Henry M. Jacocks

Natural and derivative brevetoxins : Historical background, multiplicity, and effects

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.

Brevenal is a natural inhibitor of brevetoxin action in sodium channel receptor binding assays

Abstract1. 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 Floridas 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.

Characterization of Tamulamides A and B, Polyethers Isolated from the Marine Dinoflagellate Karenia brevis

Florida red tides occur as the result of blooms of the marine dinoflagellate Karenia brevis. K. brevis is known to produce several families of fused polyether ladder compounds. The most notable compounds are the brevetoxins, potent neurotoxins that activate mammalian sodium channels. Additional fused polyether ladder compounds produced by K. brevis include brevenal, brevisin, and hemibrevetoxin B, all with varying affinities for the same binding site on voltage-sensitive sodium channels. The structure elucidation and biological activity of two additional fused polyether ladder compounds containing seven fused ether rings will be described in this paper. Tamulamide A (MW = 638.30) and tamulamide B (MW = 624.29) were isolated from K. brevis cultures, and their structures elucidated using a combination of NMR spectroscopy and high-resolution mass spectrometry. Tamulamides A and B were both found to compete with tritiated brevetoxin-3 ([(3)H]-PbTx-3) for its binding site on rat brain synaptosomes. However, unlike the brevetoxins, tamulamides A and B showed no toxicity to fish at doses up to 200 nM and did not cause significant bronchoconstriction in sheep pulmonary assays.

Synthesis, Modeling, and Biological Evaluation of Analogues of the Semisynthetic Brevetoxin Antagonist β-Naphthoyl-Brevetoxin

Brevetoxins are neurotoxic compounds produced by the dinoflagellate Karenia brevis. Extensive blooms induce neurotoxic shellfish poisoning (NSP) and asthma‐like symptoms in humans. β‐naphthoyl‐brevetoxin, the first semisynthetic brevetoxin antagonist, has been defined as the lead compound in the investigation of the mechanisms of bronchoconstriction induced by inhaled brevetoxins and relaxation or reversal of those effects by selected derivatives. In pursuit of more potent and effective brevetoxin antagonists, a series of β‐naphthoyl‐brevetoxin analogues have been synthesized. Activities were determined by competitive displacement of tritiated brevetoxin‐3 from rat brain synaptosomes and by lung resistance measurements in sheep. Additionally, preliminary computational structural studies have been performed. All analogues bound to rat brain synaptosomes with affinities similar to β‐naphthoyl‐brevetoxin but exhibited very different responses in sheep. The biological evaluations along with computational studies suggest that the brevetoxin binding site in rat brain synaptosome might be different from the ones in lung tissue and both steric and electrostatic factors contribute to the efficacy of brevetoxin antagonism.

Red tides and marine mammal mortalities: Unexpected brevetoxin vectors may account for deaths long after or remote from an algal bloom

Potent marine neurotoxins known as brevetoxins are produced by the ‘red tide’ dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.

Structure Activity Relationship of Brevenal Hydrazide Derivatives

Brevenal is a ladder frame polyether produced by the dinoflagellate Karenia brevis. This organism is also responsible for the production of the neurotoxic compounds known as brevetoxins. Ingestion or inhalation of the brevetoxins leads to adverse effects such as gastrointestinal maladies and bronchoconstriction. Brevenal shows antagonistic behavior to the brevetoxins and shows beneficial attributes when administered alone. For example, in an asthmatic sheep model, brevenal has been shown to increase tracheal mucosal velocity, an attribute which has led to its development as a potential treatment for Cystic Fibrosis. The mechanism of action of brevenal is poorly understood and the exact binding site has not been elucidated. In an attempt to further understand the mechanism of action of brevenal and potentially develop a second generation drug candidate, a series of brevenal derivatives were prepared through modification of the aldehyde moiety. These derivatives include aliphatic, aromatic and heteroaromatic hydrazide derivatives. The brevenal derivatives were tested using in vitro synaptosome binding assays to determine the ability of the compounds to displace brevetoxin and brevenal from their native receptors. A sheep inhalation model was used to determine if instillation of the brevenal derivatives resulted in bronchoconstriction. Only small modifications were tolerated, with larger moieties leading to loss of affinity for the brevenal receptor and bronchoconstriction in the sheep model.

Development of a fluorescence assay for the characterization of brevenal binding to rat brain synaptosomes.

The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs.

Red tides and marine mammal mortalities: Brevetoxicosis

Potent marine neurotoxins known as brevetoxins are produced by the ‘red tide’ dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.