Detection of Anatoxin-a and Three Analogs in Anabaena spp. Cultures: New Fluorescence Polarization Assay and Toxin Profile by LC-MS/MS

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.

Detection of Cyanotoxins in Algae Dietary Supplements

“…For E-ANA-a, the product ions were m/z 182.1178 > 98.0966, 122.0964, and 164.1068. These masses are in accordance with the results presented in previous studies which were obtained using reference standards and LC-MS/MS detection [16,47]. Moreover, to ensure proper identification, the structures were searched via the Mass Frontier TM software using their exact mass and are illustrated in Figure 4.…”

Monitoring of freshwater toxins in European environmental waters by using novel multi‐detection methods

“…Chromatographic separation was performed by gradient elution (12.5 min) starting with 2% of B for 4 min; then 70% B was held for 1 min, reduced to 2% B over 0.5 min, and held for 3 min until the next run. Given that the fragmentation pathway of each molecule is a tool often used to identify and confirm the presence of ATX-a analogues [33,47,48]. The electrospray ionization source of the 8040 mass spectrometer was operated with the following optimized values of source-dependent parameters: nebulizing gas flow, 2 L/min; Desolvation Line (DL) temperature, 300 8C; heat block temperature, 500 8C; and drying gas flow, 15 L/min.…”

An overview of diversity, occurrence, genetics and toxin production of bloom-forming Dolichospermum (Anabaena) species

“…The Dolichospermum species are able to produce various kinds of secondary metabolites, notably toxins; about 25% to 75% of cyanobacterial blooms are toxic in natural waters (Chorus and Bartram, 1999). The toxins can be classified into three different categories by chemical structure: cyclic peptides (microcystin), alkaloids (anatoxin-a, anatoxin-a (S), saxitoxin, and cylindrospermopsin) and lipopolysaccharides (LPS); and four groups according to the target organs: neurotoxins (anatoxin-a, anatoxin-a (S) and saxitoxin), hepatotoxins (microcystin), cytotoxins (cylindrospermopsin), and dermatotoxins (LPS) (Krishnamurthy et al, 1986;Chorus and Bartram, 1999;Stü ken et al, 2009;Al-Tebrineh et al, 2010;Ž egura et al, 2011;Singh and Dhar, 2013;Akcaalan et al, 2014;Corbel et al, 2014;Sanchez et al, 2014). Toxins are synthesized at all stages of cyanobacterial growth and remain mostly in the cell until their release into waters after cell lysis (Sivonen and Jones, 1999).…”

“…Under favorable conditions, Dolichospermum blooms form potential threats to the environment and human health due to their production of various kinds of potent toxins. The reported toxins produced by these species can be categorized by chemical structure into three different groups: cyclic peptides (microcystin), alkaloids (anatoxin-a, anatoxin-a(S), saxitoxin, and cylindrospermopsin) (Carmichael, 1986;Chorus and Bartram, 1999;Stü ken et al, 2009;AlTebrineh et al, 2010;Žegura et al, 2011;Singh and Dhar, 2013;Akcaalan et al, 2014;Corbel et al, 2014;Sanchez et al, 2014) and lipopolysaccharide. In recent decades, eutrophication in waters from anthropogenic activities, combined with global warming, have promoted Dolichospermum blooms to expand both in size and duration (Singh et al, 2010Koreivienė and Kasperovičienė , 2011;Huber et al, 2012;O'Neil et al, 2012;Ciré s et al, 2013;Slim et al, 2014).…”