Aphanizomenon flos-aquae (A. flos-aquae), a cyanobacterium frequently encountered in water blooms worldwide, is source of
neurotoxins known as PSPs or aphantoxins that present a major threat to the environment and to human health. Although the molecular mechanism of PSP action is well known, many unresolved questions remain concerning its mechanisms of toxicity. Aphantoxins purified from a natural isolate of A. flos-aquae DC-1 were analyzed by high-performance liquid chromatography (HPLC), the major component toxins were the gonyautoxins1 and 5 (GTX1 and GTX5, 34.04% and 21.28%, respectively) and the
neosaxitoxin (neoSTX, 12.77%). The LD50 of the
aphantoxin preparation was determined to be 11.33 μg/kg (7.75 μg
saxitoxin equivalents (STXeq) per kg) following
intraperitoneal injection of zebrafish (Danio rerio). To address the neurotoxicology of the
aphantoxin preparation, zebrafish were injected with low and high sublethal doses of A. flos-aquae DC-1 toxins 7.73 and 9.28 μg /kg (5.3 and 6.4 μg STXeq/kg, respectively) and brain tissues were analyzed by electron microscopy and RT-PCR at different timepoints postinjection. Low-dose
aphantoxin exposure was associated with
chromatin condensation, cell-membrane blebbing, and the appearance of apoptotic bodies. High-dose exposure was associated with cytoplasmic vacuolization, mitochondrial swelling, and expansion of the endoplasmic reticulum. At early timepoints (3 h) many cells exhibited characteristic features of both apoptosis and
necrosis. At later timepoints apoptosis appeared to predominate in the low-dose group, whereas
necrosis predominated in the high-dose group. RT-PCR revealed that
mRNA levels of the apoptosis-related genes encoding p53, Bax,
caspase-3, and c-Jun were upregulated after
aphantoxin exposure, but there was no evidence of
DNA laddering; apoptosis could take place by pathways independent of DNA fragmentation. These results demonstrate that
aphantoxin exposure can cause cell death in zebrafish brain tissue, with low doses inducing apoptosis and higher doses inducing
necrosis.