Tài liệu Báo cáo khoa học: Marine toxins and the cytoskeleton: a new view of palytoxin toxicity ppt

MINIREVIEW
Marine toxins and the cytoskeleton: a new view of
palytoxin toxicity
M. Carmen Louzao, Isabel R. Ares and Eva Cagide
Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain
Introduction
Palytoxin is a potent marine toxin that was first
isolated from a coelenterate tentatively identified as
Palythoa sp. This toxin was shown to be extremely
toxic to mammals, with a reported LD
50
value of just
0.45 lgÆkg
)1
after intraperitoneal injection into mice
[1]. In addition to being highly toxic, palytoxin has
large and complex structure (Fig. 1), which was deter-
mined in the 1980s [2]. This water-soluble molecule
consists of a long, partially unsaturated, aliphatic
backbone with spaced cyclic ethers and 64 chiral
centers. Examination of the structure shows that there
is, in fact, a group of different palytoxins, whose
molecular weights vary according to the species from
which they are obtained. Several biogenic origins of
palytoxins have been proposed, as these toxins have
been found not only in zooanthids but in sea anemo-
nes, polychaete worms, crabs, and herbivorous fishes,
probably due to an accumulation of the toxin by the
food chain in the organisms living close to the zoan-
thid colonies [3].
It has been reported that dinoflagellates of the genus

Ostreopsis are the most probable origin of palytoxin
[4,5]. In fact, several toxins with palytoxin-like charac-
teristics have been described and named according to
Keywords
actin filament; cytoskeleton; ostreocin-D;
Ostreopsis; ovatoxin-a; palytoxin
Correspondence
M. C. Louzao, Departamento de
Farmacologı
´
a, Facultad de Veterinaria,
Universidad de Santiago de Compostela,
Campus de Lugo, 27002 Lugo, Spain
Fax: +34 982 252 242
Tel: +34 982 252 242
E-mail:
(Received 7 July 2008, revised
12 September 2008, accepted
16 September 2008)
doi:10.1111/j.1742-4658.2008.06712.x
Palytoxin is a marine toxin first isolated from zoanthids (genus Palythoa),
even though dinoflagellates of the genus Ostreopsis are the most probable
origin of the toxin. Ostreopsis has a wide distribution in tropical and sub-
tropical areas, but recently these dinoflagellates have also started to appear
in the Mediterranean Sea. Two of the most remarkable properties of paly-
toxin are the large and complex structure (with different analogs, such as
ostreocin-D or ovatoxin-a) and the extreme acute animal toxicity. The
Na
+
⁄ K

+
-ATPase has been proposed as receptor for palytoxin. The marine
toxin is known to act on the Na
+
pump and elicit an increase in Na
+
per-
meability, which leads to depolarization and a secondary Ca
2+
influx,
interfering with some functions of cells. Studies on the cellular cytoskeleton
have revealed that the signaling cascade triggered by palytoxin leads to
actin filament system distortion. The activity of palytoxin on the actin cyto-
skeleton is only partially associated with the cytosolic Ca
2+
changes; there-
fore, this ion represents an important factor in altering this structure, but it
is not the only cause. The goal of the present minireview is to compile the
findings reported to date about: (a) how palytoxin and analogs are able to
modify the actin cytoskeleton within different cellular models; and (b) what
signaling mechanisms could be involved in the modulation of cytoskeletal
dynamics by palytoxin.
Abbreviations
F-actin, filamentous actin; G-actin, globular actin.
FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS 6067
the producing species [6]. Among the nine different
Ostreopsis species existing, five of them have been
reported as producers of toxic substances [6], but just
four of them have been named: Ostreopsis siamensis
was reported to produce ostreocin-D (Fig. 1), a potent

palytoxin analog with a LD
50
value of 0.75 lgÆkg
)1
when given intraperitoneally [4], Ostreopsis lenticularis
produces the neurotoxic ostreotoxins, Ostreopsis
mascarenensis produces mascarenotoxins, and recently,
Ostreopsis ovata has been identified as the producer of
ovatoxin-a [7].
Ostreopsis species are important components of
tropical and subtropical reef environments, but
recently these dinoflagellates also started to appear
in temperate waters, such as those from the Mediter-
ranean Sea, even producing toxic outbreaks [7,8].
Human toxicity due to palytoxin has been reported
as food poisoning (named clupeotoxism) and respira-
tory intoxications. Palytoxin enters the food chain
and accumulates mainly in fishes such as sardines,
herrings and anchovies from tropical seas, causing
neurological and gastrointestinal disturbances asso-
ciated with clupeotoxism [1]. Symptoms include a
bitter and metallic taste followed by nausea, vomit-
ing and diarrhea, with mild to acute lethargy.
Several hours later, burning sensations around the
mouth and in the extremities, impairment of sensa-
tion, muscle spasms and tremor myalgia, dyspnea
and dysphonia occur, possibly leading to death due
to myocardial injury. Recently, in Italy, exposure to
marine aerosols has been reported that may also
cause human illness, with fever being associated with

serious respiratory distress, mild dyspnea, wheezes,
and in some cases conjunctivitis [7,8]. Italian coasts
are not the only seawaters where Ostreopsis species
have appear; they have been found in the waters
around Spain and Greece as well, indicating that the
expansion in the Mediterranean Sea of these species
in recent years is becoming a potential risk for
human health in Europe.
Fig. 1. Palytoxin and ostreocin-D structures.
Palytoxin activity against the cytoskeleton M. C. Louzao et al.
6068 FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS
Distortion of the Na
+
pump
At the cellular level, a broad range of studies have
indicated that the Na
+
pump or Na
+
⁄ K
+
-ATPase is
the higher-affinity cellular receptor for palytoxin [9,10].
This protein transports three Na
+
out the cell in
exchange for two K
+
that are driven in by hydrolyzing
one molecule of ATP. This generated electrochemical

gradient is essential in maintaining electrolyte homeo-
stasis. The Na
+
⁄ K
+
-ATPase is also the specific target
for heart glycosides such as ouabain, which bind to
the pump and block it when the Na
+
-binding sites are
open to the extracellular side [10]. Nevertheless, paly-
toxin seems to convert the pump into a nonspecific ion
channel, allowing Na
+
and K
+
fluxes [9,10]. Despite
having a different interaction with the Na
+
⁄ K
+
-
ATPase, ouabain blocks palytoxin effects in several
systems, and palytoxin inhibits ouabain binding as well.
It is known that changes in ion fluxes are the imme-
diate effects of palytoxin on the cells. In particular,
increasing the Na
+
permeability leads to depolari-
zation and a secondary Ca

2+
influx [11,12] that may
lead to multiple events regulated by Ca
2+
-dependent
pathways. The mechanisms by which this rise in intra-
cellular Ca
2+
([Ca
2+
]
i
) is produced have not been
completely elucidated. Nevertheless, in excitable cells,
at least three mechanisms have been reported to be
involved in the intracellular Ca
2+
increase: (a) voltage-
dependent Ca
2+
channels activated by the initial depo-
larization of the membrane; (b) Na
+
–Ca
2+
exchanger
in reverse mode, which drives Ca
2+
into the cells
because of the intracellular Na

+
influx and membrane
depolarization; and (c) an as yet unidentified pathway
that is independent of changes in membrane potential
or changes in pH [9].
The actin cytoskeleton as a selective
target for palytoxins
Actin filaments or microfilaments are polymers of actin
that, together with a large number of actin-binding and
associated proteins, constitute the actin cytoskeleton
[13]. For a long time, it was believed that the role of
this system was mainly structural, providing the sup-
port needed to maintain the shape and organization of
the cell and the scaffolding for the actions of catalytic
molecules such as motor proteins. Nowadays, the actin
cytoskeleton is widely recognized as an enormously
dynamic structure that undergoes constant reconstruc-
tion and reorganization, which is possible because of its
ability to switch rapidly between a filamentous actin
(F-actin) polymeric form and a monomeric globular
actin (G-actin) form [14]. This dynamism enables it to
quickly remodel its structure as a consequence of cellu-
lar stimuli, and to participate in localized responses to
external agents or regional events within a cell. Many
natural compounds that have been isolated from mar-
ine sources exert their cytotoxicity by modulating
cytoskeletal properties, in particular those concerning
actin filaments [15–17]. Although this phenomenon
could seem surprising, it is not so surprising if one con-
siders the pivotal role of this structure in many cellular

functions. In eukaryotic cells, the actin cytoskeleton is
required for cell motility and surface remodeling; it is
essential for several contractile activities, such as mus-
cle contraction and the separation of daughter cells by
the contractile ring during cytokinesis; it controls cell–
cell and cell–substrate interactions, together with adhe-
sion molecules; and it actively participates in signal
transduction, cell volume regulation, secretion, and
surface receptor modulation [18–23].
Although palytoxin has been investigated as a
compound that is able to interfere with some of these
cellular functions [24–27], its effects on the actin cyto-
skeleton were not studied until few years ago. The
same is true of other palytoxin-like compounds, such
as ostreocin-D and ovatoxin-a, although in these cases
almost no information is available concerning their
biological activity. The first investigations assaying
palytoxin and ostreocin-D toxicities were performed in
freshly isolated intestinal cells (rabbit enterocytes from
the duodenum–jejunum), using fluorescent phalloidin
as an F-actin marker and laser-scanning cytometry
and confocal microscopy as techniques for analysis
[28]. There were two reasons for starting this type of
study with an intestinal model: (a) the great complexity
of the actin cytoskeleton in these cells; and (b) the
severe gastrointestinal toxicity exerted by palytoxin
in vivo [29,30]. Nanomolar concentrations (75 nm)of
palytoxin or ostreocin-D, and 4 h of incubation, were
enough to induce sharp F-actin disassembly and to
almost halve the quantity of F-actin on intestinal cells.

Interestingly, a purified extract of O. ovata that con-
tained a putative palytoxin-like compound was tested
under the same experimental system, and identical
results were obtained [6]. As previously described, the
actin filament system seems to be closely linked to
morphological cell characteristics, and therefore it
could be reasonable to expect alterations in shape.
Nevertheless, they were not observed here. Instead of
this, enterocytes retained their typical columnar mor-
phology after losing many of their microfilaments.
Similar cases using cultured cells have been reported in
the literature [31], and other cytoskeletal components
could also be participating in maintenance of the
cytoarchitecture [32,33].
M. C. Louzao et al. Palytoxin activity against the cytoskeleton
FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS 6069
The above assays were performed by incubating tox-
ins and intestinal cells in suspension. After treatment,
they were attached to the substratum with poly-
l-lysine and later analyzed. To ensure that floating
cells were sensitive to morphological modifications by
cytotoxic agents, latrunculin-A was tested with an
identical procedure to that used for palytoxin and
ostreocin-D [28]. Latrunculin-A is a toxic compound
extracted from the Red Sea sponge Negombata magni-
fica that inhibits actin polymerization and induces
morphological changes in living cells [34,35]. Latruncu-
lin-A-treated cells lost one-third of their total actin
content (reduction by 33 ± 6.7%) and underwent
rounding (Fig. 2). This fact helps to confirm that

maintenance of cells in suspension was not responsible
for the absence of shape changes when palytoxins were
probed. Another interesting feature observed in the
case of latrunculin-A was some brush border disorga-
nization in the apical region of intestinal cells that was
not found after palytoxin or ostreocin-D treatments.
New findings have demonstrated again the palytoxin
activity on the cytoskeleton of intestinal cells. This
toxin induced dose- and time-dependent F-actin dis-
ruption in cultured Caco-2 cells [36], a human carcino-
genic line that undergoes spontaneous in vitro
enterocytic differentiation [37]. Interestingly, a correla-
tion among partial F-actin breakdown after 1 h of
palytoxin treatment (100 nm), morphological altera-
tions and cell detachment from substratum was also
found in that study. In this respect, the outcome of
ostreocin-D treatment of CaCo-2 cells remains
unknown.
The impact of palytoxin action on actin filament sys-
tem is not restricted to epithelial cells. In a recent
report, Louzao et al. [12] demonstrated that palytoxins
were able also to interfere with the cytoskeleton of
neuronal cells. These assays were carried out on the
human neuroblastoma cell line BE(2)-M17, an excit-
able model previously utilized for exploring anticyto-
skeletal effects and changes in ion fluxes in response to
marine toxins [38–41]. Here, palytoxin and ostreocin-D
triggered a cascade of cytotoxic events, ranging from a
Fig. 2. Left: histograms obtained with laser-scanning cytometry, displaying a reduction in the fluorescence associated with F-actin of freshly
isolated rabbit intestinal cells incubated with palytoxin (upper) or latrunculin-A (lower) in comparison to control cells. Previous to measure-

ment, the cellular actin cytoskeleton was specifically stained with fluorescent phalloidin. Right: transmission images recorded by confocal
microscopy show the morphological differences between palytoxin (75 n
M) and latrunculin-A (10 lM) treatments after 4 h of incubation.
Arrows indicate the alterations on microvilli induced in latrunculin-A-treated cells.
Palytoxin activity against the cytoskeleton M. C. Louzao et al.
6070 FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS
rapid depolarization and cytosolic Ca
2+
increase to
cytoarchitectural restructuring. Time-dependent studies
using a 75 nm concentration of palytoxin and ostreo-
cin-D provided interesting insights into how these
toxins modify the cytoskeleton of neuroblastoma cells:
(a) the distortion of the actin cytoskeleton begins at
early stages, being detectable after 10 min of toxin
incubation; and (b) both microfilament reorganization
and morphological alterations are subsequent to the
start of F-actin disruption [12]. In agreement with the
studies using the CaCo-2 cell line, palytoxin also elicited
loss of cellular adhesion and dose- and time-dependent
F-actin disassembly in neuroblastoma cells, leading to
its entire collapse in 24 h after toxin doses of 1 nm.
Evidence from a number of systems suggests that
influx of Na
+
and ⁄ or Ca
2+
is associated with many
palytoxin-induced responses, including muscle contrac-
tion, neurotransmitter release, and oncotic death

[25,42,43]. A connection has also been found between
Ca
2+
influx and palytoxin and ostreocin-D effects on
the actin cytoskeleton. This phenomenon seems to
occur in different ways in the different cell types inves-
tigated. Ares et al. [28] found that in epithelial cells
from the rabbit duodenum–jejunum, omission of extra-
cellular Ca
2+
(nominally Ca
2+
-free medium) halved
the effect of palytoxins on F-actin disassembly. Those
data led to the idea that these toxins modified the actin
filament system of intestinal cells not only by modulat-
ing some signaling pathway activated by external
Ca
2+
, but also by acting on another, still unknown,
element. Results obtained with neuroblastoma cells
indicated that palytoxin and ostreocin-D stimulated
similar decreases in F-actin quantity, independently of
extracellular Ca
2+
entry. This effect is not related to
Ca
2+
being released from internal stores, as palytoxin
and ostreocin-D do not induce increases of Ca

2+
in
nominally Ca
2+
-free conditions [12]. On the other
hand, the presence or absence of extracellular Ca
2+
was associated with a different F-actin organization in
toxin-treated cells, which seems to suggest a new role
for this cation in palytoxin action against the actin
cytoskeleton (Fig. 3). At present, in spite of the impor-
tance of the unpolymerized actin pool for the mainte-
nance of the F-actin system, the cellular response
Fig. 3. Response induced by palytoxin and ostreocin-D on the actin cytoskeleton of neuroblastoma cells after 4 h of incubation under differ-
ent extracellular Ca
2+
conditions. Fluorescent phalloidin was utilized for labeling the cellular actin cytoskeleton before quantitative analysis
with laser-scanning cytometry and imaging with confocal microscopy.
M. C. Louzao et al. Palytoxin activity against the cytoskeleton
FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS 6071
triggered by palytoxin and ostreocin-D activity at this
level has not yet been reported. Recent studies per-
formed by Ares et al. have revealed that these marine
toxins also induce alterations in G-actin of neuroblas-
toma cells (I. R. Ares, E. Cagide, M. C. Louzao,
B. Espin
˜
a, M. R. Vieytes, T. Yasumoto and L. M.
Botana, unpublished results).
Conclusions and perspectives

This minireview compiles the latest findings on how
palytoxin and ostreocin-D are able to act on the actin
filament system within cells. A signaling pathway involv-
ing Ca
2+
influx is partially related to that activity. How-
ever, as Ca
2+
influx is not responsible for some of the
effects elicited by palytoxins on the cellular actin cyto-
skeleton, more factors must be involved. Assuming that
the Na
+
pump is the target of palytoxin, one interesting
option would be that Na
+
⁄ K
+
-ATPase per se activates
some mechanism connected to the actin cytoskeleton
after its interaction with palytoxin (or ostreocin-D, if
they share the same target). This possibility arise from
several recent studies, where has been suggested that in
addition to pumping ions, Na
+
⁄ K
+
-ATPase also acts
as a signal transducer [44,45]. In this respect, studies
carried out with ouabain, a natural blocker of the Na

+
pump and an inhibitor of palytoxin action, have pro-
vided interesting findings. Through the partial inhibition
of Na
+
⁄ K
+
-ATPase, and regardless of changes in intra-
cellular ion concentrations, ouabain-induced signaling
pathways have been recently found in several cellular
models [44–47]. It has been proposed that the ouabain-
bound Na
+
⁄ K
+
-ATPase is capable of recruiting and
activating protein tyrosine kinases through specific
protein–protein interactions [45]. On the other hand, it
should be not forgotten that the Na
+
pump could inter-
act with cytoskeletal components. Specific cytoskeletal
proteins thought to interact with Na
+
⁄ K
+
-ATPase,
either directly or indirectly, include spectrin, actin and
ankyrin [48,49], and even regulation of Na
+

pump
activity might depend on its linkage to the actin filament
system [50,51]. In any case, the study of the possible
existence of some Na
+
⁄ K
+
-ATPase-mediated signaling
mechanism involved in the modulation of actin cytoskel-
etal dynamics by palytoxins opens a new line to follow
in the future in this field of investigation.
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