Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities

Identification of nuclear receptor-mediated endocrine activities is important in a variety of fields, ranging from pharmacological and clinical screening, to food and feed safety, toxicological monitoring, and risk assessment. Traditionally animal studies such as the Hershberger and Allen-Doisy tests are used for the assessment of androgenic and estrogenic potencies, respectively. To allow fast analysis of the activities of new chemicals, food additives, and pharmaceutical compounds, high-throughput screening strategies have been developed. Here, a panel of mainly steroidal compounds, screened in different in vitro assays, was compared with two human U2-OS cell line-based CALUX® (Chemically Activated LUciferase eXpression) reporter gene assays for androgens (AR CALUX) and estrogens (ER CALUX). Correlations found between the data of these two CALUX reporter gene assays and data obtained with other in vitro screening assays measuring receptor binding or reporter gene activation (CHO cell line-based) were good (correlation coefficients (

Acute toxicity of CCl4 but not of paracetamol induces a transcriptomic signature of fibrosis in precision-cut liver slices

In rat in vivo, both paracetamol (APAP) and carbon tetrachloride (CCl4) induce liver necrosis, but long-term treatment with CCl4, in contrast to paracetamol, causes liver fibrosis. The aim of this study was to perform transcriptomic analysis to compare the early changes in mRNA expression profiles induced by APAP and CCl4 in the rat precision-cut liver slice model (PCLS) and to identify early markers that could predict fibrosis-inducing potential. Microarray data of rat PCLS exposed to APAP andCCl4was generated using a toxic dose based on decrease in ATP levels. Toxicity pathway analysis using a custom made fibrosis-related gene list showed fibrosis as one of the predominant toxic endpoints in CCl4-treated, but not in APAP-treated PCLS. Moreover, genes which have a role in fibrosis such as alpha-B crystallin, jun proto-oncogene, mitogen-activated protein kinase 6, serpin peptidase inhibitor and also the transcription factor Kruppel-like-factor-6 were up-regulated by CCl4, but not by APAP. Predicted activation or inhibition of several upstream regulators due to CCl4 is in accordance with their role in fibrosis. In conclusion, transcriptomic analysis of PCLS successfully identified the fibrotic potential of CCl4 as opposed to APAP. The application of PCLS as ex vivo model to identify early biomarkers to predict the fibrogenic potential of toxic compounds should be further explored

Application of the comparison approach to open TG-GATEs : A useful toxicogenomics tool for detecting modes of action in chemical risk assessment

Mode of action information is one of the key components for chemical risk assessment as mechanistic insight leads to better understanding of potential adverse health effects of a chemical. This insight greatly facilitates assessment of human relevance and enhances the use of non-animal methods for risk assessment, as it ultimately enables extrapolation from initiating events to adverse effects. Recently, we reported an in vitro toxicogenomics comparison approach to categorize (non-)genotoxic carcinogens according to similarities in their proposed modes of action. The present study aimed to make this comparison approach generally applicable, allowing comparison of outcomes across different studies. The resulting further developed comparison approach was evaluated through application to toxicogenomics data on 18 liver toxicants in human and rat primary hepatocytes from the Open TG-GATEs database. The results showed sensible matches between compounds with (partial) overlap in mode of action, whilst matches for compounds with different modes of action were absent. Comparison of the results across species revealed pronounced and relevant differences between primary rat and human hepatocytes, underpinning that information on mode of action enhances assessment of human relevance. Thus, we demonstrate that the comparison approach now is generally applicable, facilitating its use as tool in mechanism-based risk assessment

Application of the comparison approach to open TG-GATEs: A useful toxicogenomics tool for detecting modes of action in chemical risk assessment.

Mode of action information is one of the key components for chemical risk assessment as mechanistic insight leads to better understanding of potential adverse health effects of a chemical. This insight greatly facilitates assessment of human relevance and enhances the use of non-animal methods for risk assessment, as it ultimately enables extrapolation from initiating events to adverse effects. Recently, we reported an in vitro toxicogenomics comparison approach to categorize (non-)genotoxic carcinogens according to similarities in their proposed modes of action. The present study aimed to make this comparison approach generally applicable, allowing comparison of outcomes across different studies. The resulting further developed comparison approach was evaluated through application to toxicogenomics data on 18 liver toxicants in human and rat primary hepatocytes from the Open TG-GATEs database. The results showed sensible matches between compounds with (partial) overlap in mode of action, whilst matches for compounds with different modes of action were absent. Comparison of the results across species revealed pronounced and relevant differences between primary rat and human hepatocytes, underpinning that information on mode of action enhances assessment of human relevance. Thus, we demonstrate that the comparison approach now is generally applicable, facilitating its use as tool in mechanism-based risk assessment

Distinguishing mode of action of compounds inducing craniofacial malformations in zebrafish embryos to support dose-response modeling in combined exposures.

Knowledge on mode-of-action (MOA) is required to understand toxicological effects of compounds, notably in the context of risk assessment of mixtures. Such information is generally scarce, and often complicated by the existence of multiple MOAs per compound. Here, MOAs related to developmental craniofacial malformations were derived from literature, and assembled in a MOA network. A selection of gene expression markers was based on these MOAs. Next, these markers were verified by qPCR in zebrafish embryos, after exposure to reference compounds. These were: triazoles for inhibition of retinoic acid (RA) metabolism, AM580 and CD3254 for selective activation of respectively RA-receptor (RAR) and retinoid-X-receptor (RXR), dithiocarbamates for inhibition of lysyl oxidase, TCDD for activation of the aryl-hydrocarbon-receptor (AhR), VPA for inhibition of histone deacetylase (HDAC), and PFOS for activation of peroxisome proliferator-activated receptor-alpha (PPAR\u3b1). Next, marker gene profiles for these reference compounds were used to map the profiles of test compounds to known MOAs. In this way, 2,4-dinitrophenol matched with the TCDD and RAR profiles, boric acid with RAR, endosulfan with PFOS, fenpropimorph with dithiocarbamates, PCB126 with AhR, and RA with triazoles and RAR profiles. Prochloraz showed no match. Activities of these compounds in ToxCast assays, and in silico analysis of binding affinity to the respective targets showed limited concordance with the marker gene expression profiles, but still confirmed the complex MOA profiles of reference and test compounds. Ultimately, this approach could be used to support modeling of mixture effects based on upfront knowledge of (dis)similarity of MOA

Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index

Contains fulltext : 103595.pdf (publisher's version ) (Open Access)Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects

Structure and predicted binding mode of Org 214007-0.

<p>A) The structure of ORG 214007-0. This compound, [(-)-N-(2S,10S,14bS)]-N-(8-cyano-1,2,3,4,10,14b-hexahydro-10-methyl dibenzo<i>[c,f]</i>pyrido[1,2-<i>a</i>]azepin-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide] has a molecular weight of 430 g/mole (C₂₄H₂₃N₅OS) B) The predicted binding mode of Org 214007-0 modeled in complex with the glucocorticoid receptor and demonstrating conservation of interactions typical to steroidal glucocorticoids (Gln564, Asn570, Arg611 and Gln642).</p

Org 214007-0 behaves as a partial agonist in vitro.

<p>A) In a co-factor recruitment assay, Org 214007-0, in comparison to prednisolone, shows potent but partial recruitment of a 0.1 μM peptide presenting TIF2 -3. On the Y-axis average fluorescence counts (+/− SD) are shown. EC50 values (%CV) and percentages maximal efficacy (%CV) for Org 214007-0 versus prednisolone were 10 (7.4) nM versus 48 (3.2) nM and 67 (7.1) % vs 100% respectively. B) In THP1 cells Org 214007-0 shows partial induction of FKBP51 protein expression and C) under inflammatory conditions represses the IL-6 protein expression almost as good as prednisolone does.</p