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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.

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.

The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24 h exposure to ENN B1 at levels of 2.5–10 μmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5–1.7 μmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4 h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.

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.

Food allergy affects up to 6% of Europeans. Allergen identification is important for the risk assessment and management of the inadvertent presence of allergens in foods. The VITAL® initiative for voluntary incidental trace allergen labeling suggests protein reference doses, based on clinical reactivity in food challenge studies, at or below which voluntary labelling is unnecessary. Here, we investigated if current analytical methodology could verify the published VITAL® 2.0 doses, that were available during this analysis, in serving sizes between 5 and 500 g. Available data on published and commercial ELISA, PCR and mass spectrometry methods, especially for the detection of peanuts, soy, hazelnut, wheat, cow's milk and hen's egg were reviewed in detail. Limit of detection, quantitative capability, matrix compatibility, and specificity were assessed. Implications by the recently published VITAL® 3.0 doses were also considered. We conclude that available analytical methods are capable of reasonably robust detection of peanut, soy, hazelnut and wheat allergens for levels at or below the VITAL® 2.0 and also 3.0 doses, with some methods even capable of achieving this in a large 500 g serving size. Cow's milk and hen's egg are more problematic, largely due to matrix/processing incompatibility. An unmet need remains for harmonized reporting units, available reference materials, and method ring-trials to enable validation and the provision of comparable measurement results.

We have investigated the in vitro metabolism of pectenotoxin-2 (PTX-2) using primary hepatocytes from Wistar rats in suspension. Purified PTX-2 was rapidly metabolized. Two major and several minor oxidized PTX-2 metabolites were formed, none of which had retention times corresponding to PTX-1, -11, or −13. Hydrolysis products, such as PTX-2 seco acid, were not observed. Preliminary multi-stage LC-MS analyses indicated that the major hepatic PTX-2 metabolites resulted from the insertion of an oxygen atom at the positions C-19 to C-24, or at C-44. The rapid oxidative metabolism may explain the low oral toxicity of PTXs observed in vivo studies., Highlights • PTX-2 is rapidly metabolized in rat hepatocytes. • Two major and several minor oxidized PTX-2 metabolites were formed. • The results may explain the low oral toxicity of PTXs observed in vivo studies.

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.

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.

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, β-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.

The development and introduction of new dietary protein sources has the potential to improve food supply sustainability. Understanding the potential allergenicity of these new or modified proteins is crucial to ensure protection of public health. Exposure to new proteins may result in de novo sensitization, with or without clinical allergy, or clinical reactions through cross-reactivity. In this paper we review the potential of current methodologies (in silico, in vitro degradation, in vitro IgE binding, animal models and clinical studies) to address these outcomes for risk assessment purposes for new proteins, and especially to identify and characterise the risk of sensitization for IgE mediated allergy from oral exposure. Existing tools and tests are capable of assessing potential crossreactivity. However, there are few possibilities to assess the hazard due to de novo sensitization. The only methods available are in vivo models, but many limitations exist to use them for assessing risk. We conclude that there is a need to understand which criteria adequately define allergenicity for risk assessment purposes, and from these criteria develop a more suitable battery of tests to distinguish between proteins of high and low allergenicity, which can then be applied to assess new proteins with unknown risks. © 2017 The Authors Chemicals/CAS: immunoglobulin E, 37341-29-0

The Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) has on request of The Norwegian Food Safety Authority performed a risk assessment of furan intake in the Norwegian population based on the most recent national food consumption surveys. National occurrence data of furan concentrations in food were preferentially used in the risk assessment. When national data were lacking, VKM has used occurrence data of furan from other countries. The assessment has been performed by the VKM Panel on Food Additives, Flavourings, Processing Aids, Materials in Contact with Food and Cosmetics and the VKM Panel on Contaminants. Furan is a volatile and lipophilic compound formed in a variety of heat-treated commercial foods and contributes to the sensory properties of the product. The substance has been found in a number of foods such as coffee, canned and jarred foods including baby food containing meat and various vegetables. High concentrations of furan have been found in coffee and the presence of furan in jarred baby food and infant formulae has received much attention since such products may be the sole diet for many infants. The occurrence of furan in a variety of foods suggests that there are multiple routes of furan formation rather than a single mechanism. The Norwegian Food Safety Authority has in 2008 and 2009 collected data on furan concentrations in different food products sold on the Norwegian market (Norwegian Food Safety Authority, 2008). In 2011, the Norwegian Food Safety Authority also decided to analyse commercial porridges for infants and children sold on the Norwegian market, to see if furan could be detected in such products. The calculated furan exposures from food and beverages are based on data from the nationally representative food consumption surveys; Spedkost, Småbarnskost, Ungkost and Norkost. The consumption for each relevant food or food category in the dietary surveys were multiplied with the corresponding mean furan concentrations and totalled for each individual. The liver is the main target organ for furan toxicity both in mice and rats, but the rat is the most sensitive species. A dose-dependent increase in hepatocellular adenomas and carcinomas was observed in mice and rats, and an increase in the incidence of cholangiocarcinomas was observed in rat liver. Cholangiocarcinomas in male and female rats were the most sensitive toxicological end point observed in rodents. On the basis of the available data, VKM considers that rat cholangiocarcinomas may be relevant for assessing human risk from furan. Available in vivo data with furan indicate that a reactive metabolite, most likely cis-2-butene1,4-dial (BDA), is formed and that this metabolite can react with DNA and induce mutations. To VKM’s knowledge, no in vivo studies on genotoxicity of BDA have been performed, but BDA was found to be genotoxic in several in vitro tests. VKM therefore considers that a genotoxic mechanism in furan-induced carcinogenesis cannot be excluded and the substance was assessed as a genotoxic carcinogen. VKM used the Margin of Exposure (MOE) approach in this risk assessment. The suitability of different studies on cholangiocarcinomas for dose-response modelling was considered. The 9-month interim evaluation of a 2-year study from NTP (1993) was chosen because it demonstrates a dose-response relationship. From this study, a point of departure of 0.02 mg/kg bw/day was chosen, based on a benchmark dose lower bound (BMDL10) of 0.14 mg furan/kg bw/day and a correction factor of 7 for shorter than full life-time (2 years) study duration. For 6-, 12- and 24-month-old children, the main source of furan exposure is jarred baby food. For 4-, 9- and 13-year-old children, the major food source to the furan exposure is breakfast cereals. In adults, the major contribution to the furan exposure is coffee. The highest furan exposure was calculated for 12-month-old infants and ranged from 0.62-1.51 µg/kg bw/day. In adults the furan exposure ranged from 0.27-0.82 µg/kg bw/day. For mean exposure among infants, children and adolescents, the MOE-values ranged from 29 in 12-month-infants to 2000 in the 13-year-old adolescents. Among high consumers in these groups, the MOE-values ranged from 13 to 400. In adults, the corresponding MOE-values ranged from 59 to 74 for mean furan exposure and from 24 to 26 for high exposure. It should be noted that this risk assessment of furan contains notable uncertainties and limitations. The use of the 9-month interim study in rats including a correction factor of 7 to derive a point of departure, instead of a full life-time study (2-year) study, likely overestimates the hazard of furan. A possible over-diagnosis of the cholangiocarcinomas, due to the similarities in histopathology between cholangiofibrosis and cholangiocarcinomas in rats, may overestimate the hazard. There are also limitations in assessing food consumption and furan content in foods, leading to uncertainties in estimation of furan exposure. VKM considers that the current exposure to furan in all age groups, particularly among infants and children, is of health concern.

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 epidermal mucus protects fish against harmful environmental factors and the loss of physiological metabolites and water. It is an efficient barrier between the fish and the biosphere. The integrity of the skin mucus is thus of vital importance for the welfare and survival of the fish. Since excreted proteins and small molecules in the mucus can mirror the health status of the fish, it is a valuable matrix for monitoring stress, pathogen exposure, and nutritional effects. Several methods for sampling epidermal mucus from different fish species have previously been described, but information about their efficiency or the comparability of mucus analyses is lacking. In the present study, skin mucus from farmed Atlantic salmon was therefore sampled by three methods, including absorption or wiping with tissue paper, and scraping with a blunt blade, and the mucus proteome was analyzed by ultra-high pressure liquid chromatography high-resolution mass spectrometry. The measured protein contents, numbers, compositions and the observed data quality were compared between sampling methods. Furthermore, functional annotation and classification of the identified proteins was performed. The results showed that the three skin mucus sample types differed qualitatively as well as quantitatively. The absorbed mucus was the least tainted by proteins resulting from damage inflicted to the fish epidermis by the sampling procedure. Wiped mucus showed a better protein yield than absorbed and delivered a larger proteome of identifiable proteins, with less contamination from epithelial proteins than observed for scraped mucus. We recommend that future research of mucus should use the absorption method in cases, where it is important that the mucus is devoid of proteins from the underlying epithelium, and the wiping method, when protein yield is crucial or when the proteome of the outer epithelium is of interest.

Enniatins (ENNs) are fungal secondary metabolites that frequently occur in grain in temperate climates. Their toxic potency is connected to their ionophoric character and lipophilicity. The biotransformation of ENNs predominantly takes place via cytochrome P450 3A (CYP 3A)-dependent oxidation reactions. Possible interaction with ENNs is relevant since CYP3A4 is the main metabolic enzyme for numerous drugs and contaminants. In the present study, we have determined the kinetic characteristics and inhibitory potential of ENNB1 in human liver microsomes (HLM) and CYP3A4-containing nanodiscs (ND). We showed in both in vitro systems that ENNB1 is mainly metabolised by CYP3A4, producing at least eleven metabolites. Moreover, ENNB1 significantly decreased the hydroxylation rates of the typical CYP3A4-substrate midazolam (MDZ). Deoxynivalenol (DON), which is the most prevalent mycotoxin in grain and usually co-occurrs with the ENNs, was not metabolised by CYP3A4 or binding to its active site. Nevertheless, DON affected the efficiency of this biotransformation pathway both in HLM and ND. The metabolite formation rates of ENNB1 and the frequently used drugs progesterone (PGS) and atorvastatin (ARVS) lactone were noticeably reduced, which indicated a certain affinity of DON to the enzyme with subsequent conformational changes. Our results emphasise the importance of drug&ndash, drug interaction studies, also with regard to natural toxins.

International audience; The mycotoxin deoxynivalenol (DON) has a high global prevalence in grain-based products. Biomarkers of exposure are detectable in most humans and farm animals. Considering the acute emetic and chronic anorexigenic toxicity of DON, maximum levels for food and feed have been implemented by food authorities. The tolerable daily intake (TDI) is 1 µg/kg body weight (bw)/day for the sum of DON and its main derivatives, which was based on the no-observed adverse-effect level (NOAEL) of 100 µg DON/kg bw/day for anorexic effects in rodents. Chronic exposure to a low-DON dose can, however, also cause inflammation and imbalanced neurotransmitter levels. In the present study, we therefore investigated the impact of a 2-week exposure at the NOAEL in mice by performing behavioural experiments, monitoring brain activation by c-Fos expression, and analysing changes in the metabolomes of brain and serum. We found that DON affected neuronal activity and innate behaviour in both male and female mice. Metabolite profiles were differentiable between control and treated mice. The behavioural changes evidenced at NOAEL reduce the safety margin to the established TDI and may be indicative of a risk for human health.

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. The work presented in this paper as well as the publication of it were part of and made possible by EU COST Action 1402 ImpARAS: Improving Allergy Risk Assessment Strategy for New Food Proteins (http://imparas.eu). Paula Alvito and Ricardo Assunção acknowledge the support from National Institute of Health, Dr. Ricardo Jorge and CESAM through the Fundação para a Ciência e a Tecnologia [UID/AMB/50017/2013], through national funds, and the co-funding by the FEDER [POCI-01- 0145-FEDER-00763], within the PT2020 Partnership info:eu-repo/semantics/publishedVersion

The parasite Anisakis simplex is present in many marine fish species that are directly used as food or in processed products. The anisakid larvae infect mostly the gut and inner organs of fish but have also been shown to penetrate into the fillet. Thus, human health can be at risk, either by contracting anisakiasis through the consumption of raw or under-cooked fish, or by sensitisation to anisakid proteins in processed food. A number of different methods for the detection of A. simplex in fish and products thereof have been developed, including visual techniques and PCR for larvae tracing, and immunological assays for the determination of proteins. The recent identification of a number of anisakid proteins by mass spectrometry-based proteomics has laid the groundwork for the development of two quantitative liquid chromatography-tandem mass spectrometry methods for the detection of A. simplex in fish that are described in the present study. Both, the label-free semi-quantitative nLC-nESI-Orbitrap-MS/MS (MS1) and the heavy peptide-applying absolute-quantitative (AQUA) LC-TripleQ-MS/MS (MS2) use unique reporter peptides derived from anisakid hemoglobin and SXP/RAL-2 protein as analytes. Standard curves in buffer and in salmon matrix showed limits of detection at 1μg/mL and 10μg/mL for MS1 and 0.1μg/mL and 2μg/mL for MS2. Preliminary method validation included the assessment of sensitivity, repeatability, reproducibility, and applicability to incurred and naturally-contaminated samples for both assays. By further optimization and full validation in accordance with current recommendations the LC-MS/MS methods could be standardized and used generally as confirmative techniques for the detection of A. simplex protein in fish.

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.

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.

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.

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 maxim...

Deoxynivalenol (DON) is the most common mycotoxin in Norwegian cereals, and DON is detected in most samples of crude cereal grain and cereal food commodities such as flour, bran, and oat flakes. The Norwegian Scientific Committee for Food Safety assessed the risk for adverse effects of deoxynivalenol (DON) in different age groups of the domestic population. This review presents the main results from the risk assessment, supplemented with some recently published data. Impairment of the immune system together with reduced feed intake and weight gain are the critical effects of DON in experimental animals on which the current tolerable daily intake was established. Based on food consumption and occurrence data, the mean exposure to DON in years with low and high levels of DON in the flour, respectively, were in the range of or up to two times the Tolerable Daily Intake (TDI) in 1-year-old infants and 2-year-old children. In years with high mean DON concentration, the high (95th-percentile) exposure exceeded the TDI by up to 3.5 times in 1-, 2- , 4-, and 9-year-old children. The assessment concluded that exceeding the TDI in infants and children is of concern. The estimated dietary DON intakes in adolescent and adult populations are in the range of the TDI or below, and are not a health concern. Acute human exposure to DON is not of concern in any age group.

Wild-caught marine fish are potentially carrying parasites. Larvae of the nematode Anisakis simplex (herring or whale worm) occur in almost all commercially exploited fish stocks in temperate seas. The presence of A. simplex in fish and fish products is not only an economic concern but represents a significant consumer health risk. Anisakiasis, human infection with live larvae, can occur by consuming raw or undercooked fish while allergy symptoms can also be elicited by the presence of A. simplex proteins in processed seafood. The European Food Safety Authority (EFSA) has concluded in a scientific opinion that routine testing of seafood products for A. simplex is needed. In the present study, we have determined A. simplex proteins in farmed salmon intended for use in sushi and fish products from the Norwegian market by quantitative sandwich ELISA, immunostaining and mass spectrometry. Analytical methods detecting anisakid proteins at the single-digit milligram level appear to be sufficiently sensitive for the protection of allergic consumers. Only trace amounts (

The parasitic nematode Anisakis simplex occurs in fish stocks in temperate seas. A. simplex contamination of fish products is unsavoury and a health concern considering human infection with live larvae (anisakiasis) and allergic reactions to anisakid proteins in seafood. Protein extracts of A. simplex produce complex band patterns in gel electrophoresis and IgE-immunostaining. In the present study potential allergens have been characterised using sera from A. simplex-sensitised patients and proteome data obtained by mass spectrometry. A. simplex proteins were homologous to allergens in other nematodes, insects, and shellfish indicating cross-reactivity. Characteristic marker peptides for relevant A. simplex proteins were described.

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.

SummaryBackground Sensitization to food allergens and food allergic reactions are mostly caused by ingesting the allergen, but can also occur from exposure via the respiratory tract or the skin. Little is known about exposure to food allergens in the home environment. Objective The objective of this study was firstly to describe the frequency of detection of allergens from fish, egg, milk, and peanut in mattress dust collected from homes of 13-year-old adolescents and secondly to identify home characteristics associated with the presence of food allergen contamination in dust. Methods Food allergens were measured by dot blot analysis in mattress dust from 143 homes in Oslo, Norway. We analysed associations between home characteristics (collected by parental questionnaires and study technicians) and food allergens by multivariate regression models. Results Fish allergen was detected in 46%, peanut in 41%, milk in 39%, and egg allergen in 22% of the mattress dust samples; only three samples contained none of these allergens. All four food allergens were more frequently detected in mattresses in small dwellings (

The tremorgenic mycotoxin Thomitrem A is a secondary metabolite produced mainly by the fungus Penicillium crustosum that is frequently found on spoiled stored food and feed. Typical signs of intoxication observed in dogs after the consumption of food waste are emesis, tremors, seizures progressing to ataxia and lack of coordinated movements. How uptake of Thomitrem A relates to exposure is unknown so far since data on biotransformation and toxicokinetics are missing. In this study the toxin was therefore metabolised in an exploratory in vitro experiment by rat hepatocytes, and substrate depletion as well as the formation of hepatic metabolites were investigated. Seven metabolites were characterised by their retention times and fragmentation patterns in LC-MS/MS analysis. They were found to be products of oxidation and dehydration processes and occurred at different incubation time points, showing different signal abundance-time curve profiles. Toxicokinetic parameters were derived from the Thomitrem A depletion curve applying principles of in vitro-to-in vivo extrapolation (IVIVE). The predicted medium maximum bioavailability in rats could be of importance for the assessment of exposure in cases of intoxication if it was confirmed in vivo and in other species.

The Norwegian Food Allergy Register was established at the Norwegian Institute of Public Health in 2000. The purpose of the register is to gain information about severe allergic reactions to food in Norway and to survey food products in relation to allergen labelling and contamination. Cases are reported on a voluntary basis by first line doctors, and submitted together with a serum sample for specific IgE analysis. The register has received a total of 877 reports from 1 July, 2000 to 31 December, 2010. Two age groups, small children and young adults are over-represented, and the overall gender distribution is 40:60 males-females. The legumes lupine and fenugreek have been identified as two “new” allergens in processed foods and cases of contamination and faults in production of processed foods have been revealed. The highest frequency of food specific IgE is to hazelnuts and peanuts, with a marked increase in reactions to hazelnuts during the last three years. The Food Allergy Register has improved our knowledge about causes and severity of food allergic reactions in Norway. The results show the usefulness of population based national food allergy registers in providing information for health authorities and to secure safe food for individuals with food allergies. © 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

There is a growing awareness by consumers and food safety authorities regarding the possible presence of parasites or parasite-related potentially hazardous substances in seafood. Anisakis simplex is among the most frequently occurring parasites in wild-caught marine fish. Except for various visual inspection techniques and PCR-based methods for the detection of more or less intact worms or parasite DNA, respectively, there are at present no validated methods for the quantification of A. simplex proteins in processed fish products. This work describes the development and validation of a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification and analysis of proteins from A. simplex in seafood products. The ELISA is based on a polyclonal rabbit anti-A. simplex antibody for capture and a biotinylated conjugate of the same antibody for detection. The ELISA is specific for A. simplex and does not cross-react with other species. Recoveries ranged from 72–101% in typical food matrixes, while intra- and inter-assay precisions were

Background Fenugreek is a legume plant used as an ingredient of curry spice. Incidents of IgE-mediated food allergy to fenugreek have been reported. Coincidence with allergy to peanut, a major food allergen, seems to be common suggesting a rather high rate of cross-reactivity. Objective Characterization of fenugreek allergens using patient sera and mass spectrometry-based proteomic analysis. Methods Allergenic fenugreek proteins were detected by immunoblotting, using sera from 13 patients with specific IgE to peanut and fenugreek. IgE-binding proteins were analyzed by peptide mass fingerprinting and peptide sequencing. Results A fenugreek protein quintet in the range from 50 kDa to 66 kDa showed high IgE-affinity, the protein at 50 kDa reaching the strongest signals in all patients. Proteomic analyses allowed the classification of several fenugreek proteins to a number of allergen families. Fenugreek 7S-vicilin and 11S-legumin were partly sequenced and revealed considerable homologies to peanut Ara h 1 and Ara h 3, respectively. The presence of a fenugreek 2S albumin and pathogenesis-related (PR-10) plant pollen protein was assumed by database searching results. Conclusion In this study, individual fenugreek proteins were characterised for the first time. Observed homologies to major peanut allergens provide a molecular explanation for clinical cross-reactivity.

Celery is acknowledged as a major food allergen in Europe, and mandatory labeling for preprocessed foods has been implemented. However, no methods for the specific detection of celery protein in foods have been published. In the present study, a sandwich celery ELISA using polyclonal anticelery antibodies for capture and detection was developed and validated. The method has an LOD of 0.5 mg/kg in buffer; however, it is applicable only for the screening of food products because of extensive cross-reactivity with potato and carrot proteins. Using nanoLCion-trap MS/MS, a number of proteins in the three vegetable species were identified as candidates for causing cross-reactions due to amino acid sequence homologies. Among others, a novel patatin (Sola t 1)-like protein was detected in celery and a flavin adenine dinucleotide binding domain-containing protein (Api g 5)-like protein was identified in carrot. The utility of triple-quadrupole MS/MS for specific and quantitative analysis of celery, potato, and carrot allergens was evaluated using whole protein extracts. Several unique precursor ion-to-product ion transitions were determined for each species, suggesting the feasibility of developing an MS-based screening method to specifically detect celery allergens in foods.

Patients with peanut allergy are often serologically sensitised to other legumes. If primary peanut allergy was contracted, secondary allergies to other legume plants may develop. The cross-sensitisation implies an enhanced risk of food-related anaphylaxis for the patients even if its clinical relevance is under discussion. In the present study, 32 patients with anti-peanut immunoglobulin E-levels (IgE) of at least class 1 according to the ImmunoCap™ classification system were included. The patients had been referred to the Norwegian National Register and Reporting System for Severe Allergic Reactions to Food with histories of anaphylaxis by the consumption of legume-containing foods. The patient sera were analysed for IgE that reacted to peanut (Arachis hypogea), soybean (Glycine max), pea (Pisum sativum), lupin (Lupinus albus) and fenugreek (Trigonella foenum graecum). Immunoblot experiments were performed using extracts of in total eight different legume plants. IgE measurements and blot analyses revealed differentiated patterns of legume sensitisation. One third of the patients had anti-peanut IgE class 4 to 6. In contrast, just one patient had specific IgE higher than class 4 to respectively pea, soybean and fenugreek. However, ten patients had elevated anti-fenugreek IgE (≥class 3), significantly more than for the other legumes. The second highest levels were found for soybean, followed by pea and lupin. The immunoblots showed particularly strong IgE binding to proteins at 12, 22, 30, 36 and 50 kDa for all legumes, indicating that the major peanut allergens Ara h 1, Ara h 2 and Ara h 3 are strong candidates for causing cross-sensitivity.

Penitrem A is a potent neurotoxin produced by several species in the genus Penicillium, which primarily affects the central nervous system. The toxin has several effects on neurotransmitter release, both at the central and peripheral level, as well as on ion channels. We have evaluated the hepatic metabolism of penitrem A by rat hepatocytes and rat-liver microsomes in vitro. In addition, we have conducted an in vivo study in mice and determined metabolites in several organs. According to our results, penitrem A is extensively metabolized in the liver to at least five metabolites more hydrophilic than the parent compound.

Background Fenugreek is an ingredient in Indian-style spiced foods. Reports of adverse reactions reflect a trend toward a more international cuisine. Fenugreek allergy has not been systematically investigated so far. Objective Study the allergenicity and antigenicity of fenugreek proteins using patient sera and a newly developed polyclonal antifenugreek antibody. Methods Allergenic fenugreek proteins were identified by immunoblotting, using sera from 29 patients with specific IgE to peanut and other legumes. In addition, 2 patients were evaluated by skin prick test and open food challenge with native fenugreek powder. Spiced and flavored food products were analyzed for fenugreek by semiquantitative IgE and IgG immunoblotting. Results High levels of specific IgE to both peanut and fenugreek were seen in most sera. Fenugreek sensitization is believed to be a consequence of cross-reactivity in patients with peanut allergy. Primary fenugreek allergy was suspected in only 1 case. The fenugreek dose eliciting objective symptoms was about 2 mg in the open food challenge. Major fenugreek allergens were identified at 50, 52, and 74 kd and peanut proteins at 22, 36, and 40 kd. A specific polyclonal antifenugreek antibody was found suitable for food analysis. In a food survey, about 1/3 of the fenugreek-containing products were labeled correctly. Conclusion Fenugreek seed powder, an ingredient in spiced foods, contains several potential allergens. There is evidence for a high rate of cross-reactivity to peanut.