Effects and bioaccumulation of Cr(III), Cr(VI) and their mixture in the freshwater mussel Corbicula fluminea.

Chromium has two main oxidation states, Cr(III) and Cr(VI), that can occur simultaneously in natural waters. Current consensus holds that Cr(VI) is of high ecotoxicological concern, but regards Cr(III) as poorly bioavailable and relatively non-toxic. In this work, the effects and bioaccumulation of Cr(III), Cr(VI) and their mixture were studied using the freshwater clam Corbicula fluminea as a model organism. Mixture exposures were carried out using solutions isotopically enriched in 50Cr(III) or 53Cr(VI), allowing to quantify the contribution of each redox form to total Cr accumulation in the clams. Following exposure to individual redox forms, Cr(III) accumulated preferentially in the digestive glands and Cr(VI) in the gills of C. fluminea. In mixture exposures, both redox forms accumulated mainly in the gills; the concentration of Cr(III) in the digestive glands being much lowered compared with individual exposures. Both oxidation states affected the expression of biomarkers related to energy reserves, cellular damage and mitochondrial functioning, as well as the expression of mRNA for detoxification genes. The observed effects differed between gills and digestive glands. The present study suggests that Cr(III) is a bioavailable and biologically active elemental species deserving more consideration by the ecotoxicological community.

Early defense responses in the freshwater bivalve Corbicula fluminea exposed to copper and cadmium: Transcriptional and histochemical studies.

The aim of the present study was to measure the early effects of copper (10 and 50 µg L(-1)), cadmium (2, 10, and 50 µg L(-1)) and mixtures of these metals in the freshwater bivalve Corbicula fluminea exposed for 12 h in laboratory. Transcription levels of superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GPx), pi-class glutathione S-transferase (pi-GST), metallothionein (MT) in digestive gland and gills, and response of lysosomal system and neutral lipids in digestive gland were determined after the exposure period. Results showed that lysosomal system, neutral lipids content, and mRNA levels were modified, suggesting their early response against oxidative stress and their important role in cell integrity. The integrated biomarker response was calculated and showed that the effects of the combinations of Cu and Cd on the biomarker responses are additive. MT and pi-GST mRNA expression correspond to the largest ranges of response. As efficient biomarkers should have an early warning capacity, SOD, CAT, Se-GPx, pi-GST, MT transcripts levels, lysosomal system, and neutral lipids could be used as biomarkers of metal contamination in the aquatic environment.

SOD and CAT cDNA cloning, and expression pattern of detoxification genes in the freshwater bivalve Unio tumidus transplanted into the Moselle river.

The cDNA sequences encoding manganese superoxide dismutase (Mn-SOD) and catalase (CAT) were isolated in the freshwater bivalve Unio tumidus by reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers. Quantitative real-time PCR approach was used to evaluate the mRNA expression patterns of SOD, CAT, selenium-dependent glutathione peroxidase (Se-GPx), pi class glutathione S-transferase (pi-GST) and metallothionein (MT), in the digestive gland of Unio tumidus transplanted from a control site to four stations in the Moselle River (M1-M4), for periods of 8 and 21 days. These sites were chosen upstream and downstream of populated areas. Chemical analysis performed on sediments from the Moselle river sites did not show high levels of pollutants. Decrease of SOD, CAT, Se-GPx and MT mRNA levels were observed at M3 site after a 21-day exposure compared to control site. These results suggest inefficiency of antioxidant systems affected by cytotoxic mechanisms and confirm an environmental perturbation. Organisms transplanted at M4 site showed a strong increase of biomarkers transcription levels after 21 days of exposure. These inductions could correspond to an adaptive response to an altered environment. Our results showed that biological approaches using multibiomarkers appear as essential tools complementary to measurement of contaminants, to detect environmental degradations.

Metallothionein coding sequence identification and seasonal mRNA expression of detoxification genes in the bivalve Corbicula fluminea.

The aim of this study was to identify a metallothionein (MT) coding sequence from the freshwater bivalve Corbicula fluminea and to measure the seasonal transcriptional pattern of MT in parallel with several detoxification genes: superoxide dismutase (SOD), catalase (CAT), glutathione S-transferases (GST) and glutathione peroxidases (GPx), in the digestive gland and the gills of this bivalve during a 1-year period. We identified a C. fluminea MT complete cDNA sequence using RT-PCR and RACE-PCR. The amino acid sequence deduced from the coding sequence encodes for a protein of 73 amino acids containing 21 cysteine residues. This protein exhibits high identities and similarities with the MT sequences of numerous bivalves. MT, SOD, CAT, pi-GST and Se-GPx expression patterns did not exhibit major seasonal variations. A slight increase of MT was observed in July. Therefore, the mRNA expression of these five genes could be used as biomarkers for monitoring studies.

Glutamate cysteine ligase (GCL) in the freshwater bivalve Unio tumidus: impact of storage conditions and seasons on activity and identification of partial coding sequence of the catalytic subunit.

Glutamate cysteine ligase (GCL; EC 6.3.2.2) is the first enzyme involved in the synthesis of glutathione. A HPLC method with fluorimetric detection was used to measure GCL activity in the gills and the digestive gland of the freshwater bivalve, Unio tumidus. Storage conditions were optimized in order to prevent decrease of GCL activity and consisted in freezing the cytosolic fraction in the presence of protease (1 mM phenylmethylsulfonic fluoric acid) and gamma-glutamyltranspeptidase (1 mM L-serine borate mixture and 0.5 mM acivicin) inhibitors. Seasonal variations of activity in the digestive gland and to a lesser extent in the gills were found with activity increasing in spring compared to winter. No sex differences were revealed. The GCL coding sequence was identified using degenerated primers designed in the highly conserved regions of the catalytic subunit of GCL. The partial sequence identified encoded for 121 amino acids. The comparison of the identified partial coding sequence of U. tumidus with those available from vertebrates and invertebrates indicated that GCL sequence was highly conserved.

Integrated assessment of ceria nanoparticle impacts on the freshwater bivalve Dreissena polymorpha.

Exposures in realistic environmental conditions are essential to properly assess the effects of emerging pollutants on ecosystems. While ceria nanoparticles (nCeO2) production and use are expanding quickly, ecotoxicity studies remain very scarce. In this study, we set up experimental systems reproducing a simplified ecosystem to assess the effects of a chronic exposure to citrate-coated nCeO2 (ci-CeO2) and bare nCeO2 (ba-CeO2) on the freshwater mussel Dreissena polymorpha using an integrated multibiomarker approach. The fate of nanoparticles was tightly monitored to properly characterize the exposure. Organisms were exposed for 3 weeks and sampled weekly for biomarker analysis. Mussel filter-feeding activity resulted in significant removal of nCeO2 from the water column. At the same time, bioaccumulation was low, reaching its maximum in the first week. Mussels bioaccumulated ci-CeO2 three times more than ba-CeO2, probably due to coating-related differences in their behavior in the water column and in organisms. Meanwhile, biomarker results were integrated and synthesized using linear discriminant analysis, highlighting that pi-glutathione-S-transferase (piGST) mRNA, catalase (CAT) activity and lysosomal system were the most impacted of the seven biomarkers singled out by the discriminant analysis. These biomarker responses indicated that mussels exposed to both forms of nCeO2 were stressed and differentiate from the controls. Moreover, they responded differently to ba-CeO2 and ci-CeO2 exposure. However, biomarkers used in the experimental conditions of this study did not indicate severe nCeO2 toxicity on mussels, as cellular damage biomarkers and mussel filtering activity were left unimpaired. However, further studies are needed to investigate if the slight perturbations observed could lead to populational impacts in the long term.

Use of RNA-arbitrarily-primed PCR to detect the induction of gene expression in freshwater bivalves (Unio tumidus) transplanted into the Moselle River.

RNA-arbitrarily-primed PCR (RAP-PCR) can be used to detect changes in gene expression in organisms for which only minimal genomic information is available. In this study, RAP-PCR was used to detect modification of mRNA expression in the freshwater bivalve Unio tumidus, a mussel commonly used as a sentinel species in field studies. RNA expression was analyzed in the digestive glands of mussels from a control pond and in mussels transplanted into two sites on the Moselle River. The analysis identified a product in all animals exposed to sediments at the river station located downstream of a heavily populated area. This product was not present in animals exposed at the upstream station or at the control site. The additional PCR product was cloned and sequenced, and specific oligonucleotides for the sequence were designed to amplify cDNAs from control and transplanted mussels. A signal was obtained only with the cDNAs from animals exposed at the downstream station, confirming that the variation detected by RAP-PCR corresponds to an increase of gene expression. Chemical analysis of sediments from the control and river sites indicated that the levels of several potential pollutants were similar at the three locations and below currently accepted pollution thresholds. Our results indicate that RAP-PCR is a sensitive technique that can be applied in field studies to identify modifications in the gene expression of bioindicator species. This approach can complement chemical, biochemical and population studies to assess the impact of human activity on the ecological quality of aquatic systems.

A comparative study of the expression of CYP1A and CYP4 genes in aquatic invertebrate (freshwater mussel, Unio tumidus) and vertebrate (rainbow trout, Oncorhynchus mykiss).

CYP1A1 induction has been widely studied in vertebrates and is used as an indicator of exposure to persistent pollutants like dioxins, polychlorobiphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), especially in fish. Little work on this enzyme has been carried out in invertebrates, and results have suggested enzymatic or gene differences with the vertebrate isozyme. To date, a CYP4 isozyme has been characterized and sequenced in the marine mussel, Mytilus galloprovincialis, but no CYP1A invertebrate sequence has been found. The aim of this study was to identify CYP1A and CYP4 transcripts in the freshwater bivalve, Unio tumidus, and to compare their expression with those of the rainbow trout, Oncorhynchus mykiss, after treatment with the specific CYP1A1 and CYP4 vertebrate inducers, Aroclor 1254 and diethylhexylphthalate (DEHP), respectively. In spite of numerous amplification conditions and primer combinations tested, which had been successful in fish, no CYP1A sequence was amplified on cDNA of the digestive gland from either control and Aroclor-treated mussels. On the other hand, CYP4 transcripts were amplified in digestive glands from control and DEHP-treated mussels. As opposed to fish, no induction of this isozyme was obtained in mussels after phthalate treatment.

Identification and mRNA expression of pi-class glutathione S-transferase and selenium-dependent glutathione peroxidase in the gudgeon Gobio gobio exposed to PCB 77.

In aquatic environments some pollutants are present in water and sediments and organisms possess cellular detoxification systems to face up these xenobiotics. The gudgeon, Gobio gobio, is a freshwater benthopelagic fish that appears particularly adequate for an ecotoxicological assessment of rivers. The aim of this study was the identification of GST and GPx genes in this organism in order to develop new indicators of early exposure to xenobiotics in aquatic environments. Reverse-transcription PCR using degenerate primers and RACE-PCR allowed us to identify a selenium-dependent glutathione peroxidase (Se-GPx), belonging to the class one (GPx-1), and a pi-class glutathione S-transferase (pi-GST) cDNAs. These sequences encoded for 191 and 208 amino acids proteins respectively, they exhibit high identities and similarities with corresponding proteins in other fish and the residues essential to the enzymatic functions are highly conserved. The expression patterns of these two mRNA were established by real-time PCR in five gudgeon tissues: liver, gills, kidney, spleen and muscle. The mRNA levels of these genes were highest in the liver and their expression in the other tissues exhibited some differences. Gudgeons exposed to PCB 77 in the food exhibited an increase in pi-GST mRNA and a decrease in GPx-1 transcripts levels in the liver. However, no modification of the enzymatic activities was observed. The present study provides the first transcriptional data regarding pi-GST and GPx-1 in the gudgeon Gobio gobio.

Ecotoxicological assessment of TiO2 byproducts on the earthworm Eisenia fetida.

The increasing production of nanomaterials will in turn increase the release of nanosized byproducts to the environment. The aim of this study was to evaluate the behaviour, uptake and ecotoxicity of TiO(2) byproducts in the earthworm Eisenia fetida. Worms were exposed to suspensions containing 0.1, 1 and 10 mg/L of byproducts for 24 h. Size of TiO(2) byproducts showed aggregation of particles up to 700 µm with laser diffraction. Only worms exposed at 10 mg/L showed bioaccumulation of titanium (ICP-AES), increasing expression of metallothionein and superoxide dismutase mRNA (Real-time PCR) and induction of apoptotic activity (Apostain and TUNEL). TiO(2) byproducts did not induce cytotoxicity on cœlomocytes, but a significant decrease of phagocytosis was observed starting from 0.1 mg/L. In conclusion, bioaccumulation of byproducts and their production of reactive oxygen species could be responsible for the alteration of the antioxidant system in worms.

Molecular cloning and expression study of pi-class glutathione S-transferase (pi-GST) and selenium-dependent glutathione peroxidase (Se-GPx) transcripts in the freshwater bivalve Dreissena polymorpha.

Glutathione S-transferases (GST) and glutathione peroxidases (GPx) are essential components of cellular detoxification systems. We identified GST and GPx transcripts in the freshwater bivalve Dreissena polymorpha, their full-length coding sequences were obtained by reverse-transcription PCR using degenerated primers followed by 5' and 3' RACE-PCR (rapid amplification of cDNA ends-PCR). The cDNA identified encoded proteins of 205 and 243 amino acids corresponding respectively to a pi-class GST and a selenium-dependent GPx. The comparison of the deduced amino acid sequences with GST and GPx from other species showed that the residues essential to the enzymatic function of these two proteins are highly conserved. We studied their expression pattern in the digestive gland, the gills and the excretory system of D. polymorpha. The results showed that pi-GST mRNA expression is higher in the digestive gland than in the gills or the excretory system. Se-GPx transcripts are expressed at high, medium and very low levels in the digestive gland, the excretory system and the gills, respectively.

Induction of CYP1A1 in rat liver after ingestion of mussels contaminated by Erika fuel oils.

Polycyclic aromatic hydrocarbons (PAH) are known to be specific inducers of CYP1A1 expression in vertebrates. CYP1A1 induction has been widely studied in mammal cell cultures or in vivo, in conditions of exposure to single PAH chemicals. Here, we studied the possible transfer of PAH to rats via the food chain in environmentally-relevant conditions. Rats were fed for 2 days with PAH-contaminated mussels sampled on coasts polluted by the Erika oil-tanker wreck. CYP1A1 expression was investigated by measuring mRNA levels and EROD enzymatic activity over the 84 h following the last ingestion. CYP1A1 expression in treated rats was compared to controls fed with mussels free from PAH contamination. The results showed that ingestion of PAH-contaminated mussels induced CYP1A1 mRNA and EROD activity. Increase of transcriptional level and of EROD activity was transient with a peak within 12 h and a return to basal levels within 36 h.

Identification, sequencing and expression of selenium-dependent glutathione peroxidase transcript in the freshwater bivalve Unio tumidus exposed to Aroclor 1254.

Glutathione peroxidases (GPx) and glutathione S-transferases (GST) are essential enzymes of the cellular defense system. The aim of this work was the identification of GPx transcript in a freshwater bivalve, Unio tumidus, and the effects of Aroclor 1254 on GPx and pi-class GST (pi-GST) expression pattern. The GPx full-length coding sequence was obtained by reverse transcription PCR using degenerated primers followed by 5' and 3' rapid amplification of cDNA ends. The GPx cDNA encodes a protein of 232 amino acids. The 72nd amino acid corresponds to a selenocysteine encoded by a TGA codon. Residues essential to the enzymatic function are conserved in GPx of U. tumidus. Specific amplifications of the Se-GPx mRNA from U. tumidus were performed on the digestive gland, the excretory system and the gills. Se-GPx expression level is highest in the digestive gland. No induction of the Se-GPx was observed at the transcriptional level in the digestive gland and the excretory system of Aroclor-treated mussels, while an increase of the pi-GST mRNA level was observed in the excretory system.

cDNA cloning and expression pattern of pi-class glutathione S-transferase in the freshwater bivalves Unio tumidus and Corbicula fluminea.

Glutathione S-transferases (GSTs) are enzymes involved in major detoxification reactions of xenobiotics in many organisms. The aim of this work was the identification of GST transcripts in the freshwater bivalves Unio tumidus and Corbicula fluminea. We used degenerated primers designed in the highly conserved regions of GST to amplify the corresponding mRNA. Full-length coding sequences were obtained by 5' and 3' rapid amplification of cDNA ends. In the two species, the GST cDNAs identified encoded a protein of 205 amino acids. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzymes belong to the pi-class and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) are highly conserved. Specific amplifications of the GST mRNA from U. tumidus and C. fluminea were performed on the digestive gland, the excretory system and the gills. For each mussel, the results revealed that the pi-class GSTs are expressed at the same level in the three tissues.