Cytotoxic effect of azaspiracid-2 and azaspiracid-2-methyl ester in cultured neurons: involvement of the c-Jun N-terminal kinase.

Human poisoning by azaspiracids (AZAs) has emerged as an increasing problem in Europe in recent years. Azaspiracid-2 (AZA-2) is one of the most abundant azaspiracids in nature. Although AZA-2 was recently involved in several toxic episodes leading to human intoxications, there is no information available about its mechanism of action or its cytotoxic effect in cellular models. This paper reports on the neurotoxic effect of azaspiracid-2 and its potential cellular targets. We explore the cellular and cytotoxic effects of AZA-2 and AZA-2-methyl ester (where the carboxylic acid moiety of AZA-2 was converted to the corresponding methyl ester) in cerebellar neurons. Pharmacological tools were used to analyze the role of different signal transduction pathways in the toxicity of AZA-2. The neurotoxicity of AZA-2 and AZA-2-methyl ester was developmentally regulated, exhibiting a higher cytotoxicity in younger cells (2-3 div). After excluding several signal transduction pathways, we found that inhibition of the mitogen-activated protein kinase JNK completely prevented the cytotoxic effect of AZA-2 in neurons. Furthermore, neuronal exposure to AZA-2 or AZA-2-methyl ester caused an increase in the amount of total and phosphorylated JNK and produced nuclear accumulation of the protein. The results presented here point to a common target for AZA-1 and AZA-2 and constitute the first experimental approach to investigate the cytotoxicity of AZA-2 in vitro, establishing an initial approach to probe the mechanism of action of these group of natural toxins.