Complex Toxin Profile of French Mediterranean Ostreopsis cf. ovata Strains,Seafood Accumulation and Ovatoxins Prepurification

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg·kg⁻¹ of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg·kg⁻¹ in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell⁻¹. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.     

Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody

Palytoxin (PLTX) is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na⁺/K⁺-ATPase. Indeed, ouabain (OUA), a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na⁺/K⁺-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a K_d of 3 × 10⁻¹⁰ M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3–1.0 × 10⁻⁷ M) and possibly as a non-competitive antagonist against low PLTX concentrations (0.1–3.0 × 10⁻⁹ M). Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10⁻⁵ M). However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.     

Palytoxin-Containing Aquarium Soft Corals as an Emerging Sanitary Problem

Palytoxin (PLTX), one the most potent marine toxins, and/or its analogs, have been identified in different marine organisms, such as Palythoa soft corals, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria. Although the main concern for human health is PLTXs entrance in the human food chain, there is growing evidence of adverse effects associated with inhalational, cutaneous, and/or ocular exposure to aquarium soft corals contaminated by PLTXs or aquaria waters. Indeed, the number of case reports describing human poisonings after handling these cnidarians is continuously increasing. In general, the signs and symptoms involve mainly the respiratory (rhinorrhea and coughing), skeletomuscular (myalgia, weakness, spasms), cardiovascular (electrocardiogram alterations), gastrointestinal (nausea), and nervous (paresthesia, ataxia, tremors) systems or apparates. The widespread phenomenon, the entity of the signs and symptoms of poisoning and the lack of control in the trade of corals as aquaria decorative elements led to consider these poisonings an emerging sanitary problem. This review summarizes literature data on human poisonings due to, or ascribed to, PLTX-containing soft corals, focusing on the different PLTX congeners identified in these organisms and their toxic potential.     

First Characterization of Ostreopsis cf. ovata (Dinophyceae) and Detection of Ovatoxins during a Multispecific and Toxic Ostreopsis Bloom on French Atlantic Coast

Blooms of the benthic toxic dinoflagellate genus Ostreopsis have been recorded more frequently during the last two decades, particularly in warm temperate areas such as the Mediterranean Sea. The proliferation of Ostreopsis species may cause deleterious effects on ecosystems and can impact human health through skin contact or aerosol inhalation. In the eastern Atlantic Ocean, the toxic O. cf. ovata has not yet been reported to the north of Portugal, and the only species present further north was O. cf. siamensis, for which the toxic risk is considered low. During summer blooms of unidentified Ostreopsis species on the French Basque coast (Atlantic) in 2020 and 2021, people suffered from irritations and respiratory disorders, and the number of analyzed cases reached 674 in 2021. In order to investigate the causes, sampling was carried out during summer 2021 to (i) taxonomically identify Ostreopsis species present using a molecular approach, (ii) isolate strains from the bloom and culture them, and (iii) characterize the presence of known toxins which may be involved. For the first time, this study reports the presence of both O. cf. siamensis and O. cf. ovata, for which the French Basque coast is a new upper distribution limit. Furthermore, the presence of ovatoxins a, b, c, and d in the environmental sample and in a cultivated strain in culture confirmed the toxic nature of the bloom and allowed identifying O. cf. ovata as the producer. The present data identify a new health risk in the area and highlight the extended distribution of some harmful dinoflagellates, presumably in relation to climate change.     

Palytoxin and Analogs: Biological and Ecological Effects

Palytoxin (PTX) is a potent marine toxin that was originally found in soft corals from tropical areas of the Pacific Ocean. Soon after, its occurrence was observed in numerous other marine organisms from the same ecological region. More recently, several analogs of PTX were discovered, remarkably all from species of the dinoflagellate genus Ostreopsis. Since these dinoflagellates are also found in other tropical and even in temperate regions, the formerly unsuspected broad distribution of these toxins was revealed. Toxicological studies with these compounds shows repeatedly low LD₅₀ values in different mammals, revealing an acute toxic effect on several organs, as demonstrated by different routes of exposure. Bioassays tested for some marine invertebrates and evidences from environmental populations exposed to the toxins also give indications of the high impact that these compounds may have on natural food webs. The recognition of its wide distribution coupled with the poisoning effects that these toxins can have on animals and especially on humans have concerned the scientific community. In this paper, we review the current knowledge on the effects of PTX and its analogs on different organisms, exposing the impact that these toxins may have in coastal ecosystems.     

Lyngbyastatins 8–10, Elastase Inhibitors with Cyclic Depsipeptide Scaffolds Isolated from the Marine Cyanobacterium Lyngbya semiplena

Investigation of an extract from the marine cyanobacterium Lyngbya semiplena, collected in Tumon Bay, Guam, led to the identification of three new cyclodepsipeptides, lyngbyastatins 8–10 (1–3). The structures of 1–3 were determined by NMR, MS, ESIMS fragmentation and chemical degradation. Compounds 1–3 are closely related to lyngbyastatins 4–7. Like the latter compounds, we found 1–3 to inhibit porcine pancreatic elastase, with IC₅₀ values of 123 nM, 210 nM and 120 nM, respectively.     

Dangerous Relations in the Arctic Marine Food Web: Interactions between Toxin Producing Pseudo-nitzschia Diatoms and Calanus Copepodites

Diatoms of the genus Pseudo-nitzschia produce domoic acid (DA), a toxin that is vectored in the marine food web, thus causing serious problems for marine organisms and humans. In spite of this, knowledge of interactions between grazing zooplankton and diatoms is restricted. In this study, we examined the interactions between Calanus copepodites and toxin producing Pseudo-nitzschia. The copepodites were fed with different concentrations of toxic P. seriata and a strain of P. obtusa that previously was tested to be non-toxic. The ingestion rates did not differ among the diets (P. seriata, P. obtusa, a mixture of both species), and they accumulated 6%–16% of ingested DA (up to 420 µg per dry weight copepodite). When P. seriata was exposed to the copepodites, either through physical contact with the grazers or separated by a membrane, the toxicity of P. seriata increased (up to 3300%) suggesting the response to be chemically mediated. The induced response was also triggered when copepodites grazed on another diatom, supporting the hypothesis that the cues originate from the copepodite. Neither pH nor nutrient concentrations explained the induced DA production. Unexpectedly, P. obtusa also produced DA when exposed to grazing copepodites, thus representing the second reported toxic polar diatom.     

Characterization of intracellular and extracellular saxitoxin levels in both field and cultured Alexandrium spp. samples from Sequim Bay, Washington

Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs), intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004–2007) and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA) and an enzyme-linked immunosorbent assay (ELISA). Characterization of the PST toxin profile in the Sequim Bay isolates by pre-column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 ± 9.7 % of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region.     

Natural Benzo/Acetophenones as Leads for New Synthetic Acetophenone Hybrids Containing a 1,2,3-Triazole Ring as Potential Antifouling Agents

Marine biofouling is a natural process that represents major economic, environmental, and health concerns. Some booster biocides have been used in biofouling control, however, they were found to accumulate in environmental compartments, showing negative effects on marine organisms. Therefore, it is urgent to develop new eco-friendly alternatives. Phenyl ketones, such as benzophenones and acetophenones, have been described as modulators of several biological activities, including antifouling activity (AF). In this work, acetophenones were combined with other chemical substrates through a 1,2,3-triazole ring, a strategy commonly used in Medicinal Chemistry. In our approach, a library of 14 new acetophenone–triazole hybrids was obtained through the copper(I)-catalyzed alkyne-azide cycloaddition “click” reaction. All of the synthesized compounds were evaluated against the settlement of a representative macrofouling species, Mytilus galloprovincialis, as well as on biofilm-forming marine microorganisms, including bacteria and fungi. The growth of the microalgae Navicula sp. was also evaluated after exposure to the most promising compounds. While compounds 6a, 7a, and 9a caused significant inhibition of the settlement of mussel larvae, compounds 3b, 4b, and 7b were able to inhibit Roseobacter litoralis bacterial biofilm growth. Interestingly, acetophenone 7a displayed activity against both mussel larvae and the microalgae Navicula sp., suggesting a complementary action of this compound against macro- and microfouling species. The most potent compounds (6a, 7a, and 9a) also showed to be less toxic to the non-target species Artemia salina than the biocide Econea^®. Regarding both AF potency and ecotoxicity activity evaluation, acetophenones 7a and 9a were put forward in this work as promising eco-friendly AF agents.     

Aniquinazolines A–D,Four New Quinazolinone Alkaloids from Marine-Derived Endophytic Fungus Aspergillus nidulans

Four new quinazolinone alkaloids, namely, aniquinazolines A–D (1–4), were isolated and identified from the culture of Aspergillus nidulans MA-143, an endophytic fungus obtained from the leaves of marine mangrove plant Rhizophora stylosa. The structures of the new compounds were elucidated by spectroscopic analysis, and their absolute configurations were determined on the basis of chiral HPLC analysis of the acidic hydrolysates. The structure for 1 was confirmed by single-crystal X-ray diffraction analysis. All these compounds were examined for antibacterial and cytotoxic activity as well as brine shrimp (Artemia salina) lethality.     

Prenylated Indolediketopiperazine Peroxides and Related Homologues from the Marine Sediment-Derived Fungus Penicillium brefeldianum SD-273

Three new indolediketopiperazine peroxides, namely, 24-hydroxyverruculogen (1), 26-hydroxyverruculogen (2), and 13-O-prenyl-26-hydroxyverruculogen (3), along with four known homologues (4–7), were isolated and identified from the culture extract of the marine sediment-derived fungus Penicillium brefeldianum SD-273. Their structures were determined based on the extensive spectroscopic analysis and compound 1 was confirmed by X-ray crystallographic analysis. The absolute configuration of compounds 1–3 was determined using chiral HPLC analysis of their acidic hydrolysates. Each of the isolated compounds was evaluated for antibacterial and cytotoxic activity as well as brine shrimp (Artemia salina) lethality.     

Cyclodepsipeptides and Other O-Containing Heterocyclic Metabolites from Beauveria felina EN-135, a Marine-Derived Entomopathogenic Fungus

Bioassay-guided fractionation of a culture extract of Beauveria felina EN-135, an entomopathogenic fungus isolated from a marine bryozoan, led to the isolation of a new cyclodepsipeptide, iso-isariin D (1); two new O-containing heterocyclic compounds that we have named felinones A and B (2 and 3); and four known cyclodepsipeptides (4–7). The structures were elucidated via spectroscopic analysis, and the absolute configurations of 1 and 2 were determined using single-crystal X-ray diffraction and CD, respectively. All isolated compounds were evaluated for antimicrobial activity and brine-shrimp (Artemia salina) lethality.     

Neuritogenic and Neuroprotective Effects of Polar Steroids from the Far East Starfishes Patiria pectinifera and Distolasterias nipon

The neuritogenic and neuroprotective activities of six starfish polar steroids, asterosaponin Р₁, (25S)-5α-cholestane-3β,4β,6α,7α,8,15α,16β,26-octaol, and (25S)-5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol (1–3) from the starfish Patiria pectinifera and distolasterosides D₁–D₃ (4–6) from the starfish Distolasterias nipon were analyzed using the mouse neuroblastoma (NB) C-1300 cell line and an organotypic rat hippocampal slice culture (OHSC). All of these compounds enhanced neurite outgrowth in NB cells. Dose-dependent responses to compounds 1–3 were observed within the concentration range of 10–100 nM, and dose-dependent responses to glycosides 4–6 were observed at concentrations of 1–50 nM. All the tested substances exhibited notable synergistic effects with trace amounts of nerve growth factor (NGF, 1 ng/mL) or brain-derived neurotrophic factor (BDNF, 0.1 ng/mL). Using NB cells and OHSCs, it was shown for the first time that starfish steroids 1–6 act as neuroprotectors against oxygen-glucose deprivation (OGD) by increasing the number of surviving cells. Altogether, these results suggest that neurotrophin-like neuritogenic and neuroprotective activities are most likely common properties of starfish polyhydroxysteroids and the related glycosides, although the magnitude of the effect depended on the particular compound structure.     

Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat)

Harmful algal blooms (HABs) of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat) as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 – 10 μg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 μg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.     

Recent Updates on Marine Cancer-Preventive Compounds

The natural compounds derived from marine organisms often exhibit unique chemical structures and potent biological activities. Cancer-preventive activity is one of the rather new activities that has emerged and been extensively studied over the last decades. This review summarizes the recent updates on the marine chemopreventive compounds covering the relevant literature published in 2013–2021 and following the previous comprehensive review by Stonik and Fedorov (Marine Drugs 2014, 12, 636–671). In the current article, only the molecules having an effect on malignant transformation (or related pathway and molecules), cancer stem cells, or carcinogen-induced in vivo tumor development were considered to be “true” cancer-preventive compounds and were, therefore, reviewed. Additionally, particular attention has been given to the molecular mechanisms of chemoprevention, executed by the reported marine compounds.     

Secondary Metabolites from Penicillium pinophilum SD-272, a Marine Sediment-Derived Fungus

Two new secondary metabolites, namely, pinodiketopiperazine A (1) and 6,7-dihydroxy-3-methoxy-3-methylphthalide (2), along with alternariol 2,4-dimethyl ether (3) and l-5-oxoproline methyl ester (4), which were isolated from a natural source for the first time but have been previously synthesized, were characterized from the marine sediment-derived fungus Penicillium pinophilum SD-272. In addition, six known metabolites (5–10) were also identified. Their structures were elucidated by analysis of the NMR and mass spectroscopic data. The absolute configuration of compound 1 was determined by experimental and calculated ECD spectra. Compound 2 displayed potent brine shrimp (Artemia salina) lethality with LD₅₀ 11.2 μM.     

Mitigative Effects of PFF-A Isolated from Ecklonia cava on Pigmentation in a Zebrafish Model and Melanogenesis in B16F10 Cells

Melanin synthesis is a defense mechanism that prevents skin damage, but excessive accumulation of melanin occurs in the skin in various reactions such as pigmentation, lentigines, and freckles. Although anti-melanogenic effects have been demonstrated for various naturally occurring marine products that inhibit and control tyrosinase activity, most studies have not been extended to in vivo applications. Phlorofucofuroeckol-A (PFF-A, 12.5–100 µM) isolated from Ecklonia cava has previously been shown to have tyrosinase-mitigative effects in B16F10 cells, but it has not been evaluated in an in vivo model, and its underlying mechanism for anti-melanogenic effects has not been studied. In the present study, we evaluated the safety and efficacy of PFF-A for anti-melanogenic effects in an in vivo model. We selected low doses of PFF-A (1.5–15 nM) and investigated their mitigative effects on pigmentation stimulated by α-MSH in vivo and their related-mechanism in an in vitro model. The findings suggest that low-dose PFF-A derived from E. cava suppresses pigmentation in vivo and melanogenesis in vitro. Therefore, this study presents the possibility that PFF-A could be utilized as a new anti-melanogenic agent in the cosmeceutical industries.     

Polyoxygenated Steroids from the Octocoral Leptogorgia punicea and in Vitro Evaluation of Their Cytotoxic Activity

Five new polyoxygenated marine steroids—punicinols A–E (1–5)—were isolated from the gorgonian Leptogorgia punicea and characterized by spectroscopic methods (IR, MS, ¹H, ¹³C and 2-D NMR). The five compounds induced in vitro cytotoxic effects against lung cancer A549 cells, while punicinols A and B were the most active, with IC₅₀ values of 9.7 μM and 9.6 μM, respectively. The synergistic effects of these compounds with paclitaxel, as well as their effects on cell cycle distribution and their performance in the clonogenic assay, were also evaluated. Both compounds demonstrated significant synergistic effects with paclitaxel.     

Xishaeleganins A–D,Sesquiterpenoid Hydroquinones from Xisha Marine Sponge Dactylospongia elegans

Four new sesquiterpene hydroquinones, xishaeleganins A–D (6–9), along with eleven known related ones (12 and 14–23) were isolated from the Xisha marine sponge Dactylospongia elegans (family Thorectida). Their structures were determined by extensive spectroscopic analysis, ECD calculations, and by comparison with the spectral data reported in the literature. Compounds 7, 15, 20, and 21 showed significant antibacterial activity against Staphylococcus aureus, with minimum inhibitory concentration values of 1.5, 2.9, 5.6, and 5.6 µg/mL, which are comparable with those obtained for the positive control vancomycin (MIC: 1.0 µg/mL).     

Sesquiterpene and Acetogenin Derivatives from the Marine Red Alga Laurencia okamurai

In addition to 13 known compounds, four new bisabolane sesquiterpenes, okamurenes A–D (1–4), a new chamigrane derivative, okamurene E (5), and a new C₁₂-acetogenin, okamuragenin (6), were isolated from the marine red alga Laurencia okamurai. The structures of these compounds were determined through detailed spectroscopic analyses. Of these, okamurenes A and B (1 and 2) are the first examples of bromobisabolane sesquiterpenes possessing a phenyl moiety among Laurencia-derived sesquiterpenes, while okamuragenin (6) was the first acetogenin aldehyde possessing a C₁₂-carbon skeleton. Each of the isolated compounds was evaluated for the brine shrimp (Artemia salina) lethal assay and 7-hydroxylaurene displayed potent lethality with LD₅₀ 1.8 μM.