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Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA((R))). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA((R)) in certain labs. A fluorescence based receptor binding assay (RBA((F))) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY (R)-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA((F)). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R-2 = 0.71) with those of the RBA((F)), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA((F)) affinities expressed as% binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA((F)), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA((F)) advantages include the long-term(> 5 years) stability of the BODIPY (R)-PbTx-2 and having similar results as the commonly used RBA((R)). The RBA((F)) is cost-effective, allo

Ten fish mortality events, involving primarily Atlantic menhaden, occurred from early July through September 2000 in several bays and creeks in Delaware, USA. Two events involved large mortalities estimated at 1–2.5 million fish in Bald Eagle Creek, Rehoboth Bay. Samples from Indian Inlet (Bethany Beach), open to the Atlantic, as well as from an enclosed area of massive fish kills at nearby Bald Eagle Creek and Torque Canal were collected and sent to our laboratory for analysis. Microscopic examination of samples from the fish kill site revealed the presence of a single-cell Raphidophyte alga Chattonella cf. verruculosa at a maximum density of 1.04 × 107 cells/L. Naturally occurring brevetoxins were also detected in the bloom samples. Besides the Chattonella species, no other known brevetoxin-producing phytoplankton were present. Chromatographic, immunochemical, and spectroscopic analyses confirmed the presence of brevetoxin PbTx-2, and PbTx-3 and -9 were confirmed by chromatographic and immunochemical analyses. This is the first confirmed report in the United States of brevetoxins associated with an indigenous bloom in temperate Atlantic estuarine waters and of C. cf. verruculosa as a resident toxic organism implicated in fish kills in this area. The bloom of Chattonella continued throughout September and eventually declined in October. By the end of October C. cf. verruculosa was no longer seen, nor was toxin measurable in the surface waters. The results affirm that to avoid deleterious impacts on human and ecosystem health, increased monitoring is needed for brevetoxins and organism(s) producing them, even in areas previously thought to be unaffected. Key words: brevetoxins, Chattonella cf. verruculosa, Delaware, fish kills, harmful agal blooms. Environ Health Perspect 110:465–470 (2002). [Online 1 April 2002] http://ehpnet1.niehs.nih.gov/docs/2002/110p465-470bourdelais/abstract.html

Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and ß-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.

We developed a competitive enzyme-linked immunosorbent assay (ELISA) to analyze brevetoxins, using goat anti-brevetoxin antibodies obtained after immunization with keyhole limpet hemocyanin- brevetoxin conjugates, in combination with a three-step signal amplification process. The procedure, which used secondary biotinylated antibodies, streptavidine-horseradish peroxidase conjugate, and chromogenic enzyme substrate, was useful in reducing nonspecific background signals commonly observed with complex matrices. This competitive ELISA detected brevetoxins in seawater, shellfish extract and homogenate, and mammalian body fluid such as urine and serum without pretreatment, dilution, or purification. We investigated the application of this technique for shellfish monitoring by spiking shellfish meat with brevetoxins and by analyzing oysters from two commercial shellfish beds in Florida that were exposed to a bloom of Karenia brevis (formerly Gymnodinium breve). We performed brevetoxin analysis of shellfish extracts and homogenates by ELISA and compared it with the mouse bioassay and receptor binding assay. The detection limit for brevetoxins in spiked oysters was 2.5 µg/100 g shellfish meat. This assay appears to be a useful tool for neurotoxic shellfish poisoning monitoring in shellfish and seawater, and for mammalian exposure diagnostics, and significantly reduces the time required for analyses. Key words: antibody, brevetoxin, detection, ELISA, immunoassay, neurotoxic shellfish poisoning, PbTx, seawater, serum, shellfish, urine. Environ Health Perspect 110:179–185 (2002). [Online 17 January 2002] http://ehpnet1.niehs.nih.gov/docs/2002/110p179-185naar/abstract.html

During a Florida red tide, brevetoxins produced by the dinoflagellate Karenia brevis become aerosolized and cause airway symptoms in humans, especially in those with pre-existing airway disease (e.g., asthma). To understand these toxin-induced airway effects, we used sheep with airway hypersensitivity to Ascaris suum antigen as a surrogate for asthmatic patients and studied changes in pulmonary airflow resistance (R-L) after inhalation challenge with lysed cultures of K brevis (crude brevetoxins). Studies were done without and with clinically available drugs to determine which might prevent/reverse these effects. Crude brevetoxins (20 breaths at 100 mu g/mL; n = 5) increased R-L 128 +/- 6% (mean +/- SE) over baseline. This bronchoconstriction was significantly reduced (% inhibition) after pretreatment with the glucocorticosteroid budesonide (49%), the beta(2) adrenergic agent albuterol (71%), the anticholinergic agent atropine (58%), and the histamine H-1-antagonist diphenhydramine (47%). The protection afforded by atropine and diphenhydramine suggests that both cholinergic (vagal) and HI-mediated pathways contribute to the bronchoconstriction. The response to cutaneous toxin injection was also histamine mediated. Thus, the airway and skin data support the hypothesis that toxin activates mast cells in vivo. Albuterol given immediately after toxin challenge rapidly reversed the bronchoconstriction. Toxin inhalation increased airway kinins, and the response to inhaled toxin was enhanced after allergen challenge. Both factors could contribute to the increased sensitivity of asthmatic patients to toxin exposure. We conclude that K brevis aerosols are potent airway constrictors. Clinically available drugs may be used to prevent or provide therapeutic relief for affected individuals. Published online 2005 February 10. doi: 10.1289/ehp.7498.