Combined Effects of Lipophilic Phycotoxins (Okadaic Acid, Azapsiracid-1 and Yessotoxin) on Human Intestinal Cells Models

Abstract

Phycotoxins are monitored in seafood because they can cause food poisonings in humans. Phycotoxins do not only occur singly but also as mixtures in shellfish. The aim of this study was to evaluate the in vitro toxic interactions of binary combinations of three lipophilic phycotoxins commonly found in Europe (okadaic acid (OA), yessotoxin (YTX) and azaspiracid-1 (AZA-1)) using the neutral red uptake assay on two human intestinal cell models, Caco-2 and the human intestinal epithelial crypt-like cells (HIEC). Based on the cytotoxicity of individual toxins, we studied the interactions between toxins in binary mixtures using the combination index-isobologram equation, a method widely used in pharmacology to study drug interactions. This method quantitatively classifies interactions between toxins in mixtures as synergistic, additive or antagonistic. AZA-1/OA, and YTX/OA mixtures showed increasing antagonism with increasing toxin concentrations. In contrast, the AZA-1/YTX mixture showed increasing synergism with increasing concentrations, especially for mixtures with high YTX concentrations. These results highlight the hazard potency of AZA-1/YTX mixtures with regard to seafood intoxication.

Keywords: cytotoxicity; enterocytes; lipophilic phycotoxins; mixtures.

Figure 1

Combination index (CI) plot for binary mixtures of azaspiracid-1 (AZA-1) and yessotoxin (YTX) in four different molar ratios. CI values were calculated from fractional cytotoxicity (Fa) by computer modeling (CompuSyn) on Fa = 0.05 to 0.95. CI < 1, CI = 1 and CI > 1 indicate synergistic, additive and antagonistic effects, respectively. The vertical bar indicates the standard deviation for the three replicates. Dashed lines indicate upper and lower limit of additivity [36]. (A) Combinary index plots based on Caco-2 cells experiments; (B) Combinary index plots based on HIEC cells experiments.

Figure 2

Combination index plot for binary mixtures of okadaic acid (OA) and azaspiracid-1 (AZA-1) at four different molar ratios. CI values were calculated from fractional cytotoxicity (Fa) by computer modeling (CompuSyn) on Fa = 0.05 to 0.95. CI < 1, CI = 1 and CI > 1 indicate synergistic, additive and antagonistic effects, respectively. The vertical bar indicates the standard deviation for the three replicates. Dashed lines indicate upper and lower limit of additivity [36]. (A) Combinary index plots based on Caco-2 cells experiments; (B) Combinary index plots based on HIEC cells experiments.

Figure 3

Combination index (CI) plot for binary mixtures of okadaic acid (OA) and yessotoxin (YTX) at four different molar ratios. CI values were calculated from fractional cytotoxicity (Fa) by computer modeling (CompuSyn) on Fa = 0.05 to 0.95. CI < 1, CI = 1 and CI > 1 indicate synergistic, additive and antagonistic effects, respectively. The vertical bar indicates standards deviation between the three experiments. Dashed lines indicate upper and lower limit of additivity [36]. (A) Combinary index plots based on Caco-2 cells experiments; (B) Combinary index plots based on HIEC cells experiments.