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PubMed | Laboratory of Biochemistry and Environmental Toxicology, French National Institute for Agricultural Research, Regional Center for Research in Horticulture and Organic Agriculture and University of Piemonte Orientale
Type: | Journal: Environmental pollution (Barking, Essex : 1987) | Year: 2016

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.


PubMed | Plymouth Marine Laboratory, Laboratory of Biochemistry and Environmental Toxicology and University of Piemonte Orientale
Type: | Journal: Ecotoxicology and environmental safety | Year: 2017

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, 30gmL


Dondero F.,University of Piemonte Orientale | Banni M.,University of Piemonte Orientale | Banni M.,Laboratory of Biochemistry and Environmental Toxicology | Negri A.,University of Piemonte Orientale | And 3 more authors.
BMC Genomics | Year: 2011

Background: Mixtures of chemicals present in aquatic environments may elicit toxicity due to additive or synergistic effects among the constituents or, vice versa, the adverse outcome may be reduced by antagonistic interactions. Deviations from additivity should be explained either by the perturbations of toxicokinetic parameters and/or chemical toxicodynamics. We addressed this important question in marine mussels exposed subchronically to a binary mixture made of two wide-spread pollutants: the heavy metal nickel and the organic phosphorus pesticide Chlorpyrifos. To this aim, we carried out in tissues of Mytius galloprovincialis (Lam) a systems approach based on the evaluation and integration of different disciplines, i.e. high throughput gene expression profiling, functional genomics, stress biomakers and toxicokinetics.Results: Cellular and tissue biomarkers, viz. digestive gland lysosomal membrane stability, lysosomal/cytosol volume ratio, neutral lipid content and gill acetylcholinesterase activity were, in general, altered by either the exposure to nickel and Chlorpyrifos. However, their joint action rendered (i) an overall decrease of the stress syndrome level, as evaluated through an expert system integrating biomarkers and (ii) statistically significant antagonistic deviations from the reference model systems to predict mixture toxicity. While toxicokinetic modeling did not explain mixture interactions, gene expression profiling and further Gene Ontology-based functional genomics analysis provided clues that the decrement of toxicity may arise from the development of specific toxicodynamics. Multivariate statistics of microarray data (238 genes in total, representing about 14% of the whole microarray catalogue) showed two separate patterns for the single chemicals: the one belonging to the heavy metal -135 differentially expressed genes (DEGs) was characterized by the modulation of transcript levels involved in nucleic acid metabolism, cell proliferation and lipid metabolic processes. Chlorpyrifos exposure (43 DEGs) yielded a molecular signature which was biased towards carbohydrate catabolism (indeed, chitin metabolism) and developmental processes. The exposure to the mixture (103 DEGs) elicited a composite complex profile which encompassed the core properties of the pesticide but also a relevant set of unique features. Finally, the relative mRNA abundance of twelve genes was followed by Q-PCR to either confirm or complement microarray data. These results, in general, were compatible with those from arrays and indeed confirmed the association of the relative abundance of two GM-2 ganglioside activator genes in the development of the hyperlipidosis syndrome observed in digestive gland lysosomes of single chemical exposed mussels.Conclusion: The transcriptomic assessment fitted with biological data to indicate the occurrence of different toxicodynamic events and, in general, a decrease of toxicity, driven by the mitigation or even abolition of lysosomal responses. Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecologically relevant organisms. © 2011 Dondero et al; licensee BioMed Central Ltd.


Negri A.,University of Piemonte Orientale | Oliveri C.,University of Piemonte Orientale | Sforzini S.,University of Piemonte Orientale | Mignione F.,University of Piemonte Orientale | And 3 more authors.
PLoS ONE | Year: 2013

Global warming is a major factor that may affect biological organization, especially in marine ecosystems and in coastal areas that are particularly subject to anthropogenic pollution. We evaluated the effects of simultaneous changes in temperature and copper concentrations on lysosomal membrane stability (N-acetyl-hexosaminidase activity) and malondialdehyde accumulation (MDA) in the gill of the blue mussel Mytilus galloprovincialis (Lam.). Temperature and copper exerted additive effects on lysosomal membrane stability, exacerbating the toxic effects of metal cations present in non-physiological concentrations. Mussel lysosomal membrane stability is known to be positively related to scope for growth, indicating possible effects of increasing temperature on mussel populations in metal-polluted areas. To clarify the molecular response to environmental stressors, we used a cDNA microarray with 1,673 sequences to measure the relative transcript abundances in the gills of mussels exposed to copper (40 μg/L) and a temperature gradient (16°C, 20°C, and 24°C). In animals exposed only to heat stress, hierarchical clustering of the microarray data revealed three main clusters, which were largely dominated by down-regulation of translation-related differentially expressed genes, drastic up-regulation of protein folding related genes, and genes involved in chitin metabolism. The response of mussels exposed to copper at 24°C was characterized by an opposite pattern of the genes involved in translation, most of which were up-regulated, as well as the down-regulation of genes encoding heat shock proteins and "microtubule-based movement" proteins. Our data provide novel information on the transcriptomic modulations in mussels facing temperature increases and high copper concentrations; these data highlight the risk of marine life exposed to toxic chemicals in the presence of temperature increases due to climate change. © 2013 Negri et al.


Banni M.,Laboratory of Biochemistry and Environmental Toxicology | Negri A.,University of Piemonte Orientale | Dagnino A.,University of Piemonte Orientale | Jebali J.,Laboratory of Biochemistry and Environmental Toxicology | And 2 more authors.
Ecotoxicology and Environmental Safety | Year: 2010

In the present study, mussel (Mytilus galloprovincialis) digestive gland biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) were investigated. Mussels were exposed to a sublethal dose of B[a]P (75 nM; 19 γg/l per animal) for 24, 48 and 72 h. The following biological responses were measured in the digestive gland tissues: (1) B[a]P hydroxylase (BPH) activity, as phase I biotransformation parameter; (2) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (3) catalase (CAT) activity as potential biomarker of oxidative stress, (4) acetylcholinesterase (AChE) activity as an indication of possible neurotoxicity response. DNA damage was assessed over time using the single cell gel electrophoresis comet assay and the micronuclei test. BPH and GST activities showed an increasing trend over exposure period. CAT activity showed a symmetrical bell shape response with a maximum at 48 h. AChE activity was significantly depressed after 48 and 72 h exposure to B[a]P. Comet assay and micronuclei test in digestive gland cells suggest that B[a]P exposure induced significant DNA damage with a maximum response after 72 h exposure. © 2009 Elsevier Inc.


Mohamed B.,University of Piemonte Orientale | Mohamed B.,Laboratory of Biochemistry and Environmental Toxicology | Hajer A.,Laboratory of Biochemistry and Environmental Toxicology | Susanna S.,University of Piemonte Orientale | And 4 more authors.
Aquatic Toxicology | Year: 2014

The exposure of marine organisms to stressing agents may affect the level and pattern of gene expression. Although many studies have examined the ecological effects of heat stress on mussels, little is known about the physiological mechanisms that maybe affected by co-exposure to heat stress and environmental contaminants such as nickel (Ni). In the present work, we investigated the effects of simultaneous changes in temperature and Ni supply on lysosomal membrane stability (LMS) and malondialdehyde accumulation (MDA) in the digestive gland (DG) of the blue mussel Mytilus galloprovincialis (Lam.). To elucidate how the molecular response to environmental stressors is modulated, we employed a cDNA microarray with 1673 sequences to measure relative transcript abundances in the DG of mussels exposed to Ni along with a temperature increase. A two-way ANOVA revealed that temperature and Ni rendered additive effects on LMS and MDA accumulation, increasing the toxic effects of metal cations. Ni loads in the DG were also affected by co-exposure to 26. °C. In animals exposed only to heat stress, functional genomics analysis of the microarray data (171differentially expressed genes (DEGs)) highlighted seven biological processes, largely dominated by the up-regulation of folding protein-related genes and the down-regulation of genes involved in cell migration and cellular component assembly. Exposure to Ni at 18. °C and 26. °C yielded 188 and 262 DEGs, respectively, exhibiting distinct patterns in terms of biological processes. In particular, the response of mussels exposed to Ni at 26. °C was characterized by the up-regulation of proteolysis, ribosome biogenesis, response to unfolded proteins, and catabolic-related genes, as well as the down-regulation of genes encoding cellular metabolic processes. Our data provide new insights into the transcriptomic response in mussels experiencing temperature increases and Ni exposure; these data should be carefully considered in view of the biological effects of heat stress, particularly in polluted areas. © 2014 Elsevier B.V.


Oliveri C.,University of Piemonte Orientale | Peric L.,Ruder Boskovic Institute | Sforzini S.,University of Piemonte Orientale | Banni M.,University of Piemonte Orientale | And 4 more authors.
Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics | Year: 2014

Mollusc haemolymph proteins are known to play several important physiological roles in the immune system, heavy metal transport and the tissue distribution of lipophilic compounds. In this study, we analysed acetone-extracted proteins from mussel haemolymph by one- and two-dimensional gel electrophoresis. The proteins were identified by comparing mass spectrometry data with the invertebrate EST database, allowing us to establish the mussel haemolymph serum proteome. Extrapallial protein (EP) precursor represents the most abundant serum protein; astacin and CuZn superoxide dismutase were also detected. Slight contamination from muscle proteins, due to the sampling method, was also found. No differences were observed in the profiles obtained for male and female serum proteins. One aspect of interest was the previously reported finding that alkali-labile phosphate (ALP) from haemolymph serum may be representative of vitellogenin (vtg)-like protein content in the circulatory fluid of molluscs. In our analysis of mussel haemolymph serum, vitellogenin-like proteins were never found. To confirm these data, a typical methyl-tert-butyl-ether (MTBE) extraction, which is specific for vtg-like proteins, was performed, and the results of the electrophoretic analyses were compared with those obtained by acetonic precipitation. The results showed that the electrophoretic profiles are similar and that vtg-like proteins cannot be identified. Moreover, the main phosphoprotein present in female and male extracts is EP protein precursor. In addition, agarose gel electrophoresis demonstrates that high-molecular-weight forms of vtg-like proteins are not detectable. © 2014 Elsevier Inc.


Canesi L.,University of Genoa | Negri A.,University of Piemonte Orientale | Barmo C.,University of Genoa | Banni M.,University of Piemonte Orientale | And 4 more authors.
PLoS ONE | Year: 2011

Background: Many pesticides have been shown to act as endocrine disrupters. Although the potencies of currently used pesticides as hormone agonists/antagonists are low compared with those of natural ligands, their ability to act via multiple mechanisms might enhance the biological effect. The organophosphate Chlorpyrifos (CHP) has been shown to be weakly estrogenic and cause adverse neurodevelopmental effects in mammals. However, no information is available on the endocrine effects of CHP in aquatic organisms. In the digestive gland of the bivalve Mytilus galloprovincialis, a target tissue of both estrogens and pesticides, the possible effects of CHP on the responses to the natural estrogen 17β-estradiol (E2) were investigated. Methodology/Principal Findings: Mussels were exposed to CHP (4.5 mg/l, 72 hrs) and subsequently injected with E2 (6.75 ng/g dw). Responses were evaluated in CHP, E2 and CHP/E2 treatment groups at 24 h p.i. by a biomarker/transcriptomic approach. CHP and E2 induced additive, synergistic, and antagonistic effects on lysosomal biomarkers (lysosomal membrane stability, lysosome/cytoplasm volume ratio, lipofuscin and neutral lipid accumulation). Additive and synergistic effects were also observed on the expression of estrogen-responsive genes (GSTπ, catalase, 5-HTR) evaluated by RT-Q-PCR. The use of a 1.7K cDNA Mytilus microarray showed that CHP, E2 and CHP/E2, induced 81, 44, and 65 Differentially Expressed Genes (DEGs), respectively. 24 genes were exclusively shared between CHP and CHP/E2, only 2 genes between E2 and CHP/E2. Moreover, 36 genes were uniquely modulated by CHP/E2. Gene ontology annotation was used to elucidate the putative mechanisms involved in the responses elicited by different treatments. Conclusions: The results show complex interactions between CHP and E2 in the digestive gland, indicating that the combination of certain pesticides and hormones may give rise to unexpected effects at the molecular/cellular level. Overall, these data demonstrate that CHP can interfere with the mussel responses to natural estrogens. © 2011 Canesi et al.


Sabrine H.,Regional Research Center on Horticulture and Organic Agriculture | Afif H.,CSIC - Aula Dei Experimental Station | Mohamed B.,Laboratory of Biochemistry and Environmental Toxicology | Hamadi B.,Laboratory of Biochemistry and Environmental Toxicology | Maria H.,CSIC - Aula Dei Experimental Station
Scientia Horticulturae | Year: 2010

In this work, pea (Pisum sativum) plants exposed to increasing cadmium and copper concentrations were tested for heavy metals accumulation in flowers and for '. in vivo' pollen germination. Based on the Cd and Cu accumulation amounts in the flowers, an evaluation of the same metals effects on '. in vitro' pollen germination was achieved. Moreover, the effects of both metals on fruits number and weight and on seed set and yield at individual plant level were examined. While cadmium concentrations did not affect '. in vivo' pollen germination, only higher copper concentrations rendered a significant reduction. This is in contrast with the clear negative effect on pollen germination in vitro and might be explained by the different dynamic and bioavailability of both metals. A clear effect of Cd and Cu was observed on two important yield components ie, fruit weight and seed set. Although results obtained herein cannot give a clear cut relationship between the effect of Ca and Cu on reproductive development and its consequences on yields, they represent emerging results on the potential consequences of metals contamination on reproductive development in plants. © 2010 Elsevier B.V.


PubMed | CNRS Laboratory of Oceanic Environments and Paleo-environments (EPOC) and Laboratory of Biochemistry and Environmental Toxicology
Type: | Journal: Marine environmental research | Year: 2016

The present work aimed to assess the effects of two widespread metallic pollutants, copper and silver, along with environmentally-realistic temperature increases, on embryo-larval development of the Mediterranean mussel Mytilus galloprovincialis. First, mussel embryos upon fertilization were exposed for 48h to increasing concentrations of Cu (0.5-500g/L) and Ag (0.1-100g/L) at different temperatures (18, 20, 22 or 24C) in order to characterize toxicity of each toxicant at the different tested temperatures. Increasing concentrations of a Cu-Ag mixture were then tested in order to assess the mixture effect at different temperatures (18, 20 or 22C). Embryotoxicity was measured after 48h of exposure (D-larvae stage) considering both the percentage of abnormalities and developmental arrest in D-larvae. The results suggest that the optimum temperature for mussel larvae development is 18C (12.651.6% malformations) and beyond 20C a steep increase of abnormal larvae was observed up to 100% at 24C. Ag was more toxic than Cu with a 50% effective concentration (EC50) at 18C of 6.58g/L and 17.6g/L, respectively. Temperature increased the toxicity of both metals as proved with the EC50 at 20C at 3.86g/L and 16.28g/L for Ag and Cu respectively. Toxic unit calculation suggests additive effects of Cu and Ag in mixture at 18 and 20C. These results highlight a possible impairment of M.galloprovincialis reproduction in the Mediterranean Sea in relation to increase of both pollutants and water temperature due to global warming.

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