Induction of Apoptosis Pathways in Several Cell Lines following Exposure to the Marine Algal Toxin Azaspiracid

Abstract

Azaspiracids (AZAs) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although there have been no deaths associated with the AZA toxins, humans exposed to AZAs experience severe gastrointestinal symptoms. This toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Just recently, the AZAs have been shown to be potassium channel inhibitors. This report employed multiple human cell lines [Jurkat T lymphocytes, Caco-2 intestinal cells, and BE(2)-M17 neuroblastoma cells] in characterizing cytotoxicity and pathways of apoptosis. Cytotoxicity experiments were consistent with published literature that has shown that AZA1 is cytotoxic in both a concentration- and time-dependent manner to each cell type tested, with mean EC(50) values ranging between 1.1 and 7.4 nM. Despite the absence of morphological indices indicating apoptosis, caspase-3/7 activity was higher in all cell types treated with AZA1. Furthermore, in T lymphocytes, the most sensitive cell type, the activities of initiator caspase-2 and caspase-10 and concentrations of intracellular cytochrome c were elevated. DNA fragmentation was also observed for T lymphocytes exposed to AZA1-AZA3. Collectively, our data confirm that AZA1 was highly cytotoxic to multiple cell types and that cells exposed to AZA1 underwent atypical apoptosis, possibly in conjunction with necrotic cytotoxicity.

Isolation, Structure Elucidation, Relative LC-MS Response, and in Vitro Toxicity of Azaspiracids from the Dinoflagellate Azadinium spinosum

“…Detailed analysis of 1 H, 13 C, DEPT135, COSY, TOCSY, SELTOCSY, HSQC, and HMBC spectra of 3 showed the presence of the pentacyclic C-20−C-40 ring system, present in all other known AZAs, consistent with results of the periodate cleavage experiment. During NMR analysis of both 3 and 4, a series of minor peaks (∼3−10%) were also observed that were attributable to the corresponding 37-epimers, due to susceptibility of AZAs to epimerization in MeOH under neutral and weakly acidic conditions.…”

“…These observations are consistent with the relative configuration proposed for the C-16−C-19 region of 3 ( Figure 1). In full accord with the mass spectrometric and periodate cleavage results discussed above, detailed analysis of 1 H, 13 The chemical shifts of carbons and protons associated with the C-6−C-40 portion of 4 were similar, but not in all cases identical, to those of 1 ( Table 2) and several other AZAs. 14−17 It is apparent from an analysis of published NMR data that the chemical shifts of atoms in the vicinity of the 40-NH group and the C-20 portions of AZA are dependent on the pH of the examined NMR solutions.…”

“…The resonances of the 13 The two pairs of methylene protons at 2.03 ppm (4H, br s, H-11 and H-12) were not overlapped in the 1 H NMR spectrum by other signals and showed COSY correlations only to the olefinic proton signals centered at 5.45 (H-10) and 5.50 ppm (H-13). In the HMBC spectrum, the protons at 2.04 ppm showed correlations to the four olefinic carbons (127.8, 130.8, 131.5, and 133.4 ppm) and adjacent partner methylene carbons (33.9 and 34.1 ppm, C-11 and C-12).…”

Absorption and Effect of Azaspiracid-1 Over the Human Intestinal Barrier

“…AZA1 is highly cytotoxic to some cell lines undergoing atypical apoptosis [31]. Related to this AZAs induced cytoskeleton disorganization linking the mechanism of action to apoptotic targets that responded with irreversible kinetics [12].…”

In vitro chronic effects on hERG channel caused by the marine biotoxin azaspiracid-2

“…Actually, AZA-2 induces a significant increase of hERG channel levels on the cell surface of hERG-CHO cells. Azaspiracids are known to trigger apoptosis in many cell lines [33, 34], and therefore the alterations of surface hERG levels might be due to the apoptotic process. However, surface hERG channels are clearly increased without any evidence of annexin-V externalization, which is considered a relatively early marker of apoptosis [35, 36].…”