Lezioni, Tossicologia marina

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<