Search Results (66)

Among aquatic organisms, marine dinoflagellates are essential sources of bioactive metabolites. The benthic dinoflagellate Coolia malayensis produces metabolites that have exhibited substantial and specific cytotoxicity on cancer cells; however, isolation and identification of the purified compounds remain a challenge. This study reports C. malayensis biomass multi-step extraction plus chemical analyses for identifying compounds with antineoplastic activity. Through bio-directed fractionation, the cytotoxicity of extracts and fractions was tested on H1299 (lung), PC-3 (prostate), HeLa (cervical), and MCF-7 (breast) cancer cell lines. Dichloromethane (DCM) phase, hydroalcoholic (HYD) secondary extract, and methanolic (MET) extract showed cytotoxic effects on all cell lines. Active extracts and fractions were analyzed by HPLC-QTOF-MS, ¹H, and ¹³C NMR. Cell lines H1299 and PC-3 treated with fractions F4, F7, and DCM2-AQ-Ch sub-extract showed morphological changes resembling those observed in the apoptosis control, and no signs of necrosis were observed. The selectivity of fraction F7 was above 100 μg mL⁻¹ for healthy cells, while cytotoxic activity was observed in cancer cells. This fraction was identified as mostly fatty acids (FA) by NMR. Seventeen compounds with reported biological activities, such as antioxidant, analgesic, antiviral, and anticancer, were identified from C. malayensis extracts and fractions. Among them, the phycotoxins gambieric acid A and B, okadaic acid, and dinophysistoxin-1 were detected. Further studies are needed to reveal more significant anti-cancer potential from C. malayensis. Full article

The knowledge of the routes of excretion of the toxins accumulated by molluscs is a key step in designing methods that accelerate depuration. In this work, the excretion route, in mussels and cockles, of the main diarrhetic shellfish poisoning (DSP) toxins in Europe (okadaic acid and dinophysistoxin-2) after natural intoxication were studied. During depuration, the amounts of free toxins and their derivatives were quantified in bivalves, faeces, and water. Most toxins (>98%) were excreted through faeces as acyl derivatives (most likely 7-O-acyl esters), independent of the ratio between these derivatives and free toxins in soft tissues. The small proportion of toxins excreted into water mostly constituted the free forms of the toxins. Both species shared the same route even though they contained very different proportions of free toxins in their soft tissues. No substantial changes in this general pattern were observed during the experiment. The esters of fatty acids with 16 carbon atoms were the most abundant in both soft tissues and faeces, but they were not the same in mussels and cockles. Most of the variability in ester proportions can be attributed to the species more than to their differential excretion (water or faeces) suggesting that there are not large differences in the depuration of the different esters. Full article

Harmful algal blooms are an expanding phenomenon negatively impacting human health, socio-economic welfare, and ecosystems. Such events increase the risk of marine organisms’ exposure to algal toxins with consequent ecological effects. In this frame, the objective of this study was to investigate the ecotoxicological potential of three globally distributed dinoflagellate toxins (okadaic acid, OA; dinophysistoxin-1, DTX-1; dinophysistoxin-2, DTX-2) using Artemia franciscana as a model organism of marine zooplankton. Each toxin (0.1–100 nM) was evaluated for its toxic effects in terms of cyst hatching, mortality of nauplii Instar I and adults, and biochemical responses related to oxidative stress. At the highest concentration (100 nM), these toxins significantly increased adults’ mortality starting from 24 h (DTX-1), 48 h (OA), or 72 h (DTX-2) exposures, DTX-1 being the most potent one, followed by OA and DTX-2. The quantitation of oxidative stress biomarkers in adults, i.e., reactive oxygen species (ROS) production and activity of three endogenous antioxidant defense enzymes (glutathione S-transferase, superoxide dismutase, and catalase) showed that only DTX-2 significantly increased ROS production, whereas each toxin affected the antioxidant enzymes with a different activity profile. In general, the results indicate a negative impact of these toxins towards A. franciscana with potential consequences on the marine ecosystem. Full article

Prorocentrum, a dinoflagellate responsible for producing diarrhetic shellfish poisoning (DSP) toxins, poses significant threats to marine ecosystems, aquaculture industries, and human health. DSP toxins, including okadaic acid (OA), dinophysis toxin (DTX), and their diverse derivatives, continue to be identified and characterized. In this study, we report the isolation of four new diol esters of OA/DTX-1 from large-scale cultures of Prorocentrum lima. Their chemical structures were elucidated through comprehensive NMR and MS analyses, along with structural comparisons with the well-known OA. Notably, compound 1 featured an additional ester group within the diol unit, while compound 2 was revealed to be a C11 diol ester. The cytotoxicity of these newly isolated derivatives was evaluated against three cell lines: Neuro2a (mouse), HCT116 (human), and HepG2 (human). All diol esters exhibited cytotoxic effects, with compound 3 displaying toxicity comparable to OA. These results expand our understanding of DSP toxin diversity and provide valuable insight into the structural variations and biological activity of diol esters of OA/DTX-1. Full article

The Tam Giang-Cau Hai lagoon (TGCH) in Thua Thien Hue province (Vietnam) is a marsh/lagoon system and ranks among the largest waterbodies in Southeast Asia. It plays a significant role in terms of both socio-economic and environmental resources. However, anthropogenic stress, as well as the discharge of untreated domestic and industrial sewage with agricultural runoff from its three major tributaries, dramatically damages the water quality of the lagoon. Especially after heavy rain and flash floods, the continuous degradation of its water quality, followed by harmful algal and cyanobacterial bloom patterns (HABs), is more perceptible. In this study, several physicochemical factors, cyanotoxins (anatoxins (ATXs), saxitoxins (STXs), microcystins (MCs)), phycotoxins (STXs, okadaic acid (OA), and dinophysistoxins (DTXs)) were analyzed in water and shellfish samples from 13 stations in June 2023 from 13 stations, using enzyme-linked immunosorbent assay (ELISA) kits for the ATXs and STXs, and the serine/threonine phosphatase type 2A (PP2A) inhibition assay kit for the MCs, OA, and DTXs. The results showed for the first time the co-occurrence of freshwater cyanotoxins and marine phycotoxins in water and shellfish samples in this lagoon. Traces of ATXs and STXs were detected in the shellfish and the orders of magnitude were below the seafood safety action levels. However, toxins inhibiting the PP2A enzyme, such as MCs and nodularin (NODs), as well as OA and DTXs, were detected at higher concentrations (maximum: 130.4 μg equiv. MC-LR/kg shellfish meat wet weight), approaching the actionable level proposed for this class of toxin in shellfish (160 μg of OA equivalent per kg of edible bivalve mollusk meat). It is very important to note that due to the possible false positives produced by the ELISA test in complex matrices such as a crude shellfish extract, this preliminary and pilot research will be repeated with a more sophisticated method, such as liquid chromatography coupled with mass spectroscopy (LC-MS), in the upcoming research plan. Full article

Few works have been carried out on benthic harmful algal blooms (BHAB) species in the southern Mediterranean and no data are available for the highly dynamic Strait of Gibraltar (western Mediterranean waters). For the first time, Ostreopsis sp. 9, Prorocentrum lima and Coolia monotis were isolated in this key region in terms of exchanges between the Atlantic Ocean and the Mediterranean and subject to intense maritime traffic. Ribotyping confirmed the morphological identification of these three dinoflagellates species. Monoclonal cultures were established and the maximum growth rate and cell yield were measured at a temperature of 24 °C and an irradiance of 90 µmol photons m⁻² s⁻¹, for each species: 0.26 ± 0.02 d⁻¹ (8.75 × 10³ cell mL⁻¹ after 28 days) for Ostreopsis sp. 9, 0.21 ± 0.01 d⁻¹ (49 × 10³ cell mL⁻¹ after 145 days) for P. lima and 0.21 ± 0.01 d⁻¹ (10.02 × 10³ cell mL⁻¹ after 28 days) for C. monotis. Only P. lima was toxic with concentrations of okadaic acid and dinophysistoxin-1 measured in optimal growth conditions ranging from 6.4 pg cell⁻¹ to 26.97 pg cell⁻¹ and from 5.19 to 25.27 pg cell⁻¹, respectively. The toxin content of this species varied in function of the growth phase. Temperature influenced the growth and toxin content of P. lima. Results suggest that future warming of Mediterranean coastal waters may lead to higher growth rates and to increases in cellular toxin levels in P. lima. Nitrate and ammonia affected the toxin content of P. lima but no clear trend was noted. In further studies, we have to isolate other BHAB species and strains from Strait of Gibraltar waters to obtain more insight into their diversity and toxicity. Full article

For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels (Mytilus galloprovincialis) as sentinel organisms for the toxicity in non-bivalve invertebrates from the same area. The screening of multiple lipophilic toxins in 1150 samples has allowed reporting for the first time the presence of 13-desmethyl spirolide C, pinnatoxin G, okadaic acid, and dinophysistoxins 2 in a variety of non-traditional vectors. In general, these two emerging toxins showed the highest prevalence (12.5–75%) in most of the groups studied. Maximum levels for 13-desmethyl spirolide C and pinnatoxin G were found in the bivalves Magallana gigas (21 µg kg⁻¹) and Tellina donacina (63 µg kg⁻¹), respectively. However, mean concentrations for the bivalve group were shallow (2–6 µg kg⁻¹). Okadaic acid and dinophysistoxin 2 with lower prevalence (1.6–44.4%) showed, on the contrary, very high concentration values in specific species of crustaceans and polychaetes (334 and 235 µg kg⁻⁻¹, respectively), to which special attention should be paid. Statistical data analyses showed that mussels could be considered good biological indicators for the toxicities of certain groups in a particular area, with correlations between 0.710 (for echinoderms) and 0.838 (for crustaceans). Polychaetes could be an exception, but further extensive surveys would be needed to draw definitive conclusions. Full article

Okadaic acid (OA) and its analogues cause diarrhetic shellfish poisoning (DSP) in humans, and risk assessments of these toxins require toxicity equivalency factors (TEFs), which represent the relative toxicities of analogues. However, no human death by DSP toxin has been reported, and its current TEF value is based on acute lethality. To properly reflect the symptoms of DSP, such as diarrhea without death, the chronic toxicity of DSP toxins at sublethal doses should be considered. In this study, we obtained acute oral LD₅₀ values for OA and dinophysistoxin-1 (DTX-1) (1069 and 897 μg/kg, respectively) to set sublethal doses. Mice were treated with sublethal doses of OA and DTX-1 for 7 days. The mice lost body weight, and the disease activity index and intestinal crypt depths increased. Furthermore, these changes were more severe in OA-treated mice than in the DTX-1-treated mice. Strikingly, ascites was observed, and its severity was greater in mice treated with OA. Our findings suggest that OA is at least as toxic as DTX-1 after repeated oral administration at a low dose. This is the first study to compare repeated oral dosing of DSP toxins. Further sub-chronic and chronic studies are warranted to determine appropriate TEF values for DSP toxins. Full article

Harmful algal blooms (HABs) in coastal British Columbia (BC), Canada, negatively impact the salmon aquaculture industry. One disease of interest to salmon aquaculture is Net Pen Liver Disease (NPLD), which induces severe liver damage and is believed to be caused by the exposure to microcystins (MCs). To address the lack of information about algal toxins in BC marine environments and the risk they pose, this study investigated the presence of MCs and other toxins at aquaculture sites. Sampling was carried out using discrete water samples and Solid Phase Adsorption Toxin Tracking (SPATT) samplers from 2017–2019. All 283 SPATT samples and all 81 water samples tested positive for MCs. Testing for okadaic acid (OA) and domoic acid (DA) occurred in 66 and 43 samples, respectively, and all samples were positive for the toxin tested. Testing for dinophysistoxin-1 (DTX-1) (20 samples), pectenotoxin-2 (PTX-2) (20 samples), and yessotoxin (YTX) (17 samples) revealed that all samples were positive for the tested toxins. This study revealed the presence of multiple co-occurring toxins in BC’s coastal waters and the levels detected in this study were below the regulatory limits for health and recreational use. This study expands our limited knowledge of algal toxins in coastal BC and shows that further studies are needed to understand the risks they pose to marine fisheries and ecosystems. Full article

The successful cultivation of Dinophysis norvegica Claparède & Lachmann, 1859, isolated from Japanese coastal waters, is presented in this study, which also includes an examination of its toxin content and production for the first time. Maintaining the strains at a high abundance (>2000 cells per mL⁻¹) for more than 20 months was achieved by feeding them with the ciliate Mesodinium rubrum Lohmann, 1908, along with the addition of the cryptophyte Teleaulax amphioxeia (W.Conrad) D.R.A.Hill, 1992. Toxin production was examined using seven established strains. At the end of the one-month incubation period, the total amounts of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged between 132.0 and 375.0 ng per mL⁻¹ (n = 7), and 0.7 and 3.6 ng per mL⁻¹ (n = 3), respectively. Furthermore, only one strain was found to contain a trace level of okadaic acid (OA). Similarly, the cell quota of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged from 60.6 to 152.4 pg per cell⁻¹ (n = 7) and 0.5 to 1.2 pg per cell⁻¹ (n = 3), respectively. The results of this study indicate that toxin production in this species is subject to variation depending on the strain. According to the growth experiment, D. norvegica exhibited a long lag phase, as suggested by the slow growth observed during the first 12 days. In the growth experiment, D. norvegica grew very slowly for the first 12 days, suggesting they had a long lag phase. However, after that, they grew exponentially, with a maximum growth rate of 0.56 divisions per day (during Days 24–27), reaching a maximum concentration of 3000 cells per mL⁻¹ at the end of the incubation (Day 36). In the toxin production study, the concentration of DTX1 and PTX2 increased following their vegetative growth, but the toxin production still increased exponentially on Day 36 (1.3 ng per mL⁻¹ and 154.7 ng per mL⁻¹ of DTX1 and PTX2, respectively). The concentration of OA remained below detectable levels (≤0.010 ng per mL⁻¹) during the 36-day incubation period, with the exception of Day 6. This study presents new information on the toxin production and content of D. norvegica, as well as insights into the maintenance and culturing of this species. Full article

Dinophysis acuminata and D. acuta, which follows it seasonally, are the main producers of lipophilic toxins in temperate coastal waters, including Southern Chile. Strains of the two species differ in their toxin profiles and impacts on shellfish resources. D. acuta is considered the major cause of diarrhetic shellfish poisoning (DSP) outbreaks in Southern Chile, but there is uncertainty about the toxicity of D. acuminata, and little information on microscale oceanographic conditions promoting their blooms. During the austral summer of 2020, intensive sampling was carried out in two northern Patagonian fjords, Puyuhuapi (PUY) and Pitipalena (PIT), sharing D. acuminata dominance and D. acuta near detection levels. Dinophysistoxin 1 (DTX 1) and pectenotoxin 2 (PTX 2) were present in all net tow samples but OA was not detected. Although differing in hydrodynamics and sampling dates, D. acuminata shared behavioural traits in the two fjords: cell maxima (>10³ cells L⁻¹) in the interface (S ~ 21) between the estuarine freshwater (EFW)) and saline water (ESW) layers; and phased-cell division (µ = 0.3–0.4 d⁻¹) peaking after dawn, and abundance of ciliate prey. Niche analysis (Outlying Mean Index, OMI) of D. acuta with a high marginality and much lower tolerance than D. acuminata indicated an unfavourable physical environment for D. acuta (bloom failure). Comparison of toxin profiles and Dinophysis niches in three contrasting years in PUY—2020 (D. acuminata bloom), 2018 (exceptional bloom of D. acuta), and 2019 (bloom co-occurrence of the two species)—shed light on the vertical gradients which promote each species. The presence of FW (S < 11) and thermal inversion may be used to provide short-term forecasts of no risk of D. acuta blooms and OA occurrence, but D. acuminata associated with DTX 1 pose a risk of DSP events in North Patagonian fjords. Full article

Some strains of the dinoflagellate species Prorocentrum hoffmannianum show contrasting ability to produce diarrhetic shellfish poisoning (DSP) toxins. We previously compared the okadaic acid (OA) production level between a highly toxic strain (CCMP2804) and a non-toxic strain (CCMP683) of P. hoffmannianum and revealed that the cellular concentration of OA in CCMP2804 would increase significantly under the depletion of phosphate. To understand the molecular mechanisms, here, we compared and analyzed the proteome changes of both strains growing under normal condition and at phosphate depletion using two-dimensional gel electrophoresis (2-DE). There were 41 and 33 differential protein spots observed under normal condition and phosphate depletion, respectively, of which most were upregulated in CCMP2804 and 22 were common to both conditions. Due to the lack of matched peptide mass fingerprints in the database, de novo peptide sequencing was applied to identify the differentially expressed proteins. Of those upregulated spots in CCMP2804, nearly 60% were identified as peridinin-chlorophyll a-binding protein (PCP), an important light-harvesting protein for photosynthesis in dinoflagellates. We postulated that the high expression of PCP encourages the production of DSP toxins by enhancing the yields of raw materials such as acetate, glycolate and glycine. Other possible mechanisms of toxicity related to PCP might be through triggering the transcription of non-ribosomal peptide synthetase/polyketide synthase genes and the transportation of dinophysistoxin-4 from chloroplast to vacuoles. Full article

Toxins of the OA-group (okadaic acid, OA; dinophysistoxin-1, DTX-1) are the most prevalent in the fjords of southern Chile, and are characterized by their potential harmful effects on aquatic organisms. The present study was carried out to determine the acute toxicity of OA/DTX-1 on oxidative stress parameters in medaka (Oryzias latipes) larvae. Medaka larvae were exposed to different concentrations (1.0–30 μg/mL) of OA/DTX-1 for 96 h to determine the median lethal concentration. The LC₅₀ value after 96 h was 23.5 μg/mL for OA and 16.3 μg/mL for DTX-1 (95% confidence interval, CI was 22.56, 24.43 for OA and 15.42, 17.17 for DTX-1). Subsequently, larvae at 121 hpf were exposed to acute doses (10, 15 and 20 μg/mL OA and 5.0, 7.5 and 11.0 μg/mL DTX-1) for 96 h and every 6 h the corresponding group of larvae was euthanized in order to measure the activity levels of biochemical biomarkers (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx; and glutathione reductase, GR) as well as the levels of oxidative damage (malondialdehyde, MDA; and carbonyl content). Our results showed that acute doses caused a decrease in SOD (≈25%), CAT (≈55%), and GPx and GR (≈35%) activities, while MDA levels and carbonyl content increased significantly at the same OA/DTX-1 concentrations. This study shows that acute exposure to OA-group toxins tends to simultaneously alter the oxidative parameters that induce sustained morphological damage in medaka larvae. DTX-1 stands out as producing greater inhibition of the antioxidant system, leading to increased oxidative damage in medaka larvae. Considering that DTX-1 is the most prevalent HAB toxin in southern Chile, these findings raise the possibility of an important environmental impact on the larval stages of different fish species present in the southern fjords of the South Pacific. Full article

Okadaic acid (OA) is a marine biotoxin associated with diarrhetic shellfish poisoning (DSP), posing some threat to human beings. The oral toxicity of OA is complex, and the mechanism of toxicity is not clear. The interaction between OA and gut microbiota may provide a reasonable explanation for the complex toxicity of OA. Due to the complex environment in vivo, an in vitro study may be better for the interactions between OA and gut microbiome. Here, we conducted an in vitro fermentation experiment of gut bacteria in the presence of 0–1000 nM OA. The remolding ability of OA on bacterial composition was investigated by 16S rDNA sequencing, and differential metabolites in fermentation system with different concentration of OA was detected by LC-MS/MS. We found that OA inhibited some specific bacterial genera but promoted others. In addition, eight possible metabolites of OA, including dinophysistoxin-2 (DTX-2), were detected in the fermentation system. The abundance of Faecalitalea was strongly correlated with the possible metabolites of OA, suggesting that Faecalitalea may be involved in the metabolism of OA in vitro. Our findings confirmed the direct interaction between OA and gut bacteria, which helps to reveal the metabolic process of OA and provide valuable evidence for elucidating the complex toxicity of OA. Full article

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary. Full article