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Ciguatoxins (CTXs) and gambierones are ladder-shaped polyethers associated with ciguatera poisoning and Gambierdiscus spp. Several of these compounds contain carbonyl or hemiketal groups, which have the potential to exchange with (18)O-labeled water under acidic conditions. The effects of solvent composition and acid on the rate of exchange and on the stability of the labels at various pH values were assessed to optimize the incorporation of (18)O into Caribbean ciguatoxin-1 and -2 (C-CTX1/2), gambierone, and 44-methylgambierone. LC–HRMS results showed that (18)O-labeling occurred at the hydroxy group of the hemiketal at C-56 in C-CTX1/2, and at the hydroxy group of the hemiketal at C-4 and the ketone at C-40 in gambierones. Labeling occurred very rapidly (complete in

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.

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.

Fumonisins, a group of highly prevalent and toxic mycotoxins, are suspected to be causal agents of several diseases in animals and humans. In the animal feed industry, fumonisin esterase is used as feed additive to prevent mycotoxicosis caused by fumonisins. In humans, a popular dosage form for dietary supplements, with high patient acceptance for oral intake, is capsule ingestion. Thus, fumonisin esterase provided in a capsule could be an effective strategy against fumonisin intoxication in humans. To determine the efficacy of fumonisin esterase through capsule ingestion, two modes of application were compared using piglets in a small-scale preliminary study. The enzyme was administered intraorally (in-feed analogue) or intragastrically (capsule analogue), in combination with fumonisin B1 (FB1). Biomarkers for FB1 exposure; namely FB1, hydrolysed FB1 (HFB1) and partially hydrolysed forms (pHFB1a and pHFB1b), were measured both in serum and faeces using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and toxicokinetic parameters were calculated. Additionally, the serum sphinganine/sphingosine (Sa/So) ratio, a biomarker of effect, was determined using LC-MS/MS. A significantly higher Sa/So ratio was shown in the placebo group compared to both esterase treatments, demonstrating the efficacy of the esterase. Moreover, a significant decrease in serum FB1 area under the concentration-time curve (AUC) and an increase of faecal HFB1 AUC were observed after intraoral esterase administration. However, these effects were not observed with statistical significance after intragastric esterase administration with the current sample size.

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.

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.

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.

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.

Deoxynivalenol (DON) contamination of feed may result in reduced growth, feed refusal, immunosuppression, and health problems in swine. Piglets can be exposed to DON via placenta before birth and via milk during lactation. The extent of early-life exposure of piglets to DON is, however, not fully known. This study was therefore aimed at investigating DON uptake in sows fed with naturally contaminated diets, DON transfer across placenta during late gestation, and transfer of DON to piglets via colostrum and milk. Forty-four crossbred sows were evaluated from day 93 ± 1 of gestation until weaning of piglets and fed with feed made from naturally DON-contaminated oats at three concentration levels: (1) control (DON &lt, 0.2 mg/kg), (2) DON level 1 (1.4 mg DON/kg), and (3) DON level 2 (1.7 mg DON/kg). The transfer of DON to the piglets was evaluated in 15 sows, with repeated sampling of blood and milk from the sows and blood samples from five piglets of each litter. The piglet/sow plasma DON ratio and milk/plasma (M/P) DON ratio in sows were calculated to estimate the degree of transfer. Piglet/sow plasma ratios were 2.14 at birth, 2.30 within 12–36 h after parturition, 0.08 on day 7, 0.16 on day 21, and 0.20 at weaning. M/P ratios were 0.92, 1.11, 0.94, 1.21, and 0.90, respectively. The results indicate that DON is efficiently transferred across placenta and into milk. However, the low piglet/sow plasma ratios at mid-lactation to weaning indicate that the piglets were most strongly exposed to DON in early life, despite the high M/P ratios and efficient secretion of DON in milk throughout the entire lactation.

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.

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.

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.).

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.

Enniatins are cyclic hexadepsipeptides produced by various fungi, known to have ionophoric, antibiotic and insecticidal activity. The aim of the present study was to evaluate the intestinal absorption characteristics of enniatin B1 (ENN-B1). Using the human intestinal Caco-2 cell line, we found that the permeability of ENN-B1 in the basolateral to apical direction was 6.7× higher as compared to the permeability in the opposite direction, indicating involvement of apically located transporters. Transport of ENN-B1 in the apical to basolateral direction was increased significantly upon treatment of Caco-2 cells with the P-glycoprotein (Pgp) inhibitor verapamil and the multidrug resistance-associated protein 2 (MRP2) inhibitor MK571, but only weakly and not significantly after treatment with the breast cancer resistance protein (BCRP) inhibitor fumitremorgin C. Additionally, MDCK II cells over-expressing Pgp, MRP2 or BCRP, showed reduced sensitivity towards ENN-B1. These data demonstrate for the first time that ENN-B1 is a substrate of MRP2 and suggests that Pgp, MRP2 and possibly BCRP are involved in transport of ENN-B1 across the intestine.

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 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.

Several Lilium species are nephrotoxic in cats (Felis silvestris catus), among them Easter lilies (Lilium longiflorum). Although clinical trials have been carried out, the causative toxic phytochemicals have not yet been identified. We thus aimed to determine the toxic constituents of Easter lily flowers applying a bioassay-guided approach based on a feline kidney cell line model. The bioassay-guided fractionation traced the observed cytotoxicity to a complex mixture of compounds that were tentatively identified as steroidal glycoalkaloids of the solasodine-type, based on multiple-fragmentation ion trap and high-resolution mass spectrometry. The glycoalkaloids in the active fraction possessed trisaccharide chains, and at least 16 different congeners could be separated using liquid chromatography-mass spectrometry. The two principal compounds were solasodine trisaccharides containing two hexose and one deoxy-hexose unit. In the remaining 14 analogues, one or two of the hydroxyl groups of the second hexose from the aglycone were acetylated. In addition, some of the analogues appeared to be carbonate esters. Esterification of steroidal glycoalkaloids in plants has only been reported once and was in accordance with higher antifungal activity of the acetylated versus the parent congener. Our pilot study shows that esterification of steroidal glycoalkaloids in Lilium species might be common resulting in an array of different analogues with largely unexplored structural variability and bioactivity.

A direct, fast and sensitive LC–MS/MS method was developed to measure biomarkers for mycotoxin exposure in human urine. In total, 32 biomarkers were quantitatively or semi-quantitatively measured in 32 urine samples of Belgian volunteers using two injections. All urine samples contained deoxynivalenol-15-glucuronide, the major detoxification metabolite of deoxynivalenol, in the ng/mL range. Also deoxynivalenol-3-glucuronide and de-epoxy-deoxynivalenol-glucuronide were present in, respectively, 90 and 25 % of the samples, while deoxynivalenol was detected in 60 % of the samples, in lower concentrations. Deoxynivalenol glucuronides were the major biomarkers for deoxynivalenol exposure. Ochratoxin A was detected in 70 % of the samples in pg/mL. Citrinin and/or dihydrocitrinone were detected in 90 % of the samples, also in concentrations of pg/mL. The presence of ochratoxin A and citrinin was confirmed by a second method using sample cleanup by immunoaffinity columns, followed by LC–MS/MS. Our data show that humans are much more exposed to citrinin than realized before and suggest further work on citrinin exposure in relation with ochratoxin A exposure, as both mycotoxins are nephrotoxic.

A glutathione (GSH) adduct of the mycotoxin 4-deoxynivalenol (DON), together with a range of related conjugates, has recently been tentatively identified by LC-MS of DON-treated wheat spikelets. In this study, we prepared samples of DON conjugated at the 10- and 13-positions with GSH, Cys, CysGly, γ-GluCys and N-acetylcysteine (NAC). The mixtures of conjugates were used as standards for LC-HRMS analysis of one of the DON-treated wheat spikelet samples, as well as 19 Norwegian grain samples of spring wheat and 16 grain samples of oats that were naturally-contaminated with DON at concentrations higher than 1 mg/kg. The artificially-contaminated wheat spikelets contained conjugates of GSH, CysGly and Cys coupled at the olefinic 10-position of DON, whereas the naturally-contaminated harvest-ripe grain samples contained GSH, CysGly, Cys, and NAC coupled mainly at the 13-position on the epoxy group. The identities of the conjugates were confirmed by LC-HRMS comparison with authentic standards, oxidation to the sulfoxides with hydrogen peroxide, and examination of product-ion spectra from LC-HRMS/MS analysis. No γ-GluCys adducts of DON were detected in any of the samples. The presence of 15-O-acetyl-DON was demonstrated for the first time in Norwegian grain. The results indicate that a small but significant proportion of DON is metabolized via the GSH-conjugation pathway in plants. To our knowledge, this is the first report of in vivo conjugation of trichothecenes via their epoxy group, which has generally been viewed as unreactive. Because conjugation at the 13-position of DON and other trichothecenes has been shown to be irreversible, this type of conjugate may prove useful as a biomarker of exposure to DON and other 12,13-epoxytrichothecenes. View Full-Text

Chemical investigation of three isolates of Penicillium crustosum Thom cultures, one of which was derived from a recent dog poisoning investigation, has led to the isolation and structure elucidation of secopenitrem D (1). Penitrems A-F and roquefortine C were also present in the isolates analyzed. The structure of 1 was unambiguously assigned based on extensive 1D and 2D-NMR spectroscopic experiments, MS-aided structural studies and by comparison with structurally related congeners. Secopenitrem D lacks the C-16-C-18 ether linkage present in penitrems A-F.

2-Amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol) was isolated from the cytotoxic rice culture extract of a strain of Fusarium avenaceum , which had previously been isolated from Norwegian grain. The structural information was obtained from LC–MS/MS, GC–MS, NMR spectroscopy and high-resolution MS data. The metabolite has a striking similarity to sphinganine, an intermediate in the biosynthesis of the sphingolipids. This similarity is a major feature of the so-called sphingosine analogue toxins; the most studied being the AAL toxins and the fumonisins. 2-AOD-3-ol was found to be cytotoxic to the rat hepatoma cell line H4IIE-W and to the porcine epithelial kidney cell line PK(15) at concentrations (EC 50 ) of 16 and 24 μM, respectively. The metabolite has been found in F. avenaceum inoculated wheat that was treated to support ideal conditions for Fusarium growth, demonstrating that the fungus has the potential to produce the metabolite under field conditions, which may occur in Northern Europe.

The cytotoxicity of extracts from rice cultures of five Fusarium avenaceum strains against the porcine epithelial kidney cell-line PK-15 was investigated using the Alamar Blue assay. After the identification of known fungal metabolites, cytotoxic extracts were fractionated using semi-preparative reversed-phase HPLC and normal phase LC, and the fractions were tested for cytotoxicity. In this way, two different groups of metabolites were identified as the major cytotoxic principles of the extracts. High concentrations of enniatins, especially enniatins B and B1, inhibited the metabolic activity of PK-15 cells. Furthermore, an unidentified metabolite, produced in high amounts by a strain that produced relatively small amounts of enniatins, was also found to be cytotoxic to PK-15 cells. This study shows that enniatins, a group of cyclic depsipeptides, which have been ignored as significant contributors to the toxicity of fungal extracts, may account for most of the observed effect for F. avenaceum.

Norwegian grain samples (73 oats, 75 barley, 83 wheat) from the 2000-02 growing seasons were examined for contamination with moniliformin, and the association between the fungal metabolite and the number of kernels infected with common Fusaria was investigated. Before quantification of moniliformin using ion pairing reversed-phase high-performance liquid chromatography with diode array ultraviolet light detection, all samples were extracted using acetonitrile/water (84/16) and disposable strong anion exchange columns used for clean up. The limit of detection was 40 microg kg(-1). Moniliformin was found in 25, 32 and 76% of the barley, oats and wheat samples, respectively. The maximum concentrations of moniliformin in barley, oats and wheat were 380, 210 and 950 microg kg(-1), respectively. At the same time, the prevalence and infection level of the moniliformin-producing F. avenaceum/arthrosporioides was as high as 100 and53% on average, respectively. Moniliformin concentrations were significantly correlated to the variables grain species, growing season and infection with F. avenaceum/arthrosporioides and F. culmorum. The survey indicates that the prevalence of moniliformin in Norwegian grain is high, especially in wheat. On the other hand, field conditions in Norway do not seem to favour contamination of grain with high levels of moniliformin.

Cyanobacteria are cosmopolitan photosynthetic prokaryotes that can form dense accumulations in aquatic environments. They are able to produce many bioactive metabolites, some of which are potentially endocrine disrupting compounds, i.e., compounds that interfere with the hormonal systems of animals and humans. Endocrine disruptors represent potential risks to both environmental and human health, making them a global challenge. The aim of this study was to investigate the potential endocrine disrupting activities with emphasis on estrogenic effects of extracts from cultures of Microcystis or Planktothrix species. We also assessed the possible role of microcystins, some of the most studied cyanobacterial toxins, and thus included both microcystin-producing and non-producing strains. Extracts from 26 cyanobacterial cultures were initially screened in estrogen-, androgen-, and glucocorticoid-responsive reporter-gene assays (RGAs) in order to identify endocrine disruption at the level of nuclear receptor transcriptional activity. Extracts from selected strains were tested repeatedly in the estrogen-responsive RGAs, but the observed estrogen agonist and antagonist activity was minor and similar to that of the cyanobacteria growth medium control. We thus focused on another, non-receptor mediated mechanism of action, and studied the 17&beta, estradiol (natural estrogen hormone) biotransformation in human liver microsomes in the presence or absence of microcystin-LR (MC-LR), or an extract from the MC-LR producing M. aeruginosa PCC7806 strain. Our results show a modulating effect on the estradiol biotransformation. Thus, while 2-hydroxylation was significantly decreased following co-incubation of 17&beta, estradiol with MC-LR or M. aeruginosa PCC7806 extract, the relative concentration of estrone was increased.