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

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Abstract

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.

Highlights

► DNA strand-break levels measured in transplanted mussels in urban area vary according to the seasonal changes with the water contamination, the reproductive activity, and the water temperature. ► DNA strand breaks are higher in spring and summer compared to winter highlights the importance of the filtration activity in the water contaminants exposure of mussels. ► The MN had also a seasonal trend, whether it is less pronounced than the comet assay. ► DNA strand-break levels and MN frequencies vary in relation with PAH contamination.

Introduction

Chemical contaminants are generally present as a complex environmental mixture and at low concentration in freshwater ecosystems. They can cause subtle effects likely to increase the vulnerability of organisms to anthropogenic or natural stressors and lead to long term impairment of populations. Biomonitoring is a key process to determine the impact of pollutants on aquatic organisms. Among the biomarkers developed these past years, the measurement of DNA strand breaks with the comet assay has been shown to be a reliable biomarker of exposure, particularly in this context with the low water contaminant concentration (Frenzilli et al., 2009).

The freshwater mussel, Dreissena polymorpha, also known as the zebra mussel, is commonly used as a bioindicator organism of freshwater pollution due to its high contaminant uptake (Kraak et al., 1991, De Lafontaine et al., 2000). Zebra mussels are used for genotoxicity testing (Mersch and Johansson, 1993, Klobucar et al., 2003a, Klobucar et al., 2003b). Indeed the comet assay and MN assay are complementary genotoxicity tests, frequently used for biomonitoring in aquatic organisms such as mussels (Klobucar et al., 2003a, Klobucar et al., 2003b, Bolognesi et al., 2004a, Bolognesi et al., 2004b, Rank et al., 2005, Danellakis et al., 2011). The alkaline version of the comet assay is recommended for its sensitivity and its ability to detect several of DNA lesions induced by genotoxic compounds: DNA double strand breaks (DSB), DNA single strand breaks (SSB) and alcali-labile sites (ALS) (Singh et al., 1988). In D. polymorpha the comet assay has proved to be a useful tool to determine the genotoxic potential of a polluted environment (Binelli et al., 2007, Bourgeault et al., 2010a, Bourgeault et al., 2010b). We recently determined that gill cells were more sensitive than hemocytes to genotoxic compounds and that DNA strand breaks were persistent over 2 months in zebra mussels transplanted to contaminated sites of the Seine River basin (Bourgeault et al., 2010a, Bourgeault et al., 2010b, Vincent-Hubert et al., 2011).

The micronucleus assay detects chromosomal aberrations, which are irreversible lesions. MNs are formed by the loss of a piece of chromosome as a result of DNA double-strand breakage (clastogenic effect) or by the loss of an entire chromosome as a result of protein dysfunctions in chromosome segregation (aneugenic effect). The MN assay is widely used in ecotoxicology studies conducted with zebra mussels (Kirsch-Volders et al., 2003). Many studies (Mersch et al., 1996, Mersch and Beauvais, 1997, Klobucar et al., 2003a, Klobucar et al., 2003b, Binelli et al., 2008a, Binelli et al., 2008b) used the MN assay on zebra mussels to detect freshwater genotoxicity and they concluded that the MN induction measured in vitro was similar to that measured in vivo (≈10 percent).

Few data are available on the potential influence of different parameters on genotoxicity biomarkers. Indeed, some biotic and abiotic factors may mask the consequences of the impact of chemical contaminants on field-exposed mussels. Firstly, as observed for marine mussels, the physiological status of the organisms (ex. reproduction status), which is strongly related to water temperature, could influence the biomarkers' responses. Indeed seasonal variation was observed in DNA strand break, DNA adducts and micronuclei in marine mussels, while no data are available for zebra mussels (Skarpheoinsdottir et al., 2005, Rank et al., 2007, Villela et al., 2007, Pisanelli et al., 2009, Almeida et al., 2011). As the comet assay reflects the DNA repair capacity and the DNA lesions, it might be important to perform the assay on organisms of equivalent physiological status. Secondly, the physicochemical characteristics of water vary between seasons, which modulate the bioavailability of pollutants and therefore potentially influence the genotoxic effect. Finally, water contamination varying from season to season depending on the type of contaminants and their concentrations could be considered as one possible cause of the genotoxic effects' variability (Bolognesi et al., 2004a, Bolognesi et al., 2004b).

In the present study, biomarkers were chosen according to previous studies that had shown that sediment and suspended particulate matter were genotoxic in the Seine River estuary (Cachot et al., 2006, Vincent-Hubert et al., 2012). In the same way, DNA adducts measured in mussels were correlated with an elevated concentration of PAHs (Rocher et al., 2006). The principal sources of pollution in the Seine River basin stem from high industrial and urban activity as well as intensive agriculture. This area of France (75,000 km2) is inhabited by 16 million people, concentrated within the Paris area. This high population density leads to elevated levels of micro-contaminants in water, including polycyclic aromatic hydrocarbons (PAHs) (Meybeck, 1998, Boët et al., 1999, Blanchard et al., 2007, Tusseau-Vuillemin et al., 2007, Thévenot et al., 2009). As the flow of contaminants entering the Seine estuary flows through the Paris area, it has become important to determine the impact of chemical contaminants on aquatic organisms in this part of the Seine basin.

The aim of this study was to assess whether DNA damage measured in transplanted mussels to polluted area vary according to seasonal changes. For this purpose, a 1-year monitoring programme was conducted to measure DNA damage (DNA strand breaks, micronucleus formation) in transplanted mussels as well as temperature, reproduction status and the condition index. Mussel contamination and water contamination with PAHs were analysed. Sites were chosen in the Seine River basin, along an increasing chemical contamination gradient.

Section snippets

Chemicals and enzymes

Dispase II was purchased from Roche Diagnostic (Meylan, France) and all other reagents were purchased from Sigma-Aldrich (St-Quentin-Fallavier, France).

Mussel sampling and caging

As we need to study the urban contamination impact, the mussel sampling was realized in one site located far away from every city, not subject to urban contaminants. Zebra mussels were collected at a reference site, in the Meuse River (North-Eastern of France) in October 2008 (Cachot et al., 2006, Rocher et al., 2006, Vincent-Hubert et al., 2012

Physiological parameters

Mussel mortality, CI, GSI, and FR are represented in Table 1. The CI did not vary between seasons. The CI of mussels from the site upstream of Paris (Marnay-sur-Seine) was lower than the CI of mussels from Bougival and Triel-sur-Seine, in April and in June (p<0.05), indicating that the food quantity and/or quality was higher in urban sites. The GSI was significantly higher in April than in January and June (p<0.05) in mussels collected at the three sites, indicating that in April mussels were

Discussion

This study was set up to determine if the DNA damage measured in transplanted mussels in urban area of Paris vary according to seasonal changes. Data of only three seasons are available in this document due to mussel mortality in summer. We can try to explain this mussel mortality by the oxygen concentration present in water. The increase of water temperature induced a higher mussel oxygen demand which was unsatisfied. Indeed, the high water temperature decreased the concentration of available

Conclusion

This study highlights that the DNA strand-break levels measured in transplanted mussels in urban area vary according to the seasonal changes with the water contamination, the reproductive activity, the water temperature. The MN had also a seasonal trend, whether it is less pronounced than the comet assay.

The fact that the DNA strand breaks are higher in spring and summer highlights the importance of the filtration activity in the water contaminants exposure of mussels.

Both the genotoxicity

Acknowledgments

Aurélie Germain and Amélie Dubois contributed to the completion of this study. Cécile Michel and Adeline Bourgeault acknowledge a Ph.D. Grant from the Ile-de-France Regional Council (R2DS program). This work is supported by the PIREN-Seine research program.

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