Anatoxin-a Research Articles

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of anatoxins in cyanobacteria and drinking water.

Anatoxin-a (AN) and homoanatoxin-a (HMAN) are potent neurotoxins produced by a number of cyanobacterial species. A new, sensitive liquid chromatography/multiple tandem mass spectrometry (LC/MS(n)) method has been developed for the determination of these neurotoxins. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer in positive ion mode. The [M+H](+) ions at m/z 166 (anatoxin-a) and m/z 180 (homoanatoxin-a) were used as the precursor ions for multiple MS experiments. MS(2)bond;MS(4) spectra displayed major fragment ions at m/z 149 (AN), 163 (HMAN), assigned to [Mbond;NH(3)+H](+); m/z 131 (AN), 145 (HMAN), assigned to [Mbond;NH(3)bond;H(2)O+H](+), and m/z 91 [C(7)H(7)](+). Although the chromatographic separation of these neurotoxins is problematic, reversed-phase LC, using a C(18) Luna column, proved successful. Calibration data for anatoxin-a using spiked water samples (10 mL) in LC/MS(n) modes were: LC/MS (25-1000 microg/L), r(2) = 0.998; LC/MS(2) (5-1000(microg/L), r(2) = 0.9993; LC/MS(3) (2.5-1000 microg/L), r(2) = 0.9997. Reproducibility data (% RSD, N = 3) for each LC/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L. The detection limit (S/N = 3) for AN was better than 0.03 ng (on-column) for LC/MS(3) which corresponded to 0.6 microg/L.

P3C104 - Induction of anti-malondialdehyde antibodies in dichloroacetyl chloride-treated female MRL +/+ mice

A solid-phase extraction (SPE)–liquid chromatography (LC)–mass spectrometry (MS) method was developed to concentrate and detect nine cyanotoxins simultaneously, including six microcystins (MCs) congeners, nodularin (NOD), anatoxin-a (ATX) and cylindrospermopsin (CYN), in pure and natural waters. A dual cartridge SPE assembly was tested for the operating parameters of cyanotoxin extraction. A surrogate standard (SS), 1,9-diaminononane, was spiked in all the samples before the SPE extraction, and an internal standard (IS), 2,3,5-trimethylphenyl methyl carbamate, was spiked before LC/MS analysis. The method detection limit (MDL) was 2–100 ng/L for nine cyanotoxins in pure water and was increased by a factor of three to ten in a more complicated water matrix. The recoveries based on SS were between 83 and 104%, while those based on IS were 80–120%. The developed method was successfully employed in analyzing 33 water samples collected from eutrophic lakes, water treatment plants and distribution taps. MCs, NOD, and CYN were detected in the reservoir water, with concentrations as high as 36 μg/L. In addition, for the first time in Taiwan’s tap water, CYN was detected at concentrations as high as 8.6 μg/L. Quality control data for the field samples shows that the analytical scheme developed is appropriate for monitoring cyanotoxins.

Sensitive determination of anatoxin-a, homoanatoxin-a and their degradation products by liquid chromatography with fluorimetric detection

Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC–FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2–84.9% at concentrations of 10 μg/l. The R.S.D. values were 1.7–3.9% ( n=8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.

Quantitative Structure-activity Relationships Of (+)-anatoxin-a Derivatives

Abstract The QSAR of a series of 21 nicotinic agonists, anatoxin-a (AnTX) and derivatives, was investigated using multivariate statistical techniques. Physicochemical properties were calculated for the compounds using computational chemistry. In order to model the structures it was necessary to choose a common conformation. This was achieved by comparison of the structure of AnTX to that of (-)-cytisine, a rigid potent nicotinic agonist. The final data set of 32 molecular descriptors was examined using principal components analysis and non-linear mapping. Examination of the plots produced by these techniques demonstrated significant clustering of both chemical and activity classes. The inclusion of 6 test compounds to these models resulted in satisfactory predictions of their activity for 5 of these compounds.

Conformational studies on (+)-anatoxin-a and derivatives.

Anatoxin-a (AnTX) is a highly potent agonist acting at the nicotinic acetylcholine receptor (nAChR) and represents a valuable tool in the study of this receptor. Molecular mechanics, semi-empirical and ab initio molecular orbital energy minimization procedures were conducted to investigate the conformation of AnTX. For each minimization procedure, the s-trans enone isomer of protonated AnTX was the energetically favoured conformer due to intramolecular electrostatic interactions. Our studies are discussed in the light of previous experimental observations and conformational studies, in addition to their importance in the development of future pharmacophore models for nAChR agonist binding.

Postganglionic nicotinic action of anatoxin-a on guinea pig ileum antagonized by thymopentin.

Anatoxin-a (ANTX) is a bicyclic amine exotoxin produced by the blue green algae Anabaena flos-aquae, and has been found to cause death to livestock and waterfowl via a blockade of neuromuscular transmission and subsequent respiratory paralysis (1, 2). ANTX has been shown to be a potent nicotinic cholinergic agonist in skeletal muscle (3), Torpedo electric organ (4) and mammalian central nervous system (5), and can also induce cardiovascular effects by activating nicotinic receptors in the adrenal medulla and sympathetic ganglia (6). We studied the effect of ANTX on guinea pig ileum.KeywordsBlue Green AlgaNicotinic ReceptorAdrenal MedullaSympathetic GanglionNeuromuscular TransmissionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Nicotinic action of anatoxin-a on guinea pig ileum antagonized by thymopentin

(+)-Anatoxin-a (ANTX) stimulated guinea pig ileum contraction with a potency similar to that of acetylcholine (ACh); the stimulation was blocked by tubocurarine, hexamethonium, or atropine. Although the contraction stimulated by ANTX was blocked by atropine, no specific inhibition of the binding of [ 3H]N-methysscopolamine to ileum membranes was observed in the presence of ANTX. Furthermore, ANTX failed to stimulate the secretion of α-amylase from pancreatic acinar cells, a process that is activated by cholinergic agonists at the muscarinic receptors. When the ileum itself was stimulated by ACh, the contraction was not blocked by either hexamethonium or tubocurarine. Preincubation of the ileum with hemicholinium caused a 50% reduction in the ability of ANTX to stimulate contraction. Based upon these data, it was inferred that ANTX binds to postganglionic synaptic nicotinic receptors in the ileum, thus releasing endogenous ACh, which in turn causes ileum contraction by interacting with postsynaptic muscarinic receptors. It was also observed that thymopentin (TP-5), a pentapeptide corresponding to positions 32–36 of thymopoietin, blocked the s stimulation of ileum contraction by ANTX.

Facile detection of anatoxin-a in algal material by thin-layer chromatography with Fast Black K salt

A method for facile high-capacity screening of algal samples for anatoxin-a (ANTX-a), a potent neurotoxin of Anabaena flos-aquae, is presented. The method is based on in situ colour reaction of algal extracts containing ANTX-a on a thin-layer chromatographic plate with the diazonium reagent Fast Black K salt, and subsequent separation of the orange-red product. The product, shown to be a stable 3,3-dialkyltriazene, is derived from a reaction involving the aliphatic secondary amino group of ANTX-a. The detection limit for ANTX-a is 10 micrograms g-1 of lyophilized algal material, which is comparable to earlier methods using more complex instrumentation.

Effect of route of exposure and repeated doses on the acute toxicity in mice of the cyanobacterial nicotinic alkaloid anatoxin-a

Anatoxin-a (ANTX-a) is a potent nicotinic cholinergic alkaloid produced by some toxigenic strains of cyanobacteria. We have examined i.p. and oral ld 50 values of two ANTX-a producing Anabaena sp. and synthetic (+)-ANTX-a in outbred Swiss Webster mice. All three appeared equivalent by i.p. injection, but Anabaena flos-aquae NRC-44-1 was approximately twice as potent orally as the other two. Additionally, we were unable to demonstrate development of any biologically significant tolerance to acute ANTX-a exposure using two different repeated dose regimens.

Stability studies on the cyanobacterial nicotinic alkaloid snatoxin-A

Anatoxin-a (ANTX-a) is a potent nicotinic cholinergic bicyclic secondary amine produced by some toxigenic strains of Anabaena spp. cyanobacteria ( LD 50 = 0.20–0.25 mg/kg i.p., mouse). Studies were undertaken to examine the effects of sunlight, pH, oxygen, and copper and iron, known catalysts of nitrogen oxidation, on the stability of ANTX-a. Photolysis of this extremely potent toxin was demonstrated under conditions resembling those which occur naturally. First-order decay kinetics of ANTX-a in sunlight was both pH and light intensity dependent. In the solutions examined, which represented expected biological conditions, the half-life of ANTX-a was on the order of 1–2 hr. This compares to half-lives on the order of several days in the absence of sunlight, even in the presence of metal ions. Mouse bioassays indicate that the breakdown products of ANTX-a, by both mechanisms, are inactive. Sunlight photolysis is concluded to be a possible important detoxification route of ANTX-a.

A new procedure for the analysis and purification of naturally occurring anatoxin-a from the blue-green alga Anabaena flos-aquae

K. Harada, Y. Kimura, K. Ogawa, M. Suzuki, A. M. Dahlem, V. R. Beasley and W. W. Carmichael. A new procedure for the analysis and purification of naturally occurring anatoxin-a from the blue-green alga Anabaena flos-aquae. Toxicon 27, 1289–1296, 1989.— Anabaena flos-aquae produces at least two neurotoxins termed anatoxin-a (ANTX-a) and -a(s) [ANTX-a(s)]. ANTX-a is a potent postsynaptic depolarizing neuromuscular blocking agent. Its structure is the secondary amine, 2-acetyl-9-azabicyclo[4.2.1]non-2-ene. Known isolation and analysis methods have not always given satisfactory results. Therefore, an improved procedure was developed for both isolation and analysis of ANTX-a which involves four steps; extraction, clean-up, separation and determination. Extraction was efficiently done with 0.05 M acetic acid, and a reversed phase, ODS (octadecylsilanized) and a cation exchanger (COOH) organosilans bonded to silica gels were applied to a clean-up step. Separation and determination were then performed using two TLC and HPLC chromatographies.

Analysis of anatoxin-a by GC/ECD.

Anatoxin-a (ANTX-a), a potent cholinergic alkaloid produced by some toxigenic strains of the cyanobacterium Anabaena flos-aquae, has been responsible for death of livestock, pets, and wildlife. An increase in the frequency of cyanobacterial blooms in fresh waters of temperate North America has produced a concomitant increase in the threat posed by toxic strains of some of these microorganisms. Presently, a mouse bioassay is the general procedure used for testing the toxicity of a bloom-approximately 5 micrograms/mL ANTX-a sensitivity. A method that is over three orders of magnitude more sensitive than that bioassay is described here. It is readily capable of detecting and quantitating sublethal levels of ANTX-a. ANTX-a is isolated via solid-phase extraction (SPE), derivatized with trichloracetic anhydride, isolated a second time via SPE, and analyzed by GC/ECD. Sensitivity to 5 ng is achieved. Sample size can be as large as 10 mL. "Nontoxic" samples are shown to contain ANTX-a.

Pharmacology of Anatoxin-a, produced by the freshwater cyanophyte Anabaena flos-aquae NRC-44-1

Anatoxin-a (ANTX-A) is the neuromuscular blocking agent produced by the freshwater cyanophyte Anabaena flos-aquae (Lyngb.) de Bréb. strain NRC-44-1. Its pharmacological properties have been investigated and compared with that of synthetic ANTX-A which was derived from l-cocaine. ANTX-A is a potent depolarizing neuromuscular blocking agent possessing both muscarinic and nicotinic activity. Responses to ANTX-A on the isolated frog-rectus abdominus muscle were qualitatively similar to those obtained with acetylcholine, carbachol and decamethonium, with ANTX-A being the most potent. Neostigmine and tetrodotoxin failed to affect responses to ANTX-A. The affinity constant for ANTX-A on the frog-rectus preparation was 3·1 μM, compared to 1·0 μM for carbachol. On the chick biventer cervicis preparation ANTX-A was less potent but produced responses similar to that of decamethonium. d-Tubocurarine and hexamethonium caused a shift to the right in the dose-response curves of ANTX-A and decamethonium. On the isolated rat phrenic nerve-hemidiaphragm ANTX-A and decamethonium blocked the muscle twitch response which was not reversed by neostigmine. ANTX-A produced a depolarizing blockade on the cat sciatic nerve-anterior tibialis preparation which was similar but less than that produced by decamethonium. On the isolated guinea pig ileum, ANTX-A had a potency approximately the same as that of 1,1-dimethyl-4-phenyl piperazinium but less than that of acetylcholine.