Environmental Toxicology and Pharmacology 20 (2005) 305–312
Investigations into the cellular actions of the shellfish toxingymnodimine and analogues
Michael Dragunowa,∗, Michael Trzossb, Margaret A. Brimbleb, Rachel Camerona,Veronica Beuzenbergc, Patrick Hollandc, Doug Mountfortc
a Department of Pharmacology and The National Research Centre for Growth and Development, Faculty of Medical and Health Sciences,The University of Auckland, Auckland, New Zealand
b Department of Chemistry, University of Auckland, 23 Symonds St., Auckland, New Zealandc Cawthron Institute, 98 Halifax St., Private Bag 2, Nelson, New Zealand
Received 20 December 2004; accepted 23 February 2005Available online 12 April 2005
The effects of the shellfish toxin gymnodimine and its analogues (gymnodimine acetate, gymnodimine methyl carbonate and gymn cellular viability were tested using the Neuro2a neuroblastoma cell line. Concentrations of toxins up to 10�M had variable effects oeducing cell number as determined using the MTT assay and no effects on the expression of a number of signal transduction proTF-2, ATF-3) which are sensitive to cellular stress. However, pre-exposure of Neuro2a cells to 10�M concentrations of toxins for 24reatly sensitized these cells to the apoptotic effects of another algal toxin, okadaic acid. These results suggest that gymnodimnalogues sensitize Neuro2a cells to cytotoxins and raise the possibility that algal blooms involving the production of both okadaiolecules and gymnodimine may generate greater cytotoxicity and pose a greater public health problem. Furthermore, our stud
he Neuro2a cell line as a potentially high-throughput cellular system sensitive to the pharmacological effects of gymnodimine andnd as a potential screen for algal-derived toxins.2005 Elsevier B.V. All rights reserved.
The New Zealand coastline is a rich resource for shell-sh production and shellfish are an important traditionalood source for Maori and many other New Zealanders. Un-ortunately, worldwide each year a significant number ofases of shellfish poisoning occur. These incidents are asso-iated with natural marine biotoxins produced by microalgaeGarthwaite, 2000). Shellfish toxins are produced by free-iving microalgae, upon which filter-feeding bivalve molluscsuch as clams, mussels, oysters, or scallops feed. Shellfishoncentrate the phycotoxins in the edible tissues acting as aector transferring these toxic compounds further up the food
chain where they can be lethal to humans and carnivoresas fish and crabs. In the last few decades, incidences ofalgal blooms, both in fresh water and the sea, have beincreasingly frequent thus strict monitoring of shellfishensure consumer safety is required.
The main species of dinoflagellates that produce toare: Alexandrium spp., Gymnodinium and Pyrodinium(Ciminiello and Fattorusso, 2004). Five major classesshellfish poisoning have been identified: neurotoxic shepoisoning (NSP), diarrhetic shellfish poisoning (DSP), plytic shellfish poisoning (PSP), amnesic shellfish poiso(ASP) and ciguaterra fish poisoning (CSP). The toresponsible for these syndromes are families of compohaving similar chemical structures and effects. Chemicthey range from low molecular weight compounds to hmolecular weight, lipophilic substances. Most algal tox
306 M. Dragunow et al. / Environmental Toxicology and Pharmacology 20 (2005) 305–312
cause human illness by disrupting electrical conductanceand impeding critical physiological processes. Many bindto specific membrane receptors, leading to changes in theintracellular concentration of ions such as sodium andcalcium. Others such as the DSP poison okadaic acid arepotent inducers of apoptotic cell death and this may workthrough activation of cell death transcription factors such asc-Jun and ATF-2 (Walton et al., 1998; Woodgate et al., 1999).
The marine biotoxin gymnodimine1 was first isolatedfrom oysters (Tiostrea chilensis) collected atFoveaux Straitin the South Island of New Zealand and was found to exhibitneurotoxic shellfish poisoning with a minimum lethal dose(intraperitoneal) of 700�g/mL in the mouse bioassay (Sekiet al., 1995). Gymnodimine is produced by the dinoflagel-late,Karenia selliformis(syn.Gymnodinium selliforme) andits structure was initially elucidated by NMR spectroscopy(Seki et al., 1995) and later confirmed by X-ray crystallo-graphic analysis (Stewart et al., 1997). Gymnodimine1 hasalso recently been observed in Tunisia (Bire et al., 2002).Gymnodimine1 is a member of the spiroimine group oftoxins, which includes the spirolides, pinnatoxins and pte-riatoxins (Hu et al., 2001; Chou et al., 1996; Takada et al.,2001). Gymnodimine1 and other spiroimine toxins includ-ing gymnodimine B (Miles et al., 2000) cause a characteristicrapid death in the intraperitoneal mouse bioassay and havebeen called “fast-acting toxins” (Munday et al., 2004). Ther haveh aya (e
tion,p icali ofr sefulb icals n thes da lc
2
2
ureoD nd).T n them anH pH4 Thet on-c lventp by5 im-
purities, unknown isomers of gymnodimine, were detected atca. 4%. The crude extract was further purified by flash col-umn chromatography using silica gel (230–430 mesh) anddichloromethane/MeOH/triethylamine (10:0.2:0.2) as eluentto obtain a white solid.
2.2. General details for synthetic work
All reactions were conducted in flame- or oven-driedglassware under a dry nitrogen atmosphere unless otherwisenoted. Dichloromethane was dried over calcium hydride anddistilled prior to use. Flash chromatography was performedusing Merck Kieselgel 60 (230–400 mesh) with the indi-cated solvents. Thin layer chromatography (TLC) was car-ried out on precoated silica plates (Merck Kieselgel 60F254)and compounds were visualised by UV fluorescence or bystaining with vanillin in methanolic sulfuric acid and heat-ing. Infrared spectra were recorded with a Perkin-Elmer 1600series Fourier-transform infrared spectrometer as thin filmsbetween sodium chloride plates. Absorption maxima are ex-pressed in wave numbers (cm−1) with the following abbrevi-ations: s = strong, m = medium, w = weak and br = broad.1Hand13C NMR spectra were obtained using a Bruker AC 200Bor a Bruker AM 400 spectrometer. All chemical shifts aregiven in parts per million (ppm) downfield from tetramethyl-silane as internal standard (1H) or relative to CDCl (13C)a da let;b usinga ereo wereo
elated compounds, the spirolides, have been shown toigh oral potency with neurotoxic symptomology and mffect acetylcholine receptors in mammalian systemsGillt al., 2003).
In view of the associated public health issues the isolaurification, chemical, toxicological and pharmacolog
nvestigation of shellfish toxins is an important areaesearch. Marine toxins have also proven to be uiochemical probes for the study of a variety of biologystems. We therefore report pharmacological studies ohellfish toxin gymnodimine1 and its chemically modifienalogues gymnodimine acetate2, gymnodimine methyarbonate3 and gymnodamine4.
. Materials and methods
.1. Gymnodimine1
Gymnodimine was isolated from a laboratory cultf Karenia selliformis(syn.Gymnodinium selliformeCAW79) at the Cawthron Institute (Nelson, New Zealahe extraction and purification procedure was based oethod ofMiles et al. (2000). Prior to passage throughP-20 resin the culture was acidified with 50% HCl to.5 (approximately) and filtered (Toyo GC 90, 124 mm).
oxin was eluted off the HP-20 resin with methanol, centrated to low volume and clean up achieved by soartitioning. The purity of the isolated toxin was checked00 MHz proton NMR spectroscopy and LC–MS. Two
3ndJ values are given in hertz.1H NMR data are tabulates s, singlet; d, doublet; t, triplet; q, quartet; m, multipr, broad. High resolution mass spectra were recordedVG70-SE instrument. Electron impact (EI) spectra w
btained at 70 eV and chemical ionisation (CI) spectrabtained with ammonia as the reagent gas.
.3. Synthesis of gymnodimine acetate2
To a stirred solution of gymnodimine1(10 mg, 19.7�mol)n dichloromethane (1 mL) was added Et3N (24.7�L,77�mol), 4-(dimethylamino)pyridine (0.3 mg) and acenhydride (18.6�L, 197�mol) at 0◦C. After stirring for 1 ht room temperature, water was added and the aqueousxtracted with ether. The combined organic layers werever MgSO4 and the solvents removed under reduced pure. The residue was purified by column chromatogrdiethyl ether) to obtain gymnodimine acetate2 (6 mg, 55%s a pale yellow oil.1H NMR (CDCl3): 6.88 (br m, 1H, H-3).80 (br s, 1H, H-4); 5.44 (d,J= 11.3, 1H, H-8); 5.04 (br sH, H-18); 5.01 (dd,J1 = 12.1,J2 = 3.7, 1H, H-10); 4.06 (b, 1H, H-13); 3.96 (br s, 1H, H-16); 3.60 (br d, 1H, H-.59 and 3.35 (2× br m, 2× 1H, H-32); 2.46 (m, 2H, H-20.22–1.95, 1.77–1.42 and 1.17–1.13 (3× m, 17H, H-11, H2, H-14, H-15, H-19, H-23, H-24, H-30 and H-31); 2s, 3H, COMe); 1.95 (t,J= 1.8, 3H, H-25); 1.77 (br s, 3H-27); 1.61 (br s, 3H, H-26); 1.53 (br s, 3H, H-29); 1.09= 7.0, 3H, H-28).13C-NMR (CDCl3): 174.7, 171.5, 170.246.9, 135.0, 134.5, 132.4, 130.3, 125.5, 124.7, 124.4,1.1, 80.5, 77.5, 49.9, 46.0, 37.6, 37.2, 33.7, 31.9, 30.8,
To a stirred solution of gymnodimine1 (13 mg,25.6�mol) in dichloromethane (1 mL) was added 4-(dimethylamino)pyridine (62.6 mg, 512�mol) and methylchloroformate (29.7�L, 384�mol) at 0◦C. After stirringfor 6 h at room temperature, water was added and the aque-ous layer extracted with diethyl ether. The combined organiclayers were dried over MgSO4 and the solvents removedunder reduced pressure. The residue was purified by col-umn chromatography (diethyl ether) to obtain gymnodiminemethylcarbonate3 (5 mg, 35%) as a pale yellow oil.1H NMR(CDCl3): 6.89 (br m, 1H, H-3); 5.80 (br s, 1H, H-4); 5.48 (d,J= 11.3, 1H, H-8); 5.03 (br s, 1H, H-18); 4.87 (dd,J1 = 12.5,J2 = 3.1, 1H, H-10); 4.08 (br m, 1H, H-13); 3.96 (br s, 1H,H-16); 3.75 (s, 3H, OMe); 3.64 (m, 1H, H-7); 3.61 and 3.36(2× br m, 2× 1H, H-32); 2.46 (m, 2H, H-20); 2.21–1.95,1.80–1.41 and 1.35–1.11 (3× m, 17H, H-11, H-12, H-14, H-15, H-19, H-23, H-24, H-30 and H-31); 1.96 (t,J= 1.6, 3H,H-25); 1.81 (br s, 3H, H-27); 1.63 (br s, 3H, H-26); 1.53 (br s,3H, H-29); 1.09 (d,J= 7.0, 3H, H-28). HRMS (EI) calculatedfor C H NO M+, 565.3403; found, 565.3401.
Gymnodimine and analogues were dissolved in neat DMSOat a concentration of 1 mM. For most experiments, 1�L perwell of this stock was added to cells (in 100�L medium) toa final concentration of 10�M (1 �L per well of DMSO wasused as a vehicle to a final concentration of 1%). Further di-lutions were made in DMSO and 1�L of these dilutions wasadded to each well.
2.7. Assessment of cell number using the MTT reductionassay
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol-ium bromide] reduction is a simple colorimetric assay that canbe used to assess cell viability. Viable cells reduce MTT to aninsoluble formazan salt using mitochondrial complex1 withan accompanying colour change that directly correlates to thenumber of remaining viable cells. To each well of a 96-wellplate, 20�L of MTT stock solution (5 mg/mL in PBS, Sigma)was added at the conclusion of an experimental procedureand incubated for 90 min (37◦C, humidified 5% CO2, 95%air) to allow reduction of the MTT. The formazan reactionproduct was solubilised with 10% SDS, 0.01 M HCl (100�Lper well) and incubated overnight at 37◦C. Absorbance wasrecorded the following morning at 595 nm using a Spectra-MAX 250 plate reader (Molecular Devices) and values fromt ntrolc
2
thel ith4 andr n).F 4w omC ch-n ogy,p F-3 ffer( ed inP edo arya sh-i atr -c im-mad s-p di Le-i asq anal-y
34 47 6
.5. Synthesis of gymnodamine4
Following the reported procedure (Stewart et al., 1997)ymnodimine1 (40 mg, 78.8�mol) was dissolved in MeO1.5 mL), and AcOH (25�L) and NaCNBH3 (10 mg,59�mol) were added at 0◦C. After stirring for 5 h aoom temperature, the solvents were removed undeuced pressure. The residue was purified by columnatography (hexane/dichloromethane/MeOH/triethylam:10:2:0.1) to obtain gymnodamine4 (39 mg, 98%) as a paellow oil for which the1H NMR data were consistent wiymnodamine4 (Stewart et al., 1997). HRMS (CI) calculate
or C32H48NO4 [M+ H]+, 510.3583; found, 510.3585.
.6. Cell culture
Neuro2a murine neuroblastoma cells (ATCC CCL-1ere maintained in minimum essential medium (MEibco, Invitrogen) supplemented with 10% heat-inactivBS (Gibco, Invitrogen), sodium pyruvate (1 mM, Gib
nvitrogen), penicillin (100 U/mL, Gibco, Invitrogen) atreptomycin (10�g/mL, Gibco, Invitrogen). Confluent ceere subcultured at a ratio of 1:4 and detached with 0.
rypsin, 1 mM EDTA (Gibco, Invitrogen). Before platinrypsinised cells were centrifuged at 170×g for 5 min andesuspended in MEM. Cells were seeded in 96-well pNunc) at a density of 105 cells per mL (100�L per well).lating was carried out 24 h before the start of an ex
ment. All experiments were repeated at least two tim
reated cells were compared to those from untreated coells.
.8. Immunocytochemistry
Immunocytochemistry (ICC) was used to determineocation of proteins within fixed cells. Cells were fixed w% paraformaldehyde (PFA, 15 min, room temperature)insed in PBS containing 0.2% Triton X-100 (PBS–Tritoor protein detection, cells were incubated overnight at◦Cith primary antibody (c-Jun using antibody PC06 fralbiochem; phospho c-Jun 73 from Cell Signaling Teology, phospho c-Jun 63 from Santa Cruz Biotechnolhospho-ATF-2 from Cell Signaling Technology, and ATfrom Santa Cruz Biotechnology) diluted in immunobu
1% normal goat serum in PBS–Triton). Cells were washBS–Triton (3× 5 min) with gentle agitation and incubatvernight at 4◦C with the appropriate biotinylated secondntibody (Sigma) diluted 1:500 in immunobuffer. After wa
ng with PBS–Triton (3× 5 min), cells were incubatedoom temperature for 3 h with ExtrAvidin® peroxidaseonjugated tertiary complex (Sigma) diluted 1:500 inunobuffer. Cells were washed in PBS–Triton (3× 5 min)nd protein–antibody interactions were visualised with 3′,3-iaminobenzidine (DAB) solution (0.05% DAB, 0.1 M phohate buffer, 0.01% H2O2). After 10 min, cells were rinse
n PBS–Triton and brown staining was observed using aca DM IRB microscope. Immunocytochemical staining wuantified using the Discovery-1 microscope and imagesis system (see below).
308 M. Dragunow et al. / Environmental Toxicology and Pharmacology 20 (2005) 305–312
2.9. Hoechst 33258 nuclear staining
Hoechst 33258 (bizBenzimide) is a DNA and chromatinstain that fluoresces under UV light. It can be used to visu-alise apoptotic nuclear morphology, as cells dying by apop-tosis display either fragmented or highly condensed nuclei(Walton et al., 1998). Fixed cells were washed with PBS con-taining 0.2% Triton X-100 (PBS–Triton, Sigma) for 15 min,then incubated with Hoechst 33258 stain (8�g/mL, Sigma)for 30 min in the dark (room temperature). Cells were thenwashed with PBS–Triton (2× 5 min) and nuclear morphol-ogy was visualised under UV.
2.10. BrdU incorporation to visualise proliferating cells
BrdU labelling (incorporation into DNA) and immuno-cytochemistry were used to visualise proliferating cells.Neuro2a cells were treated with 10�M Gymnodimine acetateand 24 h later they were incubated with 10�M BrdU (Roche,60 min, 37◦C, humidified 5% CO2, 95% air,) and fixed withice-cold methanol (10 min, 4◦C) 1 h later. After aspiration ofthe methanol, cells were air-dried then rehydrated with PBS(3 min, room temperature). The cellular DNA was denaturedwith 2 M HCl (60 min, 37◦C) and washed with 0.1 M ortho-boric acid pH 8.5 (2× 5 min). Cells were then washed withP ◦B :250i ing0 do an-t ingw mt dt ffer( hed
in PBS–Triton (3× 5 min) and BrdU–antibody interactionswere visualised with DAB. After 10 min, cells were rinsed inPBS–Triton and brown staining was observed using a LeicaDM IRB microscope.
2.11. Discovery-1 automated fluorescence microscopeand image analysis system (Molecular Devices)
This is a state-of-the-art system for acquisition and analy-sis of fluorescent and brightfield images. Brightfield imagesof stress proteins (c-Jun, ATF-2, ATF-3) and BrdU-positivecells grown in 96-well plates were acquired automaticallywith laser autofocus with Discovery-1 and a cell count assaywas applied to the acquired images to determine the numbersof cells expressing stress proteins and the numbers of cellsincorporating BrdU.
2.12. Statistical analysis
One-way ANOVA’s with Tukey post-hoc comparison testswere used to evaluate dose-response studies and unpairedt-tests were used for other comparisons using the Prism4 sta-tistical program and graphing package.
3
3
ofg ed( theh ofg nb ep-
S 1 h; (ii)A
BS (3× 5 min) and incubated overnight at 4C with anti-rdU (Roche #1170 376, mouse monoclonal) diluted 1
n 0.1% BSA/PBS. Cells were washed in PBS contain.2% Triton X-100 (PBS–Triton, 3× 5 min) and incubatevernight at 4◦C with anti-mouse biotinylated secondaryibody (Sigma) diluted 1:500 in immunobuffer. After washith PBS–Triton (3× 5 min), cells were incubated at roo
emperature for 3 h with ExtrAvidin® peroxidase-conjugateertiary complex (Sigma) diluted 1:500 in immunobu1% normal goat serum in PBS–Triton). Cells were was
cheme 1. Reagents and conditions: (i) Ac2O, Et3N, CH2Cl2, DMAP, 0◦C,cOH, 0◦C, 5 h.
. Results
.1. Synthesis of gymnodimine analogues
The acetate and methyl carbonate derivativesymnodimine 2 and 3, respectively, were preparScheme 1) in order to evaluate the significance ofydroxyl group at C-10 on the biological effectsymnodimine1 implicating the involvement of hydrogeonding to this group in binding to the appropriate rec
M. Dragunow et al. / Environmental Toxicology and Pharmacology 20 (2005) 305–312 309
Fig. 1. The effects of gymnodimine (G) and gymnodimine acetate (GA) onNeuro2a cell number as determined with MTT.* Significantly different fromDMSO control group atp< 0.01.
tors. Gymnodimine acetate2 and gymnodimine methyl car-bonate3 can also be considered pro-drug forms of the actualgymnodimine toxin. Gymnodimine1was converted to its ac-etate derivative2 using acetic anhydride and triethylamine inthe presence of 4-dimethylaminopyridine. Conversion to themethyl carbonate3 derivative was carried out by treatmentwith methyl chloroformate.
In order to investigate the significance of the spiroiminemoiety in the gymnodimine structure on the biological activ-ity the spiroimine unit was selectively reduced to a spiroaminegroup using sodium cyanoborohydride (Stewart et al., 1997).
3.2. Pharmacology
Our initial studies focused upon investigating the directeffects of gymnodimine and gymnodimine acetate on the vi-ability of Neuro2a cells. Toxins were dissolved in DMSOand added to Neuro2a cells 24 h after initial plating in 96-well microplates and initially tested at concentrations of 1and 10�M. Both gymnodimine and gymnodimine acetatecaused reductions in Neuro2a cell numbers as determined bythe MTT assay compared to the 1% DMSO vehicle (Fig. 1).These effects, however, were small and tended not to be very
Fig. 2. The effects of gymnodimine (G) and gymnodimine acetate (GA) onokadaic acid-treated Neuro2a cells as determined with MTT.* Significantlydifferent from DMSO control group atp< 0.01.
robust between experiments. To search for a more robustpharmacological effect we investigated whether these twocompounds would sensitize Neuro2a cells to another morereliable toxin.
To determine whether the toxins might sensitize Neuro2acells to other stressors, we exposed Neuro2a cells for 24 hto 10�M concentrations of gymnodimine and gymnodimineacetate and then added vehicle or okadaic acid at 100 nM. Atthis concentration, okadaic acid alone produces a small de-crease in cell number caused by apoptosis (Woodgate et al.,1999). Twenty-four hours later, we performed MTT assaysto measure cell number. We found that cells pre-treated with10�M gymnodimine or gymnodimine acetate showed sig-nificantly greater vulnerability to okadaic acid (Fig. 2). Thiseffect was very robust and repeatable. Studies performed withgymnodimine methyl carbonate (Fig. 3) and gymnodamine(Fig. 4) showed similar results to those with gymnodimineand gymnodimine acetate.
Light microscopic analysis of gymnodimine acetate plusokadaic acid-treated cells showed that they exhibited manymore apoptotic cells (as determined by nuclear fragmentationand compaction, and membrane blebbing) than DMSO plusokadaic acid-treated cells (Fig. 5).
F cells a rbonateo differe
ig. 3. Left, the effects of gymnodimine methyl carbonate on Neuro2akadaic acid-treated Neuro2a cells as determined with MTT.* Significantly
s determined with MTT. Right, the effects of gymnodimine methyl caonnt from DMSO control group atp< 0.01.
310 M. Dragunow et al. / Environmental Toxicology and Pharmacology 20 (2005) 305–312
Fig. 4. Left, the effects of gymnodamine (Ga) on Neuro2a cells as determined with MTT. Right, the effects of gymnodamine on okadaic acid-treated Neuro2acells as determined with MTT.* Significantly different from DMSO control group atp< 0.01.
Fig. 5. Gymnodimine acetate (right) sensitizes Neuro2a cells to the apoptotic effects of okadaic acid (100 nM). Left, shows DMSO-treated Neuro2a cellsexposed to okadaic acid (100 nM). Cell nuclei are stained with the Hoechst DNA stain 24 h after the addition of okadaic acid. White arrow shows fragmentingapoptotic cell.
To examine the concentration-response characteristics ofthe action of gymnodimine acetate on Neuro2a cells, weexposed Neuro2a cells to 1 nM, 100 nM, 1�M and 10�Mgymnodimine acetate for 24 h and then to either sterile watervehicle or 100 nM okadaic acid. As shown inFig. 6, only10�M of gymnodimine acetate significantly sensitized cellsto okadaic acid.
To determine whether gymnodimine acetate induced pro-teins associated with cell stress in Neuro2a cells as a mech-anism for sensitization to okadaic acid, we treated Neuro2a
cells with 10�M gymnodimine acetate for 2, 8 or 24 h. Cellswere then fixed with paraformaldehyde as described in Sec-tion 2 and immunostained with antibodies to proteins that wehave found to be activated during cell stress/apoptosis. Noneof the cell stress proteins that we measured (c-Jun, ATF-2, orATF-3) were activated by gymnodimine acetate in Neuro2acells above control levels at any time-point examined (datanot shown).
We also undertook BrdU incorporation assays to deter-mine whether 10�M gymnodimine acetate had any direct
Fig. 6. Concentration-response effects of gymnodimine acetate alone (left) on Neuro2a cell number and on okadaic acid-treated Neuro2a cells (right) asdetermined with MTT.* Significantly different from DMSO control group atp< 0.01.
M. Dragunow et al. / Environmental Toxicology and Pharmacology 20 (2005) 305–312 311
Fig. 7. The effects of gymnodimine (G) and gymnodimine acetate (GA) onBrdU incorporation in Neuro2a cells. There are no statistically significantdifferences between any groups.
effect on the proliferation of Neuro2a cells. As shown inFig. 7, gymnodimine acetate did not significantly affect theincorporation of BrdU in Neuro2a cells indicating that it hadno effect on cell proliferation.
4. Discussion
The marine biotoxin gymnodimine was first isolated fromoysters (Tiostrea chilensis) collected atFoveaux Strait in theSouth Island of New Zealand and was found to exhibit neu-rotoxic shellfish poisoning with a minimum lethal dose (in-traperitoneal) of 700�g/mL in the mouse bioassay (Seki etal., 1995). The related compounds, the spirolides, have beenshown to have high oral potency with neurotoxic sympto-mology and appear to affect the acetylcholine receptors inmammalian systems (Gill et al., 2003). However, the mech-anism of action of gymnodimine is unknown.
As a first step toward understanding the mechanisms ofaction of gymnodimine, we investigated its effects on the vi-ability of a mouse Neuro2a neuroblastoma cell line. We alsotested the effects of three gymnodimine analogues in the hopeof identifying a molecule with properties different to that ofthe parent compound. We discovered that these molecules hada remarkably similar profile of action on Neuro2a cells. Allfour molecules had weak effects on Neuro2a cells alone andt fourm ion tot tionso . Tod s as-s m fors ssiono andA thata tiza-t
d itsa g the
possibility that algal blooms involving the production ofboth okadaic acid-type molecules and gymnodimine maygenerate greater toxicity and pose a greater public healthproblem. However, given that only relatively high concen-trations of gymnodimine and analogues (10�M) sensitizedcells to okadaic acid the public health implications of theseresults are presently unclear. Furthermore,Munday et al.(2004)have recently reported that although gymnodimine isvery toxic by i.p. administration, its oral toxicity is far less.In that study, they showed that the toxicity of gymnodiminemight be due to neuromuscular nicotinic receptor blockade.It is presently not clear if the sensitization to okadaic acidis specific to this molecule or whether it reflects a moregeneral phenomenon applicable to other cell stressors. Allfour gymnodimine molecules sensitized cells to okadaicacid (at similar concentrations) suggesting that regionsconserved in their structure are responsible for this activity.The ability of molecules to sensitize cells to apoptotic stimulihas the potential to be used therapeutically to augment theanti-cancer effects of chemotherapeutic agents, many ofwhich work partly by toxicity to tumour cells.
In conclusion, these studies show that gymnodimine andanalogues sensitize Neuro2a cells to okadaic acid. Althoughthe mechanisms and cellular targets responsible for these ef-fects of gymnodimine and analogues are presently not clear,the test system that we have developed provides a robustm ore,t llu-l MTTa igh-t llowf allyc ted toe
A
tionf tractC
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B R.,from
C andwan
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ended to reduce Neuro2a cell number. In contrast, allolecules produced a strong and consistent sensitizat
he toxic effects of another toxin okadaic acid. Concentraf 10�M were required to sensitize cells to okadaic acidetermine whether gymnodimine acetate induced proteinociated with cell stress in Neuro2a cells as a mechanisensitization to okadaic acid, we investigated the expref three stress-related transcription factors (c-Jun, ATF-2TF-3). None of these factors was affected suggestingctivation of these pathways is not involved in the sensi
ion to okadaic acid.These results demonstrate that gymnodimine an
nalogues act to sensitize cells to okadaic acid raisin
odel for determination of these mechanisms. Furthermhe Neuro2a cell line might provide a high-throughput cear system to screen for algal-derived toxins because thessay can be performed in a 96-well plate format at h
hroughput. The development of this screen may also aor the discovery of gymnodimine analogues with potentiytoprotective actions because the assay can be adjuslicit more or less okadaic acid toxicity.
cknowledgement
This work was supported by the New Zealand Foundaor Research, Science and Technology (Bioactives conAWX0201).
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