Citrinin Determination in Food and Food Supplements by LC-MS/MS: Development and Use of Reference Materials in an International Collaborative Study.

The development of incurred reference materials containing citrinin (CIT) and their successful application in a method validation study (MVS) in order to harmonize CIT determination in food and food supplements are demonstrated. CIT-contaminated materials made of red yeast rice (RYR), wheat flour, and Ginkgo biloba leaves (GBL), as well as food supplements made of red yeast rice (FS-RYR) and Ginkgo biloba leaves (FS-GBL), were manufactured in-house via fungal cultivation on collected raw materials. The homogeneity and stability from randomly selected containers were verified according to the ISO 13528. CIT was found to be homogenously distributed and stable in all contaminated materials, with no significant degradation during the timescale of the MVS when storage was performed up to +4 °C. Next, an MVS was organized with eighteen international laboratories using the provided standard operating procedure and 12 test materials, including three RYRs (blank, <50 µg/kg, <2000 µg/kg), two wheat flours (blank, <50 µg/kg), two GBL powders (blank, <50 µg/kg), three FS-RYRs (blank, <50 µg/kg, <2000 µg/kg), and two FS-GBLs (blank, <50 µg/kg). The results of seven CIT-incurred materials showed acceptable within-laboratory precision (RSDr) varying from 6.4% to 14.6% and between-laboratory precision (RSDR) varying from 10.2% to 37.3%. Evidenced by HorRat values < 2.0, the results of the collaborative trial demonstrated that the applied analytical method could be standardized. Furthermore, the appropriateness of producing CIT reference materials is an important step towards food and feed quality control systems and the organization of proficiency tests.

Endocrine activity of mycotoxins and mycotoxin mixtures.

Reporter gene assays incorporating nuclear receptors (estrogen, androgen, thyroid ß and PPARγ2) have been implemented to assess the endocrine activity of 13 mycotoxins and their mixtures. As expected, zearalenone and its metabolites α-zearalenol and ß- zearalenol turned out to have the strongest estrogenic potency (EC50 8,7 10-10 ± 0,8; 3,1 10-11 ± 0,5 and 1,3 10-8 ± 0,3 M respectively). The metabolite of deoxynivalenol, 3-acetyl-deoxynivalenol also had estrogenic activity (EC50 3,8 10-7 ± 1,1 M). Furthermore, most of the mycotoxins (and their mixtures) showed anti-androgenic effects (15-acetyldeoxynivalenol, 3-acetyl-deoxynivalenol and α-zearalenol with potencies within one order of magnitude of that of the reference compound flutamide). In particular, deoxynivalenol and 15-acetyl-deoxynivalenol acted as antagonists for the PPARy2 receptor. When testing mixtures of mycotoxins on the same cell systems, we showed that most of the mixtures reacted as predicted by the concentration addition (CA) theory. Generally, the CA was within the 95% confidence interval of the observed ones, only minor deviations were detected. Although these reporter gene tests cannot be directly extrapolated in vivo, they can be the basis for further research. Especially the additive effects of ZEN and its metabolites are of importance and could have repercussions in vivo.

Evaluation of a screening system for obesogenic compounds: screening of endocrine disrupting compounds and evaluation of the PPAR dependency of the effect.

Recently the environmental obesogen hypothesis has been formulated, proposing a role for endocrine disrupting compounds (EDCs) in the development of obesity. To evaluate this hypothesis, a screening system for obesogenic compounds is urgently needed. In this study, we suggest a standardised protocol for obesogen screening based on the 3T3-L1 cell line, a well-characterised adipogenesis model, and direct fluorescent measurement using Nile red lipid staining technique. In a first phase, we characterised the assay using the acknowledged obesogens rosiglitazone and tributyltin. Based on the obtained dose-response curves for these model compounds, a lipid accumulation threshold value was calculated to ensure the biological relevance and reliability of statistically significant effects. This threshold based method was combined with the well described strictly standardized mean difference (SSMD) method for classification of non-, weak- or strong obesogenic compounds. In the next step, a range of EDCs, used in personal and household care products (parabens, musks, phthalates and alkylphenol compounds), were tested to further evaluate the obesogenicity screening assay for its discriminative power and sensitivity. Additionally, the peroxisome proliferator activated receptor γ (PPARγ) dependency of the positive compounds was evaluated using PPARγ activation and antagonist experiments. Our results showed the adipogenic potential of all tested parabens, several musks and phthalate compounds and bisphenol A (BPA). PPARγ activation was associated with adipogenesis for parabens, phthalates and BPA, however not required for obesogenic effects induced by Tonalide, indicating the role of other obesogenic mechanisms for this compound.

Non-photochemical quenching kinetics during the dark to light transition in relation to the formation of antheraxanthin and zeaxanthin.

Nonlinear regression analysis (NLR) is applied to quantify the dynamic response of non-photochemical fluorescence quenching (NPQ) of Trifolium repens cv. Regal upon dark to light transition. Commonly, only steady-state levels of NPQ are evaluated, ignoring transient kinetics. Experimental NPQ kinetics are fitted best with a sum of two functions: a sigmoidal Hill function plus a transient logarithmic normal function. It is shown that not only steady-state level of NPQ, but also the speed at which steady state is reached, increased with light intensity. The question is raised which biological processes cause the induction of the components of NPQ kinetics. The NPQ kinetics are found to resemble the kinetics of antheraxanthin and zeaxanthin formation during a dark to light transition. Furthermore, both molecules are known to induce NPQ. The hypothesis is put forward that a transient phase of NPQ (0-2 min after transition) is dependent upon concentrations of antheraxanthin, whereas the saturating phase corresponds with the production of zeaxanthin. A mathematical model, based on the presented hypothesis, predicts the effect of increasing light intensity on concentrations of antheraxanthin and zeaxanthin which correspond with experimental results. Implications of the hypothesis are discussed as well as the role of NLR in evaluating chlorophyll a fluorescence kinetics.