Project description:Pharmaceutical compounds are emerging contaminants in aquatic environment due to their massive use (human and veterinary medicines, agriculture and aquaculture) and a limited removal by waste water treatment plants (WWTPs). In this work, a representative determination of ecotoxicological potential of two different NSAIDs compounds was studied in the sensitive bioindicator marine organism M. Galloprovincialis. Mussels were exposed, under regulated laboratory conditions, to Ketoprofen (KET) and Nimesulide (NIM), dosed alone at the realistic environmental concentration of 0.5Âµg/L for 14 days. Gene expression analyses of Mytilus galloprovincialis exposed to KET and NIM have been performed through a DNA microarray platform. Mussels Mytilus galloprovincialis (5 Â± 1 cm shell length) were obtained from a local farm (Numana, Ancona) and acclimatized for 10 days to laboratory conditions with aerated seawater, at 18 Â± 1 Â°C, 37 â?° salinity, pH 7.5 Â± 0.5 and oxygen saturation >94%. Mussels were distributed into three 17 L aquarium and exposed at 0.5Âµg/L to ketoprofen (KET) and nimesulide (NIM) dosed alone for 14 days. All treatments were compared to control (CTRL) containing 0.00001% of methanol. Water was changed every other day and concentration of molecules were restored. Gene transcription analyses of 12 digestive glands pools (four pools for each treatment composed by 3 digestive glands; CNTR, NIM and KET) were performed using a 8X60K Agilent oligo-DNA microarray platform GPL18667. Microarrays were synthesized in situ using the Agilent non-contact ink-jet technology including default positive and negative controls. Total RNA was isolated using Extract-all (Eurobio) procedure. RNA quality and integrity was controlled on the Agilent bioanalyzer using RNA nanochips and Agilent RNA 6000 nanoreagents (Agilent Technologies, Waldbronn, Germany). RNA concentrations were measured at 260 nm using a ND-1000 spectrophotometer (Nanodrop Technologies) using the conversion factor 1 OD = 40 mg/mL total RNA. Samples were stored at -80Â°C until further use. Gene expression profiling was performed using an Mytilus galloprovincialis oligo-DNA microarray of 59,971 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 2 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. FeatureExtraction v10.7.3.1 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

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:The invasive marine mussel Mytilus galloprovincialis has displaced the native congener Mytilus trossulus from central and southern California, but the native species remains dominant at more northerly sites that have high levels of freshwater input. To determine the extent to which interspecific differences in physiological tolerance to low salinity might explain limits to the invasive species’ biogeography, we used an oligonucleotide microarray to compare the transcriptional responses of these two species to an acute decrease in salinity. Among 6,777 genes on the microarray, 117 genes showed significant changes that were similar between species, and 12 genes showed significant species-specific responses to salinity stress. Osmoregulation and cell cycle control were important aspects of the shared transcriptomic response to salinity stress, whereas the genes with species-specific expression patterns were involved in mRNA splicing, polyamine synthesis, exocytosis, translation, cell adhesion, and cell signaling. Forty-five genes that changed expression significantly during salinity stress also changed expression during heat stress, but the direction of change in expression was typically opposite for the two forms of stress. These results (i) provide insights into the role of changes in gene expression in establishing physiological tolerance to acute decreases in salinity, and (ii) indicate that transcriptomic differences between M. galloprovincialis and M. trossulus in response to salinity stress are subtle and involve only a minor fraction of the overall suite of gene regulatory responses. Two species (Mytilus galloprovincialis, Mytilus trossulus), hypo-osmotic shock for four hours (850 mOs/kg), one control group (1000 mOs/kg) sampled at the end of the treatment exposure (850 mOs/kg), one control group (1000 mOs/kg) sampled at the beginning. Biological replicates: 6 in each treatment group, 6 in each control group. Heterologous and homologous hybridization to a microarray constructed from Mytilus californianus and Mytilus galloprovincialis sequences. A reference design that used separate pools of reference RNA for each species was employed. Reference amplified RNA (aRNA) was created for each species by pooling RNA before and after amplification. The reference pool was made up of RNA from six different samples: two base-line control samples from the beginning of the experiment, two treatment samples from the end of the four-hour hypo-osmotic exposure, and two time-control samples from the end of the four-hour exposure time. To accurately compare the transcriptomes of Mytilus galloprovincialis and M. trossulus, we chose to develop a common microarray format that could be used for both species. This microarray design consisted of probe sequences generated from the out-group species, M. californianus. M. trossulus and M. galloprovincialis are approximately 7.5 million years divergent from M. californianus, yet only 3.5 million years divergent from each other (Seed, 1992). Therefore, heterologous hybridization to the microarray allowed us to compare transcriptional responses of M. galloprovincialis and M. trossulus without the inherent sequence biases that would result from a microarray that was designed from sequences of either M. galloprovincialis or M. trossulus. A limited number of sequences (556) from ESTs from M. galloprovincialis that matched M. californianus ESTs were included on the microarray to test for the effects of sequence mismatches. Only probes that performed well for both M. galloprovincialis and M. trossulus were used in our analyses. In order to determine significant changes in expression, we conducted a two-way ANOVA, in which salinity and species were modeled as fixed effects, and focused on genes that were significant for the salinity effect or the species x temperature interaction. We ignored the species term from the ANOVA as this effect could have highlighted genes that differentially bound to probes on the microarray due to differences in sequence homology, thus not reflecting true differences in gene expression. In accordance with statistical convention, all genes with a significant species x temperature interaction were deemed not to have significant temperature effects, even if the temperature term from the ANOVA had a low P-value. All genes with FDR-corrected (Benjamini and Hochberg, 1995) P-values less than 0.05 were considered significant. Analyses were conducted in the R statistical programming environment (R Development Core Team, 2009).

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.

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Pollutants bioavailability and toxicological risk from NSAIDs to marine mussels

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