Marine Biotoxins

Abstract

This chapter revises current knowledge on marine biotoxins that are known nowadays, considering all the distinct groups based on chemical structure and lipophilic or hydrophilic characteristics. Diarrheic shellfish poisoning (GlossaryTerm

DSP

), paralytic shellfish poisoning (GlossaryTerm

PSP

), amnesic shellfish poisoning (GlossaryTerm

ASP

), azaspiracid shellfish poisoning (GlossaryTerm

AZP

), ciguatera fish poisoning (GlossaryTerm

CFP

) are some of the groups that will be reviewed in this chapter.

The recent development and application of advanced technologies from the generically defined -omics sciences coupled with bioinformatics platforms has been included in this chapter in order to understand the ecology and evolution of phytoplankton species and bloom dynamics. Dinoflagellate toxins are structurally and functionally diverse, and many present unique biological activities.

The literature and information regarding the biological activities and the potential application of these phycotoxins has been gathered in this book section.

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Marine biotoxins are natural compounds mainly produced by marine microalgae, dinoflagellates, and diatoms. A small percentage of algae produce toxins that can harm human beings and also pose a deadly risk. Toxins are nonproteinaceous metabolites with different and complex structures, physical properties, and specific mechanisms of action. In general, they have low molecular weight ranging from $250$ to $3000\mathrm{Da}$. Shellfish and fish may become contaminated with these phycotoxins due to accumulation through marine food webs. Risk to humans often results in gastrointestinal disease, loss of short-term memory, neurological disorders, paralytic effects, or even death. In addition, the threat of marine biotoxins is not only a major cause of concern on human health and food safety, but it is also detrimental to the explotation of marine resources around the world and the economy of shellfish producers and associated industries, including tourism. This chapter revises current knowledge on marine biotoxins that are known nowadays, considering all the distinct groups based on chemical structure and lipophilic or hydrophilic characteristics. Diarrheic shellfish poisoning (GlossaryTerm

DSP

), paralytic shellfish poisoning (GlossaryTerm

PSP

), amnesic shellfish poisoning (GlossaryTerm

ASP

), azaspiracid shellfish poisoning (GlossaryTerm

AZP

), ciguatera fish poisoning (GlossaryTerm

CFP

) are some of the groups that will be reviewed in this chapter. Contamination by marine biotoxins often involves more than one group of toxins and monitoring programs typically cover a range of toxins. There has been an increased effort to develop rapid and feasible screening methods for marine biotoxins. Many methods to detect toxins and phytoplankton have several technical and, sometimes, ethical limitations and generally lack an adequate validation. Functional assays, rapid screening tests, generally based on immunological methods and molecular biology techniques, among others are attractive alternatives to toxins and microalgae characterization. In this chapter, different approaches to monitor and specific detection of shellfish toxins and dinoflagellates are reviewed taking into account the availability of resources. The potential use of several techniques for marine biotoxins or phytoplankton characterization, such as biosensors, applied immunology, GlossaryTerm

PCR

, sequencing, radioisotopic, flow cytometry, metabolomics, and proteomics will be explained along the chapter. Marine phytoplankton comprised both prokaryotic and eukaryotic species that share a common ability to photosynthesize and thus thrive in the upper, euphotic zone of the worlds’ oceans. Together, the prokaryotic and eukaryotic phytoplankton support marine food webs, including valuable fisheries and play a key role in the regulation of global biogeochemical cycles. Identification and quantification of individual species remains a difficult task. The recent development and application of advanced technologies from the generically defined -omics sciences coupled with bioinformatics platforms has been included in this chapter in order to understand the ecology and evolution of phytoplankton species and bloom dynamics. Dinoflagellate toxins are structurally and functionally diverse, and many present unique biological activities. At present, marine biotoxins include an extraordinary potential source of new bioactive compounds for pharmaceutical or medical uses constituting an emergent field of interest. Sometimes, the toxin itself is directly used as a drug, although more often the toxin lead compound provides a design idea for the development of a drug molecule. The biological activities of these phycotoxins are harmful to the target organisms, since the function of the toxin is either to protect the toxic species from attack by a predator or to immobilize the potential preys. Despite the damage in the target organism, toxins have a great potential for not target organisms, in particular for therapeutic purposes in humans. For instance, the target of many marine neurotoxins is the Na${}^{+}$ channel, though the sites of interaction and, thus, the pharmacological effects differ among compounds. In this context, phycotoxins can be invaluable tools to recognize and identify ionic channels and the specific role of each channel subtype in controlling cell function, exocytosis, or the Ca${}^{2+}$-dependent release of a given neurotransmitter. The literature and information regarding the biological activities and the potential application of these phycotoxins has been gathered in this book section.

1 Marine Toxins

Marine biotoxins are produced by certain species of toxic phytoplankton and can accumulate in various marine species: fish, crustacean, or molluscs such as mussels, clams, oysters, and scallops. In shellfish, toxins mainly accumulate in the digestive glands without causing adverse effects on the shellfish itself. However, when contaminated shellfish is consumed by humans this may lead to a severe intoxication or even death, causing public health and economic problems.

Currently, five groups of marine toxins are regulated in Europe: amnesic shellfish poisoning toxins (GlossaryTerm

ASP

), paralytic shellfish poisoning toxins (GlossaryTerm

PSP

), okadaic acid (GlossaryTerm

OA

) and pectenotoxin group (GlossaryTerm

DSP

), azaspiracid group, and yessotoxin group (EC/853/2004) [1]. Another group, neurotoxic shellfish poisoning (GlossaryTerm

NSP

) toxins (e. g., brevetoxin) is regulated in United States, New Zealand, and Australia. Many cases of tetradotoxin food poisoning are reported in Southeastern Asia as well as ciguatera fish poisoning (GlossaryTerm

CFP

) that was limited to tropical and subtropical areas. Other emerging toxins (Palytoxin, cyclic imines,) are not on the radar of the European Legislation or in other regions of the world. Scientists and legislative authorities are aware of these toxins but do not have sufficient amount of information related to their activity, estimation of the toxicity and risk assessment in humans [2].

2 Lipophilic Toxins

2.1 Okadaic Acid-Group (OA) Toxins and Pectenotoxin-Group Toxin (PTXs)

GlossaryTerm

OA

-group toxins are a class of marine biotoxins that include GlossaryTerm

OA

and dinophysis toxins (GlossaryTerm

DTX

1, GlossaryTerm

DTX

2, and GlossaryTerm

DTX

3). These toxins cause Diarrhetic Shellfish Poisoning (GlossaryTerm

DSP

), which is characterized by symptoms such as abdominal pain, diarrhea, nausea, and vomiting. These toxins are produced by planktonic dinoflagellates, Dinophysis genus, and the benthic Prorocentrum genus and can be found mainly in shellfish.

They were first reported in Netherlands in 1960s [3, 4] and in Japan in 1978 [5]. Nowadays, their distribution is considered worldwide, Europe, Japan, North and South America Australia, Indonesia, and New Zealand [6].

GlossaryTerm

OA

-group toxins are heat-stable polyether compounds [7]. While GlossaryTerm

OA

and GlossaryTerm

DTX

2 only differ by the position of one methyl group in the molecule, GlossaryTerm

DTX

1 has one additional methyl group. GlossaryTerm

DTX

3 represents a wide range of derivatives of GlossaryTerm

OA

, GlossaryTerm

DTX

1, and GlossaryTerm

DTX

2 that form acylated analogues when they are esterified with saturated and unsaturated fatty acids. Acylated analogs were reported to contribute considerably to the concentration of total GlossaryTerm

OA

-group toxins [7, 8].

GlossaryTerm

OA

and GlossaryTerm

DTX

s act by inhibiting serine/threonine phosphoprotein phosphatases PP1 and PP2A.These enzymes perform the dephosphorylation of numerous proteins; this function is closely related to many essential metabolic processes in eukaryotic cells [10, 9].

Pectenotoxins (GlossaryTerm

PTX

s) frequently co-occur with the GlossaryTerm

OA

-group. The presence of GlossaryTerm

PTX

s in shellfish was discovered due to high acute toxicity in mouse bioassay (GlossaryTerm

MBA

) after intraperitoneal (GlossaryTerm

i.p.

) injections of lipophilic extracts. Animal studies indicate that they are much less potent via the oral route and they do not induce diarrhea. GlossaryTerm

PTX

s have been detected in microalgae and/or bivalve mollusks in Australia, Italy, Japan, New Zealand, Norway, Portugal, and Spain and they are produced by Dinophysis spp.

GlossaryTerm

PTX

s are cyclic polyether macrolides that include over $15$ analogs. GlossaryTerm

PTX

1 to GlossaryTerm

PTX

$5$ were originally isolated from Japanese scallops, Patinopecten yessoensis [11]. More recently, other GlossaryTerm

PTX

s were found but only four analogs (GlossaryTerm

PTX

2, GlossaryTerm

PTX

12, GlossaryTerm

PTX

11, and GlossaryTerm

PTX

13) have been identified as actual biosynthetic products of the algae. Other GlossaryTerm

PTX

s seem to be either product of the shellfish metabolism or artifacts [12].

2.2 Yessotoxin-Group Toxins (YTXs)

Yessotoxins (GlossaryTerm

YTX

s) are a group of structurally related polyether toxins produced by the dinoflagellates Protoceratium reticulatum, and Lingulodinium polyedrum, and Gonyaulax spinifera [13]. YTXs had traditionally been included within the GlossaryTerm

DSP

group but the GlossaryTerm

EU

has excluded the YTXs from the GlossaryTerm

DSP

group in 2004, and nowadays, YTXs are considered as a separate group in the current regulation [1].

YTXs was first isolated in Japan, in 1986 [14] from the digestive gland of P. yessoensis, a scallop that gave its name to the toxin. More recently, YTXs have been reported worldwide, in Korea [15], Chile [16], and New Zealand [17]. In Europe, it has been described in mollusks in Norway, Italy, Spain, and Russia [13].

YTXs are disulfated polyethers, 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 ethers [13, 14]. The presence of the sulfo-ether group makes these molecules the most polar of the lipophylic toxins. More than $90$ YTXs congeners have been reported but structures for most of them have not been determined and only $30$ have been isolated [13, 18].

There is discrepancy between the toxicity via oral administration and GlossaryTerm

i.p.

injection and the precise mechanism of action is not yet known [13]. Several studies have been carried out to clarify this, it seems clear that YTXs do not inhibit phosphatases PP1 and PP2A [19]. They modulate ${\mathrm{Ca}}^{2+}$ homeostasis in human lymphocytes [20] and produce a cytotoxic effect during in vitro experiments in various cellular models, F-actin decrease, alter cell adhesion and cause apoptotic events [6].

2.3 Azaspiracid-Group Toxins (AZAs)

Azaspiracids (GlossaryTerm

AZA

s) are a group of marine biotoxins that accumulate in shellfish and represent an emerging human health risk [21]. They were originally isolated from blue mussels from Killary Harbour (Ireland) and produced an outbreak of shellfish poisoning in the Netherlands, in 1995. Afterward, seafood contamination by GlossaryTerm

AZA

s have been reported in different coastal localizations, including Europe, North and South America, Africa, and Japan [22, 23, 24, 25, 26].

There have been several attempts to identify the GlossaryTerm

AZA

-producing organism(s). James etal [27] have detected the presence of GlossaryTerm

AZA

-1, 2, and 3 in extracts from the heterotrophic dinoflagellate Protoperidium crassipes. However, this organism is supposed to act as a vector and nowadays, Azadium spinosum is considered the organism that produces these compounds [28].

GlossaryTerm

AZA

s are nitrogen-containing polyether toxins involving a unique spiral ring assembly containing a heterocyclic amine and an aliphatic carboxylic acid moiety [27, 29, 6]. They have been identified as GlossaryTerm

AZA

1 to GlossaryTerm

AZA

5 and numerous natural analogs and artifacts of methanolic solution (methyl ester analogs). GlossaryTerm

AZA

4 and GlossaryTerm

AZA

5 seem to be a biotransformation product of primary compounds into shellfish.

GlossaryTerm

AZA

1 is highly potent and capable of producing an important degradation of the intestinal tract and finally the death of rodent. In vitro assays, GlossaryTerm

AZA

s elevate caspase activity, increase cellular concentrations of ${\mathrm{Ca}}^{2+}$ and GlossaryTerm

cAMP

, induce irreversible cytoskeletal rearrangements, deplete cellular GlossaryTerm

ATP

, inhibit neuronal ion flux and bioelectrical activity, inhibit cell–cell adhesion and stimulate cholesterol biosynthesis [21, 25]. Human symptoms of intoxication include nausea, vomiting, and stomach cramps, but thus far, no deaths have been attributed to GlossaryTerm

AZA

s [25].

3 Hydrophilic Toxins

3.1 Saxitoxin-Group Toxins (STXs)

Saxitoxin (GlossaryTerm

STX

s)-group toxins include various naturally occurring neurotoxic alkaloids that induce the so-called paralytic shellfish poisoning (GlossaryTerm

PSP

) in humans. GlossaryTerm

PSP

is characterized by symptoms varying from a slight tingling sensation or numbness around the lips to fatal respiratory paralysis [30]. The intake of toxins necessary to induce GlossaryTerm

PSP

symptoms varies greatly. This may be due to differences in susceptibility as well as a lack of precision in exposure assessments due to problems with sampling and analysis of contaminated shellfish at the time of intoxication.

They have been detected in bivalve mollusks from various areas of the world. Several decades ago, few localizations appeared to be affected by GlossaryTerm

PSP

; but nowadays they are found worldwide [31, 32, 33].

GlossaryTerm

STX

s are mainly produced by toxic dinoflagellates belonging to the genus Alexandrium: e. g., A. tamarensis, A. minutum (syn. A. excavata), A. catenella, A. fraterculus, A. fundyense, and A. cohorticula, Gymnodimium, Pyrodinium and they have also been identified in some cyanobacteria which may occur in fresh and brackish waters.

Most of GlossaryTerm

STX

-group toxins are water-soluble and heat-stable nonproteinaceous compounds whose basic structure is composed of a 3,4-propinoperhydropurine tricyclic system. Up to $57$ different GlossaryTerm

STX

analogs have been described and they can be divided into subgroups based on substituent side chain such as carbamate, sulfate, hydroxyl, hydroxybenzoate, or acetate and also decarbamoyl variants [6]. GlossaryTerm

STX

, NeoSTX, GTX1, and dc-GlossaryTerm

STX

seem to be the most toxic analogs. These neurotoxins produce a blockade of ion conductance through the voltage-gated ${\mathrm{Na}}^{+}$ channel (GlossaryTerm

VGSC

) in nerves and muscles fibers [30].

3.2 Domoic Acid

Domoic acid (GlossaryTerm

DA

) is a potent neurotoxic amino acid that accumulates in high concentrations in shellfish, anchovies, and sardines that feed on the toxic phytoplankton. GlossaryTerm

DA

can affect marine animals, seabirds, and humans via consumption of this contaminated shellfish causing amnesic shellfish poisoning (GlossaryTerm

ASP

). GlossaryTerm

DA

was first reported in Canada in 1987 when it caused various human deaths after consuming GlossaryTerm

DA

-contaminated mussels [34]. Effects on both gastrointestinal tract and nervous system were observed including vomiting, nausea, diarrhea, abdominal cramps, and hemorrhagic gastritis; neurological symptoms are headache, dizziness, vision disturbances, disorientation, loss of short-term memory, motor weakness, seizures, profuse respiratory secretions, hiccoughs, unstable blood pressure, cardiac arrhythmia, and coma. Neurological symptoms may occur after a delay of a few hours or up to $3$ days [35]. GlossaryTerm

DA

especially harms the hippocampus and amygdaloid nucleus in the brain and damages the neurons by activating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (GlossaryTerm

AMPA

) and kainate receptors, causing an influx of ${\mathrm{Ca}}^{2+}$ and cell degeneration [36, 37].

GlossaryTerm

DA

is produced by the red alga Chondria armata and also from the diatoms Pseudo-nitzschia spp. and Nitzschia [38]. It is a water-soluble and heat-stable cyclic amino acid and it is structurally very similar to another known neurotoxin, the kainic acid [39]. Several isomers of GlossaryTerm

DA

(epi-domoic acid (GlossaryTerm

epi-DA

), (domoic acid C5${}^{\prime }$-diastereomer) and isodomoic acids A, B, C, D, E, F, G, and H (GlossaryTerm

iso-DA A–H

)) have been reported as well [7]. Iso-DA A, B, and C have not been detected in shellfish tissue. GlossaryTerm

DA

transforms into GlossaryTerm

epi-DA

through long-term storage [40] and degrades and transforms to GlossaryTerm

epi-DA

and iso-DAs through exposure to ultraviolet light [41, 42, 43]. In addition, the epimerization is also accelerated by heating [44].

4 Other Toxins

4.1 Ciguatoxins-Group Toxins

Ciguatoxins (GlossaryTerm

CTX

s) are a class of marine biotoxins that cause GlossaryTerm

CFP

. GlossaryTerm

CTX

s occur in fish as a result of biotransformation of precursor gambiertoxins produced by the benthic dinoflagellate Gambierdiscus spp. They are mainly associated to tropical and subtropical areas, but recently GlossaryTerm

CTX

-group toxins were identified for the first time in fish in Europe [45]. Various congeners of GlossaryTerm

CTX

s have been identified according to differences in their molecular structure: Pacific (GlossaryTerm

P-CTX

), Caribbean (GlossaryTerm

C-CTX

), and Indian (GlossaryTerm

I-CTX

s) [46].

Symptoms of the intoxication of ciguatera in humans include gastrointestinal and neurological effects [47]. Although, Gambierdiscus toxicus has been traditionally considered the main species responsible for GlossaryTerm

CTX

s production, nowadays it is known that Gambierdiscus spp. shows a high degree of complexity and it is composed of genetically and morphologically toxin producer species. Thus, distantly related groups co-occur across geography. Ten Gambierdiscus species were reported in the Atlantic and in the Pacific region [48, 49], and other new species were found in European Atlantic waters and in the Mediterranean Sea [50].

The congeners of GlossaryTerm

CTX

s are lipophilic polyethers compounds consisting of $13$–$14$ rings fused by ether linkages into a rigid ladder-like structure. GlossaryTerm

CTX

s are colorless, tasteless, odorless, and relative heat stable, devoid of heteroatoms other than oxygen and bear few conjugated bonds. Nowadays, over $50$ chemical congeners of GlossaryTerm

CTX

s have been identified whose toxicity can vary significantly: more than 100-fold among species of Gambierdiscus spp. compared with a $2$ to 9-fold within species variation due to changing growth conditions [49]. The chemical structures of more than $20$ GlossaryTerm

P-CTX

s analogs, main GlossaryTerm

CTX

in Pacific areas, have been identified, and structural modifications are seen in both termini of the toxin molecules, mostly by oxidation. Two GlossaryTerm

CTX

s from Caribbean sea (GlossaryTerm

C-CTX

s) were first isolated by Vernoux and Lewis in 1997 [51], and then were identified structurally. Other additional congeners were identified in 2002 [52]. More recently, four Indian Ocean GlossaryTerm

CTX

s (GlossaryTerm

I-CTX

s) have been reported [53], but their structural determination remains to be established.

The GlossaryTerm

VGSC

(${\mathrm{Na}}_{\mathrm{V}}$) is the primary molecular target of GlossaryTerm

CTX

s, and their binding to the neurotoxin receptor site $5$ of ${\mathrm{Na}}_{\mathrm{V}}$ causes the opening of the ion pore, activation of the channels, and ${\mathrm{Na}}^{+}$ entrance into the cells. Membrane depolarization and functional impairment of excitable cells are produced due to ${\mathrm{Na}}^{+}$ entry into the cell. Secondary responses observed in cells exposed to GlossaryTerm

CTX

s include ${\mathrm{Ca}}^{2+}$ into the cell by reverse action of ${\mathrm{Na}}^{+}$/${\mathrm{Ca}}^{2+}$ exchangers eventually leading to muscular contraction and neurotransmitter release. ${\mathrm{Na}}^{+}$ influx also affects the entrance of water into the cell, leading to cell swelling, blebbing, and cytotoxicity [45].

4.2 Brevetoxins (BTX)

Brevetoxin-(GlossaryTerm

BTX

) group toxins are neurotoxic polyether biotoxins which can accumulate in shellfish and fish. They are primarily produced by a dinoflagellate Karenia brevis (formerly called Gymnodinium breve and Ptychodiscus brevis) first identified in the Gulf of Mexico in 1947 [54, 55, 56]. However, other algae species (Chattonella antiqua, Chattonella marina, Fibrocapsa japonica, Heterosigma akashiwo) have also been reported to produce GlossaryTerm

BTX

-like toxins [57]. GlossaryTerm

BTX

s have been described in several localizations such as North America, New Zealand, Australia, Japan and Scotland [57, 58, 59]. K. brevis or K. brevis-like species have also been reported from Japan, New Zealand, West-Atlantic, Spain, Portugal, and Greece [57]. Nevertheless, no intoxication outbreaks in humans or occurrence of GlossaryTerm

BTX

-group toxins in shellfish or fish have been reported in Europe [55].

They are grouped into types A and B based on their molecular backbone structures. GlossaryTerm

BTX

-1 (or GlossaryTerm

PbTx

-1)13 (type A) and GlossaryTerm

BTX

-2 are considered to be the parent toxins from which other GlossaryTerm

BTX

-group toxins derive [60]. GlossaryTerm

BTX

A has a backbone of $10$ fused cyclic ether rings; GlossaryTerm

BTX

B (most abundant in K. brevis) has a backbone of $11$ fused cyclic ether rings [60, 61, 62]. It is important to highlight that GlossaryTerm

BTX

-group toxins are metabolized in shellfish and fish, yielding several metabolites of GlossaryTerm

BTX

-group toxins that finally affect to consumers [55, 63].

Ion channels are the principal target of GlossaryTerm

BTX

s by binding to receptor site 5 of the GlossaryTerm

VGSC

in cell walls [60] leading to an uncontrolled ${\mathrm{Na}}^{+}$ influx into cells and depolarization of neuronal and muscle cell membranes. The response to GlossaryTerm

BTX

s in different cells, organs or in vivo models is dependent on various factors, such as the existence of different GlossaryTerm

VGSC

subtypes expressed.

4.3 Tetrodotoxin (TTXs)

GlossaryTerm

TTX

(11-[${}^3$H]-tetrodotoxin) is a neurotoxin that has been identified from taxonomically diverse marine organism. Its name derives from Tetraodontiformes, an order that includes pufferfish, ocean sunfish or mola, triggerfish, among others. However, GlossaryTerm

TTX

has been described in a diverse range of phylogenetically unrelated organism, such as newts, frogs, nematodes, starfish, crabs, mollusk, and others. It has also been suggested that GlossaryTerm

TTX

is produced by symbiotic bacteria. Actually, there has been a debate between source of production of GlossaryTerm

TTX

from animals or bacteria, and nowadays it has not been firmly established a conclusion. GlossaryTerm

TTX

may serve as an antipredator defense, offensive weapon, or for within- and between-species communication [64].

Although GlossaryTerm

TTX

is associated to tropical waters, mainly South Asia and more specifically Japan, it has been also identified in Mexico and United States [65]. In Europe, GlossaryTerm

TTX

has been reported in migrant puffer fish in Greek waters [66] and in a gastropod Charonia lampas lampas in Portugal [67].

GlossaryTerm

TTX

is a heat-stable and water-soluble heterocyclic guanidine with a highly unusual structure containing a single guanidinium moiety attached to a highly oxygenated carbon backbone that consists of a 2,4-dioxaadamantane structure with five hydroxyl groups [68].

Tetrodotoxin binds to site $1$ of the fast GlossaryTerm

VGSC

[33]. Site $1$ is located at the extracellular pore opening of the ion channel. The binding of any molecules to this site temporarily disables the function of the channel. Saxitoxin, neosaxitoxin also bind the same site although GlossaryTerm

TTX

has different affinities for the variant ${\mathrm{Na}}^{+}$ channel isoforms that confer resistance to various species [69]. GlossaryTerm

TTX

causes paralysis of voluntary muscles (including the diaphragm and intercostal muscles, stopping breathing), and reduced blood pressure, predominantly by vasodilatation. Early symptoms of this effect might include among others weakness, tingling of the lips, and dizziness [70]. Gastrointestinal symptoms are often severe and include nausea, vomiting, diarrhea, and abdominal pain. Cardiac arrhythmias may precede complete respiratory failure and cardiovascular collapse.

4.4 Palytoxin (PlTXs) and Analogs

Palytoxin (GlossaryTerm

PlTX

) is one of the most poisonous nonprotein substances known to date. GlossaryTerm

PlTX

was first isolated and purified from Palythoa toxica [71] and currently present a worldwide distribution [71, 72]. GlossaryTerm

PlTX

s have also been detected in other marine zoanthids (soft corals) of the genus Palythoa (e. g., P. tuberculosa, P. vestitus, P. mammilosa, P. carobaeorum, P. aff. Margaritae) and benthic dinoflagellates of the genus Ostreopsis (e. g., Ostreopsis siamensis, O. mascarenensis, O. ovata) [73]. Blooms of Ostreopsis spp. have also been reported in European countries such as Spain, France, Italy, and Greece [74].

The GlossaryTerm

PlTX

-group toxins are complex polyhydroxylated compounds with both lipophilic and hydrophilic areas. They are white, amorphous, hygroscopic solids. At least eight different GlossaryTerm

PlTX

analogs are known: GlossaryTerm

PlTX

, ostreocin-D, ovatoxin-A, homopalytoxin, bishomopalytoxin, neopalytoxin, deoxypalytoxin, and 42-hydroxypalytoxin [75].

The main accepted molecular action of palytoxin is blockage of the ${\mathrm{Na}}^{+}$/${\mathrm{K}}^{+}$-ATPase pump [76, 77, 78]. This binding inhibits the active transport of ${\mathrm{Na}}^{+}$ and ${\mathrm{K}}^{+}$ across the cell membrane that is essential for cell homeostasis, by transforming the pump into a nonspecific permanently open ion channel [79]. Symptoms of GlossaryTerm

PlTX

-group toxins intoxication are not well defined, but include weakness and myalgia, fever, nausea, and vomiting.

4.5 Cyclic Imines (CIs)

Cyclic imines (GlossaryTerm

CI

s) are a recently discovered group of marine biotoxins formed by: spirolides (GlossaryTerm

SPX

s), gymnodimines (GlossaryTerm

GYM

s), pinnatoxins (GlossaryTerm

PnTX

s), pteriatoxins (GlossaryTerm

PtTX

s), prorocentrolides, and spiroprorocentrimine. These toxins are macrocyclic compounds with imine- (carbon–nitrogen double bond) and spiro-linked ether moieties. They are grouped together due to that imino group acts as functioning pharmacophore [80] and the similarities in their GlossaryTerm

i.p.

, toxicity in mice [6, 81].

GlossaryTerm

SPX

s are the largest group of cyclic imines and together with GlossaryTerm

GYM

s are the best characterized. GlossaryTerm

SPX

s were described for the first time in contaminated shellfish, in 1991, in Canada [82]. These toxins are metabolites of dinoflagellates Alexandrium ostenfeldii and Alexandrium peruvianum and are sometimes found in the presence of other toxins such as GlossaryTerm

PSP

toxins [83]. Up to $14$ GlossaryTerm

SPX

s analogs have been described of which 13-desmethyl GlossaryTerm

SPX

C is the most commonly found in shellfish [81].

GlossaryTerm

GYM

s are produced by the dinoflagellates Karenia selliformes and first isolated in oysters from New Zealand [84]. GlossaryTerm

PnTX

s and GlossaryTerm

PtTX

s are the GlossaryTerm

CI

structurally and synthetically more related to GlossaryTerm

SPX

s. GlossaryTerm

PTX

s were first discovered in extracts from the digestive glands of pen shell, Pinna attenuata in China and Japan [85]. Seven GlossaryTerm

PnTX

s analogs (GlossaryTerm

PnTX

s A-G) have been characterized [81]. Pteriatoxins A, B, and C, were isolated from the Okinawan bivalve Pteria penguin in 2001 [86]. Torigoe etal [87] isolated the Prorocentrolide A for the first time from Prorocentrum lima and then the Prorocentrolide B was first isolated from P. maculosum in 1996 [88]. Finally, Spiro-prorocentrimine was isolated from a laboratory-cultured benthic Prorocentrum species of Taiwan by Lu etal [89]. At present, it is accepted that nicotinic and muscarinic acetylcholine receptors are their principal target.

4.6 Maitotoxins (MTXs) and Gambierol

Maitotoxins (GlossaryTerm

MTX

s) and gambierol are a group of water-soluble toxins produced by species of dinoflagellates of the genus Gambierdiscus, which grow on algae in tropical waters around the world. Gambierdiscus is known to produce both GlossaryTerm

MTX

and GlossaryTerm

CTX

s, which accumulate in the body of herbivorous fish and are transmitted through the tropical food chain to carnivorous species.

GlossaryTerm

MTX

is a polyketide-derived polycyclic ether integrated by four rigid polyether ladders connected by mobile hydrocarbon chains. Maitotoxin includes $32$ ether rings, $22$ methyls, $28$ hydroxyls, and $2$ sulfuric acid esters and has an amphipathic structure [90, 91, 92].

Maitotoxin activates ${\mathrm{Ca}}^{2+}$ permeable, nonselective cation channels, leading to an increase in levels of cytosolic ${\mathrm{Ca}}^{2+}$ ions. It is thought that maitotoxin leads to the formation of pores on these ion channels. Finally, a cell death cascade is activated, resulting in membrane blebbing and ultimately cell lysis [93]. Maitotoxin is known to activate cytosolic ${\mathrm{Ca}}^{2+}$-activated proteases calpain-1 and calpain-2, contributing to necrosis [94].

Gambierol was described as a potent potassium voltage-gated channel blocker that exhibits potent acute lethal toxicity against mice (minimal lethal dose: $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}$, GlossaryTerm

i.p.

) and it is suspected to participate in the symptoms of GlossaryTerm

CFP

.

5 Biotechnological Techniques Used to Study Toxic Microalgae and Marine Biotoxins

The potential use of several techniques for marine biotoxins or phytoplankton characterization are explained along the chapter. In general, the sensitivity of the analytical methods and bioassays depends on the toxins studied. Combination of several approaches, including chromatography-based techniques, to identify individual toxins and the overall toxicity of the mixture, should provide an ideal alternative. Thus, a synergistic complement can be obtained by coupling biological and or functional assays with analytical techniques based on the physicochemical properties of the toxins.

5.1 Immunological Methods

The main advantage of the immunological techniques is the variety of immunoglobulins for the specific detection of different targets. These are characterized by sensitivity, specificity, fast performing, and automation. However, the corner stone for these methods seems to be the quality of the antibody presenting cross-reactivity limitations.

An unequivocal identification of phytoplancton and detection of phycotoxins can be obtained by using a species-specific antibody against the target organism or group of toxins. Specific polyclonal or monoclonal antibodies can be reactive to one epitope and therefore more specific. Combination of some of them, allows the identification, counting, and examination of marine phytoplankton. Use of antibodies with fluorescent markers has made possible to apply different technologies, such as microscopy, flow cytometry, or flow cam. The light emitted by a labeled antibody can be detected by a fluorescent microscope, equipped with a GlossaryTerm

UV

light, a flow cytometer a confocal microscope or a flow cam. For instance, several monoclonal antibodies were obtained against different species of Alexandrium allowing the unequivocal identification of the dinoflagellate A. minutum [95]. Then, using immunofluorescence could help to identify different species in natural samples during coastal monitoring.

To discriminate and count small cells or a large number of samples, flow cytometry can be used. Designed to automate the rapid analysis and identification of cells, flow cytometry is a reliable method for the routine monitoring of the abundance of phytoplankton species, leading to an early detection of GlossaryTerm

HAB

s. This is an optimal instrument for analyzing cells ranging from $0.5$ to $20\mathrm{\mu }\mathrm{m}$ in diameter (concentrations between ${10}^6$ to ${10}^9$ cells/L [96].

In this context, a modified flow cytometer, known as FlowCam, was developed to handle larger cells. It is an integrated system combining the capabilities of flow cytometry, microscopy, imaging, and fluorescence that counts, takes images and analyses the particles that range in size from $20$ to $200$. In addition, to monitor harmful species of microalgae, other potential applications of immunodetection include studies of plankton community structure and ocean optics. Other authors used Imaging FlowCytobot that combines video and flow cytometric technology to capture images of nano- and microplankton and to measure the chlorophyll fluorescence associated with each image. The images are of sufficient resolution to identify many organisms to genus or even species level [97].

The use of immunological assays has become a promising alternative for detecting small molecules such as marine biotoxins and many kits are commercially available.

5.1.1 Enzyme-Linked Immunosorbent Assays (ELISAs)

The enzyme-labeled immunosorbent assay GlossaryTerm

ELISA

is a powerful analytical tool for natural toxins detection. Specific antibodies recognize toxins and this bound complex is quantified by labeling the free component with a reporter enzyme, usually horseradish peroxidase (GlossaryTerm

HRP

) acting as an amplifier to produce many signals. The use of the 96-well microtitre polystyrene plate, the automation and the competitive principle are the most commercial immunoassays for natural toxins.

The GlossaryTerm

ASP

GlossaryTerm

ELISA

for the determination of GlossaryTerm

DA

was approved by the GlossaryTerm

EU

as an alternative to the official chemical method GlossaryTerm

HPLC

-GlossaryTerm

UV

detection [98]. This kit is a direct competitive immunoassay where free GlossaryTerm

DA

in the sample competes with GlossaryTerm

DA

-conjugated protein coated on plastic wells for binding to antidomoic acid antibodies in the solution. Other GlossaryTerm

ELISA

tests were developed as the Ridascreen Fast GlossaryTerm

PSP

, a competitive GlossaryTerm

ELISA

for the quantitative analysis of GlossaryTerm

STX

and related toxins. A monoclonal antibody with high affinity against brevetoxin B was used to develop a competitive GlossaryTerm

ELISA

for detection of brevetoxins in molluscs [99]. Production of monoclonal antibodies for immunoassays by immunizing mice with a synthetic hapten conjugate, instead of the natural toxins, allowed the detection of Pacific ciguatoxins using a direct sandwich GlossaryTerm

ELISA

 [100, 101]. Recently, an improvement of the efficiency and simplification of a colorimetric competitive indirect immunoassay GlossaryTerm

ELISA

was carried out based on direct labeling via a covalent bond to the anti-GlossaryTerm

OA

antibody [102, 103] and a chemiluminiscent GlossaryTerm

ELISA

method for this toxin was also developed [104].

The challenge of designing antibodies with the optimal specificity and matching toxicity with toxic levels is further complicated by the structural and toxicological diversity within a toxin group together with a potential chemical conversion and biotransformation. However, since recombinant antibodies have been successfully constructed against GlossaryTerm

PlTX

 [105], similar strategies could help the challenge for complex toxins groups. Lack of standardization and transferability of in house GlossaryTerm

ELISA

tests between laboratories should be overcome by increasing the focus on validation, based on new strategies for production of high-quality antibodies. Nevertheless, a comparative evaluation of GlossaryTerm

ELISA

s and GlossaryTerm

EU

reference methods for the detection of the hydrophilic toxins GlossaryTerm

ASP

and GlossaryTerm

PSP

was carried out in many seafood products [106]. The high correlation coefficient between official and immunoassays proves that these tests can be used as screening systems in a variety of species without matrix interference. However, the best sensitive bioassay will depend on the toxins evaluated. In this context, the advantages and disadvantages of GlossaryTerm

ELISA

and cytotoxicity assays to detect GlossaryTerm

BTX

s were recently reviewed [107].

5.1.2 Phage Display Technology

The phage display technology refers to the expression of peptides, proteins, or antibodies on the surface of filamentous bacteriophage viruses. This in vitro strategy provides a valuable system for easy and rapid generation of specific antibodies fragments directed against different antigens, including difficult targets as small molecules. Semisynthetic phage display libraries were used to select recombinant antibodies against GlossaryTerm

PlTX

. Some antibodies proved high affinity for immobilized and free GlossaryTerm

PlTX

in a competitive GlossaryTerm

ELISA

with a low detection limit, a very reproducible standard curve and a wide working range [105].

5.1.3 Biosensors

Biosensors are measuring systems based on a biological recognition component with three functioning parts: a biological element, cells, microorganisms, antibodies, the transducer, capable of measuring a biomolecular interaction and converting it into a signal, and the electronics, responsible for the display of the results easily interpreted.

Most of the biosensors developed for shellfish toxins are immunosensors and the optical biosensor with surface plasmon resonance (GlossaryTerm

SPR

) is the most widely used in the large majority of reported platforms [108]. It has been incorporated into a wide range of devices to measure the analytes present in a sample. For most biomolecules, the change in response is proportional to the mass of the material bound to the surface of the sensor chip. The measure of the binding response against time is called sensorgram [109]. Small molecular weight compounds, as many drugs and toxins, are very difficult to measure. Then, an inhibition assay approach that relies upon the immobilization of analytes to the chip surface and the injection of the binding partner over the chip surface was developed. Using this assay format, the level of binding to the surface is inversely proportional to the concentration of the target analyte present in the sample. There are three key elements that determine the way in which the methods perform.

• The binding protein, often an antibody, is essential in terms of delivering a specific molecular interaction with the target analyte.

• The surface chemistry is a very important parameter in an GlossaryTerm

  SPR

  assay. Small compounds can be immobilized via the production of an analyte–protein conjugate and this, in turn, is immobilized onto the surface on a sensor chip via an amine or carboxyl coupling strategy. While this approach can be relatively straightforward, it has two main disadvantages. Firstly, during the regeneration of the chip surface, the immobilized conjugate can alter its three-dimensional structure and, thereby, bring about a different level of interaction with antibodies during subsequent interaction measurements. Secondly, the lifespan of an immobilized conjugate on a chip surface tends to be very short. The sensor chips tend to be the most expensive part of the assay, resulting in a high costper test. A much better way to produce chip surfaces is through the direct immobilization of the analyte onto the sensor surface, being much more robust and stable than the conjugate approach.

• The type of method used for sample preparation has to be compatible with this analytical approach. In general, due to the advantages of GlossaryTerm

  SPR

  over conventional immunoassays, i. e., as they are nonequilibrium based and flow through based, in many cases the requirement for a highly purified sample extract can be avoided [96].

Some examples of biosensors for GlossaryTerm

DA

, GlossaryTerm

OA

, GlossaryTerm

PSP

, or GlossaryTerm

TTX

have been developed, tested and/or validated [110, 111, 112, 113, 96]. However, one challenge for these assays is the ability to simultaneously measure multiple biotoxins in a single bioanalytical system as was performed in a Framework $7$ Project entitled CONFFIDENCE (www.conffidence.eu). Within this project, the GlossaryTerm

SPR

methods developed for GlossaryTerm

DSP

, GlossaryTerm

ASP

, and GlossaryTerm

PSP

are being combined onto a single sensor chip using a multichannel biosensor developed as part of another European project Biocop (www.biocop.org).

Comparison of GlossaryTerm

ELISA

and GlossaryTerm

SPR

biosensor technology for detection of GlossaryTerm

PSP

toxins shows that the method of choice will depend on the end-user needs. Some authors point out the reduced manual labor and simplicitiy of GlossaryTerm

SPR

biosensor compared to GlossaryTerm

ELISA

, ease of sample extraction, and superior real time semiquantitave analysis [114], although lower costs and high sensitivity are some advantages of the GlossaryTerm

ELISA

.

GlossaryTerm

DNA

-biosensors have specific probes that target only GlossaryTerm

DNA

-sequences present in the organism of interest and can be used on-site and therefore circumvent the need to return samples into the laboratory. A GlossaryTerm

DNA

-biosensor was adapted by Metfies et al. to the electrochemical detection of the toxic dinoflagellate A. ostenfeldii [115]. The technical background of this device is explained in detail in the German patent application DE 10032 042 A1 (Elektrochemischer Einwegbiosensor für die quantitative Bestimmung von Analytkonzentrationen in Flüssigkeiten). The device could facilitate the work that must be undertaken in the course of monitoring toxic algae by eliminating the need to count algae and reduce the toxins tests.

An immunosensor based on magnetic beads as support to immobilize GlossaryTerm

OA

on the surface of SPE and then to perform indirect competitive immunoassay using differential pulse voltammetry as a method of electrochemical detection [116]. Modifications of this automated flow-through amperometric immunosensor were performed for highly sensitive detection of GlossaryTerm

OA

 [117, 118]. Other sensors include the molecularly imprinted polymer (GlossaryTerm

MIP

)-based sensors that use templates to provide polymers with specific recognition properties and chemosensors based on the photoinduced electron transfer (GlossaryTerm

PET

) sensing of the toxins by synthetic fluorophores.

5.1.4 Lateral Flow Immunochromatography

Lateral flow immunochromatographic assays are based on a competitive immunoassay format, similar to the home pregnancy test and provide a qualitative indication to the presence of GlossaryTerm

OA

and analogs, and GlossaryTerm

DA

. These methods also faced the same antibody cross-reactivity challenge.

5.2 Enzymatic, Colorimetric, and Other Tests

Several studies based on the mechanism of action of GlossaryTerm

OA

-group toxins, inhibition of the enzyme PP2A, were designed and adapted. Some systems as fluorimetry or colorimetry were the most common employed [119, 120], although other as electrochemical biosensors were also reported [102, 121].

Numerous colorimetric protein phosphatase inhibition assays have been recently reviewed [102, 103]. An enzymatic colorimetric test, OkaTest, based on the GlossaryTerm

OA

-group of toxins mechanism of action, was recently internationally validated to be used as an alternative or complementary to the reference method for monitoring the GlossaryTerm

OA

toxins group in molluscs [122] according to Commission Regulations (EC) No. 2074/2005 [123] and No. 15/2011 [124].

Recently a ligand binding assay was developed to detect cyclic imine in the frame of the Atlantox project, within the Atlantic Area Operational Programme (Atlantox 2008-1/003): www.atlantox.com. This assay, under patent, is based on the mechanism of action of these toxins that act on the nicotinic acetylcholine receptor.

A fast, simple, and sensitive capillary electrophoresis-based immunoassay with electrochemical detection was developed for GlossaryTerm

STX

and analogs in shellfish. The method was based on competitive reactions between GlossaryTerm

HRP

-labeled antigen and free antigen with a limited amount of antibody [125].

The feasibility of GlossaryTerm

OA

detection in real-time by using an acoustic wave platform with a microfluidic feature and an GlossaryTerm

ELISA

-like protocol was recently reported [126].

5.3 Receptor Binding Assay (RBA)

The receptor binding assay (GlossaryTerm

RBA

) is a high-throughput method for detection of toxins in extracts of seawater and shellfish. This method is based upon the competition between radiolabeled and unlabeled toxins for available receptor sites. The radioactive toxin is displaced from its receptor by toxin present in an unknown sample, thereby, reducing the total radioactivity. Thus, the amount of radioactively labeled toxin that is displaced is proportional to the amount of toxin in the unknown sample. Then, the toxin present in an unknown sample can be quantified by comparison to a standard curve obtained using pure toxin.

GlossaryTerm

RBA

was developed from the isolation of GlossaryTerm

VGSC

obtained from animal tissues, and was basically implemented by competitive measures of radioactivity using tritiated saxitoxin ([${}^3$H]-GlossaryTerm

STX

) or brevetoxin (GlossaryTerm

PbTx

). Vieytes etal published a rapid and sensitive method that used ${\mathrm{Na}}^{+}$ channels obtained from rat brain membranes to detect GlossaryTerm

PSP

toxins. ${\mathrm{Na}}^{+}$ channels were coated onto microtiter plates and were used to develop a direct solid-phase binding assay [127]. Years later, more developed method was described by Doucette etal based on microplate scintillation technology. Good quantitative agreement of the assay with GlossaryTerm

MBA

and GlossaryTerm

HPLC

analysis of crude extracts of contaminated shellfish, as well as GlossaryTerm

PSP

toxin-producing algae, was observed [128]. Usup reported about binding properties of six saxitoxin congeners using [${}^3$H]-GlossaryTerm

STX

and a preparation rich in ${\mathrm{Na}}^{+}$ channels. In this study, EC50 values ranged from $4.38\mathrm{nM}$ for GlossaryTerm

STX

to $142\mathrm{nM}$ for GTX5 [129].

GlossaryTerm

RBA

has been refined in the last decade for the detection of GlossaryTerm

PSP

toxins. These methods suffer from limitations such as the availability of radio-labeled materials. Restrictions on the world-wide distribution of [${}^3$H]-GlossaryTerm

STX

imposed by the international Chemical Weapons Convention in 1997 served to other radiolabeled toxin development such as 11-[${}^3$H]-tetrodotoxin (GlossaryTerm

TTX

) [130, 131]. In 2004, thanks to collaborative efforts among the US Food and Drug Administration (GlossaryTerm

FDA

), International Atomic Energy Agency (GlossaryTerm

IAEA

), and the National Oceanographic and Atmospheric Administration (GlossaryTerm

NOAA

), the worldwide unavailability of ${}^3$H saxitoxin was solved temporarily. A relative stable [${}^3$H] GlossaryTerm

STX

is now distributed worldwide by GlossaryTerm

IAEA

and was used in collaborative trials of the GlossaryTerm

PSP

toxins using receptor binding assay [132]. The assay used a competition among GlossaryTerm

PSP

toxins, in shellfish extracts, and a labeled ${}^3$H GlossaryTerm

STX

diHCl for binding to GlossaryTerm

VGSC

in a rat brain membrane preparation. Quantification of binding was carried out using a microplate or traditional scintillation counter. The study focused on the ability to measure the GlossaryTerm

PSP

toxicity of samples below, near, or slightly above the regulatory limit of $800\mathrm{\mu }\mathrm{g}$ GlossaryTerm

STX

diHCl equiv/kg [1]. The correlation with the GlossaryTerm

MBA

(OMA 959.08) yielded a correlation coefficient (r2) of $0.84$, while correlation with the precolumn oxidation GlossaryTerm

HPLC

method (OMA 2005.06) yielded a (r2) of $0.92$. This method has been accepted as official method of analysis AOAC (OMA 2011.27) [133].

Similar to GlossaryTerm

PSP

toxins method; GlossaryTerm

BTX

GlossaryTerm

RBA

is a functional bioassay in which an unknown quantity of nonradiolabeled GlossaryTerm

BTX

competes with radiolabeled GlossaryTerm

BTX

[${}^3$H]GlossaryTerm

PbTx

-3, for the site $5$ receptor of GlossaryTerm

VGSC

. Although, the assay using direct detection of a radiolabeled probe, bound to rat brain membranes, is a simple and robust format of pharmacological assays; other variations have been also tested using phospholipid vesicles [134] and membranes from other animals than rats [135, 136]. More recently other study has compared various extracts of GlossaryTerm

BTX

using four independent methods: GlossaryTerm

RBA

, radioimmunoassay (GlossaryTerm

RIA

), neuroblastoma (N2A) cytotoxicity assay, and liquid chromatography/mass spectrometry (GlossaryTerm

LC-MS

). As determined by GlossaryTerm

LC

/GlossaryTerm

MS

, the GlossaryTerm

RBA

, GlossaryTerm

RIA

, and N2A cytotoxicity assay detected $73$, $83$, and $51\%$ of the total GlossaryTerm

BTX

concentration [137].

GlossaryTerm

RBA

designed for GlossaryTerm

CTX

s traditionally measures the binding competition between GlossaryTerm

CTX

in the sample and a [${}^3$H]GlossaryTerm

PbTx

standard for the ${\mathrm{Na}}^{+}$ channel receptor. The use of [${}^3$H]GlossaryTerm

PbTx

is a necessity, as nowadays no tritiated GlossaryTerm

CTX

s is available. It should to be noted that GlossaryTerm

RBA

was successfully applied for monitoring programs on ciguatera risk in three islands of French Polynesia [138, 139].

In the case of GlossaryTerm

ASP

toxins, the GlossaryTerm

RBA

quantifies GlossaryTerm

DA

activity by the competitive displacement of radiolabeled kainic acid from a cloned glutamate receptor by GlossaryTerm

DA

in a sample. In 1997, Van Dolah etal [140] reported the further development of the receptor assay using a cloned rat GLUR6 glutamate receptor. The limit of detection and selectivity of the assay were optimized through inclusion of the glutamate decarboxylase (GlossaryTerm

GLDC

) pretreatment step to eliminate potential interference due to high concentrations of endogenous glutamate in shellfish. However, as GlossaryTerm

GLDC

was difficult to obtain from suppliers, other variations were introduced [141].

Due to limitations of the standard AOAC GlossaryTerm

MBA

for estimation of toxins levels there was a need for alternative testing protocols. The relevance of GlossaryTerm

RBA

tools is high, but the need for specific equipment and licensing requirement for the use of radiolabeled compounds may lead to obvious limitations in the application of this assay. Some main disadvantages include the high dependency on the receptor source, it does not provide any information on the toxin profile, it cannot be easily automated, and it has been validated only for a few toxins. However, GlossaryTerm

RBA

is adequately sensitive and more specific than GlossaryTerm

MBA

 [45]. Attempts to modify this assay to use label other than tritiated forms has led to the development of a competitive GlossaryTerm

RBA

based upon the use of GlossaryTerm

BTX

-B2 labeled with a chemiluminescent acridinium moiety. The acridinium brevetoxin-B2 seems to be a promising alternative to the conventional radioactive ligand in order to avoid constraints associated with GlossaryTerm

RBA

 [142, 46].

6 Biotechnology Application for Phytoplankton Detection, Monitoring, and Toxins Production

Recent development and application of advanced technologies from the generically defined -omics sciences (genomics, transcriptomics, proteomics, and metabolomics) coupled with bioinformatics platforms has already provided deep and often revolutionary shifts in understanding the ecology and evolution of phytoplankton species and bloom dynamics [143].

Harmful algal blooms (GlossaryTerm

HAB

s) are natural events that have become common in coastal waters worldwide during the last years, being recurrent in European coastal waters. The toxic microalgae responsible for these blooms are mainly dinoflagellates, which display heteromorphic life cycles, including motile planktonic stages, as well as immotile benthic resting cysts [144, 145].

Monitoring of harmful species is important to understand population dynamic activities related to the exploitation of coastal areas. To distinguish species by their morphological characteristics, cells must be examined by light, epifluorescence, or electron microscopy. This can be time consuming, and requires taxonomic expertise. Moreover, in some cases, morphology-based diagnostic tests lead to misidentification due to the existence of morphologically closely related species. Molecular methods, generally based on the GlossaryTerm

DNA

probe hybridization and GlossaryTerm

PCR

techniques, have been developed for the rapid and sensitive identification of GlossaryTerm

HAB

species [145, 146, 147]. The target GlossaryTerm

DNA

genes are generally within ribosomal GlossaryTerm

RNA

operons, which are repeated in high copy number in the microalgae genome and can be genus or species specific, as 18S, 28S, 5.8S, GlossaryTerm

ITS

1, and GlossaryTerm

ITS

2 among others.

6.1 Genomics

Conventional genomics is often based upon the sequencing and annotation of whole genomes, with subsequent bioinformatics focus on the structure and function of key groups of genes. However, the ability to perform genetics experiments with microalgae has been accelerated in recent years by the availability of genome sequences. The application of emerging genomics techniques to phytoplankton research is likely to become widespread in the future.

6.1.1 Sequencing

Currently, there are more marine cyanobacterial genome sequences available than eukaryotic phytoplankton genome sequences, primarily due to differences in genome size and complexity. The average cyanobacterial genome size is $2$–$3$ million base pairs (GlossaryTerm

Mbp

), although they range up to $9\mathrm{Mbp}$. In contrast, eukaryotic genomes tend to be orders of magnitude larger [148]. Completed genome projects have focused on species with relatively small genomes, ranging from $12$ [149] up to $57\mathrm{Mbp}$ [150]. The recent availability of marine phytoplankton genome sequences has prompted a wealth of research in diverse fields including ecology, evolution, biochemistry, and biotechnology.

Concerning eukaryotic marine phytoplankton, it must be considered that it includes phylogenetic lineages resulting from primary, secondary, and tertiary endosymbiosis events. This phylogenetic and physiological diversity is translated into a wide range of potential genome sizes, structures, metabolic pathways, life histories, and evolutionary relationships. Of the four major lineages of eukaryotic phytoplankton, whole genome sequences are currently available for two; the heterokonts [150, 151] and the prasinophytes [149, 152]. Additional members of the heterokont and haptophyte lineages are in the pipeline for sequencing or draft genomes are being analyzed (e. g., the haptophyte Emiliania huxleyi; http://genome.jgi-psf.org/Emihu1/Emihu1.home.html) [148].

Genomics are more than simply genome sequencing including transcriptional analyses of both sequenced [153, 154] and unsequenced organisms [155], and also targeted metagenomics of ecologically important but uncultured phytoplankton [156].

One of the challenges in examining structural diversity in eukaryotic phytoplankton is the large number of novel genes identified in each newly sequenced genome that have no homology to genes with known or suspected functions. Often, they are similar only to genes of unknown function in closely related organisms. Depending on the genome, the number of novel genes ranges from $20$ to nearly $40\%$ highlighting the limitations of homology-based approaches. Differences in gene structure (e. g., novel splice sites, signal peptides, and untranslated regions) constitute a challenge to gene-calling algorithms designed for higher plants and metazoans. One solution has been the development of tiling arrays for sequenced genomes [148] that differs from traditional microarrays in the nature of the probes. Instead of probing for sequences of known or predicted genes that may be dispersed throughout the genome, tiling arrays probe intensively for sequences which are known to exist in a contiguous region. This is useful for characterizing regions that are sequenced, but whose local functions are largely unknown. Using tiling arrays Mock etal [154] predicted $3470$ new genes in the Thalassiosira pseudonana genome that were not previously identified using standard gene-calling algorithms. This $33\%$ of increase in the total number of genes predicted in the T. pseudonana genome suggests that gene density and number are not fully captured using standard gene-finding algorithms. Sequenced genomes also highlight the phylogenetic and metabolic diversity that characterizes marine phytoplankton. Description of new lineages, such as the uncultured picohaptophytes suggests that probably many ecologically important lineages have yet to be discovered [156].

Investment in genetic techniques both available and robust in a diverse array of marine phytoplankton will greatly help in understanding gene function, especially for the large number of hypothetical and conserved hypothetical genes [148]. New approaches have emerged and will continue to emerge that rely on genome sequence data. For example, whole genome microarrays include a range of targets such as all annotated genes or even the whole genome. New directions in this area will use genome information from diverse keystone species to create microarrays for use in characterizing environmental samples; GlossaryTerm

mRNA

sequencing approaches such as Illumina sequencing generate only short reads and can take advantage of available genomes to examine gene expression. For example, GlossaryTerm

mRNA

from two conditions is used to generate GlossaryTerm

cDNA

libraries that are sequenced to obtain quantitative gene expression profiles that can be compared.

The application of these emerging genomics techniques to phytoplankton research is likely to become widespread in the future. One challenge beyond the genomics revolution is to apply these techniques in the marine environment to examine phytoplankton physiology in situ. This will mean synthesizing many different types of data simultaneously such as real-time physical and chemical environmental variables, community species composition, gene expression profiles, and protein signatures. This system-level approach has the potential to greatly expand our understanding of how phytoplankton acts in their environment [148].

6.1.2 PCR and Real-Time PCR (qPCR)

GlossaryTerm

qPCR

is a very promising technique in terms of sensitivity, specificity, enumeration capability, and costs reduction [145, 157]. Molecular approaches, when used together with other morphological characters, may help to solve taxonomical problems. The nuclear GlossaryTerm

rDNA

internal transcribed spacer regions and the 5.8S GlossaryTerm

rRNA

gene are widely used to characterize the phylogenetic aspects of a great variety of these organisms [147, 158, 159, 160].

Even though these applications are very valuable regarding human health, the use of GlossaryTerm

PCR

and GlossaryTerm

qPCR

for the direct detection and identification as well as monitoring of toxic phytoplankton is of special interest. Several studies have been developed for simplex and multiplex detection of different groups, genus and/or species, as Alexandrium, Ostreopsis, or different cyanobacteria [161, 162, 163, 164]. It is important to highlight the fact that environmental samples often contain complex mixtures of organic matter, which may inhibit GlossaryTerm

PCR

/GlossaryTerm

qPCR

, thus an internal amplification control (GlossaryTerm

IAC

) should be included to avoid misinterpretation of false-negative results. Additionally, different strategies have been applied to overcome the problem of GlossaryTerm

PCR

/GlossaryTerm

qPCR

inhibition caused by contamination substances: the use of thermostable GlossaryTerm

DNA

polymerase, commercial nucleic acid extraction kits that remove inhibitors and facilitate purification, dilution of template GlossaryTerm

DNA

prior to GlossaryTerm

PCR

/GlossaryTerm

qPCR

assays and the use of bovine serum albumin in the GlossaryTerm

PCR

/GlossaryTerm

qPCR

assay [144, 163]. Several studies have been published applying GlossaryTerm

PCR

/GlossaryTerm

qPCR

for monitoring GlossaryTerm

HAB

worldwide, from the Mediterranean Sea to Australia [157, 164, 165, 166].

Regarding toxin quantification/detection, it is more often performed by other techniques, but different studies have also been performed applying GlossaryTerm

PCR

/GlossaryTerm

qPCR

. In 2011, Murray etal developed a method targeting the unique core gene sxtA to identify saxitoxin-producing GlossaryTerm

HAB

s in Australian marine water [165]. Their method was tested against different species and strains of Alexandrium, Ostreopsis, Gymnodinium among others. Al-Tebrineh etal also developed a GlossaryTerm

qPCR

method capable of detecting SXT producing species, but they took advantage of the higher throughput of multiplex formats of GlossaryTerm

qPCR

and also targeted the genes coding for microcystin (mcy), nodularin (nda), cylindrospermopsin (cyr) in a multiplex GlossaryTerm

qPCR

experiment to specifically detect toxin producing species [163].

6.1.3 Microarrays

In a microarray study, gene-specific probes are spotted onto precise positions on a solid surface and targeted with an unknown GlossaryTerm

DNA

or GlossaryTerm

RNA

sample. Microarrays represent the scaling up, miniaturization, and automation of well-known hybridization techniques. Recently, a FP7 European research project called MIDTAL (Microarrays for the detection of toxic algae) aimed to construct a universal microarrayfor the detection of harmful algae and their toxins (http://www.midtal.com/). Results obtained showed the third generation of the MIDTAL microarray that includes between $140$ and $163$ probes for various toxic algal species including GlossaryTerm

ASP

, GlossaryTerm

DSP

, and GlossaryTerm

PSP

producers. The designed microarray has great potential to be used as a monitoring tool for toxic algae detecting multiple species simultaneously although improvements in GlossaryTerm

RNA

extraction and tests are still needed [167]. The most obvious advantage of the microarray is its ability to identify species that cannot be identified with traditional methods, such as Pseudo-nitzschia, Alexandrium, and Heterosigma [168, 169].

6.1.4 Other Genomic Techniques

Other techniques different from sequencing and GlossaryTerm

PCR

have been also applied in phytoplankton studies. These include restriction fragment length polymorphism (GlossaryTerm

RFLP

), the first GlossaryTerm

DNA

-based technique used in phytoplankton studies; denaturing gradient gel electrophoresis (GlossaryTerm

DGGE

); single stranded conformation polymorphism (GlossaryTerm

SSCP

); random amplified polymorphic GlossaryTerm

DNA

(GlossaryTerm

RAPD

); amplified fragment length polymorphism (GlossaryTerm

AFLP

); Microsatellites or short sequence repeats (GlossaryTerm

SSR

s); molecular probes and single nucleotide polymorphisms (GlossaryTerm

SNP

s). Most of these techniques were quite useful to study phytoplankton especially when no sequences were available because they can be used without prior knowledge of the genome [170].

6.2 Transcriptomics

Transcriptomic studies gather efforts to isolate all the GlossaryTerm

RNA

transcripts, i. e., the transcriptome, from a sample of cells, and determine the presence and abundance of each particular species of gene-encoding GlossaryTerm

RNA

or GlossaryTerm

mRNA

. These studies help to elucidate genes involved in nutrient acquisition and metabolism (including those involved in synthesizing or regulating the synthesis of toxic molecules), aid in annotating genomes, and also have the potential to find novel transcripts or transcripts with unusual structures that may hint at the mechanism of gene regulation [143, 171, 172].

For many organisms with either large and/or complex genomes, transcriptome sequencing may be a more immediate and cost-effective route than whole genome sequencing to obtain ecological and evolutionary information.

An expressed sequence tag or GlossaryTerm

EST

is a short subsequence of a GlossaryTerm

cDNA

. They may be used to identify gene transcripts in gene discovery and sequence determination. The identification of GlossaryTerm

EST

s has proceeded rapidly, with approximately $74.2\mathrm{million}$ GlossaryTerm

EST

s now available in public databases (e. g., GenBank 1 January 2013, all species). The GlossaryTerm

cDNA

s used for GlossaryTerm

EST

generation are typically individual clones from a GlossaryTerm

cDNA

library. The resulting sequence is a relatively low quality fragment whose length is limited by current technology to approximately $500$–$800$ nucleotides. Because these clones consist of GlossaryTerm

DNA

that is complementary to GlossaryTerm

mRNA

, the GlossaryTerm

EST

s represent portions of expressed genes. They may be represented in databases as either GlossaryTerm

cDNA

/GlossaryTerm

mRNA

sequence or as the reverse complement of the GlossaryTerm

mRNA

, the template strand. GlossaryTerm

EST

s can be mapped to specific chromosome locations using physical mapping techniques, such as radiation hybrid mapping, Happy mapping, or GlossaryTerm

FISH

. Alternatively, if the genome of the organism that originated the GlossaryTerm

EST

has been sequenced, one can align the GlossaryTerm

EST

sequence to that genome using a computer.

GlossaryTerm

EST

analysis of the dinoflagellate A. minutum revealed $192$ genes that were differentially expressed between isolates that produced GlossaryTerm

PSP

toxins and those that did not [173]. In terms of the functional ecology of A. minutum, the $192$ genes are putative candidates for genes involved in toxin synthesis and regulation or acclimation to intracellular GlossaryTerm

PSP

toxins. The sequences in the GlossaryTerm

EST

library also suggest that GlossaryTerm

PSP

toxins generated by this alveolate did not arise from a recent gene transfer from cyanobacteria, as was previously hypothesized [173]. Other dinoflagellate GlossaryTerm

EST

sequencing projects have yielded further insights into toxin production, predator–prey interactions, genome architecture, and trophic status. As sequencing costs drop and capabilities increase, transcriptome analyses of marine phytoplankton species and strains within species will soon become not only feasible but also common. This approach has also been recently applied to A. ostenfeldii [143, 174].

6.3 Proteomics

The field of proteomics is complementary to genomics and transcriptomics since it provides additional information on gene expression and regulation. Proteomics include the determination of protein expression levels and protein–protein interaction studies. Moreover, proteomics aim to identify posttranslational modifications of proteins, as well as the organization of proteins in multiprotein complexes and their localization in tissues [172].

In the past decade, genomics began to explore the diversity, cellular evolution and adaptive abilities of marine phytoplankton. Although this has provided the community with a huge amount of information, it is not necessarily being translated into biochemical expression or phenotype. Genomics demonstrates which genes are shared, but proteomics can show clearer relationships by illustrating functional similarities and phenotypic variances. With a well-designed experiment, researchers can examine the conditions under which a protein is expressed; its cellular location, the relative quantities, and what protein–protein interactions take place [175].

Chan etal [176, 177] studied protein profiles under different environmental conditions found in Prorocentrum triestinum. He showed both constancy of protein profiles and variability of the relative abundance of some proteins under different and culture conditions. Differentially expressed protein patterns of P. triestinum indicated that P. triestinum growing under nitrogen-limited conditions possessed higher growth potential since its protein expression profiles are more similar to that of the blooming stage. These results are consistent with the observations that it is possible to rapidly trigger a bloom by the addition of a supply of nitrates. A more detailed analysis of the differential synthesis of proteins in these various stages and studies to elucidate the functions of these proteins will be critical for understanding the molecular mechanisms involved in blooming. Phytoplankton proteomic approaches that use whole genome sequences to examine protein expression by matching protein GlossaryTerm

MS

/MS spectra to those predicted from whole genome sequences are already available [150]. Cell surface proteins (GlossaryTerm

CSP

s) of Alexandrium catenella have been also studied in detail. Protein profiles at different toxin biosynthesis stages, were analyzed and $53$ differentially expressed proteins were identified. These were involved in various biological processes, nine of which might be involved in the GlossaryTerm

PST

biosynthesis of A. catenella using the quantitative proteomic approach to compare protein profiles at different toxin biosynthesis stages.

Proteomics studies should help to reveal the toxin biosynthesis mechanisms and pathways in dinoflagellates [178]. Proteomics has provided valuable information on the effects and also the mode action of the dinoflagellate metabolite yessotoxin on a human liver cell line. Also, analysis of the plastid proteome of the Florida red tide dinoflagellate K. brevis detected an electron transfer protein (plastocyanin) inherited from green algae that may contribute to ecological success in iron use. Nevertheless, much promise remains to be fulfilled in the application of proteomics to studies of GlossaryTerm

HAB

ecophysiology and bloom dynamics [179].

6.4 Metabolomics

The term metabolomics is usually defined as the comprehensive and quantitative analysis of all small molecules in a biological system [180]. Metabolomics allows monitoring and mapping the dynamic range of bioactive secondary metabolites under different conditions. The most commonly used gas chromatography-time-of-flight mass spectrometry (GlossaryTerm

GC-TOF-MS

)-based method facilitates the identification and robust quantification of a few hundred metabolites. The merging of metabolomics and metagenomics has the potential to be a particularly powerful combination in shedding light on planktonic and microbial interactions in marine ecosystems. Statistical evaluation of data provides insight into the metabolic changes within the cells as well as into the released metabolites. The knowledge of the complex patterns of chemicals released by microalgae will reveal new mechanisms for processes such as: community function, food location, or complex defensive and allelopathic interactions [181].

A great number of metabolic pathways and regulatory mechanisms could be predicted based on the genomic sequences. Nevertheless, some of the genes might not be functional in vivo. Recent technical developments make metabolomics a suitable tool to define their genes function and regulation in microalgae. Analyses of diatom genomes have suggested tight connection between a number of novel pathways and regulatory steps in diatom metabolism. However, there are still several important open questions to be addressed. Given the massive recent acceleration in genome sequencing capacities we will shortly have an enormous resource of genetic information for marine phytoplankton. Combining this information with details on their metabolic complement will greatly enhance our understanding of the metabolic processes by and how they interact with their environment, as well as the evolution of their underlying metabolic pathways [182].

Metabolomics approach has already been successfully applied to the dinoflagellate endosymbiont Symbiodinium [179]. As an emerging technique, metabolomics have not been widely applied in toxic phytoplankton studies, but some research groups are developing databases for identification of marine biotoxins using metabolomics approaches [183].

7 Potential Pharmacological Uses of Phycotoxins

In the past few decades, extensive studies have been devoted to investigate the toxicology and pharmacology of biotoxins [184, 185]. A renewed interest is seen in marine alkaloids and their analogues, including the GlossaryTerm

STX

-group toxins (GlossaryTerm

STX

s), with regards to their use as therapeutic agents or as a drug lead [186]. GlossaryTerm

STX

s bioactivity studies and molecular modeling could lead to the design of unnatural analogs with improved pharmaceutical characteristics. Recently, a group of toxins isolated from marine cone snails (genus Conus), known as conotoxins, have been shown to contain over $2000$ peptide analogs. Conotoxins are able to specifically target a broad range of ion channels and membrane receptors and are currently under investigation for possible clinical trials. In 2004, a synthetic version of a conotoxin analogue, ω-conotoxin MVIIA, also known as ziconotide (trade name Prialt) was the first marine natural product to be approved for use by the US GlossaryTerm

FDA

 [186]. Ziconotide acts by targeting N-type voltage sensitive ${\mathrm{Ca}}^{2+}$ channels and is used for the treatment of chronic pain in spinal cord injury.

Ion channels triggered by voltage changes along the cell membrane are termed voltage-gated ion channels (GlossaryTerm

VGIC

s), include channels that conduct ${\mathrm{Na}}^{+}$, ${\mathrm{Ca}}^{2+}$ and ${\mathrm{K}}^{+}$. Activation of ${\mathrm{Na}}^{+}$ or ${\mathrm{Ca}}^{2+}$ VGICs produces cell stimulation by membrane depolarization, while ${\mathrm{K}}^{+}$ channel activation results in inhibitory hyperpolarization. Toxins that bind GlossaryTerm

VGIC

s can serve as powerful molecular probes to study the channel structure and function, as well as serve as models for studying evolutionary targets and drug discovery [187]. Some marine biotoxins are classified as neurotoxins due to their ability to interact with these channels [188]. As we will discuss later in this section many marine biotoxins have affinity by one or more GlossaryTerm

VGIC

s and therefore could have an important pharmaceutical potential.

7.1 Lipophilic Toxins (LTs)

7.1.1 Okadaic Acid-Group (OA) Toxins and Pectenotoxin-Group Toxins

GlossaryTerm

OA

is a marine biotoxin that presents a great value in medical research and has facilitated the understanding of several cellular processes [189]. GlossaryTerm

OA

-group toxins have interest due to their high cytotoxic activity and the extraordinary ability to promote tumors [190, 191]. It is considered that their effect varies depending on the cell type and concentration used [192].

It has been a model for analyzing therapeutic effects of some drugs in treating neurodegenerative diseases [193]. GlossaryTerm

OA

acts as a potent neurotoxin that induces apoptotic events in various cell lines and promotes tumors in several organs. Mechanisms are dependent on cell type, the antioxidant status of each cell and the ability to metabolize GlossaryTerm

OA

 [194, 195, 196, 197, 198, 199].

GlossaryTerm

OA

-group toxins are specific inhibitors of PP1 and PP2A that are involved in the regulation of many cellular processes and modulate the extent of phosphorylation or dephosphorylation of proteins. Other cellular targets for GlossaryTerm

OA

have been suggested [189, 200, 201, 202, 203]. GlossaryTerm

OA

has been used to analyze the mechanisms by which the conjugated linoleic acids can act as antitumor agents in breast cancer cells due to its activity as an inhibitor of PP2A [189]. In addition it has been reported that the extent of GlossaryTerm

OA

-induced injuries and the toxin organotrophicity are dose-related and may be determined by the administration route. After intravenous administration, GlossaryTerm

OA

acts as a hepatotoxin with undetectable effects on the intestine but also has an impact on cytoskeletal elements at sublethal doses [204].

GlossaryTerm

PTX

s are potent cytotoxic compounds against several human cancer cell lines. They are hepatotoxic whose mechanism of action has not yet been fully elucidated [205, 206, 207]. Among other effects, the induction of apoptosis in rat hepatocytes, in salmon and p53-deficient cell lines have been reported [208].

GlossaryTerm

PTX

2 is an inhibitor of actin and cytokinesis and it is known to be used as a chemotherapeutic agent against tumors type p53 [209]. It was demonstrated that a loss of p53 sensitizes tumor cells to actin damage. GlossaryTerm

PTX

2 was first identified as a cytotoxic entity in marine sponges, which depolymerizes actin filaments and it was found to be highly effective and more potent to activate an intrinsic pathway of apoptosis in p53-deficient tumor cells compared to those with functional p53 both in vitro and in vivo. Other agents that depolymerize or knot actin filaments were also found to be toxic to p53-deficient tumors. In p53-deficient cells, GlossaryTerm

PTX

2 triggers apoptosis through mitochondrial dysfunction, and this is followed by the release of proapoptotic factors and caspase activation. Furthermore, Bax activation and Bim induction was observed only in p53-deficient cells after GlossaryTerm

PTX

2 treatment. Therefore, these results suggest that Bim triggers apoptosis by activating Bax in p53-deficient tumors upon actin damage, and that actin inhibitors may be potent chemotherapeutic agents against p53-deficient tumors.

Recent studies demonstrated that this congener do not induce diarrhea or any other signs of disease by oral administration. Body distribution of GlossaryTerm

PTX

2 and GlossaryTerm

PTX

2 seco acid (GlossaryTerm

PTX2SA

) was analyzed in mice after oral and intraperitoneally administration. Cytotoxicity assays in vitro have been the first step to the discovery of many drugs used in antitumor therapy. However, more information is needed about its toxicity to humans and their pharmacokinetics [210].

7.1.2 Yessotoxins-Group Toxins

YTXs are polyether compounds that were discovered due to their high acute toxicity during GlossaryTerm

MBA

by GlossaryTerm

i.p.

, injection for lipophilic toxin analysis of mollusks [211, 212]. There are no published reports of human poisonings caused by YTXs and no available data on chronic exposure, carcinogenicity, or genotoxicity [213]. Various authors have suggested that the target organ following GlossaryTerm

i.p.

administration is the heart [214]; however, other authors consider that YTX is involved in neurological disorders and its main target is the thymus [215]. Tereo etal have reported severe microscopic alterations in cardiac muscle [214]. Difference between these studies is not clear and has been proposed that discrepancies may be due to different purities of the toxins and the use of different strains of mice [213]. YTX administration to human lymphocytes leads to an increase in cytosolic ${\mathrm{Ca}}^{2+}$ concentration [20] and in specific activity of cyclic nucleotide phosphodiesterase $3^{\prime }$, $5^{\prime }$ [216]. Other studies have reported that cellular ${\mathrm{Ca}}^{2+}$ homeostasis is not related to its cytotoxic effect [217]. Furthermore, YTX causes dose-dependent decrease in the levels of GMPc and AMPc after incubation due to the activity of phosphodiesterases. These effects depend on ${\mathrm{Ca}}^{2+}$ and can be changed by phosphodiesterases [216, 218].

YTX induced a decrease in F-actin filaments in several cell models as cerebellar granule cells (GlossaryTerm

CGC

s), mouse fibroblast, rat L3, mouse myoblast cell lines, and GlossaryTerm

MCF

-7 human breast adenocarcinoma cells [217, 219]. However, this seems not to be a universal effect, since no effects over F-actin levels were detected in M17 neuroblastoma cell line, in rabbit enterocytes, or in human Caco-2 cells [220, 221]. YTXs cause selective disruption of the E-cadherin-catenin in epithelial cells and could potentially suppress tumor functions of E-cadherin [222]. Pathway degradation of E-cadherin is affected by YTX at long incubation times and low doses. Since E-cadherin has been linked to tumor spreading and metastasis, it has been a concern if the disruption of E-cadherin by YTX could affect tumor expansion in YTX-contaminated mollusk consumers. However, in vivo experiments demonstrated that although the resulting molecule of E-cadherin disruption had increased, YTXs did not induce any effect on E-cadherin system in in vivo experiments [223].

YTX-induced apoptotic events in different cellular lines, including cancer cells, primary cell cultures, and cell lines. There are reports related to YTX-induced apoptosis in rat hepatocytes, rat glioma cells, GlossaryTerm

HeLa

S3 cells, rat cerebellar neurons, BE(2) neuroblastoma cell line, L6 myoblast cell line, BC3H1 myoblast cell line, mouse fibroblast NIH3T3 cell line, CaCo-2 cells, GlossaryTerm

MCF

-7 cancer cells, and GlossaryTerm

HepG2

cell cultures. In this sense, YTX could be used in therapeutic applications, for instance, as an antitumor drug [224]. Thus, the European patent application EP1875906 [225] considers to use YTX as an antitumor drug, and protoceratin I, the major compound with cytotoxic activity against human tumor cell lines isolated from P. cf. reticulatum, was proved to be identical to homo YTX [226].

It has also been investigated the activity of various YTXs analogs on GlossaryTerm

MCF

-7 tumor cell lines; structural modification in homoYTX do not change the activity of the toxin, while the side chain structure of carbon $9$ seems essential. Thus, carboxiYTX and hidroxihomoYTX present less activity than YTX [227]. These data have been confirmed by kinetic studies between phosphodiesterases and YTX, hidroxiYTX and carboxiYTX, proving the lower affinity between these proteins and the last two analogs [228].

7.1.3 Azaspiracid-Group Toxins

Molecular targets of these toxins are not well known, but some studies were focused on the study of second messengers: increased intracellular ${\mathrm{Ca}}^{2+}$ and AMPc [229, 230, 231, 232], effects of reorganization of the actin cytoskeleton [233, 234, 235, 236] and effects on adhesion by activation of adhesion protein fractionation (claudins and cadherins) [234, 237, 238].

Although, symptoms observed during episodes of GlossaryTerm

AZA

s poisoning were similar to those typically observed with GlossaryTerm

DSP

, administration of toxic GlossaryTerm

AZA

s materials to mice led to great differences with the GlossaryTerm

DSP

. Mice injected with low doses of GlossaryTerm

AZA

s did not present diarrhea [239], and in a few hours develop slowly progressive paralysis, difficulty in breathing, and finally death [240]. A $200\mathrm{mg}/\mathrm{kg}$ GlossaryTerm

i.p.

dose appears to be the minimal lethal dose in mice to GlossaryTerm

AZA

1 while GlossaryTerm

AZA

3 and GlossaryTerm

AZA

2 showed a higher toxicity ($110$ and $140\mathrm{mg}/\mathrm{kg}$, respectively) [240, 241]. Damages in liver, pancreas, spleen, thymus and stomach were observed. Moreover, after administration of GlossaryTerm

AZA

sublethal doses by gavage, alterations were observed in the small intestine, affecting epithelial cells with a degree of damage progressively increasing from $4$ to $24\mathrm{h}$ after administration [242, 243]. GlossaryTerm

AZA

cytotoxicity was first observed by Flanagan etal [244] in hepatoblastoma and bladder carcinoma cells exposed to contaminated crude mussel extract. Twiner etal [235] observed that GlossaryTerm

AZA

1 causes cytotoxicity in kidney, lung and immune, neuronal, and pituitary cells. However, neurons appear to be particularly sensitive to GlossaryTerm

AZA

s, causing irreversible morphological changes with relatively short exposure times and cytotoxicity with longer exposures to these toxins [231, 236]. It also requires longer exposure times to achieve complete cytotoxicity in primary cultures of cerebellar granule cells [200]. Also, they induce apoptosis through caspase activation [210, 245].

GlossaryTerm

AZA

s are implicated in upregulation on gene expression and protein levels of low-density lipoprotein receptor (GlossaryTerm

LDLR

). This effect seems to be a response to the decreased level in intracellular cholesterol caused by GlossaryTerm

AZA

1 [25]. It was shown that T lymphocytes exposed to GlossaryTerm

AZA

1 up regulated gene expression and protein levels of GlossaryTerm

LDLR

due to decreased levels of intracellular cholesterol. Although premature to determine decisively, yet consistent with currently available in vitro data, GlossaryTerm

AZA

1 appears to be targeting a membrane protein such as a claudin, cadherin, or GlossaryTerm

LDLR

, in turn eliciting an effect on intracellular signaling molecules and the cytoskeleton, ultimately resulting in cytotoxicity.

7.2 Hydrophilic Toxins

7.2.1 Saxitoxin-Group Toxins (PSP)

GlossaryTerm

STX

-group toxins are closely related water-soluble tetrahydropurine compounds acting by interfering with GlossaryTerm

VGSC

functioning. The toxin acts from the exterior of the cells by getting access to the extracellular cavity of the channel and binding to the so-called site $1$ of the α-subunit in the ${\mathrm{Na}}^{+}$ channel [246]. Site $1$ is shaped by a short portion (SS2) of the amino acid stretches connecting the S5 and S6 trans-membrane helices in the four domains of the α subunit, giving rise to a cavity that accommodates the toxin. GlossaryTerm

STX

then forms hydrogen bonds and electrostatic interactions with the side chains of several amino acids that participate to the ion selectivity filter of the channel. The interaction of one GlossaryTerm

STX

molecule with the site $1$ of the α-subunit in the ${\mathrm{Na}}^{+}$ channel essentially plugs the channel and blocks its ion flux. The loss of ${\mathrm{Na}}^{+}$ conductance in excitable cells prevents membrane depolarization and the transmission of the action potential, representing the molecular basis of the toxic effects of GlossaryTerm

STX

. As a consequence of GlossaryTerm

VGSC

blockade, a progressive loss of neuromuscular function ensues, leading to the reported neurotoxic symptoms that can result in death by asphyxia. Skeletal analogs of GlossaryTerm

STX

with a fused-type tricyclic ring system, designated FDSTX, were synthesized as candidate ${\mathrm{Na}}^{+}$ ion channel modulators [247]. Their inhibitory activity on ${\mathrm{Na}}^{+}$ ion channels was examined by means of cell-based assay. Two of the analogs showing moderate inhibitory activity were further evaluated by the use of the patch-clamp method in cells that expressed ${\mathrm{Na}}_{\mathrm{V}}$1.4 (a GlossaryTerm

TTX

-sensitive ${\mathrm{Na}}^{+}$ channel subtype) and ${\mathrm{Na}}_{\mathrm{V}}$1.5 (a GlossaryTerm

TTX

-resistant ${\mathrm{Na}}^{+}$ channel subtype). These compounds showed moderate inhibitory activity toward ${\mathrm{Na}}_{\mathrm{V}}$1.4, and weaker inhibitory activity toward ${\mathrm{Na}}_{\mathrm{V}}$1.5. Uniquely, however, the inhibition of ${\mathrm{Na}}_{\mathrm{V}}$1.5 by one of the analogs was irreversible.

Some GlossaryTerm

STX

s have demonstrated pharmaceutical potential as a long-term anesthetic in the treatment of anal fissures and for chronic tension-type headache. The recent elucidation of the GlossaryTerm

STX

biosynthetic gene cluster in cyanobacteria and the identification of new GlossaryTerm

STX

analogs will present opportunities to further explore the pharmaceutical potential of these intriguing alkaloids [186]. A specific suite of analogs can be isolated from a single GlossaryTerm

STX

s-producing organism, which is directly a result of the evolution of genes present within the organism’s genome. Naturally occurring GlossaryTerm

STX

s can also be precursors for extracellular metabolic or chemical transformations into new analogs. Knowledge of these transformations may have important implications for the detection, toxicity, and removal of GlossaryTerm

STX

s from a contaminated source. Other medicinal uses for GlossaryTerm

STX

s may become more established by screening the bioactivity of less toxic analogs, since their use as a potential local anesthetic has long been known. The characterization of GlossaryTerm

STX

biosynthesis genes [165, 248] and their potential use in combinatorial biosynthesis, together with the constant discovery of novel analogs (either natural or transformed), is likely to expand the possibilities for their pharmaceutical use.

GlossaryTerm

STX

also has a huge pharmaceutical potential for its ability to induce anesthesia through interaction with the GlossaryTerm

VGSC

 [186]. It has been suggested that site $1$ blockers prolong the duration of anaesthesia in a synergistic manner when combined with other local anaesthetics. In spite of this, the push for GlossaryTerm

STX

to enter clinical trials has been hindered by its systematic toxicity. The use of GlossaryTerm

STX

as a slow release, prolonged anesthetic was recently demonstrated using a novel controlled release system in male rats. Liposomal formulations of GlossaryTerm

STX

, either alone or in conjunction with dexamethasone and/or bupivacaine, were able to block the sciatic nerve in rats for long periods with no damaging myotoxic, cytotoxic, or neurotoxic effects and little associated inflammation. Liposome formulations of GlossaryTerm

STX

for slow and site-directed release for prolonged anaesthesia have been postulated as a putative treatment of localized and severe joint pain.

GTX2 and GTX3 also have clinical potential and have been utilized for the treatment of anal fissures by their direct injection into both sides of the fissure. A success rate of $98\%$ with remission after $15$ and $28$ days for acute and chronic conditions respectively was observed. Both toxins have also been used in the treatment of chronic tension type headache, with $70\%$ of patients responding to treatment. These studies recognize that several GlossaryTerm

STX

s have potential as future pharmaceutical leads; therefore, more investigation is needed in this field [6].

NEO-GlossaryTerm

STX

has being developed as a local anesthetic. Doses of $100\mathrm{\mu }\mathrm{g}$ have been used by local infiltration in anesthetized adult humans without adverse effect. Wylie etal hypothesized that similar doses could cause significant respiratory, neuromuscular, and cardiovascular impairment [249].

7.2.2 Domoic Acid (ASP)

GlossaryTerm

DA

belongs to the kainoid class of compounds, which is a class of excitatory neurotransmitters. Pharmacological studies reported that GlossaryTerm

DA

isomers are not as toxic as GlossaryTerm

DA

because they bind less strongly to the kainate receptor proteins than GlossaryTerm

DA

itself; however, 5${}^{\prime }$-epi-domoic acid has a similar toxicity [35]. Mayer and Hamann [250] reviewed the available literature on pharmacological and toxicological studies with marine natural products including articles reporting on the bioactivity and/or pharmacology of $106$ marine chemicals. They included GlossaryTerm

DA

in the group of compounds that affect the immune system. Moreover they reported that GlossaryTerm

DA

at in vitro concentrations that were toxic to neuronal cells ($1\mathrm{mM}$) was shown to trigger a limited activation of rat brain microglia, an immune cell type that contributes to circa $10\%$ of the total glial population in the central nervous system, and the concomitant release of two potentially neurotoxic mediators, namely GlossaryTerm

TNF

-α and matrix metalloproteinase-9.

GlossaryTerm

DA

acts as a potent glutamate receptor agonist. Although mechanisms of domoic acid neurotoxicity are poorly defined, an excitotoxic mechanism is believed to be involved. GlossaryTerm

DA

inhibits astrocytes glutamate uptake in a dose-dependent manner, leading to accumulation of glutamate in the extracellular compartment and thus producing neurotoxicity [251].

In vitro studies have shown that, depending on its concentration, GlossaryTerm

DA

causes necrotic or apoptotic cell death in mouse cerebellar granule neurons (GlossaryTerm

CGN

). Necrosis is observed at higher concentrations of GlossaryTerm

DA

($10\mathrm{\mu }\mathrm{M}$) and involves activation of both GlossaryTerm

AMPA

/kainate and N-metyl-d-aspartic acid (GlossaryTerm

NMDA

) receptors, the latter activated by GlossaryTerm

DA

-induced glutamate release. In contrast, apoptotic damage predominates at lower concentrations of GlossaryTerm

DA

(e. g., $100\mathrm{nM}$) and is solely due to activation of GlossaryTerm

AMPA

/kainate receptors [252]. Both, necrosis and apoptosis have been shown to be mediated by oxidative stress. Upon oral administration, GlossaryTerm

DA

is poorly absorbed, has a short half-life in blood, and poorly permeates the blood–brain barrier, limiting the access of low levels of GlossaryTerm

DA

in the central nervous system, but this might not be the case during development, a period of suggested particular sensitivity to GlossaryTerm

DA

neurotoxicity. The authors evaluated if a prolonged exposure to low GlossaryTerm

DA

levels would result in neurotoxicity and/or in altered susceptibility to a subsequent exposure in cultured mouse GlossaryTerm

CGN

s. Neurons from wild-type mice and from mice lacking the glutamate cysteine ligase (GlossaryTerm

GCL

) modifier subunit (Gclm ${}^{\mathrm{-}\mathrm{/}\mathrm{-}}$) (already known to be highly susceptible to GlossaryTerm

DA

neurotoxicity) were compared. They found that prolonged exposure to low-level GlossaryTerm

DA

in vitro provides protection against toxicity of a higher GlossaryTerm

DA

acute exposure, apparently due to the ability of chronic low dose of GlossaryTerm

DA

to up-regulate glutathione (GlossaryTerm

GSH

) synthesis. Genetic conditions leading to low GlossaryTerm

GSH

levels, which already increase susceptibility to GlossaryTerm

DA

neurotoxicity, could potentially negate such protective mechanisms [252].

7.3 Other Toxins

7.3.1 Ciguatoxins (CTXs)

GlossaryTerm

CFP

is the most frequently reported seafood-toxin illness in the world. It produces gastrointestinal, cardiovascular, neurologic, and neuropsychiatric symptoms which can last from days to even months. GlossaryTerm

CTX

s derive from benthic dinoflagellates and are transferred through the food web from the algae to herbivorous and carnivorous fish, and finally to humans.

GlossaryTerm

CTX

s are among the most potent natural substances known. One of the Pacific Ocean ciguatoxins, GlossaryTerm

P-CTX

-1, poses a health risk at concentrations as low as $0.08$–$0.1\mathrm{\mu }\mathrm{g}/\mathrm{kg}$ although GlossaryTerm

CTX

s rarely accumulates in fish at levels that are lethal to humans [253]. GlossaryTerm

CTX

s activate the GlossaryTerm

VGSC

s in cell membranes, which increases ${\mathrm{Na}}^{+}$ ion permeability and depolarizes the nerve cell. This depolarization is believed to cause the array of neurological signs associated with GlossaryTerm

CFP

. Voltage-clamp studies suggested that GlossaryTerm

CTX

s cause spontaneous, and enhance evoked, action potentials by lowering activation thresholds and delayed repolarization of GlossaryTerm

VGSC

s. Several studies further elucidated cellular effects and molecular mechanisms of GlossaryTerm

CTX

s actions including selective binding and competition with GlossaryTerm

BTX

for site 5 on GlossaryTerm

VGSC

s, elevation of intracellular ${\mathrm{Ca}}^{2+}$ levels, stimulation of spontaneous and evoked neurotransmitter release from synaptosomes and motor nerve terminals, axonal and Schwann cell edema, induction of GlossaryTerm

TTX

-sensitive leakage current in dorsal root ganglion neurons and blockade of voltage-gated potassium channels [254]. Neurological symptoms are consistent with GlossaryTerm

CTX

s’ interactions with GlossaryTerm

VGSC

s; however, all the symptoms produced during GlossaryTerm

CFP

may not be exclusively due to GlossaryTerm

VGSC

s [46]. The chronic fatigue syndrome that may last for weeks or months has been related to high nitric oxide ($\mathrm{NO}$) production. Penil and muscular relaxation, as well as L-type ${\mathrm{Ca}}^{2+}$ channel activation, were shown to be GlossaryTerm

NO

-mediated via the nitric oxide synthetase pathway.

GlossaryTerm

CTX

also caused rapid hypothermia in mice, which lose their ability to regulate their own body temperature and became poikilothermic. This reduction in body core temperature was found to be due to neuroexcitatory actions of ciguatera on regions of the brain stem receiving vagal afferents and ascending pathways associated with visceral and thermoregulatory responses [255]. These authors reported a complete review on the pharmacological effects of GlossaryTerm

CTX

s, including cardiovascular and gastrointestinal effects, effects on other smooth muscle, actions on ganglionic and neuromuscular transmission and on GlossaryTerm

VGIC

, and other ${\mathrm{Na}}^{+}$-dependent actions. Despite the multiple pharmacological effects no medical applications of these toxins have been reported so far.

7.3.2 Brevetoxin-Group Toxins

GlossaryTerm

BTX

s are lipid soluble cyclic polyethers (M.W. $\approx$ $900$) and potent neurotoxins that open GlossaryTerm

VGSC

s leading to ${\mathrm{Na}}^{+}$ influx into the cell. GlossaryTerm

BTX

s exert their toxicity by interacting with neurotoxin receptor site 5 associated with domain IV of the α subunit of the GlossaryTerm

VGSC

. GlossaryTerm

BTX

s binding to tissues that contain GlossaryTerm

VGSC

s on excitable cells results in membrane depolarization, repetitive firing, and increase in ${\mathrm{Na}}^{+}$ currents [256]. At the ${\mathrm{Na}}^{+}$ channel, at least four actions of GlossaryTerm

BTX

s have been identified leading to the depolarization of excitable cell membranes: shift of the voltage dependence, inhibition of inactivation, increase of mean open times and multiple subconductance levels. A description of these actions and the mammalian GlossaryTerm

BTX

s toxicity was recently review [63].

GlossaryTerm

BTX

s exert a variety of actions in vivo affecting both the peripheral as well as the central nervous systems, probably due to their high lipid solubility and ability to penetrate the blood–brain barrier. Humans exposed to the toxin usually exhibit neurotoxic shellfish poisoning symptoms, which include nausea, difficulties in movement, and seizures [257]. Cellular effects associated with exposure to GlossaryTerm

BTX

s have been observed in the immune system of many species, although the mechanisms of action of GlossaryTerm

BTX

s on immune cells and immune competence are not well understood. Several potential mechanisms for GlossaryTerm

BTX

immunotoxicity have been suggested, including the inhibition of cathepsin, apoptosis, release of inflammatory mediators, and oxidative stress [58]. In vitro experiments have demonstrated possible GlossaryTerm

DNA

damage, chromosomal aberrations and effects on cellular growth [256, 258]. Other immune system effects include mast cell degranulation [259] and histamine release which may contribute to observed airway responses following the inhalation of aerosolized GlossaryTerm

BTX

s.

There are over $10$ different GlossaryTerm

BTX

s isolated in seawater blooms and K. brevis cultures, as well as multiple analogs and derivatives from the metabolism of shellfish and other organisms [58]. Cao etal [260], quantified the potencies and efficacies of an array of lipophilic GlossaryTerm

VGSC

s gating modifiers by measuring ${\mathrm{Na}}^{+}$ influx in murine neocortical neurons. An assay using a ${\mathrm{Na}}^{+}$-sensitive fluorescent dye to primary cultures of neurons afforded a highly quantitative assessment of the increments in neuronal ${\mathrm{Na}}^{+}$ level produced by gating modifier toxins. The relative efficacies differ substantially; the rank order of efficacy of several naturally occurring GlossaryTerm

BTX

s and semisynthetic analogs as ${\mathrm{Na}}^{+}$ channel gating modifiers was GlossaryTerm

PbTx

-1$>$ GlossaryTerm

PbTx

-desoxydioxolane$>$ GlossaryTerm

PbTx

-2$=$ GlossaryTerm

PbTx

-3$<$ GlossaryTerm

PbTx

-3α-naphthoate. K. brevis cultures also produce shorter ring structures, hemi-GlossaryTerm

BTX

s and brevenals, likely to be incomplete products of the GlossaryTerm

BTX

biosynthetic pathway. These smaller cyclic ether ring structures exhibit reduced biological activity, yet they are still consistent with a site of action common to the GlossaryTerm

BTX

s [63]. An interesting discovery was the finding that brevenal (produced both in K. brevis laboratory cultures and in the environment during K. brevis blooms), is a brevetoxin antagonist. This is a case of a toxin producing organism that also secretes its own antagonist. Brevenal is synthesized by K. brevis in significant amounts and it acts at a different receptor site on nerve cells than the brevetoxins. It is a new and important natural product which could treat chronic obstructive pulmonary disease and cystic fibrosis [58].

Taupin [261] studied the activity of four generic formulae of GlossaryTerm

BTX

derivatives – formulae I–IV – and their compounds on the growth of neurites. He reported that GlossaryTerm

BTX

derivatives stimulate neurite growth, particularly the growth of minor processes from which the axons form, on neurons in primary cultures. The activity is mediated by VGNC and the N-methyl-d-aspartate-mediated intracellular ${\mathrm{Ca}}^{2+}$ pathway. They proposed the use of GlossaryTerm

BTX

derivatives for enhancing neuronal growth and for the treatment of neurodegenerative and neurological disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis, cerebral strokes, traumatic brain, and spinal cord injuries.

7.3.3 Tetrodotoxin

GlossaryTerm

TTX

is a very well-known marine neurotoxin due to its frequent involvement in fatal food poisoning, unique chemical structure, and specific action of blocking ${\mathrm{Na}}^{+}$ channels of excitable membranes [262]. The toxicity of GlossaryTerm

TTX

relates to two factors: paralysis of skeletal muscle, including the diaphragm and intercostal muscles, leading to respiratory failure, and reduced blood pressure, predominantly due to vasodilatation. Early signs of this effect might include weakness, tingling of the lips and dizziness [70]. Pharmacological activity of GlossaryTerm

TTX

on various animals, their organs and tissues has been extensively studied, and it was found that GlossaryTerm

TTX

strongly inhibits nerve conduction and has inhibitory effects on ${\mathrm{Na}}^{+}$-${\mathrm{K}}^{+}$-ATPase activity. Recently, GlossaryTerm

TTX

has been shown to block L-type ${\mathrm{Ca}}^{2+}$ current (ICa) in canine cardiac cells [263]. These authors studied the GlossaryTerm

TTX

-sensitivity of Ca in isolated canine ventricular myocytes as a function of channel phosphorylation, extracellular pH, and the redox potential of the bathing medium using the whole cell voltage clamp technique. They demonstrated that GlossaryTerm

TTX

inhibits L-type Ca channels in the heart by binding to its selectivity filter.

GlossaryTerm

TTX

has been extensively used to elucidate the role of specific GlossaryTerm

VGSC

s subtypes in a wide range of physiological and pathophysiological processes in the nervous system. GlossaryTerm

VGSC

s play a key role in pain and GlossaryTerm

TTX

-sensitive subtypes have received much attention over the past few years because these channels have been strongly implicated in normal and pathological pain. Since GlossaryTerm

TTX

blocks this subset of GlossaryTerm

VGSC

s in a highly selective manner, this toxin may have a potential role in relieving pain [264, 265]. Nieto etal [266] evaluated the effect of the acute systemic administration of low, nontoxic, doses of GlossaryTerm

TTX

on the expression of different signs (heat hyperalgesia, mechanical- and cold allodynia) of paclitaxel-induced neuropathic pain in mice. They reported that low doses of GlossaryTerm

TTX

can be useful to prevent and treat this pain since inhibited hyperresponsiveness to thermal and mechanical stimuli. Their findings suggest that GlossaryTerm

TTX

-sensitive subtypes of ${\mathrm{Na}}^{+}$ channels play a role in the pathogenesis of chemotherapy-induced neuropathic pain.

Hagen etal [267] evaluated the analgesic activity of GlossaryTerm

TTX

administered subcutaneously in a randomized, placebo-controlled, parallel design study, in patients with moderate or severe unrelieved cancer pain. This trial suggested that GlossaryTerm

TTX

could potentially relieve moderate to severe, treatment-resistant cancer pain in a large proportion of patients, and often for prolonged periods following treatment. In a later study [268] it was assessed the long-term safety and efficacy of subcutaneous GlossaryTerm

TTX

treatment in reducing the intensity of chronic cancer-related pain. They conducted a multicenter open-label longitudinal trial, in which $30\mathrm{\mu }\mathrm{g}$ GlossaryTerm

TTX

was administered subcutaneously for $4$ days in a heterogeneous cohort of patients with persistent pain. Onset of pain relief in no more than half the patients was typically cumulative over days, and after administration ended, the analgesic effect subsided over the course of a few weeks. Toxicity was usually mild or moderate and remained so through subsequent treatment cycles, with no evidence of cumulative toxicity or tolerance. They concluded that long-term treatment with GlossaryTerm

TTX

is associated with acceptable toxicity and, in a substantial minority of patients, resulted in a sustained analgesic effect. Berde etal [70] reported that prolonged duration percutaneous sciatic nerve blockade could be achieved in rats using combinations of site $1$ ${\mathrm{Na}}^{+}$ channel toxins with either bupivacaine or epinephrine. Addition of either bupivacaine or epinephrine increases the LD${}_{50}$ of GlossaryTerm

TTX

in rats, i. e., reduces systemic toxicity from GlossaryTerm

TTX

, and also increases the potency of GlossaryTerm

TTX

by producing sensory blockade, thereby substantially improving the therapeutic index of GlossaryTerm

TTX

. Thus, combination of formulations seems desirable from the standpoint of both efficacy and safety. GlossaryTerm

TTX

, formulated as Tectin, underwent Phase III trials as an injectable systemic analgesic for chronic cancer pain. Berde etal [70] examined dose–duration relationships and sciatic nerve histology following local nerve blocks with combinations of Tectin with bupivacaine $0.25\%$ solutions, with or without epinephrine $5\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ in rats. Three-way combinations of GlossaryTerm

TTX

, bupivacaine and epinephrine produced significant prolonged sciatic nerve blockade in rats, compared to bupivacaine plain or bupivacaine $+$ epinephrine. Their studies indicate that clinically relevant combinations of Tectin with commercially available bupivacaine $0.25\%$ and epinephrine $5\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ warrant further study for prolonged-duration local anesthesia. Moreover they suggest that a single-injection approach to prolonged local anesthesia would provide good pain relief as a component of multimodal analgesic regimens, reducing postoperative opioid requirements and improving the course of postoperative recovery and acute rehabilitation.

A review by Nieto etal [269] focuses on the preclinical and clinical evidence supporting a potential analgesic role for GlossaryTerm

TTX

. In addition they compiled data on the contribution of specific GlossaryTerm

TTX

-sensitive GlossaryTerm

VGSC

s to pain. They conclude that the therapeutic use of GlossaryTerm

TTX

as an analgesic agent seems hopeful although further preclinical and clinical research is needed to clarify its potential use during painful conditions. Wang etal [270] examined the effect of GlossaryTerm

TTX

, applied topically to the rat cornea, on the duration of corneal anesthesia, administered with either proparacaine (GlossaryTerm

PPC

) or the chemical permeation enhancer octyl-trimethyl ammonium bromide (GlossaryTerm

OTAB

). The effect of test solutions on corneal healing was also studied. Combination of GlossaryTerm

TTX

and GlossaryTerm

PPC

resulted in corneal anesthesia that was $8$ to $10$ times longer in duration than that from either drug administered alone, whereas GlossaryTerm

OTAB

did not prolong anesthesia. The rate of corneal healing was moderately delayed after coadministration of GlossaryTerm

TTX

and GlossaryTerm

PPC

. They concluded that coadministration of GlossaryTerm

TTX

and GlossaryTerm

PPC

significantly prolonged corneal anesthesia, but in view of delayed corneal reepithelialization, caution is suggested in the use of the drug combination.

Bragadeeswaran etal [262] isolated three GlossaryTerm

TTX

-producing bacteria (Kytococcus sedentarius, Cellulomonas fimi and Bacillus species) from the pufferfish Arothron hispidus. Their partially purified filtrates exhibited hemolytic activity on chicken and human erythrocytes and also presented ATPase, ${\mathrm{Mg}}^{2+}$-ATPase, ${\mathrm{Na}}^{+}$ ${\mathrm{K}}^{+}$-ATPase, and AchE enzymatic activities. The authors also evaluated if the viability of leukemia cell line (P388) was adversely affected upon adding the crude microbial extracts and found a dose-dependent growth inhibition. These GlossaryTerm

TTX

-producing bacteria hold promise for the development of effective antitumor compounds.

7.3.4 Palytoxin and Analogs

GlossaryTerm

PlTX

s are a group of complex, extremely potent, and marine natural products. GlossaryTerm

PlTX

is a large (molecular weight: 2681), water-soluble polyalcohol first isolated from zoanthids (genus Palythoa) even though dinoflagellates of the genus Ostreopsis are postulated as the GlossaryTerm

PlTX

origin [77]. GlossaryTerm

PlTX

has been found in a variety of marine organisms ranging from dinoflagellates to fish and it is implicated in seafood poisoning with potential danger to public health. The most commonly reported complications of GlossaryTerm

PlTX

poisoning appears to be rhabdomyolysis, a syndrome injuring skeletal muscle, causing muscle breakdown, and leakage of large quantities of intracellular (myocyte) contents into blood plasma. Other symptoms associated with GlossaryTerm

PlTX

poisoning in humans are characterized by abdominal cramps, nausea, vomiting, diarrhea, paresthesia, bradycardia, renal failure, cyanosis, and respiratory distress [79]. The latter two precede death in fatal cases.

Toxicological studies with these compounds show repeatedly low LD${}_{50}$ values in different mammals, revealing an acute toxic effect on several organs, as demonstrated by different routes of exposure. Acute oral administration in mice induced histological and ultrastructural changes in several organs, such as liver, pancreas, cardiac, and skeletal muscle cells. The recognition of its wide distribution coupled with the poisoning effects that these toxins can have on animals, and especially on humans, have concerned the scientific community [79]. The toxin is known to affect cellular functions by selective binding to the ${\mathrm{Na}}^{+}$-${\mathrm{K}}^{+}$ ATPase and converting the pump into a nonselective channel [195, 271]. The induced ion flux alterations are reported to target pathways involved in cytoskeletal dynamics within different cellular models [77]. ${\mathrm{Na}}^{+}$-${\mathrm{K}}^{+}$ ATPase also functions as a signal transducer to relay messages from the plasma membrane to the intracellular organelles. The toxicological effects of acute oral administration of 42-OH-GlossaryTerm

PlTX

were evaluated in mice, as well as its in vitro delayed hemolytic effects [272]. The compound showed a toxicity profile comparable with that of the parent compound palytoxin (LD${}_{50}=651$ and $767\mathrm{mg}/\mathrm{kg}$, respectively), both as lethality and acute pathological effects. Also the in vitro hemolytic activity of 42-OH-GlossaryTerm

PlTX

was similar to that of GlossaryTerm

PlTX

in agreement with the common mechanism of action on the ${\mathrm{Na}}^{+}$/${\mathrm{K}}^{+}$ pump, to which the two compounds bind with the same affinity.

Franchini etal [204] used the Frog Embryo Teratogenesis Assay-Xenopus (GlossaryTerm

FETAX

) to verify the effects of GlossaryTerm

PlTX

in mortality, delayed growth, and embryo malformation. The toxicological effects of GlossaryTerm

PlTX

evaluated by GlossaryTerm

FETAX

assay revealed evident impacts on embryo mortality, teratogenesis and growth. The histological analysis of the surviving young larvae revealed structural changes compared with controls in the nervous and muscle tissue, even if some specimens did not show any significant histopathological modifications. A general reduction in the size of the main inner visceral organs (i. e., intestine, pancreas, and liver) was reported, but no morphological changes. Severe injury to the heart structure was observed in some specimens.

GlossaryTerm

PlTX

is a novel skin tumor promoter, which has been used to probe the role of different types of signaling mechanisms in carcinogenesis. The multistage mouse skin model indicates that tumor promotion is an early, prolonged, and reversible phase of carcinogenesis. Understanding the molecular mechanisms underlying tumor promotion is therefore important for developing strategies to prevent and treat cancer [273]. GlossaryTerm

PlTX

stimulates a wide range of cellular responses that are likely to play a role in carcinogenesis. Stimulation of arachidonic acid metabolism and the production of prostaglandins modulation of the epidermal growth factor receptor, and modulation of mitogen-activated protein (GlossaryTerm

MAP

) kinase cascades are the best studied [273]. GlossaryTerm

MAP

kinases are a family of serine/threonine kinases that relay a variety of signals to the cellular machinery that regulates cell fate and function. This author [274] investigated how palytoxin stimulates GlossaryTerm

MAP

kinase activity and how GlossaryTerm

MAP

kinases mediate the response of cells to GlossaryTerm

PlTX

concluding that GlossaryTerm

MAP

kinases appear to be important mediators of GlossaryTerm

PlTX

-stimulated signals. The central role of GlossaryTerm

MAP

kinases in regulating a variety of critical cellular functions, ranging from enzyme activity to gene expression and ultimately to proliferation and apoptosis, may help explain how palytoxin can stimulate the variety of effects that are characteristic of tumor promoters. Other author suggests that GlossaryTerm

PlTX

conjugates could be used as prodrugs in cancer chemotheraphy due to its high cytotoxicity in vitro indicating that targeting the prodrug to a tumor, and there releasing the toxin, selective destruction of neoplastic cells could be achieved [275]. Many findings on the involvement of GlossaryTerm

PlTX

and analogs acting on different cellular targets and modulating the actin cytoskeleton within different cellular models were published [276, 277, 77]. Louzao etal suggest that also cytoskeleton is necessary for GlossaryTerm

PlTX

activity and that this may find important applications in biomedical research, with particular interest toward the discovery and development of cytotoxic compounds providing novel antitumor therapy [77].

7.3.5 Cyclic Imines

GlossaryTerm

CI

s toxins are a very diverse group of marine biotoxins containing an imino functional group as part of its cyclic structure. In many of them, it was found that the toxicity is greatly diminished if imino group is reduced or destroyed [278]. Nowadays, there is no evidence of human poisoning caused by cyclic imines but the toxicity observed in experimental animals requires a deeper study of their activity and distribution [279]. Only the toxicity of spirolides (GlossaryTerm

SPX

s) and gymnodimines (GlossaryTerm

GYM

s) has been studied in depth. Spiroimine moiety has been established as the common pharmacophore unit of GlossaryTerm

CI

s group, although structural change in other parts of the molecules can also affect the toxicity [280]. Primary studies on mechanism of action of GlossaryTerm

CI

s suggested that it could be related to the interference on transmission of nervous impulses at neuromuscular junction level. It has been demonstrated that GlossaryTerm

SPX

s and GlossaryTerm

GYM

s act as potent antagonists of nicotinic (nAChRs) and muscarinic (mAChRs) acetylcholine receptors [281, 282, 283]. Kharrat etal [281] demonstrated that GlossaryTerm

GYM

A broadly interferes in neuromuscular transmission by directly blocking muscle-type nAChRs. GlossaryTerm

GYM

A interacts with a wide range of muscular and neuronal types of nAChRs, larger than other well characterized toxins that also act on nicotinic receptors [281]. Bourne etal [282] confirmed the interaction of GlossaryTerm

SPX

and GlossaryTerm

GYM

with several muscle and neuronal types of nAChRs by competition binding experiments against labeled α-bungarotoxin and epibatidine. No agonist effect was observed with GlossaryTerm

SPX

or GlossaryTerm

GYM

, but both toxins showed a dose-dependent antagonism in muscle- and neuronal-type receptors. The study concluded that GlossaryTerm

SPX

and GlossaryTerm

GYM

toxins display high affinity and potent antagonism but limited selectivity for the muscle type versus neuronal subtypes of nAChR [282].

GlossaryTerm

SPX

s effects on mAChRs were studied by Wandscheer etal in a human neuronal model. They observed that 13-desmethyl GlossaryTerm

SPX

C caused a dose- and time-dependent inhibition on neuroblastoma to acetylcholine extracellular stimulation. They demonstrated by competitive binding assays that the 13-desmethyl GlossaryTerm

SPX

is a competitive antagonist of mAChRs and also causes an internalization of the M3-type muscarinic receptor that might contribute to the inhibitory effect of the toxin on the ${\mathrm{Ca}}^{2+}$ response to acetylcholine stimulation [283].

7.3.6 Maitotoxins and Gambierol

Many bioactive compounds have been isolated from Gambierdiscus spp., i. e., maitotoxins (GlossaryTerm

MTX

s), gambierols, and gambieric acid. GlossaryTerm

MTX

increases intracellular ${\mathrm{Ca}}^{2+}$ and is considered one of the most potent marine biotoxins when injected intraperitoneally (ip) in mice, with an LD${}_{50}$ ($24\mathrm{h}$) of $50\mathrm{ng}/\mathrm{kg}$. However, GlossaryTerm

MTX

has no proven role in human intoxication due to its low capacity for accumulation in fish flesh and low oral potency [46]. At cellular level, GlossaryTerm

MTX

causes an increase in ${\mathrm{Ca}}^{2+}$ and ${\mathrm{Na}}^{+}$ by activating ${\mathrm{Ca}}^{2+}$-permeable nonselective cation channels, resulting in potent membrane depolarization. This causes, in excitable cells, the opening of voltage-gated ${\mathrm{Ca}}^{2+}$ and ${\mathrm{Na}}^{+}$ channels. The massive influx of ions could exceed cell buffering mechanisms affecting cell viability [284]. Due to its ability to activate ${\mathrm{Ca}}^{2+}$-permeable nonselective cation channels, GlossaryTerm

MTX

is considered a powerful tool in the study of ${\mathrm{Ca}}^{2+}$-dependent mechanisms. Another GlossaryTerm

MTX

effect is the formation of cytolytic/oncotic pores that finally conduce to cell membrane blebbing and oncotic cell death. For this reason, GlossaryTerm

MTX

could be a unique tool to explore oncotic/necrotic cell death [284].

Gambierol has moderate inhibitory activity against GlossaryTerm

VGSC

s and modified ${\mathrm{Ca}}^{2+}$ homeostasis. High concentrations of this toxin activate ${\mathrm{Na}}^{+}$ channels in human neuroblastoma cells, and, as a consequence, gambierol induces a cytosolic ${\mathrm{Ca}}^{2+}$ increment [285]. Cagide etal tested the effect of gambierol on the shape and F-actin cytoskeleton of neuroblastoma. Cells remained with spread morphology and a well-defined actin cytoskeleton, without showing evidence of alterations. Moreover, gambierol was able to produce depolarization, as well as a ${\mathrm{Ca}}^{2+}$ influx, in neuroblastoma cells and scarcely reduced their metabolism [286]. Alonso etal [287] designed and synthesized truncated skeletal analogs of gambierol with inhibitory activity against voltage-gated potassium (Kv) channels. They examined the effect of these compounds in an in vitro model of Alzheimer’s disease obtained from triple transgenic (3$\times$Tg-AD) mice, which expresses amyloid beta (Aβ) accumulation and tau hyperphosphorylation. In vitro preincubation of the cells with the compounds resulted in significant inhibition of ${\mathrm{K}}^{+}$ currents, a reduction in the extra- and intracellular levels of Aβ, and a decrease in the levels of hyperphosphorylated tau, reducing these two cellular pathologies. Thus, these compounds could be useful chemical probes for understanding the function of Kv channels and for elucidating the molecular mechanism of Aβ metabolism modulated by GlossaryTerm

NMDA

receptors. Moreover, since both Aβ peptides and phosphorylated tau proteins are supposed to interact with GlossaryTerm

NMDA

receptors and to be implicated in the glutamate enhancement of AD pathology, they could have a potential pharmacological use.

Gambieric acids isolated from the culture medium of G. toxicus were described as potent antifungal polyether compounds [46].