Okadaic Acid Content Research Articles

A study on the mechanism of the impact of phenthoate exposure on Prorocentrum lima

Extensive application of organophosphorus pesticides such as phenthoate results in its abundance in ecosystems, particularly in waterbodies, thereby providing the impetus to assess its role in aquatic organisms. However, the impact of phenthoate on marine algal physiological and proteomic response is yet to be explored despite its biological significance. In this study, we thus ought to investigate the impact of phenthoate in the marine dinoflagellate Prorocentrum lima, which is known for synthesizing okadaic acid (OA), the toxin responsible for diarrhetic shellfish poisoning (DSP). Our results showed that P. lima effectively absorbed phenthoate in seawater, with a reduction efficiency of 90.31% after 48 h. Surprisingly, the provision of phenthoate (100 and 1000 µg/L) substantially reduced the OA content of P. lima by 35.08% and 60.28% after 48 h, respectively. Meanwhile, phenthoate treatment significantly reduced the oxidative stress in P. lima. Proteomic analysis revealed that the expression level of seven crucial proteins involved in endocytosis was upregulated, suggesting that P. lima could absorb phenthoate via the endocytic signaling pathway. Importantly, phenthoate treatment resulted in the downregulation of proteins such as polyketide synthase (PKS)− 2, Cytochrome P450 (CYP450)− 1, and CYP450–2, involved in OA synthesis, thereby decreasing the OA biosynthesis by P. lima. Our results demonstrated the potential role of P. lima in the removal of phenthoate in water and exemplified the crucial proteins and their possible molecular mechanisms underpinning the phenthoate remediation by P. lima and also the regulatory role of phenthoate in restricting the OA metabolism. Collectively, these findings uncovered the synergistic mechanisms of phenthoate and P. lima in remediating phenthoate and reducing the toxic impact of P. lima.

Multi-omics analysis reveals metabolism of okadaic acid in gut lumen of rat.

Okadaic acid (OA) is an important marine lipophilic phycotoxin with various pathological properties, responsible for diarrheal shellfish poisoning events in human beings over the world. However, to date no mechanism can well explain the toxicity and symptom of OA, even diarrhea. Here, to reveal the toxic mechanism of OA to mammals, we analyzed the metabolism of OA in rat and the effects of OA exposure on the composition and function of gut bacteria using a multi-omics strategy and rRNA high-throughput technology. We found that OA exerted great effects on gut bacteria, mainly featured in heavy fluctuation of dominant genera and significant changes in the mapped bacterial function genes, including not only virulence genes of pathogenic bacteria, but also bacterial metabolism genes. In the feces of the OA-exposed group, we detected dinophysistoxin-2 (DTX-2), lespedezaflavanone F and tolytoxin, suggesting that OA could be transformed into other metabolites like DTX-2. Other metabolic biomarkers such as N-Acetyl-a-neuraminic acid, N,N-dihydroxy-L-tyrosine, nalbuphine, and coproporphyrin I and III were also highly correlated with OA content, which made the toxicity of OA more complicated and confusing. Spearman correlation test demonstrated that Bacteroides and Romboutsia were the genera most related to OA transformation, suggesting that Bacteroides and Romboutsia might play a key role in the complicated and confusing toxicity of OA. In this study, we found for the first time that OA may be converted into other metabolites in gut, especially DTX-2. This finding could not only help to reveal the complex toxicity of OA, but also have important significance for clarifying the transportation, metabolism, and environmental fate of OA in the food chain.

Multiple cooperative amplification paper SERS aptasensor based on AuNPs/3D succulent-like silver for okadaic acid quantization

Okadaic acid (OA) is the main toxic component of diarrhetic shellfish poisoning, which can cause vomiting, diarrhea, and even induce tumor formation in people who accidentally ingest it. To strengthen the front-end prevention and control of shellfish safety, a surface-enhanced Raman scattering (SERS) paper swab based on high-affinity binding and high-specific recognition performance of nucleic acid aptamer was proposed to detect OA by virtue of enhanced multistage plasmon coupling effect. Composite resonance bilayer constituted by evenly dispersed gold nanoparticles integrating with a three-dimensional (3D) succulent-like silver particle was facilely manufactured in-situ on the paper substrate. Subsequently, with the assistance of 3D nanotopography structured silver nano-octahedra connected on the signal probe, a multistage SERS signal cooperative amplification platform was formed with about 10-fold enhanced Raman intensity higher than either single-layer substrate. Owning to the unique strategy of superimposed signal enhancement, such paper SERS aptasensor possessed admirable specific recognition and sensitivity has been successfully used in the on-site quantization of OA, with a detection limit of 0.31 ng/mL. Furthermore, the construction of the SERS paper swab is of great significance to the promotion of rapid on-site analysis approach of OA content in seafood by customs, seafood market supervision departments and ordinary consumers.

Competitive near-infrared PEC immunosorbent assay for monitoring okadaic acid based on a disposable flower-like WO3-Modified screen-printed electrode

The long-term toxic effects of okadaic acid (OA) in shellfish pose a serious threat to public health, negatively impacting the development of the shellfish aquaculture industry. In this study, a novel competitive near-infrared-mediated photoelectrochemical immunosorbent assay (cNIR-PECIA) was developed for ultrasensitive and highly selective detection of OA based on NaYF4:Yb, Tm upconversion nanophosphors (UCNPs) and a flower-like WO3-modified screen-printed electrode (FL-WO3 SPE). The UCNPs function as a self-powder to convert NIR excitation into visible emissions. FL-WO3 fully utilizes the visible illumination and induces the separation of electron–hole pairs, thus generating a photocurrent. After conjugating monoclonal antibodies against OA on UCNPs (UCNPs-Ab), the bright PEC immunoprobe selectively captured OA molecules, which were then determined by a competitive indirect immunosorbent assay. Under optimal conditions, the 50% inhibitory concentration of the immunosensor was 0.09 ng mL−1. The OA concentration had a linear relationship with the antibody binding rate in the range of 0.01–60 ng mL−1 with an extremely low detection limit of 0.007 ng mL−1. Finally, the proposed cNIR-PECIA was successfully utilized to analyze OA content in mussel samples. This study affords new ideas for constructing NIR PEC sensors by using upconversion luminescent materials to match semiconductors. The superior sensing properties indicate their potential applicability in food safety analysis.

A novel high frequency SAWR based sensor combined with living cells for shellfish toxin quantitative determination

Shellfish toxin can be easily found in seafood, and bring potential harm to human health. Nowadays some analytical methods are used for shellfish toxin quantitative detection. However, there are some disadvantages in these analytical methods such as time consuming, high cost, etc. Therefore, a more suitable method is in demand. In this study, a novel method using high frequency surface acoustic wave resonator (SAWR) based sensor combined with living cells was developed for shellfish toxin continuous monitoring. ECA9706 cell lines were used as the sensing elements to establish the sensor device for DSP toxin okadaic acid (OA) quantitative monitoring. The sensor device is in serial with SAWR. SAWR output frequency was recorded for detecting characterization. The OA concentration of prepared solutions could be characterized by using SAWR sensor, and good linear relationship between sensor responses and OA concentration is achieved. Liquid chromatography tandem mass spectrometry (LC–MS/MS) is used for characterization of OA content in shellfish extract. Good linear relationship between OA content and the concentration determination provided by SAWR sensor. HEK293 cells were fixed on electrode and utilized as negative control. The results showed that ECA9706 cell-based sensor had selective responses to OA. This method is promising in shellfish toxins rapid quantitative determination.

MRP functional activity and character in the dinoflagellate Prorocentrum lima

Using laser scanning confocal microscope (LACM) and flow cytometry (FCM), we found that MK571, a specific inhibitor of multidrug resistance-associated protein (MRP), could increase the accumulation of calcein-AM in Prorocentrum lima, a DSP toxin-producing dinoflagellate. This led to the assumption that the cells of P. lima possess MRP transporters in the plasma membrane. The contents of okadaic acid (OA), a main component of DSP toxins, and MRP activities in P. lima were determined at 4, 20, 28, and 42 days after culture and at 38 day under N- and P-limited conditions. We found that both the OA level and MRP activity in P. lima increased over time; OA concentration and MRP activity conformably increased under N- and P-limited conditions compared with control counterparts. These changes in OA concentration and associated increased MRP activity suggest that MRP might be involved in OA transport in P. lima. However, MK571 and CsA, a broad-spectrum inhibitor of multidrug resistance (MDR)-associated ABC proteins, had no observable effects on OA content in P. lima. It seems that except for MDR-associated ABC proteins, other transporters like PDR might be involved in the transport of OA in P. lima.

Effect of ozonation and γ-irradiation on post-harvest decontamination of mussels (Mytillus galloprovincialis) containing diarrhetic shellfish toxins

Contamination of shellfish with diarrhetic shellfish poisoning (DSP) toxins readily occurs during algal blooms. Such phenomena raise important public health concerns and thus comprise a constant challenge to shellfish farmers, the seafood industry and health services, considering the increasing occurrence of toxic episodes around the world. To avoid the detrimental effects of such episodes, research has focused on the use of various detoxification methodologies that should be rapid, efficient, easy to apply, and will not alter the quality and sensory properties of shellfish. In the present study, both ozonation (15 mg kg−1 for 6 h) and γ-irradiation (6 kGy) were utilised in order to reduce the toxin content of contaminated shucked mussels, collected during the DSP episodes of 2007 and 2009 in Greece. DSP toxicity was monitored using the mouse bioassay (MBA) whilst the determination of toxin content of the okadaic acid (OA) group (both free and esterified forms) was carried out by LC/MS/MS analysis. Toxin reduction using γ-irradiation was in the range of 12–36%, 8–53% and 10–41% for free OA, OA esters and total OA, respectively. The appearance and texture of irradiated mussels deteriorated, pointing to a low potential for commercial use of this method. Ozonation of mussels resulted in toxin reduction in the range of 6–100%, 25–83% and 21–66% for free OA, OA esters and total OA, respectively. Reduction of OA content was substantially higher in homogenised mussel tissue compared with that of whole shucked mussels. In addition, differences detected with regard to quality parameters (TBA, sensory attributes) between ozonated and control mussels were not considerable. Even though varying percentage reductions in OA and its derivatives were achieved using ozonation under specific experimental conditions tested, it is postulated that upon optimisation ozonation may have the potential for post-harvest commercial DSP detoxification of shucked mussels.

Toxicity to medaka fish embryo development of okadaic acid and crude extracts of Prorocentrum dinoflagellates

Chronic and subchronic toxicity following exposure to the DSP (Diarrhetic shellfish poisoning) toxin okadaic acid (OA) is receiving increasing attention as a public human health biohazard. However information on ecological impacts induced by proliferation of the OA producing dinoflagellate Prorocentrum is scarce. In order to analyse the toxicity of these substances, in vivo experiments were conducted on medaka fish ( Oryzias latipes) embryos used as an experimental model. The study was focused on two strains of benthic Prorocentrum species, P. arenarium and P. emarginatum, naturally found in the Indian Ocean. Sample extracts (crude extracts, CE) were obtained from algal cultures and their toxic potential was explored. Their OA (and derivatives) content was evaluated by two methods: one based on chemical analysis using HPLC-MS, the other based on screening the inhibiting effect on protein phosphatase PP2A. P. arenarium extracts inhibit PP2A and the active toxin was confirmed as being OA by HPLC-MS. In contrast, P. emarginatum showed negative results regardless of the method used. The development of medaka fish embryos kept in medium containing pure OA or Prorocentrum CE was examined. Survival rates were reduced up to 100% depending on the concentrations used of both OA and CE of P. arenarium, while no effect was observed with CE of P. emarginatum. Anatomopathological studies of surviving embryos indicate that OA treatment resulted in significant increases in liver and digestive tract areas compared to controls. P. arenarium treated surviving embryos exhibited significant quantitative increases of global body and vitellus areas. Together, our results indicate that the toxic effects to medaka embryos development of pure OA and P. arenarium extracts containing OA are distinguishable. The differences may indicate the presence of additional toxic substance(s) (or molecules able to modulate OA impact) in the P. arenarium CE that probably are not present in P. emarginatum.

Analysis of the importance of lipid breakdown for elimination of okadaic acid (diarrhetic shellfish toxin) in mussels, Mytilus edulis: results from a field study and a laboratory experiment

Okadaic acid (OA) is a lipophilic phycotoxin, which accumulates in the digestive organs of mussels and may cause diarrhetic shellfish poisoning (DSP) in humans. Depuration of toxic mussels is a potential option for the shellfish industry to increase the availability of marketable mussels. To develop cost-effective depuration methods for DSP toxins, knowledge about the environmental conditions and physiological processes regulating the rate of depuration is essential. In this paper, the importance of lipid breakdown for elimination of OA in mussels was investigated by performing a field study and a manipulative laboratory experiment. First, total lipid content and concurrent concentration of OA in the digestive glands of farmed blue mussels, Mytilus edulis, was analysed on a monthly basis from January to June 2000. A significant positive correlation between levels of OA and lipid content was observed between January and March, when lipid levels were showing a decreasing trend. This supported a previously proposed model that breakdown of lipid stores may affect the release and elimination of this lipophilic toxin. To test this causal model, a laboratory experiment was performed. Mussels containing OA were exposed to experimental treatments (increased seawater temperature and/or food limitation) for 24 days in order to increase the energy requirements and need to use lipids as an energy source. It was predicted that mussels exposed to these treatments would have a faster elimination rate of OA compared to feeding mussels kept in ambient seawater temperature. The results showed that lipid content was significantly reduced in mussels exposed to an increased water temperature (24 °C) compared to ambient temperature (18 °C). The amount of lipids was not affected by food limitation. Although lipid content was reduced in 24 °C, the rate of depuration of OA was not faster for mussels in this treatment and no correlation was detected between lipid content and OA. Depuration rates were very similar for all treatments and followed an exponential decrease relationship ( t 1/2=8 days). Thus, the proposed model that lipid breakdown affects the mechanism of elimination of OA was not supported. Nevertheless, the observed rates of depuration provide useful information and a potential predictive tool for large-scale depuration methods of mussels. The difficulties to influence the rate of depuration of this toxin by changing the environmental conditions suggest that processes, insensitive to short-term manipulation of the external environment, regulate depuration of OA.

Morphological and toxicological variability of Prorocentrum lima clones isolated from four locations in the south-west Indian Ocean

Eight clones of the toxic dinoflagellate Prorocentrum lima (Ehrenberg) Dodge from four sites (two clones per site) on the coral reef of La Réunion, Mayotte, Europa, and Mauritius Islands in the SW Indian Ocean were isolated and cultivated under the same conditions. Morphological features of each clone, including cell size and valve and marginal pore numbers, were examined by scanning electron microscopy. The toxic potential of each clone was determined by protein phosphatase type 2A (PP2A) inhibition test and fibroblast cell line FR3T3 bioassay. Scanning electron microscopy showed that variation in morphological features of clones within and between sites was minimal and not significant. However, equivalent okadaic acid content, determined by PP2A assay, was different within and between clones isolated from the four islands. Cytotoxicity bioassay with the FR3T3 cell line confirmed the variation on global toxic potential within and between the eight P. lima clones. This test also suggested the presence of other toxic compounds without PP2A inhibiting activity in crude extracts of some clones.

Zooplankton as a potential vector of diarrhetic shellfish poisoning toxins through the food web

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 201:155-163 (2000) - doi:10.3354/meps201155 Zooplankton as a potential vector of diarrhetic shellfish poisoning toxins through the food web I. Maneiro1,*, M. Frangópulos1, C. Guisande1, M. Fernández2, B. Reguera3, I. Riveiro1 1Facultad de Ciencias del Mar, Universidad de Vigo, Aptdo. 874, 36200 Vigo, Spain 2Laboratorio de Sanidad Exterior de Vigo, European Community Reference Laboratory on Marine Biotoxins, Estación Marítima s/n, 36271 Vigo, Spain 3Instituto Español de Oceanografía, Centro Costero de Vigo, Aptdo. 1552, 36280 Vigo, Spain *E-mail: imaneiro@uvigo.es ABSTRACT: This study was carried out during an autumn bloom of Dinophysis acuminata in the Galician Rías Bajas (NW Spain). The main objective was to determine whether any of the predominant zooplankton species could ingest and transfer toxins through the pelagic food web. Grazing experiments showed that the copepods Temora longicornis and Oithona nana and the tintinnid Favella serrata fed on Dinophysis spp. whereas the copepods Acartia clausi and Euterpina acutifrons did not. However, only F. serrata seem to profit from the ingestion of the toxic dinoflagellates. Field populations of F. serrata increased whereas T. longicornis and O. nana densities declined after the toxic outbreak. Okadaic acid content found in the seston size fractions 100-200, 200-300 and 300-1000 µm showed a good correlation with F. serrata. These results confirm the hypothesis that tintinnids can transfer dinoflagellates toxins to higher trophic levels in the pelagic food web. The importance of faecal pellets with undigested dinoflagellates as an alternative pathway to transmit toxins to pelagic or benthic organisms is mentioned. KEY WORDS: Zooplankton · Dinoflagellates · Toxins · Dinophysis spp. · DSP · Ingestion rates Full article in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 201. Online publication date: August 09, 2000 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2000 Inter-Research.

Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels

The suitability of a cytotoxicity assay based on the MTT colorimetric method has been evaluated for the detection of okadaic acid in mussels. On KB cells, okadaic acid exhibited a dose-dependent cytotoxic effect, the IC 50 being inversely related to the exposure time ( IC 50 = 6.3 ng/ml, 4.0 ng/ml and 1.1 ng/ml after 24, 48 and 72 hr of contact, respectively). Using a contact time of 24 hr, the MTT cytotoxicity assay is suitable for revealing okadaic acid concentrations in mussel samples as low as 50 ng/g of digestive glands, with a sensitivity higher than that of the commercially available kits for enzyme-linked immunosorbent assay (ELISA). In the okadaic acid concentration range from 50 to 1500 ng/g of digestive glands the MTT cytotoxicity assay showed satisfactory accuracy and reproducibility. A high degree of correlation was found between the okadaic acid content of 16 naturally contaminated samples measured by the MTT cytotoxicity assay and by an ELISA.

Determination of okadaic acid content of dinoflagellate cells: A comparison of the HPLC-fluorescent method and two monoclonal antibody ELISA test kits

Total okadaic acid (okadaic acid plus methylokadaic acid) in acclimated clones of the dinoflagellates Prorocentrum hoffmannianum and P. lima was determined using the HPLC-fluorescent method, UBE ELISA test kit, and Rougier ELISA test kit. The nonokadaic acid-producing species, Amphidinium klebsii, Prorocen- trum mexicanum, P. micans, P. cassubicum, and Gambierdiscus toxicus were examined using the same methods of analysis. All three methods yielded consistent results for P. hoffmannianum which produces only okadaic acid. However, results of the three methods were not consistent for P. lima which produces both okadaic acid and methylokadaic acid. The UBE ELISA demonstrated little or no cross-reactivity with methylokadaic acid; whereas, the Rougier ELISA demonstrated varying degrees of cross-reactivity with that analog. Analyses of nonokadaic acid producing-species yielded negative results, with one exception. The Rougier ELISA demonstrated reactivity with extracts of G. toxicus. Since outbreaks of DSP may be caused by okadaic acid, methylokadaic acid, or a combination of these toxins, both ELISA kits may underestimate total toxin present in toxic shellfish.

Screening for Okadaic Acid by Immunoassay

Increasing incidences of phytoplankton blooms with the potential danger of toxin release into the food chain have necessitated the search for new diagnostic methods that can detect toxins quickly and reliably. A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantitate okadaic acid in shellfish and phytoplankton extracts. To determine the specificity of the assay, a number of toxins, such as calyculin A, brevetoxin-1, and dinophysistoxins-1, -2, and -3 were analyzed. Both dinophysistoxins-2 and -1 could be detected by the assay but in concentration ranges 10- and 20-fold higher than that for okadaic acid, respectively. Dinophysistoxin-3, calyculin A, or brevetoxin-1 could not be detected with this assay. To validate the accuracy of the method, 18 mussel and 7 phytoplankton extracts were analyzed in parallel for okadaic acid content by ELISA and liquid chromatography combined with either fluorescence or mass spectrometric detection. Very high correlation between the results was found.

Environmental effects on the production of okadaic acid from Prorocentrum hoffmannianum Faust I. temperature, light, and salinity

Clonal cultures of Prorocentrum hoffmannianum Faust (clone 882a) were grown under various temperatures, light intensities, and salinities to examine changes in growth rates and okadaic acid content. The acclimated tolerance range for growth was between 21 °C and 36 °C. With regard to light intensity, acclimated growth was possible between 2000 and 5000 lx. Growth rate was optimal at 27 °C under 5000 lx ( k = 0.53 division · day −1). Acclimated growth rate was maximum al a salinity of 34%.. Okadaic acid content changed drastically with regard to temperature and light. No trends were observed between reproduction rates and okadaic acid production. Optimum okadaic acid production (58.6 pg cell −1) was achieved at 24 °C under 2000 lx. A second peak in okadaic acid production (45 pg·cell 1) was observed at 29 °C under 4000 lx. Unlike temperature and light, salinity results showed an inverse trend between okadaic acid content and growth rate. These results suggest an increased okadaic acid content with increased environmental stress.

Maximizing okadaic acid content fromProrocentrum hoffmannianum Faust

Clonal cultures ofProrocentrum hoffmannianum Faust (clone 882a) were grown under optimal environmental conditions for maximal okadaic acid production. The environmental conditions of 25 °C and 86 µmol photon m-2 s-1 were used to cultivateP. hoffmannianum in a semi-continuous 36-L culture vessel with continuous cell suspension and pH control. Using these conditions, a 3-fold increase in harvestable biomass and okadaic acid content was observed when compared to batch culture techniques.

High performance liquid chromatography analysis of diarrhetic toxins inDinophysis spp. from the French coast

Diarrhetic shellfish poisoning has been a recurrent problem along the Brittany coast (France) since 1983. Okadaic acid (O.A.), the main toxin detected in mussels, is generally associated with the presence ofDinophysis cells in sea water. We report here the results of okadaic acid analyses by high performance liquid chromatography, on planktonic samples collected during the summer of 1991.D. sacculus, the majorDinophysis species, throughout this period, showed low okadaic acid content in raw extract, whereas toxic levels were 3- to 12-fold higher in sorted samples than in raw ones. A maximum O.A. concentration of 29.6 pg cell-1 was found inD. sacculus/D. cf. acuminata extract. Similarly, higher O.A. levels were noted in raw samples when these two species were associated. Generally speaking, variations in raw samples were similar to those in sorted samples. Nevertheless, whenD. rotundata reached a concentration equal to that ofD. sacculus in sorted samples, the O.A. level was lower.

Seasonal, geographic and individual variation of okadaic acid content in cultivated mussels in Sweden.

In Western Europe the dinoflagellate toxin, okadaic acid (OA) has been the main cause of diarrheic shellfish poisoning (DSP). Chemical determination of OA in mussels by homogenization of the hepatopancreas, extraction, purification, reaction with 9-anthryldiazomethane (ADAM), HPLC-separation, and fluorometric quantification has been used for weekly monitoring of mussel growing farms and to control harvested mussels. Within a week, substantial rises (from 0.41 to 5.4 micrograms OA/g hepatopancreas) as well as great reductions (from 7.2 to 1.8 micrograms/g hepatopancreas) were recorded. The rapid rise implies that weekly sampling is not sufficient to ensure that mussels are free from toxic levels of OA. The rapid decrease reveals that efficient toxin clearance mechanisms exist in the mussels. Substantial OA clearance occurs also at low temperatures (1.4-3 degrees C). Within a mussel growing site the OA concentrations could differ considerably between adjacent mussels (0.63 and 4.2 micrograms OA/g hepatop.) and even more between mussels grown at different depths along the same rope (0.63 and 10 micrograms OA/g hepatop.). These data emphasize the importance of sampling in studies on DST in mussels. Great differences between the different mussel growing sites were also observed. These data have been discussed with respect to the spread of the toxin by the sea, and the possibilities of reducing the exposure of the mussels to the toxic algae.