Chemical and Biological Study of Novel Aplysiatoxin Derivatives from the Marine Cyanobacterium Lyngbya sp.

Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda, whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G (1) and neo-debromoaplysiatoxin H (2) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid-liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G (1) (3.6 mg) and neo-debromoaplysiatoxin H (2) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC50) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.

Oscillatoxin I: A New Aplysiatoxin Derivative, from a Marine Cyanobacterium.

Cyanobacteria have been shown to produce a number of bioactive compounds, including toxins. Some bioactive compounds obtained from a marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) have been recognized as drug leads; one of these compounds is aplysiatoxin. We have isolated various aplysiatoxin derivatives from a M. producens sample obtained from the Okinawan coastal area. The frozen sample was extracted with organic solvents. The ethyl acetate layer was obtained from the crude extracts via liquid-liquid partitioning, then separated by HPLC using a reversed-phase column. Finally, 1.1 mg of the compound was isolated. The chemical structure of the isolated compound was elucidated with spectroscopic methods, using HR-MS and 1D and 2D NMR techniques, and was revealed to be oscillatoxin I, a new member of the aplysiatoxin family. Oscillatoxin I showed cytotoxicity against the L1210 mouse lymphoma cell line and diatom growth-inhibition activity against the marine diatom Nitzschia amabilis.

A new lyngbyatoxin from the Hawaiian cyanobacterium Moorea producens.

Lyngbyatoxin A from the marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) is known as the causative agent of "swimmer's itch" with its highly inflammatory effect. A new toxic compound was isolated along with lyngbyatoxin A from an ethyl acetate extract of M. producens collected from Hawaii. Analyses of HR-ESI-MS and NMR spectroscopies revealed the isolated compound had the same planar structure with that of lyngbyatoxin A. The results of optical rotation and CD spectra indicated that the compound was a new lyngbyatoxin A derivative, 12-epi-lyngbyatoxin A (1). While 12-epi-lyngbyatoxin A showed comparable toxicities with lyngbyatoxin A in cytotoxicity and crustacean lethality tests, it showed more than 100 times lower affinity for protein kinase Cδ (PKCδ) using the PKCδ-C1B peptide when compared to lyngbyatoxin A.

Synthesis and biological activities of simplified analogs of the natural PKC ligands, bryostatin-1 and aplysiatoxin.

Protein kinase C (PKC) isozymes play central roles in signal transduction on the cell surface and could serve as promising therapeutic targets of intractable diseases like cancer, Alzheimer's disease, and acquired immunodeficiency syndrome (AIDS). Although natural PKC ligands like phorbol esters, ingenol esters, and teleocidins have the potential to become therapeutic leads, most of them are potent tumor promoters in mouse skin. By contrast, bryostatin-1 (bryo-1) isolated from marine bryozoan is a potent PKC activator with little tumor-promoting activity. Numerous investigations have suggested bryo-1 to be a promising therapeutic candidate for the above intractable diseases. However, there is a supply problem of bryo-1 both from natural sources and by organic synthesis. Recent approaches on the synthesis of bryo-1 have focused on its simplification, without decreasing the ability to activate PKC isozymes, to develop new medicinal leads. Another approach is to use the skeleton of natural PKC ligands to develop bryo-1 surrogates. We have recently identified 10-methyl-aplog-1 (26), a simplified analog of tumor-promoting aplysiatoxin (ATX), as a possible therapeutic lead for cancer. This review summarizes recent investigations on the simplification of natural PKC ligands, bryo-1 and ATX, to develop potential medicinal leads.

Effects of the methoxy group in the side chain of debromoaplysiatoxin on its tumor-promoting and anti-proliferative activities.

Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.

Structure-activity studies on the spiroketal moiety of a simplified analogue of debromoaplysiatoxin with antiproliferative activity.

Aplog-1, a simplified analogue of tumor-promoting debromoaplysiatoxin, is antiproliferative but not tumor-promoting. Our recent study has suggested that local hydrophobicity around the spiroketal moiety is a crucial determinant for antiproliferative activity. To further clarify the structural features relevant to the activity, we synthesized two methyl derivatives of aplog-1, where a methyl group was installed at position 4 or 10 of the spiroketal moiety. 10-Methyl-aplog-1 (5) bound to the C1B domains of novel PKCs (δ, η, and θ) with subnanomolar K(i) values, approximately 10-20 times stronger than aplog-1, and markedly inhibited the growth of many human cancer cell lines, while 4-methyl-aplog-1 (4) had levels of activity similar to those of aplog-1. Interestingly, 5 showed little tumor-promoting activity unlike the tumor promoter debromoaplysiatoxin. These results suggest that 5 is a potent PKC ligand without tumor-promoting activity and could be a therapeutic lead for the treatment of cancer, like bryostatins.

Climate change: links to global expansion of harmful cyanobacteria.

Cyanobacteria are the Earth's oldest (∼3.5 bya) oxygen evolving organisms, and they have had major impacts on shaping our modern-day biosphere. Conversely, biospheric environmental perturbations, including nutrient enrichment and climatic changes (e.g. global warming, hydrologic changes, increased frequencies and intensities of tropical cyclones, more intense and persistent droughts), strongly affect cyanobacterial growth and bloom potentials in freshwater and marine ecosystems. We examined human and climatic controls on harmful (toxic, hypoxia-generating, food web disrupting) bloom-forming cyanobacteria (CyanoHABs) along the freshwater to marine continuum. These changes may act synergistically to promote cyanobacterial dominance and persistence. This synergy is a formidable challenge to water quality, water supply and fisheries managers, because bloom potentials and controls may be altered in response to contemporaneous changes in thermal and hydrologic regimes. In inland waters, hydrologic modifications, including enhanced vertical mixing and, if water supplies permit, increased flushing (reducing residence time) will likely be needed in systems where nutrient input reductions are neither feasible nor possible. Successful control of CyanoHABs by grazers is unlikely except in specific cases. Overall, stricter nutrient management will likely be the most feasible and practical approach to long-term CyanoHAB control in a warmer, stormier and more extreme world.

Lyngbya dermatitis (toxic seaweed dermatitis).

Lyngbya dermatitis is an irritant contact dermatitis caused by the blue-green alga (or cyanobacterium), Lyngbya majuscula, commonly found in tropical and temperate waters worldwide. Lesions generally appear in a bathing suit distribution minutes to hours after exposure, initially with itching or burning, evolving into a blistering eruption which eventually desquamates leaving bright red, tender erosions that resolve spontaneously in about a week. Our case is of a 13-year-old female that presented with haphazard clusters of reddish-brown vesicles and papules on her abdomen one day after swimming in rough surf conditions on the shores of Oahu, Hawaii. Histopathological examination revealed an acute irritant contact dermatitis consistent with Lyngbya dermatitis. L. majuscule, with its wealth of biologically active compounds, should be a consideration in any patient presenting with an acute irritant contact dermatitis following temperate saltwater exposure.

Dermatitis associated with exposure to a marine cyanobacterium during recreational water exposure.

BACKGROUND: Anecdotal evidence reported an outbreak of symptoms on Fraser Island during the late 1990 s similar to those expected from exposure to dermotoxins found in the cyanobacterium L. majuscula. This coincided with the presence of a bloom of L. majuscula. METHODS: Records from the Fraser Island National Parks First aid station were examined. Information on cyanobacterial blooms at Fraser Island were obtained from Queensland National Parks rangers. RESULTS: Examination of first aid records from Fraser Island revealed an outbreak of symptoms predominantly in January and February 1998. CONCLUSION: During a bloom of L. majuscula there were numerous reports of symptoms that could be attributed to dermotoxins found in L. majuscula. The other four years examined had no L. majuscula blooms and the number of L. majuscula symptoms was much reduced. These cases comprised a high percentage of the cases treated at the first aid station.

Toxins of cyanobacteria.

Blue-green algae are found in lakes, ponds, rivers and brackish waters throughout the world. In case of excessive growth such as bloom formation, these bacteria can produce inherent toxins in quantities causing toxicity in mammals, including humans. These cyanotoxins include cyclic peptides and alkaloids. Among the cyclic peptides are the microcystins and the nodularins. The alkaloids include anatoxin-a, anatoxin-a(S), cylindrospermopsin, saxitoxins (STXs), aplysiatoxins and lyngbyatoxin. Both biological and chemical methods are used to determine cyanotoxins. Bioassays and biochemical assays are nonspecific, so they can only be used as screening methods. HPLC has some good prospects. For the subsequent detection of these toxins different detectors may be used, ranging from simple UV-spectrometry via fluorescence detection to various types of MS. The main problem in the determination of cyanobacterial toxins is the lack of reference materials of all relevant toxins. In general, toxicity data on cyanotoxins are rather scarce. A majority of toxicity data are known to be of microcystin-LR. For nodularins, data from a few animal studies are available. For the alkaloids, limited toxicity data exist for anatoxin-a, cylindrospermopsin and STX. Risk assessment for acute exposure could be relevant for some types of exposure. Nevertheless, no acute reference doses have formally been derived thus far. For STX(s), many countries have established tolerance levels in bivalves, but these limits were set in view of STX(s) as biotoxins, accumulating in marine shellfish. Official regulations for other cyanotoxins have not been established, although some (provisional) guideline values have been derived for microcystins in drinking water by WHO and several countries.

Health effects of recreational exposure to Moreton Bay, Australia waters during a Lyngbya majuscula bloom.

A survey of residents in an area subject to annual toxic cyanobacterial blooms was undertaken to examine potential health effects of cyanobacteria toxins. The survey assessed the health of marine recreational water users in Deception Bay/Bribie Island area in northern Moreton Bay, Queensland, which is exposed to blooms of the nuisance and potentially harmful cyanobacterium Lyngbya majuscula. A postal survey was mailed to 5000 residents with a response rate of 27%. High numbers of people (78%) responding to the survey reported recreational water activity in Moreton Bay. Of those having marine recreational water activity, 34% reported at least one symptom after exposure to marine waters, with skin itching the most reported (23%). Younger participants had greater water exposure and symptoms than older participants. Participants with greater exposures were more likely to have skin and eye symptoms than less exposed groups, suggesting agents in the marine environment may have contributed to these symptoms. Of those entering Moreton Bay waters 29 (2.7%) reported severe skin symptoms, 12 of whom attended a health professional. Six (0.6%) reported the classic symptoms of recreational water exposure to L. majuscula, severe skin symptoms in the inguinal region. Participants with knowledge of L. majuscula were less likely to report less skin, gastrointestinal and fever and headache symptoms. In conclusion, high numbers of participants reported symptoms after exposure to waters subject to L. majuscula blooms but only a small number appeared to be serious in nature suggesting limited exposure to toxins.

Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya isolated from the Florida Everglades.

The genus of filamentous cyanobacteria, Lyngbya, has been found to be a rich source of bioactive metabolites. However, identification of such compounds from Lyngbya has largely focused on a few marine representatives. Here, we report on the pharmacology and toxicology of pahayokolide A from a freshwater isolate, Lyngbya sp. strain 15-2, from the Florida Everglades. Specifically, we investigated inhibition of microbial representatives and mammalian cell lines, as well as toxicity of the compound to both invertebrate and vertebrate models. Pahayokolide A inhibited representatives of Bacillus, as well as the yeast, Saccharomyces cerevisiae. Interestingly, the compound also inhibited several representatives of green algae that were also isolated from the Everglades. Pahayokolide A was shown to inhibit a number of cancer cell lines over a range of concentrations (IC50 varied from 2.13 to 44.57 microM) depending on the cell-type. When tested against brine shrimp, pahayokolide was only marginally toxic at the highest concentrations tested (1 mg/mL). The compound was, however, acutely toxic to zebrafish embryos (LC50=2.15 microM). Possible biomedical and environmental health aspects of the pahayokolides remain to be investigated; however, the identification of bioactive metabolites such as these demonstrates the potential of the Florida Everglades as source of new toxins and drugs.

Pathological effects of lyngbyatoxin A upon mice.

Histopathological changes induced in mice by lyngbyatoxin A were studied in connection with the occurrence of the toxin in marine turtles implicated in human intoxication. Lyngbyatoxin A showed an i.p. lethal dose 250 microg/kg in immature mice (3-week old) and most severely damaged capillaries of villi in the small intestine. Immature mice were more sensitive than matured ones and died of bleeding from the small intestines. With sublethal doses were observed erosion in the stomach, small intestine, cecum, and large intestine, as well as inflammation in the lung. Time course changes observed after p.o. administration of sublethal doses indicated severe mucus secretion and injuries to occur within 60 min in the intestine and within 24h in the stomach. Increased inflammatory cells followed these injuries. The injuries in the lung, stomach, and small intestine took a few weeks for recovery. The cause of death and the effective dose levels resembled those of aplysiatoxin poisoning.

Lyngbyabellin B, a toxic and antifungal secondary metabolite from the marine cyanobacterium Lyngbya majuscula.

Lyngbyabellin B (1) was isolated from a marine cyanobacterium, Lyngbya majuscula, collected near the Dry Tortugas National Park, Florida. This new cyclic depsipeptide displayed potent toxicity toward brine shrimp and the fungus Candida albicans. The planar structure was deduced using 1D and 2D NMR spectroscopic methods, and the stereochemistry is proposed through a combination of NMR and chiral GC/MS analysis.

Bleeding from the small intestine caused by aplysiatoxin, the causative agent of the red alga Gracilaria coronopifolia poisoning.

The cause of death by aplysiatoxin poisoning was bleeding from the small intestine in mice. The pathological changes related to the cause and progression of bleeding were studied morphologically. Bleeding from the capillaries was observed 60 min after i.p. treatment at 250 microg/kg, and this was preceded by dilatation of the lymphatic vessel and congestion of capillaries in the lamina propria from 10 min after the injection. At 100 microg/kg i.v., the target vessels were in the lung, where fibrin deposition was observed in the dilated pulmonary artery, and blood flowed out through a gap in the artery. Then, in the small intestine, similar changes appeared to have occurred, and bleeding was induced in two characteristic ways, one through deposition of fibrin in the lumen and the other via distension of the capillary wall.

Morphological observations of diarrhea in mice caused by aplysiatoxin, the causative agent of the red alga Gracilaria coronopifolia poisoning in Hawaii.

Diarrhea caused by the red alga Gracilaria coronopifolia poisoning was investigated in mice. The target site of a lethal dose was the whole small intestine where the toxin caused bleeding, resulting in hemorrhagic shock. With a sublethal dose, diarrhea appeared about 4.5 h after i.p. injection and continued for about 4 h intermittently. The site of diarrhea was the large intestine, where the submucosa first accumulated fluid from edema. Then the fluid moved into the lamina propria, the surface epithelial cells were broken and the fluid flowed into the lumen. Finally, diarrheic components apparently originating from capillaries were secreted directly into the lumen. The cecum was the main target of the diarrhea. After the diarrhea ended, the number of goblet cells was increased remarkably and many fine cracks were left on the surface of the epithelium.

Manauealides, some of the causative agents of a red alga Gracilaria coronopifolia poisoning in Hawaii.

Manauealides A-C (1-3), compounds related to debromoaplysiatoxin (5), were isolated and characterized from a red alga Gracilaria coronopifolia. Compounds 1 and 2 are presumed to be the causative toxins of G. coronopifolia food poisoning in Hawaii. Manauealide A (1) and C (3) are new macrolides, whereas manauealide B (2) is a known semisynthetic product of 5.

Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov.

Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming filamentous cyanobacterium prevalent in lakes and reservoirs of the southeastern United States, was found to produce a potent, acutely lethal neurotoxin when tested in the mouse bioassay. Signs of poisoning were similar to those of paralytic shellfish poisoning. As part of the Tennessee Valley Authority master plan for Guntersville Reservoir, the mat-forming filamentous cyanobacterium L. wollei, a species that had recently invaded from other areas of the southern United States, was studied to determine if it could produce any of the known cyanotoxins. Of the 91 field samples collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L. wollei cells-1, with the majority of samples being toxic at 500 mg kg-1. Samples bioassayed for paralytic shellfish toxins by the Association of Official Analytical Chemists method exhibited saxitoxin equivalents ranging from 0 to 58 micrograms g (dry weight)-1. Characteristics of the neurotoxic compound(s), such as the lack of adsorption by C18 solid-phase extraction columns, the short retention times on C18 high-performance liquid chromatography (HPLC) columns, the interaction of the neurotoxins with saxiphilin (a soluble saxitoxin-binding protein), and external blockage of voltage-sensitive sodium channels, led to our discovery that this neurotoxin(s) is related to the saxitoxins, the compounds responsible for paralytic shellfish poisonings. The major saxitoxin compounds thus far identified by comparison of HPLC fluorescence retention times are decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic shellfish poison C toxins being produced by L. wollei. Fifty field samples were placed in unialgal culture and grown under defined culture conditions. Toxicity and signs of poisoning for these laboratory-grown strains of L. wollei were similar to those of the field collection samples.

New saxitoxin analogues from the freshwater filamentous cyanobacterium Lyngbya wollei.

Along with decarbamoylsaxitoxin and decarbamoylgonyautoxin-2 and -3, six new saxitoxin analogues were isolated from the freshwater mat-forming filamentous cyanobacterium Lyngbya wollei collected from Guntersville Reservoir on the Tennessee River in Alabama. Their structures were determined by electrospray ionization mass spectrometry and several NMR techniques. Five of the toxins contain an acetyl moiety attached to the side chain, which is the first report of these saxitoxin analogues. In three of the toxins a hydrated ketone at C-12 was reduced to alpha-alcohol. The presence of acetate in the side chain resulted in a sevenfold to 17-fold times decrease in mouse toxicity compared to their carbamoyl counterparts, while the reduction at C-12 resulted in a complete loss of mouse toxicity.

Aplysiatoxin and debromoaplysiatoxin as the causative agents of a red alga Gracilaria coronopifolia poisoning in Hawaii.

The causative toxins of a red alga Gracilaria coronopifolia poisonings in Hawaii, which broke out in succession in September of 1994, were studied. Two major toxins were isolated from both extracts of the two original algal samples which caused separate poisonings. By spectroscopic method, these toxins were shown to be completely identical with aplysiatoxin and debromoaplysiatoxin which have previously been obtained from the sea hare and also from blue green algae. The human symptoms and the amount of these toxins in the original algal samples indicate that aplysiatoxin and debromoaplysiatoxin were the causative agents of the human poisoning incidents. This is the first reported case of the implication of aplysiatoxin and debromoaplysiatoxin in food poisoning. The existence of these toxins in the residue of algae washed in saline was confirmed by HPLC analysis. Furthermore, we observed blue-green algal parasitism on the surface of the toxic G. coronopifolia. Therefore, epiphytic organisms such as blue-green algae might be the true origin of the toxins in G. coronopifolia.