Mutagenesis, Vol. 18, No. 3, 293-298,
May 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Aneugenic potential of okadaic acid revealed by the micronucleus assay combined with the FISH technique in CHO-K1 cells
1 AFSSA, Laboratoire d’Etudes et de Recherches sur les Médicaments Vétérinaires et les Désinfectants, Unité de Toxicologie Alimentaire, BP 90203, F-35302 Fougères, France, 2 Bureau de la Recherche et des Laboratoires d’Analyses, Direction Générale de l’Alimentation, Ministère de l’Agriculture, de l’Alimentation, de la Pêche et des Affaires Rurales, 251 Rue de Vaugirard, F-75732 Paris Cedex 15, France and 3 AFSSA, Laboratoire d’Etudes et de Recherches sur l’Hygiène et la Qualité des Aliments, Unité Toxines Microbiennes, 10 Rue Pierre Curie, F-94704 Maisons Alfort Cedex, France
Okadaic acid (OA) is a major toxin involved in diarrhetic shellfish poisoning in humans and has been shown to be both a potent tumor promoter in rodent skin and stomach and an inhibitor of serine/threonine protein phosphatases, specifically PP1 and PP2A. The research on the genotoxic potential of OA amounts to only a few studies, which give conflicting results. In order to evaluate the ability of OA to induce DNA damage, the cytokinesis-block micronucleus assay was performed in the CHO-K1 cell line. A statistically significant induction of micronuclei without strong cytotoxicity was obtained after a 24 h treatment with 20 (~5-fold) and 30 nM (~10-fold) OA. Then, in order to discriminate between a clastogenic or aneugenic effect of OA, the micronucleus assay was carried out in combination with fluorescence in situ hybridization (FISH) using a (TTAGGG)n DNA probe for centromere detection. FISH analysis showed that OA mainly induced centromere-positive micronuclei (68.9% induction with 20 nM OA and 77.0% with 30 nM). Therefore, OA can be considered aneugenic. Using the same assay, biotransformation of OA was studied after a 4 h treatment with and without metabolic activation. The results show that reactive metabolites of OA were generated with a significant increase in genotoxic potential. The relationship between the different components involved in the mitotic process and OA inhibition of protein phosphatase is also discussed.
4 To whom correspondence should be addressed. Tel: +33 2 99 94 78 78; Fax: +33 2 99 94 78 80; Email: v.fessard{at}fougeres.afssa.fr