Cytotoxic effect of azaspiracid‐2 and azaspiracid‐2‐methyl ester in cultured neurons: Involvement of the c‐Jun N‐terminal kinase

Abstract

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.

Derivation of toxicity equivalency factors for marine biotoxins associated with Bivalve Molluscs

“…They were first detected 30 minutes after administration to pigs, with peak levels achieved after 4 h (Twiner, Hess, & Doucette, 2014). In humans, they cause vomiting, nausea, diarrhoea and stomach cramps within a few hours after ingestion (Klontz, Abraham, Plakas, & Dickey, 2009) AZAs target several apoptotic modulators (Botana, et al, 2014;Roman, et al, 2002;Twiner, et al, 2005), such as caspase, cytoskeleton (Vilarino, Nicolaou, Frederick, Vieytes, & Botana, 2007), cytochrome release (Twiner, Hanagriff, Butler, Madhkoor, & Doucette, 2012), c-jun-N-terminal protein kinase (JNK), calcium levels (Cao, LePage, Frederick, Nicolaou, & Murray, 2010;Vale, Wandscheer, et al, 2008), fatty acid biosynthesis (Twiner, et al, 2008). AZAs decrease cell volume mediated by potassium and chloride efflux (Vale, Nicolaou, Frederick, Vieytes, & Botana, 2010), deplete ATP (Kellmann, et al, 2009), inhibit endocytosis (Bellocci, Sala, Callegari, & Rossini, 2010) and decrease procathepsin pools in endocytosis (Sala, Bellocci, Callegari, & Rossini, 2013) (Chevallier, et al, 2015), which could explain the neurotoxicity linked to AZA (Twiner, et al, 2014).…”

Current Trends and New Challenges in Marine Phycotoxins

“…In fact, these two studies with paralytic toxins and other studies with diarrheic toxins [ 39 , 40 ] emphasize the need to review actual TEFs using toxin CRMs and the oral route for toxin administration in order to determine reliable TEFs useful to use analytical methods for neurotoxin monitoring [ 48 ]. In certain cases, TEFs have been established after measuring in vitro toxicity or cellular effects of the toxin [ 74 , 75 , 76 ]. Although these studies do not take into account neither the absorption nor the metabolism or elimination of the toxins in vivo, these data have also been taken into consideration by EFSA to establish the current TEFs [ 64 ] even when there is no approved in vitro model to evaluate the toxicity of paralytic toxins.…”

“…In certain cases, TEFs have been established after measuring in vitro toxicity or cellular effects of the toxin [ 74 , 75 , 76 ]. Although these studies do not take into account neither the absorption nor the metabolism or elimination of the toxins in vivo, these data have also been taken into consideration by EFSA to establish the current TEFs [ 64 ] even when there is no approved in vitro model to evaluate the toxicity of paralytic toxins.…”

Marine biotoxins in shellfish – Azaspiracid group ‐ Scientific Opinion of the Panel on Contaminants in the Food chain

“…More recently, AZAs have been found in scallops and crab (FSAI, 2006). Since their initial discovery in 1995, AZAs were also identified in shellfish from UK and Norway at significant levels, while trace amounts were also found in shellfish from France and Spain (Brana-Magdalena et al, 2003b), Morocco and Portugal (Taleb et al, 2006;Vale et al, 2008).…”