Proteomic analysis of anatoxin-a acute toxicity in zebrafish reveals gender specific responses and additional mechanisms of cell stress.

Anatoxin-a is a potent neurotoxin produced by several genera of cyanobacteria. Deaths of wild and domestic animals due to anatoxin-a exposure have been reported following a toxic response that is driven by the inhibition of the acetylcholine receptors at neuromuscular junctions. The consequent neuron depolarization results in an overstimulation of the muscle cells. In order to unravel further molecular events implicated in the toxicity of anatoxin-a, a proteomic investigation was conducted. Applying two-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry, we report early proteome changes in brain and muscle of zebrafish (Danio rerio) caused by acute exposure to anatoxin-a. In this regard, the test group of male and female zebrafish received an intraperitoneal (i.p.) injection of an anatoxin-a dose of 0.8µgg(-1) of fish body weight (bw) in phosphate buffered saline solution (PBS), while the control received an i.p. injection of PBS only. Five minutes after i.p. injection, brain and muscle tissues were collected, processed and analyzed with 2DE. Qualitative and quantitative analyzes of protein abundance allowed the detection of differences in the proteome of control and exposed fish groups, and between male and female fish (gender specific responses). The altered proteins play functions in carbohydrate metabolism and energy production, ATP synthesis, cell structure maintenance, cellular transport, protein folding, stress response, detoxification and protease inhibition. These changes provide additional insights relative to the toxicity of anatoxin-a in fish. Taking into account the short time of response considered (5min of response to the toxin), the changes in the proteome observed in this work are more likely to derive from fast occurring reactions in the cells. These could occur by protein activity regulation through degradation (proteolysis) and/or post-translational modifications, than from a differential regulation of gene expression, which may require more time for proteins to be synthesized and to produce changes at the proteomic level.