Anatoxin-a s

Anatoxin-a(s) is produced in both Anabaena flos-aquae [56] and Anahaena lemmer-mannii [57]. The symptoms of anatoxin-a(s) intoxication are similar to those of anatoxin-a but cause increased salivation in vertebrates hence, the similarity in the names of these compounds with the (s) added to indicate salivation [18]. The structures, however, are quite different, as are the mechanisms of action. 

Cyanobacterial species/strains Section Habitat Origin Free BMAA (P /g) Protein-associated BMAA (p,g/g) 

Myxosarcina burmensis GB-9-4 II Marine coral Marshall Islands 79 1,943 

It has been suggested that BMAA in protein may function as an endogenous neurotoxic reservoir in humans, and may slowly release the neurotoxin directly into the brain during protein metabolism. Incorporation of a nonproternaceous amino acid into a protein may have other serious impacts, such as creating proteins of aberrant function or occurrence [76]. 

Chemically, BMAA agonism seems incompatible with activity at glutamate receptors, as it lacks the characteristic dicarboxylic add structure of other excito-toxins [80]. However, the activity of BMAA is dependent on the presence of 

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An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

Strain NRC-525-17 (Canada, Saskatchewan) Anatoxin-A(S) N-hydvoxy guanidine methyl-phosphate ester, MW 252 20... 

Figure 1. Left Anatoxin-a (ANTX-A) hydrochloride. Produced by the freshwater filamentous cyanobacterium Anabaena flos-aquae NRC-44-1. Right Anatoxin-a(s). Produced by the freshwater filamentous cyanobacterium Anabaena flos-aquae NRC-525-17. Bottom Aphantoxin-I (neosaxitoxin) and Aphantoxin-II (saxitoxin) produced by certain strains of the filamentous cyanobacterium Aphantomenon flos-aquae.

Anabaena Yes Anatoxin-a Anatoxin-a(S) Microcystins Paralytic shellfish toxins... 

Onodera H, Oshima Y, Henriksen P, Yasumoto T (1997) Confirmation of anatoxin-a(s) in the cyanobacterium Anabaena lemmermanni as the cause of bird kills in Danish lakes. Toxicon 35 1645-1648... 

Two other suspected alkaloid producing cyanobacteria strains, Anabaena flos-aquae NRC-525-17 and Aphanizomenon flos-aquae NH-5, are now being studied. The toxin of flos-aquae NRC-525-17 (anatoxin-a(s)) is thought to have CNS stimulating properties (7) and that of Aph. flos-aquae NH-5 (aphantoxin) is thought to produce the paralytic shellfish poisons saxitoxin and neosaxitoxin (Fig. 1)... 

In our laboratory crude preparations of aphantoxins and anatoxin-a(s) are extracted similarly except at the final stages of purification (Fig. 2). A Bio-gel P-2 column (2.2 x 80 cm) is used for aphantoxins gel filtration and a Sephadex G-15 (2.6 x 42 cm) column for ana-toxin-(s). Both toxins are eluted with 0.1 M acetic acid at 1.5 ml/ min. Fractions of aphantoxins from Bio-gel P-2 run are spotted on thin-layer chromatography plates (Silica gel-60, EM reagents) and developed according to Buckley et al. (1976) (31). The Rf values for the aphantoxins, saxitoxin and neosaxitoxin standards (Table 1) indicates that two of the aphantoxins (i.e. I and II) are similar to saxitoxin and neosaxitoxin. 

Figure 2. Flow diagram for the extraction of Anatoxin-a(s) and Aphantoxins.

Figure 5. HPLC profile of Anatoxin-a(s) toxic peak (far-left ...

Fig. 16.2. Chemical structure of anatoxin-a(s). This image is licensed under the http //www.gnu.org/copyleft/fdl.html GNU Free Documentation License. It uses material from the http //en.wikipedia.org/wiki/Cyanotoxin Wikipedia article.

Anatoxin-a(s) is a phosphate ester of a cyclic iV-hydroxyguanidine (Fig. 16.2) [5]. It is the only natural organophosphate known and, as the synthetic parathion and malathion, irreversibly inhibits acetylcholinesterase. When this enzyme is inhibited, acetylcholine is no longer hydrolysed, the postsynaptic membrane cannot be repolarised, the nerve influx is blocked and the muscle cannot be contracted. 

In order to guarantee the water and food quality and to preserve the human health, several detection and quantification methods, each one of them having advantages and disadvantages, have been developed for microcystins and anatoxin-a(s). 

Anatoxin-a(s) was first detected by the mouse bioassay. However, the technique most commonly used for its detection is HPLC, coupled to MS detection. The irreversible inhibitory power of this toxin towards acetylcholinesterase has been described [60] and the corresponding colorimetric inhibition assay has also been developed [61-63]. To date, no antibodies towards anatoxin-a(s) have been produced. 

Our research group is working on the development of electrochemical biosensors for the detection of microcystin and anatoxin-a(s), based on the inhibition of protein phosphatase and acetylcholinesterase, respectively. These enzyme biosensors represent useful bioanalytical tools, suitable to be used as screening techniques for the preliminary yes/no detection of the toxicity of a sample. Additionally, due to the versatility of the electrochemical approach, the strategy can be applied to the detection of other cyanobacterial toxins. 

Acetylcholinesterase-based biosensor for electrochemical anatoxin-a(s) detection... 

Acetylcholinesterase inhibition has been widely used for pesticide detection [88-94], but less exploited than protein phosphatase inhibition for cyanobacterial toxin detection. Nevertheless, the anatoxin-a(s) has more inhibition power than most insecticides, as demonstrated by the higher inhibition rates [95]. In order to detect toxin concentrations smaller than usually, mutant enzymes with increased sensitivity were obtained by genetic engineering strategies residue replacement, deletion, insertion and combination of mutations. Modifications close to the active site, located at the bottom of a narrow gorge, made the entrance of the toxin easier and enhanced the sensitivity of the enzyme. 

The main drawback of acetylcholinesterase-based biosensors is the lack of selectivity because, as we mentioned, this enzyme is inhibited not only by anatoxin-a(s) but also by insecticides such as organ-ophosphorates and carbamates. This problem can be overcome by the choice of specific mutant enzymes. The combined use of mutants highly sensitive to anatoxin-a(s) and resistant to most insecticides and vice versa allows us to unambiguously discriminate between the cyanobacterial toxin and insecticides. 

It is also important to mention the use of the reactivation of the acetylcholinesterase by pyridine-2-aldoxime methochloride to discriminate between the toxin and potential insecticides [96]. Once phos-phorylated, the active site serine of the enzyme can be reactivated by powerful nucleophilic agents such as oximes. However, this reactivation is not possible if attempted too late due to the stable adduct formed by the dealkylation (aging) of the inhibitor s remaining group. When acetylcholinesterase is inhibited by anatoxin-a(s), it shows immediately the characteristics of an aged enzyme and cannot be reactivated. In this way, it is possible to distinguish between the inhibition caused by anatoxin-a(s) and the one provoked by other insecticides. 

When the amperometric biosensor was used, results correlated with those obtained by colorimetric methods. The two sensitive enzymes allowed the detection of 0.5 nM anatoxin-a(s) and the limits of detection obtained with the two insensitive mutants were 16- and 50-fold higher than those obtained with the sensitive ones. 

The developed biosensor was applied to the analysis of cyanobacterial bloom samples from freshwater lakes of Spain, Greece, France, Scotland and Denmark. Two samples from Scotland and one from Denmark irreversibly inhibit the acetylcholinesterase. The estimated concentrations were between 1.5 and 30nmol/g of dry weight, values extremely high when compared to the intraperitoneal 50% lethal dose of anatoxin-a(s) in mice (121 nmol/kg). 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

P. Henriksen, W.W. Carmichael, J.S. An and 0. Moestrup, Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds, Toxicon, 35 (1997) 901-913. 

E. Devic, D. Li, A. Dauta, P. Henriksen, G.A. Codd, J.-L. Marty and D. Fournier, Detection of anatoxin-a(s) in environmental samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases, Appl. Environ. Microbiol., 68 (2002) 4102 4106. 

F. Villate, H. Schulze, R.D. Schmid and T.T. Bachmann, A disposable acetylcholinesterase-based electrode biosensor to detect anatoxin-a(s) in water,Anal. Bioanal. Chem., 372 (2002) 322-326. 

Ammonia 368, 540 gas sensing electrodes 366 Ammonium 540-541 Amorphous silicon (a-Si) 99 Amperometric 907, 913 biosensors 359 detection 876, ell9 no sensor 930 oxygen electrodes 915 ultramicroelectrode 907 Amyloglucosidase 676 Analytical approximation 912 Anatoxin-a(s) 335 Aniline 985... 

Anatoxin-a(s)] Anabaena flos-aquae AChE (forms covalent... 

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