Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

Identification and cross-species comparison of in vitro phase I brevetoxin (BTX-2) metabolites in northern Gulf of Mexico fish and human liver microsomes by UHPLC-HRMS(/MS).

Brevetoxins (BTX) are a group of marine neurotoxins produced by the harmful alga Karenia brevis. Numerous studies have shown that BTX are rapidly accumulated and metabolized in shellfish and mammals. However, there are only limited data on BTX metabolism in fish, despite growing evidence that fish serve as vectors for BTX transfer in marine food webs. In this study, we aimed to investigate the in vitro biotransformation of BTX-2, the major constituent of BTX profiles in K. brevis, in several species of northern Gulf of Mexico fish. Metabolism assays were performed using hepatic microsomes prepared in-house as well as commercially available human microsomes for comparison, focusing on phase I reactions mediated by cytochrome P450 monooxygenase (CYP) enzymes. Samples were analyzed by UHPLC-HRMS(/MS) to monitor BTX-2 depletion and characterize BTX metabolites based on MS/MS fragmentation pathways. Our results showed that both fish and human liver microsomes rapidly depleted BTX-2, resulting in a 72-99% reduction within 1 h of incubation. We observed the simultaneous production of 22 metabolites functionalized by reductions, oxidations, and other phase I reactions. We were able to identify the previously described congeners BTX-3 and BTX-B5, and tentatively identified BTX-9, 41,43-dihydro-BTX-2, several A-ring hydrolysis products, as well as several novel metabolites. Our results confirmed that fish are capable of similar BTX biotransformation reactions as reported for shellfish and mammals, but comparison of metabolite formation across the tested species suggested considerable interspecific variation in BTX-2 metabolism potentially leading to divergent BTX profiles. We additionally observed non-enzymatic formation of BTX-2 and BTX-3 glutathione conjugates. Collectively, these findings have important implications for determining the ecotoxicological fate of BTX in marine food webs.

Atlantic Salmon Gill Epithelial Cell Line (ASG-10) as a Suitable Model for Xenobiotic Biotransformation.

Fish are exposed to xenobiotics in the water. Uptake occurs mainly through the gills, which function as an exchange point with the environment. The gills' ability to detoxify harmful compounds by biotransformation is an essential protection mechanism. The enormous numbers of waterborne xenobiotics requiring ecotoxicological assessment makes it necessary to replace in vivo fish studies with predictive in vitro models. Here, we have characterized the metabolic capacity of the ASG-10 gill epithelial cell line from Atlantic salmon. Inducible CYP1A expression was confirmed by enzymatic assays and immunoblotting. The activities of important cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes were established using specific substrates and metabolite analysis by liquid chromatography (LC) triple quadrupole mass spectrometry (TQMS). Metabolism of the fish anesthetic benzocaine (BZ) in ASG-10 confirmed esterase and acetyl transferase activities through the production of N-acetylbenzocaine (AcBZ), p-aminobenzoic acid (PABA) and p-acetaminobenzoic acid (AcPABA). Moreover, we were able to determine hydroxylamine benzocaine (BZOH), benzocaine glucuronide (BZGlcA) and hydroxylamine benzocaine glucuronide (BZ(O)GlcA) by LC high-resolution tandem mass spectrometry (HRMS/MS) fragment pattern analysis for the first time. Comparison to metabolite profiles in hepatic fractions, and in plasma of BZ-euthanized salmon, confirmed the suitability of the ASG-10 cell line for investigating biotransformation in gills.

Pharmacokinetics of a long-acting subcutaneous eprinomectin injection in semi-domesticated reindeer (Rangifer tarandus tarandus) - A pilot study.

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.

Neurotoxic Potential of Deoxynivalenol in Murine Brain Cell Lines and Primary Hippocampal Cultures.

Chronic exposure to the mycotoxin deoxynivalenol (DON) from grain-based food and feed affects human and animal health. Known consequences include entereopathogenic and immunotoxic defects; however, the neurotoxic potential of DON has only come into focus more recently due to the observation of behavioural disorders in exposed farm animals. DON can cross the blood-brain barrier and interfere with the homeostasis/functioning of the nervous system, but the underlying mechanisms of action remain elusive. Here, we have investigated the impact of DON on mouse astrocyte and microglia cell lines, as well as on primary hippocampal cultures by analysing different toxicological endpoints. We found that DON has an impact on the viability of both glial cell types, as shown by a significant decrease of metabolic activity, and a notable cytotoxic effect, which was stronger in the microglia. In astrocytes, DON caused a G1 phase arrest in the cell cycle and a decrease of cyclic-adenosine monophosphate (cAMP) levels. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α was secreted in the microglia in response to DON exposure. Furthermore, the intermediate filaments of the astrocytic cytoskeleton were disturbed in primary hippocampal cultures, and the dendrite lengths of neurons were shortened. The combined results indicated DON's considerable potential to interfere with the brain cell physiology, which helps explain the observed in vivo neurotoxicological effects.

Metabolomics workflow for quality control of differently-processed pre-cooked chicken fillets.

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.

Impact of Plasma-Activated Water Treatment on Quality and Shelf-Life of Fresh Spinach Leaves Evaluated by Comprehensive Metabolomic Analysis.

Fresh baby spinach leaves are popular in salads and are sold as chilled and plastic-packed products. They are of high nutritional value but very perishable due to microbial contamination and enzymatic browning resulting from leaf senescence. Therefore, innovative food processing methods such as plasma-activated water (PAW) treatment are being explored regarding their applicability for ensuring food safety. PAW's impact on food quality and shelf-life extension has, however, not been investigated extensively in vegetables so far. In the present study, a comprehensive metabolomic analysis was performed to determine possible changes in the metabolite contents of spinach leaves stored in a refrigerated state for eight days. Liquid chromatography high-resolution mass spectrometry, followed by stringent biostatistics, was used to compare the metabolomes in control, tap-water-rinsed or PAW-rinsed samples. No significant differences were discernible between the treatment groups at the beginning or end of the storage period. The observed loss of nutrients and activation of catabolic pathways were characteristic of a transition into the senescent state. Nonetheless, the presence of several polyphenolic antioxidants and γ-linolenic acid in the PAW-treated leaves indicated a significant increase in stress resistance and health-promoting antioxidant capacity in the sample. Furthermore, the enhancement of carbohydrate-related metabolisms indicated a delay in the senescence development. These findings demonstrated the potential of PAW to benefit food quality and the shelf-life of fresh spinach leaves.

In Vitro Glucuronidation of Caribbean Ciguatoxins in Fish: First Report of Conjugative Ciguatoxin Metabolites.

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.

Sensitivity to Dietary Wheat Gluten in Atlantic Salmon Indicated by Gene Expression Changes in Liver and Intestine.

Feed safety is a necessity for animal health and welfare as well as prerequisite for food safety and human health. Wheat gluten (WG) is considered as a valuable protein source in fish feed due to its suitability as a feed binder, high digestibility, good amino acid profile, energy density and most importantly, due to its relatively low level of anti-nutritional factors (ANFs). The main aim of this study was to identify the impact of dietary WG on salmon health by analysing growth, feed efficiency and the hepatic and intestinal transcriptomes. The fish were fed either control diet with fishmeal (FM) as the only source of protein or diets, where 15% or 30% of the FM were replaced by WG. The fish had a mean initial weight of 223 g and approximately doubled their weight during the 9-week experiment. Salmon fed on 30% WG showed reduced feed intake compared to the 15% and FM fed groups. The liver was the less affected organ but fat content and activities of the liver health markers in plasma increased with the inclusion level of WG in the diet. Gene expression analysis showed significant changes in both, intestine and liver of fish fed with 30% WG. Especially noticeable were changes in the lipid metabolism, in particular in relation to the intestinal lipoprotein transport and sterol metabolism. Moreover, the intestinal transcriptome of WG-fed fish showed shifts in the expression of a large number of genes responsible for immunity and tissue structure and integrity. These observations implied that the fish receiving WG-containing diet were undergoing nutritional stress. Overall, the study provided evidence that a high dietary level of WG can have a negative impact on the intestinal and liver health of salmon with symptoms similar to gluten sensitivity in humans.

A ranking method of chemical substances in foods for prioritisation of monitoring, based on health risk and knowledge gaps.

Chemical contaminants are present in all foods. Data on the occurrence of contaminants in foods that are often consumed or contain high contaminant concentrations are critical for the estimation of exposure and evaluation of potential negative health effects. Due to limited resources for the monitoring of contaminants and other chemical substances in foods, methods for prioritisation are needed. We have developed a straightforward semi-quantitative method to rank chemical substances in foods for monitoring as part of a risk-based food control. The method is based on considerations of toxicity, level of exposure including both occurrence in food and dietary intake, vulnerability of one or more population groups due to high exposure because of special food habits or resulting from specific genetic variants, diseases, drug use or age/life stages, and the adequacy of both toxicity and exposure data. The chemical substances ranked for monitoring were contaminants occurring naturally, unintentionally or incidentally in foods or formed during food processing, and the inclusion criteria were high toxicity, high exposure and/or lack of toxicity or exposure data. In principle, this method can be used for all classes of chemical substances that occur in foods, both unintended contaminants and deliberately added chemical substances. Foods considered relevant for monitoring of the different chemical substances were also identified. The outcomes of ranking exercises using the new method including considerations of vulnerable groups and adequacy of data and a shortened version based on risk considerations only were compared. The results showed that the resolution between the contaminants was notably increased with the extended method, which we considered as advantageous for the ranking of chemical substances for monitoring in foods.

In Vitro Toxicokinetics and Phase I Biotransformation of the Mycotoxin Penitrem A in Dogs.

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.

Biotransformation of phytoestrogens from soy in enzymatically characterized liver microsomes and primary hepatocytes of Atlantic salmon.

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.

Development and Validation of a Liquid Chromatography High-Resolution Mass Spectrometry Method for the Simultaneous Determination of Mycotoxins and Phytoestrogens in Plant-Based Fish Feed and Exposed Fish.

New protein sources in fish feed require the assessment of the carry-over potential of contaminants and anti-nutrients from feed ingredients into the fish, and the assessment of possible health risks for consumers. Presently, plant materials including wheat and legumes make up the largest part of aquafeeds, so evaluation of the transfer capabilities of typical toxic metabolites from plant-infesting fungi and of vegetable phytoestrogens into fish products is of great importance. With the aim of facilitating surveillance of relevant mycotoxins and isoflavones, we have developed and validated a multi-analyte LC-HRMS/MS method that can be used to ensure compliance to set maximum levels in feed and fish. The method performance characteristics were determined, showing high specificity for all 25 targeted analytes, which included 19 mycotoxins and three isoflavones and their corresponding aglycons with sufficient to excellent sensitivities and uniform analytical linearity in different matrices. Depending on the availability of matching stable isotope-labelled derivates or similar-structure homologues, calibration curves were generated either by using internal standards or by matrix-matched external standards. Precision and recovery data were in the accepted range, although they varied between the different analytes. This new method was considered as fit-for-purpose and applied for the analysis of customised fish feed containing wheat gluten, soy, or pea protein concentrate as well as salmon and zebrafish fed on diets with these ingredients for a period of up to eight weeks. Only mycotoxin enniatin B, at a level near the limit of detection, and low levels of isoflavones were detected in the feed, demonstrating the effectiveness of maximum level recommendations and modern feed processing technologies in the Norwegian aquaculture industry. Consequently, carry-over into fish muscle was not observed, confirming that fillets from plant-fed salmon were safe for human consumption.

Defining the targets for the assessment of IgE-mediated allergenicity of new or modified food proteins.

Many food innovations rely on the introduction and use of new or modified proteins. New or modified food proteins may lead to major health risks due to their inherent potential to cause food allergy. Currently, the pre-market allergenicity assessment for new or modified food proteins and protein sources relies on methods for identifying allergenic hazards based on characteristics of known allergens. However, there is no general consensus on the allergenicity parameters to use and the criteria that should apply for the evaluation and decisions to be made. In this paper, we propose that the strategy for allergenicity risk assessment of new or modified food proteins and the methodologies applied should be governed by the risk management questions to be answered, reflected in the information needed by risk managers to enable their informed decision making. We generated an inventory of health outcome-related assessment parameters and criteria potentially important for risk management decision-making and we discuss the implications of selecting different optional criteria (e.g. cut-off values) for what could be accepted as safe with regards to the health outcomes in the (at risk) population. The impact of these various options on both method development and risk management practices was investigated.

Transfer of Deoxynivalenol (DON) through Placenta, Colostrum and Milk from Sows to Their Offspring during Late Gestation and Lactation.

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 < 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.

Prediction of deoxynivalenol toxicokinetics in humans by in vitro-to-in vivo extrapolation and allometric scaling of in vivo animal data.

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.

Effects and biotransformation of the mycotoxin deoxynivalenol in growing pigs fed with naturally contaminated pelleted grains with and without the addition of Coriobacteriaceum DSM 11798.

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.

Role of P-glycoprotein in deoxynivalenol-mediated in vitro toxicity.

Deoxynivalenol (DON) is the most prevalent mycotoxin produced by grain-infecting Fusarium strains and frequently occurs in small cereals all over the world. After ingestion, DON is absorbed in the gut, which leads dose-dependently to critical health effects. In the present study, we have further investigated DON's previously reported affinity to the efflux transporter P-glycoprotein (Pgp) in the apical enterocyte membrane. Interaction with Pgp was studied in human colorectal adenocarcinoma (Caco-2) cells and Madin-Darby Canine Kidney wild-type (MDCKII-wt) and Pgp-overexpressing (MDCKII-MDR1) cells in different transport and cytotoxicity experiments. We found that DON was exported by Pgp and was less cytotoxic in Pgp-overexpressing cells. In the fluorometric calcein-acetoxymethylester (Calcein AM) assay DON reduced intracellular calcein retention, indicating a stimulation of Pgp-mediated efflux. In the presence of the selective Pgp inhibitors verapamil (Ver) and valspodar (PSC 833) the effect was, respectively, distinctive and significant. Verrucarol, a structural analogue of DON, was much less effective indicating the importance of the α, ß-conjugated carbonyl group in the DON molecule for Pgp interaction. Our results confirmed that Pgp might have the potential to reduce intestinal absorption of DON in vivo. Furthermore, we were able to show that DON can modulate Pgp activity in vitro.

Tissue distribution and elimination of deoxynivalenol and ochratoxin A in dietary-exposed Atlantic salmon (Salmo salar).

Post-smolt Atlantic salmon (Salmo salar) were fed standard feed with added 2 or 6 mg kg-1 pure deoxynivalenol (DON), 0.8 or 2.4 mg kg-1 pure ochratoxin A (OTA), or no added toxins for up to 8 weeks. The experiments were performed in duplicate tanks with 25 fish each per diet group, and the feed was given for three 2-h periods per day. After 3, 6 and 8 weeks, 10 fish from each diet group were sampled. In the following hours after the last feeding at 8 weeks, toxin elimination was studied by sampling three fish per diet group at five time points. Analysis of DON and OTA in fish tissues and plasma was conducted by liquid chromatography-mass spectrometry and high-pressure liquid chromatography with fluorescence detection, respectively. DON was distributed to the liver, kidney, plasma, muscle, skin and brain, and the concentrations in liver and muscle increased significantly from 3 to 8 weeks of exposure to the high-DON diet. After the last feeding at 8 weeks, DON concentration in liver reached a maximum at 1 h and decreased thereafter with a half-life (t1/2) of 6.2 h. DON concentration in muscle reached a maximum at 6 h and was then eliminated with a t1/2 = 16.5 h. OTA was mainly found in liver and kidney, and the concentration in liver decreased significantly from 3 to 8 weeks in the high-OTA group. OTA was eliminated faster than DON from various tissues. By using Norwegian food consumption data and kinetic findings in this study, we predicted the human exposure to DON and OTA from fish products through carryover from the feed. Following a comparison with tolerable daily intakes, we found the risk to human health from the consumption of salmon-fed diets containing maximum recommended levels of these toxins to be negligible.

Biotransformation of the mycotoxin enniatin B1 in pigs: A comparative in vitro and in vivo approach.

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.