Abstract
This study was set up to determine the suitability of the early life stage (ELS) alkaline comet assay for the detection of DNA strand breaks induced by genotoxicants in whole organism. This assay was performed on cells of medaka 2 days posthatch (dph). An efficient procedure for cell dissociation using enzymatic and mechanical digestion was developed. This protocol ensures 80% viability of cells and low DNA damage background. Cells from 2 dph medaka larvae were exposed in vitro to model genotoxicants, hydrogen peroxide, cadmium, and fluoranthene, followed by comet assay analysis. Results show a significant increase in the percentage of DNA damage of dissociated cells by all the tested compounds when compared to controls. The assay was also performed in vivo on medaka larvae (2 dph) exposed for 24 h to waterborne cadmium or fluoranthene. Significant induction of DNA damage levels were observed following larvae exposure to cadmium and fluoranthene at concentrations of 0.1 and 50 μM, respectively. This study demonstrates that cells of embryo life stage medaka respond to known DNA damaging agents and that the ELS comet assay may be a useful biomarker to detect DNA strand breakage in whole body of pluricellular organism induced by a range of agents. This technique may provide a sensitive, nonspecific endpoint of genotoxicity as part of ELS toxicity test.
Similar content being viewed by others
References
Sun LW, Qu MM, Li YQ, Wu YL, Chen YG, Kong ZM, Liu ZT (2004) Toxic effects of aminophenols on aquatic life using the zebrafish embryo test and the comet assay. Bull Environ Contam Toxicol 73:628–634
Kosmehl T, Hallare AV, Reifferscheid G, Manz W, Braunbeck T, Hollert H (2006) A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos. Environ Toxicol Chem 25:2097–2106
Lange M, Gebauer W, Markl J, Nagel R (1995) Comparison of testing acute toxicity on embryo of zebrafish, Brachydanio rerio and RTG-2 cytotoxicity as possible alternatives to acute fish test. Chemosphere 30:2087–2102
Lammer E, Carr GJ, Wendler JM, Rawlings JM, Belanger SE, Braunbeck T (2009) Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C 149:196–209
OECD (1992) OECD guidelines for the testing of chemicals. Section 2: effects on biotic systems test no. 210: fish, early-life stage toxicity test. Organisation for Economic Cooperation and Development, Paris
Nagel R (2002) DarT: the embryo test with the zebrafish danio rerio-a general model in ecotoxicology and toxicology. ALTEX 19:38–48
Braunbeck T, Bottcher T, Hollert H, Kosmehl T, Lammer E, Leist E, Rudolf M, Seitz N (2005) Towards an alternative for the acute fish LC50 test in chemical assessment: the fish embryo toxicity test goes multi-species—an update. ALTEX 22:87–102
McKim JM (1985) Early life stage toxicity tests. In: Rand GM, Petrocelli SR (eds) Fundamentals of aquatic toxicology: methods and applications. Hemisphere Publishing Corporation, New York
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191
Jarvis RB, Knowles JF (2003) DNA damage in zebrafish larvae induced by exposure to low-dose rate γ-radiation: detection by the alkaline comet assay. Mutat Res 541:63–69
Kammann U, Biselli S, Huhnerfuss H, Reineke N, Theobald N, Vobach M, Wosniok W (2004) Genotoxic and teratogenic potential of marine sediment extracts investigated with comet assay and zebrafish test. Environ Pollut 132:279–287
Kosmehl T, Krebs F, Braunbeck T, Hollert H (2007) Differentiation between bioavailable and total hazard potential of sediment-induced DNA fragmentation as measured by the comet assay with zebrafish embryos. J Soil Sediment 7:377–387
Kosmehl T, Hallare AV, Braunbeck T, Hollert H (2008) DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake (The Philippines) as measured by the comet assay. Mutat Res Genet Toxicol Environ Mutagen 650:1–14
Osman AGM, Mekkawy IA, Verreth J, Wuertz S, Kloas W, Kirschbaum F (2008) Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay. Environ Toxicol 23:679–687
Hyodo-Taguchi Y, Egami N (1989) Use of small fish in biomedical research, with special reference to inbred strains of medaka. In: Woodhead A (ed) Non mammalian animal models for biomedical research. CRC Press, Boca Raton
Iwamatsu T (1994) Stages of normal development in the Medaka (Oryzias latipes). Zool Sci 11:825–839
Mauthe RJ, Cook VM, Coffing SL, Baird WM (1995) Exposure of mammalian cell cultures to benzo(a)pyrene and light results in oxidative DNA damage as measured by 8-hydroxydeoxyguanosine formation. Carcinogenesis 16:133–137
Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice RR (2003) Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis 18:45–51
Olive PL, Banath JP (1995) Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent. Exp Cell Res 221:19–26
Devaux A, Pesonen M, Monod G (1997) Alkaline comet-assay in rainbow trout hepatocytes. Toxicol Vitro 11:71–79
Lee RF, Steinert S (2003) Use of single cell gel electrophoresis/comet assay for detection DNA damage in aquatic (marine and freshwater) animals. Mutat Res 544:43–64
Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Hartmann A et al (2007) Fourth International Workgroup on Genotoxicity testing: results of the in vivo comet assay workgroup. Mutat Res Genet Toxicol Environ Mutagen 627:31–35
Meintieres S, Nesslany F, Pallardy M, Marzin D (2003) Detection of ghost cells in the standard alkaline comet assay is not a good measure of apoptosis. Environ Mol Mutat 41:260–269
Singh NP (2000) A simple method for accurate estimation of apoptotic cells. Exp Cell Res 256:328–337
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutat 35:206–221
Akcha F, Vincent Hubert F, Pfhol-Leszkowicz A (2003) Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds. Mutat Res 534:21–32
De Miranda Cabral Gontijo AM, Barreto RE, Speit G, Valenzuela Reyes VA, Volpato GL, Favero Salvadori DM (2003) Anesthesia of fish with benzocaine does not interfere with comet assay results. Mutat Res Genet Toxicol Environ Mutagen 534:165–172
Babson JR, Russo-Rodriguez SE, Wattley RV, Bergstein PL, Rastetter WH, Liber HL, Andon BM, Thilly WG, Wogan GN (1986) Microsomal activation of fluoranthene to mutagenic metabolites. Toxicol Appl Pharmacol 85:355–366
Willett KL, Wassenberg D, Lienesch L, Reichert W, Di Giulio RT (2001) In vivo and in vitro inhibition of CYP1A-dependent activity in Fundulus heteroclitus by the polynuclear aromatic hydrocarbon fluoranthene. Toxicol Appl Pharmacol 177:264–271
Mitchelmore CI, Chipman JK (1998) Detection of DNA strand breaks in brouwn trout (Salmo trutta) hepatocytes and blood cells using the single cell gel electrophoresis (comet) assay. Aquat Toxicol 41:161–182
Hook SE, Lee RF (2004) Genotoxicant induced DNA damage and repair in early and late developmental stages of the gras shrimp Paleomonetes pugio embryo as measured by the comet assay. Aquat Toxicol 66:1–14
IARC (1997) Supplement: cadmium and cadmium compounds. IARC, Lyon
Cambier S, Gonzalez P, Durrieu G, Bourdineaud JP (2010) Cadmium induced genotoxicity in zebrafish at environmentally relevant doses. Ecotoxicol Environ Saf 73:312–319
Wang Y, Fang J, Leonard SS, Rao KM (2004) Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Rad Biol Med 36:1434–1443
Liu J, Qu W, Kadiiska MB (2009) Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol Appl Pharmacol 238:209–214
Risso-de Faverney C, Devaux A, Lafaurie M, Girard JP, Bailly B, Rahmani R (2001) Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygen species. Aquat Toxicol 53:65–76
Hartwing A (1994) Role of DNA repair inhibition in lead- and cadmium-induced genotoxicity: a review. Environ Health Perspect 102:45–50
Schwerdtle T, Ebert F, Thuy C, Mullenders L, Hartwing A (2010) Genotoxicity of soluble and particulate cadmium compounds: Impacts on oxidative DNA damage and nucleotide excision repair. Chem Res Toxicol 23:432–442
Mouchet F, Gauthier L, Baudrimont M, Gonzalez P, Mailhes C, Ferrier V, Devaux A (2007) Comparative evaluation of the toxicity and genotoxicity of cadmium in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test. Environ Toxicol 22:422–435
Jia X, Zhan H, Liu X (2010) Low levels of cadmium exposure induce DNA damage and oxidative stress in the liver of Oujiang colored common carp Cyprinus carpio var. color. Fish Physiol Biochem (in press)
Lacaze E, Geffard O, Bony B, Devaux A (2010) Genotoxicity assessement in the amphipod Gammarus fossarum by use of the alkaline Comet assay. Mutat Res 700:32–38
Hornung MW, Cook PM, Fitzsimmons PN, Kuehl DW, Nichols JW (2007) Tissue distribution and metabolism of benzo[a]pyrene in embryonic and larval medaka (Oryzias latipes). Toxicol Sci 100:393–405
Palmqvist A, Selck H, Rasmussen LJ, Forbes V (2003) Biotransformation and genotoxicity of fluoranthene in the deposit-feeding polychaete capitella sp.I. Environ Toxicol Chem 22:2977–2985
Sepic E, Bricelj M, Leskovsek H (2003) Toxicity of fluoranthene and its biodegradation metabolites to aquatic organism. Chemosphere 52:1125–1133
Hornung MW, Cook PM, Flynn KM, Lothenbach DB, Johnson RD, Nichols JW (2004) Use of multi-photon laser-scanning microscopy to describe the distribution of xenobiotic chemicals in fish early life stage. Aquat Toxicol 67:1–11
Woo S, Kim S, Yum S, Yim UH, Lee TK (2006) Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons. Mar Pol Bul 52:1768–1775
Billiard SM, Meyer JN, Wassenberg DM, Hodson PV, Di Giulio RT (2008) Nonadditive effects of PAHs on early vertebrate development: mechanisms and implications for risk assessment. Toxicol Sci 105:5–23
Acknowledgments
This study was supported by the GIP Seine-Aval, Haute-Normandie, and Aquitaine regions.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Morin, B., Filatreau, J., Vicquelin, L. et al. Detection of DNA damage in yolk-sac larvae of the Japanese Medaka, Oryzias latipes, by the comet assay. Anal Bioanal Chem 399, 2235–2242 (2011). https://doi.org/10.1007/s00216-010-4602-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-010-4602-y