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

Defect chemistry and oxygen transport of (La0.6Sr0.4 − xMx)0.99Co0.2Fe0.8O3 − δ, M = Ca (x = 0.05, 0.1), Ba (x = 0.1, 0.2), Sr: Part I: Defect chemistry

This paper is the first part of a two part series, where the effects of varying the A-site dopant on the defect chemistry, the diffusion coefficient and the surface catalytic properties of the materials (La0.6Sr0.4 − xMx)0.99Co0.2Fe0.8O3 − δ, M = Sr, Ca (x = 0.05, 0.1), Ba (x = 0.1, 0.2) (LSMFC) have been investigated. In part I, the findings on the defect chemistry are reported, while the transport properties are reported in part II. Substitution of Sr2+ ions with Ca2+ ions (smaller ionic radius) and Ba2+ ions (larger ionic radius) strains the crystal structure differently for each composition while keeping the average valence of the cations constant. The Ba2+ containing materials show the largest oxygen loss at elevated temperatures, while the purely Sr2+ doped material showed the smallest oxygen loss. This was reflected in the partial oxidation entropy of the materials. The measured oxygen loss was modelled with point defect chemistry models. Measurements at very low pO2 showed several phase transitions

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

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

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

In vitro toxicological characterisation of the antifungal compound soybean toxin (SBTX)

Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 μg/mL and 200 μg/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 μg/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 μg/mL or 200 μg/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but down-regulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.</p