Project description:Arsenic is an ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [AsIII] and arsenate [AsV]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses in response to the presence of arsenate and arsenite in wild type and in mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain including induction of several stress related genes and repression of energy generation processes. The responses observed in the arsB mutant strain were similar to the wild type in short term but were maintained in time while they were only transient in the wild type strain. In contrast, the responses observed in a strain lacking all arsenate reductases (the SARS12 strain) were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis. These results suggest that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, over-expression of ArsB conferred hypersentivity to nickel, copper and cadmium in an arsR mutant strain. Analysis of genome-wide gene expression patterns in response to arsenic in WT and mutants involved in arsenic detoxification in the cyanobacterium Synechocystis sp PCC 6803. Cells treated with arsenate or arsenite for 1h. Details: WT cells were treated with 1 mM arsenite for 1 h and its expression profile compared to untreated cells (grown in BG11C media). The effects of arsenate were analyzed in the same way but using a modified BG11C media that constains 15% of the normal phosphate concentration (BG11C low phosphate). A reduction in the phosphate concentration in the media is essential to detect grow inhibition after arsenate addition. In the same way that for arsenite cells were treated with 50 mM arsenate for 1 h and their expression profile was compared to untreated cells. Expression profiles of mutant strains lacking arsB gene (SARSB strain; this strain is hypersensitive to arsenite because it lacks the arsenite exporter) or arsR gene (SARSR strain; this strain expresses the arsBHC constitutively) in control conditions and in response to arsenite addition were also analyzed. In addition the expression profiles of a mutant lacking arsenate reductases (SARS12 strain that has interrupted arsC, arsI1 and arsI2 genes; this strain is hypersensitive to arsenate) were analyzed in both control conditions and after addition of 50 mM arsenate.

Project description:Direct comparison of the transcriptional patterns between male and female in the digestive gland of a natural population of the marine mussel Mytilus galloprovincialis sampled in the Bizerta Lagoon, Tunisia, across November 2007 -February March 2008 (four stages, winter peak). Background: Seasonal environmental changes may affect the physiology of Mytilus galloprovincialis (Lam.), an intertidal filter-feeder bivalve occurring commonly in Mediterranean and Atlantic coastal areas. We investigated seasonal variations in relative transcript abundance of the digestive gland and the Digestive gland (gonads) of males and females. To identify gene expression trends, we used a medium-density cDNA microarray (1.7 K probes) in dual-color competitive hybridization analyses. Results: Hierarchical clustering of digestive gland microarray data showed two main branches, distinguishing profiles associated with the “hot” months (May–August) from the other months. Genes involved in chitin metabolism, associated with mussel nutrition and digestion, showed higher expression during summer. Moreover, we found different gene expression patterns in the digestive glands of males and females during the four stages of mussel gonadal development. Microarray data from gonadal transcripts also displayed clear patterns during the different developmental phases with peak relative mRNA abundance at the ripe phase (stage III) for both sexes. Conclusion: These data showed a clear temporal pattern in gene expression profiles of mussels sampled over an annual cycle. Physiological response to thermal variation, food availability, and reproductive status across months may contribute to variation in gene expression. Test/reference design (female/male). Direct comparison of RNA extracts obtained from the Digestive gland tissue of female and male animals. Two (male, female) x four conditions (gonad developmental stage 1, stage 2, stage 3, stage 4). Dual color competitive hybridizations with label swap. Single individuals. Four biological replicates. One replicate per array.

Project description:Transcriptional profiling of the digestive gland tissue of female mussel Mytilus galloprovincialis exposed to TCDD, n-TiO2 and their binary mixture Background: Exposure of marine organisms to pollutant mixtures may affect the pattern of contaminant uptake/bioaccumulation, as well as of gene expression in the tissues. 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.We have recently shown that in the marine mussel Mytilus galloprovincialis exposure to n-TiO2, one of the most widespread type of NPs in use, in combination with 2,3,7,8-TCDD, chosen as model organic xenobiotic, can exert antagonistic or synergistic effects on different biomarkers from the molecular to the tissue level, depending on cell/tissue and type of measured response. An integrated approach involving immunhistochemical and transcriptomic analysis was employed to clarify the itteractive effects of n-TiO2 and TCDD in mussels digestive gland. In particular,TCDD bioaccumulation was evaluated utilizing specific anti-TCDD fluorescent antibodies. Moreover, immunohistochemical evaluation of antioxidant and cytoskeletal components was performed. To provide clues about how the molecular response to the investigated compounds is modulated, we used a cDNA microarray with1673 sequences. In animals exposed only to TiO2, functional genomics analysis of the microarray data (48 differentially expressed genes (DEGs)) highlighted three biological processes, largely dominated by the up-regulation of microtubule-based movement-related genes. Exposure to 2,3,7,8-TCDD yielded 49 DEGs exhibiting distinct patterns in terms of biological processes. Finally, exposure to the mixture rendered 62 GEGs characterized by the regulation of response to chemical stimulus, microtubule-based movement and intracellular signal transduction. Our data should be carefully considered in view of the biological effects of emerging pollutants, particularly in case of mixture chemicals. Transcriptional profiling of the digestive gland tissue of female mussel Mytilus galloprovincialis exposed to TCDD, n-TiO2 and their binary mixture

Project description:Transcriptional profiling of mussel (Mytilus galloprovincialis) digestive gland tissue comparing control tissue with tissue obtained from animals exposed for four days to sublethal amounts of Nickel, Chlorpyrifos, or their mixture. 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 proposed the use of 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: Stress biomarkers showed statistically significant antagonistic deviations from the reference model systems to predict mixture toxicity which are based either on simple additivity or non-interaction. While toxicokinetic modeling did not explain mixture interactions, gene expression profiling and further functional genomics analysis provided clues that the decrement of toxicity may arise from the development of specific toxicodynamics. Multivariate statistics of microarray and quantitative PCR data showed two separate patterns for the single chemicals and a composite complex profile for the mixture suggesting the occurrence of interactive molecular responses. The latter signature accounted for differentially expressed genes whose relative expression values were either in trend or in contrast with those found due to single substance treatments and a relevant set of sequences which were exclusive of the mixture gene list. Conclusion: The functional genomics assessment fits with biological data to indicate the occurrence of different toxicodynamic events and ‘in general’ a decrease of toxicity, driven by the mitigation or even abolishment of lysosomal responses such as the lipid metabolic disorder observed exclusively in single chemical-exposed samples. Furthermore, our results emphasized the importance of the application of mechanistic approaches and the power of systems assessment to study toxicological responses in ecological relevant organisms. Mussel MytArray 1.0 (Platform GPL1799)-based arrays: Three-condition experiment. Dual color competitive hybridizations. Common reference (vehicle treated animals, 0.02% Dimethyl sulfoxide); Pools of six animals. Biological replicates: 4 controls, 4 Nickel, 4 Chlorpyrifos. One replicate per array. Mussel MytArray 1.1 (Platform GPL10269)-based arrays: Four-condition experiment. Dual color competitive hybridizations. Common reference (vehicle treated animals, 0.02% Dimethyl sulfoxide); Pools of six animals. Biological replicates: 5 controls, 1 Nickel, 3 Chlorpyrifos, 5 mixture. One replicate per array.

Project description:Transcriptional profiling of mussel (Mytilus galloprovincialis) digestive gland tissue comparing control tissue with tissue obtained from animals exposed to sublethal amounts of Chlorpyrifos, animals injected into the posterior adductor muscle with 25 pico-moles 17β-estradiol (E2) and animals pre-exposed for three days to the pesticide and further injected with E2. Background: Many pesticides have been shown to act as endocrine disrupters. Although the potencies of currently used pesticides as hormone agonists/antagonists in vitro 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 possible endocrine effects of CHP in aquatic organisms. In the digestive gland of the bivalve Mytilus galloprovincialis, a target tissue for the action 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 and 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 mussel 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. Four-condition experiment. Dual color competitive hybridizations. Common reference (vehicle treated animals, 0.02% Dimethyl sulfoxide, injected with 50 μl of a solution of Artificial Sea Water containing 0.05 % ethanol); Pools of six animals. Biological replicates: 4 controls, 4 Chlorpyrifos, 4 17β-estradiol, 4 Chlorpyrifos-17β-stradiol. One replicate per array.

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