Acute oral toxicity and tissue residues of saxitoxin in the mallard (Anas platyrhynchos).

Since 2014, widespread, annual mortality events involving multiple species of seabirds have occurred in the Gulf of Alaska, Bering Sea, and Chukchi Sea. Among these die-offs, emaciation was a common finding with starvation often identified as the cause of death. However, saxitoxin (STX) was detected in many carcasses, indicating exposure of these seabirds to STX in the marine environment. Few data are available that describe the effects of STX in birds, thus presenting challenges for determining its contributions to specific mortality events. To address these knowledge gaps, we conducted an acute oral toxicity trial in mallards (Anas platyrhynchos), a common laboratory avian model, using an up-and-down method to estimate the median lethal dose (LD50) for STX. Using an enzyme-linked immunosorbent assay (ELISA), we tested select tissues from all birds and feces from those individuals that survived initial dosing. Samples with an ELISA result that exceeded approximately 10 µg 100 g-1 STX and randomly selected ELISA negative samples were further tested by high-performance liquid chromatography (HPLC). Tissues collected from mallards were also examined grossly at necropsy and then later by microscopy to identify lesions attributable to STX. The estimated LD50 was 167 µg kg-1 (95% CI = 69-275 µg kg-1). Saxitoxin was detected in fecal samples of all mallards tested for up to 48 h after dosing and at the end of the sampling period (7 d) in three birds. In those individuals that died or were euthanized <2 h after dosing, STX was readily detected throughout the gastrointestinal tract but only infrequently in heart, kidney, liver, lung, and breast muscle. No gross or microscopic lesions were observed that could be attributable to STX exposure. Given its acute toxicity, limited detectability, and frequent occurrence in the Alaska marine environment, additional research on STX in seabirds is warranted.

Delivery of local anaesthetics by a self-assembled supramolecular system mimicking their interactions with a sodium channel.

Site-1 sodium channel blockers (S1SCBs) act as potent local anaesthetics, but they can cause severe systemic toxicity. Delivery systems can be used to reduce the toxicity, but the hydrophilicity of S1SCBs makes their encapsulation challenging. Here, we report a self-assembling delivery system for S1SCBs whose design is inspired by the specific interactions of S1SCBs with two peptide sequences on the sodium channel. Specifically, the peptides were modified with hydrophobic domains so that they could assemble into nanofibres that facilitated specific binding with the S1SCBs tetrodotoxin, saxitoxin and dicarbamoyl saxitoxin. Injection of S1SCB-carrying nanofibres at the sciatic nerves of rats led to prolonged nerve blockade and to reduced systemic toxicity, with benign local-tissue reaction. The strategy of mimicking a molecular binding site via supramolecular interactions may be applicable more broadly to the design of drug delivery systems for receptor-mediated drugs.

Biocatalytic Detoxification of Paralytic Shellfish Toxins.

Small molecules that bind to voltage-gated sodium channels (VGSCs) are promising leads in the treatment of numerous neurodegenerative diseases and pain. Nature is a highly skilled medicinal chemist in this regard, designing potent VGSC ligands capable of binding to and blocking the channel, thereby offering compounds of potential therapeutic interest. Paralytic shellfish toxins (PSTs), produced by cyanobacteria and marine dinoflagellates, are examples of these naturally occurring small molecule VGSC blockers that can potentially be leveraged to solve human health concerns. Unfortunately, the remarkable potency of these natural products results in equally exceptional toxicity, presenting a significant challenge for the therapeutic application of these compounds. Identifying less potent analogs and convenient methods for accessing them therefore provides an attractive approach to developing molecules with enhanced therapeutic potential. Fortunately, Nature has evolved tools to modulate the toxicity of PSTs through selective hydroxylation, sulfation, and desulfation of the core scaffold. Here, we demonstrate the function of enzymes encoded in cyanobacterial PST biosynthetic gene clusters that have evolved specifically for the sulfation of highly functionalized PSTs, the substrate scope of these enzymes, and elucidate the biosynthetic route from saxitoxin to monosulfated gonyautoxins and disulfated C-toxins. Finally, the binding affinities of the nonsulfated, monosulfated, and disulfated products of these enzymatic reactions have been evaluated for VGSC binding affinity using mouse whole brain membrane preparations to provide an assessment of relative toxicity. These data demonstrate the unique detoxification effect of sulfotransferases in PST biosynthesis, providing a potential mechanism for the development of more attractive PST-derived therapeutic analogs.

C-H Hydroxylation in Paralytic Shellfish Toxin Biosynthesis.

The remarkable degree of synthetic selectivity found in Nature is exemplified by the biosynthesis of paralytic shellfish toxins such as saxitoxin. The polycyclic core shared by saxitoxin and its relatives is assembled and subsequently elaborated through the installation of hydroxyl groups with exquisite precision that is not possible to replicate with traditional synthetic methods. Here, we report the identification of the enzymes that carry out a subset of C-H functionalizations involved in paralytic shellfish toxin biosynthesis. We have shown that three Rieske oxygenases mediate hydroxylation reactions with perfect site- and stereoselectivity. Specifically, the Rieske oxygenase SxtT is responsible for selective hydroxylation of a tricyclic precursor to the famous natural product saxitoxin, and a second Rieske oxygenase, GxtA, selectively hydroxylates saxitoxin to access the oxidation pattern present in gonyautoxin natural products. Unexpectedly, a third Rieske oxygenase, SxtH, does not hydroxylate tricyclic intermediates, but rather a linear substrate prior to tricycle formation, rewriting the biosynthetic route to paralytic shellfish toxins. Characterization of SxtT, SxtH, and GxtA is the first demonstration of enzymes carrying out C-H hydroxylation reactions in paralytic shellfish toxin biosynthesis. Additionally, the reactions of these oxygenases with a suite of saxitoxin-related molecules are reported, highlighting the substrate promiscuity of these catalysts and the potential for their application in the synthesis of natural and unnatural saxitoxin congeners.

Paralytic shellfish poisonings resulting from an algal bloom in Nicaragua.

BACKGROUND: During an October 2005 algal bloom (i.e., a rapid increase or accumulation in the population of algae) off the coast of Nicaragua, 45 people developed symptoms of paralytic shellfish poisoning (PSP) and one person died. PSP in humans is caused by ingestion of saxitoxin, which is a neurotoxin often associated with shellfish contaminated by algal blooms. To explore the relationship between the algal bloom and human illnesses, we performed a case-control study of residents living in a coastal island. We administered a standardized clinical questionnaire, sampled locally harvested seafood and algae, and obtained urine samples for saxitoxin testing from symptomatic and asymptomatic persons. PSP case-patients were defined as island residents who developed at least one neurological symptom during the November 4-16 intoxication period. Seafood and algal samples were analyzed for saxitoxins using the receptor-binding assay and high-performance liquid chromatography. Two urine samples were analyzed for saxitoxins using a newly developed immunoassay. FINDINGS: Three shellfish and two algal samples tested positive for saxitoxins. Ten (9%) of 107 participants developed neurological symptoms during the specified time period and five required hospitalization. While 6 (67%) of 9 possible case-patients and 21 (21%) of 98 controls had eaten fish (p=0.008), all case-patients and 17 (17%) of controls had eaten clams (P<0.0001). The saxitoxin concentration in the urine of a hospitalized case-patient was 21 ng saxitoxin/g creatinine compared to 0.16 ng saxitoxin/g creatinine in the single control patient's urine. CONCLUSIONS: These findings suggest that a bloom of saxitoxin-producing algae resulted in saxitoxin accumulation in local clams and was responsible for the PSP intoxication.

Prolonged duration local anesthesia with minimal toxicity.

Injectable local anesthetics that would last for many days could have a marked impact on periprocedural care and pain management. Formulations have often been limited in duration of action, or by systemic toxicity, local tissue toxicity from local anesthetics, and inflammation. To address those issues, we developed liposomal formulations of saxitoxin (STX), a compound with ultrapotent local anesthetic properties but little or no cytotoxicity. In vitro, the release of bupivacaine and STX from liposomes depended on the lipid composition and on whether dexamethasone was incorporated. In cell culture, bupivacaine, but not STX, was myotoxic (to C2C12 cells) and neurotoxic (to PC12 cells) in a concentration- and time-dependent manner. Liposomal formulations containing combinations of the above compounds produced sciatic nerve blockade lasting up to 7.5 days (with STX + dexamethasone liposomes) in male Sprague-Dawley rats. Systemic toxicity only occurred where high loadings of dexamethasone increased the release of liposomal STX. Mild myotoxicity was only seen in formulations containing bupivacaine. There was no nerve injury on Epon-embedded sections, and these liposomes did not up-regulate the expression of 4 genes associated with nerve injury in the dorsal root ganglia. These results suggest that controlled release of STX and similar compounds can provide very prolonged nerve blocks with minimal systemic and local toxicity.

The adsorption of saxitoxin to clays and sediments in fresh and saline waters.

The adsorption of saxitoxin to Na- and Ca-montmorillonite, kaolin (crystalline and amorphous), kaolinite, Bread and Butter Creek sediment (an estuarine tidal creek), Gulf of Mexico sediment, and Santa Barbara Basin sediment in deionized water and 32 per thousand salinity simulated seawater (Instant Ocean) is reported. Adsorption was partially reversible for all cases and best described using a Freundlich isotherm. The corresponding Freundlich constants (K(F)) ranged from 8.83 x 10(3)micromol/kg to 6.76 x 10(4)micromol/kg for freshwater and 4.73 x 10(3)micromol/kg-1.11 x 10(4)micromol/kg for seawater. There is a positive linear correlation seen between the K(F) values and the cation-exchange capacity of the adsorbents. The release of saxitoxin from previously equilibrated adsorbents was determined in freshwater (0-18%) and seawater (4-53%).

Quantification of saxitoxin and neosaxitoxin in human urine utilizing isotope dilution tandem mass spectrometry.

Saxitoxin and neosaxitoxin are potent neurotoxins that can cause paralytic shellfish poisoning when consumed. A new assay is presented here to quantify saxitoxin (STX) and neosaxitoxin (NEO) in human urine samples. Sample preparation of 500-microL samples included the use of weak-cation-exchange solid-phase extraction in a multiplexed 96-well format. Extracts were preconcentrated and analyzed via 10-min hydrophilic interaction liquid chromatography followed by electrospray ionization. Protonated molecular ions were quantified via multiple reaction monitoring mode in a Qtrap mass spectrometer. The method uses novel 15N7-isotopically enriched STX and NEO internal standards. Method validation included the characterization of two enriched urine pools. The lowest reportable limits for STX and NEO were 4.80 and 10.1 ng/mL, respectively, using both quantification and confirmation ions. These two toxins were not detected in a reference range of humans who consumed seafood in the preceding 72 h, suggesting that few false positives would occur when trying to identify people exposed to STX or NEO.

Characterization of the Ag mediated surface-enhanced Raman spectroscopy of saxitoxin.

The rapid detection and quantification of saxitoxin (STX) is reported using surface-enhanced Raman spectroscopy (SERS) with a colloidal hydrosol of silver nanoparticles. Under the conditions of our experiments, the limit of detection (LD) for STX using SERS is 3 nM, with a limit of quantification (LQ) of 20 nM. It is shown that the SERS method is rapid, with spectra being collected in as little as 5 seconds total integration time for a 40 nM STX sample. In order to improve the signal-to-noise ratio, SERS spectra were generally collected with a total integration time of 1 minute (6 accumulations of 10 seconds each), with no need for extensive sample work-up or substrate preparation. Based on these results, the SERS technique shows great promise for the future detection and quantification of STX molecules in aqueous solutions.

Saxitoxin puffer fish poisoning in the United States, with the first report of Pyrodinium bahamense as the putative toxin source.

BACKGROUND: From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. METHODS: We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. RESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 microg STX equivalents (eq)/100 g tissue (action level, 80 microg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. CONCLUSIONS: Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.