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

Abstract

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.

Figure 1. Brevetoxins are based on two different structural backbones, based on what are perceived to be the two parent molecules, PbTx-2 (brevetoxin B) and PbTx-1 (brevetoxin A). All other known derivatives are based on alteration of the R-side chain, epoxidation across the double bond in the H-ring of PbTx-2, or derivatization at the C-37 hydroxyl in PbTx-2. PbTx-8, the chloromethyl ketone derivative of PbTx-2, is an artifact of chloroform extraction and subsequent phosgene conversion of PbTx-2. Common features include trans-fused polyether ring systems consisting of five- to nine-membered rings. : *Denotes likely chemical artifact from extraction.

Figure 2. Complexity of lethal components isolated from Florida red tide: the explanation for the potency of red tide events is summarized from 1980 to the present. 1981: only one specific toxin had been identified. Lethality of red tides was thought to be directly related to the concentration of toxigenic organism and the toxin it produced. Mid-1980s: characterization of multiple toxins with widely varying potencies. Lethality was thought to arise from additive effects of each toxin present. Early 1990s: the second brevetoxin structural backbone (PbTx-1) and multiple derivatives discovered and thought to be more potent than the corresponding toxins based on the PbTx-2 backbone. Early 21st century: brevenal described as the first naturally occurring antagonist to counteract the effects of brevetoxins in radioligand binding assays, in fish and mouse bioassays, and in respiratory protocols. (The pulmonary receptor for brevetoxins and brevenal may be distinct from the well-characterized brevetoxin binding site on neuronal VSSC described in the text.)

Figure 3. All active brevetoxins have several requisite features that result in complete expression of brevetoxin activities: a relatively rigid H–K ring region (G–J region in PbTx-1) thought to be involved in binding at site 5 of the VSSCs on the α-subunit (“rigid region”); an A-ring electrophile of some type, in most cases a five- or six-membered ring lactone (“head”); a several-ring “spacer” region that separates the binding region from the activity region; and a side chain (“tail”; Gawley et al. 1995).

Figure 4. (A) A model depicting the sodium-channel α-subunit three-dimensional structure and hypothesized orientation of brevetoxin “head”-down” between domains III and IV. The view from above (at far left) illustrates a possible orientation relative to the α-helices, which are shown as colored circles. (B) Diagram illustrating the putative triad of VSSC configurations and alterations produced by brevetoxins. All natural brevetoxins a) shift the activation potential, favoring the open (O) configuration at normal resting potential (thick arrow); b) produce a longer mean open time (O); c) induce subconductance states (O₁, O₂, etc.); and d) inhibit inactivation (O→I). Figure adapted from Gawley et al. (1995), Taylor (1994), Rein et al. (1994a, 1994b), and Jeglitsch et al. (1998).

Figure 5. Five new brevetoxins, based on the PbTx-2 type backbone, have been purified and characterized: PbTx-11, a toxin with a shortened side chain; PbTx-12, the only natural ketone brevetoxin known; PbTx-13 and PbTx-14, which are both believed to be extraction artifacts formed in the presence of methanol reaction with the very active exomethylene-conjugated aldehyde of PbTx-2; and PbTx-tbm, a brevetoxin PbTx-2 backbone without any side chain, a form that is prevalent in senescent cultures.

Figure 6. The brevenals are shorter trans-fused polyether molecules isolated from K. brevis. Consisting of a 6–7–6–7–7 ring motif, these materials bind with high affinity in brevetoxin receptor assays, and effectively act as inhibitors of brevetoxin binding and activity. Brevenal is the major constituent derived from cultures or the environment, with smaller amounts of brevenol (brevenal with the aldehyde reduced to the alcohol). Reduction of brevenal with tritiated borohydride can produce a new radioligand for probing polyether binding sites. Preliminary experiments indicate high-yield radioligand derivatization of brevenal. The final characterization of brevenal binding to determine if competition is truly competitive awaits completion.