Mark Poli

Purification and characterization of ciguatoxins from moray eel (Lycodontis javanicus, Muraenidae)

Viscera (48.3 kg) from moray eels (Lycodontis javanicus) collected in a ciguatera endemic area were extracted and the ciguatoxins characterized. Three major ciguatoxins, CTX-1, CTX-2 and CTX-3, were isolated and purified to homogeneity on reverse phase high performance liquid chromatography. Several minor toxins were also detected. CTX-1 (490 micrograms) was comparable by both 1H nuclear magnetic resonance (1H NMR) and mass spectroscopy (MH+ m/z = 1111) to ciguatoxin isolated previously from moray eels. CTX-2 (280 micrograms) and CTX-3 (100 micrograms) were less polar ciguatoxins not previously characterized. CTX-2 and CTX-3 differed from CTX-1 by 16 mass units, suggesting that they were less oxygenated analogues. 1H NMR revealed that the hydroxyl at C54 in CTX-1 was absent in CTX-2 and CTX-3. An additional change in the chemistry of CTX-2 compared to CTX-1 and CTX-3 was also suggested on the basis of 1H NMR, indicating that CTX-2 may arise from a different precursor to CTX-1. CTX-3 is likely to be an intermediate in the oxidation of a gambiertoxin (sodium channel toxins from Gambierdiscus toxicus) to CTX-1. The i.p. LD50 values for CTX-1, CTX-2 and CTX-3 were 0.25, 2.3 and 0.9 micrograms/kg, respectively. The signs induced in mice by the ciguatoxins were similar, except that CTX-2 and CTX-3 induced hind-limb paralysis that was absent with CTX-1. Each ciguatoxin was potent orally. CTX-1, CTX-2 and CTX-3 competitively inhibited the binding of [3H]brevetoxin-3 to voltage-dependent sodium channels with relative potencies qualitatively (but not quantitatively) comparable to mouse lethality. This study reveals that the relatively small chemical differences between CTX-1, CTX-2 and CTX-3 give rise to significant structure-activity and pharmacokinetic differences.

Neurotoxic shellfish poisoning and brevetoxin metabolites : a case study from Florida

In June of 1996, three family members were diagnosed as suffering from neurotoxic shellfish poisoning (NSP) as a result of eating shellfish harvested from Sarasota Bay, Florida. Urine from two of these patients and extracts of shellfish collected from the same location were analyzed by radioimmunoassay (RIA) and by receptor binding assay. Activity consistent with brevetoxins was present in both urine and shellfish extracts. High performance liquid chromatographic (HPLC) analysis of shellfish extracts demonstrated multiple fractions recognized by specific anti-brevetoxin antibodies, suggesting metabolic conversion of parent brevetoxins. Affinity-purification of these extracts yielded four major peaks of activity. One peak was identified by HPLC-mass spectroscopy (HPLC-MS) to be PbTx-3, which was likely produced metabolically from the dominant parent toxin PbTx-2. No PbTx-2, however, was detected. Other peaks of activity were determined to consist of compounds of apparent masses of [M + H]+ of 1018, 1034, and 1005. These higher masses are suggestive of conjugated metabolites, but their structures have yet to be determined. The material associated with these latter three peaks were recognized by both RIA and receptor binding assay, but they quantitated differently. This finding suggests that these metabolites react differently in the two assays, and this result may have important implications for seafood safety and regulation. We suggest these metabolites to be the true cause of NSP, and they should be taken into account during regulatory testing.

Strain Dependent Production of Ciguatoxin Precursors (Gambiertoxins) by Gambierdiscus-Toxicus (Dinophyceae) in Culture

Thirteen strains of Gambierdiscus toxicus isolated from Queensland (Australia), Hawaii, French Polynesia and the Virgin Islands were mass cultured and extracted for ciguatoxin. A biodetrital sample containing wild G. toxicus collected from the Republic of Kiribati was also extracted for ciguatoxin. Ciguatoxin, as characterized from moray eels, was not detected in any of the strains examined. Two Queensland strains and the wild G. toxicus produced putative ciguatoxin precursors named gambiertoxins. These gambiertoxins were less polar than ciguatoxin but produced bioassay signs in mice and in-vitro responses in isolated guinea pig atria and vas deferens which were similar (but not identical) to those produced by ciguatoxin. The gambiertoxins from cultured cells were also shown to competitively inhibit the binding of [3H]brevetoxin-3 to rat brain membranes in a dose-dependent manner. The gambiertoxins were more potent than ciguatoxin (on a per mouse unit basis) at stimulating neural elements of guinea pig atria. The two culture strains produced similar amounts of gambiertoxins, even when grown in nutrient media made from different seawater containing different concentrations of nutrients. Changes in nutrient media did not induce the other strains of G. toxicus to produce gambiertoxins. The production of these ciguatoxin precursors appears to be limited to only certain genetic strains of G. toxicus, with the majority of strains not producing these toxins. We propose that ciguatera occurs when blooms of G. toxicus strains genetically capable of producing these ciguatoxin precursors enter the marine food chain. These toxins could then become oxidatively metabolized in fishes to the major polar ciguatoxin. Wild cells produced approximately 100-fold greater quantities of gambiertoxins per cell than did the two culture strains indicating that there is considerable potential for increased production of these ciguatoxin precursors from G. toxicus in culture.

Detection of ricin by colorimetric and chemiluminescence ELISA.

A highly sensitive and specific ELISA was developed to detect ricin in biological fluids. The assay utilizes an affinity-purified goat polyclonal antibody to adsorb ricin from solution. The same antibody (biotinylated) is then used to form a sandwich, and avidin-linked alkaline phosphatase allows color development and measurement of optical density at 405 nm. Our routine assay uses a standard curve over the range of 0-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in assay buffer as well as in a 1:10 dilution of human urine or 1:50 dilution of human serum spiked with ricin. Ricin measured in spiked samples demonstrated accuracy typically within 5% of the expected value in all matrices. The coefficient of variation ranged from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml. Two variations on the routine assay were also investigated. First, lengthened incubation times and additional time for color development allowed accurate quantitation in serum dilutions as low as 1:2. Second, increased concentrations of biotinylated antibody and avidin-linked enzyme from 1:250 to 1:70 enhanced the sensitivity of the assay 10-fold, achieving a detection limit of at least 100 pg/ml (10 pg/well). The assay was also configured to a format based upon chemiluminescence, which allowed quantitation in the 0.1-1 ng/ml range, but was subject to slightly greater variability than the colorimetric assay.

Hypertension and identification of toxin in human urine and serum following a cluster of mussel-associated paralytic shellfish poisoning outbreaks.

Following four outbreaks of paralytic shellfish poisoning on Kodiak Island, Alaska, during 1994, medical records of ill persons were reviewed and interviews were conducted. Urine and serum specimens were analyzed at three independent laboratories using four different saxitoxin binding assays. High-performance liquid chromatography was used to determine the presence of specific toxin congeners. Among 11 ill persons, three required mechanical ventilation and one died. Mean peak systolic and diastolic blood pressure measurements were 172 (range 128-247) and 102 (range 78-165) mmHg, respectively, and blood pressure measurements corresponded with ingested toxin dose. All four different laboratory methodologies detected toxin in serum at 2.8-47 nM during acute illness and toxin in urine at 65-372 nM after acute symptom resolution. The composition of specific paralytic shellfish poisons differed between mussels and human biological specimens, suggesting that human metabolism of toxins had occurred. The results of this study indicate that saxitoxin analogues may cause severe hypertension. In addition, we demonstrate that saxitoxins can be detected in human biological specimens, that nanomolar serum toxin levels may cause serious illness and that human metabolism of toxin may occur. Clearance of paralytic shellfish poisons from serum was evident within 24 hr and urine was identified as a major route of toxin excretion in humans.

Identification of Caribbean ciguatoxins as the cause of an outbreak of fish poisoning among U.S. soldiers in Haiti

On 24 February 1995, six U.S. soldiers serving with the Multinational Force in Haiti became ill after eating a locally caught fish identified as the greater amberjack Seriola dumerili. The victims presented with nausea, vomiting, watery diarrhea and abdominal cramps 5-8 hr after consumption. Also present in some victims were numbness in the extremities or perioral region, bradycardia and scalp paresthesia. Patients were treated with i.v. hydration therapy and antiemetics. All recovered without sequelae over the course of 1-3 months. A portion of the cooked fish was obtained for analysis. A semipurified lipid extract was prepared according to standard methods and analyzed for the presence of Na+ channel site 5 binding activity using a brevetoxin receptor binding assay. By this assay, the fish sample contained the equivalent of approximately 20 ng Caribbean ciguatoxin/g flesh. The presence of the major Caribbean ciguatoxin (C-CTX-1) was confirmed by liquid chromatography-mass spectrometry. Using the receptor binding assay to monitor activity in TSK and PRP-1 column fractions, two minor toxins were detected in addition to C-CTX-1. One of these minor toxins was more polar, and the other less polar, than C-CTX-1. These data provide firm evidence that a family of C-CTX-1 is responsible for ciguatera in the Caribbean.

Stereostructure and Biological Activity of 42-Hydroxy-palytoxin: A New Palytoxin Analogue from Hawaiian Palythoa Subspecies

This paper reports on the analysis of the toxin content from Palythoa tuberculosa and Palythoa toxica samples collected off of the Hawaiian coast. Our work, based on in-depth high-resolution liquid chromatography-mass spectrometry analysis along with extensive NMR study, led us to structurally characterize 42-hydroxy-palytoxin, a new palytoxin congener. This toxin and palytoxin itself appeared to be the major components of toxic extract from a P. tuberculosa sample, while 42-hydroxy-palytoxin was proven by far to be the main palytoxin derivative in P. toxica. Functional studies on this new palytoxin-like compound suggest that the new palytoxin analogue and palytoxin itself present similar biological activities.

Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin. Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5-10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.

Distribution and elimination of brevetoxin PbTx-3 in rats

After i.v. administration, [3H]PbTx-3 was rapidly cleared from the blood; less than 10% remained after 1 min. Within 30 min, radiolabel distributed to skeletal muscle (69.5%), liver (18.0%), and intestinal tract (8.0%). Over 24 hr, radiolabel decreased in muscle, remained constant in liver, and increased in the intestinal tract and feces. Elimination occurred via feces (75.1%) and urine (14.4%), with 9.0% remaining in the carcass after 6 days. This distribution and elimination profile suggested that the liver was the major organ of metabolism and that biliary excretion was an important route of elimination. Thin-layer chromatography confirmed the presence of brevetoxin metabolites in fecal extracts. Skeletal muscle does not appear to be a site of metabolism, but a storage compartment, from which toxin is slowly released prior to clearance by the liver. These studies are the first demonstration of in vivo brevetoxin metabolism in mammals.

Aerosolized specific antibody protects mice from lung injury associated with aerosolized ricin exposure.

Parenteral vaccination with ricin toxoid, although protective against death after a lethal aerosol ricin challenge, only partially protects against lung lesions. Therefore, we tested whether passive protection with aerosolized specific anti-ricin IgG (goat polyclonal, affinity-purified) could protect against both lethality and lung lesions in unvaccinated mice. Healthy CD-l mice were administered antibody (Ab) by small particle aerosol. Group 1 received non-specific control Ab (2160 mg/min/m3), and groups 2 and 3 received anti-ricin IgG (960 and 3280 mg/min/m3, respectively). Each group was challenged with a lethal dose of aerosolized ricin 1 hr after Ab exposure. All group 1 (control Ab) mice developed diffuse airway epithelial necrosis, with severe interstitial edema and inflammation involving all lung lobes, and died 48-96 hr post-challenge (PC). In contrast, in groups 2 and 3 at 24 hr PC, lung lesions were absent to very mild although there was rare epithelial necrosis in the upper airways in both groups. By 48 hr PC, necrosis of the tracheal epithelium and peritracheal inflammation were noted in some group 3 mice only. By 4 days PC, lungs and airways did not differ from cage controls in most group 2 and 3 mice. Weight gain in group 2 and 3 mice paralleled that of control mice. At 14 days PC, lungs were no different in controls than in group 3 mice. However, two non-survivors in group 3 had obstructions due to proximal airway epithelial damage. All group 2 mice survived, although a mild lymphoplasmacytic perivasculitis was present at 14 days PC which was not noted in the group 3 mice.