Azaspiracids Increase Mitochondrial Dehydrogenases Activity in Hepatocytes: Involvement of Potassium and Chloride Ions

Abstract

Background: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes. Results: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10−12–1 × 10−7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K+-, Cl−-, and Na+-free media and sensitive to the specific inhibitors of KATP and hERG potassium channels, Na+/K+, ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Conclusions: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K+ and, in particular, Cl− ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

Targeting Chloride Ion Channels: New Insights into the Mechanism of Action of the Marine Toxin Azaspiracid

“…62 In this regard, it has been demonstrated that cholesterol levels control the activity of VRAC, because cholesterol depletion increases VRAC activation. 63 Although the involvement of chloride ions in the mechanism of action of azaspiracids has been formerly described, 23,64 the exact mechanism of the interaction of these toxins with chloride channels has never been elucidated; however, overexpression of VDAC proteins after exposure of neuroblastoma cells for 15 h to 15 nM AZA1 has been previously documented. 47 In this work, we demonstrated for the first time that VRAC channels are directly related to the cellular alterations caused by azaspiracids, because intracellular ATP depletion eliminated the increase in chloride channel activation observed in the cells treated with the toxin.…”

Human Health and Ocean Pollution

“…The azaspiracid group of HAB toxins also results in diarrhetic symptoms. Its mechanism of action is not yet known, but recent evidence suggests that mitochondrial dehydrogenase may be a major target of this toxin group [ 375 ].…”

Current Trends and New Challenges in Marine Phycotoxins

“…For instance, AZAs modifying hERG activity in vitro could explain at least in part the cardiotoxicity observed in rats after intraperitoneal treatment [ 213 , 214 , 215 ]. Another example could be hepatocytes’ K + and Cl - channels modification in vitro with liver affection in mice following oral exposure to these biotoxins [ 215 , 216 ]. Cytoskeletal rearrangement in the intestinal cell model could also be related to intestinal fluid accumulation in mice [ 16 , 215 ].…”