María Fraga

Multidetection of Paralytic, Diarrheic, and Amnesic Shellfish Toxins by an Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

The presence of paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP) toxins in seafood is a severe and growing threat to human health. In order to minimize the risks of human exposure, the maximum content of these toxins in seafood has been limited by legal regulations worldwide. The regulated limits are established in equivalents of the main representatives of the groups: saxitoxin (STX), okadaic acid (OA), and domoic acid (DA), for PSP, DSP, and ASP, respectively. In this study a multidetection method to screen shellfish samples for the presence of these toxins simultaneously was developed. Multiplexing was achieved using a solid-phase microsphere assay coupled to flow-fluorimetry detection, based on the Luminex xMap technology. The multidetection method consists of three simultaneous competition immunoassays. Free toxins in solution compete with STX, OA, or DA immobilized on the surface of three different classes of microspheres for binding to specific monoclonal antibodies. The IC50 obtained in the buffer was similar in single- and multidetection: 5.6 ± 1.1 ng/mL for STX, 1.1 ± 0.03 ng/mL for OA, and 1.9 ± 0.1 ng/mL for DA. The sample preparation protocol was optimized for the simultaneous extraction of STX, OA, and DA with a mixture of methanol and acetate buffer. The three immunoassays performed well with mussel and scallop matrixes displaying adequate dynamic ranges and recovery rates (around 90% for STX, 80% for OA, and 100% for DA). This microsphere-based multidetection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.

Detection of Paralytic Shellfish Toxins by a Solid-Phase Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 μg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Experimental Basis for the High Oral Toxicity of Dinophysistoxin 1: A Comparative Study of DSP

Okadaic acid (OA) and its analogues, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2), are lipophilic and heat-stable marine toxins produced by dinoflagellates, which can accumulate in filter-feeding bivalves. These toxins cause diarrheic shellfish poisoning (DSP) in humans shortly after the ingestion of contaminated seafood. Studies carried out in mice indicated that DSP poisonous are toxic towards experimental animals with a lethal oral dose 2–10 times higher than the intraperitoneal (i.p.) lethal dose. The focus of this work was to study the absorption of OA, DTX1 and DTX2 through the human gut barrier using differentiated Caco-2 cells. Furthermore, we compared cytotoxicity parameters. Our data revealed that cellular viability was not compromised by toxin concentrations up to 1 μM for 72 h. Okadaic acid and DTX2 induced no significant damage; nevertheless, DTX1 was able to disrupt the integrity of Caco-2 monolayers at concentrations above 50 nM. In addition, confocal microscopy imaging confirmed that the tight-junction protein, occludin, was affected by DTX1. Permeability assays revealed that only DTX1 was able to significantly cross the intestinal epithelium at concentrations above 100 nM. These data suggest a higher oral toxicity of DTX1 compared to OA and DTX2.

Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system

Freshwater and brackish microalgal toxins, such as microcystins, cylindrospermopsins, paralytic toxins, anatoxins or other neurotoxins are produced during the overgrowth of certain phytoplankton and benthic cyanobacteria, which includes either prokaryotic or eukaryotic microalgae. Although, further studies are necessary to define the biological role of these toxins, at least some of them are known to be poisonous to humans and wildlife due to their occurrence in these aquatic systems. The World Health Organization (WHO) has established as provisional recommended limit 1μg of microcystin-LR per liter of drinking water. In this work we present a microsphere-based multi-detection method for five classes of freshwater and brackish toxins: microcystin-LR (MC-LR), cylindrospermopsin (CYN), anatoxin-a (ANA-a), saxitoxin (STX) and domoic acid (DA). Five inhibition assays were developed using different binding proteins and microsphere classes coupled to a flow-cytometry Luminex system. Then, assays were combined in one method for the simultaneous detection of the toxins. The IC50s using this method were 1.9±0.1μg L(-1) MC-LR, 1.3±0.1μg L(-1) CYN, 61±4μg L(-1) ANA-a, 5.4±0.4μg L(-1) STX and 4.9±0.9μg L(-1) DA. Lyophilized cyanobacterial culture samples were extracted using a simple procedure and analyzed by the Luminex method and by UPLC-IT-TOF-MS. Similar quantification was obtained by both methods for all toxins except for ANA-a, whereby the estimated content was lower when using UPLC-IT-TOF-MS. Therefore, this newly developed multiplexed detection method provides a rapid, simple, semi-quantitative screening tool for the simultaneous detection of five environmentally important freshwater and brackish toxins, in buffer and cyanobacterial extracts.

The kinetic, mechanistic and cytomorphological effects of palytoxin in human intestinal cells (Caco‐2) explain its lower‐than‐parenteral oral toxicity

Palytoxin is one of the most toxic marine toxins known. Distributed worldwide, it poses a potential human health risk linked to the consumption of contaminated seafood. Despite its high parenteral toxicity, the lethal oral dose of palytoxin is several times higher than the intraperitoneal lethal dose. In the present study, we investigated the passage of palytoxin through the human intestinal barrier by employing a well‐characterized and accepted in vitro model of intestinal permeability that uses differentiated Caco‐2 cell monolayers. Trans‐epithelial electric resistance measurements showed that palytoxin disrupts the integrity of Caco‐2 monolayers at concentrations > 0.135 nm. However, confocal microscopy imaging showed that the tight‐junction protein occludin was not affected by palytoxin in the nanomolar range. This finding was supported by transmission electron microscopy imaging, where tight‐junctions appeared to be unaffected by palytoxin treatment. In addition, the nuclear envelope does not appear to be altered by high concentrations of palytoxin. However, palytoxin‐treated cells showed electron‐dense and damaged mitochondria. Toxin exposure also induced the disappearance of the differentiated Caco‐2 microvilli and organelles, as well as chromatin de‐condensation. Permeability assays showed that palytoxin could not significantly pass the Caco‐2 monolayer, despite the lack of epithelium integrity, suggesting that palytoxins would be poorly transported to blood, which may explain its lower oral toxicity. These data can help to achieve a better understanding of palytoxin poisoning. However, more studies regarding its repeated administration and chronic effects are needed.

Diarrhetic effect of okadaic acid could be related with its neuronal action: Changes in neuropeptide Y.

Okadaic acid (OA) and dinophysistoxins (DTXs) are a group of marine toxins that cause diarrheic shellfish poisoning (DSP) in humans and animals. These compounds are produced by dinoflagellates of the Prorocentrum and Dinophysis genera and can accumulate in filter-feeding bivalves, posing a serious health risk for shellfish consumers. The enteric nervous system (ENS) plays a crucial role in the regulation of the gastrointestinal tract. In addition, neuropeptides produced by ENS affects the epithelial barrier functions. In the present work we used a two-compartment human coculture model containing the SH-SY5Y neuroblastoma cell line and polarized colonic epithelial monolayers (Caco-2) to study the OA intestinal permeability. First, we have determined OA cytotoxicity and we have found that OA reduces the viability of SH-SY5Y in a dose-dependent way, even though DTX1 is 4 to 5 times more potent than OA. Besides DTX1 is 15 to 18 orders of magnitude more potent than OA in decreasing transepithelial electrical resistance (TEER) of caco-2 cells without inducing cytotoxicity. Permeability assays indicate that OA cross the monolayer and modulates the neuropeptide Y (NPY) secretion by neuroblastoma cells. This NPY also affects the permeability of OA. This offers a novel approach to establish the influence of OA neuronal action on their diarrheic effects through a cross talk between ENS and intestine via OA induced NPY secretion. Therefore, the OA mechanisms of toxicity that were long attributed only to the inhibition of protein phosphatases, would require a reevaluation.

Monitoring of freshwater toxins in European environmental waters by using novel multi-detection methods

Monitoring the quality of freshwater is an important issue for public health. In the context of the European project μAqua, 150 samples were collected from several waters in France, Germany, Ireland, Italy, and Turkey for 2 yr. These samples were analyzed using 2 multitoxin detection methods previously developed: a microsphere-based method coupled to flow-cytometry, and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The presence of microcystins, nodularin, domoic acid, cylindrospermopsin, and several analogues of anatoxin-a (ATX-a) was monitored. No traces of cylindrospermopsin or domoic acid were found in any of the environmental samples. Microcystin-LR and microcystin-RR were detected in 2 samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H2 -ATX-a and small amounts of H2 -homoanatoxin-a, whereas ATX-a and homoanatoxin-a were found in only 1 sample. These results confirm the presence and wide distribution of dihydro derivatives of ATX-a toxins in European freshwaters. Environ Toxicol Chem 2017;36:645-654.

Identification of metalliferous ecotypes of Cistus ladanifer L. using RAPD markers

Abstract The genetic diversity of Cistus ladanifer ssp. ladanifer (Cistaceae) growing on ultramafic and non-ultramafic (basic and schists) soils in the NE of Portugal was studied in order to identify molecular markers that could distinguish the metal-tolerant ecotypes of this species. Random Amplified Polymorphic DNA (RAPD) markers were used in order to estimate genetic variation and differences between populations. The RAPD dataset was analysed by means of a cluster analysis and an analysis of molecular variance (AMOVA). Our results indicate a significant partitioning of molecular variance between ultramafic and non-ultramafic populations of Cistus ladanifer, although the highest percentage of this variance was found at the intra-population level. Mantel’s test showed no relationship between inter-population genetic and geographic distances. A series of RAPD bands that could be related to heavy metal tolerance were observed. The identification of such markers will enable the use of Cistus ladanifer in phytoremediation procedures.

AFLP analysis of the pseudometallophyte Cistus ladanifer: comparison with cpSSRs and exploratory genome scan to investigate loci associated to soil variables

AimsTo assess the genetic patterns of colonisation of metalliferous areas by the pseudometallophyte Cistus ladanifer with AFLP genome scan, to compare those estimates with previous cpSSR results, and to identify loci potentially linked to tolerance to metalliferous soils.MethodsThirty-three populations were screened with AFLP markers. Statistical analyses included estimation of genetic diversity, hierarchical analysis of molecular variation and Bayesian analysis of geographical partitioning of genetic diversity. Mantel tests and Spearman’s correlation index were used to compare results obtained with AFLP and cpSSR. Generalized Estimating Equations (GEE) models were applied to correlate allele frequency distribution and soil variables (pH, Ca:Mg ratio and total contents of different trace metals).ResultsMetallicolous and non-metallicolous populations of C. ladanifer showed neither different levels of genetic diversity, nor genetic differentiation between population types. Incongruence in genetic diversity estimates between AFLP and cpSSR due to marker-specific properties was observed. Nonetheless, pairwise distance matrices computed with both markers were concordant. GEE analyses showed that the Mn total soil content has an important effect on the allele distribution in C. ladanifer. In contrast, the Ca:Mg ratio seems to have no selective effect. Moreover, we identified a particular band with a putative role in the species’ tolerance to high Mn concentrations in the soil.ConclusionsThe soil type had no influence over this species’ genetic structure. GEE showed their usefulness in revealing the association between soil variables and AFLP loci.

Absorption and Effect of Azaspiracid-1 Over the Human Intestinal Barrier.

Background: Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellates genera Azadinium and Amphidoma. These toxins cause azaspiracid poisoning (AZP), characterized by severe gastrointestinal illness in humans after the consumption of bivalve molluscs contaminated with AZAs. The main aim of the present study was to examine the consequences of human exposure to AZA1 by the study of absorption and effects of the toxin on Caco-2 cells, a reliable model of the human intestine. Methods: The ability of AZA1 to cross the human intestinal epithelium has been evaluated by the Caco-2 transepithelial permeability assay. The toxin has been detected and quantified using a microsphere-based immunoassay. Cell alterations and ultrastructural effects has been observed with confocal and transmission electron microscopy Results: AZA1 was absorbed by Caco-2 cells in a dose-dependent way without affecting cell viability. However, modifications on occludin distribution detected by confocal microscopy imaging indicated a possible monolayer integrity disruption. Nevertheless, transmission electron microscopy imaging revealed ultrastructural damages at the nucleus and mitochondria with autophagosomes in the cytoplasm, however, tight junctions and microvilli remained unaffected. Conclusion: After the ingestion of molluscs with the AZA1, the toxin will be transported through the human intestinal barrier to blood causing damage on epithelial cells.