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Imprint
Molecular characterization of toxicological properties of the marine biotoxin okadaic acid in in vitro models for the human intestinal barrier and liver
Project
Project code: BfR-LMS-08-1350-012
Contract period: 01.07.2015
- 31.01.2019
Purpose of research: Applied research
The marine biotoxin okadaic acid causes diarrhetic shellfish poisoning primarily leading to enteral injury. However, besides these well characterised acute effects, okadaic acid exhibits carcinogenic and severe cytotoxic as well as embryotoxic properties. The extent of poisoning depends on the amount of okadaic acid ingested with shellfish. Our preliminary work clearly indicates the need to differentiate between low and high doses. We suppose that low doses (lower than 50 nM) are actively detoxified in the intestinal epithelium by an efflux mechanism resulting in protection of the body against the systemic uptake of the toxin. Intoxication with higher doses leads to the destruction of the barrier function of the intestinal epithelium resulting in the uptake of okadaic acid into the blood system and transport to the liver. Here, activation into reactive metabolites can occur. The molecular interactions between the detoxification and the bioactivation shall be elucidated in this study using in vitro test systems. In subsequent stages of this project the in vivo relevance shall be investigated using different in vivo-mouse models (knockout-mice). Our investigations shall clarify the risk of an okadaic acid poisoning in the lower concentration range for humans. Additionally, unknown toxicity mechanisms of a high doses intoxication of okadaic acid are supposed to be identified.
The marine biotoxin okadaic acid is formed by algae of the genera Dinophysis and Prorocentrum. Due to its lipophilic character it accumulates in the hepatopancreas of shellfish and induces diarrheic shellfish poisoning (DSP) after consumption of highly contaminated seafood. At the molecular level, okadaic acid is a potent inhibitor of serine/threonine phosphatase 1 and 2A (PP1 and PP2A), disrupting the intracellular regulated balance of phosphorylation and dephosphorylation reactions. This affects almost all regulatory processes of the cell, but also basic cellular functions such as cell adhesion, cell motility and cytoskeletal dynamics. The destruction of the intestinal barrier is associated with the aforementioned properties of okadaic acid. Therefore, the aim of this study was to investigate the influence of okadaic acid with a model of the human intestinal barrier (Caco-2 cell line), as this seems to be the first target structure of okadaic acid. In particular, the focus should be on two different exposure scenarios: The uptake of okadaic acid amounts in the range of the legal limit value (about 20 nM), which is assumed not to induce the DSP, and high doses occurring during intoxication. The results of this study show that an active efflux mechanism exists for okadaic acid in the low-dose range from the intestinal cells out towards the intestinal lumen. This transport is mediated by ABCB1 (P-glycoprotein). At higher concentrations of okadaic acid (approx. 100 nM) the integrity of the intestinal barrier decreases. This could be demonstrated by increasing cytotoxicity and decreasing values of transepithelial electrical resistance. In order to investigate the reason of the destruction of the Caco-2-cell monolayer more closely, various tight junction proteins were investigated with regard to gene and protein expression as well as their functional properties. After 8- and 24-hour treatment of the Caco-2 cells with okadaic acid (80 and 150 nM, respectively), the genes CLDN2, CLDN4 and TJP1 were upregulated. Verification of these data by Western blot, however, showed no altered protein content for CLDN2 but an increase in CLDN4 protein content and a decrease in ZO-3 protein content. Immunofluorescence staining of ZO-1, claudin-2, claudin-4 and occludin was performed to investigate the functionality of intestinal tight junction proteins. Concentration-dependent effects of okadaic acid on the structural organization of the cytoskeleton and tight junction proteins in a food-relevant concentration were detected. The uptake of okadaic acid levels above the legally defined threshold may lead to the destruction of the barrier function of the intestinal epithelium. In the in vivo situation, this means that the okadaic acid passes through the intestinal epithelium and is transported to the liver with the portal vein blood. The liver is crucial for the metabolism of xenobiotics. Various liver homogenates from humans and rats were used to simulate the metabolism of xenobiotics. The investigation of the metabolism of okadaic acid showed a toxification of okadaic acid by an increased induction of cytotoxicity to the liver cell line HepG2. Enzymes contributing to metabolism are CYP3A4 and CYP3A5 and the corresponding rat orthologues. For human CYP1A2, okadaic acid has been shown to be toxic by increased cytotoxicity in HepG2 cells. The analysis of the formation of metabolites, however, only showed the formation of oxidized okadaic acid metabolites for the cell-free approaches with CYP3A4/3A5 and the corresponding rat orthologues, in the approach with CYP1A2 no metabolites were detected besides the parent substance. Taken together, the stronger cytotoxicity of okadaic acid by NADPH-independent enzymes and CYP1A2 and the simultaneous absence of metabolites in these approaches show that okadaic acid toxicity may be caused by the formation of reactive intermediates. It has already been described in the literature that okadaic acid can act as an inducer of lipid peroxidation. These lipid peroxides can induce free radical reactions that lead to membrane damage, interact with macromolecules such as proteins, and ultimately induce cytotoxicity. Within these investigations, however, no increased amounts of reactive oxygen species could be detected. In summary, the results of this study show a destruction of the gastrointestinal barrier in the human-relevant dose range, which can be achieved by the uptake of highly contaminated shellfish. Here, however, it is still unclear whether the destruction of the tight junction barrier can be prevented.
Section overview
Subjects
- Biotechnology
- Toxicology
