Roel van Ginkel

Isolation, structural determination and acute toxicity of pinnatoxins E, F and G.

Pinnatoxins and pteriatoxins are a group of cyclic imine toxins that have hitherto only been isolated from Japanese shellfish. As with other cyclic imine shellfish toxins, pinnatoxins cause rapid death in the mouse bioassay for lipophilic shellfish toxins, but there is no evidence directly linking these compounds to human illness. We have identified the known pinnatoxins A (1) and D (6), and the novel pinnatoxins E (7), F (8) and G (5), in a range of shellfish and environmental samples from Australia and New Zealand using LC-MS. After isolation from the digestive glands of Pacific oysters, the structures of the novel pinnatoxins were determined by mass spectrometry and NMR spectroscopy, and their LD(50) values were evaluated by ip administration to mice. Examination of the toxin structures, together with analysis of environmental samples, suggests that pinnatoxins F and G are produced separately in different dinoflagellates. Furthermore, it appears probable that pinnatoxin F (8) is the progenitor of pinnatoxins D (6) and E (7), and that pinnatoxin G (6) is the progenitor of both pinnatoxins A-C (1 and 2) and pteriatoxins A-C (3 and 4), via metabolic and hydrolytic transformations in shellfish.

Detection of tetrodotoxin from the grey side-gilled sea slug - Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand

Investigations into a series of dog poisonings on beaches in Auckland, North Island, New Zealand, resulted in the identification of tetrodotoxin (TTX) in the grey side-gilled sea slug, Pleurobranchaea maculata. The levels of TTX in P. maculata, assayed by liquid chromatography-mass spectrometry (LC-MS) ranged from 91 to 850 mg kg(-1) with a median level of 365 mg kg(-1) (n = 12). In two of the dog poisoning cases, vomit and gastrointestinal contents were found to contain TTX. Adult P. maculata were maintained in aquaria for several weeks. Levels of TTX decreased only slightly with time. While in the aquaria, P. maculata spawned, with each individual producing 2-4 egg masses. The egg masses and 2-week old larvae also contained TTX. Tests for other marine toxins were negative and no other organisms from the area contained TTX. This is the first time TTX has been identified in New Zealand and the first detection of TTX in an opisthobranch.

First report of saxitoxin production by a species of the freshwater benthic cyanobacterium, Scytonema Agardh

Saxitoxins or paralytic shellfish poisons (PSP) are neurotoxins produced by some species of freshwater cyanobacteria and marine dinoflagellates. Samples collected from the metaphyton of a drinking-water supplys pre-treatment reservoir and a small eutrophic lake in New Zealand returned positive results when screened using a Jellett PSP Rapid Test Kit. The dominant species in the sample was identified as Scytonema cf. crispum. A non-axenic clonal culture (UCFS10) was isolated from the lake. The partial 16S rRNA gene sequence shared only a 91% or less sequence similarity with other Scytonema species, indicating that it is unlikely that this genus is monophyletic and that further in-depth phylogenetic re-evaluation is required. The sxtA gene, which is known to be involved in saxitoxin production, was detected in UCFS10. Saxitoxin concentrations were determined from the lake samples and from UCFS10 using pre-column oxidation high performance liquid chromatography with fluorescence detection. Saxitoxin was the only variant detected and this was found at concentrations of 65.6 μg g⁻¹ dry weight in the lake sample and 119.4 μg g⁻¹ dry weight or 1.3 pg cell⁻¹ in UCFS10. This is the first confirmation of a saxitoxin-producing species in New Zealand and the first report of saxitoxin production by a species of Scytonema.

A sensitive assay for palytoxins, ovatoxins and ostreocins using LC-MS/ MS analysis of cleavage fragments from micro-scale oxidation

Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg⁻¹ for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg⁻¹) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of ≤ 18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL⁻¹ and the LOQ 4 ng mL⁻¹, which corresponds to 10 μg kg⁻¹ in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required.

Semisynthesis of S-desoxybrevetoxin-B2 and brevetoxin-B2, and assessment of their acute toxicities.

Brevetoxins are neurotoxins associated with blooms of marine algae such as Karenia brevis and can accumulate in the marine food chain, causing intoxication of marine animals and people consuming seafood. Brevetoxin-B2 ( 5) is a toxic metabolite produced in shellfish exposed to algae that contain brevetoxin-B ( 1). S-Desoxybrevetoxin-B2 ( 4) has been proposed as a cometabolite produced during this transformation, and while LC-MS analyses suggest its presence in shellfish, it has not yet been isolated and characterized. Studies on these materials are severely constrained by the difficulty of obtaining and purifying them from natural sources. We have developed a convenient one-pot conversion of commercially available brevetoxin-B ( 1) into S-desoxybrevetoxin-B2 ( 4), and a simple method for converting 4 into brevetoxin-B2 ( 5). Full NMR and mass-spectral characterization of 4 and 5 confirmed their structures and showed that the ratio of diastereoisomers in the synthetic 4 and 5 was similar to that observed in naturally contaminated shellfish. The LD 50 values for 4, 5, and dihydrobrevetoxin-B ( 6) by ip injection in mice were 211, 400, and 250 microg/kg, respectively. The methodology for synthesis of brevetoxin metabolites should greatly facilitate toxicological, biochemical and immunochemical studies of these substances, as well as the production of analytical standards.

Bioassay methods for detection of N-palmitoylbrevetoxin-B2 (BTX-B4)

Brevetoxins (BTXs) are a class of cyclic polyether toxins produced by the dinoflagellate Karenia brevis. These substances are subject to extensive conjugative metabolism in shellfish. BTX-B forms a conjugate with cysteine and is oxidized and reduced to yield BTX-B2, which is further modified by fatty acid addition via cysteine amide linkage to give biologically active brevetoxin metabolites. In this study, we evaluated the commonly used in vitro (ELISA, radioimmunoassay, receptor binding assay and N2A cytotoxicity assay) and in vivo mouse brevetoxin bioassays for the detection of the brevetoxin fatty acid conjugate N-palmitoylBTX-B2, and compared the results to those for dihydroBTX-B and BTX-B2. The receptor binding assay for N-palmitoylBTX-B2 showed comparable sensitivity to that for dihydroBTX-B, and an 11-fold higher sensitivity than for BTX-B2. Although the ELISA showed similarly high sensitivity to dihydroBTX-B and BTX-B2, with EC(50) values of ca. 0.26 ng/ml, it was 23 times less sensitive to N-palmitoylBTX-B2. On the other hand, the N2A cytotoxicity assay was highly sensitive to N-palmitoylBTX-B2, with an EC(50) of 0.15 ng/ml, but was 12- and 40-fold less sensitive to dihydroBTX-B and BTX-B2, respectively. The relative sensitivity of the N2A cytotoxicity assay for each of these metabolites paralleled that of the mouse bioassay (relative LD(50) values 1:20:30 for N-palmitoylBTX-B2:dihydroBTX-B:BTX-B2). We conclude that the most sensitive bioassay for dihydroBTX-B and BTX-B2 is the ELISA, whereas the N2A cytotoxicity assay is most sensitive for N-palmitoylBTX-B2.

Paralytic shellfish toxins, including deoxydecarbamoyl-STX, in wild-caught Tasmanian abalone (Haliotis rubra)

For the first time wild-caught Tasmanian abalone, Haliotis rubra, have been reported to contain paralytic shellfish toxins (PSTs). This observation followed blooms of the toxic dinoflagellate Gymnodinium catenatum. No illnesses were reported, but harvesting restrictions were enforced in commercial areas. Abalone were assayed using HPLC-FLD methodology based on AOAC official method 2005.06. An uncommon congener, deoxydecarbamoyl-STX (doSTX), was observed in addition to regulated PSTs as unassigned chromatographic peaks. A quantitative reference material was prepared from contaminated Tasmanian abalone viscera and ampouled at 54.2 μmol/L. The LD50 of doSTX via intraperitoneal injection was 1069 nmol/kg (95% confidence limits 983-1100 nmol/kg), indicating it is nearly 40 times less toxic than STX. A toxicity equivalence factor of 0.042 was generated using the mouse bioassay. Levels of PSTs varied among individuals from the same site, although the toxin profile remained relatively consistent. In the foot tissue, STX, decarbamoyl-STX and doSTX were identified. On a molar basis doSTX was the dominant congener in both foot and viscera samples. The viscera toxin profile was more complex, with other less toxic PST congeners observed and was similar to mussels from the same site. This finding implicates localised dinoflagellate blooms as the PST source in Tasmanian abalone.

Uptake, distribution and depuration of paralytic shellfish toxins from Alexandrium minutum in Australian greenlip abalone, Haliotis laevigata

Farmed greenlip abalone Haliotis laevigata were fed commercial seaweed-based food pellets or feed pellets supplemented with 8 × 10⁵ Alexandrium minutum dinoflagellate cells g⁻¹ (containing 12 ± 3.0 μg STX-equivalent 100 g⁻¹, which was mainly GTX-1,4) every second day for 50 days. Exposure of abalone to PST supplemented feed for 50 days did not affect behaviour or survival but saw accumulation of up to 1.6 μg STX-equivalent 100 g⁻¹ in the abalone foot tissue (muscle, mouth without oesophagus and epipodial fringe), which is ∼50 times lower than the maximum permissible limit (80 μg 100 g⁻¹ tissue) for PSTs in molluscan shellfish. The PST levels in the foot were reduced to 0.48 μg STX-equivalent 100 g⁻¹ after scrubbing and removal of the pigment surrounding the epithelium of the epipodial fringe (confirmed by both HPLC and LC-MS/MS). Thus, scrubbing the epipodial fringe, a common procedure during commercial abalone canning, reduced PST levels by ∼70%. Only trace levels of PSTs were detected in the viscera (stomach, gut, heart, gonad, gills and mantle) of the abalone. A toxin reduction of approximately 73% was observed in STX-contaminated abalone held in clean water and fed uncontaminated food over 50 days. The low level of PST uptake when abalone were exposed to high numbers of A. minutum cells over a prolonged period may indicate a low risk of PSP poisoning to humans from the consumption of H. laevigata that has been exposed to a bloom of potentially toxic A. minutum in Australia. Further research is required to establish if non-dietary accumulation can result in significant levels of PSTs in abalone.