549 cellular proteome changes after exposure to either titanium dioxide or carbon black particles as compared to the non-exposed controls, "Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures" (Vuong et al., 2016) [1]. Detailed methodologies on the separation of cellular proteins by 2D-GE and the subsequent mass spectrometry analyses using MALDI-TOF-TOF-MS are documented. Particle exposure-specific protein expression changes were measured via 2D-GE spot volume analysis. Protein identification was done by querying mass spectrometry data against SwissProt and RefSeq protein databases using Mascot search engine. Two-way ANOVA analysis data provided information on statistically significant A549 protein expression changes associated with particle exposures.

7.
Biochim Biophys Acta ; 1689(1): 75-82, 2004 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-15158916

RESUMEN

Inhalation of urban pollutants elevates the circulating levels of the vasoactive peptides endothelin (ET)-1 and ET-3 in rats. This effect could explain the association between episodic variations of urban pollutants and acute cardiopulmonary morbidity and mortality documented in epidemiological studies. Because the lungs are the primary source of circulating ET-1 and the main site of clearance from circulation, we investigated the response of endothelin system genes in the lungs of Fischer-344 rats after 4-h nose-only inhalation of 0.8 ppm ozone plus 49 mg/m(3) EHC-93 (Ottawa particles). The mRNA levels for preproET-1, preproET-3, endothelin-converting enzyme (ECE)-1, and ET receptor subtypes A and B were determined at 2 h, and 1, 2, 3, 7, and 14 days after exposure. The pollutants induced preproET-1 and ECE-1 (P<0.05) after 2 h, consistent with the notion of increased synthesis and conversion of the peptide ET-1 in lung endothelial cells. PreproET-3 mRNA was down-regulated at 2 h post-exposure (P<0.05), and returned to control levels by 24 h, indicating that induction of ET-3 in the lungs is not responsible for the sustained elevation of ET-3 in plasma reported after inhalation of pollutants. Our results indicate that lung endothelin system genes respond rapidly and transiently to inhalation of urban pollutants, consistent with the dynamics of urban pollutant health effects in the human population.


Asunto(s)
Contaminantes Atmosféricos/farmacología , Endotelina-1/genética , Regulación de la Expresión Génica/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Vasoconstricción , Administración por Inhalación , Animales , Humanos , Masculino , Ozono/administración & dosificación , Ozono/análisis , Ozono/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Toxicol Sci ; 88(1): 103-13, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16081523

RESUMEN

Periodic elevation of ambient particulate matter and ozone levels is linked to acute cardiac morbidity and mortality. Increased plasma levels of the potent vasoconstrictor endothelin (ET)-1, a prognostic indicator of cardiac mortality, have been detected in both animal models and humans after exposure to air pollutants. The lungs are the primary source of circulating ET-1, but the direct effects of individual air pollutants and their interaction in modulating the pulmonary endothelin system are unknown. Fischer-344 rats were exposed to particles (0, 5, 50 mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Changes in gene expression were measured using real-time reverse transcription polymerase chain reaction immediately after exposure and following 24 h recovery in clean air. Both pollutants individually increased preproET-1, endothelin converting enzyme-1, and endothelial nitric oxide synthase mRNA levels in the lungs shortly after exposure, consistent with the concomitant increase in plasma of the 21 amino acid ET-1[1-21] peptide measured by HPLC-fluorescence. PreproET-1 mRNA remained elevated 24 h after exposure to particles but not after ozone, in line with previously documented changes of the peptide in plasma. Both pollutants transiently increased endothelin-B receptor mRNA expression, while ozone decreased endothelin-A receptor mRNA levels. Coexposure to particles plus ozone increased lung preproET-1 mRNA but not plasma ET-1[1-21], suggesting alternative processing or degradation of endothelins. This coincided with an increase in the lungs of matrix metalloproteinase-2 (MMP-2), an enzyme that cleaves bigET-1 to ET-1[1-32]. Taken together, our data indicate that ozone and particulate matter independently regulate the expression of lung endothelin system genes, but show complex toxicological interaction with respect to plasma ET-1.


Asunto(s)
Contaminantes Atmosféricos/toxicidad , Endotelinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Exposición por Inhalación/efectos adversos , Pulmón/efectos de los fármacos , Ozono/toxicidad , Animales , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Endotelina-1/genética , Endotelina-1/metabolismo , Enzimas Convertidoras de Endotelina , Endotelinas/genética , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Monitoreo del Ambiente/métodos , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Organismos Libres de Patógenos Específicos
9.
Toxicol Mech Methods ; 12(3): 195-210, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-20021173

RESUMEN

Fischer 344 rats were exposed to ozone by inhalation to identify sensitive indices of acute exposure. 5-Aminosalicylic acid (5-ASA) hydroxylation in bronchoalveolar lavage (BAL), an indicator of hydroxyl radical (*OH) formation, and lipid oxidation in various regions of airways, representing oxidative stress, were measured to verify whether they can function as markers of exposure. BAL cells and supernatants taken from rats that received saline or 5-ASA (ip, 50 mg/kg) prior to ozone exposure (0, 0.4, or 0.8 ppm for 4 h) were analyzed for products of lipid oxidation. *OH formation was assessed by analysis of the BAL supernatant for 5-aminotetrahydroxybenzoic acid (5-ATHBA), a hydroxylation product of 5-ASA. The tetrahydroxy derivative of 5-ASA was higher in the BAL of ozone-treated rats than in air controls, reaching significance (p <. 05) at 0.8 ppm of ozone, The products of lipid oxidation propanal and hexanal were higher in BAL cells taken from rats exposed to ozone, reaching significance (p <. 05) at a 0.8 ppm ozone level, compared to air control animals, irrespective of whether they received saline or 5-ASA prior to ozone exposure. Increases in cholesterol levels were also seen in BAL cells after rats were exposed to ozone. However, there were no significant dose-related changes in the lipid oxidation products in BAL supernatants after exposure to ozone. Lipid oxidation products in BAL cells and 5-ATHBA in lavage exhibited the potential to serve as markers of ozone exposure. This work was supported by Health Canada (#4320105) and Toxic Substances Research Initiatives (TSRI #60).

10.
Toxicol In Vitro ; 27(4): 1287-97, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23466443

RESUMEN

We examined the utility of respiratory burst measurements in alveolar macrophages to assess adverse cellular changes following exposure to urban particles. Cells were obtained by bronchioalveolar lavage of Fisher 344 rats and exposed (0-100 µg/well) to urban particles (EHC-93, SRM-1648, SRM-1649, PM2.5), the soluble (EHC-93sol) and insoluble (EHC-93insol) fractions of EHC-93 (EHC-93tot), mineral particles (TiO(2), SiO(2)) and metal oxides (iron III oxide, iron II/III oxide, copper II oxide, nickel II oxide). The particle-induced respiratory burst was measured by chemiluminescence for 2h after the addition of particles. The cells were then stimulated with phorbol 12-myristate 13-acetate (PMA), yeast Zymosan fragments (Zymosan), or lipopolysaccharide plus interferon-gamma (LPS/IFN-γ) and the stimulant-induced respiratory burst was measured. Independently of the potential of particles to induce directly a respiratory burst, exposure to most particles attenuated the subsequent stimulant-induced burst. The notable exception was SiO(2), which produced a strong respiratory burst upon contact with the macrophages and enhanced the subsequent response to PMA or LPS/IFN-γ. Based on the degree of inhibition of the stimulant-dependent respiratory burst, particles were clustered into groups of high (SRM-1649, iron III oxide), intermediate (EHC-93tot, EHC-93insol, SRM-1648, VERP, iron II/III oxide, copper II oxide), and low (EHC-93sol, SiO(2), TiO2 and nickel II oxide) potency. Across these clusters, the potency of the particles to inhibit the stimulant-dependent respiratory burst showed poor correlation with cytotoxicity determined by XTT reduction assay.


Asunto(s)
Contaminantes Atmosféricos/toxicidad , Macrófagos Alveolares/efectos de los fármacos , Material Particulado/toxicidad , Estallido Respiratorio/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ciudades , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Masculino , Nitritos/metabolismo , Óxidos/toxicidad , Ratas , Ratas Endogámicas F344 , Acetato de Tetradecanoilforbol/farmacología , Titanio/toxicidad , Zimosan/farmacología
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