Cell Volume Decrease as a Link between Azaspiracid-Induced Cytotoxicity and c-Jun-N-Terminal Kinase Activation in Cultured Neurons

Abstract

Azaspiracids (AZAs) are a group of marine toxins recently described that currently includes 20 members. Not much is known about their mechanism of action, although the predominant analog in nature, AZA-1 targets several organs in vivo, including the central nervous system, and exhibits high neurotoxicity in vitro. AZA distribution is increasing globally with mussels being most widely implicated in AZA-related food poisoning events, with human poisoning by AZAs emerging 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 AZA-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro; 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 50mM 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 c-Jun-N-terminal kinase (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.

Ouabain‐induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells

“…We have shown JNK dephosphorylation after 6 or more hours of incubation with 1‐μM ouabain. Previously ouabain has been shown to activate JNK in HeLa and COS7 cell lines while in primary culture of mouse cerebellar cells ouabain causes a reduction of JNK activation caused by the azaspiracid toxin . Furthermore, it has been reported that JNK dephosphorylation can be caused by ERK1/2 and p38 activation, which corresponds to our results.…”

“…JNK activation is associated with excitotoxic neuronal death in stroke and epilepsy, Alzheimer's disease, Parkinson's disease and Huntington's disease . Ouabain activates JNK in HeLa and COS7 cell lines, but reduces JNK activation caused by the azaspiracid toxin in primary culture of mouse cerebellar cells …”

Comparative Effects of the Marine Algal Toxins Azaspiracid-1, -2, and -3 on Jurkat T Lymphocyte Cells

“…The cytotoxic potential of AZA1 towards various cell types is well established and has been unambiguously demonstrated using microscopy techniques (24,27,34,39), cellular protein content (27), DNA content and synthesis (27,48), release of cytosolic enzymes (31,36), and mitochondrial activity (24,31). Within the current study, the cytotoxic potential of AZA1, as well as AZA2 and AZA3, was confirmed using human Jurkat T lymphocytes as the model cell line, selected based on previous work demonstrating its high level of sensitivity to AZA1 (28).…”

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems

“…there is evidence that CIP toxins and their producers also are found in europe, the USA, and North Africa (Aasen et al, 2005a;Bire et al, 2002;Cembella and Krock, 2008;Ciminiello et al, 2006;John et al, 2003;MacKinnon et al, 2006;Marrouchi et al, 2010;Rundberget et al, 2011;touzet et al, 2008;Villar Gonzalez et al, 2006), and their presence, along with PSP to CTX also primarily in predatory fish consumed by humans (snapper, barracuda, amber jack and moray eel), and more investigations should be conducted on the role on Mtx in human intoxication.…”

“…The dinoflagellates Alexandrium ostenfeldii and Alexandrium peruvianum are the main producers of SPx, some strains of A. ostenfeldii also can produce saxitoxins -Stx (Cembella, 1998;Gribble et al, 2005;Kremp et al, 2009;MacKenzie et al, 1996), while A. peruvianum produces not only SPx (Borkman et al, 2012;tomas et al, 2012;touzet et al, 2008, 2011 but also 12-methyl gymnodimine, Stx and gonyautoxins Van Wagoner et al, 2011). SPxs were found in the early 1990s in Canada (Hu et al, 1995).…”