Hapalindoles from the Cyanobacterium ... - American Chemical Society

Article pubs.acs.org/crt

Hapalindoles from the Cyanobacterium Fischerella: Potential Sodium Channel Modulators Eva Cagide,† Paul G. Becher,‡,∥ M. Carmen Louzao,† Begoña Espiña,†,⊥ Mercedes R. Vieytes,§ Friedrich Jüttner,‡ and Luis M. Botana*,† †

Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain Institute of Plant Biology, Limnological Station, University of Zürich, 8802 Kilchberg, Switzerland § Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain ‡

ABSTRACT: Hapalindoles make up a large group of bioactive metabolites of the cyanobacterial order Stigonematales. 12-epi-Hapalindole E isonitrile, 12-epi-hapalindole C isonitrile, 12-epi-hapalindole J isonitrile, and hapalindole L from Fischerella are acutely toxic for insect larvae; however, the biochemical targets responsible for the biological activities of hapalindoles are not understood. We describe here the electron impact mass spectra of these four hapalindole congeners; their structures were confirmed by nuclear magnetic resonance spectroscopy. In combination with the presented mass spectra of 15N-labeled species and their retention times on a gas chromatography capillary column, a rapid and reliable determination should be possible in future research. The bioactivity of these hapalindoles was tested on mammalian cells focusing on their effects in the BE(2)-M17 excitable human neuroblastoma cell line. The fluorescent dye Alamar Blue was applied to monitor cytotoxicity, fura-2 to evaluate changes in the cytosolic calcium concentrations, and bis-oxonol to detect effects on membrane potential. Data showed that the hapalindoles did not affect cell viability of the neuroblastoma cells, even when they were incubated for 72 h. Neither depolarization nor initiation of calcium influx was observed in the cells upon hapalindole treatment. However, the data provide evidence that hapalindoles are sodium channel-modulating neurotoxins. They inhibited veratridine-induced depolarization in a manner similar to that of neosaxitoxin. Our data suggest hapalindoles should be added to the growing number of neurotoxic secondary metabolites, such as saxitoxins and anatoxins, already known in freshwater cyanobacteria. As stable congeners, hapalindoles may be a risk in freshwater ecosystems or agricultural water usage and should therefore be considered in water quality assessment.

■

INTRODUCTION Cyanobacterial compounds have been extensively studied since the early 1980s as potential resources of novel pharmacological compounds. Large-scale screening programs were conducted to find cyanobacterial metabolites as leads to new pharmaceutical products. In a screening program, which was initiated to find new anticancer compounds, approximately 1000 cyanobacterial strains were tested.1 Epilithic, edaphic freshwater and marine cyanobacteria turned out to be equally promising sources of new bioactive compounds. As a consequence of these efforts, a large number of new bioactive compounds with potential pharmacological significance have been isolated from cyanobacteria.2−4 Indole derivatives are the prominent group of bioactive compounds to be found in Fischerella and other strains of the Stigonematales,5−9 with hapalindoles showing toxic effects to different cell lines and organisms.10−13 However, besides a moderate RNA polymerase inhibitory activity found for 12-epihapalindole E isonitrile,14,15 physiological effects and biochemical targets have been poorly studied. 12-epi-Hapalindole J isonitrile (1), 12-epi-hapalindole C isonitrile (2), hapalindole L © XXXX American Chemical Society

(3), and 12-epi-hapalindole E isonitrile (4) from a HPLC fraction of Fischerella ATCC 43239 were shown to possess acute insecticidal activity.16 Modulation of neurophysiological processes seems to be a conceivable mechanism for this toxic effect. Inhibition of cholinesterase is a common mechanism of insecticides; however, no inhibitory activity was found for the hapalindole-containing HPLC fraction of Fischerella when it was tested on butyrylcholinesterase.17 Ion channels can also be envisaged as primary target sites. For several natural and synthetic insecticidal compounds,18−20 voltage-sensitive sodium channels are the major receptor sites.19,21−24 Sodium channels mediate the rising phase of the action potential in excitable cells, and many natural toxins exert their effect by altering this property.25−29 The paralytic shellfish toxins, such as saxitoxin (STX) and neosaxitoxin (NeoSTX), make up a group of phycotoxins produced by marine dinoflagellates and freshwater cyanobacteria.30,31 This group of toxins acts by binding to site 1 Received: May 12, 2014

A

dx.doi.org/10.1021/tx500188a | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

Table 1. Physicochemical and Chromatographic Data of Hapalindolesa GC tR (min)c compound 12-epi-hapalindole C isonitrile 12-epi-hapalindole J isonitrile 12-epi-hapalindole E isonitrile hapalindole L

absorbance λmax (nm)

emission λmax (nm)

HPLC tR (min)

DB-1301

DB-1

M+ (% intensity)

222, 282

340

9.8

9.0

6.7

304 (17)

221, 281

339

10.3

10.9

9.3

304 (52)

224, 278

339

11.2

11.4

10.5

338 (12)

220, 281

340

11.7

14.5

15.2

338 (17)

b

fragment ions (% intensity) 130 (100), 168 131 (25) 289 (100), 168 181 (35) 168 (100), 130 167 (45) 168 (100), 167 169 (17)

(67), 167 (27), (71), 167 (35), (100), 182 (64), (22), 303 (20),

a Absorption maxima in methanol, fluorescence emission maxima (excitation λ = 277 nm; MeOH), retention times (for HPLC on a CN column and GC on a DB-1301 and DB-1 capillary column), and EI molecular and fragment ions of the hapalindoles. bDetails of the CN-HPLC separation are given in ref 16. cThe GC temperature program was 200 °C for 1 min and an increase at a rate of 10 °C/min to 270 °C over 12 min for the separations on the DB-1301 column and a 25 min at 235 °C isotherm for the separations on the DB-1 column.

mmol/L CaCl2, 10 mmol/L glucose, 1.2 mmol/L MgCl2, 0.44 mmol/ L KH2PO4, and 4.2 mmol/L NaHCO3 (adjusted to pH 7.4). Growth and Extraction of Fischerella sp. Fischerella ATCC 43239 was cultivated on a mineral growth medium, the biomass extracted, and the hapalindole fraction separated by HPLC as reported previously.16 The cyanobacterial biomass was labeled with 15N by replacing the sole nitrogen source ([14N]nitrate) of the mineral growth medium36 with highly enriched 15N-labeled nitrate (98 at. %, Cambridge Isotope Laboratories, Inc., Andover, MA). Gas Chromatography and Electron Impact Mass Spectrometry. The retention times and EI (electron impact) fragmentation patterns of the hapalindoles were analyzed on a gas chromatography− mass spectrometry system (GC−MS; Fisons Instruments, GC 8000 Top, MD 800). Capillary columns [DB-1301, 30 m, 0.32 mm inside diameter, film thickness of 0.25 μm; DB-1, 30 m, 0.32 mm inside diameter, film thickness of 0.25 μm (J&W Scientific, Folsom, CA)] supplied with helium as the carrier gas (pressure of 50 kPa, split ratio of 1:15) were used for GC separations. The GC temperature programs were 200 °C for 1 min and an increase at a rate of 10 °C/min to 270 °C over 12 min for the separations on the DB-1301 column and a 25 min at 235 °C isotherm for the separations on the DB-1 column. The temperature of the injection port was 270 °C. The energy for EI ionization was 70 eV, and the detector voltage was 300 or 350 V. Mass spectra were recorded in the range of m/z 63−423. The ratio of the different hapalindole isomers in the mixture was determined by GC−MS. The concentrations of different hapalindole isomers were determined by UV spectrometry by applying a specific molar extinction coefficient of 5000.37 12-epi-Hapalindole C isonitrile, 12-epi-hapalindole E isonitrile, and hapalindole L were present in the mixture at concentrations of 19, 9.4, and 9.6 nmol/μL, respectively. 12-epi-Hapalindole J isonitrile was present at lower concentrations. Cell Line Culture. Human neuroblastoma cell line BE(2)-M17 (ATCC CRL-2267) was cultured as previously described.38,39 For hepatotoxicity studies, rat hepatocytes (cell line Clone-9, ECACC 88072203) were used as nonexcitable model cells; they were cultured as reported by Louzao and co-workers.40 For the fluorescence microscopy assays, neuroblastoma cells were seeded on 22 mm glass coverslips placed in eight-well sterile plates at a density of 1.5−5 × 104 cells/well and used after 4−7 days. For metabolic activity assays, neuroblastoma cells and hepatocytes were seeded on 96-well microplates at a density of 5 × 103 cells/well and cultured for 24 h before being treated to allow them to attach to the bottom of the microplate. Metabolic Activity Assay. Cell viability was quantified by using the AB bioassay previously set up for those cells.41 Under normal conditions, the oxidized blue nonfluorescent form of this dye is reduced when taken up by the cells to a pink fluorescent form. The amount of fluorescence is dependent on the cell number and incubation time.42−47 AB fluorescence was monitored spectrophotometrically at an emission wavelength of 590 nm (excitation wavelength of 530 nm) using an FL600 fluorescence plate reader (Bio-Tek,

of the voltage-gated sodium channel with high affinity, blocking the influx of sodium ions.31−34 On the basis of the acute insecticidal activity generated by the hapalindoles from Fischerella, we studied in more detail the action of these compounds on human neuroblastoma cells first by applying the HPLC fraction of Fischerella containing a mixture of the four identified hapalindoles. Later more detailed experiments were conducted with purified hapalindoles of this mixture. Here we report the changes in membrane potential and intracellular calcium caused by the hapalindoles in human neuroblastoma cells, providing for the first time the relationship between these compounds and their possible target on mammalian cells, the sodium channels. Because the constituents of the bioactive HPLC fraction, hapalindoles 1−4, are difficult to distinguish, we provide for these four congeners the EI mass spectra of the normal and 15 N-labeled species and a separation by GC on a capillary column to allow easy and reliable determination. The identity of these components was verified by NMR spectrometry. Understanding the responsible mechanisms behind the proven insecticidal activity is of interest and potential value for pharmacology and plant protection. Furthermore, the mode of activity in combination with tools for chemical identification is important for the assessment of the potential health hazards caused by freshwater cyanobacteria.

■

EXPERIMENTAL PROCEDURES

Materials. Ham’s F-12 medium supplemented with glutamine, Eagle’s minimum essential medium, and nonessential amino acids were purchased from Biochrom AG (Berlin, Germany). Fetal bovine serum for the neuroblastoma cell culture, trypsin-EDTA, nutrient mixture F12 Ham Kaighn’s modification, streptomycin sulfate, penicillin G potassium salt, bovine serum albumin (BSA), gentamycin, amphotericin B, and veratridine were from Sigma (Madrid, Spain). Fetal bovine serum for hepatocyte cultures was from Gibco (Barcelona, Spain). The 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 reagent grade and purchased from Sigma. Neosaxitoxin (NeoSTX) was obtained in our laboratory as previously described.35 Eight-well plates, tissue culture flasks for neuroblastoma cell cultures (25 cm2), and tissue culture plates (60 mm in diameter) for hepatocyte cultures were from Nunc (Roskilde, Denmark) and 96-well plates from Corning (Schiphol-Rijk, The Netherlands). The experimental standard salt solution used for the experiments under the microscope contained 137 mmol/L NaCl, 5 mmol/L KCl, 1 B



Article

Figure 1. EI-MS spectra of four major hapalindoles (GC−MS) present in the hapalindole mixture.

■

Winooski, VT). Cells were seeded on 96-well microplates at a density of 5 × 103 cells/well in 200 μL of culture medium. After attachment for 24 h, cells were treated with the extracts and the AB solution was added [1:10 (v/v)]. The results are expressed as the percentage of the fluorescence versus the control [means ± the standard error of the mean (SEM)]. Three replicate wells were used for each concentration of hapalindoles and the control. Measurement of Plasma Membrane Potential. The plasma membrane potential was monitored with the slow potential-sensitive fluorescent dye bis-oxonol as previously established for neuroblastoma cells.38,39 Cells were incubated in a thermostated chamber with the standard salt solution containing 5 nM bis-oxonol for 10 min at 37 °C. Fluorescence recordings were performed with a Nikon Diaphot microscope with epifluorescence optics (40× immersion fluor objective, Nikon) at an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Results are expressed as relative fluorescence.48 [Ca2+]i Measurements. Neuroblastoma cells were incubated for 10 min at 37 °C in 2 mL of the standard salt solution with 0.1% BSA containing 0.5 μM fura-2. The cells were washed three times to remove BSA and placed into a thermostated chamber at 37 °C. Measurements of the intracellular calcium concentration were made with the same microscope as described above. Fluorescence images were collected at dual-excitation wavelengths of 340 and 380 nm and an emission wavelength at 530 nm on a Lambda 10-2 Sutter Instrument Co. apparatus.38,39 Data are expressed as intracellular calcium concentrations calculated by using the method of Grynkiewicz and co-workers.49

RESULTS

The hapalindole fraction isolated from Fischerella was separated by HPLC and analyzed by GC−MS. The elution sequence of the four hapalindoles that have been identified previously by NMR spectrometry16 was identical for HPLC and GC (Table 1). Both chlorinated compounds 4 and 3 eluted after unchlorinated 2 and 1. The EI mass spectra of the hapalindoles exhibited molecular ions of medium intensity. The patterns and intensities of the fragment ions were sufficiently different to allow reliable differentiation of all four hapalindoles (Figure 1). The fragment ions of 3 corresponded to the major fragments described for fischerindole L isonitrile.50 Identical mass fragments for both compounds were as follows: 338/340 (C 21 H2335ClN 2/C21H 23 37ClN2 ), 323/325 (C 20 H2035ClN 2 / C20H2037ClN2), 303 (C21H23N2), 183 (C13H13N). The mass spectrum of chlorinated 4 also showed the fragment ion at m/z 303 that can be explained by the loss of hydrochloride. 2 and its chlorinated derivative, 4, showed further prominent ions at m/z 130, 117, and 221. The ion at m/z 117 may indicate the fragmentation of the indole moiety from these tricyclic hapalindoles. The fragmentation of the two hapalindoles was in agreement with the fragment ions (m/z 182, 168, 130) previously described for 237 and 4 (m/z 168, 130).14 All four hapalindoles shared fragment ions at m/z 168, 182, and 194. C



Article

Figure 2. EI-MS spectra of the 15N-labeled major hapalindoles present in the hapalindole mixture.

Figure 3. GC−MS chromatogram (abundance of total ions) of a crude extract from Fischerella ATCC 43239 on a DB-1301 capillary column. The compounds eluting at 9.00 (12-epi-hapalindole C isonitrile), 9.49, 10.34, 10.86 (12-epi-hapalindole J isonitrile), 11.84, and 12.72 min show molecular ions at m/z 304. The compounds eluting at 11.40 (12-epi-hapalindole E isonitrile) and 14.48 min (hapalindole L) show a molecular ion at m/z 338, and the compound eluting at 12.33 min shows a molecular ion at m/z 302.

Besides the four determined major hapalindoles, additional compounds were observed in the GC−MS chromatograms of crude extracts from Fischerella, as shown in Figure 3. The molecular ions at m/z 304 and typical fragment ions of these components were indicative of hapalindole derivatives. The compound that eluted at 9.49 min exhibited the same molecular ion [M+] as 2 and a fragmentation pattern similar to that of 2. Both compounds therefore are supposed to be isomers. Another compound, eluting at 11.84 min, is regarded as an

The nitrogen of the indole and the isonitrile moieties could be easily detected upon application of a 15N-labeled crude extract of Fischerella ATCC 43239 to the GC−MS system (Figure 2). The shift of the molecular ions from m/z 304 to 306 and from m/z 338 to 340 showed the presence of two nitrogen atoms in each of the four hapalindoles. Fragment ions with two nitrogen atoms were detected at m/z 223 for 15Nlabeled 2 and 4, at m/z 291 for 15N-labeled 1, and at m/z 305 and 325 for 15N-labeled 3. D



Article

Figure 4. (A and B) Effects of a mixture of indole derivatives on the growth of rat hepatocytes and human neuroblastoma cells, respectively (mean ± SEM; n ≥ 3). (C and D) Time courses of the effect of different concentrations of the indole derivative mixture (arrow) on human neuroblastoma cells. (C) Changes in neuroblastoma fluorescence monitored with the membrane potential-sensitive dye bis-oxonol. (D) Intracellular calcium levels registered in fura-2-loaded neuroblastoma cells (mean ± SEM; n ≥ 3).

isomer of 1, because of the identical molecular ion [M+] and similar fragment ion patterns. The compound eluting at 12.33 min from the DB-1301 column showed a molecular ion at m/z 302. A hapalindole exhibiting this molecular ion has not yet been reported. However, the fragmentation pattern was indicative of a more oxidized hapalindole derivative possibly caused by an additional double bond. It should be noted that the elution sequence for the compounds at m/z 302 (tR = 12.33 min on a DB-1301 column) and m/z 304 (tR = 12.72 min on a DB-1301 column) was different on the DB-1 capillary column (m/z 304, tR = 11.23 min; m/z 302, tR = 11.96 min), supporting the presence of a double bond in the m/z 302 compound. From experiments with hydrocarbons, it is known that components with one double bond elute before the saturated form on a DB-1301 column, but on a DB-1 capillary column, the saturated hydrocarbons elute earlier than the corresponding compounds with one double bond. The acute toxic effects on insects16 demand a better understanding of the mode of activity and assessment of the potential risk for higher animals and humans. We therefore used mammalian cell lines to study the potential targets of the hapalindoles. We first examined the hepatotoxicity of the hapalindole fraction isolated from Fischerella by using the Clone-9 hepatocyte cell line. The viability of hepatocytes was assessed using the metabolic activity marker AB. We tested the effect of a high concentration (10 μM) of the hapalindole mixture (Figure 4A), but hepatocytes showed no decrease in cell viability (maximal incubation time of 24 h). This held true also for excitable human neuroblastoma BE(2)-M17 cells even at an exposure period as long as 72 h (Figure 4B). A significant reduction of neuroblastoma cell viability was not observed.

To detect any change in the membrane potential of neuroblastoma cells caused by the indole alkaloid mixture, we applied concentrations of 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM and used the potential-sensitive fluorescent dye bis-oxonol. We found that the hapalindole mixture did not change the fluorescence or hence the membrane potential of the neuroblastoma cells (Figure 4C). Simultaneously, we studied the effect of the hapalindole mixture on the basal state of the intracellular calcium concentration ([Ca2+]i) of neuroblastoma cells. As shown in Figure 4D, the intracellular calcium concentration remained stable upon addition of the concentration range of the indole mixture. However, we observed the variations of bis-oxonol fluorescence when the application of the haplaindole mixture was combined with veratridine, an alkaloid activator of sodium channels. In these experiments, neuroblastoma cells were incubated with 10 μM hapalindoles for nearly 10 min and then 40 μM veratridine was added (Figure 5). Under these conditions, veratridine did not enhance the bis-oxonol fluorescence of the hapalindole treatment to the same extent as the control, indicating that the mixture of hapalindoles inhibited the membrane depolarization induced by veratridine. In the following experiments, we changed the procedure by first opening the sodium channels with veratridine and then added increasing concentrations of the hapalindole mixture. The addition of veratridine to neuroblastoma cells increased the bisoxonol fluorescence, indicative of a change in membrane potential, and the further addition of hapalindoles reduced this veratridine-induced fluorescence (Figure 6). As a positive control, we used NeoSTX (a known sodium channel blocker). Figure 7 shows that 600 nM NeoSTX decreased the veratridine-evoked increase in the bis-oxonol fluorescence in a E



Article

manner similar to that if the hapalindoles. In addition, NeoSTX (at 600 nM), despite being a neurotoxin, did not decrease the viability of neuroblastoma cells (Figure 8A). Hapalindoles 2−4 could be isolated and purified in sufficient amounts to test them separately. None of the compounds was cytotoxic to neuroblastoma cells at the highest concentration tested (Figure 8B). Interestingly, all of them inhibited the veratridine-induced fluorescence, though to different extents (Figure 9). Figure 10 shows the differences in potency of the hapalindoles. Fitting the data to a linear regression with GraphPad Prism version 5 yielded expected IC50 values of 4.8 μM for 2 (r2 = 0.9670), 6.7 μM for 4 (r2 = 0.9994), and 10.6 μM for 3 (r2 = 0.8754). In addition, 1 was tested under the same experimental protocols, and it was active in a manner similar to that of the other hapalindoles; however, because of the small amount available, its potency could not be determined (data not shown).

Figure 5. Time course of the inhibitory effect of 10 μM of the indole derivative mixture (first arrow) on the veratridine-induced increase in fluorescence (second arrow) in human neuroblastoma cells loaded with bis-oxonol (mean ± SEM; n ≥ 3).

■

DISCUSSION The insecticidal hapalindole fraction of the crude extract from Fischerella contained four indole derivatives as shown by LC− MS.51 After separation into pure compounds, their molecular structures were determined by NMR spectrometry.16 Because this isolation procedure is technically difficult, time-consuming, and expensive, we searched for a more rapid method for detecting and characterizing these indole derivatives and found GC−MS to be a very suitable method. The EI mass spectra were sufficiently different to allow the reliable differentiation of the four compounds. The reliability can even be improved by 15 N isotope labeling that is easy to achieve for cyanobacteria by growing them on 15N-enriched sodium nitrate. The mass shifts of fragment ions that can easily be detected support identification of a particular congener essentially. Full mass spectra for hapalindoles isolated from Fischerella have, to the best of our knowledge, not yet been published and facilitate detection of hapalindole-like alkaloids in cyanobacterial extracts without the need for compound purification. To provide a good screening for the effect of the extracts, we designed different fluorimetric approaches based on (i) measurement of the cytotoxic effect by using the metabolic activity probe Alamar Blue, (ii) changes in membrane potential detected with the dye bis-oxonol, and (iii) quantification of intracellular calcium when the cells are loaded with fura-2. With the bis-oxonol assay, no change in the membrane potential was found for different concentrations of the indole mixture when it was applied on neuroblastoma cells, because no variations in fluorescence were recorded. Simultaneously, as intracellular calcium plays an important role in cellular function and some insecticides may increment its inflow or directly act on calcium channels,19,22,52−56 the effect of the hapalindole mixture on the intracellular concentration of this ion was assayed. Likewise, the fluorescence and hence intracellular calcium concentration remained stable when the different concentrations of the hapalindole mixture were added to the neuroblastoma cells, indicating no activation of the ion channels related to those ion fluxes. Veratridine is known to selectively bind to site 2 of the sodium channels and depolarize excitable cells by opening the sodium channels and slowing their inactivation.25,27,57 We had previously developed a fluorimetric assay taking advantage of the allosteric modulations among the binding sites of sodium channels.48,58−60 Binding of veratridine is inhibited by sodium channel blockers such as paralytic shellfish toxins57−59 or local

Figure 6. Dose-dependent decrease in the fluorescence evoked by veratridine caused by the indole derivative mixture. Neuroblastoma cells were equilibrated with bis-oxonol and treated with veratridine. After the veratridine-induced response reached a steady level (100%), the indole derivative mixture (arrow) was added (mean ± SEM; n ≥ 3).

Figure 7. Dose-dependent decrease in the veratridine fluorescence increment caused by NeoSTX. Neuroblastoma cells were equilibrated with bis-oxonol and treated with veratridine. After the veratridineinduced response reached a steady level (100%), NeoSTX (arrow) was added (mean ± SEM; n ≥ 3).

F



Article

Figure 8. Effects of 600 nM NeoSTX (A) and pure hapalindoles (B) on the cell metabolism of neuroblastoma cells measured with Alamar Blue (mean ± SEM; n ≥ 3).

Figure 9. Dose-dependent decrease in the veratridine-induced depolarization caused by (A) 12-epi-hapalindole C isonitrile, (B) hapalindole L, and (C) 12-epi-hapalindole E isonitrile. Neuroblastoma cells were equilibrated with bis-oxonol and treated with veratridine. After the veratridine-induced response reached a steady level (100%), hapalindoles (arrow) were added (mean ± SEM; n ≥ 3).

anesthetics.23,61 STX blocks sodium channels by binding externally and, as a local anesthetic that can cross the membrane, inhibits the channel in a use-dependent manner by altering gating kinetics.62−65 In our hands, hapalindoles reduced the level of depolarization induced by veratridine in a dose-dependent way. Similar effects were obtained with NeoSTX, a well-known sodium channel blocker, suggesting a common mechanism of action for

both. In addition, neither indole derivatives nor NeoSTX was able to induce cell death in neuroblastoma cells incubated for as many as 72 h. These data are in agreement with previously reported data that showed sodium channel blocking toxins do not cause cellular death but prevent the cytotoxicity induced by the veratridine- and ouabain-enhanced sodium influx in neuroblastoma cells.66−73 G



Article

Figure 10. Linear regression fitting (percentage of inhibition vs log concentration) of (A) 12-epi-hapalindole C isonitrile, (B) hapalindole L, and (C) 12-epi-hapalindole E isonitrile (mean ± SEM; n ≥ 3).

systems is not understood. As produced by biofilm-forming benthic cyanobacteria, hapalindoles constitute a potential hazard especially for animals like insect larvae, mollusks, fish, or waterfowl grazing on the periphyton. In this respect, it is noteworthy that a strong correlation between the appearance of avian vacuolar myelinopathy (AVM) and the field abundance of an epiphytic growing cyanobacterium of the order Stigonematales was found.80,81 It was previously hypothesized that the ingestion of cyanobacterial neurotoxins is the causative of AVM.82 Because hapalindoles have now been shown to be neurotoxic metabolites of Stigonematales, these compounds could be considered as possible causative agents. In summary, our data indicate that the hapalindoles are acting on sodium channels having targets and mechanisms of action that are the same as or very similar to those of NeoSTX. Compound 2 turned out to be the most potent among the hapalindoles tested and 3 the weakest, although all the potencies are very close to each other. This study provides the first information about the blocking characteristics of these alkaloids on sodium channels and suggests that the toxicity of hapalindoles may be related to ion-dependent disturbances of excitable membranes. This neurophysiological activity raises concerns about water quality and potential risks for human health and animals posed by hapalindoles. The proposed molecular mechanism and the chemoanalytical data given here may assist in the monitoring or risk assessment of hapalindoles in ecosystems13 or agricultural systems.77−79 Furthermore, the blockage in neural conduction, interrupting the activity and thus preventing the propagation of action potential and neuronal communication, would be very interesting for studying a possible clinical application as local anesthetics, although a more extensive evaluation needs to be done.

To the best of our knowledge, the only previously known mode of molecular action of hapalindoles is the inhibition of RNA synthesis, though it has been pointed out that this mode of action is unlikely to fully explain the bioactive effects of these compounds.11,14,15 In this study with neuroblastoma cells, we provide the first evidence that hapalindoles inhibit the veratridine-induced depolarization in a manner similar to that of NeoSTX, although with a different potency. When the hapalindoles were tested separately, data showed that all of them (2−4) inhibited veratridine-induced depolarization. Because of the scarcity of the toxins, a linear fitting was used, in which compound 3 exhibited the lowest potency while 2 was the most potent (IC50 = 4.8 μM) derivative. These findings agree with those of other alkaloids tested on inhibition of DNA and RNA enzymes and protein biosynthesis.14,15 Quinidine and yohimbine are two examples of compounds that inhibit sodium channels in addition to having other molecular targets.74−76 Interestingly, the indole alkaloid yohimbine (C21H26N2O3) and the vinyl compound quinidine (C20H24N2O2) share structural similarities and comparable molecular masses and formulas with the tested heterocyclic hapalindoles (C21H24N2 and C21H23ClN2). Hapalindoles are produced by various species of Hapalosiphon and Fischerella and might be a risk in freshwater ecosystems or agricultural water usage. Biological effects of hapalindole-producing cyanobacteria should therefore be considered in water quality assessment. For example, Hapalosiphon and Fischerella are common in paddy fields used for growing rice and, because of the ability of N2 fixation cyanobacteria, are even considered for application as biofertilizers.77−79 Furthermore, despite the described toxicological effects of hapalindoles or Fischerella exudates on insects and fish, respectively,10,13,16,51 the ecotoxicological importance of hapalindoles and structurally related alkaloids in freshwater H



■

Article

synthesis of the hapalindoles, fischerindoles, and welwitindolinones via a redox economic approach. J. Am. Chem. Soc. 130, 17938−17954. (8) Moore, R. E., Cheuk, C., and Patterson, G. M. L. (1984) Hapalindoles: New alkaloids from the blue-green alga Hapalosiphon fontinalis. J. Am. Chem. Soc. 106, 6456−6457. (9) Moore, R. E., Cheuk, C., Yang, X. Q. G., Patterson, G. M. L., Bonjouklian, R., Smitka, T. A., Mynderse, J. S., Foster, R. S., and Jones, N. D. (1987) Hapalindoles, antibacterial and antimycotic alkaloids from the cyanophyte Hapalosiphon fontinalis. J. Org. Chem. 52, 1036− 1043. (10) Becher, P. G., and Jüttner, F. (2006) Insecticidal activity: A new bioactive property of the cyanobacterium Fischerella. Pol. J. Ecol. 54, 653−662. (11) Kim, H., Lantvit, D., Hwang, C. H., Kroll, D. J., Swanson, S. M., Franzblau, S. G., and Orjala, J. (2012) Indole alkaloids from two cultured cyanobacteria, Westiellopsis sp. and Fischerella muscicola. Bioorg. Med. Chem. 20, 5290−5295. (12) Asthana, R. K., Srivastava, A., Singh, A. P., Deepali, Singh, S. P., Nath, G., Srivastava, R., and Srivastava, B. S. (2006) Identification of an antimicrobial entity from the cyanobacterium Fischerella sp. isolated from bark of Azadirachta indica (Neem) tree. J. Appl. Phycol. 18, 33− 39. (13) Wright, A. D., Papendorf, O., Konig, G. M., and Oberemm, A. (2006) Effects of cyanobacterium Fischerella ambigua isolates and cell free culture media on zebrafish (Danio rerio) embryo development. Chemosphere 65, 604−608. (14) Doan, N. T., Rickards, R. W., Rothschild, J. M., and Smith, G. D. (2000) Allelopathic actions of the alkaloid 12-epi-hapalindole E isonitrile and calothrixin A from cyanobacteria of the genera Fischerella and Calothrix. J. Appl. Phycol. 12, 409−416. (15) Doan, N. T., Stewart, P. R., and Smith, G. D. (2001) Inhibition of bacterial RNA polymerase by the cyanobacterial metabolites 12-epihapalindole E isonitrile and calothrixin A. FEMS Microbiol. Lett. 196, 135−139. (16) Becher, P. G., Keller, S., Jung, G., Süssmuth, R. D., and Jüttner, F. (2007) Insecticidal activity of 12-epi-hapalindole J isonitrile. Phytochemistry 68, 2493−2497. (17) Becher, P. G., Baumann, H. I., Gademann, K., and Jüttner, F. (2009) The cyanobacterial alkaloid nostocarboline: An acetylcholinesterase and trypsin inhibitor. J. Appl. Phycol. 21, 103−110. (18) Raymond-Delpech, V., Matsuda, K., Sattelle, B. M., Rauh, J. J., and Sattelle, D. B. (2005) Ion channels: Molecular targets of neuroactive insecticides. Invertebr. Neurosci. 5, 119−133. (19) Bloomquist, J. R. (1996) Ion channels as targets for insecticides. Annu. Rev. Entomol. 41, 163−190. (20) von Stein, R. T., Silver, K. S., and Soderlund, D. M. (2013) Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian VoltageGated Sodium Channels. Pestic. Biochem. Physiol. 106, 101−112. (21) Narahashi, T. (1992) Nerve membrane Na+ channels as targets of insecticides. Trends Pharmacol. Sci. 13, 236−241. (22) Narahashi, T., Ginsburg, K. S., Nagata, K., Song, J. H., and Tatebayashi, H. (1998) Ion channels as targets for insecticides. Neurotoxicology 19, 581−590. (23) Wang, S. Y., and Wang, G. K. (2003) Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins. Cell. Signalling 15, 151−159. (24) Leong, D., Bloomquist, J. R., Bempong, J., Dybas, J. A., Kinne, L. P., Lyga, J. W., Marek, F. L., and Nicholson, R. A. (2001) Insecticidal arylalkylbenzhydrolpiperidines: Novel inhibitors of voltage-sensitive sodium and calcium channels in mammalian brain. Pest Manage. Sci. 57, 889−895. (25) Denac, H., Mevissen, M., and Scholtysik, G. (2000) Structure, function and pharmacology of voltage-gated sodium channels. NaunynSchmiedeberg’s Arch. Pharmacol. 362, 453−479. (26) Catterall, W. A., Goldin, A. L., and Waxman, S. G. (2005) International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol. Rev. 57, 397−409.

AUTHOR INFORMATION

Corresponding Author

*Departamento de Farmacologia,́ Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain. Telephone and fax: +34 982 822 233. Email: [email protected]. Present Addresses ∥

P.G.B.: Department of Plant Protection Biology, Chemical Ecology Unit, Swedish University of Agricultural Sciences (SLU), Box 102, 23053 Alnarp, Sweden. ⊥ B.E.: INL - International Iberian Nanotechnology Laboratory. Av. Mestre José Veiga s/n, 4715-330, Braga (Portugal) Funding

The research leading to these results has received funding from the following FEDER co-funded grants: from CDTI and Technological Funds, supported by Ministerio de Economiá y Competitividad, AGL2012-40185-CO2-01, and Conselleriá de Cultura, Educación e Ordenación Universitaria, GRC2013-016, and through Axencia Galega de Innovación, Spain, ITC20133020 SINTOX; from CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD; and from the European Union’s Seventh Framework Programme managed by REA [Research Executive Agency (FP7/2007-2013)] via Grants 265409 μAQUA, 315285 CIGUATOOLS, and 312184 PHARMASEA. The work of P.G.B. and F.J. was supported by the Swiss National Science Foundation and the Hydrobiologie-Limnologie-Stiftung für Gewässerforschung (Zürich, Switzerland). The work from B.E. was partially supported by Quadro de Referência Estratégico Nacional (QREN); Northern Regional Operational Program, Project ON2-RH-INTEGRATION. Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS STX, saxitoxin; NeoSTX, neosaxitoxin; BSA, bovine serum albumin; bis-oxonol, bis(1,3-dibutylbarbituric acid), trimethine oxonol; fura-2, fura-2 acetoxymethyl ester; AB, Alamar Blue; [M+], molecular ion; [Ca2+]i, intracellular calcium concentration; IC50, median inhibitory concentration; AVM, avian vacuolar myelinopathy

■

REFERENCES

(1) Patterson, G. M. L., Baldwin, C. L., Bolis, C. M., Caplan, F. R., Karuso, H., Larsen, L. K., Levine, I. A., Moore, R. E., Nelson, C. S., Tschappat, K. D., Tuang, G. D., Furusawa, E., Furusawa, S., Norton, T. R., and Raybourne, R. B. (1991) Antineoplastic activity of cultured blue-green algae (Cyanophyta). J. Phycol. 27, 530−536. (2) Burja, A. M., Banaigs, B., Abou-Mansour, E., Burgess, J. G., and Wright, P. C. (2001) Marine cyanobacteria: A prolific source of natural products. Tetrahedron 57, 9347−9377. (3) Haider, S., Naithani, V., Viswanathan, P. N., and Kakkar, P. (2003) Cyanobacterial toxins: A growing environmental concern. Chemosphere 52, 1−21. (4) Tan, L. T. (2013) Pharmaceutical agents from filamentous marine cyanobacteria. Drug Discovery Today 18, 863−871. (5) Chandra, A., and Johnston, J. N. (2011) Total synthesis of the chlorine-containing hapalindoles K, A, and G. Angew. Chem., Int. Ed. 50, 7641−7644. (6) Rafferty, R. J., and Williams, R. M. (2012) Total synthesis of hapalindoles J and U. J. Org. Chem. 77, 519−524. (7) Richter, J. M., Ishihara, Y., Masuda, T., Whitefield, B. W., Llamas, T., Pohjakallio, A., and Baran, P. S. (2008) Enantiospecific total I



Article

(27) Cestele, S., and Catterall, W. A. (2000) Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie 82, 883−892. (28) Vasas, G., Borbely, G., Nanasi, P., and Nanasi, P. P. (2010) Alkaloids from cyanobacteria with diverse powerful bioactivities. Mini Rev. Med. Chem. 10, 946−955. (29) Llewellyn, L. E. (2009) Sodium channel inhibiting marine toxins. Prog. Mol. Subcell. Biol. 46, 67−97. (30) Shimizu, Y. (1996) Microalgal metabolites: A new perspective. Annu. Rev. Microbiol. 50, 431−465. (31) Sivonen, K. (2000) Freshwater Cyanobacterial Neurotoxins: Ecobiology, Chemistry, and Detection. In Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection (Botana, L. M., Ed.) pp 567−581, Marcel Dekker, Inc., New York. (32) Hille, B. (1975) An essential ionized acid group in sodium channels. Fed. Proc. 34, 1318−1321. (33) Kao, C. Y., and Walker, S. E. (1982) Active groups of saxitoxin and tetrodotoxin as deduced from actions of saxitoxin analogues on frog muscle and squid axon. J. Phycol. 323, 619−637. (34) Cusick, K. D., and Sayler, G. S. (2013) An overview on the marine neurotoxin, saxitoxin: Genetics, molecular targets, methods of detection and ecological functions. Mar. Drugs 11, 991−1018. (35) Alfonso, A., Louzao, M. C., Vieytes, M. R., and Botana, L. M. (1994) Comparative study of the stability of saxitoxin and neosaxitoxin in acidic solutions and lyophilized samples. Toxicon 32, 1593−1598. (36) Jüttner, F., Leonhardt, J., and Möhren, S. (1983) Environmental factors affecting the formation of mesityloxide, dimethylallylic alcohol and other volatile compounds excreted by Anabaena cylindrica. J. Gen. Microbiol. 129, 407−412. (37) Klein, D., Daloze, D., Braekman, J. C., Hoffman, L., and Demoulin, V. (1995) New hapalindoles from the cyanophyte Hapalosiphon laingii. J. Nat. Prod. 58, 1781−1785. (38) Cagide, E., Louzao, M. C., Ares, I. R., Vieytes, M. R., YotsuYamashita, M., Paquette, L. A., Yasumoto, T., and Botana, L. M. (2007) Effects of a synthetic analog of polycavernoside A on human neuroblastoma cells. Cell. Physiol. Biochem. 19, 185−194. (39) 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. (40) Louzao, M. C., Espina, B., Vieytes, M. R., Vega, F. V., Rubiolo, J. A., Baba, O., Terashima, T., and Botana, L. M. (2008) “Fluorescent glycogen” formation with sensibility for in vivo and in vitro detection. Glycoconjugate J. 25, 503−510. (41) Espina, B., Cagide, E., Louzao, M. C., Martinez-Fernandez, M., Vieytes, M. R., Katikou, P., Villar, A., Jaen, D., Maman, L., and Botana, L. M. (2009) Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells. Biosci. Rep. 29, 13− 23. (42) O’Brien, J., Wilson, I., Orton, T., and Pognan, F. (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421− 5426. (43) Larson, E. M., Doughman, D. J., Gregerson, D. S., and Obritsch, W. F. (1997) A new, simple, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability. Invest. Ophthalmol. Visual Sci. 38, 1929−1933. (44) Abe, T., Takahashi, S., and Fukuuchi, Y. (2002) Reduction of Alamar Blue, a novel redox indicator, is dependent on both the glycolytic and oxidative metabolism of glucose in rat cultured neurons. Neurosci. Lett. 326, 179−182. (45) Vanitha, J. D., and Paramasivan, C. N. (2004) Evaluation of microplate Alamar blue assay for drug susceptibility testing of Mycobacterium avium complex isolates. Diagn. Microbiol. Infect. Dis. 49, 179−182. (46) Hamid, R., Rotshteyn, Y., Rabadi, L., Parikh, R., and Bullock, P. (2004) Comparison of alamar blue and MTT assays for high throughput screening. Toxicol. In Vitro 18, 703−710.

(47) Schreer, A., Tinson, C., Sherry, J. P., and Schirmer, K. (2005) Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout. Anal. Biochem. 344, 76−85. (48) Louzao, M. C., Vieytes, M. R., Yasumoto, T., Yotsu-Yamashita, M., and Botana, L. M. (2006) Changes in membrane potential: An early signal triggered by neurologically active phycotoxins. Chem. Res. Toxicol. 19, 788−793. (49) Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440−3450. (50) Park, A., Moore, R. E., and Patterson, G. M. L. (1992) Fischerindole L, a new isonitrile from the terrestrial blue-green alga Fischerella muscicola. Tetrahedron Lett. 33, 3257−3260. (51) Becher, P. G., and Jüttner, F. (2005) Insecticidal compounds of the biofilm-forming cyanobacterium Fischerella sp. (ATCC 43239). Environ. Toxicol. 20, 363−372. (52) Endo, M. (2006) Calcium ion as a second messenger with special reference to excitation-contraction coupling. J. Pharmacol. Sci. 100, 519−524. (53) Giorgi, C., Romagnoli, A., Pinton, P., and Rizzuto, R. (2008) Ca2+ signaling, mitochondria and cell death. Curr. Mol. Med. 8, 119− 130. (54) Gotti, C., Briscini, L., Verderio, C., Oortgiesen, M., Balestra, B., and Clementi, F. (1995) Native nicotinic acetylcholine receptors in human Imr32 neuroblastoma cells: Functional, immunological and pharmacological properties. Eur. J. Neurosci. 7, 2083−2092. (55) Millar, N. S., and Denholm, I. (2007) Nicotinic acetylcholine receptors: Targets for commercially important insecticides. Invertebr. Neurosci. 7, 53−66. (56) Cao, Z., Shafer, T. J., and Murray, T. F. (2011) Mechanisms of pyrethroid insecticide-induced stimulation of calcium influx in neocortical neurons. J. Pharmacol. Exp. Ther. 336, 197−205. (57) Manger, R., Woodle, D., Berger, A., Dickey, R. W., Jester, E., Yasumoto, T., Lewis, R., Hawryluk, T., and Hungerford, J. (2014) Flow cytometric-membrane potential detection of sodium channel active marine toxins: Application to ciguatoxins in fish muscle and feasibility of automating saxitoxin detection. J. AOAC Int. 97, 299−306. (58) Louzao, M. C., Rodriguez Vieytes, M., Garcia Cabado, A., Vieites Baptista De Sousa, J. M., and Botana, L. M. (2003) A fluorimetric microplate assay for detection and quantitation of toxins causing paralytic shellfish poisoning. Chem. Res. Toxicol. 16, 433−438. (59) Louzao, M. C., Vieytes, M. R., Baptista de Sousa, J. M., Leira, F., and Botana, L. M. (2001) A fluorimetric method based on changes in membrane potential for screening paralytic shellfish toxins in mussels. Anal. Biochem. 289, 246−250. (60) 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. (61) Hegyi, B., Komaromi, I., Nanasi, P. P., and Szentandrassy, N. (2013) Selectivity problems with drugs acting on cardiac Na+ and Ca2+ channels. Curr. Med. Chem. 20, 2552−2571. (62) Lipkind, G. M., and Fozzard, H. A. (1994) A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel. Biophys. J. 66, 1−13. (63) Yanagawa, Y., Abe, T., and Satake, M. (1987) Mu-conotoxins share a common binding site with tetrodotoxin/saxitoxin on eel electroplax Na channels. J. Neurosci. 7, 1498−1502. (64) Yanagidate, F., and Strichartz, G. R. (2007) Local anesthetics. Handb. Exp. Pharmacol., 95−127. (65) Zhang, F., Xu, X., Li, T., and Liu, Z. (2013) Shellfish toxins targeting voltage-gated sodium channels. Mar. Drugs 11, 4698−4723. (66) Manger, R. L., Leja, L. S., Lee, S. Y., Hungerford, J. M., Hokama, Y., Dickey, R. W., Granade, H. R., Lewis, R., Yasumoto, T., and Wekell, M. M. (1995) Detection of sodium channel toxins: Directed cytotoxicity assays of purified ciguatoxins, brevetoxins, saxitoxins, and seafood extracts. J. AOAC Int. 78, 521−527. (67) Manger, R. L., Leja, L. S., Lee, S. Y., Hungerford, J. M., Kirkpatrick, M. A., Yasumoto, T., and Wekell, M. M. (2003) Detection J



Article

of paralytic shellfish poison by rapid cell bioassay: Antagonism of voltage-gated sodium channel active toxins in vitro. J. AOAC Int. 86, 540−543. (68) Manger, R. L., Leja, L. S., Lee, S. Y., Hungerford, J. M., and Wekell, M. M. (1993) Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: Semiautomated assay for saxitoxins, brevetoxins, and ciguatoxins. Anal. Biochem. 214, 190−194. (69) Weiser, T. (2004) A novel toxicity-based assay for the identification of modulators of voltage-gated Na+ channels. J. Neurosci. Methods 137, 79−85. (70) Ledreux, A., Krys, S., and Bernard, C. (2009) Suitability of the Neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins). Toxicon 53, 300−308. (71) Kogure, K., Tamplin, M. L., Simidu, U., and Colwell, R. R. (1988) A tissue culture assay for tetrodotoxin, saxitoxin and related toxins. Toxicon 26, 191−197. (72) Jellett, J. F., Marks, L. J., Stewart, J. E., Dorey, M. L., WatsonWright, W., and Lawrence, J. F. (1992) Paralytic shellfish poison (saxitoxin family) bioassays: Automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay. Toxicon 30, 1143−1156. (73) Kudo, Y., Finn, J., Fukushima, K., Sakugawa, S., Cho, Y., Konoki, K., and Yotsu-Yamashita, M. (2014) Isolation of 6-deoxytetrodotoxin from the pufferfish, Takifugu pardalis, and a comparison of the effects of the C-6 and C-11 hydroxy groups of tetrodotoxin on its activity. J. Nat. Prod. 77, 1000−1004. (74) Korper, S., Wink, M., and Fink, R. H. (1998) Differential effects of alkaloids on sodium currents of isolated single skeletal muscle fibers. FEBS Lett. 436, 251−255. (75) Dessaint, J., Yu, W., Krause, J. E., and Yue, L. (2004) Yohimbine inhibits firing activities of rat dorsal root ganglion neurons by blocking Na+ channels and vanilloid VR1 receptors. Eur. J. Pharmacol. 485, 11− 20. (76) Trujillo, T. C., and Nolan, P. E. (2000) Antiarrhythmic agents: Drug interactions of clinical significance. Drug Saf. 23, 509−532. (77) Grant, I. F., Roger, P. A., and Watanabe, I. (1986) Ecosystem Manipulation for Increasing Biological N2 Fixation by Blue-Green Algae (Cyanobacteria) in Lowland Rice Fields. Biol. Agric. Hortic. 3, 299−315. (78) Gupta, A. B. (1966) Algal flora and its importance in the economy of rice fields. Hydrobiologia 28, 213−222. (79) Khan, Z. U., Tahmida Begum, Z. N., Mandal, R., and Hossain, M. Z. (1994) Cyanobacteria in rice soils. World J. Microbiol. Biotechnol. 10, 296−298. (80) Wiley, F. E., Wilde, S. B., Birrenkott, A. H., Williams, S. K., Murphy, T. M., Hope, C. P., Bowerman, W. W., and Fischer, J. R. (2007) Investigation of the link between avian vacuolar myelinopathy and a novel species of cyanobacteria through laboratory feeding trials. J. Wildl. Dis. 43, 337−344. (81) Williams, S. K., Kempton, J., Wilde, S. B., and Lewitus, A. (2007) A novel epiphytic cyanobacterium associated with reservoirs affected by avian vacuolar myelinopathy. Harmful Algae 6, 343−353. (82) Wilde, S. B., Murphy, T. M., Hope, C. P., Habrun, S. K., Kempton, J., Birrenkott, A., Wiley, F., Bowerman, W. W., and Lewitus, A. J. (2005) Avian vacuolar myelinopathy linked to exotic aquatic plants and a novel cyanobacterial species. Environ. Toxicol. 20, 348− 353.

K


Hapalindoles from the Cyanobacterium ... - American Chemical Society

Recommend Documents