Characterization of estrogen and androgen activity of food contact materials by different in vitro bioassays (YES, YAS, ERα and AR CALUX) and chromatographic analysis (GC-MS, HPLC-MS).

Endocrine active substances (EAS) show structural similarities to natural hormones and are suspected to affect the human endocrine system by inducing hormone dependent effects. Recent studies with in vitro tests suggest that EAS can leach from packaging into food and may therefore pose a risk to human health. Sample migrates from food contact materials were tested for estrogen and androgen agonists and antagonists with different commonly used in vitro tests. Additionally, chemical trace analysis by GC-MS and HPLC-MS was used to identify potential hormone active substances in sample migrates. A GC-MS method to screen migrates for 29 known or potential endocrine active substances was established and validated. Samples were migrated according to EC 10/2011, concentrated by solid phase extraction and tested with estrogen and androgen responsive reporter gene assays based on yeast cells (YES and YAS) or human osteoblast cells (ERα and AR CALUX). A high level of agreement between the different bioassays could be observed by screening for estrogen agonists. Four out of 18 samples tested showed an estrogen activity in a similar range in both, YES and ERα CALUX. Two more samples tested positive in ERα CALUX due to the lower limits of detection in this assay. Androgen agonists could not be detected in any of the tested samples, neither with YAS nor with AR CALUX. When testing for antagonists, significant differences between yeast and human cell-based bioassays were noticed. Using YES and YAS many samples showed a strong antagonistic activity which was not observed using human cell-based CALUX assays. By GC-MS, some known or supposed EAS were identified in sample migrates that showed a biological activity in the in vitro tests. However, no firm conclusions about the sources of the observed hormone activity could be obtained from the chemical results.

Conformationally constrained nicotines: polycyclic, bridged, and spiro-annulated analogues as novel ligands for the nicotinic acetylcholine receptor.

A set of novel nicotine-related, conformationally constrained compounds, including tetracyclic, bridged (4), and tricyclic, spiro-annulated (5) structures, were synthesized in a straightforward manner and optically resolved in a convenient fashion with (+)- and (-)-O,O'-di-p-toluoyltartaric acids. Absolute configurations were determined by X-ray crystallography. These compounds were evaluated for their ability to displace [(3)H]cytisine in a rat forebrain preparation and compared to (-)-nicotine. Three substances emerged with high affinity in the low nanomolar range. Moreover, one of these compounds ((+)-5b) showed not only high binding affinity (K(i) = 4.79 nM) but also significant enantioselectivity over its antipode (K(i) = 148 nM), supporting the hypothesis that conformational restraint can lead to high-affinity ligands, which are stereochemically discriminated by the nicotinic acetylcholine receptor and may feature optimum locations of the active sites of the pharmacophore.