The presence of marine biotoxins in shellfish is a serious threat for consumers and requires continuous monitoring of production areas and products. Thus far, in many countries the major test system for these toxins is the use of animal bioassays, like the mouse bioassay. In this test, mice are injected with an extract of the test sample. The presence of toxins will seriously affect the animal and often death occurs, making this test highly questionable from an ethical point of view. For this reason alternatives are required which avoid the use of animals. At the RIKILT, chemical analytical methods have been developed that allow proper identification and quantification of the toxins. Such methods have recently been developed and validated and the EU strives to implement these tests in routine monitoring, replacing the animal bioassays from 2015 onwards (EU-Regulation (EU) Nr. 15/2011). These assays, however, are expensive and do not allow detection of biotoxins that are presently unknown. We expect bioassays based on the use of in vitro models to offer a valuable alternative, allowing the detection of toxins based on their adverse effects. In vitro bioassays based on the mode of action (MOA) offer the advantage over analytical methods for being capable to detect both known and unknown compounds.
The PhD student, Marcia Bodero, exposed Caco-2 cells to known diarrhetic shellfish poisons, which was followed by microarray analysis to detect effects on mRNA expression. The bioinformatics analysis provided indications about the mode of action of the compounds. Okadaic acid (OA) and dinophysistoxin-1 (DTX-1) induced the unfolded protein response, ER stress, hypoxia and ultimately apoptosis. Another diarrhetic shellfish poison, AZA-1, mainly affected the function of mitochondria. A manuscript about this work is under review.
Importantly, this work also provided genes of which the expression can be used for detection of marine toxins. The performance of these biomarkers is presently being studied for which the performance of two methods are compared: multiplex qPCR and Luminex technology.
A fourth study aims to develop a bioassay that can detect both diahretic as well neurotoxic shellfish poisons.
|Effective start/end date||1/01/12 → 31/12/17|