This paper reviews the literature describing research performed over the past decade on the known and possible exposures and human health effects associated with Florida red tides. These harmful algal blooms are caused by the dinoflagellate, Karenia brevis, and similar organisms, all of which produce a suite of natural toxins known as brevetoxins. Florida red tide research has benefited from a consistently funded, long term research program, that has allowed an interdisciplinary team of researchers to focus their attention on this specific environmental issue—one that is critically important to Gulf of Mexico and other coastal communities. This long-term interdisciplinary approach has allowed the team to engage the local community, identify measures to protect public health, take emerging technologies into the field, forge advances in natural products chemistry, and develop a valuable pharmaceutical product. The Review includes a brief discussion of the Florida red tide organisms and their toxins, and then focuses on the effects of these toxins on animals and humans, including how these effects predict what we might expect to see in exposed people.

Symptoms consistent with inhalation toxicity have long been associated with Florida red tides, and various causal agents have been proposed. Research since 1981 has centered on a group of naturally occurring trans-fused cyclic polyether compounds called brevetoxins that are produced by a marine dinoflagellate known as Karenia brevis. Numerous individual brevetoxins have been identified from cultures as well as from natural bloom events. A spectrum of brevetoxin derivatives produced by chemical modification of the natural toxins has been prepared to examine the effects of functional group modification on physiologic activity. Certain structural features of natural and synthetic derivatives of brevetoxin appear to ascribe specific physiologic consequences to each toxin. Differential physiologic effects have been documented with many of the natural toxins and derivatives, reinforcing the hypothesis that metabolism or modification of toxin structures modulates both the specific toxicity (lethality on a per milligram basis) and potentially the molecular mechanism(s) of action. A series of naturally occurring fused-ring polyether compounds with fewer rings than brevetoxin, known as brevenals, exhibit antagonistic properties and counteract the effects of the brevetoxins in neuronal and pulmonary model systems. Taken together, the inhalation toxicity of Florida red tides would appear to depend on the amount of each toxin present, as well as on the spectrum of molecular activities elicited by each toxin. Toxicity in a bloom is diminished by the amount brevenal present.

Florida red tide brevetoxins are sodium channel neurotoxins produced by the dinoflagellate Karenia brevis. When aerosolized, the toxin causes airway symptoms in normal individuals and patients with airway disease, but systematic exposures to define the pulmonary consequences and putative mechanisms are lacking. Here we report the effects of airway challenges with lysed cultures of Karenia brevis (crude brevetoxin), pure brevetoxin-2, brevetoxin-3, and brevetoxin-tbm (brevetoxin-2 minus the side chain) on pulmonary resistance and tracheal mucus velocity, a marker of mucociliary clearance, in allergic and nonallergic sheep. Picogram concentrations of toxin caused bronchoconstriction in both groups of sheep. Brevetoxin-tbm was the least potent, indicating the importance of the side chain for maximum effect. Both histamine H 1 -and cholinergic-mediated pathways contributed to the bronchoconstriction. A synthetic antagonist, β-naphthoyl-brevetoxin-3, and brevenal, a natural antagonist, inhibited the bronchoconstriction. Only crude brevetoxin and brevetoxin-3 decreased tracheal mucus velocity; both antagonists prevented this. More importantly, picomolar concentrations of the antagonists alone improved tracheal mucus velocity to the degree seen with mM concentrations of the sodium channel blocker amiloride. Thus, Karenia brevis, in addition to producing toxins that adversely affect the airways, may be a source of agents for treating mucociliary dysfunction. Keywords bronchoconstriction; mucus transport; natural therapiesFlorida red tide is a harmful algal bloom caused by the dinoflagellate Karenia brevis (previously Gymnodinium breve). K. brevis produces at least nine structurally-related polyether brevetoxins (PbTxs) (1-3), with PbTx-2 and PbTx-3 being the predominant forms (4,5). As a ) is included in the provisional patent application filed on behalf of the University of North Carolina at Wilmington by aaiPharma; A.J.B. has provisional patents filed with respect to treatment of mucociliary diseases for synthetic and natural products derived from cultured red tide, and has an interest in any licensing that might arise from patents and final patents that are not filed yet and, thus, have not been thoroughly reviewed by the U.S. Patent and Copyright Office; J.R.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; T.A.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; I.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.G.B. has provisional patents filed with respect to treatment of mucociliary diseases for synthetic and natural products derived from cultured red tide, and has an interest in any licensing that might arise from patents and final patents not filed yet and, thus, ...

Summary1. Florida red tides produce profound neurotoxicity that is evidenced by massive fish kills, neurotoxic shellfish poisoning, and respiratory distress. Red tides vary in potency, potency that is not totally governed by toxin concentration. The purpose of the study was to understand the variable potency of red tides by evaluating the potential for other natural pharmacological agents which could modulate or otherwise reduce the potency of these lethal environmental events.2. A synaptosome binding preparation with 3-fold higher specific brevetoxin binding was developed to detect small changes in toxin binding in the presence of potential antagonists. Rodent brain labeled in vitro with tritiated brevetoxin shows high specific binding in the cerebellum as evidenced by autoradiography. Synaptosome binding assays employing cerebellum-derived synaptosomes illustrate 3-fold increased specific binding. 3.A new polyether natural product from Florida's red tide dinoflagellate Karenia brevis, has been isolated and characterized. Brevenal, as the nontoxic natural product is known, competes with tritiated brevetoxin for site 5 associated with the voltage-sensitive sodium channel (VSSC). Brevenal displacement of specific brevetoxin binding is purely competitive in nature.4. Brevenal, obtained from either laboratory cultures or field collections during a red tide, protects fish from the neurotoxic effects of brevetoxin exposure.5. Brevenal may serve as a model compound for the development of therapeutics to prevent or reverse intoxication in red tide exposures.

A new ladder-frame polyether compound containing five fused ether rings was isolated from laboratory cultures of the marine dinoflagellate Karenia brevis. This compound, named brevenal, and its dimethyl acetal derivative both competitively displace brevetoxin from its binding site in rat brain synaptosomes. Significantly, these compounds are also nontoxic to fish and antagonize the toxic effects of brevetoxins in fish. The structure and biological activity of brevenal, as well as the dimethyl acetal derivative, are described in this paper.A number of bioactive polyether compounds have been isolated from the marine dinoflagellate Karenia brevis, the organism responsible for toxic red tides along Florida's Gulf Coast. The most well-known bioactive compounds isolated from K. brevis are a family of neurotoxins called the brevetoxins (Figure 1), which consist of nine different toxins with two different structural backbones: brevetoxin-A (containing 10 fused cyclic ether rings, 5,8,6,7,9,8,8,6,6,6) 1-3 and brevetoxin-B (containing 11 fused cyclic ether rings, 6,6,6,7,7,6,6,8,6,6,6). 3-8Brevetoxins bind with high affinity to site 5 of voltage sensitive sodium channels (VSSC) in neurons. 9,10 Binding of brevetoxins to tissues containing VSSC results in membrane depolarization, repetitive firing, and increased sodium currents. 11-14 Investigation of the effect of brevetoxins on excitable membranes using voltage clamp experiments indicates that brevetoxins activate VSSC by prolonging mean open time, inhibiting channel inactivation, and shifting the channel activation potential to more negative values. 12-14 During K. brevis red tides humans are most commonly affected by brevetoxins that have been aerosolized in sea spray or bioaccumulated in shellfish. Inhaled brevetoxins cause respiratory irritation and breathing difficulties in sensitive populations. 15-17 At sufficiently high concentrations ingested brevetoxins lead to a collection of symptoms commonly referred to as neurotoxic shellfish poisoning (NSP). 18,19 NSP in humans is characterized by sensory abnormalities, cranial nerve dysfunction, gastrointestinal symptoms, and sometimes respiratory failure. 19In 1989 Prasad and Shimizu 20 isolated and described another polyether ladder compound from K. brevis cultures that contained a different structural backbone and named it hemibrevetoxin-B. Hemibrevetoxin-B contains structural features similar to brevetoxin but is about half of the size and contains only four fused cyclic ether rings (6,6,7,7). Hemibrevetoxin-B (Figure 2) showed cytotoxicity in mouse neuroblastoma cells at concentrations of 5 μM, but no fish or In this report we describe the isolation, structural characterization, and biological activity of a new ladder-frame polyether aldehyde named brevenal (Figure 3a), isolated from cultures of K. brevis. Brevenal as well as its dimethyl acetal derivative ( Figure 3b) contain five fused cyclic ether rings, have low toxicity to fish, and have a structural backbone different from both brevetoxins and hemibre...

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 microg/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.

Total synthesis of structure 1 originally proposed for brevenal, a nontoxic polycyclic ether natural product isolated from the Florida red tide dinoflagellate, Karenia brevis, was accomplished. The key features of the synthesis involved (i) convergent assembly of the pentacyclic polyether skeleton based on our developed Suzuki-Miyaura coupling chemistry and (ii) stereoselective construction of the multi-substituted (E,E)-dienal side chain by using copper(I) thiophen-2-carboxylate (CuTC)-promoted modified Stille coupling. The disparity of NMR spectra between the synthetic material and the natural product required a revision of the proposed structure. Detailed spectroscopic comparison of synthetic 1 with natural brevenal, coupled with the postulated biosynthetic pathway for marine polyether natural products, suggested that the natural product was most likely represented by 2, the C26 epimer of the proposed structure 1. The revised structure was finally validated by completing the first total synthesis of (−)-2, which also unambiguously established the absolute configuration of the natural product.

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®- 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 (R2 = 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®- PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.