Structure elucidation and biological evaluation of Maitotoxin-3, a Homologue of Gambierone, from Gambierdiscus belizeanus

toxins Article Structure Elucidation and Biological Evaluation of Maitotoxin-3, a Homologue of Gambierone, from Gambierdiscus belizeanus Andrea Boente-Juncal 1, Mercedes Álvarez 2,Álvaro Antelo 2, Inés Rodríguez 2, Kevin Calabro 3, Carmen Vale 1, Olivier P. Thomas 3and Luis M. Botana 1,* 1 Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain; [email protected] (A.B.-J.); [email protected] (C.V.) 2Laboratorio CIFGA S.A., Plaza de Santo Domingo no. 20, 5a planta, 27001 Lugo, Spain; [email protected] (M.Á.); alvar[email protected] (Á.A.); [email protected] (I.R.) 3 Marine Biodiscovery, School of Chemistry and Ryan Institute, National University of Ireland Galway (NUI Galway), University Road, H91 TK33 Galway, Ireland; [email protected] (K.C.); [email protected] (O.P.T.) *Correspondence: [email protected]; Tel.: +34-982-822-233 Received: 30 December 2018; Accepted: 28 January 2019; Published: 1 February 2019   Abstract: Gambierdiscus species are the producers of the marine toxins ciguatoxins and maitotoxins which cause worldwide human intoxications recognized as Ciguatera Fish Poisoning. A deep chemical investigation of a cultured strain of G. belizeanus, collected in the Caribbean Sea, led to the identification of a structural homologue of the recently described gambierone isolated from the same strain. The structure was elucidated mainly by comparison of NMR and MS data with those of gambierone and ascertained by 2D NMR data analyses. Gratifyingly, a close inspection of the MS data of the new 44-methylgambierone suggests that this toxin would actually correspond to the structure of maitotoxin-3 (MTX3, m/z1039.4957 for the protonated adduct) detected in 1994 in a Pacific strain of Gambierdiscus and recently shown in routine monitoring programs. Therefore, this work provides for the first time the chemical identification of the MTX3 molecule by NMR. Furthermore, biological data confirmed the similar activities of both gambierone and 44-methylgambierone. Both gambierone and MTX3 induced a small increase in the cytosolic calcium concentration but only MTX3 caused cell cytotoxicity at micromolar concentrations. Moreover, chronic exposure of human cortical neurons to either gambierone or MTX3 altered the expression of ionotropic glutamate receptors, an effect already described before for the synthetic ciguatoxin CTX3C. However, even when gambierone and MTX3 affected glutamate receptor expression in a similar manner their effect on receptor expression differed from that of CTX3C, since both toxins decreased AMPA receptor levels while increasing N-methyl-d-aspartate (NMDA) receptor protein. Thus, further studies should be pursued to clarify the similarities and differences in the biological activity between the known ciguatoxins and the new identified molecule as well as its contribution to the neurological symptoms of ciguatera. Keywords: maitotoxin-3; 44-methylgambierone; gambierone; maitotoxin; Gambierdiscus belizeanus; cytosolic calcium concentration; glutamate receptors; neurotoxicity; ciguatera Key Contribution: The 3D structure of MTX-3 found in Gambierdiscus species has finally been elucidated, and corresponds to a homologue of the recently described gambierone. Toxins 2019,11, 79; doi:10.3390/toxins11020079 www.mdpi.com/journal/toxins Toxins 2019,11, 79 2 of 19 1. Introduction Ciguatera Fish Poisoning (CFP) is a human foodborne illness caused by the ingestion of marine fish contaminated with ciguatoxins (CTX) and other structurally related ladder-shaped polyether toxins traditionally identified as maitotoxins (MTX) [ 1 ]. Ciguatera has recently become a threat to fish consumers in non-endemic regions mainly due to the expanding international trade in seafood from tropical fisheries and to the proliferation of Gambierdiscus as a consequence of ocean warming and climate change [ 2 – 4 ]. Human intoxications by ciguatera affect annually 10,000 to 500,000 people worldwide [ 4 ], although the prevalence of this disease could be higher [ 3 , 4 ]. Traditionally, CFP was thought to affect mainly tropical and sub-tropical areas but presently it has expanded worldwide [ 2 ]. In fact, the presence of Gambierdiscus was previously associated with tropical areas, but presently CFP intoxications have been identified in Europe during recent decades, especially in the Canary Islands [5,6] and Madeira [7,8]. Maitotoxins are among the largest natural non-polymeric compounds and the most toxic marine compounds identified to date [ 9 ]. These toxins are produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa [ 10 , 11 ]. Up to now, four toxin analogs designated as maitotoxin-1 (MTX1), maitotoxin-2 (MTX2), maitotoxin-3 (MTX3) and maitotoxin-4 (MTX4) isolated from different strains of these dinoflagellates have been identified in this toxin group [ 1 , 10 , 12 , 13 ]. So far, maitotoxin (MTX1) is the largest non-polymeric carbon chain molecule in nature [ 14 , 15 ]. Additionally, species of Gambierdiscus and Fukuyoa have been shown to produce other non-structurally related polyether analogs such as gambieric acids [ 16 ], gambieroxide [ 17 ], gambierol [ 18 ], and the recently identified gambierone [ 19 ]. The diversity in the chemical structures and biological activities of the molecules involved in CFP could reflect their different mechanisms of action. Thus, while MTX-like activity is associated with a massive calcium influx and rapid cell death [ 20 – 23 ], ciguatoxins and gambierone cause voltage-gated sodium channel activation at negative membrane potentials leading to cell depolarization at rest and to the disruption of peripheral and central nerve transmission [ 3 , 19 ]. Finally, gambierol acts mainly by blocking voltage dependent potassium channels [24,25]. Among the toxins involved in CFP in Australia, a putative maitotoxin-3 (p-MTX3) was frequently encountered in most species of Gambierdiscus [ 26 ]. For many years, p-MTX3 has been detected in Gambierdiscus species that showed low toxicities in functional bioassays [ 27 , 28 ], but the chemical structure of this molecule remained to be elucidated due to the low amount of biomass available. Mass spectrometry analyses of strains of Gambierdiscus revealed the presence of p-MTX3 with a major MS peak at m/z 1039.5 [ 29 ]. Presently, routine monitoring of toxins (particularly ciguatoxins) by mass spectrometry revealed the almost ubiquitous presence of this mass in positive ionization mode [ 27 , 28 , 30 ]. MTX3 was initially isolated by Holmes et al. [ 10 ] from cultures of the Gambierdiscus strain WC1/1. The molecule was depicted as di-sulfated with a MW = 1060.5 Da (for di-sodium salt) and m/z 1039.5 as the most intense peak observed in IonSpray Mass Spectrometry (ISMS). MTX3 was found to be toxic to mice [ 10 ] and despite their considerable differences in molecular size (maitotoxin-3 is about one third the MW of maitotoxin-1), both molecules showed similar in vivo activity in mice for their death-time vs. intraperitoneal dose [ 10 ]. Recently, the structure of a new maitotoxin analog, named MTX4, was identified from a strain of Gambierdiscus excentricus. MTX4 was reported to exhibit a toxic effect similar to the one of MTX1 in neuroblastoma cells [ 13 ]. The presence of the p-MTX3 with the same MS data was also described in this strain but again no chemical structure could be assigned. Initial studies indicated that MTX3 produced similar symptoms and biological effects to those elicited by MTX1 and MTX2 although with lower potency than MTX1 [ 10 ]. The reduction in potency after solvolysis of these maitotoxins led to suggests that sulfate moieties were important for the biological activity of each maitotoxin analog [10,29]. So far, five compounds produced by Gambierdiscus were found to contain at least one sulfate group: MTX1, MTX2, MTX3, gambierone and gambieroxide. The presence of a sulfate group in the molecular skeleton of maitotoxin is not synonymous of a typical maitotoxin-like biological activity. In fact, recent analysis of the biological activity of gambierone revealed that this compound did not Toxins 2019,11, 79 3 of 19 exhibit similar cellular effects to those of maitotoxin. Rather, the biological activity of gambierone resembled the effect of the voltage-gated sodium channel activator CTX3C although the biological potency of gambierone was lower than that of the synthetic ciguatoxin [ 19 ]. The biological activity of gambieroxide, the other sulfate-containing polyether compound related to maitotoxin, has not yet been assessed. During our efforts to isolate ciguatoxin from G. belizeanus we found a large chemical diversity and we decided to focus on two major metabolites with two prominent ions at m/z 1025.6 and 1039.5 in electrospray ionization (ESI)(+)-MS. The metabolite with the first mass has recently been identified as a novel ladder-shaped polyether compound named gambierone [ 19 ]. The other ion was eluted in the maitotoxin region in a typical reverse phase chromatography. We report herein the structure of the metabolite associated with the mass at m/z 1039.5. All NMR data are consistent with 44-methylgambierone (1) (Figure 1). This structure therefore represents the candidate structure for MTX3. We also report the comparative biological activities of this new metabolite and gambierone both isolated from G. belizeanus. Toxins 2019, 11, x FOR PEER REVIEW 3 of 20 gambieroxide, the other sulfate-containing polyether compound related to maitotoxin, has not yet been assessed. During our efforts to isolate ciguatoxin from G. belizeanus we found a large chemical diversity and we decided to focus on two major metabolites with two prominent ions at m/z 1025.6 and 1039.5 in electrospray ionization (ESI)(+)-MS. The metabolite with the first mass has recently been identified as a novel ladder-shaped polyether compound named gambierone [19]. The other ion was eluted in the maitotoxin region in a typical reverse phase chromatography. We report herein the structure of the metabolite associated with the mass at m/z 1039.5. All NMR data are consistent with 44- methylgambierone (1) (Figure 1). This structure therefore represents the candidate structure for MTX3. We also report the comparative biological activities of this new metabolite and gambierone both isolated from G. belizeanus. Figure 1. Structure of the metabolites isolated from Gambierdiscus belizeanus. 2. Results Gambierdiscus belizeanus was grown in 20 L bags and scaled up to 2000 L. The cells were harvested by filtration and extracted with MeOH under ultrasonication. The methanolic extract was then subjected to successive separation steps and ultimately purified by HPLC using a Reverse Phase (RP) phenylhexyl column yielding pure compound 1 (6 mg) and gambierone (2, 15 mg). 2.1. Structure Elucidation of Compound 1 Compound 1 was isolated as a white solid with the molecular formula C52H78O19S as deduced from High Resolution Mass Spectra (HRMS) data with a major ion peak at m/z 1039.4957 [M+H]+. A first inspection of the 1H NMR spectrum of 1 in CD3OD confirmed its polyketide nature with the presence of oxymethine and methyl signals (Figure S1). The 13C and HSQC NMR spectra of 1 confirmed 52 carbon signals in the form of five methyls (four singlets and one doublet), 17 methylenes (including 14 non-oxygenated, one oxygenated, and two unsaturated), 23 methines (one nonoxygenated, 18 oxygenated and four unsaturated), one ketal at δC 100.7 (C-4), one ketone at δC 211.6 (C-40), three non-protonated and oxygenated carbons and one unsaturated quaternary carbon at δC 136.0 (C-44) (Table 1, Figures S2,S3 and S6). The presence of a sulfate was demonstrated by the presence of an intense fragment at m/z 941.5299 corresponding to [M+H-H2SO4]+ The molecular formula proposed for 1 corresponded to a structural homologue of gambierone (2) and indeed the fragmentation pattern was very similar between both compounds suggesting the presence of an additional methylene in the structure of 2 compared with the one of 1. While the signals of the protons of the ladder-shaped western part of 2 were clearly maintained in 1, some differences were observed in the eastern part of the molecule. Indeed, the lack of one olefinic proton and the presence of a new methyl at δH 1.74 (s, 3H, H-52) and the unsaturated quaternary carbon were shown in the HSQC NMR spectrum (Figure S6). The position of the new methyl on the olefinic carbon C-44 was established through the key H-52/C-42, C-43 and C-44 HMBC correlations (Figure S7). The relative configuration of the C-43/C-44 double bond was deduced as E from a key H2-42/H3-52 rOe. Figure 1. Structure of the metabolites isolated from Gambierdiscus belizeanus. 2. Results Gambierdiscus belizeanus was grown in 20 L bags and scaled up to 2000 L. The cells were harvested by filtration and extracted with MeOH under ultrasonication. The methanolic extract was then subjected to successive separation steps and ultimately purified by HPLC using a Reverse Phase (RP) phenylhexyl column yielding pure compound 1(6 mg) and gambierone (2, 15 mg). 2.1. Structure Elucidation of Compound 1 Compound 1 was isolated as a white solid with the molecular formula C 52 H 78 O 19 S as deduced from High Resolution Mass Spectra (HRMS) data with a major ion peak at m/z1039.4957 [M+H] + . A first inspection of the 1 H NMR spectrum of 1 in CD 3 OD confirmed its polyketide nature with the presence of oxymethine and methyl signals (Figure S1). The 13 C and HSQC NMR spectra of 1 confirmed 52 carbon signals in the form of five methyls (four singlets and one doublet), 17 methylenes (including 14 non-oxygenated, one oxygenated, and two unsaturated), 23 methines (one non-oxygenated, 18 oxygenated and four unsaturated), one ketal at δC 100.7 (C-4), one ketone at δC 211.6 (C-40), three non-protonated and oxygenated carbons and one unsaturated quaternary carbon at δC 136.0 (C-44) (Table 1, Figures S2, S3 and S6). The presence of a sulfate was demonstrated by the presence of an intense fragment at m/z941.5299 corresponding to [M+H-H 2 SO 4 ] + The molecular formula proposed for 1 corresponded to a structural homologue of gambierone ( 2 ) and indeed the fragmentation pattern was very similar between both compounds suggesting the presence of an additional methylene in the structure of 2 compared with the one of 1 . While the signals of the protons of the ladder-shaped western part of 2 were clearly maintained in 1 , some differences were observed in the eastern part of the molecule. Indeed, the lack of one olefinic proton and the presence of a new methyl at δH 1.74 (s, 3H, H-52) and the unsaturated quaternary carbon were shown in the HSQC NMR spectrum (Figure S6). The position of the new methyl on the olefinic carbon C-44 was established through the key H-52/C-42, Toxins 2019,11, 79 4 of 19 C-43 and C-44 HMBC correlations (Figure S7). The relative configuration of the C-43/C-44 double bond was deduced as Efrom a key H2-42/H3-52 rOe. Table 1. 1H (750 MHz) and 13C (125 MHz) NMR Data for 1in CD3OD. Atom Number δH, Mult. (Jin Hz) δCn◦δH, Mult. (Jin Hz) δC 13.47, dd (11.0, 4.5) 3.42, m 67.8 27 3.51, m 77.7 2 4.10, m 69.8 28 2.19, m 1.33, m 39.8 32.00, m 1.71, dd (14.5, 10.0) 39.7 29 3.12, m 70.1 4 - 100.7 30 2.94, m 78.1 5 4.21, d (3.0) 73.0 31 1.89, m 1.54, t (11.5) 34.9 6 4.70, dd (10.0, 3.0) 77.8 32 3.76, dd (8.5, 3.5) 72.5 7 3.37, m 77.3 33 - 77.3 8 3.77, m 67.9 34 2.34, d (12.5) 2.15, d (12.5) 54.1 92.17, m 1.58, t (11.5) 37.9 35 - 143.5 10 3.35, m 79.6 36 2.54, d (14.5) 2.21, d (14.5) 43.3 11 3.79, m 82.4 37 - 79.9 12 5.64, dt (12.5, 2.0) 132.8 38 4.06, dt (10.5, 3.0) 73.1 13 5.75, dt (12.5, 2.0) 133.0 39 2.61, m 2.59, m 45.8 14 3.81, m 83.0 40 - 211.6 15 3.44, dd (5.0, 2.0) 79.9 41 2.59, m 43.9 16 1.99, m 1.49, t (12.0) 47.4 42 2.39, q (7.0) 23.3 17 - 76.6 43 5.45, t (7.5) 132.3 18 3.01, dt (11.0, 2.5) 86.8 44 - 136.0 19 1.79, m 1.62, m 25.4 45 6.33, dt (17.5, 10.5) 142.6 20 1.95, m 1.80, m 34.2 46 5.09, d (17.5) 4.90, d (11.0) 111.2 21 3.53, m 87.4 47 1.20, s 16.4 22 3.54, m 75.7 48 1.00, d (7.5) 13.5 23 1.82, m 1.64, m 32.8 49 1.19, s 16.9 24 1.92, m 1.78, m 29.5 50 4.98, br s 4.85 a118.6 25 2.19, m 35.6 51 1.13, s 20.7 26 3.11, m 85.9 52 1.74, s 11.7 aOverlapped with HOD. d: doublet, m: multiplet, t: triplet, s: singlet, br: broad. Toxins 2019,11, 79 5 of 19 Interestingly, compound 1 shared the same major ion peak at 1039.5 in ESI(+)-LRMS with the one characterizing the putative MTX3, since 1994 and recently found in other strains of Gambierdiscus [ 31 ]. Therefore, we wondered whether 44-methylgambierone ( 1 ) could be the actual structure of the long-sought p-MTX3. First, MTX3 was always assumed to be disulfated from ESI(+)-LRMS data when 1 has been proven unambiguously to contain only one sulfate group. The molecular formula was never obtained for MTX3 as only low resolution mass spectra were given with associated fragments and therefore the presence of two sulfates was only a vague assumption [ 29 ]. The fragmentation pattern obtained in our ESI(+)-LRMS analysis of 1 is in perfect agreement with the one obtained at 120V for MTX3 in the first report of this molecule and we could actually correct the proposed ions for MTX3 (Figure 2, Table 2) [ 29 ]. We are therefore very confident to propose the structure of p-MTX3 as (43E)-44-methylgambierone ( 1 ). The absolute configuration of 1 is supposed to be the same as the one described for gambierone (2) based on ECD calculations due to structure similarity [19]. Toxins 2019, 11, x FOR PEER REVIEW 5 of 20 120V for MTX3 in the first report of this molecule and we could actually correct the proposed ions for MTX3 (Figure 2, Table 2) [29]. We are therefore very confident to propose the structure of p-MTX3 as (43E)-44-methylgambierone (1). The absolute configuration of 1 is supposed to be the same as the one described for gambierone (2) based on ECD calculations due to structure similarity [19]. Figure 2. (A). ESI(+)-LRMS and (B). ESI(+)-HRMS spectra of compound 1. Table 2. Comparison between ions and fragments in positive mode for 1 and MTX3 in the literature. nd: not detected. Peaks Ion Species and Fragments Reported in ESI(+)-LRMS for: Compound 1 MTX3 by Lewis et al. [29] 1099.5 nd [M-H+2Na+K] + 1083.5 [M-H+2Na] + [M-2H+3Na] + 1077.5 [M+K] + [M-H+Na+K] + 1061.5 [M+Na] + [M-H+2Na] + 1056.5 [M+NH 4 ] + nd 1055.5 nd [M+K] + 1039.5 [M+H] + [M+Na] + 1021.5 [M+H-H 2 O] + [M+Na-H 2 O] + 1003.5 [M+H-2H 2 O] + [M+Na-2H 2 O] + 996.5 nd [M-H+Na+K-SO 3 ] + Figure 2. (A) ESI(+)-LRMS and (B) ESI(+)-HRMS spectra of compound 1. Toxins 2019,11, 79 6 of 19 Table 2. Comparison between ions and fragments in positive mode for 1 and MTX3 in the literature. nd: not detected. Peaks Ion Species and Fragments Reported in ESI(+)-LRMS for: Compound 1 MTX3 by Lewis et al. [29] 1099.5 nd [M-H+2Na+K]+ 1083.5 [M-H+2Na]+[M-2H+3Na]+ 1077.5 [M+K]+[M-H+Na+K]+ 1061.5 [M+Na]+[M-H+2Na]+ 1056.5 [M+NH4]+nd 1055.5 nd [M+K]+ 1039.5 [M+H]+[M+Na]+ 1021.5 [M+H-H2O]+[M+Na-H2O]+ 1003.5 [M+H-2H2O]+[M+Na-2H2O]+ 996.5 nd [M-H+Na+K-SO3]+ 981.5 [M+Na-SO3]+[M-H+2Na-SO3]+ 963.5 nd [M-H+2Na-H2O-SO3]+ 959.5 [M+H-SO3]+[M+Na-SO3]+ 941.5 [M+H-H2SO4]+[M+Na-H2O-SO3]+ 923.5 [M+H-H2O-H2SO4]+[M+Na-2H2O-SO3]+ 905.5 [M+H-3H2O-SO3]+[M+Na-3H2O-SO3]+ 887.5 [M+H-4H2O-SO3]+[M+Na-4H2O-SO3]+ nd: not determined. Based on MS data and their fragmentation patterns, we also propose a possible monitoring analysis for both the presence of gambierone ( 2 ) and MTX3 ( 1 ) using the following MRM transitions (Table 3). Table 3. Optimized MS/MS spectrometric parameters for the MRM transitions. Compound Maitotoxin-3 Gambierone ESI Mode Parent Ion Cone/V Fragment Ion Coll/ev Parent Ion Cone/V Fragment Ion Coll/ev +1039.5 30 233.1 30 1025.5 30 219.1 30 277.1 60 277.1 60 803.4 30 803.4 30 941.5 30 927.5 30 1021.5 4 1007.5 4 − 899.6 70 96.8 60 899.6 70 96.8 60 1037.5 70 96.8 60 1023.5 70 96.8 60 899.6 60 899.6 60 2.2. Biological Activity of MTX3 and Gambierone The biological activity of gambierone ( 2 ) was previously reported in undifferentiated human neuroblastoma cells and in cell lines expressing voltage dependent sodium channels isoforms [ 19 ]. In these biological systems 2 exhibited a moderate activity similar to the one observed for the synthetic ciguatoxin CTX3C although with lower potency. In this work, the biological activities of synthetic ciguatoxin CTX3C, MTX1, gambierone ( 2 ) and MTX3 ( 1 ) were compared in human neuronal cortical neurons previously used to evaluate the effects of other marine toxins in neuronal function [ 32 , 33 ] and in the human neuroblastoma cells used earlier [ 19 ]. As indicated in Figure 3, in vitro exposure of human cortical neurons for 5 days to 1 , 2 or CTX3C at concentrations ranging from 0.01 nM to 20 nM did not affect cellular viability in cultures of human cortical neurons. This result agrees with previous observations in primary cultures of mice cortical neurons showing no cytotoxic effect of Toxins 2019,11, 79 7 of 19 the synthetic ciguatoxin CTX3C [ 22 ]. Furthermore, it supports the previously reported similarities between the biological activities of CTX3C and 2 [19,22] , and additionally it reveals for the first time a similar activity for MTX3 and gambierone. It is worth highlighting the lack of cytotoxicity of these three compounds in human cortical neurons while MTX1 induced complete cell death after 2 h, 24 h or 5 days treatment with IC50 values below 1 nM [32]. Toxins 2019, 11, x FOR PEER REVIEW 7 of 20 Figure 3. Comparison of the effects of CTX3C, gambierone and MTX3 on cell viability in human cortical neurons. None of the toxins affected CTX0E16 cell viability at low concentrations. In this case, the maximum toxin concentration evaluated was 20 nM, and toxicity was evaluated after exposure of the cells to the different compound concentrations for 5 days in vitro. Cell viability was evaluated by the MTT assay. Results are expressed as mean ± sem of 4 independent experiments, each performed in triplicate. To complete the comparison of the biological activity of gambierone and MTX3 in the same cellular system, the effect of these compounds on the cytosolic calcium concentration ([Ca2+]c) was evaluated. Calcium homeostasis plays a main pathophysiological role in synaptic signaling and neurodegeneration [34] and a known effect of MTX1 in mice and human cortical neurons is to induce a massive calcium influx that leads to rapid neuronal death [22,33]. Therefore, the effect of gambierone and MTX3 on [Ca2+]c was evaluated. As indicated in Figure 4, exposure of differentiated human cortical neurons to either gambierone or MTX3 at 20 nM caused a small rise in the cytosolic calcium concentration. In contrast with the fast and sustained [Ca2+]c rise elicited by 5 nM MTX1 (Figure 4A), the effect of either gambierone (Figure 4B) and MTX3 (Figure 4C) was considerably shorter and smaller , thus confirming that the biological activity of MTX3 was of lower potency than that of MTX1 as previously demonstrated in vitro [10] although further investigations should be pursued to identify the role of this molecule on the neurological symptoms of human CFP [10]. Figure 3. Comparison of the effects of CTX3C, gambierone and MTX3 on cell viability in human cortical neurons. None of the toxins affected CTX0E16 cell viability at low concentrations. In this case, the maximum toxin concentration evaluated was 20 nM, and toxicity was evaluated after exposure of the cells to the different compound concentrations for 5 days in vitro . Cell viability was evaluated by the MTT assay. Results are expressed as mean ± sem of 4 independent experiments, each performed in triplicate. To complete the comparison of the biological activity of gambierone and MTX3 in the same cellular system, the effect of these compounds on the cytosolic calcium concentration ([Ca 2+ ] c ) was evaluated. Calcium homeostasis plays a main pathophysiological role in synaptic signaling and neurodegeneration [ 34 ] and a known effect of MTX1 in mice and human cortical neurons is to induce a massive calcium influx that leads to rapid neuronal death [ 22 , 33 ]. Therefore, the effect of gambierone and MTX3 on [Ca 2+ ] c was evaluated. As indicated in Figure 4, exposure of differentiated human cortical neurons to either gambierone or MTX3 at 20 nM caused a small rise in the cytosolic calcium concentration. In contrast with the fast and sustained [Ca 2+ ] c rise elicited by 5 nM MTX1 (Figure 4A), the effect of either gambierone (Figure 4B) and MTX3 (Figure 4C) was considerably shorter and smaller , thus confirming that the biological activity of MTX3 was of lower potency than that of MTX1 as previously demonstrated in vitro [ 10 ] although further investigations should be pursued to identify the role of this molecule on the neurological symptoms of human CFP [10]. The most abundant excitatory neurotransmitter receptors in the brain belong to the ionotropic class of glutamate receptors, which mediate fast synaptic transmission and readily adapt to counteract changes in neuronal activity [ 35 , 36 ]. Since activation of voltage-gated sodium channels by CTX3C and the consequent change in neuronal activity has been shown to regulate inhibition and excitation in opposite directions in mice cortical neurons [ 25 ], next, we evaluated the effect of gambierone and MTX3 on ionotropic glutamate receptors expression. In this sense, we have previously reported that chronic exposure of primary cortical neurons to CTX3C elicited long term alterations in synaptic neurotransmission leading to the down-regulation in the protein level of ionotropic glutamate receptors [ 37 ]. Therefore, in order to compare the long term effects of gambierone and MTX3 to those of CTX3C, differentiated CTX0E16 neurons were maintained in the presence of 20 nM gambierone or 20 nM MTX3 for 15 days in culture with full toxin renewal each two days, and the expression of both α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) Toxins 2019,11, 79 8 of 19 receptor subunits was evaluated. As shown in Figure 5A, exposure of human neurons to gambierone or MTX3 decreased the level of the AMPA receptor subunits by 21.31 ± 7.47% (n= 12, p= 0.0093) and 23.06 ± 8.17% (n= 12, p= 0.009), respectively. However, both compounds increased the expression of the NMDA receptors subunits by 83.45 ± 24.51% (n= 9; p= 0.0036) and 87.11 ± 26.26 (n= 7; p= 0.0051) respectively (Figure 5B). Thus, even when the activity of gambierone and MTX3 seems similar to the biological activity of CTX3C in primary mice cortical neurons [ 37 ] both compounds increased the protein expression of NMDA receptors while chronic CTX3C decreased the expression of both NMDA and AMPA receptor subunits [37]. Toxins 2019, 11, x FOR PEER REVIEW 8 of 20 Figure 4. Effect of MTX1 (A), gambierone (B) and MTX3 (C) on the cytosolic calcium concentration in human differentiated cortical neurons. A concentration of 5 nM MTX1 caused at rapid and sustained increase in [Ca2+]c while both gambierone and MTX3, at 20 nM, elicited a shorter and smaller effect on [Ca2+]c. Data represent means ± sem of 4 independent experiments in the case of gambierone and 3 independent experiments for MTX1 and MTX3. Bath application of the toxins is indicated by the arrow. The most abundant excitatory neurotransmitter receptors in the brain belong to the ionotropic class of glutamate receptors, which mediate fast synaptic transmission and readily adapt to counteract changes in neuronal activity [35,36]. Since activation of voltage-gated sodium channels by CTX3C and the consequent change in neuronal activity has been shown to regulate inhibition and excitation in opposite directions in mice cortical neurons [25], next, we evaluated the effect of gambierone and MTX3 on ionotropic glutamate receptors expression. In this sense, we have previously reported that chronic exposure of primary cortical neurons to CTX3C elicited long term alterations in synaptic neurotransmission leading to the down-regulation in the protein level of ionotropic glutamate receptors [37]. Therefore, in order to compare the long term effects of gambierone and MTX3 to those of CTX3C, differentiated CTX0E16 neurons were maintained in the Figure 4. Effect of MTX1 ( A ), gambierone ( B ) and MTX3 ( C ) on the cytosolic calcium concentration in human differentiated cortical neurons. A concentration of 5 nM MTX1 caused at rapid and sustained increase in [Ca 2+ ] c while both gambierone and MTX3, at 20 nM, elicited a shorter and smaller effect on [Ca 2+ ] c . Data represent means ± sem of 4 independent experiments in the case of gambierone and 3 independent experiments for MTX1 and MTX3. Bath application of the toxins is indicated by the arrow. Toxins 2019,11, 79 9 of 19 Toxins 2019, 11, x FOR PEER REVIEW 9 of 20 presence of 20 nM gambierone or 20 nM MTX3 for 15 days in culture with full toxin renewal each two days, and the expression of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits was evaluated. As shown in Figure 5A, exposure of human neurons to gambierone or MTX3 decreased the level of the AMPA receptor subunits by 21.31 ± 7.47% (n = 12, p = 0.0093) and 23.06 ± 8.17% (n = 12, p = 0.009), respectively. However, both compounds increased the expression of the NMDA receptors subunits by 83.45 ± 24.51% (n = 9; p = 0.0036) and 87.11 ± 26.26 (n = 7; p = 0.0051) respectively (Figure 5B). Thus, even when the activity of gambierone and MTX3 seems similar to the biological activity of CTX3C in primary mice cortical neurons [37] both compounds increased the protein expression of NMDA receptors while chronic CTX3C decreased the expression of both NMDA and AMPA receptor subunits [37]. Figure 5. Long term exposure (15 days in culture) of human differentiated cortical neurons to either gambierone or MTX3, at 20 nM, affected the protein level of glutamate receptor subunits. (A) Both compounds decreased the protein level of the AMPA receptor subunits. Western blot bands showing the expression of GluR2,3,4 subunit proteins are shown on the left panel and the corresponding quantifications of the bands are shown on the right. (B) The same treatments were performed to evaluate the effect of the compounds on the expression of NMDA receptor subunits NR2A/B. Representative western blot bands showing NR2A/B levels in differentiated CTX0E16 neurons are shown on the left panel and band intensities quantifications are shown on the right. Fifteen days exposure of human cortical neurons to 20 nM gambierone or 20 nM MTX3 increased NR2A/B levels, and effect opposite to that previously described for CTX3C in mice cortical neurons. The number of independent determinations is shown in parenthesis. ** p < 0.01 versus control. In view of the subtle differences between the chronic neuronal alterations produced by CTX3C in mice cortical neurons [37] and those produced by gambierone and MTX3 reported here the effects of both gambierone and MTX3 on cell viability were additionally evaluated at higher concentrations in human neuroblastoma cells since this cellular model was initially employed to assed the biological effects of gambierone [19]. Cell viability was evaluated using the MTT assay which evaluates the ability of the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells to reduce tetrazolium salts and form strongly colored and lipophilic formazan products which are then quantified by absorbance or fluorescence [38]. The amount of colored formazan product is in linear correlation with a healthy mitochondrial function and with the Figure 5. Long term exposure (15 days in culture) of human differentiated cortical neurons to either gambierone or MTX3, at 20 nM, affected the protein level of glutamate receptor subunits. ( A ) Both compounds decreased the protein level of the AMPA receptor subunits. Western blot bands showing the expression of GluR2,3,4 subunit proteins are shown on the left panel and the corresponding quantifications of the bands are shown on the right. ( B ) The same treatments were performed to evaluate the effect of the compounds on the expression of NMDA receptor subunits NR2A/B. Representative western blot bands showing NR2A/B levels in differentiated CTX0E16 neurons are shown on the left panel and band intensities quantifications are shown on the right. Fifteen days exposure of human cortical neurons to 20 nM gambierone or 20 nM MTX3 increased NR2A/B levels, and effect opposite to that previously described for CTX3C in mice cortical neurons. The number of independent determinations is shown in parenthesis. ** p< 0.01 versus control. In view of the subtle differences between the chronic neuronal alterations produced by CTX3C in mice cortical neurons [ 37 ] and those produced by gambierone and MTX3 reported here the effects of both gambierone and MTX3 on cell viability were additionally evaluated at higher concentrations in human neuroblastoma cells since this cellular model was initially employed to assed the biological effects of gambierone [ 19 ]. Cell viability was evaluated using the MTT assay which evaluates the ability of the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells to reduce tetrazolium salts and form strongly colored and lipophilic formazan products which are then quantified by absorbance or fluorescence [ 38 ]. The amount of colored formazan product is in linear correlation with a healthy mitochondrial function and with the cell viability [ 39 ]. As indicated in Figure 6, exposure of undifferentiated human neuroblastoma cells for 4 days to either gambierone or MTX3 at concentrations ranging from 10 to 1000 nM led to different effects on cell viability (Figure 6A). As expected from our previous work [ 19 ] gambierone did not cause cell death even at concentrations of 1 µ M while MTX3 at the highest concentration evaluated decreased cell viability by more than 70% with an IC 50 of 7.02 × 10 −7 M (95% confidence intervals from 6.09 × 10 −7 to 8.1 × 10 −7 M). In contrast, 24 hours exposure of human neuroblastoma cells to MTX1 caused complete cell death at concentrations as low as 0.01 nM (Figure 6B) while neither CTX3C, nor gambierone or MTX3 affected cell viability after 24 hours (Figure 6C). Thus, these results provide additional data indicating that MTX3 exhibits ciguatoxin-like activity rather than a maitotoxin-like toxicity. Toxins 2019,11, 79 16 of 19 4.6.6. Western Blot The protein level of glutamate receptors was evaluated in cultures of mature human cortical neurons maintained for 15 days in the presence of gambierone or MTX3, both at 20 nM, with half medium changes and toxin replacement each two days. After treatment the cells were washed three times with cold PBS and cell lysates were obtained using RIPA lysis buffer (Thermofisher), containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40 (nonyl phenoxypoliethoxylethanol), 1% sodium deoxycholate and 0.1% SDS supplemented with commercial phosphatase and protease inhibitors cocktails (Thermofisher), and stored at − 20 ◦ C when needed. Total protein concentration in cell lysates was measured in triplicate by the Bradford assay, using bovine serum albumin (BSA) as standard. For SDS-PAGE electrophoresis cell lysates samples containing 30 µ g of total protein were denaturalized in commercial 4 × Laemmli sample buffer (Bio-Rad, containing 277.8 mM Tris-HCl, pH 6.8, 4.4% SDS, 44.4% glycerol, and 0.02% bromophenol blue, supplemented with 2.5% 2-mercaptoethanol (Bio-Rad Laboratories S.A., Barcelona, Spain)) and resolved in 10% polyacrylamide gels at a constant voltage of 200 V for 38 minutes. Afterwards, proteins were transferred to PVDF membranes (10 V for 30 min) using a semi-dry transfer cell (Bio-Rad). The membranes were blocked for 1 hour with 5% BSA and incubated overnight at 4 ◦ C with primary polyclonal antibodies against AMPA receptor subunits (GluR2,3,4 at a dilution of 1:500,) or NMDA receptor subunits 2A/B (1:500) both from Thermofisher. Protein bands were detected using the Supersignal West Pico chemiluminescent substrate (Pierce, Thermo Fisher Scientific, Madrid, Spain) and the Diversity 4 gel documentation and analysis system (Syngene, Cambridge, UK). Chemiluminescence was quantified with the Diversity GeneSnap software (Syngene). β -actin (Millipore, Merck Chemicals & Life Science S.A., Madrid, Spain) was used as control for lane loading and to normalize chemiluminescence values. Each condition was analyzed in duplicate wells for each treatment. 4.6.7. Statistical Analysis All data are expressed as means ± SEM of n determinations. Statistical comparison was by Student’s ttest. pvalues < 0.05 were considered statistically significant. Supplementary Materials: The following data are available online at http://www.mdpi.com/2072-6651/11/2/ 79/s1, Figure S1: 1 H NMR spectrum of 1 at 750 MHz in CD 3 OD. Figure S2: 13 C NMR spectrum of 1 at 125 MHz in CD 3 OD. Figure S3: DEPT135 NMR spectrum of 1 at 125 MHz in CD 3 OD. Figure S4: COSY NMR spectrum of 1 at 750 MHz in CD 3 OD. Figure S5: zTOCSY NMR spectrum of 1 at 750 MHz in CD 3 OD. Figure S6: CRISIS-HSQC NMR spectrum of 1 at 750 MHz in CD 3 OD. Figure S7: HMBC spectrum of 1 at 750 MHz in CD 3 OD. Figure S8: ROESY NMR spectrum of 1 at 750 MHz in CD3OD. Author Contributions: Chemical analyses: M.Á., Á.A., K.C., I.R., O.P.T. Biological evaluation: A.B.-J., C.V., L.M.B. All the authors participated in the design of experiments and the writing and proofreading of the manuscript. Funding: Andrea Boente-Juncal is recipient of a fellowship from Ministerio de Educación, Cultura y Deporte, Spain. The research leading to these results has received funding from the following FEDER cofunded-grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). 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