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Mar 17, 2004 - Marine toxins such as brevetoxins and ciguatoxins are produced by dinoflagellates and can accumulate in seafood. These toxins affect hu...
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Chem. Res. Toxicol. 2004, 17, 572-578

Detection of Sodium Channel Activators by a Rapid Fluorimetric Microplate Assay M. C. Louzao,† M. R. Vieytes,‡ T. Yasumoto,§ and L. M. Botana*,† Departamento de Farmacologia and Departamento de Fisiologia Animal, Facultad de Veterinaria de Lugo Universidad de Santiago de Compostela, 27002 Lugo, Spain, and Japan Food Research Laboratories, Tama, Tokyo 206-0025, Japan Received November 5, 2003

Marine toxins such as brevetoxins and ciguatoxins are produced by dinoflagellates and can accumulate in seafood. These toxins affect humans through seafood consumption. Intoxication is mainly characterized by gastrointestinal and neurological disorders and, in most severe cases, by cardiovascular problems. To prevent the consumption of food contaminated with these toxins, shellfish have been tested by mouse bioassay. However, this method is expensive, timeconsuming, and ethically questionable. The objective of this study was to use a recently developed fluorimetric microplate assay to rapidly detect brevetoxins and ciguatoxins. The method is based on the pharmacological effect of brevetoxins and ciguatoxins known to activate sodium channels and involves (i) the incubation of excitable cells in 96 well microtiter plates with the fluorescent dye bis-oxonol, whose distribution across the membrane is potentialdependent, and (ii) dose-dependent cell depolarization by the toxins. Our findings demonstrate that measuring changes in membrane potential induced by brevetoxins and ciguatoxins allowed their quantitation. Active toxins could be reliably detected at concentrations in the nanomolar range. The simplicity, sensitivity, and possibility of being automated provide the basis for development of a practical alternative to conventional testing for brevetoxins and ciguatoxins.

Introduction PbTxs1 are phycotoxins produced by dinoflagellates such as Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve) and consist of at least nine structurally related, lipid soluble polycyclic ether derivatives (PbTx-1-9) (1-3). These toxins are responsible for the massive fish kills and human intoxications (known as NSP) characterized by neurological disturbances (3). The molecular target of these lipid soluble cyclic polyethers is the voltage-gated sodium channel, a fundamental transmembrane protein involved in cellular excitability (4). PbTxs bind specifically, and at high affinity, with receptor site 5 located on the R-subunit of the sodium channel (1, 2, 5). PbTxs share this binding site with the structurally related CTXs (1, 2, 6, 7). VTD, another neurotoxin, targets site 2 of the voltage-gated sodium channel for their primary action (8). CTXs are the major causative toxins of ciguatera seafood poisoning (3). CTXs accumulate in fish through the food chain, starting from the dinoflagellate Gambierdiscus toxicus (3, 9). Electrophysiological studies of the mode of action of PbTxs and CTXs identify them as specific sodium channel activators * To whom correspondence should be addressed. Tel and Fax: 34 982 252 242. E-mail: [email protected]. † Departamento de Farmacologia, Facultad de Veterinaria de Lugo Universidad de Santiago de Compostela. ‡ Departamento de Fisiologia Animal, Facultad de Veterinaria de Lugo Universidad de Santiago de Compostela. § Japan Food Research Laboratories. 1 Abbreviations: AU, arbitrary units; bis-oxonol, bis-(1,3-diethylthiobarbituric acid) trimethine oxonol; CTX, ciguatoxin; DSP, diarrheic shellfish poisoning; EMEM, minimum essential medium, eagle; LD, lethal dose; MU, mouse unit; NSP, neurotoxic shellfish poisoning; ND, not detectable; PbTx, brevetoxin; PSP, paralytic shellfish poisoning; RIA, radioimmunoassay; VTD, veratridine.

(6, 10, 11). The binding of toxins alters the channel’s function: (i) the activation voltage for channel opening shifts to a more negative value, and (ii) the inactivation of opened channels is inhibited, resulting in persistent activation (2, 12). Indeed, during the action of these phycotoxins, sodium channels remain permanently opened, which produces a continuous entry of sodium ions in most excitable cells. Sodium entry has various consequences on sodium-dependent physiological mechanisms, consisting of membrane depolarization, which, in turn, causes spontaneous and/or repetitive action potential discharges and thereby increases membrane excitability (13). The increase in membrane excitability during the action of PbTxs is responsible for the different effects exerted by these toxins on various chemical synapses and secretory cells. Selective interference with sodium channel function is therefore considered to be the primary mechanism by which many of the clinical and physiological symptoms of PbTx poisoning arise (14). Monitoring programs are in place to prevent consumption of seafood contaminated with NSP toxins. In these programs, a mouse bioassay has been used to test shellfish for the presence of PbTxs, where the regulatory limit is 20 mouse units (MU)/100 g shellfish (15). Published toxicity data for isolated PbTxs suggest that 20-100 µg of PbTx is equivalent to the regulatory limit of 20 MU/100 g. The primary advantage of mouse bioassay method is that it generally estimates the full toxic potential of the shellfish extract; however, it is ethically questionable, expensive, and time-consuming. These drawbacks have prompted an investigation of alternative methodologies. CTXs are very potent toxins (LD50 ) 0.25 µg/kg, ip, mouse), but they differ from the PbTxs (LD50 ) 500 µg/

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kg), which are more potent to fish than to mammals (16, 17). In some laboratories, cell assays for CTXs are a rapid and sensitive screening tool, especially when combined with qualitative confirmation by LC/MS (18). Despite the development of in vitro assays, in this case, the mouse bioassay also remains the most widely used assay to establish levels of CTX in extracts of fish (19). This method quantifies lethal and sublethal doses of CTX in crude extracts administered intraperitoneally to mice. However, ethical concerns remain, and the assay is not sufficiently sensitive to detect low toxicity fish in crude extracts. Both of these classes of polyether algal toxins, the CTXs and PbTxs, can be detected via their pharmacologic actions on site 5 of the voltage-gated sodium channel. We have already reported the development of screening for diarrheic shellfish poisoning (DSP) (20-23) and paralytic shellfish poisoning (PSP) (24, 25). Recently, we reported a rapid toxicity test based on a fluorimetric determination of changes in membrane potential induced by PSP toxins through application of microplate technology to increase in assay efficiency (26). The present paper describes a fluorimetric microplate assay suitable for detection of PbTxs and CTXs and potentially other site 5 toxins.

Experimental Procedures Materials. VTD, gramicidin, and PbTx-9 were obtained from Sigma (Spain). Bis-oxonol was from Molecular Probes. All other chemicals were reagent grade and purchased from Sigma and Merck (Germany). Prof. T. Yasumoto generously provided PbTx-3 and CTX-3C. Microtiter plates used for fluorescence assays were white solid plates tissue culture-treated from Costar and white clear bottom plates tissue culture-treated from Costar. The solution used in the fluorimetric microplate assay contained the following (mM): NaCl, 137; KCl, 5; CaCl2, 1; MgCl2, 1.2; KH2PO4, 0.44; NaHCO3, 4.2; glucose, 10; pH 7.4. Methods. 1. Excitable Cells. 1.1. Human Neuroblastoma Cell Line [BE(2)-M17]. The cells were grown in EMEM:Ham’s F12 (1:1) supplemented with extra glutamine, nonessential amino acids, 15% fetal bovine serum, and 50 mg/mL gentamicine and 50 µg/mL amphotericin B. Cells were grown in 25 cm2 tissue culture flasks at 37 °C in 5% CO2 and were subcultured by transferring cells released by the application of 0.1% trypsin. 2. Fluorescence Measurement. We have previously set up a microplate assay to detect toxin-induced changes in membrane potential (26). On the basis of this assay, we chose the ideal conditions to detect PbTxs and CTXs. All reactions were performed in 96 well microtiter plates in a total volume of 200 µL. The attached neuroblastoma cells were trypsinized after reaching optimum confluence. The suspension of cells was dispensed 60 000 cells/well in 96 well plates and incubated overnight (37 °C/5% CO2). Changes in membrane potential in BE(2)-M17 cells exposed to toxins were evaluated using the fluorescent probe bis-oxonol. After overnight incubation, culture medium was removed and neuroblastoma cells were exposed for 10 min in the assay solution with 4 nM of the fluorescent dye bis-oxonol whose distribution across the membrane is potential-dependent. Fluorescence was measured in a FL600 microplate fluorescence reader (Bio-Tek Instruments, Inc., VT) set at 530 nm (excitation) and 590 nm (emission). The change in membrane potential was detected after the addition of toxins. 3. Statistical Analysis. The experiments were carried out at least four times in duplicate. Results were analyzed using the Student’s t-test for paired data. A probability level of 0.05 or smaller was used for statistical significance. Results are expressed as the mean ( SD. The quantitative estimate of precision was expressed as percent coefficient of variation.

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Figure 1. Time course of changes of fluorescence in human neuroblastoma cells. First, we observed stabilization of the baseline after dye incubation. Then, addition of toxins (25 nM PbTx-3, 25 nM PbTx-9, or 25 nM CTX-3C) depolarizes neuroblastoma cells and thus increases their fluorescence. At the end of the experiment, 10 µg/mL gramicidin was added to induce complete cell depolarization. Results are expressed in AU of fluorescence ( SD (N ) 4).

Results We have previously set up a microplate assay to quantify toxins that produce PSP by detecting changes in membrane potential of neuroblastoma cells with bisoxonol (26). Procedure conditions such as the concentrations of dye, VTD, and gramicidin were chosen from this previous study. However, important modifications were made to adapt the assay for PbTxs and CTXs. After incubation of neuroblastoma cells with bis-oxonol, we found stabilization of the fluorescence in 20-30 min. To ensure baseline stabilization in the following experiments, we start registering the fluorescence of the cells in the microplate reader 30 min after the incubation with bis-oxonol. We analyzed the effect of three different sodium channel activators: two PbTxs (PbTx-3 and PbTx-9) and one CTX (CTX-3C). Figure 1 shows a time course of fluorescence intensity for 25 nM PbTx-3, 25 nM PbTx-9, and 25 nM CTX-3C. When CTX-3C is introduced into saline solution containing neuroblastoma cells incubated with bis-oxonol, the fluorescence intensity of the cells increased indicating a cell depolarization. Neither PbTx-3 nor PbTx-9 had significant effect on fluorescence. Finally, complete cell depolarization was obtained with 10 µg/mL gramicidin. On the basis of those data, it seemed difficult to detect low concentrations of PbTxs with this method. However, we did the following experiments taking advantage of the positive allosteric coupling of site 5 to the neurotoxic alkaloid binding site (site 2) on the sodium channel complex (27, 28). We examined the possible enhancement by PbTxs and CTX of VTD-dependent sodium uptake. The change in fluorescence could be detected a few minutes after the addition of CTX-3C as is shown in Figure 1. In the next set of experiments, we preincubated neuroblastoma cells for at least 10 min with PbTxs or

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Figure 3. Dose-dependent curve of percentage of enhancement VTD-induced depolarization by PbTx-3 expressed in a semilogarithmic scale. Results are expressed as the mean ( SD. Figure 2. Time course of changes of fluorescence detected in human neuroblastoma cells by using bis-oxonol. We registered the fluorescence of the cells in the microplate reader 10 min after preincubation with different concentrations of PbTx-3. Enhancement of changes in fluorescence induced by 4 µM VTD was detected during 8 min after the addition of the toxin. Results are expressed in AU of fluorescence ( SD (N ) 4). Significant differences vs VTD were found in the following concentrations of PbTx-3: 0.1, 1, 5, 10, 25, 50, and 100 nM.

CTXs to ensure that they were acting on sodium channels. Then, we added VTD and changes in fluorescence were measured for other 8-10 min. In previous studies, we found that 4 µM VTD depolarized neuroblastoma cells. This concentration was therefore used in the following experiments. PbTx-3 dose-dependent enhancement of depolarization induced by VTD is shown in Figure 2. This clear relationship between increase of VTD-induced depolarization (expressed in percentage of enhancement of fluorescence) and toxin concentration is represented in a semilogarithmic scale in Figure 3. Threshold detection of PbTx-3 was close to 0.01 nM (1.79 pg/well) with a variation coefficient lying between 44.5 and 2.1%. Precision tests showed the coefficient of variation for concentrations of PbTx-3 0.01, 0.1, 1, 5, 10, 25, 50, and 100 nM, respectively, to be 44.5, 10.2, 5.6, 6.7, 6.7, 4.0, 2.1, and 3.9%. A similarly shaped curve was obtained for PbTx-9 (Figure 5), although the sensitivity of the assay toward PbTx-9 was lower than for PbTx-3. In this case, the fluorescent microplate method is suitable for detection of 1 nM PbTx-9 (179.8 pg/well). Standard curve precision profile showed a working range (below 20% coefficient variation) covering almost the entire standard curve. Coefficients of variation at concentrations of PbTx-9 1, 10, 20, 50, and 100 nM were 36.8, 19.9, 3.3, 3.5, 3.9, 2.2, and 2.4%. Dose-dependent enhancement of VTD-induced depolarization by PbTx-9 is shown in Figure 4. The ability of an alternative neurotoxin that binds site 5 (CTX-3C) to enhance VTD-induced depolarization allowed additional confirmation of the accuracy of this method to detect toxins that activate sodium channels (Figure 6). Because of the enhanced potency of

Figure 4. Time course of changes in fluorescence obtained after preincubation with PbTx-9. Fluorescence of neuroblastoma cells was registered in the microplate reader 10 min after preincubation with different concentrations of PbTx-9. Enhancement of fluorescence induced by 4 µM VTD was detected during 8 min after the addition of the toxin. Results are expressed in AU of fluorescence ( SD (N ) 4). Significant differences vs VTD were found in the following concentrations of PbTx-9: 1, 10, 25, 50, 100, 250, and 500 nM.

CTX-3C, considerably less toxin was required to enhance depolarization induced by VTD. The threshold detection limit is close to 0.01 nM (Figures 6 and 7), and the precision (coefficient of variation) ranged from 23.5 to 1.0%. The percent coefficient of variation for concentrations of CTX-3C 0.01, 0.1, 1, 5, 10, 25, and 50 nM were 23.5, 6.8, 6.6, 5.0, 3.1, 1.3, and 1.0%.

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Figure 5. Curve of the relationship between percentage of increase in VTD-induced depolarization by PbTx-9 and toxin concentration. Results are expressed as the mean ( SD.

Figure 6. Time course of changes in fluorescence of neuroblastoma cells. Fluorescence of the cells was detected in the microplate reader 10 min after preincubation with different concentrations of CTX-3C. Enhancement of the effect produced by 4 µM VTD was registered during 8 min after the addition of the toxin. Results are expressed in AU of fluorescence ( SD (N ) 5). Significant differences vs VTD were found in the following concentrations of CTX-3C: 0.01, 0.1, 1, 5, 10, 25, and 50 nM.

Discussion There is a global concern relating to the various outbreaks occurring every year due to a different family of toxins. This increased concern is reflected in the quantity of work performed by many research groups in acquiring a good detection method (26, 29, 30). All current detection methods for PbTxs have either a biological origin or an analytical chemical origin. Some of those assays have many advantages but also have drawbacks.

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Figure 7. Dose-dependent curve of enhancement of VTDinduced depolarization by CTX-3C expressed in a semilogarithmic scale. Results are expressed as the mean ( SD.

The analytical techniques available, spectroscopy-coupled HPLC and LC-coupled MS, are techniques that require relatively expensive equipment. In general, their sensitivities are in the nanogram to microgram range (31). Methods of detection that have remained essential tools of experimentalists and public health officials alike all have their basis in receptor-ligand interactions. In particular, the interactions are based on the pharmacological event that is essential for the onset of toxicity specific binding of toxin to receptor. Shellfish toxicity monitoring is routinely carried out by mouse bioassay procedure. A second essential type of interaction has as its basis the principle of immune resistance through the interaction of toxin with antibody receptor. For that purpose, RIA and sodium channel receptor assays are developed (1, 5, 32, 33). Recent advances in fluorescent probes allow detection of toxins with a new methodology (25, 26). Related with that, techniques using living cells are interesting for detection of marine toxins and for their use in research applications (14, 25, 26, 30, 34-38) including variations of cell assays based on reporter genes (39). There is a mounting pressure to develop alternative screening test. The sodium channel receptor can be exploited and used as a receptor in simple and effective tests for PbTxs and CTXs (1, 6, 11, 32, 33, 40, 41). The fluorimetric microplate cell assay described here is based in detection of PbTxs and CTXs by the changes induced in neuroblastoma membrane potential, which is consistent with the known pharmacology of toxin:sodium channel interactions. Neuroblastoma cell depolarization is determined with the voltage sensitive fluoroprobe bisoxonol. Bis-oxonol is a slow dye that moves across membranes until it reaches electrochemical equilibrium. Cells stained with bis-oxonol exhibit circumferential ringlike patterns, typical of dominating fluorescence intensity from plasma membranes. Cell hyperpolarization will be reflected in an increased intracellular cationic dye concentration while a decreased accumulation will reflect depolarization (42). The probe decreases fluorescence intensity upon cell hyperpolarization and increases fluorescence intensity upon cell depolarization. When we

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added PbTxs or CTXs to neuroblastoma cells loaded with bis-oxonol, the increase of fluorescence was very low in agreement with published data (11). However, we found that the sensitivity of the method is increased when we exploit the positive allosteric coupling between binding sites 5 and 2 on voltage-gated sodium channels, which respectively bind polycyclic neurotoxins (e.g., PbTxs) and alkaloid neurotoxins (such as VTD) (27, 28). PbTxs equilibrate slowly and necessitate preincubation before VTD challenge. We found that PbTxs (PbTx-3 and PbTx9) (1, 2, 6, 43) enhance VTD-induced depolarization of neuroblastoma cells in a voltage-dependent manner. Results of these fluorescent microplate assays are encouraging. PbTxs could be detected in samples containing close to 0.01 nM PbTx-3 (1.79 pg/well). In addition, PbTx-3 could be used as a standard and numerical values for the test assays could be determined as PbTx-3 activity equivalents. Published toxicity data for isolated PbTxs suggest that 20-100 µg of PbTx is equivalent to the regulatory limit of 20 MU/100 g (2, 44). Concentrations of PbTxs detected by our method are lower than the amounts that cause a positive mouse bioassay, although this can vary considerably depending on the derivative involved (44). In this sense, the dose:response curve was approximately linear for another PbTx (PbTx9) at concentrations higher than for PbTx-3 (between 1 and 500 nM). PbTx-9 has a reduced binding affinity at site 5, which correlates with potency of toxin (31). Because binding is the essential first step in the onset of toxicity, the results obtained with fluorescent microplate cell assay would better reflect the composite toxicity of an unknown, because more potent forms of toxin bind more tightly to the receptor (31). No method is currently available to definitively confirm the presence or absence of PbTxs in shellfish extracts at these low concentrations. In these specific conditions, the fluorimetric microplate cell assay described here provides an alternative method to detect sodium channel activators. Even though some tests with naturally contaminated seafood must be done to confirm the utility of the method for routine assays. Bioassays (such as mouse, chicken, or mosquito bioassays) have traditionally been used to monitor fish suspected of contamination with ciguatera toxins (45). However, they have disadvantages such as low sensitivity, relatively large amounts of samples required for analysis, lack of specificity to distinguish toxins, and prolonged times required for analysis (46). Recent studies have also focused on the development of chemical methods (LC, HPLC, HPLC-MS, and NMR) for the detection and quantification of ciguatera toxins (7, 47-49). Alternative assays based on immunochemical technology have also been developed (RIA and ELISA formats and solid phase immunobead assay) (50-53). Cell culture techniques were modified to detect sodium channel activating toxins such as CTXs and PbTxs (54, 55). A new cytotoxicity assay was developed to detect CTX based on a reported gene that uses luciferase-catalyzed light generation as an end point and a microplate luminometer for quantification (56). Receptor binding assays were also used to measure CTXs (33). In this case, the binding affinity of each CTX for the sodium channel is proportional to its very low LD50 in mice (ip) indicating that the effect of CTXs likely arises from their action on sodium channels (11, 47). On the basis of that, we verified that the method that we present here is directly applicable to CTXs whose target of toxicological relevance

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is the site 5 of voltage-gated sodium channels (6, 7, 43). This assay has sufficient sensitivity for detection of CTX3C with a limit of quantitation close to 0.01 nM (2 pg/ well). By using fluorimetric microplate assay, we studied pure toxins and in this case the technique seems to be more sensitive than the mouse bioassay and cytotoxicity assays (54, 55). The method described here was not applied to naturally contaminated seafood, and so far, we do not know if there is any interference with the sample matrix components. However, it is important to point out that when we used a method similar to quantify paralytic shellfish toxins in mussels samples, we did not found important interferences (25, 26). On the basis of the studies with pure toxins, the fluorimetric microplate assay has a clear potential to quantitate PbTx and CTX bioactivity in seafood tissues. This assay offers a number of advantages in terms of rapidity and accuracy when compared with previously described cell-based methods. However, the analysis of contaminated samples must be done to ensure the important benefits that this method has in terms of effective sensitivity, reduction of incubation time, and practicality as compared with previous in vitro methodologies. Regarding the speed, a single toxin assessment can be completed in less than 1 h, and then, difficulties occasionally observed in vitro with long incubation periods are minimized. This consequently improves the screening and method ruggedness. Evaluation of toxins in a 96 well microplate format with integral automated fluorescence detection increases the throughput significantly. The interassay precision of the described procedure depends on the concentration and the toxin. Samples containing greater than 0.1 nM of toxin were measured with a coefficient of variation generally lower than 10%. In conclusion, this fluorescent microplate cell assay is specific for sodium channel activating toxins, simple, easy to use and interpret, reproducible and repeatable, and allow us to test a large number of samples in a short time. This assay shows a great promise for use in seafood safety monitoring programs. However, future research must be done to detect sodium channel-activating toxins in shellfish samples.

Acknowledgment. This work was funded with grants MCYT (INIA) CAL01-068, Xunta Galicia (PGIDIT02PXIC26101PN, PGIDT99INN26101, and PGDIDIT03AL26101PR), MCYT BMC2000-0441, SAF2003-08765-C0302, REN2001-2959-C04-03, REN2003-06598-C02-01, and FISS REMA-G03-007.

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