Karenia brevis, the harmful alga associated with red tide, produces brevetoxins (PbTxs). Exposure to these toxins can have a negative impact on marine wildlife and serious human health consequences. The elimination of PbTxs is critical to protect the marine environment and human health. TiO2 photocatalysis under 350 nm and solar irradiation leads to significant degradation of PbTxs via first order kinetics. ELISA results demonstrate TiO2 photocatalysis leads to a significant decrease in the bioactivity of PbTxs as a function of treatment time. Experiments conducted in the presence of synthetic seawater, humic material and a hydroxyl scavenger showed decreased degradation. PbTxs are highly hydrophobic and partition to organic microlayer on the ocean surface. Acetonitrile was employed to probe the influence of an organic media on the TiO2 photocatalysis of PbTxs. Our results indicate TiO2 photocatalysis may be applicable for the degradation of PbTxs.

Karenia brevis, the major harmful algal (HA) species in the Gulf of Mexico, produces a suite of brevetoxins and brevenal, a nontoxic brevetoxin antagonist. K. brevis growth is reported to be optimum at oceanic conditions, yet blooms are most problematic in coastal waters. Differences in growth rate, total brevetoxin production, brevetoxin profiles and brevenal production were evaluated among eight K. brevis clones grown at salinities of 35 and 27, but otherwise identical conditions. All measured parameters varied significantly among clones and the individual responses to decreased salinity varied as well. At 27, growth rates of four clones increased (Wilson, TXB3, SP1 and SP2), but decreased in three others (TXB4, SP3 and NBK) as compared to 35. Total brevetoxin cellular concentration varied up to ∼ten-fold among clones. For most clones (5 of 8), no significant difference in total toxin production between salinity treatments was observed; however, there was a shift in brevetoxin profiles to a higher proportion of PbTx-1 vs. PbTx-2 (in 7 of 8 clones). Brevenal production decreased in the majority of the clones (6 of 8) when grown at a salinity of 27. Results suggest that K. brevis produces more PbTx-1 and less brevenal in lower salinity waters.

During blooms of the dinoflagellate Karenia brevis, filter-feeders such as oysters and clams bioaccumulate brevetoxins, often to levels that are toxic to humans. In controlled aquarium experiments, we exposed live oysters to bloom levels of toxic K. brevis, followed by 10 weeks of exposure to non-toxic microalgae. Oysters were harvested weekly and analyzed for brevetoxins and brevetoxin metabolites to quantify toxin bioaccumulation and depuration. All of the PbTx-2 concentrated by oysters was immediately converted to a mixture of polar metabolites that were then slowly eliminated from the oysters. However, 90% of measured PbTx-3 was eliminated within two weeks of toxic exposure but without apparent biotransformation. Extracts of oysters containing high levels of PbTx-3 were toxic to mice by intraperitoneal (IP) injection. Extracts of oysters harvested after PbTx-3 had been eliminated were non-toxic despite high concentrations of PbTx-2 metabolites. Oysters collected in Florida during and after a bloom of K. brevis contained polar metabolites of PbTx-2 as well as PbTx-3, but no PbTx-2. Again, PbTx-3 concentration was a good predictor of mouse toxicity. One hundred percent conversion of PbTx-2 to polar metabolites was also accomplished in vitro by spiking oyster or clam homogenate with PbTx-2, followed by a brief incubation at room temperature. These PbTx-2 metabolites did not kill mice, either orally or by intraperitoneal injection, even at concentrations 30 times greater than toxic PbTx-3 levels.

Since January 2002, 23 cases of Puffer Fish Poisoning (PFP) were reported in four states due to saxitoxin (Quilliam et al., 2002) traced back to southern puffer fish (Sphoeroides nephelus) originating from the northern Indian River Lagoon (IRL) on Florida’s east coast (Bodager 2002). Saxitoxin was previously unknown in Florida marine waters (Landsberg et al., 2002). Because puffer fish were involved in PFP we have now routinely screened > 400 southern, 40 checkered (S. testudineus), and 40 bandtail puffer fish (S. dorsalis) for saxitoxins (STXs) statewide using the Ridascreen‚ STX ELISA kits (Usleber et al., 1991). Since April 2002 selected biota from the IRL have also been tested for STX distribution and prevalence within the food web. The geographical hot spot for STXs in the IRL is in the north from Titusville south to Melbourne. Approximately 18.1% of samples (n = 791) were below the detection limit of 1 μg STXeq/100g tissue, 27.4% were below 80μg STXeq/100g tissue, while the majority of samples, 54.5% contained moderate to high levels of STX in a range of tissues. Except for southern puffer fish, the muscle, skin, and mucus of which contain up to 5865.5 μg STXeq/100g tissue, STXs are present but below regulatory levels in the muscle of checkered puffer fish, Atlantic spadefish (Chaetodipterus faber), striped burrfish (Chilomycterus schoepfi), and porcupine fish (Diodon hystrix). Recreationally prized fish such as sheepshead (Archosargus probatocephalus), Gulf flounder (Paralichthys albigutta), southern kingfish (Menticirrhus americanus) and spotted sea trout (Cynoscion nebulosus), contained up to 35.9 μg STXeq/100g tissue in the skin and mucus. Commercially significant species such as blue crabs (Callinectus sapidus) had a maximum of 11.1 μg STXeq/100g tissue in the hepatopancreas and whole hard clams (Mercenaria sp.) a maximum of 17.2 μg STXeq/100g tissue. A maximum of 116.5 μg STXeq/100g tissue was found in polychaetes (Glycera dibranchiata), 14.2 μg STXeq/100g tissue in gastropods (Urosalpinx cinerea), 1.1 μg STXeq/100g tissue in brittle stars (Ophiothrix spiculata), and up to 4301.2μg STXeq/100g tissue in small non-harvestable whole razor clams (Ensis minor). With the exception of puffer fish, concentrations do not presently pose significant threats to public health but they indicate the significant transfer of STXs within the IRL food web. Both natural bloom samples and clonal isolates of Pyrodinium bahamense from the IRL have tested positive for STXs (Landsberg et al., 2002). On Florida’s west coast, where P. bahamense blooms are less frequent and likely reach less toxic biomass, STX concentrations are markedly lower in biota than those in the IRL. By comparison, STX concentrations in southern puffer tissues from Florida’s west coast were no higher than 2735.5 μg STXeq/100g tissue in the skin and mucus when compared with up to 10111.8 μg STXeq/100g tissue in the gut contents of southern puffers from the IRL. In addition, STX levels in west coast sheepshead and flounder are normally below our detectable limit. We still have to confirm the major transfer route of STXs from P. bahamense, the most likely source of STX, into puffer fish. Small benthic filter-feeding bivalves, a significant component of the southern puffer fish diet, are likely vectors for STX transfer and will be tested for toxicity in the upcoming months. Comparison of analytical methods is also required.

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.

Recent electrophysiological and pharmacological data indicate that dopamine enhances the activity of interneurons in the prefrontal cortex (PFC) and induces the release of GABA from these cells. We used in vivo microdialysis to examine the effects of two dopamine receptor antagonists on GABA release in the prefrontal cortex of awake, freely moving rats. Depolarization accomplished by local perfusion of potassium chloride or veratradine markedly increased extracellular GABA levels in the PFC. In contrast, local perfusion of TTX reduced extracellular GABA levels in the PFC. These data indicate that extracellular GABA is derived in part from neurons, and that extracellular levels of the inhibitory amino acid are impulse dependent. The acute administration of haloperidol weakly but significantly decreased extracellular GABA levels in the PFC; no effect of haloperidol on striatal extracellular GABA levels was observed. Systemic administration of the atypical antipsychotic drug clozapine markedly reduced extracellular GABA levels in the PFC, but did not alter striatal GABA levels. Thus, release of GABA from interneurons in the PFC is inhibited by two antipsychotic drugs. These data may suggest that different D2-like dopamine receptors are localized to pyramidal and nonpyramidal neurons in the cortex.

Abstract: Intracranial microdialysis was used to investigate the origin of extracellular γ‐aminobutyric acid (GABA) in the ventral pallidum. Changes in basal GABA levels in response to membrane depolarizers, ion‐channel blockers, and receptor agonists were determined. Antagonism of Ca2+ fluxes with high Mg2+ in a Ca2+‐free perfusion buffer decreased GABA levels by up to 30%. Inhibition of voltage‐dependent Na+ channels by the addition of tetrodotoxin also significantly decreased basal extracellular GABA concentrations by up to 45%, and blockade of Ca2+ and Na+ channels with verapamil reduced extracellular GABA by as much as 30%. The addition of either the GABAA agonist, muscimol, or the GABAB agonist, baclofen, produced a 40% reduction in extracellular GABA. GABA release was stimulated by high K+ and the addition of veratridine to increase Na+ influx. High K+‐induced release was predominately Ca2+‐dependent, whereas the effect of veratridine was potentiated in the absence of extracellular Ca2+. Both high K+‐ and veratridine‐induced elevations in extracellular GABA were inhibited by baclofen, whereas only veratridine‐induced release was antagonized by muscimol. These results demonstrate that at least 50% of basal extracellular GABA in the ventral pallidum is derived from Ca2+‐ or Na+‐dependent mechanisms. They also suggest that Na+‐dependent release of GABA via reversal of the uptake carrier can be shown in vivo.