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Toxicon 75 (2013) 27–34

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Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

Analysis of the action of gymnodimine-A and 13-desmethylspirolide C on the mouse neuromuscular system in vivo

Riadh Marrouchi a,b, Guillaume Rome a, Riadh Kharrat b, Jordi Molgó a,Evelyne Benoit a,*aCNRS, Centre de Recherche de Gif – FRC3115, Institut de Neurobiologie Alfred Fessard – FRC2118,Laboratoire de Neurobiologie et Développement – UPR3294, CNRS Bat. 32-33, 91198 Gif sur Yvette Cedex, Franceb Laboratoire de Toxines Alimentaires, Institut Pasteur de Tunis, 13 Place Pasteur, B.P. 74, 1002 Tunis-Belvédère, Tunisia

a r t i c l e i n f o

Article history:Received 28 May 2013Received in revised form 2 August 2013Accepted 6 August 2013Available online 15 August 2013

Keywords:Cyclic imine toxinsGymnodimine-A13-Desmethyl spirolide CIn vivo studiesMouse neuromuscular excitabilityNicotinic acetylcholine receptors

* Corresponding author. Tel.: þ33 (0) 1 69 82 3682 41 41.

E-mail address: benoit@inaf.cnrs-gif.fr (E. Benoit

0041-0101/$ – see front matter � 2013 Elsevier Ltdhttp://dx.doi.org/10.1016/j.toxicon.2013.08.050

a b s t r a c t

Gymnodimine-A and 13-desmethyl spirolide C are marine toxins belonging to the cyclicimine group produced by Karenia selliformis and Alexandrium ostenfeldii/peruvianum di-noflagellates, respectively. The aim of this work was to analyze the pharmacologicalproperties of both toxins (at sub-lethal doses) on neuromuscular excitability, wheninjected locally to isoflurane-anesthetized mice, using a multimodal minimally-invasivein vivo electrophysiological approach. The main effect of both toxins was a markedreversible time- and dose-dependent decrease of the compound muscle action potentialrecorded from the tail muscle in response to caudal motor nerve stimulation. The dose–response curves of gymnodimine-A and 13-desmethyl spirolide C effect on the maximalamplitude of compound muscle action potential revealed 50% inhibitory doses of 51 ng/mouse (i.e. 1.6 mg/kg or 3.3 nmol/kg mouse) and 0.18 ng/mouse (i.e. 6 ng/kg or 0.01 nmol/kgmouse), respectively. The blocking effect occurred without significant modification ofmotor nerve excitability parameters. It is concluded that the inhibition of the mousecompound muscle action potential, induced by gymnodimine-A and 13-desmethyl spi-rolide C, results from an action of these toxins at the level of the skeletal neuromuscularjunction, since both cyclic imine toxins are known to interact and block muscle-typenicotinic acetylcholine receptors. In the present in vivo study, 13-desmethyl spirolide Cwas about 300 fold more active than gymnodimine-A on equimolar basis. The presentin vivo approach, associated to recent progress in chemical synthesis of cyclic imine toxins,paves the way for more detailed structure-activity studies to obtain new and more potentsynthetic analogs.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Human poisoning by toxins (i.e. intoxination) of marineorigin is a substantial worldwide hazard that can occurindirectly by the consumption of shellfish which arecontaminated by the bioaccumulation and metabolism of

52; fax: þ33 (0) 1 69

).

. All rights reserved.

toxins. These toxins are produced mainly by dinoflagellateswhose increased frequency and wide distribution ofblooms is a major environmental and economic problemthat menaces wildlife and human health. This type ofintoxination is particularly difficult to prevent because thecontaminated organisms, which have no adverse effectsdue to toxins, seem healthy.

Intoxination by shellfish leads to at least five syndromes,including paralytic shellfish poisoning. This syndrome af-fects the peripheral nervous system and is characterized by

Fig. 1. Chemical structures of GYM-A (A) and 13-desmethyl SPX C (B).R1 ¼ Me and R2 ¼ H.

R. Marrouchi et al. / Toxicon 75 (2013) 27–3428

paresthesias, generalized numbness and progressive pa-ralysis up to respiratory muscle (for a recent review, seeWang, 2008). Among the toxins involved are the cyclicimines which constitute an emerging group of novel ma-rine macrocyclic compounds, all of which share a func-tional imine group as a part of a heterocyclic ring systemwithin their molecular framework (Krock et al., 2008).Belonging to the cyclic imine group are gymnodimines(GYMs), spirolides (SPXs), pinnatoxins (PnTxs), pteriatoxins(PtTxs), prorocentrolides and spiro-prorocentrimine. Thesecyclic imines are known as “fast-acting” toxins because, inaddition to produce similar signs of toxicity in mice injec-ted intraperitoneally (EFSA, 2010), they are characterizedby inducing rapid animal death (Otero et al., 2011).Currently, there is no regulation for any cyclic imine toxins(Marrouchi et al., 2008, 2009), and it should be noted thatno case of human poisoning by these toxins was identifiedsince their discovery (Otero et al., 2011).

GYMs and SPXs were the most studied among the cyclicimine group. The dinoflagellate species Karenia selliformis(Haywood et al., 2004), formerly named Gymnodinium sell-iforme, andAlexandrium ostenfeldii/Alexandriumperuvianum(Cembella et al., 1999, 2001; Touzet et al., 2008; Borkmanet al., 2012) have been shown to be responsible for theproduction of GYMs and SPXs, respectively. On one hand,three GYM analogs or isomers (A–C) were characterized,GYM-A being the first toxin isolated and, on the other hand,fourteen SPX analogswere identified, the 13-desmethyl SPXC being the most frequently detected in bivalve molluscs.Little is known concerning the action of cyclic imine toxinsin vivo since toxicological reports regarding these neuro-toxins are rare. Indeed, most of studies were mainly con-cerned with in vitro and acute toxicity (Otero et al., 2011). Inparticular, GYM-A and 13-desmethyl SPX C were reportedto interact with and to block nicotinic and muscarinicacetylcholine receptors both in the central and peripheralnervous systems, as well as at the neuromuscular junction(Kharrat et al., 2008; Bourne et al., 2010; Wandscheer et al.,2010; Aráoz et al., 2011; Hauser et al., 2012).

In order to improve our knowledge on the pharmaco-logical properties of cyclic imine toxins, we investigated theeffects of GYM-A and 13-desmethyl SPX C (Fig. 1) on themouse neuromuscular excitability in vivo, using theminimally-invasive electrophysiological method developedby Prof. H. Bostock (Institute of Neurology, London, En-gland). This method is particularly appropriated for long-term follow-up of the effects of sub-lethal doses of agiven toxin, in vivo, in mouse (Boërio-Guéguen et al., 2009).

2. Materials and methods

2.1. Experimental animals

Adult female Swiss mice (10.2 � 0.9 weeks of age and29.5 � 2.8 g body weight, n ¼ 35) were purchased fromJanvier Elevage (Le Genest-Saint-Isle, France) and housed atthe Gif sur Yvette campus animal facility. The experimentswere performed in accordance with the guidelines estab-lished by the French Council on animal care “Guide for theCare and Use of Laboratory Animals”: EEC86/609 CouncilDirective – Decree 2001-131, on mice under anesthesia by

means of isoflurane inhalation, and the experimental pro-tocols were approved by the French Departmental Direc-tion of Animal Protection (n� A91-453 to E. B.).

2.2. Electrophysiological recordings from the mouseneuromuscular system in vivo

Recordings from the neuromuscular system of anes-thetized mouse were obtained by means of a minimally-invasive electrophysiological method, using the Qtrac�

software written by Prof. H. Bostock (Institute of Neurology,London, England), as detailed previously (Boërio et al.,2009). Briefly, electrical stimulations were delivered tothe caudal motor nerve (at the base of the tail) by surfaceelectrodes, and the compound muscle action potential(CMAP) was recorded using needle-electrodes inserted intothe tail muscle. To study the local action of GYM-A and 13-desmethyl SPXC, intramuscular (i.m.) injections (1.25–5 mL)of phosphate buffer saline (PBS 1�) solution containing0.025–1% methanol and various doses of a given cyclicimine toxin were delivered with a 10 mL micro-syringe atthe base of mouse tail, between stimulation and groundelectrodes. Similar injections (5 mL) were also done withPBS solution containing only 1% methanol to test aneventual effect of the vehicle associated to the highest doseof cyclic imine toxins studied. Immediately after a giveninjection, on-line recordings were initiated to determinethe cyclic imine toxin and/or methanol effects on selectedexcitability parameters, such as the excitability threshold

R. Marrouchi et al. / Toxicon 75 (2013) 27–34 29

and CMAP amplitude registered continuously as a functionof time.

To better identify the underlyingmechanism(s) of actionand the duration of effect(s) of GYM-A and 13-desmethylSPX C, five different excitability tests (stimulus–response,strength–duration and current–threshold relationships, aswell as threshold electrotonus and recovery cycle; see for areview Boërio-Guéguen et al., 2009) were performedtogether before and various times (from 30 min to 12 h)after a given injection. As awhole, more than 35 parameterswere determined from these five excitability tests, andanalyzed. It is worth noting that each specific excitabilitytest provides additional and complementary informationregarding the functional status of ion channels and elec-trogenic pumps, as well as membrane properties of theneuromuscular system (Kiernan and Bostock, 2000;Lefaucheur et al., 2006; Krishnan et al., 2008).

2.3. Data analysis and statistics

The data are expressed as means � SE or � SEM of ndifferent mice. Differences between values were testedusing the parametric unpaired two-tailed t-test, two-wayANOVA, or the non parametric Mann–Whitney U-test,depending on the equality of variances estimated using theLilliefors’ test, and were considered significant whenP � 0.05. The dose–response curves were fitted usingtypical sigmoid nonlinear regression through data points.

2.4. Solutions, drugs and toxins used

GYM-A (molecular weight ¼ 507.3) and 13-desmethylSPX C (molecular weight ¼ 691) were purchased from theNational Research Council Canada (Institute for MarineBioscience, Halifax, Canada), isoflurane (AErrane�) fromBaxter S.A. (Lessines, Belgium), and PBS solution fromSigma–Aldrich (Saint Quentin Fallavier, France).

Just prior to experiments, the toxic extracts of GYM-Aand 13-desmethyl SPX C, dissolved in methanol, werediluted in PBS (1�) solution to give stock solutions con-taining 1% methanol and either 60 ng/mL of GYM-A or0.07 ng/mL of 13-desmethyl SPX C. The stock solutions wereeventually further diluted in PBS (1�) to inject final cyclicimine doses of 30–300 ng/mouse for GYM-A and 0.01–0.35 ng/mouse for 13-desmethyl SPX C. Hence, the amountof methanol injected did not exceed 1%.

3. Results

Immediately after i.m. injections of PBS solutions con-taining 0.025–1% methanol and various doses of eitherGYM-A (from 30 to 300 ng/mouse) or 13-desmethyl SPX C(from 0.01 to 0.35 ng/mouse), on-line recordings revealedthat the major effect of the two studied cyclic imine toxinswas a marked time- and dose-dependent decrease of CMAPamplitude registered continuously. This is exemplified inFig. 2A and B for CMAP recordings in response to variousstimulus pulses, before and 20 min after injections of300 ng/mouse of GYM-A and 0.35 ng/mouse of 13-desmethyl SPX C.

This effect occurred within various times, depending onthedose and type of cyclic imine toxin injected, ranging from19.3� 3.3min (n¼ 3mice) for 30 ng/mouse to 9.3� 0.9min(n ¼ 3 mice) for 300 ng/mouse of GYM-A, and from21.2 � 2.5 min (n ¼ 3 mice) for 0.01 ng/mouse to10.2 � 5.7 min (n ¼ 5 mice) for 0.35 ng/mouse of 13-desmethyl SPX C (Fig. 3). Moreover, it was completelyreversed within 3–5 h after GYM-A (125–300 ng/mouse) or13-desmethyl SPXC (0.18–0.35 ng/mouse) injections (Fig. 2AandB). Inparticular, a 81.5�19.5% (n¼ 6mice) recoverywasobtained 4.0 � 0.9 h after 13-desmethyl SPX C injectionswhen compared to control conditions, leading to a maximalCMAP amplitude not significantly different (P ¼ 0.146) tothat before injections (Fig. 2B). It is worth noting that themaximal CMAP amplitude remained stable before cyclicimine injections (Fig. 3) or before and after injections of PBSsolution containing only 1% methanol (Figs. 2C and 3). Inparticular, under these latter conditions, the maximal CMAPamplitude was not significantly affected (P ¼ 0.332), i.e.95.0� 5.6%within 57.7�13.3min (n¼ 3mice), compared tocontrol conditions. This latter result indicates that injectionsof the vehicle in which the highest doses of cyclic iminetoxins studied were dissolved had, by themselves, nomarked effect on the maximal CMAP amplitude.

The blocking effect of the two cyclic imine toxins wasquantified by establishing the dose–reponse curves, i.e. themaximal CMAP amplitude as a function of the dose of toxininjected, and determining the effective dose required toblock 50% of the maximal CMAP amplitude (ED50). Asshown in Fig. 4, the dose–response curves of GYM-A and13-desmethyl SPX C on the maximal CMAP amplituderevealed ED50 of 51 ng/mouse (i.e. 1.6 mg/kg or 3.3 nmol/kgmouse) and 0.18 ng/mouse (i.e. 6 ng/kg or 0.01 nmol/kgmouse), respectively. It thus appears that 13-desmethylSPX C was about 300 fold more efficient than GYM-A, onequimolar basis, to inhibit the CMAP.

To better identify the underlying mechanism(s) of ac-tion of GYM-A and 13-desmethyl SPXC, stimulus–response,strength–duration and current–threshold relationships, aswell as threshold electrotonus and recovery cycle, wereperformed together before and from 30 min to 12 h after agiven injection. The five different excitability tests, per-formed before and 30 min after injections of PBS solutioncontaining methanol and GYM-A (48–75 ng/mouse) or 13-desmethyl SPX C (0.09–0.35 ng/mouse) did not revealfurther effects of the two cyclic imine toxins since noapparent modification of excitability waveforms wasdetected, as illustrated in Figs. 5 and 6 for current–threshold relationship, charge–duration relationshipestablished from strength–duration testing, thresholdelectrotonus, and recovery cycle.

Furthermore, the parameters determined from these fiveexcitability tests were analyzed (Supplementary data, TableS1). It is worth nothing that most of these parameters aremore related to peripheral nerve than muscle excitabilityproperties (Bostocket al.,1998;Boërio et al., 2009). The resultsconfirm that the two cyclic imine toxins did not exert addi-tional effects sinceno significant differenceoccurredbetweentheparameters determinedbefore and30min after injectionsof PBS solution containing methanol and GYM-A (48–75 ng/mouse) or 13-desmethyl SPX C (0.09–0.35 ng/mouse).

Methanol (1%, 70 min)ControlC

1 mV

2 ms

13-desmethyl SPX C

(0.35 ng/mouse, 4 h)

13-desmethyl SPX C

(0.35 ng/mouse, 20 min)

ControlB

A Control GYM-A

(300 ng/mouse, 4 h)

GYM-A

(300 ng/mouse, 20 min)

Fig. 2. In vivo effects of i.m. injections of GYM-A (A), 13-desmethyl SPX C (B) and methanol (C) on the mouse neuromuscular system. Superimposed traces ofCMAP recorded from the tail muscle following increasing intensities of caudal motor nerve stimulation (scheme in A), before and at various times after injectionsof indicated doses of cyclic imine toxins and/or methanol.

R. Marrouchi et al. / Toxicon 75 (2013) 27–3430

4. Discussion

The present in vivo study of the multimodal excitabilityproperties of the mouse neuromuscular system points outthat the only local effect of i.m. injections of sub-lethal dosesofGYM-Aand13-desmethyl SPXC is toproduceamarkedandreversible inhibition of CMAPs recorded from the tail musclein response to caudal motor nerve stimulation. To the bestof our knowledge, this is the first report showing the efficacyof i.m. injections of cyclic imine toxins to mouse in vivo.

4.1. Comparison of GYM-A and 13-desmethyl SPX C efficacyin vivo

Thepresent results showthat13-desmethyl SPXC isabout300 fold more active than GYM-A, on equimolar basis, to

inhibit CMAPs. This is in agreement with previous toxico-logical studies regarding overall toxicity in mouse in vivo(EFSA, 2010;Otero et al., 2011). Thus, the comparison of lethaldoses producing death of 50% of mice (LD50), followingadministration of cyclic imine toxins by intraperitoneal (i.p.)injection, gavage or voluntary feeding, reveals that, whateverthe route of administration was, 13-desmethyl SPX C wassystematicallymore efficient thanGYM-A to produce toxicity(Munday et al., 2004; Kharrat et al., 2008; Munday et al.,2012a; Otero et al., 2012). In particular, following i.p. injec-tion and according to Selwood et al. (2010) andMunday et al.(2012a, 2012b), 13-desmethyl SPX C, SPX C and 20-methylSPX G are highly toxic (while SPXs A and B are about 5 and13 times less toxic), followed by PnTxs and finally by GYMs.However, it is worth noting that higher LD50 values weredeterminedbyOtero et al. (2012) for 13-desmethyl SPXC and

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Fig. 3. Time-course of the effects of GYM-A (300 ng/mouse; A, continuous line), 13-desmethyl SPX C (0.35 ng/mouse; B, continuous line) and/or methanol in-jections (1%; A and B, dotted lines) on the maximal CMAP amplitude recorded continuously as a function of time from mouse tail muscle. Values are expressedrelatively to those before injections. The arrows indicate the time of injections.

R. Marrouchi et al. / Toxicon 75 (2013) 27–34 31

20-methyl SPX G, showing lower levels of toxicity for thesetwo spirolides than those reported by Munday et al. (2012a).

The low toxicity of GYM-A and 13-desmethyl SPX Cwhen ingested, compared to that when i.p. injected, mayexplain that these compounds, as all the other cyclic iminetoxins, are of low risk to humans when acutely consumed.This is consonant with anecdotal evidence for the absence

Fig. 4. Dose-response curves of the effects of GYM-A (black circles) and 13-desmethyl SPX C (white circles) on the maximal CMAP amplitude recordedin vivo. Each value represents the mean � SEM of data obtained from 3 to 5mice, and is expressed relatively to that obtained before injections. Thecurves were calculated from typical sigmoid nonlinear regression throughdata points (r2 � 0.986). The GYM-A and 13-desmethyl SPX C doses requiredto block 50% of the maximal CMAP amplitude (ED50) were 51 and 0.18 ng/mouse, respectively.

of harmful effects in individuals consuming shellfishcontaminated with cyclic imine toxins (EFSA, 2010; Oteroet al., 2011). However, the LD50 of lipophilic shellfish ex-tracts, comprising mostly 13-desmethyl SPX C, was deter-mined to be higher after i.p. injection than after oral mouseadministration, i.e. 40 and 1 mg/kg mouse, respectively(Richard et al., 2001), which is unusual among pure cyclicimine toxins and other algal toxins.

4.2. Kinetics of in vivo effects

From a general point of view and whatever the route ofadministrationwas, all previous studies classified the cyclicimines as “fast-acting” toxins since they induced rapidonset of neurological symptoms followed by death within3–50 min (EFSA, 2010; Otero et al., 2011).

The blocking effect of i.m. injections of GYM-A and 13-desmethyl SPX C occurs quite rapidly since the CMAPamplitudewas reduced bymore than 75% about 10min aftertoxin administration. With some exceptions, the onset ofsymptoms is delayed and death occurs later when the cyclicimine toxins are orally administrated, compared to i.p. in-jection (EFSA, 2010). Although this indicates a more rapidabsorption of these toxins from the peritoneum than theintestinal tract, specific information regarding the absorp-tion, distribution, metabolism and excretion of cyclic iminetoxins in laboratory animals or humans is at present un-known for most of the members of the various familiescomposing the cyclic imine toxins. However, the systemic

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Fig. 5. Excitability waveforms recorded, in vivo, from mouse tail muscle following caudal motor nerve stimulation before (black circles) and 30 min after (whitecircles) injections of PBS solution containing methanol and 48–75 ng/mouse of GYM-A. (A) Current–threshold relationship, (B) charge–duration relationshipestablished from strength–duration testing, (C) threshold electrotonus and (D) recovery cycle. Mean � SE of data obtained from 15 (black circles) and 7 (whitecircles) different mice.

R. Marrouchi et al. / Toxicon 75 (2013) 27–3432

mouse toxicity observed following oral administration ofcyclic imine toxins clearly indicates that these toxins areabsorbed fromthe intestinal tract. This is supportedby recentstudies demonstrating that SPXswould behighlyabsorbed inthe human intestine, thus being able to enter the circulatorysystem and to reach different organs (Espiña et al., 2011).Hence, 13-desmethyl SPX C and 13,19-didesmethyl SPX Cwere reported to be detected in blood, urine and faeces ofmice as soon as 30–60 min after oral administration of sub-lethal doses of toxins,whilenoquantifiable levelswere foundafter 1–24 h (Otero et al., 2012; Alonso et al., 2013). Theselatter results strongly suggest that rapid absorption andexcretion of cyclic imine toxins occur in mammals. This mayexplain that, following i.p. or oral administration of toxins,mice either died rapidly or made a full recovery with noperceptible long-term effects (EFSA, 2010), and the presentresults showing that the blocking effect of GYM-A and 13-desmethyl SPX C on the CMAP amplitude was completelyreversed within 4 h after i.m. injections.

4.3. Type of in vivo effects

The symptoms of intoxication, similar to those reportedfor the acute toxicity of paralytic shellfish poisoning toxins,

are the same for all cyclic imine toxins and routes ofadministration. In particular, when injected intraperitone-ally to mice, GYMs (Seki et al., 1995; Munday et al., 2004;Kharrat et al., 2008) and some forms of SPXs (Hu et al.,2001; Richard et al., 2001; Munday et al., 2012a; Oteroet al., 2012) have been reported to produce neurologicalsymptoms that include hyperactivity, piloerection, hyper-extension of the back, stiffening and arching of the tail to-ward the head, tremors progressing to spasm, paralysis andextensionof thehind limbs, respiratorydistresswithmarkedabdominal breathing, tremors of the whole body and respi-ratory arrest. The present results, showing that GYM-A and13-desmethyl SPX C produce a marked inhibition of CMAPsrecorded from the mouse tail muscle and elicited by stimu-lating the caudal motor nerve, may explain some of theabove symptoms, in particular the skeletal muscle paralysis.On the other hand, other symptoms, such as hyperactivityand tremorsprogressing to spasm,are likely to result fromanaction of cyclic imine toxins on the mouse central nervoussystem. Indeed, a previous study revealed a remarkably hightoxicity of GYM-A when administered by intra-cerebroventricular injection (LD50 ¼ 0.003 mg/kg mouse),the compound being about 30–60 times less toxic whenadministered by i.p. injection (Kharrat et al., 2008).

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Fig. 6. Excitability waveforms recorded, in vivo, from mouse tail muscle following caudal motor nerve stimulation before (black circles) and 30 min after (whitecircles) injections of PBS solution containing methanol and 0.09–0.35 ng/mouse of 13-desmethyl SPX C. (A) Current–threshold relationship, (B) charge–durationrelationship established from strength–duration testing, (C) threshold electrotonus and (D) recovery cycle. Mean � SE of data obtained from 15 (black circles) and13 (white circles) different mice.

R. Marrouchi et al. / Toxicon 75 (2013) 27–34 33

Besides the CMAP inhibition, the two cyclic imine toxinsdid not modify excitability waveforms and correspondingparameters. This strongly suggests that they do not affectthe density and functional status of ion channel subtypes(mainly sodium and potassium) and electrogenic pumps,and do not produce structural alterations of paranodal andinternodal regions that could indicate subtle disorganiza-tion of the myelin sheath of the peripheral nerve, influ-encing action potential propagation (Kiernan and Bostock,2000; Lefaucheur et al., 2006; Krishnan et al., 2008).

4.4. Molecular targets

The inhibition of the CMAP amplitude by GYM-A and13-desmethyl SPXC can be explained by an in vivo action onskeletal muscle nicotinic acetylcholine receptors (a12b1d 3),which would reduce the amplitude of endplate potentialsto subthreshold levels to trigger muscle action potentials inresponse to nerve stimulation, therefore inhibiting CMAPamplitude and, consequently, neuromuscular transmission.

This is supported by in vitro studies showing that GYM-A and 13-desmethyl SPX C block endplate nicotinicacetylcholine receptors. In particular, nanomolar concen-trations of GYM-A have been reported (i) to cause a time-

and concentration-dependent inhibition of twitch tensionresponses evoked by nerve stimulation in isolated frog andmouse hemidiaphragm preparations, without affectingdirectly-elicited muscle twitch and tetanic contractions,and (ii) to reduce and completely block the amplitude ofminiature-endplate potentials and endplate potentialsrecorded intracellularly, without modifying the restingmembrane potential of muscle fibers (Kharrat et al., 2008).Similar results were obtained with lower concentrations of13-desmethyl SPX C (Aráoz et al., 2009). In addition, thetwo cyclic imine toxins exhibit an antagonist action onembryonic muscle-type (a12b1gd) of nicotinic acetylcho-line receptors incorporated to Xenopus oocyte membranesince they have been reported to dose-dependentlydecrease the peak amplitude of acetylcholine-elicitednicotinic currents, 13-desmethyl SPX C showing the high-est potency as compared to GYM-A (Bourne et al., 2010).

Indeed, available evidence indicates that cyclic iminetoxins interact in vitro, with high affinity, not only withmuscle-types, but also with neuronal-types of nicotinicacetylcholine receptors (Kharrat et al., 2008; Bourne et al.,2010; Aráoz et al., 2011; Hauser et al., 2012) and also, withmuch lower affinity, with some muscarinic acetylcholinereceptor subtypes (Wandscheer et al., 2010; Aráoz et al.,

R. Marrouchi et al. / Toxicon 75 (2013) 27–3434

2011; Hauser et al., 2012). Moreover, the presence of the six-or seven-member imine ring in the structure of GYM-A and13-desmethyl SPX C, respectively, which is one of the dis-tinguishing features of cyclic imine toxins, hasbeen reportedtobe essential for theirhigh affinity interactionwithmuscle-and neuronal-types of nicotinic acetylcholine receptors, asevidenced using X-ray crystallography (Bourne et al., 2010).

Acknowledgments

The authors thank the CNRS-DGRS cooperative programfor supporting the scientific exchange between France andTunisia. Thisworkwas supported inpartby theEuropean7thFrameProgramgrants STC-CP2008-1-555612 (Atlantox) andInterreg IVB Trans-national 2009-1/117 (Pharmatlantic).

Conflict of interest

The authors declare that they have no conflict ofinterest.

Appendix A. Supplementary data

Supplementary data related to this article can be foundat http://dx.doi.org/10.1016/j.toxicon.2013.08.050.

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