Abstract
The phycotoxin, okadaic acid (OA) and dinophysistoxin 1 and 2 (DTX-1 and -2) are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP). Data on the toxicity of the OA-group toxins show some differences with respect to the in vivo acute toxicity between the toxin members. In order to investigate whether OA and congeners DTX-1 and -2 may induce different mechanisms of action during acute toxicity on the human intestine, we compared their toxicological effects in two in vitro intestinal cell models: the colorectal adenocarcinoma cell line, Caco-2, and the intestinal muco-secreting cell line, HT29-MTX. Using a high content analysis approach, we evaluated various cytotoxicity parameters, including apoptosis (caspase-3 activation), DNA damage (phosphorylation of histone H2AX), inflammation (translocation of NF-κB) and cell proliferation (Ki-67 production). Investigation of the kinetics of the cellular responses demonstrated that the three toxins induced a pro-inflammatory response followed by cell cycle disruption in both cell lines, leading to apoptosis. Our results demonstrate that the three toxins induce similar effects, as no major differences in the cytotoxic responses could be detected. However DTX-1 induced cytotoxic effects at five-fold lower concentrations than for OA and DTX-2.
Highlights
Okadaic acid (OA) is a toxin involved in diarrhetic shellfish poisoning (DSP) syndrome, causing gastro-intestinal symptoms following ingestion of contaminated shellfish [1,2]
(A) Chemical structure of okadaic acid (OA), DTX-1 and DTX-2; (B) Dose response curves represent the viability of Caco-2 and HT29-MTX cells compared to vehicle control conditions
Our study provides in vitro results concerning the acute toxicity of DTX-1 and -2 in intestinal cells relative to OA
Summary
Okadaic acid (OA) is a toxin involved in diarrhetic shellfish poisoning (DSP) syndrome, causing gastro-intestinal symptoms following ingestion of contaminated shellfish [1,2]. Only limited studies have seriously investigated the toxic responses of the DTX-1 and DTX-2 analogues. In order to compare the toxic effects of the OA analogues, we chose to investigate the acute in vitro toxicity of OA, DTX-1 and DTX-2 in the human intestinal cell lines, Caco-2 and HT29-MTX, since the intestine is the major target of OA and its analogues. Caco-2 cells are widely used as an enterocyte model system in in vitro toxicology [27,28], whereas HT29-MTX cells have muco-secretant characteristics [27] Both cell lines were used in a proliferative state, mimicking intestinal epithelial cell turnover and to study the effect of the toxins on cell proliferation. Four suitable endpoints were selected: Ki-67 as a marker of cell cycle disruption [31], activation of caspase-3 as an apoptotic marker [32], phosphorylation of histone H2AX as marker of DNA double-strand breaks [33] and, the translocation of NF-κB as a marker of the cellular inflammatory response [34]
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