Dietary exposure to elements from the German pilot total diet study (TDS).

Dietary exposure of the German adult population to the elements aluminium, copper, mercury (and as methylmercury), manganese and lead were assessed using data from the first total diet study (TDS) in Germany. In this pilot TDS, performed 2014-2015, 246 food samples were purchased in the Berlin area, prepared 'as consumed', and subsequently analysed. Dietary exposure for the German adult population between 14 and 80 years of age was estimated by combining TDS data with individual consumption data from the German National Consumption Survey II (NVS II). Estimated mean and high-level dietary exposure values showed that none of the elements analysed exceeded toxicological reference values; neither was there an undersupply of essential elements. Assessments for methylmercury and lead in women of child-bearing age, in particular, showed no considerable elevated intake levels.

Dietary Manganese Exposure in the Adult Population in Germany-What Does it Mean in Relation to Health Risks?

Manganese is both an essential nutrient and a potential neurotoxicant. Therefore, the question arises whether the dietary manganese intake in the German population is on the low or high side. Results from a pilot total diet study in Germany presented here reveal that the average dietary manganese intake in the general population in Germany aged 14-80 years is about 2.8 mg day-1 for a person of 70 kg body weight. This exposure level is within the intake range of 2-5 mg per person and day as recommended by the societies for nutrition in Germany, Austria, and Switzerland. No information on the dietary exposure of children in Germany can be provided so far. Although reliable information on health effects related to oral manganese exposure is limited, there is no indication from the literature that these dietary intake levels are associated with adverse health effects either by manganese deficiency or excess. However, there is limited evidence that manganese taken up as a highly bioavailable bolus, for example, uptake via drinking water or food supplements, could pose a potential risk to human health-particularly in certain subpopulations-when certain intake amounts, which are currently not well defined, are exceeded.

Structure and target interaction of a G-quadruplex RNA-aptamer.

G-quadruplexes have recently moved into focus of research in nucleic acids, thereby evolving in scientific significance from exceptional secondary structure motifs to complex modulators of gene regulation. Aptamers (nucleic acid based ligands with recognition properties for a specific target) that form Gquadruplexes may have particular potential for therapeutic applications as they combine the characteristics of specific targeting and Gquadruplex mediated stability and regulation. We have investigated the structure and target interaction properties of one such aptamer: AIR-3 and its truncated form AIR-3A. These RNA aptamers are specific for human interleukin-6 receptor (hIL-6R), a key player in inflammatory diseases and cancer, and have recently been exploited for in vitro drug delivery studies. With the aim to resolve the RNA structure, global shape, RNA:protein interaction site and binding stoichiometry, we now investigated AIR-3 and AIR-3A by different methods including RNA structure probing, Small Angle X-ray scattering and microscale thermophoresis. Our findings suggest a broader spectrum of folding species than assumed so far and remarkable tolerance toward different modifications. Mass spectrometry based binding site analysis, supported by molecular modeling and docking studies propose a general Gquadruplex affinity for the target molecule hIL-6R.

Development of harmonised food and sample lists for total diet studies in five European countries.

A total diet study (TDS) is a public health tool for determination of population dietary exposure to chemicals across the entire diet. TDSs have been performed in several countries but the comparability of data produced is limited. Harmonisation of the TDS methodology is therefore desirable and the development of comparable TDS food lists is considered essential to achieve the consistency between countries. The aim of this study is to develop and test the feasibility of a method for establishing harmonised TDS food and sample lists in five European countries with different consumption patterns (Czech Republic, Finland, Germany, Iceland and Portugal). The food lists were intended to be applicable for exposure assessment of wide range of chemical substances in adults (18-64 years) and the elderly (65-74 years). Food consumption data from recent dietary surveys measured on individuals served as the basis for this work. Since the national data from these five countries were not comparable, all foods were linked to the EFSA FoodEx2 classification and description system. The selection of foods for TDS was based on the weight of food consumed and was carried out separately for each FoodEx2 level 1 food group. Individual food approach was respected as much as possible when the TDS samples were defined. TDS food lists developed with this approach represented 94.7-98.7% of the national total diet weights. The overall number of TDS samples varied from 128 in Finland to 246 in Germany. The suggested method was successfully implemented in all five countries. Mapping of data to the EFSA FoodEx2 coding system was recognised as a crucial step in harmonisation of the developed TDS food lists.

Selection and Characterization of an α6ß4 Integrin blocking DNA Aptamer.

The heterodimeric laminin receptor α6ß4 integrin plays a central role in the promotion of tumor cell growth, invasion, and organotropic metastasis. As an overproduction of the integrin is often linked to a poor prognosis, the inhibition of integrin α6ß4 binding to laminin is of high therapeutical interest. Here, we report on the combination of a cell-systematic evolution of ligands by exponential enrichment and a bead-based selection resulting in the first aptamer inhibiting the interaction between α6ß4 integrin and laminin-332. This Integrin α6ß4-specific DNA Aptamer (IDA) inhibits the adhesion of prostate cancer cells (PC-3) to laminin-332 with an IC50 value of 149 nmol/l. The Kd value concerning the aptamer's interaction with PC-3 cells amounts to 137 nmol/l. Further characterization showed specificity to α6 integrins and a half-life in murine blood plasma of 6 hours. Two truncated versions of the aptamer retained their binding capacity, but lost their ability to inhibit the interaction between laminin-332 and PC-3 cells. Confocal laser scanning microscope studies revealed that the aptamer was internalized into PC-3-cells. Therefore, in addition to the adhesion-blocking function of this aptamer, IDA could also be applied for the delivery of siRNA, microRNA or toxins to cancer cells presenting the integrin α6ß4.

SELEX of Cell-Specific RNA Aptamers.

This chapter focuses on the selection of RNA aptamers, which bind to specific cell surface components and thus can be internalized receptor mediated. Such aptamers discriminate between different tissues, e.g., detect malignant cells, and target them or induce apoptosis through drug internalization. However, before starting the selection process the choice of an ideal target can be challenging. To give an example for the selection of cell specific aptamers, we here used the interleukin-6 receptor (IL-6R) as a target, which is presented on hepatocytes, neutrophils, monocytes, and macrophages.

RAID3--An interleukin-6 receptor-binding aptamer with post-selective modification-resistant affinity.

Aptamers are an emerging class of highly specific targeting ligands. They can be selected in vitro for a large variety of targets, ranging from small molecules to whole cells. Most aptamers selected are nucleic acid-based, allowing chemical synthesis and easy modification. Although their properties make them interesting drug candidates for a broad spectrum of applications and an interesting alternative to antibodies or fusion proteins, they are not yet broadly used. One major drawback of aptamers is their susceptibility to abundant serum nucleases, resulting in their fast degradation in biological fluids. Using modified nucleic acids has become a common strategy to overcome these disadvantages, greatly increasing their half-life under cell culture conditions or even in vivo. Whereas pre-selective modifications of the initial library for aptamer selection are relatively easy to obtain, post-selective modifications of already selected aptamers are still generally very labor-intensive and often compromise the aptamers ability to bind its target molecule. Here we report the selection, characterization and post-selective modification of a 34 nucleotide (nt) RNA aptamer for a non-dominant, novel target site (domain 3) of the interleukin-6 receptor (IL-6R). We performed structural analyses and investigated the affinity of the aptamer to the membrane-bound and soluble forms (sIL-6R) of the IL-6R. Further, we performed structural analyses of the aptamer in solution using small-angle X-ray scattering and determined its overall shape and oligomeric state. Post-selective exchange of all pyrimidines against their 2'-fluoro analogs increased the aptamers stability significantly without compromising its affinity for the target protein. The resulting modified aptamer could be shortened to its minimal binding motif without loss of affinity.

Stabilized Interleukin-6 receptor binding RNA aptamers.

Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in the progression of various inflammatory diseases, such as rheumatoid arthritis and certain cancers; for example, multiple myeloma or hepatocellular carcinoma. To interfere with IL-6-dependent diseases, targeting IL-6 receptor (IL-6R)-presenting tumor cells using aptamers might be a valuable strategy to broaden established IL-6- or IL-6R-directed treatment regimens. Recently, we reported on the in vitro selection of RNA aptamers binding to the human IL-6 receptor (IL-6R) with nanomolar affinity. One aptamer, namely AIR-3A, was 19 nt in size and able to deliver bulky cargos into IL-6R-presenting cells. As AIR-3A is a natural RNA molecule, its use for in vivo applications might be limited due to its susceptibility to ubiquitous ribonucleases. Aiming at more robust RNA aptamers targeting IL-6R, we now report on the generation of stabilized RNA aptamers for potential in vivo applications. The new 2'-F-modified RNA aptamers bind to IL-6R via its extracellular portion with low nanomolar affinity comparable to the previously identified unmodified counterpart. Aptamers do not interfere with the IL-6 receptor complex formation. The work described here represents one further step to potentially apply stabilized IL-6R-binding RNA aptamers in IL-6R-connected diseases, like multiple myeloma and hepatocellular carcinoma.

German database on the occurrence of food additives: application for intake estimation of five food colours for toddlers and children.

To get a more realistic estimation of food additive intake for toddlers and children, a German database on the occurrence of food additives was created. It uses consumption data of two recent national nutrition surveys for toddlers and children in combination with qualitative information of food additive occurrence in the consumed food. The information on food additive occurrence is based on food labelling. A categorisation system was developed according to regulations to classify the foods consumed and to identify possible food additive use in the food groups. Two natural (E120, E160b) and three artificial food colours (E110, E124, E129) were chosen for an assessment of food additive intake. The percentage of food items containing one of the chosen food colours was calculated for every food group and the food groups with most items containing the additive were identified. Intake estimations were performed based on maximum permitted-use levels (MPLs). Firstly, additive use was assumed in all foods consumed (tier 2); and secondly, food additive use was assumed only for those items where labelling confirmed the use and for all foods with no labelling available (tier 2b). Intake estimations were then compared with the ADI. Most food items with at least one of the food colours were found in the food groups confectionary, desserts, fermented milk products, flavoured drinks and breakfast cereals. The tier 2b approach provided more realistic estimations, which were always below those of the tier 2 approach and below the ADI for mean exposure. Exposure for high-level consumers exceeded the ADI for two of the food additives in tier 2b. Keeping in mind that the database is only mirroring the current situation, it provides a good possibility to refine the estimation of food additive intake for toddlers and children in Germany.

Toxicology and risk assessment of acrolein in food.

Acrolein is an α,ß-unsaturated aldehyde formed by thermal treatment of animal and vegetable fats, carbohydrates and amino acids. In addition it is generated endogenously. As an electrophile, acrolein forms adducts with gluthathione and other cellular components and is therefore cytotoxic. Mutagenicity was shown in some in vitro tests. Acrolein forms different DNA adducts in vivo, but mutagenic and cancerogenous effects have not been demonstrated for oral exposure. In subchronic oral studies, local lesions were detected in the stomach of rats. Systemic effects have not been reported from basic studies. A WHO working group established a tolerable oral acrolein intake of 7.5 µg/kg body weight/day. Acrolein exposure via food cannot be assessed due to analytical difficulties and the lack of reliable content measurements. Human biomonitoring of an acrolein urinary metabolite allows rough estimates of acrolein exposure in the range of a few µg/kg body weight/day. High exposure could be ten times higher after the consumption of certain foods. Although the estimation of the dietary acrolein exposure is associated with uncertainties, it is concluded that a health risk seems to be unlikely.

Toxicology and risk assessment of 5-Hydroxymethylfurfural in food.

5-Hydroxymethylfurfural (5-HMF) as a product of the Maillard reaction is found in many foods. Estimated intakes range between 4 and 30 mg per person and day, while an intake of up to 350 mg can result from, e.g., beverages made from dried plums. In vitro genotoxicity was positive when the metabolic preconditions for the formation of the reactive metabolite 5-sulphoxymethylfurfural were met. However, so far in vivo genotoxicity was negative. Results obtained in short-term model studies for 5-HMF on the induction of neoplastic changes in the intestinal tract were negative or cannot be reliably interpreted as "carcinogenic". In the only long-term carcinogenicity study in rats and mice no tumours or their precursory stages were induced by 5-HMF aside from liver adenomas in female mice, the relevance of which must be viewed as doubtful. Hence, no relevance for humans concerning carcinogenic and genotoxic effects can be derived. The remaining toxic potential is rather low. Various animal experiments reveal that no adverse effect levels are in the range of 80-100 mg/kg body weight and day. Safety margins are generally sufficient. However, 5-HMF exposure resulting from caramel colours used as food additives should be further evaluated.