Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay

The alkaline single cell gel electrophoresis (comet) assay was applied to study genotoxic properties of two inhalation anesthetics-halothane and isoflurane-in human peripheral blood lymphocytes (PBL). The cells were exposed in vitro to either halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) at concentrations 0.1-10 mM in DMSO. The anesthetics-induced DNA strand breaks as well as alkali-labile sites were measured as total comet length (i.e., increase of a DNA migration). Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner. In experiments conducted at two different electrophoretic conditions (0. 56 and 0.78 V/cm), halothane was able to increase DNA migration to a higher extent than isoflurane. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA degradation due to cell death. For this reason a contribution of toxicity in the observed effects was examined. We tested whether the exposed PBL were able to repair halothane- and isoflurane-induced DNA damage. The treated cells were incubated in a drug-free medium at 37 degrees C for 120 min to allow processing of the induced DNA damage. PBL exposed to isoflurane at 1 mM were able to complete repair within 60 min whereas for halothane a similar result was obtained at a concentration lower by one order of magnitude: the cells exposed to halothane at 1 mM removed the damage within 120 min only partly. We conclude that the increase of DNA migration induced in PBL by isoflurane at 1 mM and by halothane at 0.1 mM was not a result of cell death-associated DNA degradation but was caused by genotoxic action of the drugs. The DNA damage detected after the exposure to halothane at 1 mM was in part a result of DNA fragmentation due to cell death.     

Genotoxicity of inhalation anaesthetics: DNA lesions generated by sevoflurane in vitro and in vivo

A moderate genotoxic activity of halothane and isoflurane applied as volatile anaesthetics has already been shown. The aim of this work was to estimate a potential genotoxicity of sevoflurane, introduced to clinical practice later than halothane and isoflurane. A genotoxic activity of all three compounds was estimated by using the comet assay in human peripheral blood lymphocytes (PBL) proliferating in vitro. We demonstrated that in contrast to the previously studied anaesthetics, sevoflurane did not induce any increase in DNA migration in the studied conditions. To estimate a genotoxic effect of a prolonged exposure to halogenated anaesthetics in vivo, PBL taken from operating room personnel (n = 29) were tested for DNA degradation and compared with those from a control non-exposed group (n = 20). No significant differences were detected between the groups. We conclude that sevoflurane does not have genotoxic properties, both in vitro and in vivo.     

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.     

The in vivo genotoxicity of cisplatin,isoflurane and halothane evaluated by alkaline comet assay in Swiss albino mice

The aim of this study was to evaluate the genotoxicity of repeated exposure to isoflurane or halothane and compare it with the genotoxicity of repeated exposure to cisplatin. We also determined the genotoxicity of combined treatment with inhalation anaesthetics and cisplatin on peripheral blood leucocytes (PBL), brain, liver and kidney cells of mice. The mice were divided into six groups as follows: control, cisplatin, isoflurane, cisplatin–isoflurane, halothane and cisplatin–halothane, and were exposed respectively for three consecutive days. The mice were treated with cisplatin or exposed to inhalation anaesthetic; the combined groups were exposed to inhalation anaesthetic after treatment with cisplatin. The alkaline comet assay was performed. All drugs had a strong genotoxicity (P < 0.05 vs. control group) in all of the observed cells. Isoflurane caused stronger DNA damage on the PBL and kidney cells, in contrast to halothane, which had stronger genotoxicity on brain and liver cells. The combination of cisplatin and isoflurane induced lower genotoxicity on PBL than isoflurane alone (P < 0.05). Halothane had the strongest effect on brain cells, but in the combined treatment with cisplatin, the effect decreased to the level of cisplatin alone. Halothane also induced the strongest DNA damage of the liver cells, while the combination with cisplatin increased its genotoxicity even more. The genotoxicity of cisplatin and isoflurane on kidney cells were nearly at the same level, but halothane caused a significantly lower effect. The combinations of inhalation anaesthetics with cisplatin had stronger effects on kidney cells than inhalation anaesthetics alone. The observed drugs and their combinations induced strong genotoxicity on all of the mentioned cells.     

Genotoxic effects of silver nanoparticles stimulated by oxidative stress in human normal bronchial epithelial (BEAS-2B) cells

Many classes of silver nanoparticles (Ag-NPs) have been synthesized and widely applied, but the genotoxicity of Ag-NPs and the factors leading to genotoxicity remain unknown. Therefore, the purpose of this study is to elucidate the genotoxic effects of Ag-NPs in lung and the role of oxidative stress on the genotoxic effects of Ag-NPs. For this, Ag-NPs were completely dispersed in medium by sonication and filtration. The Ag-NPs dispersed in medium were 43-260nm in size. We observed distinct uptake of Ag-NPs into BEAS-2B cells. The Ag-NPs aggregates were wrapped with an endocytic vesicle within the cytoplasm and nucleus of BEAS-2B cells. In the comet assay and micronucleus (MN) assay for BEAS-2B cells, Ag-NPs stimulated DNA breakage and MN formation in a dose-dependent manner. The genotoxic effect of Ag-NPs was partially blocked by scavengers. In particular, of the scavengers tested, superoxide dismutase most significantly blocked the genotoxic effects in both the cytokinesis-block MN assay and the comet assay. In the modified comet assay, Ag-NPs induced a significant increase in oxidative DNA damage. Furthermore, in the oxidative stress assay, Ag-NPs significantly increased the reactive oxygen radicals. These results suggest that Ag-NPs have genotoxic effects in BEAS-2B cells and that oxidative stress stimulated by Ag-NPs may be an important factor in their genotoxic effects.     

Genotoxic potential of marine sediments from the North Sea

The alkaline comet assay is a method for detecting DNA strand breaks and alkali labile sites in individual cells. An in vitro system was used to investigate the genotoxic potential of complex mixtures such as organic extracts of marine sediments. DNA damage was induced in leukocytes isolated from carp (Cyprius carpio) by exposure to organic sediment extracts from the North Sea or hydrogen peroxide as positive control, respectively. The minimum concentration for significant effects ranged from 1 to 40 mg sediment dry weight per milliliter assay volume. The sensitivity of the method was enhanced by using the DNA repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara C). From the results, it can be suggested that total organic carbon (TOC) as well as the different compositions of contaminants present in the sediment extracts may contribute to the genotoxic effects observed. The comet assay can be applied successfully as an in vitro bioassay for investigations on genotoxicity of marine sediment extracts.     

Time-dependent effects of sodium arsenite on DNA breakage and apoptosis observed in the comet assay

To assess genotoxic effects of sodium arsenite (NaAsO2) the single-cell gel electrophoresis (comet assay) had been conducted in various studies indicating genotoxicity. However, DNA fragmentation due to NaAsO2-induced apoptosis may constitute a bias in the interpretation of the results. Apoptotic cells can show typically large and diffuse comets, which are usually excluded during genotoxicity analysis. It is controversial whether there is a time-window in which the apoptotic process generates comets that would falsely be interpreted to be the result of genotoxic DNA damage. Therefore, we evaluated frequency histograms for single-cell measures of tail DNA (% DNA in comet tail) in 30-min intervals after incubation of mouse lymphoma L5178Y cells with sodium arsenite (NaAsO2). In parallel, we evaluated apoptosis by measuring annexin V-positive cells with flow cytometry, and visualized apoptotic cells on slides by Hoechst bisbenzimide 33258 staining. The first observed effect at 30 min after treatment was an increase in annexin V-positive cells. At about 60 min the number of cells with moderate DNA migration increased in the comet-assay analysis. After 90 min, an increase in the number of cells with high levels of DNA migration was observed, which resulted in a bimodal distribution of cells with moderate and high levels of DNA migration. Hoechst-stained apoptotic cells could only be observed at later times (> or = 120 min). This means that the treatment would have been considered to be genotoxic if analysed at 120 min even if the cells with high levels of DNA migration would have been excluded. The occurrence of annexin V-positive cells preceded the appearance of cells with moderate levels of DNA migration. We hypothesize that these cells were early apoptotic cells and not indicative of genotoxic damage. We conclude that DNA-damaging effects of NaAsO2 cannot adequately be interpreted if the comet assay is not accompanied by separate analysis of early endpoints for induction of apoptosis.     

Assessment of the genotoxicity in toad Bufo raddei exposed to petrochemical contaminants in Lanzhou Region, China

Single cell gel electrophoresis or comet assay, micronucleus (MN) test and global DNA methylation detection were used to assess the genotoxicity in toad Bufo raddei exposed to the petrochemical (mainly oil and phenol) polluted area in Lanzhou Region (LZR) comparing with a relatively unpolluted area in Liujiaxia Region (LJXR). The results from the present study indicated that DNA damage and MN frequency in toad from LZR were significantly higher than those from LJXR at the same sampling month, whereas the degree of global DNA methylation was lower, which implies that the petrochemical contaminants at environmental level in LZR were genotoxic to B. raddei. The degree of genotoxic damage was obviously related with the extent of pollution among the three sampling months in LZR. The significantly positive correlations between DNA damage and concentrations of oil and/or phenol existed in liver cells but erythrocytes, implying that liver is more suitable as a sentinel tissue for the assessment of genotoxic impact of low-level contamination. The results from both comet assay and global DNA methylation detection on liver cells showed that the genotoxicity varied significantly with oil and/or phenol concentrations, suggesting that these two methods are relatively sensitive and suitable for monitoring the genotoxicity of petrochemical pollutants on amphibians.     

Lead-induced DNA damage in Vicia faba root cells: potential involvement of oxidative stress

Genotoxic effects of lead (0-20μM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10μM. In addition, at this concentration, DNA damage time-dependently increased until 12h. Then, a decrease in DNA damages was recorded. The significant induction of micronucleus formation also reinforced the genotoxic character of this metal. Direct interaction of lead with DNA was also evaluated with the a-cellular comet assay. The data showed that DNA breakages were not associated with a direct effect of lead on DNA. In order to investigate the relationship between lead genotoxicity and oxidative stress, V. faba were exposed to lead in the presence or absence of the antioxidant Vitamin E, or the NADPH-oxidase inhibitor dephenylene iodonium (DPI). The total inhibition of the genotoxic effects of lead (DNA breakage and micronucleus formation) by these compounds reveals the major role of reactive oxygen species (ROS) in the genotoxicity of lead. These results highlight, for the first time in vivo and in whole-plant roots, the relationship between ROS, DNA strand-breaks and chromosome aberrations induced by lead.     

The protective effect exerted by ascorbic acid on DNA fragmentation of human leukocytes induced by Lachesis muta muta venom

The objective of this study was to evaluate the genotoxic and mutagenic effects of the toxins present in Lachesis muta muta's venom on human peripheral blood leukocytes and the protective potential of ascorbic acid on DNA fragmentation. The venom of L. muta muta was incubated in different concentrations (1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, and 120 µg/mL) with human blood to evaluate DNA fragmentation using the comet, agarose gel electrophoresis, and micronucleus assays. In these concentrations evaluated, the venom of L. muta muta induced genotoxicity (comet assay and agarose gel electrophoresis) and mutagenicity (micronucleus test), but they were not cytotoxic, as they did not change the rate of cell proliferation after cytokinesis blockade with cytochalasin B. The ascorbic acid significantly inhibited the genotoxicity induced by L. muta muta venom in the proportions evaluated (1:0.1 and 1:0.5, venom/ascorbic acid - w/w). Thus, future studies are needed to elucidate the protective mechanisms of ascorbic acid on the genotoxic effects induced by toxins present in snake venoms.     

Application of Euglena gracilis cells to comet assay: evaluation of DNA damage and repair

Alkaline single-cell gel electrophoresis (comet assay) enables sensitive detection of DNA damage in eukaryotic cells induced by genotoxic agents. We performed a comet assay of unicellular green alga Euglena gracilis that was exposed to genotoxic chemicals, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), benzo[a]pyrene (BAP), mitomycin C (MMC) and actinomycin D (AMD). Tail length and tail moment in migrated DNA were measured as indications of DNA damage. MNNG and BAP were found to cause concentration-dependent increases in DNA damage. The responses were more sensitive than those of human lymphocytes under the same treatment conditions. MMC and AMD showed no positive response, as reported elsewhere. The comet assays performed at specified times after treatment revealed that the DNA damaged by MNNG and gamma-ray irradiation was repaired during the initial 1h. The results clearly show that the comet assay is useful for evaluating chemically-induced DNA damage and repair in E. gracilis. Given the ease of culturing and handling E. gracilis as well as its sensitivity, the comet assay of this alga would undoubtedly prove to be a useful tool for testing the genotoxicity of chemicals and monitoring of environmental pollution.     

Estragole: a weak direct-acting food-borne genotoxin and potential carcinogen

We evaluated the genotoxicity of the food-flavouring agent estragole in V79 cells using the sister chromatid exchange (SCE) assay and the alkaline comet assay. Unexpectedly, we observed an increase in SCE without an exogenous biotransformation system (S9) and a decrease in its presence. Positive results were also observed in the alkaline comet assay without S9, indicating DNA strand breakage. To ascertain repair of damage, we performed the comet assay in V79 cells after two hours of recovery, and observed a reduction of the genotoxic response. Estragole did not produce strand breaks in plasmid DNA in vitro. We then evaluated the formation of DNA adducts in V79 cells by use of the (32)P-postlabelling assay and detected a dose-dependent formation of DNA adducts, which may be responsible for its genotoxicity. We then assayed estragole in the comet assay with two CHO cell lines, a parental AA8 cell line, and an XRCC1-deficient cell line, EM9. Results confirmed the genotoxicity of estragole without biotransformation in both cell lines, although the genotoxicity in EM9 cells compared with that in AA8 cells was not significantly different, suggesting that the XRCC1 protein is not involved in the repair of estragole-induced lesions. Estragole induces apoptosis, but only with high doses (2000μM), and after long treatment periods (24h). Overall, our results suggest that estragole, besides being metabolized to genotoxic metabolites, is a weak direct-acting genotoxin that forms DNA adducts.     

Genotoxic effects of Roundup on the fish Prochilodus lineatus

Glyphosate-based herbicides, such as Roundup, represent the most extensively used herbicides worldwide, including Brazil. Despite its extensive use, the genotoxic effects of this herbicide are not completely understood and studies with Roundup show conflicting results with regard to the effects of this product on the genetic material. Thus, the aim of this study was to evaluate the genotoxic effects of acute exposures (6, 24 and 96 h) to 10 mg L(-1) of Roundup on the neotropical fish Prochilodus lineatus. Accordingly, fish erythrocytes were used in the comet assay, micronucleus test and for the analysis of the occurrence of nuclear abnormalities and the comet assay was adjusted for branchial cells. The results showed that Roundup produces genotoxic damage in erythrocytes and gill cells of P. lineatus. The comet scores obtained for P. lineatus erythrocytes after 6 and 96 h of exposure to Roundup were significantly higher than respective negative controls. For branchial cells comet scores were significantly higher than negative controls after 6 and 24 h exposures. The frequencies of micronucleus and other erythrocyte nuclear abnormalities (ENAs) were not significantly different between Roundup exposed fish and their respective negative controls, for all exposure periods. In conclusion, the results of this work showed that Roundup produced genotoxic effects on the fish species P. lineatus. The comet assay with gill cells showed to be an important complementary tool for detecting genotoxicity, given that it revealed DNA damage in periods of exposure that erythrocytes did not. ENAs frequency was not a good indicator of genotoxicity, but further studies are needed to better understand the origin of these abnormalities.     

Use of the alkaline comet assay for the detection of transplacental genotoxins in newborn mice

Several lines of evidence show that in utero exposure to different toxicants has greater consequences than their exposure during adult life. This may be due to involvement of critical developmental stages, physiological immaturity and the long later-life span over which disease may initiate, develop and progress. The in vivo alkaline comet (single-cell gel electrophoresis) assay has been favoured by the scientific community for the evaluation of genotoxins. The objective of this study was to demonstrate the suitability of alkaline comet assay in detecting transplacental genotoxins using newborn mice. Here, we report the successful use of the comet assay in detecting multi-organ genotoxicity of known transplacental genotoxins in newborn mice. Three well known transplacental genotoxic agents, cyclophosphamide (CP), mitomycin-C (MMC) and zidovudine (AZT) were tested in pregnant Swiss mice. These compounds were administered in the late gestational period (16-20th days of pregnancy) and the comet assay was performed with lymphocytes, bone marrow, liver and kidney cells of newborn mice. Significant DNA damage was observed in all the tissues with tested transplacental genotoxins. The results of the comet assay were confirmed by the micronucleus (MN) assay of the peripheral blood of newborn mice. The results of this study provide sufficient evidence that the comet assay can be applied successfully for the detection of transplacental genotoxins in newborn mice.     

Genotoxicity of the Kishon River,Israel: the application of an in vitro cellular assay

The alkaline comet assay, a sensitive method for DNA strand breaks and alkali labile site detection in individual cells, was employed here as an ecotoxicological monitoring tool for evaluation of genotoxicity in the Kishon River, Israel. This river, the most polluted river in Israel, has recently elicited major public concern with regard to cancer incidences in people who have dived there over many years. Five water samples were collected every odd month throughout the year 2001, from four localities. The comet assay was employed on fish hepatoma cell line RTH-149. Cells were exposed for 2h, in triplicate, to Kishon water: medium (1:1) samples that were pre-adjusted for pH and salinity percentage levels. Three DNA damage parameters (comet percentages, score of damage, and cumulative tail lengths of the comet), revealed significantly higher genotoxic values in Kishon water-treated cells as compared with the controls (up to 2.4, 3.2, and 3.6-fold, respectively). Part of the sampling sites revealed higher genotoxicity than other polluted sites. The results of this study demonstrate that the comet assay with RTH-149 cells can be successfully applied to a variety of aquatic samples revealing freshwater, marine and estuary conditions. The method found to be fast, sensitive, and suitable for monitoring programs.     

Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid

Cadmium (Cd) is one of the important pollutants of soil and the genotoxicity of Cd-contaminated soil was studied in combination with imidacloprid. The single cell gel electrophoresis or comet assay was used to quantify DNA strand breaks as a measure of DNA damage induced by Cd and imidacloprid contamination in soil. The soil was artificially contaminated by Cd 2 h at 25℃ and were used in the comet assay. DNA damage was measured as the values of percentage of nuclei with tails, tail length, tail DNA, tail moment (TM), and Olive tail moment (OTM). DNA damages of root tips of Vicia faba increased after Cd treatment and there were dose-related increases in DNA damage measured as these parameters. However, the addition of imidacloprid further increased the DNA damage. These data confirmed the genotoxic effect of Cd to plants, and that the combined pollution with imidacloprid can enhance the genotoxicity of Cd.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 microg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 microg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

Genotoxicity of imidacloprid in relation to metabolic activation and composition of the commercial product

Imidacloprid is a neonicotinoid insecticide combining excellent efficiency against parasites with low toxicity for mammals. Commercially, it is co-formulated with dimethyl sulfoxide, methylpyrrolidone, propylene carbonate and mineral oil, which can modify its bioavailability and toxicological profile for humans following occupational exposure. A combined in vitro approach employing the comet assay and the micronucleus test was used to assess the genotoxicity of imidacloprid in relation to formulation, metabolic activation and exposure level. Human peripheral blood lymphocytes from unexposed healthy volunteers were treated with imidacloprid (0.2, 2 and 20 μM) and with equimolar concentrations of a commercial product, with and without addition of S9 fraction. Imidacloprid significantly increased the comet score and the frequency of micronuclei only at the highest concentration tested. DNA damage was slightly more severe with the commercial product, and was increased, though not significantly, by metabolic activation. Formation of reactive oxygen species (ROS) does not seem to be involved as a mechanism of genotoxicity, but this result may be explained by the insufficient sensitivity of the 2',7'-dichlorofluorescein diacetate assay at the test concentrations of imidacloprid. These results suggest that at concentrations<20 μM imidacloprid is not genotoxic to human lymphocytes in vitro. Nonetheless, the presence of co-formulants in the commercial product and occupational exposure, along with poor safety procedures, may present an increased risk for DNA fragmentation and chromosomal aberrations.     

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 μg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 μg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.