RESUMEN
Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.
Asunto(s)
Disruptores Endocrinos , Contaminantes Químicos del Agua , Bioensayo , Disruptores Endocrinos/análisis , Monitoreo del Ambiente , Estrógenos/análisis , Estrógenos/toxicidad , Estrona , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/toxicidadRESUMEN
The hypothesis that C60 fullerene nanoparticles (C60) exert an antagonistic interactive effect on the toxicity of benzo[a]pyrene (BaP) has been supported by this investigation. Mussels were exposed to BaP (5, 50 & 100µg/L) and C60 (C60-1mg/L) separately and in combination. Both BaP and C60 were shown to co-localize in the secondary lysosomes of the hepatopancreatic digestive cells in the digestive gland where they reduced lysosomal membrane stability (LMS) or increased membrane permeability, while BaP also induced increased lysosomal lipid and lipofuscin, indicative of oxidative cell injury and autophagic dysfunction. Combinations of BaP and C60 showed antagonistic effects for lysosomal stability, mTORC1 (mechanistic target of rapamycin complex 1) inhibition and intralysosomal lipid (5 & 50µg/L BaP). The biomarker data (i.e., LMS, lysosomal lipidosis and lipofuscin accumulation; lysosomal/cell volume and dephosphorylation of mTORC1) were further analysed using multivariate statistics. Principal component and cluster analysis clearly indicated that BaP on its own was more injurious than in combination with C60. Use of a network model that integrated the biomarker data for the cell pathophysiological processes, indicated that there were significant antagonistic interactions in network complexity (% connectance) at all BaP concentrations for the combined treatments. Loss of lysosomal membrane stability probably causes the release of intralysosomal iron and hydrolases into the cytosol, where iron can generate harmful reactive oxygen species (ROS). It was inferred that this adverse oxidative reaction induced by BaP was ameliorated in the combination treatments by the ROS scavenging property of intralysosomal C60, thus limiting the injury to the lysosomal membrane; and reducing the oxidative damage in the cytosol and to the nuclear DNA. The ROS scavenging by C60, in combination with enhanced autophagic turnover of damaged cell constituents, appeared to have a cytoprotective effect against the toxic reaction to BaP in the combined treatments.
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Fulerenos , Nanopartículas , Animales , Benzo(a)pireno/toxicidad , Fulerenos/toxicidad , Lisosomas , Modelos Animales , Nanopartículas/toxicidadRESUMEN
Temperature is a relevant abiotic factor affecting physiological performance and distribution of marine animals in natural environments. The changes in global seawater temperatures make it necessary to understand how molecular mechanisms operate under the cumulative effects of global climate change and chemical pollution to promote/hamper environmental acclimatization. Marine mussels are excellent model organisms to infer the impacts of those anthropogenic threats on coastal ecosystems. In this study, Mediterranean mussels (Mytilus galloprovincialis) were exposed to different concentrations of the metal copper (Cu as CuCl2: 2.5, 5, 10, 20, 40 µg/L) or the antibiotic oxytetracycline (OTC: 0.1, 1, 10, 100, 1000 µg/L) at increasing seawater temperatures (16 °C, 20 °C, 24 °C). Transcriptional modulation of a 70-kDa heat shock protein (HSP70) and of the ABC transporter P-glycoprotein (P-gp, encoded by the ABCB gene) was assessed along with the cAMP/PKA signaling pathway regulating both gene expressions. At the physiological temperature of mussels (16 °C), Cu and OTC induced bimodal changes of cAMP levels and PKA activities in gills of exposed animals. A correlation between OTC- or Cu- induced changes of PKA activity and expression of hsp70 and ABCB was observed. Temperature increases (up to 24 °C) altered ABCB and hsp70 responses to the pollutants and disrupted their relationship with cAMP/PKA modulation, leading to loss of correlation between the biological endpoints. On the whole, the results indicate that temperature may impair the effects of inorganic and organic chemicals on the cAMP/PKA signaling pathway of mussels, in turn altering key molecular mediators of physiological plasticity and cytoprotection.
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Cambio Climático , Animales , Ecosistema , Branquias , Mytilus , Contaminantes Químicos del AguaRESUMEN
Histidine-rich Glycoprotein (HRG) is the most abundant protein in mussel haemolymph plasma. In this study, we determined by qRT-PCR and FISH analysis the tissues involved in HRG synthesis in Mytilus galloprovincialis. The relative HRG mRNA abundance in haemocytes, digestive gland, gills, gonads, posterior adductor muscle, and mantle edge was evaluated. Immunofluorescence analysis of HRG protein distribution in the whole mussel body was performed by a specific antibody. Our data showed the highest gene expression level of HRG in the mantle edge. In particular the outer fold of the mantle edge was shown to be the site that produced the highest amount of the protein. These data indicate a possible role of this Ca2++-binding protein in shell growth. HRG was also found in many other tissues and cells in contact with the haemolymph. This may be related to the immuno-responsive role of this protein. The presence of HRG in tissues related to the feeding pathways and mucous production could indicate the potential significance of this protein into mucus associated antimicrobial action. Overall, the results demonstrate that numerous mussel tissues are involved in HRG production, some of which can release the protein into the haemolymph and others into the extrapallial fluid. These data indicate that extrapallial (EP) protein and HRG are the same protein. An annual cycle survey showed a maximum HRG mRNA as well HRG protein production in mussel tissues in summer, a season in which the animals show the greatest growth, but are more likely to be exposed to microbial pathogens.
Asunto(s)
Regulación de la Expresión Génica/genética , Glicoproteínas/metabolismo , Mytilus/metabolismo , Proteínas/metabolismo , Animales , Branquias/metabolismo , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Gónadas/metabolismo , Hemocitos/metabolismo , Hemolinfa/metabolismo , Hibridación Fluorescente in Situ , Músculos/metabolismo , Proteínas/genéticaRESUMEN
Vehicles generate particulate matter (PM) in significant amounts as their brake systems wear. These particles can influence air quality and their transport/deposition may affect the edaphic and aquatic ecosystems. As part of the LOWBRASYS H2020 project, new more eco-friendly brake disc and pad formulations were developed. PMs generated from traditional (FM1-BD1) and innovative (FM4-BD2, FMB-BD7) brake systems in bench tests were studied. The PMs' physical/chemical characteristics were preliminarily investigated. To study the possible environmental impact of the nano-micro particulate, we used a battery of ecotoxicological tests. We employed the microalga Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the bacteria Vibrio fischeri as aquatic bioindicators, while for the edaphic ecosystem we used the seeds of Lepidium sativum and Sorghum saccharatum, the nematode Caenorhabditis elegans, the earthworm Eisenia andrei and the ameba Dictyostelium discoideum. The results showed a higher sensitivity of the freshwater organisms exposed to the soluble PM fraction, with respect to the edaphic ones. FM4-BD2 brake formulation was slightly more toxic for algae (200 mg/L) than FM1-BD1 (500 mg/L). The new system FMB-BD7 particulate was not harmful for crustacean survival, and resulted weakly toxic for algal reproduction only at 500 mg/L. The particulate material per se was found to affect the algal reproduction. No toxic effects were found on nematodes, earthworms and seeds up to 1000 mg/L. However, in D. discoideum the reproduction rate was significantly reduced starting from 100 mg/L; and the lysosomal membrane stability showed a relevant alteration also at minimal concentration (0.1 mg/L). The results demonstrated a minimal risk for biodiversity of the particulates from the different brake systems and highlighted a more eco-friendly performance the new brake-pad FMB-BD7. However, the occurrence of sublethal effects should be considered as a possible contribution of the particle toxicity to the biological effects of the environmental pollution.
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Dictyostelium , Material Particulado , Contaminantes Químicos del Agua , Aliivibrio fischeri , Animales , Organismos Acuáticos , Daphnia , Ecosistema , Ecotoxicología , Material Particulado/toxicidad , Contaminantes Químicos del Agua/toxicidadRESUMEN
The effects of C60 on mTOR (mechanistic Target of Rapamycin) activity in mussel digestive gland were investigated. mTOR is a kinase that senses physiological and environmental signals to control eukaryotic cell growth. mTOR is present in two complexes: the phosphorylated mTORC1 regulates cell growth by activating anabolic processes, and by inhibiting catabolic processes (i.e. autophagy); mTORC2 also modulates actin cytoskeleton organization. Mussels were exposed to C60 (0.01, 0.1 and 1 mg/L) for 72 h. Immunocytochemical analysis using a specific antibody revealed the cellular distribution of C60 in mussel digestive gland, already at the lowest concentration. In exposed mussels, the dephosphorylation of mTORC1 and mTORC2 may explain the C60 effects, i.e. the reduction of lysosomal membrane stability, the enhancement of LC3B protein, and the increase of lysosomal/cytoplasmic volume ratio; as well the cytoskeletal alterations. No oxidative stress was observed. Multivariate analysis was used to facilitate the interpretation of the biomarker data. Finally, a low density oligo-microarray was used to understand the cellular responses to fullerene. Transcriptomics identified a number of differentially expressed genes (DEGs) showing a maximum in animals exposed to 0.1 mg/L C60. The most affected processes are associated with energy metabolism, lysosomal activity and cytoskeleton organization. In this study, we report the first data on the subcellular distribution of C60 in mussel's cells; and on the involvement of mTOR inhibition in the alterations due to nanoparticle accumulation. Overall, mTOR deregulation, by affecting protein synthesis, energy metabolism and autophagy, may reduce the capacity of the organisms to effectively grow and reproduce.
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Fulerenos/toxicidad , Mytilus edulis/fisiología , Contaminantes Químicos del Agua/toxicidad , Animales , Autofagia/efectos de los fármacos , Metabolismo Energético , Humanos , Lisosomas/metabolismo , Mytilus edulis/metabolismo , Fosforilación , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The present work aims to investigate the effects of water temperature increase on Mytilus galloprovincilis and Mytilus edulis pure larvae (PG, PE) and their hybrids (HFG, HFE). D-larvae were maintained at 18 °C or exposed to a higher temperature of 22 °C for 48 h. Initially, Embryotoxicity test was evaluated. Second, a transcriptomic analysis using a recently developed microarray platform was applied to determine the main biological processes involved in early life stages responses to temperature increase. Finally, an immunofluorescence investigation was performed to bridge the gap between transcriptomic regulation and the real changes at cellular/tissue levels. Embryotoxicity test revealed a higher sensitivity of M. edulis (PE) D-larvae as well as hybrids from females M. edulis (HFE) to temperature increase, with the highest rate of larval malformations. Transcriptomic results indicated a lack of an adequate heat shock protein (Hsp) response in PE and HFE larvae (the high expression was observed in PG larvae); the differential expression of gene involved in translation, energy metabolism and oxidative stress response may contribute to explain the observed complex alterations in the studied conditions. As revealed by immunohistochemistry, cytoskeleton proteins changes associated with a drastic decrease of Histidine-Rich Glycoprotein (HRG) may elucidate the larval abnormalities in shell development observed for PE and HFE larvae. Overall, the results indicate that each type of pure larva (PG and PE) and their respective female hybrid (HFG and HFE) react similarly to the temperature increase. Our data should be carefully considered in view of the water temperature increase in marine ecosystems and especially for the mussel's species in confluence zones.
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Mytilus , Animales , Ecosistema , Femenino , Respuesta al Choque Térmico , Larva , TemperaturaRESUMEN
The interactions between carbon-based engineered nanoparticles (ENPs) and organic pollutants might enhance the uptake of contaminants into biota. The present integrated study aimed to assess this potential 'Trojan Horse', probing the interactive effects of purpose-made multi-walled carbon nanotubes (MWCNTs), a representative ENP, and benzo[a]pyrene (BaP), a ubiquitous polycyclic aromatic hydrocarbon (PAH) pollutant, on the marine mussel Mytilus galloprovincialis. Mussels were exposed to MWCNTs and BaP either alone or in various combinations. The co-exposure of BaP with MWCNTs revealed that the presence of MWCNTs enhanced the aqueous concentrations of BaP, thereby reducing the uptake of this pollutant by mussels as evidenced by lowering BaP concentrations in the tissues. Determination of DNA damage (comet assay) showed a concentration-dependent response for BaP alone which was absent when MWCNTs were present. Global gene expression using microarray analyses indicated that BaP and MWCNTs, in combination, differentially activated those genes which are involved in DNA metabolism compared to the exposures of BaP or MWCNTs alone, and the gene expression response was tissue-specific. Mechanisms to explain these results are discussed and relate primarily to the adsorption of BaP on MWCNTs, mediated potentially by van der Waals interactions. The use of a novel approach based on gold-labeled MWCNTs to track their uptake in tissues improved the traceability of nanotubes in biological samples. Overall, our results did not indicate the 'Trojan Horse' effects following co-exposure to the contaminants and clearly showed that the adsorption of BaP to MWCNTs modified the uptake of the pollutant in marine mussels.
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Benzo(a)pireno/toxicidad , Mytilus/efectos de los fármacos , Nanotubos de Carbono/toxicidad , Animales , Ensayo Cometa , Daño del ADN , Regulación de la Expresión Génica/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidadRESUMEN
In the present study, we investigated the health status of marine mussels (Mytilus galloprovincialis) caged and deployed at three different sites on the Sardinian coastline characterized by different levels of contamination: Fornelli (F, the reference site), Cala Real (CR), and Porto Torres (PT). A new low density oligonucleotide microarray was used to investigate global gene expression in the digestive gland of mussels. Target genes were selected to cover most of the biological processes involved in the stress response in bivalve mollusks (e.g. DNA metabolism, translation, immune response, cytoskeleton organization). A battery of classical biomarkers was also employed to complement the gene expression analyses. Chemical analysis revealed higher loads of heavy metals (Pb and Cu) and total polycyclic aromatic hydrocarbons (PAHs) at PT compared to the other sites. In mussels deployed at CR, functional genomics analysis of the microarray data rendered 78 differentially expressed genes (DEGs) involved in 11 biological processes. Animals exposed at PT had 105 DEGs that were characterized by the regulation of 14 biological processes, including mitochondrial activity, adhesion to substrate, DNA metabolism, translation, metal resistance, and cytoskeleton organization. Biomarker data (lysosomal membrane stability, lysosomal/cytoplasm volume ratio, lipofuscin accumulation, metallothionein content, micronucleus frequency, and cytoskeleton alteration) were in trend with transcriptomic output. Biomarker data were integrated using the Mussel Expert System (MES), allowing defining the area in which the presence of chemicals is toxic for mussels. Our study provides the opportunity to adopt a new approach of integrating transcriptomic (microarray) results with classical biomarkers to assess the impact of pollutants on marine mussels in biomonitoring programs.
Asunto(s)
Biomarcadores/metabolismo , Monitoreo del Ambiente/métodos , Mytilus/metabolismo , Contaminantes Químicos del Agua/análisis , Animales , Estado de Salud , Italia , Contaminantes Químicos del Agua/metabolismoRESUMEN
Lysosomal membrane stability (LMS) has been used in various organisms as a very sensitive biomarker of stress. However, despite the abundance of data about regulation of the autophagic process in mammals, in the invertebrates there is only limited mechanistic understanding. Marine mussels (Mytilus galloprovincialis Lam.) are bivalve molluscs, widely used as models in ecotoxicology and as environmental bioindicators of sea water quality. In order to elucidate this fundamental process, in the present study, mussels were exposed for 3â¯days to a "priority", ubiquitous environmental contaminant, benzo[a]pyrene (B[a]P) at different concentrations (i.e. 5, 50, 100⯵g/L seawater). B[a]P accumulated in lysosomes of digestive tubule epithelial cells (digestive cells) and in enlarged lipid-rich lysosomes (autolysosomes) as detected by immunofluorescence and UV-fluorescence. B[a]P also activated the autophagic process with a marked decrease of LMS and concurrent increase in lysosomal/cytoplasmic volume ratio. Dephosphorylation of mTOR contributes to increased lysosomal membrane permeability and induced autophagy. B[a]P induced a decrease in phosphorylated (active form) mTOR. The probable role of mTOR in cell signalling and the regulation of the cellular responses to the contaminants has been also confirmed in a field study, where there was significant inactivation of mTOR in stressed animals. Statistical and network modelling supported the empirical investigations of autophagy and mTOR; and was used to integrate the mechanistic biomarker data with chemical analysis and DNA damage.
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Autofagia , Contaminantes Ambientales/toxicidad , Lisosomas/metabolismo , Mytilus/citología , Mytilus/metabolismo , Estrés Fisiológico , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia/efectos de los fármacos , Benzo(a)pireno/toxicidad , Biomarcadores/metabolismo , Inmunohistoquímica , Lisosomas/efectos de los fármacos , Modelos Estadísticos , Análisis Multivariante , Mytilus/efectos de los fármacos , Análisis de Componente Principal , Estrés Fisiológico/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidadRESUMEN
The effect of Cr(VI) as a soil contaminant on the edaphic worm Eisenia andrei was studied by evaluating the activity of Ca2+-ATPase in the intestinal mucosa. In eukaryotes, Ca2+-ATPase is a key mediator of cell signaling although comparatively little is known about its activity in earthworms. Size and anatomical constraints (i.e. small and complex) led us to develop and optimize a cyto-biochemical method to measure Ca2+-ATPase activity in earthworms. The principal site of enzyme activity was found to be the post clitellar intestinal tract; immunohistochemistry then identified plasma membrane Ca2+-ATPase (PMCA ATPase) in the apical area of the intestinal epithelium. Earthworms exposed for 28days to OECD soil contaminated with 1, 2, and 15mg/Kg Cr(VI) demonstrated about 70% inhibition of Ca2+-ATPase activity at the low Cr (VI) concentration (the half of the Italian law limit for residential areas), rising to approximately 84% inhibition at the highest concentration. Reduced enzyme activity was accompanied by decreased enzyme content and reduced lysosomal membrane stability (LMS), which is a well established early warning biomarker of stress. These data demonstrate the potential utility of Ca2+-ATPase activity as a sensitive parameter with which to detect environmental stress in earthworms.
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ATPasas Transportadoras de Calcio/metabolismo , Cromo/toxicidad , Oligoquetos/efectos de los fármacos , Oligoquetos/enzimología , Contaminantes del Suelo/toxicidad , Animales , Cromo/química , Concentración de Iones de Hidrógeno , Plomo/química , Plomo/toxicidad , Suelo/química , Contaminantes del Suelo/química , Temperatura , Factores de TiempoRESUMEN
A biomonitoring study using wild Mytilus galloprovincialis mussels sampled from six sites along the Moroccan coasts evaluated whether select biomarkers are suitable for identifying and quantifying pollution-induced stress syndrome in mussels. Lysosomal membrane stability was confirmed to be a highly sensitive biological parameter, and acetylcholinesterase activity was found a suitable biomarker of neurotoxicity. Metallothionein concentrations were in line with heavy metal concentrations detected in mussel tissues. However, malondialdehyde was not sensitive, suggesting the need for alternative biomarkers of oxidative stress. Three different approaches were used for biomarker and chemical data integration. The Integrated Biomarker Response (IBR) was suitable for classifying the stress response but did not allow to evaluate the level of stress in the organisms. The Mussel Expert System (MES) was suitable for ranking the biological effects of pollutants, also providing an indication of the evolution of the stress syndrome in the animals. Finally, the use of Principal Component Analysis (PCA) provided indication of the inorganic chemicals contributing to the detrimental biological effects.
Asunto(s)
Biomarcadores , Mytilus , Contaminantes del Agua/toxicidad , Animales , Monitoreo del Ambiente , Metales Pesados , Estrés Oxidativo , Contaminantes Químicos del AguaRESUMEN
Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new 'STressREsponse Microarray' (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P) at 5, 50, and 100 µg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a]P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a]P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in B[a]P exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota.
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Benzo(a)pireno/toxicidad , Mytilus/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Contaminantes del Agua/toxicidad , Animales , Biomarcadores , Daño del ADN/efectos de los fármacos , Exposición a Riesgos Ambientales , Monitoreo del Ambiente , Branquias/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mytilus/genéticaRESUMEN
Chromium (Cr) is one of the major and most detrimental pollutant, widely present in the environment as a result of several anthropogenic activities. In mammalian cells, Cr(VI) is known to enhance reactive oxygen species (ROS) production and to cause toxic and genotoxic effects. Less commonly investigated are the effects and mode of action of this contaminant in invertebrates, particularly in soil organisms. In this work, earthworms of the species Eisenia andrei were exposed for 1 and 3 days to various sublethal concentrations of Cr(VI) (2, 15, 30µgmL-1) using the paper contact toxicity test. In amoeboid leukocytes we investigated intracellular ROS and lipoperoxide production, oxidative DNA damage, and the effects on different cell functions. The analysis of the results shows that Cr(VI) triggered severe adverse reactions; the first events were an increase of intracellular ROS levels, generating in the cells oxidative stress conditions leading to membrane lipid peroxidation and oxidative DNA damage. Lysosomes showed relevant changes such as a strong membrane destabilization, which was accompanied by an increased catabolism of cytoplasmic proteins and accumulation of lipofuscin. With an increase in the dose and/or time of exposure, the physiological status of intracellular organelles (such as lysosomes, nucleus and mitochondria) showed further impairment and amoebocyte immune functions were adversely affected, as shown by the decrease of the phagocytic activity. By mapping the responses of the different parameters evaluated, diagnostic of (oxidative) stress events, against lysosomal membrane stability, a "health status" indicator (able to describe the stress syndrome from its early phase to pathology), we have shown that this biomarker is suitable as a prognostic test for health of earthworms. This is viewed as a crucial step toward the derivation of explanatory frameworks for prediction of pollutant impact on animal health.
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Cromo/toxicidad , Leucocitos/metabolismo , Oligoquetos/metabolismo , Estrés Oxidativo , Animales , Biomarcadores/metabolismo , Núcleo Celular/efectos de los fármacos , Daño del ADN , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Peroxidación de Lípido , Lipofuscina/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Governmental agencies (www.epa.gov/mercury) and the scientific community have reported on the high toxicity due to mercury. Indeed, exposure to mercury can cause severe injury to the central nervous system and kidney in humans. Beyond its recognized toxicity, little is known regarding the molecular mechanisms involved in the actions of this heavy metal. Mercury has been also observed to form insoluble fibrous protein aggregates in the cell nucleus. We used D. discoideum to evaluate micronuclei formation and, since mercury is able to induce oxidative stress that could bring to protein aggregation, we assessed nuclear protein carbonylation by Western Blot. We observed a significant increase in micronuclei formation and 14 carbonylated proteins were identified. Moreover, we used isotope-coded protein label (ICPL) and mass spectrometry analysis of proteins obtained by lysis of purified nuclei, before of tryptic digestion to quantify nuclear proteins affected by mercury. In particular, we examined the effects of mercury that associate a classical genotoxic assay to proteomic effects into the nucleus. The data present direct evidences for mercury genotoxicity, nuclear protein carbonylation, quantitative change in core histones, and the involvement of pseudouridine synthase in mercury toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 417-425, 2017.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Dictyostelium/efectos de los fármacos , Mercurio/toxicidad , Animales , Biblioteca de Genes , Histonas/metabolismo , Transferasas Intramoleculares/metabolismo , Pruebas de Micronúcleos , Proteínas Nucleares/química , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/genética , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacos , Hidrolisados de Proteína/química , ProteómicaRESUMEN
Eisenia andrei earthworms were exposed for 7 and 14 days to six samples of soil taken from around an abandoned lead (Pb) mine and characterized by different levels of metal contamination (S6-S1, this latter being the most contaminated soil). The organisms were analyzed for metal bioaccumulation and for biological parameters as biomarkers of stress (lysosomal membrane stability; lipofuscin lysosomal content; lysosomal/cytoplasmic volume ratio) and genotoxicity (Micronucleus frequency). Chemical analysis showed the loads of Pb, Cd, Zn, and Cu in the worms following exposure. Among the stress biomarkers, lysosomal membrane stability was significantly affected in the coelomocytes of the earthworms exposed already 7 days to different contaminated soils. Organisms exposed for 14 days to S1 showed in the cells of the chloragogenous tissue, a particularly relevant increase in lipofuscin, a biomarker of oxidative stress, and an increase in the lysosome/cytoplasm volume ratio, indicating stressful condition at the tissue level. Moreover, in the same conditions, a decrease in total body weight was observed. At the longer exposure time, the coelomocytes of worms exposed to S1, S2, and S3 (soils with higher metal concentrations) showed a significant increase in micronuclei (MNi) frequency. Expressions of the P21 and topoisomerase genes, which are involved in DNA repair, showed significant up-regulation in the cells of worms exposed to S1, S2, S3, S4 and to a less extend S6. This may indicate that the worms were only able to successfully reduce the level of DNA damage in S4 and S5 if considering MN frequency data. The biomarker data was integrated by the Earthworm Expert System, allowing an objective interpretation of the complex biological data and clearly defining the areas in which the presence of chemicals is toxic for the edaphic organisms.
Asunto(s)
Contaminación Ambiental , Metales Pesados/metabolismo , Oligoquetos/metabolismo , Contaminantes del Suelo/análisis , Animales , Biomarcadores/metabolismo , Daño del ADN , Monitoreo del Ambiente , Lisosomas/efectos de los fármacos , Metales Pesados/análisis , Minería , Oligoquetos/genética , Estrés Oxidativo , Suelo/química , Túnez , Regulación hacia ArribaRESUMEN
In this work, the toxicity of lake sediments contaminated with DDT and its metabolites DDD and DDE (collectively, DDX) was evaluated with widely used toxicity tests (i.e., Vibrio fischeri, Daphnia magna, Pseudokirchneriella subcapitata, and Lumbriculus variegatus) and with the social amoeba Dictyostelium discoideum, a model organism that is also suitable for studying pollutant-induced alterations at the molecular and cellular levels. Although the DDX concentration in the sediments was high (732.5 ppb), the results suggested a minimal environmental risk; in fact, no evidence of harmful effects was found using the different bioassays or when we considered the results of more sensitive sublethal biomarkers in D. discoideum amoebae. In line with the biological results, the chemical data showed that the concentration of DDX in the pore water (in general a highly bioavailable phase) showed a minimal value (0.0071ppb). To confirm the importance of the bioavailability of the toxic chemicals in determining their biological effects and to investigate the mechanisms of DDX toxicity, we exposed D. discoideum amoebae to 732.5ppb DDX in water solution. DDX had no effect on cell viability; however, a strong reduction in amoebae replication rate was observed, which depended mainly on a reduction in endocytosis rate and on lysosomal and mitochondrial alterations. In the presence of a moderate and transient increase in reactive oxygen species, the glutathione level in DDX-exposed amoebae drastically decreased. These results highlight that studies of the bioavailability of pollutants in environmental matrices and their biological effects are essential for site-specific ecological risk assessment. Moreover, glutathione depletion in DDX-exposed organisms is a new finding that could open the possibility of developing new pesticide mixtures that are more effective against DDT-resistant malaria vectors.
Asunto(s)
DDT/toxicidad , Dictyostelium/efectos de los fármacos , Monitoreo del Ambiente/métodos , Agua Dulce/química , Sedimentos Geológicos/química , Plaguicidas/toxicidad , Contaminantes Químicos del Agua/toxicidad , Aliivibrio fischeri/efectos de los fármacos , Animales , Disponibilidad Biológica , Chlorophyta/efectos de los fármacos , DDT/química , DDT/metabolismo , Daphnia/efectos de los fármacos , Dictyostelium/metabolismo , Plaguicidas/química , Plaguicidas/metabolismo , Pruebas de Toxicidad , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/metabolismoRESUMEN
Numerous studies on molluscs have been carried out to clarify the physiological roles of haemolymph serum proteins and haemocytes. However, little is known about the presence and functional role of the serum metabolites. In this study, Nuclear Magnetic Resonance (NMR) was used to assess whether changes of the metabolic profile of Mytilus galloprovincialis haemolymph may reflect alterations of the physiological status of the organisms due to environmental stressors, namely copper and temperature. Mussel haemolymph was taken from the posterior adductor muscle after a 4-day exposure to ambient (16 °C) or high temperature (24 °C) and in the absence or presence (5 µg/L, 20 µg/L, or 40 µg/L) of sublethal copper (Cu(2+)). The total glutathione (GSH) concentration in the haemolymph of both control and treated mussels was minimal, indicating the absence of significant contaminations by muscle intracellular metabolites due to the sampling procedure. In the (1)H-NMR spectrum of haemolymph, 27 metabolites were identified unambiguously. The separate and combined effects of exposure to copper and temperature on the haemolymph metabolic profile were assessed by Principal Component Analysis (PCA) and Ranking-PCA multivariate analysis. Changes of the metabolomic profile due to copper exposure at 16 °C became detectable at a dose of 20 µg/L copper. Alanine, lysine, serine, glutamine, glycogen, glucose and protein aliphatics played a major role in the classification of the metabolic changes according to the level of copper exposition. High temperature (24 °C) and high copper levels caused a coherent increase of a common set of metabolites (mostly glucose, serine, and lysine), indicating that the metabolic impairment due to high temperature is enforced by the presence of copper. Overall, the results demonstrate that, as for human blood plasma, the analysis of haemolymph metabolites represents a promising tool for the diagnosis of pollutant-induced stress syndrome in marine mussels.
Asunto(s)
Cobre/envenenamiento , Hemolinfa/efectos de los fármacos , Mytilus/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Contaminantes Químicos del Agua/envenenamiento , Animales , Acuicultura , Biomarcadores/metabolismo , Cobre/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Glucosa/metabolismo , Glutatión/metabolismo , Hemolinfa/metabolismo , Calor/efectos adversos , Italia , Lisina/metabolismo , Metabolómica/métodos , Mytilus/crecimiento & desarrollo , Mytilus/metabolismo , Mytilus/fisiología , Resonancia Magnética Nuclear Biomolecular , Análisis de Componente Principal , Serina/metabolismo , Distribución Tisular , Toxicocinética , Regulación hacia Arriba/efectos de los fármacos , Contaminantes Químicos del Agua/administración & dosificaciónRESUMEN
A suite of organisms from different taxonomical and ecological positions is needed to assess environmentally relevant soil toxicity. A new bioassay based on Dictyostelium is presented that is aimed at integrating slime molds into such a testing framework. Toxicity tests on elutriates and the solid phase developmental cycle assay were successfully applied to a soil spiked with a mixture of Zn, Cd, and diesel fuel freshly prepared (recently contaminated) and after 2 yr of aging. The elutriates of both soils provoked toxic effects, but toxicity was markedly lower in the aged soil. In the D. discoideum developmental cycle assay, both soils affected amoeba viability and aggregation, with fewer multicellular units, smaller fruiting bodies and, overall, inhibition of fruiting body formation. This assay is quick and requires small amounts of test soil, which might facilitate its incorporation into a multispecies multiple-endpoint toxicity bioassay battery suitable for environmental risk assessment in soils. Environ Toxicol Chem 2016;35:1413-1421. © 2015 SETAC.
Asunto(s)
Dictyostelium/efectos de los fármacos , Monitoreo del Ambiente/métodos , Gasolina/toxicidad , Metales Pesados/toxicidad , Contaminantes del Suelo/toxicidad , Bioensayo , Lisosomas/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Suelo/químicaRESUMEN
Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. In the marine mussel Mytilus galloprovincialis, exposure to nanosized titanium dioxide (n-TiO2), one of the most widespread type of NPs in use, in combination with and 2,3,7,8-tetrachlorodibenzo-p-dioxins (TCDD), chosen as model organic xenobiotic, was shown to induce significant changes in different biomarkers in hemocytes, gills and digestive gland, with distinct effects depending on cell/tissue and type of response measured. In this work, the interactive effects of n-TiO2 and TCDD at the tissue level were further investigated in mussel digestive gland using an integrated approach transcriptomics/immunohistochemistry. Mussels were exposed to n-TiO2 (100µgL(-1)) and TCDD (0.25µgL(-1)), alone and in combination, for 96h. Transcriptomic analysis identified 48-, 49- and 62 Differentially Expressed Genes (DEGs) in response to n-TiO2, TCDD and n-TiO2/TCDD, respectively. Gene Ontology (GO) term analysis revealed distinct biological processes affected in different experimental conditions. n-TiO2 mainly up-regulated cytoskeletal genes, while TCDD up-regulated endocrine and signal transduction related processes. Co-exposure induced transcriptional changes common to individual treatments, and identified a newly generated process, response to chemical stimulus. Transcription of selected genes was verified by qPCR. Moreover, expression of tubulin, as an example of target protein of interest identified by gene transcription data, was evaluated in tissue sections by immunolabelling. Tissue TCDD accumulation was evaluated by immunofluorescence with an anti-dioxins antibody. The results demonstrate both distinct and interactive effects of n-TiO2 and TCDD in mussel digestive gland at the molecular and tissue level, identify the main molecular targets involved, and underline how exposure to the n-TiO2/TCDD mixture does not result in increased TCDD accumulation and overall stressful conditions in the tissue. These represent the first data on transcriptional responses of marine invertebrates to exposure not only to n-TiO2 as a model of NP, but also to a legacy contaminant like TCDD.