Sublethal exposure of heavy metals induces micronuclei in fish, Channa punctata

Chemosphere 77 (2009) 1495–1500

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Sublethal exposure of heavy metals induces micronuclei in fish, Channa punctata Kamlesh K. Yadav, Sunil P. Trivedi * Environmental Toxicology Laboratory, Department of Zoology, University of Lucknow, Lucknow 226007, UP, India

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Article history: Received 6 July 2009 Received in revised form 6 October 2009 Accepted 9 October 2009 Available online 31 October 2009 Keywords: Micronuclei Fish Channa punctata Mercury [Hg(II)] Arsenic [As(III)] Copper [Cu(II)]

a b s t r a c t Present studies were designed to evaluate toxic potential of three common heavy metals, adequately present in agro-industrial effluents, viz. mercury, arsenic and copper using in vivo micronucleus assay in an actinopterygiian fish, Channa punctata (2n = 32). Ten days laboratory acclimatized fishes were divided into five groups. Groups I and II served as negative and positive controls, respectively and fishes of group III–V were subjected uninterrupted to sublethal concentrations (10% of 96 h LC50) of heavy metal compounds, HgCl2 (0.081 mg L 1), As2O3 (6.936 mg L 1) and CuSO4
5H2O (0.407 mg L 1) for 24, 48, 72, 96 and 168 h of exposure periods. Significant increase over and above negative control in the frequency of micronuclei was observed in fishes exposed to metal compounds. The average frequency of micronuclei in fishes exposed to Hg(II), As(III) and Cu(II) observed was 9.79, 12.03 and 8.86, respectively. It reveals that the order of induction of micronuclei frequency and toxicity was As > Hg > Cu. Findings depict genotoxic potential of these metal compounds even in sublethal concentrations. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Population explosion coupled with industrialization has led to a spurt in both resource consumption and mobilization, and consequent generation of diverse wastes including a variety of contaminants and noxious elements, viz. pesticides, ODWs (Oxygen Demanding Wastes) and heavy metals which ultimately spoil aquatic environment on their entry into it. Heavy metals viz. Hg, As and Cu, and their derivatives are widely dispersed throughout the environment as a result of fossil fuel combustion, industrial, agricultural and natural processes. Among the myriad of organic and inorganic substances released into the aquatic ecosystems, heavy metals have received considerable attention due to their toxicity and potential to bioaccumulate at various trophic levels (Blevins, 1985; Szefer et al., 1990). They not only deteriorate the physico-chemical equilibrium of the aquatic body, but also disrupt the food web and, bring about morphological, physiological and cytogenetical changes in the aquatic inhabitants. They also cause mutagenic and carcinogenic reactions in living beings. Of all the commonly occurring metallic pollutants, mercury is regarded to be most toxic. This metal made its presence felt world over when it was found instrumental in Minamata biological disaster of Japan in 1956. Mercuric chloride is the most widely used among all mercury compounds. It is a poisonous white crystalline compound, used as a reliable ingredient in antiseptics, disinfectants and preservatives, insecticides, batteries, and in metallurgical * Corresponding author. Tel.: +91 522 2740040. E-mail addresses: [email protected] (K.K. Yadav), [email protected] (S.P. Trivedi). 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.10.022

and photographic operations (Goldberg, 1996; Freeman et al., 2003). Exposure to high concentrations of mercury causes damage to nervous system, immune system, kidney and liver in human beings (Peraza et al., 1998; Rutowski et al., 1998). Weis and Weis (1997) reported teratogenic effect of inorganic mercury in killfish, Fundulus heteroclitus. Methyl mercury is known to induce chromosomal aberrations and micronuclei in the same fish (Perry et al., 1988). Among metallic pollutants, arsenic is also one of the most relevant environmental global single substance toxicants (ATSDR, 1999). Arsenic contamination of groundwater has been reported from many countries including Bangladesh, West Bengal, India, Vietnam, Argentina, China, parts of the USA (Smedley and Kinniburgh, 2002; Hossain, 2006) and now Nepal (JICA/ENPHO, 2005). It is introduced into water via weathering of rocks, minerals and ores, industrial effluents including mining wastes and atmospheric deposition (Hindmarsh and McCurdy, 1986). Human exposure takes place via contaminated water and soil as well as from food rich in arsenic, viz. garlic, marine food, etc. and occupational activities (Hughes, 2002; Rodriguez et al., 2003). It is one of the few substances known to be carcinogenic to humans through the consumption of drinking water. Wider applications of arsenic compounds, particularly arsenic trioxide, as the starting substance for the manufacture of arsenic-based pesticides, arsenic-based pharmaceuticals (Neosalvarsan), and veterinary products, decolorizing agents for glasses and enamels and wood preservatives make human beings more prone to its exposure. In spite of high toxicity, arsenic is a common contaminant in pharmaceuticals (Bohrer et al., 2005). Arsenic has long been regarded as a potential carcinogen (Hertz-Picciotto et al., 1992; Kitchin, 2001), genotoxic in both

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in vivo (Wegner et al., 2004; Chen et al., 2005; Martinez et al., 2005) and in vitro conditions (Ramirez et al., 2003; Dopp et al., 2004), besides causing chromosomal abnormalities (Wen-Chien et al., 2001). Copper, an essential trace metal for living organisms, is present in natural waters and sediments (Linder, 2001) and virtually in all media, i.e. air, water and soil (ATSDR, 1990). This metal plays a crucial role in many biological enzyme systems that catalyze oxidation/reduction reactions and have molecular oxygen as a co-substrate. However, if copper is present at relatively high concentrations in the environment, toxicity to aquatic organisms may occur. Copper sulphate commonly known as blue stone or blue vitriol is the best known and most widely used among all copper salts. It is used in agriculture, principally as a fungicide to control bacterial and fungal diseases of fruit, vegetable, nut and field crops and also used as an algaecide, herbicide, molluscicide and as a mordant in dyeing process. Copper in surface waters is a well-known environmental hazard, associated with toxicity to a variety of aquatic organisms (USEPA, 2003). High copper levels lead to an increase in the rate of free radical formation (Gwozdzinski, 1995; Bogdanova et al., 2002), teratogenicity (Stouthart et al., 1996) and chromosomal aberrations (Bhunya and Pati, 1987; Fahmy, 2000). Chronic exposure and accumulation of heavy metals by aquatic biota can result in tissue burdens that produce adverse effects not only in the exposed organisms, but also in organisms including human beings (IARC, 1993). Aquatic animals have often been used in bioassays to monitor water quality of effluents and surface waters (Carins et al., 1975; Brugs et al., 1977). Genotoxic studies on aquatic organisms exposed to polluted waters containing heavy metals have implicated DNA strand breakages (Espina and Weiss, 1995; Bolognesi et al., 1996; Pruski and Dixon, 2002) and fishes are employed as sensitive indicators for their genotoxic and mutagenic effects (Al-Sabti, 1986; Yadav and Trivedi, 2006, 2009). The micronucleus assay is a simple and sensitive assay for in vivo evaluation of genotoxic potential of xenobiotics. It is simple, reliable, sensitive, and it does not depend on any karyotypic characteristics of the test animal. Within the last decade, micronucleus (Al-Sabti, 1995) tests have played an important role in assessing exposure to water pollutants, and have proved as appropriate tools to provide an early warning of genotoxic threat to fish and other aquatic organisms, their ecosystem and finally to man. In view of the above, the present investigations were carried out to assess the genotoxic potential of HgCl2, As2O3 and CuSO4
5H2O under static renewal system by estimating the frequency of micronuclei in the kidney cells of Channa punctata (Bloch) after in vivo exposure to sublethal concentrations of aforesaid metal compounds.

2. Materials and methods 2.1. Experimental animals Healthy and live specimens of an actinopterygiian fish C. punctata (average size and weight 14 ± 1.0 cm; 30 ± 2.0 g) procured from the local lentic habitats were given prophylactic treatment by bathing in formalin (0.4%) for 15 min. Benzylkonium chloride (1 mg L 1) for 1 h and KMnO4 solution (1 mg L 1) for 1 h to keep away from any dermal infections and then were acclimatized for 10 days in large glass aquaria containing aged tap water (hardness 74.6 as CaCO3 mg L 1, alkalinity 76.35 as CaCO3 mg L 1, DO 6.91 mg L 1, COD 62.5 mg L 1, chloride 146.4 mg L 1, TDS 228.33 mg L 1, pH 7.02, and temperature 28 °C) supplied by University Bore Wells. During this period, the fishes were fed on minced goat liver and artificial fish food Tokyo. The fecal matter and other waste materials were siphoned off daily to reduce ammonia content in water. Every effort as suggested by Bennett

and Dooley (1982) was made to maintain optimal conditions during acclimatization. 2.2. Determination of sub lethal concentrations of metal compounds (LC50) The acute toxicity bioassay procedure based on standard methods (APHA et al., 1998) was conducted to determine the LC50-96 h values of mercuric chloride, arsenic trioxide, and copper sulphate pentahydrate (S.D. Fine-chem Ltd., Mumbai, India). A set of 10 fishes was randomly exposed to 10 target concentration levels of each metal compound to obtain the LC50-96 h values of the each test chemical for the species. Oxygenation of the test solution was provided with the help of aerators. The LC50 values of mercuric chloride, arsenic trioxide and copper sulphate pentahydrate were determined as 0.81, 69.36 and 4.07 mg L 1, respectively following the Trimmed Spearman–Karber Method (Hamilton et al., 1977). Based on the LC50 value, the 1/10 of LC50-96 h was estimated for each metal compound. 2.3. In vivo exposure experiment Fishes acclimatized for 10 days were divided in five groups, each group having 50 numbers of fishes. The fishes of group I and II, maintained in aged tap water were considered as negative and positive control, respectively. An intramuscular injection with single dose of Mitomycin-C (Cadila Pharmaceutical Pvt. Limited, Oncocare Division, Ahmedabad, India) @ 1 mg kg 1 body wt. was administered to group II. Fishes of groups III–V were subjected to sublethal concentrations (10% of 96 h LC50) of heavy metal compounds, HgCl2 (0.081 mg L 1), As2O3 (6.936 mg L 1) and CuSO4
5H2O (0.407 mg L 1) in a semi-static system (test water was renewed at every 24 h having same concentration of heavy metal compounds). The exposure was continued up to 168 h (seven days) and sampling was done at intervals of 24, 48, 72, 96 and 168 h at the rate of six fishes per duration. On each sampling day, the blood was immediately processed for MN assay. The blood samples were collected from the fish by caudal vein puncture technique using a heparinized syringe. 2.4. Micronucleus assay The slides were prepared by smearing one drop of blood on clean microscopic slides, fixed in methanol for 10 min and left to air-dry at room temperature and finally stained with 5% Giemsa in Sorenson buffer (pH 6.9) for 20 min. A total of 1000 erythrocytes were examined for each specimen under the light microscope. For the scoring of micronuclei, the following criteria were adopted from Fenech et al. (2003). The diameter of the MN should be less than one-third of the main nucleus. MN should be separated from or marginally overlap with main nucleus as long as there is clear identification of the nuclear boundary. MN should have similar staining as the main nucleus. 2.5. Statistical analysis The one-way analysis of variance (ANOVA), Duncan’s Multiple Range Test was employed to compare the mean differences in MN frequency between control and different exposure periods and, in between successive exposure periods. 3. Results Only micronuclei (MNi) having no connection with the main nucleus, similar in color and intensity as that of main nucleus

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and the area less than 1/3rd of the main nucleus were scored. They were recorded 1.333 ± 0.516 in group I after 24 h of exposure period and it remained almost constant and did not differ statistically for the subsequent exposure periods and on this basis data from 24 h are used to represent the whole of control values. Significantly increased levels (p < 0.05) of MNi were observed after each evaluation period in group II in comparison to group I. The maximum frequency of MNi was recorded after 96 h of exposure period. Significant differences (p < 0.05) in the frequency of MNi were also observed in between all successive sampling periods except between 48, 72 and 168 h, and between 48, 72, 96 and 168 h (Table 1, Fig. 1). Exposure to sublethal concentration of Hg(II) also causes significant induction of micronuclei over five treatment periods, i.e. 24, 48, 72, 96 and 168 h in comparison to group I. Significant levels (p < 0.05) of MNi frequency differences between successive evaluation periods were recorded except in between 48 and 96 h, 72 and 96 h, 96 and 168 h. In this group, maximum frequency (11.33 ± 1.211) of MNi was recorded after 72 h of exposure period (Table 1, Fig. 2). Significant levels (p < 0.05) of MNi frequencies at each evaluation period were also observed in group IV in comparison to group I. In this group, significant levels (p < 0.05) of MNi frequency differences between successive exposure periods were recorded except in between 72 and 168 h. The maximum frequency (15.00 ± 1.414) of MNi was recorded after 96 h of exposure period (Table 1, Fig. 3). The MNi frequencies in erythrocytes of group V were found increased throughout the exposure periods. The maximum frequency (12.00 ± 0.632) of MNi was recorded after the longest exposure period of 168 h. In this group, significant level (p < 0.05) of MNi frequencies after each exposure period and in between successive exposure periods was noticed (Table 1, Fig. 4).

Fig. 1. Metal compounds exposure response relationship of MNi frequency in peripheral erythrocytes of C. punctata for multiple exposure periods.

4. Discussion MN assay is a significant and potent tool for the assessment of genotoxicity as it is simple, reliable, sensitive, and is independent of karyotypic characteristics of the test animal. Fishes are excellent specimens for the study of the mutagenic or carcinogenic potential of contaminants present in water samples since they can metabolize, concentrate and store waterborne pollutants. They can serve

Fig. 2. Showing micronuclei induced by Hg(II) in peripheral erythrocytes of fish, C. punctata.

as useful genetic models for the evaluation of pollution in aquatic ecosystems (Mitchell and Kennedy, 1992; Park et al., 1993). The erythrocyte micronucleus test has been used with different fish

Table 1 Micronuclei frequencies in peripheral erythrocytes of C. punctata treated with Mitomycin-C (MC), mercuric chloride, arsenic trioxide and copper sulphate. Toxicants

Exposure period (h)

No. of fishes observed

No. of cells counted

No. of MN/1000

MC

Control 24 48 72 96 168

6 6 6 6 6 6

7129 6163 6174 6135 6284 6163

1.33 ± 0.516a 12.00 ± 1.265b 14.33 ± 2.251cd 15.83 ± 1.169cd 16.83 ± 1.835d 16.00 ± 0.894cd

Hg(II)

24 48 72 96 168

6 6 6 6 6

6345 6225 6404 6286 6250

8.00 ± 0.894b 9.66 ± 0.816c 11.33 ± 1.211d 10.50 ± 1.049cde 9.50 ± 1.049e

As(III)

24 48 72 96 168

6 6 6 6 6

6129 6257 6201 6200 6210

8.83 ± 0.753b 10.83 ± 1.169c 12.16 ± 1.472d 15.00 ± 1.414e 13.33 ± 0.816d

Cu(II)

24 48 72 96 168

6 6 6 6 6

6405 6230 6329 6336 6329

5.50 ± 1.049b 7.16 ± 0.753c 8.66 ± 0.516d 11.00 ± 0.894e 12.00 ± 0.632f

Values (mean ± SD) in columns with same superscript are not significantly different from each other (One Way ANOVA, Duncan’s multiple range test; p < 0.05).

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Fig. 3. Showing micronuclei induced by As(III) in peripheral erythrocytes of fish C. punctata.

Fig. 4. Showing micronuclei induced by Cu(II) in peripheral erythrocytes of fish, C. punctata.

species to monitor aquatic pollutants displaying mutagenic features (De Flora et al., 1993; Saotome and Hayashi, 2003; Pantaleao Sde et al., 2006). Rodriguez et al. (2003) have also reported the sensitivity of micronucleus test in freshwater fish species for application in field surveys. In the present study, Hg(II), As(III) and Cu(II) induced significantly (p < 0.05) higher number of MNi vis-a-vis the negative control and its frequency was recorded increased up to 96 h in groups II and IV while up to 72 and 168 h in groups III and V, respectively (Fig. 1). In the present investigation, frequencies of MNi were found increased up to 96 h in group II and then they decreased gradually; while in group III an increasing trend was recorded through out the entire exposure periods. The obtained results support the fact demonstrated by Kligerman (1982) that fish inhabiting polluted waters have greater frequencies of micronuclei. The micronuclei frequencies may vary according to the kind of pollution involved, season and the species of fish. Based on the sampling time, Hooftman and de Raat (1982) concluded that there is a time-dependent increase in MNi induction in peripheral blood of fish, an effect corroborated by the present work. Exposure of sublethal concentration of Hg(II) causes significant induction of micronuclei over five treatment periods. Genotoxic

potential of mercury has also been observed by Perry et al. (1988) in terms of micronuclei in the killfish, F. heteroclitus exposed to methyl mercury chloride. Similar observations have also been made by Nepomuceno et al. (1997) in fish Cyprinus carpio exposed to different concentrations of metallic mercury for different exposure periods. They also reported duration dependent increase in the frequencies of micronuclei induction followed by a slight stabilization and gradual decrease. Ayllon and Garcia-Vazquez (2000) have also observed significant increase in the frequency of micronuclei in fish, Poecilia latipinna, injected intraperitoneally with two different doses of mercury nitrate. In the present study, significant level (p < 0.05) of micronuclei frequency at each evaluation period was observed in fishes treated with sublethal concentration of arsenic. Both, duration and dose dependent increase in the induction of MNi in gill cells of zebra fish, Danio rerio has also been reported by Ramirez and García (2005) in response to arsenic. Vuyyuri et al. (2006) have also reported the genotoxic potential of arsenic by estimating the frequency of MNi in the buccal cells in workers occupationally exposed to this metal. Fish treated with sublethal concentration of Cu(II) also showed significant level (p < 0.05) of micronuclei frequencies after each exposure period and in between successive exposure periods. Our findings are in agreement with the earlier study carried out by Bagdonas and Vosyliene (2006) in rainbow trout, Oncorhynchus mykiss exposed to different concentrations of copper sulphate for 96 h of exposure period. Genotoxic potential of copper has also been reported by Talapatra and Banerjee (2007) in gill and kidney erythrocytes of fish, Labeo bata grown in sewage-fed fish farms having heavy metals like copper. In a study carried out without a positive control, Bhunya and Pati (1987) reported a significant dose-related increase in the incidence of micronuclei in the bone marrow cells of mice. Villela et al. (2006) have also reported the genotoxicity of copper sulphate in golden mussel, Limnoperna fortunei. They found that exposure of even 3  10 5 M concentration of CuSO4
5H2O for 24 and 48 h induced high frequencies of micronuclei and some toxicity after 48 h of exposure in haemolymph cells of golden mussel, L. fortunei. In the present investigation, frequencies of micronuclei were found increased up to 72 h in group III and up to 96 h in groups II and IV and then decreased gradually, while in group V recorded increased frequencies of micronuclei through out the entire exposure periods. Studies on the rate of micronuclei induction in various fish species showed that they generally peaked between the first and fifth days after treatment (Al-Sabti and Metcalfe, 1995; Grisolia and Cordeiro, 2000). Palhares and Grisolia (2002) also found that fishes Tilapia rendalli and Oreochromis niloticus exposed to mitomycin-C exposed for different exposure periods registered maximum frequencies of micronuclei between the third and the fifth post-inoculation days. Nepomuceno et al. (1997) suggested that the higher pollutant concentration might inhibit normal cell division, damage erythrocyte chromosome and interdict DNA duplication, thus micronuclei frequencies more or less declined. Then micronuclei frequencies trend to the smooth change and fish might promote some defensive mechanism to reduce some of metal residues in body, so as to stabilize the micronuclei frequencies relatively (Nepomuceno et al., 1997).

5. Conclusions From the study it can be concluded that aforesaid metal compounds are potential genotoxic agents and fish erythrocytes can be used for estimating the genotoxic effects of waterborne pollutants. Apart from this, fishes may serve as useful genetic models for the evaluation of pollution in aquatic ecosystems. They also

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offer several types of unique information not available from other methods: (1) early admonition of environmental damage along with hazards to the population, community, and ecosystem; (2) relationships between the individual responses of exposed organisms to pollution and the potential harm to human health based on the responses of wildlife to pollution; and (3) the development of effective measures for decontamination and remediation of freshwater bodies. Acknowledgment One of us, Kamlesh K. Yadav is thankful to CSIR, New Delhi for the award of SRF and financial support. We are grateful to Prof. Minakshi Srivastava, Head, Department of Zoology, University of Lucknow, Lucknow for providing laboratory facilities and constant encouragement for present research work. References Al-Sabti, K., 1986. Comparative micronucleated erythrocyte cell induction in three cyprinids by five carcinogenic–mutagenic chemicals. Cytobios 47, 47–54. Al-Sabti, K., 1995. An in vitro binucleated blocked hepatic cell technique for genotoxicity testing in fish. Mutat. Res. 335, 109–120. Al-Sabti, K., Metcalfe, C.D., 1995. Fish micronuclei for assessing genotoxicity in water. Mutat. Res. 343, 121–135. APHA, AWWA, WPCF, 1998. Standard methods for examination of water and wastewater, 20th ed. American Public Health Association, New York. Agency for Toxic Substances, Disease Registry (ATSDR), 1990. Toxicological profile for copper. Agency for Toxic Substances and Disease Registry, Atlanta, Georgia. Agency for Toxic Substances, Disease Registry (ATSDR), 1999. Toxicological profile for mercury: TP-93/10. Centers for Disease Control, Atlanta, Georgia. 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. Bagdonas, E., Vosyliene, M.Z., 2006. A study of toxicity and genotoxicity of copper, zinc and their mixture to rainbow trout (Oncorhynchus mykiss). Biologija 1, 8– 13. Bennett, R.O., Dooley, J.K., 1982. Copper uptake by two sympatric species of killifish Fundulus heteroclitus (L.) and F. majalis (Walbaum). J. Fish Biol. 21, 381–398. Bhunya, S.P., Pati, P.C., 1987. Genotoxicity of an inorganic pesticide, copper sulphate, in a mouse in vivo test system. Cytologia 52, 801–808. Blevins, R.D., 1985. Metal concentrations in muscle of fish from aquatic systems in East Tennessee, USA. Water Air Soil Pollut. 29, 361–371. Bogdanova, A.Y., Gassmann, M., Nikinmaa, M., 2002. Copper ion redox state is critical for its effects on ion transport pathways and met-haemoglobin formation in trout erythrocytes. Chem-Biol. Interact. 139, 43–59. Bohrer, D., Do Nascimento, P.C., Becker, E., Carvalho, L.M., Dessuy, M., 2005. Arsenic species in solutions for parenteral nutrition. J.P.E.N.J. Parenter Enteral. Nutr. 29, 1–7. Bolognesi, C., Rabboni, R., Roggiere, P., 1996. Genotoxicity biomarkers in Mytilus galloprovincialis as indicators of marine pollutant. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 113 (2), 319–323. Brugs, W.A., Cormick, J.H.M., Neiheisel, T.W., Spear, R.L., Stephan, C.E., Stokes, G., 1977. Effect of pollution on fresh water fish. J. Water Pollut. Contr. Fed. 49, 1425–1493. Carins, J., Dickson, K.L., Westlake, G.F., 1975. Biological monitoring of water and effluent quality. ASTM Publ. 607, Philadelphia. Chen, C.J., Hsu, L.I., Wang, C.H., Shih, W.L., Hsu, Y.H., Tseng, M.P., Lin, Y.C., Chou, W.L., Chen, C.Y., Lee, C.Y., Wang, L.H., Cheng, Y.C., Chen, C.L., Chen, S.Y., Wang, Y.H., Hsueh, Y.M., Chiou, H.Y., Wu, M.M., 2005. Biomarkers of exposure, effect, and susceptibility of arsenic-induced health hazards in Taiwan. Toxicol. Appl. Pharmacol. 206, 198–206. De Flora, S., Vigario, L., D’Agostini, F., Camoirano, A., Bagnasco, M., Bennecelli, C., Melodia, F., Arillo, A., 1993. Multiple biomarkers in fish exposed in situ to polluted river water. Mutat Res. 319, 167–177. Dopp, E., Hartmann, L.M., Florea, A.M., Von Recklinghausen, U., Pieper, R., Shokouhi, B., Rettenmeier, A.W., Hirner, A.V., Obe, G., 2004. Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells. Toxicol. Appl. Pharmacol. 201, 156–165. Espina, N.G., Weiss, P., 1995. DNA-repair in fish from polluted estuaries. Mar. Environ. Res. 39 (1–4), 309–312. Fahmy, M.A., 2000. Potential genotoxicity in copper sulfate treated mice. Cytologia 65, 235–242. Fenech, M., Chang, W.P., Kirsch-Volders, M., Holland, N., Bonassi, S., Zeiger, E., 2003. Human micronucleus project. HUMAN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutat. Res. 534 (1–2), 65–75.

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