Daniel G. Baden

Brevetoxicosis: Red tides and marine mammal mortalities

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.

Marine Food-Borne Dinoflagellate Toxins

Publisher Summary This chapter describes the marine food-borne dinoflagellate toxins. Toxins are substances potentially noxious to living organisms. These include poisonous substances produced by marine algae, either as byproducts of metabolism or as the necessary intermediates of metabolism for cellular growth, maintenance, or reproduction. The unifying factor in toxins is their ability to be transmitted to humans through bioaccumulation in an intermediate marine host. The intermediate accumulator of toxin, known as a “transvector,” can be divided into primary and secondary types. Dinoflagellates are foremost both in the number of known toxic species and in the potency of the toxins produced. Dinoflagellates that are responsible for the biosynthesis of many toxic compounds transmitted through the food chain are eukaryotes. The dinoflagellates are divided into two groups: those associated with red tides (the classic bloom organisms) and those associated with Ciguatera. The chapter discusses the toxinology of marine food-borne dinoflagellate toxins and describes some of the ecological factors governing the initiation and maintenance of toxic populations.

Toxicity of two toxins from the Florida red tide marine dinoflagellate, Ptychodiscus brevis

The purification and crystallization of T17, a toxin from Ptychodiscus brevis, is reported. The toxicity of this compound and a second toxin known as T34 are compared by i.v., i.p. and oral administration in mice. Both toxins produce symptoms characteristic of muscarnic stimulants; hypersalivation, rhinorrhea and excessive urination and defecation being the most commonly observed. T17, which is orally toxic, is believed to be the agent responsible for Neurotoxic Shellfish Poisoning.

The relationship of brevetoxin ‘length’ and A-ring functionality to binding and activity in neuronal sodium channels

BACKGROUNDnBrevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules.nnnRESULTSnSeveral synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states.nnnCONCLUSIONSnOur data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.

The Potential Role of Natural Tumor Promoters in Marine Turtle Fibropapillomatosis

Abstract Fibropapillomatosis (FP) in green turtles Chelonia mydas is a debilitating, neoplastic disease that has reached worldwide epizootic levels. The etiology of FP is unknown but has been linked to oncogenic viruses. Toxic benthic dinoflagellates (Prorocentrum spp.) are not typically considered tumorigenic agents, yet they have a worldwide distribution and produce a tumor promoter, okadaic acid (OA). Prorocentrum spp. are epiphytic on macroalgae and seagrasses that are normal components of green turtle diets. Here we show that green turtles in the Hawaiian Islands consume Prorocentrum and that high-risk FP areas are associated with areas where P. lima and P. concavum are both highly prevalent and abundant. The presence of presumptive OA in the tissues of Hawaiian green turtles further suggests exposure and a potential role for this tumor promoter in the etiology of FP.

High affinity binding of red tide neurotoxins to marine mammal brain

Abstract During a period of several weeks in the spring of 1996, over 200 manatees (Trichechus manatus latirostris) were found dead or dying in coastal waters or on beaches of the Florida west coast. Concurrent with this event, high densities of Gymnodinium breve, the dinoflagellate which produces the potent neurotoxin called brevetoxin, were observed in the same coastal areas. Our study demonstrates that brevetoxin binds to isolated nerve preparations from manatee brain with similar affinity as that reported for a number of terrestrial mammals. Analysis of receptor binding of tritiated brevetoxin to manatee brain, illustrates saturable specific binding, competition of specific binding by a non-radioactive toxin of the same structure, and temperature dependence of binding. Complementary studies with the red tide neurotoxin, saxitoxin, which is responsible for the intoxication syndrome paralytic shellfish poisoning, show high affinity and specific binding of this toxin to isolated nerve preparations from several marine mammals, including manatee, gray whale (Eschrichtius robustus), humpback whale (Megaptera novaeangliae), and sea lion (Zalophus californianus). These results demonstrate the specific binding of brevetoxin and saxitoxin to excitable brain tissue of marine mammals and support the hypothesis that the exposure of manatees to brevetoxin in the spring of 1996 was a factor in their stranding and death.

Crystallization and toxicology of T34: A major toxin from Florida's red tide organism (Ptychodiscus brevis)

Abstract The purification and crystallization of a major toxin from laboratory cultures of Floridas fed tide organism, Ptychodiscus brevis , is described. The crystalline toxin is soluble in acetone and chloroform, less soluble in ethyl acetate, methanol and ethanol, and is slightly soluble in water. The crystalline toxin is stable when stored in a moisture-free environment. The toxin is lethal to fish and mice, inhibits growth in three in vitro cell culture systems, and inhibits the division of fertilized sea urchin eggs. It has no effect on the formation of antibodies directed against sheep red blood cells in mice, and does not inhibit the morphological transformation of rat glioma cells from a fibroblast-like to a glial-like cell.

Binding of brevetoxins and ciguatoxin to the voltage-sensitive sodium channel and conformational analysis of brevetoxin B

The marine toxins known generically as brevetoxins, as well as their structural relative ciguatoxin, are known as polyether ladder toxins, and bind uniquely to site 5 of the voltage-sensitive sodium channel. Rat brain synaptosome binding data show similarities in binding affinity for brevetoxins having the same structural (ladder) backbone, but different affinities between brevetoxins having different backbones. Ciguatoxin has a different backbone from the brevetoxins, but binds even more strongly to the same site. Could the flexibility of the backbone be related to their relative toxicities? As part of an effort to identify the common pharmacophore for the toxins, Monte Carlo methods were used to generate conformational models of the polyether ladder toxin brevetoxin B (PbTx-2) which shows significant flexibility at the juncture of the two 7-membered rings.

Variations in major toxin composition for six clones of Ptychodiscus brevis

Extracts from six clones of Ptychodiscus brevis (formerly known as Gymnodinium breve) were analyzed by high performance liquid chromatography for the presence of brevetoxins PbTx-1, PbTx-2, and PbTx-3. Analyses indicated a wide clonal variability of the three toxin fractions in logarithmic phase cultures when normalized on a per cell basis. It appears that a much wider variability exists in toxin content for different P. brevis clones than exists in replicate extraction of multiple cultures of the diploid clone originally isolated by Wilson.

Bronchoconstriction caused by Florida red tide toxins

T17, a toxin purified from laboratory cultures of Floridas redtide organism, Ptychodiscus brevis (formerly Gymnodinium breve), produces bronchoconstriction in anesthetized artificially-ventilated guinea pigs. Bronchoconstriction, measured as a resistance to mechanical pulmonary inflation, was antagonized by atropine, but not by interruption of vagal nerve stimulation or diaphragm dissection.