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

Abstract

Okadaic acid (OA) is a shellfish toxin produced by dinoflagellates, in mussels. It is a potent tumour promoter and represents a potential threat to human health even at low concentrations. OA targets mainly the gastrointestinal tract in acute poisoning, causing diarrhoea. Therefore the present investigations were designed to study the ability of okadaic acid to induce cytotoxicity 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. 4.6 x 10(-3)-7.5 x 10(-2) microM) causes a significant reduction in cell viability. Moreover, okadaic acid inhibits 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 results obtained in these investigations is the apoptosis induced by OA in Caco-2 cells of intestinal origin, shown by DNA laddering in agarose gel electrophoresis (250-1000 base pairs). OA also induces clastogenic effects evaluated by DNA fragmentation analysis using the method of Higuchi and Aggarwal (52% for 60 ng/ml) and comet assay (increase of the frequency of comets and their tails length). Therefore, the cell death induced by OA seems clearly to be concentration-dependent after 24 h of incubation. The cytotoxic properties of okadaic acid and its ability to damage DNA result in cell death, mainly by apoptosis. Since consumption of shellfish contaminated with acceptable okadaic acid concentrations exposes colonic cells to harmful concentrations of this toxin, the possibility that OA would display its toxic effects on intestinal cells in vivo should be evaluated in human primary intestinal cells and human intestinal slices for cytotoxic effects, DNA fragmentation and apoptosis.

Combined cytotoxicity and genotoxicity of a marine toxin and seafood contaminant metal ions (chromium and cadmium)

“…OA has also been reported to induce DNA fragmentation (Traoré et al, 2001;Huynh-Delerme et al, 2003;Parameswaran et al, 2004;Pinto-Silva et al, 2005). This is partly confirmed by the present data.…”

“…This time instead of simple DNA fragmentation, apoptosis has been elicited, suggesting for these additive effects at least three mechanisms: (i) genotoxicity of OA is exaggerated and generates too much DNA lesions, leading to cell cycle arrest via a p53-independent mechanism (Traoré et al, 2001;Le Hegarat et al, 2004;Pinto-Silva et al, 2005). In the present case apoptosis generated by OA alone (Traoré et al, 1999(Traoré et al, , 2001Pinto-Silva et al, 2005) has not been observed likely because the number of cells undergoing apoptosis was too low to be detected by agarose gel electrophoresis or by acridine orange staining. (ii) Addition of genotoxic effect of OA, Cd, and Cr (Leonard et al, 2004) overcomes the repair capacity of Caco-2 cells.…”

“…(ii) Addition of genotoxic effect of OA, Cd, and Cr (Leonard et al, 2004) overcomes the repair capacity of Caco-2 cells. (iii) Combination of genotoxicity and aneuploidism of OA and epigenetic effects (Traoré et al, 2000(Traoré et al, , 2001Creppy et al, 2002;Carvalho Pinto-Silva, 2003;Le Hégarat et al, 2004) are reinforced by cytotoxicity of Cd. Both toxicants (OA and Cr) target mitochondria and lysosomes/endosomes, (Creppy et al, 2002;Traoré et al, 2000Traoré et al, , 2003Leonard et al, 2004).…”

Effects of okadaic acid on haemocytes from Mytilus galloprovincialis: A comparison between field and laboratory studies

“…The DSP toxins include okadaic acid (OA), the dinophysistoxins-1 -2, and -3 and derivative forms (Blanco et al, 2005;García et al, 2010). Okadaic acid was described to be a powerful inhibitor of phosphatases type 1 and 2A, tumour promoter and apoptosis inductor in mammal cell lines (Cabado et al, 2004;Lago et al, 2005;Lerga et al, 1999;Rossini et al, 1997;Traoré et al, 2001;Valdiglesias et al, 2010). However, information concerning the effect of the DSP toxins on bivalves needs to be improved (Carvalho Pinto-Silva et al, 2003;Flórez-Barrós et al, 2011;Galimany et al, 2008aGalimany et al, , 2008bGalimany et al, , 2008cHégaret et al, 2011;Hégaret and Wikfors, 2005;Malagoli et al, 2008).…”

Cytotoxic and Apoptotic Effects of Sprouted and Non-sprouted Lentil, Green and Yellow Split-peas

“…The LDH activity was measured by a colorimetric cytotoxicity detection kit (LDH) (Roche Diagnostics, Indianapolis, IN) according to manufacturer's instructions. The absorbance was read at 490/600 nm wavelength in microplate reader Determination of cell death by apoptosis DNA fragmentation assay: DNA fragmentation assay was performed as described by Traore et al (2001) and Wu et al (2004) with some modifications. Caco-2 cells were treated with legume extracts at 1.5, 3, 6 and 12 mg mLG 1 for 24 h. After incubation, 500 μL of lysis buffer (10 mm Tris-HCl, pH 8/5 mM EDTA/0.6% Triton X-100) was added to cells to release nuclear contents.…”