Eudesmacarbonate, a Eudesmane-Type Sesquiterpene from a Marine Filamentous Cyanobacterial Mat (Oscillatoriales) in the Florida Keys.

A new, cyclic carbonate eudesmane-type sesquiterpene, eudesmacarbonate (1), was isolated from marine filamentous cyanobacterial mats associated with apparent ingestion-related intoxications of captive bottlenose dolphins in the Florida Keys. Sequencing of 16S rDNA revealed that mats were composed of closely related Oscillatoriacean species including a previously undocumented species of Neolyngbya. The structure of 1 was elucidated by (+)-HRESIMS, 1D and 2D NMR, single-crystal X-ray diffraction, and vibrational circular dichroism data. Toxicity of 1 was assessed in the zebrafish embryo/larval model, and 1 was found to exhibit effects qualitatively similar to those observed for the known neurotoxin brevetoxin-2 and consistent with neurobehavioral impairment.

Indole Alkaloids of the Stigonematales (Cyanophyta): Chemical Diversity, Biosynthesis and Biological Activity.

The cyanobacteria are well recognized as producers of a wide array of bioactive metabolites including toxins, and potential drug candidates. However, a limited number of taxa are generally considered with respect to both of these aspects. That said, the order Stigonematales, although largely overlooked in this regard, has become increasingly recognized as a source of bioactive metabolites relevant to both human and environmental health. In particular, the hapalindoles and related indole alkaloids (i.e., ambiguines, fischerindoles, welwitindolinones) from the order, represent a diverse, and phylogenetically characteristic, class of secondary metabolites with biological activity suggestive of potential as both environmental toxins, and promising drug discovery leads. The present review gives an overview of the chemical diversity of biologically active metabolites from the Stigonematales-and particularly the so-called hapalindole-type alkaloids-including their biosynthetic origins, and their pharmacologically and toxicologically relevant bioactivities. Taken together, the current evidence suggests that these alkaloids, and the associated cyanobacterial taxa from the order, warrant future consideration as both potentially harmful (i.e., "toxic") algae, and as promising leads for drug discovery.

An Integrated Metabolomics-Based Model, and Identification of Potential Biomarkers, of Perfluorooctane Sulfonic Acid Toxicity in Zebrafish Embryos.

Known for their high stability and surfactant properties, per- and polyfluoroalkyl substances (PFAS) have been widely used in a range of manufactured products. Despite being largely phased out due to concerns regarding their persistence, bioaccumulation, and toxicity, legacy PFAS such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid continue to persist at high levels in the environment, posing risks to aquatic organisms. We used high-resolution magic angle spinning nuclear magnetic resonance spectroscopy in intact zebrafish (Danio rerio) embryos to investigate the metabolic pathways altered by PFOS both before and after hatching (i.e., 24 and 72 h post fertilization [hpf], respectively). Assessment of embryotoxicity found embryo lethality in the parts-per-million range with no significant difference in mortality between the 24- and 72-hpf exposure groups. Metabolic profiling revealed mostly consistent changes between the two exposure groups, with altered metabolites generally associated with oxidative stress, lipid metabolism, energy production, and mitochondrial function, as well as specific targeting of the liver and central nervous system as key systems. These metabolic changes were further supported by analyses of tissue-specific production of reactive oxygen species, as well as nontargeted mass spectrometric lipid profiling. Our findings suggest that PFOS-induced metabolic changes in zebrafish embryos may be mediated through previously described interactions with regulatory and transcription factors leading to disruption of mitochondrial function and energy metabolism. The present study proposes a systems-level model of PFOS toxicity in early life stages of zebrafish, and also identifies potential biomarkers of effect and exposure for improved environmental biomonitoring. Environ Toxicol Chem 2024;43:896-914. © 2024 SETAC.

High-Resolution Magic Angle Spinning (HRMAS) NMR Identifies Oxidative Stress and Impairment of Energy Metabolism by Zearalenone in Embryonic Stages of Zebrafish (Danio rerio), Olive Flounder (Paralichthys olivaceus) and Yellowtail Snapper (Ocyurus chrysurus).

Zearalenone (ZEA) is a mycotoxin, commonly found in agricultural products, linked to adverse health impacts in humans and livestock. However, less is known regarding effects on fish as both ecological receptors and economically relevant "receptors" through contamination of aquaculture feeds. In the present study, a metabolomics approach utilizing high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) was applied to intact embryos of zebrafish (Danio rerio), and two marine fish species, olive flounder (Paralichthys olivaceus) and yellowtail snapper (Ocyurus chrysurus), to investigate the biochemical pathways altered by ZEA exposure. Following the assessment of embryotoxicity, metabolic profiling of embryos exposed to sub-lethal concentrations showed significant overlap between the three species and, specifically, identified metabolites linked to hepatocytes, oxidative stress, membrane disruption, mitochondrial dysfunction, and impaired energy metabolism. These findings were further supported by analyses of tissue-specific production of reactive oxygen species (ROS) and lipidomics profiling and enabled an integrated model of ZEA toxicity in the early life stages of marine and freshwater fish species. The metabolic pathways and targets identified may, furthermore, serve as potential biomarkers for monitoring ZEA exposure and effects in fish in relation to ecotoxicology and aquaculture.

Polymethoxy-1-alkenes from Aphanizomenon ovalisporum inhibit vertebrate development in the zebrafish (Danio rerio) embryo model.

Cyanobacteria are recognized producers of a wide array of toxic or otherwise bioactive secondary metabolites. The present study utilized the zebrafish (Danio rerio) embryo as an aquatic animal model of vertebrate development to identify, purify and characterize lipophilic inhibitors of development (i.e., developmental toxins) from an isolate of the freshwater cyanobacterial species, Aphanizomenon ovalisporum.Bioassay-guided fractionation led to the purification, and subsequent chemical characterization, of an apparent homologous series of isotactic polymethoxy-1-alkenes (1-6), including three congeners (4-6) previously identified from the strain, and two variants previously identified from other species (2 and 3), as well as one apparently novel member of the series (1). Five of the PMAs in the series (1-5) were purified in sufficient quantity for comparative toxicological characterization, and toxicity in the zebrafish embryo model was found to generally correlate with relative chain length and/or methoxylation. Moreover, exposure of embryos to a combination of variants indicates an apparent synergistic interaction between the congeners. Although PMAs have been identified previously in cyanobacteria, this is the first report of their apparent toxicity. These results, along with the previously reported presence of the PMAs from several cyanobacterial species, suggest a possibly widespread distribution of the PMAs as toxic secondary metabolites and warrants further chemical and toxicological investigation.

An integrated systems-level model of ochratoxin A toxicity in the zebrafish (Danio rerio) embryo based on NMR metabolic profiling.

Ochratoxin A (OTA) is one of the most widespread mycotoxin contaminants of agricultural crops. Despite being associated with a range of adverse health effects, a comprehensive systems-level mechanistic understanding of the toxicity of OTA remains elusive. In the present study, metabolic profiling by high-resolution magic angle spinning (HRMAS) NMR, coupled to intact zebrafish embryos, was employed to identify metabolic pathways in relation to a systems-level model of OTA toxicity. Embryotoxicity was observed at sub-micromolar exposure concentrations of OTA. Localization of OTA, based on intrinsic fluorescence, as well as a co-localization of increased reactive oxygen species production, was observed in the liver kidney, brain and intestine of embryos. Moreover, HRMAS NMR showed significant alteration of metabolites related to targeting of the liver (i.e., hepatotoxicity), and pathways associated with detoxification and oxidative stress, and mitochondrial energy metabolism. Based on metabolic profiles, and complementary assays, an integrated model of OTA toxicity is, thus, proposed. Our model suggests that OTA hepatotoxicity compromises detoxification and antioxidant pathways, leading to mitochondrial membrane dysfunction manifested by crosstalk between pathways of energy metabolism. Interestingly, our data additionally aligns with a possible role of mitochondrial fusion as a "passive mechanism" to rescue mitochondrial integrity during OTA toxicity.

Identification of apparently neurotoxic metabolites from assemblages of marine filamentous cyanobacteria associated with the intoxication of captive bottlenose dolphins (Tursiops truncatus) in the Florida Keys.

Intoxications of captive bottlenose dolphins (Tursiops truncatus) in the Florida Keys have been linked to observed interactions with marine macrophytic algal and cyanobacterial communities within enclosures. Taxonomic characterization of these communities coupled, in turn, to available observational data collected during intoxication events point to a contribution of filamentous cyanobacterial assemblages comprised of members of the polyphyletic genus, Lyngbya sensu lato. To identify toxic metabolites possibly relevant to these intoxications, chemical screening for known neurotoxins from cyanobacteria, as well as other regionally relevant harmful algal bloom (HAB) taxa, was combined with toxicity testing, and subsequent bioassay-guided fractionation, employing early life stages (i.e., embryos and larvae) of zebrafish (Danio rerio) as a well-established aquatic vertebrate toxicological model. Chemical analyses did not detect (within analytical limits) any of the known algal or cyanobacterial neurotoxins. Toxicity testing, alongside bioassay-guided fractionation, however, identified several chemical fractions with a range of potentially relevant bioactivities in both zebrafish embryos and post-hatch larvae including, in particular, behavioral (e.g., aberrant swimming) and physiological (e.g., altered heart rate) endpoints indicative of possible neurotoxicity, and subsequent chemical characterization of fractions suggested a contribution of the previously identified bioactive metabolite, eudesmacarbonate, in the observed toxicity. Comparative toxicological assessment with PbTx-2, as a positive control for neurotoxicity in the zebrafish model, further supported neurotoxic activity of cyanobacterial metabolites potentially relevant, in turn, to a contribution of these metabolites to dolphin intoxications. These findings suggest, in general, that marine zoological facilities may be affected by regional HABs, and assessments of potentially toxigenic algae and cyanobacteria should be included in management strategies in these facilities.

An integrated systems-level model of the toxicity of brevetoxin based on high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) metabolic profiling of zebrafish embryos.

Brevetoxins (PbTx) are a well-recognized group of neurotoxins associated with harmful algal blooms, and specifically recurrent "Florida Red Tides," in marine waters that are linked to impacts on both human and ecosystem health including well-documented "fish kills" and marine mammal mortalities in affected coastal waters. Understanding mechanisms and pathways of PbTx toxicity enables identification of relevant biomarkers to better understand these environmental impacts, and improve monitoring efforts, in relation to this toxin. Toward a systems-level understanding of toxicity, and identification of potential biomarkers, high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) was utilized for metabolic profiling of zebrafish (Danio rerio) embryos, as an established toxicological model, exposed to PbTx-2 (the most common congener in marine waters). Metabolomics studies were, furthermore, complemented by an assessment of the toxicity of PbTx-2 in embryonic stages of zebrafish and mahi-mahi (Coryphaena hippurus), the latter representing an ecologically and geographically relevant marine species of fish, which identified acute embryotoxicity at environmentally relevant (i.e., parts-per-billion) concentrations in both species. HRMAS NMR analysis of intact zebrafish embryos exposed to sub-lethal concentrations of PbTx-2 afforded well-resolved spectra, and in turn, identification of 38 metabolites of which 28 were found to be significantly altered, relative to controls. Metabolites altered by PbTx-2 exposure specifically included those associated with (1) neuronal excitotoxicity, as well as associated neural homeostasis, and (2) interrelated pathways of carbohydrate and energy metabolism. Metabolomics studies, thereby, enabled a systems-level model of PbTx toxicity which integrated multiple metabolic, molecular and cellular pathways, in relation to environmentally relevant concentrations of the toxin, providing insight to not only targets and mechanisms, but potential biomarkers pertinent to environmental risk assessment and monitoring strategies.

Cyanobacterial LPS potentiates cadmium toxicity in zebrafish (Danio rerio) embryos.

Cyanobacteria are prevalent in the freshwater environment, reaching critical mass in harmful algal blooms. These organisms produce a variety of toxins including endotoxins such as lipopolysaccharides (LPS), which have been previously shown to decrease glutathione-S-transferase (GST) activity in zebrafish (Danio rerio) embryos. GST plays a vital role in detoxification response during oxidative stress and provides a first line of defense after toxic heavy metal insult, before increased metallothionein expression. Although some attention has focused on cyanobacterial LPS, little research has focused on effects of concurrent exposures with other toxicants. Because cyanobacterial LPS can alter detoxification enzymes including GST, we hypothesized that cyanobacterial LPS could potentiate metal toxicity. This study investigated the effects of LPS from two cyanobacterial species, Lyngbya spp. and Microcystis aeruginosa, on cadmium toxicity in zebrafish embryos. Forty-eight-hour CdCl(2) LC(50) values showed that coexposure of cadmium and Lyngbya LPS or Microcystis LPS resulted in significantly increased cadmium toxicity in comparison with cadmium alone. However, increased cadmium toxicity was not due to decreased GST activity as initially hypothesized. In concurrent Microcystis LPS-cadmium exposures, GST activity was significantly increased in comparison with control embryos at all time points and cadmium concentrations sampled. Concurrent Lyngbya LPS-cadmium exposures also resulted in increased GST activity at most exposure concentrations. These results indicate that regardless of mechanism, cyanobacterial LPS can potentiate the toxic effects of heavy metals. This represents a significant risk for aquatic organisms exposed to combinations of LPS and metals in the environment.

Comparative toxicometabolomics of perfluorooctanoic acid (PFOA) and next-generation perfluoroalkyl substances.

Owing to environmental health concerns, a number of per- and polyfluoroalkyl substances (PFAS) have been phased-out, and increasingly replaced by various chemical analogs. Most prominent among these replacements are numerous perfluoroether carboxylic acids (PFECA). Toxicity, and environmental health concerns associated with these next-generation PFAS, however, remains largely unstudied. The zebrafish embryo was employed, in the present study, as a toxicological model system to investigate toxicity of a representative sample of PFECA, alongside perfluorooctanoic acid (PFOA) as one of the most widely used, and best studied, of the "legacy" PFAS. In addition, high-resolution magic angle spin (HRMAS) NMR was utilized for metabolic profiling of intact zebrafish embryos in order to characterize metabolic pathways associated with toxicity of PFAS. Acute embryotoxicity (i.e., lethality), along with impaired development, and variable effects on locomotory behavior, were observed for all PFAS in the zebrafish model. Median lethal concentration (LC50) was significantly correlated with alkyl chain-length, and toxic concentrations were quantitatively similar to those reported previously for PFAS. Metabolic profiling of zebrafish embryos exposed to selected PFAS, specifically including PFOA and two representative PFECA (i.e., GenX and PFO3TDA), enabled elaboration of an integrated model of the metabolic pathways associated with toxicity of these representative PFAS. Alterations of metabolic profiles suggested targeting of hepatocytes (i.e., hepatotoxicity), as well as apparent modulation of neural metabolites, and moreover, were consistent with a previously proposed role of mitochondrial disruption and peroxisome proliferator-activated receptor (PPAR) activation as reflected by dysfunctions of carbohydrate, lipid and amino acid metabolism, and consistent with a previously proposed contribution of PFAS to metabolic syndrome. Taken together, it was generally concluded that toxicity of PFECA is quantitatively and qualitatively similar to PFOA, and these analogs, likewise, represent potential concerns as environmental toxicants.

Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo.

Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50mug/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed.

NMR-Based Metabolic Profiles of Intact Zebrafish Embryos Exposed to Aflatoxin B1 Recapitulates Hepatotoxicity and Supports Possible Neurotoxicity.

Aflatoxin B1 (AFB1) is a widespread contaminant of grains and other agricultural crops and is globally associated with both acute toxicity and carcinogenicity. In the present study, we utilized nuclear magnetic resonance (NMR), and specifically high-resolution magic angle spin (HRMAS) NMR, coupled to the zebrafish (Danio rerio) embryo toxicological model, to characterize metabolic profiles associated with exposure to AFB1. Exposure to AFB1 was associated with dose-dependent acute toxicity (i.e., lethality) and developmental deformities at micromolar (≤ 2 µM) concentrations. Toxicity of AFB1 was stage-dependent and specifically consistent, in this regard, with a role of the liver and phase I enzyme (i.e., cytochrome P450) bioactivation. Metabolic profiles of intact zebrafish embryos exposed to AFB1 were, furthermore, largely consistent with hepatotoxicity previously reported in mammalian systems including metabolites associated with cytotoxicity (i.e., loss of cellular membrane integrity), glutathione-based detoxification, and multiple pathways associated with the liver including amino acid, lipid, and carbohydrate (i.e., energy) metabolism. Taken together, these metabolic alterations enabled the proposal of an integrated model of the hepatotoxicity of AFB1 in the zebrafish embryo system. Interestingly, changes in amino acid neurotransmitters (i.e., Gly, Glu, and GABA), as a key modulator of neural development, supports a role in recently-reported neurobehavioral and neurodevelopmental effects of AFB1 in the zebrafish embryo model. The present study reinforces not only toxicological pathways of AFB1 (i.e., hepatotoxicity, neurotoxicity), but also multiple metabolites as potential biomarkers of exposure and toxicity. More generally, this underscores the capacity of NMR-based approaches, when coupled to animal models, as a powerful toxicometabolomics tool.

Allelopathic activity among Cyanobacteria and microalgae isolated from Florida freshwater habitats.

We evaluated allelopathic interactions between strains of Cyanobacteria and green algae isolated from south and central Florida. Allelopathy, including inhibition or stimulation of growth, was assessed by cocultivation of each of the isolated strains, as well as by evaluation of extracts prepared from the isolates. All of the strains of Cyanobacteria, and four of the six isolates of green algae, showed some allelopathic activity (i.e. inhibition or stimulation of the growth of other strains). Of these, the most pronounced activity was observed for the cyanobacterial isolate Fischerella sp. strain 52-1. In the cocultivation experiments this cyanobacterium inhibited the growth of all tested green algae and Cyanobacteria. The crude lipophilic extracts from Fischerella sp. strain 52-1 isolated from both the biomass and the culture liquid inhibited photosynthesis of the green alga Chlamydomonas sp. in a concentration- and time-dependent manner and caused extensive loss of ultrastructural cell organization. Preliminary chemical characterization of compounds extracted from Fischerella sp. strain 52-1 indicated the presence of indole alkaloids, and further characterization has confirmed that these compounds belong to the hapalindoles previously isolated from other species of Fischerella and related genera. Further chemical characterization of these compounds, and further investigation of their apparent role in allelopathy is ongoing.

Cyanobacterial toxins as allelochemicals with potential applications as algaecides, herbicides and insecticides.

Cyanobacteria ("blue-green algae") from marine and freshwater habitats are known to produce a diverse array of toxic or otherwise bioactive metabolites. However, the functional role of the vast majority of these compounds, particularly in terms of the physiology and ecology of the cyanobacteria that produce them, remains largely unknown. A limited number of studies have suggested that some of the compounds may have ecological roles as allelochemicals, specifically including compounds that may inhibit competing sympatric macrophytes, algae and microbes. These allelochemicals may also play a role in defense against potential predators and grazers, particularly aquatic invertebrates and their larvae. This review will discuss the existing evidence for the allelochemical roles of cyanobacterial toxins, as well as the potential for development and application of these compounds as algaecides, herbicides and insecticides, and specifically present relevant results from investigations into toxins of cyanobacteria from the Florida Everglades and associated waterways.

High Incidence and Levels of Ochratoxin A in Wines Sourced from the United States.

Ochratoxin A (OTA) is one of the most prevalent mycotoxin contaminants of food crops. Among the agricultural products consequently contaminated by OTA is wine. In the present study, a sample of wines sourced from the United States was assessed for OTA. Wines were primarily analyzed by high-performance liquid chromatography with fluorescence detection (HPLC-FD) coupled to a liquid-liquid extraction (LLE) technique which was developed and validated as a simplified sample preparation approach. More than 85% of the wines evaluated were found to contain OTA, at levels above the limit-of-detection (LOD = 0.1 µg L-1), and 76% were above the limit-of-quantitation (LOQ = 0.3 µg L-1) for the LLE/HPLC-FD method. More than two-thirds of the wines above the LOQ were found to exceed 1 µg L-1. Complementary analysis by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) confirmed OTA in 74% of the OTA-positive wines (i.e., >LOQ by HPLC-FD). Overall, both the occurrence and measured levels of OTA were generally high, specifically relative to previous assessments of OTA in wine, and two of the wines were above the only current (European Union) regulatory limit of two parts-per-billion (ppb, ~2 µg L-1). Possible trends with respect to geographical region and/or growing climate are noted. As the first assessment of U.S. wines in more than a decade, the overall high occurrence and levels of OTA in wine, and possible geographic and climatic trends, point to a need for regular surveillance of wines, as well as investigation of the relevant contributors to OTA occurrence toward mitigating contamination and exposure risks.

ß-Cyclodextrin Attenuates Perfluorooctanoic Acid Toxicity in the Zebrafish Embryo Model.

Perfluorooctanoic acid (PFOA) has been linked to negative health outcomes including cancer, thyroid disease, infertility, and developmental delays. ß-Cyclodextrin (ß-CD), a cyclic sugar, has been previously shown to form strong host-guest complexes with PFOA, and is proposed as a means of environmental remediation with respect to this widespread contaminant. In the present study, ß-CD was directly examined with regards to possible attenuation of the toxicity of PFOA specifically employing the zebrafish (Danio rerio) embryo model. Zebrafish embryos were exposed to various concentrations of PFOA without ß-CD, and with equimolar (1:1) and excess (2:1) molar ratios of ß-CD to PFOA, and assessed for lethality and developmental toxicity through seven days post-fertilization (dpf). Rapid onset of lethality with limited morphological abnormalities was observed at relatively low concentrations of PFOA (LC50 ≈ 50 ppm), along with effects on morphometric and neurobehavioral parameters in surviving embryos. A highly significant difference (p < 0.0001) was observed between the 2:1 treatment, and both 1:1 and PFOA only treatments, with respect to lethal concentration and apparent neurobehavioral effects, suggesting an effectively reduced toxicity of the fully complexed PFOA. In contrast, however, neither ß-CD treatment reduced developmental toxicity with respect to the morphometric endpoint (i.e., interocular distance). Whereas LC50 of PFOA alone did not change over 7 dpf, the 1:1 and 2:1 values decreased slightly over time, suggesting either delayed or alternative toxic effects on later developmental stages at presumptively lowered levels. This study, therefore, indicates ß-CD may be an effective agent to reduce toxicity of and mitigate environmental health concerns associated with PFOA, but that further study is required to elucidate the mechanism of complexation as it relates to the attenuation of toxicity.

Carotenoid glycosides from cyanobacteria are teratogenic in the zebrafish (Danio rerio) embryo model.

Toxigenicity of cyanobacteria is widely associated with production of several well-described toxins that pose recognized threats to human and ecosystem health as part of both freshwater eutrophication, and episodic blooms in freshwater and coastal habitats. However, a preponderance of evidence indicates contribution of additional bioactive, and potentially toxic, metabolites. In the present study, the zebrafish (Danio rerio) embryo was used as a model of vertebrate development to identify, and subsequently isolate and characterize, teratogenic metabolites from two representative strains of C. raciborskii. Using this approach, three chemically related carotenoids - and specifically the xanthophyll glycosides, myxol 2'-glycoside (1), 4-ketomyxol 2'-glycoside (2) and 4-hydroxymyxol 2'-glycoside (3) - which are, otherwise, well known pigment molecules from cyanobacteria were isolated as potently teratogenic compounds. Carotenoids are recognized "pro-retinoids" with retinoic acid, as a metabolic product of the oxidative cleavage of carotenoids, established as both key mediator of embryo development and, consequently, a potent teratogen. Accordingly, a comparative toxicological study of chemically diverse carotenoids, as well as apocarotenoids and retinoids, was undertaken. Based on this, a working model of the developmental toxicity of carotenoids as pro-retinoids is proposed, and the teratogenicity of these widespread metabolites is discussed in relation to possible impacts on aquatic vertebrate populations.

Metabolic profiling of zebrafish (Danio rerio) embryos by NMR spectroscopy reveals multifaceted toxicity of ß-methylamino-L-alanine (BMAA).

ß-methylamino-L-alanine (BMAA) has been linked to several interrelated neurodegenerative diseases. Despite considerable research, specific contributions of BMAA toxicity to neurodegenerative diseases remain to be fully resolved. In the present study, we utilized state-of-the-art high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR), applied to intact zebrafish (Danio rerio) embryos, as a model of vertebrate development, to elucidate changes in metabolic profiles associated with BMAA exposure. Complemented by several alternative analytical approaches (i.e., in vivo visualization and in vitro assay), HRMAS NMR identified robust and dose-dependent effect of BMAA on several relevant metabolic pathways suggesting a multifaceted toxicity of BMAA including: (1) localized production of reactive oxygen species (ROS), in the developing brain, consistent with excitotoxicity; (2) decreased protective capacity against excitotoxicity and oxidative stress including reduced taurine and glutathione; (3) inhibition of several developmentally stereotypical energetic and metabolic transitions, i.e., metabolic reprogramming; and (4) inhibition of lipid biosynthetic pathways. Matrix-assisted laser desorption time-of-flight (MALDI-ToF) mass spectrometry further identified specific effects on phospholipids linked to both neural development and neurodegeneration. Taken together, a unified model of the neurodevelopmental toxicity of BMAA in the zebrafish embryo is presented in relation to the potential contribution of BMAA to neurodegenerative disease.

A rapid method for separation and identification of microcystins using capillary electrophoresis and time-of-flight mass spectrometry.

This paper demonstrates a method for the rapid separation and identification of four microcystin (MC) variants commonly found in aquatic environments. The procedure utilizes capillary electrophoresis (CE) coupled to UV absorbance and time-of-flight mass spectrometric (TOF-MS) detectors. All four analytes were effectively separated within 6min using phosphate buffer in 50-µm ID capillaries with an applied electric field of 400V/cm. The separation of the individual compounds was optimized through the adjustment of buffer, pH, and ß-cyclodextrin content. Ultimately it was determined that, at a sufficiently high pH, all 4 compounds could be separated without the need for added cyclodextrins. The results provided accurate molecular information, assisting in the determination of compound identity. The method was then applied to environmental samples using solid phase extraction for isolation and pre-concentration. The results were comparable to those obtained by LC/MS, but with a shorter run time and lower sample and eluent consumption.

High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance of Intact Zebrafish Embryos Detects Metabolic Changes Following Exposure to Teratogenic Polymethoxyalkenes from Algae.

Techniques based on nuclear magnetic resonance (NMR) for imaging and chemical analyses of in vivo, or otherwise intact, biological systems are rapidly emerging and finding diverse applications within a wide range of fields. Very recently, several NMR-based techniques have been developed for the zebrafish as a model animal system. In the current study, the novel application of high-resolution magic angle spinning (HR-MAS) NMR is presented as a means of metabolic profiling of intact zebrafish embryos. Toward investigating the utility of HR-MAS NMR as a toxicological tool, these studies specifically examined metabolic changes of embryos exposed to polymethoxy-1-alkenes (PMAs)-a recently identified family of teratogenic compounds from freshwater algae-as emerging environmental contaminants. One-dimensional and two-dimensional HR-MAS NMR analyses were able to effectively identify and quantify diverse metabolites in early-stage (≤36 h postfertilization) embryos. Subsequent comparison of the metabolic profiles between PMA-exposed and control embryos identified several statistically significant metabolic changes associated with subacute exposure to the teratogen, including (1) elevated inositol as a recognized component of signaling pathways involved in embryo development; (2) increases in several metabolites, including inositol, phosphoryl choline, fatty acids, and cholesterol, which are associated with lipid composition of cell membranes; (3) concomitant increase in glucose and decrease in lactate; and (4) decreases in several biochemically related metabolites associated with central nervous system development and function, including γ-aminobutyric acid, glycine, glutamate, and glutamine. A potentially unifying model/hypothesis of PMA teratogenicity based on the data is presented. These findings, taken together, demonstrate that HR-MAS NMR is a promising tool for metabolic profiling in the zebrafish embryo, including toxicological applications.