Seasonal and inter-annual variability of DNA integrity in mussels Mytilus galloprovincialis: a possible role for natural fluctuations of trace metal concentrations and oxidative biomarkers.

The DNA strand fragmentation as analyzed by the Comet assay is a common biomarker widely used to assess the genotoxic potential of chemical pollutants in marine organisms. However, basal levels of DNA strand breaks can be influenced also by factors unrelated to chemical exposure, and a better knowledge on the natural variability of such response would thus be important to discriminate the effects of anthropogenic activities. In this respect, the aim of the present work was to characterize the fluctuations of DNA strand breaks in mussels, Mytilus galloprovincialis, seasonally sampled over a 3 year period from a reference site along the Adriatic coast. Parameters reflecting DNA integrity exhibited significant seasonal changes in mussels haemocytes, with considerable differences between various sampling years. Comparing such results with those previously obtained on the natural fluctuations of trace metal concentrations and oxidative stress biomarkers in mussels tissues, significant correlations were obtained between DNA strand breaks with levels of Ba, Cd, Cu, Hg, Ni, Se, activity of catalase and content of malondialdehyde. Although mechanisms remain to be demonstrated, the overall results of this work suggest the potential role of natural prooxidant factors in affecting the baseline levels of DNA integrity in mussels, and confirm that natural variability of such responses should be carefully considered when monitoring the impact of genotoxins in marine ecosystems.

Contaminant accumulation and biomarker responses in caged mussels, Mytilus galloprovincialis, to evaluate bioavailability and toxicological effects of remobilized chemicals during dredging and disposal operations in harbour areas.

Remobilization of chemicals from contaminated sediments is a major risk associated with dredging and disposal operations in harbour areas. In this work caged mussels, Mytilus galloprovincialis, were chosen as bioindicator organisms to reveal the impact and recovery of organisms from these activities in the harbour of Piombino (Tuscany, Italy) where approximately 100,000 m(3) of sediments were removed and disposed in a local confined disposal facility (CDF). Organisms were deployed before, during and after the end of operations, selecting sites differently impacted by these activities. Temporal changes in environmental bioavailability and biological effects of pollutants were assessed by integrating analyses of trace metals and polycyclic aromatic hydrocarbons (PAHs) accumulated in tissues of caged mussels with a wide array of biomarkers reflecting exposure to specific classes of pollutants and different levels of cellular unbalance or toxicity. Such biological responses included levels of metallothioneins, activity of acyl CoA oxidase (AOX) as a marker of peroxisome proliferation, oxidative stress biomarkers (content of glutathione, enzymatic activities of catalase, glutathione S-transferases, glutathione reductase, glutathione peroxidases), total oxyradical scavenging capacity (TOSC) toward peroxyl and hydroxyl radicals, lysosomal membrane stability and genotoxic effects measured as DNA strand breaks and frequency of micronuclei. Obtained results indicated that a general disturbance was already present in the whole harbour area and especially in the inner site before the beginning of operations, when caged mussels exhibited a significant accumulation of PAHs and Pb, lower TOSC values and higher levels of both lysosomal and genotoxic damages. Bioavailability of trace metals and PAHs markedly increased during dredging activities with values up to 40 microg/g for Pb and up to 2200 ng/g for PAHs in tissues of caged mussels, a significant inhibition of antioxidant efficiency and increase of oxidative damages. While bioavailability of trace metals returned to the pre-dredging values after the end of operations, the accumulation of PAHs, oxidative effects and genotoxic damages remained elevated in mussels caged in the inner area and in front of CDF. Overall this study confirmed the utility of caged mussels to assess the remobilization of chemicals from dredged sediments and the onset of potentially harmful biological effects.

Seasonal variations of exposure biomarkers, oxidative stress responses and cell damage in the clams, Tapes philippinarum, and mussels, Mytilus galloprovincialis, from Adriatic sea.

This work investigated the natural variability of several biomarkers in Tapes philippinarum and Mytilus galloprovincialis, sampled from Northern Adriatic where these organisms are important sentinel species for future environmental impact assessment. Levels of metallothioneins, peroxisomal enzymes and acetylcholinesterase, showed a significant seasonality and marked differences between clams and mussels. Among antioxidant enzymes, catalase and GST decreased during the warmer period, the latter enzyme activity resulting particularly high in clams. The total oxyradical scavenging capacity toward peroxyl radicals decreased in mussels from winter to summer, indicating a prooxidant challenge due to higher seawater temperature and intensity of light irradiance. Lysosomal membrane stability did not exhibit significant seasonal variations, while some variations were observed for DNA damages. Overall results indicated a significant influence of seasonal variability on several biomarkers and species-specific differences which should be considered to discriminate the appearance of anthropogenic disturbance.

Ecotoxicological and human health risk in a petrochemical district of southern Italy.

An ecotoxicological investigation has been carried in the petrochemical district of Priolo (Sicily, Italy), one of the largest in Europe. Results indicated a severe mercury contamination in sediments sampled near a chloro-alkali plant. A clear bioavailability of this element was demonstrated in mussels Mytilus galloprovincialis (both native and translocated) and the benthic fish Mullus barbatus, which also exhibited marked genotoxic damages. The elevated mercury concentrations in marine organisms are a serious concern for human health; according to the national average fish consumption, the provisional tolerable weekly intake (PTWI) of Hg would be easily exceeded by at least 4 to 12 fold. Such toxicological risk is of particular importance for pregnant women, being possibly involved in the elevated frequency of neonatal malformations.

Influence of the SCGE protocol on the amount of basal DNA damage detected in the Mediterranean mussel, Mytilus galloprovincialis.

Genotoxicity studies using the single cell gel electrophoresis (SCGE) assay indicate that basal levels of DNA strand breaks (SBs) in marine invertebrates are higher and more variable than those in marine vertebrates. This elevated level of DNA damage was attributed to a large number of alkali-labile sites, which are characteristic of the tightly-packaged DNA in invertebrate cells. To investigate if altering the SCGE protocol can artificially modulate high levels of SBs, SCGE experiments were performed on haemocytes from the Mediterranean mussel (Mytilus galloprovincialis) using proteinase K (PK) digestion in combination with assay buffers containing various concentrations of EDTA. In addition, the effects of Trolox (soluble antioxidant) and aurintricarboxylic acid (ATA; inhibitor of Ca(2+)/Mg(2+)-dependent nucleases) also were tested. The levels of SBs in M. galloprovincialis cells were compared with SBs in cells from a terrestrial mollusk (the snail Helix aspersa), and a teleost fish (the seabass Dicentrarchus labrax). The integrity of M. galloprovincialis DNA isolated with phenol extractions using EDTA, Trolox, and ATA was further assayed by gel electrophoresis. High SBs in mussel cells were reduced by combining EDTA with PK digestion, or using Trolox or ATA during cell processing for the SCGE assay. Snails and seabass had lower levels of SBs in the SCGE assay, and the levels were not affected by the protocol modifications. Adding EDTA, Trolox, or ATA to phenol extractions of M. galloprovincialis genomic DNA also reduced the extent of DNA fragmentation. These results suggest that the internal fluids of M. galloprovincialis may increase the basal levels of DNA SBs through oxidative and/or enzyme-mediated pathways. M. galloprovincialis is used extensively as a sentinel species for assessing the genotoxic hazard of marine pollutants. Our data suggest that the SCGE protocol should be carefully considered when assessing DNA damage in these species.