Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay

Prohormones such as dehydroepiandrosterone (DHEA) are steroid precursors that do not show hormonal activity by themselves. Abuse of these prohormones in cattle fattening is hard to prove because of strong in vivo metabolism and the difficulty to detect metabolites which are not significantly above endogenous levels. The aim of the present work was to develop an in vitro assay capable of detecting the indirect hormonal activity of prohormones that might be present in feed supplements and injection preparations. Sample extracts were incubated with a bovine liver S9 fraction in order to mimic the in vivo metabolic activation. Subsequently incubated extracts were exposed to a highly androgen-specific yeast bioassay to detect hormonal activity. Metabolic activation of DHEA, 4-androstene-3,17-dione (4-adione) and 5-androstene-3,17-diol (5-adiol) resulted in an increased androgenic activity caused by the formation of the active androgen 17ß-testosterone (17ß-T), as shown by ultra-performance liquid chromatography and time-of-flight mass spectrometry with accurate mass measurement. The developed in vitro system successfully mimics the hydroxysteroid dehydrogenase (HSD)- and cytochrome P450-mediated in vivo metabolic transitions, thus allowing assessment of both bioactivity and chemical identification without the use of animal experiments. Screening of unknown supplement samples claimed to contain DHEA resulted in successful bioactivation and positive screening results according to the androgen yeast biosenso

In silico prioritization of endocrine active substances (EAS) and their in vitro validation

In silico molecular docking can be a cheap and fast strategy to estimate the binding free energies, and consequently the dissociation constants, for a set of compounds with respect to their putative targets. Interesting targets for EAS are the ligand binding domains (LBD) of the human nuclear receptors for the sex hormones, i.e. the estrogen, androgen, progesterone, and (gluco)corticoid receptor. The Horizon 2020 project EuroMix (http://euromixproject.eu) aims to establish and disseminate new, efficient and validated strategies for the risk assessment of mixtures, while limiting the use of test animals. The present presentation deals with a part of EuroMix that is intended to set up a testing approach for mixtures of endocrine disrupting chemicals, focusing on estrogenic and anti-androgenic effects. For that purpose, a combined Adverse Outcome Pathway (AOP) was constructed, including Molecular Initiating Events, Key Events, and Adverse Outcome (reproductive dysfunction). Using this combined AOP as framework, cognate in silico and in vitro tools as well as the in vivo confirmation studies were selected, i.e. in silico: h-ER and h-AR docking; in vitro: cell-based ER and AR transcriptional activation bioassays and the H295R steroidogenesis assay; and in vivo: the Fish Sexual Development Test (FSDT, OECD Test No. 234) and a rat study, examining in (male) offspring a number of parameters, such as anogenital distance, cryptorchidism, and nipple retention. Reference chemicals were selected and in silico and in vitro testing was started, showing that when testing single compounds, there is a very good correlation between the in silico determined binding energies and the in vitro measured hormonal activities

A 155-plex High-Throughput In Vitro Coregulator Binding Assay for (Anti-) Estrogenicity Testing Evaluated with 23 Reference Compounds

To further develop an integrated in vitro testing strategy for replacement of in vivo tests for (anti-)estrogenicity testing, the ligand-modulated interaction of coregulators with estrogen receptor a was assessed using a PamChip® plate. The relative estrogenic potencies determined, based on ERa binding to coregulator peptides in the presence of ligands on the PamChip® plate, were compared to the relative estrogenic potencies as determined in the in vivo uterotrophic assay. The results show that the estrogenic potencies predicted by the 57 coactivators on the peptide microarray for 18 compounds that display a clear E2 dose-dependent response (goodness of fit of a logistic dose-response model of 0.90 or higher) correlated very well with their in vivo potencies in the uterotrophic assay, i.e., coefficient of determination values for 30 coactivators higher than or equal to 0.85. Moreover, this coregulator binding assay is able to distinguish ER agonists from ER antagonists: profiles of selective estrogen receptor modulators, such as tamoxifen, were distinct from those of pure ER agonists, such as dienestrol. Combination of this coregulator binding assay with other types of in vitro assays, e.g., reporter gene assays and the H295R steroidogenesis assay, will frame an in vitro test panel for screening and prioritization of chemicals, thereby contributing to the reduction and ultimately the replacement of animal testing for (anti-)estrogenic effects

Sub-chronic toxicity study in rats orally exposed to nanostructured silica

Synthetic Amorphous Silica (SAS) is commonly used in food and drugs. Recently, a consumer intake of silica from food was estimated at 9.4 mg/kg bw/day, of which 1.8 mg/kg bw/day was estimated to be in the nano-size range. Food products containing SAS have been shown to contain silica in the nanometer size range (i.e. 5 – 200 nm) up to 43% of the total silica content. Concerns have been raised about the possible adverse effects of chronic exposure to nanostructured silica

Prediction of the potential allergenicity of novel proteins

The potential allergenicity of novel proteins expressed in genetically modified organisms is an important item in the safety assessment of these organisms. The various components of the allergenicity assessment include the source of the protein, sequence similarity with known allergenic proteins, in vitro digestibility, sera binding tests, animal models and clinical tests. Recent developments in the field of bioinformatics studies on the sequence similarity of novel proteins with allergenic proteins, as well as in vitro digestibility, are discussed

Presence of potential allergy-related linear epitopes in novel proteins from conventional crops and the implication for the safety assessment of these crops with respect to the current testing of genetically modified crops

Mitochondria of cytoplasmic male sterile crop plants contain novel, chimeric open reading frames. In addition, a number of crops carry endogenous double-stranded ribonucleic acid (dsRNA). In this study, the novel proteins encoded by these genetic components were screened for the presence of potential binding sites (epitopes) of allergy-associated IgE antibodies, as was previously done with transgenic proteins from genetically modified crops. The procedure entails the identification of stretches of at least six contiguous amino acids that are shared by novel proteins and known allergenic proteins. These stretches are further checked for potential linear IgE-binding epitopes. Of the 16 novel protein sequences screened in this study, nine contained stretches of six or seven amino acids that were also present in allergenic proteins. Four cases of similarity are of special interest, given the predicted antigenicity of the identical stretch within the allergenic and novel protein, the IgE-binding by a peptide containing an identical stretch reported in literature, or the multiple incidence of identical stretches of the same allergen within a novel protein. These selected stretches are present in novel proteins derived from oilseed rape and radish (ORF138), rice (dsRNA), and fava bean (dsRNA), and warrant further clinical testing. The frequency of positive outcomes and the sizes of the identical stretches were comparable to those previously found for transgenic proteins in genetically modified crops. It is discussed whether novel proteins from conventional crops should be subject to an assessment of potential allergenicity, a procedure which is currently mandatory for transgenic proteins from genetically modified crops