Seafood Toxins: Classes, Sources, and
Toxicology
28
Patrizia Ciminiello, Martino Forino, and Carmela Dell’Aversano
Contents
28.1
28.2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Azaspiracids: A Recent Class of Toxins Targeting the Human Gastrointestinal
Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28.3 Brevetoxins: Typical Aerosolized Red-Tide Neurotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . .
28.4 Ciguatera: A Fitting Example of How Chemical Structure of Marine Biotoxins Is
Investigated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28.5 Cyclic Imine Toxins: A Category of Miscellaneous Emerging
Toxic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28.6 Domoic Acids: The Only Marine Biotoxins Produced by Diatoms . . . . . . . . . . . . . . . . .
28.7 Okadaic Acids: The Subtle Threat of Potent Tumor Promoters . . . . . . . . . . . . . . . . . . . . .
28.8 Palytoxins: Potent Tropical Toxins Now Rife Across the Mediterranean Basin . . .
28.9 Pectenotoxins: Potent Cytotoxic Compounds with Still Unknown Potential
Implications to Public Health in the Long Term . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28.10 Saxitoxins: The First News We Have About Seafood Toxin–Related Human
Poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28.11 Yessotoxins: Potent Toxic Compounds with Controversial Impact on
Public Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Once believed to affect only scattered areas of the world, nowadays, harmful
algal blooms (HAB) plague virtually any coastal area of the planet. Usually
caused by a pool of microalgae, HAB-related outbreaks not only harm human
health but wreak havoc on maritime economy. Over the past decades, following
the steadily increase of human poisonings, an ever-growing number of countries
have been forced to set appropriate measures in order to cope with the sanitary
P. Ciminiello (*) • M. Forino • C. Dell’Aversano
Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli “Federico II”,
Via D. Montesano, 49, Naples, Italy
e-mail: ciminiel@unina.it, forino@unina.it, dellaver@unina.it
E. Fattorusso, W. H. Gerwick, O. Taglialatela-Scafati (eds.),
Handbook of Marine Natural Products, DOI 10.1007/978-90-481-3834-0_28,
# Springer Science+Business Media B.V. 2012
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P. Ciminiello et al.
and economic predicaments brought about by HABs. Out of the thousand
microalgae reported this far, only a limited number of them have been proven
to produce marine biotoxins. Such molecules, once assumed by seafood, easily
find their way up to the table of unaware consumers. Moreover, particular
phytoplankton blooms can directly affect humans through contaminated marine
aerosol. Depending on the symptoms shown by patients, the major human
illnesses caused by toxin-contaminated seafood are commonly referred to as
paralytic, neurotoxic, amnesic, diarrhetic shellfish, and ciguatera fish poisonings
(PSP, NSP, ASP, DSP, and CFP, respectively). In this chapter, a wide-ranging
and multifaceted overview of the main marine biotoxin classes is presented.
28.1
Introduction
As healthy a food for humans as it may be, occasionally, fish and shellfish can cause
serious harm to consumers by accumulating high levels of natural marine biotoxins
produced by noxious algae. Every year, some 50,000–500,000 human intoxications
following exposure to marine biotoxins are reported worldwide with an alarming
mortality rate on a global basis [1]. Until a few decades ago, only scattered
locations seemed to be plagued by harmful algal blooms (HABs), while nowadays,
every coastal State is virtually exposed to toxic outbreaks, often induced by more
than just one toxic algal species. Out of the thousands of microscopic algal species,
only several dozens have been this far individuated as producers of potent marine
biotoxins [2, 3], frequently occurring in seawater in such small amounts as to not
pose any hazard to human health and environment as well. Nevertheless, when
toxic algal species happen to proliferate abundantly in seawater, they can be
accumulated by filter-feeding shellfish, zooplankton, fishes, crabs, and tunicates,
among others [4–6]. As a result, algal toxins find their way up through the food
chain, landing onto the table of unaware seafood consumers.
The major illnesses following assumption of toxin-contaminated shellfish are
commonly referred to as paralytic, neurotoxic, amnesic, diarrhetic shellfish, and
ciguatera fish poisonings (PSP, NSP, ASP, DSP, and CFP, respectively), according
to the symptoms shown by patients [6, 7]. Beyond these well-known classic
intoxications, also other toxin-related poisoning syndromes have been described
after exposure to azaspiracid toxins, yessotoxins, cyclic imines toxins, and
palytoxins [6, 7]. ASP aside – which are caused by diatoms – all of the other
major intoxications are basically due to blooming in the phytoplankton of dinoflagellates – microscopic, often unicellular, flagellated algae.
The HAB impact on public health grows even more alarming if it is
considered that particular phytoplankton blooms can directly affect humans either
accidentally swimming in infested waters or breathing contaminated marine aerosol.
Even though HABs often take place as massive “red tides” or blooms capable of
discoloring seawater, sometimes, high concentrations of noxious cells get noticed
only because of the harm they provoke by means of their potent biotoxins, which
28
Seafood Toxins: Classes, Sources, and Toxicology
1347
rank among the most potent toxic compounds in the world, with some of them lethal
to humans even at doses in the range of few micrograms per kilogram [8].
The role toxins play in the internal economy of their producers has not yet been
totally understood. In this regard, toxins are likely used as tool for competing for
space, for fighting predation, or even for warding off possible overgrowth of other
organisms [9].
Unfortunately, toxin-contaminated seafood neither looks nor tastes any different
from safe seafood, and no culinary treatment can guarantee inactivation of marine
biotoxins. As a consequence, fish and shellfish farms have to run costly monitoring
programs in order to check constantly the quality of their products before putting
them on the market. At the moment, there are several chemical, functional, and
biological methods able to check the occurrence of algal toxins in seafood, each
with its own shortcomings. Considering that the only common feature of all of the
known marine toxins is toxicity, it is little wonder that live-animal bioassay, such as
the mouse bioassay, is still the most widely used detection method in any regulatory
setting [10]. It is generally carried out by intraperitoneally injecting a given sample
extract into a 20-g mouse kept under observation over a certain stretch of time, with
the purpose of determining the onset of specific symptoms and eventually the timeto-death. Both symptoms and time-to-death are correlated to the kind as well as to
the amount of a toxin possibly occurring in the injected extract. Toxicity is reported
in terms of “mouse units,” which refer to the amount of toxin(s) capable of killing
a mouse in a given stretch of time. Nonetheless, the mouse bioassay, even though
suitable to the analysis of nearly every class of biotoxins, is quite controversial. In
fact, it lacks specificity as it does not allow any differentiation between the various
components within each class of toxins, and in addition, it requires both specialized
animal facilities and proficiency without mentioning that many countries across the
world have been dealing with both ethical and scientific issues regarding the
employment of live-animal assay in their regulatory settings.
Besides their noxious impacts on human health, some marine toxins also induce
severe ecological disruption through occasional widespread killing of fish, shellfish,
marine mammals, seabirds, and any other living organism linked to the marine food
web [4, 5]. Furthermore, the steady increase of HABs across the world has become
a global concern also for the havoc they wreak on the economy. In fact, in coincidence
with toxic algal proliferations, shellfish production areas as well as aquacultures are
forced to stay closed for weeks, sometimes for months. From this predicament, fishery
and tourism do not emerge unscathed either, since in the presence of HABs, governmental and competent institutions responsible for public safety have to shut down
large stretches of coast to any fishing and/or recreational activity.
Causes of the worldwide expansion of HABs are still a long way off from being
completely ascertained, but explosive growth of toxic plankton seems to be strictly
intertwined with continuous changes in weather conditions. Also, other factors,
such as variations in upwelling, temperature, transparency, turbulence, or salinity of
seawater alongside concentration of dissolved nutrients, wind, and surface illumination, are likely playing a crucial role [9].
1348
Table 28.1 Algal toxins: most common classes of marine biotoxins
Toxin class
Main algal sources
Toxicity
Structure of a representative
Azaspiracids Protoperidinium crassipes Neurotoxic
O
O
HO
O
H
O
H
H
NH
O
Me
O
O
Me
OH
Me
O
H HO
O
H
Me
Me
H
H
Me
Azaspiracid
Brevetoxins
Karenia brevis
Neurotoxic
HO
O
Me
Me
O
Me
Me
O
O
Brevetoxin-1
P. Ciminiello et al.
O
O
H
O
O
O
O
O
O
28
Gambierdiscus toxicus
Ciguatera
Me
H
H
HO Me O
H
HO
H
O H
H
H
O
H
OH
O
H
HO
H
O
O
H H
O H
H
O H
Seafood Toxins: Classes, Sources, and Toxicology
Ciguatoxins
Me
H O
H
Me
OH
H
O O
Me
OH
O
H H
H
Ciguatoxin
OH
Cyclic imines Alexandrium ostenfeldii
(spirolides)
Me
Me
N
O
Me
O
O
O
O
Me
OH
Me
HO
Me
Karenia selliformis
(gymnodimines)
13-desmethyl spirolide C
Me
Me
H
O
O
Me
O
Me
Me
N
Gymnodimine
(continued)
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Table 28.1 (continued)
Toxin class
Main algal sources
Domoic acids Pseudo-nitzschia species
Toxicity
Amnesic
Structure of a representative
Me
COOH
H
H3C
COOH
COOH
N
H
Domoic acid
Okadaic acids Dynophisis species
Prorocentrum lima
Diarrhetic tumor
promoter
Me
OH
O
HO
Me OH
O
OH
Me
O
O
O
Me
O
OH Me
Okadaic acid
P. Ciminiello et al.
O
O
Ostreopsis species
O
OH
OH
O
OH
HO
OH
OH
O
OH
Me
OH
HO
OH
OH
O
O
OH
OH
O
H2N
Me
OH
HO
OH
Me
OH
OH
OH
OH
O
OH
HO
N
H
OH
OH
O
HN
OH
Me
OH
Me
OH
HO
HO
OH
O
OH
O
O
Me
HO
OH
Me
OH
OH
O
OH
OH
HO
OH
OH
Palytoxin
Pectenotoxins Dynophisis species
Diarrhetic
O
Me
O
OH
OH
Seafood Toxins: Classes, Sources, and Toxicology
Neurotoxic tumor
promoter
28
Palytoxins
Me
O
O
O
O
O
OH
O
OH
Me
Me
OH
O
CH3
O
O
Me
Me
Pectenotoxin-2
(continued)
1351
Toxicity
Paralytic
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Table 28.1 (continued)
Toxin class
Main algal sources
Saxitoxins
Alexandrium species
Structure of a representative
O
H2N
Me
O
HN
NH
NH2
NH
OH
OH
N
H2N
Saxitoxin
Yessotoxins
Protoceratium reticulatum
HO
Me
O
H
H
O
Me
H
O
H
H
H O
NaO3SO
Me
H
O
H
H
H
O
H
O
H
Me
H OH
H
O
O Me
Me
H
Yessotoxin
O
H
P. Ciminiello et al.
NaO3SO
OH
H
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Seafood Toxins: Classes, Sources, and Toxicology
1353
Chemically, marine toxins are often large molecules and only available in small
quantities (Table 28.1). So, investigation of marine biotoxins structure usually
requires employment of sensitive NMR- and mass spectrometry–based techniques
along with HPLC analysis and sometimes combination of chemical degradation and
partial or total synthesis as well.
Etiological studies are also a challenging goal since they call for in-depth
knowledge in marine ecology and analytical chemistry alike. In this frame, once
a candidate organism is thought to be the producer of a given toxin, it needs to be
cultured with the purpose of confirming its toxigenicity. Still, some species are
difficult or totally unable to be cultured, without mentioning that not all of the
clones produce toxins in quantities sufficient for a chemical analysis.
28.2
Azaspiracids: A Recent Class of Toxins Targeting the
Human Gastrointestinal Tract
The recently discovered azaspiracids (AZAs) are toxins structurally characterized
by a polyether backbone and some unique structural features: a trispiro ring
assembly, an azaspiro ring fused with a 2,9-dioxabicyclo[3.3.1]nonane, and
a terminal carboxylic acid group (Fig. 28.1).
The first incident due to azaspiracids occurred in the Netherlands in 1995
following consumption of Irish mussels (Mytilus edulis) [11]. The symptoms of
intoxication closely resembled those associated with DSP including vomiting,
R2
O
R1
O
HO
O
H
H
O
H
H
NH
OH
R3
O
H HO
O
H
R4
O
O
O
H
Fig. 28.1 Structures of
principal azaspiracids (AZAs)
R1
R2
R3
R4
Azaspiracid-1 (AZA1)
H
H
Me
H
Azaspiracid-2 (AZA2)
H
Me
Me
H
Azaspiracid-3 (AZA3)
H
H
H
H
Azaspiracid-4 (AZA4)
OH
H
H
H
Azaspiracid-5 (AZA5)
H
H
H
OH
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P. Ciminiello et al.
severe diarrhea, stomach cramps, and headache. AZA1 was identified as the
causative toxin of the outbreak [12]. Later on, the methyl and desmethyl analogues
of AZA1, namely AZA2 and AZA3, respectively, were identified in Irish mussels
responsible for a second human intoxication [13]. Hydroxylated derivatives of
azaspiracids have also been identified in shellfish and structurally determined
[14, 15]. Up to now, approximately 20 analogues have been reported to occur
naturally in shellfish [16]. However, only two of these, AZA1 and AZA2, have been
reported to be produced by the previously unknown dinoflagellate Azadinium
spinosum [17]. Following development of liquid chromatography–mass spectrometry (LC–MS) methods for determination of azaspiracids, a wide geographical distribution of such toxins across Europe was established, including Norway, the eastern
coasts of England, Denmark, Northern France, and northwestern coasts of Spain [18].
Mice-based toxicological studies demonstrated that the major target of
azaspiracids is the gastrointestinal tract, where an extensive necrotic atrophy of
lamina propria of the villi was observed [19]. Multiple organ damages were also
observed mainly in the liver (fatty changes and single cell necrosis) and in
T and B lymphocytes in the thymus and spleen [20]. Repeated administrations of
the toxins were shown to induce lung tumors [21]. However, the limited availability
of pure azaspiracids limited the statistical value of the in vivo studies.
The mechanism of action of azaspiracids has been studied using in vitro systems.
Roman et al. indicated the cytoskeleton as an important cellular target for AZA1
and that it does not induce apoptosis [22]. In addition, AZA1 did not alter membrane potential in excitable neuroblastoma cells, suggesting that it does not produce
neurotoxic effects. In human lymphocytes, AZA1 increased cytosolic calcium and
cAMP levels and did not affect cytosolic pH. The effect of AZA2 and AZA3 on
intracellular cAMP, cytosolic calcium, and pH has also been evaluated, and the
obtained results highlighted a structure–activity relationship (SAR) among the
different azaspiracids on intracellular pH and calcium levels [23]. Risk assessment
individuated a safe level of 0.16 mg/kg of azaspiracids in shellfish. This was
adopted by the food safety authority of Ireland and proposed to the European
Food Safety Authority and Codex Alimentarius.
28.3
Brevetoxins: Typical Aerosolized Red-Tide Neurotoxins
Brevetoxins are neurotoxic polyether toxins produced by Karenia brevis (formerly
known as Gymnodinium breve and Ptychodiscus brevis). This toxic microalga has
a long history of extensive blooms in the Gulf of Mexico that initiated offshore and
subsequently carried inshore by wind and current conditions [24, 25]. These blooms
have caused massive fish kills and respiratory irritation in humans [26]. It was later
realized that the toxins in these blooms could also be passed on to humans via
shellfish to cause a syndrome named neurotoxic shellfish poisoning (NSP). In the
early 1990s, outbreaks of neurotoxic shellfish toxicity were also reported in New
Zealand and Australia and resulted in the identification of additional Gymnodinium
species producing NSP-like toxins [27]. This dinoflagellate species produces two
28
Seafood Toxins: Classes, Sources, and Toxicology
1355
types of lipid-soluble toxins: hemolytic and neurotoxic compounds [28] causing
massive fish kills, bird deaths, and marine mammal mortalities [29, 30].
Recently, neurotoxins have also been found in other fish-killing flagellate
species, Chatonella marina, C. antiqua, Fibrocapsa japonica and Heterosigma
akashiwo [31–33].
It was only in the 1960s, nearly two decades after the toxic episodes in Florida
caused by K. brevis, that a toxic fraction causing a neurological poisoning was isolated
from this dinoflagellate and contaminated shellfish [34]. Over the next years, substantial efforts were dedicated to purifying brevetoxins. However, it was not until 1979,
with the introduction of high-pressure liquid chromatography, that the primary congeners of brevetoxins were isolated [35], and within the next 5 years, their long cyclic
polyether structures were elucidated by X-ray crystallography and NMR [36, 37].
Like many of the known marine toxins, brevetoxins are tasteless, odorless, and
heat (up to 300 C) and acid stable.
Structurally, brevetoxin congeners fall into two types based on their backbone
structure (Fig. 28.2): brevetoxin B backbone (type 1) and brevetoxin A backbone (type
2) [38, 39]. The B-type backbone consists of 11 fused cyclic ether rings, while the Atype backbone is formed by ten fused cyclic ether rings, including a unique ninemembered ring in the E-position connecting the A-D and F-J moieties, thus inducing
greater flexibility than that found in the B backbone. Type 1 and type 2 toxins share
a lactone in the A ring (“head” of the molecule) and a conserved structure on the “tail”
ring, comprising three six-membered fused cyclic ether rings with conserved R-ring
substitutions. Both ends are required for the toxicity of brevetoxins. Proximal to the
three six-membered rings, there is an eight-membered ring that gives the molecule
flexibility to form a boat-chair or crown conformation.
Among brevetoxins, PbTx-2 T is the major brevetoxin produced by K. brevis
[39]. This alga also produces shorter ring structures (Fig. 28.3) with only four-fused
cyclic ether rings (7, 7, 6, 6), named hemibrevetoxins [40]. Two five-fused cyclic
ether rings (6, 7, 6, 7, 7) have also been described and named brevenals [41, 42].
Brevetoxins are depolarizing substances that open voltage-gated sodium (Na+)
ion channels in cell walls interacting with neurotoxin binding site 5 on the a-subunit
of the channel in a 1:1 stoichiometry [43]. This alters the membrane properties of
excitable cell types in ways that enhance the inward flow of Na+ into the cell,
resulting in inappropriate opening of the channel under conditions in which it is
normally closed, and it also inhibits channel inactivation. The increased membrane
permeability to sodium initially determines excitatory cellular response (including
release of neurotransmitters at some synapses), then followed by loss of cell
excitability, leading to paralysis [44].
The mouse LD50 is 170 mg/kg body weight (0.15–0.27) intraperitoneally, 94 mg/
kg body weight intravenously, and 520 mg/kg body weight orally [45]. Pathogenic
dose for humans is in the order of 42–72 mouse units.
In humans, the symptoms of NSP intoxication may occur after either inhaling
aerosol containing the toxins or as a consequence of eating contaminated seafood.
In the former case, symptoms include primarily respiratory distress, as well as eye
and nasal membrane irritation, caused principally by exposure to sea-spray aerosols
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P. Ciminiello et al.
R1O
Me
O
R2
O
"tail"
"head"
Me
Me
Me
28
O
O
O
O
O
Type 1 (brevetoxin B)
O
O
O
Me
O
27
Me
O
Me
R1O
Me
Me
Me
"head"
Type 2 (brevetoxin A)
Type
O
O
O
"tail"
O
O
O
Toxin
O
R2
O
Me
O
O
R1
O
R2
O
PbTx-1
2
H
PbTx-2
1
H
H
PbTx-3
1
H
OH
H
O
O
PbTx-5
1
COCH3
H
O
PbTx-6
1
H
H 27,28 epoxide
PbTx-7
2
H
OH
PbTx-8
1
H
Cl
PbTx-9
1
H
PbTx-10
2
H
Fig. 28.2 Structure of brevetoxins (PbTxs)
OH
OH
28
Seafood Toxins: Classes, Sources, and Toxicology
1357
OH
Me
H
H
H
O
O
O
O
H
HO
H
H
O
H
Me
hemibrevetoxin-B
Me
OH
H
H O
Me
O
H
Me
H
O
O
Me
Me
O H
H
O Me
H
OH
brevenal
Fig. 28.3 Structure of hemibrevetoxin B and brevenal
and by direct contact with toxic blooms while swimming [46]. When intoxication is
through contaminated shellfish, the symptoms are more severe than those found
when contaminated aerosol is involved and include nausea, tingling and numbness
of the perioral area, loss of motor control, and severe muscular pain [47]. There
have been no reported fatalities from NSP, although the toxin kills test mammals
when administered by various routes, including the oral one.
To date, brevetoxin-group toxins have not been reported in shellfish or fish from
Europe. However, the discovery of new algae producing brevetoxins and the
apparent trend toward expansion of algal bloom distribution suggest that this
group of toxins could also emerge in Europe. Currently, there are no regulatory
limits for these toxins in shellfish or fish in Europe.
28.4
Ciguatera: A Fitting Example of How Chemical Structure of
Marine Biotoxins Is Investigated
In subtropical and tropical regions, consumption of contaminated coral reef fishes
may cause a syndrome referred to as ciguatera, so called after the Caribbean word
“cigua” indicating small marine snails whose ingestion had long induced human
poisonings [48]. Every year, some 25,000 people are affected by ciguatera fish
poisoning (CFP) that for such a reason has long been regarded as a world health
problem [49]. The exogenous origin of the causative agent of ciguatera has been
easily ascertained because fish toxicity entwined with this poisoning has all along
fluctuated individually, seasonally, regionally, and even annually. In the late 1960s,
a Hawaiian research group isolated from moray eels a toxin – named ciguatoxin
(CTX) – that was proposed to be accumulated in fish by the food chain [50].
1358
P. Ciminiello et al.
To the aim of tracking the real producer of CTX down through the food chain,
Yasumoto et al. chose to investigate the guts of a small surgeonfish, Ctenochaetus
striatus, on the basis of the following considerations: (1) the fish was clearly
intertwined with many ciguatera outbreaks in Tahiti, (2) the fish grazes on
a limited variety of microalgae and other microorganisms, and (3) it is a favorite
prey for carnivores thus being a clear link with larger fishes in the food chain. The
outcome of Yasumoto’s studies was the identification in the guts of the analyzed
fish of a lipophilic toxin chromatographically identical with CTX [48]. In the late
1970s, in the digestive contents from C. striatus was detected the occurrence of
a dinoflagellate successively classified as Gambierdiscus toxicus that is now well
known to be the producer of ciguatera toxins [51]. This confirmed the hypothesis
that surgeon fishes obtain CTX from their diet and transfer it to carnivores through
the food chain.
Ciguatoxins are a class of more than 20 heat-stable, lipid-soluble, cyclic
polyether molecules whose chemical architecture is strictly reminiscent of that of
brevetoxins [52–57] (Fig. 28.4). The planar structure of ciguatoxin was achieved by
Yasumoto et al. who had to work on less than 1 mg of pure compound isolated from
124 kg of viscera deriving from 4 t of contaminated moray eels [52]. Despite the
very small amount of pure compound, Yasumoto succeeded in elucidating the
structure of CTX by extensive NMR analysis. Successive studies on both wild
and cultured G. toxicus allowed the isolation and structure determination of
further CTXs, such as gambierol [58], and a potent antifungal compound named
gambieric acid [59]. Yasumoto could also establish the stereochemistry of the
isolated CTXs by resorting to a combination of chiral fluorescent HPLC reagents
or anisotropic NMR reagents with chemical degradation and synthesis of partial
structures [60].
With regard to their biological activity, CTXs give rise to a wide array of
symptoms remarkably varying depending on the geographic region that can be
classified in four main categories: gastrointestinal, neurological, cardiovascular,
and general symptoms [61]. If injected intraperitoneally into a mouse, ciguatoxin
shows a DL50 of 0.45 mg/kg, while oral intake of 0.1 mg of toxin can cause illness in
humans. As previously pointed out, CTXs chemically resemble brevetoxins, of
which they exert also the same mechanism of action. In fact, CTXs activate the
voltage-sensitive Na+ channel at nM to pM concentrations thus being around
30-fold more affine than brevetoxins [62]. CTXs are particularly active on sodium
channels located along peripheral nerves (nodes of Ranvier) thus inducing
a massive influx of Na+ with cell depolarization and the onset of spontaneous
action potentials in excitable cells [63]. As a result, plasma membranes are no
longer able to preserve the internal environment and the volume of cells with
a consequent alteration of bioenergetic mechanisms. Concerning the cardiovascular
impact of CTXs, it must be underlined that once these toxins make Na+ move
intracellularly, cells start extruding sodium and taking up calcium, which is
a known intracellular trigger for muscle contraction. Indeed, an increased force of
cardiac muscle contraction is a common symptom associated to CFP. Likewise,
surge of calcium concentrations in intestinal epithelial cells induced by CTXs
28
Seafood Toxins: Classes, Sources, and Toxicology
1359
Me
H
O H
H
Me
OH
H
O
HO Me O
O
H
O
H
H
HO
O
H
H
H O
H
O H
H
O
Me
Me
H
H
O H
Me
OH
H
O
HO Me O
H
O
H
H
OH
H
O
H
HO
H
H
Me
H Me OH
H
O
O
O
H H
H
H
O
H
Me
O H
O
H
Me
Me
Gambieric acid-A
H
Me
Me
Ciguatoxin 3C
H
Me O
H
O H
O
Me
O
H
Me
HO
H O
H
H
O
H
O
Me
H O
O
O
H H
H
HO
O
O
OH
Me
H
OH
H
O
Ciguatoxin
O
H H
O
H
OH
H
H
H
H
HO
OH
OH
HO
Me
O
Me
H
O
H
H
O H
HO
O
H
H
O
H
H
H
O
H
Me
O
Me
Gambierol
H
O
Me
H
OH
Fig. 28.4 Structure of principal ciguatera-related toxins
affects important cellular ion-exchange systems thus triggering fluid secretion and
diarrhea [64].
Another important neurotoxin involved in CFP is maitotoxin (MTX, Fig. 28.5).
So far, three further analogues of MTX have been identified from G. toxicus [65].
1360
OH
H
H
Me O
O Me
Me
Me
H
Me
O
H
H
O
Me
OH
Me
OH
Me
OSO3Na
OH
O
H
H O
O
Me
Me
Me
H
O
HO
Me
H
HO
H
HO
H
OH
O
H
OH
H
H
Me
H
O
H
H
O
H
OH
H
H
O
H
OH
H
Me
OH
O
Me
OH
H
OH
HO
O
H
H
O
OH
H
O
H
O
O
Me
OH OH
O
H
H
NaO3SO
O
H
O
H
H
O
OH
H
Me
H
O H
H
OH
Me
O H
H
H
OH
Me
O
H
O
Me O
Me
H
O
H
OH
O
H
OH O
H
H
H
HO
OH
H
O
OH
H
OH
OH
OH
Maitotoxin
Fig. 28.5 Structure of maitotoxin
P. Ciminiello et al.
28
Seafood Toxins: Classes, Sources, and Toxicology
1361
With a molecular weight of 3,422, maitotoxin is by far the largest nonpolymeric
natural product of all, and apart from some proteic bacterial toxins, it is also one of
the most toxic compounds. From a chemical point of view, MTX is a ladder-shaped
polycyclic highly hydroxylated molecule. Pharmacological studies have proven
MTX a potent activator of voltage-gated calcium channels [66]. At the moment,
though, its primary target remains undefined and the molecular mechanism of
action still presents many blind spots.
28.5
Cyclic Imine Toxins: A Category of Miscellaneous
Emerging Toxic Compounds
Cyclic imine toxins constitute a heterogeneous class of marine biotoxins all featuring a macrocyclic structure with a typical imine moiety apparently being the
bioactive pharmacophore. Quite a number of toxic compounds currently belong
to this class of toxins:
• Gymnodimines [67]
• Pinnatoxins [68]
• Prorocentrolides [69]
• Pteriatoxins [70]
• Spirolides [71]
• Spiro-prorocentrimine [72]
In Fig. 28.6, representatives of each class of cyclic imines are reported. All of
these compounds are referred to as a class of emerging toxins as in-depth knowledge about them is still lacking. First and foremost, it would be necessary assessing
their real impact on human health. Cyclic imine toxins are indeed usually classified
as fast-acting toxins since they induce death within minutes from their intraperitoneal injection into mice and show an acute threshold response in mammalian
bioassays. Beyond this, though, the real toxicity of this class of toxins is still
a long way off from being clearly elucidated. This is surely due to their relatively
recent discovery – dated back to about two decades ago – with a concurrent lack of
standards, reference, or purified material, which has so far hampered thorough
toxicological evaluations. In addition, regulatory authorities have been all along
reluctant to undertake studies on the effect of such a class of compounds in the
absence of ascertained cyclic imine-related human intoxications. As a result, no
regulatory limits have been set for any of the above cyclic imines, which are at the
moment relegated to a category of compounds supposed safe for consumers until
proven otherwise [73].
All of the cyclic imine toxins with the exception of spirolides – that have been
detected worldwide – have been individuated only in scattered locations and in
taxonomically restricted group of vectors. Nonetheless, their geographical distribution is reasonably bound to spread as monitoring programs increase and sensitiveness in analytical methods improves.
Unlike pinnatoxins and pteriatoxins that have been detected exclusively in
shellfish even though thought to represent a dietary incorporation in bivalves, all
1362
O
O
N
H
O
HOOC
N
OH
N
OH
H
HO
HO
O
O
OH
O
O
O
HO
O
HO
O
O
O
OH
Gymnodimine-A
OH
O
HO
Pinnatoxin-A
OH
Prorocentrolide
HO
HO
NH2
O
N
HOOC
O
S
Pteriatoxin-A
HO
HO
O
O
H
HO3SO
HO
OH
O
Fig. 28.6 Structure of representatives of cyclic imine toxins classes
O
O
O
HO
O
Spiro-prorocentrimine
O
O
O
OH
13-desmethylspirolide C
OH
P. Ciminiello et al.
O
HO
N
N
HO
28
Seafood Toxins: Classes, Sources, and Toxicology
1363
of the other cyclic imines are strongly believed to be synthesized by either benthic
or pelagic marine dinoflagellates on account of their chemical features apparently
deriving from polyketide biosynthetic pathways common to other polyether
biotoxins [74].
Gymnodinium sp. was first regarded as culprit species of gymnodimine toxicity
[67], but successive taxonomic studies individuated the marine dinoflagellate
Karenia selliformis as the most likely producer of this group of toxins [75].
Benthic or epiphytic Prorocentrum species are the microorganisms producing
prorocentrolides [69] and spiro-prorocentrimine [72], clearly termed after their
producers.
Regarding the biological origin of spirolides, the marine dinoflagellate
Alexandrium ostenfeldii has been described as their primary, if not exclusive,
producer [76]. It is worthwhile highlighting that such a dinoflagellate is reported
to produce different kinds of toxins depending on its geographical provenance. In
fact, while Canadian [77] and Adriatic [78] strains of A. ostenfeldii were proven to
produce high levels of a number of spirolides, New Zealand strains only produced
PSP toxins [79]. Even more complex was the toxin profile of certain A. ostenfeldii
populations from Scandinavia that were shown to produce both spirolides and PSP
toxins [76].
As already described above, biological activity and mechanism of action of
cyclic imine toxins are not comprehensively understood. What seems quite plain is
that the intact imine group plays a crucial role in their toxicity as pointed out by
a dramatic decrease in biological activity upon its hydrolysis. Indeed, spirolide
E and F originating from keto amine hydrolysis are both basically inactive [80]. On
account of the similar symptomatology shown by cyclic imine toxins, a common
mode of action for all of them can be presumed. More in detail, spirolides seem to
affect calcium ions [80], while gymnodimine activates sodium channels even in
a far weaker way than other classic ion channel effectors, namely brevetoxins,
tetrodotoxin, or even saxitoxin [67]. Moreover, both transcriptional and histological
analyses of brain tissues revealed that muscarinic and nicotinic subreceptors were
upregulated following exposure to spirolides. Consequently, such receptors could
represent the spirolides’ biological target [81].
Effort should be expended to extend the knowledge on cyclic imine toxins
toxicology well beyond what is currently accessible, with the purpose of resolving
their oral toxicity and assessing the real risks they pose to human health.
28.6
Domoic Acids: The Only Marine Biotoxins Produced by
Diatoms
One morning in the summer of 1961, hundreds of crazed birds attacked the seaside
town of Capitola, California. The birds “cried like babies” as they dove into
streetlamps, crashed through glass windows, and attacked people on the ground.
Most of the birds were sooty shearwaters, a normally nonaggressive species that
feeds on small fish and comes ashore only to breed. The incident fascinated Alfred
1364
P. Ciminiello et al.
Hitchcock, who was vacationing nearby and likely inspired one of his most famous
movie, The Birds, which appeared in cinemas 2 years later.
In the winter of 1987, the agent that is now believed to be responsible for the
Capitola incident struck on the opposite shore of the continent. This time, it struck
higher on the food chain. Over a hundred people became extremely ill within hours
after dining on cultured blue mussels in restaurants around Prince Edward Island in
Canada [82]. It quickly became apparent that this was no ordinary outbreak of food
poisoning. Vomiting, cramps, diarrhea, and incapacitating headaches were
followed by confusion, loss of memory, disorientation, and (in severe cases)
seizures and coma. A few exhibited emotional volatility, with uncontrolled crying
or aggressiveness. Three elderly victims died in the hospital and one 3 months
later [83].
The causative agent of this deadly syndrome, now known as amnesic shellfish
poisoning (ASP), was identified as domoic acid [84, 85], a nonproteic amino acid
with antihelmintic activity originally isolated in 1959 from a red marine macroalga,
Chondria armata [86].
The chain-forming diatom Pseudo-nitzschia multiseries (formerly known as
Nitzschia pungens) was recognized as the causative agent of that toxic event
[87, 88]. Several further species of Pseudo-nitzschia (P. australis, P. pseudodelicatissima, P. galaxiae, P. multistriata, P. pungens, P. seriata, P. turgidula,
P. fraudulenta, P. delicatissima, P. calliantha) have successively been found to
produce domoic acid, although some species are not always toxic and there is
a considerable variability in toxicity [89–91]. These diatom species are distributed
worldwide. Consequently, accumulation of domoic acid in bivalves has also been
reported from various parts of the world [92–94]. On the other hand, domoic acid is
reported to accumulate in sea birds and marine mammals by a food-web transfer,
which can result in mass mortality of these animals [95]. Scholin et al. [96]
suggested that the origin of the domoic acid causing the deaths of sea lions on the
California coast is Pseudo-nitzschia australis. Such findings show that domoic acid
occurs widely in marine ecosystems. However, knowledge of the distribution of
domoic acid in various organisms is limited to those in temperate waters. Little is
known about accumulation of domoic acid in tropical marine organisms. It is
worthwhile emphasizing that ASP is the only shellfish poisoning produced by
a diatom.
Structurally, domoic acid (DA) is a water-soluble excitatory tricarboxylic amino
acid (Fig. 28.7), belonging to the kainoid class of compounds, structurally resembling the excitatory neurotransmitter glutamic acid. Its molecular structure was
determined in the late 1950s [86, 97–99] and then confirmed following total
synthesis [100].
Several congeners of domoic acid have been identified so far, of which three
geometrical isomers, isodomoic acids D, E, and F, and the C5’-diastereomer
were found in small amounts in both the diatom and shellfish tissue (Fig. 28.7)
[101, 102].
The biological mode of action of domoic acid results in neuronal depolarization;
the resultant short-term memory loss is symptomatic of domoic acid poisoning
28
Seafood Toxins: Classes, Sources, and Toxicology
1365
CH3
CH3
COOH
COOH
H
H3C
H3C
COOH
N
COOH
COOH
H
H
N
COOH
H
domoic acid
C5ⴕ-diastereomer
COOH
H3C
COOH
CH3
CH3
H3C
COOH
N
COOH
COOH
N
H
COOH
H
isodomoic acid D
isodomoic acid E
CH3
HOOC
COOH
CH3
N
COOH
H
isodomoic acid F
Fig. 28.7 Structure of principal domoic acids (DAs)
(reported by 25% of those affected by the Canadian amnesic shellfish poisoning
outbreak). The mechanism of DA toxicity is explained by its structural similarity
with the excitatory neurotransmitter glutamic acid but with a much stronger receptor affinity. Domoic acid binds predominantly to both kainate and AMPA subtypes
of glutamate receptor [103], resulting in depolarization of neurons. Subsequently,
the permeability to calcium ions increases [104]. This induces lesions in areas of the
brain where glutaminergic pathways are heavily concentrated, particularly in the
CA1 and CA3 regions of the hippocampus, areas responsible for learning and
memory processing [105]. The loss of memory in patients intoxicated with mussel
toxin appears to be similar to patient with Alzheimer’s disease. However, the loss of
memory in mussel-intoxicated patients was not affected by the age of patients,
whereas symptoms of Alzheimer’s disease intensify with aging and are generally
noted in older people. Further, the findings that intellect and higher cortical
functions are not influenced by DA intoxication distinguish the mussel-induced
intoxication from Alzheimer’s disease.
1366
P. Ciminiello et al.
Other clinical symptoms of ASP include abdominal cramps, vomiting, diarrhea,
incapacitating headaches, and disorientation. In the most severe case of poisoning,
patients are victims of seizures, coma, profuse respiratory secretions, unstable
blood pressure, and even death.
28.7
Okadaic Acids: The Subtle Threat of Potent Tumor
Promoters
The syndrome diarrhetic shellfish poisoning (DSP) was first discovered in the
1970s following the occurrence of a mussel intoxication in northeastern Japan
[106]. Ever since, DSP has known a steady spreading across the globe. It is
essentially associated with seafood consumption such as mussels, clams, and
scallops, which can accumulate dinoflagellate toxins in their digestive glands by
seawater filtering process. The major toxin responsible for most DSP in humans is
okadaic acid (OA) alongside a number of analogues termed dinophysistoxin-1
(DTX1) and dinophysistoxin-2 (DTX2), among others (Fig. 28.8) [107]. A number
of further congeners are also involved in DSP syndrome, but all of them are
believed to be either precursors or shellfish-modified metabolites of the active
toxins [108].
OA was primarily isolated from the marine sponges Halichondria okadai and
Halichondria melanodocia, and subsequently proven to be produced by dinoflagellates belonging to the genera Dinophysis and Prorocentrum [109, 110]. With regard
to this latter, the benthic species Prorocentrum lima is worth being mentioned
[111, 112], while among the Dinophysis species, D. acuta, D. fortii, D. acuminata,
D. norvegica, D. mitra and D. caudata must be listed [113].
Chemically, OA is a polyether compound characterized by a carboxylic acid
group and three spiro-keto ring assemblies, one of which connects a five with
a six-membered ring.
OH
O
O
O
HO
OH
R1
O
O
O
OR3
O
O
OH
R1
R2
R3
CH3
H
H
Okadaic acid (OA)
CH3
CH3
CH3
Dinophysistoxin 1 (DTX1)
H
CH3
CH3
Dinophysistoxin 2 (DTX2)
Fig. 28.8 Structure of principal okadaic acids (OAs)
R2
28
Seafood Toxins: Classes, Sources, and Toxicology
1367
Oral assumption of okadaic acid–contaminated seafood leads to the typical DSP
symptoms usually within 30 min from ingestion. Symptoms shown by intoxicated
patients are mainly gastrointestinal-like. Diarrhea, nausea, vomiting, and abdominal cramps are among the most common disturbances suffered by humans. No DSPrelated casualty has been reported so far, but sometimes remarkable morbidity has
required hospitalization. The treatment, if necessary, includes fluid replacement and
electrolyte reintegration.
OA has long been known as a potent inhibitor of Ser/Thr protein phosphatases
that represent an array of enzymes catalyzing dephosphorylation of phosphoserine
or phosphothreonine residues in eukaryotes [114]. Ser/Thr protein phosphatases are
involved in an extraordinary number of physiological processes in mammals
ranging from glycogen metabolism to coordination of cell cycle and even gene
expression. It follows that DSP toxins impact on Ser/Thr protein phosphatases
can be devastating for living organisms. More in detail, of the four major groups
of Ser/Thr protein phosphatases, OA inhibits PP2Ac at the lowest concentration
(IC50 ¼ 0.2 nM) and PP1c at the next lowest concentration (IC50 ¼ 20 nM) as
opposed to PP2B – which is slightly affected by the toxin – and to PP2C on which
no effect is detectable [115]. Inhibition of Ser/Thr protein phosphatases by OA is
known to provoke all of the gastrointestinal disorders in humans as soon as the toxin
reaches the digestive tract.
Besides, there is another far more harmful threat posed by okadaic acids (OAs)
to unaware contaminated seafood consumers: their potent tumor-promoting activity. Indeed, OA and DTX1 are classified as non-12-O-tetradecanoylphorbol-13acetate (TPA)-type tumor promoters [116]. Unlike phorbol esters which activate
protein kinase C, these two DSP toxins neither bind to phorbol ester receptors
in cell membranes nor activate protein kinase C in in vitro studies [116]. OA
and DTX1 have shown a potent tumor promotion activity on mouse skin. It appears
that by inhibiting PP2A, OA causes a general decrease of cell adhesion and
cytoskeletal reorganization resulting in an increased cell motility and invasiveness
alike [114].
Considering the alarming harmfulness of OAs to consumers, it is apparent that
a strict monitoring program needs to be run on seafood aimed at preventing possible
human intoxications. With regard to this, it needs to be emphasized that the
European Community has been recently forced to modify the official extraction
procedure for analyzing seafood. In fact, by applying the old extraction method,
yessotoxins – which in many toxic outbreaks have occurred together with DSP
toxins – and OAs were recovered in the same organic layer [106]. Therefore, the
mouse bioassay – the reference method in Europe to detect toxicity in seafood –
could not lead to any confident assessment of the toxin(s) involved. So, as these two
classes of toxins show a remarkably different toxicity on humans with, accordingly,
very different official allowance levels in shellfish (16 mg of OA and 100 mg
of yessotoxin in 100 g of mollusk, respectively), a new extraction method capable
of separating yessotoxin in an aqueous layer and OAs in a lipophilic one was
set up [117].
1368
28.8
P. Ciminiello et al.
Palytoxins: Potent Tropical Toxins Now Rife Across the
Mediterranean Basin
The lethal potency of palytoxin was well known to ancient Hawaiians who used to
smear a moss containing such toxin on their spearpoints to make them fatal.
According to an old legend, the lethal moss – known to native Hawaiians as limumake-o-Hana – started lining the walls of a tide pool near the harbor of Hana after the
ashes of an evil Shark God killed by some fishermen had been thrown into it [118]. In
the early 1960s, thanks to some local informers, P. Helfrich and J. Shupe from the
Hawaii Institute of Marine Biology succeeded in individuating the tide pool, from
which they collected some samples of the toxic moss. Fascinated by the legend and
propelled by his profound interest in marine toxins, Professor P. Scheuer from the
University of Hawaii, a pioneer of the chemistry of marine metabolites, in collaboration with P. Helfrich and R. Moore, investigated the toxic samples collected from
the legendary tide pool [119]. It was then assessed that the limu-make-o-Hana was not
a seaweed but an animal belonging to the phylum Coelenterata – possibly a new
species – assigned to the genus Palythoa [120] after which the molecule responsible
for its high toxicity was named palytoxin. After its isolation in 1971 and the
preliminary structural insights offered by Scheuer [119], it took nearly 11 years before
the correct chemical structure of palytoxin (Fig. 28.8) was disclosed [121, 122]. It is
a complex polyhydroxylated water-soluble compound, containing both lipophilic and
hydrophilic part structures. In 1982, the complete stereochemistry of palytoxin –
encompassing as many as 64 stereogenic centers in addition to eight double bonds –
was disclosed [123], and a few years later confirmed by its total synthesis [124].
Following the first report on palytoxin in 1971, many research groups from
across the world have undertaken scientific studies with the purpose of investigating
this fascinating molecule. Efforts carried out by proficient chemists have significantly contributed to identify quite a number of palytoxin analogues (Fig. 28.9).
The real biological origin of this class of toxins remains controversial. In fact,
even though several palytoxin-like compounds have been isolated from zoanthids
belonging to the genus Palythoa – such as homopalytoxin, bishomopalytoxin,
neopalytoxin, deoxypalytoxin [125], and 42-hydroxypalytoxin [126] – of late
many have defended the assumption that palytoxins are indeed produced by
microorganisms. In particular, dinoflagellates belonging to the genus Ostreopsis
seem to be the most probable biogenetic originators of palytoxins [127, 128]. In
1995, in fact, Yasumoto isolated and structurally characterized some palytoxin
analogues, named ostreocins, from extracts of the dinoflagellate O. siamensis.
The major constituent of the isolated ostreocins, accounting for 70% of the total
toxicity, resulted to be ostreocin D, whose chemical structure was assigned as
42-hydroxy-3,26-didemethyl-19,44-dideoxypalytoxin by detailed 2D NMR
analyses of both the intact compound and its ozonolysis products [129, 130].
Successively, further palytoxin-like compounds, termed mascarenotoxins, were
extracted from O. mascarenensis [131]. These toxins were confirmed as palytoxin
analogues on the basis of their mass spectrum profile and fragmentation pattern
obtained by nano-electrospray ionization quadrupole time-of-flight MS.
28
OH
O
O
H2N
OH
O
OH
OH
OH
HO
OH
OH
Me
OH
HO
R6
OH
HO
( )n
N
H
O
R1
OH
Me
HO
OH
HN
OH
Me
OH
OH
A moiety
O
OH
OH
OH
O
OH
OH
OH
8 O
9
9
HO
OH
HO
B moiety
Me
Seafood Toxins: Classes, Sources, and Toxicology
O
OH
OH
O
R2
O
O
R3
R4
OH
O
OH
HO
Me
OH
OH
OH
R5
OH
OH
OH
OH
Fig. 28.9 (continued)
1369
1
2
3
1
1
1
Ostreocin-D
Homopalytoxin
Bishomopalytoxin
Neopalytoxin
Deoxypalytoxin
42-Hydroxy-palytoxin
Me
Me
-
Me
Me
H
Me
R1
OH
OH
OH
OH
OH
H
OH
R2
Me
Me
Me
Me
Me
H
Me
R3
Fig. 28.9 Structure of palytoxin and some of its analogues
1
Palytoxin
n
OH
H
H
H
H
OH
H
R4
OH
OH
OH
OH
OH
H
OH
R5
OH
H
OH
OH
OH
OH
OH
R6
A moiety=HO
( )n
N
H
O
HN
O
O
1370
P. Ciminiello et al.
28
Seafood Toxins: Classes, Sources, and Toxicology
1371
Recently, palytoxin and some of its analogues, named ovatoxins, have been
detected in the Mediterranean O. ovata [132–134]. Ovatoxins have been identified
by high-resolution LC–MS/MS and present molecular formulae similar to
palytoxin’s, with elemental composition differences residing either in the A or
B moiety of palytoxin molecule [133, 134].
More in detail, by accurately interpreting complex HRMS/MS spectra, it was
ascertained that (1) ovatoxin-a presents two oxygen atoms less than palytoxin and
the same A moiety; (2) ovatoxin-b presents C2H4O more than ovatoxin-a, the
structural difference between the two molecules residing in the A moiety whereas
part structure B is identical; (3) ovatoxin-c presents the same A moiety as ovatoxinb and an additional oxygen atom in the B moiety; and (4) ovatoxin-d and ovatoxin-e
are isobaric compounds that present one oxygen atom more than ovatoxin-a,
located either in the B moiety (ovatoxin-d) or in the A moiety (ovatoxin-e).
Palytoxins constitute a class of extremely potent nonproteic marine biotoxins,
whose main biological target is the Na+/K+-ATPase, membrane pumps maintaining
ionic gradients critical to cell function [135]. The effect of palytoxins is basically
a conversion of these ion-specific pumps into nonselective cationic pores, thus
triggering several biological effects, some of which even life threatening.
Due to their high potency, palytoxins harm human health through diverse routes of
exposure ranging from ingestion of contaminated seafood to dermal contacts, or
inhalation of marine aerosol [136, 137]. Some fatal human poisonings attributed to
palytoxin have been reported worldwide [138, 139]; the toxin has been suggested as
the possible cause of clupeotoxism, a poorly understood syndrome caused by ingestion
of edible fish [140]. Symptoms of intoxication include vasoconstriction, hemorrhage,
ataxia, muscle weakness, ventricular fibrillation, pulmonary hypertension, ischemia,
and death. Toxicity strongly depends on the route of administration. As way of
example, palytoxin exhibits high toxicity in mammals when intravenously administered (LD50 ranging between 25 and 450 ng/kg) [141], while intragastric administration in rats shows a significantly lower toxicity (LD50 > 40 mg/kg) [141, 142].
In the late 1980s, palytoxin was also identified as a skin tumor promoter
[143, 144]. In contrast to TPA (12-O-tetradecanoylphorbol-13-acetate), palytoxin
induces neither ornithine decarboxylase in mouse skin nor HL-60 cell adhesion.
Furthermore, palytoxin neither binds to protein kinase C in vitro nor increases
ornithine decarboxylase activity in mouse skin. On the basis of such evidence,
palytoxin is classified as a non-TPA-type tumor promoter [145].
28.9
Pectenotoxins: Potent Cytotoxic Compounds with Still
Unknown Potential Implications to Public Health in the
Long Term
Pectenotoxins (PTXs) are lipophilic macrocyclic polyethers, whose chemical structures resemble okadaic acid in having cyclic ethers and a carboxyl group in the
molecule. Unlike okadaic acid, in many PTXs, the carboxyl moiety is engaged in
a macrocyclic lactone (Fig. 28.10). To date, 15 different PTXs have been isolated
1372
P. Ciminiello et al.
Me
1
O
Me
O
Me
O
O
7
O
OH
OH
OH
O
O
Me
O
O
R
O
O
Me
Me
R
C-7
C-7
CH2OH
R Pectenotoxin 1 (PTX1)
R Pectenotoxin 2 seco acid (PTX2SA)
CH3
R Pectenotoxin 2 (PTX2)
S 7-epi-Pectenotoxin 2 seco acid (PTX2SA)
CHO
R Pectenotoxin 3 (PTX3)
CH2OH
S Pectenotoxin 4 (PTX4)
COOH
R Pectenotoxin 6 (PTX6)
COOH
S Pectenotoxin 7 (PTX7)
Fig. 28.10 Structures of principal pectenotoxins (PTXs)
and characterized from a range of source organisms [146]. PTXs in filter-feeding
organisms originate from dietary microalgae. Up to now, only the genus Dinophysis
(e.g., D. acuta, D. fortii, D. acuminata, and D. caudata) has been implicated in
contamination of shellfish with PTXs [146]. However, only PTX-2 and the seco
acids of PTX-2 (PTX-2SA and epi-PTX-2SA) have been isolated from phytoplankton, while the other compounds have been detected only in shellfish samples.
Therefore, it has been supposed that an oxidation occurs in the hepatopancreas of
shellfish, and that many PTXs are products of shellfish metabolism after ingestion
of PTX-producing microalgae [146, 147].
Pectenotoxins in shellfish are always accompanied by okadaic acids and/or
yessotoxins and are co-extracted with them. So, initially, pectenotoxins have been
grouped together with okadaic acids and yessotoxins in the DSP class. However,
animal studies have indicated that pectenotoxins do not induce diarrhea and they
are much less toxic than okadaic acid by oral administration [148]. In addition,
unlike many DSPs which are potent phosphatase inhibitors, PTX-1 and PTX-6 are
inactive against PP-1 and PP2A [149]. Thus, PTXs are currently considered as
a separate group of toxins.
Since PTXs often co-occur with other phycotoxins in shellfish, no toxic episodes
in humans can be unequivocally related to them, and therefore, there is no information about their toxicity to humans. It has been shown that PTXs are potently
cytotoxic [150] and cause necrosis to hepatocytes [151]. Nothing is known of the
chronic toxicology of PTXs or the potential implications to public health in the long
term.
28
Seafood Toxins: Classes, Sources, and Toxicology
1373
28.10 Saxitoxins: The First News We Have About Seafood
Toxin–Related Human Poisoning
One of the first recorded fatal cases of human poisoning after eating shellfish
contaminated with dinoflagellate toxins happened in 1793, when Captain George
Vancouver and his crew landed in British Columbia in an area now known as
Poison Cove [152]. He noted that for local Indian tribes, it was taboo to eat shellfish
when seawater became phosphorescent due to dinoflagellate blooms. However, the
link between toxic shellfish and dinoflagellates was only ascertained right before
World War II, when Sommer et al. studied toxic outbreaks that occurred in the
San Francisco Bay between 1920 and 1937 [153, 154].
The causative toxins of these events are alkaloids, referred to as paralytic
shellfish poisoning (PSP) toxins.
Historically, PSP incidents are associated with dinoflagellates, such as
Alexandrium [155] – the first to be identified as PSP toxin producers – Pyrodinium
and Gymnodinium species [156]. Besides, with developments in technology and
research on marine algae, more species and classes of microorganism have been
found to produce these toxins. Marine bacteria such as Moraxella [157] and
Alteromonas tetraodonis [158] and freshwater cyanobacteria such as
Aphanizomenon flos-aquae, Anabaena circinalis, Lyngbya wollei, Cylindrospermopsis raciborskii and Protogonyaulax [159] have all been found to produce
or influence the production of these toxins in algae. Infection of Ostreopsis
lenticularis by Pseudomonas species was also found to affect the production of
toxins [160].
The parent compound of this class of toxins is saxitoxin (STX), of which over
29 congeners are currently known [161]. Chemically, they are tetrahydropurine
derivatives whose structures vary by different combination of hydroxyl and sulfate
substitutions at four sites on the molecule. Based on substitutions at R4, the
saxitoxins can be subdivided into four groups: (1) the neurotoxic and highly potent
carbamate toxins which include the nonsulfated saxitoxins (STX) and neosaxitoxin
(NEO), and gonyautoxins (GTX1-GTX4) which are singly sulfated and more lethal
than the nonsulfated carbamate toxins; (2) weakly toxic N-sulfocarbamoyl toxins
(B1, B2, C1–C4) which are the least toxic to mammals of all the PSP toxins;
(3) decarbamoyl (dc-) analogues which are thought to arise from the metabolism of
dinoflagellate toxins within the shellfish; and (4) deoxydecarbamoyl (do-) toxins
that have been detected until now only in Australian populations of G. catenatum
[162] (Fig. 28.11).
Recently, the use of hydrophilic interaction liquid chromatography–mass spectrometry (HILIC–MS) followed by in-depth NMR investigation allowed to eluci~
date the structure of new analogues: 11b-hydroxy-N-sulfocarbamoyl
saxitoxin and
the unusual 11,11-dihydroxy saxitoxin and 11,11-dihydroxy-N-sulfocarbamoyl
saxitoxin [163, 164].
Saxitoxins are classified as fast-acting neurotoxins as PSP symptoms develop
fairly rapidly, within 0.5–2 h after ingestion of contaminated shellfish, depending
on the amount of toxin consumed [165]. In humans, the peripheral nervous system
1374
P. Ciminiello et al.
R4
R1
NH
N
H2N
NH2
NH
N
OH
11
OH
R3
R2
STX = saxitoxin
NEO = neosaxitoxin
GTX = gonyautoxin
Carbamate
toxins
O NH2
R4
N-Sulfocarbamoyl
toxins
O NHSO3−
Decarbamoyl
toxins
Deoxydecarbamoyl
toxins
OH
H
R1
R2
R3
H
H
H
STX
GTX5, B1
dcSTX
OH
H
H
NEO
GTX6, B2
dcNEO
H
H
OSO3−
GTX2
C1
dcGTX2
doGTX2
H
OSO3−
H
GTX3
C2
dcGTX3
doGTX3
OH
H
OSO3−
GTX1
C3
dcGTX1
OH
OSO3− H
GTX4
C4
dcGTX4
H
H
OH
11αOH-STX
11αOH-dcSTX
H
OH
H
11βOH-STX
11βOH-dcSTX
OH
H
OH
11αOH-NEO
11αOH-dcNEO
OH
OH
H
11βOH-NEO
11βOH-dcNEO
O
O
doSTX
Fig. 28.11 Structures of principal paralytic shellfish poisoning (PSP) toxins
is affected, with symptoms ranging from tingling of the tongue and lips, followed
by a numbness spreading toward the extremities, to vomiting, pain, diarrhea, loss of
coordination, and breathing difficulty. In severe cases, ataxia, muscle weakness,
and respiratory paralysis can occur. Symptoms can turn into coma or death, but
recovery is generally complete, with no lasting side effects, when respiratory
support is provided within 12 h of exposure. In unusual cases, because of the
weak hypotensive action of the toxins, death may occur from cardiovascular
collapse despite respiratory support.
Saxitoxins are potent, reversible blockers of voltage-activated sodium channels on excitable cells [166], but, due to the differences in charge state and
substitution groups to the basic STX structure, they bind with different affinities
to site 1 of sodium channels, resulting in different toxicities [167]. Thus, health
risks can be reliably assessed just if the level of each toxin is individually
determined.
28
Seafood Toxins: Classes, Sources, and Toxicology
1375
28.11 Yessotoxins: Potent Toxic Compounds with Controversial
Impact on Public Health
Yessotoxins (YTXs) represent a class of lipophilic polyether compounds, including
a number of analogues that have been detected in shellfish and/or phytoplankton.
The parent compound of this class of toxins is yessotoxin (YTX), isolated for the
first time in 1986 from the scallops Patinopecten yessoensis that were implicated in
a diarrhetic shellfish poisoning episode in Japan [168]. The first unicellular organism identified as a producer of YTX was the dinoflagellate Protoceratium
reticulatum (Claparède and Lachmann) B€
utschli 1885 [169]. Subsequently, YTX
has been found in cells of P. reticulatum from different places in Japan [170–172];
in the Adriatic Sea in Italy [173]; in Nova Scotia, Canada [174]; in Norway [175];
and in Spain [176, 177]. Other confirmed producers of YTXs are the dinoflagellates
Lingulodinium polyedrum (Stein) Dodge [177, 178], and Gonyaulax spinifera
Dodge [179]. Some authors suggested that the real producers of YTXs are bacteria
associated with the dinoflagellates; however, to our knowledge, there is no solid
evidence for this [180].
YTX production within and among dinoflagellate species tested to date is highly
variable. As for the YTXs profile in the producing organisms, the data on the
relative amounts are not so numerous, and all refer to cultures of P. reticulatum.
The data reported in the literature concur that YTX is by far the major toxin
produced by P. reticulatum. HomoYTX and a great number of other YTX analogues were sometimes shown to be present in the cultures, but in much lesser
quantities [181]. There are, however, two reports in which the major toxin of
P. reticulatum cultures is homoYTX [182, 183]. About the other two microalgae
which were reported to produce YTXs, cultures of Lingulodinium polyedrum were
shown to contain YTX [177], while Gonyaulax spinifera cultures were reported to
produce unspecified YTXs identified by ELISA analyses [179].
Going to the contaminated shellfish, the YTX profile dramatically changes. YTX
(or homoYTX) is no more the dominant toxin, but other analogues, such as
hydroxylated and carboxylated derivatives, originating from metabolic oxidation
within the mollusk, take the scene [181].
Chemically, yessotoxins are polycyclic ether compounds, structurally closely
related to brevetoxins and ciguatoxins (Fig. 28.12).
The structure of YTX was established first [168, 184] and gave the basic
framework to elucidate the structures of the other YTXs. It is a disulfated polyether,
with a characteristic ladder shape formed by 11 adjacent ether rings of different
sizes and a terminal acyclic unsaturated side chain consisting of nine carbons and
two sulfate esters.
Since the initial discovery of YTX from Japanese scallops, a significant number
of analogues, including the homoyessotoxins (homoYTXs), have been identified in
toxic shellfish and/or algal cultures from different countries, suggesting the spread
of this toxin worldwide [181]. Although the structure of some of them are still
unknown [185], for many of them, full structure determination was carried out by
NMR and/or liquid chromatography coupled with mass spectrometry (LC–MS).
1376
P. Ciminiello et al.
HO
OSO3H
H
O
H
O
Me
OH
H
H
H
H O
HO3SO
HO3SO
Me
H
O
H
H
H
O
O
H
H OH
H
H
O
H
O
R3
O
Me
H
O Me
Me
H
Adriatoxin (ATX)
H
H O
R1
( )n
R2
Me
O
H
H
H
O
H O H
H
H
O
Me
H
O
H
H OH
H O H
R1
R2
Yessotoxin (YTX)
OSO3H
OSO3H
45-Hydroxyyessotoxin (45-hydroxyYTX)
OSO3H
OSO3H
45,46,47-Trinoryessotoxin (NorYTX)
OSO3H
OSO3H
Homoyessotoxin (homoYTX)
OSO3H
OSO3H
45-Hydroxyhomoyessotoxin (45-OHhomoYTX)
OSO3H
OSO3H
OH
H
O
H
Me
O Me
Me
R3
Me
Me
Me
Me
Me
n
OH
1
OH
OH
45
1
O OH
H
1
OH
2
OH
45
2
OH
1-Desulfoyessotoxin (1-dsYTX)
Carboxyyessotoxin (carboxyYTX)
Carboxyhomoyessotoxin(carboxyhomoYTX)
Fig. 28.12 (continued)
OH
OSO3H
OSO3H
OSO3H
OSO3H
OSO3H
Me
Me
Me
OH
1
OH
COOH
1
OH
COOH
2
28
Seafood Toxins: Classes, Sources, and Toxicology
1377
Noroxohomoyessotoxin (NoroxohomoYTX)
OSO3H
OSO3H
Noroxoyessotoxin (NoroxoYTX)
OSO3H
OSO3H
44,55-Dihydroxyyessotoxin
OSO3H
OSO3H
1-Desulfocarboxyhomoyessotoxin
OH
OSO3H
4-Desulfocarboxyhomoyessotoxin
OSO3H
OH
45-Hydroxycarboxyyessotoxin
OSO3H
OSO3H
Me
O
Me
O
2
2
HO 55
OH
44 OH
Me
Me
Me
Me
OH
1
COOH
2
OH
COOH
2
OH
COOH
45
1
OH
Fig. 28.12 Structures of principal yessotoxins (YTXs)
There is evidence that some YTX analogues identified, such as hydroxylated and
carboxylated derivatives, largely result from metabolism of yessotoxin in the
shellfish after ingestion [186].
Yessotoxin and its analogues were at beginning included within the diarrhetic
shellfish poisoning (DSP) group mainly because, following the standard procedure
of mouse bioassay, they are extracted together with the DSP toxins okadaic acid
(OA) and dinophysistoxins (DTXs), when they co-occur in contaminated shellfish.
However, their toxic activities are significantly different; in fact, YTX and its
analogues do not induce diarrhea and, compared to OA, show a much lower (four
orders of magnitude) potency for the inhibition of protein phosphatase 2A [187]. On
the contrary, their cardiotoxic effects have been demonstrated in mice after intraperitoneal (i.p.) and oral exposure of very high doses of YTX [188]. For these
reasons, YTXs are not anymore included in the list of DSP toxins.
The toxicological studies carried out on YTX revealed that it is more toxic than
any DSP toxin, when intraperitoneally injected, since the dose inducing 50% of
mice lethality is very low (100 mg/kg) [188]; on the contrary, its oral toxicity is
weaker, as deduced by considering that the oral dose of 1 mg/kg – which is ten
times the lethal dose by intraperitoneal injection – does not kill the mice [187, 189].
Thus, on account of their diverse relative harmfulness, the European Food Safety
Authorities have established an allowance level for yessotoxin in shellfish which is
almost tenfold as high as that set for DSP toxins (16 mg of okadaic acid and 100 mg
of yessotoxin in 100 g of mollusk, respectively) [117]. Accordingly, the EU set up
a new protocol of extraction capable of separating OAs and YTXs in lipophilic and
hydrophilic layer, respectively [117], as already described in the okadaic acids
section of this chapter. Nevertheless, the recent finding of desulfocarboxyhomoYTXs in Adriatic mussels [190] raises some additional concerns, due to the
fact that these two new desulfoyessotoxins are unexpectedly recovered together
1378
P. Ciminiello et al.
with OAs in the lipophilic layer, so that the purpose of the European new protocol
fails. Hence, a further overhaul of the EU control procedure would be urgently
needed.
The chemical structure of YTX resembles that of brevetoxins, which are known
to interfere with the voltage-sensitive sodium channel [191]; this finding suggested
a possible interaction between YTX and cellular ion channels. Recently, however, it
has been observed that YTX does not interact with sodium channels nor induces any
competitive displacement of brevetoxins from site 5 of sodium channels [192]. It
has been proposed that YTX may interact with calcium channels inducing an uptake
of calcium in human lymphocytes [193, 194].
Another molecular effect elicited by YTX is the disruption of the E-cadherin
system in epithelial cells [195, 196]. Finally, studies on immune cells point to
phosphodiesterases as an intracellular target for YTX [197].
Although no human intoxication is known to have been caused by consumption
of shellfish contaminated by YTXs, the widespread occurrence of these compounds
in shellfish, sometimes at high levels, arouses an increasing interest in studying
YTX toxicity. Unfortunately, data on the toxicity by oral ingestion for most YTX
analogues are lacking; this appears particularly critical for some compounds, such
as desulfoYTXs, where the lack of a sulfate group decreases their hydrophilicity in
comparison to YTXs. So the biomembranes permeability and, consequently, the
toxicity level by oral ingestion could be greatly affected. An additional issue related
to the presence of desulfoYTXs in contaminated shellfish comes from two toxicological studies. In 1990, Terao et al. [187] found that totally desulfated YTX,
differently from YTX, did not affect the heart but caused severe fatty degeneration
and intracellular necrosis in the liver and pancreas. Finally, a recent report [198]
provided evidence that desulfated YTX interacts with transmembrane helix
domains.
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