Mutagenesis vol. 19 no. 2 pp. 85-90,
March 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Evaluation of in vivo genotoxicity of cypermethrin in Drosophila melanogaster using the alkaline Comet assay
1Embryotoxicology Section and 2Developmental Toxicology Section, Industrial Toxicology Research Centre, PO Box 80, M.G. Marg, Lucknow, 226 001 Uttar Pradesh, India
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The single cell gel electrophoresis (SCGE) assay, also known as the Comet assay, is one of the most promising genotoxicity tests developed in recent years to measure and analyse DNA damage in single cells. The present study was undertaken to assess the in vivo genotoxicity of the synthetic pyrethroid cypermethrin in brain ganglia and anterior mid gut of Drosophila melanogaster. Freshly emerged first instar larvae (22 ± 2 h) were placed in different concentrations of cypermethrin (0.0004, 0.0008, 0.002, 0.2 and 0.5 p.p.m.) mixed in standard Drosophila food and allowed to grow. At 96 ± 2 h, brain ganglia and anterior midgut from control and treated larvae were dissected out, single cell suspensions were prepared and a Comet assay was performed. Our results revealed a significant dose-dependent increase in DNA damage in the cells of brain ganglia and anterior midgut of D.melanogaster exposed to cypermethrin as compared with controls (P < 0.05 at 0.002 p.p.m.; P < 0.001 at 0.2 and 0.5 p.p.m.). The present study shows in vivo genotoxicity of cypermethrin even at very low concentrations, which proves D.melanogaster as a model for in vivo genotoxicity assessment using the Comet assay.
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The single cell gel electrophoresis (SCGE or Comet assay) is one of the most promising and upcoming genotoxicity tests. It is less resource intensive than the conventional genotoxic techniques and permits both qualitative and quantitative assessment of DNA damage in any eukaryotic cell population. The simplicity and sensitivity of the Comet assay has resulted in a rapid and widespread progression of this technique in many areas, e.g. environmental monitoring (Cavallo et al., 2002
; Rajaguru et al., 2002), in vivo and in vitro genotoxicity testing (Anderson et al., 1996, 2001; Dhawan et al., 2002) and epidemiological and biomonitoring studies in human populations exposed occupationally, environmentally or clinically (Bajpayee et al., 2002; Marczynski et al., 2002; Mohankumar et al., 2002). This test procedure has been recommended in the Committee on Mutagenicity Guidelines of the UK Department of Health (COM) for determining mutagenicity of chemicals (Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment, 2000) as an in vitro genotoxicity assay using tissues other than haematopoietic tissue.
One of the advantages of the Comet assay for in vivo genetic toxicology studies is the ability to use any tissue (or different tissues) as the target for evaluation of chemical genotoxicity and some physiological conditions (Sasaki et al., 2000). Investigators have used the assay to monitor age-dependent DNA damage in hepatocytes of rats (Martelli et al., 2002) and the induction and persistence of DNA damage in somatic (Anderson et al., 1997) and germ cells (Anderson et al., 1997; Olsen et al., 2001) of chemically treated rodents. The Comet assay has now become an acceptable tool for environmental biomonitoring and has been successfully applied in assessing soil pollution using earthworms (Salagovic et al., 1996), in freshwater planaria (Guecheva et al., 2001), in various organs of medaka (Oryzias latipes) for evaluation of levels of DNA damage (Tice, 1995), in zebra mussels (Pavlica et al., 2001), in Mytilus (Pruski and Dixon, 2002), in freshwater Hydra (Devaux and Larno, 1999) for aquatic toxicology and also in yeast cells (Miloshev et al., 2002).
Over the past decade, issues of animal use and care in toxicology research and testing have become one of the fundamental concerns for both science and ethics. Emphasis has been given to the use of alternatives to mammals in testing, research and education. Drosophila melanogaster is the most widely used insect model because of its well-elucidated genetics and developmental biology. Moreover, the use of Drosophila has been recommended by the European Centre for the Validation of Alternative Methods (ECVAM), whose goal is to promote the scientific and regulatory acceptance of alternative methods which reduce, refine or replace the use of laboratory animals (Festing et al., 1998; Benford et al., 2000). In recent years, Drosophila has evolved into a model organism in toxicological studies (Gaivao et al., 1999; Kar Chowdhuri et al., 1999, 2001; Nazir et al., 2001, 2003a,b,c; Mukhopadhyay et al., 2002a,b, 2003). The present study was therefore undertaken to evaluate the usefulness of D.melanogaster as an in vivo model for assessment of genotoxicity of a synthetic pyrethroid insecticide, cypermethrin, using the alkaline Comet assay.
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Materials and methods |
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Fly strain
The fly and larvae of wild-type D.melanogaster (Oregon R+) were cultured at 24 ± 1°C on standard Drosophila food containing agar, corn meal, brown sugar and yeast.
Chemicals
Agarose, low melting point agarose, ethidium bromide and collagenase were obtained from Sigma Chemical Co. (St Louis, MO). Phosphate-buffered saline (PBS) Ca2+- and Mg2+-free, Trypan blue and ethyl methanesulphonate (EMS) were procured from Hi-Media Pvt. Ltd (Mumbai, India). Technical grade cypermethrin (purity 98.5%) was a kind gift from Aimco Pesticides Ltd (Mumbai, India). All other chemicals were obtained locally and were of analytical reagent grade.
Pesticide
Cypermethrin dissolved in dimethyl sulphoxide (DMSO) (0.3% final concentration) was added to standard Drosophila food in five different concentrations: 0.5 (1/100 of recommended agricultural dose), 0.2 [maximum residue level (MRL) of cypermethrin in fruits; Chaddha, 1992], 0.002 (1/100 of MRL value), 0.0008 (1/250 of MRL value) and 0.0004 (1/500 of MRL value) p.p.m.
Experimental design
Freshly emerged first instar larvae (22 ± 2 h) were transferred to standard Drosophila food containing different concentrations of cypermethrin and were allowed to grow on it. Control larvae were grown on standard Drosophila food without cypermethrin. At 96 ± 2 h, the larvae were removed from the food and washed with 50 mM sodium phosphate buffer. Brain ganglia and the anterior region of the midgut from 50 larvae were explanted in Poels’ salt solution (PSS) (Lakhotia and Mukherjee, 1980) and collected separately in 1.5 ml microcentrifuge tube. A single cell suspension of the tissues was then prepared by the method of Howell and Taylor (1968) with some modifications. PSS in the microcentrifuge tube was replaced with collagenase (0.5 mg/ml in PBS, pH 7.4) and kept for 15 min at 24°C. The cells were then passed through nylon mesh (60 µm). Collagenase was removed by washing the cell suspension three times with PBS. The cells were finally suspended in 80 µl of PBS.
Positive control
EMS, a well-known mutagen (Bilbao et al., 2002), was used as a positive control in the present study. Single cell suspensions were prepared as described above from brain ganglia and anterior midgut tissues of the larvae (96 ± 2 h stage) grown on normal food. After washing three times in PBS, the cells were suspended in 300 µl of PBS containing 5 mM EMS and incubated for 1 h at 24°C. The cells were then washed three times in PBS and processed for the Comet assay.
Viability assay
The cells were checked for viability before the start of the experiment using Trypan blue dye (Pool-Zobel et al., 1993).
Comet assay
Slides were prepared in duplicate according to the method of Bajpayee et al. (2002) with some modifications and were finally immersed in freshly prepared chilled lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris pH 10.0 and 1% Triton X-100, pH 10) for 2 h. After lysis, the slides were placed in a horizontal gel electrophoresis tank (Life Technologies, Gaithersburg, MD) filled with fresh, chilled electrophoresis solution (1 mM Na2EDTA and 300 mM NaOH, pH > 13). The slides were left in this solution for 10 min to allow DNA unwinding. Electrophoresis was conducted for 15 min at 0.7 V/cm and 300 mA at 4°C using a power supply from Techno Source Pvt. Ltd (Mumbai, India). All the steps from slide preparation onwards were performed under dimmed light to avoid additional DNA damage. Following electrophoresis, Tris buffer (0.4 M Tris pH 7.5) was added dropwise to neutralize excess alkali and this was repeated three times. Slides were then stained with ethidium bromide (20 µg/ml, 75 µl/slide) for 10 min in the dark. They were dipped once in chilled distilled water to remove excess ethidium bromide and subsequently coverslips were placed over them. The slides were stored in a dark, humidified chamber and analysed within 3–4 h.
Slides were analysed using an image analysis system (Kinetic Imaging, Liverpool, UK) attached to a fluorescent microscope (Leica, Germany). The images were captured by CCD camera and transferred to a computer and analysed using Komet 5.0 software. The parameters taken were tail DNA (%), tail length (estimated leading edge from the nucleus; µm) and tail moment (arbitrary units) (Olive et al., 1990, 1992). The tail moment is defined as the distance between the centre of mass of the tail and the centre of mass of the head, in micrometres, multiplied by the percentage of DNA in the tail. This number was then compared with the total DNA content. Images from 50 cells (25 from each slide) were analysed.
Statistical analysis
The values were compared using Student’s t-test. Prior to analysis, homogeneity of variance and normality assumptions concerning the data were tested. Significance was ascribed at P < 0.05. Data for the tail moment were statistically analysed and are presented in the figure as box-and-whisker plots, which represent the range (minimum and maximum), medians and 75th percentile.
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Results |
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Cell viability measured at the time of the experiment always exceeded 95% in all the treatment groups. The alterations incorporated in the present study from the conventional Comet assay are shown in Table I.
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Drosophila larvae exposed to EMS and to different concentrations of cypermethrin showed a significant increase in DNA damage in the cells of brain ganglia and anterior mid gut (Tables II and III).
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In brain ganglia, a dose-dependent increase was observed in all the comet parameters, i.e. tail moment (TM; arbitrary unit), tail length (µm) and tail DNA (%). As is evident from Table II, a statistically significant increase in all parameters was observed in these cells at the three higher concentrations of cypermethrin (P < 0.001 at 0.002, 0.2 and 0.5 p.p.m.). TM data are also presented as box-and- whisker plots (Figure 1A) and distribution of cells (%) (Figure 2B). Of the total cells observed, 64% were in the category <2.0 TM at 0.0004 p.p.m. cypermethrin concentration. However, in the highest treatment group (0.5 p.p.m.) only 4% cells were in the category <2.0 TM, while 32% were in the category <6.0 TM, reflecting the severity of DNA damage in this group.
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A similar dose-dependent increase in DNA damage was also observed in cells of the anterior midgut of larvae exposed to cypermethrin (Table III and Figures 1B and 2B).
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Discussion |
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Issues relating to the experimental procedure, data analysis and presentation of the results have been discussed (Tice et al., 2000
). However, the Comet assay technique still requires some modification and standardization under different experimental conditions and/or using different experimental materials.
In the present study, the Comet assay was applied to evaluate the in vivo genotoxic potential of cypermethrin in D.melanogaster. Due to the relatively small cell size, low melting point agarose was used at 1.5% (0.75% final concentration), in contrast to the 1% (0.5% final concentration) generally used and recommended (Tice et al., 2000; Bajpayee et al., 2002; Bilbao et al., 2002; Dhawan et al., 2002). A major modification made in the present study in the composition of the lysing solution as compared with that of Bilbao et al. (2002) for Drosophila was removing DMSO, which at 10% is usually added to scavenge radicals generated by the iron released from haemoglobin (Singh et al., 1988). No such heme groups are present in Drosophila. In the present study, no scorable cells could be detected when slides were placed in lysing solution containing DMSO as used conventionally. Our earlier study had shown that a dietary concentration of over 0.3% DMSO was cytotoxic to D.melanogaster (Nazir et al., 2003a). Although Bilbao et al. (2002) in their study used 2 h lysis and 20 min unwinding and electrophoresis of neuroblast cells of Drosophila, we optimized the experimental conditions and reduced the times of unwinding and electrophoresis to 10 and 15 min, respectively, resulting in an improvement in performance of the assay.
Cypermethrin, a pyrethroid insecticide, is a neurotoxicant, affecting the central nervous system (Smith and Soderlund, 1998), and has been shown to increase the cGMP content in mammalian brain (Abassy et al., 1983; Ruight, 1985). The anterior region of the midgut is one of the first tissues to come into contact with the chemical when the organism takes in the contaminated food. Cypermethrin has been reported to be genotoxic in mouse spleen and bone marrow (Amer et al., 1993). In the present study, DNA damage was observed in brain ganglia cells and the anterior midgut of Drosophila. Earlier, we reported the vulnerability of these tissues to cypermethrin as reflected by rapid heat shock protein 70 (hsp70) induction, an indicator of cytotoxicity (Mukhopadhyay et al., 2002b).
The present study shows the usefulness of the modified method for the Comet assay for the evaluation of in vivo genotoxicity in D.melanogaster. However, further validation of the modified method with further genotoxicants will be needed.
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Acknowledgements |
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The authors wish to thank Prof. P.K.Seth (Director, ITRC) for his keen interest throughout the study. I.M. was supported by a Council of Scientific and Industrial Research Senior Research Fellowship (grant no. 31/29/129/2002 EMR-1). M.B. was supported by a Council of Scientific and Industrial Research Junior Research Fellowship (grant no. 31/29/123/2001 EMR-1). Financial assistance from the Department of Biotechnology, Government of India, New Delhi (grant no. BT/PRO 390/R&D/12/030/96) to D.K.C. and Council of Scientific and Industrial Research, New Delhi (HRD/YS-99/PROJECT/2001) to A.D. is gratefully acknowledged. ITRC Communication no. 2248.
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3To whom correspondence should be addressed. Tel: +91 522 2213618; Fax: +91 522 2228227; Email: dhawanalok{at}hotmail.com
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-
Abassy,M.A., Eldefrawi,M.E. and Eldefrawi,A.T. (1983) Pyrethroid action on nicotinic acetylcholine receptor/channel. Pestic. Biochem. Physiol., 19, 299–308.[CrossRef]
Amer,S.M., Ibrahim,A.A. and el-Sherbeny,K.M. (1993) Induction of chromosomal aberrations and sister chromatid exchange in vivo and in vitro by insecticide cypermethrin. J. Appl. Toxicol., 13, 341–345.[Medline]
Anderson,D., Dhawan,A., Yu,T.W. and Plewa,M.J. (1996) Investigation of bone marrow and testicular cells in vivo using the comet assay. Mutat. Res., 370, 159–174.[CrossRef][ISI][Medline]
Anderson,D., Dobrzynka,M.M., Jackson,L.I., Yu,T.W. and Brinkworth,M.H. (1997) Somatic and germ cell effects in rats and mice after treatment with 1,3-butadiene and its metabolites, 1,2-epoxybutane and 1,2,3,4-diepoxybutane. Mutat. Res., 391, 233–242.[Medline]
Anderson,D., Dhawan,A., Yardley-Jones,A., Ioannides,C. and Webb,J. (2001) Effect of antioxidant flavonoids and a food mutagen on lymphocytes of a thalassemia patient without chelation therapy in the comet assay. Teratog. Carcinog. Mutagen., 21, 165–174.[Medline]
Bajpayee,M., Dhawan,A., Parmar,D., Pandey,A.K., Mathur,N. and Seth,P.K. (2002) Gender related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay. Mutat. Res., 520, 83–91.[ISI][Medline]
Benford,D.J., Hanley,B.A., Bottrill,K. et al. (2000) Biomarkers as predictive tools in toxicity testing. Altern. Lab. Anim., 28, 119–131.
Bilbao,C., Ferreiro,J.A., Comendador,M.A. and Sierra,L.M. (2002) Influence of mus201 and mus308 mutations of Drosophila melanogaster on the genotoxicity of model chemicals in somatic cells in vivo measured with the comet assay. Mutat. Res., 503, 11–19.[ISI][Medline]
Cavallo,D., Tomao,P., Marinaccio,A., Perniconi,B., Setini,A., Palmi,S. and Iavicoli,S. (2002) Evaluation of DNA damage in flight personnel by comet assay. Mutat. Res., 516, 148–152.[ISI][Medline]
Chaddha,D.S. (1992) Cypermethrin. In Chaddha,D.S. (ed.), Prevention of Food Adulteration Act and Rules, 1952 Amended in 1992. Associated Chamber of Commerce and Industry of India, New Delhi, pp. 114–127.
11. Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (2000) Guidance on a Strategy for Testing of Chemicals for Mutagenicity. Department of Health, London, p. 21.
Devaux,A. and Larno,V. (1999) Genotoxicity assessment in the freshwater hydra using the comet assay. Comet Assay Workshop, Slovakia, May 29–June 2.
Dhawan,A., Anderson,D., de Pascual-Teresa,S., Santos-Buelga,C., Clifford,M.N. and Ioannides,C. (2002) Evaluation of the antigenotoxic potential of monomeric and dimeric flavanols and black tea polyphenols against heterocyclic amine-induced DNA damage in human lymphocytes using the comet assay. Mutat. Res., 515, 39–56.[ISI][Medline]
Festing,M.F.W., Baumans,V., Combes,D.R., Halder,M., Hendriksen,F.M., Howard,B.R., Lovell,D.P., Moore,G.J., Overend,P. and Wilson,M.S. (1998) Reducing the use of laboratory animals in biomedical research: problems and possible solutions. Altern. Lab. Anim., 26, 283–301.
Gaivao,I., Sierra,L.M. and Comendador,M.A. (1999) The w/w+ SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds. Mutat. Res., 440, 139–145.[ISI][Medline]
Guecheva,T., Henriques,J.A. and Erdtmann,B. (2001) Genotoxic effect of freshwater planarian in vivo studied with the single cell gel test (Comet assay). Mutat. Res., 497, 19–27.[Medline]
Howell,S.L. and Taylor,K.W. (1968) Potassium ions and the secretion of insulin by islets of langerhans incubated in vitro. Biochem. J., 108, 17–24.[ISI][Medline]
Kar Chowdhuri,D., Saxena,D.K. and Viswanathan,P.N. (1999) Effect of hexachlorocyclohexane (HCH), its isomers and metabolites on Hsp70 expression in transgenic Drosophila melanogaster. Pestic. Biochem. Physiol., 63, 15–25.
Kar Chowdhuri,D., Nazir,A. and Saxena,D.K. (2001) Effect of three chlorinated pesticides on hsr
stress gene in transgenic Drosophila melanogaster. J. Biochem. Mol. Toxicol., 15, 173–186.[CrossRef][ISI][Medline]
Lakhotia,S.C. and Mukherjee,T. (1980) Specific activation of puff 93D of Drosophila melanogaster by benzamide and the effect of benzamide on the heat shock induced puffing activity. Chromosoma, 81, 125–136.[CrossRef][ISI][Medline]
Marczynski,B., Rihs,H.P., Rossbach,B., Holzer,J., Angerer,J., Scherenberg,M., Hoffmann,G., Burning,T. and Wilhelm,M. (2002) Analysis of 8-oxo-7, 8-dihydro-2' deoxyguanosine and DNA strand breaks in white blood cells of occupationally exposed workers: comparison with ambient monitoring, urinary metabolites and enzyme polymorphisms. Carcinogenesis, 23, 273–281.
Martelli,A., Carrozzino,R., Mattioli,F., Bucci,G., Lamarino,G. and Brambilla,G. (2002) DNA damage in tissues of rat treated with potassium canrenoate. Toxicology, 171, 95–103.[CrossRef][ISI][Medline]
Miloshev,G., Mihaylov,I. and Anachkove,B. (2002) Application of the single cell gel electrophoresis on yeast cells. Mutat. Res., 513, 69–74.[Medline]
Mohankumar,M.N., Janani,S., Prabhu,B.K., Vivek Kumar,P.R. and Jeevanram,R.K. (2002) DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay. Mutat. Res., 520, 179–187.[ISI][Medline]
Mukhopadhyay,I., Nazir,A., Mahmood,K., Saxena,D.K., Das,M., Khanna,S.K. and Kar Chowdhuri,D. (2002a) Toxicity of argemone oil: effect on hsp70 expression and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. Cell Biol. Toxicol., 18, 1–11.[Medline]
Mukhopadhyay,I., Nazir,A., Saxena,D.K. and Kar Chowdhuri,D. (2002b) Toxicity of cypermethrin: hsp70 as a biomarker of exposure in transgenic Drosophila. Biomarkers, 7, 501–510.[CrossRef][ISI][Medline]
Mukhopadhyay,I., Saxena,D.K., Bajpai,V.K. and Kar Chowdhuri,D. (2003) Argemone oil induced cellular damage in the reproductive tissues of transgenic Drosophila melanogaster: protective role of 70 kDa heat shock protein. J. Biochem. Mol. Toxicol., 17, 223–233.[CrossRef][ISI][Medline]
Nazir,A., Mukhopadhyay,I., Saxena,D.K. and Kar Chowdhuri,D. (2001) Chlorpyrifos induced hsp70 expression and effect on reproductive performance in transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. Arch. Environ. Contam. Toxicol., 41, 443–449.[CrossRef][ISI][Medline]
Nazir,A., Mukhopadhyay,I., Saxena,D.K. and Kar Chowdhuri,D. (2003a) Evaluation of No Observed Adverse Effect Level (NOAEL) of solvent dimethyl sulphoxide in Drosophila melanogaster: a developmental, reproductive and cytotoxicity study. Toxicol. Mech. Methods, 13, 147–152.[CrossRef]
Nazir,A., Mukhopadhyay,I., Saxena,D.K. and Kar Chowdhuri,D. (2003b) Evaluation of toxic potential of captan: Induction of hsp70 and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9. J. Biochem. Mol. Toxicol., 17, 98–107.[Medline]
Nazir,A., Saxena,D.K. and Kar Chowdhuri,D. (2003c) Induction of hsp70 in transgenic Drosophila: biomarker of exposure against phthalimide group of chemicals. Biochim. Biophys. Acta, 1621, 218–225.[Medline]
Olive,P.L., Banath,J.P. and Durand,R.E. (1990) Heterogeneity in variation induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat. Res., 122, 86–94.[ISI][Medline]
Olive,P.L., Wlodek,D., Durand,R.E. and Banath,J.P. (1992) Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp. Cell Res., 198, 259–260.[CrossRef][ISI][Medline]
Olsen,A.K., Bjortuft,H., Wiger,R., Holme,J., Seeberg,E., Bjoras,M. and Brunborg,G. (2001) Highly efficient base excision repair (BER) in human and rat male germ cells. Nucleic Acids Res., 29, 1781–1790.
Pavlica,M., Klobucar,G.I., Mojas,N., Erben,R. and Papes,D. (2001) Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutat. Res., 490, 209–214.[ISI][Medline]
Pool-Zobel,B.L., Guigas,C., Klein,R.G., Neudecker,C.H., Renner,H.W. and Schmezer,P. (1993) Assessment of genotoxic effects by lindane. Food Chem. Toxicol., 31, 271–283.[CrossRef][ISI][Medline]
Pruski,A.M. and Dixon,D.R. (2002) Effects of cadmium on nuclear integrity and DNA repair efficiency in the gill cells of Mytilus edulis L. Aquat. Toxicol., 57, 127–137.[CrossRef][ISI][Medline]
Rajaguru,P., Vidya,L., Baskarasethupathi,B., Kumar,P.A., Palanivel,M. and Kalaiselvi,K. (2002) Genotoxicity evaluation of polluted groundwater in human peripheral blood lymphocytes using the comet assay. Mutat. Res., 517, 29–37.[Medline]
Ruight,G.S.F. (1985) Pyrethroids. In Kerkut,G.A. and Gilbert,L.I. (eds), Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon Press, New York, NY, Vol. 12, pp. 183–262.
Salagovic,J., Gilles,J., Verschaeve,L. and Kalina,I. (1996) The comet assay for the detection of genotoxic damage in the earthworms: a promising tool for assessing the biological hazards of polluted sites. Folia Biol. (Praha), 42, 17–24.
Sasaki,Y.F., Sekihashi,K., Izumiyama,F., Nishidate,E., Saga,A., Ishida,K. and Tsuda,S. (2000) The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from IARC monographs and U.S. NTP carcinogenicity database. Crit. Rev. Toxicol., 30, 629–799.[ISI][Medline]
Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184–191.[CrossRef][ISI][Medline]
Smith,J.J. and Soderlund,D.M. (1998) Action of the pyrethroid insecticide cypermethrin on rat brain IIa sodium channel expressed in Xenopus oocytes. Neurotoxicology, 19, 823–832.[Medline]
Tice,R.R. (1995) The single cell gel/comet assay: a microgel electrophoretic technique for the detection of DNA damage and repair in individual cells. In Phillips,D.H. and Venitt,S. (eds), Environmental Mutagenesis. Bios Scientific Publishers, Oxford, pp. 315–339.
Tice,R., Agurell,E., Anderson,D., Burlinson,B., Hartmann,A., Kobayashi,H., Miyamae,Y., Rojas,E., Ryu,J.C. and Sasaki,Y.F. (2000) Single cell gel/Comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen., 35, 206–221.[CrossRef][ISI][Medline]
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