Structure Elucidation and Biological Evaluation of Maitotoxin-3, a Homologue of Gambierone, from Gambierdiscus belizeanus

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/z 1039.4957 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: 44-methylgambierone; Gambierdiscus belizeanus; ciguatera; cytosolic calcium concentration; gambierone; glutamate receptors; maitotoxin; maitotoxin-3; neurotoxicity.

Figure 1

Structure of the metabolites isolated from Gambierdiscus belizeanus.

Figure 2

(A) ESI(+)-LRMS and (B) ESI(+)-HRMS spectra of compound 1.

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.

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.

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.

Figure 6

Effect of high concentrations of gambierone and MTX3 on cell viability in undifferentiated human neuroblastoma cells. (A) Four days exposure of human neuroblastoma cells to either gambierone or MTX3 differentially affected cell viability. At concentrations of 100 and 1000 nM MTX3 significantly decreased cell viability while gambierone, at the same concentrations, did not alter mitochondrial function. (B) 24 hours exposure of neuroblastoma cells to MTX1 evoked complete cell death at 0.1 nM. (C) Exposure of neuroblastoma cells to synthetic ciguatoxin CTX3C, gambierone or MTX3 for 24 hours did not affect cell viability. Data are mean ± sem of 3 to 4 independent experiments, each performed in triplicate wells. ** p < 0.01; *** p < 0.005 vs. control.