Effect of storage and cooking on the fatty acid profile of omega-3 enriched eggs and pork meat marketed in Belgium.

The fatty acids (FA) profile was determined in n-3 enriched (Columbus™) Belgian eggs and pork in order to evaluate to what extent the n-3 fatty acids, which are very sensitive to oxidation, are resistant to storage or cooking. In standard eggs or pork, no change of the fatty acid profile was observed after storage or cooking without culinary fat, as well as in Columbus™ eggs and pork after storage. Some cooking processes (eggs in custard and meat in oven) induced a slight significant loss of n-3 fatty acids in Columbus™ eggs or pork (11.1% in fat from eggs cooked in custard vs. 15.3% in raw Columbus™ eggs and 11.0% in fat from oven cooked meat vs. 11.6% in raw Columbus™ meat). As expected, when Columbus™ pork is cooked with culinary fat, its fatty acid profile is modified according to the nature of the fat used.

A review of dietary and non-dietary exposure to bisphenol-A.

Due to the large number of applications of bisphenol-A (BPA), the human exposure routes are multiple. We aimed to review shortly the food and non-food sources of BPA, and to evaluate their contribution to the human exposure. Food sources discussed here include epoxy resins, polycarbonate and other applications, such as paperboard and polyvinylchloride materials. Among the non-food sources, exposures through dust, thermal paper, dental materials, and medical devices were summarized. Based on the available data for these exposure sources, it was concluded that the exposure to BPA from non-food sources is generally lower than that from exposure from food by at least one order of magnitude for most studied subgroups. The use of urinary concentrations from biomonitoring studies was evaluated and the back-calculation of BPA intake seems reliable for the overall exposure assessment. In general, the total exposure to BPA is several orders of magnitude lower than the current tolerable daily intake of 50 µg/kg bw/day. Finally, the paper concludes with some critical remarks and recommendations on future human exposure studies to BPA.

Validation of a two-plate microbiological method for screening antibiotic residues in shrimp tissue.

Microbiological inhibition screening tests could play an important role to detect residues of antibiotics in the different animal food products, but very few are available for the aquaculture products in general, and for shrimps in particular. A two-plate microbiological method to screen shrimp for residues of the most commonly used antibiotics has been developed and validated according to criteria derived from the European Commission Decision 2002/657/CE. Bacillus subtilis was used as a sensitive strain to target antibiotics. Culture conditions on Petri plates (pH of medium) were selected to enhance the capacity of antibiotic detection. Antibiotic residues were extracted from shrimps using acetonitrile/acetone (70/30, v/v) before application on Petri plates seeded with B. subtilis. The method was validated using spiked blank tissues as well as antibiotic treated shrimps with enrofloxacin and tetracycline, two antibiotics often found to be used in shrimp production. For tetracyclines and (fluoro)quinolones, the detection capability was below the maximum residue limit (MRL), while it was around the MRL for sulfonamides. The specificity of the microbiological screening was 100% in all cases while the sensitivity and accuracy was 100% in almost all cases. The capacity of the method to detect contaminated samples was confirmed on antibiotic treated shrimps, analyzed in parallel with a confirmatory method (Liquid Chromatography coupled to mass spectrometry (LC-MS)).

LC-MS/MS multi-analyte method for mycotoxin determination in food supplements.

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in food supplements is presented. The analytes included A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin), aflatoxins (aflatoxin-B(1), aflatoxin-B(2), aflatoxin-G(1) and aflatoxin-G(2)), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B(1), fumonisin-B(2) and fumonisin-B(3)), ochratoxin A, zearalenone, beauvericin and sterigmatocystin. Optimization of the simultaneous extraction of these toxins and the sample pretreatment procedure, as well as method validation were performed on maca (Lepidium meyenii) food supplements. The results indicated that the solvent mixture ethyl acetate/formic acid (95:5, v/v) was the best compromise for the extraction of the analytes from food supplements. Liquid-liquid partition with n-hexane was applied as partial clean-up step to remove excess of co-extracted non-polar components. Further clean-up was performed on Oasis HLB cartridges. Samples were analysed using an Acquity UPLC system coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer equipped with an electrospray interface operated in the positive-ion mode. Limits of detection and quantification were in the range of 0.3-30 ng g(-1) and 1-100 ng g(-1), respectively. Recovery yields were above 60% for most of the analytes, except for nivalenol, sterigmatocystine and the fumonisins. The method showed good precision and trueness. Analysis of different food supplements such as soy (Glycine max) isoflavones, St John's wort (Hypericum perforatum), garlic (Allium sativum), Ginkgo biloba, and black radish (Raphanus niger) demonstrated the general applicability of the method. Due to different matrix effects observed in different food supplement samples, the standard addition approach was applied to perform correct quantitative analysis. In 56 out of 62 samples analysed, none of the 23 mycotoxins investigated was detected. Positive samples contained at least one of the toxins fumonisin-B(1), fumonisin-B(2), fumonisin-B(3) and ochratoxin A.

Quantitative methods for food allergens: a review.

The quantitative detection of allergens in the food chain is a strategic health objective as the prevalence of allergy continues to rise. Food allergenicity is caused by proteins either in their native form or in forms resulting from food processing. Progress in mass spectrometry greatly opened up the field of proteomics. These advances are now available for the detection and the quantification of traces of allergenic proteins in complex mixtures, and complete the set of biological tests used until now, such as ELISA or PCR. We review methods classified according to their ability to simultaneously quantify and identify allergenic proteins and underline major advances in the mass-spectrometric methods.

Food flavonoid aryl hydrocarbon receptor-mediated agonistic/antagonistic/synergic activities in human and rat reporter gene assays.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor mediating the adverse effects of dioxins and polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the genetic-, time-, dose-, species- and tissue-dependent AhR-mediated agonistic/antagonistic activities of three food flavonoids: quercetin, chrysin and genistein. To that end, four stably transfected cell lines were used in cell-based luciferase reporter gene assays: three lines were transformed with the ptKLuc vector harbouring four dioxin-responsive elements (DREs) upstream of the thymidine kinase promoter and the luciferase gene (HepG2-Luc, T-47D-Luc and H4IIE-ULg). The fourth is a patented cell line transformed with a different construct: H4IIE DR-CALUX((R)). Both H4IIE cells were compared for their genetic construction. Human hepatoma (HepG2-Luc) and human breast tumour (T-47D-Luc) cells were compared for tissue-dependent effects. Rat hepatoma (H4IIE-ULg) and human hepatoma (HepG2-Luc) cells were compared for species-dependent activities. We concluded that quercetin, chrysin and genistein act in a time-, dose-, species- and tissue-specific way. For example, genistein displayed agonistic activities when exposed to rat hepatoma cells during 6h but not after 24h. Flavonoids displayed agonistic/antagonistic activities in human breast tumour cells, depending on the exposure time, while in human hepatoma cells, only antagonistic activities of flavonoids were measured. In addition, we report, in all the cells, a synergy between an isoflavone and two food contaminants; the 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene, a PAH. In rat cells, this synergy occurred when cells were exposed to flavonoids and contaminant for 6h, while it was observed in human cells only after 24h.

Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements using high performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector.

High performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector (HPLC/UV-FLD) has been used to set up a method to detect the 15(+1) EU priority polycyclic aromatic hydrocarbons (PAHs) in food supplements covering the categories of dried plants and plant extracts excluding oily products. A mini validation was performed and the following parameters have been determined: limit of detection, limit of quantification, precision, recovery and linearity. They were in close agreement with quality criteria described in the Commission Regulation (EC) No 333/2007 concerning the PAH benzo[a]pyrene in foodstuffs, except the not fluorescent cyclopenta[c,d]pyrene for which the UV detection leads to a higher limit of detection. Analysis of twenty commercial food supplements covering mainly the class of dried plants was performed to evaluate their PAHs contamination levels and to test the applicability of the method to various plant matrices. Fifty percent of analyzed samples showed concentration exceeding 2 microgkg(-1) for one or more PAHs.

Solubilisation and binding characteristics of a recombinant beta2-adrenergic receptor expressed in the membrane of Escherichia coli for the multianalyte detection of beta-agonists and antagonists residues in food-producing animals.

The number of substances with beta-agonistic activity, illegally introduced in meat production or in sports doping as anabolic or beta-blocking agents is increasing. Analytical methods suited for their multianalyte detection are thus necessary. In this perspective, receptor assays were developed. The research activities undertaken in this study describe the solubilisation of a recombinant human beta(2)-adrenergic receptor produced in the inner membrane of genetically modified Escherichia coli, using the detergent n-dodecyl-beta-d-maltoside. Its potential to detect the presence of beta-agonists or beta-blockers in biological samples was evaluated. The solubilised beta(2)-adrenergic receptor retained its binding affinity in a radio-receptor assay based on the competition for the binding to receptors between a ligand (beta-agonist or antagonist) and the radioligand [(125)I]iodocyanopindolol. The IC(50) values ranged from 5+/-1 x 10(-8) M (clenbuterol) to 8+/-2 x 10(-6) M (isoxsuprine) for the beta-agonists tested and from 1.5+/-0.2 x 10(-10) M (carazolol) to 1.2+/-0.2 x 10(-5) M (metoprolol) for the beta-blockers tested. It was shown to have a lower limit of detection than a radio-receptor assay using the solubilised beta(2)-adrenoceptor expressed in a mammalian cell line. The solubilised recombinant human beta(2)-adrenoreceptor expressed in E. coli would be a useful tool to develop non radioactive multianalyte screening methods.

Comprehensive two-dimensional gas chromatography with isotope dilution time-of-flight mass spectrometry for the measurement of dioxins and polychlorinated biphenyls in foodstuffs. Comparison with other methods.

A comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC x GC-TOF-MS) experimental setup was tested for the measurement of seven 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs), ten 2,3,7,8-substituted polychlorinated dibenzofurans (PCDFs), four non-ortho-polychlorinated biphenyls (PCBs), eight mono-ortho-PCBs, and six indicator PCBs (Aroclor 1260) in foodstuff samples. A 40m RTX-500 (0.18mm I.D., 0.10 microm df) was used as the first dimension (1D) and a 1.5 m BPX-50 (0.10mm I.D., 0.10 microm df) as the second dimension (2D). The GC x GC chromatographic separation was completed in 45 min. Quantification was performed using 13C-label isotope dilution (ID). Isotope ratios of the selected quantification ions were checked against theoretical values prior to peak assignment and quantification. The dynamic working range spanned three orders of magnitude. The lowest detectable amount of 2,3,7,8-TCDD was 0.2 pg. Fish, pork, and milk samples were considered. On a congener basis, the GC x GC-ID-TOF-MS method was compared to the reference GC-ID high resolution mass spectrometry (HRMS) method and to the alternative GC-ID tandem-in-time quadrupole ion storage mass spectrometry (QIST-MS/MS). PCB levels ranged from low picogram (pg) to low nanogram (ng) per gram of sample and data compared very well between the different methods. For all matrices, PCDD/Fs were at a low pg level (0.05-3 pg) on a fresh weight basis. Although congener profiles were accurately described, RSDs of GC x GC-ID-TOF-MS and GC-QIST-MS/MS were much higher than for GC-ID-HRMS, especially for low level pork and milk. On a toxic equivalent (TEQ) basis, all methods, including the dioxin-responsive chemically activated luciferase gene expression (DR-CALUX) assay, produced similar responses. A cost comparison is also presented.

Enhancement of steroid receptor-mediated transcription for the development of highly responsive bioassays.

We have previously generated several transformed human mammary cell lines for the detection of steroid receptor-mediated activities and used these cell lines to detect and characterize steroid hormone (ant)agonistic compounds. In this report, we describe the specific optimization procedures used to enhance receptor-mediated transcription through the human glucocorticoid, progesterone and androgen receptors, respectively. Sodium arsenite-induced chemical stress leads to a substantial and specific increase in the glucocorticoid receptor-mediated transcription, resulting in maximal stimulations of more than 2000-fold by the agonist dexamethasone. Similarly, a combined treatment with forskolin (an activator of adenylate cyclase) and trichostatin A (an inhibitor of histone deacetylases) leads to a synergistic enhancement of progesterone or androgen stimulation, resulting in a maximal induction of more than 200-fold or about 100-fold, respectively. The enhanced responses to specific steroids are mediated by the corresponding nuclear receptor. We show that by using these enhanced transcriptional stimulation protocols, it is possible to detect lower amounts of steroid hormones without substantially affecting the relative biological activities of various agonists. Finally, the application of these enhanced reporter cell assays to real biological samples from meat-producing animals is evaluated, and some validation parameters are presented.

DR-CALUX((R)) screening of food samples: evaluation of the quantitative approach to measure dioxin, furans and dioxin-like PCBs.

European legislation laid down maximum tolerable levels of dioxin in feed and food as well as analytical method requirements. In order to face with large monitoring programs, it was foreseen in the EU strategy to integrate screening methods, using either a qualitative (screening) approach, or a quantitative approach. In this study, dioxin results obtained using the Dioxin Responsive Chemical-Activated LUciferase gene eXpression (DR-CALUX((R))) cell-based assay (quantitative approach), were compared with gas chromatography-high resolution mass spectrometry (GC-HRMS) analyses data. Instead of using World Health Organization-toxic equivalent factor (WHO-TEF), the comparison was based on the assessment of relative effective potencies (REPs) for each congener of the 17 toxic 2,3,7,8-polychlorodibenzo-p-dioxins/furans (PCDD/Fs) and 12 dioxin-like polychlorobiphenyls (DL-PCBs). According to published data, DR-CALUX((R))-REP evaluated here appear similar to WHO-TEF for PCDD/Fs while lower values were observed for DL-PCBs. We analyzed two "home made" contaminated fat samples, displaying both the same WHO-toxic equivalent quantities (WHO-TEQ) concentration (12pgWHO-TEQg(-1)). They were spiked with either a low or a high amount of DL-PCBs. In both cases, the DR-CALUX((R)) measured concentration (picogram 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) eq.g(-1)) corresponded to the PCDD/Fs WHO-TEQ concentration only. A good agreement was nevertheless found between the DR-CALUX((R)) measurements and the recalculated DR-CALUX((R))-TEQ contents (using DR-CALUX((R))-REP instead of WHO-TEF), demonstrating that the observed response was due, in both cases, to the addition of the responses of the standards added to the fat. By contrast, in real contaminated samples (feed or cod liver samples), DR-CALUX((R)) measured concentrations were similar to WHO-TEQ GC-HRMS measured concentrations. But, depending on the PCDD/Fs and DL-PCBs congener content, the DR-CALUX((R)) measured concentrations were either lower or higher than calculated DR-CALUX((R))-TEQ contents, demonstrating that possible co-extracted contaminants contributed to the CALUX response. Owing to these divergences, the quantitative determination of dioxin-like content in food and feed using CALUX as screening method is questionable, except for samples displaying constant congener patterns, in which cases, correction factors could be applied.

Use of reporter cell lines for detection of endocrine-disrupter activity.

We have studied stable transformed human mammary cell lines with highly inducible steroid receptor-mediated luciferase reporter gene expression. Cells responding specifically to glucocorticoids, progestagens, androgens, or estrogens are described and characterized. The use of this high-throughput, cell-based assay for analysis of steroid (ant)agonists is reported. Systematic characterization of endocrine-disrupting activity on human receptors and in a human-cell system is interpreted for a selection of xenobiotics. We show that the phytoestrogens apigenin and genistin have progestagenic and androgenic activity, respectively. Finally, application of cell-based assays to the analysis of environmental samples is discussed.

Recombinant human estrogen, androgen and progesterone receptors for detection of potential endocrine disruptors.

This work reports the binding capacity of various chemicals (so-called endocrine disruptors) to recombinant human steroid receptors (hERalpha, hPR and hAR). The tested chemicals are organochlorine insecticides (DDT and its metabolites, methoxychlor, aldrin, dieldrin, chlordecone, lindane, trichlorobenzene), estrogenic insecticides (endosulfan, toxaphene, nonachlor), herbicides (alachlor and atrazine), fungicides (benomyl and vinclozolin), industrial chemicals (nonylphenol, bisphenol A, diphenylphtalate), antioxidants (butylated hydroxyanisol) and some phytoestrogens. Except for phytoestrogens, most of the tested chemicals (DDT and its metabolites, aldrin, alpha- and beta-endosulfan, toxaphen, trans-nonachlor) show higher affinities for hPR than for hERalpha, indicating that the interaction with the progesterone receptor could contribute to the endocrine-disrupting effects imputed to these chemicals. We propose to use binding assays using recombinant human steroid receptors as screening tools for the detection of endocrine disruptors in various samples.

Multiresidue determination of (fluoro)quinolone antibiotics in swine kidney using liquid chromatography-tandem mass spectrometry.

New antibiotics were recently developed, among which are the (fluoro)quinolones. This paper presents an analytical method which allows the determination of 11 (fluoro)quinolones in swine kidneys: norfloxacin, ofloxacin, cinoxacin, oxolinic acid, nalidixic acid, flumequine, enrofloxacin, enoxacin, ciprofloxacin, danofloxacin and marbofloxacin. The procedure involves a rapid and efficient pre-treatment by solid-phase extraction (recoveries 83-98%), followed by the sensitive and selective determination of all compounds in a single run using LC-ESI-MS-MS. Multiple reaction monitoring (MRM) was used for selective detection of each (fluoro)quinolone. Quinine was selected as internal standard. The accuracy of the method, expressed as recovery, was between 89 and 109%; the repeatability had a maximum RSD lower than 15%. The limits of detection (LOD) were much lower than the respective Maximum Residue Limits (MRL)/4.

Adaptation to multiday ozone exposure is associated with a sustained increase of bronchoalveolar uric acid.

The phenomenon of ozone tolerance is described, but the underlying mechanisms remain unknown. We tested whether adaptation to multiday ozone exposure was related to an upregulated pulmonary antioxidant defence. Six calves were exposed to 0.75 ppm ozone, 12 h day(-1) for seven consecutive days. Pulmonary function tests and bronchoalveolar lavage (BAL) were performed before, after the first (D1), third (D3) and seventh (D7) exposure. Differential cell count, total proteins, 8-epi-PGF2alpha, glutathione and uric acid were determined in BAL. Dynamic lung compliance and arterial oxygen tension were significantly decreased and lung oedema impaired pulmonary function on D1. By repeating ozone exposures, progressive functional adaptation occurred. Ozone induced a significant increase of BAL neutrophil percentage on D1. On D3 and D7, neutrophil percentage was progressively decreased, but remained significantly elevated. BAL total proteins were significantly increased on D1 and decreased progressively until D7. 8-Epi-PGF2alpha was significantly increased on D1 and was returned to baseline on D3 and D7, whilst glutathione significantly increased on D3 and returned to baseline on D7. Uric acid was increased ten-fold on D1. On D3, uric acid was increased six-fold and was persistently elevated at D7. This study suggests that ozone adaptation of functional and inflammatory variables is accompanied with sustained BAL uric acid elevation.