DNA Damage In Mussels Research Articles

Comet assay in ecogenotoxicology: Applications in Mytilus sp

The comet assay is a sensitive technique to detect DNA damage caused by exposure to genotoxic chemical and physical agents and is widely used in ecotoxicology. The assay has been applied in aquatic species, mainly fish and bivalves, in field biomonitoring programs and in experimental studies. The aim of the present study was to retrieve and review the published evidence to define the role of the comet assay in the assessment of genotoxic pollutants. The study focused on the application of the test in Mytilus sp, used as a sentinel organism. Twenty-one biomonitoring studies, carried out in wild and in transplanted mussels, were evaluated. An increase of the comet parameters in animals from polluted areas with respect to the controls was observed in the majority of the studies with a large variability (frequency ratio:1.2–14.5) associated with types and extent of exposure to pollutants. Three studies out of 21 reported a lack of response. Heavy metals, polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and pesticides were the main types of chemicals detected in sediments and/or mussel tissues from polluted areas. Twenty-nine laboratory studies were retrieved showing the sensitivity of the comet assay in detecting DNA damage in mussels exposed to the most relevant pollutants and mixtures of relevant contaminants, such as pharmaceuticals, anti-fouling agents or crude oil. The comet test also appeared to be a suitable approach to detect the genotoxic effects of nanoparticles. In some studies problems in the interpretation of data or discrepancies between the results from different laboratories were noted. Critical steps in experimental protocol and characterization of pollution, environmental variables such as temperature, salinity, food availability, physiological and pathological status of the animals are important factors which should be controlled and considered in the analysis of the results.

Biomarkers of oxidatively induced DNA damage in dreissenid mussels: A genotoxicity assessment tool for the Laurentian Great Lakes.

Activities of fast growing human population are altering freshwater ecosystems, endangering their inhabitants and public health. Organic and trace compounds have a high potential for adverse impacts on aquatic organisms in some Great Lakes tributaries. Toxic compounds in tissues of organisms living in contaminated environments change their metabolism and alter cellular components. We measured oxidatively induced DNA damage in the soft tissues of dreissenid mussels to check on the possible contaminant-induced impact on their DNA. The animals were obtained from archived samples of the National Oceanic and Atmospheric Administration (NOAA) Mussel Watch Program. Mussels were collected from the harbor of Ashtabula River in Ohio, and a reference area located at the Lake Erie shore. Using gas chromatography-tandem mass spectrometry with isotope dilution, we identified and quantified numerous oxidatively modified DNA bases and 8,5'-cyclopurine-2'-deoxynucleosides. We found significant differences in the concentrations of these potentially mutagenic and/or lethal lesions in the DNA of mussels from the harbor as compared to the animals collected at the reference site. These results align NOAA's data showing that elevated concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals were found in mussels within the harbor as compared to mussels collected in the reference site. The measured DNA lesions can be used as biomarkers for identifying DNA damage in mussels from polluted and reference sites. Such biomarkers are needed to identify the bioeffects of contaminants in affected organisms, as well as whether remedial actions have proven successful in reducing observed toxic effects.

Assessment of the effect of environmental pollution in a marina on caged mussels using chemical and genotoxic analysis

ABSTRACTAn integrated approach using the contamination levels and DNA damage in mussels (Mytilus galloprovincialis) was applied in order to assess the chemical contamination in a marina (Eastern coastline of Aegean Sea). Mussels, which were harvested from a reference site (Foca), were transplanted into a marina situated along the coast of Izmir Bay. The transplanted mussels were collected at the 14th, 30th and 60th day of the experimental period. Polycyclic aromatic hydrocarbon (PAH) levels (27–51 ng g−1 wet weight) detected in the mussels were similar to the levels detected in other coastal areas of the Mediterranean Sea. The marina’s sediment was found to be contaminated with PAHs (∑PAH = 25 µg g−1) of pyrolytic origin and may become a source of pollution and a threat to the marine environment. In order to assess the DNA damage, the haemolymph and gill cells of the mussels were used for the comet analysis and considered as an indicator of exposure to genotoxic chemicals including 16 PAH compounds and metals. The highest levels of DNA damage expressed as %Tail-DNA (%T-DNA) were observed at the end of the experiment (21.5% T-DNA). The correlation analyses conducted between 2-, 3-, 4-ring PAHs in mussels and %T-DNA in haemolymph and gill cells showed a significant positive correlation. This investigation confirmed that transplanted mussel can be a useful tool to determine PAH contamination in marinas.

Assessment of the genotoxic potential along the Danube River by application of the comet assay on haemocytes of freshwater mussels: The Joint Danube Survey 3

In this study we assessed the level of genotoxic pollution along the Danube River by measuring the level of DNA damage in the haemocytes of freshwater mussels of Unio sp. (Unio pictorum/Unio tumidus) and Sinanodonta woodiana. The comet assay was used for the assessment of DNA damage. The research was performed on 34 out of 68 sites analysed within the Joint Danube Survey 3 — the world's biggest river research expedition of its kind in 2013. During research, 2285 river kilometres were covered with an average distance of 68km between the sites. The complex data set on concentrations of various substances present in water, suspended particulate matter and sediment on investigated sites gave the opportunity to identify the groups of xenobiotics which mostly affect the studied biomarker — DNA damage. The highest levels of DNA damage were recorded in the section VI (Panonnian Plain), which is under the impact of untreated wastewater discharges. Both positive and negative influences of the large tributaries on the level of genotoxicity in the Danube River were evident. Significant correlation in response was detected between the studied species of freshwater mussels. The level of DNA damage in mussels correlated with concentrations of compounds from the group of hazardous priority substances (polycyclic aromatic hydrocarbons), persistent organic pollutants (dioxins) and emerging pollutants (Oxazepam, Chloridazon-desphenyl).

Evaluation of single-cell gel electrophoresis data: Combination of variance analysis with sum of ranking differences

Specimens of the mussel Mytilus galloprovincialis were collected from five sites in the Boka Kotorska Bay (Adriatic Sea, Montenegro) during the period summer 2011–autumn 2012. Three types of tissue, haemolymph, digestive gland were used for assessment of DNA damage. Images of randomly selected cells were analyzed with a fluorescence microscope and image analysis by the Comet Assay IV Image-analysis system. Three parameters, viz. tail length, tail intensity and Olive tail moment were analyzed on 4200 nuclei per cell type. We observed variations in the level of DNA damage in mussels collected at different sites, as well as seasonal variations in response. Sum of ranking differences (SRD) was implemented to compare use of different types of cell and different measure of comet tail per nucleus. Numerical scales were transferred into ranks, range scaling between 0 and 1; standardization and normalization were carried out.SRD selected the best (and worst) combinations: tail moment is the best for all data treatment and for all organs; second best is tail length, and intensity ranks third (except for digestive gland). The differences were significant at the 5% level. Whereas gills and haemolymph cells do not differ significantly, cells of the digestive gland are much more suitable to estimate genotoxicity. Variance analysis decomposed the effect of different factors on the SRD values. This unique combination has provided not only the relative importance of factors, but also an overall evaluation: the best evaluation method, the best data pre-treatment, etc., were chosen even for partially contradictory data.The rank transformation is superior to any other way of scaling, which is proven by ordering the SRD values by SRD again, and by cross validation.

Fluctuating estuarine conditions are not confounding factors for the Comet assay assessment of DNA damage in the mussel Mytilus edulis

The Comet assay is finding increasing application as a biomarker assay for the genotoxic potential of contaminants in field transplantation experiments involving mussels. Especially in estuaries, habitats that are of particular concern, environmental variables, such as salinity, can vary significantly. Although hinted at in the literature, there is a lack of clarification as to whether changes in salinity or emersion-induced hypoxia have the potential to alter background DNA damage in mussels, thus masking the extent of potential genotoxic effects following exposure to environmental contaminants. The present study exposed Mytilus edulis in the laboratory to static salinities (25, 50, 75, and 100 %) for 72 h. Mussels were also subjected to simulated tidal cycles, including periods of emersion, for 72 h. None of these treatments resulted in a significant change in the level of DNA damage expressed as % tail DNA. These experiments demonstrate that salinity, within the limits of the concentrations tested, and temporary emersion are not confounding factors for Comet assay data derived from M. edulis.

The anthropogenic impact on water quality of the river Danube in Serbia: Microbiological analysis and genotoxicity monitoring

The aim of this work was to examine the impact of urban wastewaters on the water quality of the Danube River in Serbia. Samples of water and sediments for microbiological analysis and genotoxicity monitoring were collected from 6 sites during spring and/or autumn 2010. Sanitary analysis, i.e. enumeration of total and fecal coliforms and intestinal enterococci, indicated moderate to critical fecal contamination, while organic load assessment (oligotroph to heterotroph ratio, index of phosphatase activity) revealed the category of moderately polluted water. Mercury-resistant bacteria were detected in all water samples, with high numbers at locations positioned downstream of Belgrade. There was no correlation of the microbiological parameters of the sediment and water samples. Genotoxicity monitoring, performed by the comet assay on hemocytes of mussels Sinanodonta woodiana, indicated a significant increase of DNA damage in mussels collected from the studied sites compared with the control group.

Monitoring of the impact of Prestige oil spill on Mytilus galloprovincialis from Galician coast

In November 2002, the Prestige oil tanker was wrecked in front of Galician coast (NW of Spain), spilling near 63,000 tons of heavy oil until February 2003. Contamination produced was very extensive (70% of Galician beaches were reached by the oil) but heterogeneous, alternating intensely affected zones with neighbour locations where the repercussion was minimal. The objective of this study was to monitor sea environment contamination caused by Prestige oil spill during an 11-month period (August 2003–June 2004, nine samplings) in two locations of Galician coast with different geographical properties (Lira and Ancoradoiro beaches), by means of chemical determination of total polycyclic aromatic hydrocarbons (TPAH) in seawater, and using as exposure biomarker TPAH content in mussel ( Mytilus galloprovincialis) tissues, and as effect biomarker DNA damage in mussel gills, evaluated by the comet assay. In addition, recovery ability of the mussels was determined after a 7-day stay in the laboratory. TPAH contents in seawater were very high in the earliest samplings, but then they maintained below 200 ng L − 1, similar to reference seawater. However, TPAH levels in mussel tissues were more variable: they increased again from January 2004, probably due to the adverse meteorological conditions that turned over the sea bottom and dispersed the oil accumulated in sediments. In most samplings, these levels decreased during the recovery stage. DNA damage in oil-exposed mussels was significantly higher than in reference mussels, both before and after the recovery phase, but they did not differ to one another. Comet tail length was slightly reduced during the recovery stage, indicative of a certain DNA repair in exposed mussels. This study showed up the importance of monitoring sea contamination events during an extended time, not only in evaluating the presence of the contaminants in the environment but also in determining their bioaccumulation and their effect on the exposed organisms.

Monitoring DNA damage in indigenous blue mussels ( Mytilus edulis) sampled from coastal sites in Denmark

Damage to DNA detected by use of the single-cell gel electrophoresis (comet) assay was monitored in blue mussels, Mytilus edulis, sampled from coastal waters in Denmark. Mussels from five locations in Køge Bay, an area receiving wastewater from many industries and municipalities, were collected five times during 1999 and six times in 2001. In 1999, both gill cells and haemolymph cells were examined, and sediments were sampled on three dates from the same five locations. In the autumn of 1999, mussels were also collected at six reference sites without known pollution. Results showed a significantly higher level of DNA damage in gill cells compared with haemolymph cells. Because of this, only gill cells were sampled for the monitoring in 2001. Levels of DNA damage, expressed as tail moments, were significantly higher for the mussels in Køge Bay when compared with levels of DNA damage in mussels from the non-polluted coastal areas. No clear seasonal variation was demonstrated. Analysis of the correlation between chromium, nickel, cadmium and mercury in sediments and tail moments in haemolymph and gill cells from the five sites showed a statistically significant positive correlation between tail moments and chromium, nickel and cadmium ( P < 0.01). The overall conclusion was that the comet assay on blue mussels could be useful for screening of genotoxic pollution in marine waters.

Evaluation of PAH bioaccumulation and DNA damage in mussels ( Mytilus galloprovincialis) exposed to spilled Prestige crude oil

We analyzed the hydrocarbon composition of the Prestige oil as it reached the shores, its solubility in sea water, its bioaccumulation, and the genotoxic damage associated to oil exposure, using Mytilus galloprovincialis as sentinel organism. Mussels were exposed to two oil volumetric ratios (1:500 and 2:500) for 12 days. Great concentrations of total polycyclic aromatic hydrocarbons (TPAH) have been obtained, being in general higher in the samples from the dose of 1:500, both in sea water (55.14 vs. 41.96 μg/l) and mussel tissue (16,993.80 vs. 17,033.00 μg/kg), probably due to the great tendency of these compounds to link to particles in water. Comet assay results reflected an increase in the DNA damage associated to oil exposure, higher in the mussels exposed to the higher aqueous TPAH content. In the view of our results, the importance of the evaluation of biodisponibility, bioaccumulation and DNA damage in the assessment of the effects of xenobiotic pollutants to marine environments could be highlighted.

Effects of temperature on baseline and genotoxicant-induced DNA damage in haemocytes of Dreissena polymorpha

The potential application of the Comet assay for monitoring genotoxicity in the freshwater mussel Dreissena polymorpha was explored and a preliminary investigation was undertaken of the baseline levels of DNA damage in mussel haemocytes of animals kept at different temperatures. In addition, in vitro cell sensitivity against genotoxicants was assessed in relation to increasing temperatures. The mussels were kept at four different constant temperatures (4, 18, 28 and 37 °C) for 15 h. The haemocytes withdrawn were treated in vitro with melphalan, as a model genotoxic compound, or sodium hypochlorite, a common water disinfectant capable of producing mutagenic/carcinogenic by-products, at the established temperatures for 1 h. The data obtained in vivo, in cells directly withdrawn from the mussels showed a significant ( P<0.001, Student’s t test) inter-individual variability, probably due to genetic and epigenetic factors and an increasing amount of DNA damage at increasing temperature. Mussel haemocytes showed a clear dose–response effect after in vitro melphalan treatment. Hypochlorite treatment also significantly increased DNA migration: the damage was temperature dependent, with a similar increase at 4 and 28 °C and a minimum level at 18 °C. This study demonstrates the potential application of the Comet assay to haemocytes of D. polymorpha. However, these findings suggest that temperature could alter both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants in in vitro conditions. Therefore, more information is needed about seasonal variations and the natural background levels of DNA damage in mussels living in the wild, before they are used for the monitoring of genotoxic effects in aquatic environments.

Elevation of cytochrome P450-immunopositive protein and DNA damage in mussels (Mytilus edulis) transplanted to a contaminated site

Mytilus edulis were collected from a reference site (Port Quin) and an urban/industrial contaminated site (New Brighton) in the UK during June 1999. Levels of PCBs (Σ7 congeners) and CB-138 were determined to be, respectively, 21 fold and 16 fold higher in the mussel digestive glands from New Brighton. Levels of CYP1A-immunopositive protein were 1.5 fold higher (P<0.05) at the polluted site but the levels of DNA strand breaks were 1.3 fold higher (P<0.05) at the reference site. Mussels from Port Quin were placed in cages at both sites and both transplanted and indigenous populations sampled in September (13 weeks). Mussels transplanted from the reference site to the industrial site, reported elevated levels of CYP1A-immunopositive protein (1.4 fold; P<0.05) and higher levels of DNA damage (1.2 fold; P<0.05) compared to caged populations at the reference site and a PCB loading similar to the populations from the polluted site. Moreover, transplanted mussels had DNA damage 1.8 fold greater (P<0.05) than indigenous mussels at the transplant site. These changes were small but significant when compared to the observed temporal changes in the indigenous populations.

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.

DNA integrity and total oxyradical scavenging capacity in the Mediterranean mussel, Mytilus galloprovincialis: a field study in a highly eutrophicated coastal lagoon

In Mediterranean coastal lagoons, the combination of human impact and wide variability of natural environmental factors can lead to upsets in ecosystem homeostasis resulting in biodiversity decline. Oxidative damage has been causally linked to various kinds of environmental stress, both natural and artificial, the result being impairment of cellular functions. DNA damage and the efficiency of antioxidant defences in Mytilus galloprovincialis from the highly eutrophicated Orbetello Lagoon (Tuscany, Italy) were investigated, respectively by the single cell gel electrophoresis (or Comet test) and the total oxyradical scavenging capacity assay. Results showed significantly higher levels of DNA damage in mussels collected from the inner parts of the lagoon compared to specimens from more external sites. Specimens with the lower genetic integrity also exhibited a reduced efficiency in neutralizing three potent cellular oxidizing species, namely peroxyl radicals (ROO ), hydroxyl radicals ( OH) and peroxynitrite (HOONO), suggesting the involvement of reactive oxygen species in mediating the genetic damage. The analyzed biological parameters also showed a seasonal variability with a minimum of both DNA integrity and antioxidant scavenging efficiency during the warm months and an opposite trend in winter. The potential of analyzed techniques is discussed for the assessment of both anthropogenic and natural disturbance.

Influence of sunlight on DNA damage in mussels exposed to polycyclic aromatic hydrocarbons

DNA single-strand break levels were compared in mussels deployed in San Diego Bay. Mussels were deployed at two locations, a reference site and at a location known to have high concentrations of PAHs. Two vertically oriented lines of mussels were deployed at each location for a period of 32 days. Mussels were attached to the lines at depths of 0.3, 1 and 3 m. One line was fully exposed to ambient sunlight, the other was shaded from sunlight to examine the role of PAH light activation on the expression of DNA damage in hemocytes and whole organism growth. Damage and growth measurements were made on days 0, 5, 14, and 32. Temperatures at the stations varied only by ≈0.5 °C. Hydrocarbon levels at the high PAH station were on average six times higher than the reference station. Slower growth and higher DNA damage was observed in cells from light exposed mussels at the PAH contaminated site. These effects decreased with depth. High levels of DNA damage could be induced with low intensity ultraviolet light exposure of cells from depurated or shaded mussels exposed to PAHs. DNA damage in mussels from the high impact site could be reduced to baseline levels after 24 h depuration in the dark. These results confirm previous findings showing increased DNA damage in PAH and sunlight exposed mussels, and demonstrate the significant impact of photoactivation on the health of these organisms.

Tissue dose, DNA adducts, oxidative DNA damage and CYP1A-immunopositive proteins in mussels exposed to waterborne benzo[ a]pyrene

A collaborative study was performed on Mediterranean mussels ( Mytilus galloprovincialis) exposed to a wide dose-range (0.5–1000 ppb) of benzo[ a]pyrene (B[ a]P). We selected this model polycyclic aromatic hydrocarbon in order to confirm the formation of a specific DNA adduct, previously detected in gill DNA, and to clarify the in vivo effects of this mutagenic chemical requiring host-metabolism in mussels. B[ a]P concentration reached consistently higher values in the digestive gland than in other analyzed tissues of mussels exposed to B[ a]P for 2 or 3 days. With the exception of some values at 1000 ppb of B[ a]P, DNA adduct levels increased significantly with the dose in gills and digestive gland and ranged from 0.054 to 0.789 adducts per 10 8 nucleotides (mean values per dose-point). Conversely, more complex dose-response relationships were found by detecting in parallel the levels of an oxidative DNA lesion (8-OHdG) and of CYP1A-immunopositive proteins (the latter measured in the digestive gland only). Overall, the formation of DNA adducts, the evidence of oxidative DNA damage, and changes in CYP1A-immunopositive protein levels support the hypothesis that B[ a]P can induce DNA damage in mussels through a number of different molecular mechanisms.

DNA damage in mussels at sites in San Diego Bay

Identification and assessment of introduced and other toxicants is crucial to any comprehensive study of contaminants within the marine environment. The relationship between DNA single-strand breaks and the exposure of marine organisms to environmental contaminants was examined at sites in San Diego Bay, CA. A comprehensive assessment of the extent and consequences of marine environmental contamination in the area of Naval Station San Diego was conducted in the summer of 1995. The study addressed contamination sources, distributions, concentrations, transport, sediment–water exchange, biological effects, and degradation. The biological effects portion of the study (this paper) included contaminant bioaccumulation, organismal growth, and the determination of DNA single-strand breaks using the Comet assay. DNA damage was determined in hemocytes collected from deployed and resident mussels, Mytilus edulis, at six stations in and around the Naval Station San Diego. Deployed mussels were exposed on station for ∼30 days in plastic mesh bags, placed 1 m above the bottom. Hemocyte samples were collected on days 0, 12, and 32. It was found that stations exhibiting the extremes of contaminant exposure, both highest and lowest concentrations, were easily identified using growth and DNA damage measurements. Sediment chemistry and bioaccumulation data indicated, Hg, Cu, and Zn, to be the most notable contaminants. The Comet assay, and in particular germ cell DNA damage determinations, were found to respond rapidly to station contaminants. Results from this study and an earlier 1993 study suggest that the non-sediment associated effects observed at one station may have been the result of the photoactivation of accumulated PAHs.