RESUMEN
Exposure to xenobiotic such as benzo[a]pyrene (B[a]P) induces metabolic changes, which have a considerable impact on the cellular response. Nevertheless, we are just in the beginning to reach an understanding of these processes. In this study, a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach was applied to distinguish the metabolic changes that bladder epithelia cells undergo upon B[a]P exposure. To closely reflect the epithelia cell conditions in vivo, freshly isolated primary porcine urinary bladder epithelial cells (PUBEC) were utilized for the current study. An untargeted metabolomics approach was used to characterize the time- (6 h, 24 h, 48 h) and dose-dependent (0.5 µM, 5 µM, 10 µM B[a]P) changes in the metabolome of PUBEC upon B[a]P exposure, which led to the profiling of more than 200 metabolites that differed significantly between control and exposed samples. Multivariate analysis of the data highlighted that in the experimental setup/model used other than the exposure concentration, it is the exposure time which seems to be most important for distinguishing between different groups and hence may have a bigger role in B[a]P-mediated toxicity but may be specific for cell model used and hence requires further investigations. Further, enrichment and pathway analysis using MetaboAnalyst highlighted that exposure to B[a]P mainly alters the cellular amino acid metabolism. Particularly, 1-pyrroline-5-carboxylic acid (P5C), an intermediate of the cycling of the amino acid proline, was identified as a differentially altered metabolite at all concentrations and exposure times used in the experiment. An increase in the activity of proline dehydrogenase/proline oxidase (PRODH/POX), which oxidizes proline to P5C, was also observed, further supporting our metabolomic data. Our findings contribute to an improved knowledge about the reprogramming of metabolism which is a fundamental element of the cellular response to B[a]P and draw attention to the role of proline in this context.
Asunto(s)
Benzo(a)pireno/toxicidad , Contaminantes Ambientales/toxicidad , Células Epiteliales/efectos de los fármacos , Metaboloma/efectos de los fármacos , Prolina/metabolismo , Vejiga Urinaria/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Metabolómica , Cultivo Primario de Células , Prolina Oxidasa/metabolismo , Porcinos , Factores de Tiempo , Vejiga Urinaria/metabolismoRESUMEN
A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.
Asunto(s)
Calcio/metabolismo , Contaminantes Ambientales/toxicidad , Hierro/metabolismo , Óxido Nítrico/fisiología , Dibenzodioxinas Policloradas/toxicidad , Proteoma/metabolismo , Calmodulina/metabolismo , Fraccionamiento Celular , Línea Celular , Núcleo Celular/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Homeostasis , Humanos , Electroforesis en Gel de Poliacrilamida Nativa , Proteómica , Vejiga Urinaria/citologíaRESUMEN
Epidemiological studies suggest that environmental exposure to airborne particulate matter may promote cardiovascular diseases; however, it is not clear whether this observation actually reflects exposure to nanosized particles in the environment. In the present study, the human endothelial cell line EA.hy926 was exposed to pure carbon black and, to mimic exposure to diesel exhaust, carbon black loaded with benzo[a]pyrene to ascertain effects of these particles on the cell proteome and metabolom. Particular emphasis was laid on an extended exposure period (14 days) and a low particle concentration (100 ng/mL). While ROS production essentially remained unaffected, exposure of the cells to the particles resulted in a significantly enhanced cell proliferation. Evaluation of the obtained proteomic and phosphoproteomic data revealed modulations of proteins involved in catalytic processes and cytoskeleton maintenance. The bioinformatic evaluation of the data revealed the possible involvement of the transcription factor peroxisome proliferator-activated receptor gamma. The further analysis of the cytoskeleton indicated changes of the cell motility, which is in agreement with an observed increase in the cellular migration and invasion, and macroscopic changes of the cytoskeleton of the exposed cells.
Asunto(s)
Benzo(a)pireno/toxicidad , Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Metabolómica , Proteómica , Hollín/toxicidad , Calcio/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Biología Computacional , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patología , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Metabolómica/métodos , Estrés Oxidativo/efectos de los fármacos , Proteómica/métodos , Especies Reactivas de Oxígeno/metabolismo , Integración de Sistemas , Factores de TiempoRESUMEN
There is a need of guidance on how local irritancy data should be incorporated into risk assessment procedures, particularly with respect to the derivation of occupational exposure limits (OELs). Therefore, a board of experts from German committees in charge of the derivation of OELs discussed the major challenges of this particular end point for regulatory toxicology. As a result, this overview deals with the question of integrating results of local toxicity at the eyes and the upper respiratory tract (URT). Part 1 describes the morphology and physiology of the relevant target sites, i.e., the outer eye, nasal cavity, and larynx/pharynx in humans. Special emphasis is placed on sensory innervation, species differences between humans and rodents, and possible effects of obnoxious odor in humans. Based on this physiological basis, Part 2 describes a conceptual model for the causation of adverse health effects at these targets that is composed of two pathways. The first, "sensory irritation" pathway is initiated by the interaction of local irritants with receptors of the nervous system (e.g., trigeminal nerve endings) and a downstream cascade of reflexes and defense mechanisms (e.g., eyeblinks, coughing). While the first stages of this pathway are thought to be completely reversible, high or prolonged exposure can lead to neurogenic inflammation and subsequently tissue damage. The second, "tissue irritation" pathway starts with the interaction of the local irritant with the epithelial cell layers of the eyes and the URT. Adaptive changes are the first response on that pathway followed by inflammation and irreversible damages. Regardless of these initial steps, at high concentrations and prolonged exposures, the two pathways converge to the adverse effect of morphologically and biochemically ascertainable changes. Experimental exposure studies with human volunteers provide the empirical basis for effects along the sensory irritation pathway and thus, "sensory NOAEChuman" can be derived. In contrast, inhalation studies with rodents investigate the second pathway that yields an "irritative NOAECanimal." Usually the data for both pathways is not available and extrapolation across species is necessary. Part 3 comprises an empirical approach for the derivation of a default factor for interspecies differences. Therefore, from those substances under discussion in German scientific and regulatory bodies, 19 substances were identified known to be human irritants with available human and animal data. The evaluation started with three substances: ethyl acrylate, formaldehyde, and methyl methacrylate. For these substances, appropriate chronic animal and a controlled human exposure studies were available. The comparison of the sensory NOAEChuman with the irritative NOAECanimal (chronic) resulted in an interspecies extrapolation factor (iEF) of 3 for extrapolating animal data concerning local sensory irritating effects. The adequacy of this iEF was confirmed by its application to additional substances with lower data density (acetaldehyde, ammonia, n-butyl acetate, hydrogen sulfide, and 2-ethylhexanol). Thus, extrapolating from animal studies, an iEF of 3 should be applied for local sensory irritants without reliable human data, unless individual data argue for a substance-specific approach.
Asunto(s)
Contaminantes Ocupacionales del Aire/toxicidad , Irritantes/toxicidad , Exposición Profesional/análisis , Contaminantes Ocupacionales del Aire/química , Contaminantes Ocupacionales del Aire/farmacocinética , Ojo/efectos de los fármacos , Humanos , Irritantes/química , Irritantes/farmacocinética , Nivel sin Efectos Adversos Observados , Exposición Profesional/efectos adversos , Sistema Respiratorio/efectos de los fármacos , Medición de Riesgo , Umbral Sensorial , Solubilidad , Valores Limites del UmbralRESUMEN
The recent introduction of the La(3+) precipitation method for the enrichment of phosphoproteins allows a gel-based analysis of these posttranslationally modified proteins. However, if this method is applied to cell lysates stored in urea-containing lysis buffer for an extended period of time, incomplete phosphoprotein recovery is observed. We ascribe this effect to the presence of urea in the lysis buffer. To overcome this problem various strategies were tested, where cell lysates stored at least for one year were utilized. By applying an optimized protocol approximately 250 proteins could be observed following separation by 2DE.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Lantano/química , Fosfoproteínas/aislamiento & purificación , Tiourea/química , Urea/química , Precipitación Química , Células Endoteliales/química , Humanos , Concentración de Iones de Hidrógeno , Fosfoproteínas/análisis , Replegamiento ProteicoRESUMEN
Benzo[a]pyrene (BaP), a five-ring polycyclic aromatic hydrocarbon, is a well-recognized environmental pollutant. Coal-processing waste products, petroleum sludge, asphalt, creosote, and tobacco smoke, all contain high levels of BaP. Exposure to BaP elicits many adverse biological effects, including tumor formation, immunosuppression, teratogenicity, and hormonal effects. In addition to the genetic damage caused by BaP exposure, several studies have indicated the disruption of protein-protein signaling pathways. However, contrary to the large number of studies on BaP-induced DNA damage, only few data have been gathered on its effects at the protein level. This review highlights all proteomic studies to date used for assessing the toxicity of BaP and its metabolites in various organ systems. It will also give an overview on the role proteomics may play to elucidate the mechanisms underlying BaP toxicity.
Asunto(s)
Benzo(a)pireno/metabolismo , Benzo(a)pireno/toxicidad , Contaminantes Ambientales/toxicidad , Animales , Benzo(a)pireno/química , Daño del ADN , Exposición a Riesgos Ambientales , Peces , Humanos , Ratones , Órganos en Riesgo , Proteómica , RatasRESUMEN
More than 90 % of all bladder cancers are transitional cell carcinomas arising from the cells lining the inside of the hollow organ (uroepithelium). Cell cultures from primary urinary bladder epithelial cells (PUBEC) of pigs were established to assess the uptake, intracellular concentration, and subcellular distribution of the environmental pollutant benzo(a)pyrene (BaP). During treatment of the cells with 0.5 µM BaP for up to 24 h, intracellular concentration of BaP increased without saturation but with marked differences between various PUBEC pools. Analysis of BaP uptake by laser scanning microscopy indicated that BaP is rapidly partitioned into the cell membrane, while only a slight but significant increase in BaP fluorescence intensity was observed in the cytosol and nucleus. Spectrofluorometric quantification of BaP in PUBEC using ex situ calibration revealed a strong accumulation of BaP, leading to intracellular concentrations ranging from 7.28 to 35.70 µM in cells exposed to 0.5 µM BaP and from 29.9 to 406.64 µM in cells exposed to 10 µM BaP. These results were confirmed by gas chromatographic mass spectrometric analysis. Apoptotic cell nuclei were assessed by TUNEL analysis to see whether BaP exposure at the given concentrations results in a toxic effect. While apoptotic cells were barely detectable in control epithelial cells, there was a marked elevation in apoptosis in the BaP-exposed cells. In conclusion, a comprehensive study on uptake and quantification of BaP in epithelial cells from pig bladder is reported for the first time. The study may be helpful in understanding the pattern of BaP uptake and distribution in bladder and its possible implication in bladder cancer development.
Asunto(s)
Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Células Epiteliales/efectos de los fármacos , Urotelio/efectos de los fármacos , Animales , Benzo(a)pireno/metabolismo , Carcinógenos/metabolismo , Línea Celular , Fragmentación del ADN , Cromatografía de Gases y Espectrometría de Masas , Etiquetado Corte-Fin in Situ , Indicadores y Reactivos , Cinética , Microscopía Confocal , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , Porcinos , Urotelio/citologíaRESUMEN
The simultaneous analysis of a wide array of proteins may provide valuable information on the activation and suppression of cellular systems at different stages of the exposure-disease continuum. In this review, results of proteomic studies in the field of toxicology are covered, focusing on the effects of chemical carcinogens. So far, alterations of highly abundant proteins have been identified which, irrespective of the wide differences in study design and technologies used, can be grossly assigned to three functional classes: proteins related to cellular stress response, inflammation, and stimulation of the immune system. It is obvious that the observed protein alterations are not causal factors in the development of chemically induced cancer but rather reflect common reactions to cellular perturbations. In order to gain deeper insights into the process of chemical carcinogenesis, the previously applied "shotgun" analyses have to be abandoned in favour of targeted proteomic approaches focusing on the accurate identification and quantification of selected proteins. Advanced analytical techniques such as selective reaction monitoring (SRM) and multiple reaction monitoring (MRM) offer this opportunity. If toxicoproteomic research moves into that direction and takes advantage of such techniques it will have the potential to contribute to the elucidation of chemical carcinogenesis.
Asunto(s)
Pruebas de Carcinogenicidad/métodos , Carcinógenos/toxicidad , Proteínas de Neoplasias/análisis , Neoplasias/inducido químicamente , Neoplasias/metabolismo , Proteoma/análisis , Proteómica/métodos , Animales , Humanos , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
Of the numerous animal models available for proteomic studies only a small number have been successfully used in understanding human biology. To date, rodents have been widely employed in proteomic and genomic studies but often these models do not truly mimic the relevant human conditions. On the other hand, the pig shows similarity in size, shape and physiology to human and has been used as a major mammalian model for many studies concerning xenotransplantation, cardiovascular diseases, blood dynamics, nutrition, general metabolic functions, digestive-related disorders, respiratory diseases, diabetes, kidney and bladder diseases, organ-specific toxicity, dermatology and neurological sequelae. With the substantially improved knowledge of the structure and function of the pig genome in the last two decades it has been found that this animal shares a high sequence and chromosomal structure homology with humans. Nevertheless, in comparison to other available model organisms, very little work has been devoted to pig proteomics until recently. Keeping this in mind, the present review will highlight some of the advantages and disadvantages of pig as a model system for proteomic studies.
Asunto(s)
Modelos Animales de Enfermedad , Proteómica , Porcinos/fisiología , Animales , Humanos , Porcinos/metabolismoRESUMEN
Epithelial tissue lining the inner side of the urinary bladder is the most common target for bladder cancer-related diseases. Bladders of freshly slaughtered pigs were utilised for a comprehensive analysis of the proteome and phosphoproteome of bladder epithelial cells. Following protein separation by 2-D gel electrophoresis and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) the first proteome and phosphoproteome maps of pig urinary bladder epithelial cells (PUBEC) were established. A total of 120 selected protein spots were identified. By using the La(3+) enrichment method further developed in our laboratory we identified 31 phosphoproteins with minimal contamination by non-phosphopeptides. The 2-DE map of pig urothelial cells may prove as a useful tool for studies on uroepithelial biology, and the analysed phosphoproteins expression pattern, together with the whole cell proteome, will be helpful for identifying the proteins involved in bladder-related diseases.
Asunto(s)
Fosfoproteínas/análisis , Proteoma/análisis , Vejiga Urinaria/química , Urotelio/química , Animales , Células Cultivadas , Electroforesis en Gel Bidimensional , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos , Vejiga Urinaria/citología , Vejiga Urinaria/metabolismo , Urotelio/citología , Urotelio/metabolismoRESUMEN
Various CBB-based methods for staining proteins separated by 2-D gel electrophoresis were compared with regard to sensitivity and resolution. A modified Kang's CBB staining protocol, which we have modified, includes phosphoric acid in a concentration of 8% instead of the original 2%. This proved to be the best approach. Protein amounts as low as 2 ng and approximately 2300 spots in the gel can be detected by employing this protocol. The modified procedure takes less time to carry out. Moreover, this practice is more sensitive and resolves more protein spots than most protocols reported to date and is compatible with subsequent mass spectrometric analysis.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Espectrometría de Masas/métodos , Proteoma/química , Colorantes de Rosanilina/química , Coloración y Etiquetado/métodos , Extractos Celulares/química , Línea Celular Tumoral , Coloides/química , Humanos , Concentración de Iones de Hidrógeno , Ácidos Fosfóricos/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37 degrees C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH(3))(3)Bi] at rather low concentrations of bismuth (0.2 to 1 mumol kg(-1) [dry weight]). An increase of bismuth up to 2 to 14 mmol kg(-1) (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h(-1) kg(-1) (dry weight) to 2,100 pmol h(-1) kg(-1) (dry weight) in human feces samples and from approximately 5 pmol h(-1) kg(-1) (dry weight) to 120 pmol h(-1) kg(-1) (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH(3))(3)Bi.
Asunto(s)
Tracto Gastrointestinal/microbiología , Metales/metabolismo , Adulto , Animales , Biotransformación , Ciego/microbiología , Heces/química , Heces/microbiología , Humanos , Inactivación Metabólica , Intestino Grueso/microbiología , Intestino Delgado/microbiología , Masculino , Ratones , Ratones Endogámicos C3HRESUMEN
Epidemiological studies have indicated that exposure of humans to inorganic arsenic in drinking water is associated with the occurrence of bladder cancer. The mechanisms by which arsenic induces this malignancy are still uncertain; however, arsenic metabolites are suspected to play a pivotal role. The aim of the present study was the investigation of uptake capabilities of human urothelial cells (UROtsa) compared with primary human hepatocytes (phH) as well as the intracellular distribution of the arsenic species. Additionally, we were interested in the cyto- and genotoxic potential (comet assay, radical generation) of the different arsenic compounds in these two cell types. Our results show that UROtsa cells accumulate higher amounts of the arsenic species than the phH. Differential centrifugation revealed that the arsenic compounds are preferentially distributed into nuclei and ribosomes. After 24-h exposure, arsenic is mainly found in the ribosomes of UROtsa cells and in the nuclei and mitochondria of phH. In contrast to the pentavalent arsenic species, the trivalent species induced a 4- to 5-fold increase of DNA damage in hepatocytes. Radical generation, measured by thiobarbituric acid reactive substances, was more pronounced in hepatocytes than in urothelial cells. In summary, the uptake of arsenic compounds appears to be highly dependent upon cell type and arsenic species. The nonmethylating urothelial cells accumulate higher amounts of arsenic species than the methylating hepatocytes. However, cyto- and genotoxic effects are more distinct in hepatocytes. Further studies are needed to define the implications of the observed accumulation in cellular organelles for the carcinogenic activity of arsenic.
Asunto(s)
Arsenicales/metabolismo , Hepatocitos/metabolismo , Urotelio/citología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayo Cometa , Hepatocitos/efectos de los fármacos , Humanos , Fracciones Subcelulares/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
Benzo[a]pyrene (B[a]P), a well-known polyaromatic hydrocarbon, is known for its lung carcinogenicity, however, its role in bladder cancer development is still discussed. Comparative two-dimensional blue native SDS-PAGE analysis of protein complexes isolated from subcellular fractions of 0.5 µM B[a]P-exposed cells indicated a differential regulation of proteins involved in carbohydrate, fatty acid, and nucleotide metabolism, suggesting a possible metabolic flux redistribution. It appeared that B[a]P exposure led to a repression of enzymes (fructose-bisphosphate aldolase A, glucose-6-phosphate isomerase, lactate dehydrogenase) involved in glycolysis, and an up-regulation of proteins (glucose-6-phosphate 1-dehydrogenase, 6-phosphogluconolactonase) catalyzing the pentose phosphate pathway and one carbon metabolism (10-formyltetrahydrofolate dehydrogenase, bifunctional purine biosynthesis protein). Untargeted metabolomics further supported the proteomic data, a lower concentration of glycolytic metabolite was observed as compared to glutamine, xylulose and fatty acids. The analysis of the glutathione and NADPH/NADP+ content of the cells revealed a significant increase of these cofactors. Concomitantly, we did not observe any detectable increase in the production of ROS. With the present work, we shed light on an early phase of the metabolic stress response in which the urothelial cells are capable of counteracting oxidative stress by redirecting the metabolic flux from glycolysis to pentose phosphate pathway.
Asunto(s)
Benzo(a)pireno/farmacología , Metaboloma , Metabolómica , Vía de Pentosa Fosfato/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/metabolismo , Biomarcadores , Línea Celular Tumoral , Daño del ADN , Metabolismo Energético , Glucólisis/efectos de los fármacos , Humanos , Redes y Vías Metabólicas , Metabolómica/métodos , NADP/metabolismo , Estrés Oxidativo , Proteómica/métodos , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Crystalline silica has been classified as a human carcinogen, but there is still considerable controversy regarding its fibrogenic and carcinogenic potential. In the present study, we investigated the genotoxic potential of bentonite particles (diameter < 10 microm) with an a-quartz content of up to 6% and different chemical modifications (alkaline, acidic, organic). Human lung fibroblasts (IMR90) were incubated for 36 h, 48 h, or 72 h with bentonite particles in concentrations ranging from 1 to 15 microg/cm2. Genotoxicity was assessed using the micronucleus (MN) assay and kinetochore analysis. The generation of reactive oxygen species (ROS) caused by bentonite particles via Fenton-like mechanisms was measured acellularly using electron spin resonance (ESR) technique and intracellularly by applying an iron chelator. Our results show that bentonite-induced genotoxic effects in human lung fibroblasts are weak. The formation of micronuclei was only slightly increased after exposure of IMR90 cells to an acidic sample of bentonite dust with a quartz content of 4-5% for 36 h (15 microg/cm2), 48 h (5 microg/cm2), and 72 h (1 microg/cm2), to an alkaline sample with a quartz content of 5% for 48 h and 72 h (15 microg/cm2), and to an acidic bentonite sample with 1% quartz for 72 h (1 microg/cm2). Native (untreated) and organic activated bentonite particles did not show genotoxic effects in most of the experiments. Also, bentonite particles with a quartz content < 1% were negative in the micronucleus assay. Generation of ROS measured by ESR was dependent on the content of transition metals in the sample but not on the quartz content or the chemical modification. Reduction of MN after addition of the iron chelator 2,2'-dipyridyl showed that ROS formation also occurs intracellularly. Altogether, we conclude that the genotoxic potential of bentonite particles is generally low but can be altered by the content of quartz and available transition metals.
Asunto(s)
Bentonita/toxicidad , Cinetocoros , Pulmón/efectos de los fármacos , Pruebas de Micronúcleos , Cuarzo/análisis , Bentonita/análisis , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Fibroblastos/efectos de los fármacos , Humanos , Radical Hidroxilo , Quelantes del Hierro/farmacología , Cinetocoros/efectos de los fármacos , Especies Reactivas de OxígenoRESUMEN
Physiological modifications of intracellular Ca2+ ([Ca2+]i) levels trigger and/or regulate a diversity of cellular activities (e.g. neurotransmitter release, synaptic plasticity, muscular contraction, cell proliferation), while calcium overloads could result in cytotoxicity. Previously, we have shown that trimethyltin chloride (Me3SnCl; TMT) modulates calcium homeostasis in cervix adenocarcinoma (HeLa S3) cells [Florea, A.-M., Dopp, E., Büsselberg, D., 2005. TMT induces elevated calcium transients in HeLa cells: types and levels of response. Cell Calcium 37, 252-258]. Here we compare [Ca2+]i-changes induced by trimethyltin chloride in neuroblastoma SY5Y and HeLa S3 cells using calcium-sensitive dyes (fluo-4/AM (fluo-4) and rhod-2/AM (rhod-2)) and laser scanning microscopy (LSM). TMT-induced calcium elevations in neuroblastoma SY5Y as well as in HeLa S3 cells. [Ca2+]i rose to a sustained plateau or to transient spikes. Overall, the detected averaged increase of the maximum calcium elevation were: 0.5 microM approximately 125.6%; 5 microM approximately 130.1%; 500 microM approximately 145% in HeLa S3 cells and 0.5 microM approximately 133.3%; 5 microM approximately 136.1%; 500 microM approximately 147.1% in neuroblastoma SY5Y cells. The calcium rise derived from internal stores did not significantly depend on the presence of calcium in the external solution: approximately 109% (no calcium added) versus approximately 117% (2 mM calcium; 5 microM TMT) in HeLa cells. This difference was similar in neuroblastoma SY5Y cells, were approximately 127% versus approximately 136% increase (5 microM TMT) were measured. Staining of calcium stores with rhod-2 showed a TMT-induced [Ca2+]i-decrease in the stores followed by an increase of the calcium concentration in the nuclei of the two cell lines tested. Our results suggest that toxic effects in human tumour cells after exposure to trimethyltin compounds might be due to an elevation of [Ca2+]i.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Homeostasis/efectos de los fármacos , Espacio Intracelular/metabolismo , Compuestos de Trimetilestaño/toxicidad , Adenocarcinoma/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Humanos , Espacio Intracelular/efectos de los fármacos , Microscopía Confocal , Neuroblastoma/metabolismoRESUMEN
Phthalic acid esters such as di(2-ethylhexyl)phthalate (DEHP) are widely used as plasticizers in PVC products manufactured for commercial, medical, and consumer purposes. Humans are exposed to phthalates originating, e.g., from blood storage bags, tubing materials, and from food-wrapping. While xenoestrogenic and chronic toxic effects of phthalates have been extensively discussed, there is little data on genotoxic effects in human cells. The alkaline comet assay was used to detect single-strand breaks and alkali labile sites of DNA after incubation of human nasal mucosal cells (n = 11) and peripheral lymphocytes (n = 11) with mono(2-ethylhexyl)phthalate (MEHP), the principal hydrolysis product of DEHP. MEHP showed a dose-dependent enhancement of DNA migration both in human mucosal cells and in lymphocytes. This effect indicates a genotoxic potential of MEHP in human mucosal cells. It confirms previous data obtained on the effect of MEHP on lymphocytes.
Asunto(s)
Daño del ADN , Ácidos Ftálicos/toxicidad , Adulto , Ensayo Cometa , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Femenino , Humanos , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Pruebas de Mutagenicidad , Mucosa Nasal/citología , Mucosa Nasal/efectos de los fármacosRESUMEN
The carcinogenic potential of xenobiotics and possible confounders are often difficult to differentiate in in vivo studies. In contrast, in vitro studies allow investigation of the impact of carcinogens on human target cells under standardized conditions. The aim of the present study is to demonstrate whether three-dimensional mini organ-cultures (MOCs) of human inferior nasal turbinate epithelia may represent a useful model to study genotoxic effects of xenobiotics in vitro. Culture of mini organs was performed by cutting 1mm3 pieces from fresh specimens of inferior nasal turbinates. After a period of 5-6 days the specimens were fully covered with epithelium. On days 7, 9, and 11 of culture, intact MOCs from 25 tissue donors were incubated with dimethyl sulfoxide (DMSO) as a negative control, or with mono(2-ethylhexyl) phthalate (MEHP), benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). On days 7 and 11, MOCs were analyzed by the alkaline Comet assay to detect DNA-single-strand breaks, alkali-labile sites and incomplete excision-repair sites. DNA migration after single exposure of non-cultivated fresh specimens was also analyzed. In order to detect regimen-specific effects, DNA fragmentation after single exposure of intact MOCs was compared with that of cells after separation of MOCs on day 7 of culture and consecutive exposure of individual cells. Significant DNA migration as a measure of DNA single-strand breaks, alkali-labile sites and incomplete excision repair sites, was found after electrophoresis due to single and triple exposure of MOCs to MEHP, BPDE and MNNG. Triple exposure of MOCs compared to single exposure revealed no difference after exposure to DMSO or MEHP, and an increased migration after exposure to BPDE and MNNG. When single exposure of isolated cells from fresh specimens was compared with that of intact MOCs, DMSO and MNNG had no significantly different effect, whereas exposure to MEHP or BPDE caused a reduced migration in cells from MOCs. When exposure of isolated cells harvested from MOCs was compared with exposure of intact MOCs, MEHP and BPDE caused a significantly lower DNA migration in intact MOCs. MOCs provide an in vitro model suitable for the assessment of genotoxic effects of environmental pollutants both after single or repetitive exposure. Due to the intact structure of the exposed mucosa this model may be a helpful tool in mimicking the in vivo situation in ecogenotoxicology studies.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Daño del ADN/efectos de los fármacos , Dietilhexil Ftalato/análogos & derivados , Dietilhexil Ftalato/toxicidad , Metilnitronitrosoguanidina/toxicidad , Cornetes Nasales/citología , Adulto , Ensayo Cometa , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Masculino , Técnicas de Cultivo de ÓrganosRESUMEN
The studies described in this paper deal with a sequence of cellular events induced by the environmental toxicant benzo[a]pyrene (B[a]P) that were investigated in primary urinary bladder epithelia cells (PUBEC) from pigs by using a proteomic approach. Two-dimensional (2DE) gel electrophoresis unveiled the differences in protein expression between cells exposed to 0.5 µM B[a]P for 24 h and control cells. Twenty-five differentially expressed proteins involved in DNA repair, mitochondrial dysfunction, and apoptosis were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). These findings were supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by a time-dependent increase in histone H2A.x (H2AX) phosphorylation upon B[a]P exposure. On the other hand, the expression of voltage-dependent anion channel 2 (VDAC2), cathepsin D (CTSD), heat shock protein 27 (HSP27), and heat shock protein 70 (HSP70) hinted to apoptosis occurring through the intrinsic apoptotic mitochondrial pathway. Taken together, these data suggest that B[a]P is capable of inducing DNA damage in urinary bladder epithelial cells at low concentrations during a short exposure period, thus eventually leading to cell death by apoptosis. BIOLOGICAL SIGNIFICANCE: Epidemiological studies have indicated PAHs as potential candidates for initiating bladder cancer development, although the precise risk is still unknown (Kaufman et al. (2009)). In recent years, the understanding of the metabolic capacity of urothelial cells has broadened continuously; i.e. a wide range of xenobiotic metabolizing cytochrome P450 enzymes (CYP) were detected in urothelial cells from humans and animals (Roos et al., 2006; Guhe et al., 1996), thus indicating that urothelial cells are not only passively exposed to reactive metabolites but also actively by intracellularly producing reactive intermediates that can induce cancer. Moreover, small quantities of non-metabolized B[a]P and its hydroxylated derivatives have been identified in blood and urine (Rossella et al. (2009)). Thus, it appears plausible that B[a]P, a highly lipophilic compound, is taken up by the urothelium and metabolically activated to carcinogenic intermediates in these cells. In our previous studies with primary uroepithelial cells isolated from freshly slaughtered pigs we demonstrated the ability of these cells for a strong uptake of B[a]P and its conversion to the oxidative metabolite (3-OH-B[a]P) (Verma et al. (2012)). The present study is a continuation of this previous work exhibiting the effects of B[a]P exposure on cellular functions of PUBEC. The results indicated caspase-dependent apoptosis induced by B[a]P due to DNA damage (possibly lethal double-strand breaks as indicated by H2AX phosphorylation). Taken together, these studies provide strong evidence for the ability of B[a]P to act as a bladder carcinogen.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzo(a)pireno/toxicidad , Daño del ADN , Células Epiteliales/metabolismo , Proteoma/metabolismo , Vejiga Urinaria/metabolismo , Animales , Catepsina D/metabolismo , Células Cultivadas , Células Epiteliales/patología , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Histonas/metabolismo , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos , Factores de Tiempo , Vejiga Urinaria/patología , Canal Aniónico 2 Dependiente del Voltaje/metabolismoRESUMEN
Posttranslational modification (PTM) of proteins, particularly phosphorylation, is a key element in the regulation of cell functions. In many signal transduction processes, PTM is a pivotal step. Various analytical methods have been proposed for the identification of phosphoproteins; however, most of these methods require sophisticated equipment. Here we present an easily applicable method of phosphoprotein enrichment. This method is based on single-step precipitation by lanthanum chloride and allows subsequent protein identification by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF-MS). The method proved its suitability for the isolation of phosphoproteins from frozen tissue and cultured cells samples after cell lysis in various buffer systems (urea/thiourea and EGTA/EDTA). The tests revealed that the isolation of phosphoproteins can be achieved with high efficiency even from complex protein mixtures. Our results indicate that lanthanum-based enrichment of phosphoproteins can be a useful tool in phosphoproteomic studies.