- Email: [email protected]
Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions
Aquatic Toxicology 78S (2006) S93–S98
Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions Claudia Bolognesi a,∗ , Emanuela Perrone a , Paola Roggieri a , Daniela M. Pampanin b , Andrea Sciutto a a
Environmental Carcinogenesis Unit, National Institute for Research on Cancer, L.go Rosanna Benzi, 10, 16132 Genoa, Italy b IRIS – International Research Institute of Stavanger AS, Mekjarvik 12, N-4070 Randaberg, Norway
Abstract The aim of the present study was to standardize and to assess the predictive value of the cytogenetic analysis by MN test in fish erythrocytes as a biomarker for marine environmental contamination. MN frequency baseline in erythrocytes was evaluated in a number of fish species from a reference area (S. Teresa, La Spezia Gulf) and genotoxic potential of a number of common chemical contaminants and mixtures was determined in fish experimentally exposed in aquarium under controlled conditions. Fish (Scophthalmus maximus) were exposed for 3 weeks to 50 ppb of single chemicals (dialkyl phthalate, bisphenol A, tetrabromodiphenyl ether), 30 ppb nonylphenol and mixtures (North Sea oil and North Sea oil with alkylated phenols). Chromosomal damage was determined as micronuclei (MN) frequency in fish erythrocytes. Nuclear anomalies such as blebbed, notched and lobed nuclei were also recorded. Significant increase in MN frequency was observed in erythrocytes of fish exposed to bisphenol A and tetrabromodiphenylether. Chemical mixture North Sea oil + alkylated phenols induced the highest MN frequency (2.95 micronucleated cells/1000 cells compared to 1 MNcell/1000 cells in control animals). The study results revealed that micronucleus test, as an index of cumulative exposure, appears to be a sensitive model to evaluate genotoxic compounds in fish under controlled conditions. © 2006 Elsevier B.V. All rights reserved. Keywords: Scophthalmus maximus; Xenobiotics; MN frequency; Biomarker; Fish erythrocytes
1. Introduction Coastal areas are increasing at risk from toxic contamination from land based discharges, coastal industries and shipping. A wide number of common marine pollutants are studied only for acute effects and their long term adverse environmental hazard is not known. A comprehensive assessment of the real toxic and genotoxic potential for these compounds in marine organisms is needed. The use of biological markers assists in the identification of causal relationship between the exposure to toxic contaminants and increased risk of effects on individuals and populations that may lead to decreased ecological integrity. It has been demonstrated that fish may act as a sentinel organism in genetic toxicology studies. Many fish species can transform organic xenobiotics by efficient enzymatic systems producing by-products linking to critical macromolecules, such as proteins or DNA (Collier and Varanasi, 1991; Gravato and Santos, 2002).
∗
Corresponding author. Tel.: +39 010 5600215. E-mail address: [email protected] (C. Bolognesi).
0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.02.015
DNA and chromosomal damage are the most important critical events following the exposure to carcinogenic and/or genotoxic agents. Chromosomal damage, as a result of inefficient or incorrect DNA repair, is expressed during the cell division and represents an index of accumulated genotoxic effects. Chromosomal effects could be measured as macrolesions in cells exposed to genotoxic agents. Analysis of chromosomal aberrations is difficult in fish because fish chromosomes are generally small in size and high in number. The micronucleus test is the most applied technique to evaluate chromosomal damage in different organisms (Anderson et al., 1994; Bolognesi et al., 1999, 2004; Campana et al., 2003; Al-Sabti and Metcalfe, 1995). Micronuclei (MN) arise from chromosomal fragments or whole chromosomes that are not incorporated into daughter nuclei at mitosis. MN are small fragments of chromatin separated from the main cell nucleus which are index of chromosomal breaking or mitotic spindle dysfunctions (Schmid, 1975). This test has the advantage that it can be applied in interphase to any proliferating cell population regardless of the karyotype. MN can be analysed in different fish cell types such as peripheral erythrocytes, gill, kidney, hepatic and fin cells (Al-Sabti and Metcalfe, 1995; Arkhipchuk and Garanko, 2005). Branchial epithelium, as the
S94
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
primary target for all the water-borne contaminants, showed high background levels of MN and a high sensitivity for the cytogenetic effects induced by environmental contaminants. However gill cells are not easily obtained and prepared for the MN analysis. The application of MN assay in fish hepatocytes has primary limitations because of the low mitotic index of liver cells. The use of peripheral erythrocytes avoids the complex procedures associated with cell preparation and animal sacrifice. In addition high mitotic rate of hematopoietic tissues provides a rapid response to genotoxic exposure, revealed as chromosomal damage in peripheral blood. Micronucleus test in fish erythrocytes has been applied to evaluate the genotoxic impact of environmental pollutants under laboratory and field conditions, revealing interesting correlations with the exposure to a number of chemical and physical agents (Al-Sabti and Metcalfe, 1995; Schultz et al., 1993; Gustavino et al., 2001). The analysis of the baseline MN frequencies obtained by different authors from the literature shows a large interspecies variability, ranging from 0 to 13. This scientific evidence can be related to an interspecies difference in metabolic competency and DNA repair mechanisms as well as in the MN expression. However the large majority of papers report data ranging from 0 to 1. A significant difference (up to 1–2 orders of magnitude) in the MN baseline frequency was recorded in the same species from different authors e.g. Cyprinus carpio (Landolt and Kocan, 1983; Al-Sabti, 1986; Nepomuceno et al., 1997; Llorente et al., 2002; Grisolia and Starling, 2001; Gustavino et al., 2001) or Oncorhynchus mykiss (Schultz et al., 1993; Castano et al., 1998; Ayllon and Garcia-Vazquez, 2001). This observation outlines the importance to standardize and intercalibrate the experimental procedure for MN assay with special reference to the criteria of scoring in order to compare data from different labs. The aim of this study is to standardize and to assess the predictive value of the cytogenetic analysis by MN test in fish erythrocytes as a biomarker for marine environmental contamination. MN frequency baseline in erythrocytes was evaluated in different fish species and the genotoxic potential of a number of common chemical contaminants and mixtures was determined in fish exposed under controlled conditions. 2. Materials and methods Samples of fish belonging from different species from a reference area (S. Teresa, La Spezia Gulf) were collected in order to evaluate the baseline for MN and other nuclear anomalies. Fish samples were collected from the same site with the same size and weight and they are acclimatized in the lab for 7 days. Turbot (Scophthalmus maximus) was chosen for the experimental study in aquarium. Groups of animals were exposed in aquarium under controlled conditions for a period of 3 weeks. In a first experiment carried out during the period September–October 2002 the fish were exposed to mixtures of chemicals: (a) 0.5 ppm North sea crude oil-Statfjord; (b) 0.5 ppm of North sea crude oil spiked with 0.1 ppm of a mixture of alkylated phenols; (c)
30 ppb of nonylphenol and (d) controls. The exposure was carried out using a continuous flow system designed for performing studies of chronic exposures of marine organisms to mixtures of poorly water soluble substances. Details about the exposure are reported by Sundt et al. (2006). In a second experiment carried out on May 2003 the animals were exposed to 50 ppb of single chemicals (dialkyl phthalate, bisphenol A, tetrabromodiphenylether) and controls. At least six individuals/experimental group were used to evaluate the genotoxic effects induced by the treatment. Blood was collected from the caudal vein and immediately smeared on clean glass slides, dried overnight, fixed with methanol for 20 min and stained with acridine orange. About 4000 erythrocytes per animal were analysed by a fluorescence microscope under 1000× magnification. Only clearly isolated MN were counted as such and other nuclear anomalies were recorded separately. MN scoring followed prior established criteria. MN was defined as small round or oval intracytoplasmic bodies with a diameter 1/5–1/20 of the main nucleus and on the same optical plane as the major nucleus. The MN was not linked or connected to the main nucleus. Cells with more than four MN were discarded to exclude apoptotic phenomena. Nuclear anomalies, chromatin buds, lobes, invaginations, vacuoles, were recorded separately. Cells showing abnormal morphological forms (degenerated cells), induced by cytotoxic events have to be discarded. 2.1. Statistical analysis Differences between groups were evaluated by nonparametric Mann–Whitney U-test. The relationship between MN frequency and nuclear evaginations or buds was evaluated using regression analysis. All the data were analysed using SPSS statistical software package (SPSS 13.0 for Windows). 3. Results Table 1 and Fig. 1 show the baseline frequencies for MN and other nuclear abnormalities in different fish species from a reference area (S. Teresa, La Spezia Gulf). The MN frequency ranges between 0 and 2.0 with a high interspecies and interindividual variability, as it has been commonly observed for different biomarkers. The large interindividual variability associated to the low baseline frequency for this biomarker confirming the need for the scoring of a consistent number of cells (at least 4000 cells) in an adequate number of animals for each study point. A correlation was observed between MN frequency and nuclear evaginations or buds (R2 0.585, p < 0.016, n = 24) (Fig. 2) suggesting the importance for recording this anomaly in order to improve the information obtained with this assay. No correlation was found between MN frequency and nuclear invaginations, parameter mainly associated to cytotoxicity. This observation outlines the importance to standardize and intercalibrate the experimental procedure for MN assay with
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
S95
Fig. 1. Spontaneous frequency of micronucleated erythrocytes/1000 cells and cells with nuclear buds/1000 cells in fish from different species.
Table 1 Spontaneous frequency of micronuclei and other nuclear abnormalities in fish erythrocytes belonging to different species from a reference area (S. Teresa, La Spezia Gulf, Italy) Species
Boops boops Dicentrarchus labrax Mullus barbatus Pagellus mormyrus Sargus sargus Seriola dumerili Serranus cabrilla Sparus auratus Sphyraena sphyraena Trachurus trachurus
MN cellsa /1000 cells
Cells with NAb /1000 cells
Mean (S.D.)
Invaginations Mean (S.D.)
BUDS, Mean (S.D.)
1.75 (0.35) 0.75 (0.64) 0.33 (0.57) 0.4 (0.72) 0.25 (0.35) 0.38 (0.18) 0.0 (0.0) 0.12 (0.18) 0.25 (0.35) 0.25 (0.32)
15.6 (5.03) 1.5 (0.40) 2.63 (0.93) 3.83 (2.76) 8.75 (2.47) 1.62 (0.53) 1.08 (0.80) 6.75 (4.95) 1.87 (1.24) 3.0 (2.16)
1.62 (0.54) 0.0 (0.0) 0.16 (1.42) 0.58 (0.63) 0.37 (0.53) 0.12 (0.18) 0.0 (0.0) 0.50 (0.71) 0.62 (0.88) 1.25 (0.74)
Five fish for each species were analysed. a MN cells = micronucleated cells/1000 cells. b Cells with NA = cells with nuclear abnormalities.
special reference to the scoring criteria in order to compare data from different labs. Fig. 3A shows the results obtained in a first experiment carried out under controlled conditions. Statistically significant increase in MN frequency as well as in nuclear abnormalities was observed in erythrocytes from fish treated with bisphenol A (p < 0.005) and tetrabromodiphenylether (p < 0.001), no effect of dialkylphthalate was recorded (the mean MN frequency is lower with respect to the control value). Frequencies of MN in peripheral fish erythrocytes from groups of fish treated with nonylphenol, North sea oil and North sea oil + alkylated phenols are shown in Fig. 3B. Highest effects were observed in animals exposed to chemical mixtures (2.95 MN/1000 cells compared to 1 MN/1000 cells in control animals). A large interindividual variability in MN frequency was observed in nonylphenol treated animals, where the results did not reach the statistical significance. A negligible level of nuclear abnormalities was recorded in fish erythrocytes from fish exposed to chemical compounds or mixtures considered in our study. 4. Discussion
Fig. 2. Correlation between micronucleated erythrocytes/1000 cells and cells with nuclear buds/1000 cells in fish from different species (R2 0.585, p = 0.016).
The micronucleus test in peripheral erythrocytes provides a feasible approach to monitor the effects of environmental genotoxic agents in fish. A large variability in spontaneous MN frequency in different fish species was evident from the analysis of the scientific literature. The large majority of papers report a very low range of MN frequency, but they show also a large range of variability in the frequency from the same species. The low baseline MN frequency represents the main limitation for the MN test applied in fish erythrocytes, requiring the scoring of a large number of cells from a consistent number of animals.
S96
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
Fig. 3. Micronucleated erythrocytes (MNcells/1000 cells) from fish (Scophthalmus maximus) treated for a period of 3 weeks with (A) dialkylphthalate (50 ppb), bisphenol A (50 ppb), tetrabromodiphenylether (50 ppb) and controls; (B) nonylphenol (30 ppb), North sea crude oil (NSA), North Sea crude oil spiked with 0.1 ppm of a mixture of alkylated phenols and controls.
The lack of uniform criteria of scoring for MN and other nuclear abnormalities is the main critical factor responsible for the large variability of MN frequency in the same species affecting the sensitivity of the assay and preventing the comparison of the results from different labs. Our data on spontaneous MN induction in different species from a reference area show a low range of frequency and a large variability. Interestingly the frequency of nuclear evaginations is positively correlated to the MN frequency, suggesting the importance to record also this parameter. A number of papers recently appeared in the scientific literature reporting an association between the frequency of nuclear anomalies and the exposure to genotoxic agents (Ayllon and Garcia-Vazquez, 2000; Pacheco and Santos, 2002). A comprehensive application has been proposed for the MN assay in human lymphocytes, including a set of parameters of genotoxicity and citotoxicity: MN as markers of chromosomal breakage and/or loss, nucleoplasmic bridges as markers of chromosomal rearrangements, nuclear evaginations or buds as markers of gene amplification, cellular necrosis and apoptosis (Fenech et al., 1999). The different frequencies of these anomalies are related to specific genotoxic events associated to the different mechanisms of action of the carcinogenic/mutagenic agents. The application of MN in its comprehensive mode in fish erythrocytes needs the expression and standardization of specific criteria for the different nuclear anomalies. It’s also very impor-
tant the evaluation of the percent of degenerated cells, directly associated to cytotoxic events. The micronucleus test in fish erythrocytes was widely validated in lab after exposure to different agents. A dose response increase in MN frequency was observed after exposure to a range of doses of X-rays (Schultz et al., 1993; Gustavino et al., 2001). The MN test in fish erythrocytes responded positively to a large number of experimental carcinogens, such as aflatoxins, benzidine, ethylmethanesulfonate, methylcholantrene, chlorinated hydrocarbons (Al-Sabti and Metcalfe, 1995), cyclophosphamide (Ayllon and Garcia-Vazquez, 2000) and also to the most common carcinogenic environmental pollutants, such as polycyclic aromatic hydrocarbons (PAH) (Al-Sabti and Metcalfe, 1995; Pacheco and Santos, 2002), pesticides (Grisolia, 2002) and heavy metals (Al-Sabti and Metcalfe, 1995). The chemical compounds and mixtures tested in the present study are among the most diffused in the aquatic environment. The dialkyl and alkyl/aryl phthalates are high production synthetic chemicals and ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Several studies had indicated that some phthalates are tumorigenic in mammals inducing peroxisomal proliferation, but it has been observed that the increase in radical production in rat liver is not enough to give rise to a biologically significant degree of DNA damage (Elliott and Elcombe, 1987). Tetrabromodiphenylether is a representative compound belonging to the class of brominated flam retardants, demonstrating genetic effects in mammalian systems (Helleday et al., 1999). Bisphenol A is applied in the manufacture of polycarbonate plastic and epoxy resins as protective coating on food containers for composites and sealants in dentistry. This compound was poorly investigated about its genotoxic activity; it did not show mutagenic effects in the umu test system (Chen et al., 2002). Nonylphenol is one of the most widely used alkylphenols, applied to make non-ionic surfactants used as emulsifying wetting, dispersing or stabilizing agents in industrial, agricultural and domestic consumer products. Despite their wide diffusion and persistence in the environment, the potential toxicological properties of these compounds are at present time largely unknown. Studies carried out with nonylphenol ethoxylate did not reveal any increase of chromosomal aberrations in Allium cepa, nor of MN frequency in mice (Grisolia et al., 2004). Oil spill causes significant quantities of discharges into the marine environment and several studies have indicated genotoxic damage on aquatic organisms (Hamoutene et al., 2002; Taban et al., 2004; Parry et al., 1997; Harvey et al., 1999; Barˇsien˙e and Barˇsyt˙e Lovejoy, 2000; Barˇsien˙e, 2002; Pietrapiana et al., 2002; Barˇsien˙e et al., 2004; Frenzilli et al., 2004). Elevated levels of MN frequency were also detected in flounder (Platychthys flesus) 8 months after oil spill from Butinge oil terminal in the Baltic Sea (Barˇsien˙e et al., 2004).
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
The application and validation of a sensitive biomarker susceptible to the effects of chemical mixtures is relevant in biomonitoring aquatic pollution. Our results show positive genotoxic effects, measured as MN frequency in erythrocytes from fish exposed to bisphenol A, tetrabromodiphenylether, North Sea oil, and to a mixture of North Sea oil associated to alkylated phenols. In conclusion the results of this study add further evidence about the application of micronucleus assay in fish erythrocytes as an useful parameter in the assessment of genotoxic pollutants and as a valuable biomarker in the field, in monitoring coastal areas and polluted water reservoirs. An improvement of the sensitivity of the method could be reached through the expression and standardization of specific criteria for scoring of MN and other nuclear anomalies. Acknowledgement This work was supported by The European Commission (Research Directorate General, Environment Program-Marine Ecosystems) through the BEEP project “Biological Effects of Environmental Pollution in Marine Coastal Ecosystems” (contract EVK3-CT2000-00543). References Al-Sabti, K., 1986. Clastogenic effects of five carcinogenic mutagenic chemicals on the cells of the common carp, Cyprinus carpio L. Comp. Biochem. Physiol. 85, 5–9. Al-Sabti, K., Metcalfe, C.D., 1995. Fish micronuclei for assessing genotoxicity in water. Mutat. Res. 343, 121–135. Anderson, S.L., Hose, J.E., Knezovich, J.P., 1994. Genotoxic and developmental effects in sea urchins are sensitive indicators of effects of genotoxic chemicals. Environ. Toxicol. Chem. 13, 1033–1041. Arkhipchuk, V.V., Garanko, N.N., 2005. Using the nucleolar biomarker and the micronucleus test on in vivo fish fin cells. Ecotoxicol. Environ. Saf. 62, 42–52. Ayllon, F., Garcia-Vazquez, E., 2000. Induction of micronuclei and other nuclear abnormalities in european minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test. Mutat. Res. 467, 177–186. Ayllon, F., Garcia-Vazquez, E., 2001. Micronuclei and other nuclear lesions as genotoxicity indicators in rainbow trout Oncorhynchus mykiss. Ecotoxicol. Environ. Saf. 49, 221–225. Barˇsien˙e, J., Barˇsyt˙e Lovejoy, D., 2000. Environmental genotoxicity in Klaip˙eda port area. Int. Rev. Hydrobiol. 85, 663–672. Barˇsien˙e, J., 2002. Genotoxic impacts in Klaip˙eda marine Port and B¯uting˙e oil terminal areas (Baltic Sea). Mar. Environ. Res. 54, 475–479. ˇ Barˇsien˙e, J., Lazutka, J., Syvokien˙ e, J., Dedonyt˙e, V., Rybakovas, A., Bjornstad, A., Andersen, O.K., 2004. Analysis of micronuclei in blue mussels and fish form the Baltic and the North Seas. Environ. Toxicol. 19, 365–371. Bolognesi, C., Landini, E., Roggieri, P., Fabbri, R., Viarengo, A., 1999. Genotoxicity biomarkers in the assessment of heavy metal effects in mussels: experimental studies. Environ. Mol. Mutagen. 33 (4), 287–292. Bolognesi, C., Frenzilli, G., Lasagna, C., Perrone, E., Roggieri, P., 2004. Genotoxicity biomarkers in Mytilus galloprovincialis: wild versus caged mussels. Mutat. Res. 552, 187–196. Campana, M.A., Panzeri, A.M., Moreno, V.J., Dulour, F.N., 2003. Micronuclei induction of Rana catesbiana tadpoles by the pyrethroid insecticide lambda-cyhalothrin. Genet. Mol. Biol. 26, 99–103.
S97
Castano, A., Carbonell, G., Carballo, M., Fernandez, C., Boleas, S., Tarazona, J.V., 1998. Sublethal effects of repeated intraperitoneal cadmium injections on rainbow trout (Oncorhynchus mykiss). Ecotoxicol. Environ. Saf. 41, 29–35. Chen, M.Y., Ike, M., Fujita, M., 2002. Acute toxicity, mutagenicity, and estrogenicity of bisphenol-A and other bisphenols. Environ. Toxicol. 17 (1), 80–86. Collier, T.K., Varanasi, U., 1991. Hepatic activities of xenobiotic metabolizing enzymes and biliary levels of xenobiotics in English sole (Parophrys vetulus) exposed to environmental contaminants. Arch. Environ. Contam. Toxicol. 20 (4), 462–473. Elliott, B.M., Elcombe, C.R., 1987. Lack of DNA damage or lipid peroxidation measured in vivo in the rat liver following treatment with peroxisomal proliferators. Carcinogenesis 8 (9), 1213–1218. Fenech, M., Crott, J., Turner, J., Brown, S., 1999. Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide. Mutagenesis 14 (6), 605– 612. Frenzilli, G., Scarcelli, V., Del Barga, I., Nigro, M., Forlin, L., Bolognesi, C., Sturve, J., 2004. DNA damage in eelpout (Zoarces viviparus) from G¨oteborg harbour. Mutat. Res. 552, 187–195. Gravato, C., Santos, M.A., 2002. Juvenile sea bass liver P450, EROD induction, and erythrocytic genotoxic responses to PAH and PAH-like compounds. Ecotoxicol. Environ. Saf. 51 (2), 115–127. Grisolia, C.K., 2002. A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides. Mutat. Res. 518, 145–150. Grisolia, C.K., Bilich, M.R., Formigli, L.M., 2004. A comparative toxicologic and genotoxic study of the herbicide arsenal, its active ingredient imazapyr, and the surfactant nonylphenol ethoxylate. Ecotoxicol. Environ. Saf. 59 (1), 123–126. Grisolia, C.K., Starling, F.L.R.M., 2001. Micronuclei monitoring of fishes from Lake Paranoa, under influence of sewage treatment plant discharges. Mutat. Res. 491, 39–44. Gustavino, B., Scornajenghi, K.A., Minissi, S., Ciccotti, E., 2001. Micronuclei induced in erythrocytes of Cyprinus carpio (telostei, pisces) by X-rays and colchicine. Mutat. Res. 494, 151–159. Hamoutene, D., Payne, J.F., Rahimtula, A., Lee, K., 2002. Use of Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons. Mar. Environ. Res. 54, 471–474. Harvey, J.S., Lyons, B.P., Page, T.S., Stewart, C., Parry, J.M., 1999. An assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in selected invertebrate and vertebrate species. Mutat. Res. 441, 103–114. Helleday, T., Tuominen, K.L., Bergman, A., Jenssen, D., 1999. Brominated flame retardants induce intragenic recombination in mammalian cells. Mutat. Res. 439, 137–147. Landolt, M.L., Kocan, R.M., 1983. Fish cell cytogenetics: a measure of the genotoxic effects of environmental pollutants. In: Nriagu, J.O. (Ed.), Aquatic Toxicology. Wiley, New York, pp. 335–352. Llorente, M.T., Martos, A., Castano, A., 2002. Detection of cytogenetic alterations and blood cell changes in natural populations of carp. Ecotoxicology 11, 27–34. Nepomuceno, J.C., Ferrari, I., Spano, M.A., Centeno, A.J., 1997. Detection of micronuclei in peripheral erythrocytes of Cyprinus carpio exposed to metallic mercury. Environ. Mol. Mutagen. 30, 293–297. Pacheco, M., Santos, M.A., 2002. Naphthalene and -naphthoflavone effects on Anguilla anguilla L. hepatic metabolism and erythrocytic nuclear abnormalities. Environ. Int. 28, 285–293. Parry, J.M., Harvey, J.S., Lyons, B.P., 1997. The application of genetic toxicology in the analysis of the consequences of a major marine pollution incident. Mutat. Res. 379 (Suppl. 1), S91. Pietrapiana, D., Modena, M., Guidetti, P., Falugi, C., Vacchi, M., 2002. Evaluating the genotoxic damage and hepatic tissue alterations in demersal fish species: a case study in the Ligurian Sea (NW-Mediterranean). Mar. Pollut. Bull. 44, 238–243.
S98
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
Schmid, W., 1975. The micronucleus test. Mutat. Res. 31, 9–15. Schultz, N., Norrgren, L., Grawe, J., Johannison, A., Medhage, O., 1993. Micronucleus frequency in circulating erythrocytes from rainbow trout (Oncorhynchus mykiss) subjected to radiation, an image analysis and flow cytometric study. Comp. Biochem. Physiol. 105C, 207– 211.
Sundt, R.C., Pampanin, D.M., Larsen, B.K., Brede, C., Herzke, D., Bjornstad, A., Andersen, O.K., 2006. The BEEP Stavanger workshop: mesocosm exposures. Aquat. Toxicol. 78S, S5–S12. Taban, I.G., Bechmann, R.K., Torgrimsen, S., Baussant, T., Sanni, S., 2004. Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay. Mar. Environ. Res. 58, 701–705.
Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions Claudia Bolognesi a,∗ , Emanuela Perrone a , Paola Roggieri a , Daniela M. Pampanin b , Andrea Sciutto a a
Environmental Carcinogenesis Unit, National Institute for Research on Cancer, L.go Rosanna Benzi, 10, 16132 Genoa, Italy b IRIS – International Research Institute of Stavanger AS, Mekjarvik 12, N-4070 Randaberg, Norway
Abstract The aim of the present study was to standardize and to assess the predictive value of the cytogenetic analysis by MN test in fish erythrocytes as a biomarker for marine environmental contamination. MN frequency baseline in erythrocytes was evaluated in a number of fish species from a reference area (S. Teresa, La Spezia Gulf) and genotoxic potential of a number of common chemical contaminants and mixtures was determined in fish experimentally exposed in aquarium under controlled conditions. Fish (Scophthalmus maximus) were exposed for 3 weeks to 50 ppb of single chemicals (dialkyl phthalate, bisphenol A, tetrabromodiphenyl ether), 30 ppb nonylphenol and mixtures (North Sea oil and North Sea oil with alkylated phenols). Chromosomal damage was determined as micronuclei (MN) frequency in fish erythrocytes. Nuclear anomalies such as blebbed, notched and lobed nuclei were also recorded. Significant increase in MN frequency was observed in erythrocytes of fish exposed to bisphenol A and tetrabromodiphenylether. Chemical mixture North Sea oil + alkylated phenols induced the highest MN frequency (2.95 micronucleated cells/1000 cells compared to 1 MNcell/1000 cells in control animals). The study results revealed that micronucleus test, as an index of cumulative exposure, appears to be a sensitive model to evaluate genotoxic compounds in fish under controlled conditions. © 2006 Elsevier B.V. All rights reserved. Keywords: Scophthalmus maximus; Xenobiotics; MN frequency; Biomarker; Fish erythrocytes
1. Introduction Coastal areas are increasing at risk from toxic contamination from land based discharges, coastal industries and shipping. A wide number of common marine pollutants are studied only for acute effects and their long term adverse environmental hazard is not known. A comprehensive assessment of the real toxic and genotoxic potential for these compounds in marine organisms is needed. The use of biological markers assists in the identification of causal relationship between the exposure to toxic contaminants and increased risk of effects on individuals and populations that may lead to decreased ecological integrity. It has been demonstrated that fish may act as a sentinel organism in genetic toxicology studies. Many fish species can transform organic xenobiotics by efficient enzymatic systems producing by-products linking to critical macromolecules, such as proteins or DNA (Collier and Varanasi, 1991; Gravato and Santos, 2002).
∗
Corresponding author. Tel.: +39 010 5600215. E-mail address: [email protected] (C. Bolognesi).
0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.02.015
DNA and chromosomal damage are the most important critical events following the exposure to carcinogenic and/or genotoxic agents. Chromosomal damage, as a result of inefficient or incorrect DNA repair, is expressed during the cell division and represents an index of accumulated genotoxic effects. Chromosomal effects could be measured as macrolesions in cells exposed to genotoxic agents. Analysis of chromosomal aberrations is difficult in fish because fish chromosomes are generally small in size and high in number. The micronucleus test is the most applied technique to evaluate chromosomal damage in different organisms (Anderson et al., 1994; Bolognesi et al., 1999, 2004; Campana et al., 2003; Al-Sabti and Metcalfe, 1995). Micronuclei (MN) arise from chromosomal fragments or whole chromosomes that are not incorporated into daughter nuclei at mitosis. MN are small fragments of chromatin separated from the main cell nucleus which are index of chromosomal breaking or mitotic spindle dysfunctions (Schmid, 1975). This test has the advantage that it can be applied in interphase to any proliferating cell population regardless of the karyotype. MN can be analysed in different fish cell types such as peripheral erythrocytes, gill, kidney, hepatic and fin cells (Al-Sabti and Metcalfe, 1995; Arkhipchuk and Garanko, 2005). Branchial epithelium, as the
S94
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
primary target for all the water-borne contaminants, showed high background levels of MN and a high sensitivity for the cytogenetic effects induced by environmental contaminants. However gill cells are not easily obtained and prepared for the MN analysis. The application of MN assay in fish hepatocytes has primary limitations because of the low mitotic index of liver cells. The use of peripheral erythrocytes avoids the complex procedures associated with cell preparation and animal sacrifice. In addition high mitotic rate of hematopoietic tissues provides a rapid response to genotoxic exposure, revealed as chromosomal damage in peripheral blood. Micronucleus test in fish erythrocytes has been applied to evaluate the genotoxic impact of environmental pollutants under laboratory and field conditions, revealing interesting correlations with the exposure to a number of chemical and physical agents (Al-Sabti and Metcalfe, 1995; Schultz et al., 1993; Gustavino et al., 2001). The analysis of the baseline MN frequencies obtained by different authors from the literature shows a large interspecies variability, ranging from 0 to 13. This scientific evidence can be related to an interspecies difference in metabolic competency and DNA repair mechanisms as well as in the MN expression. However the large majority of papers report data ranging from 0 to 1. A significant difference (up to 1–2 orders of magnitude) in the MN baseline frequency was recorded in the same species from different authors e.g. Cyprinus carpio (Landolt and Kocan, 1983; Al-Sabti, 1986; Nepomuceno et al., 1997; Llorente et al., 2002; Grisolia and Starling, 2001; Gustavino et al., 2001) or Oncorhynchus mykiss (Schultz et al., 1993; Castano et al., 1998; Ayllon and Garcia-Vazquez, 2001). This observation outlines the importance to standardize and intercalibrate the experimental procedure for MN assay with special reference to the criteria of scoring in order to compare data from different labs. The aim of this study is to standardize and to assess the predictive value of the cytogenetic analysis by MN test in fish erythrocytes as a biomarker for marine environmental contamination. MN frequency baseline in erythrocytes was evaluated in different fish species and the genotoxic potential of a number of common chemical contaminants and mixtures was determined in fish exposed under controlled conditions. 2. Materials and methods Samples of fish belonging from different species from a reference area (S. Teresa, La Spezia Gulf) were collected in order to evaluate the baseline for MN and other nuclear anomalies. Fish samples were collected from the same site with the same size and weight and they are acclimatized in the lab for 7 days. Turbot (Scophthalmus maximus) was chosen for the experimental study in aquarium. Groups of animals were exposed in aquarium under controlled conditions for a period of 3 weeks. In a first experiment carried out during the period September–October 2002 the fish were exposed to mixtures of chemicals: (a) 0.5 ppm North sea crude oil-Statfjord; (b) 0.5 ppm of North sea crude oil spiked with 0.1 ppm of a mixture of alkylated phenols; (c)
30 ppb of nonylphenol and (d) controls. The exposure was carried out using a continuous flow system designed for performing studies of chronic exposures of marine organisms to mixtures of poorly water soluble substances. Details about the exposure are reported by Sundt et al. (2006). In a second experiment carried out on May 2003 the animals were exposed to 50 ppb of single chemicals (dialkyl phthalate, bisphenol A, tetrabromodiphenylether) and controls. At least six individuals/experimental group were used to evaluate the genotoxic effects induced by the treatment. Blood was collected from the caudal vein and immediately smeared on clean glass slides, dried overnight, fixed with methanol for 20 min and stained with acridine orange. About 4000 erythrocytes per animal were analysed by a fluorescence microscope under 1000× magnification. Only clearly isolated MN were counted as such and other nuclear anomalies were recorded separately. MN scoring followed prior established criteria. MN was defined as small round or oval intracytoplasmic bodies with a diameter 1/5–1/20 of the main nucleus and on the same optical plane as the major nucleus. The MN was not linked or connected to the main nucleus. Cells with more than four MN were discarded to exclude apoptotic phenomena. Nuclear anomalies, chromatin buds, lobes, invaginations, vacuoles, were recorded separately. Cells showing abnormal morphological forms (degenerated cells), induced by cytotoxic events have to be discarded. 2.1. Statistical analysis Differences between groups were evaluated by nonparametric Mann–Whitney U-test. The relationship between MN frequency and nuclear evaginations or buds was evaluated using regression analysis. All the data were analysed using SPSS statistical software package (SPSS 13.0 for Windows). 3. Results Table 1 and Fig. 1 show the baseline frequencies for MN and other nuclear abnormalities in different fish species from a reference area (S. Teresa, La Spezia Gulf). The MN frequency ranges between 0 and 2.0 with a high interspecies and interindividual variability, as it has been commonly observed for different biomarkers. The large interindividual variability associated to the low baseline frequency for this biomarker confirming the need for the scoring of a consistent number of cells (at least 4000 cells) in an adequate number of animals for each study point. A correlation was observed between MN frequency and nuclear evaginations or buds (R2 0.585, p < 0.016, n = 24) (Fig. 2) suggesting the importance for recording this anomaly in order to improve the information obtained with this assay. No correlation was found between MN frequency and nuclear invaginations, parameter mainly associated to cytotoxicity. This observation outlines the importance to standardize and intercalibrate the experimental procedure for MN assay with
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
S95
Fig. 1. Spontaneous frequency of micronucleated erythrocytes/1000 cells and cells with nuclear buds/1000 cells in fish from different species.
Table 1 Spontaneous frequency of micronuclei and other nuclear abnormalities in fish erythrocytes belonging to different species from a reference area (S. Teresa, La Spezia Gulf, Italy) Species
Boops boops Dicentrarchus labrax Mullus barbatus Pagellus mormyrus Sargus sargus Seriola dumerili Serranus cabrilla Sparus auratus Sphyraena sphyraena Trachurus trachurus
MN cellsa /1000 cells
Cells with NAb /1000 cells
Mean (S.D.)
Invaginations Mean (S.D.)
BUDS, Mean (S.D.)
1.75 (0.35) 0.75 (0.64) 0.33 (0.57) 0.4 (0.72) 0.25 (0.35) 0.38 (0.18) 0.0 (0.0) 0.12 (0.18) 0.25 (0.35) 0.25 (0.32)
15.6 (5.03) 1.5 (0.40) 2.63 (0.93) 3.83 (2.76) 8.75 (2.47) 1.62 (0.53) 1.08 (0.80) 6.75 (4.95) 1.87 (1.24) 3.0 (2.16)
1.62 (0.54) 0.0 (0.0) 0.16 (1.42) 0.58 (0.63) 0.37 (0.53) 0.12 (0.18) 0.0 (0.0) 0.50 (0.71) 0.62 (0.88) 1.25 (0.74)
Five fish for each species were analysed. a MN cells = micronucleated cells/1000 cells. b Cells with NA = cells with nuclear abnormalities.
special reference to the scoring criteria in order to compare data from different labs. Fig. 3A shows the results obtained in a first experiment carried out under controlled conditions. Statistically significant increase in MN frequency as well as in nuclear abnormalities was observed in erythrocytes from fish treated with bisphenol A (p < 0.005) and tetrabromodiphenylether (p < 0.001), no effect of dialkylphthalate was recorded (the mean MN frequency is lower with respect to the control value). Frequencies of MN in peripheral fish erythrocytes from groups of fish treated with nonylphenol, North sea oil and North sea oil + alkylated phenols are shown in Fig. 3B. Highest effects were observed in animals exposed to chemical mixtures (2.95 MN/1000 cells compared to 1 MN/1000 cells in control animals). A large interindividual variability in MN frequency was observed in nonylphenol treated animals, where the results did not reach the statistical significance. A negligible level of nuclear abnormalities was recorded in fish erythrocytes from fish exposed to chemical compounds or mixtures considered in our study. 4. Discussion
Fig. 2. Correlation between micronucleated erythrocytes/1000 cells and cells with nuclear buds/1000 cells in fish from different species (R2 0.585, p = 0.016).
The micronucleus test in peripheral erythrocytes provides a feasible approach to monitor the effects of environmental genotoxic agents in fish. A large variability in spontaneous MN frequency in different fish species was evident from the analysis of the scientific literature. The large majority of papers report a very low range of MN frequency, but they show also a large range of variability in the frequency from the same species. The low baseline MN frequency represents the main limitation for the MN test applied in fish erythrocytes, requiring the scoring of a large number of cells from a consistent number of animals.
S96
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
Fig. 3. Micronucleated erythrocytes (MNcells/1000 cells) from fish (Scophthalmus maximus) treated for a period of 3 weeks with (A) dialkylphthalate (50 ppb), bisphenol A (50 ppb), tetrabromodiphenylether (50 ppb) and controls; (B) nonylphenol (30 ppb), North sea crude oil (NSA), North Sea crude oil spiked with 0.1 ppm of a mixture of alkylated phenols and controls.
The lack of uniform criteria of scoring for MN and other nuclear abnormalities is the main critical factor responsible for the large variability of MN frequency in the same species affecting the sensitivity of the assay and preventing the comparison of the results from different labs. Our data on spontaneous MN induction in different species from a reference area show a low range of frequency and a large variability. Interestingly the frequency of nuclear evaginations is positively correlated to the MN frequency, suggesting the importance to record also this parameter. A number of papers recently appeared in the scientific literature reporting an association between the frequency of nuclear anomalies and the exposure to genotoxic agents (Ayllon and Garcia-Vazquez, 2000; Pacheco and Santos, 2002). A comprehensive application has been proposed for the MN assay in human lymphocytes, including a set of parameters of genotoxicity and citotoxicity: MN as markers of chromosomal breakage and/or loss, nucleoplasmic bridges as markers of chromosomal rearrangements, nuclear evaginations or buds as markers of gene amplification, cellular necrosis and apoptosis (Fenech et al., 1999). The different frequencies of these anomalies are related to specific genotoxic events associated to the different mechanisms of action of the carcinogenic/mutagenic agents. The application of MN in its comprehensive mode in fish erythrocytes needs the expression and standardization of specific criteria for the different nuclear anomalies. It’s also very impor-
tant the evaluation of the percent of degenerated cells, directly associated to cytotoxic events. The micronucleus test in fish erythrocytes was widely validated in lab after exposure to different agents. A dose response increase in MN frequency was observed after exposure to a range of doses of X-rays (Schultz et al., 1993; Gustavino et al., 2001). The MN test in fish erythrocytes responded positively to a large number of experimental carcinogens, such as aflatoxins, benzidine, ethylmethanesulfonate, methylcholantrene, chlorinated hydrocarbons (Al-Sabti and Metcalfe, 1995), cyclophosphamide (Ayllon and Garcia-Vazquez, 2000) and also to the most common carcinogenic environmental pollutants, such as polycyclic aromatic hydrocarbons (PAH) (Al-Sabti and Metcalfe, 1995; Pacheco and Santos, 2002), pesticides (Grisolia, 2002) and heavy metals (Al-Sabti and Metcalfe, 1995). The chemical compounds and mixtures tested in the present study are among the most diffused in the aquatic environment. The dialkyl and alkyl/aryl phthalates are high production synthetic chemicals and ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Several studies had indicated that some phthalates are tumorigenic in mammals inducing peroxisomal proliferation, but it has been observed that the increase in radical production in rat liver is not enough to give rise to a biologically significant degree of DNA damage (Elliott and Elcombe, 1987). Tetrabromodiphenylether is a representative compound belonging to the class of brominated flam retardants, demonstrating genetic effects in mammalian systems (Helleday et al., 1999). Bisphenol A is applied in the manufacture of polycarbonate plastic and epoxy resins as protective coating on food containers for composites and sealants in dentistry. This compound was poorly investigated about its genotoxic activity; it did not show mutagenic effects in the umu test system (Chen et al., 2002). Nonylphenol is one of the most widely used alkylphenols, applied to make non-ionic surfactants used as emulsifying wetting, dispersing or stabilizing agents in industrial, agricultural and domestic consumer products. Despite their wide diffusion and persistence in the environment, the potential toxicological properties of these compounds are at present time largely unknown. Studies carried out with nonylphenol ethoxylate did not reveal any increase of chromosomal aberrations in Allium cepa, nor of MN frequency in mice (Grisolia et al., 2004). Oil spill causes significant quantities of discharges into the marine environment and several studies have indicated genotoxic damage on aquatic organisms (Hamoutene et al., 2002; Taban et al., 2004; Parry et al., 1997; Harvey et al., 1999; Barˇsien˙e and Barˇsyt˙e Lovejoy, 2000; Barˇsien˙e, 2002; Pietrapiana et al., 2002; Barˇsien˙e et al., 2004; Frenzilli et al., 2004). Elevated levels of MN frequency were also detected in flounder (Platychthys flesus) 8 months after oil spill from Butinge oil terminal in the Baltic Sea (Barˇsien˙e et al., 2004).
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
The application and validation of a sensitive biomarker susceptible to the effects of chemical mixtures is relevant in biomonitoring aquatic pollution. Our results show positive genotoxic effects, measured as MN frequency in erythrocytes from fish exposed to bisphenol A, tetrabromodiphenylether, North Sea oil, and to a mixture of North Sea oil associated to alkylated phenols. In conclusion the results of this study add further evidence about the application of micronucleus assay in fish erythrocytes as an useful parameter in the assessment of genotoxic pollutants and as a valuable biomarker in the field, in monitoring coastal areas and polluted water reservoirs. An improvement of the sensitivity of the method could be reached through the expression and standardization of specific criteria for scoring of MN and other nuclear anomalies. Acknowledgement This work was supported by The European Commission (Research Directorate General, Environment Program-Marine Ecosystems) through the BEEP project “Biological Effects of Environmental Pollution in Marine Coastal Ecosystems” (contract EVK3-CT2000-00543). References Al-Sabti, K., 1986. Clastogenic effects of five carcinogenic mutagenic chemicals on the cells of the common carp, Cyprinus carpio L. Comp. Biochem. Physiol. 85, 5–9. Al-Sabti, K., Metcalfe, C.D., 1995. Fish micronuclei for assessing genotoxicity in water. Mutat. Res. 343, 121–135. Anderson, S.L., Hose, J.E., Knezovich, J.P., 1994. Genotoxic and developmental effects in sea urchins are sensitive indicators of effects of genotoxic chemicals. Environ. Toxicol. Chem. 13, 1033–1041. Arkhipchuk, V.V., Garanko, N.N., 2005. Using the nucleolar biomarker and the micronucleus test on in vivo fish fin cells. Ecotoxicol. Environ. Saf. 62, 42–52. Ayllon, F., Garcia-Vazquez, E., 2000. Induction of micronuclei and other nuclear abnormalities in european minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test. Mutat. Res. 467, 177–186. Ayllon, F., Garcia-Vazquez, E., 2001. Micronuclei and other nuclear lesions as genotoxicity indicators in rainbow trout Oncorhynchus mykiss. Ecotoxicol. Environ. Saf. 49, 221–225. Barˇsien˙e, J., Barˇsyt˙e Lovejoy, D., 2000. Environmental genotoxicity in Klaip˙eda port area. Int. Rev. Hydrobiol. 85, 663–672. Barˇsien˙e, J., 2002. Genotoxic impacts in Klaip˙eda marine Port and B¯uting˙e oil terminal areas (Baltic Sea). Mar. Environ. Res. 54, 475–479. ˇ Barˇsien˙e, J., Lazutka, J., Syvokien˙ e, J., Dedonyt˙e, V., Rybakovas, A., Bjornstad, A., Andersen, O.K., 2004. Analysis of micronuclei in blue mussels and fish form the Baltic and the North Seas. Environ. Toxicol. 19, 365–371. Bolognesi, C., Landini, E., Roggieri, P., Fabbri, R., Viarengo, A., 1999. Genotoxicity biomarkers in the assessment of heavy metal effects in mussels: experimental studies. Environ. Mol. Mutagen. 33 (4), 287–292. Bolognesi, C., Frenzilli, G., Lasagna, C., Perrone, E., Roggieri, P., 2004. Genotoxicity biomarkers in Mytilus galloprovincialis: wild versus caged mussels. Mutat. Res. 552, 187–196. Campana, M.A., Panzeri, A.M., Moreno, V.J., Dulour, F.N., 2003. Micronuclei induction of Rana catesbiana tadpoles by the pyrethroid insecticide lambda-cyhalothrin. Genet. Mol. Biol. 26, 99–103.
S97
Castano, A., Carbonell, G., Carballo, M., Fernandez, C., Boleas, S., Tarazona, J.V., 1998. Sublethal effects of repeated intraperitoneal cadmium injections on rainbow trout (Oncorhynchus mykiss). Ecotoxicol. Environ. Saf. 41, 29–35. Chen, M.Y., Ike, M., Fujita, M., 2002. Acute toxicity, mutagenicity, and estrogenicity of bisphenol-A and other bisphenols. Environ. Toxicol. 17 (1), 80–86. Collier, T.K., Varanasi, U., 1991. Hepatic activities of xenobiotic metabolizing enzymes and biliary levels of xenobiotics in English sole (Parophrys vetulus) exposed to environmental contaminants. Arch. Environ. Contam. Toxicol. 20 (4), 462–473. Elliott, B.M., Elcombe, C.R., 1987. Lack of DNA damage or lipid peroxidation measured in vivo in the rat liver following treatment with peroxisomal proliferators. Carcinogenesis 8 (9), 1213–1218. Fenech, M., Crott, J., Turner, J., Brown, S., 1999. Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide. Mutagenesis 14 (6), 605– 612. Frenzilli, G., Scarcelli, V., Del Barga, I., Nigro, M., Forlin, L., Bolognesi, C., Sturve, J., 2004. DNA damage in eelpout (Zoarces viviparus) from G¨oteborg harbour. Mutat. Res. 552, 187–195. Gravato, C., Santos, M.A., 2002. Juvenile sea bass liver P450, EROD induction, and erythrocytic genotoxic responses to PAH and PAH-like compounds. Ecotoxicol. Environ. Saf. 51 (2), 115–127. Grisolia, C.K., 2002. A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides. Mutat. Res. 518, 145–150. Grisolia, C.K., Bilich, M.R., Formigli, L.M., 2004. A comparative toxicologic and genotoxic study of the herbicide arsenal, its active ingredient imazapyr, and the surfactant nonylphenol ethoxylate. Ecotoxicol. Environ. Saf. 59 (1), 123–126. Grisolia, C.K., Starling, F.L.R.M., 2001. Micronuclei monitoring of fishes from Lake Paranoa, under influence of sewage treatment plant discharges. Mutat. Res. 491, 39–44. Gustavino, B., Scornajenghi, K.A., Minissi, S., Ciccotti, E., 2001. Micronuclei induced in erythrocytes of Cyprinus carpio (telostei, pisces) by X-rays and colchicine. Mutat. Res. 494, 151–159. Hamoutene, D., Payne, J.F., Rahimtula, A., Lee, K., 2002. Use of Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons. Mar. Environ. Res. 54, 471–474. Harvey, J.S., Lyons, B.P., Page, T.S., Stewart, C., Parry, J.M., 1999. An assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in selected invertebrate and vertebrate species. Mutat. Res. 441, 103–114. Helleday, T., Tuominen, K.L., Bergman, A., Jenssen, D., 1999. Brominated flame retardants induce intragenic recombination in mammalian cells. Mutat. Res. 439, 137–147. Landolt, M.L., Kocan, R.M., 1983. Fish cell cytogenetics: a measure of the genotoxic effects of environmental pollutants. In: Nriagu, J.O. (Ed.), Aquatic Toxicology. Wiley, New York, pp. 335–352. Llorente, M.T., Martos, A., Castano, A., 2002. Detection of cytogenetic alterations and blood cell changes in natural populations of carp. Ecotoxicology 11, 27–34. Nepomuceno, J.C., Ferrari, I., Spano, M.A., Centeno, A.J., 1997. Detection of micronuclei in peripheral erythrocytes of Cyprinus carpio exposed to metallic mercury. Environ. Mol. Mutagen. 30, 293–297. Pacheco, M., Santos, M.A., 2002. Naphthalene and -naphthoflavone effects on Anguilla anguilla L. hepatic metabolism and erythrocytic nuclear abnormalities. Environ. Int. 28, 285–293. Parry, J.M., Harvey, J.S., Lyons, B.P., 1997. The application of genetic toxicology in the analysis of the consequences of a major marine pollution incident. Mutat. Res. 379 (Suppl. 1), S91. Pietrapiana, D., Modena, M., Guidetti, P., Falugi, C., Vacchi, M., 2002. Evaluating the genotoxic damage and hepatic tissue alterations in demersal fish species: a case study in the Ligurian Sea (NW-Mediterranean). Mar. Pollut. Bull. 44, 238–243.
S98
C. Bolognesi et al. / Aquatic Toxicology 78S (2006) S93–S98
Schmid, W., 1975. The micronucleus test. Mutat. Res. 31, 9–15. Schultz, N., Norrgren, L., Grawe, J., Johannison, A., Medhage, O., 1993. Micronucleus frequency in circulating erythrocytes from rainbow trout (Oncorhynchus mykiss) subjected to radiation, an image analysis and flow cytometric study. Comp. Biochem. Physiol. 105C, 207– 211.
Sundt, R.C., Pampanin, D.M., Larsen, B.K., Brede, C., Herzke, D., Bjornstad, A., Andersen, O.K., 2006. The BEEP Stavanger workshop: mesocosm exposures. Aquat. Toxicol. 78S, S5–S12. Taban, I.G., Bechmann, R.K., Torgrimsen, S., Baussant, T., Sanni, S., 2004. Detection of DNA damage in mussels and sea urchins exposed to crude oil using comet assay. Mar. Environ. Res. 58, 701–705.