Michael J. Twiner

Azaspiracid Shellfish Poisoning: A Review on the Chemistry, Ecology, and Toxicology with an Emphasis on Human Health Impacts

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 μg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.

Eosinophilia and biotoxin exposure in bottlenose dolphins (Tursiops truncatus) from a coastal area impacted by repeated mortality events

Bottlenose dolphins (Tursiops truncatus) inhabiting coastal waters in the northern Gulf of Mexico have been impacted by recurrent unusual mortality events over the past few decades. Several of these mortality events along the Florida panhandle have been tentatively attributed to poisoning from brevetoxin produced by the dinoflagellate Karenia brevis. While dolphins in other regions of the Florida coast are often exposed to K. brevis blooms, large-scale dolphin mortality events are relatively rare and the frequency and magnitude of die-offs along the Panhandle raise concern for the apparent vulnerability of dolphins in this region. We report results from dolphin health assessments conducted near St. Joseph Bay, Florida, an area impacted by 3 unusual die-offs within a 7-year time span. An eosinophilia syndrome, manifested as an elevated blood eosinophil count without obvious cause, was observed in 23% of sampled dolphins. Elevated eosinophil counts were associated with decreased T-lymphocyte proliferation and increased neutrophil phagocytosis. In addition, indication of chronic low-level exposure to another algal toxin, domoic acid produced by the diatom Pseudo-nitzschia spp., was determined. Previous studies of other marine mammal populations exposed recurrently to Pseudo-nitzschia blooms have suggested a possible link between the eosinophilia and domoic acid exposure. While the chronic eosinophilia syndrome could over the long-term produce organ damage and alter immunological status and thereby increase vulnerability to other challenges, the significance of the high prevalence of the syndrome to the observed mortality events in the St. Joseph Bay area is unclear. Nonetheless, the unusual immunological findings and concurrent evidence of domoic acid exposure in this sentinel marine species suggest a need for further investigation to elucidate potential links between chronic, low-level exposure to algal toxins and immune health.

Transcriptional profiling and inhibition of cholesterol biosynthesis in human T lymphocyte cells by the marine toxin azaspiracid.

Azaspiracid-1 (AZA-1) is a marine biotoxin reported to accumulate in shellfish from several countries, including eastern Canada, Morocco, and much of western Europe, and is frequently associated with severe gastrointestinal human intoxication. As the mechanism of action of AZA-1 is currently unknown, human DNA microarrays and qPCR were used to profile gene expression patterns in human T lymphocyte cells following AZA-1 exposure. Some of the early (1 h) responding genes consisted of transcription factors, membrane proteins, receptors, and inflammatory genes. Four- and 24-h responding genes were dominated by genes involved in de novo lipid biosynthesis of which 17 of 18 involved in cholesterol biosynthesis were significantly up regulated. The up regulation of synthesis genes was likely in response to the ca. 50% reduction in cellular cholesterol, which correlated with up regulated protein expression levels of the low-density lipoprotein receptor. These data collectively detail the inhibition of de novo cholesterol synthesis, which is the likely cause of cytotoxicity and potentially a target pathway of the toxin.

An extract of Hydrilla verticillata and associated epiphytes induces avian vacuolar myelinopathy in laboratory mallards.

Avian vacuolar myelinopathy (AVM) is a neurological disease affecting bald eagles (Haliaeetus leucocephalus), American coots (Fulica americana), waterfowl, and other birds in the southeastern United States. The cause of the disease is unknown, but is thought to be a naturally produced toxin. AVM is associated with aquatic macrophytes, most frequently hydrilla (Hydrilla verticillata), and researchers have linked the disease to an epiphytic cyanobacterial species associated with the macrophytes. The goal of this study was to develop an extraction protocol for separating the putative toxin from a hydrilla‐cyanobacterial matrix. Hydrilla samples were collected from an AVM‐affected reservoir (J. Strom Thurmond Lake, SC) and confirmed to contain the etiologic agent by mallard (Anas platyrhynchos) bioassay. These samples were then extracted using a solvent series of increasing polarity: hexanes, acetone, and methanol. Control hydrilla samples from a reference reservoir with no history of AVM (Lake Marion, SC) were extracted in parallel. Resulting extracts were administered to mallards by oral gavage. Our findings indicate that the methanol extracts of hydrilla collected from the AVM‐affected site induced the disease in laboratory mallards. This study provides the first data documenting for an “extractable” AVM‐inducing agent.