Abstract
Guanitoxin (GNT) is a potent neurotoxin produced by freshwater cyanobacteria that can cause the deaths of wild and domestic animals. Through reports of animal intoxication by cyanobacteria cells that produce GNT, this study aimed to investigate the bio-accessibility of GNT in simulated solutions of the gastrointestinal content in order to understand the process of toxicosis promoted by GNT in vivo. Dissolution tests were conducted with a mixture of Sphaerospermopsis torques-reginae (Cyanobacteria; Nostocales) cultures (30%) and gastrointestinal solutions with and without proteolytic enzymes (70%) at a temperature of 37 °C and rotation at 100 rpm for 2 h. The identification of GNT was performed by LC-QqQ-MS/MS through the transitions [M + H]+ m/z 253 > 58 and [M + H]+ m/z 253 > 159, which showed high concentrations of GNT in simulated gastric fluid solutions (p-value < 0.001) in comparison to simulated solutions of intestinal content. The gastric solution with pepsin promoted the stability of GNT (p-value < 0.05) compared to the simulated solution of gastric fluid at the same pH without the enzyme. However, the results showed that GNT is also available in intestinal fluids for a period of 2 h, and solutions containing the pancreatin enzyme influenced the bio-accessibility of the toxin more compared to the intestinal medium without enzyme (p-value < 0.05). Therefore, the bio-accessibility of the toxin must be considered both in the stomach and in the intestine, and may help in the diagnosis and prediction of exposure and risk in vivo through the oral ingestion of GNT-producing cyanobacteria cells.
Highlights
Guanitoxin (GNT) [1] (formerly Anatoxin-a(s)) is a potent natural neurotoxin produced by freshwater cyanobacteria [2,3]
The results presented in this study came from cultures of Sphaerospermopsis torques-reginae (ITEP-24), with a cell concentration of 3.29 × 106
The bio-accessibility of the toxin was measured by high-performance liquid chromatography-tandem triple-quadrupole mass spectrometry (HPLC-QqQ-MS/MS) with multiple reaction monitoring (MRM) using the transitions [M + H] + m/z 253 > 58 and m/z 253 > 159 (Scheme 1)
Summary
Guanitoxin (GNT) [1] (formerly Anatoxin-a(s)) is a potent natural neurotoxin produced by freshwater cyanobacteria [2,3]. The result of the irreversible inactivation of AChE is the accumulation of acetylcholine in the synaptic clefts, causing cholinergic hyperstimulation that, in most cases, is lethal for organisms [6,7,8]. GNT has been associated with the death of domestic and wild animals that accidentally consumed water containing cyanobacterial cells [5,6,8,9]. The lethal dose (LD50) of GNT was determined in mice to comprise a range from 20 μg/kg to 40 μg/kg, with a survival time of 10 to 30 min, and it is considered ten times more toxic than other cyanotoxins of the same class [3,5,10]. Other LD50 and inhibitory concentration (IC50) values were observed in fish, Cladocera, and insects, showing symptoms of intoxication common to those observed in mammals [11,12,13,14,15]
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