Comparative study of Domoic Acid and Okadaic Acid induced-chromosomal abnormalities in the Caco-2 cell line

Abstract

Okadaic Acid (OA) the major diarrheic shellfish poisoning (DSP) toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA), the major Amnesic Shellfish Poisoning (ASP) toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN) arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA) and domoic acid (DA) to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH) using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.

Figure 1:

(a) Control cells 24h following the addition of Cytochalasin B showing binucleated cells, (400X), (b) MN in binucleated cells treated by Domoic acid, (100 ng/ml); (c) MN in mononucleated cells treated by okadaic acid, (60 ng/ml). Cells are stained with Giemsa (arrows indicate micronuclei)

Figure 2:

MN frequency in Caco-2 cells exposed to OA. Data are expressed as mean ± SD. *indicates significant differences as compared to negative control (P<0.05).

Figure 3:

Micronuclei (MN) frequency in Caco-2 cells exposed to DA. Data are expressed as mean ± SD. *indicates significant differences as compared to negative control (P<0.05).

Figure 4:

FISH analysis in Caco-2 cells after 24h exposure to OA. MN=number of micronucleated cells; CEN+= MN containing one or more centromeric signals (percentage); CEN−= MN containing no centromeric signal (percentage); MMC= Mytomicin C (clastogenic compound). Results presented are yielded by 3 independent experiments.

Figure 5:

FISH analysis in Caco-2 cells after 24h exposure to DA. MN= number of micronucleated cells; CEN+= MN containing one or more centromeric signals (percentage); CEN−= MN without a centromeric signal (percentage); MMC= Mytomicin C (clastogenic compound). Results are means of 3 independent experiments.