Caribbean Ciguatoxin-1 stability under strongly acidic conditions: Characterisation of a new C-CTX1 methoxy congener.

The recent emergence of ciguatera in the eastern Atlantic, particularly in the Canary Islands (Spain) and Madeira (Portugal) prompted the development and implementation of liquid chromatography tandem-mass spectrometry (LC/MS-MS) methods for the detection of ciguatoxins in fish. The complexity of fish tissue matrices, low concentrations of ciguatoxins in hazardous fish, and the scarcity of ciguatoxin standards present challenging issues for successful implementation of routine ciguatoxin analysis. A laboratory reference material of Caribbean Ciguatoxin-1 (C-CTX1), which was previously confirmed in fish responsible for ciguatera outbreaks in the Canary Islands, was used to assess the toxin's stability under strongly acidic conditions and solvent systems commonly used in LC-MS/MS. It was observed that strongly acidic conditions caused the transformation of C-CTX1 to a C56 methoxy congener, C-CTX1-Me. C-CTX1 was structurally characterised by LC-MS/MS and fragmentation pathways are presented showing the same fragmentation pattern as C-CTX1-Me. These results suggest that the use of strongly acidic conditions during sample pretreatment for C-CTX analysis, might produce significant artefacts, and risks failing to detect the presence of C-CTX1.

Elucidation and partial NMR assignment of monosulfated maitotoxins from the Caribbean.

Compounds similar to maitotoxin (MTX) have been isolated from several laboratory strains of the dinoflagellate Gambierdiscus spp. from the Caribbean. Mass spectral results suggest that these compounds differ from MTX by the loss of one sulfate group and, in some cases, the loss of one methyl group with the addition of one degree of unsaturation. NMR experiments, using approximately 50 nmol of one of these compounds, have demonstrated that the 9-sulfo group of MTX is still present, suggesting that these compounds are 40-desulfo congeners of MTX.

An Updated Review of Ciguatera Fish Poisoning: Clinical, Epidemiological, Environmental, and Public Health Management.

Ciguatera Fish Poisoning (CFP) is the most frequently reported seafood-toxin illness in the world. It causes substantial human health, social, and economic impacts. The illness produces a complex array of gastrointestinal, neurological and neuropsychological, and cardiovascular symptoms, which may last days, weeks, or months. This paper is a general review of CFP including the human health effects of exposure to ciguatoxins (CTXs), diagnosis, human pathophysiology of CFP, treatment, detection of CTXs in fish, epidemiology of the illness, global dimensions, prevention, future directions, and recommendations for clinicians and patients. It updates and expands upon the previous review of CFP published by Friedman et al. (2008) and addresses new insights and relevant emerging global themes such as climate and environmental change, international market issues, and socioeconomic impacts of CFP. It also provides a proposed universal case definition for CFP designed to account for the variability in symptom presentation across different geographic regions. Information that is important but unchanged since the previous review has been reiterated. This article is intended for a broad audience, including resource and fishery managers, commercial and recreational fishers, public health officials, medical professionals, and other interested parties.

Flow cytometric-membrane potential detection of sodium channel active marine toxins: application to ciguatoxins in fish muscle and feasibility of automating saxitoxin detection.

Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems.

Aquatic food accounts for over 40% of global animal food products, and the potential contamination with toxins of algal origin--marine biotoxins--poses a health threat for consumers. The gold standards to assess toxins in aquatic food have traditionally been in vivo methods, i.e., the mouse as well as the rat bioassay. Besides ethical concerns, there is also a need for more reliable test methods because of low inter-species comparability, high intra-species variability, the high number of false positive and negative results as well as questionable extrapolation of quantitative risk to humans. For this reason, a transatlantic group of experts in the field of marine biotoxins was convened from academia and regulatory safety authorities to discuss future approaches to marine biotoxin testing. In this report they provide a background on the toxin classes, on their chemical characterization, the epidemiology, on risk assessment and management, as well as on their assumed mode of action. Most importantly, physiological functional assays such as in vitro bioassays and also analytical techniques, e.g., liquid chromatography coupled mass spectrometry (LC-MS), as substitutes for the rodent bioassay are reviewed. This forms the basis for recommendations on methodologies for hazard monitoring and risk assessment, establishment of causality of intoxications in human cases, a roadmap for research and development of human-relevant functional assays, as well as new approaches for a consumer directed safety concept.

Federal seafood safety response to the Deepwater Horizon oil spill.

Following the 2010 Deepwater Horizon oil spill, petroleum-related compounds and chemical dispersants were detected in the waters of the Gulf of Mexico. As a result, there was concern about the risk to human health through consumption of contaminated seafood in the region. Federal and Gulf Coast State agencies worked together on a sampling plan and analytical protocols to determine whether seafood was safe to eat and acceptable for sale in the marketplace. Sensory and chemical methods were used to measure polycyclic aromatic hydrocarbons (PAHs) and dispersant in >8,000 seafood specimens collected in federal waters of the Gulf. Overall, individual PAHs and the dispersant component dioctyl sodium sulfosuccinate were found in low concentrations or below the limits of quantitation. When detected, the concentrations were at least two orders of magnitude lower than the level of concern for human health risk. Once an area closed to fishing was free of visibly floating oil and all sensory and chemical results for the seafood species within an area met the criteria for reopening, that area was eligible to be reopened. On April 19, 2011 the area around the wellhead was the last area in federal waters to be reopened nearly 1 y after the spill began. However, as of November 9, 2011, some state waters off the Louisiana coast (Barataria Bay and the Delta region) remain closed to fishing.

Assessing the incidence of ciguatera fish poisoning with two surveys conducted in Culebra, Puerto Rico, during 2005 and 2006.

BACKGROUND: Although ciguatera fish poisoning (CFP) is the most common seafood intoxication worldwide, its burden has been difficult to establish because there are no biomarkers to diagnose human exposure. OBJECTIVE: We explored the incidence of CFP, percentage of CFP case-patients with laboratory-confirmed ciguatoxic meal remnants, cost of CFP illness, and potential risk factors for CFP. METHODS: During 2005 and again during 2006, we conducted a census of all occupied households on the island of Culebra, Puerto Rico, where locally caught fish are a staple food. We defined CFP case-patients as persons with gastrointestinal symptoms (abdominal pain, vomiting, diarrhea, or nausea) and neurological symptoms (extremity paresthesia, arthralgia, myalgia, malaise, pruritus, headache, dizziness, metallic taste, visual disturbance, circumoral paresthesia, temperature reversal, or toothache) or systemic symptoms (e.g., bradycardia) within 72 hr of eating fish during the previous year. Participants were asked to save fish remnants eaten by case-patients for ciguatoxin analysis at the Food and Drug Administration laboratory in Dauphin Island, Alabama (USA). RESULTS: We surveyed 340 households during 2005 and 335 households during 2006. The estimated annual incidence of possible CFP was 4.0 per 1,000 person-years, and that of probable CFP was 7.5 per 1,000 person-years. One of three fish samples submitted by probable case-patients was positive for ciguatoxins. None of the case-patients required respiratory support. Households that typically consumed barracuda were more likely to report CFP (p = 0.02). CONCLUSIONS: Our estimates, which are consistent with previous studies using similar case findings, contribute to the overall information available to support public health decision making about CFP prevention.

Ciguatera fish poisoning on the West Africa Coast: An emerging risk in the Canary Islands (Spain).

Ciguatera fish poisoning (CFP) is endemic in certain tropical and subtropical regions of the world. CFP had not been described on the West Africa Coast until a 2004 outbreak in the Canary Islands. In 2008-2009, two additional outbreaks of ciguatera occurred. Individuals afflicted had consumed lesser amberjack (Seriola rivoliana) captured from nearby waters. Caribbean ciguatoxin-1 (C-CTX-1) was confirmed in fish samples by LC-MS/MS. Ciguatoxic fish in this region may pose a new health risk for the seafood consumer.

Advances in monitoring and toxicity assessment of brevetoxins in molluscan shellfish.

Herein, we describe advancements in monitoring of brevetoxins in molluscan shellfish, with respect to exposure management and control of neurotoxic shellfish poisoning (NSP). Current knowledge of the fate of brevetoxins in molluscan shellfish, and the toxic potency of brevetoxin metabolites, is presented. We review rapid assays for measuring composite brevetoxins, and methodology for measuring constituent brevetoxins, in contaminated shellfish. The applicability of in vitro methods for estimating brevetoxin burden and composite toxicity in shellfish is assessed. Specific and measurable biomarkers of brevetoxin exposure and toxicity in shellfish, and of human intoxication, are described. Their utility in regulatory monitoring of toxic shellfish and in clinical diagnosis of NSP is evaluated.

Ciguatera: a public health perspective.

Ciguatera fish poisoning is a seafood-borne illness caused by consumption of fish that have accumulated lipid-soluble ciguatoxins. In the United States, ciguatera is responsible for the highest reported incidence of food-borne illness outbreaks attributed to finfish, and it is reported to hold this distinction globally. Ciguatoxins traverse the marine food web from primary producers, Gambierdiscus spp., to commonly consumed fish in tropical and subtropical regions of the world. Ciguatoxins comprise 12 known congeners among Caribbean and tropical Atlantic fish and 29 reported congeners among Pacific fish. Expanding trade in fisheries from ciguatera-endemic regions contributes to wider distribution and increasing frequency of disease among seafood consumers in non-endemic regions. Ciguatoxins produce a complex array of gastrointestinal, neurological and cardiological symptoms. Treatment options are very limited and supportive in nature. Information derived from the study of ciguatera outbreaks has improved clinical recognition, confirmation, and timely treatment. Such studies are equally important for the differentiation of ciguatoxin profiles in fish from one region to the next, the determination of toxicity thresholds in humans, and the formulation of safety limits. Analytical information from case and outbreak investigations was used to derive Pacific and Caribbean ciguatoxin threshold contamination rates for adverse effects in seafood consumers. To these threshold estimates 10-fold safety factors were applied to address individual human risk factors; uncertainty in the amount of fish consumed; and analytical accuracy. The studies may serve as the basis for industry and consumer advisory levels of 0.10ppb C-CTX-1 equivalent toxicity in fish from the tropical Atlantic, Gulf of Mexico, Caribbean, and 0.01ppb P-CTX-1 equivalent toxicity in fish from Pacific regions.

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.

Biomarkers of neurotoxic shellfish poisoning.

Urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) were examined for biomarkers of brevetoxin intoxication. Brevetoxins were concentrated from urine by using solid-phase extraction (SPE), and analyzed by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine extracts were fractionated by LC, and fractions analyzed for brevetoxins by ELISA. In subsequent LC-MS/MS analyses, several brevetoxin metabolites of B-type backbone were identified, with elution profiles consistent with those of ELISA. The more abundant brevetoxin metabolites in urine were characterized structurally by LC-MS/MS. With the exception of BTX-3, brevetoxin metabolites in urine differed from those found in shellfish and in shellfish meal remnants. Proposed structures of these major urinary metabolites are methylsulfoxy BTX-3, 27-epoxy BTX-3, and reduced BTX-B5. BTX-3 was found in all specimens examined. BTX-3 concentrations in urine, as determined by LC-MS/MS, correlated well with composite toxin measurements by ELISA (r(2)=0.96). BTX-3 is a useful biomarker for confirmation of clinical diagnosis of NSP.

An electrochemiluminescence-based competitive displacement immunoassay for the type-2 brevetoxins in oyster extracts.

A new competitive electrochemiluminescence-based immunoassay for the type-2 brevetoxins in oyster extracts was developed. The assay was verified by spiking known amounts of PbTx-3 into 80% methanol extracts of Gulf Coast oysters. We also provide preliminary data demonstrating that 100% acetone extracts, aqueous homogenates, and the clinical matrixes urine and serum can also be analyzed without significant matrix interferences. The assay offers the advantages of speed ( 2 h analysis time); simplicity (only 2 additions, one incubation period, and no wash steps before analysis); low limit of quantitation (conservatively, 50 pg/mL = 1 ng/g tissue equivalents); and a stable, nonradioactive label. Due to the variety of brevetoxin metabolites present and the lack of certified reference standards for liquid chromatography-mass spectrometry confirmation, a true validation of brevetoxins in shellfish extracts is not possible at this time. However, our assay correlated well with another brevetoxin immunoassay currently in use in the United States. We believe this assay could be useful as a regulatory screening tool and could support pharmacokinetic studies in animals and clinical evaluation of neurotoxic shellfish poisoning victims.

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.

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.

On the use of neuro-2a neuroblastoma cells versus intact neurons in primary culture for neurotoxicity studies.

Neuroblastoma cell lines have been used extensively to screen novel compounds for neurotoxic properties and associated mechanisms. Such transformed cell lines often display morphological, developmental, and signaling characteristics that are substantially different from the parental cell type. Consequently, the response of neuroblastoma cells to toxin exposure may differ from that of neurons. An appreciation of the pharmacological and functional differences between neurons and neuron-like cell lines is therefore essential when interpreting data derived from neuroblastoma-based assays. We have compared the effects of several neurotoxins on Ca2+ homeostasis and cell viability in cerebellar granule neurons (CGN) and a neuroblastoma cell line (Neuro-2a). To explore the mechanisms underlying differential sensitivity of intact neurons and neuroblastoma cells to neurotoxins, we also compared CGN and Neuro-2a cells for expression of voltage-gated sodium channels (VGSC) and N-methyl-D-aspartate receptors (NMDAR). Cytotoxic potency in neurons was several orders of magnitude greater for Caribbean-ciguatoxin-1 (C-CTX-1) than either domoate (Dom) or brevetoxin-2 (PbTx-2). In addition, the cytotoxic potency of C-CTX-1 was two orders of magnitude greater in CGN than in Neuro-2a cells. The effect of C-CTX-1 and Dom on calcium homeostasis was compared in fluo-3 loaded neurons. Dom caused an elevation in intracellular calcium ([Ca2+]i) at concentrations that paralleled the concentration/response relationship for cytotoxicity in CGN. Conversely, C-CTX-1 did not elevate [Ca2+]i within the dynamic concentration range for cell death. The discordance of the concentration/response relationships for C-CTX-1 induced cytotoxicity and [Ca2+]i elevation suggests that acute C-CTX-1 cytotoxicity may involve mechanisms other than Ca2+ load. C-CTX-1-induced elevation of [Ca2+]i in neurons was dependent on activation of NMDAR and the reverse mode of operation of the Na+/Ca2+ exchanger. These data demonstrate that, although C-CTX-1, domoate, and PbTx-2 share the ability to produce neurotoxicity and mobilize calcium, their respective molecular targets and mechanisms of neurotoxicity differ. Neuro-2a cells that were not pretreated with veratridine and ouabain were insensitive to C-CTX-1 and glutamatergic agonists. VGSC expression was 20-fold lower in Neuro-2a cells than in CGN, whereas NMDARs were not expressed in these neuroblastoma cells. It is therefore likely that the enhanced sensitivity of CGN, relative to Neuro-2a cells, to neurotoxins is a consequence of pronounced differences in VGSC and NMDAR expression. These results underscore the need to exercise caution in interpreting negative cytotoxicity data derived from the use of neuroblastoma cell lines.

Use of two detection methods to discriminate ciguatoxins from brevetoxins: application to great barracuda from Florida Keys.

In Florida (USA), numerous cases of human ciguatera fish poisoning, as well as neurotoxic shellfish poisoning following consumption of local seafood products, have been reported. By using in parallel, the sodium channel receptor binding assay (RBA), and the ouabain/veratridine-dependent cytotoxicity assay (N2A assay), we established criteria to identify, detect, and quantify ciguatoxins in fish extracts, with a brevetoxin as internal standard. Results showed that the Caribbean ciguatoxin C-CTX-1 exhibited an 8-fold higher potency in the RBA than brevetoxins and, a 440 and 2300-fold higher potency in the N2A assay than PbTx-1 and PbTx-3, respectively. Moreover, a sensitivity comparison between assays revealed that the N2A assay was more sensitive (12-fold) for ciguatoxin analysis, whereas the RBA was more sensitive (3-24-fold) for brevetoxins analysis. Based on the relative potency between toxins and the opposite sensitivity of both assays we have used the RBA and the N2A assay to screen great barracuda (Sphyraena barracuda) collected from the Florida Keys for ciguatoxins and brevetoxins. Fish extract analysis showed a sodium channel-dependent activity consistent with the presence of ciguatoxins, and not brevetoxins. Among 40 barracudas analyzed, 60% contained ciguatoxin levels in their liver measurable by the N2A assay with the most toxic fish containing 2.1ppb C-CTX-1 equivalents.

Biomonitoring of ciguatoxin exposure in mice using blood collection cards.

Ciguatera is a human food poisoning caused by consumption of tropical and subtropical fish that have, through their diet, accumulated ciguatoxins in their tissues. This study used laboratory mice to investigate the potential to apply blood collection cards to biomonitor ciguatoxin exposure. Quantitation by the neuroblastoma cytotoxicity assay of Caribbean ciguatoxin (C-CTX-1) spiked into mice blood was made with good precision and recovery. The blood collected from mice exposed to a sublethal dose of Caribbean ciguatoxic extract (0.59 ng/g C-CTX-1 equivalents) was analyzed and found to contain detectable toxin levels at least 12 h post-exposure. Calculated concentration varied from 0.25 ng/ml at 30 min post-exposure to 0.12 ng/ml at 12 h. A dose response mice exposure revealed a linear dose-dependent increase of ciguatoxin activity in mice blood, with more polar ciguatoxin congeners contributing to 89% of the total toxicity. Finally, the toxin measurement in mice blood exposed to toxic extracts from the Indian Ocean or from the Pacific Ocean showed that the blood collection card method could be extended to each of the three known ciguatoxin families (C-CTX, I-CTX and P-CTX). The low matrix effect of extracted dried-blood samples (used at 1:10 or 1:20 dilution) and the high sensitivity of the neuroblastoma assay (limit of detection 0.006 ng/ml C-CTX-1), determined that the blood collection card method is suitable to monitor ciguatoxin at sublethal doses in mice and opens the potential to be a useful procedure for fish screening, environmental risk assessment or clinical diagnosis of ciguatera fish poisoning in humans or marine mammals.