Patent Publication Number: US-2011065138-A1

Title: Quantitative Method For Detecting Yessotoxins In Fishery Products On Based On The Activation That The Toxin Produces In Cellular Phosphodiesterases And Therapeutic Use Of This Activation

Description:
The present invention describes the detection and quantification of yessotoxins in vitro with respect to their ability for activating phosphodiesterase enzymes, one of the cellular targets of these toxins. It also describes therapeutic applications derived from the yessotoxin-phosphodiesterase bond. 
     Marine phycotoxins are substances produced by algae which represent a serious public health problem. These toxins accumulate in mollusks and fish in such a manner that when they are ingested by man they produce food poisoning. Phycotoxin classification is carried out in five large groups with respect to the type of intoxication they produce: paralyzing toxins (PSP), diarrhetic toxins (DSP), neurotoxic toxins (NSP), ciguatera toxins (CFP) and amnesic toxins (ASP) (Van Dolah, 2000). There are other groups of phycotoxins which produce different symptoms and which are not included in the former groups, which are: pectenotoxins (PTXs), azaspiracids and yessotoxins (YTXs) (Van Dolah, 2000). Initially, PTXs and YTXs were grouped with the DSPs, since they are lipophilic toxins which usually coexist in toxic episodes. However, in the last decade, they have been considered as a different group since they do not induce diarrhea, their oral toxicity is low and their molecules are different (Draisci, Lucentini et al., 2000). Within these last groups, yessotoxins (hereinafter, YTXs) represent a serious economic problem due to their recent and ubiquitous presence, and due to the absence of a sensitive and specific method for detecting them. 
     YTXs have been detected in Japan, Europe, New Zealand and Chile, and they are produced by the  Protoceratium reticulatum  and  Lingulodinium polyedrum  ( Gonyaulax polyedra ) dinoflagellates. These toxins accumulate in marine mollusks and are heat stable, and therefore they are not destroyed during the cooking of marine products. Their absorption, from the digestive tube, is low and therefore they are not toxic when ingested orally, although histopathological modifications have been detected in the liver and pancreas after an oral administration of YTXs in rats (Terao, Ito et al., 1990). However, after an intraperitoneal injection these toxins give rise to important cardiotoxic effects and show a high lethal potency (Draisci, Lucentini et al., 2000). These intraperitoneal effects must be taken into account because, as has been mentioned, YTXs coexist with DSP toxins, and when detecting the latter in bioassays they may produce interferences which lead to detecting false positives. For this reason, when preparing fishery product extract for monitoring the presence of these toxins, it is necessary to perform additional extractions with organic solvents which separate YTXs and DSP toxins. Although these modifications imply the extraction of a large amount of fatty acids, which may also give rise to false positives (Yasumoto, Murata et al., 1984). 
     The detection methods for phycotoxins in samples coming from marine product extracts are classified in assay methods and analytical methods. Assay methods are those which provide a value for the total toxin content by measuring a single biological or biochemical response which encompasses the activity of all the toxins present in the sample. Analytical methods are those in which separation, identification and individual quantification of the toxins in the sample with respect to an instrumental response is performed. The first include in vivo assays in rats or mice, and in vitro assays, amongst which must be stressed the enzymatic inhibition assays, cell assays, receptor assays, etc. In these cases, toxicity determination is performed with respect to a dose-response curve obtained with one of the representative toxins of each group. Quantification of the response is performed, amongst others, by means of calorimetric, fluorimetric, luminescence, polarized fluorescence methods, or by determining ligand-receptor interactions in real time with biosensors. The second are in vitro assays requiring a prior calibration of the instrumental equipment with standards of known concentrations of each toxin. These assays include chemical methods such as high performance liquid chromatography (HPLC), mass spectrometry or capillary electrophoresis. In general, the instrumental chemical methods are used when it is necessary to identify and quantify each one of the toxins present in a sample. However, in monitoring or health inspection programs a greater relevance is given to knowledge of the potential overall toxicity and therefore assay methods, also called functional methods, are used (Fernández, Míguez et al., 2002). 
     There are several liquid chromatography-mass spectrometry (Draisci, Palleschi et al., 1999; Goto, Igarashi et al., 2001) and fluorescence HPLC (Ramstad, Larsen et al., 2001; Yasumoto and Takizawa, 1997) analytical methods for detecting YTXs in contaminated mollusks. Within the in vitro functional assays for detecting these toxins there are two recent ones: the E-cadherin detection assay (Pierotti, Malaguti et al., 2003; Rossini, 2002) and the caspase activation assay (Malaguti, Ciminello et al., 2002). However, the only officially accepted method is the mouse bioassay, according to Commission Decision 2002/225/EEC of 15 Mar., 2002. This decision provides that the maximum level of YTXs in a mollusk sample is 1 mg of YTXs per Kg of mollusk flesh. The bioassay consists in observing, for 24 hours, three mice that have been intraperitoneally inoculated with an extract equivalent to 5 grams of mollusk digestive gland, where YTXs usually accumulate, or to 25 grams of whole mollusk. Since the maximum amount of YTX allowed could induce death by intraperitoneal administration within 6 hours, this assay has been modified by shortening the observation time and introducing additional extractions in the method. (Yasumoto, Murata et al., 1984). These extractions allow separating DSP, PTX and azaspiracid toxins from YTXs, although they imply extracting a large amount of fatty acids. If two of the three inoculated mice die, it is considered that there is YTX in the extract. This technique implies sacrificing animals, does not provide an exact value of toxin concentration, is hardly reproducible, gives rise to false positives and needs an additional extraction process in order to be able to discriminate the presence of other toxins. Other biological methods mentioned for detecting YTXs are slow methods with which results are not obtained in less than 24-48 hours and they require a careful process of toxin extraction. Furthermore, they are not based on a specific and unique characteristic of YTXs, since other DSPs are detected along with these toxins. 
     Several studies have been performed in order to determine the cellular target and, therefore, the mechanism of action of YTXs. YTX has a lipophilic molecule consisting of eleven rings with ether groups bound to an unsaturated side chain and to two sulfonic esters.  FIG. 1  shows some of the natural analogs of YTX which are differentiated in the side chain substituents, although recently more than 50 natural derivatives have been described the structure of which has not yet been identified. It has been observed in in vitro studies with YTX that it can produce apoptosis, although its potency is less than that of okadaic acid, a DSP toxin that usually occurs associated to YTX (Leira, Alvarez et al., 2001); and, in contrast to what occurs with okadaic acid, YTX does not inhibit cellular phosphatases (Draisci, Lucentini et al., 2000). YTX has a cytotoxic effect because it inhibits growth in human hepatocellular carcinoma cells (HEP-G2), and it may thus be used as an antitumor drug. It has also been described that YTX modifies cytosolic calcium levels in lymphocytes (De la Rosa, L. A., Alfonso, A. et al., 2001), and increases the calcium flow induced by maitotoxin in these cells (De la Rosa, L. A., Alfonso, A. et al., 2001). It has recently been observed that YTX decreases intracellular levels of cyclic adenosine monophosphate (cAMP) second messenger through the activation of cellular phosphodiesterases (PDEs), which are the enzymes which destroy cAMP, suggesting that these enzymes may be one of the cellular targets of YTXs (Alfonso, de la Rosa et al., 2003). On the other hand, it has been observed that YTX is a histamine release inhibitor, activated by immunological stimulus, in rat mastocytes, and it may thus be used as an antiallergic or antiasthmathic drug. 
     In mammalian cells there are about 11 families of PDE with different isoforms (Houslay and Adams, 2003). These enzymes regulate and maintain cAMP and cyclic guanosine monophosphate (cGMP) levels constant, the latter being second messengers necessary for cell functioning involved in numerous vital functions (Soderling and Beavo, 2000). From the pharmaceutical point of view, these enzyme families are very important since their modulation is involved in treating diseases such as asthma, rheumatoid arthritis and cancer (Houslay and Adams, 2003). For this reason, describing the natural or synthetic molecules which affect PDEs and methods for studying the activity of these molecules, which may be applied in HTS (high throughput screening) protocols, are very important tools for discovering new treatments against these diseases. In this sense, describing the inhibitory effect of YTXs on tumor cell growth and the modulation they produce on histamine release are two signs of the pharmacological importance of these molecules for their possible therapeutic application. 
     Making use of the recent description of the YTX mechanism of action, the present invention develops methods for detecting these toxins in extracts from fishery products, based on their specific affinity for cellular PDEs. These are functional assays in which the presence of other toxins the mechanism of action of which is different does not interfere, i.e., they do not act on PDEs, but they coexist with YTX in toxic episodes. Furthermore, false positives and animal sacrifices are prevented and the contamination monitoring process in said products is expedited, since results on the exact concentration of the toxin can be obtained in 1-2 hours. 
     The invention set forth describes three uses based on the discovery that YTX is a PDE activator and involves the conversion of this activation into a measurable signal. 
     Use 1: Method for Determining PDE-YTX Biomolecular Bonds Using an Affinity Sensor. 
     Determining molecular interactions in real time using a biosensor is a new technique the application of which is extending into different research fields (Hide, Tsutsui et al., 2002; Lee, Mozsolits et al., 2001; Mariotti, nunni et al., 2002; Tsoi and Yang, 2002). A biosensor is used which is an equipment detecting molecular reactions between a biologically active molecule, called a ligand, and another molecule it binds to, called a receptor. The ligand is bound to the support surface of the equipment, generally a cuvette or a plate. Created on the support surface where the bonds occur is an electromagnetic field, called an evanescent field, which is extremely sensitive to changes in mass. The biosensor transforms the changes in mass occurring on the support surface due to the ligand-receptor bond into an electrical signal. Commercial biosensor models which can be used for this method are those marketed by the Biacore or Thermo Labsystems companies. In the present invention, the PDEs function as the ligand and samples with YTX, which acts as a receptor, are added thereto. The signal in the biosensor will be larger or smaller depending on the amount of toxin adhered to the PDEs and, therefore, depending on the YTX present in the sample. 
     Cellular PDEs which function as ligands bond to the support surface. Known concentrations of YTX, which acts as a receptor, are subsequently added on this surface. The technique works well using planar surfaces or surfaces formed by a matrix. The PDE-YTX bond follows kinetics which adjust to an equation of pseudo-first order from which a constant is obtained which is called the apparent binding rate (Rap), and which is different for each concentration of toxin. A calibration line is drawn with the Rap and toxin concentration data. A test sample (an extract of fishery products) is added on the surface, its Rap is calculated and the YTX concentration in the test sample can be obtained by placing this value on the calibration line. 
    
    
     EMBODIMENT OF THE INVENTION 
     The method is carried out at a temperature between 22 and 37° C. 
     a.—A solution of PDEs at a concentration between 0.1-0.24 mg/mL at pH 7.7 is added onto an activated double compartment surface. These enzymes bind to the support surface by means of non-dissociable covalent bonds. The active groups the PDEs did not bind to were then blocked with different blocking solutions (BSA, Etanolamine, Tris-HCl . . . ). 
     b.—A solution with YTX at a known concentration is added to one compartment. The other compartment is used as a blank and the toxin solvent is added into it. The association kinetics between the PDEs and YTX are recorded for 15 minutes. 
     c.—The ligand-receptor dissociation is then carried out by washing both compartments with buffer solution at pH 7.7, thus dissociating YTX from the PDEs. 
     d.—The compartments are regenerated with an acid or base solution in order to completely remove YTX. Thus the PDEs will be accessible for a new addition of YTX. 
     e.—Steps b, c and d are repeated with 5 different concentrations of YTX. 
     f.—The apparent binding rates (Rap) are obtained from the association kinetics for each concentration of toxin. The plotting of Rap values against YTX concentrations follows a linear fit with a regression coefficient greater than 0.9. A line is thus obtained with which the concentration of YTX in a sample can be obtained if its Rap is known. 
     g.—An extraction of the meat of the fishery product to be studied is performed. This extraction is performed following Decision 2002/225/EEC of 15 Mar., 2002 or any other official method (D.O.G.A., 1986) for determining maximum levels and analysis methods for certain marine toxins present in different fishery products. An aliquot of the extract (test sample) is taken and is added on the PDE bound to the surface. Association kinetics are obtained from which its Rap is calculated. By placing this test Rap value on the regression line obtained, the YTX concentration present in the sample can be determined. 
       FIG. 2  shows the graphic profile of the steps to be taken in this method, from surface activation to adding the test sample. The regression line is shown in  FIG. 3  with the Rap values against known concentrations of YTX. 
     Use 2: Method for Determining PDE Activation by YTX Using a Fluorescent Molecule. 
     A usual way of detecting cellular PDE activity is to observe their ability to destroy cAMP. There is a fluorescent derivative of cAMP, anthraniloyl-cAMP (excitation wavelength: 350 nm, emission wavelength: 445 nm), the fluorescence of which decreases as it degrades. The decrease of fluorescence over time can be expressed as the destruction rate of cAMP. In the presence of PDEs, the destruction rate increases, and if these enzymes are activated, the degradation rate will be even greater. In the present invention the degradation rate of the fluorescent indicator anthraniloyl-cAMP in the presence of PDEs is determined and its variation when samples with YTX are added is studied. Fluorescence is read with a fluorimeter that is prepared for reading microtitration plates. The destruction rate is determined in the presence of several known concentrations of YTX. The representation of the destruction rate against toxin concentration follows a linear fit with a regression coefficient greater than 0.9. A regression line is thus obtained in which the destruction rate value obtained with a sample from fishery products (test sample) can be transformed into YTX concentration. 
     Embodiment of the Invention 
     The method is carried out in a microtitration plate in a temperature range between 22 and 37° C. and the fluorescence is measured at an excitation wavelength of 360 nanometers and an emission wavelength of 460 nanometers. 
     There are four types of wells and each one of them is carried out in duplicate. 
     WELLS A: Wells for calculating the cAMP concentration. Anthraniloyl-cAMP (fluorescent indicator) is added thereto at 5 concentrations between 2 and 10 μM. 
     WELLS B: Control wells with 8 μM of fluorescent indicator and enzymes. 
     WELLS C: Calibration wells with 8 μM of fluorescent indicator, enzymes and known concentrations of YTX. 
     WELLS D: Test sample wells with 8 μM of fluorescent indicator, enzymes and samples from an extract of any fishery product. 
     a.—Test buffer (10 mM Tris HCl+1 mM CaCl 2  pH 7.4) is added in all the wells for a final incubation volume of 100 μL, and the corresponding amount, depending on the type of well, of anthraniloyl-cAMP. A first reading is performed for 2 minutes. 
     b.—Between 2 and 5 μg of PDEs are added in wells B, C and D and a new reading is performed for 2 minutes. 
     c.—YTX at a known concentration or a sample from a fishery product is added to wells C and D. YTX at concentrations between 0.1 and 10 μM is added. The samples from an extract are obtained following Decision 2002/225/EEC of 15 Mar., 2002 or any other official method (D.O.G.A., 1986) for determining maximum levels and analysis methods for certain marine toxins present in different fishery products. 
     d.—After these additions the plate is shaken and successive fluorescence measurements are performed for 15 minutes, acquiring data every minute. 
     e.—A line is obtained with a regression coefficient greater than 0.999, by plotting the fluorescence data obtained with wells A against the concentration of indicator for each well. 
     f.—The fluorescence data for the rest of the wells in an anthraniloyl-cAMP concentration is transformed using the previous line in an anthraniloyl-cAMP concentration. The amount of indicator destroyed per unit of time, i.e. the destruction rate of AMPc, is obtained from the cAMP concentration at toxin addition time zero and from the concentration after 10 minutes. 
     g.—The destruction rate data obtained with wells B is considered as a control destruction rate. 
     h.—A YTX concentration standard line is obtained by plotting the destruction rate data of wells C against the YTX concentration. The YTX in that sample is determined by substituting on this line the destruction rate obtained in wells D. 
       FIG. 4  represents the fluorescence units calibration line against a concentration of anthraniloyl-cAMP.  FIG. 5  represents the standard line for destruction rates of cAMP against YTX concentration for a standard assay. 
     Use 3. Use of Phosphodiesterases as a Therapeutic Target of YTX and Compounds which Induce the Activation thereof. 
     a.1—Use of YTX as an inhibitor of the immunological activation of mastocytes and basophils. 
     Immunological activation of mastocytes and basophils requires a temporary increase of cAMP. This increase is indispensable for cell response activation (Botana and MacGlashan, 1994). PDE activation cancels this initial cAMP peak, and therefore prevents cell activation. In the presence of YTX, i.e. with activated PDEs, cell response will be inhibited. The inhibiting effect can be used in antiallergic or antiasthmatic therapeutic strategies, these being two pathologies in which mastocytes play a predominant role (Metcalfe, Baram, D. et al., 1997). The present use describes the quantification of the inhibition that YTX produces on cell activation induced by immunological stimulus in mastocytes in rats. Cell response inhibition can be determined according to different protocols described in the literature. One in which the response is quantified according to the histamine released by mastocytes in rats into the extracellular medium is set forth below (Alfonso, Cabado, A. G. et al., 2000; Estévez, Vieytes, et al., 1994). 
     Embodiment of the Invention 
     a.—The rats are sensitized 15 days before conducting the experiment. Each rat is subcutaneously injected with 1 mL of physiological serum with 150 mg of ovalbumin and 10 9    Bordetella pertussis  bacteria. 
     b.—Mastocytes are extracted from the chest and abdomen of a sensitized rat. The two populations are mixed and a cell suspension is obtained. 
     c.—The cell suspension is preincubated for 10 minutes with various concentrations of YTX and subsequently incubated 10 minutes in the presence of 5 mg/mL of ovalbumin. 
     d.—The reaction is stopped in cold conditions and the released histamine is separated from the histamine remaining in the cells by means of centrifugation. 
     e.—The supernatant is removed with the histamine released into the medium and the cells are cleaved with hydrochloric acid and ultrasound in order to release the histamine not sensitive to the action of the stimulus. 
     f.—Both mediums are deproteinized with trichloroacetic acid. 
     g.—The histamine is finally quantified, converting it into a fluorescent molecule by reaction in a base medium with o-phthalic dialdehyde. The reaction is stopped with hydrochloric acid and the fluorescence is read at 360 nm excitation and 460 nm emission. 
       FIG. 6  shows the percentage of inhibition of histamine release induced by ovalbumin in the presence of several concentrations of YTX. 
     a.2.—Use of YTX as a Neoplasic Cell Proliferation Inhibitor. 
     Neoplasic cell growth inhibition is an indicator of antitumor activity widely used to describe antineoplasic properties of new drugs. It has been found that YTX is cytotoxic for human hepatocellular carcinoma cells, and it has further been described that this toxin induces apoptosis (programmed cell death) in neuroblastoma cells (Leira, Alvarez et al., 2001), which all indicates that YTX is susceptible to being used as an antitumor drug. The ability of YTX as a cytotoxic drug for hepatic carcinoma tumor cells is quantified in the present use. Cell growth inhibition can be determined according to different protocols described in the literature. One of these protocols is set forth below in which the response is quantified in the HEP-G2 cell line by means of crystal violet staining and subsequent acetylation. 
     Embodiment of the Invention 
     a.—HEP-G2 cells are seeded on a microtitration plate with a density of 10000 cells per well. They are incubated for 24 hours with growth medium at 37° C. and 5% CO 2 . 
     b.—Different concentrations of YTX are added and it is incubated for 48 hours at 37° C. and 5% CO 2 . 
     c.—10 μL of 11% glutaraldehyde are added to fix the cells and it is incubated for 15 minutes. It is washed 3-4 times with distilled water. 
     d.—A 0.1% solution of crystal violet is added and the plate is shaken for 15 minutes. 
     e.—The dye is removed by washing with distilled water and it is subsequently dried. 
     f.—10% acetic acid is added and shaking is maintained for 15 minutes. 
     g.—Absorbance is read in a spectrophotometer at 595 nanometers. 
     h.—It was found with this protocol that 10 μM of YTX induce cell growth inhibition of about 82+/−1%. 
     LITERATURE 
     
         
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