Abstract
Anatoxin-a (ATX) is a potent neurotoxin produced by several species of Anabaena spp. Cyanobacteria blooms around the world have been increasing in recent years; therefore, it is urgent to develop sensitive techniques that unequivocally confirm the presence of these toxins in fresh water and cyanobacterial samples. In addition, the identification of different ATX analogues is essential to later determine its toxicity. In this paper we designed a fluorescent polarization (FP) method to detect ATXs in water samples. A nicotinic acetylcholine receptor (nAChR) labeled with a fluorescein derivative was used to develop this assay. Data showed a direct relationship between the amount of toxin in a sample and the changes in the polarization degree of the emitted light by the labeled nAChR, indicating an interaction between the two molecules. This method was used to measure the amount of ATX in three Anabaena spp. cultures. Results indicate that it is a good method to show ATXs presence in algal samples. In order to check the toxin profile of Anabaena cultures a LC-MS/MS method was also developed. Within this new method, ATX-a, retention time (RT) 5 min, and three other molecules with a mass m/z 180.1 eluting at 4.14 min, 5.90 min and 7.14 min with MS/MS spectra characteristic of ATX toxin group not previously identified were detected in the Anabaena spp. cultures. These ATX analogues may have an important role in the toxicity of the sample.
Highlights
IntroductionATX is a potent neurotoxic alkaloid produced by the cyanobacterium Anabaena flos-aquae
Freshwater toxins can be divided into three groups including microcystins and nodularin that produces hepatotoxic effects, anatoxin-a (ATX), homoanatoxin-a (HATX) and saxitoxins with neurotoxic effects and cylindrospermopsin that inhibits protein synthesis and induce oxidative stress [1].ATX is a potent neurotoxic alkaloid produced by the cyanobacterium Anabaena flos-aquae. other species like Anabaena planctonica, Oscillatoria acuminata, Aphanizomenon gracile and Cylindrospermun stagnale were described as ATX producers [2,3]
The toxin-receptor interactions were measured as the fluorescence polarization (FP) variation of a membrane-nicotinic acetylcholine receptors (nAChR)-F conjugate in the presence of different concentrations of ATX
Summary
ATX is a potent neurotoxic alkaloid produced by the cyanobacterium Anabaena flos-aquae. Other species like Anabaena planctonica, Oscillatoria acuminata, Aphanizomenon gracile and Cylindrospermun stagnale were described as ATX producers [2,3]. ATX (Figure 1) has a semi-rigid bicyclic secondary amine structure, 2-acetyl-9-azabicyclo[4:2:1]non-2-ene (C10H15NO). Both ATX and HATX produce rapid death of animals by respiratory paralysis and acute asphyxia, since these alkaloids are potent agonists of nicotinic acetylcholine receptors (nAChR) [4,5,6,7]. Besides the acute mouse death, under a high dose, ATX can act as a tumor promoter, and may cause cytotoxic or teratogenic effects after continuous low dose intake [9]
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