DNA breaks and cell cycle arrest induced by okadaic acid in Caco-2 cells, a human colonic epithelial cell line

Okadaic acid (OA) is a shellfish toxin produced by dinoflagellates, in muss els. It is a potent tumour promoter and represents a potential threat to hu man health even at low concentrations. OA targets mainly the gastrointestin al tract ill acute poisoning, causing diarrhoea. Therefore the present inve stigations were designed to study the ability of okadaic acid to induce cyt otoxicity and DNA lesions in a human colonic cell line (Caco-2). Incubation of Caco-2 cells with OA (3.75-60 ng/ml, i.e 4.6x10(-3)-7.5x10(-2) muM) cau ses a significant reduction in cell viability. Moreover, okadaic acid inhib its protein and DNA synthesis with, respectively, IC50 of 16 and 6.5 ng/ml after 24 h incubation. It also provokes cell cycle arrest, characterised by an increase in the number of S phase cells, correlated with a significant decrease in G0/G1 phase cells at high concentration. One of the main result s obtained in these investigations is the apoptosis induced by OA in Caco-2 cells of intestinal origin, shown by DNA laddering in agarose gel electrop horesis (250-1000 base pairs), OA also induces clastogenic effects evaluate d by DNA fragmentation analysis using the method of Higuchi and Aggarwal (5 2% for 60 ng/ml) and comet assay (increase of the frequency of comets and t heir tails length). Therefore, the cell death induced by OA seems clearly t o be concentration-dependent after 24 h of incubation. The cytotoxic proper ties of okadaic acid and its ability to damage DNA result in cell death, ma inly by apoptosis. Since consumption of shellfish contaminated with accepta ble okadaic acid concentrations exposes colonic cells to harmful concentrat ions of this toxin, the possibility that OA would display its toxic effects on intestinal cells in vivo should be evaluated in human primary intestina l cells and human intestinal slices for cytotoxic effects, DNA fragmentatio n and apoptosis.