First Identification of Palytoxin-Like Molecules in the Atlantic Coral Species Palythoa canariensis.

Palytoxin (PLTX) is a complex marine toxin produced by Zoanthids (Palyhtoa), dinoflagellates (Ostreopsis), and cyanobacteria (Trichodesmium). Contact with PLTX-like compounds present in aerosols or marine organisms has been associated with adverse effects on humans. The worldwide distribution of producer species and seafood contaminated with PLTX-like molecules illustrates the global threat to human health. The identification of species capable of palytoxin production is critical for human safety. We studied the presence of PLTX analogues in Palythoa canariensis, a coral species collected in the Atlantic Ocean never described as a PLTX-producer before. Two methodologies were used for the detection of these toxins: a microsphere-based immunoassay that offered an estimation of the content of PLTX-like molecules in a Palythoa canariensis extract and an ultrahigh-pressure liquid chromatography coupled to an ion trap with a time-of-flight mass spectrometer (UPLC-IT-TOF-MS) that allowed the characterization of the toxin profile. The results demonstrated the presence of PLTX, hydroxy-PLTX and, at least, two additional compounds with PLTX-like profile in the Palythoa canariensis sample. The PLTX content was estimated in 0.27 mg/g of lyophilized coral using UPLC-IT-TOF-MS. Therefore, this work demonstrates that Palythoa canariensis produces a mixture of PLTX-like molecules. This is of special relevance to safeguard human health considering Palythoa species are commonly used for decoration by aquarium hobbyists.

Ovatoxin-a, A Palytoxin Analogue Isolated from Ostreopsis cf. ovata Fukuyo: Cytotoxic Activity and ELISA Detection.

This study provides the first evaluation of the cytotoxic effects of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a), the major toxin produced by Ostreopsis cf. ovata in the Mediterranean Sea. Its increasing detection during Ostreopsis blooms and in seafood highlights the need to characterize its toxic effects and to set up appropriate detection methods. OVTX-a is about 100 fold less potent than PLTX in reducing HaCaT cells viability (EC50 = 1.1 × 10(-9) M vs 1.8 × 10(-11) M, MTT test) in agreement with a reduced binding affinity (Kd = 1.2 × 10(-9) vs 2.7 × 10(-11) M, saturation experiments on intact cells). Similarly, OVTX-a hemolytic effect is lower than that of the reference PLTX compound. Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting the lack of a hydroxyl group at C44 as a critical feature for PLTXs cytotoxic effects. A sandwich ELISA developed for PLTX detects also OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate manner (Bias = 0.3%), also in O. cf. ovata extracts and contaminated mussels. Although in vitro OVTX-a appears less toxic than PLTX, its cytotoxicity at nanomolar concentrations after short exposure time rises some concern for human health. The sandwich ELISA can be a viable screening method for OVTXs detection in monitoring program.

Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish.

Despite ethical and technical concerns, the in vivo method, or more commonly referred to mouse bioassay (MBA), is employed globally as a reference method for phycotoxin analysis in shellfish. This is particularly the case for paralytic shellfish poisoning (PSP) and emerging toxin monitoring. A high-performance liquid chromatography method (HPLC-FLD) has been developed for PSP toxin analysis, but due to difficulties and limitations in the method, this procedure has not been fully implemented as a replacement. Detection of the diarrhetic shellfish poisoning (DSP) toxins has moved towards LC-mass spectrometry (MS) analysis, whereas the analysis of the amnesic shellfish poisoning (ASP) toxin domoic acid is performed by HPLC. Although alternative methods of detection to the MBA have been described, each procedure is specific for a particular toxin and its analogues, with each group of toxins requiring separate analysis utilising different extraction procedures and analytical equipment. In addition, consideration towards the detection of unregulated and emerging toxins on the replacement of the MBA must be given. The ideal scenario for the monitoring of phycotoxins in shellfish and seafood would be to evolve to multiple toxin detection on a single bioanalytical sensing platform, i.e. 'an artificial mouse'. Immunologically based techniques and in particular surface plasmon resonance technology have been shown as a highly promising bioanalytical tool offering rapid, real-time detection requiring minimal quantities of toxin standards. A Biacore Q and a prototype multiplex SPR biosensor have been evaluated for their ability to be fit for purpose for the simultaneous detection of key regulated phycotoxin groups and the emerging toxin palytoxin. Deemed more applicable due to the separate flow channels, the prototype performance for domoic acid, okadaic acid, saxitoxin, and palytoxin calibration curves in shellfish achieved detection limits (IC20) of 4,000, 36, 144 and 46 µg/kg of mussel, respectively. A one-step extraction procedure demonstrated recoveries greater than 80% for all toxins. For validation of the method at the 95% confidence limit, the decision limits (CCα) determined from an extracted matrix curve were calculated to be 450, 36 and 24 µg/kg, and the detection capability (CCß) as a screening method is ≤10 mg/kg, ≤160 µg/kg and ≤400 µg/kg for domoic acid, okadaic acid and saxitoxin, respectively.

Stereoisomers of 42-hydroxy palytoxin from Hawaiian Palythoa toxica and P. tuberculosa: stereostructure elucidation, detection, and biological activities.

Palytoxin ranks among the most potent marine biotoxins. Its lethality was well known to native Hawaiians that used to smear a "moss" containing the toxin on their spears to cause instant death to their victims. Human intoxications due to exposure to palytoxin and to its many congeners have been reported worldwide. Currently, palytoxins constitute the main threat to public health across the Mediterranean Sea. In the present work we report on the isolation and stereostructural determination of a new palytoxin analogue from a Hawaiian Palythoa tuberculosa sample. This new toxin is a stereoisomer of 42-hydroxypalytoxin isolated from Palythoa toxica. The whole absolute configuration of this latter toxin is also reported in the paper. Interestingly, the two 42-hydroxypalytoxins do not share the same biological activity. The stereoisomer from P. tuberculosa showed cytotoxicity toward skin HaCaT keratinocytes approximately 1 order of magnitude lower than that of 42-hydroxypalytoxin from P. toxica and about 2 orders of magnitude lower than that of palytoxin itself. This finding holds the prospect of interesting structure-activity relationship evaluations in the future.

Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Characterization of palytoxin binding to HaCaT cells using a monoclonal anti-palytoxin antibody.

Palytoxin (PLTX) is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA), a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a K(d) of 3 × 10-10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3-1.0 × 10-7 M) and possibly as a non-competitive antagonist against low PLTX concentrations (0.1-3.0 × 10-9 M). Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10-5 M). However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.

The stretch-activated channel blocker Gd(3+) reduces palytoxin toxicity in primary cultures of skeletal muscle cells.

Palytoxin (PLTX) is one of the most toxic seafood contaminants ever isolated. Reports of human food-borne poisoning ascribed to PLTX suggest skeletal muscle as a primary target site. Primary cultures of mouse skeletal muscle cells were used to study the relationship between Ca(2+) response triggered by PLTX and the development of myotoxic insult. Ca(2+) imaging experiments revealed that PLTX causes a transitory intracellular Ca(2+) response (transient phase) followed by a slower and more sustained Ca(2+) increase (long-lasting phase). The transient phase is due to Ca(2+) release from intracellular stores and entry through voltage-dependent channels and the Na(+)/Ca(2+) exchanger (reverse mode). The long-lasting phase is due to a massive and prolonged Ca(2+) influx from the extracellular compartment. Sulforhodamine B assay revealed that the long-lasting phase is the one responsible for the toxicity in skeletal muscle cells. Our data analyzed, for the first time, pathways of PLTX-induced Ca(2+) entry and their correlation with PLTX-induced toxicity in skeletal muscle cells. The cellular morphology changes induced by PLTX and the sensitivity to gadolinium suggest a role for stretch-activated channels.

Harmful dinoflagellate Ostreopsis cf. ovata Fukuyo: detection of ovatoxins in field samples and cell immunolocalization using antipalytoxin antibodies.

Ostreopsis cf. ovata, a benthic dinoflagellate often blooming along the Mediterranean coasts, has been associated with toxic events ranging from dyspnea to mild dermatitis. In late September 2009, an Ostreopsis cf. ovata bloom occurred in the Gulf of Trieste (Northern Adriatic Sea; Italy), causing pruritus and mild dermatitis in beachgoers. An integrated study was initiated to characterize Ostreopsis cells by light and confocal microscopy, PCR techniques, immunocytochemistry, and high resolution liquid chromatography-mass spectrometry (HR LC-MS). The presence of Ostreopsis cf. ovata of the Atlantic/Mediterranean clade was unambiguously established by morphological and genetic analyses in field samples. Several palytoxin-like compounds (ovatoxin-a,-b,-c,-d,-e) were identified by HR LC-MS, ovatoxin-a being the most abundant (45-64 pg/cell). Surprisingly, no palytoxin was detected. For the first time, monoclonal and polyclonal antipalytoxin antibodies revealed the intracellular cytoplasmic localization of ovatoxins, suggesting their cross-reactivity with these antibodies. Since harmful dinoflagellates do not always produce toxins, the immunocytochemical localization of ovatoxins, although qualitative, can provide an early warning for toxic Ostreopsis cells before their massive diffusion and/or concentration in seafood.

Antibody characterization and immunoassays for palytoxin using an SPR biosensor.

Palytoxin (PLTX), a polyether marine toxin originally isolated from the zoanthid Palythoa toxica, is one of the most toxic non-protein substances known. Fatal poisonings have been linked to ingestion of PLTX-contaminated seafood, and effects in humans have been associated with dermal and inhalational exposure to PLTX containing organisms and waters. Additionally, PLTX co-occurrence with other well-characterized seafood toxins (e.g., ciguatoxins, saxitoxins, tetrodotoxin) has hindered direct associations of PLTX to seafood-borne illnesses. There are currently no validated methods for the quantitative detection of PLTX(s). As such, a well-characterized, robust, specific analytical technique is needed for the detection of PLTX(s) in source organisms, surrounding waters, and clinical samples. Surface plasmon resonance (SPR) biosensors are ideally suited for antibody characterization and quantitative immunoassay detection. Herein, we describe a newly developed SPR assay for PLTX. An anti-mouse substrate was used to characterize the kinetic values for a previously developed monoclonal anti-PLTX. The characterized antibody was then incorporated into a sensitive, rapid, and selective PLTX assay. Buffer type, flow rate, analyte-binding time, and regeneration conditions were optimized for the antibody-PLTX system. Cross-reactivity to potentially co-occurring seafood toxins was also evaluated. We show that this optimized assay is capable of measuring low- to sub-ng/mL PLTX levels in buffer and two seafood matrices (grouper and clam). Preliminary results indicate that this SPR biosensor assay allows for (1) rapid characterization of antibodies and (2) rapid, sensitive PLTX concentration determination in seafood matrices. Method development information contained herein may be broadly applied to future PLTX detection and/or antibody characterization efforts.

Quantitative detection of staphylococcal enterotoxin B by resonant acoustic profiling.

A rapid and sensitive detection of staphylococcal enterotoxin B (SEB) was developed using a novel acoustic sensing technique: Resonant Acoustic Profiling (RAP), which utilizes high-frequency piezoelectric quartz resonators for monitoring biomolecular interactions. An automated four-channel instrument consisting of acoustic sensors covalently conjugated with anti-SEB antibodies was used. As the samples flowed across control and active sensors simultaneously, binding was measured as a change in the resonant frequency. The lower limit of detection (LLOD) for the label free direct format was 25 ng/mL. Detection sensitivity was increased by adding mass sequentially to the captured SEB on the sensor in the form of sandwich antibodies and biotin-avidin-based gold nanoparticles. The LLOD for the mass enhanced formats were 5 and 0.5 ng/mL of SEB, respectively. The lowest sensitivity corresponds to 1.3 fM in a 75 microL sample. The total assay time including the enhancement steps was less than 10 min. SEB was detected in both neat urine and PBS buffer-spiked samples, with linear correlations between resonant frequency signals and SEB concentrations (R(2) of 0.999 and 0.998, respectively). No significant cross-reactivity was observed with homologue toxins SEA, SED, and TSST, but some cross-reactivity was observed with the closely related toxin SEC(1) when we used a polyclonal antibody in the assay. SEC(1) cross-reactivity was not observed when a SEB-specific monoclonal antibody was employed in the assay. Thus the specificity of the assay presented here was dependent on the quality of the antibodies used. In addition to detection, we evaluated RAP's ability to measure the toxin in unknown samples rapidly by measuring the initial binding rate of the interaction, thereby further shortening the assay time to 6 min.

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.

Identification of the RNA N-glycosidase activity of ricin in castor bean extracts by an electrochemiluminescence-based assay.

A simple electrochemiluminescence-based assay for RNA N-glycosidase activity has been modified to permit its use with authentic extracts of Ricinus communis (castor beans) and Abrus precatorius (jequirity seeds)--the natural sources of ricin and abrin. Modifications include the addition of an RNase inactivator to the reaction mixture, elimination of a signal-enhancing monoclonal antibody, and optimization of the incubation temperature. Concurrent testing with two substrates provides a diagnostic tool enabling castor bean toxins to be differentiated from a larger selection of N-glycosidase toxins than was previously examined.

A Novel Sensitive Cell-Based Immunoenzymatic Assay for Palytoxin Quantitation in Mussels.

The marine algal toxin palytoxin (PLTX) and its analogues are some of the most toxic marine compounds. Their accumulation in edible marine organisms and entrance into the food chain represent their main concerns for human health. Indeed, several fatal human poisonings attributed to these compounds have been recorded in tropical and subtropical areas. Due to the increasing occurrence of PLTX in temperate areas such as the Mediterranean Sea, the European Food Safety Authority (EFSA) has suggested a maximum limit of 30 µg PLTX/kg in shellfish meat, and has recommended the development of rapid, specific, and sensitive methods for detection and quantitation of PLTX in seafood. Thus, a novel, sensitive cell-based ELISA was developed and characterized for PLTX quantitation in mussels. The estimated limits of detection (LOD) and quantitation (LOQ) were 1.2 × 10-11 M (32.2 pg/mL) and 2.8 × 10-11 M (75.0 pg/mL), respectively, with good accuracy (bias = 2.5%) and repeatability (15% and 9% interday and intraday relative standard deviation of repeatability (RSDr), respectively). Minimal interference of 80% aqueous methanol extract allows PLTX quantitation in mussels at concentrations lower than the maximum limit suggested by EFSA, with an LOQ of 9.1 µg PLTX equivalent/kg mussel meat. Given its high sensitivity and specificity, the cell-based ELISA should be considered a suitable method for PLTX quantitation.

Toxicity and pathophysiology of palytoxin congeners after intraperitoneal and aerosol administration in rats.

Preparations of palytoxin (PLTX, derived from Japanese Palythoa tuberculosa) and the congeners 42-OH-PLTX (from Hawaiian P. toxica) and ovatoxin-a (isolated from a Japanese strain of Ostreopsis ovata), as well as a 50:50 mixture of PLTX and 42-OH-PLTX derived from Hawaiian P. tuberculosa were characterized as to their concentration, composition, in-vitro potency and interaction with an anti-PLTX monoclonal antibody (mAb), after which they were evaluated for lethality and tissue histopathology after intraperitoneal (IP) and aerosol administration to rats. Once each preparation was characterized as to its toxin composition by LC-HRMS and normalized to a total PLTX/OVTX concentration using HPLC-UV, all four preparations showed similar potency towards mouse erythrocytes in the erythrocyte hemolysis assay and interactions with the anti-PLTX mAb. The IP LD50 values derived from these experiments (0.92, 1.93, 1.81 and 3.26 µg/kg, for the 50:50 mix, 42-OH-PLTX, PLTX, and ovatoxin-a, respectively) were consistent with published values, although some differences from the published literature were seen. The aerosol LD50 values (0.063, 0.045, 0.041, and 0.031 µg/kg for the 50:50 mix, 42-OH PLTX, PLTX, and ovatoxin-a, respectively) confirmed the exquisite potency of PLTX suggested by the literature. The tissue histopathology of the different toxin preparations by IP and aerosol administration were similar, albeit with some differences. Most commonly affected tissues were the lungs, liver, heart, salivary glands, and adrenal glands. Despite some differences, these results suggest commonalities in potency and mechanism of action among these PLTX congeners.

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.

Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods.

Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). PLTX outbreaks are usually associated with Indo-Pacific waters, however their recent repeated occurrence in Mediterranean-European Atlantic coasts demonstrate their current worldwide distribution. Human sickness and fatalities have been associated with toxic algal blooms and ingestion of seafood contaminated with PLTX-like molecules. These toxins represent a serious threat to human health. There is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. We have developed an immuno-detection method for PLTX-like molecules based on the use of microspheres coupled to flow-cytometry detection (Luminex 200™). The assay consisted of the competition between free PLTX-like compounds in solution and PLTX immobilized on the surface of microspheres for binding to a specific monoclonal anti-PLTX antibody. This method displays an IC50 of 1.83 ± 0.21 nM and a dynamic range of 0.47-6.54 nM for PLTX. An easy-to-perform extraction protocol, based on a mixture of methanol and acetate buffer, was applied to spiked mussel samples providing a recovery rate of 104 ± 8% and a range of detection from 374 ± 81 to 4430 ± 150 µg kg(-1) when assayed with this method. Extracts of Ostreopsis cf. siamensis and Palythoa tuberculosa were tested and yielded positive results for PLTX-like molecules. However, the data obtained for the coral sample suggested that this antibody did not detect 42-OH-PLTX efficiently. The same samples were further analyzed using a neuroblastoma cytotoxicity assay and UPLC-IT-TOF spectrometry, which also pointed to the presence of PLTX-like compounds. Therefore, this single detection method for PLTX provides a semi-quantitative tool useful for the screening of PLTX-like molecules in different matrixes.

A revisited hemolytic assay for palytoxin detection: Limitations for its quantitation in mussels.

Palytoxin (PLTX) and its analogues have been detected as seafood contaminants associated with a series of human foodborne poisonings. Due to a number of fatalities ascribed to the ingestion of PLTX-contaminated marine organisms, the development of methods for its detection in seafood has been recommended by the European Food Safety Authority (EFSA). Due to its feasibility, the spectrophotometric hemolytic assay is widely used to detect PLTX in different matrices, even though a standardized protocol is still lacking. Thus, on the basis of available assay procedures, a new standardized protocol was set up using purified human erythrocytes exposed to PLTX (working range: 3.9 × 10(-10)-2.5 × 10(-8) M) in a K(+)-free phosphate buffered saline solution, employing a 5 h incubation at 41 °C. An intra-laboratory characterization demonstrated its sensitivity (limit of detection, LOD = 1.4 × 10(-10) M and quantitation, LOQ = 3.4 × 10(-10) M), accuracy (bias = -0.8%), repeatability (RSDr = 15% and 6% for intra- and inter-day repeatability, respectively) and specificity. However, the standardized method seems not to be suitable for PLTX quantitation in complex matrices, such as mussel (Mytilus galloprovincialis) extracts, at least below the limit suggested by EFSA (30 µg PLTXs/Kg shellfish meat). Thus, the hemolytic assay for PLTX quantitation in seafood should be used only after a careful evaluation of the specific matrix effects.

Sensitive and specific colorimetric ELISAs for Staphylococcus aureus enterotoxins A and B in urine and buffer.

We present here simple, sensitive and accurate colorimetric capture ELISAs for staphylococcal enterotoxins A and B. Standard curves were linear over the range 0.5-1 ng/mL, and toxins could be accurately measured at 0.5 ng/mL in assay buffer or 0.1 ng/mL in human urine. Cross-reactivity between serotypes was negligible. Detection in serum was complicated by the presence of specific antibodies to SE's in most normal sera. These assays offer a viable, cost-effective method for analysis of these ubiquitous toxins. Further, their sensitivity in undiluted urine makes them ideal candidates for evaluating human exposure.

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.

In vivo and in vitro effects of 42-hydroxy-palytoxin on mouse skeletal muscle: structural and functional impairment.

Palytoxins (PLTXs) are known seafood contaminants and their entrance into the food chain raises concern about possible effects on human health. The increasing number of analogs being identified in edible marine organisms complicates the estimation of the real hazard associated with the presence of PLTX-like compounds. So far, 42-OH-PLTX is one of the few congeners available, and the study of its toxicity represents an important step toward a better comprehension of the mechanism of action of this family of compounds. From this perspective, the aim of this work was to investigate the in vivo and in vitro effect of 42-OH-PLTX on skeletal muscle, one of the most sensitive targets for PLTXs. Our results demonstrate that 42-OH-PLTX causes damage at the skeletal muscle level with a cytotoxic potency similar to that of PLTX. 42-OH-PLTX induces cytotoxicity and cell swelling in a Na(+)-dependent manner similar to the parent compound. However, the limited Ca(2+)-dependence of the toxic insult induced by 42-OH-PLTX suggests a specific mechanism of action for this analog. Our results also suggest an impaired response to the physiological agonist acetylcholine and altered cell elasticity.