Effects of neurotoxin - anatoxin-a on common carp (Cyprinus carpio L.) innate immune cells in vitro

The aim of this study was to determine if cyanoneurotoxin - anatoxin-a (ANTX-a) alters the essential functions of innate immune cells such as free radicals generation in phagocytic cells and phagocytosis.

In the experiments pure ANTX-a was used at concentrations of 0.01, 0.05, 0.1 and 1 µg/ml RPMI-1640 medium. Phagocytes were isolated from carp blood and pronephros. Relative changes in intracellular total free radical presence in fish phagocytes were monitored using a fluorescent probe, dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ) which detects hydrogen peroxide (H2O2), nitric oxide (NO), peroxyl radical and peroxynitrite anion. Phagocytic activity of fish leukocytes was analyzed with a Vybrant phagocytosis assay kit.

The H2O2 level generated in response to ANTX-a at the highest used concentration was significantly suppressed in pronephros but not in blood phagocytes. Moreover, it was observed that generation of superoxide radicals and nitrite formation was significantly increased in blood and pronephros phagocytes after incubation with lower concentrations of the neurotoxin. The phagocytosis of fish leukocytes was significantly reduced at the two highest used toxin concentrations (0.1 and 1 µg/ml medium).

This findings suggests that ANTX-a could change innate immunity and reduced adaptive immunity after stress induced by cyanobacterial blooms.