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Dichlorodiphenyldichloroethylene (DDE), the primary persistent metabolite of dichlorodiphenyltrichloroethane (DDT), has toxic effects on cells, but its dose-dependent impact on mitochondrial proteins involved in mitochondrial fusion and fission processes associated with cell viability impairment has not yet been analysed. Mitochondrial fusion and fission processes are critical to maintaining the mitochondrial network and allowing the cell to respond to external stressors such as environmental pollutants. Fusion processes are associated with optimizing mitochondrial function, whereas fission processes are associated with removing damaged mitochondria. We assessed the effects of different DDE doses, ranging between 0.5 and 100 µM, on cell viability and mitochondrial fusion/fission proteins in an in vitro hepatic cell model (human hepatocarcinomatous cells, HepG2); the DDE induced a decrease in cell viability in a dose-dependent manner, and its effect was enhanced in conditions of coincubation with dietary fatty acids. Fusion protein markers exhibited an inverted U-shape dose-response curve, showing the highest content in the 2.5-25 µM DDE dose range. The fission protein marker was found to increase significantly, leading to an increased fission/fusion ratio with high DDE doses. The low DDE doses elicited cell adaption by stimulating mitochondrial dynamics machinery, whereas high DDE doses induced cell viability loss associated with mitochondrial dynamics to shift toward fission. Present results are helpful to clarify the mechanisms underlying the cell fate towards survival or death in response to increasing doses of environmental pollutants.
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Mitochondrial impairments in dynamic behavior (fusion/fission balance) associated with mitochondrial dysfunction play a key role in cell lipotoxicity and lipid-induced metabolic diseases. The present work aimed to evaluate dose- and time-dependent effects of the monounsaturated fatty acid oleate on mitochondrial fusion/fission proteins in comparison with the saturated fatty acid palmitate in hepatic cells. To this end, HepG-2 cells were treated with 0, 10 µM, 50 µM, 100 µM, 250 µM or 500 µM of either oleate or palmitate for 8 or 24 h. Cell viability and lipid accumulation were evaluated to assess lipotoxicity. Mitochondrial markers of fusion (mitofusin 2, MFN2) and fission (dynamin-related protein 1, DRP1) processes were evaluated by Western blot analysis. After 8 h, the highest dose of oleate induced a decrease in DRP1 content without changes in MFN2 content in association with cell viability maintenance, whereas palmitate induced a decrease in cell viability associated with a decrease mainly in MFN2 content. After 24 h, oleate induced MFN2 increase, whereas palmitate induced DRP1 increase associated with a higher decrease in cell viability with high doses compared to oleate. This finding could be useful to understand the role of mitochondria in the protective effects of oleate as a bioactive compound.
Asunto(s)
Dinaminas/genética , GTP Fosfohidrolasas/genética , Enfermedades Metabólicas/genética , Dinámicas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/genética , Ácido Oléico/metabolismo , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ácidos Grasos Monoinsaturados/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/toxicidad , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/patología , Dinámicas Mitocondriales/genética , Ácido Oléico/farmacología , Palmitatos/metabolismo , Palmitatos/farmacologíaRESUMEN
4-Nonylphenol (4-NP) is an emerging environmental pollutant widely diffused in waters and sediments. It mainly derives from the degradation of alkyl phenol ethoxylates, compounds commonly employed as industrial surfactants. 4-NP strongly contaminates foods and waters for human use; thus, it displays a wide range of toxic effects not only for aquatic organisms but also for mammals and humans. After ingestion through the diet, it tends to accumulate in body fluids and tissues. One of the main organs where 4-NP and its metabolites are concentrated is the liver, where it causes, even at low doses, oxidative stress and apoptosis. In the present study, we analyzed the effects of 4-NP on a human hepatic cell line (HepG2) to deepen the knowledge of its cytotoxic mechanism. We found that 4-NP, in a range of concentration from 50 to 100 µM, significantly reduced cell viability; it caused a partial block of proliferation and induced apoptosis with activation of caspase-3 and overexpression of p53. Moreover, 4-NP induced-apoptosis seemed to involve both an ER-stress response, with the appearance of high level of GRP78, CHOP and the spliced XBP1, and a dysregulation of mitochondrial physiology, characterized by an overexpression of main markers of mitochondrial dynamics. Our data support the idea that a daily consumption of 4-NP-contaminated foods may lead to local damages at the level of gastrointestinal system, including liver, with negative consequences for the organ physiology.
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Apoptosis/efectos de los fármacos , Citotoxinas/toxicidad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Fenoles/toxicidad , Chaperón BiP del Retículo Endoplásmico , Células Hep G2 , Humanos , Hígado/patología , Mitocondrias Hepáticas/patologíaRESUMEN
1,1,1-trichloro-2,2-bis (p-chlorophenyl)-ethane (DDT) and its main metabolite 1,1-Dichloro-2,2-bis (p, p'-chlorophenyl) ethylene (DDE) act as endocrine disruptors in humans and wildlife. Immunomodulatory functions have also been attributed to both xenobiotics. DDT was banned in the 1970s due to its toxicity, but it is still produced and used for indoor residual spraying with disease vector control purposes. Due to their persistence and lipophilic properties, DDT and DDE can bioaccumulate through the food chain, being stored in organisms' adipose depots. Their endocrine disruptor function is mediated by agonist or antagonist interaction with nuclear receptors. Present review aimed to provide an overview of how DDT and DDE exposure impacts reproductive and immune systems with estrogen-disrupting action in humans and wildlife. Studies showing DDT and DDE impact on mitochondrial function and apoptosis pathway will also be reviewed, suggesting the hypothesis of direct action on mitochondrial steroid receptors.
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DDT/toxicidad , Diclorodifenil Dicloroetileno/toxicidad , Disruptores Endocrinos/toxicidad , Contaminantes Ambientales/toxicidad , Insecticidas/toxicidad , Mitocondrias/efectos de los fármacos , Animales , Animales Salvajes , Humanos , Mitocondrias/metabolismo , Receptores de Esteroides/metabolismoRESUMEN
Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the α-gliadin peptide 31-43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31-43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31-43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31-43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis.
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Enfermedad Celíaca/enzimología , Enfermedad Celíaca/metabolismo , Proteínas de Unión al GTP/metabolismo , Transglutaminasas/metabolismo , Adolescente , Adulto , Anticuerpos , Autoanticuerpos/inmunología , Enfermedad Celíaca/inmunología , Dieta Sin Gluten , Fibroblastos/metabolismo , Gliadina/inmunología , Voluntarios Sanos , Humanos , Péptidos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Piel/metabolismo , Adulto JovenRESUMEN
Auto-antibodies to the ubiquitous enzyme type-2 transglutaminase (TG2) are a specific hallmark of celiac disease (CD), a widely diffused, multi-factorial disease, affecting genetically predisposed subjects. In CD an inflammatory response, at the intestinal level, is triggered by diet consumption of gluten-containing cereals. Intestinal mucosa displays various degrees of atrophy and hyperplasia, with consequent global intestinal dysfunction and other relevant extra-intestinal symptoms. Through deamidation of specific glutamines of gluten-derived gliadin peptides, TG2 strongly enhances gliadin immunogenicity. In addition, TG2 cross-linking activity may generate complexes between TG2 itself and gliadin peptides, and these complexes seem to cause the auto-immune response by means of an apten-carrier-like mechanism of antigen presentation. Anti-TG2 antibodies can be early detected in the intestinal mucosa of celiac patients and are also abundantly present into the serum, thus potentially reaching other organs and tissues by blood circulation. Recently, the possible pathogenetic role of auto-antibodies to TG2 in CD has been investigated. Here, we report an overview about the genesis of these antibodies, their specificity, their modulating ability toward TG2 enzymatic or non-enzymatic activities and their biological effects exerted by interacting with extracellular TG2 or with cell-surface TG2. We also discuss the auto-immune response occurring in CD against other TG members (i.e. type 3 and type 6) and analyze the occurrence of anti-TG2 antibodies in other auto-immune CD-related diseases. Data now available let us to suppose that, even if antibodies to TG2 do not represent the triggering molecules in CD, they could be important players in disease progression and manifestations.
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Autoanticuerpos/metabolismo , Enfermedad Celíaca/patología , Proteínas de Unión al GTP/inmunología , Transglutaminasas/inmunología , Animales , Autoanticuerpos/inmunología , Biocatálisis , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/metabolismo , Epítopos/inmunología , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Gliadina/química , Gliadina/metabolismo , Humanos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/química , Transglutaminasas/metabolismoRESUMEN
Mitochondria play a crucial role in energetic metabolism, signaling pathways, and overall cell viability. They are in the first line in facing cellular energy requirements in stress conditions, such as in response to xenobiotic exposure. Recently, a novel regulatory key role of microRNAs (miRNAs) in important signaling pathways in mitochondria has been proposed. Consequently, alteration in miRNAs expression by xenobiotics could outcome into mitochondrial dysfunction, reactive oxygen species overexpression, and liberation of apoptosis or necrosis activating proteins. The aim of this review is to show the highlights about mitochondria-associated miRNAs in cellular processes exposed to xenobiotic stress in different cell types involved in detoxification processes or sensitive to environmental hazards in marine sentinel organisms and mammals.
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Monitoreo del Ambiente , Mamíferos/fisiología , MicroARNs , Mitocondrias/efectos de los fármacos , Xenobióticos/toxicidad , Animales , Mitocondrias/fisiología , Estrés Oxidativo , Especies Reactivas de Oxígeno , Especies CentinelaRESUMEN
The presence of illicit drugs in the aquatic environment represents a new potential risk for aquatic organisms, due to their constant exposure to substances with strong pharmacological activity. Currently, little is known about the ecological effects of illicit drugs. The aim of this study was to evaluate the influence of environmental concentrations of cocaine, an illicit drug widespread in surface waters, on the skeletal muscle of the European eel (Anguilla anguilla). The skeletal muscle of silver eels exposed to 20â¯ngâ¯L-1 of cocaine for 50â¯days were compared to control, vehicle control and two post-exposure recovery groups (3 and 10â¯days after interruption of cocaine). The eels general health, the morphology of the skeletal muscle and several parameters indicative of the skeletal muscle physiology were evaluated, namely the muscle whole protein profile, marker of the expression levels of the main muscle proteins; cytochrome oxidase activity, markers of oxidative metabolism; caspase-3, marker of apoptosis activation; serum levels of creatine kinase, lactate dehydrogenase and aspartate aminotransferase, markers of skeletal muscle damages. Cocaine-exposed eels appeared hyperactive but they showed the same general health status as the other groups. In contrast, their skeletal muscle showed evidence of serious injury, including muscle breakdown and swelling, similar to that typical of rhabdomyolysis. These changes were still present 10â¯days after the interruption of cocaine exposure. In fact, with the exception of the expression levels of the main muscle proteins, which remained unchanged, all the other parameters examined showed alterations that persisted for at least 10â¯days after the interruption of cocaine exposure. This study shows that even low environmental concentrations of cocaine cause severe damage to the morphology and physiology of the skeletal muscle of the silver eel, confirming the harmful impact of cocaine in the environment that potentially affects the survival of this species.
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Anguilla/fisiología , Cocaína/toxicidad , Músculo Esquelético/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Aspartato Aminotransferasas , Cocaína/análisis , L-Lactato Deshidrogenasa , Pruebas de ToxicidadRESUMEN
Mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been suggested to play a key role in insulin resistance development. Reactive oxygen species (ROS) production and lipid accumulation due to mitochondrial dysfunction seemed to be important mechanisms leading to cellular insulin resistance. Moreover, mitochondria are functionally and structurally linked to ER, which undergoes stress in conditions of chronic overnutrition, activating the unfolded protein response, which in turn activates the principal inflammatory pathways that impair insulin action. Among the nutrients, dietary fats are believed to play key roles in insulin resistance onset. However, not all dietary fats exert the same effects on cellular energy metabolism. Dietary omega 3 polyunsaturated fatty acids (PUFA) have been suggested to counteract insulin resistance development by modulating mitochondrial bioenergetics and ER stress. In the current review, we summarized current knowledge on the role played by mitochondrial and ER stress in inflammation and insulin resistance onset, focusing on the modulation role of omega 3 PUFA on these stress pathways. Understanding the mechanisms by which omega 3 PUFA modulates cellular metabolism and insulin resistance in peripheral tissues may provide additional details on the potential impact of omega 3 PUFA on metabolic function and the management of insulin resistance in humans.
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Estrés del Retículo Endoplásmico/efectos de los fármacos , Ácidos Grasos Omega-3/metabolismo , Resistencia a la Insulina/fisiología , Mitocondrias/metabolismo , Animales , Estrés del Retículo Endoplásmico/fisiología , HumanosRESUMEN
Brown adipose tissue (BAT) with its thermogenic function due to the presence of the mitochondrial uncoupling protein 1 (UCP1), has been positively associated with improved resistance to obesity and metabolic diseases. During recent years, the potential influence of environmental pollutants on energetic homoeostasis and obesity development has drawn increased attention. The purpose of this review is to discuss how regulation of BAT function could be involved in the environmental pollutant effect on body energy metabolism. We mainly focused in reviewing studies on animal models, which provide a better insight into the cellular mechanisms involved in this effect on body energy metabolism. The current literature supports the hypothesis that some environmental pollutants, acting as endocrine disruptors (EDCs), such as dichlorodiphenyltrichoroethane (DDT) and its metabolite dichlorodiphenylethylene (DDE) as well as some, traffic pollutants, are associated with increased obesity risk, whereas some other chemicals, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), had a reverse association with obesity. Noteworthy, the EDCs associated with obesity and metabolic disorders impaired BAT mass and function. Perinatal exposure to DDT impaired BAT thermogenesis and substrate utilization, increasing susceptibility to metabolic syndrome. Ambient particulate air pollutions induced insulin resistance associated with BAT mitochondrial dysfunction. On the other hand, the environmental pollutants (PFOS/PFOA) elicited a reduction in body weight and adipose mass associated with upregulation of UCP1 and increased oxidative capacity in brown-fat mitochondria. Further research is needed to better understand the physiological role of BAT in response to exposure to both obesogenic and anti-obesogenic pollutants and to confirm the same role in humans.
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Alpha-gliadin peptide 31-43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31-43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31-43 internalization by Caco-2 cells. In this study, we used a chemical cross-linker to block peptide 31-43 on cell surface proteins, and pulled-down peptide-proteins complexes using antibodies raised against peptide 31-43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31-43 in Coomassie-stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31-43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31-43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31-43 is able to interact with a membrane-like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31-43 internalization by cells.
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Endocitosis/fisiología , Gliadina/metabolismo , Anticuerpos/inmunología , Células CACO-2/metabolismo , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/fisiopatología , Recuento de Células , Proteínas de Unión al GTP , Gliadina/toxicidad , Células HEK293/metabolismo , Humanos , Inmunidad Innata/inmunología , Inmunidad Innata/fisiología , Mucosa Intestinal/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Receptores de Superficie Celular/fisiología , TransglutaminasasRESUMEN
pH-sensitive vesicles used as drug delivery systems (DDSs) are generally composed of protonable copolymers. The disaggregation of these nanoparticles (NPs) during drug release implies the dispersion of positively charged cytotoxic polyelectrolytes in the human body. To alleviate such issue, we synthesised A(BC)n amphiphilic block copolymers with linear (n = 1) and branched (n = 2) architectures to obtain pH-sensitive vesicles capable of releasing drugs in acidic conditions via controlled swelling instead of disaggregation. We obtained this feature by fine-tuning the relative amount of pH-sensitive and hydrophobic monomers. We studied pH-driven swelling by measuring NPs size in neutral and acidic conditions, the latter typical of tumours or inflamed tissues (pHâ¼6) and lysosomes (pHâ¼4.5). Dynamic light scattering (DLS) and zeta potential data provided useful indications about the influence of architecture and chemical composition on NPs swelling, stability and polycation release. Results demonstrated that vesicles made of linear copolymers with â¼22-28% in mol of protonable monomers in the 'BC' block swelled more than other species following a pH change from pH 7.4 to pH 4.5. We finally evaluated the cytotoxicity of vesicles composed of linear species, and paclitaxel (PTX) release from the latter in both cancer and normal cells.
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Antineoplásicos Fitogénicos/química , Paclitaxel/química , Polietilenglicoles/química , Polímeros/química , Antineoplásicos Fitogénicos/metabolismo , Antineoplásicos Fitogénicos/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Paclitaxel/metabolismo , Paclitaxel/farmacología , Polietilenglicoles/metabolismo , Polietilenglicoles/farmacología , Polímeros/metabolismo , Polímeros/farmacologíaRESUMEN
Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.
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Autoanticuerpos/farmacología , Enfermedad Celíaca/inmunología , Fibroblastos/efectos de los fármacos , Proteínas de Unión al GTP/inmunología , Gliadina/farmacología , Fragmentos de Péptidos/farmacología , Transglutaminasas/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Transporte Biológico , Estudios de Casos y Controles , Enfermedad Celíaca/genética , Enfermedad Celíaca/patología , Dermis/citología , Dermis/efectos de los fármacos , Dermis/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas de Unión al GTP/genética , Expresión Génica , Gliadina/síntesis química , Glútenes/química , Glútenes/inmunología , Voluntarios Sanos , Humanos , Masculino , Fragmentos de Péptidos/síntesis química , Cultivo Primario de Células , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/genéticaRESUMEN
The release of antibiotics into the environment can result in antibiotic resistance (AR) spread, which in turn can seriously affect human health. Antibiotic resistant bacteria have been detected in different aquatic environments used as drinking water source. Water disinfection may be a possible solution to minimize AR spread but conventional processes, such as chlorination, result in the formation of dangerous disinfection by-products. In this study advanced oxidation processes (AOPs), namely H2O2/UV, TiO2/UV and N-TiO2/UV, have been compared with chlorination in the inactivation of an AR Escherichia coli (E. coli) strain in surface water. TiO2 P25 and nitrogen doped TiO2 (N-TiO2), prepared by sol-gel method at two different synthesis temperatures (0 and -20°C), were investigated in heterogeneous photocatalysis experiments. Under the investigated conditions, chlorination (1.0 mg L(-1)) was the faster process (2.5 min) to achieve total inactivation (6 Log). Among AOPs, H2O2/UV resulted in the best inactivation rate: total inactivation (6 Log) was achieved in 45 min treatment. Total inactivation was not observed (4.5 Log), also after 120 min treatment, only for N-doped TiO2 synthesized at 0°C. Moreover, H2O2/UV and chlorination processes were evaluated in terms of cytotoxicity potential by means of 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenylte-trazolium colorimetric test on a human-derived cell line and they similarly affected HepG2 cells viability.
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Desinfección/métodos , Escherichia coli/fisiología , Purificación del Agua/métodos , Farmacorresistencia Microbiana , Halogenación , Peróxido de Hidrógeno , Oxidación-Reducción , Rayos UltravioletaRESUMEN
The aim of this study was to verify if the freshwater safety values established from the European Community (1998) and the Italian Ministry of Health (2001) for cadmium (44.5nM/L in drinking water and 178nM/L in sewage waters) were safe for amphibians, since at these same concentrations cadmium induced endocrine disruption in the newt Triturus carnifex. Adult male specimens of T. carnifex were exposed daily to cadmium (44.5nM/L and 178nM/L as CdCl2, nominal concentrations), respectively, during 3- and 9-months; at the same time, control newts were exposed to tap water only. The accumulation of cadmium in the skin, liver and kidney, the levels of metallothioneins in the skin and the liver, the expression of metallothionein mRNA in the liver, as well as the presence of histological alterations and of apoptosis in the target organs were evaluated. The 9-months exposure induced cadmium accumulation in all the tissues examined; moreover, histological changes were observed in all the tissues examined, irrespective of the dose or the time of exposure. Apoptosis was only detected in the kidney, whereas metallothioneins and metallothionein mRNA did not increase. This study demonstrates that the existing chronic water quality criterion established for cadmium induces in the newt T. carnifex cadmium accumulation and histological alterations in the target organs examined. Together with our previous results, showing that, at these same concentrations, cadmium induced endocrine disruption, the present results suggest that the existing chronic water quality criterion for cadmium appears to be not protective of amphibians.
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Apoptosis/efectos de los fármacos , Cadmio/toxicidad , Exposición a Riesgos Ambientales , Regulación de la Expresión Génica/efectos de los fármacos , Metalotioneína/análisis , Triturus/fisiología , Animales , Cadmio/metabolismo , Cloruro de Cadmio , Disruptores Endocrinos/toxicidad , Italia , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Metalotioneína/genética , Piel/efectos de los fármacos , Triturus/metabolismo , Contaminantes Químicos del Agua/toxicidadRESUMEN
We intended to verify the safety of the freshwater values established for cadmium by the European Community and the Italian Ministry of Health in drinking water (5 µg/L) and sewage waters (20 µg/L). Therefore, we chronically exposed the newt Triturus carnifex to 5 µg/L and 20 µg/L doses of cadmium, respectively, during 3 and 9 months and verified the effects on the adrenal gland. We evaluated the serum concentrations of adrenocorticotropic hormone (ACTH), corticosterone, aldosterone, norepinephrine, and epinephrine. During the 3-month exposure, both doses of cadmium decreased ACTH and corticosterone serum levels and increased aldosterone and epinephrine serum levels. During the 9-month exposure, the 5 µg/L dose decreased ACTH and increased aldosterone and epinephrine serum levels; the 20 µg/L dose decreased norepinephrine and epinephrine serum levels, without affecting the other hormones. It was concluded that (1) chronic exposure to the safety values established for cadmium disrupted the adrenal gland activity and (2) the effects of cadmium were related both to the length of exposure and the dose administered. Moreover, our results suggest probable risks to human health, due to the use of water contaminated by cadmium.
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Enfermedades de las Glándulas Suprarrenales/inducido químicamente , Glándulas Suprarrenales/efectos de los fármacos , Intoxicación por Cadmio/etiología , Cadmio/toxicidad , Tasa de Supervivencia , Triturus/fisiología , Contaminantes Químicos del Agua/envenenamiento , Animales , Relación Dosis-Respuesta a DrogaRESUMEN
BACKGROUND: Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2) activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. METHODS AND PRINCIPAL FINDINGS: We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins), three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. CONCLUSIONS: Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here identified in this study were already known as TG2 substrates, we can also suppose that transamidating activity and differential phosphorylation of the same targets may represent a novel regulatory mechanism whose relevance in celiac disease pathogenesis is still unexplored.
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Anticuerpos/farmacología , Enfermedad Celíaca/enzimología , Proteínas de Unión al GTP/inmunología , Mucosa Intestinal/metabolismo , Fosfoproteínas/metabolismo , Transglutaminasas/inmunología , Anticuerpos/inmunología , Western Blotting , Células CACO-2 , Biología Computacional , Electroforesis en Gel Bidimensional , Humanos , Procesamiento de Imagen Asistido por Computador , Proteína Glutamina Gamma Glutamiltransferasa 2 , ProteómicaRESUMEN
Anti-tissue transglutaminase (tTG) antibodies are specifically produced in the small-intestinal mucosa of celiac disease (CD) patients. It is now recognized that these antibodies, acting on cell-surface tTG, may play an active role in CD pathogenesis triggering an intracellular response via the activation of different signal transduction pathways. In this study, we report that anti-tTG antibodies, both commercial and from a CD patient, induce a rapid Ca(2+) mobilization from intracellular stores in Caco-2 cells. We characterized the mechanism of Ca(2+) release using thapsigargin and carbonylcyanide-p-trifluoromethoxyphenylhydrazone, which are able to deplete specifically endoplasmic reticulum and mitochondria of Ca(2+), respectively. Our data highlight that both pathways of calcium release were involved, thus indicating that the spectrum of cellular responses downstream can be very wide. In addition, we demonstrate that the increased Ca(2+) level in the cells evoked by anti-tTG antibodies was sufficient to activate tTG, which is normally present as a latent protein due to the presence of low Ca(2+) and to the inhibitory effect of GTP/GDP. Herein, we discuss the importance of intracellular tTG activation as central in the context of CD pathogenesis.
Asunto(s)
Autoanticuerpos/farmacología , Señalización del Calcio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Transglutaminasas/metabolismo , Células CACO-2 , Calcio/metabolismo , Enfermedad Celíaca/enzimología , Enfermedad Celíaca/inmunología , Membrana Celular/enzimología , Citoplasma/enzimología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Proteínas de Unión al GTP , Homeostasis , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/antagonistas & inhibidores , Transglutaminasas/inmunologíaRESUMEN
BACKGROUND: Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca(2+) homeostasis and tTG activity. METHODS/PRINCIPAL FINDINGS: We studied Ca(2+) homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31-43 and 57-68 rapidly mobilized Ca(2+) from intracellular stores. Specifically, peptide 31-43 mobilized Ca(2+) from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57-68 mobilized Ca(2+) only from mitochondria. We also found that gliadin peptide-induced Ca(2+) mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31-43, but not peptide 57-68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31-43, but not of peptide 57-68, induces the expression of both genes. CONCLUSIONS: By inducing Ca(2+) mobilization from the ER, peptide 31-43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31-43 and 57-68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin-tTG crosslinking in enterocytes and specialized antigen-presenting cells.
Asunto(s)
Calcio/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Gliadina/farmacología , Fragmentos de Péptidos/farmacología , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Biotina/análogos & derivados , Biotina/metabolismo , Western Blotting , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células CACO-2 , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática/efectos de los fármacos , Proteínas de Unión al GTP/agonistas , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica/efectos de los fármacos , Gliadina/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Proteína Glutamina Gamma Glutamiltransferasa 2 , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual , Transglutaminasas/genéticaRESUMEN
Human bones, recovered from excavations, are an important biological archive of information. In particular, the analysis of the collagen fraction is useful for paleodietary reconstruction, via light stable isotopes, and for (14)C dating. Generally, collagen extraction procedures do not prevent loss of integrity of proteins. As a consequence, information about the state-of-remains preservation is unavailable. Here we describe a "soft" nondestructive CH(3)COOH-based method to recover collagen from archaeological bones, and also to obtain material for successive isotopic analyses. Our isotopic measurements on the extracts indicate that the CH(3)COOH-based method of extraction may be routinely employed in the context of paleodiet studies. In addition, we propose that biochemical characterization by denaturant electrophoresis and Western blot on CH(3)COOH extracts may be used as a bone collagen quality indicator.