Parent Toxin Research Articles

Deoxynivalenol Oligoglycosides: New “Masked” Fusarium Toxins Occurring in Malt, Beer, and Breadstuff

The co-occurrence of deoxynivalenol-3-glucoside with its parent toxin, deoxynivalenol, has been recently documented in many cereal-based foods, especially in those produced by enzyme-catalyzed processes. The presence of this masked mycotoxin in the human diet has become an issue of health concern, mainly because of its assumed bioavailability. A selective immunoaffinity-based preconcentration strategy, followed by ultrahigh-performance liquid chromatography coupled with high-resolution orbitrap mass spectrometry, revealed that, in addition to the most common deoxynivalenol-3-glucoside, also oligoglycosylated deoxynivalenols with up to four bound hexose units were present in cereal-based products. The structure, origination, and fate of these deoxynivalenol conjugates during malt/beer production and bread baking have been thoroughly investigated. Special attention has been paid to the changes of deoxynivalenol conjugates enabled by industrial glycosidase-based enzymatic preparations. To the authors' best knowledge, this is the first study documenting the complexity of masked deoxynivalenol issue.

Occurrence of different trichothecenes and deoxynivalenol-3-β-d-glucoside in naturally and artificially contaminated Danish cereal grains and whole maize plants

Fusarium mycotoxins such as deoxynivalenol (DON) can occur in cereals conjugated to glucose and probably also to other sugars. These conjugates, which are often referred to as "masked mycotoxins", will not be detected with routine analytical techniques. Furthermore, it is suspected that the parent toxin may again be released after hydrolysis in the digestive tracts of animals and humans. Today, our knowledge of the occurrence of these compounds in cereal grains is limited. In this paper, a LC-MS/MS method for the simultaneous determination of DON, deoxynivalenol-3-β-D-glucoside (DON-3-glucoside), 3 acetyl-DON, nivalenol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin in naturally (n = 48) and artificially (n = 30) contaminated cereal grains (wheat, barley, oat, rye triticale) is reported. The method has also been applied to whole fresh maize plant intended for production of maize silage (n = 10). The samples were collected from the harvest years 2006-2010, The results show that DON-3-glucoside and DON co-occurred in cereal grains and, especially in several of the highly contaminated samples, the concentration of the glucoside can be relatively high, corresponding to over 37% of the DON concentration. The DON-3-glucoside levels in both the naturally and in the artificially grain inoculated with Fusarium were second only to DON, and were generally higher than those of the other tested trichothecenes, which were found at low concentrations in most samples, in many cases even below the detection limit of the method. This argues for the importance of taking DON-3-glucoside into account in the ongoing discussion within the European Community concerning exposure re-evaluations for setting changed values for the tolerable intake for DON. Our results indicate that, in the naturally contaminated grains and in the Fusarium infested cereal grains (winter and spring wheat, oat, triticale), the concentration level of DON-3-glucoside is positively correlated to the DON content. When the DON concentration is high, then the content of DON-3-glucoside will most probably also be high and vice versa.

Synthesis of deoxynivalenol-3-ß-D-O-glucuronide for its use as biomarker for dietary deoxynivalenol exposure

Trichothecene mycotoxins are prevalent toxic secondary metabolic products of several fungal species and pose a serious threat to human and animal health. Deoxynivalenol (DON) is known to undergo rapid metabolisation after uptake. The formed glucuronides are urinary excreted and could therefore serve as possible biomarkers for daily uptake measurement. So far human exposure to the major toxin DON was estimated from dietary average intake or by measurement of the parent toxin after hydrolysis. These approaches are indirect and time-consuming. Due to the clear demand for a direct determination method and lack of an available reference substance we synthesised DON-3-O-ö-D-glucuronide. The Königs-Knorr procedure using acetobromo-α-D-glucuronic acid methyl ester as glucuronyl-donor was optimised to produce the target compound in mg scale allowing subsequent characterisation via nuclear magnetic resonance and LC-MS/MS.

Botulinum neurotoxin D-C uses synaptotagmin I/II as receptors and human synaptotagmin II is not an effective receptor for type B, D-C, and G toxins

Botulinum neurotoxins (BoNTs) are classified into seven types (A-G), but multiple subtype and mosaic toxins exist. These subtype and mosaic toxins share a high sequence identity, and presumably the same receptors and substrates with their parental toxins. Here, we report that a mosaic toxin, type D-C (BoNT/D-C), uses different receptors from its parental toxin BoNT/C. BoNT/D-C, but not BoNT/C, binds directly to the luminal domains of synaptic vesicle proteins synaptotagmin (Syt) I and II, and requires expression of SytI/II to enter neurons. The SytII luminal fragment containing the toxin-binding site can block the entry of BoNT/D-C into neurons and reduce its toxicity in vivo in mice. We also found that gangliosides increase binding of BoNT/D-C to SytI/II and enhance the ability of the SytII luminal fragment to block BoNT/D-C entry into neurons. These data establish SytI/II, in conjunction with gangliosides, as the receptors for BoNT/D-C, and indicate that BoNT/D-C is functionally distinct from BoNT/C. We further found that BoNT/D-C recognizes the same binding site on SytI/II where BoNT/B and G also bind, but utilizes a receptor-binding interface that is distinct from BoNT/B and G. Finally, we also report that human and chimpanzee SytII has diminished binding and function as the receptor for BoNT/B, D-C and G owing to a single residue change from rodent SytII within the toxin binding site, potentially reducing the potency of these BoNTs in humans and chimpanzees.

The Impact of the Fusarium Mycotoxin Deoxynivalenol on the Health and Performance of Broiler Chickens

The aim of the present experiment was to investigate the effects of feeding grains naturally contaminated with Fusarium mycotoxins on morphometric indices of jejunum and to follow the passage of deoxynivalenol (DON) through subsequent segments of the digestive tract of broilers. A total of 45 1-d-old broiler chickens (Ross 308 males) were randomly allotted to three dietary treatments (15 birds/treatment): (1) control diet; (2) diet contaminated with 1 mg DON/kg feed; (3) diet contaminated with 5 mg DON/kg feed for five weeks. None of the zootechnical traits (body weight, body weight gain, feed intake, and feed conversion) responded to increased DON levels in the diet. However, DON at both dietary levels (1 mg and 5 mg DON/kg feed) significantly altered the small intestinal morphology. In the jejunum, the villi were significantly (P < 0.01) shorter in both DON treated groups compared with the controls. Furthermore, the dietary inclusion of DON decreased (P < 0.05) the villus surface area in both DON treated groups. The absolute or relative organ weights (liver, heart, proventriculus, gizzard, small intestine, spleen, pancreas, colon, cecum, bursa of Fabricius and thymus) were not altered (P > 0.05) in broilers fed the diet containing DON compared with controls. DON and de-epoxy-DON (DOM-1) were analyzed in serum, bile, liver, feces and digesta from consecutive segments of the digestive tract (gizzard, cecum, and rectum). Concentrations of DON and its metabolite DOM-1 in serum, bile, and liver were lower than the detection limits of the applied liquid chromatography coupled with mass spectrometry (LC-MS/MS) method. Only about 10 to 12% and 6% of the ingested DON was recovered in gizzard and feces, irrespective of the dietary DON-concentration. However, the DON recovery in the cecum as percentage of DON-intake varied between 18 to 22% and was not influenced by dietary DON-concentration. Interestingly, in the present trial, DOM-1 did not appear in the large intestine and in feces. The results indicate that deepoxydation in the present study hardly occurred in the distal segments of the digestive tract, assuming that the complete de-epoxydation occurs in the proximal small intestine where the majority of the parent toxin is absorbed. In conclusion, diets with DON contamination below levels that induce a negative impact on performance could alter small intestinal morphology in broilers. Additionally, the results confirm that the majority of the ingested DON quickly disappears through the gastrointestinal tract.

Effects of Milling and Baking Technologies on Levels of Deoxynivalenol and its Masked Form Deoxynivalenol-3-Glucoside

The co-occurrence of the major Fusarium mycotoxin deoxynivalenol (DON) and its conjugate deoxynivalenol-3-glucoside (DON-3-Glc) has been documented in infected wheat. This study reports on the fate of this masked DON within milling and baking technologies for the first time and compares its levels with those of the free parent toxin. The fractionation of DON-3-Glc and DON in milling fractions was similar, tested white flours contained only approximately 60% of their content in unprocessed wheat grains. No substantial changes of both target analytes occurred during the dough preparation process, i.e. kneading, fermentation, and proofing. However, when bakery improvers enzymes mixtures were employed as a dough ingredient, a distinct increase up to 145% of conjugated DON-3-Glc occurred in fermented dough. Some decrease of both DON-3-Glc and DON (10 and 13%, respectively, compared to fermented dough) took place during baking. Thermal degradation products of DON, namely norDON A, B, C, D, and DON-lactone were detected in roasted wheat samples and baked bread samples by means of UPLC-Orbitrap MS. Moreover, thermal degradation products derived from DON-3-Glc were detected and tentatively identified in heat-treated contaminated wheat and bread based on accurate mass measurement performed under the ultrahigh mass resolving power. These products, originating from DON-3-Glc through de-epoxidation and other structural changes in the seskviterpene cycle, were named norDON-3-Glc A, B, C, D, and DON-3-Glc-lactone analogically to DON degradation products. Most of these compounds were located in the crust of experimental breads.

Chimeric δ-endotoxins of Bacillus thuringiensis with increased activity against Helicoverpa armigera

We selected 50 crystal (Cry) toxins specific to lepidopteran insects. Phylogenetic relationships of whole toxins as well as individual domains revealed that Cry1Jb and Cry1Ac are distantly placed but related to reduced cross-resistance in Helicoverpa armigera. Multiple alignments of Cry1Jb and Cry1Ac amino acid sequences showed significant differences in the composition and length of the loops of domains II and III. This was further confirmed by the superpositioning of 3D structures. cry1Jb and cry1Ac genes cloned in expression vectors were overexpressed in Escherichia coli Castellani & Chalmers, and proteins were harvested. Insect bioassays revealed that the wild-type Cry1Jb and Cry1Ac proteins showed differential specificity to H. armigera (Hubner), Spodoptera litura (Fab.) and Earias vittella F. Chimeric genes were constructed by exchanging the domains between cry1Jb and cry1Ac and overexpressed in BL21(DE3). Substitution of domain III of Cry1Jb with that of Cry1Ac enhanced its activity against H. armigera 7.8-fold. Bioassay of the parental and chimeric toxins against S. litura revealed no activity even at the discriminative dose of 100 μg/ml, but a significant difference in growth was observed. The present results, along with previous domain swapping experiments, suggest that protein engineering not only reveals the mechanism by which endotoxins work, but also generates novel toxins with enhanced toxicity/broader specificity.

Isolation and molecular cloning of beta-neurotoxins from the venom of the scorpion Centruroides suffusus suffusus

This communication reports the identification and characterization of two new toxins from the venom of the scorpion Centruroides suffusus suffusus, named: CssVIII and CssIX, according to the original nomenclature of toxins previously described for this scorpion. The isolation was obtained by means of two chromatographic steps, and a cDNA library was used to fully identify their precursors. CssVIII and CssIX contain signal peptides of 19 and 17 amino acid residues, and mature peptides of 66 and 65 residues, respectively. Intracranial injections into mice of both purified toxins showed toxicity results similar to those found for toxins CssII and CssIV. Additionally, they compete with the parent toxin CssIV, in binding and displacement experiments, conducted with brain synaptosomes showing nanomolar affinities. These results strongly support the conclusion that they are new β-neurotoxins and certainly would be of the interest of researchers in the field of venomics for studying sodium channels.

Effects of Mutations Within Surface-Exposed Loops in the Pore-Forming Domain of the Cry9Ca Insecticidal Toxin of Bacillus thuringiensis

The pore-forming domain of Bacillus thuringiensis insecticidal Cry toxins is formed of seven amphipathic α-helices. Because pore formation is thought to involve conformational changes within this domain, the possible role of its interhelical loops in this crucial step was investigated with Cry9Ca double mutants, which all share the previously characterized R164A mutation, using a combination of homology modeling, bioassays and electrophysiological measurements. The mutations either introduced, neutralized or reversed an electrical charge carried by a single residue of one of the domain I loops. The ability of the 28 Cry9Ca double mutants to depolarize the apical membrane of freshly isolated Manduca sexta larval midguts was tested in the presence of either midgut juice or a cocktail of protease inhibitors because these conditions had been shown earlier to greatly enhance pore formation by Cry9Ca and its R164A single-site mutant. Most mutants retained toxicity toward neonate larvae and a pore-forming ability in the electrophysiological assay, which were comparable to those of their parental toxin. In contrast, mutants F130D, L186D and V189D were very poorly toxic and practically inactive in vitro. On the other hand, mutant E129A depolarized the midgut membrane efficiently despite a considerably reduced toxicity, and mutant Q192E displayed a reduced depolarizing ability while conserving a near wild-type toxicity. These results suggest that the conditions found in the insect midgut, including high ionic strength, contribute to minimizing the influence of surface charges on the ability of Cry9Ca and probably other B. thuringiensis toxins to form pores within their target membrane.

Delivering Drugs Into the Brain: Barriers and Possibilities

Delivering Drugs Into the Brain: Barriers and Possibilities

Naturally contaminated shellfish samples: quantification of diarrhetic shellfish poisoning toxins in unhydrolysed and hydrolysed extracts and cytotoxicity assessment

Contamination of shellfish from the Portuguese coast with diarrhetic shellfish poisoning (DSP) toxins is a recurrent event, with most of the commercial bivalves contaminated with high percentages of esters of okadaic acid (OA) and dinophysistoxin-2 (DTX2). This report describes the quantification of DSP toxins in unhydrolysed and hydrolysed extracts of several cockle and mussel samples naturally contaminated and the evaluation of their cytotoxicity profiles in V79 cells. The quantification of the acyl esters in the shellfish samples involved the cleavage of the ester bond through alkaline hydrolysis and the release of the parent toxins OA and DTX2. Unhydrolysed and hydrolysed extracts were then analyzed by liquid chromatography (LC) coupled with mass spectrometry (MS) for the detection and quantification of DSP toxins. The cytotoxicity of the analysed extracts was evaluated using the MTT reduction assay and compared with the cytotoxicity presented by different concentrations of OA standard (1-100 nM). OA exhibited marked cytotoxic effects and decreased cell viability in a dose dependent mode, with an IC₅₀ of 27 nM. The cytotoxicity pattern of unhydrolysed extracts was clearly dependent on the concentration of free toxins. Moreover, the cytotoxicity of the esterified toxins present was revealed after their conversion into free toxins by alkaline hydrolysis. For the hydrolysed extracts of cockles and mussels, the cytotoxicity presented was mainly related to the concentration of OA and DTX2.

Possible mechanism for the foodweb transfer of covalently bound microcystins

Microcystins (MCs) are cyanobacterial toxins that inhibit protein phosphatases 1 and 2A (PP1, PP2A) within an animal through both reversible and covalent interactions. Only MCs that have accumulated in animal tissue in reversible interactions are currently considered when estimating risk to higher trophic levels and humans through food web exposure. However, the majority of MCs is likely covalently bound to target proteins in tissues and these MCs are not quantified or included in these assessments. These covalently bound MCs may be made bioavailable in the digestive system of a consumer through the digestion of their attached protein phosphatase. Three common digestive enzymes, pepsin, chymotrypsin, and trypsin, did not digest cyclic MC-LR and MC-LY, but were very active against a control peptide with typical linkages and standard amino acids in “L” conformation, supporting the possibility for MC-peptide formation during gut passage. To test if digestion products could be biologically active in the consumer, four predicted MC-peptides were synthesized and assayed for activity against PP1 by the protein phosphatase inhibition assay (PPIA). All four MC-peptides were active against PP1 and comparably half (58%) as inhibitory as the parent toxin. This in vitro study demonstrated that MCs covalently bound to proteins may represent a reservoir of potential toxicity for consumers.

Activity of Bacillus thuringiensis hybrid protein against a lepidopteran and a coleopteran pest

The use of Cry proteins from Bacillus thuringiensis are an important strategy for biological control. Recently it has been demonstrated that Cry hybrid proteins (by domain swapping) resulted in improved toxicities in comparison with parental proteins. Here, an SN1917 hybrid toxin was constructed and tested against Colombian pest insects Tecia solanivora (Lepidoptera: Gelechiidae), a severe potato pest, and Hypothenemus hampei (Coleoptera: Scolytidae), which attacks coffee crops. The SN1917 protoxin had a concentration causing 50% mortality (LC(50)) of 392 ng cm(-2), and SN1917 toxin showed an LC(50) of 201 ng cm(-2) against T. solanivora first instar larvae. The two parental toxins (Cry1B and Cry1I) used to make this new hybrid protein caused a mortality of 60% and 52%, respectively. Unfortunately, H. hampei first instar larvae proved to be resistant to the toxin. We conclude that SN1917 is an option for biological control and resistance management of T. solanivora. Implications for H. hampei are discussed.

Neuro-exocytosis: botulinum toxins as inhibitory probes and versatile therapeutics☆

For the fundamental process of quantal neurotransmitter release, a consensus is being reached on the recycling pathways for transmitter-containing, small synaptic vesicles (SSVs), and major inroads have been made into deciphering the multiple steps of regulated exocytosis. These advances arose from the identification of approximately 80 proteins in SSVs, elucidation of the structures of pertinent macromolecular complexes, utilisation of different serotypes (A-G) of botulinum neurotoxin (BoNT) together with transgenic mice lacking key genes. Hence, converging evidence continues to emerge for the sequential formation of complexes between the three SNAREs (SNAP-25, syntaxin and VAMP) and their regulatory proteins (complexins, Munc18), as well as for the Ca2+ triggering of membrane fusion/exocytosis via its sensor, synaptotagmin. Moreover, molecular data gained on BoNTs have been translated into Clinical Medicine with type A now being applied worldwide for effectively treating >100 human conditions due to overactivity of nerves supplying various muscles or glands. A recent advance is the successful engineering of a chimera from two BoNTs to acquire the capability of re-targeting a more active moiety to sensory neurons, with resultant inhibition of the release of a pain mediator. Encouragingly, this novel recombinant protein blocks the exocytotic response triggered by a stimulant (capsaicin) of nociceptive C fibres that activates their vanilloid receptors, a feat not possible for either parental toxin. Reaching this landmark has generated optimism for designing further variants of such a versatile therapeutic for normalising the hyper-activity of particular cell types, especially those underlying the many cases of chronic pain that do not respond to existing drugs.

Toxin composition of a Prorocentrum lima strain isolated from the Portuguese coast

Microalgae of the genus Dinophysis and Prorocentrum are known producers of okadaites, responsible for the human syndrome known as diarrhetic shellfish poisoning (DSP). In temperate regions, only species from the genus Dinophysis are commonly held responsible for shellfish contamination. This is probably related to the different ecological strategies of the two genera, namely the planktonic nature of Dinophysis versus the benthic/epiphytic nature of toxic Prorocentrum species. In recent years, the threat of global warming has drawn attention to the study of benthic toxic microalgae in southern European waters. Here we present results on the toxin production and toxin profile of a Prorocentrum lima strain isolated from the Portuguese coast. This strain, IO66-01, presented a mean growth rate of 0.49 divisions d −1, not common in temperate strains, and only comparable with tropical strains. The parent toxins found were okadaic acid (OA) and dinophysistoxin-1 (DTX1). The major diol esters were D8- and D9- congeners of both OA and DTX1.

Temperature and base requirements for the alkaline hydrolysis of okadaite's esters

Portuguese bivalves are recurrently contaminated with okadaic acid (OA) and dinophysistoxin-2 (DTX2), found mainly in esterified forms. Throughout the years different conditions have been reported in the literature for releasing the parent toxins through an alkaline hydrolysis step, in order to simplify their detection by HPLC-FLD or LC–MS. In order to clearly understand toxin stability and reaction end-point the binominous temperature/time course and base concentration were studied using naturally contaminated bivalve samples. The results showed a strong temperature dependence of the reaction. At 60 °C and 70 °C the hydrolysis was fast, and 40 min were sufficient for maximal recovery of OA and DTX2, while at 40 °C and 50 °C it was only complete after 100 min and 60 min, respectively. At room temperature the reaction was slow and incomplete even after 2 h. Stability of OA and DTX2 in semi-purified bivalve matrix at 70 °C for 2 h was demonstrated. Concentrations of sodium hydroxide lower than 2.5 M, corresponding to a final incubation concentration of 0.23 M, resulted in incomplete release of parent toxins, demonstrating that high concentrations are needed when taking into account the dilution in the supernatant extract.

Detection of Diarrheic Shellfish Poisoning and Azaspiracids Toxins in Moroccan Mussels: Comparison of LC-MS Method with the Commercial Immunoassay Kit

Diarrheic shellfish poisoning (DSP) is a recurrent gastrointestinal illness in Morocco, resulting from consumption of contaminated shellfish. In order to develop a rapid and reliable technique for toxins detection, we have compared the results obtained by a commercial immunoassay-“DSP-Check” kit” with those obtained by LC-MS. Both techniques are capable of detecting the toxins in the whole flesh extract which was subjected to prior alkaline hydrolysis in order to detect simultaneously the esterified and non esterified toxin forms. The LC-MS method was found to be able to detect a high level of okadaic acid (OA), low level of dinophysistoxin-2 (DTX2), and surprisingly, traces of azaspiracids 2 (AZA2) in mussels. This is the first report of a survey carried out for azaspiracid (AZP) contamination of shellfish harvested in the coastal areas of Morocco. The “DSP-Check” kit was found to detect quantitatively DSP toxins in all contaminated samples containing only OA, provided that the parent toxins were within the range of detection and was not in an ester form. A good correlation was observed between the two methods when appropriate dilutions were performed. The immunoassay kit appeared to be more sensitive, specific and faster than LC-MS for determination of DSP in total shellfish extract.

New Esters of Okadaic Acid in Seawater and Blue Mussels (Mytilus edulis)

Marine algal toxins of the okadaic acid (OA) group can occur as diol esters and sulfated diol esters in algae and as fatty acid esters in shellfish. Several of these ester forms have been identified, but the most common procedure for detecting OA group toxin esters is by measuring the increase in parent toxin after alkaline hydrolysis. Use of this alkaline hydrolysis method led to the discovery of high levels of conjugates of OA and dinophysistoxins-2 (DTX2) in seawater and of OA, DTX1, and DTX2 in blue mussel hepatopancreas (HP) from Flødevigen, Norway, during a bloom of Dinophysis spp. In the water sample, a C 8-diol ester, a C 9-diol ester, and a previously undescribed C 8-triol ester of OA were characterized using HPLC-MS (2), -MS (3), and -MS (4) in combination with various derivatization procedures. Palmitic acid (16:0) ester derivatives of these diol/triol esters were found in mussel HP and characterized using HPLC-MS (2), -MS (3), and -MS (4). To the authors' knowledge, hybrid diol-fatty acid esters of OA have not been previously described. Mass spectral analysis showed the presence of two forms of hybrid esters: one with the fatty acid conjugated to the 7-OH of the OA moiety and the other with the fatty acid conjugated to the OH group in the "diol" moiety. In the water sample, the C 8-diol ester was the most abundant, whereas in the mussels, the 16:0-C 9-diol hybrid ester was most abundant, and only minor amounts of the 16:0-C 8-diol hybrid ester were detected, suggesting that C 8- and C 9-diol esters of OA may be metabolized differently in blue mussels. 7- O-acyl esters of OA, DTX1, and DTX2 are thought to contribute to shellfish toxicity by being hydrolyzed in the human stomach to the parent toxins, and the newly characterized hybrid esters are likely to contribute similarly.

Characterization of New Formalin-Detoxified Botulinum Neurotoxin Toxoids

Antigenicities of several formalin-detoxified botulinum neurotoxin preparations were measured by inhibition and sandwich enzyme-linked immunosorbent assay (ELISA), and immunogenicity was studied in mice. The toxoids were derived primarily from the serotype A 150-kDa neurotoxin protein, while one toxoid was derived from the naturally occurring 900-kDa toxin-hemagglutinin complex. Antigenicity was severely compromised in two commercially available toxoids. A variety of new toxoids were synthesized in-house by optimizing formaldehyde reaction conditions. Three of the resulting toxoids were found to be antigenically identical to the native toxin, as measured by inhibition ELISA, in spite of showing a reduction of toxicity by more than 100,000-fold. Sandwich ELISAs indicated that the in-house toxoids were two- to threefold less antigenic than the neurotoxin compared to commercial toxoids, which were about 100-fold less antigenic. Mice were immunized twice, on day 0 and day 14. By day 28, relatively high toxin-specific immunoglobulin G (IgG) titers were detected in animals that had received any of the in-house toxoids, with greater than 99% being IgG1 and the remainder being IgG2. These immunized mice remained asymptomatic after being challenged with 50 to 1,000,000 50% lethal dose (LD(50)) units of the 900-kDa neurotoxin. In contrast, animals immunized with several different batches of commercially available toxoids did not develop measurable toxin-specific antibody titers. However, these mice survived neurotoxin challenges with 2 LD(50) units but died when challenged with 6 LD(50) units. Neutralizing titers measured from pools of sera generated with the in-house toxoid preparations ranged from 2.5 to 5 U/ml. In terms of predicting immunogenicity, inhibition ELISAs comparing each formalin toxoid to the parent toxin provided good insight for screening the new toxoids as well as for estimating their relative in vivo potencies. Inhibition ELISA data indicate that those toxoids that most closely resemble the native toxin are highly immunogenic and protective. The superior quality of these new toxoids makes them useful tools for continued use in ELISA development and for antitoxin production.

Profiles and levels of fatty acid esters of okadaic acid group toxins and pectenotoxins during toxin depuration. Part II: Blue mussels ( Mytilus edulis) and flat oyster ( Ostrea edulis)

Bivalve molluscs accumulate toxins of the okadaic acid (OA) and pectenotoxin (PTX) groups, which are frequently found in Dinophysis spp. Transformation of the OA-group toxins into fatty acid ester derivatives (often designated “DTX3”) is common in many bivalve species but the degree to which these toxins are transformed vary between species, and is also depending on the parent toxin involved. In this paper, detailed profiles and levels of fatty acid esters of OA, DTX1, DTX2 and PTX2 SA were studied in blue mussels ( Mytilus edulis) and European flat oysters ( Ostrea edulis), collected during a bloom of Dinophysis spp. and after 3 and 6 weeks of depuration. Analysis of samples by HPLC-MS/MS and HPLC-MS 2 revealed some differences in identity and abundance of fatty acid moieties of the OA-group esters between species, but the 16:0 fatty acid esters dominated in both oysters and mussels, which is in accordance with the free fatty acid profiles in these species. A wider range of PTX2 SA-esters were detected compared to esters of the OA-group toxins in both mussels and oysters, and in oysters, both 14:0, 18:4 and 20:5 fatty acid side chains were more common than 16:0. OA-group toxins were esterified to a larger degree in oysters (83–93%) compared to mussels (21–41%), and in mussels a higher proportion of OA was esterified compared to DTX1 and DTX2. Contrary to what was observed for OA-group toxins, PTX2 SA was esterified to a larger degree in mussels (81%) compared to oysters (64%). Calculations of depuration rates for all individual esters of each parent compound showed that the esters of DTX1 depurated significantly slower from both mussels and oysters compared to esters of OA, DTX2 and PTX2 SA, but overall the depuration rates of esters of both toxin group were highly similar for both species. This indicated that differences in depuration rates are not causing the large species-specific differences in levels and profiles of these toxins. Instead, the results for the OA-group toxins suggested that a higher rate of esterification in oysters is the main factor causing the observed differences in the proportion of esters to free toxin. For PTX2 SA, large differences in ester profiles and a higher proportion of esters of PTX2 SA in mussels compared to oysters suggested differential assimilation and metabolic rate processes for the PTXs compared to OA-group toxins between these species. Hence, although produced by the same Dinophysis species, conclusions about the dynamics of one toxin group based on results from the other group should be avoided in future studies.