Micronuclei and other nuclear abnormalities in mussels (Mytilus galloprovincialis) as biomarkers of cyto‐genotoxic pollution in mediterranean waters

Abstract

Environmental genotoxicity and cytotoxicity along the Spanish Mediterranean coast was investigated through the determination of levels of micronuclei (MN) and other nuclear abnormalities (NAs) such as nuclear buds (NB) and binucleated cells (BN) in gills of wild mussels, Mytilus galloprovincialis, from 17 study sites. The results obtained were studied in relation to the exposure to main pollutants (metals, PAHs, PCBs and DDTs), gill antioxidant enzyme activities and environmental variables (temperature and salinity). The highest MN and NB levels were found in mussels from metal-polluted sites, such as Cartagena (MN: 11.6‰, NB: 4.6‰) and Portman (MN: 8.0‰, NB: 3.5‰), where genotoxicity seemed to be related to the oxidative stress generated by exposure to metals. High frequencies of MN and NB in mussels from Algeciras 1 (MN: 8.6‰, NB: 4‰) and of MN (7.2‰) in mussels from Manilva also indicated genotoxicity, though not attributable to the pollutants analysed. In contrast, MN levels at sites highly polluted by organic contaminants such as Barcelona, Vallcarca, Tarragona and Valencia, were lower than expected (ranging from 3.8 to 5.8‰). On the other hand, evidences of cytotoxicity indicated by the high frequencies of BN found in mussels from Vallcarca, Barcelona, and Cartagena (3.9, 3.4, and 2.3‰, respectively) appeared to be specifically related to the accumulation of organic pollutants. Positive correlations found between MN and NB frequencies, and also between both NAs and water temperature, suggested that they were related genotoxic events, and that this environmental factor is able to influence their levels and has to be taken into account in the application of these biomarkers in field studies.

The mussel caging approach in assessing biological effects of wastewater treatment plant discharges in the Gulf of Finland (Baltic Sea)

“…Genotoxic chemicals induce DNA damage by directly reacting with the DNA molecule or through increased formation of ROS (Fernandez et al, 2011). The function of the antioxidant system is essential to prevent the formation of the OH À anion, the most toxic ROS, which readily reacts with the DNA molecule leading to various forms of DNA damage such as fragmentation of the DNA (Fernandez et al, 2011), as observed in the present study. The observed increased activity of the antioxidant enzymes indicated excessive formation of ROS at both exposed sites, even more so at H2, where also the highest level of DNA damage was detected.…”

DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

“…Epithelial-like gill cells are sensitive to micronucleus formation and the frequency of cells with MN or with other NA, a complementary parameter of chromosomal damage, is widely used to measure genotoxic effects in marine mussels [43], [59]. The frequency of both MN and NA significantly increased in the mussels exposed to 50–200 nM doses of combined metals, and reached values comparable to those detected in metal polluted sites along the Spanish Mediterranean coast [60] and higher than those registered in the industrial area of the Venice lagoon [43]–[44].…”

Mussel micronucleus cytome assay

“…The use of caged mussels allows robust study designs that control for age, and stage of sexual maturity, of the animals, thus reducing experimental variability in field studies. Seasonal differences in the MN frequency were also observed in many studies and a number of physical parameters related to season, such as salinity, temperature, pH and food availability, have been identified 34,35,41,47,67,73 . Water temperature was shown to have a direct effect on the mitotic rate and consequently on the extent of MN expression 34,35 .…”

“…In field studies, however, the collection of hemolymph from the posterior adductor muscle with a hypodermic needle allows repeated samplings without killing the animal, but this depends on how much hemolymph is extracted. The technique validated to evaluate MN frequency in gill cells is the most frequently applied in different species of marine [21][22][23][25][26][27][30][31][32][33][45][46][47][65][66][67][68][69][70][71] and freshwater bivalves 43,[57][58][59] and can be applied in the field as well as laboratory studies; however, in field studies, samples should be transferred to the laboratory within 24 h. The main limitation for using gill cells in large-scale biomonitoring studies is that cell preparation requires enzymatic treatment, washes and incubations in buffers to obtain single-cell suspensions. An alternative protocol, involving the direct spreading of cells from gill arches on slides, was proposed and applied in a number of biomonitoring studies in the Baltic and North Seas 28,34,35,[72][73][74][75][76][77][78][79][80] .…”