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Cyclic imines are a class of lipophilic shellfish toxins comprising gymnodimines, spirolides, pinnatoxins, portimines, pteriatoxins, prorocentrolides, spiro-prorocentrimine, symbiomines and kabirimine. They are structurally diverse, but all share an imine moiety as part of a bicyclic ring system. These compounds are produced by marine microalgal species and are characterized by the rapid death that they induce when injected into mice. Cyclic imines have been detected in a range of shellfish species collected from all over the world, which raises the question as to whether they present a food safety risk. The European Food Safety Authority (EFSA) considers them to be an emerging food safety issue, and in this review, the risk posed by these toxins to shellfish consumers is assessed by collating all available occurrence and toxicity data. Except for pinnatoxins, the risk posed to human health by the cyclic imines appears low, although this is based on only a limited dataset. For pinnatoxins, two different health-based guidance values have been proposed at which the concentration should not be exceeded in shellfish (268 and 23 µg PnTX/kg shellfish flesh), with the discrepancy caused by the application of different uncertainty factors. Pinnatoxins have been recorded globally in multiple shellfish species at concentrations of up to 54 times higher than the lower guidance figure. Despite this observation, pinnatoxins have not been associated with recorded human illness, so it appears that the lower guidance value may be conservative. However, there is insufficient data to generate a more robust guidance value, so additional occurrence data and toxicity information are needed. [ABSTRACT FROM AUTHOR]

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A–D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced 'unknown' compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts. [ABSTRACT FROM AUTHOR]

Paralytic shellfish poisoning is a worldwide problem induced by shellfish contaminated with paralytic shellfish toxins. To protect human health, a regulatory limit for these toxins in shellfish flesh has been adopted by many countries. In a recent study, mice were dosed with saxitoxin and tetrodotoxin mixtures daily for 28 days showing toxicity at low concentrations, which appeared to be at odds with other work. To further investigate this reported toxicity, we dosed groups of mice with saxitoxin and tetrodotoxin mixtures daily for 21 days. In contrast to the previous study, no effects on mouse bodyweight, food consumption, heart rate, blood pressure, grip strength, blood chemistry or hematology were observed. Furthermore, no histological findings were associated with dosing in this trial. The dose rates in this study were 2.6, 3.8 and 4.9 times greater, respectively, than the highest dose of the previous study. As rapid mortality in three out of five mice was observed in the previous study, the deaths are likely to be due to the methodology used rather than the shellfish toxins. To convert animal data to that used in a human risk assessment, a 100-fold safety factor is required. After applying this safety factor, the dose rates used in the current study were 3.5, 5.0 and 6.5 times greater, respectively, than the acute reference dose for each toxin type set by the European Union. Furthermore, it has previously been proposed that tetrodotoxin be included in the paralytic shellfish poisoning suite of toxins. If this were done, the highest dose rate used in this study would be 13 times the acute reference dose. This study suggests that the previous 28-day trial was flawed and that the current paralytic shellfish toxin regulatory limit is fit for purpose. An additional study, feeding mice a diet laced with the test compounds at higher concentrations than those of the current experiment, would be required to comment on whether the current paralytic shellfish toxin regulatory limit should be modified. [ABSTRACT FROM AUTHOR]

Regulatory limits for toxins in shellfish are required to ensure the health of consumers. However, these limits also impact the profitability of shellfish industries making it critical that they are fit for purpose. Since human toxicity data is rarely available, the setting of regulatory limits is dependent on animal data which can then be extrapolated for use in the assessment of human risk. The dependence on animal data to keep humans safe means that it is critical that the toxicity data used is robust and of high quality. Worldwide, the protocols used in toxicity testing are varied, making it hard to compare results and adding confusion over which results better reflect the true toxicity. In this study, we look at the effect of mouse gender, i.p. dose volume, mouse body weight and feeding protocols (both acute and sub-acute) on the toxicity of saxitoxin. This allowed the effect of different variables used in toxicity testing to be understood and showed that the feeding protocol used in both acute and sub-acute studies greatly influenced the toxicity of saxitoxin in mice. Therefore, the adoption of a standard protocol for the testing of shellfish toxins is recommended. [ABSTRACT FROM AUTHOR]

Fungal endophytes in perennial ryegrass are essential to New Zealand's pastoral system due to anti-insect effects. However, endophytes also produce compounds which can be detrimental to animals. Furthermore, as these toxins have been detected in the milk and fat of animals grazing common-toxic (containing lolitrem B) or AR37 endophyte-infected herbage they could enter the human food chain. To assess the risk to human health mice were fed for 90 days with three dose rates of lolitrem B and of AR37. Parameters indicative of animal health were measured as well as chemical, hematological and histological analysis of samples collected on day 90. Since endophyte toxin residues have been detected in milk, they could be transferred from mother to offspring via breast milk. To evaluate possible effects on reproduction two complete generations of mice were fed lolitrem B or AR37. At the dose rates given no adverse effects were observed in either study. The 100-fold safety factor to allow the use of animal data in human health assessments was applied and by considering the concentrations of lolitrem B or AR37 metabolites which could be ingested by a consumer it is highly unlikely that they pose any risk to human health. [ABSTRACT FROM AUTHOR]

Identifying compounds responsible for the observed toxicity of the Gambierdiscus species is a critical step to ascertaining whether they contribute to ciguatera poisoning. Macroalgae samples were collected during research expeditions to Rarotonga (Cook Islands) and North Meyer Island (Kermadec Islands), from which two new Gambierdiscus species were characterized, G. cheloniae CAWD232 and G. honu CAWD242. Previous chemical and toxicological investigations of these species demonstrated that they did not produce the routinely monitored Pacific ciguatoxins nor maitotoxin-1 (MTX-1), yet were highly toxic to mice via intraperitoneal (i.p.) injection. Bioassay-guided fractionation of methanolic extracts, incorporating wet chemistry and chromatographic techniques, was used to isolate two new MTX analogs; MTX-6 from G. cheloniae CAWD232 and MTX-7 from G. honu CAWD242. Structural characterization of the new MTX analogs used a combination of analytical chemistry techniques, including LC–MS, LC–MS/MS, HR–MS, oxidative cleavage and reduction, and NMR spectroscopy. A substantial portion of the MTX-7 structure was elucidated, and (to a lesser extent) that of MTX-6. Key differences from MTX-1 included monosulfation, additional hydroxyl groups, an extra double bond, and in the case of MTX-7, an additional methyl group. To date, this is the most extensive structural characterization performed on an MTX analog since the complete structure of MTX-1 was published in 1993. MTX-7 was extremely toxic to mice via i.p. injection (LD50 of 0.235 µg/kg), although no toxicity was observed at the highest dose rate via oral administration (155.8 µg/kg). Future research is required to investigate the bioaccumulation and likely biotransformation of the MTX analogs in the marine food web. [ABSTRACT FROM AUTHOR]

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis. [ABSTRACT FROM AUTHOR]

Regulatory limits for shellfish toxins are required to protect human health. Often these limits are set using only acute toxicity data, which is significant, as in some communities, shellfish makes up a large proportion of their daily diet and can be contaminated with paralytic shellfish toxins (PSTs) for several months. In the current study, feeding protocols were developed to mimic human feeding behaviour and diets containing three dose rates of saxitoxin dihydrochloride (STX.2HCl) were fed to mice for 21 days. This yielded STX.2HCl dose rates of up to 730 µg/kg bw/day with no effects on food consumption, growth, blood pressure, heart rate, motor coordination, grip strength, blood chemistry, haematology, organ weights or tissue histology. Using the 100-fold safety factor to extrapolate from animals to humans yields a dose rate of 7.3 µg/kg bw/day, which is well above the current acute reference dose (ARfD) of 0.5 µg STX.2HCl eq/kg bw proposed by the European Food Safety Authority. Furthermore, to reach the dose rate of 7.3 µg/kg bw, a 60 or 70 kg human would have to consume 540 or 630 g of shellfish contaminated with PSTs at the current regulatory limit (800 µg/kg shellfish flesh), respectively. The current regulatory limit for PSTs therefore seems appropriate. [ABSTRACT FROM AUTHOR]

Understanding the toxicity and production rates of the various secondary metabolites produced by Gambierdiscus and cohabitating benthic dinoflagellates is essential to unravelling the complexities associated with ciguatera poisoning. In the present study, a sulphated cyclic polyether, gambierone, was purified from Gambierdiscus cheloniae CAWD232 and its acute toxicity was determined using intraperitoneal injection into mice. It was shown to be of low toxicity with an LD50 of 2.4 mg/kg, 9600 times less toxic than the commonly implicated Pacific ciguatoxin-1B, indicating it is unlikely to play a role in ciguatera poisoning. In addition, the production of gambierone and 44-methylgambierone was assessed from 20 isolates of ten Gambierdiscus, two Coolia and two Fukuyoa species using quantitative liquid chromatography–tandem mass spectrometry. Gambierone was produced by seven Gambierdiscus species, ranging from 1 to 87 pg/cell, and one species from each of the genera Coolia and Fukuyoa, ranging from 2 to 17 pg/cell. The production of 44-methylgambierone ranged from 5 to 270 pg/cell and was ubiquitous to all Gambierdiscus species tested, as well as both species of Coolia and Fukuyoa. The relative production ratio of these two secondary metabolites revealed that only two species produced more gambierone, G. carpenteri CAWD237 and G. cheloniae CAWD232. This represents the first report of gambierone acute toxicity and production by these cohabitating benthic dinoflagellate species. While these results demonstrate that gambierones are unlikely to pose a risk to human health, further research is required to understand if they bioaccumulate in the marine food web. [ABSTRACT FROM AUTHOR]

Epoxyjanthitrems I–IV (1–4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichloë festucae var. lolii. Although structures for epoxyjanthitrems I–IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests. [ABSTRACT FROM AUTHOR]

Gambierdiscus and Fukuyoa species have been identified in Aotearoa/New Zealand's coastal waters and G. polynesiensis, a known producer of ciguatoxins, has been isolated from Rangitāhua/Kermadec Islands (a New Zealand territory). The warming of the Tasman Sea and the waters around New Zealand's northern subtropical coastline heighten the risk of Gambierdiscus proliferating in New Zealand. If this occurs, the risk of ciguatera fish poisoning due to consumption of locally caught fish will increase. Research, including the development and testing of sampling methods, molecular assays, and chemical and toxicity tests, will continue. Reliable monitoring strategies are important to manage and mitigate the risk posed by this emerging threat. The research approaches that have been made, many of which will continue, are summarised in this review. [ABSTRACT FROM AUTHOR]

Epichloë endophytes have been used successfully in pastoral grasses providing protection against insect pests through the expression of secondary metabolites. This approach could be extended to other plant species, such as cereals, reducing reliance on pesticides. To be successful, the selected endophyte must express secondary metabolites that are active against cereal insect pests without any secondary metabolite, which is harmful to animals. Chanoclavine is of interest as it is commonly expressed by endophytes and has potential insecticidal activity. Investigation of possible mammalian toxicity is therefore required. An acute oral toxicity study showed the median lethal dose of chanoclavine to be >2000 mg/kg. This allows it to be classified as category 5 using the globally harmonized system of classification and labelling of chemicals, and category 6.1E using the New Zealand Hazardous Substances and New Organisms (HSNO) hazard classes, the lowest hazard class under both systems of classification. A three-week feeding study was also performed, which showed chanoclavine, at a dose rate of 123.9 mg/kg/day, initially reduced food consumption but was resolved by day seven. No toxicologically significant effects on gross pathology, histology, hematology, or blood chemistry were observed. These experiments showed chanoclavine to be of low toxicity and raised no food safety concerns. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) is a potent neurotoxin associated with human poisonings through the consumption of pufferfish. More recently, TTX has been identified in bivalve molluscs from diverse geographical environments, including Europe, and is therefore recognised as an emerging threat to food safety. A recent scientific opinion of the EFSA Panel on Contaminants in the Food Chain recognised the need for further data on the acute oral toxicity of TTX and suggested that, since saxitoxin (STX) and TTX had similar modes of action, it was possible that their toxicities were additive so could perhaps be combined to yield one health-based guideline value. The present study determined the toxicity of TTX by various routes of administration. The testing of three different mixtures of STX and TTX and comparing the experimentally determined values to those predicted on the basis of additive toxicity demonstrated that the toxicities of STX and TTX are additive. This illustrates that it is appropriate to treat TTX as a member of the paralytic shellfish group of toxins. Since the toxicity of TTX was found to be the same as STX by feeding, a molar toxicity equivalence factor of 1.0 for TTX can be applied. [ABSTRACT FROM AUTHOR]