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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.

The plant parasitic fungus Claviceps purpurea sensu lato produces sclerotia containing toxic ergot alkaloids and uncharacterized indole diterpenoids in grasses including cereals. The aim of this study was to detect as many peptide ergot alkaloids and indole diterpenoids in ergot sclerotia as possible by using a liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS) approach and applying filtering of diagnostic fragment ions for data extraction. The sample set consisted of 66 Claviceps sclerotia from four different geographic locations in southeastern Norway as well as Saskatchewan, Canada. The host plants included both wild grasses and important cereal grains such as rye. DNA sequencing showed that the sclerotia were from three Claviceps species, i.e., Claviceps purpurea sensu stricto (s.s.), Claviceps humidiphila, and Claviceps arundinis (former C. purpurea genotypes G1, G2, and G2a, respectively). All sclerotia from cereal grains were from C. purpurea s.s. Diagnostic fragment filtering was based on detecting specific product ions in MS/MS data sets that are well-conserved across the different ergot alkaloid subgroups and indole diterpenoids of the paspaline/paxilline type. The approach extracted mass spectra from 67 peptide ergot alkaloids (including C-8 epimers and lactam variants) and five indole diterpenoids. In addition, three clavines were detected by using targeted analysis. The sum of the peak areas for ergot alkaloids, which have been assigned as "major" analogues by the European Food Safety Authority (ergometrine, ergosine, ergotamine, α-ergocryptine, ergocornine, ergocristine, and their 8-S epimers), accounted for at least 50% of the extracted total ergot alkaloid metabolome. Univariate and multivariate statistical analyses showed that several of the alkaloids were specific for certain species within the C. purpurea species complex and could be used as chemotaxonomic markers for species assignment.

Juveniles are considered as one of the most vulnerable population groups concerning mycotoxins and their modified forms. The weaning stage is a particularly vulnerable period in the life of mammals, reflected in intestinal and immune dysfunction. The current study investigated the toxicokinetic (TK) characteristics of zearalenone (ZEN), zearalenone-14-glucoside (ZEN14G), and zearalenone-14-sulfate (ZEN14S) in weaned (4-week-old) piglets, by means of oral and intravenous administration of equimolar doses, i.e., 331, 500, and 415 μg/kg bodyweight, respectively. Plasma and urine were sampled pre- and post-administration and were quantitatively analyzed for ZEN, ZEN14G, ZEN14S, and in vivo metabolites by liquid chromatography-high-resolution mass spectrometry. Tailor-made TK models were elaborated to process data. A statistical comparison of the results was performed with TK data obtained in a previously reported study in pigs of 8 weeks of age. Additionally, porcine plasma protein binding was determined to support TK findings. The TK results for ZEN, ZEN14G, and ZEN14S, obtained in 4- and 8-week-old pigs, revealed significant age-related differences, based on differences in intestinal permeability, body fat content, gastrointestinal transit time, and biotransformation, with a special emphasis on an increased absorbed fraction of ZEN14G, i.e., 94 vs 61% in 4- compared to 8-week-old pigs. Since the growing pig has been reported to be a suitable pediatric animal model for humans concerning TK processes, these results may contribute to refine the risk assessment concerning modified ZEN forms in juvenile animals and humans.

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.

The contents and profiles of small molecules in a food can provide information about quality-related properties. Processing methods and deterioration during storage, e.g. from bacterial proliferation and degradation, might also lead to changes in the metabolome, which can be determined by mass spectrometry-based metabolomics. By measuring as many metabolites as possible in differently treated pre-cooked chicken fillets in an untargeted approach, we studied individual and combined effects of vacuum packaging (VP), soluble gas stabilisation (SGS), high pressure processing (HPP), and microwave volumetric heating (MW) on the quality and shelf-life of the finished product. The extensive dataset was processed using an optimised workflow of consecutive software tools with stringent statistical analysis to prevent over-interpretation, which is an inherent risk of metabolomics data. Our results showed the predominant influence of VP on storage quality since SGS, HPP, and MW did not have the potential to extent shelf-life.

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,β-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.

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.

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&ndash, 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&ndash, 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.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB−. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3β-hydroxysteroid dehydrogenase (3β-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3β-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB− had an opposite effect on 3β-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11β1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB− extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.

Deoxynivalenol (DON) is one of the most prevalent Fusarium mycotoxins in grain and can cause economic losses in pig farming due to reduced feed consumption and lower weight gains. Biodetoxification of mycotoxins using bacterial strains has been a focus of research for many years. However, only a few in vivo studies have been conducted on the effectiveness of microbial detoxification of fusariotoxins. This study was therefore aimed at investigating the effect of a feed additive containing the bacterial strain Coriobacteriaceum DSM 11798 (the active ingredient in Biomin® BBSH 797) on growth performance and blood parameters, as well as uptake and metabolism of DON, in growing pigs. Forty-eight crossbred (Landrace-Yorkshire/Duroc-Duroc) weaning pigs were fed with pelleted feed made from naturally contaminated oats, with DON at four concentration levels: (1) control diet (DON < 0.2 mg kg−1), (2) low-contaminated diet (DON = 0.92 mg kg−1), (3) medium-contaminated diet (DON = 2.2 mg kg−1) and (4) high-contaminated diet (DON = 5.0 mg kg−1) and equivalent diets containing DSM 11798 as feed additive. During the first 7 days of exposure, pigs in the highest-dose group showed a 20–28% reduction in feed intake and a 24–34% reduction in weight gain compared with pigs in the control and low-dose groups. These differences were levelled out by study completion. Towards the end of the experiment, dose-dependent reductions in serum albumin, globulin and total serum protein were noted in the groups fed with DON-contaminated feed compared with the controls. The addition of DSM 11798 had no effect on the DON-related clinical effects or on the plasma concentrations of DON. The ineffectiveness of the feed additive in the present study could be a consequence of its use in pelleted feed, which might have hindered its rapid release, accessibility or detoxification efficiency in the pig’s gastrointestinal tract.

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.

The dietary exposure to the mycotoxin deoxynivalenol (DON) can be assessed by human biomonitoring (HBM). Here, we assessed the relation between dietary DON intake and the excretion of its major metabolite DON-15-glucuronide (DON15GlcA) through time, in an everyday situation. For 49 volunteers from the EuroMix biomonitoring study, the intake of DON from each meal was calculated and the excretion of DON and its metabolites was analyzed for each urine void collected separately throughout a 24-h period. The relation between DON and DON15GlcA was analyzed with a statistical model to assess the residence time and the excreted fraction of ingested DON as DON15GlcA (fabs_excr). Fabs_excr was treated as a random effect variable to address its heterogeneity in the population. The estimated time in which 97.5% of the ingested DON was excreted as DON15GlcA was 12.1 h, the elimination half-life was 4.0 h. Based on the estimated fabs_excr, the mean reversed dosimetry factor (RDF) of DON15GlcA was 2.28. This RDF can be used to calculate the amount of total DON intake in an everyday situation, based on the excreted amount of DON15GlcA. We show that urine samples collected over 24 h are the optimal design to study DON exposure by HBM.

Oat (Avena sativa L.) ranks seventh in the world cereal production and is considered to be an important source for many valuable components of nutritional and biological importance, i.e. proteins, fats, carbohydrates, fibre, minerals and vitamins. Because of these properties the amount of oat used for human consumption has increased progressively during the last years. Unfortunately, the quality of this grain crop is often compromised by mycotoxin contamination, which is relatively ubiquitous despite efforts to control the problem. Therefore, it is important to investigate the distribution pattern of mycotoxins and their conjugated derivatives in contaminated oat grains. For this purpose we have developed a state-of-the-art multi-mycotoxin high-resolution mass spectrometry method and analysed oat samples for their content of the most important mycotoxins commonly occurring in Norwegian cereal grain. Quantitative mapping of selected Fusarium free and modified mycotoxins was performed in fractions collected during processing trials consisting of dehulling and sequential pearling. Both the derivative free mycotoxins and their metabolites were mainly present in the hulls compared to the oat kernel, thus dehulling resulted in a significant reduction of the total mycotoxin load, followed by some further reduction by pearling. Furthermore, free and modified mycotoxins were unevenly distributed in relation to each other throughout the grain fractions, showing a shift towards glucosidated forms, such as deoxynivalenol-3-glucoside and HT-2-3-glucoside in the oat kernel, which highlights potential food safety concerns associated with in planta modified mycotoxins.

The aim of this study was to determine the toxicokinetic characteristics of ZEN and its modified forms, α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalenone-14-glucoside (ZEN14G), and zearalenone-14-sulfate (ZEN14S), including presystemic and systemic hydrolysis in pigs. Crossover pig trials were performed by means of intravenous and oral administration of ZEN and its modified forms. Systemic plasma concentrations of the administered toxins and their metabolites were quantified and further processed via tailor-made compartmental toxicokinetic models. Furthermore, portal plasma was analyzed to unravel the site of hydrolysis, and urine samples were analyzed to determine urinary excretion. Results demonstrate complete presystemic hydrolysis of ZEN14G and ZEN14S to ZEN and high oral bioavailability for all administered compounds, with further extensive first-pass glucuronidation. Conclusively, the modified-ZEN forms α-ZEL, β-ZEL, ZEN14G, and ZEN14S contribute to overall ZEN systemic toxicity in pigs and should be taken into account for risk assessment.

The ability to identify drivers responsible for algal community shifts is an important aspect of environmental issues. The lack of long-term datasets, covering periods prior to these shifts, is often limiting our understanding of drivers responsible. The freshwater alga, Gonyostomum semen (Raphidophyceae), has significantly increased distribution and mass occurrences in Scandinavian lakes during the past few decades, often releasing a skin irritating slime that causes discomfort for swimmers. While the alga has been extensively studied, long-term data from individual lakes are often absent or greatly limited and drivers behind this species' success are still not clear. However, if specific and persistent taxa biomarkers for G. semen could be detected in dated sediment cores, long-term data would be improved and more useful. To test for biomarkers, we examined the pigment composition of several G. semen strains in culture. Further, dated sediment core samples from Lake Lundebyvann, Norway, were used to test the pigments' suitability as biomarkers in paleolimnological studies. Modifications to a common analysis allowed for the successful detection of the polar xanthophyll heteroxanthin and the non-polar chlorophyll a, as well as several other algal pigments by using high performance liquid chromatography-photometric diode arrays (HPLC-PDA). Heteroxanthin was confirmed by liquid chromatography-mass spectrometry (LC-MS) and detected by HPLC-PDA in all examined G. semen strains, along with chlorophyll a. Using HPLC-PDA, we also identified and confirmed the presence of the biomarker, xanthophyll heteroxanthin, in sediment core samples up to 60 years of age. The specificity of this xanthophyll was also tested by examining a wide range of algal strains from common Norwegian phytoplankton species. Heteroxanthin was not detected in any species commonly occurring in significant amounts in Norwegian lakes. We therefore conclude that heteroxanthin is a suitable pigment biomarker for G. semen and that this pigment can be successfully used for paleolimnological studies.

Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by LC-MS, but their identities were not confirmed due to a lack of standards. We have synthesized DON-GSH conjugates in alkaline solution. The major products 2 and 5 were isolated and their structures determined by mass spectrometry and NMR spectroscopy as GSH adducts at C-13 and C-10 (via epoxide and Michael addition, respectively) of 1. Other Michael addition products were also tentatively identified by LC-MS. Concentrations of 2 and 5 were determined by quantitative NMR and are suitable for use as quantitative standards for LC-MS studies of plant and animal metabolism of 1. LC-MS showed that in the presence of human glutathione S-transferases of the alpha and mu classes, the reaction of DON and GSH proceeded with a half-life of 17 h, identical with the rate of the uncatalyzed reaction rate, indicating an absence of catalysis.

This study determined the presence, levels and co-occurrence of mycotoxins in fish feeds in Kenya. Seventy-eight fish feeds and ingredients were sampled from fish farms and fish feed manufacturing plants and analysed for 40 mycotoxins using high-performance liquid chromatography-high resolution mass spectrometry. Twenty-nine (73%) mycotoxins were identified with 76 (97%) samples testing positive for mycotoxins presence. Mycotoxins with the highest prevalences were enniatin B (91%), deoxynivalenol (76%) and fumonisin B1 (54%) while those with the highest maximum levels were sterigmatocystin (&lt, 30.5&ndash, 3517.1 &micro, g/kg), moniliformin (&lt, 218.9&ndash, 2583.4 &micro, g/kg) and ergotamine (&lt, 29.3&ndash, 1895.6 &micro, g/kg). Mycotoxin co-occurrence was observed in 68 (87%) samples. Correlations were observed between the fumonisins, enniatins B and zearalenone and its metabolites. Fish dietary exposure estimates ranged between &lt, 0.16 and 43.38 &micro, g/kg body weight per day. This study shows evidence of mycotoxin presence and co-occurrence in fish feeds and feed ingredients in Kenya. Fish exposure to these levels of mycotoxins over a long period of time may lead to adverse health effects due to their possible additive, synergistic or antagonist toxic effects. Measures to reduce fish feed mycotoxin contamination should be taken to avoid mycotoxicosis in fish and subsequently in humans and animals through residues.

The tremorgenic mycotoxin penitrem A is produced by Penicillium species as a secondary metabolite on moldy food and feed. Dogs are sometimes exposed to penitrem A by consumption of spoiled food waste or fallen fruit. The lipophilic toxin crosses the blood-brain barrier and targets neuroreceptors and neurotransmitter release mechanisms in the central and peripheral nervous systems. Typical symptoms of penitrem A intoxication are periodical or continuous tremors, which can be passing, persistent or lethal, depending on the absorbed dose. There is presently no information on the biotransformation and toxicokinetics of penitrem A in dogs. The aim of the present study was therefore to identify potential metabolites of the toxin by performing in vitro biotransformation assays in dog liver microsomes. Analyses by liquid chromatography coupled to high-resolution mass spectrometry led to the provisional identification of eleven penitrem A phase I metabolites, which were tentatively characterized as various oxidation products. Furthermore, elimination parameters determined in in vitro assays run under linear kinetics were used for in vitro-to-in vivo extrapolation of the toxicokinetic data, predicting a maximal bioavailability of more than 50%. The metabolite profile detected in the in vitro assays was similar to that observed in the plasma of an intoxicated dog, confirming the predictive capability of the in vitro approach.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungi. Once ingested, ZEN may be absorbed and metabolised to α- and β-zearalenol (α-ZOL, β-ZOL), and to a lesser extent α- and β-zearalanol (α-ZAL, β-ZAL). Further biotransformation to glucuronide conjugates also occurs to facilitate the elimination of these toxins from the body. Unlike ZEN and its metabolites, information regarding the estrogenic activity of these glucuronide conjugates in various tissues is lacking. ZEN-14-O-glucuronide, α-ZOL-14-O-glucuronide, α-ZOL-7-O-glucuronide, β-ZOL-14-O-glucuronide and β-ZOL-16-O-glucuronide, previously obtained as the major products from preparative enzymatic synthesis, were investigated for their potential to cause endocrine disruption through interference with estrogen receptor transcriptional activity. All five glucuronide conjugates showed a very weak agonist response in an estrogen responsive reporter gene assay (RGA), with activity ranging from 0.0001% to 0.01% of that of 17β-estradiol, and also less than that of ZEN, α-ZOL and β-ZOL which have previously shown estrogenic potencies of the order 17β-estradiol>α-ZOL>ZEN>β-ZOL. Confirmatory mass spectrometry revealed that any activity observed was likely a result of minor deconjugation of the glucuronide moiety. This study confirms that formation of ZEN and ZOL glucuronides is a detoxification reaction with regard to estrogenicity, serving as a potential host defence mechanism against ZEN-induced estrogenic activity.

Employees at grain elevators and compound feed mills are exposed to large amounts of grain dust during work, frequently leading to airway symptoms and asthma. Although the exposure to grain dust, microorganisms, β-1→3-glucans and endotoxins has been extensively studied, the focus on the mycotoxin content of grain dust has previously been limited to one or few mycotoxins. Our objective was therefore to screen settled grain dust from grain elevators and compound feed mills for fungal metabolites by LC/MS-MS and explore differences between work places, seasons and climatic zones. Seventy fungal metabolites and two bacterial metabolites were detected. Trichothecenes, depsipeptides, ergot alkaloids, and other metabolites from Fusarium, Claviceps, Alternaria, Penicillium, Aspergillus, and other fungi were represented. The prevalence of individual metabolites was highly variable, and the concentration of each metabolite varied considerably between samples. The prevalence and concentration of most metabolites were higher in grain elevators compared to compound feed mills. Differences between seasons and climatic zones were inconclusive. All samples contained multiple mycotoxins, indicating a highly complex pattern of possible inhalational exposure. A mean exposure of 20 ng/m3 of fungal metabolites was estimated, whereas a worst case scenario estimated as much as 10 ?g/m3. Although many of these compounds may be linked to toxicological and immunological effects through experimental or epidemiological studies, it still remains to be determined whether the detected concentrations implicate adverse health outcomes when inhaled.

Source at http://dx.doi.org/10.3390/md15060148 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.

Wild ruminants, including deer species (cervids) have incorrectly been regarded as refractory to yew (Taxus) intoxication. This assumption has been based upon anecdotal observations of individual deer browsing on yew over time without apparent adverse effect. A single case of yew intoxication was reported in a free-ranging Norwegian moose (Alces alces) in 2008. The current report describes five additional cases of yew toxicosis in moose, seven in roe deer (Capreolus capreolus) and two in reindeer (Rangifer tarandus tarandus), all in Norway. The animals were found dead during the winter, close to or within gardens containing yew plants showing signs of browsing. Gross findings included lung congestion and edema, thoracic and pericardial effusion, bilateral heart dilatation, epi- and endocardial hemorrhage, and enlarged (congested) spleen. Yew plant remnants were detected in the rumen of all animals with the exception of a single moose. Histology revealed multifocal acute myocardial degeneration and necrosis with hemorrhage in roe deer, but not in the two other species. A qualitative high performance liquid chromatography-ion trap mass spectrometry analysis was used to tentatively identify five major Taxus alkaloids (taxines) in crude yew extracts and in heart and liver samples from the moose cases. All five major taxines were detected with good signal/noise ratio in tissue samples from the four moose with visible ruminal yew content, whereas lower levels of taxines were detected in the moose without visible ruminal yew content. Possible differences in interspecies tolerance to taxines and role of individual protective adaptation are discussed.

Enniatins (ENNs) are hexadepsipeptidic mycotoxins produced by Fusarium species. They occur in mg/kg levels in grain from Northern climate areas. Major ENNs such as ENN B and B1 have shown considerable cytotoxicity in different in vitro test systems. To adequately assess exposure and in vivo toxicity the toxicokinetic properties need to be investigated. The present study describes the metabolism of ENN B1 both in vitro and in vivo in pigs, comparing metabolites found in vitro in experiments with liver microsomes from different pig strains to those found in the plasma of pigs after single oral or intravenous application of ENN B1. Metabolites of hepatic biotransformation were tentatively identified and characterised by high performance liquid chromatography coupled to ion trap and high-resolution mass spectrometry, as well as chemical derivatisations. Kinetic parameters of metabolite formation and elimination were determined. Metabolite formation was higher when ENN B1 was absorbed from the gut compared to intravenous administration indicating pre-systemic metabolism of ENN B1 after oral uptake. The in vitro approach resulted in the detection of ten ENN B1 metabolites, while six were detected in in vivo samples. The putative ENN B1 metabolites were products of hydroxylation, carbonylation, carboxylation and oxidative demethylation reactions.

Commercial immunoaffinity columns (IACs) are today available for all major mycotoxins. However, manufacturers give usually no or very limited information on the epitope, i.e. the specific part of the toxin molecule that binds to the antibody. 4-Deoxynivalenol (DON) is a trichothecene mycotoxin that is produced by plant pathogenic field fungi and is regulated in many countries worldwide. DON was shown to be biotransformed via different metabolic pathways, and thus many different biotransformation products may be found in different products or organisms. In addition, several structurally similar mycotoxins may co-occur with DON. We compared five commercial IACs for their retention of a range of DON derivatives modified in the C-3, C-8, C-10, C-13 or C-15 positions, as well as nivalenol (NIV) and T-2 tetraol. The DON-derivatives were deepoxy-DON, DON 3-, 8- and 15-O-β-d-glucuronides, 3- and 15-O-acetyl-DON, DON-3-O-β-d-glucoside, 10- and 13-cysteinyl-adducts of DON, and the 13-mercaptoethanol and 10,13-dimercaptoethanol adducts of DON. The C-3 derivatives and deepoxy-DON were retained by most of the columns. Only one of the five IACs retained C-15 and C-8 derivatives, but it did not retain C-3 derivatives or deepoxy-DON. The antibodies in two of the IACs bound C-10 conjugates, but C-13 derivatives were not retained by any of the columns. This study shows that all of the antibodies in commercial IACs bind a range of DON derivatives, especially those that are modified at C-3. NIV was retained by three of the columns, and T-2 tetraol was partially retained by one IAC.

This is the pre-peer reviewed version of the following article: Øya, E., Afanou, K.A., Malla, N., Uhlig, S., Rolen, E., Skaar, I., ... Holme, J.A. (2017). Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species. Indoor Air: International Journal of Indoor Environment and Health, 28(1), 28-39. https://doi.org/10.1111/ina.12426, which has been published in final form at https://doi.org/10.1111/ina.12426. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory‐related diseases. We characterized X‐ray‐inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, β‐glucan and protease content/activity. The pro‐inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS‐2B) and THP‐1 monocytes and phorbol 12‐myristate 13‐acetate (PMA)‐differentiated THP‐1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin‐degrading protease that was either of the metallo‐ or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of β‐glucans were found mainly in hyphae particle preparations. PMA‐differentiated THP‐1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2. Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP‐9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro‐inflammatory potential.

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'. Copyright © 2014 John Wiley & Sons, Ltd.

Enniatins, a large group of cyclodepsipeptides, are widely distributed contaminants of different crops intended for human and animal consumption. Enniatin B is one of the principal analogues in species of the genus Fusarium, known to have ionophoric, antibiotic, and insecticidal activity. Regardless of considerable cytotoxic effects observed in vitro, enniatins have been characterised as compounds with low acute toxicity in vivo. The biotransformation of enniatin B has previously been elucidated in liver microsomes, and 12 different metabolites (M1 to M12) have been reported. In order to provide a better basis for understanding the potential toxic effects in humans and animals, different samples (eggs, livers, plasma) from two different feeding studies have been analysed for the presence of enniatin B and its hepatic metabolites. The earlier reported metabolite M11, and a novel metabolite (designated M13), were dominant in liver samples from enniatin B exposed broilers. The peak area corresponding to the sodiated molecular ion of M11 was approximately 2.5 times larger than that of parent enniatin B in liver samples collected after one week of exposure. The same metabolites were also present in serum samples. In egg samples, only metabolites M13 and M4 were detected. The comparison of mass spectrometric data of M13 and enniatin B suggested that M13 is a monohydroxylated metabolite. The hepatic biotransformation of enniatin B was also investigated in vitro in chicken microsomes demonstrating good correlation with the metabolite profiles in the chicken samples. The results of the present study demonstrated an extensive biotransformation of enniatin B in vivo confirming previously reported in vitro data.

In this pilot survey the levels of various mycotoxin biomarkers were determined in third trimester pregnant women from eastern Croatia. First void urine samples were collected and analysed using a "dilute and shoot" LC-ESI-MS/MS multi biomarker method. Deoxynivalenol (DON) and its metabolites: deoxynivalenol-15-glucuronide and deoxynivalenol-3-glucuronide were detected in 97.5% of the studied samples, partly at exceptionally high levels, while ochratoxin A was found in 10% of the samples. DON exposure was primarily reflected by the presence of deoxynivalenol-15-glucuronide with a mean concentration of 120 μg L(-1), while free DON was detected with a mean concentration of 18.3 μg L(-1). Several highly contaminated urine samples contained a third DON conjugate, tentatively identified as deoxynivalenol-7-glucuronide by MS/MS scans. The levels of urinary DON and its metabolites measured in this study are the highest ever reported, and 48% of subjects were estimated to exceed the provisional maximum tolerable daily intake (1 μg kg(-1) b.w.).

Conjugation with the biologically relevant thiol glutathione is one of the metabolic pathways for the mycotoxin deoxynivalenol (DON) in wheat. The occurrence of putative DON–cysteine conjugates has also been shown in wheat, likely in part as a result of degradation of the glutathione conjugates. It was reported that thiols react in vitro with DON at two positions: reversibly at C-10 of the α,β-unsaturated ketone and irreversibly at C-13 of the epoxy group. We synthesized pure DON–cysteine adducts and made analytical standards using quantitative NMR experiments. Compounds were characterized using NMR and LC–HRMS/MS and tested in vitro for toxicity. Cysteine conjugates were much less toxic than DON at the same concentration, and LC–HRMS analysis demonstrated that there was no detectable metabolism of the conjugates in human monocytes or human macrophages.

Biosynthesis of the black perithecial pigment in the filamentous fungus Fusarium graminearum is dependent on the polyketide synthase PGL1 (oPKS3). A seven-membered PGL1 gene cluster was identified by over-expression of the cluster specific transcription factor pglR. Targeted gene replacement showed that PGL1, pglJ, pglM and pglV were essential for the production of the perithecial pigment. Over-expression of PGL1 resulted in the production of 6-O-demethyl-5-deoxybostrycoidin (1), 5-deoxybostrycoidin (2), and three novel compounds 5-deoxybostrycoidin anthrone (3), 6-O-demethyl-5-deoxybostrycoidin anthrone (4) and purpurfusarin (5). The novel dimeric bostrycoidin purpurfusarin (5) was found to inhibit the growth of Candida albicans with an IC50 of 8.0 +/− 1.9 μM. The results show that Fusarium species with black perithecia have a previously undescribed form of 5-deoxybostrycoidin based melanin in their fruiting bodies.

A study was carried out on 43 malting barley samples collected in 2013 across the Umbria region (central Italy) to determine the incidence of the principal mycotoxigenic fungal genera, to identify the Fusarium species isolated from the grains, and to detect the presence of 34 fungal secondary metabolites by liquid chromatography-high-resolution mass spectrometry. The multimycotoxin-method development involved the evaluation of both a two-step solvent and QuEChERS protocol for metabolite extraction. The former protocol was selected because of better accuracy, which was evaluated on the basis of spike-recovery experiments. The most frequently isolated fungal species belonged to the genera Alternaria and Fusarium. The predominant Fusarium species was F. avenaceum, followed by F. graminearum. HT-2 toxin was the most frequently detected mycotoxin, followed by enniatin B, enniatin B1, T-2 toxin, and nivalenol. As a consequence of the observed mixed fungal infections, mycotoxin co-occurrence was also detected. A combination of mycological and mycotoxin analyses allowed the ability to obtain comprehensive information about the presence of mycotoxigenic fungi and their contaminants in malting barley cultivated in a specific geographic area.

α- and β-zearalenol (α-ZOL and β-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of α-ZOL and β-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by α-ZOL and β-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities.

A new natural ergopeptine, ergosedmine, was isolated from sclerotia of the ascomycete Claviceps purpurea . Its structure was elucidated by 1D- and 2D-NMR spectroscopy and mass spectrometry as N -( N -lysergyl)-cyclo(isoleucyl-phenylanalyl-prolyl). Partial epimerization of ergosedmine into ergosedminine was evident during purification and in DMSO- d 6 . According to common ergot alkaloid nomenclature ergosedmine belongs to the ergoannines, one of the four subgroups of ergopeptines. The sclerotia were obtained from reed feather grass ( Calamagrostis arundinacea ). Phylogenetic analyses using ITS- and beta-tubulin sequences of pure culture isolates obtained from the C. purpurea sclerotia demonstrated that the ergosedmine-producing strain belongs to the genotype G2, which is associated with wet and shady habitats.

A new selective method for the determination of the bioactive amine gizzerosine in poultry feed is presented based on hydrophilic interaction chromatography coupled to electrospray ionisation and tandem quadrupole mass spectrometry. The protonated molecular ions were selected for fragmentation and scanning of the two major product ions using the mass analyser in the multiple reaction monitoring mode. The analytical methodology included acid hydrolysis of feed sample aliquots and desalting and crude clean-up of hydrolysates using cation-exchange chromatography on a polymeric solid-phase extraction sorbent. Mean overall recoveries (n = 9) were 68–82% in spike-recovery experiments. The limit of detection was 0.25 mg kg−1. The method was applied to the analysis of fish meal and fish meal-containing poultry feed samples. Although gizzerosine could not be detected in any of the samples, it was found in fish meal made from sand eel after heating to 130 °C for 24 h.

This paper reports the mass spectra, obtained after electron ionisation (EI) at 70 eV, of 34 trichothecenes and five culmorin compounds after acylation with pentafluoropropionic anhydride. The derivatised fungal metabolites were separated by gas chromatography, and the mass spectra were obtained by scanning of a single quadrupole mass filter in the scan range m/z 200-900. The fragmentation pathways of three trichothecenes (triacetyl-deoxynivalenol, 4,15-diacetoxy-scirpenol, T-2 toxin) have been studied in more detail by linked scan - high-resolution mass spectrometry. The most common trichothecenes are today more often routinely analysed using LC/MS-based methodologies. However, EI-MS may give complementary structural information, and the data that are summarised in this article may help to identify analogues of one of the largest class of mycotoxins, the tricothecenes, as well as culmorin compounds that are commonly co-produced by Fusarium culmorum and F. graminearum in cultures or naturally contaminated samples.

The skin mucus of fish is in permanent contact with the aquatic environment. Data from the analysis of the chemical composition of skin mucus could potentially be used for monitoring the health status of the fish. Knowledge about mucus composition or change in composition over time could also contribute to understanding the aetiology of certain diseases. The objective of the present study was the development of a workflow for non-invasive sampling of skin mucus from farmed salmon (Salmo salar) for the targeted and untargeted detection of small metabolites. Skin mucus was either scraped off, wiped off using medical wipes, or the mucus&rsquo, water phase was absorbed using the same type of medical wipes that was used for the wiping method. Following a simple filtration step, the obtained mucus samples were subjected to hydrophilic interaction chromatography coupled to high-resolution mass spectrometry. Post-acquisition processing included the targeted analysis of 86 small metabolites, of which up to 60 were detected in absorbed mucus. Untargeted analysis of the mucus samples from equally treated salmon revealed that the total variation of the metabolome was lowest in absorbed mucus and highest in the scraped mucus. Thus, future studies including small-molecule metabolomics of skin mucus in fish would benefit from a sampling regime employing absorption of the water phase in order to minimize the bias related to the sampling step. Furthermore, the absorption method is also a less invasive approach allowing for repetitive sampling within short time intervals.

Taxa of the Alternaria infectoria species group are the predominant Alternaria spp. found in cereals in Northern Europe. While several pyrones have been isolated from A. infectoria and described as taxonomical markers for species identification, information about the bioactivity of metabolites from the fungus is missing. Bioassay-guided fractionation of rice culture extracts from several strains of A. infectoria linked the observed toxicity of the extracts in MRC-5 cells to free fatty acids, i.e. linoleic acid and alpha-linolenic acid. The fungus also produced a cytotoxic pyrone, which upon isolation and NMR spectroscopic analysis was identified as a mixture of phomenins A and B (approximately 10:1), which have not previously been isolated from an Alternaria species.

A strain of a yet unidentified Fusarium sp. produced in addition to enniatins A1, B and B1 a number of minor enniatins. The strain formed a well supported clade with strains identified as Fusarium acuminatum (Gibberella acuminata) in phylogenetic analyses using the TEF-1alpha gene sequences. Two of the minor enniatins were easily recognised as hydroxylated species on the basis of their fragment ion spectra. The hydroxylation could be traced to one of the amino acid moieties using multiple-stage ion trap mass spectrometry. Different approaches for acetylation of the isolated compounds and complete hydrolysis supported the elucidation of the amino acid moiety as 3-hydroxy-2-methylamino-butyric acid, which is equivalent with N-methyl-threonine. The primary structures of the two enniatins were tentatively determined to be cyclo[Hiv-N-Me-Val-Hiv-N-Me-Val-Hiv-N-Me-Thr] and cyclo[Hiv-N-Me-Leu-Hiv-N-Me-Val-Hiv-N-Me-Thr]. The two depsipeptides represent new analogues and were named enniatin P1 and P2, respectively.

Most Fusarium species are capable of producing mycotoxins that may cause adverse effects on human or animal health. The most commonly studied Fusarium mycotoxins include trichothecenes, zearalenone and fumonisins. However, it seems that nearly all of the most prevalent Fusarium species infecting grains are also capable of producing other toxic metabolites. The existing studies, although exiguous, have clearly demonstrated that other toxic metabolites of Fusarium spp. are also present in our foods and feeds, occasionally at very high levels. It is apparent that since mycotoxins, including these 'other' metabolites, are natural toxins, they cannot be completely eliminated from food and feed chains. However, scientific studies are needed to determine their true significance. Thus, the mechanism and level of toxicity as well as presence and concentration levels will have to be fully clarified. In this paper, we briefly review the prevalence of the dominant Fusarium species contaminating maize and small-grain cereals worldwide, and the current knowledge on the biological activity as well as the natural occurrence of their selected less-known toxic metabolites. Additionally, the significance of these 'other' Fusarium mycotoxins is discussed.

Four major alkaloids in the extracts from sclerotia of Claviceps purpurea , picked from wild grasses, have been identified as lactam (non-cyclol) ergot alkaloids. The structural information was obtained from ion trap MS and NMR spectroscopy. The data for one of the lactam ergot alkaloids were coinciding with ergocristam [ N -(lysergyl-valyl)-cyclo(phenylalanyl-prolyl)]. The structural information of two further lactam alkaloids was suggestive of either α- or β-ergocryptam [ N -(lysergyl-valyl)-cyclo(leucyl-prolyl) or N -(lysergyl-valyl)-cyclo(isoleucyl-prolyl)] and ergoannam [ N -(lysergyl-leucyl)-cyclo(leucyl-prolyl) or N -(lysergyl-isoleucyl)-cyclo(isoleucyl-prolyl)]. The constitution of the fourth lactam ergot alkaloid corresponded to N -(lysergyl-isoleucyl)-cyclo(phenylalanyl-prolyl), a new ergopeptam, which has not been described before. Additionally, the cyclol-analogue of the new ergopeptam was detected in the extracts and has been identified on the basis of its product ion spectrum from fragmentation of [M + H] + . The study described in this paper shows that lactam ergot alkaloids may not only be minor products of ergopeptine biosynthesis, as has been suggested hitherto, but may be major biosynthetic endproducts for some ergot strains. This is also the first report demonstrating the production of an ergot alkaloid that contains isoleucine as the second amino acid, i.e. the N -(lysergyl-isoleucyl)-moiety, by parasitic, naturally growing C. purpurea . This unusual type of ergot alkaloid has so far only been found in saprophytic cultures of C. purpurea .

2-Amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol) belongs to the class of sphingosine analogue toxins, which is a class of toxins that is known among several fungal genera and some lower marine animals. Many sphingosine analogues have been shown to inhibit ceramide synthase through structural similarities with the substrates of the enzyme. The adverse effects of some analogues, e.g. the fumonisins, are a result of ceramide synthase inhibition. The primary aim of this work was to generate basic knowledge on the toxicity of 2-AOD-3-ol in vitro. The human hepatocarcinoma cell line Hep G2 and horse erythrocytes were used in order to achieve this aim. The second aim of this work was to gain insight into the capabilities of the fungus Fusarium avenaceum, a common contaminant of grain in Scandinavia, to produce the toxin under laboratory conditions on different grain species, and in the field. The metabolite was found to be cytotoxic in several assays (lower µM-range) that measure general cytotoxicity (Alamar Blue" assay, Neutral Red uptake, LDH leakage), but did not inhibit ceramide synthase as measured by the increase of the sphinganine/sphingosine ratio. The cellular membrane was likewise not found to be a primary target of the molecule. Strains of F. avenaceum produced up to 6.3 mg/g of 2-AOD-3-ol on wheat grain under controlled conditions in the laboratory, while only low amounts (

Yucca schidigera juice in doses of 1.5 g (63 mg sapogenin) and 3.0 g (126 mg sapogenin) per kg live weight was administered intraruminally to 30 lambs for 21 days to investigate whether the saponins in Y. schidigera were toxic to lambs and whether they could cause hepatogenous photosensitisation. Twelve lambs died or had to be euthanised. The main pathological findings in the diseased lambs were acute tubular necrosis in the kidneys, dehydration and watery content in the gastrointestinal tract. Fifteen lambs were euthanised at the end of the study, and the main pathological findings in dosed animals were accumulation of homogeneous pale PAS-positive material in the hepatocytes. There was a rise in serum creatinine and urea concentrations in the lambs with renal lesions the day before they died. Major Y. schidigera-related saponins were found in the liver and kidney samples from all lambs that were dosed with Y. schidigera juice. The results of the present study demonstrate that un-hydrolysed saponins can be absorbed from the gastrointestinal tract. The possible role of saponins in causing nephrotoxicity is discussed.

The Fusarium species complex found on small-grain cereals in Northern Europe is largely dominated by F. avenaceum, while other important species include F. tricinctum, F. poae, F. culmorum and F. graminearum. The dominance of F. avenaceum has in recent years initiated extensive analytical activity in Norway and Finland in order to gain insight into the contamination of grain with secondary metabolites related to the fungus. Of these, moniliformin is the most studied compound with regard to toxicity. However, the data from analytical surveys indicate that field conditions in Northern Europe do not favour production of the metabolite. Instead, enniatins are regularly found in ppm-concentrations in grain, especially wheat and barley, while the bio-production of a range of other F. avenaceum related metabolites has so far barely been investigated. This paper summarises the results from mycological and chemical analyses of grain samples from Norway and Finland for major Fusarium species and F. avenaceum-related secondary metabolites.

3-Hydroxy acids are constituents of the lipid A part of lipopolysaccharides and may potentially be used as chemical markers of endotoxin. While commercial enzymatic assays, such as the widely used Limulus amebocyte lysate (LAL) assay, commonly detect merely the water-soluble fraction of the bioactive endotoxin, the chemical approach aims to estimate the total amount of endotoxin present in a sample. Our objective was to develop a simple method for quantitative profiling of 3-hydroxy fatty acids in occupational and environmental samples based on detection with HPLC-MS/MS. We included eleven 3-hydroxy fatty acids (3-hydroxyoctanoic acid to 3-hydroxyoctadecanoic acid) in the HPLC-MS/MS based method, which involved base hydrolysis of filter samples using 1M sodium hydroxide and removal of the base as well as concentration of the fatty acids using solid-phase extraction on a functionalized polystyrene-divinylbenzene polymer. Recovery trials from spiked glass fiber filters, using threo-9,10-dihydroxyhexadecanoic acid as internal standard, gave an overall recovery of 54-86% for 3-hydroxy fatty acids of medium chain length (3-hydroxynonanoic to 3-hydroxypentadecanoic acid). 3-Hydroxyoctanoic acid and the longer chain fatty acids were more problematic yielding overall spike recoveries of 11-39%. While the 3-hydroxy fatty acid profile of pure lipopolysaccharides was dominated by 3-hydroxydecanoic, 3-hydroxydodecanoic and 3-hydroxytetradecanoic acid the aqueous phase from drilling mud contained in addition relatively high amounts of 3-hydroxyoctanoic and 3-hydroxynonanoic acid. Endotoxin activity as measured by the LAL assay was reasonably correlated (R(2)=0.54) to the sum of 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid and 3-hydroxytetradecanoic acid in these samples.

Conjugation of deoxynivalenol (DON) with sulfur compounds is recognized as a significant reaction pathway, and putative DON–glutathione (DON–GSH) conjugates have been reported in planta. To understand and control the reaction of trichothecenes with biologically important thiols, we studied the reaction of DON, T-2 tetraol, and de-epoxy-DON with a range of model thiols. Reaction conditions were optimized for DON with 2-mercaptoethanol. Major reaction products were identified using HRMS and NMR spectroscopy. The results indicate that thiols react reversibly with the double bond (Michael addition) and irreversibly with the epoxide group in trichothecenes. These reactions occurred at different rates, and multiple isomers were produced including diconjugated forms. LC-MS analyses indicated that glutathione and cysteine reacted with DON in a similar manner to the model thiols. In contrast to DON, none of the tested mercaptoethanol adducts displayed toxicity in human monocytes or induced pro-inflammatory cytokines in human macrophages.

The cytotoxicity of methanolic extracts from rice cultures of 53 Fusarium avenaceum strains, which had been isolated from different host organisms in Northern Europe, Canada and Australia/New Zealand, was investigated in a rat hepatoma (H4IIE-W), porcine epithelial kidney (PK-15), foetal feline lung fibroblast, dog lymphoblast (D3447), and a human hepatocarcinoma (Hep G2) cell line using the Alamar Blue™ assay. All extracts were screened for known fungal metabolites using high-performance liquid chromatography with photodiode array and mass spectrometric detection, and both known and unknown metabolites were semi-quantified. Known metabolites that were determined in the cultures include acuminatopyrone, 2-amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol), antibiotic Y, aurofusarin, chlamydosporol, chlamydospordiol, enniatins, fusarin A and C, and moniliformin. Multiple regression analysis was used in order to relate fungal metabolites to the cytotoxicity of the extracts. Separate linear regression models were constructed for each cell line. Eleven different fungal metabolites were related to the cytotoxicity ( P 0.05 ). Out of these, nine metabolites were siginificantly related to the cytotoxicity in only one of the five models, while two, namely enniatins and 2-AOD-3-ol, were significant contributors in three or four regression models, respectively. This paper describes how multiple regression analysis may be applied for the assignment of bioactivity/toxicity to the constituents of a multi-component mixture.

The enniatins A, A1, B, B1, B2 and B3 were purified from hexane-extracts of Fusarium avenaceum rice cultures, using semi-preparative HPLC, after precipitation of lipids. Their toxicity, as well as the toxicity of the related fungal metabolite beauvericin (Bea) and the trichothecenes deoxynivalenol (DON) and T-2 toxin, was tested in two cell lines of human origin (hepatocellular carcinoma-line Hep G2 and fibroblast-like foetal lung cell line MRC-5) by using the BrdU and Alamar Blue assays. All the compounds evoked toxicity in the in vitro assays at the concentrations tested. The MRC-5 cell line in combination with the BrdU assay resulted in the lowest inhibitory concentrations (IC(50)) for exposure with enniatins in the range 0.8 microM (enniatin A) to 3.6 microM (enniatin B). The cytotoxicity of DON in the BrdU assay was comparable to the cytotoxicity of enniatins A, B and Bea in a multiple regression model, while DON was significantly more cytotoxic than the enniatins in the Alamar assay. This study indicates that enniatins, fungal metabolites that are commonly found in grain in Northern Europe, may have an underestimated toxic potential.

Norwegian grain samples (73 oats, 75 barley, 80 wheat) from the 2000 to 2002 growing seasons were examined for contamination with five different enniatins and the association between the found concentrations and the prevalence or infection level with several common Fusarium species investigated. Enniatin B was the fungal metabolite with the highest prevalence (100%) and the highest maximum concentration (5800 μg/kg, wheat). The maximum concentration of all five enniatins together in a single sample was 7400 μg/kg (wheat). Enniatin concentrations were correlated with several independent variables, among them grain species. Beauvericin was only sporadically detected in barley and wheat and at concentrations just above the limit of detection of 3 μg/kg, while amounts up to 120 μg/kg were found in oats. The likelihood of detecting enniatin A1 as well as the concentrations of enniatins B and B1 could be mainly related to infection with Fusarium avenaceum/arthrosporioides, and the likelihood of detecting beauvericin could be related to infection with Fusarium poae. This survey indicates that the prevalence of enniatins A1, B and B1 in Norwegian grain is high, and that enniatin B concentrations of above 1000 μg/kg are common in barley and wheat.

The secondary metabolites of 24 isolates of Fusarium avenaceum from Norwegian cereals and grown on rice have been characterized. Moniliformin (MON), enniatins (ENNs), and beauvericin (BEA) were analyzed by high-performance liquid chromatography. Porcine kidney epithelial cells (PK15, American Type Culture Collection) were used to study the cytotoxicity of MON in the extracts. The following metabolites were produced by all isolates, ranked by concentration in rice cultures: ENN-B, MON, ENN-B1, and ENN-A. BEA was produced by eight isolates. The productions of BEA and ENN-A were significantly correlated, as was the case with ENN-B and ENN-B1. MON production was correlated neither to any of the other toxins nor to toxicity.