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DNA breaks and cell cycle arrest induced by okadaic acid in Caco-2 cells, a human colonic epithelial cell line
A Traoré1, I Baudrimont, S Ambaliou
1Laboratory of Toxicology and Applied Hygiene, Faculty of Pharmaceutical Sciences, University Victor Segalen Bordeaux 2, 146, rue Léo-Saignat, 33076 Bordeaux, France.
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.
Insights
Okadaic acid (OA), a shellfish toxin, causes cell death and DNA damage in human intestinal cells. Further research is needed to understand its in vivo effects on human intestinal cells.
Area of Science:
- Toxicology
- Cell Biology
- Gastroenterology
Background:
- Okadaic acid (OA) is a potent shellfish toxin produced by dinoflagellates.
- It poses a threat to human health, primarily affecting the gastrointestinal tract and causing diarrhea.
- OA is a known tumor promoter, raising concerns about its effects even at low concentrations.
Purpose of the Study:
- To investigate the cytotoxic effects of okadaic acid (OA) on human colonic cells (Caco-2).
- To assess the ability of OA to induce DNA lesions and apoptosis in these cells.
- To establish the concentration-dependent relationship between OA exposure and cellular damage.
Main Methods:
- Incubation of Caco-2 cells with varying concentrations of OA (3.75-60 ng/ml).
- Assessment of cell viability, protein synthesis, and DNA synthesis inhibition using IC50 values.
- Analysis of cell cycle arrest, DNA laddering (apoptosis), DNA fragmentation (Higuchi and Aggarwal method), and comet assay.
Main Results:
- OA significantly reduced Caco-2 cell viability and inhibited protein and DNA synthesis.
- OA induced cell cycle arrest, with an increase in S phase cells and a decrease in G0/G1 phase cells at higher concentrations.
- Apoptosis was confirmed by DNA laddering, and clastogenic effects were evident through DNA fragmentation and comet assay, both showing concentration-dependent increases.
Conclusions:
- Okadaic acid exhibits cytotoxic properties and induces DNA damage, leading to cell death primarily through apoptosis in human colonic cells.
- The observed effects are concentration-dependent, highlighting the risk associated with consuming contaminated shellfish.
- Further in vivo studies using human primary intestinal cells and tissue are recommended to evaluate OA's toxicity in a more relevant physiological context.