RESUMO
The identification of molecules whose biological activity can be properly modulated by light is a promising therapeutic approach aimed to improve drug selectivity and efficacy on the molecular target and to limit the side effects compared to traditional drugs. Recently, two photo-switchable diastereomeric benzodiazopyrrole derivatives 1RR and 1RS have been reported as microtubules targeting agents (MTAs) on human colorectal carcinoma p53 null cell line (HCT 116 p53-/-). Their IC50 was enhanced upon Light Emitting Diode (LED) irradiation at 435 nm and was related to their cis form. Here we have investigated the photo-responsive behavior of the acid derivatives of 1RR and 1RS, namely, d1RR and d1RS, in phosphate buffer solutions at different pH. The comparison of the UV spectra, acquired before and after LED irradiation, indicated that the transâcis conversion of d1RR and d1RS is affected by the degree of ionization. The apparent rate constants were calculated from the kinetic data by means of fast UV spectroscopy and the conformers of the putative ionic species present in solution (pH range: 5.7-8.0) were modelled. Taken together, our experimental and theoretical results suggest that the photo-conversions of trans d1RR/d1RS into the corresponding cis forms and the thermal decay of cis d1RR/d1RS are dependent on the presence of diazonium form of d1RR/d1RS. Finally, a photo-reaction was detected only for d1RR after prolonged LED irradiation in acidic medium, and the resulting product was characterized by means of Liquid Chromatography coupled to High resolution Mass Spectrometry (LC-HRMS) and Nuclear Magnetic Resonance (NMR) spectroscopy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/terapia , Fotoquimioterapia , Pirróis/farmacologia , Cromatografia Líquida , Neoplasias Colorretais/patologia , Compostos de Diazônio/química , Compostos de Diazônio/farmacologia , Células HCT116 , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Pirróis/químicaRESUMO
Fifty-five strains of Ostreopsis were collected in the Mediterranean Sea and analyzed to characterize their toxin profiles. All the strains were grown in culture under the same experimental conditions and identified by molecular PCR assay based on the ITS-5.8S rDNA. A liquid chromatography-high resolution multiple stage mass spectrometry (LC-HRMSn) approach was used to analyze toxin profiles and to structurally characterize the detected toxins. Despite morphological and molecular characterization being consistent within the species O. cf. ovata, a certain degree of toxin variability was observed. All the strains produced ovatoxins (OVTXs), with the exception of only one strain. Toxin profiles were quite different from both qualitative and quantitative standpoints: 67% of the strains contained OVTX-a to -e, OVTX-g, and isobaric PLTX, in 25% of them only OVTX-a, -d, -e and isobaric PLTX were present, while 4% produced only OVTX-b and -c. None of the strains showed a previously identified profile, featuring OVTX-f as dominant toxin, whereas OVTX-f was a minor component of very few strains. Toxin content was mostly in the range 4-70 pg/cell with higher levels (up to 238 pg/cell) being found in strains from the Ligurian and South Adriatic Sea. Structural insights into OVTX-b, -c, -d, and -e were gained, and the new OVTX-l was detected in 36 strains.
Assuntos
Dinoflagellida , Toxinas Marinhas , Cromatografia Líquida , Mar Mediterrâneo , Espectrometria de Massas em TandemRESUMO
Anecdotal reports exist of aquarium hobbyists that experienced severe respiratory distress and/or skin injury following cleaning operation of home aquaria containing Palythoa sp. soft corals. Hundreds of cases of respiratory illness and/or dermatitis have been recorded in proximity to the sea concomitantly with algal blooms of Ostreopsis spp. in the Mediterranean area. Both Palythoa spp. and Ostreopsis spp. contain congeners of palytoxin, a highly potent toxin whose inhalation hazard is however unknown. In this study, we demonstrate the presence of high levels of palytoxins (palytoxin and hydroxypalytoxin) in both soft coral and seawater from a home marine aquarium involved in the poisoning of a whole family. Due to the high toxin levels found in seawater, a procedure for a rapid and efficient determination of palytoxin in seawater was setup. A comparison of symptoms of Palythoa- and Ostreopsis-related inhalatory poisonings showed many similarities including fever, respiratory distress, nausea, and flu-like symptoms. From the chemical and symptomatological data reported herein it is reasonable to hold palytoxins responsible for respiratory disorders following inhalation. Although the exact mechanism through which palytoxin congeners exert their inhalatory toxicity is still unknown, this represents a step toward demonstrating that palytoxin congeners exert toxic effects through inhalation both in natural environments and in the surroundings of private and public aquaria.
Assuntos
Acrilamidas/análise , Acrilamidas/intoxicação , Antozoários/química , Dinoflagellida/química , Acrilamidas/administração & dosagem , Administração por Inalação , Adolescente , Adulto , Animais , Venenos de Cnidários/análise , Venenos de Cnidários/intoxicação , Exposição Ambiental/efeitos adversos , Humanos , Piranos/análise , Piranos/intoxicação , Água do Mar/química , Extração em Fase SólidaRESUMO
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 Ostreopsis cf. ovata in the Mediterranean Sea. Its increasing detection during Ostreopsis 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 (Kd = 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 O. cf. ovata 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.
Assuntos
Dinoflagellida/química , Toxinas Marinhas/toxicidade , Acrilamidas , Animais , Bivalves/química , Linhagem Celular , Venenos de Cnidários , Ensaio de Imunoadsorção Enzimática , Humanos , Toxinas Marinhas/isolamento & purificação , Mar Mediterrâneo , Alimentos Marinhos , Frutos do Mar , Testes de ToxicidadeRESUMO
Blooms of benthic dinoflagellates of the genus Ostreopsis (mainly O. cf. ovata and occasionally O. cf. siamensis) represent a serious concern for humans in the Mediterranean area, due to production of palytoxin-like compounds listed among the most potent marine toxins known. In this work, six strains of Ostreopsis sp. from Cyprus Island were analyzed through an integrated approach based on molecular, chemical, and eco-toxicological methods. Cypriot Ostreopsis sp. was found to be a species distinct from O. cf. ovata and O. cf. siamensis, belonging to the Atlantic/Mediterranean Ostreopsis spp. clade. Some variability in toxin profiles emerged: three strains produced ovatoxin-a (OVTX-a), OVTX-d, OVTX-e, and isobaric palytoxin, so far found only in O. cf. ovata; the other three strains produced only new palytoxin-like compounds, which we named ovatoxin-i, ovatoxin-j1, ovatoxin-j2, and ovatoxin-k. The new ovatoxins present the same carbon skeleton as ovatoxin-a, differing primarily in an additional C2H2O2 moiety and an unsaturation in the region C49-C52. Other minor structural differences were found, including the presence of a hydroxyl group at C44 (in OVTX-j1 and OVTX-k) and the lack of a hydroxyl group in the region C53-C78 (in OVTX-i and OVTX-j1). The toxin content of the analyzed Ostreopsis sp. strains was in the range 0.06-2.8 pg cell(-1), definitely lower than that of a Ligurian O. cf. ovata strain cultured under the same conditions. Accordingly, an eco-toxicological test on Artemia salina nauplii demonstrated that Ostreopsis sp. presents a very low toxicity compared to O. cf. ovata. The whole of these data suggest that Ostreopsis sp. from Cyprus Island poses a relatively low risk to humans.
Assuntos
Dinoflagellida/química , Toxinas Marinhas/química , Toxinas Marinhas/toxicidade , Animais , Artemia/efeitos dos fármacos , Cromatografia Líquida , Dinoflagellida/classificação , Dinoflagellida/metabolismo , Ilhas , Toxinas Marinhas/metabolismo , Espectrometria de Massas , Estrutura MolecularRESUMO
The new benthic toxic dinoflagellate, Ostreopsis fattorussoi sp. nov., is described from the Eastern Mediterranean Sea, Lebanon and Cyprus coasts, and is supported by morphological and molecular data. The plate formula, Po, 3', 7â³, 6c, 7s, 5â´, 2'''', is typical for the Ostreopsis genus. It differs from all other Ostreopsis species in that (i) the curved suture between plates 1' and 3' makes them approximately hexagonal, (ii) the 1' plate lies in the left half of the epitheca and is obliquely orientated leading to a characteristic shape of plate 6â³. The round thecal pores are bigger than the other two Mediterranean species (O. cf. ovata and O. cf. siamensis). O. fattorussoi is among the smallest species of the genus (DV: 60.07 ± 5.63 µm, AP: 25.66 ± 2.97 µm, W: 39.81 ± 5.05 µm) along with O. ovata. Phylogenetic analyses based on the LSU and internal transcribed spacer rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. Ostreopsis fattorussoi produces OVTX-a and structural isomers OVTX-d and -e, O. cf. ovata is the only other species of this genus known to produce these toxins. The Lebanese O. fattorussoi did not produce the new palytoxin-like compounds (ovatoxin-i, ovatoxin-j1 , ovatoxin-j2 , and ovatoxin-k) that were previously found in O. fattorussoi from Cyprus. The toxin content was in the range of 0.28-0.94 pg · cell-1 . On the Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18°C-31.5°C), with peaks in June and August.
Assuntos
Dinoflagellida/classificação , Chipre , DNA de Algas/genética , Dinoflagellida/genética , Dinoflagellida/ultraestrutura , Líbano , Mar Mediterrâneo , Filogenia , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Paralytic shellfish poisoning (PSP) is a serious human illness caused by the ingestion of seafood contaminated with saxitoxin and its derivatives (STXs). These toxins are produced by some species of marine dinoflagellates within the genus Alexandrium. In the Mediterranean Sea, toxic Alexandrium spp. blooms, especially of A. minutum, are frequent and intense with negative impact to coastal ecosystem, aquaculture practices and other economic activities. We conducted a large scale study on the sxt gene and toxin distribution and content in toxic dinoflagellate A. minutum of the Mediterranean Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques. We developed a new qPCR assay for the estimation of the sxtA1 gene copy number in seawater samples during a bloom event in Syracuse Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 × 10° sxtA1 gene copy number per reaction. The linear correlation between sxtA1 gene copy number and microalgal abundance and between the sxtA1 gene and STX content allowed us to rapidly determine the STX-producing cell concentrations of two Alexandrium species in environmental samples. In these samples, the amount of sxtA1 gene was in the range of 1.38 × 10(5) - 2.55 × 10(8) copies/L and the STX concentrations ranged from 41-201 nmol/L. This study described a potential PSP scenario in the Mediterranean Sea.
Assuntos
Dinoflagellida/patogenicidade , Monitoramento Ambiental/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Saxitoxina/genética , Intoxicação por Frutos do Mar , Dinoflagellida/genética , Ecossistema , Marcadores Genéticos , Humanos , Mar Mediterrâneo , Microalgas/genética , Saxitoxina/toxicidade , Água do Mar/parasitologia , Intoxicação por Frutos do Mar/parasitologiaRESUMO
Palytoxins from Ostreopsis cf. ovata (a putative palytoxin and ovatoxins) are emerging toxins in the Mediterranean basin and are not yet regulated, although there is evidence that they can accumulate in seafood and thus enter the human food chain. This poses serious concerns for human health, because palytoxin itself is among the most potent marine toxins known. In 2009, the European Food Safety Authority (EFSA) announced the need for optimization of efficient analytical methods for detecting palytoxins and for preparing standards. Herein, we propose a procedure including a one-step extraction, solid-phase-extraction (SPE) clean-up, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) detection of individual palytoxins in mussels. The method enabled efficient chromatographic separation of individual compounds, including structural isomers, with good sensitivity, reproducibility, and linearity in a large dynamic range (14-1000 ng mL(-1) in matrix). As a result, the putative palytoxin from Ostreopsis cf. ovata was identified as an isomer of palytoxin itself and re-named isobaric palytoxin. The whole procedure (sample preparation and LC-HRMS analysis) proved able to detect palytoxins in both spiked and natural mussel samples at levels as low as 70 µg kg(-1) in crude mussel extracts and 15 µg kg(-1) after SPE clean-up. Although full validation of the method is currently prevented by the unavailability of palytoxin(s) certified standards and reference material, this study constitutes a first step towards achieving this.
Assuntos
Acrilamidas/análise , Bivalves/química , Cromatografia Líquida/métodos , Toxinas Marinhas/análise , Espectrometria de Massas/métodos , Frutos do Mar/análise , Animais , Venenos de Cnidários , Contaminação de Alimentos/análise , Inocuidade dos AlimentosRESUMO
Blooms of the benthic dinoflagellate Ostreopsis cf. ovata are a concern in the Mediterranean Sea, since this species produces a wide range of palytoxin-like compounds listed among the most potent marine toxins. This study focused on two analogs of palytoxin found in cultures of six strains of O. cf. ovata isolated from the south of Catalonia (NW Mediterranean Sea). In addition to some already known ovatoxins, our strains produced two minor compounds, ovatoxin-g and the so far called putative palytoxin, whose structures had not been elucidated before. Insufficient quantity of these compounds impeded a full nuclear magnetic resonance (NMR)-based structural elucidation; thus, we studied their structure in crude algal extracts through liquid chromatography-electrospray ionization high-resolution mass spectrometry(n) (LC-ESI-HRMS(n)) in positive ion mode. Under the used MS conditions, the molecules underwent fragmentation at many sites of their backbone and a large number of diagnostic fragment ions were identified. As a result, tentative structures were assigned to both ovatoxin-g and the putative palytoxin, the latter being identified as a palytoxin isomer and re-named as isobaric palytoxin.
Assuntos
Acrilamidas/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Dinoflagellida/química , Éteres Cíclicos/isolamento & purificação , Água do Mar , Espectrometria de Massas por Ionização por Electrospray/métodos , Venenos de Cnidários , Mar Mediterrâneo , Estrutura Molecular , Água do Mar/análise , Água do Mar/microbiologiaRESUMO
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 Ca(2+) is detected when palytoxin binds to the pump. It has been demonstrated that the increase of cytosolic free Ca(2+) 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-Ca(2+) complex in aqueous solution by NMR- and molecular modeling-based approach. We identified two specific regions of palytoxin where Ca(2+) 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.
Assuntos
Acrilamidas/química , Cálcio/química , Cátions/química , ATPase Trocadora de Sódio-Potássio/química , Venenos de Cnidários , Espectroscopia de Ressonância Magnética , Modelos Moleculares , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Since the late 1990s, a respiratory syndrome has been repetitively observed in humans concomitant with Ostreopsis spp. blooms (mainly O. cf. ovata) in the Mediterranean area. Previous studies have demonstrated that O. cf. ovata produces analogues of palytoxin (ovatoxins and a putative palytoxin), one of the most potent marine toxins. On the basis of the observed association between O. cf. ovata blooms, respiratory illness in people, and detection of palytoxin complex in algal samples, toxic aerosols, containing Ostreopsis cells and/or the toxins they produce, were postulated to be the cause of human illness. A small scale monitoring study of marine aerosol carried out along the Tuscan coasts (Italy) in 2009 and 2010 is reported. Aerosols were collected concomitantly with O. cf. ovata blooms, and they were analyzed by both PCR assays and LC-HRMS. The results, besides confirming the presence of O. cf. ovata cells, demonstrated for the first time the occurrence of ovatoxins in the aerosol at levels of 2.4 pg of ovatoxins per liter of air. Given the lack of toxicological data on palytoxins by inhalation exposure, our results are only a first step toward a more comprehensive understanding of the Ostreopsis-related respiratory syndrome.
Assuntos
Dinoflagellida/química , Monitoramento Ambiental/métodos , Toxinas Marinhas/análise , Acrilamidas/análise , Acrilamidas/química , Aerossóis/análise , Venenos de Cnidários , Dinoflagellida/genética , Dinoflagellida/isolamento & purificação , Itália , Biologia Marinha , Toxinas Marinhas/química , Água do MarRESUMO
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 Palythoa tuberculosa sample. This new toxin is a stereoisomer of 42-hydroxypalytoxin isolated from Palythoa toxica. 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 P. tuberculosa showed cytotoxicity toward skin HaCaT keratinocytes approximately 1 order of magnitude lower than that of 42-hydroxypalytoxin from P. toxica 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.
Assuntos
Acrilamidas/farmacologia , Antozoários/química , Toxinas Marinhas/química , Acrilamidas/química , Animais , Cromatografia Líquida de Alta Pressão , Venenos de Cnidários/química , Venenos de Cnidários/farmacologia , Havaí , Humanos , Queratinócitos/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Mar Mediterrâneo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Piranos/química , Piranos/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.
Assuntos
Dinoflagellida/genética , Expressão Gênica/genética , Saxitoxina/análogos & derivados , Saxitoxina/genética , Dinoflagellida/metabolismo , Família Multigênica/genética , Neurotoxinas/genética , Neurotoxinas/metabolismo , RNA Mensageiro/genética , Saxitoxina/metabolismo , Intoxicação por Frutos do Mar/genética , Intoxicação por Frutos do Mar/metabolismoRESUMO
Cyanobacteria in water supplies are considered an emerging threat, as some species produce toxic metabolites, cyanotoxins, of which the most widespread and well-studied are microcystins. Consumption of contaminated water is a common exposure route to cyanotoxins, making the study of cyanobacteria in drinking waters a priority to protect public health. In drinking water treatment plants, pre-oxidation with chlorinated compounds is widely employed to inhibit cyanobacterial growth, although concerns on its efficacy in reducing cyanotoxin content exists. Additionally, the effects of chlorination on abundant but less-studied cyanometabolites (e.g. cyanopeptolins whose toxicity is still unclear) remain poorly investigated. Here, two chlorinated oxidants, sodium hypochlorite (NaClO) and chlorine dioxide (ClO2), were tested on the toxic cyanobacterium Microcystis aeruginosa, evaluating their effect on cell viability, toxin profile and content. Intra- and extracellular microcystins and other cyanometabolites, including their degradation products, were identified using an untargeted LC-HRMS approach. Both oxidants were able to inactivate M. aeruginosa cells at a low dose (0.5 mg L-1), and greatly reduced intracellular toxins content (>90%), regardless of the treatment time (1-3 h). Conversely, a two-fold increase of extracellular toxins after NaClO treatment emerged, suggesting a cellular damage. A novel metabolite named cyanopeptolin-type peptide-1029, was identified based on LC-HRMSn (n = 2, 3) evidence, and it was differently affected by the two oxidants. NaClO led to increase its extracellular concentration from 2 to 80-100 µg L-1, and ClO2 induced the formation of its oxidized derivative, cyanopeptolin-type peptide-1045. In conclusion, pre-oxidation treatments of raw water contaminated by toxic cyanobacteria may lead to increased cyanotoxin concentrations in drinking water and, depending on the chemical agent, its dose and treatment duration, also of oxidized metabolites. Since the effects of such metabolites on human health remain unknown, this issue should be handled with extreme caution by water security agencies involved in drinking water management.
RESUMO
Cyanobacteria produce a plethora of structurally diverse bioactive secondary metabolites, including cyanotoxins which pose a serious threat to humans and other living organisms worldwide. Currently, a wide variety of mass spectrometry-based methods for determination of microcystins (MCs), the most commonly occurring and studied class of cyanotoxins, have been developed and employed for research and monitoring purposes. The scarcity of commercially available reference materials, together with the ever-growing range of mass spectrometers and analytical approaches, make the accuracy of quantitative analyses a critical point to be carefully investigated in view of a reliable risk evaluation. This study reports, a comparative investigation of the qualitative and quantitative MCs profile obtained using targeted and untargeted liquid chromatography-mass spectrometry approaches for the analyses of cyanobacterial biomass from Lake Kastoria, Greece. Comparison of the total MCs content measured by the two approaches showed good correlation, with variations in the range of 3.8-13.2%. In addition, the implementation of an analytical workflow on a hybrid linear ion trap/orbitrap mass spectrometer is described, based on combining data-dependent acquisition and a powerful database of cyanobacterial metabolites (CyanoMetDB) for the annotation of known and discovery of new cyanopeptides. This untargeted strategy proved highly effective for the identification of MCs, microginins, anabaenopeptins, and micropeptins. The systematic interpretation of the acquired fragmentation patterns allowed the elucidation of two new MC structural variants, MC-PrhcysR and MC-Prhcys(O)R, and proposal of structures for two new microginins, isomeric cyanostatin B and MG 821A, and three isomeric micropeptins at m/z 846.4715, 846.4711 and 846.4723.
Assuntos
Cianobactérias , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida/métodos , Fluxo de Trabalho , Cianobactérias/metabolismo , Microcistinas/química , Oligopeptídeos/metabolismoRESUMO
Marine toxins have a significant impact on seafood resources and human health. Up to date, mainly based on bioassays results, two genera of toxic microalgae, Gambierdiscus and Fukuyoa have been hypothesized to produce a suite of biologically active compounds, including maitotoxins (MTXs) and ciguatoxins (CTXs) with the latter causing ciguatera poisoning (CP) in humans. The global ubiquity of these microalgae and their ability to produce (un-)known bioactive compounds, necessitates strategies for screening, identifying, and reducing the number of target algal species and compounds selected for structural elucidation. To accomplish this task, a dereplication process is necessary to screen and profile algal extracts, identify target compounds, and support the discovery of novel bioactive chemotypes. Herein, a dereplication strategy was applied to a crude extract of a G. balechii culture to investigate for bioactive compounds with relevance to CP using liquid chromatography-high resolution mass spectrometry, in vitro cell-based bioassay, and a combination thereof via a bioassay-guided micro-fractionation. Three biologically active fractions exhibiting CTX-like and MTX-like toxicity were identified. A naturally incurred fish extract (Sphyraena barracuda) was used for confirmation where standards were unavailable. Using this approach, a putative I/C-CTX congener in G. balechii was identified for the first time, 44-methylgambierone was confirmed at 8.6 pg cell-1, and MTX-like compounds were purported. This investigative approach can be applied towards other harmful algal species of interest. The identification of a microalgal species herein, G. balechii (VGO920) which was found capable of producing a putative I/C-CTX in culture is an impactful advancement for global CP research. The large-scale culturing of G. balechii could be used as a source of I/C-CTX reference material not yet commercially available, thus, fulfilling an analytical gap that currently hampers the routine determination of CTXs in various environmental and human health-relevant matrices.
Assuntos
Ciguatera , Ciguatoxinas , Dinoflagellida , Animais , Humanos , Ciguatoxinas/toxicidade , Ciguatoxinas/análise , Toxinas Marinhas/análise , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
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.
Assuntos
Dinoflagellida/química , Toxinas Marinhas/química , Toxinas Marinhas/toxicidade , Acrilamidas/química , Acrilamidas/isolamento & purificação , Acrilamidas/toxicidade , Animais , Cromatografia Líquida , Venenos de Cnidários , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Toxinas Marinhas/isolamento & purificação , Espectrometria de Massas , Camundongos , Testes de Toxicidade AgudaRESUMO
Ovatoxin-a is the main toxin produced by Ostreopsis ovata, a benthic dinoflagellate that has bloomed massively across the Mediterranean basin over the past years, inflicting both human and environmental suffering. Ovatoxin-a has recently been isolated from cultures of O.â ovata and structurally identified as an analogue of palytoxin: in comparison with palytoxin, ovatoxin-a lacks three hydroxy groups at the 17-, 44- and 64-positions, but features an extra hydroxy functionality at the 42-position. Herein we report on the NMR-based elucidation of the stereochemistry of ovatoxin-a, which includes 7 stereogenic double bonds and 62 asymmetric carbon atoms. Understanding the full stereochemistry of ovatoxin-a is a step towards the elucidation of its mechanism of action on a molecular level.
Assuntos
Dinoflagellida/química , Toxinas Marinhas/química , Acrilamidas/química , Cromatografia Líquida de Alta Pressão , Venenos de Cnidários , Espectroscopia de Ressonância Magnética , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/toxicidade , Espectrometria de Massas , Modelos Moleculares , Conformação Molecular , EstereoisomerismoRESUMO
Currently, the benthic dinoflagellate Ostreopsis cf. ovata represents a serious concern to human health in the whole Mediterranean basin due to the production of palytoxin congeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d/-e), listed among the most potent marine toxins. High resolution liquid chromatography-mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain of Ostreopsis cf. ovata collected at Portonovo (Italy) in 2008 is reported herein. Toxin profile was different from those previously reported for other O. cf. ovata, both qualitatively and quantitatively. For the first time, ovatoxin-a did not dominate the toxin profile, and a new palytoxin congener, here named ovatoxin-f, was detected. Ovatoxin-f and its elemental formula present C(2)H(4) more than ovatoxin-a. HR CID MS(n) experiments allowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region not previously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile of the analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of the Mediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, the presence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.
Assuntos
Dinoflagellida/química , Toxinas Marinhas/química , Cromatografia Líquida de Alta Pressão , Dinoflagellida/classificação , Toxinas Marinhas/isolamento & purificação , Espectrometria de Massas , Mar Mediterrâneo , Microalgas/química , Água do Mar/químicaRESUMO
Mass appearances of the toxic dinoflagellate genus Ostreopsis are known to cause dangerous respiratory symptoms in humans exposed to aerosols. The outbreaks can appear in shallow marine waters of temperate regions around the globe. We followed a massive bloom event on a public beach on the northern Adriatic coast near Rovinj, Croatia. We identified the responsible species and the produced toxins as well as the dynamics of the event with respect to environmental conditions. Ostreopsis cf. ovata appeared in masses from September through October 2010 on a public beach near Rovinj, Croatia but stayed undetected by public health organizations. Respiratory symptoms were observed whenever humans were exposed to substrate samples containing large numbers of Ostreopsis cells. During the mass abundance of O. cf. ovata also exposure to the aerosols on the beach evoked respiratory symptoms in humans. Our measurements showed high cell abundances and high toxin contents with a stable relative contribution of putative Palytoxin and Ovatoxins a-e. Artificial beach structures proved to dramatically reduce settling of the observed Ostreopsis biofilm. Blooms like those reported herein have a high potential to happen undetected with a high potential of affecting the health of coastal human populations. Increased monitoring efforts are therefore required to understand the ecology and toxicology of those bloom events and reduce their negative impact on coastal populations.