ToxSci Advance Access published online on October 8, 2009
Toxicological Sciences, doi:10.1093/toxsci/kfp246
Cell volume decrease as a link between azaspiracid-induced cytotoxicity and c-jun-N-terminal kinase activation in cultured neurons
* Departamento de Farmacología, Facultad de Veterinaria, USC, Lugo, Spain
Department of Chemistry and The Skaggs Institute for Chemical Biology. The Scripps Research Institute, La Jolla, California
Department of Chemistry and Biochemistry, University of California, San Diego. La Jolla, California
Departamento de Fisiología, Facultad de Veterinaria, USC, Lugo, Spain
* Corresponding author: Luis M. Botana. Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela. Campus Universitario s/n, 27002, Lugo, Spain. Tel.: 34-982 252 242 ; FAX : 34-982 252 242. E-mail address: Luis.Botana{at}lugo.usc.es
Received July 10, 2009; revision received October 2, 2009; accepted October 5, 2009
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Abstract |
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Azaspiracids are a group of marine toxins recently described that currently includes 20 analogues. Not much is known about their mechanism of action, although the predominant analogue in nature, azaspiracid-1 (AZA-1) targets several organs in vivo, including the central nervous system and exhibits high neurotoxicity in vitro. Azaspiracid distribution is increasing globally with mussels being most widely implicated in azaspiracid-related food poisoning events and human poisoning by azaspiracids has emerged as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for azaspiracid-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro (div), however it did not modify membrane potential in mature neurons. Furthermore, in immature cells AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50 mM K+. Preincubation of the neurons with 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-Acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride or ouabain before addition of AZA-1 decreased the AZA-1 induced neurotoxicity and the increase in phosphorylated JNK caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin.
Key Words: Azaspiracid; AZA-1; cytotoxicity; cerebellar granule cell; cell volume; c-Jun-N-terminal kinase.