In vivo effects of Aphanizomenon flos-aquae DC-1 aphantoxins on gas exchange and ion equilibrium in the zebrafish gill

Aphantoxins, neurotoxins or paralytic shellfish poisons (PSPs) generated by Aphanizomenon flos-aquae, are a threat to environmental safety and human health in eutrophic waters worldwide. The molecular mechanisms of neurotoxin function have been studied; however, the effects of these neurotoxins on oxidative stress, ion transport, gas exchange, and branchial ultrastructure in fish gills are not fully understood. Aphantoxins extracted from A. flos-aquae DC-1 were detected by high-performance liquid chromatography. The major ingredients were gonyautoxins 1 and 5 and neosaxitoxin, which comprised 34.04%, 21.28%, and 12.77% of the total, respectively. Zebrafish (Danio rerio) were administered A. flos-aquae DC-1 aphantoxins at 5.3 or 7.61 μg saxitoxin equivalents (eq)/kg (low and high doses, respectively) by intraperitoneal injection. The activities of Na+-K+-ATPase (NKA), carbonic anhydrase (CA), and lactate dehydrogenase (LDH), ultrastructural alterations in chloride and epithelial cells, and reactive oxygen species (ROS) and total antioxidative capacity (T-AOC) were investigated in the gills during the first 24 h after exposure. Aphantoxins significantly increased the level of ROS and decreased the T-AOC in zebrafish gills from 3 to 12 h post-exposure, suggesting an induction of oxidative stress and inhibition of antioxidant capacity. Reduced activities of NKA and CA demonstrated abnormal ion transport and gas exchange in the gills of aphantoxin-treated fish. Toxin administration also resulted in increased LDH activity and ultrastructural alterations in chloride and epithelial cells, suggesting a disruption of function and structure in zebrafish gills. The observed abnormalities in zebrafish gills occurred in a time- and dose-dependent manner. These findings demonstrate that aphantoxins or PSPs may inhibit ion transport and gas exchange, increase LDH activity, and result in ultrastructural damage to the gills through elevations in oxidative stress and reduced antioxidant capacity. These effects of aphantoxins in the gills of zebrafish suggest an induction of respiratory toxicity. The parameters investigated in this study may be also considered as biomarkers for studying aphantoxin/PSP exposure and cyanobacterial blooms in nature.