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Lezioni, Tossicologia marina Appunti scolastici Premium

Appunti in inglese per l'esame di Tossicologia marina della professoressa Tubaro. Gli argomenti che vengono trattati sono i seguenti: la parte di tossicologia generale e la parte di tossicologia speciale-marina, principalmente sugli Algae Shellfish poisonings.

Esame di Tossicologia marina docente Prof. A. Tubaro

Anteprima

ESTRATTO DOCUMENTO

MARINE TOXICOLOGY

Toxic algae (microalgae) are microscopic unicellular organisms that live in water bodies. The major part of

algae are producers on the food chain and without them all the rest of the food chain couldn't exist. There

are also food for molluscs like oysters, mussels and clams but also for crustacean and fish larvae.

Occasionally, these algae can grow very fast leading to blooms, so a copious proliferation that we usually

observe in summer on the surface of lake and little water bodies, but also into the sea. These blooms can

cause the phenomenon of eutrophication. Very famous blooms are the red tides, because of some algae

contain red pigments. The term “tide” is not really appropriate because it is not a real tide; also the color

can change, thus also the term “red” is inappropriate. Usually these big proliferations of planktonic algae

are useful for aquaculture, but in some cases they cause serious effects on environment, on human health

and on the commerce of aquaculture's products. There are now known about 5000 species of marine algae,

but just 300 of them can reach high concentration (>1 million cells/L) and only 80 can produce powerful

toxins. Some toxins are very powerful and 500-1000 ug can be lethal for a person; however, different toxins

have different power (and different LD50)

Red tides were very well known in the past. They were described in an opera of Exodus, that associated the

red tide to loss of blood. In 1793 Captain George Vancouver reported first algae poisoning during a trip in

British Columbia. Indigenous people didn't eat molluscs when water was lighting because of the presence of

bioluminescent algae. Both in Canada and USA there are endemic area for paralytic shellfish poisoning. You

can find some advertisement telling 'no collect shellfish' and so on.

Cycle of an algae. Cysts can germinate only during certain times of the year with warmer temperatures and

increased light stimulating germination. The cyst breaks open and a swimming cell emerge. The cell

reproduces by simple division within a few days (or hatching). If conditions remain optimal cells will

continue to divide, reproducing exponentially. A single cell can produce several hundreds of cells within a

couple of weeks. If other single cells reproduce similarly, then the toxicity in shellfish may occur. When

nutrients are gone, growth stops and gametes are formed. Two gametes join to form one cell, which

develops into a zygote and then into a cyst. The cyst fall to the ocean bottom and is capable of germination

after years.

There are 2 main types of algal proliferation: 1) that causes toxic effects in human beings, but they can

affect other organisms such as fishes; 2) that doesn't cause toxic effects in human beings. For example,

Noctiluca scintillans and Gonyaulax polygramma are not toxic for humans but they can cause death in

invertebrates and fishes subsequently to the oxygen depletion. Karenia mikimotoi (a dinoflagellate) and

Prymnesium parvum can cause serious damages in an aquaculture but these 2 species are not toxic for

humans. In the case of P. parvum, fishes are suffocated by the presence of a foam, produced by this

microalgae, that obstructs the gills; fish can not breath anymore. Also Karenia brevis can cause massive

fishes deaths: it is spread along the New Zealand and the Florida coast. If some people recognize a bloom of

this, have to inform immediately those responsible. Pfiesteria piscidica produces some toxins, in particular

two that affect seriously fishes. One is neurotoxic, the other one is dermotoxic and it can provoke dermatitis

with visible wounds. A massive death of pelicans and cormorants was detected along the west coast of

America and was attributed to Pseudonitzschia diatoms; they produced domoic acid, that passed through

the food chain until seabirds.

One dish of mussels can cause illness or even death if they contain the right toxin. Mussels filter water

continuously and all the compounds inside can be accumulated for this reason in shellfish you can have

huge amount of toxins. They can also transform toxins in compounds easily metabolized for them, but not

fom us.

Algae and Shellfish Poisoning

usually, main vectors of this intoxication are molluscs. During the uptake of oxygen they open the valves

allowing the passage of big amount of water through the gills. The now filtered particulate passes to the

stomach, where the digestion begins. The substances useful for the mollusc are transferred into the

digestive gland. Filtering a lot of water, the mollusc will be exposed to anything is in the water: virus,

bacteria, toxic algae and so on. Since the principal food of mollusc is phytoplankton, if the filtered algae

produce toxins, the latter can be accumulated. As long the mollusc eat phytoplankton, the contamination

will be higher, moreover during an algae bloom. At the end of the bloom they start to excrete toxins until

they are not in the body anymore. In USA algae poisonings cover 7,4% of the overall marine intoxications.

Algae toxins represent a problem all over the world and there are more than 60000 cases of poisoning per

year. Although bacterial and viral intoxications in shellfish are more common, algae poisoning can be surely

more powerful, sometimes lethal, than them. When cooking mussels, some toxins are thermostable so they

are not eliminated. to recognize shellfish poisonings is quite difficult so proper data on the number of cases

are not available. We don't know actually the true percentage of sick people; for example, since ciguatera is

not lethal but it causes just acute diarrhea, a lot of cases are not registered.

Of the (about) 80 species that produce toxins, most of them belong to the taxon of Pyrrophyta, Dinofiaceae

class, commonly known as Peridineae or dinoflagellates. Toxins responsible of human intoxications are

produced mainly by two types of unicellular organisms: dinoflagellates and diatoms [and cyanobacteria].

Algae toxins are secondary metabolites that are produced only in certain taxonomic groups of microalgae.

The role of these metabolites is still not clear, maybe they can act as deterrent for predators. Production of

these metabolites can vary according to environmental factors. They are usually thermostable – so not well

destroyed with boiling – and passing through the food chain they can cause intoxications. If they find

optimal environmental conditions (nutrients, oxygen, temperature) in the sea they can bloom and be

filtered by shellfish. Biomonitoring systems allow for the block of harvest and commerce of contaminated

products (shellfish in aquaculture, for instance). Developing countries without monitoring system and

dependent from the sea for their feeding can have sanitary problems.

But, is there a real increase of algae toxins? Well, there are some reason because the answer can be NO.

Probably, a concomitance of factors can lead to a detected increase of algae poisonings:

Increase of scientific knowledge of toxic species;

• Increase on using coastal water for aquaculture;

• Increase of eutrophication;

• Unusual climate conditions;

• Transport through ballast waters of dinoflagellates and molluscs;

• Better monitoring system.

A continuous check of the quality of water is needed. When monitoring an aquaculture there can be found

several algae, more or less toxic, but the real thing to do is the analysis of cultivated molluscs because they

are the products that will be finish in the consumers' dish.

Ostreopsis ovata produces palytoxin, in particular ovatoxin. Environmental problem →

List of poisoning. Since 2000 maybe there is also palytoxin in the Adriatic sea, produced by bloom of

Ostreopsis (ovatoxin is an analog of palytoxin).

DSP – Diarrhetic Shellfish Poisoning

DSP is the main problem in Italy and is one of the most frequent poisoning. It was discovered in Japan in

1978 by Prof. Yasumoto. The last outbreak in Italy was in September 2010, in Turin and Aosta with more

than 300 cases of contamination caused by mussels coming from Trieste. In 99% of cases you have diarrhea

with a massive loss of water. Other symptoms are nausea, abdominal pain, vomit, weakness and chills.

Symptoms come out after 30 minutes up to some hours, rarely after 8-12 hours. Intensity of symptoms

depends on the assumed dose (at least 40 ug of OA or 36 of DTX1).

DSP is caused by okadaic acid and dinophysistoxins (DTX). There are three main types of DTXs identified

with numbers: DTX1, DTX2, DTX3. These 4 compounds are very similar and they differ only for the presence

of different substitutes in 3 positions. Once, DSP toxins were included with yessotoxins and pectenotoxins,

since they were extracted with OA and DTXs. Dinophysis fortii produces only OA and it was the responsible

for the first DSP outbreak in Italy. D. norvegica, D. acuta, D. acuminata and D. caudata produce mainly

OA; ??????? CHIEDO ALLA PROF PERCHE' NELL'ELENCO COMPAIONO PIU' VOLTE LE SPECIE. Prorocentrum

lima is sometimes (and not surely) responsible of DSP. Production of DTXs and OA depends on

environmental factors and on the geographic area.

The mechanism of action includes several compartment. The most quoted mechanism is the inhibition of

proteinphosphatase 1 and 2A (PP1 and PP2A). There is an increase on the phosphorylation of proteins that

control the secretion of Na from enterocytes; and of element of the cytoskeleton, that involved structural

modification of cells. The main result is the entrance of water in the gut, leading to diarrhea. In the case of

DTX1 there is also tumor promotion. Keep in mind that promoter needs an initiator, otherwise it cannot

work; moreover the promotion has to be continuous, thus with continuous exposures. The main target of

DTXs is the small intestine but in few cases can be involved even the liver. The thresholds for commerce are

160 ug of toxins (OA, DTXs and derivatives) per kg of mollusc meat.

The diagnosis of DSP is based on the anamnesis (the medical history) of patients. There are not available yet

specific diagnostic test. The minimum dose that provokes diarrhea in adult is 40 ug OA or 36 ug DTX1. The

diagnosis can be confused with microbial or azaspiracid intoxication that cause diarrhea too. For example,

the first case of DSP in Italy was confused with an azaspiracid poisoning. However, the treatment is the

same, i.e. the supply of minerals and water that the patient massively loses. The prognosis lasts 3-4 days,

without having pharmacological treatment. There is no a specific antidote, since the main symptom is

diarrhea. With regards to the promotion of tumor, OA and DTX1 are considered just weak promoters and is

still unknown their real role in this process. However, promoters need to be assumed several times

continuously and it is very rare, with the exception of countries that base their feeding on shellfish, that

someone eats several times a week mussels or clams. Microcystin is a stronger tumor promoter compared

to OA.

OA better inhibits PP2A than PP1; the contrary is valid for DTX1.

The DSP is the main problem in Italy and in Spain along the coast (here also PSP, that can be lethal). DSP is

spread all over the world but the majority of cases are reported in Europe and in Japan, but there are some

new cases in USA. The first recognized case in Italy was in 1989, along the Emilia Romagna coast. It is likely

that beforehand there were some cases more, not recognized as DSP. Since 1989 there haven't been more

cases until 2010, when 300 people were intoxicated. From 1989 monitoring systems are doing their work;

there are spread along the Italian coast, with particular regard of Liguria, Adriatic sea and Sardinia.

People get, contract the intoxication eating molluscs like mussels (Mytilus galloprovincialis), clams (Tapes

decussatus, Venus gallina) and oysters (Ostrea edulis). Molluscs are not affected by the toxins and they

accumulate them in the digestive gland. There is no morphological change in a mollusc full of toxins.

Mytilus galloprovincialis is one of the most contaminated species. They are cultivated in ropes and they can

accumulate huge amount of toxins, mainly produced by Dynophysiaceae. This group moves as the

thermocline moves, in 10 cm. When a toxins or the putative presence of a toxin in a mollusc are recognized,

it starts a procedure that forbids the sale of the cultivated products and monitors the situation even after

the sale of products. These procedures are also ruled by the EU. Although these precautions, is not

uncommon to see people self-harvesting clams or other molluscs in the beach, and the intoxication can

derive also from these practices.

Toxins have to be individuated also at low concentration, even lower than 2 ppb. Some toxins have not a

chromophore, a part of the molecule by which it can be recognized; moreover there can be other

substances in the sample, covering the one we are trying to study. The optimal test should be rapid, able to

analyze a lot of sample in a brief time. It's important to have a zero point, a white sample, in order to

establish the quantity from which the toxin can be recognized. There are three main kinds of analyses:

Chemical analysis. HPLC is relatively slow and not used anymore, substituted by LC-MS (Liquid

• Chromatography – Mass Spectrometry), that is very rapid, although the cost, and it can recognize 1

ng of substance in 1 g of tissue. Moreover, there can be evaluate the presence of different toxins

together. Since 2011 LC-MS is operative in almost all European countries and it is preferred also to

some biological tests.

Biochemical analysis are classified into structural assays and functional assays. In the structural

• assays there is an interaction between a part of the toxin and a part of another molecule – for

instance, in the immune-enzymatic assay, the binding site of antibodies. The signal that comes out

is proportional to the amount of toxin in the sample. The most used structural assay is undoubtedly

ELISA. This test has a sensibility of 0,1 ug per g of tissue; they are rapid and easy to use. There are a

wide range of commercially available kit, but the disadvantage is that actually ELISA can recognize

just OA, DTX1 and DTX2. Another disadvantage of ELISA is the development of false positive,

because the part of toxin that bind the other molecule can be shared with other substances.

Functional assays rely on a biochemical action of the toxin and the amount of detected toxin is

usually proportional to its toxicity in vivo because this phosphatase binds with the same affinity. The

most used functional assay is that of inhibition of PP2A (more details below). The sensibility is 50

ng/g of tissue, and they are also available in commercial kits. Another, but not widely used,

biochemical test is the radio-immunological test, that involves the utilization of radionuclides.

Biological tests. The mouse bioassay has a sensibility of 200 ng/g tissue and it is cheap, rapid and

• easy; unfortunately it can produce false positive and recognized other 'lipophilic toxins', that can be

accumulated in the studied tissue. The mouse bioassay can be compared to an acute toxicity test.

In vivo test has the pro and con to show the overall effect of the substance, resulting from the

action of all the toxins, not only one. Advantages of mouse bioassay are: 1) it gives a measure of the

total toxicity, that correlate the response of the animal to the toxin; 2) it doesn't need particular

purification procedure of mussels neither particular machine tools; 3) it can reveal also other toxic

compound; 4) the toxicity detected is comparable to that on human. There are also many

disadvantage: 1) you need an animal house; 2) animals have not to be stressed and all in the same

condition (same weight, age, sex, etc.); 3) possible interference caused by substances extract with

the wanted toxins (false positive and false negative); 4) variability of the response between the

laboratories, and for this reason is needed an intercalibration among labs to assess the variability;

5) less precise than analytical method and less sensibility.

Otherwise one can carry out an in vitro test of cytotoxicity; most of time the cytotoxicity is too high

and you cannot recognize the effect of a toxin from the one of another toxin.

DTX3 is usually the product of transformation of DTX1, DTX2 and OA that mussels try to detoxify, so it is not

present in algae but just a product of mussels metabolism! In vitro, DTX3 cannot be more metabolized. In

USA there has been developed a quickly immuno-based test, usable directly after the harvest of mussels on

the ship.

Acute toxicity tests must be performed under the same environmental condition:

T 22 +/- 1 °C;

 Relative humidity 60-70%

 Light-dark cycle of 12 hours. Differences in length of days and quantity of light can provoke

 hormonal interference;

Controlled quality of administered water and food;

 Litter;

Rodents are kept in the animal house for one week before the test. They are divided for sex, possible not

more of 3 individuals per cage, to avoid fight between males. You can build up a self-made calibration curve

(death time and concentration of toxin on the axis), using logically the same animals (same sex, age,

standard and laboratory conditions); that curve will be utilized just for the actual test. In order to get results

as best as possible is useful to carry out several analysis in combo, as LC-MS and mouse bioassay

(quantitative and qualitative evaluation). The ideal way to monitoring is to combine method of rapid assay

for screening like biochemical assay (functional) and after use a LC-MS to confirm results.

Prophylaxis. What happens if the tests are positive to DSP? The first thing to do immediately is to stop the

harvest and commerce of mussels from that area. Molluscs are periodically checked from the sanitary

authority, according to regulatory programs, both European and national. The most used method is LC-MS,

this is validated from the EU and labs have to use this in a ring analysis to consolidate data. Mouse bioassay

was formally descarded in 2010, but it is still used for other correlated purposes. If 2 or 3 treated animals

died after the mouse bioassay, the assumption was that molluscs were contaminated with OA, DTXs or

pectenotoxins in amount over the one permitted. There is suggested by the protocol to perform further

extraction with different solvent, in order to identify the presence of other putative toxins.

The maximum amount of OA, DTXs, pectenotoxins and azaspiracids in molluscs is establish 160 ug/kg of

mussels meat. For yessotoxins the limit is 1 mg/kg. Other lipophilic toxins can be found using mouse

bioassay. The toxicological characterization is not yet completely clear: under investigation are

pectenotoxins and yessotoxins.

Yessotoxins

They don't induce diarrhea; they are produced by Gonyaulax grindlei (now Protoceratium reticulatum), G.

polyedra (now Lingolodinium polyedrum), G. spinifera and they are extremely toxic per parenteral way, but

practically no toxic if assumed by oral administration in mouse.

Pectenotoxins

There are 9 different types of pectenotoxins. They are produced by D.fortii and D.caudata. The limit for

commerce is 160 ug/kg mussel meat, detected with LC-MS for DSP.

PP2A Inhibition assay – A functional method for diarrhetic shellfish toxins detection

DSP is caused by OA, DTX1 and DTX2, which are accumulated in bivalve molluscs. To avoid poisonings, EU

has established a threshold for the commercialization to 160 ug/kg of mussel meat. Methods to detect

these toxins, with an always increasing precision, are needed. One is the PP2A inhibition assay, a functional

assay based on the mechanism of action of DSP toxins. OA (okadaic acid) is the main DSP toxin and it is

an inhibitor of PP2A. These enzymes are involved

in the dephosphorylation of different protein,

which are involved in the regulation of many

cellular processes. When PP2A is inhibited there

is an increased phosphorylation degree of

proteins that control the secretion of electrolytes

through intestinal epithelium. This electrolytes

disequilibrium leads to big amount of water in

the intestinal lumen, that induces diarrhea. DTX3

cannot directly inhibit this enzyme (PP2A),

because this toxin is inactive if not hydrolyzed. However, hydrolysis can occur at gastrointestinal level and

DTX3 can induce diarrhea.

At pH 8-8.4 the PP2A can dephosphorylate an artificial substrate named p-NNP (p-nitrophenyl phosphate).

When this enzyme acts, p-NPP is transformed into p-nitrophenol (p-NP) and this reaction can be measured

by a spectrophotometer. OA can block this reaction avoid the synthesis of p-nitrophenol. Different

previously known concentrations of OA are used to build up a calibration curve plotting the absorbance

against the OA concentrations. Using this calibration you can measure the concentration of unknown

samples!

OA inhibits PP2a → PP2A cannot dephosphorylate n-NPP → no synthesis of n-NP

Here the pipeline to carry out a PP2A assay:

1. Collection and preparation of shellfish sample;

2. Shellfish meat extraction;

3. Dilution of shellfish extraction;

4. Preparation of OA reference solutions;

5. Preparation of enzyme and substrate solutions;

6. Performance of the assay;

7. Data analysis.

Collection and preparation of sample. Is needed a big amount of shellfishes, like 2 or 3 kg. Shellfish are

cleaned up with freshwater, then opened with a knife. The abductor muscle is cut and the meat is washed

again in order to remove sand and other undesirable material. There is now a passage on a sieve for 5

minutes, in order to drain the tissues (meat). After this, with a minipinner the meat is homogenized and the

sample is ready.

Shellfish meat extraction. 1 g of meat is extracted using 4 ml of 90% aqueous methanol. With an Ultra-

Turrax the content of the test tube is homogenized (1 min x 14000 rpm). Now it comes centrifugation at

3000 g for 10 minutes, and the supernatant is collected to carry out the assay. The extract need to be

hydrolyzed in order to liberate OA, DTX1 and DTX2 from their esters, which will inhibit PP2A.

Dilution of shellfish extract. Different solutions of the sample extracts have to be prepared, in order to have

a good probability that at least one of them will fall in the central part of the calibration curve (linear):

[A]: 50 ul extract + 950 ul of buffer (40 mM Tris/HCl pH 8,4)

• [B]: 500 ul [A] + 250 ul buffer

• [C]: 500 ul [B] + 1000 ul buffer

• [D]: 500 ul [C] + 1000 ul buffer

The buffer contains Mg, that is needed by the PP2A to function.

Preparation of OA reference solutions. Using a stock solution of OA (the standard reference compound) in

EtOH, 10 OA reference solutions are prepared by dilution adding the Tris/HCl buffer. Concentration range is

from 0 to 2,222 ng/ml. In this way, reference solutions that will be used in the assay are obtained.

Preparation of enzyme and substrate solutions. PP2A is isolated from the skeletal mass of rabbits, or

produced by recombinant bacteria. The concentration of PP2A will be 0,05 U/ml. The final concentration of

the substrate, p-NPP, will be of 141 mM.

Performance of the assay. In each well of the 96-well plate we have to add:

50 ul of the OA reference solution [for the calibration curve] or extract solution [for the analysis] (in

• the blank → 50 ul of Tris buffer);

50 ul of the enzyme solution (in the blank → 50 ul of Tris buffer);

• 100 ul of Tris buffer;

• 50 ul of substrate solution.

Now is time to shake the well plate for 1 minute with the microplate shaker. Then, incubate the plate for 1

hour at 36 °C, and finally read the absorbance with the spectrophotometer. Each determination is made in

duplicate. If the color in the well is an intense yellow, there is a lower concentration of toxin; if the color in

the well is a bland yellow, more transparent, there is a higher concentration of toxin.

Analysis of data

Mean Abs (absorbance) values for the blank well is subtracted from the mean values of the other

• wells;

The net Abs value for the well containing 0 ng/ml OA represent the 100% of enzyme activity

• (control);

Calculation of the % residual enzyme activity; ratio between Abs of the test well and Abs of control;

• OA calibration curve: % residual enzyme activity of OA reference solutions is plotted against OA

• concentration of these solutions;

Calculation of OA concentration in samples: interpolation of the enzyme activity of the sample from

• the linear interval of the calibration curve obtained from the OA reference solutions, taking into

account the sample preparation and dilution;

Toxins concentration: expressed in mg of OA equivalent/ kg of meat;

• LOQ (limit of quantification): 64 ug OA equivalent/kg meat.

We will not consider all the point of the curve but just the middle part where we have a linear situation of

the calibration curve. There are also to consider all the dilutions made in the assay. OA equivalents means

that the assay cannot discriminate from all possible toxins (so, in these equivalents are comprised also DTX1

and DTX2). The LOQ – Limit of Quantification, namely the lowest toxic concentration is 64 ug of OA

equivalent/kg meat.

Table on slide.

Palytoxin

Algal toxins are secondary metabolites of algae, produced under some specific conditions; they are not toxic

for algae but for other organisms. These metabolites are accumulated in seafood and can induce some

poisonings in human. The vectors with which human can contract intoxication are mainly mussels, fishes

and crustaceans. Mussels accumulate toxins through filtration; fishes that eat mussels undergo the process

of bioaccumulation. Toxins are colorless, odorless and thermostable, and thus really difficult to detect.

Palytoxin is a complicated molecule from a chemical point of view. It is considered one of the most toxic

compound present in nature of non-protein origin. It was identified for the first time in 1971 in several soft

corals of Hawaii, known and Palythoa and Zooanthus species, that are used as decorative elements in

aquariums. This is one of the most problematic issue related to human intoxications. Palytoxin is not only

synthesized by soft corals but also from dinoflagellates of the genera Ostreopsis and Trichodesmium. These

are unicellular algae, originally diffused in the tropics but afterword identified also in more temperate areas.

Researchers were very curious about the presence of palytoxin in algae and corals and they found that this

toxin was present also in Trichodesmium spp., that is a cyanobacteria, and maybe the link between coral

and algae. There are different exposure routes, each one with specific symptomatology:

Oral exposure: GI problems, muscle cramps, myalgia, cardiac dysfunctions, respiratory stress,

• cyanosis, death. This kind of exposure is associated with the ingestion of contaminated seafood, the

oral exposure is the most dangerous;

Cutaneous exposure: edema, erythema, urticarial rush, itch, numbness and swelling, neurological

• problems as paresthesia and metallic taste. This exposure is associated to the handling of Palythoa

corals or to seawater contact during Ostreopsis blooms;

Aerosol: rhinorrhea, cough, respiratory distress, fever, conjunctivitis, dermatitis. It is associated with

• aerosolized water during Ostreopsis blooms and during cleaning of aquariums containing Palythoa

species. Ostreopsis produces palytoxin and through the action of waves people can inspire aerosols.

The most part of case of contamination was not investigated chemically but information were obtained just

on observation of symptoms and on the knowledge of vectors; palytoxin was chemically identified in only 2

cases. Only few cases were ascribed unequivocally to palytoxin. In Philippines there was a case in 1988:

after 15 hours from the ingestion of a crab a person died. Another case in Japan in 1987: a person

consumed a fish (Scarus ovifrons) and died after 4 days. The last one case of death in Madagascar, in 1999: a

woman died after have eaten a sardin-like fish. In Japan (2000) 11 people on 33 eating a fish soup were

intoxicated and hospitalized. In the Mediterranean sea no intoxication after ingestion of contaminated

seafood have been occurred. However, huge amounts of palytoxin and analogs have been found in mussels,

echinoderms ad crustaceans. Moreover, signs of toxicity were recovered.

Experimental studies have been done in order to study the toxicity of palytoxin. The first report on in vivo

toxicity of PLTX was achieved using a semi-purified toxin, because the procedure of extraction and

purification were not well developed yet; in 1981 the exact chemical formula was obtained. Lots of in vivo

studies were performed after parenteral and oral administration of the toxin in different animal models.

However, due to the limited availability of PLTX the majority of study was carried out in mice. In vitro studies

have been performed in order to identify the mechanism of action. The most useful data are those that

come from the oral exposure studies. The first LD50 per os was obtained in mice and it was equal to 510

ug/kg of body weight according to the OECD 425 pipeline. OECD pipelines are established from EU in order

to limit the use of treated animals in the study of toxic compound; the OECD 425 is the one to follow in case

of PLTX and it provides some statistical procedures that involve the use of only 2 animals. The LD50 is

calculated then by an algorithm. The lethality in mice starts from 600 ug/kg per os (oral exposure);

according to a new study on acute oral toxicity of PLTX the LD50 is of 767 ug/kg. Symptoms are mainly

spasm (86%), paralysis (80%) and respiratory problems (70%). The ultrastructural analysis has revealed a

fibrillar disorganization and aggregation of mitochondria in skeletal and cardiac muscle cells. Also NOAEL

and LOAEL are necessary to establish the limit. Seven days of PLTX administration caused lethality and toxic

effects at doses >30 mg/kg/die (LOAEL); the NOAEL was estimated to be equal to 3 mg/kg/die. At

histological level, macroscopic alterations were observed at GI level (gastric ulcers and intestinal fluid

accumulation); pulmonary level (severe inflammation, locally associated with necrosis); myocardial level

(hypereosinophilia and fiber separation). Lungs, heart, liver and GI trait are the main PLTX targets. However,

these studies are not so common since a very limited amount of the substance studied, palytoxin, is

available.

The high toxicity of PLTX is related to the mechanism of action. PLTX transforms the K-Na ATPase in a

cationic channel and the gradient is not maintained anymore. The K-Na ATPase is a transmembrane pump

which is essential to maintain cellular homeostasis; it serves to transfer 3 Na ions out of the cell and 2 K ions

into the cell, in a cyclic process that exploits the hydrolysis of ATP. PLTX bond with a subunit of the ATPase

changes it from a transmembrane pump to a non-specific cationic channel; as a consequence, a consistent

ionic imbalance at cellular level is induced. The great amount of sodium ions into the cell provokes ROS

production, cell mass reduction, mitochondrial dysfunctions and an increase of calcium in the cell: this

latter effect leads to membrane and cytoskeleton damages.

In vitro effects are distinct according to excitable cells (neurons, muscle and cardiac cells) and non-excitable

cells. In excitable cells, the calcium increase is mediated by the reverse functioning of the Na-Ca exchanger

and by the activation of voltage-gated calcium channels. Calcium dependent toxic (and dramatic) effects

are: 1) disruption of the cardiac excitation-contraction coupling; 2) Uncontrolled muscle contraction; 3)

uncontrolled neurotransmitter release. In non-excitable cells, the big amount of intracellular sodium

induces cytoplasm acidification due to the reverse functioning of the Na-H proton pump, and the overload

of protons leads to mitochondrial dysfunction (the electrochemical gradient is not maintained anymore)

and ROS production. In this case, effects are more sodium-dependent instead of calcium-dependent (keep

in mind that in both situation all begin with an overload of sodium). The state of non-homeostasis induces a

necrotic cell death. Thus, palytoxin is able to block the cardiac beating.

There are many palytoxin-like compound; omopalytoxin,

neopalytoxin, 42-hydroxypalytoxin, etc. according to different

substitutes in several positions. The molecule of the leader

compound – palytoxin – has 64 chiral centers, so the number

of stereoisomers is really exponential. Each of these analogs

could have different toxicity. 42-hydroxypalytoxin is completely

equal to palytoxin but a hydroxyl group on position 42. It was

initially identified in Palythoa toxica. In Palythoa tuberculosa, a sister of Palythoa toxica, produces a 42-

hydroxypalytoxin with a conformational change in C50. The 42-OH-PLTX has a lethality in mice starting from

300 ug/kg per os. The LD50 is 651 ug/kg, overlapping the LD50 of palytoxin; also the symptoms and the in

vivo cytotoxicity are very similar to those induced by PLTX. However, the conformational inversion on C50

reduce the cytotoxicity of the Palythoa tuberculosa 42-OH-PLTX of almost 2 orders of magnitude.

Ostreocin-D is produced by Ostreopsis ovata. Generally, this toxin is less toxic than PLTX. In vitro studies

revealed that Ost-D is less toxic than PLTX by 3 orders of magnitude.

For ovatoxin-a no complete toxicological evaluations have been carried out so far; however, ovatoxin-a

should be less toxic of 2 orders of magnitude compared to PLTX.

EC50 is a parameter used to compare the cytotoxicity of compounds and indicates the concentration that

causes the same effect in 50% of the treated cells.

These test are important to establish the limit of this toxins in the seafood. PLTX is still not regulated but

there is a suggested limit from EFSA and FDA of 30 ug of PLTX equivalents (PLTX + Ost-D) / kg of mussel

meat. However, new data regarding the repeated oral toxicity of PLTX should be considered to revise this

limit. Moreover the toxic potential of the new PLTX analogs should be evaluated to deeply characterize the

real risk on human health. The method employed to detect palytoxin in seafood should be rapid, sensitive,

specific and user friendly. There are chemical, biological and antibody-based methods available to detect

PLTX. The two official methods employed are LC-MS and HR (high resolution) LC-MS (chemical) and the

mouse bioassay. Mouse bioassay and LC-MS are thus the most commonly used. LC-MS is difficult to

interpret because we could have a lot of interference. For mouse bioassay is the same: the extraction

procedure can extract other lipophilic toxins, so the toxicity can be due also by other toxins.

The ELISA (Enzyme Linked ImmunoSorbent Assay) assay is very sensitive and specific and uses antibodies;

it is very easy to use and requires a basic lab equipment. It is a colorimetric assay, so the color is

proportional related to the concentration of analyte: the more intense the color the higher the

concentration of analyte (do not confuse with PP2A inhibition assay!). Antibodies are linked to the plastic of

the wells of the plate. Antibodies are able to capture (so called capturing antibodies) analyte molecules.

There are 2 types of ELISA. In the competitive ELISA, only one antibody is employed; there is a competition

between the sample antigen and the enzyme-linked antigen, that produces the colorimetric reaction. In the

sandwich ELISA, the antigen is detected by 2 antibodies. In this case we have 2 kind of detection:

Direct immunodetection: the antibody is conjugated with an enzyme system;

• Indirect immunodetection: a secondary antibody recognizes the detecting antibody (that bind the

• antigen) and is conjugated with the enzyme system. In this case we use 3 antibodies: the first is

attached to the well surface, the second recognizes the antigen bound to the first antibody and the

third has the purpose to amplify the signal. The latter (an unspecific antibody) is conjugated with an

enzyme which leads to the colorimetric reaction.

Commonly used enzyme are the HRP – Horse Radish Peroxidase and AKP – Alkaline Phosphatase. The

substrate that gives the colorimetric reaction is TMB, a chromogen that become blue when reacting with

the enzyme. Antibodies can be monoclonal or polyclonal. The monoclonal antibodies recognize all the same

epitope of the antigen because they are produced from clones of a single plasma cell. Polyclonal antibodies

recognize different parts (epitopes) of the antigen.

A newly developed sandwich ELISA assay for PLTX is now developed. Main features:

Very sensitive (LOD = 1,1 ng/ml and LOQ = 2,2 ng/ml)

• Repeatable

• Accurate (bias = 2,1%)

• Specific for PLTX and analogs (ovatoxin-a, 42-OH-PLTX, PLTX-biot)

Monoclonal antibodies obtained in mice are used as capturing agents; the recognition of palytoxin and

analogs is get from polyclonal antibodies obtained in rabbit. The third antibody will be thus an anti-rabbit

antibody conjugated with the HRP enzyme. Procedure:

1. Preparation of the sample (mussels, algae or seawater);

2. Extraction of the toxins (performed using methanol or butanol);

3. ELISA assay (performed in parallel with the calibration curve);

4. Interpretation of data (extrapolation of PLTX content in the sample based on the calibration curve).

The hemolytic assay is another method used to quantify palytoxin, exploiting its ability to hemolyze

erythrocytes of different species. The hemolytic activity of palytoxin was discovered casually by Habermann,

that after incubation of red blood cell with the toxin forgot the experiment; on his return he found

erythrocytes hemolyzed. PLTX is a potent but slow hemolysin, in pigs, rats, mice, rabbits, guinea pigs and

human erythrocytes. The hemolytic assay is based on the skill

of PLTX to convert the Na-K ATPase into

an unspecific cationic channel, leading to

a late lysis of red blood cells. The release

of hemoglobin can be measured with a

spectrophotometer. The release of

hemoglobin was found to be time,

temperature and PLTX concentration

dependent. If the T increases from 37 °C

to 42 °C, the hemolysis occurs faster.

Under 15 °C there is no hemolysis even

after 8 hours of incubation at high

concentration. PLTX induces hemolysis

after 4 hours of incubation of the samples with the toxin and no differences were observed from samples

incubated for 6 hours or more. The presence of borates (boric acid oe sodium tetraborate 5,0x10-6 M) and

calium ions (>2,0x10-5 M) in the buffer solution is reported to increase hemolysis induced by PLTX, due to

its ability to promote interaction between the toxin and the Na-K ATPase. This assay shows pros and cons.

Advantages:

No sterile conditions;

• Easy to perform;

• Rapid;

• Sensible;

• Specific.

Disadvantages:

Variability depending on the species of origin of the erythrocytes;

• Possible interindividual variability depending on sex, age and other factors;

• Marked matrix effects.

Mice and rats are in fact more sensible to PLTX activity.

Apart these disadvantages, the hemolytic assay is one of the most promising method to recognize and

quantify PLTX; for this reason, it would be useful a characterization in order to detect unequivocally PLTX all

over the world with a standardized pipeline. A general and uniform protocol is still lacking for this assay and

it has never been characterized as screening tool to quantify PLTX in shellfish. Practically, erythrocytes are

purified:

Samples of erythrocytes are diluted in a red blood cell preservation solution;

• Samples are centrifuged ffor 10 minutes at 4°C and 2400 rpm;

• After the centrifuge, the surnatants are removed;

• Red blood cells pellet is washed 3 times with centrifuge;

• The final pellet is resuspended with 1:10 dilution in the red blood cell preservation solution;

• Samples are now stored at 4°C in red blood cell preservation solution (PBS + 1 mM EDTA + 5 mM of

• glucose).

After purification the assay can be carried out. Samples are put in the wells of the plate (125 ul) with 125 ul

of 0,1% v/v of tween 20. and a certain concentration of the toxin. Tween is a buffer that forms a complex

with the membrane cholesterol of erythrocytes, leading to the lysis. The negative control is composed of

125 ul of red blood cell suspension and 125 ul of K-free D-PBS, without the toxin. The percentage of

hemolysis is calculated by a formula:

[(Abs of treated samples – Abs of negative control)/ (Abs positive control – Abs negative control)] x 100.

A calibration curve has to be constructed in order to extrapolate the amount of toxin in unknown samples.

The resulting parameters are:

EC50 = 6,2 x 10-9 M;

• Working range = 3,91 x 10-10 to 2,5 x 10-8 M

• LOD (Limit of Detection) = 2,36 x 10-10 M (6,3 ug/kg);

• LOQ = 6,09 x 10-10 M (16,3 ug/kg)

This method is repeatable, accurate and specific. The low intense color is index of a lower concentration of

PLTX in the sample. The hemolytic assay cannot be used so far in quantification of PLTX in mussels at level

below the maximum limit suggested by EFSA of 30 ug of PLTX/ kg of mussel meat.

AZP – Azaspiracid Shellfish Poisoning

AZP is quite new: it was identified in 1995 after an intoxication characterized by symptoms similar to those

of DSP occurred in the Netherlands. This poisoning was caused by mussels coming from Killary Harbor in

Ireland. Chemical analysis were performed and there was a positive response for DSP with low

concentration of okadaic acid and analogs, but too low to explain this intoxication; in fact a new toxin was

found and named killarytoxin. Three years later more complete chemical analyses were carried out and the

name of the new toxin recovered was azaspiracid, due to its particular structure. Azaspiracid is a lipophilic

toxin produced by Azadinium spinosum, a very little dinoflagellate. This toxin can be accumulated in edible

seafood like mussels, oysters, clams, scallops, snails, crabs and sponges. So far, human AZP were exclusively

due to contaminated mussels coming from Ireland. Symptoms are very similar to those of DSP and of a

generic bacterial intoxication: nausea, vomit, diarrhea. Almost surely, the incidence of this intoxication is

underestimated because it can be confused easily with DSP. DSP and AZP differ in that AZP symptoms

appear after few hours and remain for 2-5 days. At now, AZA (azaspiracid) has been detected along the

western coastline of Europe, especially along the Norwegian coast, and also in Chile. However, cases of AZP

are limited:

8 cases, Netherlands 1995;

• 24 cases, Ireland 1997;

• 10 cases, Italy 1998;

• 16 cases , UK 2000;

• 30 cases, France 1998; 219 cases, France 2008 (last intoxication).

All these cases are due to ingestion of mussels coming only from Ireland. In 2008 began a monitoring

program and since that moment outbreaks have not been recognized anymore. In 1995 anything was

known about azaspiracid, neither the producers, and the chemical structure was identified recently. AZA is

present in plankton. Initially, the heterotrophic dinoflagellate Protoperidinium crassipes was believed to be

the main producer; another dinoflagellate was discovered to produce this toxin in 2007, Azadinium

spinosum, prey of Protoperidinium crassipes. Thus, the producer is now believed to be A.spinosum.

Afterward, some other species of Azadinium genus were discovered in Italy, Chile, Korea, Denmark, Ireland,

Scotland, Argentina, France and Mexico. Not all the species are known to produce azaspiracid. Recently,

another Azadinium species (Azadinium dexteroporum) able to produce AZA (mainly AZA3 and AZA7, analogs

of the lead compound azaspiracid) was found in the gulf of Naples.

The name azaspiracid was given to the toxin because of the presence of a cyclic amino group called AZA

group, of a 3-rings spiranic group and of a carboxyl residue (that provokes acidity). Up to now more than 30

analogs of azaspiracid have been characterized. Different substitutions on side chains determine different

azaspiracid molecules. From a toxicological point of view they are classified with a number in order of

discovery. AZA1, AZA2 and AZA3 are actually the most studied; AZA4 and AZA5 are now under investigation.

EU imposed an admissible threshold in mussels of 160 ug of AZA1 equivalent (AZA1, 2, 3) / kg of mussel

meat. Most of 30 analogs of AZA metabolites of AZA1 and AZA2 biotransformation in mussels and/or

artifacts due to mussels conservation. Recent studies demonstrate that already after 24 hours it is possible

to find AZA1 and AZA2 metabolites in mussels (AZA 3, 5, 8, 11, 12, 17, 19). Other metabolites are detected

after some days: AZA 4, 10 and 21 after 2 days, AZA23 after 3 days, AZA9 after 6 days.

Notably, in tissue samples at T > 100°C AZA are prone to degradation. Cooking at high T could perhaps

degrade AZA and analogs. However, no information are available regarding the resulting toxicity of most of

these compounds. Among these analogs, the main products due to mussels bioconversion are AZA17 and

AZA19, which take origin respectively from AZA3 and AZA6. These analogs are known for their toxicity

arising problems for public health, since they are not regulated by EU.

According to data obtained by in vivo experiments and on epidemiology, GI apparatus seems to be the

target organ of these toxins. Although experiments have been performed from 20 years ( in vitro and in

vivo), the mechanism of action of AZA is still unknown, as the intracellular pathways and the molecular

targets: we know just symptoms!

In in vitro studies, multiple effects have been observed on various cell lines (monocytes, lung cells,

enterocytes, lymphocytes), including cell death both by necrosis and apoptosis, alteration of cytoskeleton

and decrease of cells volume. In vivo studies in mice by intraperitoneal and oral administration have shown

multi-organ damages (liver, pancreas, intestine, lungs, stomach, spleen and thymus). Very unspecific

damages have been observed in different cell models:

AZA reduces cell viability even at very low concentration, by the activation of apoptotic processes;

• AZA reduces ATP levels, cell adhesion, cell volume and activates some kinases.;

• AZA is able to alter intracellular levels of calcium, cAMP and pH: these effects might modulate

• apoptosis induction;

AZA can modulate ionic fluxes through plasma membrane, especially in neurons (modulating

• bioelectric activity) and cardiomyocytes (modulating K fluxes on hERG channels, responsible for the

auto-generation of the membrane potential in these cells).

However, in vivo studies show no damages at cardiac level.

Firsts in vivo studies aimed to evaluate the differences between AZA and okadaic acid. AZA and OA have a

completely different chemical structure. After few hours, mice develops a progressive paralysis of legs and

difficulties on breathing because of the block of muscular lung system. With low doses of injected AZA, mice

doesn't develop diarrhea; furthermore, instead of OA, AZA does not inhibit proteinphosphatase. According

to these findings, the molecular mechanism of AZA could be really different to that of OA. After

intraperitoneal administration, the minimum lethal doses were identified:

AZA1: 200 ug/kg of body weight, TEF = 1

• AZA2: 110 ug/kg of body weight, TEF = 1,8

• AZA3: 140 ug/kg of body weight, TEF = 1,4

The limited toxicological information does not allow the setting of robust toxic equivalence factor (TEF) for

AZA analogs. TEF are number that give us an idea on the potency of an analogs in respect to the reference

compound. AZA4 and AZA5, which are products of a metabolic bioconversion, have been found to be

significantly less toxic than the main toxins AZA1, 2, 3. TEF of AZA4 and AZA5 are 0,4 and 0,2. These TEF

values are not so reliable because the studies were done on a little number of mice.

After per os exposure (the most likely exposure route for human), AZA1 was demonstrated to cause

widespread organ damages, mainly to intestine (villi erosion, necrosis of the lamina propria), liver

(hepatocytes vacuolisation, liver hypertrophy, accumulation of fatty acid and mild inflammation) and lung

(interstitial pneumonia). Based on these effects, it can be hypothesized that liver and lungs can be the

target organs of this toxin after oral administration.

In 2001, the FSAI – Food Safety Authority of Ireland performed the first AZA risk assessment in shellfish

using LC-MS. Mussels were collected from the accident site. They established a putative concentration that

had led to symptoms to 5,7-10,7 ug AZA/g hepatopancreas, but they didn't take into account the cooking

stability of AZA and the analogs presence in hepatopancreas. In America, FDA sets the action level at 0,16

ppm of AZA equivalents. In Europe a different threshold is suggested according to a revision of the FSAI limit

including the now-available data on toxicity. The FSAI revision led to an estimate of 113,4 ug AZA per a 60 kg

person. Based on this limit, EU imposed in 2001 the regulatory level to 160 ug AZA equivalents/kg of

shellfish meat. Considering the new insights on this issue, in 2008 EFSA revised the limit to 30 ug of AZA1

equivalents/kg of shellfish meat. According to EFSA, the acute reference dose (ArfD) id of 0,2 ug of AZA1

equivalents/kg body weight, corresponding to 12 ug of AZA1 equivalents for a 60 kg person.

Since there are no information on the mechanism of action, no functional assay is available. EFSA suggested

the LC-MS/MS as reference method to detect AZA. Mouse bioassay was however frequently used, but at

the end of 2014 this method should be completely substituted by LC-MS/MS.

Brevitoxins

Brevitoxins are neurotoxic polyethers produced by dinoflagellates belonging to the Karenia genera, in

particular by Karenia brevis. Karenia brevis blooms have occurred in the gulf of Mexico and along the

southern east coast of the USA, mainly in Florida. These blooms (also called Florida red tides) consist on red

tides, where the water is brown/red colored. There are 2 different structural backbones of these molecules:

A backbones show 10 trans-fused cyclic rings, while B backbones 11. Brevitoxins can also present an

additional structure where the lacton ring is opened. Depending on the backbone and the substitutions,

there are 15 different brevitoxins: they all derived from brevitoxins 1 (A backbone) and 2 (B backbone).

Karenia brevis can produce other brevitoxins-related compounds as hemibrevitoxins and brevenals.

Hemibrevitoxins are incomplete products of brevitoxins. Brevenals are instead antagonist of brevitoxins.

These metabolites are synthesized by shellfish and have been identified and structurally characterized; it is

now know that they are involved in marine mammals toxicity. Filter-feeding shellfish can accumulate these

toxins and act as vectors, leading to human and animals intoxications. For aquatic species there is a high

mortality: fishes, aquatic birds, sea turtle, crabs, lion's mane jellyfish, dolphins and manatees are affected.

Massive mortality of fish in Japan, China and Australia was caused by blooms of Chantonella marina, which

is a brevitoxins producer.

Human can be exposed through different routes. Ingestion of contaminated foodstuffs is a risk factor of NSP

– Neurotoxic Shellfish Poisoning, that presents GI and neurological symptoms: nausea, diarrhea, vomit,

headache, tingling in the face, difficulty in speaking, paresthesia, loss of coordination and bradycardia.

Brevitoxins can be also aerosolized from the water by the action of wind and waves; inspiring this aerosol

can cause skin irritation, conjunctivitis, rhinorrhea, bronchoconstriction, cough. People can get intoxication

also through cutaneous exposure. However, like other toxin poisonings, the oral route is the most

dangerous.

Until now, poisonings have occurred mainly in Florida and Texas, with 4 major outbreaks. Fortunately no

lethality has been reported; cases of NSP were not so common. The intoxication caused by brevitoxins

shows symptoms that appear after 30 minutes and last 2-3 days.

A lot of studies were focused on ADME of brevitoxins, finding that liver is the main target. After intravenous

administration, 70% of the toxin was concentrated in skeletal muscle, 18% in liver and 8% in intestinal tract;

lower concentrations were recovered in heart, kidneys, brain, spleen, testes and lungs. After intratracheal

instillation, the toxin was accumulated in liver, skeletal muscle and GI tract. Skeletal muscle might be a

storage compartment for the toxin. After intraperitoneal injection in mice, 39% of the toxin was bound to

components in mouse plasma; the 7% of the toxin in the blood was associated with serum albumin. Large

amount of toxin was associated with plasma high-density lipoproteins (HDLs). An in vitro study on possible

bonds of brevitoxin to human serum albumin have found covalent and non-covalent interactions between

them with the formation of an adduct made up from one or two toxin molecules and one HSA molecule.

The elimination happens mainly through urine and feces; there is no more toxin in the body after 3-4 days

after exposure. Summarizing, brevitoxins are accumulated mainly in the skeletal muscle tissue and undergo

to hepatic metabolism and biliary excretion. The tissue distribution and elimination patterns of brevitoxin

metabolites however depend on the structural features resulting from different reaction occurring in

shellfish.

The molecular targets of brevitoxin are the voltage-gated sodium channels in excitable cells. VGSCs are

transmembrane proteins formed by an α-subunit and one or more β-subunits. The α-subunit is composed

of 4 homologous domains, consisting of 6 transmembrane α-helices connected by internal and external

polypeptide loops. S5 and S6 helices form the ion-conducting pore and the S4 helix works as a voltage

sensor. These channel exist in 3 states: activated, deactivated and inactivated.

In case of membrane depolarization,

the VGSC opens its gate allowing an

intracellular flux of sodium ions

(activated state). When the

transmembrane potential rises, the

channel inactivates by closing the gate

and sodium ions flux is blocked and

membrane repolarization occurs until

the normal resting potential is

restablished. At this point, the channel

returns into its deactivated state with

closed gate. Brevitoxin acts on S5 with a

''head-down'' orientation, with the A

ring (see structure) extending towards

the intracellular opening of the gate and the J or K ring near the extracellular surface of the pore. Brevitoxin

bond to the VGSC lead to:

1. A shift in the voltage dependence of activation to a lower membrane potential. In presence of toxin,

the channel opens under conditions in which it is normally closed;

2. An inhibition of channel inactivation;

3. An increase in mean open times, allowing big amount of sodium ions to enter in the cells.

These effects lead to the alteration of membrane properties of the excitable cells, and this represents the

basis of the toxic effects of this group of toxins. Several studies were also performed in vivo to establish the

acute toxicity. After a single administration, LD50 of brevitoxin 2 were: 200 ug/kg intraperitoneal; 200 ug/kg

intravenous; 6600 ug/kg oral. Even after a single administration, LD50 of brevitoxin 3 were: 170 ug/kg

intraperitoneal; 94 ug/kg intravenous; 520 ug/kg oral. Most of studies have been performed with brevitoxin

2 and 3 because they are more available.

After single intraperitoneal administration, mice treated with brevitoxin 3 presented a syndrome

characterized by hypersalivation, lachrymation, excessive urination and defecation. These symptoms

appeared 30 minutes after administration. Mice treated orally with brevitoxin 3 presented tremors,

muscular contraction, breathless and death, that are completely different symptoms compared to those of

the intraperitoneal route. Furthermore, these symptoms appeared 5 hours after administration. Rats, after

inhalation of 500 ug of brevitoxin 3/m3/2h/day for 5 consecutive days shown reduction of body weight, no

macroscopic or microscopic lesions, no inflammation nor immune system dysfunctions.

A NOEL (No Observable Effect Level) for brevitoxin in human has not been established yet, although toxicity

occurs in nanomolar concentration range. FDA suggested an admissible level for brevitoxin in shellfish of 0,8

ppm of brevitoxin 2 equivalents. It is clear that the consumption of even a few contaminated shellfish may

result in poisoning and that the severity of the disease may be different upon many factors including dose,

body weight, underlying medical condition, age.

Since the '80s, monitoring systems in Florida have been employed. There is no specific treatment for NSP.

This poisoning shares several symptoms with other poisonings and its diagnosis can be a challenge. To verify

the diagnosis, analysis on meal remnants or samples taken from the same harvesting area should be

performed. The analysis of urine sample can be used to confirm the presence of brevitoxin. Analgesics, fluid

replacement and respiratory support are putative primary solutions. GI decontamination can be performed

with activated charcoal. A potential employment of brevenals as therapeutic agents might be considered in

the treatment of NSP.

PSP – Paralytic Shellfish Poisoning

PSP Is one of the most dangerous intoxication that can be recovered and it is widespread all over the world.

It was the first intoxication by shellfish discovered. In Italy there were several intoxications, but no one felt

sick. Poisoned people developed just some irritation on lips and on the mouth. The amount of toxin was

very low, considering the fact that if the toxin is concentrated in only one mussel it can kill a person. This

intoxication has a very high rate of mortality, until 22% (1 person on 5). The mean around the world is

however 6%. Having different analogs with different toxicities, this family of compound can cause a variety

of effects. The oral exposure at concentration from 2 to 1500 ug/kg can provoke a paralytic intoxication. The

lethal dose in an adult man is of 1-2 mg. This poisoning is characterized by neurological symptoms, which

come out after 30 minutes from the consumption of contaminated shellfishes. In a less severe intoxication

the main symptoms are: perioral paresthesia (around the mouth), with inflammations of lips and tongue,

and some gastrointestinal symptoms (they are less common). In an intermediate poisoning the paresthesia

spreads on the face, in the hairs, in the harms and in the legs, where it shown off also muscular weakness.

Subsequently, the patient undergoes to an intorbidimento generale. Severe intoxications provoke

neurological effects, like double visions, ataxia, vertigo, confusion; the patient feels a sensation of slightness

and he has difficulty in the assessment of weight. Subsequently comes the paralysis of limbs, and it can

spread to the respiratory muscles leading to a respiratory block and finally death. During the poisoning, the

patient remains conscious. Unspecific GI symptoms are nausea, vomit, diarrhea, abdominal cramps.

Diagnosis. If the patient goes in dyspnoea he needs a respiratory support immediately. Up to now, specific

tests are not available. Apart hypotension, symptoms are tantamount to these of the tetrodotoxin in puffer

fish; saxitoxin and tetrodotoxin have a quite similar structure and the same mechanism of action.

Prophylaxis. The monitoring relies on AOAC LC and other LC-MS methods (chemical assays) and on the

AOAC Mouse bioassay. The latter consists in an injection of an acidic extract of molluscs on mice. The death,

characterized by neurological symptoms, comes after 5-7 minutes (if there is saxitoxin). The sensibility of

this test is equal to 400 ug of STX per kg of mussel meat. There are some promising receptor assays, useful

for dosing toxins that act on the sodium channels, based on the tenet that the affinity of a toxin for its

receptor is proportional to its toxicity in vivo. These tests are still in phase of assessment.

Saxitoxin is an alkaloid positively charged with more than 30 analogs, among which

neo-saxitoxin and gonyautoxins 1, 2, 3 and 4.

Saxitoxins (STX), more than 6 compounds

• Decarbamoyl-STX, more than 6 compounds

• Sulphodecarbamoyl-STX, more than 6 compounds

• Deoxydecarbamoyl-STX, more than 6 compounds

These compounds are produced by several microalgae, like Alexandrium tamarense, Alexandrium minutum,

Gymnodinium catenatum, Pyrodinium bahamense. These microalgae are endemic of North America, but

they are spreading all over the oceans, with a sporadic presence in the Mediterranean sea. There have been

some cases in Italy, for example in Emilia Romagna; from this case it began a monitoring program to avoid

the risk of intoxications, with good results. The presence of this algae is ascertained even in the gulf of

Trieste, where they cannot bloom because of the environmental conditions.

Saxitoxin acts blocking the voltage-dependent sodium channel, and thus stopping the transmission of

nervous impulse. The bond between STX and the sodium channel does not allow the membrane potential

to spread, with effect on the transmission of the nervous stimuli and the muscular contraction. For its

toxicity, STX is included in the convention of Paris of 1993 on armi di distruzione di massa. It cannot be sold

without special permit; even the laboratory use is extremely controlled.

Main vectors are edible molluscs, that accumulate toxins without having effects. Only one shellfish can

accumulate an amount enough to kill a person. Different species have different ability on accumulating and

excreting these toxins. Even other organisms, that eat shellfishes, can work as vectors, like crabs, puffer

fishes, codfishes, herrings and salmons. Some other important species die before they reach high

concentration.

The limit established by the law is 800 ug of STX equivalent per kg of mollusc meat.


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DETTAGLI
Corso di laurea: Biologia ambientale
SSD:
Università: Trieste - Units
A.A.: 2015-2016

I contenuti di questa pagina costituiscono rielaborazioni personali del Publisher stefano.scilipoti di informazioni apprese con la frequenza delle lezioni di Tossicologia marina e studio autonomo di eventuali libri di riferimento in preparazione dell'esame finale o della tesi. Non devono intendersi come materiale ufficiale dell'università Trieste - Units o del prof Tubaro Aurelia.

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