Identification of Palytoxin–Ca²⁺ Complex by NMR and Molecular Modeling Techniques

More than 40 years after its isolation, the understanding of how palytoxin interacts with biological systems has yet to be fully determined. The Na⁺,K⁺-ATPase pump constitutes a molecular receptor for palytoxin that is able to convert the pump into an open channel, with consequent loss of cellular K⁺ and remarkable rise of cytosolic Na⁺ levels. In addition, a slight permeability to Ca²⁺ is detected when palytoxin binds to the pump. It has been demonstrated that the increase of cytosolic free Ca²⁺ concentration gives rise to downstream events ultimately leading to cell death. The widely accepted recognition of the dependence of important cellular events on calcium ion concentration propelled us to investigate the occurrence of palytoxin–Ca²⁺ complex in aqueous solution by NMR- and molecular modeling-based approach. We identified two specific regions of palytoxin where Ca²⁺ is preferentially coordinated. This study constitutes the first characterization of a calcium complex with palytoxin and, as such, is expected to support the investigation of the toxin molecular bioactivity

Ovatoxin-a, A Palytoxin Analogue Isolated from <i>Ostreopsis</i> cf. <i>ovata</i> Fukuyo: Cytotoxic Activity and ELISA Detection

This study provides the first evaluation of the cytotoxic effects of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a), the major toxin produced by <i>Ostreopsis</i> cf. <i>ovata</i> in the Mediterranean Sea. Its increasing detection during <i>Ostreopsis</i> blooms and in seafood highlights the need to characterize its toxic effects and to set up appropriate detection methods. OVTX-a is about 100 fold less potent than PLTX in reducing HaCaT cells viability (EC₅₀ = 1.1 × 10^–9 M vs 1.8 × 10^–11 M, MTT test) in agreement with a reduced binding affinity (<i>K</i>_d = 1.2 × 10^–9 vs 2.7 × 10^–11 M, saturation experiments on intact cells). Similarly, OVTX-a hemolytic effect is lower than that of the reference PLTX compound. Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting the lack of a hydroxyl group at C44 as a critical feature for PLTXs cytotoxic effects. A sandwich ELISA developed for PLTX detects also OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate manner (Bias = 0.3%), also in <i>O.</i> cf. <i>ovata</i> extracts and contaminated mussels. Although in vitro OVTX-a appears less toxic than PLTX, its cytotoxicity at nanomolar concentrations after short exposure time rises some concern for human health. The sandwich ELISA can be a viable screening method for OVTXs detection in monitoring program

Stereoisomers of 42-Hydroxy Palytoxin from Hawaiian <i>Palythoa toxica</i> and <i>P. tuberculosa</i>: Stereostructure Elucidation, Detection, and Biological Activities

Palytoxin ranks among the most potent marine biotoxins. Its lethality was well known to native Hawaiians that used to smear a “moss” containing the toxin on their spears to cause instant death to their victims. Human intoxications due to exposure to palytoxin and to its many congeners have been reported worldwide. Currently, palytoxins constitute the main threat to public health across the Mediterranean Sea. In the present work we report on the isolation and stereostructural determination of a new palytoxin analogue from a Hawaiian <i>Palythoa tuberculosa</i> sample. This new toxin is a stereoisomer of 42-hydroxypalytoxin isolated from <i>Palythoa toxica</i>. The whole absolute configuration of this latter toxin is also reported in the paper. Interestingly, the two 42-hydroxypalytoxins do not share the same biological activity. The stereoisomer from <i>P. tuberculosa</i> showed cytotoxicity toward skin HaCaT keratinocytes approximately 1 order of magnitude lower than that of 42-hydroxypalytoxin from <i>P. toxica</i> and about 2 orders of magnitude lower than that of palytoxin itself. This finding holds the prospect of interesting structure–activity relationship evaluations in the future

Isolation and Structure Elucidation of Ovatoxin-a, the Major Toxin Produced by Ostreopsis ovata

Since 2005, the benthic dinoflagellate Ostreopsis cf. ovata has bloomed across the Mediterranean basin, provoking serious toxic outbreaks. LC/MS studies have identified a number of palytoxin-like compounds, termed ovatoxins, along with trace amounts of putative palytoxin as the causative agents of the O. cf. ovata-related human sufferings. So far, any risk assessment for ovatoxins as well as establishment of their allowance levels in seafood has been prevented by the lack of pure toxins. The present paper reports on the isolation, NMR-based structural determination, and preliminary mouse lethality evaluation of ovatoxin-a, the major toxic compound contained in O. cf. ovata extracts. Availability of pure ovatoxin-a will open the double prospect of fully evaluating its toxicity and preparing reference standards to be employed in LC/MS quantitative analyses. Elucidation of ovatoxin-a’s complex structure will ultimately herald the understanding of the molecular bases of ovatoxins bioactivity

MAPKs activation and toxin-induced COX-2, TNF-α, IL-8 and IκBα mRNA expression upon p38 MAPK and JNK inhibition.

<p>(<b>A</b>) Western blot analysis of MAPK phosphorylation in macrophages incubated 4 h with the <i>O</i>. cf. <i>ovata</i> toxin extract (final OSTRTX concentration 2 ng/ml and 20 ng/ml) (lane 2, 3), 2 ng/ml PLTX (lane 4) or the vehicle (lane 1). 20 µg of whole cell extracts were separated by SDS-PAGE on 8% gel and submitted to western immunoblot with phospho-specific antibodies against ERK1/2, JNK and p38 MAPK. Actin was stained as a loading control. (<b>B</b>) Total RNA was extracted from macrophages pre-incubated 1 h with 1 µM p38 inhibitor (ip38) or 1 µM and 5 µM JNK inhibitor (iJNK), and then exposed 4 h to 2 ng/ml palytoxin. In parallel, macrophages were left untreated and then exposed to PLTX or to the vehicle alone. RNA was amplified with gene-specific primers. Expression data, normalized to the housekeeping B2M gene, were analyzed by the 2^−ΔΔCT method and referred to the value obtained in control cells (CTR). The boxplots show the results of 3 independent experiments, run in duplicate. Asterisks indicate statistical significance versus cells receiving only PLTX (*<i>p</i><0.05; **<i>p</i><0.01).</p

IκB-α mRNA levels in human macrophages exposed to PLTX and to the OSTRTX extract.

<p>Evaluation of IκB-α mRNA levels by quantitative Real-Time PCR assay. Total RNA, extracted from primary human macrophages exposed 4 h to PLTX, to the <i>O</i>. cf. <i>ovata</i> toxin extract (final OSTRTX concentration 2 ng/ml and 20 ng/ml) or to the vehicle (i.e. 0.05% MetOH), was amplified with IκB-α gene-specific primers. Expression data, normalized to the housekeeping B2M gene, were analysed by the 2^−ΔΔCT method and referred to the value obtained in untreated cells (CTR). The boxplots show the results of 8 and 5 independent experiments performed with PLTX and the OSTRTX extract, respectively. Asterisks indicate statistical significance versus cells receiving the vehicle alone (*<i>p</i><0.05; **<i>p</i><0.01).</p

Structure of palytoxin.

<p>Cleavage between C-8 and C-9 occurs in HR LC-MS and MS² experiments and divides the molecule in two moieties, A-side and B-side.</p

IκBα and p65 protein levels in cells treated with the vehicle, the OSTRTX extract and PLTX.

<p>(<b>A</b>) IκBα protein levels were determined in whole cell lysates obtained from macrophages incubated 4 h with the vehicle (lane 1), the OSTRTX extract (lanes 2–3) or PLTX (lane 4). Protein extracts (15 µg) were resolved by SDS-PAGE on 8% gel and then submitted to Western immunoblotting. Blots were probed with an anti-IκBα (upper panel) and an anti-p65 (RelA) antibody to check for protein loading (lower panel). (<b>B</b>) intracellular content and subcellular distribution of p65 (RelA) in PLTX- versus vehicle-treated cells was assessed by immunoblotting analysis of whole (15 µg, lanes 1–2), cytosolic (15 µg, lanes 3–4) and nuclear (10 µg, lane 5–6) extracts. Whole cell extracts were obtained by lysing cells in SDS buffer. In parallel, cells were sub-fractionated by extraction in Buffer A (BUFF A, cytosolic proteins) followed by Buffer B (nuclear proteins, BUFF B), the residual material, containing insoluble proteins, including those associated with cytoskeletal structures, was solubilized in SDS-PAGE sample buffer and run in parallel (lanes 7–8). (<b>C</b>) Approaches to inhibit p65 <i>in vitro</i> degradation during extraction in native conditions. Cell pellets, containing an equivalent number of macrophages, were lysed in SDS buffer (SDS BUFF, lane 1), Buffer A (BUFF A, lane 2) or Buffer A supplemented with palytoxin (BUFF A+PLTX, lane 3). An equal volume of SDS sample buffer was added to all tubes and comparable volumes of the resulting protein extracts were resolved by electrophoresis and immunoblotted with an anti p65 antibody.(<b>D</b>) cell pellets as in C were lysed in SDS buffer (lane 1), Buffer A (lane 2) and Buffer A further supplemented with lysososmal protease inhibitors (BUFF A PLUS, lane 3) and submitted to SDS-PAGE and immunoblotting with an anti p65 antibody.</p

COX-2, IL-8 and TNF-α mRNA levels in human macrophages exposed to PLTX and to the OSTRTX extract.

<p>Evaluation of COX-2, IL-8 and TNF-α mRNA levels by quantitative Real-Time PCR assay. Total RNA, extracted from primary human macrophages exposed 4 h to PLTX (2 ng/ml), to the <i>O</i>. cf. <i>ovata</i> toxin extract (final OSTRTX concentration 2 ng/ml and 20 ng/ml) or the vehicle (i.e. 0.05% MetOH), was amplified with gene-specific primers. Expression data, normalized to the housekeeping B2M gene, were analyzed by the 2^−ΔΔCT method and referred to the value obtained in untreated cells (CTR). The boxplots show the results of 8 and 5 independent experiments performed with PLTX and the OSTRTX extract, respectively. Asterisks indicate statistical significance versus cells receiving the vehicle alone (*<i>p</i><0.05; **<i>p</i><0.01).</p

Inhibition of PLTX-induced COX-2, TNF-α, and IL-8 mRNA expression upon NF-κB inhibition.

<p>Total RNA was extracted from primary human macrophages pre-incubated 1 h with 50 µM andrographolide, an NF-κB inhibitor molecule (iNF-κB), and then exposed 4 h to PLTX (2 ng/ml). In parallel, macrophages were treated with PLTX or the vehicle alone. RNA was amplified with gene-specific primers. IκBα and UbC mRNA levels were used as positive and negative control, respectively. Expression data, normalized to the housekeeping B2M gene, were analysed by the 2^−ΔΔCT method and referred to the value obtained in untreated cells (CTR). The boxplots show the results of 5 independent experiments. Asterisks indicate statistical significance versus cells receiving PLTX (*<i>p</i><0.05; **<i>p</i><0.01).</p