Optimization of ciguatoxin extraction method from blood for Pacific ciguatoxin (P-CTX-1).

Ciguatera diagnosis relies on clinical observations associated with a recent consumption of fish. Although needed, direct confirmation of exposure in subjects showing ciguatera disease symptoms is currently unavailable. We previously reported that ciguatoxins were measurable in the blood of mice exposed to extracts of Pacific ciguatoxins isolated from Gambierdiscus polynesiensis, and of Indian Ocean or Caribbean Sea ciguatoxins, isolated from fish. Although highly efficient for extracting spiked purified Caribbean-CTX-1, the methanolic extraction method previously described is found here to yield only 6% recovery of spiked Pacific-CTX-1 (P-CTX-1). We report in this short communication a substantially modified method for ciguatoxin extraction from both dried and fresh blood. With this method, toxin measurement is directly accomplished in acetonitrile deproteinated whole fresh blood or phosphate buffer solution (PBS) eluted dried blood using the N2A cell-based assay. Spike studies using increasing concentrations of purified ciguatoxins reveal linear (r2 above 0.87 for all toxins) and overall efficient toxin recoveries (62%, 96%, and 96% from fresh blood and 75%, 90%, and 74% from dried blood, for C-CTX-1, P-CTX-3C, and P-CTX-1, respectively). Comparative blood matrix analysis for P-CTX-1 recovery shows increased recovery of ciguatoxin activity from whole fresh blood than from dried blood, greater by 20% in P-CTX-1 spiked mice blood and by over 85% in P-CTX-1 exposed mouse blood. In conclusion, both Caribbean and Pacific ciguatoxins can be readily extracted from blood using this modified method; however, in the case of P-CTX-1 we find that fresh blood is optimal.     

Isolation and characterisation of Indian Ocean ciguatoxin.

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean.     

Repeat exposure to ciguatoxin leads to enhanced and sustained thermoregulatory, pain threshold and motor activity responses in mice: relationship to blood ciguatoxin concentrations

Ciguatera is a common illness in tropical and subtropical regions that manifests in complex and long-lived symptoms which are more severe in subsequent exposures. This study measures central and peripheral neurologic signs, in parallel with blood toxin levels, in mice exposed once or twice (at 3 days interval) to a sublethal dose of ciguatoxin P-CTX-1 (0.26ng/g via i.p.). Mice were implanted with radiotransmitters to monitor motor activity and core temperature. A single exposure to ciguatoxin elicited an immediate and transient decrease in motor activity and temperature, and subsequent long-lasting thermoregulatory dysfunction resulting in stabilized body temperature around 36.0 degrees C with no observable circadian rhythm. The hypothermic response and the reduced activity were enhanced with a second exposure with 30% of the mice dying within 7h. Measurement of the peripheral nervous system by the tail flick assay revealed increased latency with a single ciguatoxin exposure, and a greater effect following the second exposure. Toxin was measurable in blood up to 3 days following the first exposure; at the 1h time point the concentrations were significantly elevated after a second exposure. These findings indicate an early response to ciguatoxin manifest in a central response to lower body temperature and reduce motor activity and a more persistent effect on the peripheral system leading to spinal heat antinociception and delayed fever-like response. The greater neurological response to a second ciguatoxin exposure was associated with elevated concentrations of ciguatoxin in the blood solely over the first hour of exposure. In conclusion, a single exposure to toxin exerts a significant neurological response which may be enhanced with subsequent exposure.     

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.     

Depuration Kinetics and Growth Dilution of Caribbean Ciguatoxin in the Omnivore Lagodon rhomboides: Implications for Trophic Transfer and Ciguatera Risk

Modeling ciguatoxin (CTX) trophic transfer in marine food webs has significant implications for the management of ciguatera poisoning, a circumtropical disease caused by human consumption of CTX-contaminated seafood. Current models associated with CP risk rely on modeling abundance/presence of CTX-producing epi-benthic dinoflagellates, e.g., Gambierdiscus spp., and are based on studies showing that toxin production is site specific and occurs in pulses driven by environmental factors. However, food web models are not yet developed and require parameterizing the CTX exposure cascade in fish which has been traditionally approached through top-down assessment of CTX loads in wild-caught fish. The primary goal of this study was to provide critical knowledge on the kinetics of C-CTX-1 bioaccumulation and depuration in the marine omnivore Lagodon rhomboides. We performed a two-phase, 17 week CTX feeding trial in L. rhomboides where fish were given either a formulated C-CTX-1 (n = 40) or control feed (n = 37) for 20 days, and then switched to a non-toxic diet for up to 14 weeks. Fish were randomly sampled through time with whole muscle, liver, and other pooled viscera dissected for toxin analysis by a sodium channel-dependent MTT-based mouse neuroblastoma (N2a) assay. The CTX levels measured in all tissues increased with time during the exposure period (days 1 to 20), but a decrease in CTX-specific toxicity with depuration time only occurred in viscera extracts. By the end of the depuration, muscle, liver, and viscera samples had mean toxin concentrations of 189%, 128%, and 42%, respectively, compared to fish sampled at the start of the depuration phase. However, a one-compartment model analysis of combined tissues showed total concentration declined to 56%, resulting in an approximate half-life of 97 d (R² = 0.43). Further, applying growth dilution correction models to the overall concentration found that growth was a major factor reducing C-CTX concentrations, and that the body burden was largely unchanged, causing pseudo-elimination and a half-life of 143–148 days (R² = 0.36). These data have important implications for food web CTX models and management of ciguatera poisoning in endemic regions where the frequency of environmental algal toxin pulses may be greater than the growth-corrected half-life of C-CTX in intermediate-trophic-level fish with high site fidelity.     

A rapid, simplified enzyme immunoassay stick test for the detection of ciguatoxin and related polyethers from fish tissues

Y. Hokama A rapid, simplified enzyme immunoassay stick test for the detection of ciguatoxin and related polyethers from fish tissues. Toxicon23, 939–946. 1985. — A simplified and rapid enzyme immunoassay stick test is presented in this study. The salient feature of this test is the use of a coating (Liquid Paper) on the stick to adsorb the lipid ciguatoxin and its related polyether toxins onto the stick. This rapid solid phase stick test has been able to differentiate clinically implicated fishes (14 samples) from non-toxic (60 samples) with P < 0.005. Comparison of the slick test with the enzyme immunoassay procedure with the positive fish samples demonstrated a good correlation. All samples were positive by both procedures. Further comparison of the stick test, enzyme immunoassay and the mouse bioassay with unknown fish samples showed good relationships between the three procedures. Examination of 71 Seriola dumerili, a fish generally implicated in ciguatera poisoning, with the stick test procedure showed that 89% of the fishes were non-toxic and thus consumed without any incidence of ciguatera poisoning. Analysis of graded concentrations of ciguatoxin, in ng/ml of methanol, showed a typical immunological precipitation pattern. The stick test as conceived is simple and rapid, while retaining its sensitivity and specificity to detect ng levels of ciguatoxin and its related polyether toxins. It is suggested that the stick test will be valuable in the screening of ciguatoxin and related polyether toxins in contaminated fish tissues.     

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding.

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for > 90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and < 900 Da were suspected to contribute to the overall toxicity.     

Characterization of the developmental toxicity of Caribbean ciguatoxins in finfish embryos

Since oviparous fishes mobilize fat stores to produce eggs, we investigated the potential for deposition of gonadal ciguatoxins to the oil laden yolk sacs which nourish developing embryos, and characterized the effects of these toxins on finfish development. Results showed that ciguatoxins are more concentrated in the egg mass (0.18 ng/g) of a toxic fish than in the muscle (<0.04 ng/g). We used a microinjection technique in a Japanese medaka (Oryzias latipes) developmental fish model to mimic the maternal route of toxin exposure to finfish embryos. We describe the developmental effects of two preparations isolated from Caribbean great barracuda (Sphyraena barracuda): a highly purified toxin (C-CTX-1), and ciguatoxins extracted from the flesh of a toxic fish. C-CTX-1 induced a significant decrease in heart rate after four days, which did not persist with further development. Crude extracts from ciguatoxic fish flesh induced hyperkinetic twitching and severe spinal deformities. These effects were observed in embryos receiving as little as 5 pg/egg, and were consistently found in embryos receiving doses exceeding 10 pg/egg. The occurrence of twitching and spinal deformities increased in both frequency and severity with dose. Larvae suffering from spinal abnormalities were unable to orient themselves, and could not feed, resulting in mortality. The greater distribution of toxin to eggs as compared to flesh suggests that fish with low to moderate (0.5 ppb) flesh toxin levels would maternally transfer detrimental amounts of ciguatoxins to their offspring.     

Studies on the mode of action of ciguateric toxins

The effects of ciguatoxin, scaritoxin and maitotoxin, the main toxins involved in ciguatera fish poisoning, has been studied in pentobarbital anaesthetized cats. Intraveinous injections of increasing doses of these toxins (5 to 160 μg/kg of partially purified samples) evoked respiratory and cardiovascular disturbances: hyperventilation at low doses and respiratory depression leading to respiratory arrest at high doses; bradycardia and troubles of the atrioventricular conduction at low doses, arrhythmias and ventricular tachycardia with transient hypertension at sublethal doses, and falling arterial pressure leading to complete heart failure at high doses.The mode of action of ciguatoxin has been studied by testing the preventive efects of pharmacological compounds such as hexamethonium, atropine, propanolol and phentolamine and by proceeding to bilateral adrenalectomy. The results have indicated both central and peripheral effects. Cholinergic and also α - adrenergic actions were pointed out.     

Ability of some plant extracts, traditionally used to treat ciguatera fish poisoning, to prevent the in vitro neurotoxicity produced by sodium channel activators.

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators.     

Isolation of two toxins from a parrotfish Scarus gibbus

E. Chungue, R. Bagnis, N. Fusetani and Y. Hashimoto. Isolation of two toxins from a parrotfish Scarus gibbus. Toxicon15, 89–93, 1977.—Clinical and epidemiological observations suggested that at least two toxins are involved in poisoning from ingestion of the parrotfish Scarus gibbus. In the present study, the parrotfish flesh collected from the Gambier Islands was extracted by the method adopted for extraction of ciguatoxin and the crude toxin obtained was fractionated by column chromatography using silicic acid and DEAE-cellulose. Two fat-soluble toxins were separated by DEAE-cellulose column chromatography. The polar one was very similar to ciguatoxin and the other different from ciguatoxin. The latter toxin (SG-1) was further purified by repeated gel filtration on Sephadex LH-20 to give an oily substance with a lethality to mice of 0·03 mg per kg. It showed some similarity to ciguatoxin in biological and chemical properties, but an apparent dissimilarity in chromatographic behavior. The significance of this toxin in the ciguatera phenomenon is briefly discussed.     

Characterisation of multiple Caribbean ciguatoxins and congeners in individual specimens of horse-eye jack (Caranx latus) by high-performance liquid chromatography/mass spectrometry.

We studied the variation in toxin profiles of purified extracts of 10 individual specimens and two pools of ciguateric Caranx latus. High-performance liquid chromatography/mass spectrometry (HPLC/MS) identified in all individual samples at least seven Caribbean ciguatoxins (C-CTXs) comprising C-CTX-1 and its epimer C-CTX-2 ([M+H](+) m/z 1141.58), and five new C-CTX congeners with pseudo-molecular ions at m/z 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57. In some samples, additional C-CTX isomers were detected with [M+H](+) ions at m/z 1141.58 (two), 1143.60 (one) and 1157.57 (two). The two low-toxic pools contained only four to six ciguatoxins. The comparison in relative proportions of four different mass classes ([M+H](+) at m/z 1141, 1143, 1157 and 1127) showed that the group at m/z 1157 increased (2-20%) with flesh toxicity. More than 80% of group m/z 1141 comprised C-CTX-1, C-CTX-2 and their isomer C-CTX-1a whose level in this group correlated with fish toxicity. Contrary to low-toxic fishes, high-risk specimens had C-CTX-1 levels <50% and were subjected to large losses of activity on purification indicating that unstable ciguatoxins were present. A possible conversion of C-CTX-1 into C-CTX-1a was identified when flesh was cooked, without changes in toxicity. In conclusion, HPLC/MS characterised 12 C-CTXs accumulated by C. latus at variable levels.     

Ciguatera Poisoning: Clinical and Immunological Aspects

Abstract

Ciguatera poisoning refers to a type of fish intoxication following consumption of fishes contaminated with ciguatoxin (CTX). The latter toxin originates in a dinoflagellate designated Gambierdiscus toxicus, passes through herbivorous and then to carnivorous fishes along the food chain, ultimately to man. Ciguatera poisoning is unlike tetrodotoxin or scombroid fish poisonings. The Eormer is due to a toxin isolated From puffer fish and the latter to histamine-like degradation products due to bacterial spoilage of fish fissues. Ten minutes Lo 24 hr after consumption of a CTX contaminated fish, clinical symptoms involving the gastroinlestinal, neurological and cardiovascular systems are manifested. The neurological symptoms may persist for munths in some individuals. Ciguatera poisoning is rarely fatal (less than 0.5%). Therapy at best is symptomatic with no specific drugs. Detection in fisti tissues depends on the mouse test Eollowing extraction of the toxin in organic solvents and more recently an immunological approach using specific anti-CTX monoclonal antibody. The stick test is rapid, simple, and specific and requires nu laboratory equipment. It is hoped to adapt the stick test for wide scale use in screening fishes for markets in the endemic areas of the tropical regions.     

Occurrence of a ciguatoxin-like substance in the Spanish mackerel (Scomberomorus commersoni)

A lipid-soluble toxin, similar to ciguatoxin as isolated by Scheueret al. (1967), has been found in the flesh of the Spanish mackerel, Scomberomorus commersoni, caught in Queensland. The ciguatoxin-like substance was experimentally characterized by examination of specific biological and chromatographic properties of the lipid-soluble extract from a pooled sample of flesh from Spanish mackerel. Flesh from specimens known to have caused S. commersoni poisoning in humans was confirmed as toxic by cat bioassay. A toxin was extracted from S. commersoni which yielded, on partial purification, a clear, oily substance with an ld₅₀ i.p. to mice of 0.72 mg/kg, and which had chromatographic properties similar to those of classical ciguatoxin. However, the R_f value on thin-layer chromatography plates was lower for S. commersoni toxin than for classical ciguatoxin. This is the first record of a ciguatoxin-like substance experimentally identified in S. commersoni, a pelagic fish that occurs throughout Queensland coastal waters. The majority of toxic S. commersoni are caught between latitudes 24° and 26°S.     

Methylmercury exposure, mercury levels in blood and hair, and health status in Swedes consuming contaminated fish

Levels of total mercury in blood cells ranging 8–390 ng/g (in one case 1100) were found in 162 Swedes who consumed fish containing 0.3–7 mg Hg/kg. Levels above 100 ng/g were seen only in subjects 40–80 years of age, levels above 200 ng/g only in persons who consumed fish containing about 1 mg Hg/kg or more. 20 subjects eating fish containing about 0.04 mg Hg/kg ranged 8–45 ng/g blood cells, and 22 subjects eating commercially available fish 3–57 ng/g. Long-term exposure to 4 μg Hg as methylmercury/kg body weight/day - as estimated from fish intake records - corresponded to a blood cell mercury level of about 300 ng/g. After the end of exposure, biologic half-time of mercury in blood cells ranged 58–87 days in 4 subjects, while the corresponding figure was 164 days in one individual. A screening for signs and symptoms of methylmercury poisoning in 86 of the exposed subjects revealed no clearcut case of poisoning. Some subjects of symptoms in a high-mercury (82–1100 ng/g blood cells) group as compared to a low-mercury (12–75 ng/g) group.     

A rapid hemolysis assay for the detection of sodium channel-specific marine toxins

Current methods of detection for fish and shellfish biotoxins in monitoring and research purposes are either labor intensive, expensive, require specialized techniques or all of the above. This paper reports on the development of a fairly sensitive, rapid, and inexpensive assay which detects the presence of compounds that affect the sodium channel. It is based on the principles of the mouse neuroblastoma tissue culture assay for sodium channel specific-biotoxins using red blood cells (RBCs) from the red tilapia (Sarotherodon mossambicus). This assay has the potential to complement the use of live animal bioassay testing for marine toxins. Veratridine, a sodium channel activator and ouabain, an inhibitor of Na(+)/K(+) ATPase, both react with the tilapia RBCs by affecting the permeability of the cell's membrane. Saxitoxin (STX), its analogs, and tetrodotoxin (TTX) can inhibit the action of veratridine and ouabain leaving the cell morphologically normal. By sequencing the addition of veratridine and ouabain, with either the extracted samples, saxitoxin, tetrodotoxin, or ciguatoxin (CTX-a sodium channel activator) to the RBCs a sodium channel antagonist or activator can be detected. Results using pure concentrations of a sodium channel-specific toxin could be detected to inhibit hemolysis at a concentration of 0.3 microg/ml STX, 3.5 microg/ml for neo-STX, 3.0 microg/ml for GTX, and 5.0 microgl for TTX in the presence of ouabain and veratridine. CTX was detected at a concentration of 50 microg/ml. The RBCs from the red tilapia was used due to the fish's ability to osmoregulate its internal environment to survive in both fresh and saltwater. In addition, with growing opposition to live animal testing, this assay has been designed as a non-lethal means of testing for sodium channel affecting marine toxins. No test animals are sacrificed and blood may be drawn from the same fish for continued sample testing.     

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 0.10 ppb C-CTX-1 equivalent toxicity in fish from the tropical Atlantic, Gulf of Mexico, Caribbean, and 0.01 ppb P-CTX-1 equivalent toxicity in fish from Pacific regions.     

Morphological abnormalities and sensorimotor deficits in larval fish exposed to dissolved saxitoxin

The dietary uptake of one suite of dinoflagellate-produced neurotoxins, that are commonly called paralytic shellfish poisoning (PSP) toxins, is known to cause acute fish kills. However, little is known about the effects of dissolved phase exposure and the potential sublethal effects of this route of exposure on early developmental stages of fish. Toxin exposure during early development is of particular concern because the embryos and larvae of some marine fish species may be unable to actively avoid the dissolved toxins that algal cells release into the water column during harmful algal blooms. Here we use the zebrafish (Danio rerio) as a model experimental system to explore the sublethal effects of a dissolved PSP toxin, saxitoxin (STX), on early development in fish, including sensorimotor function, morphology, and long-term growth and survival. Aqueous phase exposures of 229 +/- 7 microg STX eq. l(-1) caused reductions in sensorimotor function as early as 48 h postfertilization (hpf) and paralysis in all larvae by 4 days postfertilization (dpf). Rohon-Beard mechanosensory neurons appeared to be more sensitive to STX than dorsal root ganglion neurons at this dose. Additionally, exposure to 481 +/- 40 microg STX eq. l(-1) resulted in severe edema of the eye, pericardium, and yolk sac in all exposed larvae by 6 dpf. The onset of paralysis in STX-exposed larvae was stage-specific, with older larvae becoming paralyzed more quickly than younger larvae (5 h at 6 dpf as compared to 8 and 46 h for 4 and 2 dpf larvae, respectively). When transferred to clean water, many larvae recovered from the morphological and sensorimotor effects of STX. Thus, the sublethal effects of the toxin on larval morphology and behavior were reversible. However, zebrafish exposed to STX transiently during larval development (from 2 to 4 dpf) had significantly reduced growth and survival at 18 and 30 days of age. Collectively, these data show that (1) dissolved phase STX is bioavailable to fish embryos and larvae, (2) the toxin is a paralytic with potencies that are stage-specific for fish larvae, (3) the observed toxicological effects of STX exposure are reversible, and (4) a short-term toxin exposure can negatively impact the survival of fish several weeks later. Dissolved algal toxins may therefore have important sublethal effects on vulnerable species of fish.     

Negative inotropic and arrhythmic effects of high doses of ciguatoxin on guinea-pig atria and papillary muscles

Ciguatoxin, the toxin present in fish responsible for ciguatera, at doses equal or above the maximum positive inotropic dose in atria (greater than 0.15 mouse units/ml) induced arrhythmias in atria and papillary muscles stimulated at 1 Hz and dose-dependent negative inotropy in atria. Negative inotropy was enhanced by ouabain or by an increase in stimulation to 3 Hz, little affected by procaine or increasing Ringer [Ca2+] and reversed by lidocaine and tetrodotoxin (TTX). Ciguatoxin caused negative inotropy associated with cell depolarisation in 1.2 mM Ca2+-Ringer and additionally caused signs of Ca overload in 3.2 mM Ca2+-Ringer. Ciguatoxin induced transient after-contractions and contracture in atria which were common in 3.2 mM but not 1.2 mM Ca2+-Ringer and which were enhanced by ouabain. TTX and lidocaine abolished after-contractions and contracture while procaine was less effective. Extrasystoles consisting of short bursts of 1-2 extra contractions per sec were seen in atria and papillary muscles within 45 min of ciguatoxin being added. The effect was observed in 3.2 mM but seldom in 1.2 mM Ca2+-Ringer and was absent when low doses of propranolol or TTX were added prior to ciguatoxin. Flutter was observed in a few papillary muscles after ciguatoxin. These results suggest that the toxic effects of ciguatoxin stem from its direct action of opening myocardial Na+ channels. Extrasystoles appeared to result mainly from its effect on neural Na+ channels causing an increased release of noradrenaline from the nerves associated with the myocardium.     

Chronic Phase Lipids in Sera of Several Chronic Diseases Reacting with MAB–CTX (Antibody to Ciguatoxin)

The membrane immunobead assay results on the acetone lipid fraction of serum from chronic fatigue syndrome (CFS) patients (60 samples) and normal individuals (with no clinical CFS or other disease symptoms) showed significant differences with 4 exceptions (4 normals showed 1:40 and 1:80 titres). This represented approximately 10.8% of the normal samples, with 3 samples at 1:20, the majority of the CFS titred 1:40 through 1:160. This represented 95.0% of the samples. The small numbers of hepatitis patients and chronic ciguatera fish poisoning patients also had titres of 1:40 to 1:80 in all of the serum samples examined. The weights of the lipids in mg/ml serum essentially are very similar, except 1 or 2 of CFS and hepatitis B showed values at the upper level. Comparison of sexes showed 65% females and 35% men with CFS, representing a ratio of approximately 2:1 (female/male). It is concluded that certain disease conditions and environmental exposures to deleterious factors (toxin, chemicals, microorganisms) trigger the release of lipids (probably by the liver) with similar epitopes to ciguatoxin, and that they react with MAb–CTX. We designate these lipids as “chronic phase lipids” comparable to “acute phase protein” in inflammatory and traumatic diseases.