Deliverable 9.1 - Report on mixtures and implementation strategy in Europe – Assessment of chemical mixtures under consideration of current and future regulatory requirements and scientific approaches

This report gives an overview on the regulatory processes and requirements for risk assessment of chemical mixtures, identifies gaps in the European legislation and summarises potential approaches for the health risk assessment of chemical mixtures

Diverse roles of androgen receptor (AR) domains in AR-mediated signaling

Androgens control male sexual development and maintenance of the adult male phenotype. They have very divergent effects on their target organs like the reproductive organs, muscle, bone, brain and skin. This is explained in part by the fact that different cell types respond differently to androgen stimulus, even when all these responses are mediated by the same intracellular androgen receptor. To understand these tissue- and cell-specific readouts of androgens, we have to learn the many different steps in the transcription activation mechanisms of the androgen receptor (NR3C4). Like all nuclear receptors, the steroid receptors have a central DNA-binding domain connected to a ligand-binding domain by a hinge region. In addition, all steroid receptors have a relatively large amino-terminal domain. Despite the overall structural homology with other nuclear receptors, the androgen receptor has several specific characteristics which will be discussed here. This receptor can bind two types of androgen response elements (AREs): one type being similar to the classical GRE/PRE-type elements, the other type being the more divergent and more selective AREs. The hormone-binding domain has low intrinsic transactivation properties, a feature that correlates with the low affinity of this domain for the canonical LxxLL-bearing coactivators. For the androgen receptor, transcriptional activation involves the alternative recruitment of coactivators to different regions in the amino-terminal domain, as well as the hinge region. Finally, a very strong ligand-induced interaction between the amino-terminal domain and the ligand-binding domain of the androgen receptor seems to be involved in many aspects of its function as a transcription factor. This review describes the current knowledge on the structure-function relationships within the domains of the androgen receptor and tries to integrate the involvement of different domains, subdomains and motifs in the functioning of this receptor as a transcription factor with tissue- and cell-specific readouts

Overview on legislation and scientific approaches for risk assessment of combined exposure to multiple chemicals: the potential EuroMix contribution

This article reviews the current legislative requirements for risk assessment of combined exposure to multiple chemicals via multiple exposure routes, focusing on human health and particularly on food-related chemicals. The aim is to identify regulatory needs and current approaches for this type of risk assessment as well as challenges of the implementation of appropriate and harmonized guidance at international level. It provides an overview of the current legal requirements in the European Union (EU), the United States and Canada. Substantial differences were identified in the legal requirements for risk assessment of combined exposure to multiple chemicals and its implementation between EU and non-EU countries and across several regulatory sectors. Frameworks currently proposed and in use for assessing risks from combined exposure to multiple chemicals via multiple routes and different durations of exposure are summarized. In order to avoid significant discrepancies between regulatory sectors or countries, the approach for assessing risks of combined exposure should be based on similar principles for all types of chemicals. OECD and EFSA identified the development of harmonized methodologies for combined exposure to multiple chemicals as a key priority area. The Horizon 2020 project “EuroMix” aims to contribute to the further development of internationally harmonized approaches for such risk assessments by the development of an integrated test strategy using in vitro and in silico tests verified for chemical mixtures based on more appropriate data on potential combined effects. These approaches and testing strategies should be integrated in a scientifically based weight of evidence approach to account for complexity and uncertainty, to improve risk assessment

Preliminary molecular genetic analysis of the Receptor Interacting Protein 140 (RIP140) in women affected by endometriosis

BACKGROUND: Endometriosis is a complex disease affecting 10–15% of women at reproductive age. Very few genes are known to be altered in this pathology. RIP140 protein is an important cofactor of oestrogen receptor and many other nuclear receptors. Targeting disruption experiments of nrip1 gene in mice have demonstrated that nuclear receptor interacting protein 1 gene (nrip1), the gene encoding for rip140 protein, is essential for female fertility. Specifically, mice null for nrip1 gene are viable, but females are infertile because of complete failure of mature follicles to release oocytes at ovulation stage. The ovarian phenotype observed in mice devoid of rip140 closely resembles the luteinized unruptured follicle (LUF) syndrome that is observed in a high proportion of women affected of endometriosis or idiopathic infertility. Here we present a preliminary work that analyses the role of NRIP1 gene in humans. METHODS: We have sequenced the complete coding region of NRIP1 gene in 20 unrelated patients affected by endometriosis. We have performed genetic association studies by using the DNA variants identified during the sequencing process. RESULTS: We identified six DNA variants within the coding sequence of NRIP1 gene, and five of them generated amino acid changes in the protein. We observed that three of twenty sequenced patients have specific combinations of amino-acid variants within the RIP140 protein that are poorly represented in the control population (p = 0.006). Moreover, we found that Arg448Gly, a common polymorphism located within NRIP1 gene, is associated with endometriosis in a case-control study (59 cases and 141 controls, p(allele positivity test )= 0.027). CONCLUSION: Our results suggest that NRIP1 gene variants, separately or in combinations, might act as predisposing factors for human endometriosis

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue

<p>Abstract</p> <p>Background</p> <p>The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. Based on a phylogenetic analysis of DNA-binding domains, we define two conserved groups of orthologous NR2E genes: the NR2E1 subclass, which includes <it>C. elegans nhr-67, Drosophila tailless </it>and <it>dissatisfaction</it>, and vertebrate Tlx (NR2E2, NR2E4, NR2E1), and the NR2E3 subclass, which includes <it>C. elegans fax-1 </it>and vertebrate PNR (NR2E5, NR2E3). PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members.</p> <p>Results</p> <p>We show that NR2E3 subclass member FAX-1, unlike NHR-67 and other NR2E1 subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We use site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 of the DNA-binding domain. This amino acid position is part of the "P box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. The relaxed specificity allows FAX-1 to bind a much larger repertoire of half-sites than NHR-67. While NR2E1 class proteins bind both monomeric and dimeric sites, the NR2E3 class proteins bind only dimeric sites. The presence of a single strong site adjacent to a very weak site allows dimeric FAX-1 binding, further increasing the number of dimeric binding sites to which FAX-1 may bind <it>in vivo</it>.</p> <p>Conclusion</p> <p>These findings identify subclass-specific DNA-binding specificities and dimerization properties for the NR2E1 and NR2E3 subclasses. For the NR2E1 protein NHR-67, Asp-19 permits binding to AAGTCA half-sites, while Asn-19 permits binding to AGGTCA half-sites. The apparent conservation of DNA-binding properties between vertebrate and nematode NR2E receptors allows for the possibility of evolutionarily-conserved regulatory patterns.</p

PKCε Stimulated Arginine Methylation of RIP140 for Its Nuclear-Cytoplasmic Export in Adipocyte Differentiation

Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that plays roles in diverse metabolic processes including fat accumulation in adipocytes. Previously we identified three methylated arginine residues in RIP140, which rendered its export to the cytoplasm; but it was unclear what triggered RIP140 arginine methylation.In this study, we determined the activated PKCepsilon as the specific trigger for RIP140 arginine methylation and its subsequent export. We identified two PKCepsilon-phosphorylated residues of RIP140, Ser-102 and Ser-1003, which synergistically stimulated direct binding of RIP140 by 14-3-3 that recruited protein arginine methyl transferase 1 to methylate RIP140. The methylated RIP140 then preferentially recruited exportin 1 for nuclear export. As a result, the nuclear gene-repressive activity of RIP140 was reduced. In RIP140 null adipocyte cultures, the defect in fat accumulation was effectively rescued by the phosphorylation-deficient mutant RIP140 that resided predominantly in the nucleus, but less so by the phospho-mimetic RIP140 that was exported to the cytoplasm.This study uncovers a novel means, via a cascade of protein modifications, to inactivate, or suppress, the nuclear action of an important transcription coregulator RIP140, and delineates the first specific phosphorylation-arginine methylation cascade that could alter protein subcellular distribution and biological activity