anatoxin-a(s)- natural organophosphorus anticholinesterase agent

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A rewiew about Natural Organophosphorus Anticholinesterase Agent - Anatoxin-A(s)


  • Mil. Med. Sci. Lett. (Voj. Zdrav. Listy) 2011, vol. 80, p. 129-139ISSN 0372-7025




    Jiri Patocka1....., Ramesh C. Gupta2, Kamil Kuca3

    1 Department of Radiology and Toxicology, Faculty of Health and Social Studies, University of South Bohemiaesk Budjovice, esk Budjovice, Czech Republic2 Breathitt Veterinary Center, Toxicology Department, Murray State University, Hopkinsville, KY, USA3 Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec Krlov, CzechRepublic

    Received 1th July 2011.Revised 22th August 2011.Published 9th September 2011.


    Anatoxin-a(s) is a guanidinemethyl phosphate ester isolated from the freshwater cyanobacterium (blue-green algae) Anabaena flos-aquae strain NRC 52517. Previous work has shown anatoxin-a(s) to be a potentirreversible inhibitor of electric eel acetylcholinesterase (AChE, EC Anatoxin-a(s) has been shownto be an active site-directed inhibitor of AChE, which is resistant to reactivation by oximes because of theenzyme-oxime adduct formation. In vivo pretreatment with physostigmine and high concentrations ofpyridine 2-aldoxime methochloride (2-PAM) were the only effective antagonists against a lethal dose ofanatoxin-a(s). Anatoxin-a(s) is very toxic and it is produced by cyanobacteria during its blooms. Purifiedtoxin has an LD50 (i.p) of approximately 20-50 g/kg body weight in mice. Toxicoses associated withcholinesterase-inhibiting anatoxin-a(s) have been observed in humans, animals, birds and fish. Anatoxin-a(s) induces clinical signs of hypercholinergic preponderance, such as salivation, lacrimation, urinaryincontinence, defecation, convulsion, fasciculation, and respiratory arrest.

    Key words: natural organophosphate; anatoxin-a(s); anticholinesterase; chemistry; pharmacology;

    toxicology; blue-green algae; neurotoxin


    Acetylcholinesterase inhibiting (anti-ChE)organophosphorus compounds(OP) are a group

    of chemicals such as nerve agents andpesticides that are synthesized and used forvarious purposes, including human andveterinary medicine (Bajgar, 2004; Gupta,2006, 2009; Satoh and Gupta, 2010). OPs aredenoted with a general formula (Figure 1),proposed by Gerhard Schrader in 1937.Basically, these OPs with insecticidal propertiesare esters of phosphoric acid (Figure 1,Schrader, 1950). This implies that a biologicallyactive OP compound contains an oxygen orsulphur, two similar or dissimilar side chains

    University of South Bohemia eskBudjovice, Faculty of Health and SocialStudies, Department of Radiology andToxicology, Matice kolsk 17,370 01 esk Budjovice, Czech

  • bound to the phosphorus atom, and an organicor inorganic acid residue (leaving group, x):

    R1 and R2 are capable of almost infinitevariation. They may represent alcohols,phenols, thiols, amides, alkyl or aryl groups

    Patocka et al.: Anatoxin-a(s): Natural organophosphorus anticholinesterase agent


    Anatoxin a(s) A natural organophosphate

    Cyanobacteria, formerly called "blue-greenalgae", are simple and primitive photosyntheticmicroorganisms, which widely occur in fresh,brackish and salt waters. George Francis firstreported a toxigenic bloom in Nature in 1878, a"poisonous Australian lake" with "a thick scumlike green oil paint" and vividly described acuteintoxications of sheep, horses, dogs, and pigs(Puschner and Roegner, in press). Analysis ofarchaeological evidence coupled with anevolving understanding of modern blooms havebegun to implicate the role of cyanotoxinpoisoning to more widespread mammaliandie -offs dating back to the Pleistocene age, i.e.about 150,000 years BC (Braun and Pfeiffer,2002). A controversial hypothesis about the roleof cyanobacteria in the various mass extinctionevents has begun to emerge (Castle and Rodgers,2009). By now, 40 different genera and 2000species of cyanobacteria are known and 80species of them are known to produce potenttoxins responsible for a wide array of humanillnesses, aquatic mammal and bird morbidityand mortality, and extensive fish kills. Thesecyanotoxins act as neurotoxins or hepatotoxinsand are structurally and functionally diverse,and many are derived from unique biosyntheticpathways (Patocka, 2001; van Apeldoorn et al.,2007; Aroz et al., 2010; Humbert, 2009:Puschner and Roegner, 2012).

    Figure 1. Common Structure of Organophosporous Compounds

    attached directly to phosphorus. A common Xradical may be halogene, cyanide, thiocyanate,etc. An OP compound becomes a strong AChEinhibitor and consequently more toxic when Sis replaced by O.

    Anatoxins are mainly produced bycyanobacteria in the Anabaena genus (Beltranand Neilan, 2000), but also by other genera, suchas Plantkothrix, Oscillatoria, Microcystis,Aphanizomenon, Cylindorspermum, andPhormidium (Sivonen and Jones, 1999).Anatoxins are neurotoxins and can be dividedinto anatoxin-a, homoanatoxin-a, and anatoxin-a(s). Anatoxin-a(s) is a natural OP, which has aunique N-hydroxyguanidine methylphosphateester with a chemical structure (CAS RegistryNo. 103170-78-1,(5S)-2-amino-1-((hydroxy-methoxypho-sphinyl) oxy)-N,N-dimethyl-4,5-dihydro-1H-imida-zole-5-methanamin; Mol Wt., 252daltons) as shown in Figure 2. Anatoxin-a(s) hasbeen commonly detected in the United States(Monserrat et al., 2001) and Europe (Henriksenet al., 1997). Anatoxin-a(s) acts as a non-competitive potent irreversible inhibitor of AChEactivity, preventing it from hydrolyzing theneurotransmitter acetylcholine (Mahmood andCarmichael, 1986). The letter "s" corresponds tothe characteristic symptom of salivation causedin vertebrates, mainly in doses close to LD50levels of 20-40 g/kg (Matsunaga et al., 1989;Falconer, 1998). As a consequence, acetylcholine(ACh) remains available to bind membranereceptors, resulting in continuous musclestimulation. When respiratory muscles are alsoaffected, death may ensue by respiratory failureand brain hypoxia (Matsunaga et al., 1989,Carmichael and Falconer, 1993).

  • Isolation

    Anatoxin-a(s) is produced by Anabaena flos-aquae clone NRC 525-17 and can be purifiedfrom lyophilized cells. Purification procedureinvolved extraction with 1.0 M acetic acid:ethanol (80:20), column chromatography(Sephadex G-15 and CM-Sephadex C-25) andhigh performance liquid chromatography.Purified toxin has a repored LD50 (i.p) ofapproximately 20-50 g/kg body weight in mice(Mahmood and Carmichael, 1986; Falconer,1998).

    Synthesis and Biosynthesis

    The alkyl chain of anatoxin-a(s) (cyclicguanidines), which can be used as an intermediatein the total synthesis of anatoxin-a(s), wassynthesized recently in both racemic andenantiomerically pure forms (Moura and Pinto,2010). C-2, C-4, C-5 and C-6 of the anatoxin-a(s) are derived from the guanido carbon, C-5,C-4 and C-3 of L-arginine, respectively, and thethree O,N-methyl groups originate from thetetrahydrofolate C1 pool. Erythro-4-Hydroxy-l-arginine, a minor constituent of the cyanophyte,may be a precursor in the biosynthesis (Moore etal., 1992).

    Anticholinesterase properties

    The in vitro inhibition of electric eel acetyl-cholinesterase (AChE, E.C. and horse serumbutyrylcholinesterase (BuChE, E.C. by

    anatoxin-a(s) was time- and concentration-dependent. The inhibition of electric eel AChEfollows first order kinetics, indicative of irreversibleinhibition. The irreversibility of electric eel AChEinhibition was confirmed by a plot of Vmax versustotal enzyme concentration (Mahmood andCarmichael, 1987).

    The kinetics of inhibition of ChE by anatoxin-a(s) supports the theory that signs of intoxication areprimarily due to ChE inhibition. Assays of serumChE of rats in an acute toxicity study showed acomplete inactivation of the enzyme at doses of 350and 600 g anatoxin-a(s)/kg body weight. It wasconcluded that anatoxin-a(s) may be acting as ananti-ChE, thereby causing toxicity (Mahmood andCarmichael, 1987). Cook et al. (1988) comparedanatoxin-a(s) to paraoxon, physostigmine andpyridostigmine for effects on brain ChE after i.p.injection into mice. The duration of clinical signs inmice injected with anatoxin-a(s) was comparable tothat of paraoxon and sign persisted longer than withcarbamates. Anatoxin-a(s) did not inhibit brain ChEactivity, suggesting that this toxin is unable to crossthe blood-brain barrier. Experiments of Cook and hisco-workers (1989a) validated that anatoxin-a(s) isstrictly a peripheral ChE inhibitor. When rats wereinjected (i.p) with 1.5, 3.0 or 9.0 g/kg of anatoxin-a(s) or with 800 g/kg of paraoxon, unlike paraoxon,anatoxin-a(s) did not cause detectable inhibition ofChE in the central nervous system, but did causeinhibition of ChE in blood.

    Mechanism of action

    Hyde and Carmichael (1991) investigated theinteraction of anatoxin-a(s) with AChE and


    Patocka et al.: Anatoxin-a(s): Natural organophosphorus anticholinesterase agent

    Figure 2. Chemical structure of anatoxin-a(s).

  • suggested that this natural OP is an irreversibleactive-site directed inhibitor of AChE. Somesimilarities have been noted between anatoxin-a(s) and the synthetic anti-ChE OP. Thereversibility of cholinesterase inhibition inplasma, red blood cells, and diaphragm in micegiven anatoxin-a(s) was characterized by Cookand his co-workers (1991) and was comparedwith the effects of two known ChE inhibitors, theOP compound paraoxon and the carbamatecompound pyridostigmine bromide. The timerequired for recovery from anatoxin-a(s)-inducedinhibition of ChE in plasma, red blood cells, anddiaphragm was similar to or longer than that withparaoxon and longer than that withpyridostigmine. Based on the duration ofanatoxin-a(s) induced clinical signs and ChEinhibition in mice, anatoxin-a(s) appears to be anin vivo irreversible


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