Ad A. C. M. Peijnenburg

Distribution, elimination, and toxicity of silver nanoparticles and silver ions in rats after 28-day oral exposure.

We report the results of a 28-day oral exposure study in rats, exposed to <20 nm noncoated, or <15 nm PVP-coated silver nanoparticles ([Ag] = 90 mg/kg body weight (bw)), or AgNO(3) ([Ag] = 9 mg/kg bw), or carrier solution only. Dissection was performed at day 29, and after a wash-out period of 1 or 8 weeks. Silver was present in all examined organs with the highest levels in the liver and spleen for all silver treatments. Silver concentrations in the organs were highly correlated to the amount of Ag(+) in the silver nanoparticle suspension, indicating that mainly Ag(+), and to a much lesser extent silver nanoparticles, passed the intestines in the silver nanoparticle exposed rats. In all groups silver was cleared from most organs after 8 weeks postdosing, but remarkably not from the brain and testis. Using single particle inductively coupled plasma mass spectrometry, silver nanoparticles were detected in silver nanoparticle exposed rats, but, remarkably also in AgNO(3) exposed rats, hereby demonstrating the formation of nanoparticles from Ag(+)in vivo that are probably composed of silver salts. Biochemical markers and antibody levels in blood, lymphocyte proliferation and cytokine release, and NK-cell activity did not reveal hepatotoxicity or immunotoxicity of the silver exposure. In conclusion, oral exposure to silver nanoparticles appears to be very similar to exposure to silver salts. However, the consequences of in vivo formation of silver nanoparticles, and of the long retention of silver in brain and testis should be considered in a risk assessment of silver nanoparticles.

Presence of Nano-Sized Silica during In Vitro Digestion of Foods Containing Silica as a Food Additive

The presence, dissolution, agglomeration state, and release of materials in the nano-size range from food containing engineered nanoparticles during human digestion is a key question for the safety assessment of these materials. We used an in vitro model to mimic the human digestion. Food products subjected to in vitro digestion included (i) hot water, (ii) coffee with powdered creamer, (iii) instant soup, and (iv) pancake which either contained silica as the food additive E551, or to which a form of synthetic amorphous silica or 32 nm SiO(2) particles were added. The results showed that, in the mouth stage of the digestion, nano-sized silica particles with a size range of 5-50 and 50-500 nm were present in food products containing E551 or added synthetic amorphous silica. However, during the successive gastric digestion stage, this nano-sized silica was no longer present for the food matrices coffee and instant soup, while low amounts were found for pancakes. Additional experiments showed that the absence of nano-sized silica in the gastric stage can be contributed to an effect of low pH combined with high electrolyte concentrations in the gastric digestion stage. Large silica agglomerates are formed under these conditions as determined by DLS and SEM experiments and explained theoretically by the extended DLVO theory. Importantly, in the subsequent intestinal digestion stage, the nano-sized silica particles reappeared again, even in amounts higher than in the saliva (mouth) digestion stage. These findings suggest that, upon consumption of foods containing E551, the gut epithelium is most likely exposed to nano-sized silica.

Characterization of Translocation of Silver Nanoparticles and Effects on Whole-Genome Gene Expression Using an In Vitro Intestinal Epithelium Coculture Model

Applications of nanoparticles in the food sector are eminent. Silver nanoparticles are among the most frequently used, making consumer exposure to silver nanoparticles inevitable. Information about uptake through the intestines and possible toxic effects of silver nanoparticles is therefore very important but still lacking. In the present study, we used an in vitro model for the human intestinal epithelium consisting of Caco-2 and M-cells to study the passage of silver nanoparticles and their ionic equivalents and to assess their effects on whole-genome mRNA expression. This in vitro intestine model was exposed to four sizes of silver nanoparticles for 4 h. Exposure to silver ions was included as a control since 6-17% of the silver nanoparticles were found to be dissociated into silver ions. The amount of silver ions that passed the Caco-2 cell barrier was equal for the silver ion and nanoparticle exposures. The nanoparticles induced clear changes in gene expression in a range of stress responses including oxidative stress, endoplasmatic stress response, and apoptosis. The gene expression response to silver nanoparticles, however, was very similar to that of AgNO(3). Therefore, the observed effects of the silver nanoparticles are likely exerted by the silver ions that are released from the nanoparticles.

Allermatch™, a webtool for the prediction of potential allergenicity according to current FAO/WHO Codex alimentarius guidelines

BackgroundNovel proteins entering the food chain, for example by genetic modification of plants, have to be tested for allergenicity. Allermatch™ http://allermatch.org is a webtool for the efficient and standardized prediction of potential allergenicity of proteins and peptides according to the current recommendations of the FAO/WHO Expert Consultation, as outlined in the Codex alimentarius.DescriptionA query amino acid sequence is compared with all known allergenic proteins retrieved from the protein databases using a sliding window approach. This identifies stretches of 80 amino acids with more than 35% similarity or small identical stretches of at least six amino acids. The outcome of the analysis is presented in a concise format. The predictive performance of the FAO/WHO criteria is evaluated by screening sets of allergens and non-allergens against the Allermatch databases. Besides correct predictions, both methods are shown to generate false positive and false negative hits and the outcomes should therefore be combined with other methods of allergenicity assessment, as advised by the FAO/WHO.ConclusionsAllermatch™ provides an accessible, efficient, and useful webtool for analysis of potential allergenicity of proteins introduced in genetically modified food prior to market release that complies with current FAO/WHO guidelines.

Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities.

Recently we constructed yeast cells that either express the human estrogen receptor α or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3′-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.

An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

BackgroundIn vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively.ResultsThe study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.ConclusionsUntargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.

Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE – binding linear epitopes of allergens

BackgroundTransgenic proteins expressed by genetically modified food crops are evaluated for their potential allergenic properties prior to marketing, among others by identification of short identical amino acid sequences that occur both in the transgenic protein and allergenic proteins. A strategy is proposed, in which the positive outcomes of the sequence comparison with a minimal length of six amino acids are further screened for the presence of potential linear IgE-epitopes. This double track approach involves the use of literature data on IgE-epitopes and an antigenicity prediction algorithm.ResultsThirty-three transgenic proteins have been screened for identities of at least six contiguous amino acids shared with allergenic proteins. Twenty-two transgenic proteins showed positive results of six- or seven-contiguous amino acids length. Only a limited number of identical stretches shared by transgenic proteins (papaya ringspot virus coat protein, acetolactate synthase GH50, and glyphosate oxidoreductase) and allergenic proteins could be identified as (part of) potential linear epitopes.ConclusionMany transgenic proteins have identical stretches of six or seven amino acids in common with allergenic proteins. Most identical stretches are likely to be false positives. As shown in this study, identical stretches can be further screened for relevance by comparison with linear IgE-binding epitopes described in literature. In the absence of literature data on epitopes, antigenicity prediction by computer aids to select potential antibody binding sites that will need verification of IgE binding by sera binding tests. Finally, the positive outcomes of this approach warrant further clinical testing for potential allergenicity.

Adequacy of methods for testing the safety of genetically modified foods

1315 into GM food technology, including study of its potential effects on health. These responses reflect an appropriately cautious approach towards the science of genetic modification. They reflect the real concern expressed by both “singleinterest groups” and a wider public. These anxieties may seem odd, even irrational, given that GM foods were introduced in the USA without any sign of consumer anxiety. Why? Because Europe now lives in a post-BSE (bovine spongiform encephalopathy) age, one in which society has learned that the epidemic of BSE was brought on by unchecked industry-driven changes in farming practices and that the denials of risk by government and scientific authorities were worthless. That concern is now spreading beyond the UK. Recognition of this deeply ingrained public scepticism about food technology has led Monsanto to rethink its entire GM-food strategy. The comments by Lachmann, Sykes, and Gosden are therefore disappointing because they reflect a failure to understand the new, and apparently unwelcome, dialogue of accountability that needs to be forged between scientists and the public. Risks are not simply questions of abstract probabilities or theoretical reassurances. What matters is what people believe about these risks and why they hold those beliefs. Ewen and Pusztai’s data are preliminary and non-generalisable, but at least they are now out in the open for debate, as are the results, also published in today’s Lancet, of Brian Fenton and colleagues. Only by welcoming that debate will the standard of public conversation about science be raised. Berating critics rather than engaging them—and criticising reports of research, as the Royal Society did with the Pusztai data,before those data were reviewed and published in the proper way—will only intensify public scepticism about science and scientists.

Subacute effects of hexabromocyclododecane (HBCD) on hepatic gene expression profiles in rats.

Hexabromoyclododecane (HBCD), used as flame retardant (FR) mainly in textile industry and in polystyrene foam manufacture, has been identified as a contaminant at levels comparable to other brominated FRs (BFRs). HBCD levels in biota are increasing slowly and seem to reflect the local market demand. The toxicological database of HBCD is too limited to perform at present a solid risk assessment, combining data from exposure and effect studies. In order to fill in some gaps, a 28-day HBCD repeated dose study (OECD407) was done in Wistar rats. In the present work liver tissues from these animals were used for gene expression profile analysis. Results show clear gender specificity with females having a higher number of regulated genes and therefore being more sensitive to HBCD than males. Several specific pathways were found to be affected by HBCD exposure, like PPAR-mediated regulation of lipid metabolism, triacylglycerol metabolism, cholesterol biosynthesis, and phase I and II pathways. These results were corroborated with quantitative RT-PCR analysis. Cholesterol biosynthesis and lipid metabolism were especially down-regulated in females. Genes involved in phase I and II metabolism were up-regulated predominantly in males, which could explain the observed lower HBCD hepatic disposition in male rats in this 28-day study. These sex-specific differences in gene expression profiles could also underlie sex-specific differences in toxicity (e.g. decreased thyroid hormone or increased serum cholesterol levels). To our knowledge, this is the fist study that describes the changes in rat hepatic gene profiles caused by this commonly used flame retardant.

Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights.

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5μM DON for 3, 6 and 24h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4μM DON for 6 and 24h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs.