Françoise Vincent-Hubert

Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[a]P and a combination of B[a]P and Cd

The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10μg/L), B[a]P (10μg/L) or the two combined chemicals (10μg/L+1μg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl(2) or CdCl(2)+B[a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10μg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[a]P (10μg/L) induced an early increase of DNA damage in gill cells (after 10h and 1 day), while in both gill cells and haemocytes, B[a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd+B[a]P mixture decreased the DNA-damaging effect of Cd and B[a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[a]P did not induce any cytogenetic effect. B[a]P, Cd and Cd+B[a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[a]P, which makes them more suitable for future bio-monitoring studies.

Genotoxicity assessment and detoxification induction in Dreissena polymorpha exposed to benzo(a)pyrene

The use of DNA adduct analysis has previously focused on the use of marine organisms for biomonitoring, whereas similar investigations in freshwater organisms are sparse. In that context, we have investigated the relevance of DNA adducts as biomarkers of genotoxicity in the freshwater mussels Dreissena polymorpha. The objective of the present study is to determine the stability of DNA adducts induced by benzo[a]pyrene (B[a]P) in zebra mussels. Mussels were exposed to dissolved B[a]P (10-100 µg/l) for 4 days. Afterwards, mussels were kept in clean water for 28 days and DNA adduct levels were subsequently measured in two different organs, the digestive glands and the gills, using the (32)P-postlabelling technique. In parallel, the expression of genes involved in the detoxification system was assessed by qPCR (catalase, superoxide dismutase, glutathione S transferase, HSP70, aryl hydrocarbon receptor, P glycoprotein). We observed a higher level of DNA adducts in the digestive glands compared to the gills. Moreover, in gills, the level of DNA adduct was dependent on the B[a]P concentration. The levels of adducts tended to decrease in both organs after 28 days in clean water. In addition, an early induction of HSP70, PgP, AHR and SOD mRNA levels was noticed in the gills compared to the digestive glands. CAT and GST gene expression increased from 12h exposure in both organs. A higher gene expression level of those genes was observed in the gills, except for AHR and CAT genes. Data converge towards the fact that DNA adducts hence represent a very promising biomarker of B[a]P contamination and potentially of exposure to polycyclic aromatic hydrocarbons. In addition, for the first time in this study, B[a]P detoxification system was characterised in D. polymorpha.

Combined eukaryotic and bacterial community fingerprinting of natural freshwater biofilms using automated ribosomal intergenic spacer analysis.

Biofilms are complex communities playing an important role in aquatic ecosystems. Automated ribosomal intergenic spacer analysis (ARISA) has been used successfully to explore biofilm bacterial diversity. However, a gap remains to be filled as regards its application to biofilm eukaryotic populations. The aim of this study is to use ARISA to detect eukaryotic population shifts in biofilm. We designed a new set of primers to focus specifically on the ITS1-5.8S-ITS2 region of diatoms and tested it on natural biofilms. Additionally, we tested universal primers, used previously to perform ARISA on fungal communities. Cloning and sequencing showed that the universal primer set amplified various eukaryotes, whereas the new set was diatom specific. The new set amplified a wider variety of diatoms. Therefore, the universal set is appropriate to study the general eukaryotic population shifts in biofilms, whereas the new set is more appropriate to study diatoms specifically. We used both primer sets, along with a bacterial set, to study the population shifts in natural river biofilms. Principal component analysis of the ARISA fingerprints revealed seasonal shifts that did not coincide for bacterial and eukaryotic communities. Therefore, the use of both eukaryotic and bacterial primers provides a useful insight to assess microbial succession in biofilms.

Seasonal and PAH impact on DNA strand-break levels in gills of transplanted zebra mussels.

Genotoxicity endpoints are useful tools to biomonitor the physicochemical and biological quality of aquatic ecosystems. A caging study on the freshwater bivalve Dreissena polymorpha was planned to run over four seasons in the Seine River basin in order to assess whether DNA damage measured in transplanted mussels to polluted area vary according to seasonal changes. Three sites were chosen along the Seine River, one upstream from Paris and two downstream, corresponding to a chemical gradient of water contamination. The DNA strand break (comet assay) and chromosomal damage (micronucleus test) were measured in caged mussels at each site and in winter, spring and summer, along with PAH water contamination, PAH bioaccumulation, the mussel condition index (CI), the gonado-somatic index (GSI) and the filtration rate (FR). The level of DNA strand break measured in winter was low and increased in spring, concomitantly with FR and GSI. Over the same period, micronucleus (MN) frequency and PAH bioaccumulation decreased significantly in caged mussels, with both parameters positively correlated to each other. DNA strand-break levels and MN frequencies showed inter-site variations corresponding to the chemical contamination gradient. These two genotoxicity endpoints usefully complement each other in field studies. These results show that the MN test and comet assay, when applied to gill cells of caged zebra mussels, are sensitive tools for freshwater genotoxicity monitoring.

Mutagenicity and genotoxicity of suspended particulate matter in the Seine river estuary

Highly mutagenic compounds such as some PAHs have been identified in surface waters and sediments of the Seine river estuary. Suspended particulate matter (SPM) represents a dynamic medium that may contribute to the exposure of aquatic organisms to toxic compounds in the water column of the estuary. In order to investigate major sources of mutagenic contaminants along the estuary, water samples were taken at 25 m downstream of the outlet of an industrial wastewater-treatment plant (WWTP). SPM samples were analyzed for their genotoxicity with two short-term tests, the Salmonella typhimurium mutagenicity assay (TA98+S9 mix) and the comet assay in the human HepG2 cell line. Sampling sites receiving effluents from a chemical dye industry and WWTP showed the highest mutagenic potencies, followed by petrochemical industries, petroleum refinery and pulp and paper mills. These data indicate that frame-shift mutagens are present in the Seine river estuary. Furthermore, the comet assay revealed the presence of compounds that were genotoxic for human hepatocytes (HepG2 cells). We also observed a high level of mutagenic potency in the sediment of the lower estuary (3 × 10⁴ revertants/g). The source of mutagenic and genotoxic compounds seems to be associated with various types of effluents discharged in the Seine river estuary. Both test systems resulted in the same assessment of the genotoxicity of particulate matter, except for three of the 14 samples, underlying the complementarity of bioassays.

Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river.

The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution.

Detection of 8-oxodG in Dreissena polymorpha gill cells exposed to model contaminants.

Genotoxic end-points are routinely measured in various sentinel organisms in aquatic environments in order to monitor the impact of water pollution on organisms. As a first step towards the evaluation of oxidative DNA damage (8-oxodG) in organisms exposed to chemical water pollution, we have optimized the association between the comet assay and the hOGG1 enzyme for use on zebra mussel (Dreissena polymorpha) gill cells by in vitro exposure to H₂O₂. Firstly, we observed that in vitro exposure of D. polymorpha gill cells to benzo[a]pyrene (B[a]P, 98.4nM) induced an increase of the Olive Tail Moment (OTM) in both the comet-hOGG1 and comet-Fpg assays, indicating that B[a]P causes oxidative DNA damage. By contrast, methylmethane sulfonate (MMS, 33μM) only induced an increase of the Fpg-sensitive sites, indicating that MMS caused alkylating DNA damage and confirming that hOGG1 does not detect alkylating damage. Thus, the hOGG1 enzyme seems to be more specific towards oxidative DNA damage, such as 8-oxodG than Fpg. Secondly, as was observed in vitro, the in vivo exposure of D. polymorpha to B[a]P (24.6 and 98.4nM) increased oxidative DNA damage in gill cells, whereas only Fpg-sensitive sites were detected in mussels exposed to MMS (240μM). These results show that the comet-hOGG1 assay detects oxidative DNA lesions induced in vitro by H₂O₂ and in vivo with BaP. The comet-hOGG1 assay will be used to detect oxidative DNA lesions (8-oxodG) in mussels exposed in situ.

cDNA cloning and expression of two Ki-ras genes in the flounder, Platichthys flesus, and analysis of hepatic neoplasms.

The screening of a flounder cDNA library with a partial sequence of ras gene from flounder (exons 1 and 2) allowed the isolation of two complete cDNA sequences (ras1 and ras2) highly homologous to human Ki-rasb genes. ras1 and ras2 sequences have an homology of 77.3% indicating that they represent two distinct genes, which differ particularly in their 3 regions. ras1 and ras2 intron 1 sequencing revealed an homology of only 50%, confirming that they represent two different genes. Both genes encode for a 188 amino-acid protein, a size characteristic of Ki-rasb proteins. ras1 protein has the stronger homology to the human Ki-rasb protein (99% identity) and ras2 presents a 85.5% of homology. Two transcripts of respectively 2 and 2.8 kb were identified by northern blots with either ras1 or 2 probes. Preneoplastic and neoplastic livers collected from 14 flounder did not present any mutation on the ras2 gene.

DNA adduct formation and induction of detoxification mechanisms in Dreissena polymorpha exposed to nitro-PAHs

Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 μM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 μM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 μM. Surprisingly, at the higher concentration (500 μM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 μM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha.

Metallothionein mRNA induction is correlated with the decrease of DNA strand breaks in cadmium exposed zebra mussels

We have previously shown that cadmium (Cd) and benzo[a]pyrene (BaP) induced early DNA damages in zebra mussels, and that the level of DNA strand breaks (SB) returned to a basal level after 3 days of exposure to Cd. The aim of the present study was to go further in the mechanisms of Cd and BaP detoxification. For that purpose, expression of genes encoding for metallothionein (MT), aryl hydrocarbon receptor (AHR), P-gp, catalase, glutathione S-transferase and heat shock protein 70 (HSP70) proteins have been measured using RT-qPCR. Data reported here show that Cd is a strong inducer of MT and HSP70 genes, and that BaP is a strong inducer of P-gp and AHR genes. Exposure to Cd and BaP resulted in moderate changes in antioxidant enzymes mRNA. Since the increase of MT mRNA occurred when the DNA SB level returned to its basal level, we can suggest that MT is implicated in cadmium detoxification.