RESUMO
Ciguatoxins (CTX) are potent marine neurotoxins, which can bioaccumulate in seafood, causing a severe and prevalent human illness known as ciguatera poisoning (CP). Despite the worldwide impact of ciguatera, effective disease management is hindered by a lack of knowledge regarding the movement and biotransformation of CTX congeners in marine food webs, particularly in the Caribbean and Western Atlantic. In this study we investigated the hepatic biotransformation of C-CTX across several fish and mammalian species through a series of in vitro metabolism assays focused on phase I (CYP P450; functionalization) and phase II (UGT; conjugation) reactions. Using liquid chromatography high-resolution mass spectrometry to explore potential C-CTX metabolites, we observed two glucuronide products of C-CTX-1/-2 and provided additional evidence from high-resolution tandem mass spectrometry to support their identification. Chemical reduction experiments confirmed that the metabolites were comprised of four distinct glucuronide products with the sugar attached at two separate sites on C-CTX-1/-2 and excluded the C-56 hydroxyl group as the conjugation site. Glucuronidation is a novel biotransformation pathway not yet reported for CTX or other related polyether phycotoxins, yet its occurrence across all fish species tested suggests that it could be a prevalent and important detoxification mechanism in marine organisms. The absence of glucuronidation observed in this study for both rat and human microsomes suggests that alternate biotransformation pathways may be dominant in higher vertebrates.
Assuntos
Ciguatoxinas/metabolismo , Peixes/metabolismo , Glucuronídeos/metabolismo , Animais , Biotransformação , Região do Caribe , Ciguatera/etiologia , Ciguatera/metabolismo , Cadeia Alimentar , Humanos , Microssomos Hepáticos/metabolismo , Ratos Wistar , Alimentos Marinhos/intoxicaçãoRESUMO
An amendment to this paper has been published and can be accessed via the original article.
RESUMO
BACKGROUND: Sphingolipids are structural components and signaling molecules in eukaryotic membranes, and many organisms produce compounds that inhibit sphingolipid metabolism. Some of the inhibitors are structurally similar to the sphingolipid biosynthetic intermediate sphinganine and are referred to as sphinganine-analog metabolites (SAMs). The mycotoxins fumonisins, which are frequent contaminants in maize, are one family of SAMs. Due to food and feed safety concerns, fumonisin biosynthesis has been investigated extensively, including characterization of the fumonisin biosynthetic gene cluster in the agriculturally important fungi Aspergillus and Fusarium. Production of several other SAMs has also been reported in fungi, but there is almost no information on their biosynthesis. There is also little information on how widely SAM production occurs in fungi or on the extent of structural variation of fungal SAMs. RESULTS: Using fumonisin biosynthesis as a model, we predicted that SAM biosynthetic gene clusters in fungi should include a polyketide synthase (PKS), an aminotransferase and a dehydrogenase gene. Surveys of genome sequences identified five putative clusters with this three-gene combination in 92 of 186 Fusarium species examined. Collectively, the putative SAM clusters were distributed widely but discontinuously among the species. We propose that the SAM5 cluster confers production of a previously reported Fusarium SAM, 2-amino-14,16-dimethyloctadecan-3-ol (AOD), based on the occurrence of AOD production only in species with the cluster and on deletion analysis of the SAM5 cluster PKS gene. We also identified SAM clusters in 24 species of other fungal genera, and propose that one of the clusters confers production of sphingofungin, a previously reported Aspergillus SAM. CONCLUSION: Our results provide a genomics approach to identify novel SAM biosynthetic gene clusters in fungi, which should in turn contribute to identification of novel SAMs with applications in medicine and other fields. Information about novel SAMs could also provide insights into the role of SAMs in the ecology of fungi. Such insights have potential to contribute to strategies to reduce fumonisin contamination in crops and to control crop diseases caused by SAM-producing fungi.
Assuntos
Fumonisinas , Fusarium , Fungos , Fusarium/genética , Família Multigênica , EsfingolipídeosRESUMO
Deoxynivalenol (DON) is a trichothecene mycotoxin that is produced by several species of Fusarium, which may infect grain crops. DON, as well as other type-B trichothecenes, contain an α,ß-unsaturated carbonyl group that may react with sulfhydryl groups in, for example, amino acids and peptides. Such conjugates have been shown to occur in plants. Nucleophilic addition of thiols to the conjugated double bond in DON afforded several isomeric reaction products, and the thermodynamically favored isomers of DON-10-cysteine and DON-10-glutathione have been prepared and characterized previously. This study reports the preparation and characterization of the kinetically favored DON-10-cysteine isomer. We subsequently studied and compared the rate of the deconjugation reaction of the two DON-10-cysteine isomers and the thermodynamically favored DON-10-glutathione adduct. The deconjugation rate of the thermodynamically favored thiol conjugates was slow with half-lives of weeks even at pH 10.7, while the kinetically favored DON-10-cysteine isomer deconjugated within a few hours, affording free DON. We adapted a simple and rapid oxidation protocol in which the sulfide linkage was oxidized to a sulfoxide or sulfone that, when treated with the base, rapidly eliminated the adducted thiol as its sulfenate or sulfinate to afford free DON. The deconjugation reactions of the sulfoxides and sulfones of thermodynamically favored DON-10-thiols were complete within hours or minutes at pH 10.7, respectively. The increase in deconjugation rates for the kinetically favored DON-10-cysteine were less dramatic. Oxidation of sulfides to sulfoxides is known to occur in vivo, and thus, our data show that thiol-conjugated DON might become bioavailable via sulfide oxidation followed by elimination to regenerate DON. The oxidation-elimination approach could also be useful for the indirect quantification of DON-10-thiol conjugates in plant and animal tissues.
Assuntos
Micotoxinas/química , Compostos de Sulfidrila/química , Tricotecenos/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , OxirreduçãoRESUMO
Age-related differences in toxicokinetic processes of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON3G) were studied. DON3G [55.7 µg/kg bodyweight (BW)] and an equimolar dose of DON (36 µg/kg BW) were administered to weaned piglets (4 weeks old) by single intravenous and oral administration in a double two-way cross-over design. Systemic and portal blood was sampled at different time points pre- and post-administration and plasma concentrations of DON, DON3G and their metabolites were quantified using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) methods. Data were processed using tailor-made compartmental toxicokinetic (TK) models to accurately estimate TK parameters. Results were statistically compared to data obtained in a previous study on 11-week-old pigs using identical experimental conditions. Significant age-related differences in intestinal and systemic exposure to both DON and DON3G were noted. Most remarkably, a significant difference was found for the absorbed fraction of DON3G, after presystemic hydrolysis to DON, in weaned piglets compared to 11-week-old piglets (83% vs 16%, respectively), assumed to be mainly attributed to the higher intestinal permeability of weaned piglets. Other differences in TK parameters could be assigned to a higher water/fat body ratio and longer gastrointestinal transit time of weaned piglets. Results may further refine current risk assessment concerning DON and DON3G in animals. Additionally, since piglets possibly serve as a human paediatric surrogate model, results may be extrapolated to human infants.
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Glucosídeos/farmacocinética , Tricotecenos/farmacocinética , Administração Intravenosa , Administração Oral , Animais , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Feminino , Glucosídeos/administração & dosagem , Glucosídeos/toxicidade , Masculino , Suínos , Distribuição Tecidual , Tricotecenos/administração & dosagem , Tricotecenos/toxicidade , DesmameRESUMO
Ciguatera poisoning is linked to the ingestion of seafood that is contaminated with ciguatoxins (CTXs). The structural variability of these polyether toxins in nature remains poorly understood due to the low concentrations present even in highly toxic fish, which makes isolation and chemical characterization difficult. We studied the mass spectrometric fragmentation of Caribbean CTXs, i.e., the epimers C-CTX-1 and -2 (1 and 2), using a sensitive UHPLC-HRMS/MS approach in order to identify product ions of diagnostic value. We found that the fragmentation of the ladder-frame backbone follows a characteristic pattern and propose a generalized nomenclature for the ions formed. These data were applied to the structural characterization of a pair of so far poorly characterized isomers, C-CTX-3 and -4 (3 and 4), which we found to be reduced at C-56 relative to 1 and 2. Furthermore, we tested and applied reduction and oxidation reactions, monitored by LC-HRMS, in order to confirm the structures of 3 and 4. Reduction of 1 and 2 with NaBH4 afforded 3 and 4, thereby unambiguously confirming the identities of 3 and 4. In summary, this work provides a foundation for mass spectrometry-based characterization of new C-CTXs, including a suite of simple chemical reactions to assist the examination of structural modifications.
Assuntos
Ciguatera/prevenção & controle , Ciguatoxinas/isolamento & purificação , Peixes , Alimentos Marinhos/análise , Animais , Região do Caribe , Cromatografia Líquida de Alta Pressão/métodos , Ciguatoxinas/química , Conformação Molecular , Espectrometria de Massas em Tandem/métodosRESUMO
Microcystins are cyclic heptapeptides from cyanobacteria that are potent inhibitors of protein phosphatases and are toxic to animals and humans. At present, more than 250 microcystin variants are known, with variants reported for all seven peptide moieties. While d-glutamic acid (d-Glu) is highly-conserved at position-6 of microcystins, there has been only one report of a cyanobacterium (Anabaena) producing microcystins containing l-Glu at the variable 2- and 4-positions. Liquid chromatography-mass spectrometry analyses of extracts from Planktothrix prolifica NIVA-CYA 544 led to the tentative identification of two new Glu-containing microcystins, [d-Asp3]MC-ER (12) and [d-Asp3]MC-EE (13). Structure determination was aided by thiol derivatization of the Mdha7-moiety and esterification of the carboxylic acid groups, while 15N-labeling of the culture and isotopic profile analysis assisted the determination of the number of nitrogen atoms present and the elemental composition of molecular and product-ions. The major microcystin analog in the extracts was [d-Asp3]MC-RR (1). A microcystin with an unprecedented high-molecular-mass (2116 Da) was also detected and tentatively identified as a sulfide-linked conjugate of [d-Asp3]MC-RR (15) by LC-HRMS/MS and sulfide oxidation, together with its sulfoxide (16) produced via autoxidation. Low levels of [d-Asp3]MC-RW (14), [d-Asp3]MC-LR (4), [d-Asp3,Mser7]MC-RR (11), [d-Asp3]MC-RY (17), [d-Asp3]MC-RF (18), [d-Asp3]MC-RR-glutathione conjugate (19), and [d-Asp3]MC-RCit (20), the first reported microcystin containing citrulline, were also identified in the extract, and an oxidized derivative of [d-Asp3]MC-RR and the cysteine conjugate of 1 were partially characterized.
Assuntos
Cianobactérias/química , Microcistinas/química , Isótopos de Nitrogênio/química , Nitrogênio/química , Anabaena/química , Toxinas Bacterianas/química , Cromatografia Líquida/métodos , Oxirredução , Planktothrix , Compostos de Sulfidrila/química , Sulfetos/química , Espectrometria de Massas em Tandem/métodosRESUMO
Deoxynivalenol (DON) is the most prevalent mycotoxin in cereals worldwide. It can cause adverse health effects in humans and animals, and maximum levels in food and feed have been implemented by food authorities based on risk assessments derived from estimated intake levels. The lack of human toxicokinetic data such as absorption, distribution, and elimination characteristics hinders the direct calculation of DON plasma levels and exposure. In the present study, we have, therefore, used in vitro-to-in vivo extrapolation of depletion constants in hepatic microsomes from different species and allometric scaling of reported in vivo animal parameters to predict the plasma clearance [0.24 L/(h × kg)] and volume of distribution (1.24 L/kg) for DON in humans. In addition, we have performed a toxicokinetic study with oral and intravenous administration of DON in pigs to establish benchmark parameters for the in vitro extrapolation approach. The determined human toxicokinetic parameters were then used to calculate the bioavailability (50-90%), maximum concentration, and total exposure in plasma, and urinary concentrations under consideration of typical DON levels in grain-based food products. The results were compared to data from biomonitoring studies in human populations.
Assuntos
Microssomos Hepáticos/efeitos dos fármacos , Modelos Biológicos , Tricotecenos , Administração Oral , Animais , Área Sob a Curva , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Grão Comestível/química , Feminino , Contaminação de Alimentos/análise , Humanos , Técnicas In Vitro , Injeções Intravenosas , Masculino , Microssomos Hepáticos/metabolismo , Valor Preditivo dos Testes , Ratos , Especificidade da Espécie , Sus scrofa , Toxicocinética , Tricotecenos/sangue , Tricotecenos/toxicidadeRESUMO
The peroxisome proliferator-activated receptors (PPARs) function as ligand-activated transcription factors that convert signals in the form of lipids to physiological responses through the activation of metabolic target genes. Due to their key roles in lipid and carbohydrate metabolism, the PPARs are important drug targets. However, for several of the PPAR drugs currently in use, adverse side effects have been reported. In an effort to identify compounds from marine organisms that may serve as molecular scaffolds for the development of novel and safer PPAR-targeting drugs, we performed a bioassay-guided screening of organic extracts made from organisms supplied by the Norwegian Biobank of Arctic Marine Organisms (Marbank). Among several interesting hits, we identified two poorly described isomeric oxo-fatty acids from the microalgae Chaetoceros karianus for which we provide the first evidence that they might display dual specificity towards human PPARα and PPARγ. Principal component analysis showed that C. karianus stood out from other Chaetoceros species, both with respect to the metabolic profile and the PPAR activity. The isolation of these compounds holds the potential of uncovering a PPAR pharmacophore with tunable activity and specificity.
Assuntos
Diatomáceas/química , Ácidos Graxos/química , Ácidos Graxos/farmacologia , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Humanos , Isomerismo , Ligantes , Metaboloma/efeitos dos fármacos , Microalgas/químicaRESUMO
RATIONALE: The biological activities most commonly associated with indole-diterpenoids are tremorgenicity in mammals and toxicity in insects through modulation of ion channels. The neurotoxic effects of some analogues are the cause of syndromes such as 'ryegrass staggers' and 'Paspalum staggers' in cattle and sheep. Our purpose was to obtain and interpret mass spectra of some pure Claviceps-related indole-diterpenoids (paspaline, paspalinine, paxilline, paspalitrems A and B) to facilitate identification of related compounds for which standards were not available. METHODS: C. paspali-infected Paspalum dilatatum as well as C. purpurea sclerotia obtained from infected Phalaris arundinacea were extracted and the extracts separated via liquid chromatography. Low- and high-resolution mass spectra were then obtained of known and potentially unknown indole-diterpenoids. RESULTS: At least 20 different indole-diterpenoids were detected in the C. paspali extract with molecular masses ranging from 405 Da (C28H40NO) to 517 Da (C32H40NO5). The C. purpurea sclerotia were shown to contain several indole-diterpenoids with molecular masses ranging from 405 Da (C28H40NO) to 419 Da (C28H38NO2). CONCLUSIONS: This study demonstrates for the first time that C. purpurea may also produce indole-diterpenoids. This might explain why grazing of Phalaris spp. is occasionally connected with a tremorgenic syndrome in cattle, called 'phalaris staggers'.
Assuntos
Claviceps/química , Diterpenos/química , Indóis/química , Espectrometria de Massas/métodos , Diterpenos/análise , Análise de Fourier , Indóis/análise , Phalaris/microbiologiaRESUMO
Ciguatera poisoning (CP) is endemic to several subtropical and tropical regions and is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The recent discovery of Caribbean CTXs (C-CTXs) in Gambierdiscus spp. isolated from the Caribbean resulted in the identification of a precursor analogue, C-CTX5, that is reduced into C-CTX1. C-CTX5 has two reducible sites, a ketone at C-3 and hemiketal at C-56. Chemical reductions of C-CTX5 into C-CTX3/4 resulted in two peaks in the LC-HRMS chromatograms with a ratio that differed markedly from that observed in fish extracts and the reduction of C-CTX1 isolated from fish. Reduction of C-CTX5 should have produced four diastereoisomers of C-CTX3/4, prompting a more detailed study of the reduction products. LC-HRMS with a slow gradient was used to separate and detect the four stereoisomers of C-CTX3/4, and to determine the distribution of these analogues in naturally contaminated fish tissues and following chemical reduction of isolated analogues. The results showed that in naturally contaminated fish tissues C-CTX1/2 is a mixture of two diastereoisomers at C-3 and that C-CTX3/4 is a mixture of two pairs of diastereoisomers at C-3 and C-56. The data suggests that there is variability in the enzymatic reduction at C-3 and C-56 of C-CTXs in reef fish, leading to variations in the ratios of the four stereoisomers. Based on these findings, a naming convention for C-CTXs is proposed which aligns with that used for Pacific CTX congeners and will aid in the identification of the structure and stereochemistry of the different CTX analogues.
Assuntos
Ciguatera , Ciguatoxinas , Dinoflagellida , Animais , Ciguatoxinas/toxicidade , Ciguatoxinas/química , Ciguatera/epidemiologia , Peixes , Região do Caribe , Dinoflagellida/químicaRESUMO
This study aimed to prove the validity of a mixture of chemicals, including salts, small organic molecules, mucin, and α-amylase, as saliva surrogate ("artificial saliva") for assessing leakage of methacrylate monomers and other constituents from dental materials. To achieve this, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA), diurethane dimethacrylate (UDMA), bisphenol A glycerolate dimethacrylate (BisGMA), diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO), bisphenol A (BPA), and five homologues of ethoxylated bisphenol A dimethacrylate (BisEMA EO2-6) in unstimulated and artificial saliva, and compared their concentrations in the two saliva media following either spiking with a mixture of the compounds or incubation of test specimens of printed biomaterials. Test specimens were immersed in unstimulated/artificial saliva, incubated at 37 °C for 24 h, and saliva aliquots were extracted with methanol and subsequently analyzed by LC-MS/MS. The method was validated with regard to matrix effects, linearity, selectivity, lower limits of quantification (LLOQ), precision, bias and combined measurement uncertainty (u'). The performance characteristics of the method were comparable for unstimulated and artificial saliva samples. The combined u' for individual chemicals at a concentration of 10 × LLOQ were within the range of 5.3-14 % for unstimulated saliva and 6.9-16 % for artificial saliva, except for the BisEMA homologues. Combined u' for the latter were 27-74 % in unstimulated saliva, and 27-79 % in artificial saliva. There was no detectable release of BPA from the test specimens, and the TPO concentrations were mainly below the LLOQ. TEGDMA and UDMA were detected in the highest quantities, and at comparable concentrations in the unstimulated and artificial saliva. For all BisEMA homologues, the release was higher in unstimulated saliva than in artificial saliva. The study showed that the artificial saliva model can be a suitable replacement for native saliva, but might underestimate leakage of more lipophilic methacrylates.
Assuntos
Compostos Benzidrílicos , Resinas Compostas , Fenóis , Saliva , Humanos , Saliva/química , Cromatografia Líquida , Resinas Compostas/análise , Resinas Compostas/química , Saliva Artificial/análise , Espectrometria de Massas em Tandem , Metacrilatos/química , Ácidos Polimetacrílicos/química , Polietilenoglicóis/química , Teste de MateriaisRESUMO
Metabolomic analysis has been explored to search for disease biomarkers in humans for some time. The application to animal species, including fish, however, is still at the beginning. In the present study, we have used targeted and untargeted metabolomics to identify metabolites in the plasma of Atlantic salmon (Salmo salar) challenged with Piscine orthoreovirus (PRV-1), aiming to find metabolites associated with the progression of PRV-1 infection into heart and skeletal muscle inflammation (HSMI). The metabolomes of control and PRV-1-infected salmon were compared at three time points during disease development by employing different biostatistical approaches. Targeted metabolomics resulted in the determination of affected metabolites and metabolic pathways, revealing a substantial impact of PRV-1 infection on lipid homeostasis, especially on several (lyso)phosphatidylcholines, ceramides, and triglycerides. Untargeted metabolomics showed a clear separation of the treatment groups at later study time points, mainly due to effects on lipid metabolism pathways. In a subsequent multi-omics approach, we combined both metabolomics datasets with previously reported proteomics data generated from the same salmon plasma samples. Data processing with DIABLO software resulted in the identification of significant metabolites and proteins that were representative of the HSMI development in the salmon.
RESUMO
The tobacco alkaloid nicotine is known for its activation of neuronal nicotinic acetylcholine receptors. Nicotine is consumed in different ways such as through conventional smoking, e-cigarettes, snuff or nicotine pouches. The use of snuff has been associated with several adverse health effects, such as inflammatory reactions of the oral mucosa and oral cavity cancer. We performed a metabolomic analysis of nicotine-exposed THP-1 human monocytes. Cells were exposed to 5 mM of the alkaloid for up to 4 h, and cell extracts and medium subjected to untargeted liquid chromatography high-resolution mass spectrometry. Raw data processing revealed 17 nicotine biotransformation products. Among these, cotinine and nornicotine were identified as the two major cellular biotransformation products. The application of multi- and univariate statistical analyses resulted in the annotation, up to a certain level of identification, of 12 compounds in the cell extracts and 13 compounds in the medium that were altered by nicotine exposure. Of these, four were verified as methylthioadenosine, cytosine, uric acid, and L-glutamate. Methylthioadenosine levels were affected in both cells and the medium, while cytosine, uric acid, and L-glutamate levels were affected in the medium only. The effects of smoking on the pathways involving these metabolites have been previously demonstrated in humans. Most of the other discriminating compounds, which were merely tentatively or not fully identified, were amino acids or amino acid derivatives. In conclusion, our preliminary data suggest that some of the potentially adverse effects related to smoking may also be expected when nicotine is consumed via snuff or nicotine pouches.
Assuntos
Espectrometria de Massas , Metabolômica , Monócitos , Nicotina , Humanos , Nicotina/metabolismo , Nicotina/análogos & derivados , Metabolômica/métodos , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Espectrometria de Massas/métodos , Células THP-1 , Cotinina/análogos & derivados , Cotinina/metabolismo , Cromatografia Líquida/métodos , Metaboloma/efeitos dos fármacos , Ácido Glutâmico/metabolismoRESUMO
In May-June 2019, the microalga Chrysochromulina leadbeateri caused a massive fish-killing event in several fjords in Northern Norway, resulting in the largest direct impact ever on aquaculture in northern Europe due to toxic algae. Motivated by the fact that no algal toxins have previously been described from C. leadbeateri, we set out to investigate the chemical nature and toxicity of secondary metabolites in extracts of two strains (UIO 393, UIO 394) isolated from the 2019 bloom, as well as one older strain (UIO 035) isolated during a bloom in Northern Norway in 1991. Initial LC-DAD-MS/MS-based molecular networking analysis of the crude MeOH extracts of the cultivated strains showed that their profiles of small organic molecules, including a large number of known lipids, were very similar, suggesting that the same class of toxin(s) were likely the causative agents of the two harmful algal bloom (HAB) events. Next, bioassay-guided fractionation using the RTgill-W1 cell line and metabolomics analysis pointed to a major compound affording [M + H]+ ions at m/z 1399.8333 as a possible toxin, corresponding to a compound with the formula C67H127ClO27. Moreover, our study unveiled a series of minor analogues exhibiting distinct patterns of chlorination and sulfation, together defining a new family of compounds, which we propose to name leadbeaterins. Remarkably, these suspected toxins were detected in situ in samples collected during the 2019 bloom close to Tromsø, thereby consistent with a role in fish kills. The elemental compositions of the putative C. leadbeateri ichthyotoxins strongly indicate them to be long linear polyhydroxylated polyketides, structurally similar to sterolysins reported from a number of dinoflagellates.
Assuntos
Proliferação Nociva de Algas , Toxinas Marinhas , Noruega , Toxinas Marinhas/toxicidade , Toxinas Marinhas/química , Toxinas Marinhas/análise , Estuários , Animais , Espectrometria de Massas em Tandem , Haptófitas/químicaRESUMO
Brevetoxins (BTX) are a group of marine neurotoxins produced by the harmful alga Karenia brevis. Numerous studies have shown that BTX are rapidly accumulated and metabolized in shellfish and mammals. However, there are only limited data on BTX metabolism in fish, despite growing evidence that fish serve as vectors for BTX transfer in marine food webs. In this study, we aimed to investigate the in vitro biotransformation of BTX-2, the major constituent of BTX profiles in K. brevis, in several species of northern Gulf of Mexico fish. Metabolism assays were performed using hepatic microsomes prepared in-house as well as commercially available human microsomes for comparison, focusing on phase I reactions mediated by cytochrome P450 monooxygenase (CYP) enzymes. Samples were analyzed by UHPLC-HRMS(/MS) to monitor BTX-2 depletion and characterize BTX metabolites based on MS/MS fragmentation pathways. Our results showed that both fish and human liver microsomes rapidly depleted BTX-2, resulting in a 72-99% reduction within 1 h of incubation. We observed the simultaneous production of 22 metabolites functionalized by reductions, oxidations, and other phase I reactions. We were able to identify the previously described congeners BTX-3 and BTX-B5, and tentatively identified BTX-9, 41,43-dihydro-BTX-2, several A-ring hydrolysis products, as well as several novel metabolites. Our results confirmed that fish are capable of similar BTX biotransformation reactions as reported for shellfish and mammals, but comparison of metabolite formation across the tested species suggested considerable interspecific variation in BTX-2 metabolism potentially leading to divergent BTX profiles. We additionally observed non-enzymatic formation of BTX-2 and BTX-3 glutathione conjugates. Collectively, these findings have important implications for determining the ecotoxicological fate of BTX in marine food webs.
RESUMO
Ciguatera poisoning (CP) is a severe seafood-borne disease, caused by the consumption of reef fish contaminated with Caribbean ciguatoxins (C-CTXs) in the Caribbean and tropical Atlantic. However, C-CTXs have not been identified from their presumed algal source, so the relationship to the CTXs in fish causing illness remains unknown. This has hindered the development of detection methods, diagnostics, monitoring programs, and limited fundamental knowledge on the environmental factors that regulate C-CTX production. In this study, in vitro and chemical techniques were applied to unambiguously identify a novel C-CTX analogue, C-CTX5, from Gambierdiscus silvae and Gambierdiscus caribaeus strains from the Caribbean. Metabolism in vitro by fish liver microsomes converted algal C-CTX5 into C-CTX1/2, the dominant CTX in ciguatoxic fish from the Caribbean. Furthermore, C-CTX5 from G. silvae was confirmed to have voltage-gated sodium-channel-specific activity. This finding is crucial for risk assessment, understanding the fate of C-CTXs in food webs, and is a prerequisite for development of effective analytical methods and monitoring programs. The identification of an algal precursor produced by two Gambierdiscus species is a major breakthrough for ciguatera research that will foster major advances in this important seafood safety issue.
Assuntos
Ciguatera , Ciguatoxinas , Dinoflagellida , Animais , Ciguatoxinas/toxicidade , Região do Caribe , PeixesRESUMO
Reindeer (Rangifer tarandus tarandus) are exposed to the pathogenic parasitic nematode Elaphostrongylus rangiferi during grazing. The severity of disease is dose-dependent. Prophylactic anthelmintic treatment is needed to improve animal health and reindeer herding sustainability. Herds are traditionally only gathered once during the summer, requiring a drug with a persistent effect. In this study we investigated the suitability of long-acting eprinomectin, given as a single subcutaneous injection at 1 mg/kg bodyweight in adult reindeer and calves. Plasma and faeces concentrations were determined using ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS). Plasma concentrations remained above the presumed effect level of 2 ng/mL for 80 days, demonstrating the drug's potential. Pharmacokinetic parameters were compared to other species using allometric scaling. Calves and adults had slightly different profiles. No viable faecal nematode eggs were detected during treatment. Eprinomectin was measurable in the reindeer faeces up to 100 days, which is of environmental concern.
Assuntos
Rena , Animais , Rena/parasitologia , Projetos Piloto , Ivermectina , Injeções SubcutâneasRESUMO
Recent identification of 6PPD-quinone as the chemical causing acute toxicity in coho salmon has led to substantial concern regarding the toxicity of this contaminant for other aquatic species. Environmental occurrence of 6PPD-quinone is probably high, because it is an oxidation product of a common tire rubber additive. Research on 6PPD-quinone toxicity in fish has revealed a rather unusual pattern, with closely related species exhibiting responses ranging from extreme sensitivity to no effect. Of 11 previously studied fish species, 6PPD-quinone was toxic to four. The species-specific toxicity of 6PPD-quinone complicates urgently needed environmental risk assessment. We investigated the acute toxicity of 6PPD-quinone in Atlantic salmon and brown trout alevins (sac fry). These species have previously not been tested for sensitivity to 6PPD-quinone. The fish were exposed in static conditions in eight treatments with initial concentrations ranging from 0.095 to 12.16 µg/L. Fish were observed for 48 h, and changes in concentrations of 6PPD-quinone were monitored throughout the experiment. No mortalities or substantial changes in behavior were recorded in either Atlantic salmon or brown trout. This provides an important first step in assessing effects of 6PPD-quinone on these economically and culturally highly important species. Environ Toxicol Chem 2022;41:3041-3045. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
Assuntos
Salmo salar , Animais , Borracha , Truta/fisiologia , Especificidade da Espécie , QuinonasRESUMO
Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) or high-resolution MS (LC-HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC-MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard's reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC-MS/MS and LC-HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1-GRT derivatization strategy mitigates many of the shortcomings of current LC-MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes.