Characterization of polar brevetoxin derivatives isolated from Karenia brevis cultures and natural blooms.

Several novel brevetoxin derivatives were isolated and identified in Karenia brevis cultures and natural blooms by using solid phase extraction (SPE) and LC/MS(MS) techniques. These analogs were more polar compared with previously described brevetoxins, and were poorly extractable by conventional non-polar solvent (chloroform) partitioning. Brevetoxin analogs were structurally confirmed as hydrolyzed (open A-ring) forms of brevetoxins PbTx-1, PbTx-7, PbTx-2, and PbTx-3, and of oxidized PbTx-1 and PbTx-2. Some of these open A-ring derivatives were in greater abundance than their non-hydrolyzed counterparts. All were in much greater abundance in bloom water filtrate compared with cell-rich fractions. Open A-ring compounds were cytotoxic in mouse neuroblastoma (N2a) cell assay. In the K. brevis bloom-exposed Eastern oyster, brevetoxin metabolites with opened A rings were identified (e.g., open-ring cysteine-PbTx conjugates), contributing to their overall toxin burden.     

Lipophilic toxicity from the marine dinoflagellate Karenia brevisulcata: use of the brevetoxin neuroblastoma assay to assess toxin presence and concentration.

A method for assessing the presence and concentration of a major toxin produced by Karenia brevisulcata has been developed, based on the neuroblastoma assay for brevetoxins. This cytotoxicity assay, coupled with observations of the dose-response relationship and of changes in cellular morphology during the assay gave a characteristic toxin 'signature'. This signature was consistent whether the toxicity was assessed in extracts of shellfish, seawater samples, uni-algal cultures of K. brevisulcata, or in partially purified toxin samples. Using this method it was possible to confirm the presence of this toxicity during a mixed Karenia bloom, and infer that K. brevisulcata toxin (KBT) was the probable cause of the fish and shellfish mortality observed at that time. The neuroblastoma assay thus provides a solution to the problem of confirming the presence of KBT in suspected K. brevisulcata events, even in the absence of a known toxin structure.     

Brevetoxins, like ciguatoxins, are potent ichthyotoxic neurotoxins that accumulate in fish.

Brevetoxins and ciguatoxins are closely related potent marine neurotoxins. Although ciguatoxins accumulate in fish to levels that are dangerous for human consumption, live fish have not been considered as potential sources of brevetoxin exposure in humans. Here we show that, analogous to ciguatoxins, brevetoxins can accumulate in live fish by dietary transfer. We experimentally identify two pathways leading to brevetoxin-contaminated omnivorous and planktivorous fish. Fish fed with toxic shellfish and Karenia brevis cultures remained healthy and accumulated high brevetoxin levels in their tissues (up to 2675 ng g(-1) in viscera and 1540 ng g(-1) in muscle). Repeated collections of fish from St. Joseph Bay in the Florida panhandle reveal that accumulation of brevetoxins in healthy fish occurs in the wild. We observed that levels of brevetoxins in the muscle of fish at all trophic levels rise significantly, but not to dangerous levels, during a K. brevis bloom. Concentrations were highest in fish liver and stomach contents, and increased during and immediately following the bloom. The persistence of brevetoxins in the fish food web was followed for 1 year after the K. brevis bloom.     

Confirmation of brevetoxin metabolism in cockle, Austrovenus stutchburyi, and greenshell mussel, Perna canaliculus, associated with New Zealand neurotoxic shellfish poisoning, by controlled exposure to Karenia brevis culture.

We examined metabolism of PbTxs in New Zealand cockle, Austrovenus (A.) stutchburyi, and greenshell mussel, Perna (P.) canaliculus, by means of liquid chromatography coupled with tandem mass spectrometry. PbTx-2, PbTx-3 and BTX-B5 were detected in Karenia (K.) brevis culture medium in the ratio of ca. 50:2:5. The amounts of PbTx-3 and BTX-B5 were greatly increased in both seawater and shellfish exposed to K. brevis cultures or supernatant prepared by disruption of K. brevis under appropriate condition, while those of PbTx-2 were decreased. Some PbTx-2 was present in P. canaliculus, but not in A. stutchburyi. Low levels of BTX-B1 were detected in A. stutchburyi, but not P. canaliculus. Levels of PbTx-3 and BTX-B5 were highest immediately after exposure and then declined rapidly in both shellfish. BTX-B1 increased in concentration after exposure, and was then gradually eliminated from A. stutchburyi. Three successive exposures of A. stutchburyi to K. brevis cultures resulted in similar initial levels of PbTx-3 and BTX-B5, while BTX-B1 accumulated after each dose. In P. canaliculus, initial levels of PbTx-3 were similar, while PbTx-2 and BTX-B5 accumulated after each dose. PbTx-3 and BTX-B5 are proposed to be suitable markers for monitoring shellfish toxicity after a red tide event.     

Confirmation of brevetoxin metabolism in the Eastern oyster (Crassostrea virginica) by controlled exposures to pure toxins and to Karenia brevis cultures.

Previously, we analyzed Eastern oysters (Crassostrea virginica) naturally exposed to a Karenia brevis red tide and found that brevetoxins (PbTx) are rapidly accumulated and metabolized. Several metabolites were isolated and later identified, including a cysteine-PbTx conjugate (MH(+): m/z 1018) and its sulfoxide product (m/z 1034). In the present study, we confirm and extend those findings by examining PbTx metabolism and elimination in oysters exposed to pure toxins (PbTx-2 and -3) under controlled conditions. Waterborne PbTx-3 was rapidly accumulated, but not metabolized, in the oyster and was largely eliminated within 2 weeks after exposure. In contrast, PbTx-2 was accumulated and rapidly metabolized. Metabolites of PbTx-2 included the reduction product PbTx-3 (m/z 897), and the cysteine conjugates (m/z 1018 and 1034) isolated previously from the field samples. Levels of the metabolite PbTx-3 in PbTx-2-exposed oysters were highest immediately after exposure and declined at a rate similar to parent PbTx-3 in PbTx-3-exposed oysters. Cysteine-PbTx persisted for 8 weeks after exposure. The same metabolites were confirmed in oysters exposed to laboratory cultures of K. brevis. PbTx metabolites contribute to neurotoxic shellfish poisoning (NSP) and should be included in analytical protocols for monitoring shellfish toxicity after a K. brevis red tide event.     

Monitoring of brevetoxins in the Karenia brevis bloom-exposed Eastern oyster (Crassostrea virginica).

Brevetoxin uptake and elimination were examined in Eastern oyster (Crassostrea virginica) exposed to recurring blooms of the marine alga Karenia brevis in Sarasota Bay, FL, over a three-year period. Brevetoxins were monitored by in vitro assays (ELISA, cytotoxicity assay, and receptor binding assay) and LC-MS, with in vivo toxicity of shellfish extracts assessed by the traditional mouse bioassay. Measurements by all methods reflected well the progression and magnitude of the blooms. Highest levels recorded by mouse bioassay at bloom peak were 157 MU/100g. Oysters were toxic by mouse bioassay at levels >or=20 MU/100g for up to two weeks after bloom dissipation, whereas brevetoxins were measurable by in vitro assays and LC-MS for several months afterwards. For the structure-based methods, summed values for the principal brevetoxin metabolites of PbTx-2 (cysteine and cysteine sulfoxide conjugates), as determined by LC-MS, were highly correlated (r(2)=0.90) with composite toxin measurements by ELISA. ELISA and LC-MS values also correlated well (r(2)=0.74 and 0.73, respectively) with those of mouse bioassay. Pharmacology-based cytotoxicity and receptor binding assays did not correlate as well (r(2)=0.65), and were weakly correlated with mouse bioassay (r(2)=0.48 and 0.50, respectively). ELISA and LC-MS methods offer rapid screening and confirmation, respectively, of brevetoxin contamination in the oyster, and are excellent alternatives to mouse bioassay for assessing oyster toxicity following K. brevis blooms.     

Effects of the dinoflagellate Karenia brevis on larval development in three species of bivalve mollusc from Florida.

The effects of Karenia brevis (Wilson clone) on larval survival and development of the northern quahog, Mercenaria mercenaria, eastern oyster, Crassostrea virginica and bay scallop, Argopecten irradians, were studied in the laboratory. Larvae were exposed to cultures of whole and lysed cells, with mean total brevetoxin concentrations of 53.8 and 68.9 microgL(-1), respectively. Survival of early (3-day-old) larvae was generally over 85% for all shellfish species at K. brevis densities of 100 cells ml(-1) or less, and not significantly different between whole and lysed culture. At 1000 cells ml(-1), survival was significantly less in lysed culture than whole culture for both M. mercenaria and C. virginica. Survival of late (7-day-old) larvae in all three species was not significantly affected by K. brevis densities of 1000 cells ml(-1) or less. At 5000 cells ml(-1), however, survival was reduced to 37%, 26% and 19% for A. irradians, M. mercenaria and C. virginica, respectively. Development of C. virginica and M. mercenaria larvae was protracted at K. brevis densities of 1000 cells ml(-1). These results suggest that blooms of K. brevis, and particularly their associated brevetoxins, may have detrimental consequences for Florida's shellfisheries by disrupting critical larval processes. Special attention should be paid to blooms of K. brevis where these shellfish occur naturally or where aquaculture and restoration activities are either ongoing or planned.     

Comparative effects of the toxic dinoflagellate Karenia brevis on clearance rates in juveniles of four bivalve molluscs from Florida, USA.

The effects of Karenia brevis (Gymnodiniales, Gymnodiniaceae) on the feeding activity of juveniles of four species of bivalve mollusc were examined in the laboratory to assess the potential impacts on these important shellfish populations from Florida. Clearance rates were determined under short-term (one hour) static and long-term (two days) flow-through conditions using both whole and lysed cultures of K. brevis. Under short-term conditions, the bay scallop, Argopecten irradians, was the most sensitive species, exhibiting a 79% reduction in clearance rate at 1000 cells ml(-1) of whole K. brevis culture compared to the control (no K. brevis). The eastern oyster, Crassostrea virginica, was the least responsive, showing a 38% reduction in clearance rate between the same treatments. The green mussel, Perna viridis, and the northern quahog, Mercenaria mercenaria, displayed intermediate responses. Similar results were also observed during long-term exposures to a continuous supply of K. brevis. Bay scallops showed a significant decline in clearance rate at 100 cells ml(-1) after 24h exposure; clearance rate of oysters was not affected by K. brevis at this concentration. No mortality was observed for any species during these brief exposures. The prospect for recovery of bay scallop populations in Florida estuaries where they were once abundant may be hampered by recurring blooms of K. brevis. Reduced clearance rates in M. mercenaria at high K. brevis densities could translate into poor growth of cultured Florida hard clams. On the other hand, P. viridis, which also showed reduced clearance rates at high K. brevis concentrations, might be negatively impacted by K. brevis blooms, thereby affecting their ability to spread into estuaries hampered by recurring toxic algal blooms.     

Brevetoxin-induced neural insult in the retrosplenial cortex of mouse brain

Brevetoxins (polyether breve toxins; PbTx) are polyether neurotoxins produced by the marine dinoflagellate Karenia brevis, an organism associated with red tide blooms in the Gulf of Mexico and along the Atlantic coast from Florida to North Carolina. Brevetoxin-3 (PbTx-3) is a major component of the array of brevetoxins found in marine aerosols measured along red tide affected beaches. Humans exposed to aerosolized brevetoxins for short periods of time often suffer a variety of adverse health effects. It was consequently of interest to assess the potential for aerosolized brevetoxin to produce a neurotoxic response. Female BALB/c mice were exposed nose-only for 2 consecutive days to PbTx-3 aerosol, with a 2-h exposure on the first day and a 4-h exposure on the second day. The average PbTx-3 exposure concentrations on days 1 and 2 were 312 +/- 113 mug brevetoxin 3/m3 and 278 +/- 24 mug brevetoxin 3/m3, respectively. The brevetoxin-containing aerosol had a mass median aerodynamic diameter of 0.92 mum with a geometric standard deviation of 1.38. Coronal sections of mouse brains were evaluated for neuronal damage using both silver and Fluoro-Jade B staining to identify degenerating neuronal elements. PbTx-3 inhalation exposure produced neuronal degeneration in the posterior cingulate/retrosplenial cortex of mice as evidenced by silver-positive degenerating neurons in this region. No staining was found in other regions of the PBTx-3-exposed mouse brains or in brains of control, sham-exposed mice. The existence of a neurotoxic insult in PbTx-3-exposed mice was confirmed using Fluoro-Jade B to label degenerating neurons. Fluro-Jade-positive neurons were observed in the retrosplenial cortex of PBTx-3 exposed, but not control, mice. These results suggest that subacute exposure to PbTx-3 for 2 days is sufficient to induce neuronal degeneration in a discrete region of the mouse cerebral cortex.     

Inhalation toxicity of brevetoxin 3 in rats exposed for 5 days

Brevetoxins are potent neurotoxins produced by the marine dinoflagellate Karenia brevis. Exposure to brevetoxins may occur during a K. brevis red tide when the compounds become aerosolized by wind and surf. This study assesses possible adverse health effects associated with short-term inhalation exposure to brevetoxin 3. Male F344/Crl/Br rats were exposed to 500 microg brevetoxin 3/m3 by nose-only inhalation for 0.5 or 2 h/d for 5 consecutive days. Control rats were sham exposed for 2 h to vehicle. Calculated deposited brevetoxin doses were 8.3 and 33 microg/kg/d for the low- and high-dose groups, respectively. At the termination of exposures, only body weights of the high-dose group (Group B) were significantly below control values. By immunohistochemistry (IHC), small numbers of splenic and peribronchiolar lymphoid tissue macrophages stained positive for brevetoxin, while nasal mucosa, liver, and brain were IHC negative for brevetoxin. No gross or microscopic lesions were observed in any tissue examined. There was no biochemical evidence of cytotoxicity or inflammation in bronchoalveolar lavage fluid. Alveolar macrophages showed some evidence of activation following brevetoxin exposure. Humoral-mediated immunity was suppressed in brevetoxin-exposed rats as indicated by a >70% reduction in splenic plaque-forming cells in brevetoxin-exposed animals compared to controls. Results suggest that the immune system may be a target of toxicity following brevetoxin inhalation. Future studies will focus on identification of a no-effect level and mechanisms underlying brevetoxin-induced immune suppression.     

A cellular target for the lipophilic toxins from Karenia brevisulcata.

Crude lipophilic toxin from Karenia brevisulcata has been shown to be toxic to mammalian neuroblastoma (Neuro2A) cells in culture. This toxicity is partially antagonised by the addition of saxitoxin. The dose-response curves of saxitoxin acting to antagonise the action of K. brevisulcata toxin and of brevetoxin were examined and they displayed similar EC50 values. These results suggest that at least some of the effect on Neuro2A cells of the lipophilic toxicity found in K. brevisulcata results from an interaction with the mammalian voltage-dependent sodium channel.     

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.     

Brevetoxin metabolism and elimination in the Eastern oyster (Crassostrea virginica) after controlled exposures to Karenia brevis.

The metabolism and elimination of brevetoxins were examined in the Eastern oyster (Crassostrea virginica) following controlled exposures to Karenia brevis cultures in the laboratory. After a 2-day exposure period ( approximately 62 million cells/oyster), elimination of brevetoxins and their metabolites was monitored by using liquid chromatography/mass spectrometry (LC/MS). Composite toxin in oyster extracts was measured by in vitro assay (i.e. cytotoxicity, receptor binding, and ELISA). Of the parent algal toxins, PbTx-1 and PbTx-2 were not detectable by LC/MS in K. brevis-exposed oysters. PbTx-3 and PbTx-9, which are accumulated directly from K. brevis and through metabolic reduction of PbTx-2 in the oyster, were at levels initially (after exposure) of 0.74 and 0.49 microg equiv./g, respectively, and were eliminated largely within 2 weeks after dosing. PbTx-7 and PbTx-10, the reduced forms of PbTx-1, were non-detectable. Conjugative brevetoxin metabolites identified previously in field-exposed oysters were confirmed in the laboratory-exposed oysters. Cysteine conjugates of PbTx-1 and PbTx-2, and their sulfoxides, were in the highest abundance, as apparent in LC/MS ion traces, and were detectable for up to 6 months after dosing. Composite toxin measurements by in vitro assay also reflected persistence (up to 6 months) of brevetoxin residues in the oyster. Levels of cysteine conjugates, as determined by LC/MS, were well correlated with those of composite toxin, as measured by ELISA, throughout depuration. Composite toxin levels by cytotoxicity assay were well correlated with those by receptor binding assay. Cysteine-PbTx conjugates are useful LC/MS determinants of brevetoxin exposure and potential markers for composite toxin in the Eastern oyster.     

Characterization of brevetoxin metabolism in Karenia brevis bloom-exposed clams (Mercenaria sp.) by LC-MS/MS

Brevetoxin metabolites were identified and characterized in the hard clam (Mercenaria sp.) after natural exposure to Karenia brevis blooms by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Principal brevetoxins BTX-1 and BTX-2 produced by K. brevis were not detectable in clams. Metabolites of these brevetoxins found in clams included products of oxidation, reduction, hydrolysis and amino acid/fatty acid conjugation. Of highest abundance were cysteine and taurine conjugates. We also found glutathione, glycine-cysteine, and γ-glutamyl-cysteine conjugates. A series of fatty acid derivatives of cysteine-brevetoxin conjugates were also identified.     

Identification of a Key Residue for Oligomerisation and Pore-Formation of Clostridium perfringens NetB

Necrotic enteritis toxin B (NetB) is a β-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis.     

Characterization of Florida red tide aerosol and the temporal profile of aerosol concentration

Red tide aerosols containing aerosolized brevetoxins are produced during the red tide bloom and transported by wind to coastal areas of Florida. This study reports the characterization of Florida red tide aerosols in human volunteer studies, in which an asthma cohort spent 1 h on Siesta Beach (Sarasota, Florida) during aerosolized red tide events and non-exposure periods. Aerosol concentrations, brevetoxin levels, and particle size distribution were measured. Hourly filter samples were taken and analyzed for brevetoxin and NaCl concentrations. In addition, the aerosol mass concentration was monitored in real time. The results indicated that during a non-exposure period in October 2004, no brevetoxin was detected in the water, resulting in non-detectable levels of brevetoxin in the aerosol. In March 2005, the time-averaged concentrations of brevetoxins in water samples were moderate, in the range of 5-10 μg/L, and the corresponding brevetoxin level of Florida red tide aerosol ranged between 21 and 39 ng/m³. The temporal profiles of red tide aerosol concentration in terms of mass, NaCl, and brevetoxin were in good agreement, indicating that NaCl and brevetoxins are components of the red tide aerosol. By continuously monitoring the marine aerosol and wind direction at Siesta Beach, we observed that the marine aerosol concentration varied as the wind direction changed. The temporal profile of the Florida red tide aerosol during a sampling period could be explained generally with the variation of wind direction.     

Effects of brevetoxins on murine myeloma SP2/O cells: aberrant cellular division

Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.     

Remarkable difference in accumulation of paralytic shellfish poisoning toxins among bivalve species exposed to Pyrodinium bahamense var. compressum bloom in Masinloc Bay, Philippines.

Seasonal variation of bivalve toxicity was monitored in association with the abundance of the toxic dinoflagellate Pyrodinium bahamense var. compressum in Masinloc Bay, Luzon Island. Among 7 species of bivalve, 6 species became toxic during a bloom of the dinoflagellate. However, remarkable difference in the toxicity was observed among the species. The toxicity of thorny oyster Spondylus squamosus was the highest among the species, showing more than 30 times that of safety consumption level after the peak bloom of the dinoflagellate, while other bivalve species showed much lower toxicity. The toxicity of thorny oyster decreased under absence of the dinoflagellate, but this species maintained a considerably high toxicity throughout a year. Similar trend was observed in penshell Atrina vexillum in a small scale, indicating that these species in the bay are not safe for human consumption almost throughout a year. The toxicity of green mussel Perna viridis increased to significant level during a bloom, but it decreased rapidly when the dinoflagellate disappeared. Toxin analysis of cultured and natural cells showed typical toxin profile of the dinoflagellate. Toxin profile of all the bivalve species reflected the characteristic toxin profile of the dinoflagellate.     

Brevetoxin binding: molecular pharmacology versus immunoassay

Brevetoxin PbTx-3 isolated from Florida's red tide dinoflagellate Ptychodiscus brevis has been produced recently in tritiated form by reductive tritiation of brevetoxin PbTx-2. Tritiated PbTx-3 has been used as a specific probe in competitive radioimmunoassays developed to detect brevetoxins in food sources, and this probe has also been utilized to characterize the brevetoxin binding component in rat brain synaptosomes. Brevetoxins PbTx-2 and PbTx-3, possessing the same structural backbone (type-1) as the tritiated probe, and PbTx-1 and PbTx-7, possessing a second structural backbone (type-2), have been compared quantitatively in their individual abilities to competitively displace tritiated PbTx-3 from its specific binding site in each assay. Type-1 toxins displaced labeled probe with ED50 values of 20-22 nM and 12-17 nM in radioimmunoassay and synaptosomes, respectively. Type-2 toxins displaced labeled probe with ED50 values of 92-93 nM and 3.5-4.1 nM in RIA and synaptosomes, respectively. Synaptosome assays reflect potency of each toxin examined, while radioimmunoassay reflects structural similarities to the immunizing toxin PbTx-3.