Identification of Portimine B, a New Cell Permeable Spiroimine That Induces Apoptosis in Oral Squamous Cell Carcinoma.

Spiroimines are a class of compounds produced by marine dinoflagellates with a wide range of toxicity and therapeutic potential. The smallest of the cyclic imines, portimine, is far less toxic than other known members in several animal models. Portimine has also been shown to induce apoptosis and reduce the growth of a variety of cancer cell lines at low nanomolar concentrations. In an effort to discover new spiroimines, the current study undertook a metabolomic analysis of cultures of cyclic imine-producing dinoflagellates, and a new analog of portimine was discovered in which the five-membered cyclic ether is open. Further scrutiny with human oral cavity squamous cell carcinoma (OCSCC) cell lines revealed that the open ring congener was less potent than portimine A but could still lead to the accumulation of apoptotic gene transcripts, fragment genomic DNA, and reduce cancer cell proliferation in the range of 100-200 nM.

Production of extracellular superoxide and hydrogen peroxide by five marine species of harmful bloom-forming algae.

Harmful bloom-forming algae include some of the most prolific microbial producers of extracellular reactive oxygen species (ROS). However, the taxonomic diversity of ROS production, the underlying physiological mechanisms and ecophysiological roles of ROS cycling are not completely characterized among phytoplankton taxa that form harmful algal blooms (HABs). This study examines the extracellular production of the ROS superoxide and hydrogen peroxide by five marine HAB species: Chattonella marina, Heterosigma akashiwo, Karenia brevis, Pseudo-nitzschia sp. and Aureococcus anophagefferens. All species produced extracellular superoxide and hydrogen peroxide. Rates of ROS production per cell spanned several orders of magnitude and varied inversely with cell density, suggesting a potential signaling role for extracellular ROS. ROS production was also detected in the spent media of all cultures except K. brevis, indicating the presence of cell-free ROS-generating constituents, such as enzymes or metabolites, which could be further investigated as molecular targets for tracking ROS production in laboratory and field settings. Finally, ratios of superoxide to hydrogen peroxide production could not be accounted for by superoxide dismutation alone, except in the case of K. brevis, indicating a diversity of ROS production and degradation pathways that may ultimately help illuminate the functions of HAB-derived ROS.

Biosynthetic Studies of 13-Desmethylspirolide C Produced by Alexandrium ostenfeldii (= A. peruvianum): Rationalization of the Biosynthetic Pathway Following Incorporation of (13)C-Labeled Methionine and Application of the Odd-Even Rule of Methylation.

Understanding the biosynthesis of dinoflagellate polyketides presents many unique challenges. Because of the remaining hurdles to dinoflagellate genome sequencing, precursor labeling studies remain the only viable way to investigate dinoflagellate biosynthesis. However, prior studies have shown that polyketide chain assembly does not follow any of the established processes. Additionally, acetate, the common precursor for polyketides, is frequently scrambled, thus compromising interpretation. These factors are further compounded by low production yields of the compounds of interest. A recent report on the biosynthesis of spirolides, a group belonging to the growing class of toxic spiroimines, provided some insight into the polyketide assembly process based on acetate labeling studies, but many details were left uncertain. By feeding (13)C methyl-labeled methionine to cultures of Alexandrium ostenfeldii, the producing organism of 13-desmethylspirolide C, and application of the odd-even methylation rule, the complete biosynthetic pathway has been established.

Chemical analysis of Karenia papilionacea.

One of the most widely studied organisms responsible for Harmful Algal Blooms (HABs) is the marine dinoflagellate Karenia brevis. This organism produces neurotoxic compounds known as brevetoxins. A related dinoflagellate, Karenia papilionacea, has been reported to occasionally co-bloom with K. brevis but has received little attention as a possible toxin producing species. Therefore, our aim was to investigate the toxin profile for K. papilionacea. A toxic fraction was identified using a cell based cytotoxicity assay and the toxin was isolated and identified as the ladder frame polyether brevetoxin-2 (PbTx-2) using mass spectrometry (MS) and nuclear magnetic resonance (NMR). Toxin production in K. papilionacea increased in response to hypoosmotic stress, as previously observed in K. brevis.

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype.

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north-eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra-strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large-scale biogeographic patterns in this species.

Phylogenetic relationships, morphological variation, and toxin patterns in the Alexandrium ostenfeldii (Dinophyceae) complex: implications for species boundaries and identities.

Alexandrium ostenfeldii (Paulsen) Balech and Tangen and A. peruvianum (Balech and B.R. Mendiola) Balech and Tangen are morphologically closely related dinoflagellates known to produce potent neurotoxins. Together with Gonyaulax dimorpha Biecheler, they constitute the A. ostenfeldii species complex. Due to the subtle differences in the morphological characters used to differentiate these species, unambiguous species identification has proven problematic. To better understand the species boundaries within the A. ostenfeldii complex we compared rDNA data, morphometric characters and toxin profiles of multiple cultured isolates from different geographic regions. Phylogenetic analysis of rDNA sequences from cultures characterized as A. ostenfeldii or A. peruvianum formed a monophyletic clade consisting of six distinct groups. Each group examined contained strains morphologically identified as either A. ostenfeldii or A. peruvianum. Though key morphological characters were generally found to be highly variable and not consistently distributed, selected plate features and toxin profiles differed significantly among phylogenetic clusters. Additional sequence analyses revealed a lack of compensatory base changes in ITS2 rRNA structure, low to intermediate ITS/5.8S uncorrected genetic distances, and evidence of reticulation. Together these data (criteria currently used for species delineation in dinoflagellates) imply that the A. ostenfeldii complex should be regarded a single genetically structured species until more material and alternative criteria for species delimitation are available. Consequently, we propose that A. peruvianum is a heterotypic synonym of A. ostenfeldii and this taxon name should be discontinued.

Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay.

This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7-40% from log phase growth to late log - early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20-31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60-90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC-MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity.

Effects of Karenia brevis on clearance rates and bioaccumulation of brevetoxins in benthic suspension feeding invertebrates.

Blooms of the toxic alga Karenia brevis occur along coastlines where sessile suspension feeding invertebrates are common components of benthic communities. We studied the effects of K. brevis on four benthic suspension feeding invertebrates common to the coast of the SE United States: the sponge Haliclona tubifera, the bryozoan Bugula neritina, the bivalve Mercenaria mercenaria, and the tunicate Styela plicata. In controlled laboratory experiments, we determined the rate at which K. brevis was cleared from the seawater by these invertebrates, the effect of K. brevis on clearance rates of a non-toxic phytoplankton species, Rhodomonas sp., and the extent to which brevetoxins bioaccumulated in tissues of invertebrates using an enzyme-linked immunosorbent assay (ELISA). All four invertebrate species cleared significant quantities of K. brevis from seawater, with mean clearance rates ranging from 2.27 to 6.71 L g h⁻¹ for H. tubifera and S. plicata, respectively. In the presence of K. brevis, clearance rates of Rhodomonas sp. by B. neritina and S. plicata were depressed by 75% and 69%, respectively, while clearance rates by H. tubifera and M. mercenaria were unaffected. Negative effects of K. brevis were impermanent; after a recovery period of 13 h, B. neritina and S. plicata regained normal clearance rates. All four invertebrates accumulated high concentrations of brevetoxin after a 4h exposure to K. brevis, but when animals were transferred to filtered seawater for 15 h after exposure, brevetoxin concentrations in the tissues of H. tubifera and B. neritina decreased by ∼80%, while there was no change in toxin concentration in the tissues of S. plicata and M. mercenaria. High cell concentrations of K. brevis may cause a suppression of clearance rates in benthic suspension feeding invertebrates, resulting in a positive feedback for bloom formation. Also, high concentrations of toxin may accumulate in the tissues of benthic suspension feeding invertebrates that may be transferred to higher-level consumers.

QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION FOR COCHLODINIUM FULVESCENS (DINOPHYCEAE), A HARMFUL DINOFLAGELLATE FROM CALIFORNIA COASTAL WATERS(1).

Harmful blooms formed by species of the dinoflagellate Cochlodinium have caused massive fish kills and substantial economic losses in the Pacific Ocean. Recently, prominent blooms of Cochlodinium have occurred in central and southern California (2004-2008), and Cochlodinium cells are now routinely observed in microscopical analysis of algal assemblages from Californian coastal waters. The first documented economic loss due to a Cochlodinium bloom in California occurred in Monterey Bay and resulted in the mortality of commercially farmed abalone. Increasing occurrences of Cochlodinium blooms, the fact that these cells preserve poorly using standard techniques, and the difficulty of identifying preserved specimens using morphological criteria make Cochlodinium species prime candidates for the development of a quantitative real-time polymerase chain reaction (qPCR) approach. The 18S rDNA gene sequenced from Cochlodinium cells obtained from California coastal waters, as well as GenBank sequences of Cochlodinium, were used to design and test a Molecular Beacon(®) approach. The qPCR method developed in this study is species specific, sensitive for the detection of C. fulvescens that has given rise to the recent blooms in the eastern Pacific Ocean, and spans a dynamic abundance range of seven orders of magnitude. Initial application of the method to archived field samples collected during blooms in Monterey Bay revealed no statistically significant correlations between gene copy number and environmental parameters. However, the onset of Cochlodinium blooms in central California was consistent with previously reported findings of correlations to decreased surface temperature and increased inputs of nitrogenous nutrients.

AFLP fingerprinting shows that a single Prymnesium parvum harmful algal bloom consists of multiple clones.

Due to slow rates of molecular evolution, DNA sequences used to identify and build phylogenies of algal species involved in harmful algal blooms (HABs) are generally invariant at the intraspecific level. This means that it is unknown whether HAB events result from the growth of a single clone, a few dominant clones, or multiple clones. This is true despite the fact that several physiological and demographic traits, as well as toxicity, are known to vary across clones. We generated AFLP fingerprints from a set of 6 clonal isolates, taken from a bloom of Prymnesium parvum at a striped bass mariculture facility. This new haptophyte bloom was recently implicated in fish kills at several sites in the United States. The AFLP fragments were highly reproducible and showed that all isolates were distinguishable due to abundant AFLPs unique to single isolates. These results demonstrate that blooms can be genetically diverse outbreaks and indicate that AFLP can be a powerful molecular tool for characterizing and monitoring this diversity.

The new carotenoid pigment moraxanthin is associated with toxic microalgae.

The new pigment "moraxanthin" was found in natural samples from a fish mortality site in the Inland Bays of Delaware, USA. Pure cultures of the species, tentatively named Chattonella cf. verruculosa, and natural samples contained this pigment as a dominant carotenoid. The pigment, obtained from a 10 L culture of C. cf. verruculosa, was isolated and harvested by HPLC and its structure determined from MS and 1D- and 2D-NMR. The data identified this pigment as a new acylated form of vaucheriaxanthin called moraxanthin after the berry like algal cell. Its presence in pure cultures and in natural bloom samples indicates that moraxanthin is specific to C. cf. verruculosa and can be used as a marker of its presence when HPLC is used to analyze natural blooms samples.

Structure and relative potency of several karlotoxins from Karlodinium veneficum.

The karlotoxins are a family of amphidinol-like compounds that play roles in avoiding predation and in prey capture for the toxic dinoflagellate Karlodinium veneficum. The first member of the toxin group to be reported was KmTx 1 (1), and here we report an additional five new members of this family (3-7) from the same strain. Of these additional compounds, KmTx 3 (3) differs from KmTx 1 (1) in having one less methylene group in the saturated portion of its lipophilic arm. In addition, 64-E-chloro-KmTx 3 (4) and 10-O-sulfo-KmTx 3 (5) were identified. Likewise, 65-E-chloro-KmTx 1 (6) and 10-O-sulfo-KmTx 1 (7) were also isolated. Comparison of the hemolytic activities of the newly isolated compounds to that of KmTx 1 shows that potency correlates positively with the length of the lipophilic arm and is disrupted by sulfonation of the polyol arm.

Structure and biosynthesis of amphidinol 17, a hemolytic compound from Amphidinium carterae.

Amphidinol 17 (AM17; 1), a novel amphidinol, has been isolated from a Bahamas strain of Amphidinium carterae. This new congener contains the signature hairpin region and a Delta(6) polyene arm, whereas the polyol arm is distinct from those of other amphidinols. The pattern of acetate incorporation in 1 was directly determined by feeding a single labeled substrate, [2-(13)C]acetate. While the highly conserved regions within the amphidinol family of AM17 have exhibited identical occurrences of cleaved acetates to other amphidinols for which the biosynthesis has been explored, the polyol arm for AM17 displays a higher degree of nascent chain processing that shows similarities to amphidinolide biosynthesis. AM17 exhibited an EC(50) of 4.9 microM in a hemolytic assay using human red blood cells but displayed no detectable antifungal activity.

Sterols and fatty acids of three harmful algae previously assigned as Chattonella.

Sterol and fatty acid compositions were determined for three harmful algal species previously classified in the genus Chattonella (Raphidophyceae): the new genus Chloromorum toxicum (ex Chattonella cf. verruculosa), Verrucophora farcimen (Dictyochophyceae), previously Chattonella aff. verruculosa, and Verrucophora verruculosa (=Pseudochattonella verruculosa) previously Chattonella verruculosa. The major fatty acids of C. toxicum were 14:0, 16:0, 18:1n-9, 18:4n-3 and 20:5n-3, and those of the Verrucophora strains were. 14:0, 16:0, 18:0, 18:4n-3, 18:5n-3 and 22:6n-3. C. toxicum contained the 24beta-ethyl sterols, poriferasterol and clionasterol, as its major sterols. For comparison, the stereochemistry of the 24-ethyl sterols of two raphidophytes, Chattonella marina and Heterosigma akashiwo, was determined to be 24alpha and 24beta, respectively. Both Verrucophora strains contained the 27-nor sterol occelasterol as the only detected sterol. This was the first time occelasterol has been found in algae.

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS.

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.

New fish-killing alga in coastal Delaware produces neurotoxins.

Ten fish mortality events, involving primarily Atlantic menhaden, occurred from early July through September 2000 in several bays and creeks in Delaware, USA. Two events involved large mortalities estimated at 1-2.5 million fish in Bald Eagle Creek, Rehoboth Bay. Samples from Indian Inlet (Bethany Beach), open to the Atlantic, as well as from an enclosed area of massive fish kills at nearby Bald Eagle Creek and Torque Canal were collected and sent to our laboratory for analysis. Microscopic examination of samples from the fish kill site revealed the presence of a single-cell Raphidophyte alga Chattonella cf. verruculosa at a maximum density of 1.04 x 10(7) cells/L. Naturally occurring brevetoxins were also detected in the bloom samples. Besides the Chattonella species, no other known brevetoxin-producing phytoplankton were present. Chromatographic, immunochemical, and spectroscopic analyses confirmed the presence of brevetoxin PbTx-2, and PbTx-3 and -9 were confirmed by chromatographic and immunochemical analyses. This is the first confirmed report in the United States of brevetoxins associated with an indigenous bloom in temperate Atlantic estuarine waters and of C. cf. verruculosa as a resident toxic organism implicated in fish kills in this area. The bloom of Chattonella continued throughout September and eventually declined in October. By the end of October C. cf. verruculosa was no longer seen, nor was toxin measurable in the surface waters. The results affirm that to avoid deleterious impacts on human and ecosystem health, increased monitoring is needed for brevetoxins and organism(s) producing them, even in areas previously thought to be unaffected.

A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shellfish, and mammalian body fluid.

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.