Research on Marine Toxins in Taiwan

Some health issues of marine toxins in Taiwan are introduced here. From 1986 to 2002, the causative agents of seafood poisoning incidents have been reported to be tetrodotoxin (TTX, 30 outbreaks), paralytic shellfish poisons (PSP, 3 outbreaks), grass carp bile toxins (sporadically), ciguateric toxins (about 1 outbreak per year), excess dose of vitamin A (2 outbreaks), histamine (more than 5 outbreaks), pyropheophorbide a (1 outbreak), fish roe of Ascrossochelius paradoxus (1 outbreak), and allergens (sporadically). Among them, health issue of TTX in Taiwan is the most concerned problem. Therefore, TTX‐containing animals have been found to include puffer, goby, xanthid crab, gastropod, starfish, and flatworm. The dried dressed fish fillets produced from the non‐toxic puffers have been found to be adulterated by toxic puffers. The SDS‐PAGE and PCR methods for identifying species of puffers and their products have been developed. PSP was distributed in the purple clam, xanthid crab, and gastropod in Taiwan. The toxic alga Alexandrium minutum appeared in December–March. The toxin production of alga was affected by a variety of nutritional, environmental, and physiological factors. Most shellfish possessed high resistance to PSP, but the susceptibility of shellfish to the toxic alga was quite different depending on species. The food safety of animal bile juice is another important issue in Taiwan. The toxicity of bile juice was in the order of grass carp ? chicken ? snake. The major component of grass carp bile powder was 5α‐cyprinol sulfate (more than 90%) and those of other animals are bile salts. The toxic potencies of 5α‐cyprinol sulfate and 5α‐cyprinol induced acute kidney failure, but bile salts induced chronic liver dysfunction. Other marine toxins including diarrheic shellfish poisons (DSP), neurotoxic shellfish poisons (NSP), and amnesic shellfish poisons (ASP) are also very important from the health viewpoint in the world, though no poisonings due to those toxins have so far occurred in Taiwan.     

Flavonoid compounds from the red marine alga Alsidium corallinum protect against potassium bromate‐induced nephrotoxicity in adult mice

Potassium bromate (KBrO₃), an environmental pollutant, is a well‐known human carcinogen and a potent nephrotoxic agent. Currently, natural products have built a well‐recognized role in the management of many diseases induced by pollutants. As potent natural sources of bioactive compounds, marine algae have been demonstrated to be rich in novel secondary metabolites with a broad range of biological functions. In this study, adults male mice were orally treated for 15 days with KBrO₃ (0.5 g/L) associated or not with extract of Alsidium corallinum, a red Mediterranean alga. In vitro study demonstrated that algal extract has antioxidant efficacy attributable to the presence of flavonoids and polyphenols. Among these, Liquid chromatography–mass spectrometry analysis showed A. corallinum is rich in kaempferol, apigenin, catechin, and quercetin flavonoids. In vivo study showed that supplementation with the alga significantly prevented KBrO₃‐induced nephrotoxicity as indicated by plasma biomarkers (urea, uric acid, and creatinin levels) and oxidative stress related parameters (malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione, vitamin C, hydrogen peroxide, protein oxidation products) in kidney tissue. The corrective effect of A. corallinum on KBrO₃‐induced kidney injury was also supported by molecular and histopathological observations. In conclusion, it was established that the red alga, thanks to its bioactive compounds, effectively counteracts toxic effects of KBrO₃ and could be a useful coadjuvant agent for treatment of this pollutant poisonings. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1475–1486, 2017.     

Identification of polycavernoside A as the causative agent of the fatal food poisoning resulting from ingestion of the red Alga Gracilaria edulis in the Philippines

Outbreaks of seaweed poisonings are widely spread over the pacific area. Fatal glycosidic macrolides, polycavernosides, and potent tumor promoters, aplysiatoxins, have been previously isolated from edible seaweed. During 2002-2003, three fatal poisoning incidents occurred resulting from ingestion of two edible red alga, Acanthophora specifera and Gracilaria edulis, in Philippines causing eight deaths among 36 patients. Analytical methods for polycavernosides and aplysiatoxins were first developed, and the causative toxin from G. edulis, collected during the second poisoning event on December 2, 2002, was then investigated. The semipurified toxic fraction obtained from this alga based on mouse bioassay was applied to LC-diode array detection (LC-DAD) and LC/electrospray-MS (LC/ESI-MS) analyses. Both LC-DAD and LC/MS chromatograms of this fraction suggested the presence of polycavernoside A (PA) by comparison with the authentic PA. The amount of PA in the alga was estimated as 84 and 72 nmol/kg, using the standard calibration curves for LC-DAD and for LC/ESI-MS in single ion monitoring (SIM) mode, respectively. Other polycavernoside congeners, A2, A3, and B2, and aplysiatoxin and debromoaplysiatoxin were less than the detection limit (2 nmol/kg alga, signal-to-noise ratio: 3) by LC/ESI-MS SIM analysis. In ESI-MS/MS, authentic polycavernosides showed the daughter ions corresponding to a sequential loss of fucosylxylose residues. These fragmentations were applied to LC/ESI-MS/MS for polycavernosides in selective reaction monitoring (SRM) mode. On SRM mass chromatograms, the toxic fraction from the alga showed the peaks corresponding to PA, supporting the identification of PA as the cause of poisoning of G. edulis in Philippines.     

Toxic marine dinoflagellates in Singapore waters that cause seafood poisonings

1. The present paper reviews the toxic dinoflagellates found in Singapore waters that produce toxins that can accumulate through marine food chains to cause seafood poisonings. 2. Singapore waters contain dinoflagellate species linked to three types of seafood poisoning: paralytic shellfish poisoning, diarrhetic shellfish poisoning (DSP) and ciguatera. 3. Paralytic shellfish poisoning and DSP occur by eating bivalve shellfish contaminated with saxitoxins and okadaic acid analogues, respectively. Shellfish accumulate these toxins from filter feeding on a number of species of (mostly) planktonic dinoflagellates. 4. In contrast, benthic species of dinoflagellates of the genus Gambierdiscus produce the ciguatoxins that are bioaccumulated into finfish to cause ciguatera. 5. Paralytic shellfish poisoning and DSP are the major concern for local and regionally produced seafood. To the best of our knowledge, ciguatera poisoning in Singapore only originates from imported reef fish.     

Preclinical antitoxic properties of Spirulina (Arthrospira)

Context: Spirulina (Arthrospira) exerts a wide spectrum of pharmacological activities which are mainly attributed to its antioxidant effect. However, Spirulina has also been reported (both in preclinical and in clinical scenarios) to exhibit other bioactive effects, including an antitoxic potential.

Objective: We performed a systematic review of the literature, conducted in TOXNET, PubMed/MEDLINE, and Science Direct-Scopus; all available years were included. Searching criteria included the effects of Spirulina on experimental poisonings from arsenic, cadmium, carbon tetrachloride, deltamethrin, fluoride, hexachlorocyclohexane, iron, lead, lindane, and mercury.

Results: In all cases, it was established that the blue-green alga, and its isolated compounds, effectively counteracted these pollutants toxic effects on the exposed organisms. Some molecular mechanisms are proposed, although they have not been fully elucidated yet.

Conclusion: Spirulina could be a useful coadjuvant agent within clinical practice for treatment of these or other pollutants poisonings.     

First evidences of PAMAM dendrimer internalization in microorganisms of environmental relevance: A linkage with toxicity and oxidative stress

Abstract

This article reports novel results on the toxic mechanisms of action of amine- and hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimers toward microorganisms of environmental relevance, namely a cyanobacterium of the genus Anabaena and the green alga Chlamydomonas reinhardtii. We used PAMAM ethylenediamine core dendrimers from generations G2 to G4, which displayed a positive charge, measured as ζ-potential, in culture media. All amine-terminated and most remarkably the G4 hydroxyl-terminated dendrimer inhibited the growth of both microorganisms. The effect on the growth of the green alga was significantly higher than that on the cyanobacterium. With concentrations expressed in terms of molarity, there was a clear relationship between dendrimer generation and toxicity, with higher toxicity for higher generation. Hormesis was observed for hydroxyl-terminated dendrimers at low concentrations. The cationic dendrimers and G4-OH significantly increased the formation of reactive oxygen species (ROS) in both organisms. ROS formation was not related with the chloroplast or photosynthetic membranes and photosystem II photochemistry was unaffected. Cell damage resulted in cytoplasm disorganization and cell deformities and was associated to an increase in ROS formation and lipid peroxidation in mitochondria in the green alga; cell wall and membrane disruption with apparent loss of cytoplasmic contents was found in the cyanobacterium. It was determined for the first time that cationic PAMAM dendrimers were quickly and largely internalized by both organisms. These results warn against the generalization of the use of dendrimers, which may pose significant risk for the environment and particularly for primary producers which are determinant for the health of natural ecosystems.     

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.     

Two enone fatty acids isolated from Gracilaria verrucosa suppress the production of inflammatory mediators by down-regulating NF-κB and STAT1 activity in lipopolysaccharide-stimulated RAW 264.7 cells

Gracilaria verrucosa is a common marine red alga that has anti-oxidant and anti-cancer properties. Recently, we reported that anti-inflammatory constituents of G. verrucosa operate through an unknown mechanism. For this reason, we isolated two enone fatty acids from G. verrucosa and investigated their molecular mechanism in LPS-stimulated RAW264.7 cells. We found that the two compounds inhibited the production of inflammatory markers (nitric oxide, TNF-α, and IL-6) in a dose-dependent manner. We next studied the effects of G. verrucosa compounds on LPS-induced signaling pathways. The two compounds suppressed NF-κB reporter activity by interfering with nuclear translocation of NF-κB and suppressed JAK/STAT (p-STAT1) signaling. These results suggest that G. verrucosa inhibits the production of inflammatory mediators (NO, TNF-α, and IL-6) by suppressing the activation of NF-κB and the phosphorylation of STAT1.     

Mycetism: A Review of the Recent Literature

Approximately 100 of the known species of mushrooms are poisonous to humans. New toxic mushroom species continue to be identified. Some species initially classified as edible are later reclassified as toxic. This results in a continually expanding list of toxic mushrooms. As new toxic species are identified, some classic teachings about mycetism no longer hold true. As more toxic mushrooms are identified and more toxic syndromes are reported, older classification systems fail to effectively accommodate mycetism. This review provides an update of myscetism and classifies mushroom poisonings by the primary organ system affected, permitting expansion, as new, toxic mushroom species are discovered.     

Toxicity of the Diatom Genus Pseudo-nitzschia (Bacillariophyceae): Insights from Toxicity Tests and Genetic Screening in the Northern Adriatic Sea

Diatoms of the genus Pseudo-nitzschia H.Peragallo are known to produce domoic acid (DA), a toxin involved in amnesic shellfish poisoning (ASP). Strains of the same species are often classified as both toxic and nontoxic, and it is largely unknown whether this difference is also genetic. In the Northern Adriatic Sea, there are virtually no cases of ASP, but DA occasionally occurs in shellfish samples. So far, three species—P. delicatissima (Cleve) Heiden, P. multistriata (H. Takano) H. Takano, and P. calliantha Lundholm, Moestrup, & Hasle—have been identified as producers of DA in the Adriatic Sea. By means of enzme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with UV and visible spectrum detection (HPLC-UV/VIS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), we reconfirmed the presence of DA in P. multistriata and P. delicatissima and detect for the first time in the Adriatic Sea DA in P. galaxiae Lundholm, & Moestrup. Furthermore, we attempted to answer the question of the distribution of DA production among Pseudo-nitzschia species and strains by sequencing the internal transcribed spacer (ITS) phylogenetic marker and the dabA DA biosynthesis gene and coupling this with toxicity data. Results show that all subclades of the Pseudo-nitzschia genus contain toxic species and that toxicity appears to be strain dependent, often with geographic partitioning. Amplification of dabA was successful only in toxic strains of P. multistriata and the presence of the genetic architecture for DA production in non-toxic strains was thus not confirmed.     

Human risk associated with palytoxin exposure

Palytoxin (PTX) was first isolated from the zoanthid Palythoa toxica. Evaluation of PTX toxicity using various animal models determined that PTX was extremely potent through intravenous, intraperitoneal, and intratracheal exposure. PTX was less potent by direct intragastric exposure. PTX also caused significant, non-lethal effects through dermal and ocular exposure. PTX and PTX-like compounds have now been found in additional zoanthid species, red alga, a sea anemone, and several dinoflagellates. PTXs are found throughout certain reef associated food webs, including in fish and crabs responsible for human illness and death. Many of the organisms found to contain PTXs in the environment are also sold in the home aquarium trade, and recent evidence suggests poisonings have occurred through exposure to these organisms. Due to co-occurrence with other seafood toxins, such as ciguatoxins, saxitoxins, and tetrodotoxin, it has been difficult to assess the true risk of PTX poisoning through seafood consumption in humans, but limited cases have been well documented, some involving human fatalities. Recent evidence also suggests that humans are negatively impacted through PTX exposure by inhalation and dermal routes. Continued research into the distribution and occurrence of PTX and PTX-like compounds both in seafood and marine organisms sold in the aquarium trade appears warranted.     

Poisoning by Gyromitra esculenta--a review

Gyromitra esculenta (Pers.: Fr.) Fr. and a few other mushrooms have caused severe poisonings and even deaths in humans. Clinical data are characterized primarily by vomiting and diarrhoea, followed by jaundice, convulsions and coma. Gastrointestinal disorders distinguish this poisoning. Frequent consumption can cause hepatitis and neurological diseases. The species of concern are mainly G. esculenta and G. gigas (Kromb.) Cooke (non Phill.). Nevertheless, recent advances in chromatography, biochemistry and toxicology have established that other Ascomycetes species also may prove toxic. Gyromitrin (acetaldehyde methylformylhydrazone, G) and its homologues are toxic compounds that convert in vivo into N-methyl-N-formylhydrazine (MFH), and then into N-methylhydrazine (MH). The toxicity of these chemicals, which are chiefly hepatotoxic and even carcinogenic, has been established through in vivo and in vitro experiments using animals, cell cultures and biochemical systems. When we consider the chemical nature and the reactivity of these natural compounds, we suggest that chemical and biochemical mechanisms may explain their intrinsic biological activity.     

Pinnatoxins and spirolides in Norwegian blue mussels and seawater

Fast-acting cyclic imines belonging to the pinnatoxin and pteriatoxin group of toxins were originally identified in shellfish of the genera Pinna and Pteria in Japan, after food poisoning events in China linked to consumption of Pinna spp. Recently, a range of new and known pinnatoxin analogs has been identified in shellfish, sediment, and seawater samples from Australia and New Zealand. Although the structurally closely-related spirolide toxins are better known, and have a worldwide distribution including Norway and other parts of Europe, the presence of pinnatoxins has not been reported in European waters or shellfish. Here we report results from a survey of Norwegian blue mussels for the presence of pinnatoxins and spirolides, by LC-MS/MS analysis of extracts obtained as part of Norway’s routine monitoring programme for regulated algal toxins during late autumn and early winter 2009. Spirolides and pinnatoxin G were widespread (pinnatoxin G (1), spirolide C (2), iso-spirolide C (3), 13-desmethylspirolide C (4), 13,19-didesmethylspirolide C (5), and 20-methylspirolide G (6) were detected in 69%, 13%, 60%, 22%, 33%, and 77%, respectively, of the shellfish samples) and, although levels were generally low, concentrations of up to 115 μg/kg of pinnatoxin G (1) and 226 μg/kg of 13-desmethylspirolide C (4) were detected. We also analyzed stored extracts from passive sampling disks deployed as part of a separate study in autumn 2007. All the stored extracts contained 20-methylspirolide G (which predominated at most locations), most contained pinnatoxin G (73%) and 13,19-didesmethylspirolide C (67%), but iso-spirolide C (36%) and 13-desmethylspirolide C (52%) were also detected in many of the samples. These results suggest that pinnatoxins may be much more widespread than previously suspected, and indicate that they or related compounds could be responsible for sporadic incidents of rapid-onset symptoms during mouse bioassays of shellfish in Europe and elsewhere. The toxicological significance of these levels of pinnatoxins and spirolides is at present unclear. However, although pinnatoxins appear to be less toxic than spirolides by intraperitoneal injection in the mouse bioassay, recently published preliminary toxicological data indicate that pinnatoxins may be as much as an order of magnitude more toxic than spirolides by oral ingestion via food.     

海藻中两种新化学成分的分离和结构鉴定

目的：研究中国南海褐藻Sargassum vachellianum和绿藻Ulva lactuca的化学成分。 方法：应用各种色谱技术进行分离纯化,用MS,IR,¹HNMR,¹³CNMR（DEPT）,HMQC和HMBC鉴定化合物。 结果：分离并鉴定了2个新化合物,化合物1 vachellin为11-甲基-Δ^1,5-3,7,10-三羰基-2,4,8,9-四氮-环十一二烯,化合物2 lactucasterol为胆甾-28-甲基-23,24-环丙烷-Δ⁵-4-酮。 结论：1和2为新化合物。     

Chemical constituents of the red alga Laurencia tristicha

Six new sesquiterpenes, 10-hydroxy-epiaplysin (1), 10-hydroxy-aplysin (2), 10-hydroxy-debromoepiaplysin (3), aplysin-9-ene (4), epiaplysinol (5) and debromoepiaplysinol (6), together with 13 known compounds (7–19), have been isolated from the red alga Laurencia tristicha. The structures of 1–6 were determined by spectroscopic methods including IR, EI-MS, HREI-MS, and 1D and 2D NMR techniques. All compounds were obtained from this species for the first time and were tested for cytotoxic activities against several human cancer cell lines including lung adenocarcinoma (A549), stomach cancer (BGC-823), hepatoma (Bel 7402), colon cancer (HCT-8) and HeLa cell lines. Compound 6 showed selective cytotoxicity against HeLa cell line with IC₅₀ 15.5 μM, cholest-5-en-3β,7α-diol (14) was toxic to all tested cell lines with IC₅₀ values of 16.8, 5.1, 0.5, 0.5, and 0.3 μM, respectively, and other compounds were inactive (IC₅₀>10 μg/ml).     

Pesticide abuse in Europe: effects on the Cinereous vulture (Aegypius monachus) population in Spain

A survey was carried out to investigate incidents of pesticide poisoning of the Cinereous vulture (Aegypius monachus) in Spain during the period 1990–2006. A total of 241 incidents affecting 464 vultures were investigated to establish their causes: approved use, misuse, or deliberate abuse. Other factors studied were compounds, other species affected by the incident, the mode of application, spatial and temporal variation and reasons for the pesticide abuse involved. Approved use was responsible for only a minor fraction (1.3%) of the incidents whereas up to 98% of the investigated incidents were intentional poisonings. Pesticide mortality mainly affects adult individuals (83%) and the implications of this for population dynamics could be important. Eleven different compounds were involved in these incidents although three compounds accounted for up to 88% of the poisoning cases: carbofuran, aldicarb, and strychnine. Most of the pesticide kills seem to be related to the illegal control of predators. Given the minor impact of labeled-use pesticides, currently approved pesticide use does not represent a problem for the Cinereous vulture. Nevertheless, availability of highly toxic pesticides may exacerbate illegal use. As a few compounds, mainly granular insecticides, are responsible for most pesticide kills, stronger regulation and control of these in the EU could result in a decrease of mortality related to pesticide abuse in several endangered species without a significant effect on agriculture.     

Paralytic toxins in three new gastropod (Olividae) species implicated in food poisoning in southern Taiwan.

The toxins in the new gastropods Oliva miniacea, O. mustelina and O. nirasei implicated in a food paralytic poisoning incident in South Taiwan in February 2002 were studied. It was found that the three species of gastropods contained moderate amounts of toxin in edible portion only, and the highest toxicity score was 18 MU/g for O. miniacea, 10 MU/g for O. mustelina, and 27 MU/g for O. nirasei. The toxin was partially purified from the toxic specimens of each species by ultrafiltration using a YM-1 membrane, followed by chromatography on Bio-Gel P-2 column. Analyses by HPLC, GC-MS and LC-MS showed that the toxin from O. miniacea, O. nirasei and O. mustelina contained TTX, and related compounds 4-epi TTX and anhydro-TTX. The paralytic shellfish poisons were not found.     

Dual Effects of Ginkgo biloba Leaf Extract on Human Red Blood Cells

Abstract: Extracts from the leaves of Ginkgo biloba have been used in Chinese medicine for thousands of years. Today, various standardized preparations from G. biloba leaf extract have been developed. G. biloba leaf extract, which contains flavonoids and terpenoids as the major biologically active components, has become one of the most popular and commonly used herbal remedies due to its wide spectrum of beneficial effects on health. In this study, we investigated the effects of G. biloba leaf extract on the properties of human red blood cells in the presence and absence of amyloid peptide (Aβ25‐35), peroxide and hypotonic stress. The results suggest that G. biloba leaf extract has a dual action, both protective and disruptive, on red blood cells, depending on whether an exogenous stress is present. G. biloba leaf extract has a protective role on red blood cells against Aβ‐ and hypotonic pressure‐induced haemolysis, peroxide‐induced lipoperoxidation, as well as glutathione consumption and methaemoglobin formation. On the other hand, G. biloba leaf extract also exhibited damage to red blood cells by increasing cell fragility, changing cellular morphology and inducing glutathione consumption and methaemoglobin formation, especially when applied at high doses. These anti‐ and pro‐oxidative activities of polyphenolic substances are thought to be involved in the dual function of G. biloba leaf extract. The results of this study suggest that high doses of herbal remedies and dietary supplements can be toxic to cells.     

Nangallenes A and B,halogenated nonterpenoid C15-acetogenins from the Bornean red alga Laurencia nangii

Two new halogenated nonterpenoids C₁₅-acetogenins, nangallenes A-B (1–2), together with two known halogenated compounds itomanallene A (3) and 2,10-dibromo-3-chloro-α-chamigrene (4), were isolated and identified from the organic extract of the marine red alga Laurencia nangii Masuda collected from the coastal waters in Semporna, Borneo. Their structures were established by means of spectroscopic analysis including IR, high-resolution electrospray ionization mass spectrometry (HRESI-MS), and 1D and 2D NMR techniques. All these metabolites were submitted for the antifungal assay against four species of selected marine fungi. Compounds 1–4 showed potent activity against Haliphthoros sabahensis and Lagenidium thermophilum.