Maitotoxin: An Enigmatic Toxic Molecule with
Useful Applications in the Biomedical Sciences
Juan G. Reyes, Claudia Sánchez-Cárdenas, Waldo Acevedo-Castillo,
Patricio Leyton, Ignacio López-González, Ricardo Felix,
MaríaA. Gandini, Marcela B. Treviño, and Claudia L. Treviño
Many marine invertebrates produce potent toxins, turning themselves poisonous as a defense strategy
against predators. In contrast, other organisms can become poisonous by accumulating toxins from their
own prey. Dinoagellates are aquatic photosynthetic microbial eukaryotes, and some species produce
highly toxic metabolites. These dinoagellate toxins bioaccumulate up the food chain in various con-
sumer organisms. Many lter-feeding organisms such as bivalves accumulate such toxins with no appar-
ent adverse effects on them1 but causing intoxication when ingested by their predators, including sh and
marine mammals, and ultimately also when humans consume contaminated seafood.2
Four major groups of dinoagellate toxins have been described, namely, saxitoxins, ladder-shaped poly-
ether compounds, long-chain polyketides, and macrolides.2 The dinoagellate species Gambierdiscus
toxicus produces several potent polyether toxins, some of which were initially identied in connection
with a common type of food poisoning called ciguatera, caused by consumption of certain contaminated
tropical and subtropical sh. Ciguatera involves a combination of gastrointestinal, neurological, and
cardiovascular disorders. The two most common toxin classes associated with ciguatera are ciguatoxin
(CTx) and maitotoxin (MTx), and they are among the most lethal natural substances known to man.2
23.1 Introduction .................................................................................................................................. 677
23.2 History .......................................................................................................................................... 678
23.3 Natural Origin .............................................................................................................................. 679
23.4 Toxicology .................................................................................................................................... 679
23.5 Synthesis ...................................................................................................................................... 680
23.5.1 Biosynthesis of Ladder-Shaped Polyether Compounds .................................................. 680
23.5.2 Chemical Synthesis of Maitotoxin .................................................................................. 681
23.6 Mechanisms of Action ................................................................................................................. 683
23.6.1 MTx and Plasma Membrane Channel Activation ........................................................... 683
23.6.2 MTx Interaction with Membranes ................................................................................... 683
23.7 MTx Bioactivity and Applications ............................................................................................... 684
23.7.1 Insulinotropic Actions of MTx ........................................................................................ 685
23.7.2 MTx as Interleukin-1β Secretagogue and Oncotic Death Inducer .................................. 686
23.7.3 MTx and Sperm Physiology ............................................................................................ 687
23.8 Final Remarks .............................................................................................................................. 688
Acknowledgments .................................................................................................................................. 689
References .............................................................................................................................................. 689
678 Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection
Most of the neurological symptoms of ciguatera are caused by CTx, which exert their effects due
primarily to the activation of voltage-gated sodium channels, causing cell membrane depolarization.1
MTx displays diverse pharmacological activities, which seem to be derived from its ability to activate
Ca2+-uptake processes in a variety of cell types.3 MTx is the largest and most toxic known nonbiopoly-
meric toxin, with a molecular weight of 3422 Da. MTx is a very interesting compound given its extremely
potent biological activity, and it has been used as a powerful pharmacological tool for the elucidation of
Ca2+-dependent cellular processes.
The discovery of MTx is closely related to the characterization of ciguatera, a food-borne illness caused
by the consumption of sh contaminated with certain dinoagellate toxins. Ciguatera symptoms can
vary with the geographic origin of the contaminated sh. Gastrointestinal symptoms—such as diarrhea,
vomiting, and abdominal pain—occur rst, usually within 24 h of eating implicated sh. Neurological
symptoms may occur at the same time or may follow several days later and include thermal sense inver-
sion, characterized by the feeling of receiving an electric shock when touching cold water, pain and
weakness in the lower extremities, and circumoral and peripheral paresthesia.
This mode of poisoning was called “ciguatera” after cigua, a snail commonly occurring in the
Caribbean Sea.4,5 Ciguatera occurs worldwide in tropical and subtropical regions causing 20,000–50,000
victims per year. The agents causing this condition bioaccumulate in sh as they are transferred up the
food chain, to be nally consumed by humans. Ancient references to toxic diseases conditions similar
to “ciguatera” are found in Homer’s Odyssey (ca. 800 BC), in reports of a pandemic occurring in China
(600 BC), and in the chronicles written by Pedro Martir de Anglería in 1555.6
MTx was discovered in 1965 by Bagnis, who reported that the human symptoms caused by inges-
tion of contaminated herbivorous sh were different from those caused by carnivorous sh, primarily
involving gastrointestinal discomfort and less neurological disorders. The molecule responsible for these
symptoms was discovered upon examination of the toxic constituents in the surgeonsh Ctenochaetus
striatus and was named after the Tahitian namesake of this species—“maito.”7 MTx is a polyketide-
derived polycyclic ether consisting of four extended fused-ring systems termed polyether ladders (molec-
ular formula C164H256O68S2Na2) (Figure 23.1).
HH H H H
Me Me Me
FIGURE 23.1 Structure of MTx. (Taken from Treviño, C.L. et al., Maitotoxin: A unique pharmacological tool for eluci-
dating Ca2+-dependent mechanisms, in: Botana, L.M. (ed.), Seafood and Freshwater Toxins: Pharmacology, Physiology
and Detection, 2nd edn., CRC Press, Boca Raton, F L, pp. 503–516, 2008.) The grey region was utilized for the theoretical
molecular dynamics calculations in t he section M Tx interaction with membranes.
679Maitotoxin: An Enigmatic Toxic Molecule with Useful Applications in the Biomedical Sciences
23. 3 Natural Origin
The marine dinoagellate G. toxicus is a single-celled phytoplanktonic organism, and it may be found on
the surface of algae in tropical waters worldwide. Among other toxins, it produces CTx and MTx, which
accumulate through the food chain as carnivorous sh consume contaminated herbivorous reef sh.
MTx toxin accumulates primarily in the liver and viscera of shes, but not in their esh.8 Higher concen-
trations of toxins can be found in large predatory sh such as barracuda, grouper, amberjack, snapper,
and shark. Because the sh industry has no borders and marine products are shipped to many countries,
ciguatera sh poisoning can occur almost anywhere. People who live in or travel to endemic areas should
avoid consumption of barracuda or moray eel, should be cautious with grouper and red snapper, and are
advised to enquire about local sh associated with ciguatera. Since there is no reliable way to “decon-
taminate” or even to distinguish contaminated sh by smell or appearance, it is wise to avoid eating the
viscera of any reef sh and to prevent consumption of large predacious reef sh.9,10.
The temperatures of the northern Caribbean and extreme southeastern Gulf of Mexico have been
predicted to increase 2.5°C–3.5°C during the next years.11 Higher temperatures favor G. toxicus growth12
and are also likely to alter sh migration patterns. Ciguatera outbreaks have been correlated with sea-
surface temperature increases in the south Pacic Ocean.13
After Yasumoto discovered in 1977 that the dinoagellate G. toxicus was responsible for producing
MTx, he cultivated this organism for 10 years in order to have enough material to isolate this toxin and
to determine its structure.14,15 For some years, MTx was commercially available for experimentation and
was used to study Ca2+ dynamics in diverse cell types (see Section 23.7). Having this tool commercially
available again would certainly continue to be useful for research purposes.
Human ingestion of seafood contaminated with toxins produced by marine phytoplankton10,16,17 can
cause a variety of diseases. These toxins can have a wide range of acute and chronic health effects not
just in humans but also in other animal species. Given that these compounds are tasteless, odorless, and
heat and acid stable, conventional food testing methods fail to detect and destroy them in contaminated
Ciguatera can cause mild to severe symptoms lasting from a few days and up to years. Around 400
species of sh are considered to be ciguateric and contain distinct combinations and quantities of toxins.
Classical ciguateric symptoms include gastrointestinal and neurological disorders, abdominal cramps,
diarrhea, nausea, vomiting, temperature reversal, and itching. The toxins can even be passed on to a fetus
or to a newborn child, via placental or breast milk transmission, respectively.9,10
Most of the neurological symptoms of ciguatera are due to CTx, while MTx is considered to be less
important in the generation of ciguatera symptoms given that it is less concentrated in shes. However, it
should be noted that the observed toxicity differences could also be the result of chemical modications
of the toxins possibly occurring as they pass through the food chain, and it is therefore difcult to estab-
lish a direct relationship between the various symptoms and a particular toxin. Diagnosis of ciguatera is
based solely on the presence of the general symptoms in correlation to patients with a recent history of
In contrast to the lipid-soluble CTxs, MTx is water soluble, and it apparently does not accumulate in
the esh of shes but rather in organs such as the liver.18 MTx has a very low oral potency as compared
to its high lethality when injected intraperitoneally (i.p.), and pure MTx is even more toxic than CTx.
For example, in mice, CTx is lethal at 0.45 μg/kg i.p. and MTx at a dose of 0.15 μg/kg i.p. However, the
precise lethal dose depends on the mouse strain, the sample source, and even the sample preparation pro-
cedure, as MTx binds to glass and plastic and thus its exact concentration may be underestimated. Mice
injected i.p. with MTx display reduced body temperature, piloerection, dyspnea, progressive paralysis,
slight tremors or convulsions, and long death times. High doses of MTx produce CTx-like symptoms,
such as gasping with convulsions and shorter death times.
680 Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection
Three different MTx molecules have been isolated from a variety of strains of G. toxicus. Injection
of MTx-1 and MTx-2 in mice exerted similar symptoms, except that MTx-2 exhibited shorter death
times. MTx-3 induced additional symptoms such as intense gasping that ameliorated near death; how-
ever, further purication of MTx-3 by HPLC reduced the gasping symptoms, suggesting that additional
bioactive components may be present in the crude preparation.19 The death times produced by MTx-1
and MTx-3 were very similar. Desulfonation of MTx (solvolysis) reduces the toxicity of all three forms
Efforts to develop a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) to
detect CTx have been made over the past few years, such as the Hokama enzyme immunoassay stick
test.21 There is a commercial kit called Cigua-Check® that may be used by sherman or restaurants to
prevent ingestion of contaminated sh. Unfortunately, this kit only detects CTx. Detection can be con-
rmed by nding CTx and MTx in contaminated sh samples by high-performance liquid chromatog-
raphy and mass spectrometry, although this process is costly and not widely available in high risk areas,
such as small islands.
To date, there is no antidote for ciguatera, but medications such as amitriptyline have been used to
diminish some of the symptoms of chronic ciguatera, including fatigue and paresthesias. There are sev-
eral palliative remedies including medicinal teas used in both the Indo-Pacic and West Indies regions.
However, none of these treatments have been properly standardized to provide effective treatment.9,10
Patients are also advised to avoid alcohol, nuts, and nut oil for at least 6 months after the intoxication in
order to avoid reappearance of symptoms.
23. 5 Synthesis
23.5.1 Biosynthesis of Ladder-Shaped Polyether Compounds
The structural similarities among dinoagellate-produced polyether ladder toxins including breve-
toxins, CTxs, yessotoxins, and MTxs strongly suggest that their biosynthetic pathways may share
similar strategies. In the case of brevetoxins, experimental studies on their biosynthetic pathways are
starting to unveil the biochemical reactions involved in the production of such complex polyethers.
Based on tracing studies using radiolabeled carbon precursors, it is now clear that polyketides in
general are assembled from acetyl-CoA or malonyl-CoA precursors; they a lso contain carbon units
derived from methionine and from some la rger carbon unit precursors, such as glycolate.22 The car-
bon chain formed from the aforementioned precursors appears to be interrupted by C-1 deletions of
acetate-der ived carbon atoms. Although a Favorskii-like rearrangement has been suggested to be
involved in the ring size reduction observed in polyether ladder toxins, such proposed catalytic mech-
anism (which in vitro takes place with halogenated compounds and under strong basic conditions)
has not yet been unequivocally demonstrated. In this biosynthetic pathway, and similarly to bacterial
polyether synthesis, polyketide synthases appear to play a n important role. This family of enzymes
catalyzes the synthesis of complex natural products from precursors such as acetyl-CoA, propionyl-
CoA, and methylmalonyl-CoA through a biosynthetic strategy resembling the one used for fatty acid
(FA) biosynthesis.23 Thus, polyketides are built by a series of successive condensations of simple
precursors, decarboxylations, reductions, and rea rrangements. However, unlike FA biosynthesis, in
which the FA chain is subjected to the whole series of intermediate reactions, the partial chemical
processing of intermediaries in polyketide biosynthesis can give rise to a complex pattern of func-
tional groups associated to the polyether chain. Furthermore, different dinoagellate species can use
different combinations of starter CoA-activated precursors and chain-extension substrates during
polyketide biosynthesis, which at least in some cases involve the generation of specic chiral centers
and cyclization. Despite the proposed existence of similar polyether ladder biosynthetic pathways in
different dinoagellate species, suggested by their products’ structural similarities.22, 24 Dening the
specic reactions of any such pathway has proven a very difcult task. This challenge stems from
the diversity of chemica l structures found in this family of compounds, from the diversity of species
within this phylum of alveolate euka ryotes, and from the particularly complex genetic organization
681Maitotoxin: An Enigmatic Toxic Molecule with Useful Applications in the Biomedical Sciences
observed in these organisms, including multigene phylogenies, diverse plastid endosymbiotic rela-
tionships, and chromatin conguration.22 ,25 –28 It has been suggested that for polyether ladder toxins,
the par ticipating acetate units can enter the tricarboxylic acid (TCA) cycle, and some TCA interme-
diate could be the actual precursor involved in the condensation process.29 In fact, to the best of our
knowledge, there a re thus far no reports of specic studies on MTx biosynthesis. Further more, it is
likely that the toxins isolated from the organs of herbivorous shes represent a chem ically modied
version of the toxins originally synthesized by the dinoagellates.3 0–32
The cyclization process in these compounds is probably associated to the formation of an epox-
ide intermediate presumably catalyzed by epoxidases and epoxide hydrolases.33, 34 The accompany-
ing polyepoxide process proposed for marine polyether ladder toxins has clear precedents in several
reports on epoxide and cycle formation during antibiotic polyether biosynthesis.35 Although the pro-
cess whereby 10 or more epoxides are formed and then coordinated to a polyepoxide cascade to yield
polyether ladder toxins still remains unclear, an oxidase-catalyzed tandem epoxide formation and
epoxide protection, followed by a hydrolase-catalyzed epoxide opening and cycle rea rrangement, is
23.5.2 Chemical Synthesis of Maitotoxin
The chemical structures of marine toxins in general—and MTx in particular—are very interesting and
represent a formidable challenge for organic synthesis.36 The appearance in the literature of the struc-
ture of brevetoxin-B (the rst marine polycyclic ether to be isolated and characterized37) awoke a large
interest in synthetic organic chemists. The structure of brevetoxin-B is characterized by several fused
polycyclic ethers containing ether rings with 6, 7, and 8 units, in addition to 23 chiral centers, certainly
a daring task for chemical synthesis. It contains 32 fused ether rings, 28 hydroxyl groups, 21 methyl
groups, 98 chiral centers, and 2 sulfates.3,38 –4 0
The efforts of chemical synthesis have been directed toward developing new methods that
allow synthesis of only part of these molecules by initially building structural fragments of these
ladder-shaped polyether toxins. Great progress in the synthesis of several marine polycyclic ethers
has been accomplished since the year 2000.41,42 They designed a remarkable synthetic strategy involv-
ing an efcient iterative method for the stereoselective construction of transfused polycyclic ethers
based on induced reductive cyclization of β-alcoxy-acrylate by samarium iodide (SmI2). In the case
of the synthesis of the ring systems of MTx, the stereoselectivity was accomplished by state transition
chelation (Figure 23.2).42– 46
Oxepane ring formation also was stereoselectively synthesized through this reaction generating a sin-
gle product with an 84% yield (Figure 23.3).
FIGURE 23.2 Transition state of the cyclization induced by SmI2. (From Sakamoto, Y. et al., Org. Lett., 3, 2749, 2001;
Nakata, T., Chem. Rec., 10, 159, 2010.)
FIGURE 23.3 Synthesis of polycyclic et hers based on cyclizat ion induced by SmI2. (From Nakata, T., Chem. Rec., 10,
682 Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection
Moreover, the trans-membered fused polycyclic 6,7,6 and 6,7,7,6 ethers were iteratively synthesized
using this cyclization induced by SmI2 strategy (Figure 23.4).42
Additionally, in 1998, Dr. Nicolaou’s research group (La Jolla, CA) reported the complete synthe-
sis of brevetoxin-A, another ladder-shaped polyether compound, the rst stereoselective synthesis
of pyrans involving the opening of epoxides with a hydroxyl group. This synthesis route has the
particularity of overcoming the natural preference for cyclization to an unwanted product 5-exo by
placing one C−C bond adjacent to the epoxy fragment. Thus, under these conditions, the structure
shown in Figure 23.5 exclusively undergoes ring closure to produce a 6-endo by pyran system instead
of 5-exo product. The selectivity observed is attributed to the π orbital stabilization generated by a
carbon atom next to the electron-decient transition state provoking an endo attack; this effect would
be absent during the exo attack.47
Using similar synthetic strategies, this same research group has accomplished the stereoselective syn-
thesis of ladder-shaped portions of a large part of MTx.40,41,48–50 Altogether, between Dr. Nakata’s and Dr.
Nicolaou’s group, 29 out of the 32 rings of MTx have been chemically synthesized.
Although thus far no complete chemical synthesis has been achieved for MTx, complete synthesis has
been successful for some of the other ladder-shaped polycyclic ester toxins. New methods such as bio-
mimetic cascades, cross-coupling reactions catalyzed by palladium, and radical reactions could provide
additional tools for approaching the laboratory synthesis of this type of compounds.
FIGURE 23.4 Examples of ethers synthesized by iterative strategy induced cyclization 6,7,6 and 6,7,7,6 membered poly-
cyclic ethers. (From Nakata, T., Chem. Rec., 10, 159, 2010.)
FIGURE 23.5 Reactions involved for the formation of cyclic ethers. (From Nicolaou, K. et al., J. Am. Chem . Soc., 108,
2468, 1986; Nicolaou, K.C. et al., J. Am . Chem . Soc., 130, 7466, 2008.)
683Maitotoxin: An Enigmatic Toxic Molecule with Useful Applications in the Biomedical Sciences
23.6 Mechanisms of Action
23.6.1 MTx and Plasma Membrane Channel Activation
After the early work of Dr. Yasumoto’s group,51–53 it became clear that MTx acted on mammalian cells
inducing entry of external Ca2+, and based on pharmacological criteria, it was proposed that MTx activated
voltage-gated Ca2+ (Cav) channels.54–57 Consistent with this proposal, MTx-induced Ca2+ entry was absent
in broblast that lacked these channels.55 However, even this early work on the mechanisms of action of
MTx pointed out some peculiar aspects of the toxin’s effects, such as a delay of about 2 min in the induction
of Ca2+ entry observed in certain cell types55 or the reported inhibition of Na+-K+-ATPase induced by the
toxin.58 Furthermore, it was soon reported that MTx was able to induce inositol triphosphate (IP3) release
independently of its action on Ca2+ channels.59 These results were corroborated by Gusovsky et al.60–63 in
several cell lines, showing also that MTx activated phosphoinositide (PI) breakdown in a Ca2+-dependent
manner but it was not inhibited by Cav channel blockers, suggesting the activation of phospholipase C
(PLC) or the activation of other Ca2+ channels. The fact that MTx action on PI breakdown was not mim-
icked by Ca2+ ionophores supported the notion that MTx activated PLC directly. However, in the work of
Gusovsky et al.64 using HL60 cells, it became clear that MTx action on PI turnover was associated to a Ca2+-
induced activation of PLC, rather than a direct PLC activation by MTx. Furthermore, the work of Pin et al.65
suggested that the effects of MTx on gamma-aminobutyric acid (GABA) release in striatal neurons were
associated to a Ca2+-dependent Na+ inux, introducing the notion of an additional cellular target for MTx.
Similar results of a Ca2+-dependent Na+ inux was reported by Sladescek et al.66 The pleiotropic effects and
action mechanisms of MTx on different cells was reviewed by Hamilton and Perez.67
Although the effects of MTx were pleiotropic, they all seemed to involve an intracellular Ca2+ increase.
The fact that MTx activated cation channels was clear from direct voltage-clamp current measurements
(e.g., [38,68–81]). Some of the proposed channels were Cav and also nonselective cation channels such as
store-operated channels (SOCs). Furthermore, in some cells such as skin broblasts, MTx seems to activate
large conducting channels82,83 leading to cell lysis. Hence, the action of MTx would be highly dependent on
the channel expression prole of each cell type and also on the MTx concentration used (e.g., ). Due to
scope limitations in this review, we have excluded a discussion of all cellular effects downstream of the MTx
interaction with membranes, on which a wealth of data is undoubtedly available in the literature.
The ability of MTx to activate Cav and other cationic channels posed the question as to whether
the toxin was activating channels by inserting itself in the membrane and perturbing the phospholipid
membrane structure (see MTx interaction with membranes in the following texts) or whether it had spe-
cic interactions with the proteins forming the channels. Murata et al.85 reported that either a removal
of the sulfated residues from MTx or a hydrogenation of the molecule decreased (by several orders of
magnitude) its ability to induce Ca2+ entry or PI breakdown in insulinoma or glioma cells. When these
MTx derivatives were used together with intact MTx, they acted as blockers of the MTx effect. The work
of Konoki et al.86 describing inhibition of MTx-induced Ca2+ entry in glioma C6 cells by brevetoxin
and synthetic fragments of MTx (corresponding to a sulfated portion, rings EF-GH, and rings LM-NO)
strongly suggested that MTx acted on specic sites to activate Ca2+ entry in these cells. Interestingly,
the sulfated fragment of MTx (EF-GH rings, and hence, a portion expected to remain in the aqueous
phase or at the lipid–water interphase) was more potent in inhibiting MTx effects on Ca2+ uptake than
the LM-NO rings. Recent work by Oishi et al.87 clearly showed that an articial ladder-shaped heptacy-
clic polyether was the most potent substance described to date capable of inhibiting MTx-induced Ca2+
inux in glioma C6 cells, strongly suggesting the existence of a specic MTx binding site in these cells.
However, this work did not give insight into the molecular mechanisms of action of MTx.
23.6.2 MTx Interaction with Membranes
The insertion of MTx in membranes was rst suggested by Konoki et al.,88 with rings
RSTUVWXYZA′B′C′D′E′F′ inserted in and spanning the phospholipid bilayer and rings
ABCDEFGHIJKLMNOPQ —containing the sulfated parts of the molecule—staying in the aqueous
684 Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection
environment. This work was later cited by Murata et al.,89 Nicolau et al.,90 and Nicolau and Aversa91
to discuss the properties of MTx. This association of MTx with phospholipid membranes was deduced
from the structural properties of MTx and by analogy to the properties of other ladder-shaped polyether
toxins.89 However, to the best of our efforts, we were unable to nd the specic model or program used
to estimate MTx distribution in phospholipid membranes. Thus, in order to theoretically estimate the
stability of the relatively hydrophobic portion of MTx in a phosphatidylcholine phospholipid membrane,
we performed force eld parameterization of rings P–F′ and molecular dynamics as shown in the follow-
ing texts. The molecular structure of MTx was taken from Nicolau and Frederic4 0 (Figure 23.1). Partial
parameterization with the CHARMM force eld was accomplished taking different sections of the mol-
ecule from ring P to F′ using the CCPN web applications (http://www.ccpn.ac.uk/). The 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer was built with visual molecular dynamics (VMD
1.9).92 The mentioned fragment of MTx was distributed in the center of the bilayer, and the program not
(just) another molecular dynamics (NAMD) allowed to iteratively locate the molecule to acquire a con-
formation giving steady values of potential energy parameters (http://www.ks.uiuc.edu/Research/vmd/).
This modeling clearly shows that rings P–F′ and tail were stable within the lipid bilayer (Figures 23.6
and 23.7), and this conformation is likely restricted and modied by the more hydrophilic portion of MTx
(see also Murata et al.89). This distribution of the hydrophobic portion of MTx is consistent with the pro-
posed role of this part of MTx (or similar portions of ladder-shaped polyether toxins) in the interaction
with integral membrane α-helices.89,93 However, these results do not exclude that the hydrophilic por-
tion of MTx, anchored in the membrane by the hydrophobic portion, could also interact externally with
channels86 or, as we could cautiously propose, that both mechanisms could be responsible for activation
of different types of channels in biological membranes.
23.7 MTx Bioactivity and Applications
Ca2+ is an ubiquitous secondary intracellular messenger responsible for mediating a multitude of cellular
responses as diverse as proliferation, development, contraction, secretion, and fertilization. Ca2+ action is
quite simple: cells at rest have an intracellular concentration of ∼100 nM but are activated when this level
rises to ∼1 μM. However, the universality of Ca2+ as an intracellular messenger depends on its remark-
able capacity to create a wide range of spatial and temporal signals.
FIGURE 23.6 The initial position of MTx in the 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) membrane
was set with the plane of the molecule parallel to the POPC/water inter phase. This position and conformation as well as the
environment were then allowed to drif t with molecula r dyna mics calculations toward steady states with minimal energy
conformations. Water molecules: oxygen = red, hydrogen = white. Phospholipids: light blue large chains. MTx: darker blue
at the center of the bilayer. (http://www.ibt.unam.mx/server/ PRG.base?tipo:doc,dir:PRG.curriculum,par:ctrevino)
685Maitotoxin: An Enigmatic Toxic Molecule with Useful Applications in the Biomedical Sciences
MTx has attracted much attention given its powerful bioactivity involving disruption of Ca2+ homeo-
stasis. MTx is not only one of the most potent toxins, but it also possesses multiple activities that appear
to be linked to elevation of intracellular Ca2+ concentration. Thus, the toxin serves as a versatile tool for
studies on cellular events associated with intracellular Ca2+ changes that are of particular interest, includ-
ing hormone secretion, programmed cell death activation, and fertilization.
23.7.1 Insulinotropic Actions of MTx
Although some hormones such as insulin-like growth factor and adiponectin have hypoglycemic effects,94
insulin has long been considered the only hypoglycemic agent in mammals. Insulin is synthesized and
secreted by pancreatic β-cells located in specialized structures, the islets of Langerhans. In general, β-cells
adjust insulin secretion to the prevailing blood glucose levels by a process called glucose-stimulated insu-
lin secretion (GSIS). Inside pancreatic β-cells, glucose metabolism induces insulin secretion by altering
the cellular array of messenger molecules. ATP is particularly important given its role in regulating cation
channel activity dependent upon its hydrolysis.
t= 20 ps
5,000 10,000 15,000 20,000 25,000 30,000 35,000
t= 40 ps t= 60 ps
FIGURE 23.7 (a) Plot of energy (kcal/mol) versus the molecular simulation steps (1 ps corresponds to 500 steps). Each
curve corresponds to the different components of potential energy: binding energy (blue line), angles (red line), electro-
static energy (green line), and kinetic energy (orange line). (b) Spatial distribution of MTx in the 1,2-dioleoyl-sn-glycero-3-
phosphocholine (DOPC) membrane at 20, 40, and 60 ps. From the potential energy curves and the molecular images, it can
be seen that this portion of MTx is stably located within the hydrophobic portion of the phospholipid bilayer at 40 ps, with
a location a nd dynamics that certainly would be restricted by the hydrophilic portion that is expected to be located at the
water/phospholipid interface. (See also Murata, M. et al., Bull. Chem. Soc. Jpn., 81, 307, 2008.)
686 Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection
ATP-dependent K+ (KATP) channels play a key role in insulin secretion. Under euglycemic conditions,
KATP channels are maintained in an open state, resulting in K+ efux and thus clamping the resting mem-
brane potential close to −70 mV. When glucose is elevated, ATP levels increase and displace bound ADP
on KATP channels, which results in channel closure. These events lead to a small membrane depolariza-
tion that activates voltage-dependent Ca2+ channels, which trigger Ca2+ inux and raise the intracellular
Ca2+ concentration, thus promoting insulin secretion.95,96
Several reports have shown that some members of the transient receptor potential (TRP) channel
family, which mediate nonselective cationic currents (NSCCs), are expressed, and might contribute to
pancreatic β-cell function. Although the role of TRP channels in β-cells remains largely enigmatic, these
channels may provide an alternative for the depolarizing background membrane conductance required
for the cells to depolarize upon KATP channel closure.96 Indeed, it has been reported that thermosensitive
TRPM2, TRPM4, and TRPM5 channels control insulin secretion levels by sensing intracellular Ca2+
increase, NAD+ metabolites, or hormone receptor activation.97
In addition to glucose, insulin secretion may be regulated by diverse chemical messengers such as
neurotransmitters and hormones,96 as well as by exogenous substances such as toxins that act on ion
channels. Hence, some peptide toxins present in the venom of marine organisms may affect NSCCs and
serve as potential insulinotropic agents. For example, it has been shown that the activity of TRPV1, a
channel that modulates insulin secretion in β-cells, is affected by crude cell-free extracts obtained from
marine invertebrates.98,99 Interestingly, one of these extracts has shown insulinotropic activity.100
By activating NSCCs, MTx has also shown insulinotropic activity in insulinoma cells. The time
course of these currents is very similar to that evoked by incretin hormones such as glucagon-like pep-
tide-1 (GLP-1), which stimulate glucose-dependent insulin secretion by activating cAMP-mediated sig-
naling pathways.101 Likewise, NSCCs in insulinoma cells can be attenuated by application of a Ca2+
SOC blocker SKF 96365, suggesting a contribution of the mammalian TRP-related channels in these
currents.102 The ability to activate NSCCs in insulin-secreting cells stresses the role of MTx as a helpful
tool for the analysis of ion channels and insulin secretion.103 Likewise, the role of MTx as a novel blood
glucose-lowering agent remains an interesting topic for future research.
23.7.2 MTx as Interleukin-1β Secretagogue and Oncotic Death Inducer
Most inammatory reactions are mediated by cytokines, including IL-1, IL-6, TNF-α, and TGF-β. The
term interleukin-1 (IL-1) refers to two cytokines, IL-1α and IL-1β, which are the master cytokines of
local and systemic inammation.104,105 In particular, IL-1β is primarily synthesized in activated macro-
phages as an immature protein that remains cytosolic until converted through proteolytic cleavage by
caspase-1 into its mature active form, which can then be exported outside the cell.
Given its ability to induce cell death secondary to its disruption of Ca2+ homeostasis, MTx is likely to
trigger innate immune responses and inammation in vivo. Indeed, it has been suggested that the toxic
effect of MTx during shellsh seafood poisoning may involve a component mediated by secretion of
proinammatory cytokine IL-1β. In line with this, Verhoef and coworkers106 reported that MTx induces
a biphasic release of IL-1β from bacterial lipopolysaccharide-primed macrophages. At subnanomolar
concentrations, MTx induced mature IL-1β release via a mechanism that can be blocked by high extra-
cellular K+ or nominally zero extracellular Ca2+. MTx may therefore represent an exceptional tool for
studying specic components of the innate immune response and/or the physiology of inammatory
effector cells such as monocytes, macrophages, and neutrophils. One representative example of this
type of application is the work by Mariathasan and colleagues.107 These authors found that cryopyrin is
responsible for assembly of the so-called inammasome, a cytosolic complex of proteins that activates
caspase-1 to process the proinammatory cytokine IL-1β. Cryopyrin is essential for inammasome acti-
vation in response to signaling pathways triggered by specic bacterial infections as well as by MTx.
It is worth mentioning here that there are several human diseases caused by different mutations in the
cryopyrin gene, including familial cold autoinammatory syndrome, Muckle–Wells syndrome, as well as
chronic infantile neurological cutaneous and articular syndrome.108 Mutations in the cryopyrin gene are
associated with gain of function leading to an enhanced and faster production of IL-1β. In this scenario, MTx
could be used as a probe to study possible mechanisms of release and implications of IL-1β overproduction.
687Maitotoxin: An Enigmatic Toxic Molecule with Useful Applications in the Biomedical Sciences
Likewise, the second phase of IL-1β release induced by MTx from macrophages occurs at nanomolar
concentrations.106 In this case, MTx produces secretion of unprocessed IL-1β, which is indicative of cell
lysis. Interestingly, cell death induced by MTx shares some elements involved in the signaling cascade
activated by stimulation of purinergic receptors of the P2Z/P2X7 type.82,83,109 As discussed earlier, MTx
initially activates Ca2+-permeable channels and then induces the formation of large cytolytic/oncotic
pores (COPs) that allow molecules <800 Da to enter the cell. These effects are similar to those observed
upon activation of P2Z/P2X7 receptors in a variety of cell types, raising the intriguing possibility that
MTx and P2Z/P2X7 receptor stimulation activate a common cytolytic pore.
Given the high permeability of the MTx-induced channels for Ca2+ transport and the structural simi-
larity of MTx with palytoxin—a marine peptide toxin that converts the plasmalemmal Na+/K+-ATPase
(NKA) pump into a channel—it has been proposed that MTx may activate another member of the P-type
ATPase family, specically the plasmalemmal Ca2+-ATPase (PMCA) pump. The results obtained by
Sinkins and colleagues110 are consistent with this idea and suggest that MTx binds to PMCA and con-
verts the pump into a Ca2+-permeable nonselective cation channel. Therefore, MTx could be used as a
cell death inducer to unveil some of the molecular mechanisms involved in this process. For instance,
whether or not the channel mode of operation of the PMCA plays a role in pathological cell death could
be an interesting possibility for future investigations.
23.7.3 MTx and Sperm Physiology
Fertilization is fundamental for the preservation of life by sexual reproduction. The ability of the sperm
and the oocyte to recognize, adhere to, and fuse with each other is a crucial aspect of fertilization. All
these processes are largely determined by nicely orchestrated ionic uxes.111 Hence, it is well known that
raises in intracellular Ca2+ play crucial roles in sperm functions such as capacitation, motility, and the
The acrosome reaction is a secretory process triggered in sperm by components of the outer layer of
the egg, and in many species it must occur before the sperm can fertilize the egg. However, the mecha-
nisms responsible for increasing intracellular Ca2+ and resulting in the biochemical events that trigger
the acrosome reaction are not fully understood. Two different types of Ca2+ channels have been proposed
to participate in mammalian sperm acrosome reaction: one necessary for a fast transient change in Ca2+
levels and another needed to sustain an elevated intracellular Ca2+ concentration. The membrane path-
way responsible for the rst phase of Ca2+ entry seems to belong to the Cav channel family, while the
sustained Ca2+ inux may be carried through a store depletion-operated pathway.112,113 Interestingly, it
has been reported that several TRP channels are expressed in sperm and may be important for the sus-
tained Ca2+ entry that drives the acrosome reaction.113–115
Evidence obtained in our laboratory indicated that MTx activates a Ca2+ inux that induces the
mammalian acrosome reaction. The data initially suggested that the actions of MTx were comparable
to those of other agents that promote a sustained increase in intracellular Ca2+ and drive the mam-
malian sperm acrosome reaction, including the physiological ligands of the zona pellucida (ZP).116
More recently, however, we found differences in the acrosome reaction induced by MTx and the ZP
in human and mouse sperm. Our data indicated that the acrosome reaction induced by the physiologi-
cal ligands and by MTx occurred through distinct pathways.117 By using specic PLC antagonists,
the participation of a PLC-dependent signaling pathway in the ZP-induced acrosome reaction was
conrmed. In contrast, the use of PLC in hibitors blocked the acrosome reaction induced by MTx in
mouse but not in human sperm, unveiling species-specic variants of the acrosome reaction induced
by the toxin.
Lastly, MTx has also been instrumental in unveiling of some of the mechanisms of the spermato-
genic cell regulation exerted by Sertoli cells. We have previously shown that glucose and lactate, two
substrates secreted by Sertoli cells toward the adluminal compartment in the seminiferous tubules,
can modulate the activity of MTx-sensitive Ca2+ channels in enzymatically dissociated rat spermato-
cytes and spermatids.118 By inducing changes in intracellular Ca2+, both substrates can activate a Ca2+/
calmodulin-dependent protein kinase that results in the phosphorylation of MTx-sensitive channels.
We have recently developed in our laboratory a methodology to study Ca2+ signaling in spermatogenic
688 Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection
cells by preparing slices of seminiferous tubules. This methodology has the advantage of preserving
tissue architecture and intercellular connections,119 and we demonstrated that Ca2+ signaling differs in
dissociated spermatogenic cells compared to spermatogenic cells inside the tubules. Here, we show
that MTx induces a generalized Ca2+ increase when applied to this seminiferous tubule slice (STS)
preparation, which can be used to further study spermatogenic cell Ca2+ dynamics in a physiological
environment that preserves cell interactions within the tubule (Figure 23.8). The knowledge generated
using this approach could have relevant implications for the understanding of the physiological process
of spermatogenic cell regulation by Sertoli cells, as well as the hormonal control that they may exert on
spermatogenesis, which is not possible to study in vitro.
23.8 Final Remarks
MTx has inspired vast experimentation by organic a nd biological researchers due to its st ructura l
complexity and intricate mode of action. However, k nowledge of both its organic and biochemi-
cal synthesis, as well as of its biological ta rget(s), rema ins incomplete. The current lack of com-
mercially available MTx underscores the importance for achieving its organic synthesis, so t hat it
can become readily available again for studies on Ca2+ dyna mics in different systems and also to
help identify its putative receptor(s). This would lead to a better understanding of the molecular
mechanisms involved in MTx action, which in tur n may help explain the apparent discrepancies in
its functional modalities. Knowledge in this area could also help to nd appropriate treatment or an
antidote for ciguatera.
T (seg)(d) (e)
FIGURE 23.8 STSs are obtained as reported. Briey, the Tunica albuginea is removed from mice testis and the semi-
niferous tubules are mechanically dispersed with tweezers in a Petri dish containing Ringer solution (in mM: 125NaCl,
2.5KCl, 2CaCl2, 1MgCl2, 1.25NaH2PO4, 26NaHCO3, 12 glucose, gassed with 5% CO2, 95% O2, adjusted to pH 7.4). The
dispersed tubules are embedded in agar (low melting point, 3%) to form a cube that is mounted on the plate of a vibratome,
and 160 μM thick slices are obtained. STSs were loaded with uo 4-AM (20 μM) immobilized with a nylon mesh, placed on
the stage of a microscope, and continuously per fused (2 mL/min) with gassed physiological solution at room temperature.
Fluorescence images were acquired (for equipment deta ils, see ) every second with an exposure/illumination time of
10 ms for a total of 10 min (600 images). Pseudocolored uorescence image obta ined from the recording of STS before
(a) and after addition of 50 nM MTx (b and c). Fluorescence traces obta ined from three different cells in the STS shown
in (a) and (d). Fluorescence image (black and white) of the same STS after incubation with uo 4-AM (e). (From Sánchez-
Cárdenas, C. et al., Biol. Reprod., 87, 92, 2012.)
689Maitotoxin: An Enigmatic Toxic Molecule with Useful Applications in the Biomedical Sciences
The authors would like to thank José Luis de la Vega, Yoloxochitl Sánchez, and Shirley Ainsworth for
technical assistance. This work was supported by grants DGAPA-UNAM (IN202212 to CT, IN217210 to
ILG) and CONACyT (99333 to CT, 84362 to ILG) and Fondecyt (1110267 to JGR).
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