S.: Aneugenic potential of okadaic acid revealed by the micronucleus assay combined with the FISH technique in CHO-K1 cells

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 cyto-toxicity 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 meta-bolites of OA were generated with a significant increase in genotoxic potential. The relationship between the different components involved in the mitotic process and OA inhibi-tion of protein phosphatase is also discussed