Study of permeability of the marine toxin azaspiracid-1 over in vitro human intestinal model

Phycotoxins are natural products responsible of different intoxications in humans, especially when harmful algae blooms (HABs) occur. Azaspiracid poisoning (AZP)[1] in humans is characterized by gastrointestinal illness due to the ingestion of shellfish that accumulated azaspiracids (AZAs). These lipophilic toxins are produced by some dinoflagellates from Azadinium and Amphidoma genera. Although multiple cellular damages have been described, the mechanism of action of azaspiracid-group toxins is still unknown. In order to establish levels of absorption of these toxins, the permeability of azaspiracid-1 (AZA-1) through the in vitro human intestinal model of Caco-2 cells was studied. These cells are able to spontaneously differentiate to enterocytes, expressing the brush border enzyme activities and representing a reliable model of the human intestine. The transport experiments were performed in the insert/well configuration with differentiated Caco-2 monolayer [2]. The permeability of Caco-2 monolayers to the toxin was evaluated after incubation of 10 nM and 100 nM AZA-1 at several times, and then the amount of toxin of both insert and well compartments was quantified by union competition immunoassay with Luminex 200^TM analyzer [3]. The pass of AZA-1 from inserts to wells revealed a time and dose-dependent absorption by Caco-2 monolayer. Those results indicate that AZA-1 would be absorbed in the human intestine, thus being able to enter the circulatory system and to reach and affect different organs.

Acknowledgements: The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012 – 40185-CO2 – 01, AGL2014 – 58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013 – 016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union's Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007 – 2013) under grant agreement 312184 PHARMASEA.

Keywords: Azaspiracid-1, Caco-2, intestinal permeability, Luminex.

References:

[1] Twiner MJ, Rehmann N, Hess P, Doucette GJ. Azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts. Mar Drugs 2008; 6: 39 – 72

[2] Fernandez DA, Louzao MC, Vilarino N, Fraga M, Espina B, Vieytes MR, Botana LM. Evaluation of the intestinal permeability and cytotoxic effects of cylindrospermopsin. Toxicon 2014; 91: 23 – 34

[3] Fraga M, Vilarino N, Louzao MC, Botana LM. Sensitivity improvement of an immuno-detection method for azaspiracids based on the use of microspheres coupled to a flow-fluorimetry system. International Meeting on Marine Research (IMMR), Peniche, Portugal; 2014; doi:10.3389/conf.fmars.2014.02.00166