Effect-based in vitro bioassays for lipophilic marine biotoxins : a new strategy to replace the mouse bioassay

Marine biotoxins in fish and shellfish can cause a number of adverse health effects in consumers, such as diarrhoea, amnesia, and death by paralysis. Worldwide, there are monitoring programs for testing shellfish on a regular basis. In some countries, testing is performed by using the so-called mouse bioassay (MBA), an assay raising both ethical and practical concerns because of animal distress and shortcomings in respect to specificity. The MBA may result in both false negatives and false positives. A false negative does not protect the consumers as anticipated and the high amounts of false positives encountered when applying the MBA lead to unnecessary closures of extraction areas, damaging local economies. A full ban of the MBA or its total replacement by analytical chemical methods has failed because these detection methods are unable to detect all toxin analogues and newly emerging toxins and will thus result in false negatives by definition. To fully replace the MBA, there is a clear need for new functional animal-free in vitro assays with specific endpoints that are able to detect both the known and yet unknown marine biotoxins. In Europe a method based on LC-MS/MS has been developed as an alternative for the MBA and is now the reference method for lipophilic marine biotoxins (LMBs) and used in the routine monitoring. However, as outlined above safety is not fully guaranteed when relying only on such a method and, as a result, the MBA is still used for surveillance purposes. The aim of the work presented in this thesis was to develop a new strategy to fully replace the MBA for detecting LMBs without the risk of missing a contaminated sample that can lead to an intoxication. This was achieved by combining effect-based bioassays and a mass spectrometry analysis, including the official EU-RL method. Chapter 1 addresses the safety issues of the marine biotoxins produced by algae, corals and bacteria and summarises the current legislations and recommendations and the methods of detection. In Chapter 2, the neuro-2a bioassay, a cell-based in vitro bioassay that was previously shown to be sensitive for several hydrophilic and lipophilic marine biotoxins, was studied for its ability to screen seafood products for the presence of lipophilic marine biotoxins. All (regulated) LMBs and their analogues were tested, and the neuro-2a bioassay outcomes showed that all these LMBs could be detected at low concentrations. Next, blank and contaminated sample extracts were prepared and tested, showing that matrix effects led to false positive screening outcomes. Therefore, the standard extraction procedure for LMBs with methanol was modified by introducing a clean-up step with n-hexane before further extraction on the SPE-column. First, the possible recovery losses due to this extra n-hexane wash step were assessed, showing that the n-hexane did not lead to recovery losses of the LMBs and that the matrix effect was successfully removed. Finally, the applicability of the neuro-2a bioassay was assessed by testing a broad range of shellfish samples contaminated with various LMBs, including diarrhoeic shellfish poisoning (DSP) toxins. The samples were also analysed by LC-MS/MS. Overall, the neuro-2a bioassay showed screening outcomes that were well in line with the toxin levels as determined by the EU-RL LC-MS/MS reference method. In chapter 3, a study with DNA microarrays was performed to explore the effects of two diarrhoeic and one azaspiracid shellfish toxin, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole genome mRNA expression of undifferentiated intestinal Caco-2 cells. In this chapter the whole genome mRNA expression was analysed in order to reveal the possible modes of action of these toxins and to select genes that can be used as potential markers in new additional bioassays for the detection and identification of these LMBs. It was observed that OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1 induce similar toxic effects. Biological interpretation of the microarray data showed that both compounds induced endoplasmic reticulum (ER) stress, hypoxia, and unfolded protein response (UPR). The gene expression profile of AZA-1 resulted in a different expression profile and showed increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. In chapter 4, twelve marker genes were selected from the previous study and five were used to develop a multiplex qRT-PCR method. This multiplex qRT-PCR method is able to detect three toxin profiles, i.e. a OA/DTX, AZA/YTX and PTX profile. The multiplex capacity of this qRT-PCR is limited to five genes. The use of a multiplex magnetic bead-based assay was explored, allowing the use of all twelve selected marker genes and two reference genes. This 14-plex also resulted in clear profiles with sometimes higher induction factors as obtained by the 5-plex qRT-PCR method. As a result, contaminated samples could easily be distinguished from the blank samples, showing the expected profiles. These multiplex assays can thus detect these LMBs in shellfish samples and the obtained profile indicates the toxin-type present. However, compared with the neuro-2a bioassay, this assay has been shown adequate so far for only a limited number of LMBs (not all LMBs have been tested), and it is more laborious, time consuming and expensive. It should be used in cases were suspect screening outcomes from the neuro-2a bioassay cannot be explained by the toxin levels as measured with the EU-RL LC-MS/MS reference method. In chapter 5, the neuro-2a bioassay as an initial screening assay was combined with the EU-RL LC-MS/MS method for confirmation and it was investigated whether this combination is able to replace the MBA for the detection and quantification of LMBs. Samples that were tested previously in the MBA (in Chile) were used. It turned out that all samples that tested positive in the MBA were also suspect in the neuro-2a bioassay and most of these samples were confirmed to be positive for the presence of LMBs by LC-MS/MS analysis. The results confirm that the combination of the neuro-2a bioassay for screening and the EU-RL LC-MS/MS method for confirmation, is a promising alternative for the unethical MBA. The data even strongly indicated that the MBA alone probably led to false positives and the unnecessary closure of extraction areas or withdrawal of products from the market, a problem not encountered when using the neuro-2a assay in combination with LC-MS/MS. In chapter 6, a fully integrated testing strategy was presented for replacing the MBA, enabling the detection of the hydrophilic marine biotoxins. The steps and methods are discussed, and some points of attention and further developments required are addressed. Taking all together it is concluded that the proposed strategy contributes to a future with a complete animal free alterative testing strategy replacing the MBA.