ARTICLE pubs.acs.org/crt
Comparative Cytotoxicity of Gambierol versus Other Marine Neurotoxins E. Cagide,† M. C. Louzao,† B. Espi~na,† I. R. Ares,† M. R. Vieytes,‡ M. Sasaki,§ H. Fuwa,§ C. Tsukano,§ Y. Konno,§ M. Yotsu-Yamashita,|| L. A. Paquette,^ T. Yasumoto,# and L. M. Botana*,† Departamento de Farmacología and ‡Departamento de Fisiología Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain § Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan ^ Evans Chemical Laboratories, The Ohio State University, Columbus, Ohio 43210, United States # Japan Food Research Laboratories, Tama Laboratory, Tokyo 206-0025, Japan
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ABSTRACT: Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine organisms contaminated with those toxins results in seafood poisonings. In many cases, the lack of toxic material turns out to be an obstacle to make the toxicological investigations needed. In this study, we evaluate the cytotoxicity of several marine toxins on neuroblastoma cells, focusing on gambierol and its effect on cytosolic calcium levels. In addition, we compared the effects of this toxin with ciguatoxin, brevetoxin, and gymnocin-A, with which gambierol shares a similar ladder-like backbone, as well as with polycavernoside A analogue 5, a glycosidic macrolide toxin. For this purpose, different fluorescent dyes were used: Fura-2 to monitor variations in cytosolic calcium levels, Alamar Blue to detect cytotoxicity, and Oregon Green 514 Phalloidin to quantify and visualize modifications in the actin cytoskeleton. Data showed that, while gambierol and ciguatoxin were successful in producing a calcium influx in neuroblastoma cells, gymnocin-A was unable to modify this parameter. Nevertheless, none of the toxins induced morphological changes or alterations in the actin assembly. Although polycavernoside A analogue 5 evoked a sharp reduction of the cellular metabolism of neuroblastoma cells, gambierol scarcely reduced it, and ciguatoxin, brevetoxin, and gymnocin-A failed to produce any signs of cytotoxicity. According to this, sharing a similar polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins, whose effects may be being underestimated.
’ INTRODUCTION Gambierol is a ladder-shaped polyether obtained along with ciguatoxin congeners from the marine dinoflagellate Gambierdiscus toxicus.1 This toxin shows a lethal dose against mice about 5080 μg kg1 by intraperitoneal injection and 150 μg kg1 by oral consumption, and the neurological symptoms resemble those shown by ciguatoxins.2 There is little information about the biological effects of gambierol, although the biogenetic origin and pathological effects suggest that gambierol may be also responsible of ciguatera poisoning.2,3 So far, two possible targets have been postulated for gambierol: low concentrations of the toxin inhibit voltage-gated potassium currents;4,5 and high concentrations activate sodium channels leading to a calcium inflow in human neuroblastoma cells.6 Calcium is the most common signal transduction in cells (ranging from bacteria to specialized neurons); therefore, the control of intracellular calcium concentration is a major factor in cellular homeostasis for all cell types. The aim of this study was to gain a deeper understanding regarding the mechanisms responsible for the cytosolic calcium [Ca2þ]i increment evoked by gambierol in the micromolar range. r 2011 American Chemical Society
In addition, the structural complexity and interesting biological properties of polycyclic ether compounds make them some of the most fascinating products found in the marine environment. Ciguatoxin-3C (CTX-3C), a polyether molecule belonging to the ciguatoxins group responsible for the seafood poisoning called ciguatera,7 is produced by the same marine dinoflagellate as gambierol. This poisoning is one of the most widespread seafood poisonings, resulting from the consumption of fish that have accumulated CTXs through the marine food chain.8 CTXs are a group of toxins that bind to site 5 on the voltage-gated sodium channel and alter the channel function. The toxin binding changes the activation voltage for channel opening to a more negative value and inhibits the inactivation of opened channels, resulting in persistent activation.913 Brevetoxins (PbTx) share a similar ladder-shaped backbone with CTXs and produces the toxicity by binding to the same receptor in cells.14,15 Received: January 26, 2011 Published: April 25, 2011 835
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Figure 1. Time courses of the effect of different blockers (first arrow) on the cytosolic calcium increment of neuroblastoma cells induced by 30 μM gambierol (second arrow). Mean ( SEM (n = 3).
Gymnocin-A is a novel polycyclic ether recently isolated from Karenia mikimotoi, a close dinoflagellate to the producer of brevetoxins, Karenia brevis,16 and also structurally related to ciguatoxins, brevetoxins, and gambierol.1722 Though its mechanism of action has not been determined, it showed in vitro cytotoxicity against a murine P388 lymphocytic leukemia cell line (EC50 = 1.3 μM).16 Another new toxin related to two fatal human incidents is polycavernoside A,23,24 which is a glycosidic macrolide first isolated from the red alga Polycavernosa tsudai (Gracilaria edulis).23 Despite its potency and interesting structure, very little is known about it. Here, we use a synthetic analogue, named analogue 5, which maintains the macrocyclic core of the original polycavernoside A.25 On the basis of the similar structure of these compounds, in this work we tried to compare the effects evoked by these toxins; therefore, the polycyclic backbone could confer any characteristics that could be related to the ligandreceptor interaction.
’ EXPERIMENTAL PROCEDURES Materials. The group of Dr. M. Sasaki generously provided the synthetic gambierol26,27 and gymnocin-A16,17,19,21 and Dr. T. Yasumoto the CTX3C and brevetoxins. The synthetic analogue of polycavernoside A, compound 5, was synthesized by Dr. Paquette.25 Ham’s F12 supplemented with glutamine, Eagle’s minimum essential medium (EMEM), and nonessential amino acids were purchased from Biochrom AG (Berlin, Germany). Fetal bovine serum (FBS) for neuroblastoma cell culture, trypsin-EDTA, nutrient mixture F-12 Ham Kaighn’s modification, streptomycin sulfate salt, penicillin G potassium salt, bovine serum albumine (BSA), gentamycin, amphotericin B, and veratridine were from Sigma (Madrid, Spain). Fluorescent dyes bis-(1,3-dibutylbarbituric acid) trimethine oxonol (bis-oxonol) and fura-2 acetoxymethyl ester (fura-2) were purchased from Molecular Probes (Leiden, The Netherlands). Alamar Blue (AB) was from Biosource International (Nivelles, Belgium). All other chemicals were of reagent grade and purchased from Sigma (Madrid, Spain). 836
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Figure 2. Images recorded with confocal microscopy showing F-actin disposition in the control (A) or in cells treated with 30 μM gambierol for 4 h (B). Transmission images showing the morphology of the same cells in each case are displayed in the file below (C, D). Long-term effect of 30 μM gambierol on the metabolic activity of neuroblastoma cells (E). Mean ( SEM (n = 3). *P < 0.05. Mean ( SEM of 3 experiments. Twenty-five square centimeter tissue culture flasks for neuroblastoma cell culture and 60 mm tissue culture plates for hepatocytes culture were from Nunc (Roskilde, Denmark), and 96 well plates were from Corning (Schiphol-Rijk, The Netherlands). Experimental standard salt solution used for the microscope experiments contained (in mmol/L) the following: NaCl 137, KCl 5, CaCl2 1, glucose 10, MgCl2 1.2, KH2PO4 0.44, and NaHCO3 4.2, pH 7.4. Cell Line Culture. The cells used for experimentation were the excitable human neuroblastoma cell line BE(2)-M17 (ATCC Number CRL-2267), cultivated in EMEM/Ham’s F12 supplemented with glutamine, nonessential amino acids, gentamicine, amphotericin B/ and fetal bovine serum as previously described.6,2832 Neuroblastoma cells were grown in 25 cm2 tissue culture flasks in a humidified atmosphere containing 5% CO2 at 37 C and were subcultured by transferring cells released by the application of trypsinEDTA. Cells destined for the fluorescence microscope assays were seeded on 22 mm glass coverslips at a density of 1.(55) 104 cells/ well and used after 47 days. When the metabolic activity assay was performed, neuroblastoma cells were seeded on 96 well microplates at a density of 5 103 cells/well and were cultured for 24 h before treatment in order to allow them to attach to the bottom of the microplate. Measurement of Plasma Membrane Potential. Plasma membrane potential was monitored with the slow potential-sensitive fluorescent dye bis-oxonol as was previously established for neuroblastoma cells.6 Cells were incubated in a thermostatted chamber with the standard salt solution containing 5 nM bis-oxonol for 10 min at 37 C. Fluorescence recordings were performed at the wavelengths 490 nm (excitation) and 530 nm (emission) with a Nikon Diaphot microscope with epifluorescence optics (Nikon 40x immersion fluor objective). Results are expressed as relative fluorescence. [Ca2þ]i Measurements. Neuroblastoma cells were incubated for 10 min at 37 C in 2 mL of standard salt solution with 0.1% BSA containing 0.5 μM fura-2. After dye loading, the cells were washed three times to remove BSA, and the glass coverslips were inserted into the thermostatted chamber at 37 C. Calcium measurements were
made with the same microscope described above. Fluorescent images were collected by dual excitation wavelengths at 340 and 380 nm, and a wavelength emission at 530 nm with Lambda 102 Sutter Instrument Co., equipment. Data are expressed as intracellular calcium concentration ([Ca2þ]i), calculated by using the method of Grynkiewicz et al.33 Cytoskeleton Labeling. Cells were preincubated with each toxin for 4 h. After this time, they were fixed in 4% formaldehyde solution and permeabilized using Triton X-100. Fluorescent filamentous actin (F-actin) staining with Oregon Green 514 Phalloidin was performed as described in Ares et al.34 The samples mounted on slides were analized with a laser-scanning cytometer (LSC; CompuCyte, Cambridge, MA) for measuring the F-actin quantity. The maximum green pixel was the fluorescent parameter analyzed with this technique. Imaging of F-actin distribution as well as cytomorphology were captured through the C1 laser confocal system (Nikon Instruments Europe B.V., The Netherlands). An argon laser light source (at 488 nm) was utilized for exciting the fluorescent phalloidin with both detection methods. Metabolic Activity Assay. Cellular metabolism was quantified by using the AlamarBlue (AB) bioassay previously set up for those cells.28,30 In normal conditions the oxidized blue nonfluorescent form of this dye is reduced into the cells to a pink fluorescent form, varying proportionately with cell number and time.35 AB fluorescence was monitored spectrophotometrically at 530 nm excitation and 590 nm emission wavelengths, using a FL600 fluorescence plate reader (Bio-Tek, Vermont, U.S.A.). Cells were seeded in 96 well microplates at a density of 5000 cells/well in 200 μL of culture medium. After 24 h of cell attachment, cells were treated with the toxins, and AB was added (1:10 v/v). The results are expressed as a percentage of the mean fluorescence value versus control, considered as the 100% viability, ( SEM. Three replicate wells were used for each control and concentrations of toxin tested per microplate. Statistical Analysis. All the experiments were carried out at least three times. Results were analyzed using the Student’s t test for paired data where appropriate. A probability level of 0.05 or smaller was used for statistical significance. Results are expressed as the mean ( SEM. 837
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Figure 3. F-actin disposition in the control (A, G) or in cells treated with PbTx-3 (B), PbTx-9 (C), and CTX-3C (H) for 4 h. The morphology of the same cells in each case are displayed in the file below by the transmission images (D, E, F, I, and J). Long-term effect of 250 nM brevetoxin-3 (K) and 25 nM CTX-3C (L) on the metabolic activity of neuroblastoma cells. Mean ( SEM (n = 3).
’ RESULTS Gambierol is a polycyclic ether toxin isolated from Gambierdiscus toxicus, the same marine dinoflagellate causative of ciguatera fish poisoning.1 We have previously reported that high concentrations of this toxin activate sodium channels in human neuroblastoma cells, and as a consequence, gambierol induces a cytosolic calcium increment.6 Since there was shown the importance of voltage-gated calcium channels and the Naþ-Ca2þ exchanger in the gambierolstimulated Ca2þ influx, we deeply investigated mechanisms responsible for the elevation of intracellular calcium in neuroblastoma cells by using the fluorescent dye fura-2
First, we incubated the cells during nearly 10 min with the preferential T-type Ca2þ channel blocker mibefradil (5 μM),3639 and then, 30 μM gambierol was added. As can be observed in Figure 1A, treatment with this blocker resulted in a decrease of the gambierol-evoked calcium entry. Since L-type Ca2þ channels seemed to be involved in the cytosolic calcium increase triggered by gambierol,6 we combined 5 μM mibefradil and 20 μM nifedipine to detect the influence of T- and L-type voltage-gated calcium channels on the effect produced by the toxin, but the reduction of the gambierol evoked calcium increment was not greater when the cells were incubated with both blockers than with mibefradil alone (Figure 1B). 838
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and the morphology (Figure 2C and D) of neuroblastoma cells, but no effect was observed in both parameters when gambierol was added to the cells for 4 h. In addition, ion fluxes may be related to cell death; therefore, we measured the cellular metabolism of BE(2)-M17 cells with Alamar Blue. Figure 2E shows a small but significant reduction in the metabolism of the cells starting at 36 h of incubation with this toxin. PbTx-3, PbTx-9, and CTX-3C are toxins known to bind to site 5 of voltage-gated sodium channels that trigger a sodium overload, though a depolarization of the cells, as well as cytosolic calcium increase, occurs. We had previously confirmed that 25 nM PbTx-3, 25 nM PbTx-9, and 25 nM CTX-3C depolarize neuroblastoma cells.6,32 Since those effects resemble those of gambierol, we compared the action of these toxins on the cytoskeleton. Similar to gambierol, the profiles revealed by the fluorescence of the Oregon Green 514 phalloidin bound to F-actin in control cells (Figure 3A and G) and those one incubated with PbTx-3 (Figure 3B), PbTx-9 (Figure 3C), and CTX-3C (Figure 3H) showed no differences. In all these cases, the distribution pattern of the fluorescence in the treated cell was almost the same as the control ones, indicating no effect on the F-actin level. Furthermore, control cells visualized with confocal microscopy presented a well-defined actin cytoskeleton and spread morphology. After exposition to CTX-3C or brevetoxin, neuroblastoma cells did not show evidence of alteration either in their F-actin organization or in their morphological pattern (Figure 3DF and I and J). Moreover, the cells did not show any variations in their metabolic activity even when incubated for 72 h with the toxins (Figure 3K and L). On the basis of the similar structure, we also checked the effect of gymnocin-A. In order to detect any changes of the membrane potential of neuroblastoma cells caused by this toxin, we applied increasing concentrations until 13 μM was reached and used the potential-sensitive fluorescent dye bis-oxonol. We found that gymnocin-A did not change the fluorescence and hence the membrane potential of the neuroblastoma cells (Figure 4A). Simultaneously, we studied the effect on the basal state of intracellular calcium concentration ([Ca2þ]i) of neuroblastoma cells. As shown in Figure 4B, the intracellular calcium concentration remained stable upon the addition of the concentration range of gymnocin-A. In addition, confocal images revealed an intact F-actin system in untreated neuroblastoma cells (Figure 5A) and also in those exposed to gymnocin-A (Figure 5B). Likewise, cells without (Figure 5C) or with toxin (Figure 5D) do not display apparent differences in their morphological characteristics as is revealed in transmission pictures when the cells were treated with gymnocin-A for 4 h. Even when we registered the metabolic activity with Alamar Blue for a time as long as 72 h, Figure 5E shows that gymnocin-A induces no effect on the growth of neuroblastoma cells. Another toxin that induces a cytosolic calcium increase and a depolarization of neuroblastoma cells is polycavernoside A analogue 5,29 though its structure is quite different from that of the previous ones. Since almost nothing is known about its mechanism of action, we tested its effect on the metabolic rate of neuroblastoma cells. Figure 6 shows that 12 μM polycavernoside A analogue 5 produced a significantly high reduction in metabolic activity, as opposed to that of the other toxins tested.
Figure 4. Time courses of the effect of several concentrations of gymnocin-A (arrow) in human neuroblastoma cells. (A) Changes in neuroblastoma plasma membrane potential monitored with the fluorescent dye bis-oxonol. (B) Intracellular calcium levels registered in furaloaded neuroblastoma cells. Mean ( SEM (n = 3).
Our previous study pointed out that gambierol was inducing the NaþCa2þ exchanger to act in reverse mode and thus contributing to the maintenance of the calcium accumulation stimulated by gambierol; therefore, we considered this calcium entry pathway by incubating the cells with 0.5 μM 20 ,40 -dichlorobenzamil, a NaþCa2þ exchanger inhibitor, in addition to 5 μM mibefradil. As can be seen in Figure 1C, treatment of the cells with both blockers produced a greater reduction of the gambierol-induced increment in [Ca2þ]i. Furthermore, combination of the T- and L-type voltagegated calcium channel blockers mibefradil (5 μM) and nifedipine (20 μM) along with the NaþCa2þ exchanger inhibitor 0.5 μM 20 ,40 -dichlorobenzamil (Figure 1D) was equally effective in reducing Ca2þ influx stimulated by gambierol in human neuroblastoma cells. In order to confirm the influence of the NaþCa2þ exchanger, we tested the effect of the NaþCa2þ exchanger (reverse mode) inhibitor KB-R7943 (5 μM)4043 on the [Ca2þ]i increase induced by 30 μM gambierol since in order to restore the altered [Naþ]i homeostasis, the NaþCa2þ exchanger can function to cause Ca2þ accumulation through a reversed mode of operation (Naþ efflux with Ca2þ influx).40,44,45 Figure 1E shows that there is a significant decrease in the calcium inflow with this inhibitor, though it was not completely abolished. Since an ionic disorder has been related with changes in the volume of the cells and in the cytoskeleton structure,46,47 we checked the gambierol effect on the F-actin system (Figure 2A and B)
’ DISCUSSION Besides being important primary producers, and therefore an important part of the food chain, dinoflagellates are also known 839
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Figure 5. Images recorded with confocal microscopy showing F-actin disposition in the control (A) or in cells treated with gymnocin-A (B) for 4 h. The morphology of the same cells in each case are displayed in the file below by the transmission images (C, D). Long-term effect of gymnocin-A (E) on the metabolic activity of neuroblastoma cells. Mean ( SEM (n = 3).
for producing toxins, particularly when they occur in large numbers, called algal blooms. These marine toxins have drawn wide interest because of their economical impact and disastrous effect upon the fisheries and shellfish industry as well as public health in many parts of the world. One of the most interesting groups of marine toxins, from structural and pharmacological points of view, are polyether compounds, which are characterized by a continuous array of trans-fused O-heterocyclic rings. Gambierol is a new polyether compound, which has been reported to induce a cytosolic calcium increase.6 Deepening our understanding of this activity in human BE(2)-M17 neuroblastoma cells, we observed that preincubation of cells mibefradil reduced gambierol-induced calcium influx, indicating that T-type voltage-gated calcium channels are also involved in this action and not only L- or N-type Ca2þ channels,6 though combining mibefradil and nifedipine does not produce an additive response. On the contrary, combining mibefradil with the NaþCa2þ exchanger inhibitor 20 ,40 -dichlorobenzamil resulted in an important reduction of the gambierol-induced increment in [Ca2þ]i, and combination of the T- and L-type voltage-gated calcium channel blockers mibefradil and nifedipine along with this NaþCa2þ exchanger inhibitor also resulted in a complete abolishment of the Ca2þ influx stimulated by gambierol in human neuroblastoma cells. The great importance of the NaþCa2þ exchanger was confirmed by using KB-R7943 as a specific inhibitor of the NaþCa2þ exchanger in reverse mode. These data corroborate the hypothesis that gambierol induces a sodium current acting as a partial agonist of sodium channels6,48 and as a consequence a calcium influx due to the NaþCa2þ exchanger working in reverse mode and to voltage-gated calcium channels. We tried to compare the effects produced by gambierol with other polyether compounds. These molecules generally present a great diversity in size, with widely varying functional groups and different toxicological and chemical characteristics, and potent
Figure 6. Effect of polycavernoside A analogue 5 on the metabolic activity of neuroblastoma cells. Mean ( SEM (n = 3).
biological activities. In fact, gambierol, CTX-3C, and PbTx-3 and -9 have been reported to be successful in producing a membrane depolarization and a subsequent calcium influx, though here we show that gymnocin-A was not. Recently, it has been hypothesized that gambierol binds to a new site in voltage-gated potassium channels that could interfere with sodium channels.49 Although we do not detect the same effect with gambierol as with gymnocin-A at the concentrations tested, taking into account their similarities in structure, it would be very interesting to check if they share a common behavior. Since cell volume is closely dependent on [Ca2þ]i and the ions fluxes,50 we tested the effect of gambierol on the morphology and F-actin cytoskeleton of neuroblastoma cells, but they remained 840
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Chemical Research in Toxicology with a spread morphology and a well-defined actin cytoskeleton, without showing evidence of alteration either in their morphology pattern or in their actin cytoskeleton. This lack of effect is the same as the result of treating the cells with ciguatoxin as well as with brevetoxins and gymnocin-A. Interestingly, the cells showed a slight reduction in the metabolism of the cells starting at 36 h of incubation with gambierol, while the metabolic rate showed no decrease when incubating with CTX-3C, PbTx-3, or gymnocin-A. A detection assay based on the cytotoxicity induced by the ionic imbalance generated by ciguatoxins in neuroblastoma cells is usually employed,51 but veratridine and oubain are used to detect cytotoxicity. Since the ionic imbalance induced by these toxins by themselves is not high enough to produce cell death, probably it is also not high enough to evoke a change in cell volume, or morphology of the cells, at least at the incubation times tested. However, polycavernoside A analogue 5, which is an example of a new glycosidic macrolide isolated from the red alga Gracilaria edulis and related to two fatal human incidents has a very different structure compared to that of the previous ones.23,24 Although this is a toxin with a still unknown target,29 it produces a sharp reduction in the metabolic activity of neuroblastoma cells, indicating that polycavernoside A analogue 5 would be a potent cytotoxin, which could be indicative of the effect produced by the natural polycavernoside A. Therefore, while gambierol, ciguatoxin, and brevetoxins were successful in producing depolarization, as well as a calcium influx, gymnocin-A was unable to modify any of those parameters in neuroblastoma cells. In addition, the actin cytoskeleton seems not to be an early target for any of these toxins since none of them induced morphological changes or alterations in the actin assembly, though gambierol seemed to induce a reduction in viability of neuroblastoma cells at long time periods, and polycavernoside A analogue 5 turned out to be a potent cytotoxin. According to this, sharing the same polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins to understand the possible implications that these toxins may have in intoxications, especially in situations where they can coexist, since they may occur in similar areas.
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’ REFERENCES (1) Satake, M., Murata, M., and Yasumoto, T. (1993) Gambierol: a new toxic polyether compound isolated from the marine dinoflagellate Gambierdiscus toxicus. J. Am. Chem. Soc. 115, 361–362. (2) Ito, E., Suzuki-Toyota, F., Toshimori, K., Fuwa, H., Tachibana, K., Satake, M., and Sasaki, M. (2003) Pathological effects on mice by gambierol, possibly one of the ciguatera toxins. Toxicon 42, 733–740. (3) Fuwa, H., Kainuma, N., Tachibana, K., Tsukano, C., Satake, M., and Sasaki, M. (2004) Diverted total synthesis and biological evaluation of gambierol analogues: elucidation of crucial structural elements for potent toxicity. Chem.—Eur. J. 10, 4894–4909. (4) Cuypers, E., Abdel-Mottaleb, Y., Kopljar, I., Rainier, J. D., Raes, A. L., Snyders, D. J., and Tytgat, J. (2008) Gambierol, a toxin produced by the dinoflagellate Gambierdiscus toxicus, is a potent blocker of voltage-gated potassium channels. Toxicon 51, 974–983. (5) Ghiaroni, V., Sasaki, M., Fuwa, H., Rossini, G. P., Scalera, G., Yasumoto, T., Pietra, P., and Bigiani, A. (2005) Inhibition of voltagegated potassium currents by gambierol in mouse taste cells. Toxicol. Sci. 85, 657–665. (6) Louzao, M. C., Cagide, E., Vieytes, M. R., Sasaki, M., Fuwa, H., Yasumoto, T., and Botana, L. M. (2006) The sodium channel of human excitable cells is a target for gambierol. Cell Physiol. Biochem. 17, 257–268. (7) Lewis, R. J., and Holmes, M. J. (1993) Origin and transfer of toxins involved in ciguatera. Comp. Biochem. Physiol., Part C: Comp. Pharmacol. 106, 615–628. (8) Yasumoto, T. (2005) Chemistry, etiology and food chain dynamics of marine toxins. Proc. Jpn. Acad. 81(B), 43–51. (9) Baden, D. G., and Mende, T. J. (1982) Toxicity of two toxins from the Florida red tide marine dinoflagellate, Ptychodiscus brevis. Toxicon 20, 457–461. (10) Catterall, W. A., and Gainer, M. (1985) Interaction of brevetoxin A with a new receptor site on the sodium channel. Toxicon 23, 497–504. (11) Gallagher, J. P., and Shinnick-Gallagher, P. (1985) Effects of crude brevetoxin on membrane potential and spontaneous or evoked end-plate potentials in rat hemidiaphragm. Toxicon 23, 489–496. (12) Poli, M. A., Mende, T. J., and Baden, D. G. (1986) Brevetoxins, unique activators of voltage-sensitive sodium channels, bind to specific sites in rat brain synaptosomes. Mol. Pharmacol. 30, 129–135. (13) Louzao, M. C., Vieytes, M. R., Yasumoto, T., and Botana, L. M. (2004) Detection of sodium channel activators by a rapid fluorimetric microplate assay. Chem. Res. Toxicol. 17, 572–578. (14) Lombet, A., Bidard, J. N., and Lazdunski, M. (1987) Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltagedependent Naþ channel. FEBS Lett. 219, 355–359. (15) Gawley, R. E., Rein, K. S., Kinoshita, M., and Baden, D. G. (1992) Binding of brevetoxins and ciguatoxin to the voltage-sensitive sodium channel and conformational analysis of brevetoxin B. Toxicon 30, 780–785. (16) Sasaki, M., Tsukano, C., and Tachibana, K. (2002) Studies toward the total synthesis of gymnocin A, a cytotoxic polyether: a highly convergent entry to the F-N ring fragment. Org. Lett. 4, 1747–1750. (17) Tsukano, C., and Sasaki, M. (2003) Total synthesis of gymnocin-A. J. Am. Chem. Soc. 125, 14294–14295. (18) Tanaka, K., Itagaki, Y., Satake, M., Naoki, H., Yasumoto, T., Nakanishi, K., and Berova, N. (2005) Three challenges toward the assignment of absolute configuration of gymnocin-B. J. Am. Chem. Soc. 127, 9561–9570. (19) Tsukano, C., Ebine, M., and Sasaki, M. (2005) Convergent total synthesis of gymnocin-A and evaluation of synthetic analogues. J. Am. Chem. Soc. 127, 4326–4335. (20) Simpson, G. L., Heffron, T. P., Merino, E., and Jamison, T. F. (2006) Ladder polyether synthesis via epoxide-opening cascades using a disappearing directing group. J. Am. Chem. Soc. 128, 1056–1057. (21) Sasaki, M., and Fuwa, H. (2008) Convergent strategies for the total synthesis of polycyclic ether marine metabolites. Nat. Prod. Rep. 25, 401–426.
’ AUTHOR INFORMATION Corresponding Author
*Tel: 34 982 822 233. Fax: 34 982 252 242. E-mail: luis.botana@ usc.es. Funding Sources
This work was funded by the following FEDER cofounded-grants: Contract grant sponsor, EU VIIth Frame Program; contract grant numbers, 211326-CP (CONffIDENCE), 265896 BAMMBO, 265409 μAQUA, and 262649 BEADS. Contract grant sponsor: the Atlantic Area Programme (Interreg IVB Trans-national); contract grant numbers, 2008-1/003 (Atlantox) and 2009-1/117 (Pharmatlantic). Contract grant sponsor, Ministerio de Ciencia y Tecnología, Spain; contract grant numbers, REN2003-06598-C02-01, AGL200760946/ALI, SAF2009-12581 (subprogram NEF), AGL200913581-CO2-01, TRA2009-0189, and AGL2010-17875. Contract grant sponsor, Xunta de Galicia, Spain; contract grant numbers, GRC 2010/10 and PGIDT07CSA012261PR, PGDIT 07MMA006261PR, PGIDIT (INCITE) 09MMA003261PR, 2009/053, and 2009/XA044 (Consell. Educacion), 2008/CP389 (EPITOX, Consell. Innovacion e Industria, programa IN.CI.TE.), and 10PXIB261254 PR. 841
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Chemical Research in Toxicology
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dx.doi.org/10.1021/tx200038j |Chem. Res. Toxicol. 2011, 24, 835–842
