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.</p

Detection and profiling of diarrheic marine biotoxins in shellfish by mRNA analysis of exposed Caco-2 cells using qRT-PCR and multiplex magnetic bead-based assays

The mouse bioassay for the detection of marine biotoxins in shellfish products is 40 years old and still in use. A full ban or total replacement of this in vivo test has been postponed because of the fear that current chemical-based detection methods could miss a new emerging toxin. In order to fully replace the mouse bioassay, more efforts are needed in the search for functional assays with specific endpoints. Gene expression elicited by diarrheic shellfish poisons (DSP) in Caco-2 cells allowed us to determine three “DSP profiles”, i.e., OA/DTX, AZA-YTX, and PTX profiles. Twelve marker genes were selected to represent the three profiles. qRT-PCR is relatively cheap and easy, and although its multiplex capacity is limited to 5 genes, this was sufficient to show the three expected profiles. The use of the multiplex magnetic bead-based assay was an even better alternative, allowing the detection of all 12 selected marker genes and 2 reference genes, and resulting in clear profiles with for some genes even higher induction factors than obtained by qRT-PCR. When analyzing blank and contaminated shellfish samples with the multiplex magnetic bead-based assay, the contaminated samples could easily be distinguished from the blank samples, and showed the expected profiles. This work is one step further towards the final replacement of the mouse bioassay. We suggest to combine the neuro-2a bioassay for screening with detection by analytical chemical analyses and with the multiplex magnetic bead-based assay for confirmation of known and unknown toxins.</p

Sulfated polymers in biological mineralization: a plausible source for bio-inspired engineering

Biomineralization leads to the formation of inorganic crystals with unique, ordered, refined shapes that are regulated by specific macromolecules. This process has been a source of inspiration for exploring novel approaches to the fabrication of inorganic-based surfaces and interfaces. Among those macromolecules, sulfated polymers, referred to as proteoglycans, have not received enough attention, although there is increasing evidence of their widespread occurrence in biominerals. Here we examine the available information on the nature, distribution and possible role of sulfated polymers in biomineralization, and highlight new directions to stimulate further research activities

Molecular epidemiology and genetic diversity of Listeria monocytogenes isolates from a wide variety of ready-to-eat foods and their relationship to clinical strains from listeriosis outbreaks in Chile

Listeria monocytogenes is a pathogen transmitted through food that can cause severe infections in high-risk groups such as pregnant women, elderly, young children and immunocompromised individuals. It is a ubiquitous bacterium that can survive in harsh conditions, such as dry environments, at low temperatures, in brine conditions and at low pH values. It also has the capacity to form biofilms, which makes it particularly successful even in colonizing surfaces within food processing plants. This study analyzed the presence of L. monocytogenes in ready-to-eat food (RTE) such as sausage, cheese, fresh salads, and other types of raw food. 850 samples of refrigerated and packaged food collected in 2008 and 2009 were analyzed. It was found that 25% of these samples were contaminated with L. monocytogenes strains. Serotyping and virulence genes detection by polymerase chain reaction (PCR) identified that strains belonging to serotype 4b, and containing one or more genes encoded by pathogenicity island (LIPI-1), were significantly associated with specific food types. Furthermore, using pulse field gel electrophoresis (PFGE), it was possible to associate isolates from cheese with strains from clinical cases of listeriosis outbreaks that occurred during the same time period within the same geographic regions. In addition, a strong correlation was observed between isolates from frozen seafood and from clinical strains obtained from sporadic cases of listeriosis. In agreement with reports described in other countries, our results shown that Chilean strains of L. monocytogenes from food products include the most virulent serotypes, encoding for the main virulence genes of the LIPI-1, and were clonally related to clinical isolates from sporadic cases and outbreaks of listeriosis. In conclusion, we show that Chilean isolates of L. monocytogenes from RTE and raw food products can cause disease in humans, representing a public health risk that justifies permanent surveillance

Whole genome mRNA transcriptomics analysis reveals different modes of action of the diarrheic shellfish poisons okadaic acid and dinophysis toxin-1 versus azaspiracid-1 in Caco-2 cells

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, 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. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish.</p

A strategy to replace the mouse bioassay for detecting and identifying lipophilic marine biotoxins by combining the neuro-2a bioassay and LC-MS/MS analysis

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.</p

Marine biotoxins and associated outbreaks following seafood consumption : Prevention and surveillance in the 21st century

Marine biotoxins are mostly produced by phytoplankton. Proliferation of algae producing marine biotoxins, also known as harmful algal bloom (HAB), occurs worldwide. Such event depends on environmental conditions, including temperature, water pH/salinity, current patterns and anthropogenic nutrient input. Marine biotoxins can accumulate in seafood products and as such present a threat to consumers.This paper reviews and compiles up-to-date literature on reported human intoxications following exposure to marine biotoxins through seafood consumption. The review includes a discussion about prevention of such outbreaks and surveillance programs to identify possible limitations and approaches for limiting the impact of HABs on human health. It is concluded that marine biotoxins represent a threat to human health as thousands of poisonings following consumption of seafood contaminated with marine biotoxins were reported in the 21st century, emphasizing the need for carrying on/developing surveillance programs to detect the presence of HABs, and for development, validation and implementation of sensitive high-throughput methods for detecting these biotoxins in seafood to protect consumers. Regarding the possible presence of unknown toxins and general lack of standards for many known toxins, in vitro effect-based bioassays may play an important role in the monitoring for biotoxins

Screening for the presence of lipophilic marine biotoxins in shellfish samples using the neuro-2a bioassay

The neuro-2a bioassay is considered as one of the most promising cell-based in vitro bioassays for the broad screening of seafood products for the presence of marine biotoxins. The neuro-2a assay has been shown to detect a wide array of toxins like paralytic shellfish poisons (PSPs), ciguatoxins, and also lipophilic marine biotoxins (LMBs). However, the neuro-2a assay is rarely used for routine testing of samples due to matrix effects that, for example, lead to false positives when testing for LMBs. As a result there are only limited data on validation and evaluation of its performance on real samples. In the present study, the standard extraction procedure for LMBs was adjusted by introducing an additional clean-up step with n-hexane. Recovery losses due to this extra step were less than 10%. This wash step was a crucial addition in order to eliminate false-positive outcomes due to matrix effects. Next, the applicability of this assay was assessed by testing a broad range of shellfish samples contaminated with various LMBs, including diarrhetic shellfish toxins/poisons (DSPs). For comparison, the samples were also analysed by LC-MS/MS. Standards of all regulated LMBs were tested, including analogues of some of these toxins. The neuro-2a cells showed good sensitivity towards all compounds. Extracts of 87 samples, both blank and contaminated with various toxins, were tested. The neuro-2a outcomes were in line with those of LC-MS/MS analysis and support the applicability of this assay for the screening of samples for LMBs. However, for use in a daily routine setting, the test might be further improved and we discuss several recommended modifications which should be considered before a full validation is carried out